Pathogen inactivation
in cellular blood products by
photodynamic treatment
PROEFSCHRIFT
ter verkrijging van
de graad van Doctor aan de Universiteit Leiden,
op gezag van Rector Magnificus prof.mr. P.F. van der Heijden,
volgens besluit van het College voor Promoties
te verdedigen op woensdag 12 mei 2010
klokke 11:15 uur
door
Laurence Liliane Trannoy
geboren te Soissons (Frankrijk)
in 1973
Promotiecommissie
Promotor:
Prof.dr. A. Brand
Co-promotor:
Dr. J.W.M. Lagerberg (Sanquin Research, Amsterdam)
Overige leden:
Prof.dr. F.H.J. Claas
Prof.dr. F. Koning
Prof.dr. H.J. Zaaijer (Amsterdam Medisch Centrum)
The research described in this thesis was conducted at the Sanquin Blood Bank
Southwest Region, at the department of Immunohematology and Blood
Transfusion and at the department of Molecular and Cellular Biology of the Leiden
University Medical Center, Leiden. The studies were financed by ZonNW (grant
nr. 28-24140).
ISBN: 978-90-9025274-2
Layout and cover design: L. Trannoy
Printed by: Ipskamp Drukkers B.V.
Publication of this thesis was financially supported by Sanquin Blood Bank,
Buchem B.V., Beckman Coulter B.V., and Greiner Bio-One B.V.
Voor mijn liefsten:
Jan, Félix et Laura
3
ABBREVIATIONS
1
O2 = singlet oxygen
AAMI = alternatively activated macrophages
AlPcS4 = Tetrasulfonated aluminum
phthalocyanine
APC = Antigen presenting cells
ATCC = American Type Culture Collection
ATP = Adenosine triphosphate
BFU-E = Burst forming unit of erythrocytes
BM = Bone marrow
BVDV = Bovine viral diarrhoea virus
CB = Cord blood
cb = Cytochalasine B
CBSC = Cord blood stem cells
CFU = Colony forming unit
CFU-GEM = Mixed CFU of granulocytes,
monocytes and erythrocytes
CFU-GM = CFU of granulocytes and
monocytes
CFU-Mk = CFU of megakaryocytes
CPV = Canine parvovirus
DAT = Direct gel agglutination test
DIP = Dipyridamole
DMMB = Dimethyl-methylene blue
G- = gram negative
G+ = gram positive
GSH = reduced glutathione
GvHD = Graft-versus-host disease
Hb = Hemoglobin
Hct = Hematocrit
HBV = Hepatitis B virus
HCV = Hepatitis C virus
HES = Hydroxy-ethyl starch
4
HIV = Human immunodeficiency virus
HLA = Human leukocytes antigens
HPC = Hematopoietic progenitor cells
HTLV = Human T-lymphoma virus
IAGT = Indirect Antiglobulin Test
LPS = Lipopolysaccharide
MB = Methylene blue
MNC = Mononuclear cells
MLR = Mixed lymphocytes reaction
NaP = sodium phosphate buffer
NAT = Nucleic acid test
NHFTR = non-hemolytic febrile transfusion
reactions
PBS = Phosphate-buffered saline
PC = Platelet concentrate
PDT = Photodynamic treatment
PHA = Phytohemagluttin
PPV = Porcine parvovirus
PRV = Pseudorabies virus
RBC = Red blood cells
RBCC = Red blood cell concentrate
ROS = Reactive oxygen species
SD = solvent/detergent
SAG-M = Saline Adenine Glucose Mannitol
T50 = half lifespan
TCID50 = Tissue Culture Infectious Dose
(50%)
Tri-P(4) = mono-phenyl-tri-(N-methyl-4pyridyl)-porphyrin chloride
VSV = Vesicular stomatitis virus
WBC = White blood cells
WNV = West-Nile virus
CONTENTS
Chapter I
Introduction
Chapter II
Relation between K+ leakage and damage to Band 3 in
photodynamically treated red cells.
7
37
Chapter III Selective protection of red blood cells against photodynamic damage by the Band 3 ligand dipyridamole.
53
Chapter IV Positively charged porphyrins: a new series of photosensitizers for pathogen inactivation in red blood cell
concentrates.
69
Chapter V
Differential sensitivities of pathogens in red blood cell
concentrates to Tri-P(4)-photoinactivation.
93
Chapter VI Photodynamic treatment with Tri-P(4) for pathogen
inactivation in cord blood stem cell products.
113
Chapter VII Impact of photodynamic treatment with porphyrin Tri-P(4)
on the immunomodulatory capacity of white blood cellscontaining red blood cell products.
135
Chapter VIII Summarizing discussion
159
Nederlandse samenvatting
171
Résumé en Français
177
Dankwoord - Remerciements
185
Curriculum vitae
187
List of publications
189
5
6
CHAPTER I
INTRODUCTION
1. BLOOD TRANSFUSION SAFETY
Blood product safety is a major priority of blood suppliers. Transmission of viruses
or other infectious agents can have deleterious effects for the transfused patients.
To maximize the safety of blood products, various procedures are initiated
starting as from the moment a person is willing to donate blood.1
RECRUITMENT AND SELECTION OF VOLUNTEERS
Before each blood donation, donors must fill a questionnaire as a control of good
health and faithfulness. The questionnaire is meant to exclude high-risk behavior
individuals, potential sick donors, and travelers to endemic areas for infections.
Donors must be willing to give blood without any kind of remuneration. Almost all
studies have shown that unpaid donors are safer than paid donors.2,3 Indeed, the
latter failed more frequently than non-remunerated donors to mention illness or
lifestyle involving risks.4
SCREENING FOR INFECTION
The presence of antigens or antibodies against Human Immunodeficiency virus
(HIV) 1 and 2, Human T-lymphoma virus (HTLV) 1 and 2, Hepatitis C virus (HCV),
Hepatitis B virus (HBV) and the presence of Treponema pallidum (Syphilis) is
determined in donor blood samples. A major concern in the determination of
antibodies is the window period. This is the period in which the presence of
7
Chapter I
infection is not detectable yet because the amount of either antibodies and/or
antigens is below the detection limit. To shorten the window phase, more
advanced techniques such as nucleic acid test (NAT) are used to detect the
presence of viral genetic material from HIV, HCV5,6, West-Nile Virus (WNV; In
North America)7,8 and HBV (Japan)9.
Furthermore, besides these serological and molecular tests, additional
precautions are taken.
Plasma is kept in quarantine for at least 6 months until the donor, coming back for
a new donation, is tested again for infection.10 If the donor is healthy and testing
for infectious disease markers is negative, the plasma donation is released for
transfusion. Platelet concentrates (PC) are cultured to detect bacterial
contamination.11
For cellular blood products that are destined to high-risk patients like severely
immunocompromised patient, pregnant women, or neonates, detection of not
routinely tested viruses such as Cytomegalovirus (CMV) and human B19 Parvovirus is performed to avoid, sometimes even fatal, transfusion complications.
2. THE PERMANENT THREAT OF PATHOGEN CONTAMINATION
A wide list of pathogens is permanently threatening the blood supply, whereas no
screening test is available or readily available.
New viruses or pathogens are emerging. Since the bird flu virus (H5N1) was
discovered to be able to infect humans12, with fatal outcome in children and
elders, increased attention was necessary to ensure the safety of the blood
supply. Although it is not yet known if this virus can be transmitted through blood
transfusion, travelers in regions where this virus spread out, were subjected to a
temporarily deferral for at least 3 weeks as recommended by WHO.13,14
Another example of an emerging virus is WNV, which emerged in 1999 for the
first time in North America where it became endemic.15 In 2002, the virus was
discovered to be transmitted through blood transfusion.16,17 Since 2003, routine
detection for this virus was implemented for all blood donations in the USA.18,19 In
8
Introduction
UK, the National Blood Service performs a test for this virus in donors who
donates within 28 days of a visit to the USA or Canada. In the Netherlands, such
donors are temporarily suspended for donation during 60 days.
Another potential danger for blood transfusion is prions. Prions are pathogenic
proteins which can lead to Creutzfeldt-Jakob disease and its variant (CJD or
vCJD).20. It is still unclear if prions are transmissible through blood transfusion.21,22
Direct evidences were only obtained in animal experiments.23 In addition, no test
is available to detect contamination of an individual with prions. To counterpart a
potential epidemic, leukodepletion of blood components was implemented, as
prions may be associated to leukocytes24; although this declaration was denied by
other researchers.25 A deferral strategy was introduced to exclude blood donors
who had received blood transfusion before 1980 or who resided in UK for longer
than 6 months.
New viruses are still discovered, thanks to advanced molecular techniques.
SenV26, TTV27, and Hepatitis G virus28 were found to be transmissible through
blood transfusion; however, no disease is associated with these infections.29
HTLV-3 and HTLV-4 were recently discovered in rural Cameroon but it is not
known yet if they can cause diseases.30
People travel more, especially from western countries to developing countries
where some diseases are endemic such as malaria-like diseases caused by
infection with parasites Plasmodium or Babasia and Chagas disease caused by
infection with parasite Trypanosoma cruzi. Infections with these pathogens are
mostly asymptomatic, and blood donors which have journeyed in these regions
are temporarily deferred for donations.31 However, a case of a very long
incubation period before the appearance of detectable anti-Plasmodium
antibodies was reported.32 Moreover, parasites carriers such as mosquitoes can
be transported to other, non endemic-countries by plane, exposing airport
workers to infection risks.33
Besides the efforts made to minimize risks for virus and tropical parasite
transmission, bacterial contamination of blood products remains a challenge.
9
Chapter I
About 17-22% of all reported transfusion-related fatalities in USA were caused by
transfusion of contaminated blood products with bacteria.34 The frequency of
clinical apparent sepsis through transfusion amounts 1 in 15.000 platelet units34
and 1 in 10.4 million red blood cell (RBC) units35. Only PCs are cultured for bacteria
but the standard techniques available to detect the presence of bacteria last
several days. If the post-donation cultured sample is negative, PC can be released
with the mention “negative to date”. However, if the cultured sample appeared to
be positive after that time, hospital employees are informed of the bacterial
contamination and asked to inform about transfusion reactions. In addition, the
red blood cell concentrate (RBCC) originated from the same donor is recalled and
discarded.
However, this strategy cannot completely prevent the transfusion of a
contaminated product and served rather to determine the frequency of bacterial
contamination in related blood products. Other attempts to reduce
contamination by diversion of the first 20 ml of blood withdrawal and
improvement of skin disinfection at time of donation have been introduced. As
the majority of contaminating bacteria were shown to originate from skin flora36,
this new regulation has been shown to reduce the frequency of bacterial
contaminated blood products.37
In summary, stringent selection of donors, deferral strategies and the use
of highly sensitive tests make that blood transfusion has never been so safe. In
industrialized countries, residual risks of infection through blood transfusion
amount lower than 1 in 205.000 for HBV, 1 in 270.000 units for HCV and 1 in 3.1
million units for HIV, and almost inexistent for HTLV and Treponema pallidum.38-40
However, the safety of blood products is not only challenged by the well known
viral infections mentioned above, but also by increased donor travel, patients
health conditions, and the time of infection of the blood donor. Emerging
pathogens raise the concern as tests are either not available or supplementary
tests must be required to ensure a pathogen-free product for high-risk patients.
Infection can develop in an unexpected way (long incubation period). Bacterial
contamination tests last too long. Considering the wide range of pathogens, it
may be unrealistic to implement test for each of them. Increased costs and time
10
Introduction
consuming leading to longer storage of labile blood cells prior to release may have
enormous consequences on the blood supply. Finally, it is clear that, even though
blood transfusions are presently considered to be very safe, continuous efforts
are necessary to keep it safe.
3. UMBILICAL CORD BLOOD: A PARTICULAR BLOOD PRODUCT
Umbilical cord blood cells can be used for hematopoietic stem cell transplantation
and tissue engineering e.g. megakaryocyte and RBC expansion.
Normally, during the gestation, placenta acts as a filter that protects the fetus
from opportunistic infections. That is why cord blood is in principle considered as
free from infections.41 However, this filter does not always work perfectly and
contamination from mother to child occurs.42-47
To ensure the safety of cord blood transplants, routine viral detection is
performed in maternal blood. Tests for the detection of HIV1/2, HCV, HBV,
HTLV1/2, Treponema pallidum are standard. A recall after 6 months helps to
determine if the umbilical cord blood donor (the baby) has not developed any
congenital diseases [source Eurocord]. A major problem with umbilical cord blood
donation is the high rate of bacterial contamination. Contamination takes
principally place at delivery and during the cord blood collection. Depending on
the sample size and the method of testing, up to 13% of cord blood stem cell
(CBSC) products have been found to be positive in bacterial cultures.48,49 No case
of sepsis or bacterial contamination related complications were reported after
stem cell transplantation. This is probably due to the low bacteria titers (1-10
CFU/ml) contaminating cord blood product. As cord blood products are stored at
very low temperature (-190°) until transplantation purposes, bacteria cannot
grow to clinically significant levels. However, bacterial contamination of cord
blood products may limit expansion protocols. Cord blood hematopoietic
progenitor cells culture and expansion may create conditions in which low
numbers of contaminating bacteria may reach levels of clinical significance,
compromising cell growth and exposing immuno-compromised patients to high
11
Chapter I
risks of infection. Therefore for such purposes, pathogen-free cord blood products
are required.
Efforts to minimize the risks of bacterial contamination, for example by stringent
disinfection and intensive monitoring of clean areas, did not reduce the rate of
microbial contamination.50 In addition, the addition of antibiotics in vitro
appeared to be unsuccessful and gave a new opportunity for contamination.51 In
general, the use of antibiotics is preferably avoided, considering the development
of bacteria resistance to this kind of antimicrobial agents.52 Recently, the FDA
guidelines and Netcord FACT Standards requested the release of pathogen-free
CBSC products for unrelated transplantation.53 In case of contamination, the
product should be discarded. This may be a problem in case the cord blood is
from a related donor, intended for transplantation e.g. to a diseased sibling and
for patients from ethnic minorities. Indeed, the cord blood bank has an advantage
on bone marrow donations because it may help to reduce the shortage in stem
cell products for non-Caucasian patients. Mothers originating from immigrant
populations are easily reachable through regular maternity controls whereas
those persons are largely less represented among blood and bone marrow
donation institutions. Some of the cord blood donations from ethnic minorities
may have a particular HLA typing. The discard strategy because of contamination
may lead to the loose of these valuable products. As for common blood products,
pathogen inactivation may represent an approach to circumvent the problem of
microbial contamination.
4. PATHOGEN INACTIVATION
Since pathogens involved in transfusion-transmitted infection are very
heterogeneous, a non-specific approach of inactivation of a broad range of
pathogens would be desired. The ideal method to ensure the production of
pathogen-free blood products should be a method that inactivates at once a
broad range of pathogens in blood products. Such method of decontamination is
commonly but often wrongly referred to as sterilisation. Sterilisation is a process
that destroys or removes all microorganisms (pathogenic or not) from an object or
12
Introduction
a solution. A sterilized item cannot support life in any form. Despite platelets and
RBCs do not contain a nucleus and thus are not able to reproduce themselves,
they anyway have an active respiratory metabolism thereby they are living cells,
forming the essential therapeutic components of the blood products. Treatment
of cellular blood products to inactivate pathogens must be applied in such a way
that the therapeutic effect of the blood cells such as platelets and RBCs remain
intact. Therefore, the common sterilization procedures are not suitable for PC and
RBCC. However, pathogen inactivation procedures that will have a broad
antimicrobial activity while preserving all blood components will represent a
proactive approach to blood safety. The challenge of pathogen inactivation is to
reduce maximally the greatest number of potential pathogens in blood without
significantly compromising the cellular or protein constituents or introducing
some new toxicity, carcinogenicity, or teratogenicity.54
PRINCIPLES OF PATHOGEN INACTIVATION TECHNIQUES
I.
METHODS OF EVALUATION
The key requirement in the evaluation of the efficacy of pathogen inactivation
strategies is to show that the procedure is effective in reducing the risk of
infection by pathogens.
Potential infectivity is determined by several measures. Genomic equivalents refer
to the number of viral particles detected by molecular techniques. The tissue
culture infectious dose (50%) TCID50 refers to the dose of viral particles in a tissue
culture assay capable of infecting 50% of susceptible cells or animals. The term
TCID50 log10 reduction is usually used to define the degree of pathogen reduction.
Although the “acceptable” log10 reduction number for a given organism is not
standardized, experience suggests the fewer than 4 log reductions may be
insufficient and greater than 6 log10 reduction is ordinarily sufficient.54 However,
in practice a 6 log10 reduction order is not always possible to demonstrate
because of the limitations in inocula for some specimens; thereby a maximal of 5
log10 reduction can be demonstrated. As no all conceivable organisms can be
tested, different classes of organisms are selected to cover the range of potential
13
Chapter I
pathogens. Model viruses are used in place of pathogens that cannot be cultured
in vitro (table 1).
Virus
Group
RNA/DNA
Size
(nm)
Model
for
Lipid-env.
DNA
100200
HBV,
CMV
Duck Hepatitis B virus Hepadnaviridae Lipid-env.
RNA
(DHBV)
40-45
HBV
Bovine viral diarrhorea Flaviviridae
virus (BVDV)
Lipid-env.
RNA
37-50
HCV,
WNV
Porcine Parvovirus (PPV)
Canine Parvovirus (CPV)
Non lipidenv. DNA
18-26
Human
ParvoB19
Lipid-env.
RNA
100
HIV
Virus
Family
Pseudorabies virus (PRV)
Herpesviridae
Vesicular
virus (VSV)
Parvoviridiae
Stomatitis Rhabdoviridae
Table 1: Viruses, their properties and their use as model
II.
CHEMICAL TREATMENTS
The combination of organic solvent and detergent (SD) is widely used for
pathogen inactivation. The combination most often used is TNBP (tri-(n-butyl)phosphate) with Triton-X100.55 TNBP acts as an organic solvent which removes
lipids from the membranes, while Triton X-100 is a non-ionic detergent which
stabilizes TNBP and disrupts lipid bilayers for easier extraction of lipids. Solvent
and detergent must be removed because the chemical reaction is non-selective.
The detergent keeps the lipids from the membranes in suspension in order to be
removed before the final product is transfused.
Lipid-envelop viruses as HIV, HBV and HCV or bacteria are sensitive to this
treatment and are inactivated in an irreversible way. Non-lipid enveloped viruses
14
Introduction
like Hepatitis A virus (HAV) and human B19 parvo-virus are insensitive to the SDtreatment. This chemical treatment is only suitable for pathogen inactivation in
plasma products. As the SD treatment disintegrates plasma membranes, it is not
suitable for pathogen inactivation of cellular blood components. Therefore,
alternative methods to inactivate pathogens without affecting the therapeutic
blood cells were developed. For the treatment of RBC, Pen 110 and Frangible
Anchor Linker Effector compounds S-303 are under investigation.
Pen 110 is an alkylating ethyleneamine oligomer of low-molecular weight, highly
water-soluble, and positively charged, diffusing readily through cell membranes
and having a high selectivity for nucleic acid guanine bases. The disruption of
nucleic acid replication forms the biochemical basis for pathogen reduction by the
Inactine Pen 110 process.56,57 Pen 110 is capable of inactivating a broad spectrum
of viruses, bacteria, protozoa, and mycoplasma in RBCC56,58-62 RBC membrane
integrity seems to be maintained in Pen 110-treated RBCs. Hemolysis was less
than 1% after 42 days of storage at 4°C. In vivo RBC recovery after autologous
transfusion of treated RBCs in healthy subjects was comparable with the
untreated control group.62 Alteration of RBC surface antigens was not detected
and neoantigens formation was not identified during subsequent testing.
However, phase III clinical trials were halted when antibody formation was
reported in repeatedly transfused patients. All other clinical studies have been
suspended.54
S-303 (N,N-bis(2-chloroethyl)-2-aminoehtyl-3-[(acridin-9-yl)amino] propionate
dihydrochloride) is an alkylating agent derived from a quinacrine mustard and
belongs to a class of compounds called frangible anchor-linked-effectors (FRALE).
The molecule consists of three components: an acridine ring, a planar compound
that reversibly intercalates with double-stranded and single-stranded DNA and
RNA; a bi-functional alkylating moiety that crosslinks DNA and RNA strands; and
an alkyl ester linker that hydrolyses when exposed to neutral pH.63 The compound
is rapidly taken up by cells, bacteria and viruses. S-303 molecule becomes active
as a result of pH change attendant upon admixture with the blood component
and induces DNA and RNA cross-linkage inhibiting pathogen replication and
transcription. Extracellular reactions are minimized by the inclusion of a
15
Chapter I
glutathione quenching agent. The alkyl ester linker spontaneously degrades
releasing S-303 that diffuses outside of cell and is absorbed by an appropriate
compound.64 S-303 binds to other proteins and cell membranes as well as to
nucleic acids, and up to 20% can potentially remain bound to the surface or
contained within the RBCs.54 S-303 has demonstrated pathogen inactivation of a
wide range of viruses, bacteria, and protozoa.65 No unexpected toxicities have
been described. Assays for RBC storage lesions (extracellular potassium (K+)
leakage, plasma free hemoglobin (Hb), adenosine triphosphate (ATP), 2,3diphosphoglycerate (2-3 DPG), glucose, and lactate) are comparable to control
RBCs. The RBC function appears to be normal, and in-vivo survival studies with
radioactive chromate labeling comply with the required standard of at least 75%
recovery at 24 hours. However, as seen with Inactine Pen110 treatment, phase III
clinical trials were halted when antibodies to residual RBC bound S-303 were
discovered in 2 subjects.66 Additional studies have revealed that 1% of patients
and healthy donors that had never been exposed to S-303 had naturally occurring
antibodies that reacted with S-303 treated RBC. Modifications have been made to
the S-303 treatment process to reduce the amount of RBC bound S-303 in
attempts to eliminate immunoreactivity and immunogenicity. Preliminary finding
indicate that RBCs from the modified S-303 treatment process were crossmatchcompatible with the anti-S-303 antibodies formed after exposure to the original S303 formulation as well as the anti– S-303 antibodies found in the patients and
donors never exposed to S-303. The antibodies do not appear to impair RBC
survival or to pose any clinical problem.67
PHOTOCHEMICAL TREATMENT
In photochemical treatment, light is used as a source of energy for intermolecular
reactions, which consists of a reaction between the light-excited molecule and a
different non-excited molecule. A good example of intermolecular reaction is the
photoaddition of furocoumarins to pyrimidines.68 By the use of psoralens (family
of furocoumarins), photochemical treatment can be applied in cellular blood
components. Psoralens are molecules that can intercalate into DNA base pairs.
Upon activation by UVA-light (320 – 400 nm), formation either of monofunctional
16
Introduction
adducts (single strand) or bifunctional adducts (interstrand cross-links) with DNA
takes place. These adducts block the replication and transcription machinery and
cause cell death.69
INTERCEPT, the S-59 amotosalen system, can currently be applied in PC.70 It has
been evaluated in clinical trials which, albeit not unequivocally, demonstrated
comparable platelet transfusion requirements and posttransfusion platelet
increments between photochemically treated platelets and control platelets.71
Application in plasma is currently under investigation.70 Functional quality of
plasma proteins (factor VII, factor VIII, von Willebrand factors, metalloprotease,
fibrinogen, protein C, protein S, and antithrombin) decreased slightly but were
considered still sufficient for therapeutic use.72 Antibodies against neo-antigens
have not been detected thus far. Clinical trials are ongoing.54
Platelet and plasma are substantially transparent above 300 nm, but hemoglobin
present in RBC has several absorption bands in the range 300-630 nm. Therefore
photoactivated compounds for use in RBCC should exhibit significant absorption
outside this spectrum (i.e. at longer wavelengths) and for this reason psoralens
like S-59 cannot be used for pathogen inactivation in RBCC treatment.
PHOTODYNAMIC TREATMENT
A special form of photochemical reactions is photosensitization. Photosensitized
reactions can occur when a mixture of at least two compounds is irradiated: one
compound absorbs the light but the other is chemically altered. The term
photodynamic refers to oxygen-requiring photosensitized reactions in biological
systems.73
In photodynamic reactions, oxygen (O2) acts as an intermediate transporting
electrons or energy between the light-absorbing molecule and the altered
molecule.73 Figure 1 shows a schematic overview of the successive reactions
involved in a photodynamic process. The light absorbing molecule is referred to as
the photosensitizer (P). The altered molecule is termed a substrate (S). After
absorption of light, the photosensitizer is transformed from its singlet electronic
ground state (1P0) to an electronically excited state (1P*) (equation 1). Via
17
Chapter I
intersystem crossing, the short-lived singlet excited stated is converted to the
triplet excited state (3P*) (equation 2) which has lower energy than the singlet
excited state, but the lifetime of the triplet excited state is much longer (typically
> 500 ns).74
There are two mechanisms by which the triplet excited state photosensitizer can
react with biomolecules: type I and type II reactions.
In a type I reaction, 3P* can either abstract an electron from, or donate an
electron to a substrate molecule (S). This reaction produces radical forms of both
the photosensitizer and the substrate (equations 3). The substrate radicals (S1- )
can react with other oxygen, to give oxidized forms of the substrate (equation 4).
The photosensitizer radicals (P+ ) can react with oxygen to give superoxide radical
(O2-ͻ; equation 5) which can rapidly oxidize cellular targets (S2) (equation 6).
z
z
Type II reaction can occur when the energy difference between the triplet excited
state and the ground state of the photosensitizer exceeds 94.5 kJ/mol. 3P* can
then directly transfer its energy to ground state oxygen (3O2), thereby generating
the highly reactive singlet oxygen (1O2) (equation 7).
The photosensitizer returns to its ground state without being chemically altered
and is able to repeat the process of energy transfer to oxygen many times. Singlet
oxygen can react with a variety of cellular components such as amino acids
(cysteine, histidine, tryptophan, tyrosine and methionine), nucleoside (mainly
guanine) and unsaturated lipids.75 Because of the short lifetime of 1O2, damages
are induced at, or close to, the sites where the photosensitizer is activated.
Figure 1: Schema of type I and type II photodynamic reactions
18
Introduction
5. PHOTOSENSITIZERS USED FOR PATHOGEN INACTIVATION OF RBC
OPTIMAL PHOTOSENSITIZER PROPERTIES
Photosensitizers used for pathogen inactivation should have a high selectivity for
the pathogens so that the pathogens are inactivated without altering the quality
of cells. For RBC containing blood products, the sensitizer should absorb light of a
long wavelength (> 600 nm) to avoid absorption by hemoglobin. An extra
advantage of the use of long wavelength light is the greater penetration of the
light in tissues and cell containing suspensions. During the past 15 years, a
number of photosensitizers have been studied for their ability to inactivate
pathogens in cellular blood products.
PHENOTHIAZINES
Phenothiazinium photosensitizers are efficient producers of singlet oxygen. The
Phenothiazinium dye Methylene Blue (MB) is positively charged and planar
molecule with affinity for nucleic acids and the surfaces of viruses.76,77 Upon UV
light exposure, most enveloped viruses are inactivated; however, non-enveloped
viruses are more resistant to treatment. As the photosensitizer is easily reduced
by biological systems to a neutral species MB has a low antibacterial and
antiprotozoa activity.78,79 Because of its virucidal activities, MB has been used in
Europe for approximately 15 years for pathogen inactivation of single unit plasma.
Millions of MB single units of plasma have been transfused without unexpected
adverse outcome.80
Methylation of this dye results in Di-Methyl-Methylene Blue (DMMB) which is
considerably more bactericidal in-vitro than MB and less susceptible to
reduction.77 DMMB (Fig.3) is a more hydrophobic dye with a permanent positive
charge and can pass through the cellular plasma membrane.81 The photosensitizer
intercalates into DNA82 and is a highly efficient photobactericide against a wide
range of bacteria and against both extra- and intracellular viruses and nonenveloped species upon light activation at wavelength >600 nm.83,84 However, up
to one-half of an added DMMB is associated with RBCs. This binding is responsible
19
Chapter I
for the photoinduced hemolysis.76. The use of quinacrine which inhibits
photosensitizer binding to RBC membranes was shown to enhance VSV
inactivation and to diminish hemolysis of photo-treated RBCs.82 However, DMMB
exhibited (dark) toxicity85 and post-treatment removal of the molecules e.g. by
filtration is required before transfusion.
RIBOFLAVIN
Riboflavin is a natural compound, a proven and accepted non toxic agent known
as vitamin B2 an essential dietary requirement in humans. The Riboflavin
molecule is a polycyclic planar molecule with a sugar side chain that confers water
solubility. The planar portion of the molecule is capable of intercalating into the
bases of DNA and RNA of pathogens, and absorbing visible light (max absorption
at 475 nm and 444 nm) or UV light. Light-activated, intercalated riboflavin oxidizes
guanine in nucleic acids, a process preventing replication of the pathogen’s
genome.86 Especially type I reaction (formation of radicals through electron
transfer) are involved in the photoreaction of Riboflavin.87
Upon light activation, the photosensitizer breaks down to secondary
photosensitizers Lumichrome and Lumiflavin.88,89 The advantages of these
photoproducts are that they are also naturally generated in situ by light
transmitted through the skin and eyes and are naturally excreted by the human
body.90
The particularity of Riboflavin is that this photosensitizer is sensitive to UV light
and blue light (250 - 500 nm) and not to red light (> 600 nm), therefore Riboflavin
seems not very, suitable for pathogen inactivation of RBCC. In contrast, Riboflavin
was shown to be effective in inactivating pathogens in this blood product. A
reduction of 4 to 7 log10 of extra- and intracellular HIV, BVDV, and PRV was
obtained after treatment with Riboflavin. The non-enveloped PPV which has a
tight-capsid, was more resistant. The antibacterial efficacy of Riboflavin was
demonstrated with the inactivation of S. aureus, E. coli, K. pneumoniae, and Y.
enterocolitica.91 However, for this, it was necessary to reduce the RBCC
20
Introduction
hematocrit to 30-37% instead of normally used 60% before the suspension was
subjected to visible light (O = 450 nm).64,86,91 92,93
PORPHYRINS
Porphyrins are a class of deeply colored red or purple pigments, of natural or
synthetic origin, which vary in their substituent around the same basic skeleton.
The latter is an aromatic macrocyclic ring consisting of four pyrrole rings, linked
together by four methane bridges (Fig.2). Porphyrins are probably the most
important class of compounds in biological systems because of their central role
in photosynthesis, biological oxidation and reduction, and oxygen transport.74,94
The interest for porphyrins in clinical use rose when a mixture of porphyrin, called
Hematoporphyrin derivatives (HpD), and later a purified form of HpD known as
Photofrin was found to localize in tumour cells, making it useful in detecting and
treating cancers.95-97To improve therapies and diminish side effects, in particularly
long lasting skin photosensitivity, the development of new photosensitizers was
necessary. In photodynamic treatment of cancer, red light (>600 nm) is usually
used because long wavelength enter deeper in tissue. Since porphyrins have only
a small absorption band in this region, research was focused on the synthesis of
new photosensitizers with a strong absorption in the red.98 This resulted to a
second generation of photosensitizers, among which phthalocyanines and mesosubstituted porphyrins appeared to be promising for RBCC decontamination.
Figre 2. Basic skeleton of porphyrin
21
Chapter I
I.
Phthalocyanines
Phthalocyanines are porphyrin derivatives with an increased aromatic structure.
They absorb intensively in the visible region with a maximum absorption around
670-680 nm and to a lesser extend in the near-ultraviolet region (see spectrum,
Fig.4).74 Therefore phthalocyanines are suitable for application in RBCC. The most
effective molecules in inactivating pathogens were silicon phthalocyanine (Pc4)
and chloro-aluminium sulfonated phthalocyanine (AlPcS4) (Fig.3).
Pc4 is a hydrophobic molecule which targets the lipid envelope of pathogens.
Formulated into liposomes to maximize delivery into viruses with minimum
distribution to RBCs, Pc4 was able to inactivate HIV-1 is three forms: cell-free
virus, cell-associated virus and virus in latency infected cells, and to inactivate
both enveloped and non-enveloped viruses.99,100 AlPcS4 is a water-soluble
derivative which is efficient in cell-free lipid-enveloped virus inactivation, but not
capable of inactivating intracellular virus.101,102 However, with both
phthalocyanines RBC damage was apparent which required the addition of
antioxidant such as vitamin E or a mixture of antioxidants containing either
tocopherol succinate and carnitine or Trolox, Mannitol and glutathione to
diminish the extensive hemolysis, K+ efflux and binding of IgG to phototreated
RBCs.100,103-105
II.
Meso-substituted porphyrins
The growing interest in the meso-substituted porphyrins began when besides
their photosensitizing properties106, their capacity to intercalate into DNA was
discovered.107 Their potential antimicrobial effect was shown by Merchat et
al.108,109; especially cationic meso-substituted porphyrin were able to inactivate
both Gram-positive and Gram-negative bacteria.
Meso-substituted porphyrins absorb radiant energy intensively in the UV region,
at their Soret band (400-410 nm) and to a lesser extent in the long visible bands
called the Q bands (580 – 650 nm) (see spectrum, Fig.4).74 Again their capacity to
absorb red light make these porphyrins suitable for application in RBCC.
22
Introduction
In this thesis, 5 different meso-substituted porphyrins, from which the most
powerful was the mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin chloride (TriP(4)) (Fig.3) were studied for their pathogenical potential in RBCs (chapter 3).
AlPcS4
DMMB
Tri-P(4)
Figure 3. Main photosensitizers used in this thesis
23
Chapter I
Figure 4. Absorption spectrum in Methanol of Hemoglobin and photosensitizers TriP(4) and AlPcS4.
6. CURRENT STATUS OF PATHOGEN INACTIVATION OF RBC
Many different techniques were studied to inactivate microorganisms in RBCC.
Most of the treatments are able to induce a considerable reduction of viable
pathogens. However, too much damage to the RBCs, such as hemolysis, RBC
aggregation105, K+ leakage, enhanced transbilayer mobility of lipids110 and IgG
binding111 are induced by pathogen inactivation techniques, especially the one
based on the use of light. To reduce the damage to the RBCs, various antioxidants
had to be added to the RBC suspension before treatment.100,104,105 Such additives
may have side-effects and may have to be removed before transfusion.
Besides the direct damage to RBCs, several treatments have shown to induce
antibodies against either the agent added to the suspension or to the treated
RBCs. Despite encouraging in-vitro results concerning the efficacy of S-303 and
P110 treatments to inactivate a broad spectrum of pathogens while preserving invitro RBC qualities (Hb, ATP, 2-3 DPG level and storage time) and in-vivo survival,
unexpected results in clinical trials showed up which resulted to their suspension.
24
Introduction
The future of both S-303 and PEN110 as pathogen inactivation methods for RBCC
is uncertain until the immunogenicity is understood and resolved.54 For
Riboflavin, extended animal studies are required to ensure the absence of
neoantigenicity to Riboflavin and its breakdown product before proceeding
clinical studies.64
7. OUTLINE OF THIS THESIS
This thesis describes different studies regarding the photo-decontamination of
RBC and Cord-blood-derived stem cell products. Besides the inactivation of
pathogens, major efforts were taken to preserve the therapeutic quality of the
treated blood products. By using the photosensitizer DMMB, we tried to identify
the target for RBC damage induced by photodynamic treatment (PDT). Based on
this, we evaluated the possibility to protect the RBCs against photo-damage
without reducing the virucidal capacity of the photo-treatment (Chapters 2 and
3). A series of meso-substituted porphyrins were tested for their potential to
inactivate viruses in RBCC. From the series, one photosensitizer Tri-P(4) stood out
as a very promising compound to be applied in pathogen inactivation in RBCC
(Chapters 4 and 5). Photosensitization with Tri-P(4) was further tested in Cordblood derived stem cell products (Chapter 6). Since the treatment did not seem to
affect the viability of leukocytes, the potential immunomodulatory effect on
leukocytes was investigated (Chapter 7).
8. REFERENCES
1. Meulenbroek A.J. Van bloed tot geneesmiddel, gezond vertrouwen. Sanquin ed.
Amsterdam: Artoos Nederland; 2002.
2. Kalibatas V. Payment for whole blood donations in Lithuania: the risk for
infectious disease markers. Vox Sang. 2008 Apr;94(3):209-15.
3. van der Poel CL. Remuneration of blood donors: new proof of the pudding? Vox
Sang. 2008 Apr;94(3):169-70.
25
Chapter I
4. Boltho Massarelli V., Van Aken W, Genetet B. Ethics of transfusion: the Council of
Europe's view. Transfusion Medicine 2004; An European Course on Blood
transfusion:11-39.
5. Koppelman MHGM, Sjerps MC, Reesink HW, Cuypers HTM. Evaluation of COBAS
AmpliPrep nucleic acid extraction in conjunction with COBAS AmpliScreen HBV
DNA, HCV RNA and HIV-1 RNA amplification and detection. Vox Sang.
2005;89(4):193-200.
6. Council of Europe Health.Deptmt. K-FB. Pathogen inactivation of labile blood
products. Strasbourg: Council of Europe; 2001. Report No.: ISBN 92-871-4560-1.
7. Laino C. ASH: Screening of blood supply for West Nile Virus justified By past
Prevention Success 2004. Available from www.docguide.com/news/content.nsf.
8. Pealer LN, Marfin AA, Petersen LR, Lanciotti RS, Page PL, Stramer SL, Stobierski
MG, Signs K, Newman B, Kapoor H, et al. Transmission of West Nile virus through
blood transfusion in the United States in 2002. N.Engl.J.Med. 2003 Sep
25;349(13):1236-45.
9. Yoshikawa A, Gotanda Y, Itabashi M, Minegishi K, Kanemitsu K, Nishioka K.
Hepatitis B NAT virus-positive blood donors in the early and late stages of HBV
infection: analyses of the window period and kinetics of HBV DNA. Vox Sang.
2005;88(2):77-86.
10. Roback JD, Caliendo AM, Newman JL, Sgan SL, Saakadze N, Gillespie TW, Lane TA,
Kurtzberg J, Hillyer CD. Comparison of cytomegalovirus polymerase chain
reaction and serology for screening umbilical cord blood components.
Transfusion 2005;45(11):1722-8.
11. Hillyer CD, Josephson CD, Blajchman MA, Vostal JG, Epstein JS, Goodman JL.
Bacterial contamination of blood components: risks, strategies, and regulation:
joint ASH and AABB educational session in transfusion medicine. Hematology
2003;575-89.
12. Huai Y, Xiang N, Zhou L, Feng L, Peng Z, Chapman RS, Uyeki TM, Yu H. Incubation
period for human cases of avian influenza A (H5N1) infection, China.
Emerg.Infect.Dis. 2008 Nov;14(11):1819-21.
13. Likos AM, Kelvin DJ, Cameron CM, Rowe T, Kuehnert MJ, Norris PJ. Influenza
viremia and the potential for blood-borne transmission. Transfusion 2007
Jun;47(6):1080-8.
26
Introduction
14.
Epidemic and Pandemic alert and response - Recommendation concerning SARS
and blood transfusion safety 2003. Available from www.WHO.int.
15. Wüllenweber J, Cassens U, Sica S, Sibrowski W, Herrmann M. West Nile Virus - an
Emerging Transfusion-Transmissible Pathogen. Transf. Med. Hemoth.
2004;31(6):373-7.
16. Bianco C. West Nile Virus transmission by blood transfusion and transplantation.
Blood Therapies in Medicine 2003;3(3):78-83.
17. Harrington T, Kuehnert MJ, Kamel H, Lanciotti RS, Hand S, Currier M,
Chamberland ME, Petersen LR, Marfin AA. West Nile virus infection transmitted
by blood transfusion. Transfusion 2003;43(8):1018-22.
18. Biggerstaff BJ, Petersen LR. Estimated risk of transmission of the West Nile virus
through blood transfusion in the US, 2002. Transfusion 2003;43(8):1007-17.
19. Hollinger FB, Kleinman S. Transfusion transmission of West Nile virus: a merging
of historical and contemporary perspectives. Transfusion 2003;43(8):992-7.
20. Haltia M. Human prion diseases. Ann. Med. 2000 Oct;32(7):493-500.
21. Goodnough LT, Hewitt PE, Silliman CC. Transfusion Medicine: Joint ASH and AABB
Educational Session. Hematology 2004 Jan 1;2004(1):457-72.
22. Dorsey K, Zou S, Schonberger LB, Sullivan M, Kessler D, Notari E, Fang CT, Dodd
RY. Lack of evidence of transfusion transmission of Creutzfeldt-Jakob disease in a
US surveillance study. Transfusion 2009 May;49(5):977-84.
23. Houston F, Foster JD, Chong A, Hunter N, Bostock CJ. Transmission of BSE by
blood transfusion in sheep. Lancet 2000;356(9234):999-1000.
24. Seghatchian J. nvCJD and leucodepletion: an overview. Transfus Sci 2000;22(12):47-8.
25. Turner ML. Variant Creutzfeldt-Jakob disease and blood transfusion. Curr. Opin.
Hematol. 2001 Nov;8(6):372-9.
26. Akiba J, Umemura T, Alter HJ, Kojiro M, Tabor E. SEN virus: epidemiology and
characteristics of a transfusion-transmitted virus. Transfusion 2005;45(7):1084-8.
27
Chapter I
27. Okamura A, Yoshioka M, Kubota M, Kikuta H, Ishiko H, Kobayashi K. Detection of
a novel DNA virus (TTV) sequence in peripheral blood mononuclear cells. J. Med.
Virol. 1999;58(2):174-7.
28. Hadlock KG, Foung SK. GBV-C/HGV: a new virus within the Flaviviridae and its
clinical implications. Transfus. Med. Rev. 1998;12(2):94-108.
29. Viazov S, Ross RS, Varenholz C, Lange R, Holtmann M, Niel C, Roggendorf M. Lack
of evidence for an association between TTV infection and severe liver disease. J.
Clin. Virol. 1998 Dec;11(3):183-7.
30. Mahieux R, Gessain A. [New human retroviruses: HTLV-3 and HTLV-4]. Med. Trop.
2005 Nov;65(6):525-8.
31. Kitchen AD, Chiodini PL. Malaria and blood transfusion. Vox Sang. 2006
Feb;90(2):77-84.
32. Kitchen A, Mijovic A, Hewitt P. Transfusion-transmitted malaria: current donor
selection guidelines are not sufficient. Vox Sang. 2005 Apr;88(3):200-1.
33. Thellier M, Lusina D, Guiguen C, Delamaire M, Legros F, Ciceron L, Klerlein M,
Danis M, Mazier D. Is airport malaria a transfusion-transmitted malaria risk?
Transfusion 2001;41(2):301-2.
34. Wagner SJ. Transfusion-transmitted bacterial infection: risks, sources and
interventions. Vox Sanguinis 2004 Apr;86(3):157-63.
