Micron 39 (2008) 461–470
www.elsevier.com/locate/micron
Activation of mononuclear bone marrow cells treated in vitro with
a complex homeopathic medication
Beatriz Cesar a, Ana Paula R. Abud a, Carolina C. de Oliveira a, Francolino Cardoso b,
Waldemiro Gremski a, Juarez Gabardo a, Dorly de Freitas Buchi a,*
a
Departamento de Biologia Celular, Setor de Ciências Biológicas, Universidade Federal do Paraná (UFPR), Curitiba, PR, Brazil
b
Instituto de Tecnologia do Paraná-TECPAR, Curitiba, PR, Brazil
Received 16 November 2006; received in revised form 6 February 2007; accepted 7 February 2007
Abstract
Canova is a Brazilian homeopathic medication with immunomodulatory properties, recommended for patients where the immune system is
depressed. Previous studies demonstrated that Canova induces up-regulation in numbers of leukocytes. The bone marrow microenvironment is
composed of growth factors, stromal cells, extracellular matrix and progenitor cells that differentiate into mature blood cells. We now report the
effect of in vitro administration of the medication on the mononuclear differentiation of the bone marrow cell. Swiss mice femurs were dissected
cleaned and the cells of the marrow were flushed. The cells were plated, treated or not, incubated for different times and processed for light,
transmission and scanning electron, and confocal microscopy analysis. Bone marrow cells showed an enhanced proliferation in vitro in response to
Canova medication and Canova plus M-CSF and an increase was also observed in the numbers of the cell niches and ring-shaped nuclei cells.
Confocal and transmission and scanning electron microscopy showed the stages of monocyte maturation, with resting and activated cells. With
Canova treatment there was a marked increase in cell size, which is mainly attributable to the augmented cytoplasm, an increase in the number of
mitochondria, expansion of the RER and an enlarged Golgi. The response to Canova treatment indicates that it influences mononuclear
differentiation and activation of bone marrow progenitor and stromal cells.
# 2007 Elsevier Ltd. All rights reserved.
Keywords: Bone marrow cells; Stromal cells; Mononuclear cells; Activation; Canova medication
1. Introduction
Great strides in developing western medicine in the last
century have being made. Countless diseases were eliminated
or controlled through advances in immunology, parasitology,
and the discovery of antibiotic drugs and vitamins. Conventional Western, or Allopathic, medicine has achieved significant leaps in preserving both the quality and longevity of
life, and it is important to recognize this contribution. However,
conventional medicine is a wide label, and not all conventional
medicine is beneficial. While in many cases symptoms are
relieved, cured or prevented many allopathic medicines can do
a lot of harm to the body. The symptoms of a disease, for
* Corresponding author at: UFPR, Centro Politécnico, SCB, Depto de Biologia Celular, sala 215, Laboratório de Estudo de Células Inflamatórias e
Neoplásicas, Jardim das Américas, Curitiba, CEP 81531-980 PR, Brazil.
Tel.: +55 41 3361 1770; fax: +55 41 3361 1568.
E-mail addresses: [email protected], [email protected] (D.d.F. Buchi).
0968-4328/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.micron.2007.02.005
homeopathy, are the body’s attempt to cure itself. By contrast,
conventional medicine often works by suppressing the body’s
natural reactions. In the homeopathic method, the immune
system of the body is stimulated to support the healing system.
This practice accomplishes the equilibrium or homeostasis of
the whole body.
Canova (CA) is a Brazilian complex homeopathic medicine
produced from Aconitum napellus, Thuya occidentalis, Bryonia
alba, Lachesis muta and Arsenicum album. CA is found in
homeopathic drugstores and is indicated for patients whose
immune system appears to be depressed. Clinical observation of
these patients confirmed the success of this treatment. It seems to
enhance the individual’s own immunity to trigger a particular
immunologic response against several pathological conditions.