35. Andreu G, Morel P, Forestier F, Debeir J, Rebibo D, Janvier G, Herve P.
Hemovigilance network in France: organization and analysis of immediate
transfusion incident reports from 1994 to 1998. Transfusion 2002
Oct;42(10):1356-64.
36. de Korte D, Marcelis JH, Soeterboek AM. Determination of the degree of bacterial
contamination of whole-blood collections using an automated microbe-detection
system. Transfusion 2001 Jun;41(6):815-8.
37. de Korte D, Curvers J, de Kort WL, Hoekstra T, van der Poel CL, Beckers EA,
Marcelis JH. Effects of skin disinfection method, deviation bag, and bacterial
screening on clinical safety of platelet transfusions in the Netherlands.
Transfusion 2006 Mar;46(3):476-85.
28
Introduction
38. Kluter H. The Struggle for Safer Blood: Pathogen Inactivation of Cellular Blood
Preparations. Blood Therapies in Medicine 2002;2(2):42-7.
39. Dodd RY, Notari EP, Stramer SL. Current prevalence and incidence of infectious
disease markers and estimated window-period risk in the American Red Cross
blood donor population. Transfusion 2002 Aug;42(8):975-9.
40. Stramer SL. Viral diagnostics in the arena of blood donor screening. Vox Sang.
2004 Jul;87 Suppl 2:180-3.
41. Koi H, Zhang J, Parry S. The mechanisms of placental viral infection. Ann. N.Y.
Acad. Sci. 2001 Sep;943:148-56.
42. Van Geertruyden JP, Thomas F, Erhart A, D'Alessandro U. The contribution of
malaria in pregnancy to perinatal mortality. Am. J. Trop. Med. Hyg. 2004 Aug
1;71(suppl):35-40.
43. Saback FL, Gomes SA, de Paula VS, da Silva RR, Lewis-Ximenez LL, Niel C. Agespecific prevalence and transmission of TT virus. J. Med. Virol. 1999;59(3):318-22.
44. Pahwa S. Human immunodeficiency virus infection in children: nature of
immunodeficiency, clinical spectrum and management. Pediatr. Infect. Dis. J.
1988;7(Suppl):61-71.
45. Linder N, Sirota L, Aboudy Y, German B, Lifshits T, Barnea BS, Lieberman B,
Mendelson E, Barzilai A. Placental transfer of maternal rubella antibodies to fullterm and preterm infants. Infection 1999;27(3):203-7.
46. Zufferey J, Hohlfeld P, Bille J, Fawer CL, Blanc D, Pinon JM, Vaudaux B. Value of
the comparative enzyme-linked immunofiltration assay for early neonatal
diagnosis of congenital Toxoplasma infection. Pediatr. Infect. Dis. J.
1999;18(11):971-5.
47. Alpert SG, Fergerson J, Noel LP. Intrauterine West Nile virus: ocular and systemic
findings. Am. .J. Ophthalmol. 2003 Oct;136(4):733-5.
48. Honohan A, Olthuis H, Bernards AT, van Beckhoven JM, Brand A. Microbial
contamination of cord blood stem cells. Vox Sang. 2002;82(1):32-8.
49. Kogler G, Callejas J, Hakenberg P, Enczmann J, Adams O, Daubener W, Krempe C,
Gobel U, Somville T, Wernet P. Hematopoietic transplant potential of unrelated
cord blood: critical issues. J. Hematother. 1996;5(2):105-16.
29
Chapter I
50. Cassens U, Ahlke C, Garritsen H, Krakowitzky P, Wullenweber J, Fischer RJ, Peters
G, Sibrowski W. Processing of peripheral blood progenitor cell components in
improved clean areas does not reduce the rate of microbial contamination.
Transfusion 2002;42(1):10-7.
51. Stroncek DF, Fautsch SK, Lasky LC, Hurd DD, Ramsay NK, McCullough J. Adverse
reactions in patients transfused with cryopreserved marrow. Transfusion
1991;31(6):521-6.
52. Engelhard D. Bacterial and fungal infections in children undergoing bone marrow
transplantation. Bone Marrow Transplant. 1998 Apr;21 Suppl 2:S78-80.:S78-S80.
53. Netcord, Foundation for the Accreditation of Cellular Therapy. International
Standards for Cord Blood Collection, Processing, Testing, Banking, Selection, and
Release. 2006 Dec. Report No.: 3rd edition.
54. Bryant BJ, KLEIN HG. Pathogen inactivation: the definitive safeguard for the blood
supply. Arch. Pathol. Lab Med. 2007 May;131(5):719-33.
55. Horowitz B, Lazo A, Grossberg H, Page G, Lippin A, Swan G. Virus inactivation by
solvent/detergent treatment and the manufacture of SD-plasma. Vox Sang.
1998;74 Suppl 1:203-6.
56. Lazo A, Tassello J, Jayarama V, Ohagen A, Gibaja V, Kramer E, Marmorato A, BilliaShaveet D, Purmal A, Brown F, et al. Broad-spectrum virus reduction in red cell
concentrates using INACTINEtm PEN110 chemistry. Vox Sang. 2002;83(4):313-23.
57. Purmal A, Valeri CR, Dzik W, Pivacek L, Ragno G, Lazo A, Chapman J. Process for
the preparation of pathogen-inactivated RBC concentrates by using PEN110
chemistry: preclinical studies. Transfusion 2002;42(2):139-45.
58. Zavizion B, Serebryanik D, Chapman J, Alford B, Purmal A. Inactivation of Gramnegative and Gram-positive bacteria in red cell concentrates using INACTINE
PEN110 chemistry. Vox Sang. 2004 Oct;87(3):143-9.
59. Zavizion B, Pereira M, de Melo JM, Serebryanik D, Mather TN, Chapman J, Miller
NJ, Alford B, Bzik DJ, Purmal A. Inactivation of protozoan parasites in red blood
cells using INACTINE PEN110 chemistry. Transfusion 2004 May;44(5):731-8.
60. Zavizion B, Purmal A, Chapman J, Alford B. Inactivation of mycoplasma species in
blood by INACTINE PEN110 process. Transfusion 2004 Feb;44(2):286-93.
30
Introduction
61. Mather T, Takeda T, Tassello J, Ohagen A, Serebryanik D, Kramer E, Brown F, Tesh
R, Alford B, Chapman J, et al. West Nile virus in blood: stability, distribution, and
susceptibility to PEN110 inactivation. Transfusion 2003;43(8):1029-37.
62. AuBuchon JP, Pickard CA, Herschel LH, Roger JC, Tracy JE, Purmal A, Chapman J,
Ackerman S, Beach KJ. Production of pathogen-inactivated RBC concentrates
using PEN110 chemistry: a Phase I clinical study. Transfusion 2002;42(2):146-52.
63. Allain JP, Bianco C, Blajchman MA, Brecher ME, Busch M, Leiby D, Lin L, Stramer
S. Protecting the Blood Supply From Emerging Pathogens: The Role of Pathogen
Inactivation. Transf. Med. Rev. 2005 Apr;19(2):110-26.
64. Benjamin RJ. Red blood cell pathogen reduction: in search of serological
agnosticism. Transfusion 2006 Sep;1(1):222-6.
65. Corash L. Helinx technology for inactivation of infectious pathogens and
leukocytes in labile blood components: from theory to clinical application.
Transfus. Apher. Sci. 2001;25(3):179-81.
66. Benjamin RJ, McCullough J, Mintz PD, Snyder E, Spotnitz WD, Rizzo RJ, Wages D,
Lin JS, Wood L, Corash L, et al. Therapeutic efficacy and safety of red blood cells
treated with a chemical process (S-303) for pathogen inactivation: a Phase III
clinical trial in cardiac surgery patients. Transfusion 2005;45(11):1739-49.
67. Conlan MG, Garratty G, Castro G, Erickson A, Schott MA, Arndt P, Cook D, Corash
L, Stassinopoulos A. Antibodies to S-303 Treated RBC Prepared with the Original
Treatment Process (OSRBC) for Pathogen Inactivation Do Not React with RBC
Prepared with a Modified S-303 Treatment Process (MSRBC). ASH Annual
Meeting Abstracts 2005 Nov 16;106(11):430.
68. Coyle J.D., Hill R.R., oberts D.R. Light, chemical change and life: a source book in
photochemistry. The Open University Press ed. Milton Keynes: 1988.
69. Gasparro FP, Dall'Amico R, Goldminz D, Simmons E, Weingold D. Molecular
aspects of extracorporeal photochemotherapy. Yale J. Biol. Med. 1989
Nov;62(6):579-93.
70. Wollowitz S. Fundamentals of the psoralen-based Helinx technology for
inactivation of infectious pathogens and leukocytes in platelets and plasma.
Semin. Hematol. 2001;38(4 Suppl 11):4-11.
31
Chapter I
71. Janetzko K, Lin L, Eichler H, Mayaudon V, Flament J, Kluter H. Implementation of
the INTERCEPT Blood System for Platelets into routine blood bank manufacturing
procedures: evaluation of apheresis platelets. Vox Sang. 2004 May 1;86(4):23945.
72. Ciaravino V, McCullough T, Cimino G, Sullivan T. Preclinical safety profile of
plasma prepared using the INTERCEPT Blood System. Vox Sang. 2003
Oct;85(3):171-82.
73. Tappeiner H.V., Jesionek A. Therapeutische versuche mit fluoreszierenden
stoffen. Muench.Med.Wochenschr. 1903;7:2042-4.
74. Milgrom L.R. The colours of life: an introduction to the chemistry of porphyrins
and related compounds. New York: 1997.
75. van Steveninck J., Dubbelman T.M.A.R., Verweij H. Photodynamic membrane
damage. In: Kessel D & Dougherty TV, editor. Porphyrin Photosensitization. New
York: Plenum Press; 1983. p. 227-40.
76. Wagner SJ. Virus inactivation in blood components by photoactive phenothiazine
dyes. Transfus. Med. Rev. 2002;16(1):61-6.
77. Wainwright M. Pathogen inactivation in blood products. Curr. Med. Chem. 2002
Jan;9(1):127-43.
78. Wainwright M, Phoenix DA, Laycock SL, Wareing DR, Wright PA.
Photobactericidal activity of phenothiazinium dyes against methicillin-resistant
strains of Staphylococcus aureus. FEMS Microbiol.Lett. 1998 Mar 15;160(2):17781.
79. Wainwright M, Phoenix DA, Marland J, Wareing DR, Bolton FJ. A study of
photobactericidal activity in the phenothiazinium series. FEMS Immunol
Med.Microbiol. 1997 Sep;19(1):75-80.
80. Mohr H. Methylene blue and thionine in pathogen inactivation of plasma and
platelet concentrates. Transfus. Apher. Sci. 2001 Dec;25(3):183-4.
81. Paardekooper M, Van den Broek PJ, de Bruijne AW, Elferink JG, Dubbelman TM,
van Steveninck J. Photodynamic treatment of yeast cells with the dye toluidine
blue: all-or-none loss of plasma membrane barrier properties. Biochim. Biophys.
Acta 1992 Jul 8;1108(1):86-90.
32
Introduction
82. Jori G, Brown SB. Photosensitized inactivation of microorganisms. Photochem.
Photobiol. Sci. 2004 May;3(5):403-5.
83. Wagner SJ, Skripchenko A. Comparison of the sensitivity of Duck Hepatitis B and
Vesicular Stomatitis VIrus to photoinactivation by DMMB. Transfusion
2000;40(SP230):99S.
84. Wagner SJ, Skripchenko AA, Robinette D, Mallory DA, Hirayama J, Cincotta L,
Foley J. The use of dimethylmethylene blue for virus photoinactivation of red cell
suspensions. In: Vyas BF, editor. Advances in Transfusion Safety: Dev Biol. 1999
ed. Basel, Karger; 1999. p. 125-9.
85. Wainwright M, Phoenix DA, Rice L, Burrow SM, Waring J. Increased cytotoxicity
and phototoxicity in the methylene blue series via chromophore methylation. J.
Photochem. Photobiol. B: Biology 1997 Oct;40(3):233-9.
86. Corbin F. Pathogen inactivation of blood components: current status and
introduction of an approach using riboflavin as a photosensitizer. Int. J. Hematol.
2002 Aug;76 Suppl 2:253-7.
87. Douki T, Cadet J. Modification of DNA bases by photosensitized one-electron
oxidation. Int. .J. Radiat. Biol. 1999 May;75(5):571-81.
88. Wainwright M. The emerging chemistry of blood product disinfection. Chemical
Society Reviews 2002;31(2):128-36.
89. Dodd RY. Pathogen inactivation: mechanisms of action and in vitro efficacy of
various agents. Vox Sang. 2002 Aug;83 Suppl 1:267-70.
90. Schuyler R. Use of riboflavin for photoinactivation of pathogens in blood
components. Transfus. Apher. Sci. 2001 Dec;25(3):189-90.
91. Reddy HL, Doane S, McLean R., McDaniel S., Dawson L. Reduction of Virus in Red
Blood Cell Suspensions with Riboflavin and Light. Transfusion 2002;42(S9):16S.
92. Goodrich RP, Murthy KK, Doane SK, Fitzpatrick CN, Morrow LS, Arndt PA, Reddy
HL, Buytaert-Hoefen KA, Garratty G. Evaluation of potential immune response
and in vivo survival of riboflavin-ultraviolet light-treated red blood cells in
baboons. Transfusion 2008 Oct 14.
33
Chapter I
93. McAteer MJ, Tay-Goodrich BH, Doane S, Hansen E, McBurney LL, Stewart L.
Photoinactivation of virus in packed red blood cell units using Riboflavin and
visible light. Transfusion 2000;40(SP231):99S.
94. Rest A. Porphyrins and phthalocyanines. In: Coyle J.D., Roberts D.R., Hill R.R.,
editors. Light, chemical change and life: a source book in photochemistry. Milton
Keynes: The Open University Press; 1988. p. 43-51.
95. Policard A. Etude sur les aspects offerts par des tumeurs expérimentales
examinées à la lumière de Wood. C. R. Soc. Biol. (Paris) 1924;91(1423):1424.
96. Inaguma M, Hashimoto K. Porphyrin-like fluorescence in oral cancer: In vivo
fluorescence spectral characterization of lesions by use of a near-ultraviolet
excited autofluorescence diagnosis system and separation of fluorescent extracts
by capillary electrophoresis. Cancer 1999 Dec 1;86(11):2201-11.
97. Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D, Korbelik M, Moan J,
Peng Q. Photodynamic therapy. J. Natl. Cancer Inst. 1998 Jun 17;90(12):889-905.
98. Dougherty TJ. A brief history of clinical photodynamic therapy development at
Roswell Park Cancer Institute. J. Clin. Laser Med. Surg. 1996 Oct;14(5):219-21.
99. Ben Hur E, Oetjen J, Horowitz B. Silicon phthalocyanine Pc 4 and red light causes
apoptosis in HIV-infected cells. Photochem. Photobiol. 1997 Mar;65(3):456-60.
100. Ben-Hur E, Chan WS, Yim Z, Zuk MM, Dayal V, Roth N, Heldman E, Lazo A, Valeri
CR, Horowitz B. Photochemical decontamination of red blood cell concentrates
with the silicon phthalocyanine PC 4 and red light. Developments in Biological
Standardization 2000;102:149-55.
101. Margolis-Nunno H, Ben-Hur E, Gottlieb P, Robinson R, Oetjen J, Horowitz B.
Inactivation by phthalocyanine photosensitization of multiple forms of human
immunodeficiency virus in red cell concentrates. Transfusion 1996;36(8):743-50.
102. Horowitz B, Rywkin S, Margolis-Nunno H, Williams B, Geacintov N, Prince AM,
Pascual D, Ragno G, Valeri CR, Huima-Byron T. Inactivation of viruses in red cell
and platelet concentrates with aluminum phthalocyanine (AIPc) sulfonates. Blood
Cells 1992;18(1):141-9.
103. Ben-Hur E, Rywkin S, Rosenthal I, Geacintov NE, Horowitz B. Virus inactivation in
red cell concentrates by photosensitization with phthalocyanines: protection of
34
Introduction
red cells but not of vesicular stomatitis virus with a water-soluble analogue of
vitamin E. Transfusion 1995;35(5):401-6.
104. Rywkin S, Lenny L, Goldstein J, Geacintov NE, Margolis-Nunno H, Horowitz B.
Importance of type I and type II mechanisms in the photodynamic inactivation of
viruses in blood with aluminum phthalocyanine derivatives. Photochem.
Photobiol. 1992;56(4):463-9.
105. Ben-Hur E, Barshtein G, Chen S, Yedgar S. Photodynamic treatment of red blood
cell concentrates for virus inactivation enhances red blood cell aggregation:
protection with antioxidants. Photochem. Photobiol. 1997;66(4):509-12.
106. Diamond I, Granelli SG, McDonagh AF. Photochemotherapy and photodynamic
toxicity: Simple methods for identifying potentially active agents. Biochem. Med.
1977 Apr;17(2):121-7.
107. Fiel RJ, Howard JC, Mark EH, Datta Gupta N. Interaction of DNA with a porphyrin
ligand: evidence for intercalation. Nucleic Acids Res 1979 Jul 11;6(9):3093-118.
108. Merchat M, Bertolini G, Giacomini P, Villanueva A, Jori G. Meso-substituted
cationic porphyrins as efficient photosensitizers of gram-positive and gramnegative bacteria. J. Photochem. Photobiol. B, Biology 1996;32(3):153-7.
109. Merchat M, Spikes JD, Bertoloni G, Jori G. Studies on the mechanism of bacteria
photosensitization by meso-substituted cationic porphyrins. J. Photochem.
Photobiol. B, Biology 1996;35(3):149-57.
110. Deuticke B, Henseleit U, Haest CW, Heller KB, Dubbelman TM. Enhancement of
transbilayer mobility of a membrane lipid probe accompanies formation of
membrane leaks during photodynamic treatment of erythrocytes. Biochim.
Biophys. acta 1989;982(1):53-61.
111. Sato Y, Kamo S, Takahashi T, Suzuki Y. Mechanism of free radical-induced
hemolysis of human erythrocytes: hemolysis by water-soluble radical initiator.
Biochemistry 1995;34(28):8940-9.
35
36
CHAPTER II
RELATION BETWEEN K+ LEAKAGE
AND DAMAGE TO BAND 3 IN
PHOTODYNAMICALLY TREATED
RED CELLS
Laurence L. Trannoy, Anneke Brand and Johan W. M. Lagerberg
Blood Bank Leiden-Haaglanden, Leiden, the Netherlands
Adapted from: Photochemistry and Photobiology, 2002, 75(2): 167–171
37
Chapter II
ABSTRACT
Potassium (K+) leakage is one of the first events that appear after
photosensitization of RBCs. This event may subsequently lead to colloid osmotic
hemolysis. The aim of our study was to determine which photodynamically
induced damage is responsible for increased membrane cation permeability. This
was done by studying the effect of DMMB-mediated PDT on different membrane
transport systems. Inhibition of band 3 activity (anion transport) showed a
comparable light dose dependency as PDT-induced K+ leakage, whereas glycerol
transport activity was inhibited only at higher light doses. Dipyridamole (DIP), an
inhibitor of anion transport, protects band 3 against DMMB-induced damage, and
prevents the increase in cation permeability of the membrane. Damage to
glycerol transport was partially reduced when PDT was performed in the presence
of DIP. Because DIP has no affinity for the glycerol transporter, this protection
might result from the reduced photodamage to band 3. These results support the
hypothesis that band 3 might be involved in glycerol transport. Glucose transport
was not affected by DMMB-mediated PDT. The present results are the first to
show a causal relationship between DMMB-mediated photodamage to band 3
and increased cation permeability of RBCs.
38
K+ leakage and damage to Band 3 in red cells
INTRODUCTION
In spite of continuing improvement in laboratory tests to decrease the window
phase and so to reduce the possibility of viral transmission, viral and bacterial
contamination remains a major problem of the blood transfusion services.1 The
increasing costs cannot be met by many countries outside the western world. It is
therefore necessary to investigate new strategies to improve the safety of blood
component transfusion. Inactivation of the infectious pathogens may present an
alternative method of ensuring pathogen-free blood. A heating or solvent–
detergent treatment is already included in the processing of plasma products.
These approaches have been shown to reduce the risk of virus transmission2,3;
however, because of the fragility of the cells, they are not applicable to cellular
blood products. The use of PDT as a method for pathogen inactivation in cellular
blood products such as RBCC is being explored because various lipid-enveloped
and non-enveloped viruses, gram-positive and gram-negative bacteria can be
inactivated.4-9 PDT is based on the use of light-activated agents, photosensitizers.
The photosensitizers studied for pathogen inactivation in RBCC usually belong to
the group of phthalocyanines10 or to the group of phenothiazine dyes, such as
MB11 and DMMB.12 On light activation, these molecules generate reactive oxygen
species such as singlet oxygen (1O2), which are able to react rapidly with
substrates present in their environment. Pathogens can be inactivated completely
by PDT, but all photosensitizers tested so far also induce damage to RBCs.13-17 A
better understanding of the molecular mechanisms behind photodynamically
induced damage is needed to improve the selectivity of the photodynamic
decontamination technique and to reduce, or if possible to avoid, damage to
RBCs. Increased permeability of the membrane to cations, as measured by K+
leakage, is one of the first events that appear after photosensitization of RBCs. It
is believed that this increased permeability will lead ultimately to colloid
osmotic.9,14-17 It is as yet unclear which photodynamically induced damage is
responsible for the increased membrane permeability. Dubbelman et al.18 have
shown that inhibition of several carrier-mediated transport systems for glucose,
glycerol and sulfate precedes K+ leakage after protoporphyrin-mediated PDT.
Based on the dose-squared relationship between fluence and photohemolysis, it
39
Chapter II
has been suggested that hemolysis results from damage to the anion transport
protein of the RBC, band 3.19 Recently, VanSteveninck et al. 20 have shown that
the band 3 ligand dipyridamole (DIP) protects RBCs against DMMB-mediated PDT.
This protection was selective for RBCs; DIP had no effect on the virucidal efficacy
of DMMB. The aim of the present study was to gain a better insight into the
molecular mechanisms behind DMMB-mediated photodynamically induced K+
leakage in RBCs. As it is likely that damage to a membrane-spanning protein could
be involved in K+ leakage, the kinetics of photodynamically induced K+ leakage and
inhibition of carrier-mediated transport of glucose, glycerol and sulfate were
compared. Because DIP is known to protect against DMMB-mediated K+ leakage,
the effect of DIP on the photoinhibition of the various transport systems was also
investigated. Based on both the kinetics of inhibition and the effect of DIP, an
attempt was made to identify the critical target for DMMB induced K+ leakage.
MATERIALS AND METHODS
MATERIALS
RBCC were provided by the Red Cross Blood Bank Leiden-Haaglanden (Leiden, The
Netherlands) after consent of human volunteer donors. The erythrocytes were
centrifuged shortly after collection, washed three times with phosphate-buffered
saline (PBS) containing 30 mM sucrose and resuspended at 10% hematocrit (Hct)
in the same buffer. Sucrose was added to prevent photohemolysis.21 DMMB was
obtained from Sigma-Aldrich, Zwijndrecht, the Netherlands. Stock solutions of 1
mM were made in 50 mM sodium phosphate buffer (NaP), pH 7.4, and stored at
4°C. DIP (2,6-bis(diethanolamino)-4,8-dipiperidino-[5,4-d] pyrimidine) (SigmaAldrich) was stored as a 200 mM stock solution in dimethylsulfoxide (DMSO) at
4°C. Sodium [35S] sulfate (Na235SO4) and D-[U-14C-glucose] were obtained from
Amersham, ’s-Hertogenbosch, the Netherlands. All other chemicals were also
purchased from Sigma-Aldrich.
40
K+ leakage and damage to Band 3 in red cells
PHOTODYNAMIC TREATMENT
Illumination was performed with a 300 W halogen lamp (Philips, Brussel,
Belgium). The light passed a water and red filter to transmit only light with O
above 600 nm and to avoid heating of the sample. RBC suspensions were
illuminated in 3 cm diameter culture dishes (Greiner, Alphen a/d Rijn, the
Netherlands) containing 1 mL of RBC suspension (10% Hct). Irradiance was 10
mW/cm2, as measured with an IL400A photometer equipped with an SEL033/F/U
detector (International Light, Newbury, MA).
RBC suspensions were incubated with 100 μM DIP in the dark. After 5 min, 15 μM
DMMB was added to the suspension. After another 5 min of incubation in the
dark, illumination was started. All treatments were performed at a constant
temperature of 20°C under continuous shaking (150 cycles/min). In control
experiments, the same volumes of DMSO and NaP were added, without DIP and
DMMB, respectively. These samples were illuminated as described previously.
POTASSIUM LEAKAGE AND HEMOLYSIS
K+ leakage from RBCs was determined immediately after illumination, by means of
a flame photometer (Clinical Flame Photometer 410C; Corning, Halstead, UK).
Hemolysis was determined by measuring the optical density of the supernatant at
540 nm using a Novaspec II spectrophotometer (Pharmacia Biotech, Weesp, the
Netherlands). Both K+ and Hb release were expressed as a percentage of the total
efflux evoked by lysis of the cells in distilled water.
BAND 3 ACTIVITY
Band 3 activity was measured by the influx of Na235SO4 as described previously.22
Briefly, after PDT, cells were centrifuged and resuspended (10% Hct) in PBS plus 1
mM Na2SO4. The suspensions were incubated with 0.5 μCi/mL Na235SO4 at 37°C.
After 0, 10, 20 and 30 min of incubation, samples were taken and washed twice
with PBS to remove free Na235SO4. After precipitating the Hb with 5%
trichloroacetic acid, radioactivity in the cells was determined by means of liquid
scintillation counting (Tri-Carb 4000, Packard Instrument Co., Downers Grove, IL).
41
Chapter II
Band 3 activity of PDT-treated cells is expressed as percentage of control (cells
without DIP or DMMB). Because DIP inhibits band 3 activity strongly, it must be
removed before anion uptake can be measured. Therefore, cells incubated with
DIP were centrifuged and resuspended to a 2% Hct solution in PBS containing 30
mM sucrose. After overnight incubation at 4°C, the cells were washed twice and
resuspended in PBS plus 1 mM Na2SO4. Band 3 activity was determined as
described previously.
GLUCOSE TRANSPORT
Efflux of glucose was measured as described by Harris23 and Sen and Widdas24,
with slight modifications. After illumination, cells were resuspended in buffer
containing 135 mM NaCl, 10 mM KCl, 10 mM Tris and 100 mM glucose
(Trisbuffered saline [TBS]–glucose), pH 7.4, at 10 % Hct. Cells were loaded with
0.25 μCi/mL D-[U-14C-glucose] for 5 min at room temperature. The labeled cells
were washed twice with TBS–glucose to remove free 14C-glucose. After
resuspension of the 14C-glucose–loaded cells in 180 mM NaCl plus 12.5 mM
glucose at 10% Hct, the efflux of 14C-glucose was determined. For this, samples
were taken every 30 s and centrifuged immediately for 10 s at 13.000 rpm. The
amount of radioactivity in the supernatant was determined by means of liquid
scintillation counting. Efflux was calculated as percentage of the total amount of
14
C-glucose taken up. Glucose transport activity is expressed as percentage of the
activity of control (cells without DIP and sensitizer). The specificity of the glucose
transport activity after DMMB-PDT was evaluated by incubating cells with the
glucose transport inhibitor Cytochalasine B (cb; 50 μM) for 10 min prior to loading
with 0.25 μCi/mL D-[U-14C-glucose]. After washing, cells were resuspended in
buffer plus 50 μM cb to maintain the inhibition during the measurement.
GLYCEROL TRANSPORT
Glycerol transport was measured by the hemolysis technique as described by
Naccache and Sha’afi.25 Briefly, RBCs were centrifuged and resuspended in 300
mM glycerol (2% Hct). Hemolysis was followed by the decreased scattering of the
suspension, as measured at 620 nm (DU-64 spectrophotometer; Beckman,
42
K+ leakage and damage to Band 3 in red cells
Fullerton, CA). Nonspecific glycerol transport was measured in the presence of 2
mM CuSO4, which completely inhibits the facilitated diffusion of glycerol.26 Active
glycerol transport of the treated cells is expressed as percentage of the activity in
control (cells without DIP and sensitizer).
STATISTICS
All experiments were performed three to five times with blood from different
donors. Paired t-tests were used to determine the significance of differences
between two groups. A p-value < 0.05 was considered significant. Means and
standard deviations are reported.
RESULTS
EFFECT OF DMMB-MEDIATED PDT ON MEMBRANE PERMEABILITY
Photo-oxidation of human RBCs with DMMB resulted in increased permeability of
the membrane toward cations, as measured by the leakage of K+. This effect was
detected already at low light doses. At 3 J/cm2, the leakage amounted to 20% (Fig.
1). None of the light doses used induced any hemolysis. The K+ leakage was
inhibited by 80% when DIP was present in the cell suspension during the PDT with
DMMB.
Figure 1. Effect of DIP on DMMB-mediated
+
photodynamically induced K leakage.
Ten percent erythrocyte suspensions in PBS and 30
mM sucrose were incubated in the dark with (U,S)
or without ({,z) 100 μM DIP. After 5 min, DMMB
(15 μM) was added (z,S) and the cells were
incubated in the dark for another 5 min. In control
cells ({), neither DIP nor DMMB was present. The
RBC suspensions were exposed to light doses of 0 to
2 +
9 J/cm . K leakage is expressed as percentage of the
total efflux evoked by lysis of cells in distilled water.
43
Chapter II
EFFECT OF DMMB-MEDIATED PDT ON BAND 3 ACTIVITY
DIP is known to inhibit band 3 activity strongly. To study the effect of PDT on band
3 activity, DIP should be removed from the cells before the transport of anions
can be measured. Therefore, cells containing DIP were subjected to extensive
washing. This washing procedure resulted in complete restoration of anion
transport activity in DIP-treated cells. The washing procedure had no effect on the
band 3 activity of control cells (without DIP). Band 3 activity was inhibited strongly
by DMMB-mediated PDT (Fig. 2). At 3 J/cm2, a 30% inhibition of anion transport
was observed, which increased to over 60% at 9 J/cm2. When phototreatment was
performed in the presence of DIP, even at a light dose of 9 J/cm2, band 3 activity
was not inhibited, and was maintained at the level of control cells (cells without
DMMB or DIP).
Figure 2. Protective effect of DIP against DMMBinduced inhibition of band 3 activity.
Ten percent erythrocyte suspensions in PBS and
30 mM sucrose were incubated in the dark with
(U,S) or without (z) 100 μM DIP. After 5 min,
DMMB (15 μM) was added (z,S), and the cells
were incubated in the dark for another 5 min.
The RBC suspensions were exposed to light
2
doses of 0 to 9 J/cm . Samples treated with DIP
were washed before band 3 activity was
measured. Band 3 activity is given as percentage
of control cells in which neither DIP nor DMMB
were present.
EFFECT OF DMMB-MEDIATED PDT ON GLUCOSE TRANSPORT ACTIVITY
Incubation of the RBCs with DIP, which is known to bind with low affinity to the
erythrocyte glucose transporter27, has a small, but significant (P< 0.05), inhibitory
effect on the glucose transport activity. In contrast to the results obtained with
protoporphyrin, DMMB-mediated photosensitization of red cells did not affect the
activity of the glucose transporter (Fig. 3). Even at light doses that induced 90%
44
K+ leakage and damage to Band 3 in red cells
leakage of K+ (9 J/cm2), glucose transport activity was not inhibited significantly.
The efflux as measured at this high light dose was not because of nonspecific
leakage of glucose. Cytochalasine B, an inhibitor of the Glut-1 glucose
transporter28, inhibited glucose exchange in treated cells to an extent comparable
to that in control cells, suggesting that the protein conformation was not affected
by DMMB-PDT.
Figure 3. Effect of DMMB-PDT on the glucose transport of RBCs.
Ten percent erythrocyte suspensions in PBS and 30 mM sucrose were incubated in
the dark for 5 min with DIP only (DIP), with DIP and DMMB (DIP & DMMB) or with
DMMB only (DMMB). [DIP] = 100 μM, [DMMB] = 15 μM. Glucose transport of the
2
treated cells was measured immediately after light exposure of 9 J/cm . Control for
specificity of the activity measurement was performed with the use of 50 mM
Cytochalasine B (cb) after incubation with DMMB in the dark or exposed to a light
2
dose of 9 J/cm . Glucose transport activity is given as percentage of control in which
neither DIP nor DMMB were present. ** P < 0.05.
EFFECT OF DMMB-MEDIATED PDT ON GLYCEROL TRANSPORT ACTIVITY
After an initial lag phase, glycerol transport was inhibited rapidly by DMMBmediated photosensitization (Fig. 4). In contrast to what was observed with band
3 activity, DIP only partially protected against DMMB-mediated photodamage to
the glycerol transport. DIP itself had no effect on glycerol transport activity. The
45
Chapter II
measured transport activity after DMMB-PDT was carrier mediated, as checked
with the use of CuSO4, a specific blocker of the carrier. Passive transport was
measured in this case and was negligible in comparison with the active transport.
After 6 J/cm2, the active transport was equal to the passive transport, suggesting a
total inhibition of the carrier by DMMB-PDT (data not shown).
Figure 4. Partial protective effect of DIP against
DMMB-induced inhibition of glycerol transport.
Ten percent erythrocyte suspensions in PBS and 30
mM sucrose were incubated in the dark with (U,S)
or without ({,z) 100 μM DIP. After 5 min, DMMB
(15 μM) was added (z,S) and the cells were
incubated in the dark for another 5 min. In control
cells ({), neither DIP nor DMMB were present. The
RBC suspensions were exposed to light doses of 0 to
2
9 J/cm . Measurement of glycerol transport activity
occurred immediately after light exposure from 0 to
2
9 J/cm . Glycerol transport activity is given as
percentage of control.
DISCUSSION
A damaged band 3 has been suggested as being responsible for various forms of
RBC lysis, including immunolysis29, hyperthermic lysis30, glycerol lysis31 and
osmotic lysis.32,33 Based on the observation that photohemolysis obeys a secondorder power dependence on light dose, and on the great potency of eosin
isothiocyanate to sensitize photohemolysis, Pooler suggested a role in
photohemolysis of band 3 protein, which exists as a dimer in the membrane.19
Recently, VanSteveninck et al.20 showed that the band 3 ligand DIP could protect
RBCs against photodynamically induced K+ leakage and delayed hemolysis. This
again suggested an involvement of damage to band 3 in increased cation
permeability and photohemolysis. Based on the comparable light dose
dependency and response to DIP, we were able to show a direct correlation
between DMMB-mediated damage to band 3 and K+ leakage. Already, at a light
dose of 3 J/cm2, there was considerable K+ leakage and inhibition of band 3
46
K+ leakage and damage to Band 3 in red cells
activity, as measured by decreased sulfate transport. Both K+ leakage and
inhibition of band 3 activity were completely prevented when illumination was
performed in the presence of DIP. These results suggest strongly that DMMBmediated photodamage to band 3 is indeed a causal factor in inducing K+ leakage.
The study of the transport systems for glucose and glycerol showed that they
were not involved in the onset of DMMB-photodynamically induced K+ leakage.
Glucose transport was not affected by the PDT. Neither the rate of glucose efflux
nor the ability of cytochalasin B to inhibit glucose transport was decreased. The
facilitated diffusion of glycerol was inhibited by DMMB-mediated PDT. However,
an involvement of this transport system in K+ leakage is highly unlikely because of
the difference in light dose dependency. At a light dose of 3 J/cm2, K+ leakage was
already initiated, whereas glycerol transport was not affected. Notwithstanding
the fact that DIP has no known preference for the glycerol transporter,
photoinhibition of glycerol lysis was prevented partially by the presence of DIP in
the illumination mixture. This was not caused by a general stabilization of the red
cell membrane, because addition of DIP after the PDT had no effect on the
inhibition of glycerol transport (data not shown). An alternative explanation for
the protective effect of DIP could be the involvement of band 3 in glycerol
transport as previously suggested.31,34,35 Indeed, it has been observed that
erythrocytes from patients suffering from systemic lupus erythomatosus have a
decreased glycerol transport activity, which correlated with a decreased quantity
of band 3 protein in the membrane.34 Further evidence for the involvement of
band 3 in glycerol transport is provided by the observation that the classical band
3 transport inhibitor 4.49-diisothiocyanostilbene-2,29-disulfonate also inhibits
glycerol transport.31,35 Not all band 3 inhibitors influence glycerol transport
activity.
From our results it is clear that DIP has no effect on the glycerol transport activity
(Fig. 3), which is in agreement with the finding that phenylglyoxal and 4.49dinitrostilbene-2,29-disulfonate, which bind at the same location in band 3 as
DIP36, also had no effect on glycerol transport activity.35 We hypothesize that the
observed inhibition of glycerol transport activity by DMMB-mediated PDT is, at
least partly, because of damage to band 3, and that DIP prevents such damage. In
conclusion, our results are the first to show a direct correlation between DMMB47
Chapter II
mediated photodamage to band 3 and K+ leakage. It remains to be elucidated
whether this is specific for DMMB or is a general phenomenon for all
photosensitizers, and whether band 3 is the only critical target. Also, it has to be
established which photodamaged residues in band 3 are responsible for the
leakage of potassium.
REFERENCES
1. Moor AC, Dubbelman TM, VanSteveninck J, Brand A. Transfusion-transmitted
diseases: risks, prevention and perspectives. Eur. J. Haemotol. 1999;62(1):1-18.
2. Mannucci PM, Colombo M. Virucidal treatment of clotting factor concentrates.
Lancet 1988;2(8614):782-5.
3. Gomperts ED. Procedures for the inactivation of viruses in clotting factor
concentrates. Am. J. Hematol. 1986;23(3):295-305.
4. Corash L. Inactivation of viruses, bacteria, protozoa, and leukocytes in platelet
concentrates. Vox Sang. 1998;74(Suppl 2):173-6.
5. Nitzan Y, Wexler HM, Finegold SM. Inactivation of anaerobic bacteria by various
photosensitized porphyrins or by hemin. Curr. Microbiol. 1994;29(3):125-31.
6. Mohr H, Bachmann B, Klein-Struckmeier A, Lambrecht B. Virus inactivation of blood
products by phenothiazine dyes and light. Photochem. Photobiol. 1997;65(3):441-5.
7. Rywkin S, Ben-Hur E, Reid ME, Oyen R, Ralph H, Horowitz B. Selective protection
against IgG binding to red cells treated with phthalocyanines and red light for virus
inactivation. Transfusion 1995;35(5):414-20.
8. Sieber F, O'Brien JM, Gaffney DK. Merocyanine-sensitized photoinactivation of
enveloped viruses. Blood Cells 1992;18(1):117-27.
9. Smetana Z, Ben-Hur E, Mendelson E, Salzberg S, Wagner P, Malik Z. Herpes simplex
virus proteins are damaged following photodynamic inactivation with
phthalocyanines. J. Photochem. Photobiol. B 1998;44(1):77-83.
10. Moor AC, van der Veen A, Dubbelman TM, VanSteveninck J, Brand A. Photodynamic
sterilization of red cells and its effect on contaminating white cells: viability and
mechanism of cell death. Transfusion 1999;39(6):599-607.
48
K+ leakage and damage to Band 3 in red cells
11. Perrotta PL, Baril L, Tead C, Chapman J, Dincecco D, Buchholz DH, Snyder EL. Effects
of methylene blue-treated plasma on red cells and stored platelet concentrates.
Transfusion 1999;39(1):63-9.
12. Wagner SJ, Skripchenko A, Robinette D, Mallory DA, Hirayama J, Cincotta L, Foley J.
The use of dimethylmethylene blue for virus photoinactivation of red cell
suspensions. Dev. Biol. Stand. 2000;102:125-9.
13. Moor AC, Wagenaars-van Gompel AE, Brand A, Dubbelman MA, VanSteveninck J.
Primary targets for photoinactivation of vesicular stomatitis virus by AIPcS4 or Pc4
and red light. Photochem. Photobiol. 1997;65(3):465-70.
14. Rywkin S, Ben-Hur E, Malik Z, Prince AM, Li YS, Kenney ME, Oleinick NL, Horowitz B.
New phthalocyanines for photodynamic virus inactivation in red blood cell
concentrates. Photochem. Photobiol. 1994;60(2):165-70.
15. Skripchenko AA, Robinette D, Wagner SJ. Comparison of methylene blue and
methylene violet for photoinactivation of intracellular and extracellular virus in red
cell suspensions. Photochem. Photobiol.1997;65(3):451-5.
16. Wagner SJ, Skripchenko A, Robinette D, Foley JW, Cincotta L. Factors affecting virus
photoinactivation by a series of phenothiazine dyes. Photochem. Photobiol.
1998;67(3):343-9.
17. Wagner SJ, Skripchenko A, Robinette D, Mallory DA, Cincotta L. Preservation of red
cell properties after virucidal phototreatment with dimethylmethylene blue
[published erratum appears in Transfusion 1998 Oct;38(10):996]. Transfusion
1998;38(8):729-37.
18. Dubbelman TM, de Goeij AF, van Steveninck J. Protoporphyrin-induced
photodynamic effects on transport processes across the membrane of human
erythrocytes. Biochim. Biophys. Acta 1980;595(1):133-9.
19. Pooler JP. A new hypothesis for the target in photohemolysis: dimers of the band 3
protein. Photochem. Photobiol. 1986;43(3):263-6.
20. VanSteveninck J, Trannoy LL, Besselink GAJ, Dubbelman TMAR, Brand A, Korte Dd,
Verhoeven AJ, Lagerberg JWM. Selective Protection of erythrocytes against
photodynamic damage by the band3 ligand Dipyridamol. Transfusion
2000;40(11):1330-6.
21. Dubbelman TM, de Bruijne AW, Christianse K, van Steveninck J. Hypertonic
cryohemolysis of human red blood cells. J. Membr. Biol. 1979;50(3-4):225-40.
49
Chapter II
22. Knauf PA, Rothstein A. Chemical modification of membranes. I. Effects of sulfhydryl
and amino reactive reagents on anion and cation permeability of the human red
blood cell. J. Gen. Physiol. 1971;58(2):190-210.
23. Harris EJ. An analytical study of the kinetics of glucose movement in human
erythrocytes. J. Physiol. 1964;173:344-53.
24. Sen AK, Widdas WF. Determinations of the temperature and pH dependence of
glucose transfer across the human erythrocyte membrane meaured by glucose exit.
J. Physiol. 1962;160:392-403.
25. Naccache P, Sha'afi RI. Patterns of nonelectrolyte permeability in human red blood
cell membrane. J. Gen. Physiol. 1973;62(6):714-36.
26. Carlsen A, Wieth JO. Glycerol transport in human red cells. Acta Physiol. Scand.
1976;97(4):501-13.
27. Hellwig B, Joost HG. Differentiation of erythrocyte-(GLUT1), liver-(GLUT2), and
adipocyte- type (GLUT4) glucose transporters by binding of the inhibitory ligands
cytochalasin B, forskolin, dipyridamole, and isobutylmethylxanthine. Mol.
Pharmacol. 1991;40(3):383-9.
28. Vasianin SI, Vinogradova NA. [Transport of monosaccharides in model systems:
inhibition of sugar transport in frog muscle fibers, treated with glutaraldehyde]
Transport monosakharidov v modelnykh sistemakh: ingibirovanie transporta
sakharov v myshechnykh voloknakh liagushki, obrabotannykh glutaral'degidom.
Tsitologiia 1989;31(7):856-60.
29. Kannan R, Labotka R, Low PS. Isolation and characterization of the hemichromestabilized membrane protein aggregates from sickle erythrocytes. Major site of
autologous antibody binding. J. Biol. Chem. 1988;263(27):13766-73.
30. Lepock JR, Frey HE, Bayne H, Markus J. Relationship of hyperthermia-induced
hemolysis of human erythrocytes to the thermal denaturation of membrane
proteins. Biochim. Biophys. Acta 1989;980(2):191-201.
31. Sauer A, Kurzion T, Meyerstein D, Meyerstein N. Kinetics of hemolysis of normal
and abnormal red blood cells in glycerol-containing media. Biochim. Biophys. Acta
1991;1063(2):203-8.
32. Salhany JM, Rauenbuehler PB, Sloan RL. Alterations in pyridoxal 5'-phosphate
inhibition of human erythrocyte anion transport associated with osmotic hemolysis
and resealing. J. Biol. Chem 1987;262(33):15974-8.
50
K+ leakage and damage to Band 3 in red cells
33. Sato Y, Kamo S, Takahashi T, Suzuki Y. Mechanism of free radical-induced hemolysis
of human erythrocytes: hemolysis by water-soluble radical initiator. Biochemistry
1995;34(28):8940-9.
34. Hashimoto N, Fujita S, Yokoyama T, Ozawa Y, Kingetsu I, Kurosaka D, Sabolovic D,
Schuett W. Cell electrophoretic mobility and glycerol lysis of human erythrocytes in
various diseases. Electrophoresis 1998;19(7):1227-30.
35. Zou CG, Agar NS, Jones GL. Haemolysis of human and sheep red blood cells in
glycerol media: the effect of pH and the role of band 3. Comp. Biochem. Physiol. A
Mol. Integr. Physiol. 2000;127(3):347-53.
36. Falke JJ, Chan SI. Molecular mechanisms of band 3 inhibitors. 3. Translocation
inhibitors. Biochemistry 1986;25(24):7899-906.
51
52
CHAPTER III
SELECTIVE PROTECTION OF RBCS
AGAINST PHOTODYNAMIC
DAMAGE BY THE BAND 3 LIGAND
DIPYRIDAMOLE
John vanSteveninck1, Laurence L. Trannoy1,2, Geert A.J. Besselink3, Tom M.A.R.
Dubbelman4, Anneke Brand2, Dirk de Korte3, Arthur J. Verhoeven3,
and Johan W.M. Lagerberg4
Departments of Molecular Cell Biology, Leiden University Medical Center; 2
Immunohematology & Blood bank, the Red Cross Bloodbank Leiden-Haaglanden,
Leiden; 3 Department of Transfusion Technology, CLB, Sanquin Blood Supply
Foundation, Amsterdam; 4 Boston Clinics PDT BV, Leiden
1
In memoriam to John van Steveninck
Adapted from Transfusion 2000; 40; 1330-1336
53
Chapter III
ABSTRACT
BACKGROUND: All studied photosensitizers for virus inactivation impair RBCs. To
reduce damage to the RBCs without affecting virucidal activity, selective
protection of the RBCs is necessary. The ability of the band 3 ligand, dipyridamole
(DIP), to react with singlet oxygen and to increase the selectivity of
photodecontamination was investigated.
STUDY DESIGN AND METHODS: Solutions of DIP were illuminated in the presence
of AlPcS4 and DMMB. Solutions of amino acids, RBCs, and vesicular stomatitis
virus (VSV) in RBC suspensions were photodynamically treated in the presence or
absence of DIP.
RESULTS: Illumination of a solution of DIP in the presence of AlPcS4 or DMMB
resulted in changes in the optical spectrum of DIP. The photooxidation of DIP was
inhibited by azide and augmented by deuterium oxide, which suggests the
involvement of singlet oxygen. Photooxidation of amino acids and photodamage
to RBCs was strongly reduced in the presence of DIP. In contrast,
photoinactivation of VSV in RBC suspensions was only slightly affected by DIP.
CONCLUSION: DIP can improve the specificity of photodynamic decontamination
of RBCC, thereby increasing the practical applicability of this
photodecontamination method.
54
Selective protection by Dipyridamole
INTRODUCTION
The safety of blood transfusion components is a major issue in transfusion
medicine practice. Improved donor screening and the implementation of
diagnostic screening of blood samples have substantially reduced the risk of virus
transmission by transfusion. However, mainly because of window-period
donations, residual risk exists.1 For plasma components, the risk has been reduced
considerably by the application of virucidal treatments such as heating or
solvent/detergent (SD) treatment.2,3 These procedures, however, are not
applicable to cellular blood components, because of the fragility of the cells. The
use of light-activated agents, photosensitizers, is widely studied for the
inactivation of viruses in cellular blood components, especially RBCC.4-6 Upon
activation of the photosensitizer with visible light, various reactive oxygen species
are formed. Although photosensitizers can mediate both type I (charge transfer,
radicals) and type II (energy transfer, singlet oxygen [1O2]) reactions, most
photosensitizers are considered to act mainly via 1O2. It is able to inactivate a
broad range of enveloped viruses.4 However, as 1O2 is not selective and can be
generated in all locations where sensitizer molecules are present, phototreatment
can also damage RBCs. This means that photosensitizers with high affinity for virus
particles must be used to protect the RBCs from photodynamic damage.
Promising results have been obtained with phenothiazine dyes, such as MB and
DMMB6,10,11 , and with certain phthalocyanines.12-14 However, all photosensitizers
tested so far also induce damage to the RBCs, as detected by an increased
permeability of the membrane to cations. To minimize the damage to RBCs
induced by photodynamic pathogen inactivation, we searched for chemicals that
bind preferentially to the RBC membrane and react readily with 1O2. An obvious
way to achieve effective binding of candidate scavengers is the use of band 3
ligands. Band 3 constitutes the anion transporter of the RBC, and, with about a
million copies per cell, it is the most abundant protein on the RBC membrane. An
additional argument for using scavengers that bind to band 3 is the proposed
involvement of band 3 damage in photohemolysis.15 Band 3 is widely studied and
many ligands are known. A well-known ligand for band 3 is DIP (Fig. 1). DIP binds
ŶŽŶĐŽǀĂůĞŶƚůLJ͕ǁŝƚŚŚŝŐŚĂĨĨŝŶŝƚLJ;<сϭ͘ϮʅDͿƚŽďĂŶĚϯĂŶĚǁŝƚŚƐƚŽŝĐŚŝŽŵĞƚƌLJ
55
Chapter III
of one molecule of DIP per band 3 dimer. The inhibition of anion transport is not
due to binding of DIP to the anion-binding side, but to blocking of the anion
channel.16 Besides its use as ligand for band 3, DIP is widely used in the treatment
of cardiovascular diseases because of its vasodilating activity and its ability to
inhibit platelet aggregation.17 It has previously been reported that DIP also exerts
an inhibitory effect on lipid peroxidation18,19 and possesses activity as a scavenger
of superoxide anion, hydroxyl radical, and peroxyl radical.18,20 These properties
make DIP a promising candidate for selective protection of RBCs against
photodynamic damage. The aim of the present study is to investigate whether DIP
is able to react with photodynamically formed 1O2, thereby protecting substrates
against photodynamic damage. In addition, the possibility of using DIP for
selective protection of RBCs against virucidal PDT was investigated. For this we
used AlPcS4 and DMMB, both of which are photosensitizers whose role in virus
inactivation in RBC suspensions is well studied.
Figure 1. Structure of dipyridamole
MATERIALS AND METHODS
MATERIALS
DIP (2,6-bis(diethanolamino)-4,8-dipiperidino-[5,4-d]pyrimidine) was purchased
(Sigma-Aldrich BV, Zwijndrecht, the Netherlands). Stock solutions (20 mM) were
prepared in ethanol. AlPcS4 was obtained from Porphyrin Products (Logan, UT).
VSV (San Juan strain) and the host cells A549 were provided by the departments
of Virology and Lung Diseases respectively (Leiden University Medical Center,
Leiden, the Netherlands). A549 cells were cultured in medium (RPMI 1640)
56
Selective protection by Dipyridamole
supplemented with 10-percent fetal bovine serum (Gibco BRL, Breda, the
Netherlands). All other chemicals were obtained from Sigma-Aldrich and were of
the highest purity available. Heparinized human blood, provided by the regional
Red Cross Blood Bank (Leiden-Haaglanden, the Netherlands), was centrifuged
shortly after collection. The RBCs were washed three times with 150 mM NaCl and
20 mM HEPES, pH 7.6, and resuspended at 2-percent Hct in the same buffer.
PHOTODYNAMIC TREATMENT
For all illuminations, the light source used was a slide projector with a 150 W lamp
(HLX 64640, Xenophot, Osram, Germany).21 To avoid light absorption by Hb and
DIP, a cutoff filter, transmitting only light with a wavelength above 590 nm, was
used in all experiments. Both AlPcS4 (maximum absorption at 675 nm) and DMMB
(maximum absorption at 652 nm) strongly absorb in this region. The irradiance
was 350 W per m2, as measured with a photometer (IL1400A, International Light,
Newburyport, MA) equipped with a detector (SELO33, International Light). All
illuminations were performed at a constant temperature of 20°C under
continuous mixing.
Solutions of DIP ;ϭϬ ʅMͿ ǁĞƌĞ ŝůůƵŵŝŶĂƚĞĚ ŝŶ ƚŚĞ ƉƌĞƐĞŶĐĞ ŽĨ ϭϬ ʅM AlPcS4.
Absorption spectra of DIP after different illumination times were recorded with a
spectrophotometer (DU-64, Beckman, Fullerton, CA). Suspensions of RBCs (2%
Hct) either with or without VSV (105 infectious particles/mL) were incubated with
100 ʅM DIP ĂƚϮϬΣŝŶƚŚĞĚĂƌŬ͘ĨƚĞƌϱŵŝŶƵƚĞƐ͕ϭʅM AlPcS4 or DMMB was added
to the suspension. After another 5 minutes of incubation, the suspensions were
illuminated for different periods.
OXYGEN CONSUMPTION
Oxygen consumption during illumination was measured with an oxygen monitor
(YSI, Yellow Springs, OH) equipped with a Clark-type electrode. A 3-mL sample of
histidine (2 mM in 150 mM NaCl/1% Triton-X100/20 mM HEPES, pH 7.6) was
ŝůůƵŵŝŶĂƚĞĚ ŝŶ ƚŚĞ ƉƌĞƐĞŶĐĞ ŽĨ Ϯ͘ϱ ʅM AlPcS4 Žƌ Ϭ͘ϱ ʅM DMMB plus various
amounts of DIP at a constant temperature of 20°C under continuous mixing. The
rate of oxygen consumption was calculated from the initial slope of the curve.
57
Chapter III
POTASSIUM LEAKAGE AND DELAYED HEMOLYSIS
K+ leakage from RBCs was determined immediately after illumination by means of
a flame photometer (Clinical Flame Photometer 410C, Corning, Halstead, UK). To
study delayed hemolysis, RBCs were treated with a light dose inducing minimal K+
leakage. The light dose used with DMMB was 75 kJ per m2, and that used with
AlPcS4 was 200 kJ per m2. After treatment, the cells were centrifuged,
resuspended in fresh buffer, and stored in the dark at room temperature. After
different storage times, released Hb was determined by measuring the OD of the
supernatant at 540 nm using a DU-64 spectrophotometer. Both K+ and Hb release
were expressed as a percentage of the total efflux evoked by lysis of the cells in
distilled water.
VSV INFECTIVITY ASSAY
Assay of the infectivity of VSV by an endpoint dilution assay was performed as
described previously.14 In short, VSV samples were serially diluted (10 times),
inoculated into A549 cell cultures in 96-well microtiter plates (Greiner, Alphen a/d
Rijn, the Netherlands), and incubated for 72 hours. The cytopathology of the cells
was scored in eight well replicates for each dilution. Quantification of the virus
titer was performed according to the Spearman-Karber method.22
STATISTICS
All experiments were performed at least four times with blood from different
donors. Values are expressed as mean ± standard error.
RESULTS
PHOTOOXIDATION OF DIP
It has previously been shown that oxidation of DIP with hydroxyl radicals results in
a changed optical spectrum of DIP.23 To investigate whether 1O2 also can induce
58
Selective protection by Dipyridamole
oxidation of DIP, a solution of DIP ;ϭϬʅM) was illuminated in the presence of 10
ʅM AlPcS4.
After application of different light doses, the optical spectrum of DIP was
recorded. The characteristic yellow color of DIP (absorption maximum at 410 nm)
disappeared gradually upon illumination, while at 270 nm a new absorption
maximum appeared (Fig. 2). Illumination, also with white light, in the absence of
AlPcS4 had no effect on the optical spectrum of DIP, which indicates that DIP does
not act as a photosensitizer. The photodynamically induced degradation of DIP
was slowed by the addition of 1 mM sodium azide (Fig. 3). When the buffer was
made in deuterium oxide (D2O) instead of in H2O, photodynamic degradation was
strongly augmented (Fig.3). Comparable results were obtained with DMMB (2
ʅM) as photosensitizer (results not shown).
Figure 2. Effect of photooxidation on the spectral properties of DIP.
A solution of DIP (10 μM in 150 mM NaCl/10 mM Hepes, pH 7.6) was illuminated in
the presence of 10 μM AlPcS4. After 0 ({), 20 (U), 40 (V), 60 (z) and 100 (S)
2
kJ/m , the optical spectrum of DIP was recorded using a Beckman DU64
spectrophotometer.
59
Chapter III
Figure 3. Effects of sodium azide and deuterium oxide on the photooxidation of
dipyridamole.
A solution of DIP (10 μM in 150 mM NaCl/10 mM Hepes, pH 7.6) was illuminated in
the presence of 10 μM AlPcS4. ({): no extra additions, (S): 1 mM sodium azide
added, (z): H2O replaced by D2O.
EFFECT OF DIP ON THE PHOTOOXIDATION OF HISTIDINE
Illumination of a 2 mM solution of histidine in the presence of AlPcS4 ;Ϯ͘ϱʅM) or
DD;Ϭ͘ϱʅM) resulted in a rapid decrease in the oxygen content of the buffer.
The rate of oxygen consumption in the absence of DIP ĂŵŽƵŶƚĞĚƚŽϰϴцϰʅM of
O2 per minute for AlPcS4 and to ϱϲ ц ϲ ʅM of O2 per minute for DMMB. The
inclusion of increasing amounts of DIP in the illumination mixture resulted in a
decreasing rate of photooxidation of histidine (Fig. 4). Comparable results were
obtained when the amino acids tryptophan, methionine, and tyrosine or the DNA
base guanosine was used as substrate. Illumination in the absence of substrate, in
either the presence or absence of DIP, induced no significant oxygen
consumption. In addition, no changes in the optical properties of DIP were
60
Selective protection by Dipyridamole
observed, which indicates that, under these conditions of low sensitizer
concentration and high DIP concentration, DIP was not significantly oxidized.
Figure 4. Effect of dipyridamole on the photooxidation of histidine.
A histidine solution (2 mM in 150 mM NaCl/10 mM Hepes/1% Triton-X100,
pH 7.6) was illuminated in the presence of 2.5 μM AlPcS4 ({) or 0.5 μM
DMMB (z) with increasing concentrations of DIP. The initial oxygen
consumption during illumination was recorded using a Clark-type electrode.
The oxygen consumption rates obtained in the absence of DIP were
normalized to 100%.
EFFECT OF DIP ON PHOTODYNAMICALLY INDUCED DAMAGE TO RBCS
To investigate whether the presence of DIP can protect RBCs against
photodynamic damage, RBC suspensions (2% Hct) were incubated in the dark for
5 minutes with DIP before photosensitizer was added. As can be seen from Fig. 5,
both AlPcS4 and DMMB induced damage to the RBCs upon illumination, as
reflected by the increased concentration of extracellular K+. Neither illumination
without photosensitizer nor incubation in the dark with photosensitizer induced
any K+ leakage. Incubation of the RBCs with DIP strongly reduced the
61
Chapter III
photodynamically induced K+ leakage (Fig. 5). However, the magnitude of this
reduction differed strongly with the photosensitizer used. The protection factor,
defined as the ratio between the light doses needed to induce 50-percent K+
leakage in the presence and in the absence of DIP, for AlPcS4 amounted to 1.5,
while that with DMMB was 4.5. The effect of DIP on delayed hemolysis induced by
PDT is depicted in Fig. 6. In the absence of DIP, both PDTs induced complete
hemolysis 10 hours after their use. When illumination was conducted in the
presence of DIP, hemolysis was strongly delayed. With AlPcS4 as sensitizer,
complete hemolysis was obtained 24 hours after PDT. With DMMB, the protective
effect of DIP was even more pronounced: 30 hours after PDT, hemolysis
amounted to less than 10 percent. Because of its lipophilic nature, DIP can be
inserted between phospholipids.24 The insertion of molecules in the membrane
results in membrane expansion, which can result in protection of the RBC against
osmotic hemolysis.25,26 To investigate the involvement of membrane expansion in
the protection, DIP was added to the RBC suspension after the PDT. In this case,
DIP had no effect on delayed hemolysis (not shown). These results imply that it is
very likely that the protective effect of DIP is due to its scavenging properties.
Figure 5. Effect of dipyridamole on photodynamically
induced potassium leakage
from RBCs.
Two percent-erythrocyte suspensions were incubated in
the dark without (open symbols) or with 100 μM DIP
(closed symbols). After 5 min,
the suspensions were exposed to light in the presence
of 1 μM AlPcS4 ({,z) or 1 PM
DMMB (U,S).
62
Selective protection by Dipyridamole
Figure 6. Effect of dipyridamole on photodynamically induced delayed hemolysis.
Two percent-erythrocyte suspensions were incubated in the dark without (open
symbols) or with 100 μM dipyridamole (closed symbols). After 5 min, the
suspensions were exposed to light in the presence of 1 μM AlPcS4 (‫ړ‬,‫ )ڗ‬or 1 PM
2
2
DMMB (‫ټ‬,‫)ٻ‬. After a light dose of 200 kJ/m or 75 kJ/m respectively, the cells were
washed, resuspended in fresh PBS (without dipyridamole or sensitizer) and stored at
room temperature in the dark.
EFFECT OF DIP ON THE PHOTOINACTIVATION OF VSV IN RBC
SUSPENSIONS
From the above, it is clear that DIP strongly protects RBCs against photodamage.
To investigate whether DIP can increase the specificity of a photody-namic
virucidal treatment, the effect of DIP on the photoinactivation of VSV was
investigated. For this, VSV was spiked (105 infectious particles/mL) in 2 percent
RBC suspensions. The suspensions were incubated in the dark for 5 minutes with
DIP before the photosensitizer was added. After different light doses, samples
were taken to assay for viral infectivity. As can be seen from Fig. 7, both AlPcS4and DMMB-mediated photoinactivation of VSV was only slightly affected by DIP.
When measured directly after the treatment, the light doses needed for 5 log
inactivation of VSV induced only very limited leakage of potassium out of the
RBCs; this indicated the potential use of these photosensitizers for
photodecontamination of RBCC.
63
Chapter III
Figure 7. Effect of dipyri-damole on photodynamic inactivation of VSV.
5
VSV (10 infectious particles / mL) in 2 % erythrocyte suspensions, were incubated
without (open symbols) or with 100 μM DIP (closed symbols). After 5 min, the
suspensions were exposed to light in the presence of 1 μM AlPcS4 ({,z) or 1 PM
DMMB (U,S).
DISCUSSION
The cardiovascular drug DIP is known to be a scavenger of superoxide anions,
hydroxyl radicals, and peroxyl radicals.18,20 The present study is the first to show
that DIP can also react with 1O2, the most important reactive oxygen species
formed upon PDT. With illumination of a solution of DIP ;ϭϬʅM) in the presence
of AlPcS4 ;ϭϬʅMͿŽƌDD;ϮʅM) as photosensitizer, the optical spectrum of DIP
changes (Fig. 2), and this indicates a chemical reaction between DIP and a
photogenerated reactive species. The involvement of 1O2 in this reaction was
demonstrated by both the inhibitory effect of the 1O2 scavenger azide and the
faster oxidation in D2O (Fig. 3). Because of the much longer lifetime of 1O2 in D2O
than in H2O, reactions involving 1O2 are strongly accelerated in D2O.27 The changes
in the optical spectrum of DIP differ from those obtained with DIP exposed to
hydroxyl radicals, which indicates the formation of different reaction products.23
The exact nature of the reaction product(s) and the possible intermediates
remains to be elucidated. DIP is able to protect various substrates (histidine,
64
Selective protection by Dipyridamole
tryptophan, methionine tyrosine, and guanosine), which are known to be oxidized
by 1O2,28,29 against photooxidation (Fig. 4). Under the conditions used in these
experiments, no change in the optical spectrum of DIP could be observed. This
suggests that, under certain circumstances; that is, a high concentration of DIP
and a low concentration of 1O2; DIP reacts with 1O2 without being chemically
changed. This physical interaction can most likely be attributed to the amine
groups present in DIP, which are well known physical quenchers of 1O2.30 The
following mechanism for the reactions of DIP with 1O2 is suggested:
DIP + 1O2
[DIP---O2]
DIP + O2
+ 1O2
oxidized DIP
The present study proposed to investigate whether the cardiovascular drug DIP is
able to selectively protect RBCs against damage induced by virucidal PDT. DIP
binds with high affinity to the anion transport protein, band 3, the most abundant
protein on the RBC membrane. This means that the concentration of 1O2
scavengers in the vicinity of the RBC is higher than in the rest of the solution,
which makes feasible the selective protection of RBCs against PDT. It was shown
that DIP strongly protects RBCs against damage induced by PDT. The conditions
used in these experiments are quite similar to those used in the photooxidation of
histidine, which suggests that the protection of RBCs is due to the physical
quenching of 1O2 by DIP. The extent of RBC protection by DIP strongly depends on
the sensitizer used to induce the RBC damage. With DMMB as photosensitizer,
the protection by DIP was much stronger than with AlPcS4. A feasible explanation
for these differences in protection by DIP could be the localization of the
photosensitizers. For effective scavenging, the activated photosensitizer and DIP
should be in proximity to each other. DIP is known to bind in the hydrophobic
interior of band 3.16 Because of its hydrophilic nature it is very unlikely that AlPcS4
will be located in proximity to DIP.
65
Chapter III
DMMB is much more hydrophobic and possibly is located in the same
microenvironment as DIP, which makes effective 1O2 scavenging feasible. To study
the effect of DIP on the inactivation of viruses, we used the model virus, VSV,
which was shown to be as sensitive to photoinactivation as HIV.10 In contrast to
the very strong protection found with RBCs, DIP caused hardly any decrease in the
AlPcS4- or DMMB-mediated photoinactivation of VSV. Because it has been shown
that DIP binds with low affinity (950-1150 M–1) to lipid bilayers,24 the binding of
DIP to VSV cannot be excluded. But because DIP does not interfere with the
photoinactivation of VSV, it must be concluded that the binding of DIP is not in
the proximity of the critical target for virus photoinactivation. The present results
were obtained by illumination of a model virus in a severely diluted RBC
suspension (2% Hct). Further research is focused on the effect of DIP on the
photoinactivation of other viruses, including HIV-1/2 and HCV, in a more
concentrated RBC suspension. In addition, the effect of DIP on the shelf-life of
photodynamically decontaminated RBCC must be investigated. Thus, the present
study shows that DIP has the potency to become a very valuable tool to increase
the specificity of the pathogen inactivation of RBCC, thereby increasing the
practical applicability of this photodecontamination method.
REFERENCES
1. Moor AC, Dubbelman TM, vanSteveninck J, et al. Transfusion-transmitted diseases:
risks, prevention and perspectives. Eur. J. Haematol. 1999;62:1-18.
2. Mannucci PM, Colombo M. Virucidal treatment of clotting factor concentrates. Lancet
1988;2:782-5.
3. Gomperts ED. Procedures for the inactivation of viruses in clotting factor concentrates.
Am. J. Hematol. 1986;23:295-305.
4. Ben-Hur E, Moor AC, Margolis-Nunno H, et al. The photodecontamination of cellular
blood components: mechanisms and use of photosensitization in transfusion medicine.
Transfus. Med. Rev. 1996;10:15-22.
5. Moor AC, van der Veen A, Wagenaars-van Gompel AE, et al. Shelf-life of
photodynamically sterilized RBC concentrates with various numbers of white cells.
Transfusion 1997;37:592-600.
66
Selective protection by Dipyridamole
6. Wagner SJ, Skripchenko A, Robinette D, et al. Preservation of red cell properties after
virucidal phototreatment with dimethylmethylene blue. Transfusion 1998;38:729-37.
7. Spikes JD. Porphyrins and related compounds as photodynamic sensitizers. Ann. N Y
Acad. Sci. 1975;244:496-508.
8. Michaeli A, Feitelson J. Reactivity of singlet oxygen toward amino acids and peptides.
Photochem. Photobiol. 1994;59:284-9.
9. Redmond RW, Gamlin JN. A compilation of singlet oxygen yields from biologically
relevant molecules. Photochem. Photobiol. 1999;70:391-475.
10. Skripchenko A, Robinette D, Wagner SJ. Comparison of Methylene blue and methylene
violet for photoinactivation of intracellular and extracellular virus in red cell
suspensions. Photochem. Photobiol. 1997;65:451-5.
11. Wagner SJ, Skripchenko A, Robinette D, et al. Factors affecting virus photoinactivation
by a series of phenothiazine dyes. Photochem. Photobiol. 1998;67:343-9.
12. Rywkin S, Ben-Hur E, Malik Z, et al. New phthalocyanines for photodynamic virus
inactivation in red blood cell concentrates. Photochem. Photobiol. 1994;60:165-70.
13. Smetana Z, Ben-Hur E, Mendelson E, et al. Herpes simplex virus proteins are damaged
following photodynamic inactivation with phthalocyanines. J. Photochem. Photobiol. B
1998;44:77-83.
14. Moor AC, Wagenaars-van Gompel AE, Brand A, et al. Primary targets for
photoinactivation of vesicular stomatitis virus by AlPcS4 or Pc4 and red light.
Photochem. Photobiol. 1997;65:465-70.
15. Pooler JP. A new hypothesis for the target in photohemolysis: dimers of the band 3
protein. Photochem. Photobiol. 1986;43:263-6.
16. Falke JJ, Chan SI. Molecular mechanisms of band 3 inhibitors. 2. Channel blockers.
Biochemistry 1986;25:7895-8.
17. FitzGerald GA. Dipyridamole. N. Engl. J. Med. 1987;316:1247-57.
18. Iuliano L, Violi F, Ghiselli A, et al. Dipyridamole inhibits lipid peroxidation and
scavenges oxygen radicals. Lipids 1989;24:430-3.
19. Nepomuceno MF, Alonso A, Pereira da Silva L, et al. Inhibitory effect of dipyridamole
and its derivatives on lipid peroxidation in mitochondria. Free Radic. Biol. Med.
1997;23:1046-54.
67
Chapter III
20. Iuliano L, Pedersen JZ, Rotilio G, et al. A potent chainbreaking antioxidant activity of
the cardiovascular drug dipyridamole. Free Radic. Biol. Med. 1995;18:239-47.
21. Dubbelman TM, De Goeij AF, vanSteveninck J. Protoporphyrin-induced photodynamic
effects on transport processes across the membrane of erythrocytes. Biochim.
Biophys. Acta 1980;595:133-9.
22. Spearman C. The method of right and wrong cases (constant stimuli) without ‘Gauss’
formulae. Br. J. Psychol. 1908;2:227-42.
23. Iuliano L, Pratico D, Ghiselli A, et al. Reaction of dipyridamole with the hydroxyl radical.
Lipids 1992;27:349-53.
24. Nassar PM, Almeida LE, Tabak M. Binding of dipyridamole to phospholipid vesicles: a
fluorescence study. Biochim. Biophys. Acta 1997;1328:140-50.
25. De Bruijne A W, van Steveninck J. The influence of dimethylsulfoxide on the red cell
membrane. Biochem. Pharmacol. 1974;23:3247-58.
26. Lagerberg JW, Williams M, Moor AC, et al. The influence of merocyanine 540 and
protoporphyrin on physicochemical properties of the erythrocyte membrane. Biochim.
Biophys. Acta 1996;1278:247-53.
27. Rodgers MA. Solvent-induced deactivation of singlet oxygen: additivity relationships in
nonaromatic solvents. J. Am. Chem. Soc. 1983;105:6201-5.
28. Schothorst AA, vanSteveninck J, Went LN, et al. Photodynamic damage of the red
blood cell membrane caused by protoporphyrin in protoporphyria and in normal red
blood cells. Clin. Chim. Acta 1972;39:161-70.
29. Dubbelman TM, de Goeij AF, vanSteveninck J. Photodynamic effects of protoporphyrin
on human red blood cells. Nature of the cross-linking of membrane proteins. Biochim.
Biophys. Acta 1978;511:141-51.
30. Young RH, Martin RL. On the mechanism of quenching of singlet oxygen by amines. J.
Am. Chem. Soc. 1972;94:5183-5.
68
CHAPTER IV
POSITIVELY CHARGED
PORPHYRINS: A NEW SERIES OF
PHOTOSENSITIZERS FOR
PATHOGEN INACTIVATION IN RED
BLOOD CELL CONCENTRATES
Laurence L. Trannoy1, Johan W.M. Lagerberg2, Tom M.A.R. Dubbelman3, Hans J.
Schuitmaker3, and Anneke Brand1
1
Department of Research and Education, Sanquin Blood Supply Foundation, Blood
Bank South West1,5, Leiden, the Netherlands; 2Sanquin Research at CLB,
Amsterdam, the Netherlands; 3PhotoBioChem n.v., Leiden, the Netherlands
Adapted from TRANSFUSION 2004; 44:1186-1196.
69
Chapter IV
ABSTRACT
BACKGROUND: PDT could be a way to inactivate pathogens in RBCC. The
objective of this study was to characterize the virucidal activity and RBCdamaging activity of a series of cationic porphyrins. Using the most efficacious
photosensitizer, various in-vitro human RBC quality parameters and in-vivo RBC
survival in Rhesus monkeys were evaluated.
STUDY DESIGN AND METHODS: RBCC, spiked with 5 log10 of extracellular VSV,
were treated with porphyrins (25 μM) and red light (100 W/m2) and essayed for
virucidal activity. In-vitro RBC quality variables were assessed during 5 weeks of
storage in various additive solutions. In-vivo survival was investigated with
autologous RBCs in Rhesus monkeys.
RESULTS: Tri-P(4) was by far the best sensitizer of a series tested, giving the least
hemolysis under conditions that resulted in 5 log10 kill of extracellular VSV. Under
our experimental conditions, the percentage hemolysis in treated cells was 5.1%
± 1.1% after five weeks of storage in SAG-M compared to 1.9% ± 1.1% in the
untreated control. Storage in AS-3 resulted in hemolysis of 2.3% ± 1.9%. With the
exception of IgG binding and potassium leakage, quality parameters of RBCs
remained unchanged after photodynamic treatment. Addition of reduced
glutathione (GSH) during treatment reduced IgG binding. The 24-hour recovery
and the half-life span T50 of treated RBCs in Rhesus monkeys were satisfactory.
CONCLUSION: Porphyrin Tri-P(4) may be a suitable photosensitizer for
decontamination of RBCC. However, further exploration to optimize the method
is necessary to reach clinically acceptable goals.
70
Porphyrins: Photosensitizers for pathogen inactivation in RBC
INTRODUCTION
Blood transfusions in developed countries are generally considered to be safe,
however recent reports revealed that maximal vigilance is still needed and that
considerable efforts are required to maintain optimal safety of blood transfusion
practices.
The emergence of new viruses, such as the WNV in the North American continent
or TTV in Asia,1,2 the potential risk of transmission of variant PrPsc,3,4 the bacterial
contamination of blood products responsible for sepsis,5,6 the reduction but not
the elimination of the window-periods with increasing sensitivity of viral and
bacterial testing,7,8 and in less developed countries, the lack of adequate and
systematic viral and bacterial screening4 promote the development of new
strategies to increase the safety of the blood products.9
Chemical and photodynamical pathogen inactivation technologies are the most
recent developments for the reduction of pathogen transmission via blood
transfusions. The chemical Pen 11010,11 and Frangible Anchor Linker Effector
compounds S-30312 are under investigation for the decontamination of RBCC.
These compounds cause irreversible alkylation of nucleophilic groups in
macromolecules, including nucleic acids, rendering pathogens noninfectious and
inhibiting white blood cells (WBC) proliferation.13,14 However, both chemicals are
toxic and must be removed before transfusion of the RBCC. PDT relies on the
activation of a light-sensitive agent (photosensitizer) in an oxygen-rich
environment, resulting in the generation of reactive oxygen species of which
singlet oxygen is the most important. Singlet oxygen can react with proteins, lipids
and nucleic acid. Because of its short lifetime, damage is localized at the binding
site of the photosensitizer. The efficacy and selectivity of the photodynamic
treatment depend on the physical and chemical properties of the photosensitizers
used. Various photosensitizers, including phenothiazines DMMB and MB15-17,
phthalocyanines18-21, merocyanines20, riboflavin (vitamin B2)22, benzoporphyrin
derivative monoacid ring A BPD-MA and, photofrin23 have been tested for their
virucidal and bactericidal activities and, their abilities to preserve the functional
integrity of the therapeutic RBCs. All dyes tested so far induced considerable RBC
damage under conditions inactivating 5 log10 of enveloped viruses. To reduce RBC
71
Chapter IV
damage, scavengers such as dipyridamol, and vitamin E derivatives (Trolox,
vitamin E succinate),15 reduced glutathione (GSH), mannitol, or cremophore19,21
were added to the RBCC.
The aim of our study was to find a photosensitizer that efficiently inactivates
viruses without RBC damage. We focused on a series of positively charged
porphyrin derivatives which have been shown to have a strong photo-bactericidal
potential.24,25 From this series, mono-phenyl-tri(N-methyl-4-pyridyl) porphyrin, TriP(4), gave the most promising results and was investigated in more detail. Various
RBC variables were investigated after storage in various additive solutions. In
addition, to evaluate the in-vivo survival of Tri-P(4)-treated RBCs, a limited study,
not meant as a formal preclinical study, was performed using two Rhesus
monkeys.
MATERIAL AND METHODS
STORAGE SOLUTIONS
AS-3 (Haemonetics Co, Braintree, MA) contains 70 mM NaCl, 61.1 mM glucose,
2.22 mM adenine, 2 mM citric acid, 20 mM Na3citrate and 15.5 mM Na2HPO4.
SAG-M (Baxter, Utrecht, the Netherlands) contains 150 mM NaCl, 1.25 mM
adenine, 50 mM glucose and 29 mM mannitol. SAG-S and SAG-D have the same
composition as SAG-M, except that mannitol is replaced by 29 mM sucrose or by
29 mM dextran-1000, respectively. These two storage solutions were
experimental. The chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
OTHER CHEMICAL
Stock solution of 1 mM GSH (Boehringer-Mannheim, Germany) was made in PBS
and stored at 2-6°C. Dipyridamol (DIP; 2,6-bis(diethanolamino)-4,8-dipiperidino[5,4-d] pyrimidine) (Sigma-Aldrich, St. Louis, MO) was stored as a 200 mM stock
solution in dimethylsulfoxide (DMSO) at 2-6°C. Radioactive disodium-51chromate
(51Cr) was purchased from NEN, Boston, MA.
72
Porphyrins: Photosensitizers for pathogen inactivation in RBC
DISPOSABLES
EDTA- and serum- tubes were purchased from Vacuette, Greiner Bio-One, Austria.
Polystyrene culture dishes with a diameter of 9 cm and 11-mL sterile polystyrene
test tubes were obtained from Greiner, Alphen a/d Rijn, the Netherlands.
MODEL VIRUS AND HOST CELLS
VSV (San Juan strain) was kindly provided by the Department of Virology at the
Leiden University Medical Center (LUMC, Leiden, the Netherlands). A549 cells for
the VSV infectivity assay were kindly provided by the department of Lung
Diseases, LUMC.
SENSITIZERS
The following porphyrins (Fig. 1) (obtained from Mid-Century, Posen, IL, USA),
were studied: meso-tetra-(N-methyl-4-pyridyl)-porphyrin chloride (Tetra-P(4)),
trans-diphenyl-di-(N-methyl-4-pyridyl)-porphyrin chloride (Trans-P(4)), cisdiphenyl-di-(N-methyl-4-pyridyl)-porphyrin chloride (Cis-P(4)), triphenyl-mono-(Nmethyl-4-pyridyl)-porphyrin chloride (Mono-P(4)), tetra-(N-2-hydroxy-ethyl-4pyridyl)-porphyrin chloride (TNH-P(4)), tetra(4-N, N, N-trimethylanilinium)porphyrin chloride (TAP). Photosensitizer mono-phenyl-tri-(N-methyl-4pyridyl)-porphyrin chloride (Tri-P(4)) was obtained from Porphyrin Systems,
Luebeck, Germany. Aluminum phthalocyanine tetrasulfonate (AlPcS4) was
purchased from Porphyrin Products (Logan, Utah, USA). Stock solutions (1 mM)
were prepared either in 50 mM sodium-phosphate buffer (NaP) pH 7.4
(Tetra-P(4), Tri-P(4), TAP, TNH-P(4) and AlPcS4) or in DMSO (Trans-P(4), Cis-P(4),
Mono-P(4)). Solution in Na-phosphate buffer were sterilized by filtering through a
0.22 μm filter and stored at 4 oC. Sensitizer concentration, after a 10-fold dilution
in absolute ethanol, was measured fluorimetrically with a Perkin Elmer LS50B
Luminescence Spectrometer. The concentration was determined using a linear
calibration curve.
73
Chapter IV
R4
+
Tetra-P(4): R1=R2=R3=R4= N -CH3
+
Tri-P(4): R1=R2=R3= N -CH3, R4= CH
+
Trans-P(4): R1=R3= N -CH3, R2=R4= CH
N
HN
R3
+
Cis-P(4): R1=R2= N -CH3, R3=R4= CH
R1
NH
N
+
Mono-P(4): R1= N -CH3, R2=R3=R4= CH
+
TNH-P(4): R1=R2=R3=R4= N -CH2-CH2-OH
+
TAP: R1=R2=R3=R4= C-N -(CH3)3
R2
Figure 1: Structures of the positively charged
porphyrins used.
RED BLOOD CELL CONCENTRATES
After written consent blood from volunteer donors (500 ± 50 mL) was collected in
73 mL citrate-phosphate-dextrose adenine (CPDA) in quadruple polyvinylchloride
bags (NPBI, Emmer-Compascuum, the Netherlands). After extraction of the buffycoat, the Hct of the RBCC was adjusted to 60 ± 4% with the storage solution SAGM.26 In Europe, the allowed maximum shelf life of CPDA-RBCC in SAG-M is limited
to 35 days.
PHOTODYNAMIC TREATMENT OF RBCC
Porphyrins were added to RBCC to give a final concentration of 25 μM. Where
indicated, DIP or GSH were added to RBCC before illumination. The RBC
suspensions were thoroughly mixed. Samples of 6 mL were transferred to 9 cmpolystyrene culture dishes and agitated at room temperature on a horizontal
reciprocal shaker (60 cycles/min, GFL, Burgwedel, Germany) for 5 min in the dark.
The resulting thickness of the cell layer was 1 mm. The dishes were illuminated
74
Porphyrins: Photosensitizers for pathogen inactivation in RBC
from above with a 300 W halogen lamp (Philips, Eindhoven, the Netherlands). To
avoid heating of the samples, the light passed through a 1-cm water filter. A cutoff filter (Kodak, wratten nr. 23A, Rochester, NY), only transmitting light > 600 nm,
was used in all experiments. The irradiance at the cell layer was 100 W/m2, as
measured with an IL1400A photometer equipped with a SEL033 detector
(International Light, Newburyport, MA, USA).
Cells illuminated without sensitizer were used as control because illumination
alone was shown to have no effect on RBC function or virus inactivation. Cells
with sensitizer kept in the dark were used as dark control.
VSV INFECTIVITY ASSAY
Stock solutions of VSV were added to RBCC so that the volume of the spike was
less than 10% of the total volume of RBCC. The number of extracellular virus
particles was approximately 105/mL and was our maximum starting titer of
inoculum. PDT was performed as described above.
Infectivity of VSV was assayed as previously described.27 In short, RBC samples
taken after various light doses were centrifuged for 5 min at 500 x g. The
supernatants were serially diluted (10x) and added to A549 cell cultures in 96 well
microtiter plates. After 72-hours incubation the cytopathology of the cells was
scored in 8-well replicates for each dilution. Quantification of the virus titer was
performed according to the Spearman-Karber method,28 expressing the quantity
of virus infecting 50% of the cells (TCID50). Results are expressed as percentage of
the control.
RBC QUALITY VARIABLES
Hemolysis was determined by measuring the absorbance of free Hb at 540 nm.
Cell supernatants were obtained by centrifugation of diluted (10x) RBCs (in PBS) at
12000 x g for 3 min. Absorbance was measured using a DU-64 spectrophotometer
(Beckman, Fullerton, CA). This method was shown to be as sensitive as the
hemiglobincyanide method.29
75
Chapter IV
Potassium leakage from RBCs was measured using a flame photometer (Model
410C, Corning, Halstead, UK). Both K+ leakage and hemolysis were expressed as a
percentage of the total concentration.
ATP content of the cells was determined by adding firefly lantern extract after
lysis of the cells in H2O, and dilution with a glycine buffer (25 mM gly-gly, 20 mM
MgCl2, pH 7.5). The luminescence was measured using a Model 1251 luminometer
(Bio-Orbit, Turku, Finland), as described before.21
Measurement of RBC lactate and glucose content of the medium were performed
according to the manufacturer’s instructions (Procedure 735 and 16-UV
respectively, Sigma Diagnostics, St. Louis, MO).