Previous studies demonstrated that CA activates macrophages
both in vivo and in vitro. It was observed that the in vitro
production of tumor necrosis factor-a (TNFa) by macrophages is
significantly diminished when the medicine is administrated
(Piemonte and Buchi, 2002). NADPH oxidase activity was
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B. Cesar et al. / Micron 39 (2008) 461–470
increased as well as that of inducible nitric oxide synthase
(iNOS), consequently producing reactive oxygen species (ROS)
and nitric oxide (NO), respectively (De Oliveira et al., 2006). CA
stimulates an increase of the endosomal/lysosomal system as
well as the phagocytic activity of macrophages when interacted
with Saccharomyces cerevisiae and Trypanosoma cruzi epimastigotes (Lopes et al., 2006). The modulatory effects of CA were
also observed both in vivo and in vitro in experimental infection
by Leishmania amazonensis, controlling infection progression
and limiting its dissemination (Pereira et al., 2005). Moreover, it
is neither toxic nor mutagenic (Seligmann et al., 2003). Similarly,
the improvement in immune response of CA-treated mice was
demonstrated in studies with Sarcoma 180. A reduction in
sarcoma size was observed and a significant infiltration of
lymphoid cells, granulation tissue and fibrosis occurred,
surrounding the tumor. All animals from the treated group
survived, and in 30% of them a total regression of the tumor was
shown. The treatment with CA increased total numbers of
leukocytes. Among lymphocytes, T CD4, B and NK cells
increased (Sato et al., 2005). These results suggested a direct or
indirect action of the CA on hematopoiesis. So the bone marrow
cells were treated and processed for light, transmission and
scanning electron, and confocal microscopy, and also for flow
cytometry. All microscopy techniques showed that monocytic
lineage (CD11b) and stromal cells (adherent cells) were activated
by treatment. Canova also increased cell clusters over adherent
cells, suggesting areas of proliferation and differentiation (Abud
et al., 2006).
The hematopoietic microenvironment influences the growth
and differentiation of hematopoietic cells. The adherent cell
layers elaborate soluble factors and deposit an extracellular
matrix, which in turn influences hematopoietic proliferation and
differentiation. The differentiation of monocytic cells (monoblasts, promonocytes and monocytes) and the differentiation of
monocytes into macrophages are supported by macrophage
colony-stimulating factor (M-CSF). M-CSF stimulates predominantly the proliferation of progenitors committed to macrophage lineages (Kurosaka et al., 1999; Sweet and Hume, 2003;
Hassan et al., 1994; Shima et al., 1995; Imada et al., 2005).
CA medication has been successfully used in clinics and
several experimental studies have been carried out to examine
the medication’s biological activity and mechanisms of
activation of macrophages, although more experiments are
necessary to complete the knowledge on the action of Canova.
Mononuclear phagocytes, represented by monocytes, tissue
macrophages, dendritic cells (DCs), microglia and osteoclasts,
maintain tissue homeostasis and provide a first line of defense
against invading pathogens. Thus, the aim of this study is
further characterize the in vitro effects of CA of mononuclear
cultured cells of mice bone marrow.
2. Materials and methods
2.1. Animals
Male Swiss mice from the Rockefeller lineage (8–12 weeks
old) were used. The animals had free access to food and water.
All recommendations of the National Law (No. 6.638,
November, 5, 1979) for scientific management of animals
were observed and the Institutional Animal Care Committee of
the Federal University of Paraná approved all related practices.
Experiments were carried out in the Laboratory of Research in
Neoplasic and Inflammatory Cells, which has a management
program for residues.
2.2. Canova medication
Canova (CA) is a commercial medicine that represents a new
form of immunomodulatory therapy and follows Hahnemann’s
ancient homeopathic techniques. Canova is an aqueous,
colorless and odorless solution produced and sold in Brazilian
authorized drugstores. Mother tinctures are purchased from
authorized agencies indicated by the Brazilian Health Ministry.
These agencies assure the quality (endotoxin free) and physicochemical composition of its products. Starting from the original
mother tincture (in the case of a plant this is an ethanolic
extract) several dinamizations – succussion (shaking) and
dilution in distilled water – are performed. Decimal dilutions
(dH) are prepared. The final commercial product Canova, is
composed of 11 dH Aconitum napellus (Ranunculaceae),
19 dH Thuya occidentalis (Cupresaceae), 18 dH Bryonia alba
(Curcubitaceae), 19 dH Arsenicum album (arsenic trioxide),
18 dH Lachesis muta (Viperidae) and less than 1% ethanol in
distilled water (www.canovadobrasil.com.br). In our experiments, we used the commercial product purchased from
Canova do Brasil. The CA was always vigorously shaken, a
process called succussion, immediately before each treatment.
2.3. M-CSF
The L929 cells were seeded in culture bottles (150 cm2), to a
density of 1 106 cells per bottle, and cultivated in DMEM
(Dulbeccos Modified Eagle’s Medium) rich in glucose (Chem
Sigma Co., St Louis, MO, USA), supplemented with 5% of FSB
(fetal bovine serum) (Chem Sigma Co.). The conditioned
medium was gathered after 7 days, at which period the cells had
arrived at confluence. The above mentioned conditioned
medium containing the produced M-CSF was centrifuged to
eliminate the cells in the conserved suspension and maintained
at 20 8C until the moment of its use. It can be conserved under
these conditions for more than six months. Once defrosted, the
aliquots were preserved at 0 8C to avoid degradation of M-CSF,
due to an excess of cycles of freeze-warming.
2.4. Bone marrow preparation
Mice were anesthetized with Ether and cervical dislocation
was used to the animal’s euthanasia. Femurs were dissected and
cleaned. Epiphyses were removed and the marrow was flushed
with DMEM containing 10% FBS with 1 mg/ml ciprofloxacin
(Sigma Pharma), and purified by passing 1.077 Ficoll-hypaque
(FH) (Chem Sigma Co.). This product is a solution of Ficoll and
sodium diatrizoate adjusted density of 1.077. When blood is
overlaid on this reagent and the solution is centrifuged,
B. Cesar et al. / Micron 39 (2008) 461–470
mononuclear cells concentrate at the plasma-reagent interface
(Boyum, 1968). For the FH method, 10 ml of the flushed bone
marrow was layered onto 3 ml of the FH solution in a sterile
15 ml centrifuge tube. The tube was capped and then centrifuged
in a tabletop centrifuge at 1500 rpm for 40 min at ambient
temperature (21 25 8C). A diffuse band of leukocytes
(mononuclear cells) formed above the erythrocytes and
polymorphonuclear cells, which were seen together in pellet.
This diffuse band layer of cells was aseptically removed with a
pipette and transferred to a sterile 15 ml centrifuge tube. The cells
were washed with PBS due toxicity of the Ficoll to cells.
Mononuclear cells were counted in a Neubauer chamber and
suspended in DMEM with 10% SFB supplemented with 1 mg/ml
ciprofloxacin and 4 mM L-glutamine (Chem Sigma Co.).
2.5. Liquid culture
Cells were adjusted to a concentration of 2.5 105 ml1,
and plated on 24 well culture plates (for adherent and nonadherent cell experiments) with glass cover slips and
maintained at 37 8C under a 5% CO2 atmosphere for 48, 72
and 96 h. All experiments were performed at least three times in
quadruplicate and four treatment groups were examined. The
control did not receive any treatment, because our previous
results demonstrated no statistical differences between the
control group and the ethanolic aqueous solution group. The
cells from the Canova group were treated with 20% Canova.
The M-CSF group received 30% M-CSF and in the Canova plus
M-CSF (CA + M) group the cells were treated with 30% MCSF and 20% CA. The CA and the CA + M groups also
received a 1% CA dose administered daily to the culture, after
24 h of the previous treatment.
2.6. Morphological assay
Cells (2.5 105) were plated into culture plates with cover
slips for morphological analysis (Buchi and Souza, 1992). They
were maintained as described above. After 48, 72 and 96 h, they
were rinsed with phosphate buffer solution (PBS), fixed in
Bouin, stained with Giemsa, dehydrated and mounted with
Entellan1. Adhered cells were observed by light microscopy
using a Nikon Eclipse E200 microscope, which detected
structural characteristics of lymphocytes, resident macrophages, activated macrophages, cell niches and ring-shaped
nuclei cells. For each cover slip, 100 cells were counted from
the total cells plated (2.5 105). Ten cover slips for each
treatment and time were observed and counted. Mean data
obtained in percentage was transformed as described in the item
statistical analysis.