Lipid peroxidation was essayed by measuring the generation of thiobarbituric acid
reactive species as described by Miller et al.30 Intracellular GSH was measured
according to Sedlak and Lindsay.31 RBC deformability was measured as described
previously.32
Anion transport through band 3 was measured by the influx of Na235SO4 as
described previously.33 Briefly, after PDT, cells were centrifuged and resuspended
(10% Hct) in PBS plus 1 mM Na2SO4 and 0.5 μCi/mL Na235SO4 at 37°C. After 0, 5,
10, 15, 20 and 30 min incubation at 37°C, samples were washed twice with icecold PBS to remove free Na235SO4. After precipitating the Hb with 5%
trichloroacetic acid, radioactivity in the cells was determined by means of liquid
scintillation counting (Tri-Carb 4000, Packard Instrument Co., Downers Grove, IL).
AGGLUTINATION AND IGG BINDING
The direct gel agglutination test (DAT) was performed on human RBCs and
monkey RBCs using DiaMed-ID micro typing system LISS/Coombs (DiaMed SA,
Cressier sur Morat, Zwitserland) according to manufacturer’s instructions. The
test includes antibodies against the four human IgG sub-classes and C3d.
IgG binding to human-RBCs was analyzed by flow cytometry. Briefly, RBCs were
washed twice with PBS / 0.5% Bovine Serum Albumin (BSA) and resuspended to
5% Hct in the same buffer. A 50 μL sample was incubated with 50 μL of diluted
(20x) FITC-conjugated F(ab’)2 fragment of rabbit anti-human IgG (gamma-chains)
or FITC-conjugated F(ab’)2 fragment of rabbit Ig (negative control) (DAKO,
76
Porphyrins: Photosensitizers for pathogen inactivation in RBC
Glostrup, Denmark) for 30 min at 4°C. Labeled cells were washed with PBS/0.5%
BSA before analysis on the Epics XL (Beckman Coulter, Mijdrecht, the
Netherlands). A total of 20.000 events were analyzed, and mean fluorescence
intensity (MFI) of the FITC signal (FL1) was determined. The indirect antiglobulin
test (IAT) was performed using plasma-free RBCs (washed 6 times in SAG-M) and
autologous plasma, which was photodynamically treated separately. IgG binding
was analyzed macroscopically and by flow cytometry.
STORAGE OF HUMAN RBCC AFTER PHOTODYNAMIC TREATMENT
After illumination of RBCC, Hct 60% in SAG-M, 5-mL samples were transferred to
11-mL sterile polystyrene test tubes. After centrifugation at 500 x g for 10 min the
supernatant was removed. Cells were resuspended to 60% Hct in SAG-M, SAG-S,
SAG-D or AS-3. The RBC suspensions were stored in tubes in the dark at 2-6°C.
These conditions were chosen for convenience when working with small samples.
RBC quality was assayed after 1, 7, 21 and 35 days of storage.
STATISTICS
All in-vitro experiments were performed at least 4 times with blood from different
donors. Values are expressed as mean ± standard deviations (SD). The p values
were calculated using student’s t-tests. A p value less than 0.05 were defined as
significant.
RHESUS MONKEY SURVIVAL STUDIES
All procedures were performed in accordance with the guidelines of the Animal
Care and Use Committee installed by Dutch law. In a pilot study, the effect of PDT
on monkey RBCs was determined with respect to VSV kill, RBC quality (K+ leakage
and hemolysis) and 51Cr uptake. In-vivo survival studies were carried out using two
Rhesus monkeys (Macaca mulatta). Monkey RBCs were prepared from 20 mL of
freshly drawn blood, collected after ketamine-anaesthesia in 2.5 mL CPDA. CPDA
blood was centrifuged at 500 x g for 5 minutes, and the plasma and buffy-coat
77
Chapter IV
were discarded. The RBCs were resuspended in SAG-M to a Hct of 60 ± 4% and
photodynamically treated as described above.
In-vivo survival was investigated after autologous transfusion of either fresh or 35
days stored RBCC. (Table 1). RBCs were stored in AS-3 in polystyrene tubes at 4°C
during 5 weeks. Twenty four hours before transfusion, RBCs were labeled with
51
Cr. Unbound radioactivity was removed by washing in excess volume with AS-3.
A 1-mL sample was removed before transfusion and used to determine the
labeling efficiency and the remaining Tri-P(4) concentration (excitation 424 nm,
emission 659 nm). Labeling efficiency was shown to be 87 ± 9% in both untreated
and PDT-treated RBCs. Freud et al.34 have shown that until RBCs are committed to
hemolysis, 51Cr leakage does not occur during storage of PDT-treated RBCs. For
parallel in-vitro analysis during storage, control and Tri-P(4)-treated monkey RBCs
were washed and resuspended to 60% Hct in AS-3.
To determine the 24-hour recovery, blood samples in EDTA were drawn 30 min
and 1, 2, 4, 8 and 24 hours after transfusion. To determine the life span of the
transfused RBCs, samples were drawn at Day 2 and Day 3 after transfusion and
thereafter twice a week until the radioactivity was decreased to less than 45% of
the initial value. The Hb, Hct, Mean cell volume (MCV), RBC, WBC and platelet
numbers, creatinin, total bilirubin, LDH and haptoglobin were measured before
and after transfusion by standard procedures. Urine samples were recovered
during 24 hours after transfusion. The 24-hour recovery and the RBC survival,
expressed as T50, were calculated according to the ICSH guidelines.35
All radioactivity counts were corrected for Hct, decay and elution of 51Cr, which
was taken to be 1% per day.
Exp nr.
Monkey A
Monkey B
1
RBC, non-treated (control)
RBC, PDT-treated
< 24 hrs
2
RBC, PDT-treated
RBC, non-treated (control)
< 24 hrs
3
RBC, non-treated (control)
RBC, PDT-treated
35 days
4
RBC, PDT-treated
RBC, non-treated (control)
35 days
Table 1: Experimental design of the in-vivo RBC survival studies
78
Storage time
Porphyrins: Photosensitizers for pathogen inactivation in RBC
RESULTS
HUMAN RBC
INITIAL SCREENING OF PHOTOSENSITIZERS
VSV inactivation. All sensitizers tested were capable of inactivating extracellular
VSV. However, the light dose needed for 5-log10 VSV kill varied strongly (Fig. 2A).
Tri-P(4) appeared to be the most efficient sensitizer, requiring a light dose of 90
kJ/m2 (15 min at 100 W/m2). The dose required for Mono-P(4) was fourfold
higher. All other sensitizers required an intermediate light dose varying from 120
to 240 kJ/m2.
RBC damage. A light dose needed to kill 5-log10 VSV was used to study the effect
of the different porphyrins on RBC integrity. Figure 2B shows the RBC hemolysis
after storage of treated cells in SAG-M. Virucidal treatment with Tri-P(4) induced
the least hemolysis, 5.1 ± 1.1% after 35 days of storage. Virucidal treatment with
TAP, TNH and Tetra-P(4) induced intermediate levels of hemolysis (mean 16.13 ±
0.81% ), whereas Trans-P(4), Cis-P(4) and Mono-P(4) induced very high levels of
hemolysis. Further experiments were restricted to Tri-P(4). RBCs were
photodynamically treated with 25 μM Tri-P(4) and a light dose of 90 kJ/m2 (15
min, 100 W/m2). RBCs were stored for 5 weeks at 4-6°C.
HEMOLYSIS AND POTASSIUM LEAKAGE: COMPARISON OF VARIOUS
ADDITIVE SOLUTIONS
After 35 days of storage in tubes, hemolysis of control cells amounted to 1.9 ±
1.1% and was independent of the additive solution used. Storage of Tri-P(4)treated RBCs in SAG-S or SAG-D resulted in lower hemolysis as compared to SAGM (Fig. 3). Hemolysis was the lowest after storage in AS-3 (2.3 ± 1.9%). After
storage in SAG-S, SAG-D and AS-3, there was no significant difference between
treated cells and controls. When stored in SAG-M, SAG-S, and SAG-D, Tri-P(4)
alone (dark control) resulted in a slight, but not significant, increase in hemolysis.
79
Chapter IV
Potassium leakage during storage was substantially higher for Tri-P(4)-treated
cells than for the controls and was independent of the storage solution (Fig. 4).
We observed a rapid increase in extracellular K+ 1 day after illumination, followed
by a gradual increase to about 60% after 35 days of storage. In the controls, K+
leakage was around 40% after 35 days. The presence of the scavengers GSH (32
mM) or DIP (125 μM) during PDT had no effect on the extent of K+ leakage (data
not shown).
80
light only;
+), Tetra-P(4), 240 kJ/m2
2
% hemolysis
% hemolysis
Infectious particles
(% of control)
Figure 2: (A) Infectivity of VSV after
PDT. RBCC spiked with 5-log10
extracellular VSV were illuminated
with red light (light intensity: 100
2
W/m ) in the presence of 25 μM
Tetra-P(4) (S), Tri-P(4) (z), TNH-P(4)
({), TAP (V), Trans-P(4) (T), Cis-P(4)
(¡), Mono-P(4) (U). Results are expressed as percentage of un-treated
control. (B) RBC hemolysis after
virucidal treatment. RBCC with 25 μM
porphyrins were illu-minated with a
light dose giving 5-log10 kill of VSV.
After treatment, the cells were stored
for 35 days at 4 to 6°C. Control (red
(S), Tri-P(4), 90 kJ/m (z), TNH-P(4),
2
2
120 kJ/m ({), TAP, 180 kJ/m (V),
2
Trans-P(4), 150 kJ/m (T), Cis-P(4),
2
2
190 kJ/m (¡), Mono-P(4), 360 kJ/m
(U). Results are expressed as
percentage of total Hb. Error bars
were omitted for reason of clarity.
Porphyrins: Photosensitizers for pathogen inactivation in RBC
Figure 3. Role of the storage solution on RBC hemolysis.
RBCC, Hct 60% in SAG-M, were treated with red light only (+), 25μM Trip-(4) only ({) or with 25μM
2
Tri-P(4) plus red light (90 kJ/m , z). After treatment, cells were resuspended in SAG-M, SAG-S, SAGD or AS-3 and stored for 35 days at 2-6°C. Hemolysis is expressed as percentage of total Hb. Data
presented are the average ± SD of 4 to 6 experiments. Significant differences are indicated with * (p
value < 0.05).
Figure 4: Potassium leakage after PDT with Tri-P(4).
2
RBCC, Hct 60% in SAG-M, were treated with red light only (90 kJ/m ; open symbols)
2
or 25 μM Tri-P(4) plus red light (90 kJ/m , filled symbols). After treatment cells were
resuspended in SAG-M ({;z), SAG-S (V;T), SAG-D (U;S) or AS-3 (‘;¡) and
stored for 35 days at 4 to 6°C. Data presented are average ± SD of four to six
experiments. Significant differences are indicated with * (p value < 0.05).
81
Chapter IV
OTHER RBC VARIABLES
RBC quality variables after storage in SAG-M are summarized in Table 2. After 35
days of storage, the cellular ATP content and extracellular glucose levels
decreased and the lactate concentration increased to comparable levels in both
control and Tri-P(4) treated cells. The GSH concentration in treated cells was
slightly decreased after 35 days of storage. The photodynamic dose used in these
experiments did not induce any detectable lipid peroxidation, changes in
deformability of the cells or changes in anion transport through band 3. The use of
different additive solutions had no effect on any of the mentioned variables (data
not shown).
ATP
(μmol/g Hb)
Glucose
(mg/mL)
Lactate
(mol/L)
GSH
(μmol/g Hb)
1
3.8 r 0.4
4.1 r 0.5
12.3 r 2.2
1.5 r 0.3
5
1.8 r 0.3
2.3 r 0.5
23.0 r 4.0
1.5 r 0.2
1
4.0 r 0.5
3.5 r 1.5
12.1 r 3.9
1.6 r 0.1
Week
Control
Tri-P(4)
5
1.6 r 0.3
2.4 r 0.5
23.9 r 1.4
1.2 r 0.3
Table 2: RBC quality after photodynamic treatment with Tri-P(4) and red light * and stored in SAG-M
during 5 weeks (mean ± sd).
2
2
* Light dose of 90 kJ/m which corresponds to an illumination time of 15 min at 100 W/m
IgG BINDING AND AGGLUTINATION
Virucidal treatment of RBCC, Hct 60% in SAG-M with Tri-P(4) resulted in binding of
IgG to RBCs. The extent of the binding was light dose dependent and increased
with illumination time. IgG binding after treatment at 90 kJ/m2 was very low,
sometimes undetectable by DAT (Table 3) and flow cytometry (Fig. 5A). The MFI
of untreated cells was 4.7 ± 0.6, that of treated cells with 90 kJ/m2 was 5.1 ± 0.7
and with 360 kJ/m2 the MFI was 8.2 ± 2 (Fig. 5A). PDT with AlPcS4 (2.5 μM, 360
kJ/m2) was previously described to induce IgG binding19 and was used as a positive
control. Both Tri-P(4)-induced IgG binding measured by flow cytometry and
82
Porphyrins: Photosensitizers for pathogen inactivation in RBC
128
agglutination in the DAT could be prevented by the presence of GSH (32 mM)
during the illumination. Addition of GSH after PDT did not reduce or prevent IgG
binding or agglutination (Fig. 5B and Table 3). The presence of the singlet oxygen
scavenger DIP (125 μM) during illumination did not prevent IgG binding or
agglutination.
128
360 kJ/m2
AlPcS4
GSH during PDT
A
B
GSH post PDT
Events
Events
360 kJ/m2
Tri-P(4)
10 0
+ Tri-P(4)
0
0
90 kJ/m2
Tri-P(4)
10 1
10 2
FL1 LOG
10 3
10 4
10 0
10 1
10 2
FL1 LOG
10 3
10 4
Figure 5: (A) Binding of IgG to RBCs after PDT with Tri-P(4). RBCC were treated in the presence of 25
2
2
2
μM Tri-P(4) with a light dose of 90 kJ/m or 360 kJ/m . RBCC treated with AlPcS4 (2.5 μM, 360 kJ/m )
served as positive control. One representative experiment of a total of seven experiments is shown.
(B) Effect of GSH on the Tri-P(4)-induced binding of IgG to RBCs. RBCC were treated with 25 μM Tri2
P(4) with a light dose of 360 kJ/m . GSH (32 mM) was added to the RBCC either before or after the
illumination. After treatment cells were analyzed for IgG binding by flow-cytometry, as described in
Material and Methods. One representative experiment of a total of three experiments is shown. The
untreated sample is shown in solid gray.
To unravel the process of the photodynamically induced IgG binding, plasma and
plasma-free RBCs were treated with Tri-P(4) (light dose 360 kJ/m2) separately. No
IgG binding was detectable after phototreatment of the plasma-free RBCs. Using a
cross-match reaction (IAT), i.e. plasma added to RBCs, it was shown that both TriP(4)-treated plasma and Tri-P(4)-treated plasma-free-RBCs were required for IgG
binding and to induce agglutination. The IAT remained negative when a single
treated component, RBCs or plasma, was added to an untreated one (Table 3).
83
Chapter IV
DAT
Untreated
RBCC†
0 (n = 7)
2
90 kJ/m
360 kJ/m
0 / + (n = 4 / 3)
2
+ GSH* during PDT
4+ (n = 7)
IAT
0 (n = 3)
+ GSH* after PDT
4+ ( n = 3)
Washed RBCs‡
Untreated
2
PDT (360 kJ/m )
2
PDT (360 kJ/m ) + GSH*
Plasma
Untreated
2
PDT (360 kJ/m )
2
PDT (360 kJ/m ) + GSH*
0
0
0
0
2+
0
0
0
0
* [GSH ] = 32 mM
†RBCC, Hct 60% in SAG-M
‡ Washed 6 times in SAG-M (= plasma-free RBCs)
Table 3: DAT and IAT score after PDT with Tri-P(4) and red light
MONKEY RBCS
IN VITRO QUALITY
Tri-P(4) treatment of monkey RBCC spiked with VSV, using the same conditions as
for human RBCC, resulted also in a 5-log10 kill of VSV. RBC hemolysis was assayed
after 35 days of storage in AS-3. AS-3 was selected because it was shown to
maintain human RBC integrity better than SAG-M (Fig. 3). After 35 days of
storage, the hemolysis of untreated monkey RBCC was 2.7%, 1.5-fold higher than
with human RBCC. After PDT with Tri-P(4), hemolysis amounted to 4.2% after 35
days of storage. This was twofold higher than observed with human RBCC in AS-3.
After 1 week of storage, the level of photodynamically induced K+ leakage was as
high as in human-RBCC after 35 days of storage. After PDT with Tri-P(4) at 90
kJ/m2, no IgG binding was found on monkey RBCs in the DAT using anti-Hu-IgG
and C3.
84
Porphyrins: Photosensitizers for pathogen inactivation in RBC
IN VIVO SURVIVAL
The 24-hr recovery of autologous RBCs is shown in Table 4. After PDT, the 24-hr
recovery of fresh RBCs was in the same range as for non-treated cells (85%). After
5 weeks of storage, the 24-hr recovery of treated RBCs was approximately 11%
lower than that of untreated stored cells. The values for T50 showed a large
variation, which was independent of the treatment. The residual Tri-P(4)
concentration in the transfusion products was about 1-2 μM. No clinical
symptoms suggesting transfusion-related adverse effects were observed.
Blood samples taken 30 min, 1 hour and 24 hours after transfusion were analysed
for chemical and hematological values. Values for number of WBC, RBC and
platelets, serum creatinin, total bilirubin and LDH were all in the normal range and
there were no differences between PDT-treated and untreated transfusions (data
not shown). Haptoglobin concentration was increased in some samples 24 hours
after transfusion, but this was not associated with the treatment method of RBCs.
Twenty-four hours after transfusion, urine analysis (51Cr, bilirubin, protein)
showed no change with the values obtained before the RBC infusions (data not
shown).
Transfusion product
Fresh RBCC
Monkey A
Monkey B
Stored RBCC
Monkey A
Monkey B
24-hr recovery (%)
Untreated
PDT
82
90
87
80
81
62
73
68
T50 (days)
Untreated
PDT
16
22
18
19
23
15
26
20
Table 4: 24-hour recovery and T50 of Tri-P(4)-treated monkey RBCs.
85
Chapter IV
DISCUSSION
Positively charged porphyrins are known to inactivate bacteria.24,36 Our study has
shown that they are also able to inactivate extracellular VSV. VSV is a lipid
enveloped, single stranded RNA virus which has been found to be as sensitive to
PDT with Pc4 and red light as HIV and is therefore a useful virus to study PDT.37 All
positively charged porphyrins tested were able to inactivate extracellular VSV
spiked in RBCC (Fig. 2a), however the compounds differed greatly by their
potential to preserve RBC integrity during storage. Under conditions that induced
a 5-log10 inactivation of extracellular VSV, the hydrophobic compounds Trans-P(4),
Cis-P(4) and Mono-P(4) caused a very high percentage of cell lysis after 35 days of
storage. Because of their hydrophobic nature, these compounds most likely
penetrated the bilayer of the RBCs. The hydrophilic compounds Tetra-P(4), TAP
and TNH-P(4) showed a moderate percentage of lysed cells. The amphiphilic TriP(4) showed the least hemolysis and could therefore be a suitable photosensitizer
for pathogen inactivation in of RBCC.
Tetra-, Tri- and Di-P(4)’s have been shown to intercalate in isolated calf thymus
DNA.38,39 Although this property may contribute to the selective killing of viruses,
the exact mechanism by which the porphyrins inactivate viruses still remains to be
explored.
RBC integrity after Tri-P(4)-mediated phototreatment was influenced by the
composition of the storage solution. Storage of treated cells in SAG-S, SAG-D or
AS-3 resulted in much less hemolysis as compared to storage in SAG-M.
Comparable results were recently obtained by Besselink et al. 40 using the
photosensitizer DMMB. The presence of high concentrations of impermeable
macromolecules in SAG-S (29 mM sucrose), SAG-D (29 mM dextran 1000) and AS3 (22 mM citrate) compensate for the osmotic activity of intracellular Hb.41-42
RBC metabolic function and deformability remained intact after PDT with Tri-P(4),
whereas K+ leakage during storage was increased. We have previously shown that
K+ leakage induced by PDT with DMMB was related to damage to band 3 and that
both band 3 damage and K+ leakage could be reduced by scavenger DIP.43 Tri-P(4)
seems to have a different mechanism, since K+ leakage is induced without
affecting band 3 activity, and the addition of DIP did not reduce K+ leakage. The
86
Porphyrins: Photosensitizers for pathogen inactivation in RBC
mechanism underlying K+ leakage induced by Tri-P(4) phototreatment remains
unclear. Further investigations focusing on channels involved in K+ concentration
regulation such as the Na+-K+ pump, Gàdos channel and the K+Cl- co-transporter
might help to unravel the mechanism behind the K+ leakage.
IgG binding was induced by PDT with Tri-P(4). However the extent of IgG binding
at virucidal light dose (90 kJ/m2) was very low and was sometimes not detectable
(Table 3). IgG binding induced by PDT is a known phenomenon which can be
prevented by the addition of the scavenger GSH during illumination. GSH can
prevent IgG binding by prevention formation of disulfide bridges.19 In agreement
with previous findings of Rywkin et al.19 the addition of GSH after the
phototreatment was unable to reverse IgG binding or agglutination. Addition of
DIP did not prevent IgG binding. To ensure cell survival and to avoid problems
with compatibility testing, it may be necessary to add GSH or cysteine during the
PDT with Tri-P(4). Both GSH and cysteine are natural constituents of the blood and
therefore might not have to be removed before transfusion.
The cross-match IAT showed agglutination only when both plasma-free RBCs and
plasma were photo-treated. This suggests that both the RBC membrane and
plasma proteins were modified by PDT. Remarkably, there was no agglutination
when Tri-P(4)-treated plasma-free RBCs were incubated with untreated plasma.
This indicates that Tri-P(4) does not induce formation of neoantigens reacting
with naturally occurring auto-antibodies. This however does not preclude that
phototreated RBC membrane alterations may cause new antibody formation
upon transfusion.
Based on the promising in-vitro results on one side and the considerable efforts to
translate our experimental set-up for application in humans on the other, we
evaluated the in-vivo survival of Tri-P(4) treated RBCs in 2 Rhesus monkeys. The
number of animals was kept to a minimum by performing a cross-over study. The
study was not meant to obtain statistic relevance but to evaluate whether
proceeding with Tri-P(4) was worthwhile. Both the 24-hour recovery and the long
term survival were satisfactory after transfusion of phototreated fresh RBCs. In
accordance with previous reports,44 we found monkey RBCs to be more fragile
than human RBCs. Storage of monkey RBCC resulted in higher hemolysis as
compared to human RBCC after the same storage interval. Moreover, the
87
Chapter IV
experimental conditions storing RBCC in tubes were suboptimal compared to the
Blood Bank conditions in which RBCC are stored in bags. After 5 weeks of storage,
the 24-hour recovery was lower compared to fresh RBCs and recovery was
approximately 10% reduced by Tri-P(4)-mediated PDT (Table 4). The T50 however,
varied in our study from 15 to 26 days, and was not clearly associated with either
the storage or the PDT. The true impact of PDT on in-vivo survival after storage is
difficult to interpret as suboptimal storage condition, using tubes instead of bags,
were used.
Recent publication by Vitex and Cerus45,46 have reported the formation of
autoantibodies in patients receiving Pen-110- or S-303-treated RBCs and Phase III
clinical trials have been abandoned. These results were unexpected and not
foreseen despite extensive animal studies. Our study showed that treatment with
Tri-P(4) preserved the structure and functionality of RBCs but resulted in a slight
elevation of hemolysis after 5 weeks of storage, an acceleration of K+ leakage,
and a weak induction of IgG binding, which could be prevented by GSH. Although
it seems that neoantigens reacting with naturally occurring IgG antibodies are not
formed after Tri-P(4)-mediated PDT, to predict the immunogenicity of Tri-P(4)treated RBCs in human trials in-vivo, further research is required to elucidate the
cause of this event.
The results obtained in this study form an acceptable basis for further
investigations on the use of Tri-P(4) for pathogen inactivation in RBCC. Further
research will focus on the inactivation of viruses more relevant to transfusion
medicine, such as HIV and hepatitis viruses. Also the inactivation of intracellular
viruses has to be studied. It might well be possible that for inactivation of other
more resistant viruses, a more stringent photoinactivation is needed, which might
result in more RBC damage. Therefore, more insight in both the mechanism of
pathogen inactivation and RBC damage after phototreatment with Tri-P(4) is of
utmost importance.
In conclusion, although we realize that the step from “bench” experiments to a
functioning pathogen inactivation system in a blood-bank setting is a big one, TriP(4)-mediated phototreatment might become a suitable method to inactivate
pathogens in cellular blood products.
88
Porphyrins: Photosensitizers for pathogen inactivation in RBC
REFERENCES
1. Charrel RN, de Lamballerie X, Durand JP et al. Prevalence of antibody against West Nile
virus in volunteer blood donors living in southeastern France. Transfusion
2001;41:1320-1.
2. Dai CY, Yu ML, Chuang WL et al. The molecular epidemiology and clinical significance of
TT virus (TTV) infection in healthy blood donors from southern Taiwan. Transfus.
Apher. Sci. 2001;24:9-15.
3. Roddie PH, Turner ML, Williamson LM. Leucocyte depletion of blood components.
Blood Rev. 2000;14:145-56.
4. Moor AC, Dubbelman TM, VanSteveninck J et al. Transfusion-transmitted diseases:
risks, prevention and perspectives. Eur. J. Haemotol. 1999;62:1-18.
5. Kuehnert MJ, Roth VR, Haley NR et al. Transfusion-transmitted bacterial infection in
the United States, 1998 through 2000. Transfusion 2001;41:1493-9.
6. Perez P, Bruneau C, Chassaigne M et al. Multivariate analysis of determinants of
bacterial contamination of whole-blood donations. Vox Sang. 2002;82:55-60.
7. Ruzzenenti MR, De Luigi MC, Bruni R et al. PCR testing for HCV in anti-HCV negative
blood donors involved in the so called HCV +ve post-transfusion hepatitis. Transfus.
Sci. 2000;22:161-4.
8. Liu H, Shah M, Stramer SL et al. Sensitivity and specificity of human T-lymphotropic
virus (HTLV) types I and II polymerase chain reaction and several serologic assays in
screening a population with a high prevalence of HTLV-II. Transfusion 1999;39:118593.
9. Epstein JS, Vostal JG. FDA approach to evaluation of pathogen reduction technology.
Transfusion 2003;43:1347-50.
10. Purmal A, Valeri CR, Dzik W et al. Process for the preparation of pathogen-inactivated
RBC concentrates by using PEN110 chemistry: preclinical studies. Transfusion
2002;42:139-45.
11. AuBuchon JP, Pickard CA, Herschel LH et al. Production of pathogen-inactivated RBC
concentrates using PEN110 chemistry: a Phase I clinical study. Transfusion
2002;42:146-52.
89
Chapter IV
12. Cook D. In vivo analysis of packed red blood cells treated with S-303 to inactivate
pathogens. Blood 1998;92:10 (suppl.1)
13. Fast LD, DiLeone G, Edson CM et al. PEN110 treatment functionally inactivates the
PBMNCs present in RBC units: comparison to the effects of exposure to gamma
irradiation. Transfusion 2002;42:1318-25.
14. Grass JA, Hei DJ, Metchette K et al. Inactivation of leukocytes in platelet concentrates
by photochemical treatment with psoralen plus UVA. Blood 1998;91:2180-8.
15. VanSteveninck J, Trannoy LL, Besselink GAJ et al. Selective protection of erythrocytes
against photodynamic damage by the band3 ligand Dipyridamol. Transfusion
2000;40:1330-6.
16. Wagner SJ, Skripchenko A, Robinette D et al. Preservation of red cell properties after
virucidal phototreatment with dimethylmethylene blue. Transfusion 1998;38:729-37.
17. Wagner SJ. Virus inactivation in blood components by photoactive phenothiazine dyes.
Transfus. Med. Rev. 2002;16:61-6.
18. Rywkin S, Ben-Hur E, Malik Z et al. New phthalocyanines for photodynamic virus
inactivation in red blood cell concentrates. Photochem. Photobiol. 1994;60:165-70.
19. Rywkin S, Ben-Hur E, Reid ME et al. Selective protection against IgG binding to red cells
treated with phthalocyanines and red light for virus inactivation. Transfusion
1995;35:414-20.
20. Lagerberg JW, Uberriegler KP, Krammer B et al. Plasma membrane properties involved
in the photodynamic efficacy of merocyanine 540 and tetrasulfonated aluminum
phthalocyanine. Photochem. Photobiol. 2000;71:341-6.
21. Moor AC, van der Veen A, Wagenaars-van Gompel AE et al. Shelf-life of
photodynamically sterilized red cell concentrates with various numbers of white cells.
Transfusion 1997;37:592-600.
22. McAteer MJ, Tay-Goodrich BH, Doane S et al. Photoinactivation of virus in packed red
blood cell units using Riboflavin and visible light. Transfusion 2000;40:99S
23. North J, Neyndorff H, Levy JG. Photosensitizers as virucidal agents. J. Photochem.
Photobiol. B 1993;17:99-108.
24. Merchat M, Spikes JD, Bertoloni G et al. Studies on the mechanism of bacteria
photosensitization by meso- substituted cationic porphyrins. J. Photochem. Photobiol.
B 1996;35:149-57.
90
Porphyrins: Photosensitizers for pathogen inactivation in RBC
25. Minnock A, Vernon DI, Schofield J et al. Photoinactivation of bacteria. Use of a cationic
water-soluble zinc phthalocyanine to photoinactivate both gram-negative and grampositive bacteria. J. Photochem. Photobiol. B 1996;32:159-64.
26. Novotny VM, van Doorn R, Rozier Y et al. Transfusion results of filtered and
subsequently stored random platelet suspensions prepared from buffy coats. Vox
Sang. 1992;63:23-30.
27. Moor AC, Wagenaars-van Gompel AE, Brand A et al. Primary targets for
photoinactivation of vesicular stomatitis virus by AIPcS4 or Pc4 and red light.
Photochem. Photobiol. 1997;65:465-70.
28. Spearman C. The method of right and wrong cases ('constan stimuli') without Gauss's
formulae. Br. J. Psychol. 1908;2:227-42.
29. Rowan RM, AZ, OWVA et al. Recommendations for reference method for
haemoglobinometry in human blood (ISCH standard 1995) and specifications for
international haemiglobincyanide standard (4th edition). J. Clin. Pathol. 1996;49:271-4.
30. Miller DM, Spear NH, Aust SD. Effects of deferrioxamine on iron-catalyzed lipid
peroxidation. Arch. Biochem. Biophys. 1992;295:240-6.
31. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl
groups in tissue with Ellman's reagent. Anal. Biochem. 1968;25:192-205.
32. de Bruijne AW, van Steveninck J. The influence of dimethylsulfoxide on the red cell
membrane. Biochem. Pharm. 1974;23:3247-58.
33. Knauf PA, Rothstein A. Chemical modification of membranes. I. Effects of sulfhydryl
and amino reactive reagents on anion and cation permeability of the human red blood
cell. J. Gen. Physiol. 1971;58:190-210.
34. Freud A, Canfi A, Ben-Hur E. Validity of chromium-51 labelling of human erythrocytes
as an assay for their survival after photodynamic therapy in vitro for blood sterilization.
Int J Radiat Biol 1993;63:651-3.
35. Moroff G, Sohmer PR, Button LN. Proposed standardization of methods for
determining the 24-hour survival of stored red cells. Transfusion 1984;24:109-14.
36. Merchat M, Bertolini G, Giacomini P et al. Meso-substituted cationic porphyrins as
efficient photosensitizers of gram-positive and gram-negative bacteria. J. Photochem.
Photobiol. B 1996;32:153-7.
91
Chapter IV
37. Margolis-Nunno H, Ben-Hur E, Gottlieb P et al. Inactivation by phthalocyanine
photosensitization of multiple forms of human immunodeficiency virus in red cell
concentrates. Transfusion 1996;36:743-50.
38. Sari MA, Battioni JP, Dupre D et al. Interaction of cationic porphyrins with DNA:
importance of the number and position of the charges and minimum structural
requirements for intercalation. Biochemistry 1990;29:4205-15.
39. Munson BR, Fiel RJ. DNA intercalation and photosensitization by cationic meso
substituted porphyrins. Nucleic Acids Research 1992;20:1315-9.
40. Besselink GA, Ebbing IG, Hilarius PM et al. Composition of the additive solution affects
red blood cell integrity after photodynamic treatment. Vox Sang. 2003;85:183-9.
41. Pooler JP. The kinetics of colloid osmotic hemolysis. II. Photohemolysis. Biochim.
Biophys. Acta 1985;812:199-205.
42. Besselink GA, Ebbing IG, Hilarius PM et al. Composition of the additive solution affects
red blood cell integrity after photodynamic treatment. Vox Sang. 2003;85:183-9.
43. Trannoy LL, Brand A, Lagerberg JW. Relation between K+ leakage and damage to Band
3 in photodynamically treated red cells. Photochem. Photobiol. 2002;75:167-71.
44. Rowe AW. Primates: models for red cell transfusion studies--cryopreservation and
survival of transfused red cells in primates. J. Med. Primatol. 1994;23:415-25.
45. V.I.Technologies I. Vitex halts enrollment in Pase III chronic trial of the Inactine
Pathogen Reduction System for Red Blood cells. www vitechnologies com 17-11-2003.
46. Cerus Co, Baxter International Inc. Baxter And Cerus Halt Red Blood Cell Clinical Trials
For Investigational Pathogen Inactivation System. www cerus com/Press releases 4-92003.
92
CHAPTER V
DIFFERENTIAL SENSITIVITIES OF
PATHOGENS IN RED BLOOD CELL
CONCENTRATES TO TRI-P(4)PHOTOINACTIVATION
Laurence L. Trannoy,1 Fokke G. Terpstra,2 Dirk de Korte,3 Johan W.M. Lagerberg,3
Arthur J. Verhoeven,3 Anneke Brand,1 and Frank A. C. van Engelenburg.2
1
Department of Research and Education, Sanquin Blood Bank Southwest, Leiden,
the Netherlands; 2Sanquin Virus Safety Services, Sanquin Research, Amsterdam,
the Netherlands; 3Department of Blood Cell Research, Sanquin Research,
Amsterdam, the Netherlands.
Adapted from Vox Sanguinis; 2006; 91, 111–118
93
Chapter V
ABSTRACT
Background and Objectives: PDT with the cationic porphyrin Tri-P(4) was
previously shown to effectively inactivates VSV in RBCC with limited damage to
RBC. The aim of this study was to determine the pathogen inactivating capacity of
PDT with Tri-P(4) for a broader range of pathogens and the associated effect on in
vitro red blood cell quality.
Materials and methods: A series of viruses and bacteria were spiked in 60% RBCC.
Pathogen inactivation were determined after PDT with 25 μM Tri-P(4) and red
light up to 360 kJ/m2. Human immunodeficiency virus (HIV)-infected cells were
evaluated for cell death induction, and treated RBCs for induction of hemolysis
and ATP content.
Results: For the lipid enveloped viruses bovine viral diarrhoea virus (BVDV), HIV
and pseudorabies virus (PRV), the gram positive (G+) bacterium Staphylococcus
aureus and the gram negative (G-) bacteria Pseudomonas aeruginosa and Yersinia
enterolitica, 5 log10 or more inactivation was measured after 60 min of PDT with
Tri-P(4). The required treatment time to achieve this level of inactivation was four
times longer than required for VSV in the previous study. For cell-associated HIV
only 1.7 log10 of inactivation was found, despite clear induction of cell death of
HIV-infected cells. The non-enveloped virus canine parvovirus (CPV) was
completely resistant to the treatment. PDT with Tri-P(4) for 60 min of RBCC and
subsequent storage in AS-3 resulted in 4% of hemolysis after 35 days of storage.
ATP content of untreated and treated RBCs declined with similar kinetics during
storage.
Conclusion: PDT with Tri-P(4) for 60 min of RBCC inactivates a wide range of
pathogens, but not cell-associated HIV and a non-enveloped virus and
compromises RBC quality. This impairs PDT with Tri-P(4) as a suitable method for
pathogen inactivation in RBCC. Therefore, further improvements of the treatment
to potentiate pathogen inactivation and to preserve RBC integrity will be required
to generate an effective treatment to decontaminate RBCC.
94
Pathogen inactivation in RBC
INTRODUCTION
Risks associated with blood transfusion have dramatically decreased by the
implementation of careful donor selection and sensitive tests. Since the
introduction of nucleic acid tests (NAT) for Hepatitis C virus (HCV) and HIV, the risk
of transfusion-associated transmission of these viruses has decreased to less than
one in a million donations, although low levels of viraemia were missed in a
recent case of HIV transmission through transfusion of RBCC.1,2 Furthermore,
there is a residual risk of transmission of viruses that are not regularly screened,
such as human parvovirus B19 and human cytomegalovirus.3,4 The emergence of
new viruses and increased donor travel has resulted in an increased awareness of
the risks associated with blood transfusion.5-7
The safety of cellular blood products is not only challenged by emerging viral
infections, but also by common bacterial infections, as RBCC are not routinely
tested for bacterial contamination. The prevalence of bacterial transmission
through RBC transfusion is reported by the French Hemovigilance network to
amount to 1.6% (73 / 4706).8 Also reports from the British Serious Hazards of
Transmission and the US FDA indicate that bacterial contamination of RBCC,
mainly with Yersinia Enterolitica and Pseudomonas spp, is a major cause of
morbidity and mortality.9,10
Implementation of individual tests to detect a wider range of pathogens
responsible for morbidity and mortality seems unrealistic. A strategy involving
pathogen inactivation technologies may offer better perspectives, further
improving the safety of transfusion products. Pathogen inactivation technologies
based on the use of UV-light, as developed for platelets are very promising 11.
However, this technology is not applicable to RBCC, as the technologies based on
the use of light are hampered by the fact that hemoglobin (Hb) strongly absorbs
between 300-600 nm. Therefore, pathogen inactivation technologies for RBCC can
use either light-independent chemical compounds or photodynamic compounds
that can be activated with light at wavelengths above 600 nm. The chemical
approach uses S-30312 or Inactine 13, which have a high affinity for nucleic acid,
inducing irreversible alkylation. RBCC treated with these compounds showed
95
Chapter V
acceptable in-vitro quality and in-vivo survival. However, transfused patients
unexpectedly produced antibodies against the treated RBCs, resulting in the
postponement of the initiated clinical trials.14,15
The photodynamic approach relies on the use of light-absorbing molecules which,
upon activation with light at the appropriate wavelength, produce singlet oxygen,
causing oxidative damage to target cells. The resulting damage occurs in the close
vicinity of the photosensitizer and can lead to inactivation of the microorganism.16 Several photosensitizers have been investigated for their ability to
inactivate pathogens in RBCC; however most of them caused unacceptable RBC
damage.17-21
Recently, we showed that PDT of RBCC with the positively charged porphyrin TriP(4) resulted in 5 log10 reduction of the model virus VSV with only limited damage
to subsequently stored RBCs.22 This was considered worthy of further
investigations. In this article, we extend the previous study by investigating the
effect of PDT with Tri-P(4) on a series of viruses and bacteria. HIV-infected cells
were used to study the inactivation of cell-associated HIV and to study the effect
of the treatment on infected cells. Finally, we evaluated the effect of PDT dosage,
needed for optimal pathogen inactivation, on in-vitro RBC quality.
MATERIAL AND METHODS
REAGENTS AND PHOTOSENSITIZER
Photosensitizer Mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin chloride (Tri-P(4))
was kindly provided by Buchem N.V. (Apeldoorn, the Netherlands). Stock
solutions of 1 mM were prepared in PBS pH 7.4 (Leiden University Medical Center
(LUMC) pharmacy, Leiden, the Netherlands) and stored in the dark at 2-6 °C. RBC
storage solutions SAG-M and AS-3 were obtained from Baxter Fenwal (Utrecht,
the Netherlands) and Haemonetics Co. (Braintree, MA), respectively. SAG-M
contains 150 mM NaCl, 1.25 mM adenine, 50 mM glucose, and 29 mM mannitol.
AS-3 contains 70 mM NaCl, 61.1 mM glucose, 2.22 mM adenine, 2 mM citric acid,
20 mM Na3citrate, and 15.5 mM Na2HPO4.
96
Pathogen inactivation in RBC
PREPARATION OF RBCC
Whole blood was collected using a quadruple CPD / SAG-M blood bag system
(Compoflex; Fresenius Hemocare, Emmer Compascuüm, the Netherlands). After
overnight storage at room temperature, the whole blood unit was centrifuged at
2900 x g for 8 min and separated into RBC, plasma, and buffy coat with
Compomat G4 (Fresenius Hemocare). SAG-M was transferred from one of the
satellite bags to the RBC. The RBC suspension was leucodepleted by passage over
a BioR leucocyte-reduction filter (Fresenius Hemocare, Cavezzo, Italy). The volume
of the resulting suspension was 270-320 mL with a hematocrit (Hct) of about 60%.
BACTERIA
Pseudomonas aeruginosa (G-, isolated from a patient) was kindly provided by the
Central Bacteriological Laboratory (Haarlem, the Netherlands). Yersinia
enterolitica (G-, ATCC #9610) and Staphylococcus aureus (G+, ATCC #2593) were
kindly provided by the Department of Medical Microbiology of the LUMC, Leiden,
the Netherlands. Bacteria were grown in 10 mL of brain heart infusion broth
(Oxoid Ltd., Hampshire, UK) at 35 °C. After 18 hours, bacteria were harvested by
centrifugation at 2000 x g for 10 min, washed twice with PBS and finally
resuspended in PBS to an optical density of 0.7 at 650 nm corresponding to
approximately 108 CFU/mL.
VIRUSES AND CELL LINES
A series of lipid-enveloped and non-lipid enveloped viruses were used as models
for the major blood-borne viruses (Table 1). Pseudorabies virus (PRV), strain
Aujeszki Bartha K61 (Duphar, Weesp, the Netherlands), was cultivated and
titrated on PD5 swine kidney cells (Duphar). Bovine viral diarrhoea virus (BVDV),
strain NADL (VR-534, ATCC, Manassas, VA, USA), was cultivated on MDBK cells
(CCL-22, ATCC) and titrated on EBTr cells (Animal Sciences Group, Lelystad, the
Netherlands). HIV, strain HTLV-IIIB (National Cancer Institute, Bethesda, MD,
USA), was cultivated on H9 cells (National Cancer Institute) and titrated on MT2
cells (Wellcome, Beckenham, Kent, UK). Canine parvovirus (CPV), strain 780916
97
Chapter V
(State University Rotterdam, Rotterdam, the Netherlands), was cultivated and
titrated on A72 cells (State University Rotterdam). For BVDV and PRV nonconcentrated-serum containing culture supernatants served as inocula for spiking
studies. For CPV and extracellular HIV (E-HIV) concentrated serum-free inocula
were used. For cell-associated HIV (CA-HIV) chronically HIV-infected H9 cells
(National Cancer Institute, Bethesda, MD) were used. CA-HIV contains all forms of
the virus present during its life cycle: provirally infected cells, and intracellular,
membrane bound and cell-free virus particles.