2.7. Transmission electron microscopy
Differences among the groups were mainly found after 96 h
of culture. Thus, after bone marrow mononuclear cultures
maintained at 37 8C, 5% CO2 for 96 h, non-adherent cells were
harvested and centrifuged to form a pellet; the adherent cells
were gently scraped off with a ‘‘rubber policeman’’ and
463
collected by centrifugation. Both adherent and non-adherent
cells were fixed for 60 min in 2% glutaraldehyde, 4%
paraformaldehyde, 5 mM CaCl2, in 0.1 M cacodylate buffer
(pH 7.2); post-fixed in 1% osmium tetroxide (OsO4),
dehydrated in acetone and embedded in Epon. Ultrathin
sections were stained with lead citrate and uranyl acetate and
observed with a Jeol-JEM 1200 EX II transmission electron
microscope at the Electron Microscopy Center of the Federal
University of Paraná. A GATAN CCD camera and GATAN
digital micrograph software were used to obtain the digital
images.
2.8. Scanning electron microscopy
Differences among the groups were mainly found after 96 h
of culture. Thus, cells (2.5 105), cultivated for 96 h, were
fixed with 2.5% glutaraldehyde (0.1 M cacodylate buffer, pH
7.2), washed and post-fixed in 1% OsO4 for 30 min in the dark
at room temperature (Buchi et al., 1993). After washing, the
cells were dehydrated using increasing ethanol concentrations.
Cells were CO2 critical point dehydrated, metalized and
observed using a JEOL JSM-6360 LV SEM scanning electron
microscope in the Electron Microscopy Center at the Federal
University of Paraná.
2.9. Confocal microscopy
Surface markers for CD 11b were characterized for
adherent cells (2.5 105). All antibodies used were from a
mouse lineage panel specific for bone marrow, purchase from
BD Pharmingen. As this protocol requires various washing
steps, we incubated the cells for 96 h to form a complete
monolayer on the plate. This assures that at the end of the
process it still had adherent cells. Immunostaining was
performed according to standard protocols using commercially available antibodies CD11b (Mac-1) for monocyte/
macrophage cells. The cells were maintained on ice, blocked
with 1% PBS/BSA (bovine serum albumin) and incubated
with 1 mg biotinylated antibody in 1% PBS/BSA for 40 min
(Pearce et al., 2004). After washing, they were fixed in 2%
paraformaldehyde for 30 min and the aldehyde radicals
were blocked with 0.1 M glycine in PBS. The cells were
incubated with phycoerythrin (PE) labelled secondary
antibody in PBS for 40 min. The nuclei were stained with
300 nM DAPI (4,6-diamidino-2-phenylindole, dihydroxychloride) (Molecular Probes, Eugene, OR, USA), which
was added 15 min before cell observation. The cells were
washed with PBS, mounted with fluormont-G and the
fluorescence was analyzed with a Radiance 2001 laser
scanning confocal microscope (BIO-RAD) coupled to an
Eclypse E-800 (Nikon).
2.10. Statistical analysis
Percentage data, obtained
from
pffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ffi light microscopy analysis,
were transformed into x þ 0:5 consequently presenting a
normal distribution. These data were submitted to variance
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B. Cesar et al. / Micron 39 (2008) 461–470
analysis (ANOVA) with a factorial diagram to determine the
statistical significance. The Tukey test was performed when the
effects of interaction was significant. The level of significance
was taken at (*p < 0.05, **p < 0.01). Data are representative of
three independent experiments.
3. Results
3.1. Morphological assay
The mononuclear cells were characterized as lymphocytes,
cell niches, ring nuclei cells, and resident and activated
macrophages (Fig. 1). To better express the results, all
morphological assays are summarized in Table 1. The cell
density increased in all groups along the culture time and this
increase is higher with M-CSF, CA + M and Canova
treatments, respectively.
3.2. Lymphocytes
The lymphocytes were characterized by their classical
morphology; rounded small nuclei with condensed chromatin
and a thin cytoplasm. A lower cell proliferation was constant in
all treatment groups and of culture time. In the CA + M and MCSF groups, the cultures had a significantly decrease in the
numbers of lymphocytes when compared with the control at the
end of 96 h treatment. Canova treatment also significantly
decreased the number of lymphocytes, but these did not
drastically disappear with time, as in the CA + M and M-CSF
groups (Table 1; Graph 1).
3.3. Resident macrophages
Resident macrophages were characterized by the classical
fibroblast-like morphology with a central nucleus, little
Fig. 1. Microphotographs of control and treated cultures. After 96 h, mononuclear cells were fixed and stained with Giemsa. (a) Control cells; (b and c) Canova
treated cells. In the control group they have a resting cell (RC), mostly round, with fewer membrane extensions, and a classical fibroblast-like aspect. The Canova and
M-CSF groups showed activated cell (AC), cell niches (CN) and ring-shaped nuclei cells (RN). Original magnification with 40 objective for (a and b) and 100
objective for (c).
B. Cesar et al. / Micron 39 (2008) 461–470
465
Table 1
Morphological analysis of adherent cells
Lymphocytes
Treatment/time
Control
Canova
Canova + M-CSF
M-CSF
48 h
0.7979
0.7679
0.7199
0.7316
a (A)
b (A)
c (A)
c (A)
72 h
0.7151
0.7273
0.7204
0.7107
ab (B)
a (B)
ab (A)
b (B)
96 h
0.7177
0.7152
0.7071
0.7071
a
a
a
a
Resident macrophages
Treatment/time
Control
Canova
Canova + M-CSF
M-CSF
48 h
0.8381
0.8332
0.6343
0.7770
a (A)
a (A)
b (A)
a (A)
72 h
0.8279
0.8520
0.6004
0.7109
a (A)
a (A)
c (AB)
b (A)
96 h
0.6873
0.5307
0.5271
0.4924
a (B)
b (B)
b (B)
b (B)
Active macrophages
Treatment/time
Control
Canova
Canova + M-CSF
M-CSF
48 h
0.0153
0.0518
0.3194
0.1709
c (C)
c (B)
a (B)
b (C)
72 h
0.1555
0.1044
0.3507
0.2674
b (B)
b (B)
a (B)
a (B)
96 h
0.2974
0.4488
0.4491
0.4703
b (A)
a (A)
a (A)
a (A)
Cell niches
Treatment/time
Control
Canova
Canova + M-CSF
M-CSF
48 h
0.7071
0.7071
0.7085
0.7136
b (A)
b (A)
ab (A)
a (B)
72 h
0.7071
0.7085
0.7083
0.7169
b (A)
b (A)
b (A)
a (B)
96 h
0.7071
0.7091
0.7107
0.7283
b (A)
b (A)
b (A)
a (A)
Ring nuclei cells
Treatment/time
Control
Canova
Canova + M-CSF
M-CSF
48 h
0.7137
0.7244
0.7252
0.7123
b (A)
a (A)
a (A)
b (A)
72 h
0.7107
0.7157
0.7265
0.7089
b (A)
a (AB)
a (A)
b (A)
96 h
0.7071
0.7112
0.7201
0.7119
b (A)
ab (B)
a (A)
ab (A)
(B)
(B)
(A)
(B)
Bone marrow cells were differentiated by morphological cell characteristics.
The % mean data were analyzed with ANOVA with a factorial diagram and
Tukey test. Results are expressed as transformed number, representative of three
independent experiments. (CA = Canova; CA + M = Canova plus M-CSF;
M = M-CSF). Different treatments we analyzed over different times. Capital
letters indicate comparison within the line (time of culture) and small letters
within of the column (treatments). The same letters indicate that the there is no
difference between them and different letters indicate a statistical difference
between them.
Graph 1. Number of lymphocytes among adherent cells. Different treatments
were analyzed over the different times. Capital letters indicate comparison
within the line (time of culture) and small letters within the column treatments).
The same letters indicate that the there is no difference between them and
different letters indicate statistical difference between them.