Virus
Virus family Virus group
Genome type Size [nm]
Model for
PRV
Herpes
Lipid enveloped
dsDNA
100-200
Large lipid env. dsDNA viruses
(Hepatitis B virus,
Cytomegalovirus)
Lipid enveloped
ssRNA
37-50
Hepatitis C virus,
BVDV Flavi
West-Nile virus
HIV
Retro
Lipid enveloped
2 ssRNA
100
(Relevant virus)
CPV
Parvo
Non-lipid
enveloped
ssDNA
18-26
Human parvovirus B19
Table 1: Viruses, their properties and use as model
LIGHT SOURCE AND EXPERIMENTAL SET-UP
A 300 W halogen lamp (Philips, Eindhoven, the Netherlands) combined with a cutoff filter (Kodak, wratten nr. 23A, Rochester, NY) only transmitting light > 600 nm,
was used as source of red light. To avoid heating of the samples, the red light was
passed through a 1-cm thick water layer. RBCC was treated in tissue culture grade
Petri dishes (Greiner, Alphen a/d Rijn, the Netherlands) placed under the red light
98
Pathogen inactivation in RBC
source on a shaker. The fluence rate at the level of the Petri dish was adjusted to
100 W/m2 as measured with an IL1400A radiometer with a SEL033 detector
(International Light, Newburyport, MA, USA).
PHOTODYNAMIC TREATMENT
Stock solutions of bacteria and viruses were 10-fold diluted in the RBCC. To
maintain the original Hct, RBCC were centrifuged at 2000 x g for 10 min.
Subsequently, 10% of the total RBCC volume was removed from the supernatant
and the same volume of solutions containing pathogens was added to RBCC. TriP(4) was added to the spiked RBCC to a final concentration of 25 μM. The
suspension was incubated in the dark for either 5 min (for viruses) or 30 min (for
bacteria) under continuous mixing. Subsequently, the suspensions were
transferred to Petri dishes (Greiner). The resulting thickness of the cell layer was 1
mm. The dishes were illuminated, under continuous agitation (100-120 RPM) for
various periods of time to determine pathogen inactivation kinetics. For each
sample, a separate Petri dish was used. The maximum illumination time was 60
min, corresponding to a light dose of 360 kJ/m2. As a negative control (or light
control), RBCC were illuminated without addition of pathogens and
photosensitizer for the maximum illumination time tested. As a positive control
(or dark control), RBCC were spiked with pathogens and photosensitizer and
subsequently were kept in the dark for the maximum illumination time tested.
INACTIVATION OF BACTERIA
The number of viable bacteria in samples collected was determined with limiting
dilution assays, according to a modified version of the method described by Miles
and Misra.23 Samples were serially 10-fold diluted in PBS. Each dilution was plated
on an iso-sensitest agar (Oxoid Ltd, Hampshire, UK) plate. Staphylococcus aureus
and Pseudomonas aeruginosa were incubated at 35°C for at least 18 h and
Yersinia enterolitica was incubated at room temperature (20-25°C) for at least 36
h. After the incubation, the colonies on the agar plates were counted and colony
forming units (CFU) were calculated for each sample tested. Clearance factors (CF)
were calculated by the formula: CF=log10 (total amount of bacteria spiked as
99
Chapter V
derived from the inoculum sample / total amount of bacteria recovered from the
treated sample).
INACTIVATION OF VIRUSES
Infectivity was measured with TCID50 assays and bulk culture tests. For TCID50
assays, three-fold serial dilutions of samples were prepared in culture media and
50 μL (or 0.5 mL for HIV) volumes were tested in eight replicates. To detect small
amounts of virus, samples were tested in duplicate bulk culture tests using 175
cm2 flasks. BVDV, CPV, and PRV cultures were inspected microscopically for
cytopathic effects at 6, 7, and 5 days post-infection (dpi), respectively. HIV
cultures were inspected microscopically twice a week for formation of syncytia
until 21 dpi. Prior to testing the samples were 27-fold diluted with culture
medium and centrifuged at 1000 x g for 10 min to remove the RBCs. HIV-infected
H9 cells were isolated from the RBCC with Ficoll-Paque density gradient
centrifugation and tested in a co-culture assay with MT2 cells as previously
described.24 Virus titres were calculated by the Spearman-Kärber method, and
expressed as TCID50 / mL which define the reciprocal dilution that is able to infect
50% of the inoculated cultures. If all cultures were negative, the titre was
considered to be less than 1/total test volume (mL). Reduction factors (RF) were
calculated by the formula: RF = log10 (total amount of virus spiked as derived from
the reference sample / total amount of virus recovered from the treated sample).
APOPTOSIS OF CHRONICALLY HIV INFECTED H9 CELLS
HIV-infected H9 cells were isolated from the RBCC samples using Ficoll-Paque
density centrifugation, diluted with culture medium and incubated at 37°C. After
24 h, the cells were resuspended in HEPES binding buffer (20 mM HEPES, 132 mM
NaCl, 6 mM KCl, 1 mM MgSO4, 1.2 mM KHPO4, 0.5% albumin, and 1 mM CaCl2, pH
7.4). The cells were labelled with AnnexinV and propidium iodine (PI) (ApoptestTMFitc kit, VPS Diagnostics, Hoeven, the Netherlands) during 10 min on ice, washed
once with binding buffer and analysed in BD flow cytometer (Facscan) (Becton
Dickinsons, Erembodegem, Belgium). As a positive control for apoptosis induction,
incubation with 200 μM etoposide (Sigma-Aldrich, Saint-Louis, MI) for 16 h was
100
Pathogen inactivation in RBC
used; as a negative control, untreated HIV-infected H9 cells and non-infected H9
cells incubated in culture medium only were used.
RBC QUALITY ASSESSMENT
Photodynamically treated RBCC, without pathogens, were washed twice and
resuspended in AS-3 to a final Hct to 60%. The washed RBCs were stored in 15 mL
tubes (Greiner) at 2-6°C for 35 days. Total Hb was measured with a Coulter Act 10
instrument (Beckman Coulter, Mijdrecht, the Netherlands). Free Hb in the
supernatant was measured with an Anthos SHT3 spectrophotometer (Anthos
Labtec b.v., Heerhugowaard, the Netherlands) at 415 nm. From these values, the
percentage hemolysis was calculated after correction for Hct. ATP content of the
cells was determined using enzymatic reaction with hexokinase and glucose in
neutralized perchloric acid extracts. NADPH production was measured at 340 nm
in an Anthos SHT3 spectrophotometer and the concentration was calculated
based on the absorption coefficient of NADPH.
RESULTS
PDT INDUCED BACTERIAL INACTIVATION
Photodynamic treatment with Tri-P(4) for 60 minutes resulted in > 6.4 log10
inactivation of the G+ bacteria S. aureus (Table 2). High levels of inactivation (5 6 log10, but not complete) were obtained for G- bacteria P. aeruginosa and Y.
enterolitica. No inactivation (<1 log10) of the bacteria tested was observed in the
dark controls.
PDT INDUCED VIRUS INACTIVATION
Photodynamic treatment with Tri-P(4) for 60 minutes resultedшϰ͘ϲ
in ůŽŐ
10
inactivation of PRV and > 5.5 log10 inactivation of BVDV and extracellular (E)-HIV
(Table 3). In contrast to these cell-free lipid-enveloped viruses, the inactivation of
cell-associated (CA)-HIV was limited to approximately 2 log10, while the nonenveloped CPV was completely resistant to the treatment. With exception of PRV,
101
Chapter V
no inactivation (<1 log10) of viruses was observed in the dark controls, indicating
that the observed viral inactivation was due to the combined effect of Tri-P(4) and
light. For PRV, a reduction of approximately 1.2 log10 was observed in the dark
controls.
Mean clearance factor ± standard deviation (log10)*
Inoculated
Bacteria
(log10
CFU/mL)
10 min
15 min
30 min
60 min
S.aureus
7.4 ± 0.2
2.8 ± 1.2
NA
5.8 ± 0.8
>6.4 ± 0.2
P.aeruginosa
7.8 ± 0.4
NA
1.2 ± 0.9
3.5 ± 1.1
4.8 ± 0.6
0.1 ± 0.6
Y.enterolitica
7.8 ± 0.2
NA
1.5 ± 0.3
2.7 ± 0.2
5.9 ± 0.4
0.3 ± 0.5
Dark
†
§
0.2 ± 0.4
Table 2: Inactivation of bacteria by photodynamic treatment with 25 μM Tri-P(4) and red light
* The mean ± standard deviation of 2 - 4 independent experiments with RBCC units from different
†
donors is presented. No bacteria were detected in all replicates tested. The limit of detection used
§
was 1 log10 CFU/mL. Dark controls: RBCC units were inoculated with bacteria and 25 μM Tri-P(4),
and subsequently incubated for 60 min in the dark. NA: Not applied
PDT INDUCED CELL DEATH OF HIV-INFECTED H9 CELLS
To further investigate the resistance of CA-HIV to PDT, we determined by FACS
analysis Annexin V and PI straining of the HIV-infected H9 cells treated with PDT.
No increase of Annexin V and PI straining of HIV-infected H9 cells was observed
after 15 min of treatment (Fig. 1). However a clear increase of Annexin V and PI
staining was observed after 30 min of treatment (p=0.001 as compared to 15
min), and approximately 90% of the cells stained positive after 60 min of
treatment. The staining of the untreated and dark controls of HIV-infected H9
cells was similar. HIV infection by itself already causes a certain degree of cell
death. Comparing uninfected and HIV-infected H9 cells, we found 12.5% and 35 %
positive for Annexin V and PI, respectively, while both type cells were even
sensitive to etoposide (88% and 74% positive for Annexin V and PI, respectively)
(data not shown).
102
Pathogen inactivation in RBC
Virus
Exp.
*
Inoculated
(Total TCID50 )
Reduction factor ± standard deviation (log10)
шϰ͘ϴцϬ͘Ϯ
шϰ͘ϴцϬ͘Ϯ
шϱ͘ϴцϬ͘Ϯ
шϰ͘ϲцϬ͘Ϯ
6.13
1.5 ± 0.4
шϮ͘ϴцϬ͘ϯ
>5.5 ± 0.3
-0.4 ± 0.4
6.25
1.7 ± 0.3
шϯ͘ϬцϬ͘Ϯ
>5.6 ± 0.2
-0.1 ± 0.4
6.80
3.0 ± 0.4
шϰ͘ϯцϬ͘ϯ
6.62
2.7 ± 0.4
>5.0 ± 0.2
>6.0 ± 0.2
-0.2 ± 0.3
8.22
1.1 ± 0.4
чϭ͘ϭцϬ͘ϯ
чϭ͘ϴцϬ͘ϯ
0.4 ± 0.4
8.12
чϬ͘ϴцϬ͘ϰ
чϭ͘ϬцϬ͘ϯ
чϭ͘ϳцϬ͘ϯ
чϬ͘ϮцϬ͘ϯ
7.56
0.1 ± 0.4
0.0 ± 0.4
0.1 ± 0.4
0.1 ± 0.4
7.14
-0.4 ± 0.3
-0.4 ± 0.3
-0.2 ± 0.4
-0.4 ± 0.3
8.15
10
B
10
A
10
B
10
A
10
B
10
A
10
B
10
A
10
B
10
шϰ͘ϵцϬ͘ϮΘ
чϳ͘ϮцϬ͘Ϯ
Dark
†
30 min
A
60 min
§
15 min
1.4 ± 0.3
PRV
7.86
BVDV
E-HIV
CA-HIV
CPV
шϰ͘ϲцϬ͘ϮΘ
чϳ͘ϬцϬ͘Ϯ
хϱ͘ϳцϬ͘ϮΘ
чϲ͘ϮцϬ͘Ϯ
1.1 ± 0.4
0.1 ± 0.3
Table 3: Inactivation of viruses by photodynamic treatment with 25 μM Tri-P(4) and red light
*
§
Each virus was tested in duplicate using RBCC units derived from two different donors. These
2
†
samples were also tested in 175 cm culture flasks. Dark controls: RBCC units were inoculated with
viruses and 25 μM Tri-P(4), and subsequently incubated for 60 min in the dark.
RBC IN VITRO QUALITY
We studied the in-vitro quality of RBCs after PDT. After PDT with the highest
tested light dose (60 min of illumination) of 60% RBCC in SAG-M, the treated cells
were washed twice and subsequently stored in AS-3 for a total period of 35 days.
After 0, 7, 21, and 35 days samples were collected and tested for hemolysis and
ATP content. The hemolysis of the treated RBCs gradually increased with time up
103
Chapter V
to 4 ± 2.6% after 35 days of storage (Fig. 2A). The hemolysis in untreated and dark
controls however remained below 1% after 35 days of storage. The ATP content of
untreated, treated RBCs, and dark controls declined with similar kinetics during
storage (Fig. 2B).
100
90
% cell death
80
70
60
50
40
30
20
10
0
untreated
15 min PDT
30 min PDT
60 min PDT
dark control
HIV-infected H9 cells
Figure 1. Induction of cell death of HIV-infected H9 cells by photodynamic treatment
(PDT) with 25 μM Tri-P(4) and red light.
HIV-infected H9 cells were spiked in RBCC and illuminated for 0, 15, 30, or 60
minutes. A dark control was collected after incubation of spiked RBCC with 25 μM
Tri-P(4) for 60 minutes, but not illuminated. Cells were analyzed for Annexin V
expression and PI uptake after incubation at 37°C for 24 h. % of cell death expressed
the total % of Annexin V positive plus Annexin V / PI double positive cells.
104
Pathogen inactivation in RBC
Figure 2. Hemolysis (A) and ATP content
(B) of RBCs stored in AS-3 for 35 days.
After PDT with Tri-P(4) and illumination
with red light for 60 min, samples were
washed and suspended in AS-3 as
described in Material and Methods. (o)
Untreated samples; (Ƒ) dark controls;
(Ɣ) photodynamically treated samples.
Mean with standard deviation of 4
independent experiments are shown.
DISCUSSION
To extent our previous study which showed few damage to RBCs after
photodynamic inactivation of VSV with Tri-P(4) 22, a broader range of bacteria and
viruses were tested and the subsequent damage induced to RBCs was evaluated.
We showed that both G+ and G- bacteria were sensitive to PDT with Tri-P(4). The
clearance factors obtained – approximately 5 log10 – is likely to be sufficient to
significantly increase the safety of RBCC for bacterial contamination, because
treatment will be performed directly after production and before storage; and the
initial contamination of blood products is very low, at the sensitivity level of
culturing systems, which is 1-10 CFU/mL.
105
Chapter V
We found that the viruses tested vary widely in their sensitivity to PDT with TriP(4) of RBCC. To reach 5 log10 of inactivation of cell-free HIV, we had to use a
fourfold higher light dose than required for 5 log10 of inactivation of VSV.22 VSV
has been used as a model virus for HIV, because both viruses are lipid-enveloped
RNA viruses of medium size and are equally sensitive to PDT with the
phthalocyanine Pc4.25 As the virucidal spectrum of PDT is dependent on the
selected photosensitizer, the illumination system, and the treatment solution, it is
not surprising to encounter in our study differences in sensitivities between VSV
and HIV. Cell-associated HIV was clearly less sensitive to PDT with Tri-P(4) than
cell-free HIV. This difference can be explained by a lack of diffusion of Tri-P(4) into
cells and the notion that HIV can be shedded into endosomes in a infectious
form.26 Tri-P(4) is positively charged photosensitizer and it is known that charged
photosensitizers are in general less effective in intracellular inactivation than
neutral photosensitizers.27 From preliminary data we know that Tri-P(4) does not
enter the cell, indicating that intracellular HIV is probably not exposed to the PDTmediated reactive oxygen species.
To further understand the difference in sensitivity to PDT with Tri-P(4) between
cell-free and cell-associated HIV, we determined whether the treatment induce
cell death in HIV-infected H9 cells. Although the treatment clearly induces cell
death, this is not enough to stop effectively the virus production. While the cells
undergo the process cell death – measured after 24 h 90% of the cells stain
positive for Annnexin V and PI - the virus already produced in the cells and not
reached by the PDT with Tri-P(4), can still be excreted and subsequently
contaminate the RBCC.
The sensitivities of BVDV and PRV to PDT with Tri-P(4) of RBCC were similar to that
of cell-free HIV. In dark controls no inactivation was found, except for PRV,
indicating that there was no direct inactivating effect of Tri-P(4). For PRV a mean
inactivation of 1.2 log10 was observed. This inactivation is probably due to antialpha-galactosyl natural antibodies present in normal human serum - 10 to 15% of
human plasma is still present in RBCC - resulting in neutralization of PRV, which
was produced on swine kidney cells.28
106
Pathogen inactivation in RBC
In contrast to the lipid-enveloped viruses tested, no inactivation after PDT with
Tri-P(4) of RBCC of the non-enveloped Canine parvovirus (CPV) was found. Within
the Parvoviridae family, however, various sensitivities to PDT were reported.
Porcine parvovirus (PPV) was found to be resistant to PDT with MB, while human
parvovirus B19 was found to be sensitive.29 This remarkable difference in
sensitivity might be explained by differences in the composition of the capsid of
PPV and human-B19. In our study, we used CPV that like PPV belongs to the
parvovirus genus, while human-B19 belongs to erythrovirus genus.30 Possibly, by
using CPV as model for human-B19, the inactivation of human-B19 after PDT with
Tri-P(4) of RBCC is underestimated.
The PDT with Tri-P(4) results in more than 5 log10 reduction of the lipid-enveloped
viruses extracellular-HIV, BVDV and PRV. Assuming that these data are relevant
for the major blood-borne infections HIV, HBV and HCV, the treatment may be
particularly useful for preventing viral transmission of RBCC derived from window
blood donations. To illustrate the contribution of PDT with Tri-P(4) to viral safety
of RBCC, a simple calculation of the residual risk can be made. The 95% detection
limit for viral screening in RBCC donations is estimated to be 183 gEq/mL for HIV
NAT, 126 gEq/mL for HCV NAT and 10000 IU/mL for HbSAg test.31,32 Assuming that
RBCC contain 15 mL of plasma and all virus is present in the plasma fraction of the
RBCC, the maximal viral load in RBCC is 3.103 gEq of HIV, 2 .103 gEq of HCV and
1,5.105 IU of HBV. Assuming that the genome-equivalent (or IU) to infectiousparticle ratio is 1000 for HIV and 10 for HCV and HBV33, we can estimate the
presence of maximally 3 infectious particles of HIV, 200 infectious particles of HCV
and 15000 infectious particles of HBV per RBCC. A 5 log10 reduction would result
in a residual infectivity per RBCC of 0.00003 infectious particle of HIV, 0.002
infectious particle of HVC and 0.15 infectious particle of HBV. This calculation
illustrates that the risk of transmitting the infection by transfusion of a treated
RBCC in case of a window donation is significantly reduced compared to untreated
RBCC. However, considering the titers of non-routinely tested viruses, which may
amount to 106 to 1012 IU/mL, it is clear that the conditions used in our study to
inactivate these viruses would fail to achieve total inactivation.
107
Chapter V
Recent studies investigating the mechanism of pathogen killing found that, with
the yeast C. albicans, the addition of 10% PBS enhanced influx of the
photosensitizer into the yeast, potentiating the photosensitizing effect.34 This
mechanism might also be applicable for bacteria as suggested by Merchat et al.35
It would be interesting to investigate the conditions upon which bacteria,
leukocytes, and viruses, but not the RBCs, are permeable to the photosensitizer,
increasing the Tri-P(4)-photoinactivation efficiency.
In our previous studies, we showed that PDT with Tri-P(4) for 15 min was sufficient
for 5 log10 inactivation of VSV inducing only a slight increase in hemolysis of
subsequently stored RBCs.22 However, treatment up to 60 min was required to
inactivate substantially bacteria and viruses tested in this study, and this clearly
increased RBC damage. The hemolysis in treated RBCC remained below 0.8% after
8 days of storage, but increased to 4% after 35 days of storage. The relatively high
values for hemolysis can partially be explained due to suboptimal storage of the
treated RBCs in tubes and lower values would be expected when the samples
would be stored in bags. The latter was for practical reasons not possible. The
hemolysis in untreated samples and dark controls was about 3-4 times higher
than during storage under optimal conditions in bags. In contrast to the
hemolysis, PDT with Tri-P(4) did not appeared to induce more ATP loss in AS-3
stored RBCs.
In conclusion we showed that PDT with Tri-P(4) of RBCC inactivates a wide range
of pathogens, potentially closing the window period for routinely tested viruses.
In Western countries, however, incidence of window donation is so rare that the
benefit for increasing the blood safety is very small. Further research must include
the use of particular incubation media, which could enhance the virucidal and
bacterial effect of the photodynamic treatment, the protection of the RBCs as well
as the improvement of storage media to better preserve RBC integrity. These
many problems must be solved before Tri-P(4)-mediated PDT can be considered
as a feasible approach to inactivate pathogens in RBC products.
108
Pathogen inactivation in RBC
REFERENCES
1. Barbara J. Why 'Safer than ever' may not be quite safe enough. Transfus. Med.
Hemother. 2004 Jan;31(S-1):2-10.
2. Delwart EL, Kalmin ND, Jones TS, Ladd DJ, Foley B, Tobler LH, Tsui RCP, Busch MP.
First report of human immunodeficiency virus transmission via an RNA-screened
blood donation. Vox Sang. 2004 Apr 1;86(3):171-7.
3. Cesaire R, Kerob-Bauchet B, Bourdonne O, Maier H, Amar KO, Halbout P, Dehee A,
Desire N, Dantin F, Bera O, et al. Evaluation of HTLV-I removal by filtration of blood
cell components in a routine setting. Transfusion 2004 Jan;44(1):42-8.
4. Nichols WG, Price TH, Gooley T, Corey L, Boeckh M. Transfusion-transmitted
cytomegalovirus infection after receipt of leukoreduced blood products. Blood 2003
May 15;101(10):4195-200.
5. Wüllenweber J, Cassens U, Sica S, Sibrowski W, Herrmann M. West Nile Virus - an
Emerging Transfusion-Transmissible Pathogen. Transfus. Med. Hemother.
2004;31(6):373-7.
6. Seifried E., Mueller M.M. European Parliamentary Public Hearing on The impact of
SARS and Other emerging pathogens on transfusion medicine. Blood Therapies in
Medicine 2003;3(3):109-10.
7. Rox JM, Eis-Hubinger AM, Muller J, Vogel M, Kaiser R, Hanfland P, Daumer M. First
human immunodeficiency virus-1 group O infection in a European blood donor. Vox
Sang. 2004 Jul;87(1):44-5.
8. Andreu G, Morel P, Forestier F, Debeir J, Rebibo D, Janvier G, Herve P.
Hemovigilance network in France: organization and analysis of immediate
transfusion incident reports from 1994 to 1998. Transfusion 2002 Oct;42(10):135664.
9. Stainsby D., Cohen H., Jones H. Haemovigilance in the UK - the Shot scheme. Blood
banking and transfusion medicine 2004;2(1):27-30.
10. Wagner SJ. Transfusion-transmitted bacterial infection: risks, sources and
interventions. Vox Sang. 2004 Apr 1;86(3):157-63.
11. Allain JP, Bianco C, Blajchman MA, Brecher ME, Busch M, Leiby D, Lin L, Stramer S.
Protecting the Blood Supply From Emerging Pathogens: The Role of Pathogen
Inactivation. Transfus. Med. Rev. 2005 Apr;19(2):110-26.
109
Chapter V
12. Cook D. In vivo analysis of packed red blood cells treated with S-303 to inactivate
pathogens. Blood 1998;92:10 (suppl.1)(503a).
13. Purmal A, Valeri CR, Dzik W, Pivacek L, Ragno G, Lazo A, Chapman J. Process for the
preparation of pathogen-inactivated RBC concentrates by using PEN110 chemistry:
preclinical studies. Transfusion 2002;42(2):139-45.
14. Cerus Co, Baxter International Inc. Baxter And Cerus Halt Red Blood Cell Clinical
Trials For Investigational Pathogen Inactivation System. Message to: Cerus Co.,
Baxter International Inc. In: www.cerus.com/Press releases. 2003 Sep 4.
15. V.I.Technologies I. Vitex halts enrollment in Pase III chronic trial of the Inactine
Pathogen Reduction System for Red Blood cells. Message to: Inc. V.I.echnologies.
In: www.vitechnologies.com. 2003 Nov 17.
16. Malik Z, Hanania J, Nitzan Y. Bactericidal effects of photoactivated porphyrins--an
alternative approach to antimicrobial drugs. J. Photochem. Photobiol. B 1990
May;5(3-4):281-93.
17. VanSteveninck J, Trannoy LL, Besselink GAJ, Dubbelman TMAR, Brand A, Korte Dd,
Verhoeven AJ, Lagerberg JWM. Selective Protection of erythrocytes against
photodynamic damage by the band3 ligand Dipyridamol. Transfusion
2000;40(11):1330-6.
18. Rywkin S, Ben-Hur E, Malik Z, Prince AM, Li YS, Kenney ME, Oleinick NL, Horowitz B.
New phthalocyanines for photodynamic virus inactivation in red blood cell
concentrates. Photochem.Photobiol 1994;60(2):165-70.
19. Wagner SJ. Virus inactivation in blood components by photoactive phenothiazine
dyes. Transfus. Med. Rev. 2002;16(1):61-6.
20. Lagerberg JWM, Uberriegler KP, Krammer B, VanSteveninck J, Dubbelman TM.
Plasma membrane properties involved in the photodynamic efficacy of
merocyanine 540 and tetrasulfonated aluminum phthalocyanine. Photochem.
Photobiol. 2000;71(3):341-6.
21. North J, Neyndorff H, Levy JG. Photosensitizers as virucidal agents. J. Photochem.
Photobiol. B 1993;17(2):99-108.
22. Trannoy L.L., Lagerberg J.W.M., Dubbelman T.M.A.R., Schuitmaker J.J, Brand A.
Positively charged porphyrins: a new series of photosensitizers for sterilization of
RBCs. Transfusion 2004 Aug 1;44(8):1186-96.
110
Pathogen inactivation in RBC
23. Miles A.A., Misra S.S. The estimation of the bactericidal power of the blood. J.
Hygiene 1938;38:732-49.
24. Borkow G, Barnard J, Nguyen TM, Belmonte A, Wainberg MA, Parniak MA. Chemical
barriers to human immunodeficiency virus type 1 (HIV-1) infection: retrovirucidal
activity of UC781, a thiocarboxanilide nonnucleoside inhibitor of HIV-1 reverse
transcriptase. J. Virol. 1997 Apr 1;71(4):3023-30.
25. Ben-Hur E, Chan WS, Yim Z, Zuk MM, Dayal V, Roth N, Heldman E, Lazo A, Valeri CR,
Horowitz B. Photochemical decontamination of red blood cell concentrates with the
silicon phthalocyanine PC 4 and red light. Developments in Biological
Standardization 2000;102:149-55.
26. Gould SJ, Booth AM, Hildreth JE. The Trojan exosome hypothesis. Proc. Natl. Acad.
Sci. U.S.A 2003 Sep 16;100(19):10592-7.
27. Margolis-Nunno H, Ben-Hur E, Gottlieb P, Robinson R, Oetjen J, Horowitz B.
Inactivation by phthalocyanine photosensitization of multiple forms of human
immunodeficiency virus in red cell concentrates. Transfusion 1996;36(8):743-50.
28. Hayashi S, Ogawa S, Takashima Y, Otsuka H. The neutralization of pseudorabies
virus by anti-alpha-galactocyl natural antibody in normal serum. Virus Res. 2004
Jan;99(1):1-7.
29. Mohr H, Bachmann B, Klein-Struckmeier A, Lambrecht B. Virus inactivation of blood
products by phenothiazine dyes and light. Photochem. Photobiol. 1997;65(3):441-5.
30. Käsermann F., Kempf C, Boschetti N. Strengths and limitations of the model virus
concept. PDA. J. Pharm. Sci. Technol. 2004 Sep;58(5):244-9.
31. Koppelman MHGM, Sjerps MC, Reesink HW, Cuypers HTM. Evaluation of COBAS
AmpliPrep nucleic acid extraction in conjunction with COBAS AmpliScreen HBV
DNA, HCV RNA and HIV-1 RNA amplification and detection. Vox Sang.
2005;89(4):193-200.
32. Yoshikawa A, Gotanda Y, Itabashi M, Minegishi K, Kanemitsu K, Nishioka K. Hepatitis
B NAT virus-positive blood donors in the early and late stages of HBV infection:
analyses of the window period and kinetics of HBV DNA. Vox Sang. 2005;88(2):7786.
33. Weusten JJ, van Drimmelen HA, Lelie PN. Mathematic modeling of the risk of HBV,
HCV, and HIV transmission by window-phase donations not detected by NAT.
Transfusion 2002 May;42(5):537-48.
111
Chapter V
34. Lambrechts SA, Aalders MC, Van Marle J. Mechanistic study of photodynamic
inactivation of Candida albicans by a cationic porphyrin. Antimicrob. Agents
Chemother. 2005 May;49(5):2026-34.
35. Merchat M, Spikes JD, Bertoloni G, Jori G. Studies on the mechanism of bacteria
photosensitization by meso- substituted cationic porphyrins. J. Photochem.
Photobiol. B 1996;35(3):149-57.
112
CHAPTER VI
PHOTODYNAMIC TREATMENT
WITH TRI-P(4) FOR PATHOGEN
INACTIVATION IN CORD BLOOD
STEM CELL PRODUCTS
Laurence L. Trannoy1, Yvette van Hensbergen1, Johan W.M. Lagerberg2 and
Anneke Brand1
1
Department of Research and Education, Sanquin Blood Bank Southwest, Leiden,
the Netherlands; 2Department of Blood Cell Research, Sanquin Research,
Amsterdam, the Netherlands
Adapted from Transfusion; 2008; 48(12): 2629-37
113
Chapter VI
ABSTRACT
Background: Hematopoietic stem cell transplants and culture of hematopoietic
progenitor cells (HPC) require pathogen free conditions. The application of a
method of pathogen inactivation in RBCC using PDT was investigated for the
decontamination of cord blood stem cell (CBSC) products.
Study design and methods: CBSC products, spiked with G+ and G- bacteria, were
treated with PDT using Tri-P(4) and red light. After PDT, in-vitro and in-vivo
evaluation of the CBSC functions were performed.
Results: PDT of CBSC products resulted in the inactivation of the bacteria, with
Staphylococcus aureus being the most resistant. Complete decontamination was
achieved when CBSC products were contaminated with low titers of bacteria. PDT
had no effect on leukocyte viability, the ex-vivo expansion potential of the
progenitor cells and their capacity to differentiate to various hematopoietic cell
lineages. However, PDT reduced the engraftment of human CBSC in NOD-scid
mice; affecting particularly the B-cell lineage engraftment.
Conclusion: Pathogen inactivation of CBSC with Tri-P(4)-mediated PDT is feasible
at contamination level up to 10-20 cfu/mL and can be considered when ex-vivo
expansion culture is anticipated. However this treatment is not recommended for
transplantation purposes at this time. Further investigations may elucidate why
engraftment is diminished.
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Pathogen inactivation in cord blood stem cell products
INTRODUCTION
The use of HPC products is increasing and expanding from hematopoietic
transplantation to tissue engineering. To overcome the current limitations of the
use of cord blood transplantation in adult patients, such as delayed hematopoietic
reconstitution because of the low number of stem cells, ex-vivo expansion of CBSC
is widely explored.1-4
HPC products can harbor microorganisms, which in low levels (< 20 cfu/mL) may
not be of clinical relevance in transplantation. However, HPC culture and
expansion may create conditions in which low numbers of contaminating bacteria
may reach levels of clinical significance. The contamination rate of HPC products
varies with the graft source. Bacterial contamination of mobilized peripheral
blood HPCs is lower compared to bone marrow harvests (2.1 % and 3.5 %
respectively)5, while up to 13% of CBSC products has been found positive in
bacterial cultures, depending on the sample size and the method of testing.6,7
Recently, the FDA guidelines and Netcord FACT standards requested the release
of pathogen-free CBSC products for transplantation. All contaminated products
are discarded, what may form a serious problem in case of cord blood
transplantation with a family donor. To minimize the risks of bacterial
contamination at the time of cord blood collection, efforts were made, i.e. by
stringent disinfection and intensive monitoring of clean areas. However, these
efforts did not reduce the rate of microbial contamination.8 The addition of
antibiotics in-vitro appeared to be unsuccessful and even introduced new
contamination.9 In general, the use of antibiotics is discouraged because of the
risk of development of bacterial resistance.10
To overcome these problems, we evaluated whether pathogen inactivation by
PDT with a cationic porphyrin, mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin
(Tri-P(4)) could be suitable for CBSC products. This method was originally
developed for RBCC, where it successfully inactivates non-cell bound lipidenveloped viruses, G+ and G- bacteria.11,12 Based on RBCC experiments, we first
tested and optimized the PDT conditions for CBSC application.11 Subsequently, we
115
Chapter VI
studied the potency of PDT to inactivate the G+ bacterium Staphylococcus aureus
and the G-bacteria Pseudomonas aeruginosa and Streptococcus agalactiae, since
especially this latter bacterium is frequently found in CBSC products (6.25 %).6
Furthermore, we evaluated the effect of PDT on the CBSC viability and HPC
functions.
MATERIAL AND METHODS
MATERIALS
Photosensitizer Tri-P(4) was kindly provided by Buchem b.v., Apeldoorn, the
Netherlands. Stock solutions of 1 mM were prepared in PBS and stored in the dark
at 2-6°C.
PBS (pH 7.4), Polymixin-B, Ficoll gradient and Hydroxy-ethyl starch (HES) were
provided by the Leiden University Medical Center (LUMC) Pharmacy, Leiden, the
Netherlands.
Cipoxin and Fungizone were provided by Bristol-Meyers-Squibb, Woerden, the
Netherlands.
IMDM medium was purchased from Invitrogen, Breda, the Netherlands. Penicillin
and streptomycin (pen/strep) were purchased from Bio-Whittaker (Verviers,
Belgium). Human serum albumin Cealb® and AB heparin plasma were provided by
Sanquin, Amsterdam, the Netherlands. Thrombopoietin was kindly provided by
Amgen, Thousand Oaks, CA, USA. DNAse, Human transferring saturated with
FeCl3.H2O were purchased from Sigma-Aldrich (Saint-Louis, MI, USA).
Phycoerythrin-conjugated (PE) anti-human CD3, CD14, CD15, CD19, CD33, CD34,
CD41, CD56 and isotype-matched mouse IgG2a monoclonal antibodies and
fluorescein-conjugated (FITC) anti-human CD45, CD61, CD62p and isotypematched mouse IgG1 monoclonal antibodies, apoptosis detection kit containing
Annexin V-FITC and binding buffer, IOTest3 lysis solution and the Flow-count
fluoropheres were purchased from Beckman Coulter, Mijdrecht, the Netherlands.
Rat anti-mouse CD45-FITC and rat IgG2b-FITC were purchased from PharMingen,
San Diego, CA, USA.
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Pathogen inactivation in cord blood stem cell products
StemSep CD34 positive selection kit, Methocult GF H4434, MegaCult and alkaline
phophastase detection system were provided by StemCell technologies, Grenoble,
France.
MICROORGANISMS
VSV, St Juan strain, cultivated in baby hamster kidney cells and the host cells A549
were kindly provided by the departments of Virology and Lung diseases
respectively, LUMC, Leiden, the Netherlands.
Pseudomonas aeruginosa (isolated from a patient) was kindly provided by the
Central Bacteriological Laboratory, Haarlem, the Netherlands. Staphylococcus
aureus (ATCC #2593) and Streptococcus agalactiae (ATCC #13813) were kindly
provided by the department of Medical Microbiology, LUMC, the Netherlands.
Bacteria were grown in 10 ml of brain heart infusion broth (Oxoid Ltd, Hampshire,
UK) at 35°C, 5% CO2. After 18 hours, bacteria were harvested by centrifugation at
20000 gmin, washed twice with PBS and resuspended in PBS to an optical density
of 0.7 at 650 nm, corresponding to about 108 colony forming units (CFU) per mL.
Aerobic hemo-cultures bottles were provided by Organon Teknika, Boxtel, the
Netherlands.
CORD BLOOD PROCESSING
After consent cord blood (CB) was collected in-utero before delivery of the
placenta. All newborns had a gestational age > 36 weeks. Blood was drawn from
the umbilical vein into a closed system (MacoPharma, Tourcoing, France)
containing 25 mL citrate-phosphate-dextrose anticoagulant solution and
processed within 36 hours. HES was added to the CB at a volume ratio of 1:5.
After a soft spinning at 500 gmin the stem cell-rich supernatant was separated
from the residual erythrocyte fraction and subsequently the leukocytes were
concentrated by spinning at 4000 gmin. The supernatant HES-plasma and
erythrocyte fraction were used to dilute the concentrated cells to a final white
blood cell concentration of 20.106 cells per mL and a Hct of 15-20 %. These values
were chosen as standard for the present investigation.
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Chapter VI
LIGHT SOURCE
For all illuminations, the light source was a 300 W halogen lamp (Philips,
Eindhoven, NL) combined with a cut-off filter (Kodak, wratten nr. 23A, Rochester,
NY) only transmitting light > 600 nm. The fluence rate at the level of the Petri dish
was adjusted to 100 W per m2, as measured with an IL1400A radiometer with a
SEL033 detector (International Light, Newburyport, MA). To avoid heating of the
samples, the light was passed through a 1-cm thick water layer. The temperature
did never exceed 25°C.
PHOTODYNAMIC TREATMENT
Tri-P(4) was added to the spiked CBSC to a final concentration of 50 μM, unless
otherwise indicated. Six mL of CBSC samples were transferred into 9 cm diameter
Petri dishes (tissue culture grade, Greiner, Alphen a/d Rhijn, the Netherlands),
resulting in a cell layer thickness of 1 mm. The dishes were illuminated under
continuous agitation (100-120 rpm) for various periods to determine pathogen
inactivation kinetics. The maximum illumination time was 60 min, corresponding
to a light dose of 360 kJ/m2. Two controls for the treatment were used, a light
control and a dark control. The light control corresponded to CBSC illuminated
without addition of photosensitizer and pathogens. The dark control
corresponded to CBSC spiked with photosensitizer and pathogens but kept in the
dark.
VIRAL INACTIVATION
Stock solutions of VSV were diluted 10-fold in CBSC to obtain approximately 105
extracellular viral particles per mL. Prior to PDT, samples with virus and
photosensitizer were incubated in the dark for 5 min under continuous mixing.
Infectivity of VSV was studied using Tissue Culture Infectious Dose 50% (TCID50), as
described before.13 The TCID50 is defined as the reciprocal dilution that is able to
infect 50% of the inoculated cultures. Briefly, CBSC samples spiked with VSV were
centrifuged at 2500 gmin. Subsequently, supernatants were 10-fold serially
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Pathogen inactivation in cord blood stem cell products
diluted, inoculated into A549 cell cultures in 96-wells microtiter plates (Greiner)
and incubated for 72 hours. The cytopathology of the cells was scored in 8 well
replicates for each dilution. Quantification of the virus titer was performed
according to the Spearman-Karber method13. Virus survival is expressed as
percentage of inocula.
BACTERIAL INACTIVATION
Bacterial suspensions were 10-fold diluted in CBSC to obtain a high titer of
contamination (107 cfu per mL). Low titer contamination (0.1-20 cfu per mL) was
obtained by a 107 and 108 fold dilution in PBS before the 10-fold dilution in the
CBSC. Prior to PDT, samples with bacteria were incubated with Tri-P(4) in the dark
for 30 min under continuous mixing.
The number of viable bacteria was determined with limiting dilution assays,
according to a modified version of the method described by Miles and Misra.14
Samples were 10-fold serially diluted in PBS. Each solution was plated on isosensitest agar and incubated at 35°C, 5% CO2. After at least 18 h, the colonies on
the agar plates were counted and the numbers of cfu were calculated for each
sample tested. Bacterial survival is given as percentage of inocula.
Contamination of whole CBSC samples (6 mL) spiked with low titer of bacteria (<
20 CFU/mLl) was tested in 2 standard aerobic hemo-culture bottles. The bottles
were incubated up to 7 days at 35°C using the BacT/Alert continuous monitoring
blood culture system (Organon Teknika, Boxtel, the Netherlands).
FLOW CYTOMETRIC ANALYSIS
After PDT application of CBSC, samples were washed 4 times with PBS at 1500
gmin to remove the photosensitizer. Cells were incubated with anti-human
monoclonal antibodies for 20 min at room temperature. After red cell lysis with
lysis solution IOTest3, samples were analyzed on a four-laser cytometer EpicsXLMCL flow cytometer using Expo32 software (Beckman Coulter, Mijndrecht, the
Netherlands). For the determination of CD34 expression, a total number of 75000
119
Chapter VI
counts was used and for all other cell types 10000 counts. Quantification of cell
concentrations was performed by using Flow-count fluorospheres.
For the 24 hour cell viability assessment, CBSC were washed to remove the
photosentizer and subsequently resuspended in autologous HES-plasma and kept
overnight at 2-6°C. Cell viability was assayed using Annexin V and binding buffer.
Unstained cells and cells labeled with isotype-matched mouse IgG2a-PE and IgG1FITC of irrelevant specificity were used as negative controls.
HPC COLONY ASSAYS OF CBSC
After PDT application and removal of the photosensitizer, CBSC were assayed in
duplicate in semi-solid cultures for their number of CFU of granulocytes and
monocytes (CFU-GM), burst forming units of erythrocytes (BFU-E), mix of those
units (CFU-GEM) and CFU of megakaryocytes (CFU-Mk). For CFU-GM, CFU-GEM
and BFU-E, 5.103 of leukocytes were plated and cultured in MethoCult GF H4434
(StemCell technologies, Grenoble, France). Hematopoietic colonies were scored
after 14 days of culture at 37°C, 5% CO2. Results are expressed as number of
colonies per 5x103 of plated cells.
For CFU-Mk, 1x105 of leukocytes were plated and cultured in MegaCult (StemCell
technologies). Megakaryocyte colonies containing more than 3 cells were scored
after 14 days at 37 °C, 5 % CO2. To differentiate the CFU-Mk to non-CFU-Mk, cells
were dehydrated and stained immune-histochemically with anti-human CD41
antibody and an alkaline phosphatase detection system (StemCell technologies).