Graph 2. Number of activated macrophages among adherent cells. Different
treatments were analyzed over the different times. Capital letters indicate
comparison within the line (time of culture) and small letters within the
column treatments). The same letters indicate that the there is no difference
between them and different letters indicate statistical difference between
them.
cytoplasm with few extensions, resulting in an elongated and
not so spread appearance. These cells have a small, condensed
and ‘‘kidney’’ shaped nuclei. Although the absence of growth
factors, after 96 h resident macrophages stayed adhered to the
glass coverslip, although their number decreased afterwards
(Table 1; Fig. 1).
3.4. Activated macrophages
Their classical morphology, an increased membrane
ruffling, increased spreading, and a large and euchromatic
nucleus characterized activated macrophages. These morphological characteristics are known to be typical of activated
macrophages. In the control group, they were mostly round,
Graph 3. Number of cell niches among adherent cells. Different treatments
were analyzed over the different times. Capital letters indicate comparison
within the line (time of culture) and small letters within the column treatments).
The same letters indicate that the there is no difference between them and
different letters indicate statistical difference between them.
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B. Cesar et al. / Micron 39 (2008) 461–470
with fewer membrane extensions. The addition of Canova, MCSF and CA + M to the cultures increased cell spreading and
cell density. CA + M gave the highest number of characteristic
activated macrophage morphology, followed by M-CSF,
Canova and control group, which had the lowest. At 96 h
CA, CA + M and M gave the same amount of activated cells
(Table 1; Fig. 1; Graph 2).
3.6. Ring-shaped nuclei cells
3.5. Cell niches
3.7. Transmission electron microscopy (TEM)
Cell niches were associated with the adherent layer in small
foci over the stromal cells. The M-CSF group showed a higher
presence of cell niches and it was increased with culture time.
In the control group, these clusters were not observed and
Canova and CA + M groups evidenced an intermediate number
of these cells (Table 1; Fig. 1; Graph 3).
3.7.1. Non-adherent cells
The majority of cells found in the culture supernatant was
round and had a large nucleus containing a large nucleolus.
There were cells with a resident and activated morphology. The
same had a nucleus with condensed chromatin, sparse
mitochondria and few organelles. The activated cells had a
These cells exhibited constricted ring-shaped nuclei with a
wide cytoplasmic center. The Canova and CA + M groups had
the higher number of these cells. This number decreased with
time for all groups except the Canova plus M-CSF treated group
(Table 1; Fig. 1).
Fig. 2. Transmission electron-micrographs of control and treated cultures. (a) Supernatant control cells with resting morphology, nucleus with abundant
heterochromatin and a moderate amount of cytoplasm. (b–d) Supernatant and adherent Canova treated cells with activated morphology, nucleus with many
euchromatin, and fingerlike projections, a Golgi apparatus is present in the juxtanuclear region, rough surfaced endoplasmic reticulum and elongated mitochondria.
RN, Ring-shaped nucleus; N, nucleus; M, mitochondria; RER, rough endoplasmic reticulum; G, golgi apparatus.
B. Cesar et al. / Micron 39 (2008) 461–470
nucleus with abundant euchromatin and an evident nucleolus.
Aggregates of free ribosomes and rough endoplasmic
reticulum were prominent. Many elongated mitochondria
were dispersed in the cytoplasm. Many finger-like projections
were seen at the cell surface, some of them contacting other
cell. The Golgi apparatus showed great number stack of
membranes. All these morphologic characteristics show an
intense cell metabolism. These results were found in the all
groups (Fig. 2).
3.7.2. Adherent cells
The adherent cells were characterized by an increase in cell
size, with augmented cytoplasm. The characteristics of resident
and activated cells are as cited above, as it can be seen in Fig. 2.
The activated cells contained numerous electron-dense
granules, surrounded by membranes, which were larger than
those observed in non-adherent cells. The Golgi apparatus was
composed of few stacks of membranes in control cells; in
Canova and CA + M groups an enlarged Golgi zone was
apparent. The adhered adjacent cells developed elaborate
cytoplasm projections, similar to those described for nonadherent cells. The Canova plus M-CSF group and Canova
treated group showed more finger-like projections connecting
the cells when compared with the Control group. The space
between two cells in adhesion areas contained irregularly
distributed extra-cellular particles, constituting a septate-like
zone (Fig. 3).