Results are expressed as number of colonies per 1x105 of plated cells.
CD34+ EXPANSION AND DIFFERENTIATION INTO MEGAKARYOCYTES
After PDT application and removal of the photosensitizer, CBSC were resuspended
in autologous HES-plasma and kept overnight at 2-6°C. The next day, CD34+ cells
were isolated and expanded. Therefore mononuclear cells (MNC) were isolated
by centrifugation on Ficoll density gradient and subsequently, CD34+ cells were
isolated using MS separation columns (MiniMacs, MiltenyivBiotec, Amsterdam,
NL) and StemSep CD34 positive selection kit according manufacturer instructions.
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Pathogen inactivation in cord blood stem cell products
Cell concentration in the CD34 positive fraction was determined using a counting
chamber. The purity of the CD34+ cell suspension was 90 % ± 5 % as determined
by flow cytometry. The cells were then subsequently cultured at 1x105 cells per
mL in IMDM supplemented with 20% (v/v) AB heparin plasma, 0.34% (v/v) human
serum albumin Cealb®, 0.5 mg per mL human transferring saturated with
FeCl3.H2O, 1% (v/v) penicillin and streptomycin and 50 ng per mL Thrombopoietin.
Cultures were performed in 24-well plates (Greiner, Alphen a/d Rhijn, the
Netherlands) for 10 days at 37°C, 5% CO2. Cell concentrations were determined at
day 3, 7 and 9 of culture using a counting chamber. Culture medium was
refreshed with TPO at day 7, without TPO at day 9. At day 10 the expansion rate
was determined as follows:
Total number of harvested cells
Expansion rate =
Total number of CD34+ cells seeded.
The differentiation potential of the progenitor cells into megakaryocytes was
evaluated by flow cytometric measurements of stem cells (CD34+, CD61-),
megakaryocyte progenitors (CD61+) and megakaryocyte activation state (CD41+,
CD62p+).
CBSC TRANSPLANTATION
Human CBSC were transplanted into NOD-CB17-Scid (NOD-scid) mice to assay
bone marrow (BM) reconstitution after PDT.
Female 5-6 weeks old mice were obtained from Charles River (France) and were
maintained in animal facility of the LUMC (Leiden, the Netherlands). Animals were
housed in micro-isolator cages in laminar flow racks and were given autoclaved
food and acidified water containing 0.07 mg per mL Polymixin-B, 0.09 mg per mL
cipoxin and 0.1 mg per mL Fungizone. All animal experiments were approved by
the animal committee of the LUMC.
Seven to 10 weeks old mice were treated with whole body irradiation with a
single sub-lethal dose of 3.5 Gy, 4-24 hours before transplantation. The irradiated
recipients were divided into 2 groups. Each group of 3 mice received CBSC from
the same CB donor either as light control or after PDT with Tri-P(4) for 60 min.
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Chapter VI
Before transplantation, samples were washed and kept overnight as described
above. The next day, red cells were lysed with sterile IOTest3 lysis solution for 10
min at room temperature. Subsequently, the leukocytes were washed twice with
PBS and resuspended in PBS until a viable CD34+ cell concentration of 5x105 cells
per mL. Mice were transplanted by intravenous injection in the tail with
approximately 100.000 of CD34+ cells [range: 71250 - 112500 cells]. The total
number of CD45+ cells injected was in average 40.4 x 106 cells [range: 0.92 x 106 –
73.1 x 106 cells]. To avoid clotting, 0.13 mg per mL of DNAse was added to the
syringe.
After 7 weeks, mice were sacrificed with CO2 and BM was flushed from the
femurs. The level of chimerism in recipient mice was determined by flow
cytometric assessment of the percentage of human-CD45+ cells in murine BM and
peripheral blood. Therefore, after red cell lysis, suspensions of BM cells were
incubated with FITC-conjugated anti-human CD45 or mouse IgG1 monoclonal
antibodies and PE-conjugated rat anti-mouse CD45 and rat IgG2b (PharMingen,
San Diego, CA, USA) for 30 min at room temperature. Within the human-CD45
positive population, the proportion of myeloid cells was determined by using antihuman CD34, CD33 and CD41 while the proportion of lymphoid cells was
determined by using anti-human CD3 and CD19.
STATISTICAL ANALYSIS
For statistical analysis of the differences in cell viability one-way Analysis of
Variance (ANOVA) was used. To determine differences in Log inactivation of
microorganisms and to compare quantitative measurements between light
controls and PDT samples in HPC cultures and transplanted mice, paired
Student’s-t-test or Mann-Whitney test was used. All data are depicted as Mean ±
Standard Deviation (SD). P values < 0.05 were considered as statistically
significant.
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Pathogen inactivation in cord blood stem cell products
RESULTS
EFFECT OF PDT ON VSV INACTIVATION
% survival
PDT of CBSC products with 25 μM Tri-P(4) for 60 min resulted in a 3.6 log10 ± 1.3
inactivation of VSV. With 50 μM Tri-P(4), the reduction was 5.4 ± 0.3 log10 (Fig. 1).
In light and dark controls, no reduction in viral load was observed during the
treatment (data not shown).
A proper decontamination procedure should inactivate at least 5 log10 of the
added microorganism, therefore all other experiments were performed with 50
μM Tri-P(4).
1000
100
10
1
0.1
0.01
0.001
0.0001
0.00001
0
15
30
45
Illumination time (min)
60
Figure 1. Inactivation kinetic of
VSV in CBSC with PDT using
25μM (Ŷ) and 50μM (Ɣ) TriP(4).
Results are expressed as % of
viral
survival
(logarithmic
scale). 10% less survival
corresponds
to
1
log10
inactivation. Mean ± SD of 3
independent experiments are
shown.
Asterisks
indicate
statically significant differences
(P < 0.001) between the two
concentrations tested after 60
min of treatment as calculated
with the student’s-t-test.
EFFECT OF PDT ON BACTERIAL INACTIVATION
High load spike experiments (107 CFU/mL) were performed to determine the
kinetics of Tri-P(4)-mediated PDT to inactivate G- and G+ bacteria (Fig. 2). The
bacterial load of the studied bacteria decreased with increasing illumination time.
After 60 min of treatment, S. aureus (G+) load was reduced by 3.3 log10 ± 1.1 and
P. aeruginosa (G-) load by 4.2 log10 ± 0.8. S. agalactiae (G-) was shown to be very
123
Chapter VI
sensitive to PDT as within 30 min of illumination a 6.5 log10 ± 0.2 reduction was
obtained. In light and dark controls, no reduction in bacterial load was observed
during the treatment (data not shown). Low load spike experiments of CBSCs
were performed to determine the presence of residual bacteria in the treated
products (table 1). All CBSC treated with PDT were found negative up to 7 days of
culture. All samples treated with light only (no Tri-P(4)), became positive within 24
hours of culture, confirming the microbial contamination of the samples.
% survival
1000
100
10
1
0.1
0.01
0.001
0.0001
0.00001
0
Bacteria
15
30
45
Illumination time (min)
N
Mean initial titer
CFU/ mL (range)
P. aeruginosa
5
7,52 (0,9 - 17,5)
S. aureus
3
1,93 (0,1 - 3,7)
S. agalactiae
4
3,96 (0,4 - 10,6)
60
Figure 2.Inactivation kinetic of bacteria in high load
spiked CBSC using 50 μM
Tri-P(4).
(Ŷ) S. aureus; (Ɣ) P. aeruginosa, (Ÿ) S. agalactiae.
Results are expressed as
percentage of bacterial
survival
(logarithmic
scale). Mean ± SD of 3 to
5 independent experiments are shown. At
values < 0.001 no colony
was detected.
Status of duplicated hemo-cultures in Bact-alert
(culture duration ± sd)
Light control
positive
(18,5 h ± 1,92)
positive
(15,67 h ± 0,29)
positive
(17,12 h ± 2,81)
Table 1: Inactivation of low titer of bacteria in CBSC by PDT using 50μM Tri-P(4).
124
PDT
negative
(7 days)
negative
(7 days)
negative
(7 days)
Pathogen inactivation in cord blood stem cell products
EFFECT OF PDT ON CELL VIABILITY
The effect of PDT on the viability of the various cell types in the CBSC was
analyzed 24 hours after treatment. As can be seen from Fig. 3, PDT has no effect
on the viability of the hematopoietic precursor cells (CD34+). Also for the other
leukocyte subsets present in the CBSC product, no increase in the number of
apoptotic cells was observed after PDT.
Figure
3.
Apoptosis
of
leukocytes in CBSC 24 hours
after PDT.
Light controls (black bars), dark
controls (hatched bars), PDT
(gray bars). Results are given as
% of Annexin V positive cells
within the indicated cell
population. Mean +/- SD of 7
independent experiments are
showed. Statistics differences
were analyzed using one-way
ANOVA. All p values were >
0.05.
EFFECT OF PDT ON IN-VITRO HPC DIFFERENTIATION
To examine the effect of PDT on the in-vitro growth of myeloid and
megakaryocytic progenitor cells, semi-solid HPC colony assays were performed. As
can be seen from Fig. 4, PDT did not affect the formation of erythroid colonies
(BFU-e) and myeloid / megakaryocytic colonies (CFU-GM, CFU-GEM and CFU-Mk).
125
Chapter VI
BFU-E Light control
PDT
CFU-GM Light control
PDT
CFU-GEM
Light control
PDT
CFU-Mk
Light control
PDT
0
20
40
60
number of colonies
Figure 4. Effect of PDT on HPC differentiation in colony assays.
Results are expressed in total numbers of BFU-E, CFU-GM and CFU-GEM per
3
5
5.10 cells plated and numbers of CFU-MK per 1.10 cells plated. Statistic
differences between light controls and PDT samples were determined using
the paired student’s-t-tests. All p values were > 0.05.
EFFECT OF PDT ON EX-VIVO EXPANSION AND DIFFERENTIATION OF
CD34+ CELLS INTO MEGAKARYOCYTES
Since photodynamic treatment may in particular be considered for ex-vivo
expansion of CBSC, we examined the effect of PDT on this assay. As shown in Fig.
5A, there were no difference in expansion rate between cells treated with PDT
and cells treated with light only (p = 0,78). To determine the effect of the HPC
differentiation into megakaryocytes, different subsets of the expanded population
were examined (Fig. 5B). The proportion of progenitor megakaryocytes, as
determined by labeling with CD61, was similar for light controls and treated cells.
Also the remaining number of progenitor stem cells CD34+ / CD61- after
differentiation was the same in both groups. In addition, the activation state of
the megakaryocytes in the expanded cultures as determined by the double
expression of CD41 and CD62p+ on the cells after differentiation was not affected
by PDT (Fig. 5C).
126
Pathogen inactivation in cord blood stem cell products
B
50
120
40
100
% of total cells
Expansion rate
A
30
20
10
0
60
40
20
0
Light
control
PDT
progenitor
stem cells
progenitor
Mk
CD41
C
80
Light control
CD62p
PDT
Figure 5. Effect of PDT on ex-vivo HPC expansion and differentiation into
megakaryocytes.
(A) Ex-vivo expansion; (B) Differentiation of CD34+ into megakaryocytes. Results
give the % of progenitor stem cells (CD34+, CD61- cells) and of progenitor
megakaryocytes (CD61+) from the total number of cells in expanded culture. Light
controls (black bars), PDT (gray bars). Mean ± SD of 5 independent experiments are
shown. Statistic differences between light controls and PDT samples were
determined using the paired student’s-t-test; all p values were > 0.05; (C) Activation
state of megakaryocytes in expanded cultures: pictures representatives of 5
independent experiments with similar results.
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Chapter VI
EFFECT OF PDT ON IN-VIVO ENGRAFTMENT
As the results from all in-vitro assays did not show a negative effect of PDT on
CBSC, we tested in-vivo the effect of PDT on the CBSC engraftment potential (Fig.
6). Only 50% (6 out of 12) of the mice from the PDT group engrafted against 67%
(8 out of 12) in the control group (p=0.68). In addition, in engrafting mice, the
percentage of human CD45+ cells detected in murine BM was significantly lower
in the PDT group than in the control group. The reduction in CD45+ cells is
accompanied by a dramatic loss in the number of CD19+ cells. No significant
change was observed in the proportion of other lymphoid cells (CD3+ cells) or
myeloid cells (CD34+, CD33+ and CD41+ cells).
% of BM cells
100
80
60
40
20
0
CD45 CD34 CD19
CD3
CD33 CD41
Figure 6. Effect of PDT on BM reconstitution after transplantation of human-CBSCs in
NOD-scid mice.
Results showed the percentage (and means) of human-derived cells in the murine
BM from engrafted mice (>10% Hu-CD45+ cells) 7 weeks after transplantation of 4
independent cord blood donors. In the light control group (ż) 8 mice from 12
engrafted, in the PDT group (Ŷ) 6 mice from 12 engrafted (p=0.68; Fisher Exact test).
Means are indicated by horizontal lines, light controls by dotted lines, PDT by plain
¥
lines. P values were determined using paired student-t-test or Mann Whitney test.
128
Pathogen inactivation in cord blood stem cell products
DISCUSSION
We investigated the potential of Tri-P(4)-mediated PDT to decontaminate CBSC in
order to overcome the risks due to pathogenic contamination in ex-vivo and invitro applications.
Our results showed that model virus VSV and bacteria could be inactivated in
CBSC (Fig. 1 and 2). However, in comparison with application in RBCC11,12, the
treatment required a 2-fold higher Tri-P(4) concentration. The need for increase in
Tri-P(4) concentration may be explained by the difference in product contents.
CBSC contains plasma proteins which are nearly absent in the tested SAG-M
containing RBCC. These proteins, in particularly albumin, quench most of the
singlet oxygen generated by the action of light on the photosensitizer.15 HES,
which is also present in CBSC had only a dilution effect in plasma and did not
affect the PDT efficacy (data not shown).
Surprisingly, G+ S. aureus appeared to be more resistant to PDT inactivation as
compared to the G- bacteria tested. This result is in contrast with the high
sensitivity of the G+ bacteria found after PDT with porphyrins in saline solutions
and RBCC.11,12,16,17 Thereby, interesting suggestions can be made about the
binding site of Tri-P(4) on bacteria. Indeed, macrophages and platelets, which are
present in high amounts in CBSC products can bind to bacteria via negatively
charged teichoic acid components which are very abundant on the G+ bacterial
wall.18,19 The cells may compete with the positively charged photosensitizer TriP(4) for binding to bacteria through the teichoic acids, thereby reducing the
efficacy of Tri-P(4) to inactivate G+ bacteria in CBSC. However, despite these
limitations, we showed that pathogen inactivation in CBSC using PDT is feasible
for low dose of contaminants, more representative for the actual contamination
of CB (table 1).6
In our studies, results from all in-vitro assays demonstrated that PDT did not
affect HPC functions. Growth of myeloid and megakaryocytic progenitor cells and
HPC expansion after PDT did not differ from the light controls. These results
supported the use of PDT to decontaminate CBSC for subsequent use as
129
Chapter VI
transplant to reconstitute the BM. Therefore, to determine the effect of PDT on
the CBSC engraftment and the recovery of hematopoietic cell system in-vivo, we
performed xeno-transplantation experiments in NOD-scid mice.20
To our surprise, PDT application of CBSC products resulted in a reduced number of
engrafted mice and a lower number of human-CD45+ in their BM as compared to
the light control group. The reduction in engraftment was accompanied by a
dramatic reduction in the number of CD19+ cells and a tendency to a lower
number of myeloid cells, although the latter being not statically significant. From
these results, it is clear that PDT application of CBSC impaired the HPC
engraftment and hematopoietic recovery in NOD/Scid mice.
The diminished engraftment observed 42-49 days after transplantation could be
the result of an impaired homing of the photodynamically treated cells (Fig. 6).
Possibly, the cells may need an elongated recovery time to regain their migratory
capacity, to settle into the BM microenvironment, or to proliferate and
differentiate properly.21 Moreover, measurements up to 120 days after
transplantation in the BM, spleen and peripheral blood would give more insights
in the level of PDT-induced engraftment inhibition.
Although the mechanism of human HPC homing and engraftment in NOD/Scid
mice is still not completely understood22, it is well accepted to observe skewing in
B-cell development in the mouse BM after transplantation of cord blood-derived
HPC in NOD/Scid models.23-27 Correspondingly, we found this predominance in B
cell development in the mouse BM from our light control group. It has been
shown by Fibbe et al. and Hogan et al. that the preferential B-cell engraftment
was not related to the presence of committed or relatively mature B cells in the
graft.23,24 Therefore, it is unlikely that the dramatic decrease in B-cell numbers in
mouse BM is explained by a selective effect of PDT on the B-cell precursors or
committed stem cells. Many factors have been described to influence migration
and engraftment of stem cells, such as cell cycle, cytokines, chemokines,
expression of adhesion molecules and cell-cell interaction.21,28-33 Further research
should reveal whether PDT interacts with one of these factors, thereby
contributing to the reduced engraftment potential. In conclusion, PDT with Tri130
Pathogen inactivation in cord blood stem cell products
P(4) is able to decontaminate CBSC without affecting in-vitro cell viability and
growth. Engraftment capabilities are however altered.
REFERENCES
1. Sasayama N, Kashiwakura I, Tokushima Y, Xu Y, Wada S, Murakami M, Hayase Y, Takagi
Y, Takahashi TA. Expansion of megakaryocyte progenitors from cryopreserved
leukocyte concentrates of human placental and umbilical cord blood in short-term
liquid culture. Cytotherapy 2001;3:117-26.
2. Encabo A, Mateu E, Carbonell U, Minana MD. CD34+CD38- is a good predictive marker
of cloning ability and expansion potential of CD34+ cord blood cells. Transfusion
2003;43:383-9.
3. Proulx C, Boyer L, Hurnanen DR, Lemieux R. Preferential Ex Vivo Expansion of
Megakaryocytes from Human Cord Blood CD34(+)-Enriched Cells in the Presence of
Thrombopoietin and Limiting Amounts of Stem Cell Factor and Flt-3 Ligand. J
Hematother Stem Cell Res. 2003;12:179-88.
4. Gluckman E. Current status of umbilical cord blood hematopoietic stem cell
transplantation. Exp. Hematol. 2000;28:1197-205.
5. Kamble R, Pant S, Selby GB, Kharfan-Dabaja MA, Sethi S, Kratochvil K, Kohrt N, Ozer H.
Microbial contamination of hematopoietic progenitor cell grafts incidence, clinical
outcome, and cost-effectiveness: an analysis of 735 grafts. Transfusion 2000;45:874-8.
6. Honohan A, Olthuis H, Bernards AT, van Beckhoven JM, Brand A. Microbial
contamination of cord blood stem cells. Vox Sang. 2002;82:32-8.
7. Kogler G, Callejas J, Hakenberg P, Enczmann J, Adams O, Daubener W, Krempe C, Gobel
U, Somville T, Wernet P. Hematopoietic transplant potential of unrelated cord blood:
critical issues. J. Hematother. 1996;5:105-16.
8. Cassens U, Ahlke C, Garritsen H, Krakowitzky P, Wullenweber J, Fischer RJ, Peters G,
Sibrowski W. Processing of peripheral blood progenitor cell components in improved
clean areas does not reduce the rate of microbial contamination. Transfusion
2002;42:10-7.
9. Stroncek DF, Fautsch SK, Lasky LC, Hurd DD, Ramsay NK, McCullough J. Adverse
reactions in patients transfused with cryopreserved marrow. Transfusion 1991;31:5216.
131
Chapter VI
10. Engelhard D. Bacterial and fungal infections in children undergoing bone marrow
transplantation. Bone Marrow Transplant. 1998;21:S78-80
11. Trannoy L.L., Lagerberg J.W.M., Dubbelman T.M.A.R., Schuitmaker J.J, Brand A.
Positively charged porphyrins: a new series of photosensitizers for sterilization of RBCs.
Transfusion 2004;44:1186-96.
12. Trannoy LL, Terpstra FG, de Korte D, Lagerberg JWM, Verhoeven AJ, Brand A, van
Engelenburg FAC. Differential sensitivities of pathogens in red cell concentrates to TriP(4)-photoinactivation. Vox Sang. 2006;91:111-8.
13. Spearman C. The method of right and wrong cases ('constan stimuli') without Gauss's
formulae. Br. J. Psychol 1908;2:227-42.
14. Miles A.A., Misra S.S. The estimation of the bactericidal power of the blood. J. Hygiene
1938;38:732-49.
15. Kanofsky JR. Quenching of singlet oxygen by human plasma. Photochem. Photobiol.
1990;51:299-303.
16. Lambrechts SA, Aalders MC, Verbraak FD, Lagerberg JW, Dankert JB, Schuitmaker JJ.
Effect of albumin on the photodynamic inactivation of microorganisms by a cationic
porphyrin. J. Photochem. Photobiol. B 2005;79:51-7.
17. Merchat M, Bertolini G, Giacomini P, Villanueva A, Jori G. Meso-substituted cationic
porphyrins as efficient photosensitizers of gram-positive and gram-negative bacteria. J.
Photochem. Photobiol. B 1996;32:153-7.
18. Greenberg JW, Fischer W, Joiner KA. Influence of lipoteichoic acid structure on
recognition by the macrophage scavenger receptor. Infect. Immun. 1996;64:3318-25.
19. Majcherczyk PA, Rubli E, Heumann D, Glauser MP, Moreillon P. Teichoic acids are not
required for Streptococcus pneumoniae and Staphylococcus aureus cell walls to trigger
the release of tumor necrosis factor by peripheral blood monocytes. Infect. Immun.
2003;71:3707-13.
20. Bonnet D, Bhatia M, Wang JC, Kapp U, Dick JE. Cytokine treatment or accessory cells
are required to initiate engraftment of purified primitive human hematopoietic cells
transplanted at limiting doses into NOD/SCID mice. Bone Marrow Transplant.
1999;23:203-9.
21. Guenechea G, Segovia JC, Albella B, Lamana M, Ramirez M, Regidor C, Fernandez MN,
Bueren JA. Delayed engraftment of nonobese diabetic/severe combined
132
Pathogen inactivation in cord blood stem cell products
immunodeficient mice transplanted with ex vivo-expanded human CD34(+) cord blood
cells. Blood 1999;93:1097-105.
22. Wulf-Goldenberg A, Eckert K, Fichtner I. Cytokine-pretreatment of CD34(+) cord blood
stem cells in vitro reduces long-term cell engraftment in NOD/SCID mice. Eur. .J Cell
Biol. 2008;87:69-80.
23. Fibbe WE, Noort WA, Schipper F, Willemze R. Ex vivo expansion and engraftment
potential of cord blood-derived CD34+ cells in NOD/SCID mice. Ann. N Y Acad. Sci.
2001;938:9-17.
24. Hogan CJ, Shpall EJ, McNulty O, McNiece I, Dick JE, Shultz LD, Keller G. Engraftment
and development of human CD34(+)-enriched cells from umbilical cord blood in
NOD/LtSz-scid/scid mice. Blood 1997;90:85-96.
25. Ballen KK, Valinski H, Greiner D, Shultz LD, Becker PS, Hsieh CC, Stewart FM,
Quesenberry PJ. Variables to predict engraftment of umbilical cord blood into
immunodeficient mice: usefulness of the non-obese diabetic--severe combined
immunodeficient assay. Br. J. Haematol. 2001;114:211-8.
26. Noort WA, Wilpshaar J, Hertogh CD, Rad M, Lurvink EG, Luxemburg-Heijs SA,
Zwinderman K, Verwey RA, Willemze R, Falkenburg JH. Similar myeloid recovery
despite superior overall engraftment in NOD/SCID mice after transplantation of human
CD34(+) cells from umbilical cord blood as compared to adult sources. Bone Marrow
Transplant. 2001;28:163-71.
27. Locatelli F, Maccario R, Comoli P, Bertolini F, Giorgiani G, Montagna D, Bonetti F, De
Stefano P, Rondini G, Sirchia G, et al. Hematopoietic and immune recovery after
transplantation of cord blood progenitor cells in children. Bone Marrow Transplant.
1996;18:1095-101.
28. Ahmed F, Ings SJ, Pizzey AR, Blundell MP, Thrasher AJ, Ye HT, Fahey A, Linch DC, Yong
KL. Impaired bone marrow homing of cytokine-activated CD34+ cells in the NOD/SCID
model. Blood 2004;103:2079-87.
29. Zhai QL, Qiu LG, Li Q, Meng HX, Han JL, Herzig RH, Han ZC. Short-term ex vivo
expansion sustains the homing-related properties of umbilical cord blood
hematopoietic stem and progenitor cells. Haematologica 2004;89:265-73.
30. Graf T. Differentiation plasticity of hematopoietic cells. Blood 2002;99:3089-101.
31. Gothot A, van der Loo JC, Clapp DW, Srour EF. Cell cycle-related changes in
repopulating capacity of human mobilized peripheral blood CD34(+) cells in non-obese
diabetic/severe combined immune-deficient mice. Blood 1998;92:2641-9.
133
Chapter VI
32. Springer TA. Traffic signals on endothelium for lymphocyte recirculation and leukocyte
emigration. Annu. Rev. Physiol 1995;57:827-72.
33. Peled A, Kollet O, Ponomaryov T, Petit I, Franitza S, Grabovsky V, Slav MM, Nagler A,
Lider O, Alon R, et al. The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and
VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration
and engraftment of NOD/SCID mice. Blood 2000;95:3289-96.
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CHAPTER VII
IMPACT OF PHOTODYNAMIC
TREATMENT WITH MESOSUBSTITUTED PORPHYRIN ON
THE IMMUNOMODULATORY
CAPACITY OF WHITE BLOOD
CELL-CONTAINING RED BLOOD
CELL PRODUCTS
Laurence L. Trannoy*1, Dave L. Roelen2, Karin M. Koekkoek2, Anneke Brand1,2
1
Department of Research and Education, Sanquin Blood Bank South-west, Leiden,
the Netherlands; 2Department of Immunohematology and Blood Transfusion,
Leiden University Medical Center, Leiden, the Netherlands
Accepted for publication in Photochemistry and Photobiology
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Chapter VII
ABSTRACT
After transfusion, the presence of contaminating white blood cells (WBC) in blood
components may result in either deleterious or positive immunological responses.
We have previously reported that PDT with meso-substituted mono-phenyl-tri-(Nmethyl-4-pyridyl)-porphyrin (Tri-P(4)) and red light can inactivate pathogens in
RBC products. The present study explored the effect of PDT on contaminating
WBC in RBC products with varying hematocrit (Hct). After PDT, we evaluated
adaptive and innate immunomodulation through allogeneic and mitogenic
stimulation. PDT resulted in decreased T cell proliferation which was more
pronounced with lower Hct. Dark effect of porphyrin Tri-P(4) was remarkable on
antigen-presenting cells affecting expression of co-stimulatory molecules
CD80/CD86. Finally, cytokine profile after PDT revealed a mixed Th1/Th2 type
response while surface antigen expression supported the development of
alternatively activated macrophages (AAMI or type 2 macrophages) instead of
dendritic cells. In conclusion, PDT with Tri-P(4) altered proliferation, allostimulation, cell surface antigen expression and cytokine profiles of the cells.
These results suggest that PDT may be potentially useful in preventing transfusion
associated Graft-versus-Host Disease and alloimmunisation. It seems worthwhile
to further explore PDT-induced immunomodulation to optimize conditions which
may result in allo-tolerance by AAMI.
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Impact of PDT with porphyrins on WBC-containing RBC
INTRODUCTION
Risks associated with blood transfusion has dramatically decreased since the
implementation of careful donor selection and the development of sensitive
diagnostic tests, however a certain risk for complications by contaminating
pathogens still remains.1,2
Not only are viruses, bacteria and protozoa considered as pathogenic, allogeneic
WBC present in blood components can also be recognized as pathogenic
contaminants, inducing immune responses in the recipient, sometimes with
deleterious effects. Such WBC-induced complications of blood transfusion include
transfusion associated Graft-versus Host disease (GvHD), transfusion related
immunosuppression, which can result in enhanced susceptibility to post-operative
infection and multiple-organ-dysfunction syndrome, non-hemolytic febrile
transfusion reactions (NHFTR) and immunization to Human leukocyte antigens
(HLA).3 Although the mechanism is yet to be unraveled, the presence of allogeneic
WBC in transfusion products can be beneficial reducing the risk of allograft
rejection and of spontaneous recurrent abortion.3
A number of approaches have been implemented to minimize the pathogenic role
of contaminating WBC. Leukodepletion of blood components by filtration has
resulted in a reduction in the frequency of NHFTR’s and HLA immunisation.2
Gamma irradiation is effective in preventing WBC proliferation but does not
impair the stimulatory capabilities of these cells.4 Some of these techniques,
aimed to inactivate pathogen in blood products, such as Mirasol-PathogenReduction technology5 and Inactine technology using Pen 1106 have been shown
to also reduce the proliferative capacity of the WBC as well as their ability to
present antigens. Furthermore, it has been shown that photodynamic treatment
(PDT) can have immunomodulatory effects in various experimental systems. Invivo studies showed that PDT with verteporfin was effective in alleviating immune
pathology in murine models of arthritis, contact hypersensitivity, experimental
allergic encephalomyelitis and retention of allogeneic skin grafts.7,8 PDT of
peripheral blood mononuclear cells with a rhodamine derivative TH9402 may
induce tolerogenic dendritic cells (DC)9, whereas treatment with extracorporeal
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Chapter VII
therapy using 8-methoxypsoralen was found to reverse GvHD.10 Investigations in
the mechanism involved in immunomodulatory PDT have shown that PDT
selectively eliminate certain cell populations, disrupt T cell response by affecting
APCs by lowering expression of key cell surface molecules, including MHC I and II,
CD80, CD86 and CD54 and modulate the cytokine network.11-13
PDT utilizes mainly porphyrin and phthalocyanine-based photosensitizers which
accumulates somewhat selectively in rapidly dividing cells such as activated cells
of the immune system.14-16 Exposure to sub-lethal doses of PDT may result in the
modification of cell surface receptor expression levels and cytokine release and
consequently influence cell behaviour.8
Mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin chloride (Tri-P(4)) is a positively
charged amphiphilic porphyrin that can be activated with red light to generate
singlet oxygen responsible for oxidative damage to vicinal molecules. We have
previously demonstrated that this photosensitizer was superior to other similar
porphyrin photosensitizers in its action to efficiently inactivate a wide range of
viruses and bacteria with limited damage to RBC.17,18 PDT with Tri-P(4) was also
shown to have limited effects on cord blood derived stem cells and progenitor
cells.19 These observations raised the question as to whether this treatment may
be able to modulate WBC present in RBC product to reduce immunological posttransfusion reactions or to enhance beneficial tolerizing effects of blood
transfusion.
We therefore explored the potential immunological effects of PDT with Tri-P(4) on
contaminating WBC in RBC products with varying Hct. For this, we evaluated the
effect of the treatment on in-vitro adaptive and innate immunological
parameters, such as proliferation in response to mitogens and to allogeneic
stimulator cells, the ability to stimulate proliferation of healthy untreated
responder cells, survival, expression of constitutive and inducible cell surface
antigens and production of soluble mediators (cytokines).
138
Impact of PDT with porphyrins on WBC-containing RBC
MATERIALS AND METHODS
MATERIALS
Photosensitizer Tri-P(4) (mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin chloride)
was kindly provided by Buchem b.v., Apeldoorn, the Netherlands. Stock solutions
of 1 mM were prepared in PBS and stored in the dark at 2-6°C. PBS (pH 7.4) and
Ficoll gradient were provided by the Leiden University Medical Center (LUMC)
Pharmacy, Leiden, the Netherlands. SAG-M is a red cell storage solution
containing 150 mM NaCl, 1.25 mM adenine, 50 mM glucose and 29 mM mannitol
(Fresenius Hemocare, Emmer-Compascuum, the Netherlands). IMDM and RPMIglutamine was purchased from Invitrogen, Breda, the Netherlands. Penicillin and
streptomycin (pen/strep) were purchased from Bio-Whittaker (Verviers, Belgium).
Pools of 40 individual AB sera, screened for the absence of HLA antibodies and
disturbance in mixed lymphocyte reaction (MLR), were obtained from Sanquin
Blood Bank Southwest (Rotterdam, the Netherlands). Phytohemagluttin (PHA),
ůŝƉŽƉŽůLJƐĂĐĐŚĂƌŝĚĞ ;>W^Ϳ ĂŶĚ ɴ-mercapto-ethanol were purchased from SigmaAldrich (Zwijndrecht, the Netherlands). Phycoerythrin (PE)-conjugated anti-human
CD14, CD16, CD33, CD54, CD80, CD83, CD86, HLA-DR monoclonal antibodies
(MoAb), PE-conjugated isotype-matched mouse IgG2a MoAb, fluorescein (FITC)conjugated anti-human CD45 MoAb, FITC-conjugated isotype-matched mouse
IgG1 MoAb, propidium iodine (PI), IOTest3 lysis solution and the Flow-count
fluorospheres were purchased from Beckman Coulter (Mijdrecht, the
Netherlands).
BLOOD PRODUCTS AND PROCESSING
Whole blood derived buffy coats were provided by healthy HLA-typed donors
from Sanquin Blood bank supply (Leiden, the Netherlands). The buffy coats were
washed twice at 5000 gmin with SAG-M to remove plasma. After the last wash,
the supernatant and the WBC layer were transferred into a new tube and
centrifuged at 8500 gmin. The WBC pellet was resuspended in 10 mL SAG-M for
counting. Subsequently, the WBC and SAG-M were added to the washed RBC to
139
Chapter VII
obtain a final product with a WBC concentration 20.106 cell / mL and a Hct of 20%
r 4% or 55% r5%. Hct and cell concentrations were measured by Act 10 blood
analyzer (Beckman Coulter, Mijdrecht, the Netherlands).
The final product obtained is referred to as WBC-enriched RBC (WBC-RBC). All
samples were DNA typed at low resolution for the loci HLA-A, -B, -C, -DRB1 and DQB1 by polymerase chain reaction /sequence-specific oligonucleotide using a
reverse dot-blot method.20 HLA typing was performed at the national reference
laboratory for histocompatibility testing (LUMC, the Netherlands).
LIGHT SOURCE
For all illuminations, the light source was a 300 W halogen lamp (Philips,
Eindhoven, the Netherlands) combined with a cut-off filter (Kodak, wratten nr.
23A, Rochester, NY) only transmitting light > 600 nm. The fluence rate at the level
of the Petri dish was adjusted to 100 W per m2, as measured with an IL1400A
radiometer with a SEL033 detector (International Light, Newburyport, MA). To
avoid heating of the samples, the light was passed through a 1-cm thick water
layer. The temperature did never exceed 25°C.
PHOTODYNAMIC TREATMENT
Tri-P(4) was added to the blood components to a final concentration of 25 μM.
The WBC-RBC suspensions were thoroughly mixed. Subsequently 3 mL samples
were transferred into 6 cm diameter petri dishes (tissue culture grade, Greiner,
Alphen a/d Rhijn, the Netherlands), resulting in a cell layer thickness of 1 mm. The
dishes were agitated at room temperature on a horizontal circular shaker (75
rpm) for 5 min in the dark and subsequently illuminated under continuous
agitation for 30 min corresponding to a light of 180 kJ/m2. The light control
corresponded to WBC-RBC illuminated without addition of photosensitizer. The
dark control corresponded to WBC-RBC spiked with photosensitizer but kept in
the dark during the whole procedure (including preincubation and illumination
time).
140
Impact of PDT with porphyrins on WBC-containing RBC
MITOGENIC STIMULATION
To study proliferation, mononuclear cells (MNC) were isolated from WBC-RBC
immediately after PDT by centrifugation on Ficoll density gradient and
subsequently, the cells were resuspended in culture medium containing RMPIGlutamine supplemented with 10% human serum and 100 μg/mL Pen/strep. The
cells (106 cell/mL) were stimulated with 1 μg/mL PHA in U-bottom 96-well plates.
Cell proliferation was quantified by incubating the cells during the last 18 h of 4ĚĂLJ ĐƵůƚƵƌĞƐ ǁŝƚŚ ϭ ʅŝ ΀3H]thymidine. Before being pulsed, supernatants were
collected and stored at –ϰϬΣ͘ WƌŽůŝĨĞƌĂƚŝŽŶ ǁĂƐ ŵĞĂƐƵƌĞĚ ĂƐ ΀3H]thymidine
incorporation by liquid scintillation spectroscopy using a beta plate (Wallac,
Turku, Finland). Results are expressed as % of light control.
MIXED LYMPHOCYTE REACTION
To study allo-reactivity, MNC isolated from WBC-RBC after PDT by Ficoll isolation,
as described above, were co-cultured with MNC isolated from a completely HLAmismatched buffy coat (allogeneic cells). Equal numbers of responder and
stimulator cells (100 μL of 106 cells/mL) were cultured in U-bottom 96-well plates
(Costar, Cambridge, MA, USA). Stimulator cells were obtained by irradiation at
3000 Rad. Tri-P(4)-treated cells, controls and allogeneic cells were used both as
responders and as stimulators. As a control for spontaneous proliferation, the
responder and stimulator cells were cultured in medium only. Cell proliferation
was quantified by incubating the cells during the last 18 h of 5-day cultures with 1
ʅŝ ΀3H]thymidine (Amersham International, Amersham, UK). Assays were
performed in triplicate. Before being pulsed, supernatants were collected and
stored at –40°C. Proliferation was measured as described above. Results are
expressed as % of light control.
LPS STIMULATION
After PDT, WBC-RBC was washed 4 times at 5000 gmin to remove the
photosensitizer and, subsequently diluted 10-fold with IMDM supplemented with
100 μg/mL Pen/strep and 50μM beta-mercapto-ethanol. For stimulation with LPS,
141
Chapter VII
cell suspension (150 μl/well) were plated in triplicate in plate-bottom plate
(Costar, Cambridge, MA, USA) and 50 μl of LPS (final concentration 250 pg/mL) or
culture medium was added. After 20 h of incubation at 37°C, 5% CO2, samples
were pooled then centrifuged at 2700 gmin. Supernatant was harvested and
frozen at -40°C until use. Supernatant from non-LPS treated cell suspensions were
harvested and frozen to be used as reference values before stimulation.
CYTOKINE ANALYSIS
Harvested supernatants from MLR and LPS stimulation assays were tested for a
total of 17 cytokines at once (Interleukin-1beta (IL-ϭɴͿ͕/>-2, IL-4, IL-5, IL-6, IL-7, IL8, IL-10, IL-12p70, IL-13, IL-17, Granulocyte-colony stimulating factor (G-CSF),
Granulocyte-monocyte-CSF (GM-CSF), Interferon-gamma (IFN-ɶͿ͕ ŵŽŶŽĐLJƚĞƐ
chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-ϭɴ ;D/WϭɴͿ ĂŶĚ ƚƵŵŽƌ ŶĞĐƌŽƐŝƐ ĨĂĐƚŽƌ-alpha (TNF-ɲͿ ǁŝƚŚ ƚŚĞ ŝŽ-Plex human Cytokine
panel following the manufacturer’s description. Samples were analyzed using a
Bio-Plex array reader equipped with Bio-Plex software (Bio-Rad Laboratories,
Veenendaal, the Netherlands).
IMMUNOPHENOTYPING
After PDT application of WBC-RBC, samples were washed 4 times with PBS at
1500 gmin to remove the photosensitizer. Cells were incubated with anti-human
MoAb for 20 min at room temperature. After red cell lysis with lysis solution
IOTest3, samples were analyzed on a four-laser cytometer EpicsXL-MCL flow
cytometer using Expo32 software (Beckman Coulter, Mijndrecht, the
Netherlands).
CELL VIABILITY
Using PI labeling and flow cytometry, cell viability was assayed in cell cultures
from MLR at day 1, 4 and 6; day 0 being the day of input. Unstained cells and cells
labeled with PE-conjugated isotype-matched mouse IgG2a and FITC-conjugated
IgG1 of irrelevant specificity were used as negative controls.
142
Impact of PDT with porphyrins on WBC-containing RBC
STATISTICS
For statistical analysis, Repeated measured Analysis of Variance (ANOVA) or
Friedman test were used when appropriate, depending on the type of
distribution. P values are from comparison with light controls and determined by
Tukey’s or Newman-Keuls post-tests as indicated in legends. P values < 0.05 were
considered as statistically significant. All data are expressed as Mean ± Standard
Deviation (sd).
RESULTS
EFFECT OF PDT ON PROLIFERATION
After PDT application, MNC were tested for their capacity to proliferate in
response to PHA (Fig.1A), and to allogeneic stimulation (Fig.1B). PDT induced a
significant reduction in proliferation when samples were treated in low Hct level.
After stimulation with PHA, no significant difference was observed between light
and dark controls. However, after PDT at low Hct level and allogeneic stimulation,
we observed a significant increase in proliferation in dark controls as compared to
light controls. The spontaneous proliferation of the photo-treated cells and dark
control cells did not differ from the light controls (not shown).
EFFECT OF PDT ON THE ABILITY OF MNC TO PRESENT ANTIGEN
After PDT application, MNC were tested for their capacity to present antigen and
thereby stimulate a
proliferative response in untreated HLA-mismatched allogeneic cells. As can be
seen from Fig.1C, the proliferative response induced by photodynamically treated
stimulator cells was significantly reduced by approximately 50% of the light
controls when the cells were treated in low Hct level. In high Hct level, no
difference between the samples was seen. In the dark samples, no difference in
the proliferative response was seen as compared to light controls. The
spontaneous proliferation of the irradiated stimulator cells did not differ between
the samples (not shown).
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Chapter VII
Figure 1. Proliferation capacity of MNC after
PHA stimulation (A), and allogeneic
stimulation (B) after PDT of WBC-RBC in low (a
20%) and high (a 50%). (C) Capacity of phototreated MNC to stimulate proliferation in
MLR.
Proliferation is expressed as % of light control.
Mean with standard deviation are shown.
Light controls (black bars), Dark samples (gray
bars), PDT samples (white bars). Statistic
differences between the three groups of
samples within each Hct level were performed
using ANOVA and Tukey-post tests.
Comparisons of each group between the two
Hct levels were performed using student’s ttest. (*) p values < 0.05; (**) p values < 0.01.
144
Impact of PDT with porphyrins on WBC-containing RBC
THE EFFECT OF PDT ON CELL VIABILITY
Immediately after PDT of WBC-RBC, MNC were isolated by Ficoll centrifugation.
Cell viability was determined during MLR at day 1, day 4 and day 6 of culture to
check that the proliferation inhibition induced by PDT may not be due to excessive
apoptosis induction. PDT did not affect the overall cell viability. The % of PIpositive cells was 10-15% at day 1 and increased to 25-30% at day 6 of the MLR
culture and was similar to untreated controls. There was no difference in nonviable cells in cultures where photodynamically treated cells were used as
responders or in cultures where the treated cells were used as stimulator cells. In
addition, no difference was seen between light controls and dark controls (data
not shown).