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3.8. Scanning electron microcopy (SEM) and confocal
microscopy
The majority of adhered cells presented macrophages
morphological characteristics. This fact was confirmed by
the SEM results and almost all cells from the Canova treated
group were activated with many projections. The higher cell
density in Canova and CA + M groups, constituting a
monolayer with a greater number of more spread cells. In
the control group, there were fewer and smaller cells (Fig. 4).
These observations were confirmed by CD11b positive
staining, observed by confocal microscopy. Almost all adherent
cells, from all groups, were positively stained with CD11b.
Clusters of CD11b receptors were found on the surface of most
cells (Fig. 5).
4. Discussion
Abud et al. (2006) showed that the treatment did not modify
the expression of the analyzed surface markers, as well as
cytokines production. All microscopy techniques showed that
the monocytic lineage (CD11b+) and the stromal cells (adherent
cells) were activated by treatment. This was expected since
adherent cells were mostly macrophages and CA is a
macrophage activator (Piemonte and Buchi, 2002; Sato
et al., 2005). Each investigator showed that after CA treatment,
the total number of leukocytes increased, suggesting a direct or
Fig. 3. Transmission electronmicrographs of adherent treated cells. (a and b) Show the space between two cells in adhesion areas having extra-cellular particles,
irregularly distributed constituting a septate-like zone. Inserts shown in (c and e), with higher magnification, adhesion areas and in (d) a coated pit.
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B. Cesar et al. / Micron 39 (2008) 461–470
Fig. 4. Scanning electronmicrographs of adherent bone marrow treated cells. (a and b) Give a general view of control cultures with less and smaller cells, with (c and
d) a general view of Canova treated cultures with higher cell density, almost all cells being activated with many projections, constituting a monolayer.
indirect action of the Canova medication on hematopoiesis. A
role for CA in regulating macrophage activation and
inflammation was supported by in vitro and in vivo studies,
in which CA enhanced macrophage phagocytic activity was
showed against non-infective forms (Lopes et al., 2006). An
immune response, increasing the number of leukocytes and
improving the tumoricidal activity of CA-treated mice, was
demonstrated in studies with Sarcoma 180 (Sato et al., 2005). A
specific population of cells was now separated from bone
marrow. Bone marrow-derived mononuclear cells constitute a
Fig. 5. Confocal microscopy of bone marrow cells. The adherent cells were stained with CD11b antibodies (red) and DAPI (blue), fixed and observed using confocal
microscopy. The majority of adherent cells was monocyte/macrophage, positive for CD11b. (a) Shows the control culture and (b) the Canova treated group. Bar
30 mm.
B. Cesar et al. / Micron 39 (2008) 461–470
population of myeloid and lymphoid cells that can proliferate/
differentiate (Bender et al., 2004) in response to specific
stimuli, such as M-CSF, which was used as a positive control.
We found few activated macrophages in the control group
compared to the CA group (Graph 2). The Canova treatment
rapidly differentiated the bone marrow resident macrophages
into activated cells. Committed cells are capable to promptly
adhere, thus the activated cells found in the control group may
be explained by the fact that the differentiated macrophages
were not removed as described by others (Lin et al., 2001). The
pronounced macrophage spreading observed after 96 h in CA
and CA + M groups was a typical morphological characteristic
of activated cells, different from that of the control group,
which had a small number of adherent cells after 4 days. These
results corroborate with these of Dexter et al. (1976), which
showed that after 72 h culture of mice bone marrow, the
majority of adherent cells was phagocytic mononuclear cells
with numerous cytoplasmic extensions, and an overall tendency
to spread. Our electron microscopy analysis showed these
alterations to occur in macrophages, which increased their
cytoplasm volume, the mitochondria number, and the RER, and
enlarged the Golgi apparatus. Occasionally, the contact
processes of two adjacent macrophages alter the plasma
membranes, the cell surfaces and the intercellular regions, as
can be seen in Fig. 3. It can be observed that the cytoplasmic
extensions modified the contact region, which had septate
junctions that possibly function as adhesive elements to
maintain the stroma structure. These results confirm previous
studies in which Canova medicine altered the distribution of
proteins related to adhesion and spreading in treated macrophages (Piemonte and Buchi, 2002).