EFFECT OF PDT ON THE RELEASE OF CYTOKINES BY ALLO-ACTIVATED
PHOTO-TREATED RESPONDER CELLS IN MLR
The cytokine pattern produced in MLR by photodynamically treated MNC used as
responders was determined. As the effect of PDT on the proliferation was
dependent on the Hct level of the treated product, a similar relationship was
observed with the cytokines released during the cultures. When MNC were
treated in low Hct level (<30), a significant negative correlation was observed
between proliferation and the release of IL-4, IL-10, IL-13, IL-12, TNF-D, MIP-ϭɴ
and INF-J (Fig.2); the lower the proliferation, the more cytokines were released. In
contrast, the release of IL-2 was positively correlated with proliferation; the lower
the proliferation, the lower IL-2 was released. In high Hct level (>50), no significant
change in cytokine release in PDT samples was seen as compared with light
controls. Moreover, for IL-ϭɴ͕/>-5, IL-6, IL-7, IL-8, IL-17, G-CSF, GM-CSF, MCP-1, no
significant difference with light controls was observed at both Hct levels (data not
shown). Finally, no difference in cytokine pattern between dark and light controls
was seen (data not shown).
145
Chapter VII
Figure 2.Correlation between proliferation and cytokine release in MLR wherein MNC
were used as responders after PDT of WBC-RBC in Hct <30 (circles) or in Hct > 50 (squares).
Both level of released cytokine and proliferation rate are expressed as % of light control.
Only PDT samples are represented. Results shown are mean of triplicates of 6 independent
experiments, each one represented by one dot. Correlations were indicated by Pearson r
and p value. Fit lines were established by linear regression.
EFFECT OF PDT ON THE RELEASE OF CYTOKINES BY TRI-P(4)-TREATED
STIMULATOR CELLS IN MLR
The cytokine pattern produced in MLR by allogeneic cells after antigen
presentation by Tri-P(4)-treated MNC was determined. In concordance with the
proliferation results, the level of cytokine released did not correlate with the Hct
level of the product during PDT. However, as can be seen from Fig.3, significant
negative correlations between proliferation and the release of IL-ϭɴ͕ />-6, INF-J,
TNF-D, MIP-1E and GM-CSF were observed. Striking was that the cytokine levels
mostly exceeded the levels of light controls (> 100%) when proliferation was the
146
Impact of PDT with porphyrins on WBC-containing RBC
most inhibited, except for IL-2 showing a significant positive correlation with
proliferation. In addition, no difference in cytokine pattern between dark and light
controls was seen (data not shown).
Figure 3. Correlation between proliferation and cytokine release in MLR wherein MNC were
used as stimulators after PDT of WBC-RBC.
Both level of released cytokine and proliferation rate are expressed as % of light control.
Only PDT samples are represented. Results shown are mean of triplicates of 5 or 6
independent experiments, each one represented by one dot. Correlations were indicated by
Pearson r and p value. Fit lines were established by linear regression.
147
Chapter VII
EFFECT OF PDT ON LPS-STIMULATED CELLS
After PDT application, WBC-RBCs were stimulated with LPS for 20 hours, after
which cytokine production and antigen expression were determined. As can be
seen in Fig.4, in dark and PDT samples, a significant reduction of nearly 50% in
release of IL-8 and MIP-ϭɴ ĂƐ ĐŽŵƉĂƌĞĚ ƚŽ ůŝŐŚƚ ĐŽŶƚƌŽůƐ ǁĂƐ ŽďƐĞƌǀĞĚ ;ŐƌĂƉŚ
Fig.4) whereas for the other cytokines tested no difference with the light controls
was observed (frame in Fig.4).
The proportion of cells expressing surface antigen were determined and given in
Fig.5. PDT induced an increase in numbers of CD14, HLA-DR and CD83 expressing
cells as compared to light and dark controls. The increased in the amount of HLADR and CD83 positive cells was only observed after LPS stimulation, while the
increased in the amount of CD14 positive cells was LPS independent. Moreover,
four hours after PDT application and before LPS stimulation, the amount of CD80
and CD86 expressing cells decreased significantly as compared to light controls as
well in the dark samples as in PDT samples. No PDT effect and LPS effect was
observed in the amounts of CD33, CD54 and CD16 expressing cells (Fig.5).
Moreover, alteration in the stimulation capabilities of APC was associated with
increased production of cytokines suggestive of active inhibition. Investigation of
the innate immunity of WBC in treated RBC products using LPS showed that TriP(4) alone affect the APC response.
Changes in cellular expression of cell surface markers were determined by
measuring mean fluorescence intensities (MFI) of each marker and given in Table
1. The MFI of CD14 and HLA-DR expressions increased upon PDT as compared to
light and dark controls and both were LPS independent. In contrast, MFI of CD83
expression increased upon PDT only without LPS, while after LPS stimulation, MFI
remained similar between the three groups of samples. No significant change in
MFI of CD33, CD16, CD54, CD80 and CD86 expressions were observed after PDT as
compared to light and dark controls.
148
Impact of PDT with porphyrins on WBC-containing RBC
IL-ϭɴ
IL-2
IL-4
IL-6
IL-7
IL-10
IL-12
IL-17
INF-g
TNF-a
MCP-1
G-CSF
GM-CSF
Light control
Dark
PDT
568 ± 699
566 ± 644
568 ± 642
23 ± 18
23 ± 14
20 ± 10
56 ± 47
45 ± 29
41 ± 23
6578 ± 3734 6780 ± 5186 7123 ± 2896
3.1 ± 1.9
2.8 ± 1.8
2.6 ± 0.9
119 ± 102
137 ± 162
133 ± 119
3.1 ± 2.5
4.2 ± 3.7
3.0 ± 1.6
27 ± 9.0
25 ± 9.4
23 ± 6.2
150 ± 116
175 ± 113
161 ± 88
203 ± 217
214 ± 204
210 ± 127
856 ± 984
716 ± 538 1190 ± 906
1004 ± 922 687 ± 474
778 ± 446
145 ± 142
134 ± 94
118 ± 67
Figure 4. Effect of PDT on the release
of cytokines in LPS-stimulated cells.
Cytokine releases are expressed in
pg/mL. Mean ± sd of 6 independent
experiments are shown. Asterisks
indicates statistical differences with
the light controls as determined by
ANOVA and Tukey post-test (*) p
values < 0.05; (**) p values < 0.01.
Graph legend: Light controls (black
bars), dark controls (gray bars); PDT
(white bars).
DISCUSSION
PDT with Tri-P(4) and red light has been shown to inactivate a number of
pathogens including viruses and bacteria in RBCC18 and cord blood products.19 As
contaminating WBC in RBCC are implicated as a causative factor for an immune
response after transfusion3,21, we were interested to determine the effect of TriP(4)-mediated PDT on these cells.
149
Chapter VII
PDT with Tri-P(4) had no effect on the spontaneous proliferation and cell viability
of the treated WBCs. However, photodynamically treated WBCs showed impaired
proliferation after stimulation with PHA and upon co-culture with allogeneic
stimulator cells. The degree of impairment of proliferative capacity was increasing
when WBC had been exposed to PDT in lower Hct. Although cytokine production
was reduced at a certain level of proliferation inhibition, stronger proliferation
inhibition resulted in less decrease of cytokine production (Fig.2). PDT also
impaired the capacity of antigen-presenting cells (APC) to stimulate the T-cell
proliferation in MLR, however, this effect was less influenced by Hct. Moreover,
the reduction in the stimulation capabilities of APC was associated with increased
production of cytokines suggestive of active inhibition. We also found by using LPS
that the APC response was affected by the presence of Tri-P(4) only, in the
absence of light.
Medium
Light control
HLA-DR
CD83
CD14
CD33
CD16
CD54
CD80
CD86
107 ± 89
30 ± 12
729 ± 234
39 ± 12
32 ± 12
87 ± 95
62 ± 98
69 ± 29
Dark
LPS
PDT
114 ± 63 210 ± 139*
33 ± 7.6
41 ± 6.7*
†
886 ± 271 1016 ± 404
39 ± 17
35 ± 16
31 ± 7.4
31 ± 6.6
106 ± 130
76 ± 57
42 ± 29
45 ± 19
97 ± 73
110 ± 55
Light control
136 ± 129
26 ± 12
773 ± 187
52 ± 24
32 ± 8.4
215 ± 272
34 ± 3.7
59 ± 48
Dark
PDT
99 ± 73 208 ± 157*
26 ± 11
28 ± 8.7
†
709 ± 249 1174 ± 411
49 ± 29
48 ± 20
31 ± 12
40 ± 23
147 ± 178 166 ± 160
27 ± 6.7
41 ± 7.4
52 ± 27
66 ± 24
Table 1: Intensities of cell surface marker expression (MFI) on WBC after PDT
Data indicated are Mean Fluorescence Intensity (MFI) ± SD of 8 independent experiments.
Statistic differences were determined with ANOVA and Newman-Keuls post tests or with Friedman
test depending on the type of distribution. Tests were performed in respective medium. Superscript
symbols shown indicate a significant difference with light controls (p < 0.05): (*) from ANOVA and
(†) from Friedman test.
150
Impact of PDT with porphyrins on WBC-containing RBC
Figure 5. Effect of PDT on the cell surface expression on LPS-stimulated WBC-RBC.
Cell surface antigen expression is expressed as percentage of positive cells from the CD45+
population. Day 0 corresponds to un-stimulated WBC-RBC 4 hours after PDT. Medium and LPS
corresponded to WBC-RBC cultured after PDT without and with LPS respectively for 20 hours.
Light controls (black bars), Dark controls (gray bars), PDT (white bars). Mean ± sd of 8 independent
experiments are shown. Asterisks indicates statistical differences with the light controls in their
respective culture media as determined by ANOVA and Tukey post-test; (*) p values < 0.05; (**) p
values < 0.01; n.d. not detectable.
The dark effect of the photosensitizer Tri-P(4) on APCs was compatible with our
observations that Tri-P(4) binds non covalently but differentially to leukocytes
with a preference for monocytes/macrophages and granulocytes (L.L. Trannoy,
unpublished). We observed that release of both chemoattractants IL-8 and MIP1β were reduced as well in dark and in PDT samples. It has been reported that,
151
Chapter VII
the release of these cytokines have in common a signaling pathway.22,23 Further
studies are required to identify the membrane receptor which may interact with
Tri-P(4) disrupting simultaneously the release of IL-8 and MIP-ϭɴ͘DŽƌĞŽǀĞƌ, the
expression of co-stimulatory molecules CD80 and CD86 on APC were also reduced
both in dark and PDT samples (Fig.5). These results suggest that Tri-P(4) affects
the antigen presentation functionality of APC by interacting directly with
monocytes / macrophages in the absence of light.
The Hct of the blood product influences the photodynamic effect on T cells.
Increasing the Hct caused higher pigmentation of the blood product and may
hinder proper light penetration to the WBC. Thereby PDT may affect differently
the WBCs. High doses and low doses PDT has been shown to generate different
results as high doses resulted in necrotic or apoptotic cells, while low doses PDT
or sublethal doses PDT may result in the modification of cell surface receptor
expression and consequently influence cell activities.8,24
Another possible explanation is the capacity of RBC to scavenge reactive oxygen
species (ROS).25 Tri-P(4) is a porphyrin that remains extracellular by binding
exclusively to cell membrane of living and resting cells (unpublished results, L.L.
Trannoy). Illumination of porphyrin Tri-P(4) at appropriate wavelength generates
ROS, in particularly singlet oxygen (1O2) which reacts with substances in close
vicinity. ROS is also a natural product, produced by neutrophils in host defense to
kill invading pathogens and involved in intercellular and intracellular signaling of
homeostasis, cell proliferation and differentiation and inflammatory and immune
responses.26 Generation of ROS by light activation of Tri-P(4) can cause lipid and
amino-acid peroxidation, affecting cell membrane associated targets, and may
produce a number of effects on the immune cells. RBC may play a protective role
by scavenging PDT-generated extracellular ROS and varying the Hct of RBC may
affect the degree of photo-damage induced by ROS. Further research would
determine whether illumination with a lower light dose in the absence of RBC may
show the same effects on WBC after PDT with Tri-P(4).
In our study, T-cell response and IL-2 release correlated with the Hct level of the
photodynamically treated WBC-RBC products, as seen after PDT when Tri-P(4)152
Impact of PDT with porphyrins on WBC-containing RBC
treated cells were used as responders in MLR and after PHA stimulation. This
indicated that T-cells are target for ROS-mediated PDT and that the presence of
RBC during PDT application may protect the T cells against the damaging effect of
singlet oxygen. Possible targets of PDT may be the TCR and CD28 signaling
pathways which were found to be sensitive to oxidative stress induced e.g. by BSO
(DL-buthionine-(S,R)-sulfoximine) depleting intracellular antioxidant reduced
glutathione 27 and after PUVA treatment.28 Both treatments result in T-cell
hyporesponsiveness.
Our results showed that PDT with Tri-P(4) act both on APCs and on T cells, on one
hand through a dark effect of Tri-P(4) down-regulating the costimulatory
molecules CD80 and CD86 and on the other hand through a photodynamic effect
inhibiting the responder and stimulatory potentials of WBC in MLR. Therefore we
suggest that the immunomodulatory effect of PDT may result in an impaired
interaction between T-cells and APC. Blockage of the CD80/CD86-CD28 costimulation was shown to result in T cell hyporesponsiveness and in generation of
alternatively activated macrophages (AAMI), also referred to as type 2
macrophages 29-31 The induction of this type of macrophages is supported on one
hand by the PDT-induced increase in cell number and cellular expression of HLADR and CD14 on APC, and on the other hand by the refractoriness of CD80 / CD86
up-regulation to LPS stimulation (Table 1). These cellular events are partly
characteristic of AAMI as demonstrated by Xu 32 and Verreck.30 As these cells are
unable to prime T-cells, they may contribute to the PDT-induced T-cell
hyporesponsiveness.31,33 An additional indication pointing toward the involvement
of APC after PDT is the fact that un-stimulated WBC-RBC cultures of photo-treated
cells showed an up-regulation of CD83 which was refractory to LPS stimulation
(Table 1). Indeed, monocytes are precursors of dendritic cells (DC) and
macrophages, with each cell type having the capability to convert into the other
until late in differentiation / maturation process.34 Hence, PDT seems to activate
monocytes to differentiate into DC as suggested by the high expression of CD83
and HLA-DR (Table 1); however, LPS stimulation skews this polarization favoring
type 2 macrophage development (lower CD83, higher CD14 expression).30
Finally, at a level of PDT-induced proliferation reduction up to 40%, it appeared
that the allo-stimulated cells released increasing cytokine levels proportionally to
153
Chapter VII
the degree of inhibition of proliferation; cytokine levels exceeding even these in
light control cultures (Fig.3). These results strongly suggest an immunomodulatory
event. Moreover, the cytokine profile revealing a mixed Th1/Th2 type response,
suggests that several types of APC may be synergically or simultaneously involved.
In conclusion, in addition to inactivating pathogens, PDT with Tri-P(4) may be able
to steer the immune response to T-cell hyporesponsiveness by functionally
affecting both T cells and APC. These dual effects may present an advantage on
gamma irradiation which was shown to inhibit proliferation of the MNC without
affecting the antigen-presenting capabilities of the irradiated cells.35
Consequently, we may consider the possibility of using PDT with Tri-P(4) to
deviate an immune response towards tolerance or to prevent undesired
transfusion related immunological reactions.
Acknowledgments--This work was supported by ZonNW (grant nr. 28-24140), the
Netherlands. The authors thank Aine Honohan for her technical supports and her
helpful English editing. The authors thank also Johan Lagerberg for the critical
review of this article.
REFERENCES
1. Barbara J. Why 'Safer than ever' may not be quite safe enough. Transfus. Med.
Hemother. 2004 Jan;31(S-1):2-10.
2. Andreu G, Morel P, Forestier F, Debeir J, Rebibo D, Janvier G, Herve P.
Hemovigilance network in France: organization and analysis of immediate
transfusion incident reports from 1994 to 1998. Transfusion 2002 Oct;42(10):135664.
3. Blajchman MA, Dzik S, Vamvakas EC, Sweeney J, Snyder EL. Clinical and molecular
basis of transfusion-induced immunomodulation: summary of the proceedings of a
state-of-the-art conference. Transfus. Med. Rev. 2001 Apr;15(2):108-35.
4. Fast LD. Control of immune responses induced by the transfer of allogeneic white
blood cells during transfusion. Transfusion 2005 Aug;45(s2):44s-50s.
154
Impact of PDT with porphyrins on WBC-containing RBC
5. Fast LD, DiLeone G, Li J, Goodrich RP. Functional inactivation of white blood cells by
Mirasol treatment. Transfusion 2006 Apr;46(4):642-8.
6. Fast LD, DiLeone G, Edson CM, Purmal A. PEN110 treatment functionally inactivates
the PBMNCs present in RBC units: comparison to the effects of exposure to gamma
irradiation. Transfusion 2002;42(10):1318-25.
7. Obochi MO, Ratkay LG, Levy JG. Prolonged skin allograft survival after
photodynamic therapy associated with modification of donor skin antigenicity.
Transplantation 1997 Mar 27;63(6):810-7.
8. Ratkay LG, Waterfield JD, Hunt DW. Photodynamic therapy in immune (nononcological) disorders: focus on benzoporphyrin derivatives. BioDrugs. 2000
Aug;14(2):127-35.
9. Broady R, Yu J, Levings MK. Pro-tolerogenic effects of photodynamic therapy with
TH9402 on dendritic cells. J. Clin. Apher. 2008;23(2):82-91.
10. Heshmati F. Mechanisms of action of extracorporeal photochemotherapy. Transfus.
Apheresis. Sci. 2003 Aug;29(1):61-70.
11. Hunt DW, Chan AH. Influence of photodynamic therapy on immunological aspects
of disease - an update. Expert Opin. Investig. Drugs 2000;9(4):807-17.
12. Gollnick SO, Liu X, Owczarczak B, Musser DA, Henderson BW. Altered expression of
interleukin 6 and interleukin 10 as a result of photodynamic therapy in vivo. Cancer
Res. 1997;57(18):3904-9.
13. King DE, Jiang H, Simkin GO, Obochi MO, Levy JG, Hunt DW. Photodynamic
alteration of the surface receptor expression pattern of murine splenic dendritic
cells. Scand. J. Immunol. 1999;49(2):184-92.
14. Jiang H, Granville DJ, McManus BM, Levy JG, Hunt DW. Selective depletion of a
thymocyte subset in vitro with an immunomodulatory photosensitizer. Clin.
Immunol. 1999;91(2):178-87.
15. Hunt DW, Jiang H, Granville DJ, Chan AH, Leong S, Levy JG. Consequences of the
photodynamic treatment of resting and activated peripheral T lymphocytes.
Immunopharm. 1999;41(1):31-44.
16. Jiang H, Granville DJ, North JR, Richter AM, Hunt DW. Selective action of the
photosensitizer QLT0074 on activated human T lymphocytes. Photochem.
Photobiol. 2002 Aug;76(2):224-31.
155
Chapter VII
17. Trannoy L.L., Lagerberg J.W.M., Dubbelman T.M.A.R., Schuitmaker J.J, Brand A.
Positively charged porphyrins: a new series of photosensitizers for sterilization of
RBCs. Transfusion 2004 Aug 1;44(8):1186-96.
18. Trannoy LL, Terpstra FG, de Korte D, Lagerberg JWM, Verhoeven AJ, Brand A, van
Engelenburg FAC. Differential sensitivities of pathogens in red cell concentrates to
Tri-P(4)-photoinactivation. Vox Sang. 2006;91(2):111-8.
19. Trannoy LL, van Hensbergen Y, Lagerberg JW, Brand A. Photodynamic treatment
with mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin for pathogen inactivation in
cord blood stem cell products. Transfusion 2008 Dec;48(12):2629-37.
20. Verduyn W, Doxiadis II, Anholts J, Drabbels JJ, Naipal A, D'Amaro J, Persijn GG,
Giphart MJ, Schreuder GM. Biotinylated DRB sequence-specific oligonucleotides.
Comparison to serologic HLA-DR typing of organ donors in eurotransplant. Hum.
Immunol. 1993 May;37(1):59-67.
21. Vamvakas EC. Meta-analysis of randomized controlled trials of the efficacy of white
cell reduction in preventing HLA-alloimmunization and refractoriness to randomdonor platelet transfusions. Transfus. Med. Rev. 1998;12(4):258-70.
22. Lakshmanan U, Porter AG. Caspase-4 Interacts with TNF Receptor-Associated Factor
6 and Mediates Lipopolysaccharide-Induced NF-{kappa}B-Dependent Production of
IL-8 and CC Chemokine Ligand 4 (Macrophage-Inflammatory Protein-1 ). J.
Immunol. 2007 Dec 15;179(12):8480-90.
23. Lucchi NW, Moore JM. LPS induces secretion of chemokines by human
syncytiotrophoblast cells in a MAPK-dependent manner. J. Reprod. Immunol. 2007
Feb;73(1):20-7.
24. Moor AC. Signaling pathways in cell death and survival after photodynamic therapy.
J. Photochem. Photobiol. B 2000;57(1):1-13.
25. Fonseca AM, Porto G, Uchida K, Arosa FA. Red blood cells inhibit activation-induced
cell death and oxidative stress in human peripheral blood T lymphocytes. Blood
2001;97(10):3152-60.
26. Fialkow L, Wang Y, Downey GP. Reactive oxygen and nitrogen species as signaling
molecules regulating neutrophil function. Free Radic. Biol. Med. 2007 Jan
15;42(2):153-64.
27. Gringhuis SI, Papendrecht-van der Voort EA, Leow A, Nivine Levarht EW, Breedveld
FC, Verweij CL. Effect of redox balance alterations on cellular localization of LAT and
156
Impact of PDT with porphyrins on WBC-containing RBC
downstream T-cell receptor signaling pathways. Mol. Cell Biol. 2002 Jan;22(2):40011.
28. Legitimo A, Consolini R, Di S, Bencivelli W, Mosca F. Psoralen and UVA light: an in
vitro investigation of multiple immunological mechanisms underlying the
immunosuppression induction in allograft rejection. Blood Cells Mol. Dis29(1):2434.
29. Fujihara M, Takahashi TA, Azuma M, Ogiso C, Maekawa TL, Yagita H, Okumura K,
Sekiguchi S. Decreased inducible expression of CD80 and CD86 in human monocytes
after ultraviolet-B irradiation: its involvement in inactivation of allogenecity. Blood
1996;87(6):2386-93.
30. Verreck FA, de Boer T, Langenberg DM, van der ZL, Ottenhoff TH. Phenotypic and
functional profiling of human proinflammatory type-1 and anti-inflammatory type-2
macrophages in response to microbial antigens and IFN-gamma- and CD40Lmediated costimulation. J. Leukoc. Biol. 2006 Feb;79(2):285-93.
31. Tzachanis D, Berezovskaya A, Nadler LM, Boussiotis VA. Blockade of B7/CD28 in
mixed lymphocyte reaction cultures results in the generation of alternatively
activated macrophages, which suppress T-cell responses. Blood 2002;99(4):146573.
32. Xu W, Schlagwein N, Roos A, van den Berg TK, Daha MR, van Kooten C. Human
peritoneal macrophages show functional characteristics of M-CSF-driven antiinflammatory type 2 macrophages. Eur. J. Immunol. 2007 Jun;37(6):1594-9.
33. Haugen TS, Nakstad B, Skjonsberg OH, Lyberg T. CD14 expression and binding of
lipopolysaccharide to alveolar macrophages and monocytes. Inflammation
1998;22(5):521-32.
34. Palucka KA, Taquet N, Sanchez_Chapuis F, Gluckman JC. Dendritic cells as the
terminal stage of monocyte differentiation. J. Immunol. 1998;160(9):4587-95.
35. Fast LD, DiLeone G, Edson CM, Purmal A. Inhibition of murine GVHD by PEN110
treatment. Transfusion 2002;42(10):1326-32.
157
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SUMMARIZING CONCLUSION
Blood transfusions in developed countries are generally considered to be safe,
however recent events illustrated that blood transfusions still need a maximum of
vigilance and that considerable efforts are required to maintain optimal safety of
blood transfusion practices. The emergence of new virus, such as the WNV in the
North American continent or TTV in Asia,1,2 the potential risk of transmission of
variant PrPsc 3,4, the bacterial contamination of blood products responsible for
sepsis,5,6 the reduction but not the elimination of the window-periods despite the
increased sensitivity of the tests,7,8 promote the development of new strategies to
increase the safety of the blood products. Chemical and photodynamic pathogen
inactivation technologies are the most recent developments for the reduction of
pathogen transmission via transfusion of cellular blood products.
This thesis describes the potential of PDT to decontaminate RBCC and CBSC.
Besides the inactivation of pathogens, the viability and functions of RBCs and
leukocytes, including hematopoietic progenitor stem cells, were evaluated.
FINDING A SUITABLE PATHOGEN PHOTOINACTIVATION METHOD
The original idea to inactivate pathogens in blood products is to add and activate
an agent that targets pathogens but leaves the RBCs unharmed. Since RBCs
contain no nucleic acids, photosensitizers binding to DNA or RNA were, at least in
theory, the most promising photosensitizers to investigate. Photosensitization
reaction resulting from the impact of light on the chemical agent leads to
disruption of DNA and RNA strands. This results in the incapacity of pathogens to
159
Chapter VIII
reproduce and infect new cells. Theoretically, RBCs lacking nucleic acids remain
therefore unaffected.
However, all photosensitizers tested so far for pathogen photoinactivation also
induce damage to RBCs. Since a photosensitizer needs to bind to a target to
induce damage, it is obvious, that the binding of the photosensitizers is not
restricted to nucleic acids, but that binding to membranes also occurs. Minimizing
photodamage to RBCs can be achieved by using more pathogen-specific
photosensitizers or by specific protection of the cells. For this latter, more inside
in the molecular mechanisms behind photodynamically induced damage is
needed. A possible mechanism of photodamage and the specific protection of
RBCs are described in chapters 2 and 3. The quest for more pathogen-specific
photosensitizers is described in chapters 4 and 5.
PROTECTION OF RED BLOOD CELLS
Upon activation of the photosensitizer with visible light, various reactive oxygen
species (ROS) are formed. ROS can inactivate a broad range of pathogens.
However, as ROS are not selective and can be generated in all locations where
sensitizer molecules are present, phototreatment can also damage RBCs.
A better understanding of the molecular mechanisms behind photodynamicallyinduced damage is needed to improve the selectivity of the photodynamic
decontamination technique and to avoid damage to RBCs. Increased permeability
of the membrane to cations, as measured by K+ leakage, is one of the first events
that appear after photosensitization of RBCs. This damage results subsequently in
delayed RBC hemolysis. In chapter 2, we used the photosensitizer DMMB to get
more insight behind the PDT-mediated potassium leakage. As it is likely that
damage to a membrane protein could be involved in K+ leakage, the kinetics of
photodynamically induced K+ leakage and inhibition of several transport systems
were investigated. Inhibition of band 3 activity by DMMB-mediated PDT showed a
comparable light dose dependency as PDT-induced K+ leakage, whereas glycerol
transport activity was inhibited only at higher light doses. Glucose transport was
not affected by DMMB-mediated PDT. Dipyridamole (DIP), an inhibitor of anion
160
Conclusion
transport, protected band 3 against DMMB-induced damage, and prevented the
increase in cation permeability of the membrane. These results suggest that there
is a direct correlation between DMMB-mediated photodamage to band 3 and K+
leakage.
Band 3 constitutes the anion transporter of the RBC and, with about a million
copies per cell, it is the most abundant protein on the RBC membrane. DIP binds
non-covalently, with high affinity to band 3 and has been reported to possess
activity as a scavenger of various ROS. An obvious way to minimize the damage to
RBCs induced by photodynamic pathogen inactivation would be to use DIP during
PDT. In chapter 3, we showed that DIP can react with 1O2 which is the main ROS
formed by PDT. The presence of DIP during PDT with photosensitizers DMMB and
AlPCS4 resulted in a significant protection of the RBCs detected by decreased K+
leakage and hemolysis. In contrast to the strong protection of RBCs by DIP, hardly
any effect on the photoinactivation of the model virus VSV was observed (chapter
3). This indicates that DIP has the potential to increase the specificity of the
pathogen inactivation of RBC concentrates, thereby increasing the practical
applicability of this treatment. However, the results were obtained under
experimental conditions illuminating a model virus in diluted RBC suspension.
Further research should focus in the effect of DIP on the photoinactivation of
other viruses, including HIV, HCV, in a more concentrated RBC suspension. In
addition, the effect of DIP on the self-life of phototreated RBC concentrates needs
further investigation.
Another way to protect RBC from PDT-induced hemolysis is to choose the optimal
RBC suspension media. In this case the protection is indirect: the damaging agent
is not inactivated but the effect of damage is suppressed. Studies have shown that
solution in which phototreatment was performed could influence the amount of
hemolysis induced by PDT.9,10 Less hemolysis was found when illumination was
performed solutions with high osmotic strength as SAG-M. The solution in which
the RBCs are stored after PDT has a much more pronounced effect on hemolysis.
As shown in chapter 4, RBC hemolysis was reduced in SAG-S, SAG-D and AS-3 as
compared to the standard RBC additive solution SAG-M. The presence of
impermeable solutes, such as sucrose (SAG-S), dextran-100 (SAG-D), and citrate
161
Chapter VIII
(AS-3) prevented RBC hemolysis after PDT by counterbalancing the osmotic
activity of hemoglobin. RBC integrity was best maintained when illumination was
performed in the high osmotic additive solution SAG-M followed by storage in AS3.
PATHOGEN INACTIVATION WITH TRI-P(4) IN RBC PRODUCT:
ADVANTAGES AND DRAWBACKS
In our search to find a photosensitizer that efficiently inactivates pathogens
without inducing RBC damage, we focused on a series of meso-substituted
positively charged porphyrins. These photosensitizers have been shown to have a
strong bactericidal potential.11,12 From this series photosensitizer Tri-P(4) stood
out as the best porphyrin suitable for pathogen inactivation in RBCC (chapter 4).
Our preliminary studies have shown that PDT of RBC with the amphiphilic
photosensitizer Tri-P(4) gives the most satisfactory results combining low
hemolysis with efficient virus kill. In contrast, hydrophobic cationic porphyrins
such as Trans-P(4), Cis-P(4) and mono-P(4) were supposed, like DMMB, to bind to
RBC and were found to induce a rapid potassium efflux. Hydrophilic porphyrin
derivatives tetra-P(4), TAP, TNH-P(4) and AlPcS4 (chapter 3) may not bind to RBCs
but showed a moderate cell lysis.
When illuminated in SAG-M and stored in AS-3, parameters for RBC quality after
PDT with Tri-P(4) were satisfactory as shown by in-vitro measurements and in-vivo
autologous RBC survival experiments in Rhesus monkeys. To extent the study, we
investigated the pathogen-inactivating capacity of PDT with Tri-P(4) for a broader
range of pathogens (chapter 5). From this study, it became clear that for a >5 log
inactivation of various enveloped viruses, and gram positive and gram negative
bacteria, a light dose of 360 kJ/m2 was required. This implies a four times longer
illumination time than required for the inactivation of VSV (chapter 5). These
more rigid illumination conditions clearly increased RBC damage.
Despite these promising results, pathogen inactivation with Tri-P(4) presents
many drawbacks. Firstly, the efficacy of PDT with Tri-P(4) to inactivate the nonenveloped CPV is low. Non-enveloped viruses are known to be very resistant to
162
Conclusion
chemical and photochemical treatments. However, as discussed in chapter 5,
there is a remarkable difference in sensitivity to PDT within the parvoviridae
families. Possibly, by using CPV as a model for human parvovirus B19, the
inactivation of parvovirus B19 after PDT with Tri-P(4) is underestimated. Further
testing evaluating the inactivation of human parvo B19 and of other nonenveloped viruses such as Hepatitis A virus in RBC using PDT with Tri-P(4) may
clarify this issue.
More worrying is that Tri-P(4) is unable to inactivate intracellular viruses (chapter
5). In contrast to neutral photosensitizers, cationic dyes do not penetrate the
membrane bilayer as shown by the low cytotoxic effects of PDT with Tri-P(4) on
WBC (chapter 6). Recently, Lambrechts et al. have demonstrated that under
hypotonic conditions the influx of the photosensitizer into the yeast Candida
albicans was enhanced, thereby potentiating the photosensitizing effect of TriP(4).13 it remains to be elucidated whether there are conditions upon which
bacteria, viruses and infected leukocytes, but not the RBCs, become permeable to
the photosensitizer, thereby increasing the Tri-P(4) photoinactivation efficiency.
Furthermore, a broader range of pathogen to inactivate in RBCC including fungi,
parasites, and antibiotic resistant bacterial strains remain to be investigated.
Studies with skin infection models have shown that fungi as well fungal spores are
sensitive to PDT with Tri-P(4).14,15 This raises the question whether RBCC
environment provides the satisfactory conditions for an efficient fungicidal effect.
Moreover, besides bacteria, bacterial endotoxins can also be responsible for fatal
post-transfusion reactions.16 The impact of PDT on endotoxin release from
photoinactivated bacteria need also to be investigated.
Another major concern about the suitability Tri-P(4) for photodecontamination of
RBCC is the increase of IgG binding to the RBC membrane upon PDT. This is a
problem also observed with other photosensitizer a.o DMMB and AlPcS4, and so
far only observed with human cells (chapter 4). Binding of IgG is normally a sign of
senescence of RBC which are subsequently removed from the circulation by
macrophages. It can be speculated that PDT-induced IgG binding was the cause of
a lower in-vivo recovery and a shorter RBC survival after transfusion. The
163
Chapter VIII
mechanism behind the process of PDT-induced IgG binding and whether these
coated cells are phagocytized is still unknown. We have found that photodamage
of both plasma proteins and RBC membrane is required to induce this IgG binding.
This suggests a more complex phenomenon than simply the induction of a
senescence process exposing phosphatidyl serine onto the RBC membrane. To
circumvent interference with transfusion-related diagnostic tests, it is of great
importance to prevent IgG binding to RBCs during pathogen inactivation
application. A possibility to do this is the addition of GSH to the RBC suspensions.
GSH can prevent IgG binding by preventing the formation of sulfur bridges
between cysteine amino acids. GSH has an advantage over other scavengers as it
is a natural constituent of the blood and therefore might not have to be removed
before transfusion. However, when considering the up-scaling of the phototreatment from small blood aliquots, as used in our studies, to full RBCC units,
questions raise regarding the transfusion safety of photo-treated RBCs. With
higher RBC volume, a greater amount of GSH would be required. The toxicity of
large amount of GSH transfused intravenously needs to be determined. This is a
subject of highest importance because the development of antibodies against
RBCs treated with pathogen inactivation techniques is a major project killer.
Despite the satisfactory in-vitro results and the in-vivo survival of treated RBCs in
animals, Phase III clinical studies with S-303 and Pen 110 systems were halted
because of the formation of antibodies against neo-antigens on the treated RBCs.
Therefore, the future of these pathogen inactivation methods, including Tri-P(4)mediated PDT, is uncertain until the immunogenicity is completely understood
and resolved.
OTHER PERSPECTIVES FOR PDT WITH PORPHYRIN TRI-P(4)
The amphiphilic character of photosensitizers was reported to promote cell
uptake and better intracellular targeting.17 However, the cytotoxicity studies
performed with such photosensitizers were often conducted with tumor cell lines
cells while relatively little detailed information exists regarding non-transformed
cells, especially cells of the immune system.18
164
Conclusion
Although porphyrin Tri-P(4) is amphiphilic and was reported to bind to DNA19,20,
there seems to be very limited uptake of Tri-P(4) by intact cells. As mentioned
above, Tri-P(4) is unable to inactivate intracellular virus, most likely because the
porphyrin is not taken up by the cells. We also observed no cytototoxic effect
upon PDT with Tri-P(4) of leukocytes present in treated RBCs. This observation
had lead to conduct photoinactivation studies in CBSC products (chapter 6) and to
investigate potential immunomodulating effects of PDT with Tri-P(4) (chapter 7).
Bacterial contamination of CBSC is a major problem. It has been reported that up
to 13% of CBSC products have been found positive in bacterial cultures.21,22
Recently, the FDA guidelines and Netcord FACT standards requested the release
of pathogen-free CBSC products for transplantation. All contaminated products
have to be discarded. Because of the low number of stem cells, protocols for exvivo expansion of CBSC are developed. The presence of contaminants is a critical
issue for such cultures which present ideal conditions for bacterial growth.
PDT with Tri-P(4) of CBSC resulted in the inactivation of spiked virus (VSV), the G+
S. aureus and, the G- P. aeruginosa and S. agalactiae (chapter 6). However, in
comparison with application in RBCs, the treatment required a twofold higher TriP(4) concentration. This may be explained by the difference in product
composition. CBSC contains plasma proteins which are nearly absent in RBCs.
These proteins, in particularly albumin, quench most of the singlet oxygen
generated by the action of light on the photosensitizer. Also the presence of high
number of macrophages and platelets may influence the bacterial inactivation. G+
bacteria are known to be readily sensitive to PDT when treated in saline or in
RBCC containing SAG-M. However when treated in CBSC, the sensitivity of G+
bacteria to PDT decreased and became even lower than this of G- bacteria.
Macrophages and platelets, which are abundant in CBSC, can bind to G+ bacteria
via the teichoic acid components. The cells may compete with Tri-P(4) for binding
to bacteria through the teichoic acid, thereby reducing the efficacy of Tri-P(4) to
inactivate G+ bacteria.
PDT with Tri-P(4) had no effect on the viability of hematopoietic progenitor stem
cells nor on their ability to differentiate and form colonies in-vitro. Also the exvivo expansion and differentiation of CD34-positive cells isolated from
photodynamically treated CBSC into megakaryocytes was not affected. Despite
165
Chapter VIII
these excellent in-vitro results apparently not affecting lineage-committed cells,
transplantation of photo-treated stem cells in NOD/scid mice showed a lower
engraftment and hematopoietic recovery as compared to untreated cells. The
diminished engraftment could be the result of an impaired homing of the
photodynamically treated cells. Many factors have been described to influence
migration and engraftment of stem cells, such as cell cycle, cytokines,
chemokines, expression of adhesion molecules and cell-cell interaction.23-29
Further research is needed to reveal whether PDT interacts with one of these
critical factors, thereby contributing to the reduced engraftment potential.
As in CBSCs, PDT with Tri-P(4) had no effect on the viability of leukocytes in RBC
products (chapter 7). However, PDT does affect their functions. Alterations in
proliferation, in allo-stimulation, in cell surface antigen expression and cytokine
profiles are critical events in Tri-P(4)-phototreated cells. These changes result in
an interesting issue, which is steering the immune response to T-cell
hyporesponsiveness. For this, a combined effect of dark properties of Tri-P(4)
which is binding to antigen-presenting cells (APC) and the effect of
photodynamically-generated ROS on T cells was required. Binding of Tri-P(4)
resulted in down-regulation of CD80/CD86, up-regulation of HLA-DR and CD14
and in lower release of chemoattractants IL-8 and MIP-1beta. These results may
support the formation of alternatively-activated macrophages capable of inducing
T-cell hyporesponsiveness. In addition, impairment of the T cell response to alloantigens upon PDT with Tri-P(4) was strongly dependent on the amount of RBC.
The lesser RBCs were present, the more the T cell proliferation was inhibited. The
reason of this correlation may be that the increased RBC concentration reduces
the amount of light that reached the leukocytes during PDT. Therefore, by
adapting the light dose, PDT can modulate the interaction between T-cells and
APC. Such modulation can have advantages in establishing appropriate treatment
conditions for prevention of transfusion-associated Graft-versus-Host disease and
alloimmunization, or for induction of tolerance in a recipient. However, it has
been suggested that RBCs have a role as modulators of T-cell growth and survival
and thereby may contribute to immunomodulating effects of transfusion.30,31
Therefore the effect of the presence of RBC during PDT with Tri-P(4) of leukocyte
products may not be underestimated. Further research is required to establish if
166
Conclusion
adjusting the light dose may result in same immunomodulating effects as a
variation in concentration of RBC.
In conclusion, due to the particular properties of porphyrin Tri-P(4), many
purposes are conceivable for PDT with Tri-P(4) from pathogen inactivation to
immunomodulation. The great advantage of this cationic amphiphilic dye is the
modulation of its effect by adjusting the environment. PDT with Tri-P(4) can then
be applied for various goals.
At first, PDT with Tri-P(4) appeared to be a good method for pathogen inactivation
in stem cell containing products, including those derived from à priori
contaminated areas, for development of hematopoietic as well as regenerative
therapies. Besides cord blood products, PDT with Tri-P(4) may be applied to
decontaminate other sources of hematopoietic progenitor cells (e.g. bone marrow
or menstrual blood).32,33
However, the original purpose to apply photodynamic treatment to enhance
safety of RBCs is hampered by the photodynamically-induced damage to RBC. This
can only be solved by providing adequate protection to RBC and this led to too
many concerns regarding the therapeutic quality of the treated blood products.
Therefore, it is unlikely that PDT with Tri-P(4) will be used for pathogen
inactivation in RBC. Nevertheless, the studies with RBC revealed another potential
of PDT with Tri-P(4) for dividing cell populations and for modulation of alloimmune responses, including tolerance induction.
REFERENCES
1. Charrel RN, de Lamballerie X, Durand JP, Gallian P, Attoui H, Biagini P, de Micco P.
Prevalence of antibody against West Nile virus in volunteer blood donors living in
southeastern France. Transfusion 2001;41(10):1320-1.
2. Dai CY, Yu ML, Chuang WL, Wang CS, Lin ZY, Chen SC, Hsieh MY, Wang LY, Tsai JF,
Chang WY. The molecular epidemiology and clinical significance of TT virus (TTV)
infection in healthy blood donors from southern Taiwan. Transfus. Apher. Sci.
2001;24(1):9-15.
3. Roddie PH, Turner ML, Williamson LM. Leucocyte depletion of blood components.
Blood Rev. 2000;14(3):145-56.
167
Chapter VIII
4. Moor AC, Dubbelman TM, VanSteveninck J, Brand A. Transfusion-transmitted
diseases: risks, prevention and perspectives. Eur. J. Haemotol. 1999;62(1):1-18.
5. Kuehnert MJ, Roth VR, Haley NR, Gregory KR, Elder KV, Schreiber GB, Arduino MJ,
Holt SC, Carson LA, Banerjee SN, et al. Transfusion-transmitted bacterial infection in
the United States, 1998 through 2000. Transfusion 2001;41(12):1493-9.
6. Perez P, Bruneau C, Chassaigne M, Salmi LR, Noel L, Allouch P, Audurier A, Gulian C,
Janus G, Boulard G, et al. Multivariate analysis of determinants of bacterial
contamination of whole-blood donations. Vox Sang. 2002;82(2):55-60.
7. Ruzzenenti MR, De Luigi MC, Bruni R, Giannini G, Bo A, Valbonesi M, Barresi R,
Torretta F, Gianotti P, Bruzzone B. PCR testing for HCV in anti-HCV negative blood
donors involved in the so called HCV +ve post-transfusion hepatitis. Transfus. Sci.
2000;22(3):161-4.
8. Liu H, Shah M, Stramer SL, Chen W, Weiblen BJ, Murphy EL. Sensitivity and
specificity of human T-lymphotropic virus (HTLV) types I and II polymerase chain
reaction and several serologic assays in screening a population with a high
prevalence of HTLV-II. Transfusion 1999;39(11-12):1185-93.
9. Besselink GA, Ebbing IG, Hilarius PM, De_Korte D, Verhoeven AJ, Lagerberg JW.
Composition of the additive solution affects red blood cell integrity after
photodynamic treatment. Vox Sang. 2003;85(3):183-9.
10. Wagner SJ, Skripchenko A, Thompson-Montgomery D. Use of a flow-cell system to
investigate virucidal dimethylmethylene blue phototreatment in two RBC additive
solutions. Transfusion 2002;42(9):1200-5.
11. Merchat M, Spikes JD, Bertoloni G, Jori G. Studies on the mechanism of bacteria
photosensitization by meso- substituted cationic porphyrins. J. Photochem.
Photobiol. B 1996;35(3):149-57.
12. Minnock A, Vernon DI, Schofield J, Griffiths J, Parish JH, Brown ST. Photoinactivation
of bacteria. Use of a cationic water-soluble zinc phthalocyanine to photoinactivate
both gram-negative and gram-positive bacteria. J. Photochem. Photobiol. B 1996
Feb;32(3):159-64.
13. Lambrechts SA, Aalders MC, Van Marle J. Mechanistic study of photodynamic
inactivation of Candida albicans by a cationic porphyrin. Antimicrob Agents
Chemother 2005 May;49(5):2026-34.
168
Conclusion
14. Smijs TG, Van Der Haas RN, Lugtenburg J, Liu Y, De Jong RL, Schuitmaker HJ.
Photodynamic Treatment of the Dermatophyte Trichophyton Rubrum and its
Microconidia with Porphyrin Photosensitizers. Photochem.Photobiol. 2004 Apr 1.
15. Smijs TG, Schuitmaker HJ. Photodynamic inactivation of the dermatophyte
Trichophyton rubrum. Photochem. Photobiol. 2003;77(5):556-60.
16. Vasconcelos E, Seghatchian J. Bacterial contamination in blood components and
preventative strategies: an overview. Transfus. Apher. Sci. 2004 Oct;31(2):155-63.
17. Paquette B, Boyle RW, Ali H, MacLennan AH, Truscott TG, van Lier JE. Sulfonated
phthalimidomethyl aluminum phthalocyanine: the effect of hydrophobic
substituents on the in vitro phototoxicity of phthalocyanines. Photochem.
Photobiol. 1991 Mar;53(3):323-7.
18. Hunt DW, Chan AH. Influence of photodynamic therapy on immunological aspects
of disease - an update. Expert Opin. Investig. Drugs 2000;9(4):807-17.
19. Munson BR, Fiel RJ. DNA intercalation and photosensitization by cationic meso
substituted porphyrins. Nucleic Acids Research 1992;20(6):1315-9.
20. Sari MA, Battioni JP, Dupre D, Mansuy D, Le_Pecq JB. Interaction of cationic
porphyrins with DNA: importance of the number and position of the charges and
minimum structural requirements for intercalation. Biochemistry 1990;29(17):420515.
21. Honohan A, Olthuis H, Bernards AT, van Beckhoven JM, Brand A. Microbial
contamination of cord blood stem cells. Vox Sang. 2002;82(1):32-8.
22. Kogler G, Callejas J, Hakenberg P, Enczmann J, Adams O, Daubener W, Krempe C,
Gobel U, Somville T, Wernet P. Hematopoietic transplant potential of unrelated
cord blood: critical issues. J. Hematother. 1996;5(2):105-16.
23. Guenechea G, Segovia JC, Albella B, Lamana M, Ramirez M, Regidor C, Fernandez
MN, Bueren JA. Delayed engraftment of nonobese diabetic/severe combined
immunodeficient mice transplanted with ex vivo-expanded human CD34(+) cord
blood cells. Blood 1999 Feb 1;93(3):1097-105.
24. Ahmed F, Ings SJ, Pizzey AR, Blundell MP, Thrasher AJ, Ye HT, Fahey A, Linch DC,
Yong KL. Impaired bone marrow homing of cytokine-activated CD34+ cells in the
NOD/SCID model. Blood 2004 Mar 15;103(6):2079-87.
169
Chapter VIII
25. Zhai QL, Qiu LG, Li Q, Meng HX, Han JL, Herzig RH, Han ZC. Short-term ex vivo
expansion sustains the homing-related properties of umbilical cord blood
hematopoietic stem and progenitor cells. Haematologica 2004 Mar;89(3):265-73.
26. Graf T. Differentiation plasticity of hematopoietic cells. Blood 2002 May
1;99(9):3089-101.
27. Gothot A, van der Loo JC, Clapp DW, Srour EF. Cell cycle-related changes in
repopulating capacity of human mobilized peripheral blood CD34(+) cells in nonobese diabetic/severe combined immune-deficient mice. Blood 1998 Oct
15;92(8):2641-9.
28. Springer TA. Traffic signals on endothelium for lymphocyte recirculation and
leukocyte emigration. Annu. Rev. Physiol 1995;57:827-72.
29. Peled A, Kollet O, Ponomaryov T, Petit I, Franitza S, Grabovsky V, Slav MM, Nagler A,
Lider O, Alon R, et al. The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and
VLA-5 on immature human CD34(+) cells: role in transendothelial/stromal migration
and engraftment of NOD/SCID mice. Blood 2000 Jun 1;95(11):3289-96.
30. Fonseca AM, Porto G, Uchida K, Arosa FA. Red blood cells inhibit activation-induced
cell death and oxidative stress in human peripheral blood T lymphocytes. Blood
2001;97(10):3152-60.
31. Arosa FA, Pereira CF, Fonseca AM. Red blood cells as modulators of T cell growth
and survival. Curr. Pharm. Des 2004;10(2):191-201.
32. Padley D, Koontz F, Trigg ME, Gingrich R, Strauss RG. Bacterial contamination rates
following processing of bone marrow and peripheral blood progenitor cell
preparations. Transfusion 1996;36(1):53-6.
33. Patel AN, Park E, Kuzman M, Benetti F, Silva FJ, Allickson JG. Multipotent menstrual
blood stromal stem cells: isolation, characterization, and differentiation. Cell
Transplant. 2008;17(3):303-11.
170
NEDERLANDSE SAMENVATTING
HET INACTIVEREN VAN PATHOGENEN IN
CELLULAIRE BLOED PRODUCTEN MET
FOTODYNAMISCHE BEHANDELING
INTRODUCTIE: BLOEDPRODUCTEN EN ZIEKTEVERWEKKERS
De veiligheid van bloedtransfusie is een van de prioriteit van de bloedbank. Om
veiligheid te garanderen is een reeks procedures geïmplementeerd. Ten eerste
dient het doneren van bloed een vrijwillige, onbetaalde activiteit te zijn. Het
donorbloed wordt vervolgens getest op de aanwezigheid van belangrijke
ziekteverwekkers, zoals Hepatitis B virus (HBV), Hepatitis C virus (HCV), Human
Immunodeficiency Virus (HIV), Human T-cell leucemia virus (HTLV) en Treponema
pallidum.
In geindustrialiseerde landen bedraagt de frequentie van geïnfecteerde
bloeddonaties ongeveer 1 op 270.000 voor HCV, 1 op 205.000 voor HBV en 1 op
3,1 miljoen voor HIV. De frequenties zijn nog veel lager voor HTLV en Treponema
palladium.
Ondanks deze lage frequenties, kan het risico dat een geïnfecteerde bloeddonatie
aan een patiënt wordt overgedragen niet uitgesloten worden. Ten eerste komt
het voor dat, wanneer een donor zeer recentelijk geïnfecteerd is, de
ziekteverwekker nog niet detecteerbaar is in zijn bloed omdat de concentratie van
het pathogeen of de hoeveelheid antilichamen tegen de ziekteverwekker nog te
laag is. Ten tweede wordt de bloedvoorziening voortdurend bedreigd door
nieuwe opkomende ziekteverwekkers, bijvoorbeeld het vogelgriep virus (H5N1)
en het West-Nijl virus. Mensen reizen steeds meer en gaan steeds vaker naar
tropische landen waar infecties zeer vaak voorkomen met parasieten,
verantwoordelijk voor malaria of Chagas ziekte. Bovendien duurt het testen van
bloeddonaties op bacteriële contaminatie te lang om dit op tijd, dus voor de
171
Samenvatting
transfusie, op te merken. Ten slotte is er niet voor alle bekende ziekteverwekkers
een test beschikbaar.
Gezien het brede scala aan ziekteverwekkers is het niet realistisch om alle
bloeddonaties op alle micro-organismen te testen. Dit zou leiden tot hogere
kosten en langere verwerking van het bloed, wat uiteindelijk enorme gevolgen
zou hebben voor de bloedvoorziening in ziekenhuizen.
Om de veiligheid van de bloedtransfusie te vergroten, zijn technieken ontwikkeld
om ziekteverwekkers te inactiveren zonder dat de kwaliteit van het bloedproduct
achteruit gaat. Zulke technieken worden “Pathogene Inactivatie Technieken”
genoemd. Deze zijn vergelijkbaar met sterilisatietechnieken, met het grote
verschil dat, bij sterilisatieprocessen alle componenten, inclusief de cellen,
geïnactiveerd of vernietigd worden. Deze totale vernietiging is niet toepasbaar op
cellulaire bloedproducten, omdat de bloedcellen noodzakelijk zijn voor het
therapeutische effect van een bloedtransfusie. Om deze reden is binnen dit
promotieonderzoek gewerkt aan een methode, waarmee met behulp van
fotodynamische behandeling, specifiek de ziekteverwekkers in cellulaire
producten worden geïnactiveerd.
HET PRINCIPE VAN FOTODYNAMISCHE BEHANDELING OM
PATHOGENEN TE INACTIVEREN
De fotodynamische reactie betreft de activering van een lichtgevoelige molecuul
(fotosensitizer) door belichting met rood licht in de aanwezigheid van zuurstof. Als
de fotosensitizer eenmaal is geactiveerd, dan gaat deze chemische reacties aan
met andere moleculen, waardoor zich zeer reactieve deeltjes vormen. Deze zeer
reactieve toxische deeltjes zijn verantwoordelijk voor oxidatieve schade aan
talloze moleculen die aanwezig zijn op virussen, bacteriën en bloedcellen. De
eigenschappen van de fotosensitizer bepalen de effectiviteit en de selectiviteit
van de fotodynamische behandeling (PDT voor PhotoDynamic Treatment).
DOEL VAN HET ONDERZOEK
In het onderzoek beschreven in dit proefschrift richtte onze pathogene inactivatie
techniek zich voornamelijk op bloedproducten die rode bloedcellen bevatten
172
Pathogene inactivatie in cellulaire bloed producten met fotodynamische behandeling
zoals rode bloedcel concentraten (RBC) en navelstrengbloed. Het doel van het
onderzoek was een fotosensitizer te vinden waarmee micro-organismen
geïnactiveerd worden zonder de bloedcellen te beschadigen. Alle bekende
fotosensitizers die in staat waren om verschillende pathogenen te inactiveren,
waren ook schadelijk voor de rode bloedcellen. In hoofdstukken 2 en 3 hebben
wij studies uitgevoerd met als doel meer inzicht te krijgen in het mechanisme
achter de fotodynamische schade aan rode bloedcellen, wat vervolgens moest
leiden tot het beter beschermen van deze cellen. De zoektocht naar meer
specifieke fotosensitizers is beschreven in hoofdstukken 4 en 5. In hoofdstukken
6 en 7 beschreven wij de toepassing van pathogene inactivering op
navelstrengbloed en op witte bloedcellen.
SCHADE AAN RBC MEMBRAAN DOOR PDT MET DMMB EN SELECTIEVE
BESCHERMING
In hoofdstuk 2 wordt een studie beschreven naar het schadelijke effect van PDT
met dimethyl-methylene blauw (DMMB) op de rode bloedcellen. DMMB is een
fotosensitizer die bindt aan rode bloedcellen, waardoor de cellen direct
blootgesteld worden aan de schadelijke effecten van de oxidatieve deeltjes. We
hebben een eiwit, zogenaamd Band 3, geïdentificeerd waarop fotodynamische
schade plaatstvindt. Band 3 transporteert anionen door het rode
bloedcelmembraan. Als de activiteit van Band 3 verstoord is, kan de rode bloedcel
zijn osmotische regulatie niet meer garanderen. De cellen zwellen op, waardoor
ze vervolgens kapot gaan. Die wordt hemolyse genoemd.
In hoofdstuk 3 hebben wij een manier gevonden om Band 3 selectief te
beschermen tegen fotodynamische schade door PDT met DMMB. Als
Dipyridamole toegevoegd wordt aan het bloedproduct, is Band 3 beschermd
tegen het schadelijke effect van PDT met DMMB. Dipyridamole is een molecuul
dat aan Band 3 bindt en de schadelijke oxidatieve deeltjes vangt. Dipyridamole
werkt eigenlijk als een schild en beschermt Band 3 zonder de inactivatie van
virussen door PDT met DMMB te verminderen.
Helaas bleek echter dat PDT met DMMB ook op andere plekken van de rode
bloedcellen schade aanricht, waardoor de kwaliteit van de rode bloedcellen zeer
173
Samenvatting
moeilijk in stand te houden bleek. Vandaar dat wij op zoek zijn gegaan naar
andere fotosensitizers.
DE ZOEKTOCHT NAAR EEN NIEUWE FOTOSENSITIZER: TRI-P(4)
In hoofdstuk 4 is een reeks nieuwe fotosensitizers getest en geëvalueerd op hun
potentieel om virussen te inactiveren. Tevens zijn hun effecten op de kwaliteit van
de rode bloedcellen geëvalueerd. De nieuwe fotosensitizers bestaan uit derivaten
van porfyrines. Porphyrines zijn macrocyclische molecules (zeer grote cyclus) met
vier pyrrole moleculen (fig.1). Van de 7 verschillende geteste porfyrines, was TriP(4) de fotosensitizer die virussen het beste kon doden met beperkte schade aan
rode bloedcellen. Tests in resusapen wezen uit dat de PDT-benhandelde rode
bloecellen na transfusie net zoveel kans op overleving hadden als de
onbehandelde cellen. Het vervolgonderzoek hierop is beschreven in hoofdstuk 5,
waar PDT met Tri-P(4) getest werd op een breed scala aan micro-organismen die
voornamelijk relevant zijn bij bloed transfusie. Deze studie heeft laten zien dat
modelvirussen voor HCV, HBV en HIV geïnactiveerd konden worden. Echter PDT
met Tri-P(4) scoorde onvoldoende bij het inactiveren van intracellulaire virussen
en Parvo-virussen (waarvan de menselijke variant B19 veroorzaakt de vijfde
ziekte, of anders genoemd erythema infectiosum). Parvo-virussen zijn bekend om
hun hoge resistentie tegen chemische behandelingen. Bacteriën, vaak betrokken
bij rode bloedcel contaminatie zoals Yersinia enterolitica,
waren ook gevoelig voor PDT met Tri-P(4). In vergelijking
met DMMB, brengt Tri-P(4) minder schade toe aan rode
bloedcellen. Desalniettemin leidt fotodynamische schade
door Tri-P(4) toch tot een vermindering van de
therapeutische kwaliteit van het bloedproduct.
Vanwege de al geringe kans op overdracht van microorganismen via bloedtransfusie, blijkt de ontsmetting
methode via PDT met Tri-P(4) geen toegevoegde waarde Figure 1: basische
vorm van
te hebben in het toenemen van de transfusionelle
porphyrine
veiligheid. Dit vanwege de beperking van de behandeling
om alle virussen te kunnen inactiveren en de schade, desondanks niet te
verwaarlozen, aan de rode bloed cellen. Daarom is het besloten om deze techniek
174
Pathogene inactivatie in cellulaire bloed producten met fotodynamische behandeling
van pathogen inactivatie niet in te voeren voor de ontsmetting van rode bloed cel
concentraten.
ONTSMETTING VAN NAVELSTRENGBLOED
Het gebruik van navelstrengbloed berust op een hoge concentratie
bloedstamcellen, waardoor het een geschikt alternatief is voor
beenmergtransplantatie, voornamelijk bij kinderen. De bloedstamcellen worden
ook gekweekt voor nieuwe therapeutische doeleinden. Bij afname van het
navelstrengbloed wordt het product vaak vervuild met bacteriën, wat gevaarlijk
kan zijn voor de getransplanteerde patiënt en het kweken van de stamcellen zelf
kan belemmeren.
Zoals beschreven in hoofdstuk 6 inactiveert PDT met Tri-P(4) alle bacteriën in
vervuilde navelstrengbloedproducten. De stamcellen overleven deze
ontsmettingsbehandeling. Helaas bleek uit studies in muizen dat de kwaliteit van
het navelstrengbloed als transplantaat vermindert, waarschijnlijk doordat er toch
fotodynamische schade aan de cellen plaatsvindt, die de cel migratie naar de
beenmerg zou hebben aangetast. De stamcellen konden echter wel verder
gekweekt worden tot andere bloedcellen, zoals thrombocyten. Doordat tijdens
het kweken van deze cellen, het gebruik van antibiotica steeds bemoeilijkt wordt
omdat bacteriën resistentie ontwikkelen tegen antibiotica, zou PDT met Tri-P(4)
een interessante alternatieve behandeling zijn voor de ontsmetting van overige
stamcel bevattende producten zoals de beenmerg en het menstruatiebloed. Met
deze andere bronnen van stamcellen wordt onderzoek gedaan naar het herstel
van weefsels en organen.
DE MODULATIE VAN HET IMMUUNSYSTEEM MET PDT EN TRI-P(4)
In hoofdstuk 7 is een studie beschreven naar het effect van PDT met Tri-P(4) op
de witte bloedcellen die aanwezig zijn in rode bloedcelconcentraten. De transfusie
van witte bloedcellen geeft bijwerkingen doordat het lichaam van de
getransfuseerde patiënt als “vreemd” wordt herkend en aangevallen wordt door
de witte bloedcellen van de donor. Een andere bijwerking is de aanmaak van
antilichamen door de patiënt tegen de getransfuseerde witte bloedcellen. Zulke
antilichamen kunnen problemen geven bij een latere bloedtransfusie.
175
Samenvatting
Daarentegen, na een transplantatie (in het bijzonder niertransplantatie) bevordert
de transfusie van witte bloedcellen de acceptatie van het transplantaat door het
lichaam van de getransplanteerde patiënt.
Deze studie richtte zich op de bestudering van het effect van PDT met Tri-P(4) op
het modulatiepotentieel van de witte bloedcellen. Onze resultaten laten zien dat
PDT met Tri-P(4) het opwekken van de ontstekingsreacties remt. De sterkte van
de remming is afhankelijk van de hoeveelheid rode bloedcellen die aanwezig zijn
in het bloedproduct. De cellen werken als een schild tegen fotodynamische
schade aan witte bloedcellen. De resultaten gaven sterke aanwijzingen dat de
immune respons van donor witte bloedcellen gemoduleerd kon worden met PDT
en Tri-P(4). Verder onderzoek is nodig om te bepalen of PDT met Tri-P(4) nieuwe
ontwikkelingen zal brengen in het voorkomen van afstotingsreacties na
orgaantransplantatie of het aanmaken van antilichamen na bloedtransfusie.
CONCLUSIE
Onze studies hebben laten zien dat de fotosensitizer Tri-P(4) veel beter is dan alle
andere geteste fotosensitizers om pathogenen te inactiveren in rode
bloedcelproducten. Echter, ondanks de veelbelovende resultaten van
fotodynamische ontsmetting van rode bloedcelproducten, zou de resulterende
therapeutische kwaliteit van het bloedproduct toch te veel reden voor
bezorgdheid opleveren bij transfusiespecialisten. Vandaar dat verder onderzoek
naar het gebruik van PDT met Tri-P(4) voor pathogene inactivatie in rode bloedcel
concentraten niet voortgezet zal worden. Desalniettemin hebben onze studies
verschillende toepassingen voor de fotodynamische behandeling met Tri-P(4) op
bloed producten naar voren gebracht. De pathogeen inactiverende werking van
Tri-P(4) zou wel gebruikt kunnen worden in andere bronnen van stamcellen zoals
menstruatiebloed en beenmerg, gezien het geringe effect op stamcellen.
Daarnaast zouden de effecten van de behandeling op de witte bloed cellen
gunstig kunnen zijn om transfusiegerelateerde immune reacties te voorkomen of
om tolerantie na orgaan- of celtransplantatie te bevorderen.
176
RÉSUMÉ EN FRANÇAIS
INACTIVATION PAR LE TRAITEMENT
PHOTODYNAMIQUE DES AGENTS
PATHOGENIQUES CONTENUS DANS LES
PRODUITS SANGUINS CELLULAIRES
INTRODUCTION : LES DONS SANGUINS ET LES AGENTS INFECTIEUX
La production de produits sanguins sains est l'une des priorités des banques de
sang. Cela passe par une série de procédures qui commence par le don de sang
volontaire et non-rémunéré. Le don sanguin est ensuite testé, entre autres, pour
la présence d’agents infectieux tels que les virus d’hépatite B et C (VHB, VHC), le
virus d’immunodéficience acquise (VIH), le virus T-lymphotropique humain (HTLV)
causant divers cancers et la bactérie Treponema pallidum responsable de la
syphilis.
Dans les pays industrialisés, le nombre de dons infectieux est actuellement très
faible, de l’ordre de 1 sur 205.000 pour le VHB, 1 sur 270.000 pour le VHC, 1 sur
3.1 million pour le VIH. La fréquence est encore plus faible pour le HTLV et le
Treponema pallidum. Bien que ces chiffres soient très faibles, le danger de
transfuser un don sanguin infecté par un agent pathogène persiste.
Tout d’abord, un individu récemment infecté peut faire un don de sang contaminé
alors que la présence de l’agent infectieux n’est pas encore détectable, la
concentration de l'agent pathogène ou d'anticorps dans le sang étant encore trop
faible. Ensuite, les banques de sang sont confrontées à une menace permanente
de nouvelles infections, comme par exemple l’émergence de nouveaux virus tels
que celui de la grippe aviaire (H5N1) ou du virus du Nil occidental. Par ailleurs, les
gens voyagent de plus en plus dans les pays tropicaux, s’exposant à des infections
parasitaires responsables des maladies comme la malaria ou la maladie de
Chagas. Il y a encore trop de contaminations bactériennes des produits sanguins
qui ne sont pas détectées à temps à cause de la longue durée des tests de culture.
177
Résumé
Pour finir, il n’y a pas de tests de dépistage disponibles pour tous les agents
pathogéniques connus. Au regard de la gamme d’agents infectieux susceptibles de
contaminer le sang, il serait inconcevable que chaque don soit testé pour tous les
agents pathogéniques. Ceci générerait une hausse des coûts de production et un
processus de préparation plus long, ayant des conséquences énormes sur
l’approvisionnement des dons sanguins aux centres hospitaliers.
Afin d’assurer la sécurité des transfusions sanguines, de nouvelles technologies
ont été développées. Elles ont pour but d’inactiver les agents pathogéniques sans
que la qualité du produit sanguin ne soit altérée. Ces techniques sont appelées
« Techniques d’inactivation pathogénique ». Elles sont comparables à des
techniques de stérilisation, avec la grande différence que, dans le processus de
stérilisation, tous les composants, incluant les cellules sanguines, sont détruits,
alors que ceci n’est pas envisageable pour les produits sanguins cellulaires. Les
cellules sanguines sont en effet indispensables à l’effet thérapeutique de la
transfusion sanguine. C’est pour cette raison qu’au cours de ces travaux de
recherche, nous avons travaillé à une méthode, basée sur le traitement
photodynamique, qui inactive spécifiquement les agents pathogéniques dans les
produits sanguins cellulaires.
LE PRINCIPE DU TRAITEMENT PHOTODYNAMIQUE POUR INACTIVER
LES AGENTS PATHOGENIQUES
Le traitement photodynamique (TPD) implique l’excitation par la lumière rouge
d’une molécule photosensible (photosensibilisateur) en présence d’oxygène. Le
photosensibilisateur excité réagit avec d’autres molécules, produisant ainsi des
espèces chimiques toxiques qui oxydent de nombreuses molécules présentes sur
les agents pathogéniques ou les cellules sanguines. Les propriétés du
photosensibilisateur déterminent l’efficacité et la sélectivité du TPD.
BUT DE LA RECHERCHE
Le développement de notre technique de photo-décontamination se concentrait
essentiellement sur les produits sanguins labiles tels les concentrés de globules
rouges et le sang placentaire. Notre objectif était de trouver un
photosensibilisateur capable d’inactiver les agents pathogéniques sans
178
Inactivation par le TPD des agents pathogéniques contenus dans les produits sanguins
endommager les cellules sanguines. Jusqu’alors, tous les photosensibilisateurs
susceptibles d’avoir une bonne activité anti-pathogénique endommageaient aussi
les globules rouges. Les chapitres 2 et 3 décrivent les travaux effectués pour
mieux comprendre les mécanismes impliqués dans l’endommagement des
globules rouges par le TPD afin de pouvoir mieux les protéger. Les chapitres 4 et 5
décrivent la découverte d’un nouveau photosensibilisateur qui serait plus
spécifique pour les agents pathogéniques que pour les globules rouges. Enfin, les
chapitres 6 et 7 décrivent l’application du TPD pour la décontamination du sang
placentaire et ses effets sur les globules blancs.
L’ENDOMMAGEMENT DES GLOBULES ROUGES PAR TPD AVEC LE
PHOTOSENSIBILISATEUR DMMB ET PROTECTION SELECTIVE.
Le photosensibilisateur DMMB (di-méthyle-méthylène bleu) est un agent
chimique capable d’inactiver une grande gamme d’agents viraux, bactériens et
parasitaires. Cependant le DMMB se fixe sur les globules rouges, exposant
directement les cellules aux effets néfastes des espèces chimiques oxydantes.
Nous avons identifié une protéine, appelée Bande 3, comme étant la molécule à
laquelle le DMMB se fixe et qui serait endommagée par le TPD (chapitre 2). La
protéine Bande 3 assure l’échange de molécules tel que le chlorure et le
bicarbonate à travers la membrane plasmique des globules rouges. Quand
l’activité de Bande 3 est perturbée, voire inhibée, le globule rouge n’est plus en
état de réguler son état osmotique. Il finit alors par éclater. C’est le phénomène
d’hémolyse. Dans le chapitre 3, nous décrivons une manière de protéger la
protéine Bande 3 contre les dommages photodynamiques causés par le TPD avec
le DMMB. Pour cela, du dipyridamole est ajouté aux produits sanguins. Cet agent
chimique se fixe sur la protéine Bande 3 et la protège en contrant les espèces
chimiques oxydantes générées par le TPD. Tout cela se fait sans réduction de
l’efficacité du DMMB à inactiver les agents viraux.
Cependant, les dommages causés aux globules rouges par le TPD avec le DMMB
sont très étendus. La qualité des globules rouges est très difficile à maintenir en
état après le traitement. C’est pourquoi, nous sommes allés à la recherche de
nouveaux photosensibilisateurs.
179
Résumé
TRI-P(4), UN NOUVEAU PHOTOSENSIBILISATEUR, TRES PROMETTEUR
Comme décrit dans le chapitre 4, de nouveaux photosensibilisateurs ont été
testés. Ils sont tous dérivés de porphyrines, des molécules macrocycliques (cycle
de grande taille) tétra-pyrroliques (formées de quatre molécules de pyrrole)
(fig.1). Ces dérivés de porphyrines ont été testés pour déterminer leur potentiel à
inactiver les agents viraux et bactériens. Parallèlement, leurs effets sur les
globules rouges ont été étudiés. Parmi les 7 molécules différentes testées, la
porphyrine Tri-P(4) s’est avérée être supérieure aux autres par sa plus grande
efficacité à inactiver le virus tout en causant le moins de dommages aux globules
rouges. Des globules rouges de singes Rhésus ont été sujets au TPD avec le TriP(4) puis re-transfusés aux animaux. La chance de survie des cellules traitées au
Tri-P(4) était comparable à celles des cellules non traitées. La suite des études est
décrite dans le chapitre 5, où une gamme plus large d’agents pathogéniques a été
traitée avec le TPD et le Tri-P(4). L’étude a montré que les agents particulièrement
importants en transfusion sanguine comme le VHB, VHC et le VIH étaient
sensibles au traitement et pouvaient être rendus inactifs mais cela nécessitait une
durée de traitement 4 fois plus longue. Cependant, le TPD avec le Tri-P(4) n’était
pas capable d’inactiver les virus intracellulaires (ceux qui ont infecté des globules
blancs), ni les Parvovirus (dont la variante humain B19 est responsable de
l’érythème infectieux aigu, autrement dit la cinquième maladie). Ces virus sont
connus pour être les plus résistants aux traitements chimiques. En revanche, les
bactéries se sont avérées être très sensibles au TPD avec le Tri-P(4). Comparé au
DMMB, le Tri-P(4) cause moins de dommages aux globules
rouges. Néanmoins, ces dommages contribuent à une
diminution de la qualité thérapeutique du produit sanguin.
Compte tenu de la faible fréquence de transfusion de sang
contaminé, la technique de décontamination utilisant le
TPD avec le Tri-P(4) ne semble pas apporter de valeur
ajoutée à l’accroissement de la sécurité transfusionnelle, de Figure 1: Squelette
par sa capacité limitée à inactiver tous les virus et aux
de base de
dégâts, malgré tout non négligeables, causés aux globules
porphyrine
rouges. Par conséquent, il a été décidé que le traitement d’inactivation
180
Inactivation par le TPD des agents pathogéniques contenus dans les produits sanguins
pathogénique par le TPD avec le Tri-P(4) ne serait pas appliqué dans les banques
de sang.
DECONTAMINATION DU SANG PLACENTAIRE
Le sang placentaire provient du cordon ombilical après la naissance du nouveauné. Ce produit contient une quantité importante de cellules souches (cellules de
sang primitives encore indifférenciées) capable de restituer un système
immunitaire après transplantation chez un patient. Les cellules souches issues du
sang placentaire sont aussi mises en culture pour développer de nouveaux
intérêts thérapeutiques. Cependant, pendant la récolte du sang placentaire, le
produit est souvent contaminé par des bactéries. D’une part, celles-ci peuvent
être dangereuses pour le patient qui reçoit le produit transplanté. D’autre part,
les bactéries trouvent dans les cultures cellulaires les conditions idéales pour leur
croissance, empêchant la culture saine des cellules souches.
Comme décrit dans le chapitre 6, le TPD avec Tri-P(4) est capable d’inactiver les
bactéries contaminantes sans affecter la survie des cellules souches.
Malheureusement, des études chez les souris ont montré que les cellules souches
traitées au TPD ont une capacité réduite à reconstituer un système immunitaire
après transplantation. Ceci est probablement dû aux effets photodynamiques qui
ont malgré tout causé quelques dommages aux cellules souches affectant leur
migration vers la moelle osseuse. Cependant, les cellules souches traitées au TPD
avec Tri-P(4) pouvaient être mises en culture pour produire d’autres cellules
sanguines. Comme pendant leurs cultures, l’utilisation d’antibiotiques devient de
plus en plus limitée à cause du développement de souches bactériennes
résistantes, le TPD avec le Tri-P(4) pourrait être une méthode intéressante pour la
décontamination des autres sources de cellules souches comme la moelle osseuse
et les menstrues. Ces autres sources de cellules souches contribuent à la
recherche sur la thérapie régénérative pour la reconstitution d’organes ou de
tissus.
181
Résumé
LA MODULATION DU SYSTEME IMMUNITAIRE PAR LE TPD AVEC TRIP(4)
Dans le chapitre 7, nous décrivons une étude effectuée pour étudier l’effet du
TPD avec le Tri-P(4) sur les globules blancs restés dans les concentrés de globules
rouges obtenus après centrifugation. La transfusion des globules blancs peut avoir
des effets néfastes chez le patient. Par exemple, après transfusion sanguine, les
globules blancs contenus dans le don sanguin reconnaissent les tissus du patient
comme étrangers et les attaquent. Ou encore, le patient produit des anticorps
dirigés contre les globules blancs transfusés. Ces anticorps peuvent présenter des
problèmes de compatibilité aux cours des prochaines transfusions sanguines. En
revanche, il a été démontré que la transfusion des globules blancs à un patient
transplanté peut favoriser l’acceptation de la greffe.
Nous avons étudié le potentiel du TPD avec le Tri-P(4) à moduler la fonction des
globules blancs. Nos résultats ont montré que ce traitement inhibait les réactions
inflammatoires et que le niveau d’inhibition était fortement dépendant de la
quantité de globules rouges présents pendant le TPD. Les globules rouges
agissaient en fait comme un bouclier contre les effets néfastes du TPD. Les effets
induits par le TPD sur les fonctions immunitaires des globules blancs se
produisaient par un mécanisme complexe qui affecte toutes les sortes de cellules
(celles qui initient l’inflammation et celles qui répondent aux stimuli). Des
recherches plus approfondies permettraient de déterminer si le TPD avec le TriP(4) serait efficace pour empêcher des réactions de rejet après transplantation ou
pour empêcher la formation d’anticorps après transfusion.
CONCLUSION
Nos études ont montré que le photosensibilisateur Tri-P(4) est supérieur à tous les
autres photosensibilisateurs testés pour inactiver les agents pathogéniques dans
les produits sanguins de globules rouges. Cependant, malgré les résultats très
prometteurs concernant la décontamination des concentrés de globules rouges, la
qualité thérapeutique du produit sanguin après le traitement photodynamique
reste préoccupante. C’est la raison pour laquelle les recherches de
développement du TPD avec la porphyrine Tri-P(4) pour l’inactivation
182
Inactivation par le TPD des agents pathogéniques contenus dans les produits sanguins
pathogénique dans les concentrés de globules rouges ne seront pas poursuivies.
Néanmoins, les études effectuées avec le photosensibilisateur Tri-P(4) ont permis
de découvrir d’autres potentiels. Grace à sa capacité à inactiver les bactéries, tout
en épargnant les cellules souches, le TPD avec le Tri-P(4) pourrait être fortement
utile pour la décontamination d’autres sources de cellules souches comme celles
des menstrues ou de la moelle osseuse. Par ailleurs, ses effets sur les globules
blancs pourraient permettre la prévention des effets néfastes liés à la transfusion
sanguine, et la promotion de la tolérance après transplantation d’organes ou de
cellules.
183
184
DANKWOORD
Wetenschap is teamwork. Dit proefschrift was niet mogelijk geweest zonder de
hulp en ondersteuning van velen.
Ten eerste, speciale dank ben ik verschuldigd aan Anneke en Johan. Ik bedank ze
voor hun expertise, adviezen, geduld en hun trouwe ondersteuning.
Aan alle leden van O&O. Het was zeer gezellig in de nieuwbouw met de
gloednieuwe laboratoria. Jullie hebben allemaal bijgedragen aan mijn onderzoek,
van het geven van simpele tips tot het uitvoeren van experimenten, zonder de
broodnodige ontspanning tijdens de lunch te vergeten.
Aan alle leden van NBB. Ik bedank jullie voor de hulp en de prettige samenwerking
voor het verkrijgen van stamcel eenheden. Speciale dank ben ik verschuldigd aan
Jacqueline. Dankzij haar en Peter van MCB, ben ik aan mijn wetenschappelijke
carrière begonnen bij de bloedbank en het LUMC.
Aan Karin, Els, Jolien, Liesbeth en Yvonne van de afdeling IHB. Ik bedank ze voor
hun bijdrage in het uitvoeren van experimenten. In het bijzonder dank aan Karin,
dankzij haar inzet, ervaring en grenzeloze enthousiasme, heb ik veel genoten en
veel geleerd van de samenwerking met haar. Verder nog bedankt aan Dave voor
het delen van zijn expertise en ideeën.
Aan Tom en Hans van MCB uit het Sylvius lab, de voormalige groep van Prof. John
van Steveninck die helaas veel te vroeg is vertrokken. John was de oorspronkelijke
bedenker van het idee achter mijn onderzoek. Dit boekje is ook in blijvende
herinnering aan hem en zijn wetenschappelijke inbreng. Met Tom en Hans heb ik
een bijzonder avontuur beleefd met het ontstaan van PhotoBioChem. Dit
avontuur was van korte duur, maar toch bedank ik hun voor de intensieve
samenwerking en de bijzondere momenten en ervaringen.
Dank aan Threes en Saskia. PDT en Tri-P(4) heeft ons bijen gebracht. Ik hoop dat
het avontuur met PDT voor ons nog niet over is.
Tot slot wil ik graag al mijn andere vrienden bedanken voor hun oprechte
interesse. Dat is mede dankzij deze “bewonderaars” wat mijn gestimuleerd heeft
om dit proefschrift af te maken.
185
Remerciements
Merci à Marie. Enfin, ce chapitre de ma vie est fini et c’est sans aucun doute grâce
au dernier petit coup de pouce qu’elle m’a donné.
Merci à mes sœurs Pascale, Annie et Nathalie, qui m’ont permis de faire des
études et qui m’ont toujours soutenue. Sans le savoir, leur solidarité envers moi
et leurs expériences de vie m’ont permis d’accomplir ce magnifique chapitre de
ma vie. Merci à ma mère, pour tout l’amour et surtout la liberté qu’elle m’a
donnée pour en arriver là. Aussi un grand merci à ma nièce Léa et ma cousine
Christelle pour leur aide pour le résumé en français. Par la même, merci aussi à
ma copine et ancienne camarade d’université Séverine. Si tous les francophones
comprennent le sujet de ma thèse, ce sera grâce à elles!
Ook bedankt aan mijn schoonfamilie. Ze zorgden voor de broodnodige
ontspanning en gezelligheid.
Aan mijn lieve Jan. Hij heeft altijd zonder te twijfelen gezegd “jij gaat
promoveren“. Hij gaf me alle ruimte en tijd, een luisterend oor en adviezen om
eindelijk deze promotie af te maken. Hij is zo geduldig geweest. Hij was van een
ongelofelijke grote steun. Dat is niet uit te drukken met één woord als bedankt.
Dankzij hem is dit belangrijke hoofdstuk van mijn leven af, maar wij hebben nog
zoveel te beleven samen met onze prachtige kindjes Félix en Laura. Ik kijk uit naar
deze mooie momenten.
Tot ziens allemaal,
Salut à tous et à toutes,
Laurence
186
CURRICULUM VITAE
Laurence Trannoy werd op 17 april 1973 geboren te Soissons in Frankrijk. Na het
behalen van haar Baccalaureaat aan het André Malraux lyceum te Montereaufault-Yonne in 1991 begon ze de studie Cellulaire en Moleculaire Biologie aan de
Université Pierre et Marie Curie te Parijs. Tijdens haar studie deed ze mee aan een
Erasmus uitwisselingsprogramma. Ze vertrok in 1994 gedurende een jaar om
Medische Biologie aan de UVA te studeren. Het studiejaar werd afgerond met een
stage bij de afdeling Celbiologie en Cytologie van de UVA onder leiding van Dr. C.L.
Woldringh. In 1995 ging ze terug naar Parijs om af te studeren en zich te
specialiseren in Immunologie aan de Université René Descartes. De
afstudeerstage werd uitgevoerd in het onderzoeksinstituut Sanquin (voorheen
CLB) te Amsterdam in de afdeling Auto-immuunziekten, onder leiding van Prof. E.
Hack en Dr. E. Eldering. In september 1996 behaalde ze haar Franse
doctoraalexamen, het zgn. DEA.
In 1999 begon ze aan haar promotie onderzoek over inactiveren van pathogenen
in cellulaire bloed producten met behulp van fotodynamische behandeling bij
Sanquin Bloedbank Zuidwest, de afdeling Moleculaire Celbiologie en de afdeling
Immunohematologie en Bloedtransfusie van het LUMC te Leiden. Dit onderzoek
werd begeleid door Prof. Dr. A. Brand, Dr. T.M.A.R. Dubbelman, Dr. J.W.M.
Lagerberg en Dr. D.L. Roelen.
Sinds mei 2009 is ze werkzaam als wetenschappelijk onderzoeker op de afdeling
Dermatologie van het LUMC. Ze ontwikkelt een fotodynamische therapie om met
schimmel geïnfecteerde huid te behandelen.
187
188
LIST OF PUBLICATIONS
J. van Steveninck, L.L. Trannoy, G.A. Besselink, T.M.A.R. Dubbelman, A.Brand, D.
de Korte, A.J. Verhoeven and J.W. Lagerberg. Selective protection of RBCs against
photodynamic damage by the Band 3 ligand Dipyridamole. Transfusion 2000; 40:
1330-6
L.L.Trannoy, A. Brand and J.W. Lagerberg. Relation between K+ leakage and
damage to Band 3 in photodynamically treated red cells. Photochem. Photobiol.
2002; 75: 167-71
L.L. Trannoy, J.W. Lagerberg, T.M.A.R. Dubbelman, H.J. Schuitmaker and A. Brand.
Positively charged porphyrins: a new series of photosensitizers for sterilization of
RBCs. Transfusion 2004; 44: 1186-96
L.L.Trannoy, F.G. Terpstra, D. de Korte, J.W. Labergerg, A.J. Verhoeven, A. Brand
and F.A van Engelenburg. Differential sensitivities of pathogens in red cell
concentrates to Tri-P(4)-photoinactivation. Vox Sanguinis 2006; 91: 111-18
L.L.Trannoy, Y. van Hensbergen, J. W. Lagerberg and A. Brand. Photodynamic
treatment with Tri-P(4) for pathogen inactivation in cord blood stem cell products.
Transfusion 2008; 48:2629-37
L.L. Trannoy, D.L. Roelen, K.K. Koekkoek and A. Brand. Impact of photodynamic
treatment with Meso-substituted porphyrin on the immunomodulatory capacity
of white blood cell-containing red blood cell products. Photochem. Photobiol.
2010, 86: 223-30
189