It was suggested that M-CSF is a requirement for normal
macrophage function (Sweet and Hume, 2003). This raises an
important issue with regard to the many in vitro studies that
have been performed on this cell type. Since, it is normally
present in vivo and because its absence has gross effects on
normal cell functions, M-CSF should be present in the culture
medium during in vitro studies on macrophage function. Thus,
the Canova group, despite not having received the M-CSF, had
these characteristics, namely, a higher number of macrophages
with an active morphology. As macrophages can produce a
large amount of cytokines and growth factors and since these
cells are the targets of Canova, a partial supply of the necessary
molecules may be occurring with this treatment, indicating that
the treated stromal cells supplied the needed factors necessary
for survival.
Initially, the CA + M group presented the lower number of
resident macrophages, probably because of the differentiation
process of activated macrophages. It seems that the medicine
and the M-CSF acted with synergy accelerating the differentiation process, in such a way that the M-CSF and the medicine
alone took more time to give the same effort.
Although, there was a reduction of lymphocytes number in
the control group, it was further reduced by the addition of MCSF or CA + M. The CA group also significantly decreased the
lymphocyte number, but these cells did not drastically
disappear, as seeing in the CA + M and M-CSF groups. At
469
the same time when there was a decrease in the lymphocytes
number, it increased the number of cell niches in the M-CSF
group. In contrast, it was not observed in the Ca + M group
(Graph 3). Considering that stromal cells produce many
cytokines that mediate lymphocyte binding, inducing adhesion
between them and the stromal cells (Miyake et al., 1991), we
can suggest that the set of cytokines produced by M-CSF and by
CA are different. We found an increase in cellular niches in the
M-CSF group, and in the Ca group and Ca + M, the formation
of niches was diminished suggesting that the soluble factors
induced by CA are not promoting this type of adhesion in the
stromal cells. On the other hand, the ring shaped nuclei cells
that can be considered precursor cells (Biermann et al., 1999),
were present and maintained their number in the culture in the
Ca + M group (Table 1) suggesting therefore that the treatment
stimulates the maintenance and proliferation of precursor cells
of the myieloid lineage.
The fundamental role of the marrow microenvironment in
hematopoietic regulation has been demonstrated by several in
vitro and in vivo studies, which showed that marrow stromaL
components can regulate and influence the production of young
and mature hematopoietic cells (Holyoake, 1996; Long et al.,
1990). Some cells with ring-shaped nuclei were described these
cell types in mouse bone marrow as monocytes-myeloid
precursor cells (Biermann et al., 1999). Studies indicated that
these unusually shaped nuclei are more deformable than
spheroid nuclei, facilitating passage through the blood vessel
endothelial lining and a rapid migration through tissue
interstitial spaces (Olins and Olins, 2005).
We now present evidence for an effect of Canova on
expansion of mononuclear murine cell bone marrow, when
cultured with or without M-CSF, emphasising that Canova is
a non-toxic medication. Human therapeutic applications of
living cells generated by cell culture are of great current
interest in a number of clinical settings. One such application
that has just been tested clinically is the ex vivo production of
hematopoietic cells for treatment of patients after high-dose
chemotherapy and/or radiotherapy. Because of cell plasticity,
bone marrow cells have the ability to repopulate injured
livers, promote angiogenesis, affect cardiac and skeletal
muscles, and neurons (Dahlke et al., 2006; Yokoyama et al.,
2006; Orlic et al., 2001; Ferrari et al., 1998; Mezey et al.,
2000). Current bone marrow transplantation practices are
generally effective for restoring the hematopoietic system
after chemotherapy, but suffer from an extremely high cost,
limited donor availability, and high patient morbidity and
mortality. The feasibility of collection, ex vivo cultureexpansion and intravenous infusion of human bone marrowderived progenitor stromal cells, has therefore been proposed
to augment or replace life-saving stem cell transplantation
procedures. Thus far, much effort has been focused on the
development of biological processes, which can generate
potentially therapeutic cells.
Acknowledgements
We thank TECPAR, SETI and CAPES for financial support.
470
B. Cesar et al. / Micron 39 (2008) 461–470
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