From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
B LO 0 D
XLI,
VOL.
NO.
of
Colony-forming
Yernenite
Jews
By Un Mintz
DECREASE
IN
for
reasons,
is
in
people
tendency
to
various
Genetic
neutropenia
without
a special
The
locyte
genetic
present
study
colony
formation
neutropenic
and
Conditioned
of
We
the
the
From
Tel
of
Aviv,
determine
cells
the
Genetics,
Yemenite
Jews,4’5
the
potential
for
Yemenite
efficiency
using
of
our
fibroblasts
in
was
granu-
with
Jews
marrow
vitro
used
cells
assay.6’2
as
a
source
formation.
Weizmann
Beilinson
infections.”2
described.
from
cloning
embryo
colony
in
been
to
granulocytes,
recurrent
and
also
patients
human
D,
origin,3
marrow
tested
for
of
Medicine
Inst
Hospital,
itute
Tel
of
Aviv
Science,
Rehovot,
University
and
Medical
the
School,
Israel.
Submitted
August
Supported
by
Un
have
from
Department
Department
bone
Neutropenia
neutrophil
with
has
undertaken
required
circulating
African
nonneutropenic
medium
inducer6’2
of
associated
infection,
by
Genetic
logic
abnormalities
were
observed
in the
granulocytes
in bone marrow
smears
or in
colonies
formed
in vitro. The results
indicate that the neutropenia
in these patients
was not due to a deficiency
of granulocyte
colony-forming
cells.
It is suggested
that
the neutropenia
is due to a defect
in the
release
mechanism
of mature
granulocytes
from
the bone
marrow
to the peripheral
blood.
usually
of
in the
and Leo Sachs
NUMBER
was
neutropenia.
from
THE
Cells
With
The frequency
of normal
granulocyte
colony-forming
cells
in the bone
marrow
of
Yemenite
Jews with genetic,
absolute
neutropenia
and no special
tendency
to infection
has been
studied
with
conditioned
medium
from
human
embryo
fibroblasts.
Cells from these neutropenic
patients
gave
an average
of about
twice
the number
of
granulocyte
colonies
as bone marrow
cells
from nonneutropenic
patients.
No morpho-
A
1973
JUNE
Granulocyte
Marrow
LT1_
of Hematology
6
Normal
Bone
The Journal
Mintz,
University
30, 1972;
a grant
M.D.:
Medical
Biology,
and
Head,
the
Resident,
School,
October
revised
from
Talisman
Department
Tel
Aviv,
Department
of
20,
1972;
Foundation,
of
Israel.
Genetics,
New
Medicine
Leo
accepted
Sachs,
Weizmann
D,
October
York,
Beilinson
Ph.D.:
Institute
25,
1972.
N.Y.
Hospital,
Meyerhof
of
Tel
Aviv
Professor
Science,
of
Rehovot,
Israel.
©
Blood,
1973
Vol.
by
Grune
41,
No.
& Stratton,
Inc.
6 (June),
1973
745
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
746
MINTZ
MATERIALS
Neutropenic
SACHS
METHODS
Patients
Yemenite
and
AND
AND
Jewish
Hasharon
patients
hospitals
(Tel
attending
the hematologic
Aviv University
Medical
follow-up
clinics
of
School)
were
selected
the following
criteria
: leukopenia
of less than
4000
cells/cu
than
2000
cells/cu
mm, no history
of recurrent
infections
drugs, no lymphadenopathy
or hepatosplenomegaly,
and no
of immune
or collagen
disease.
The bone
marrow
aspirations
part of the hematologic
checkup.
Conditioned
Medium
From
Human
Embryo
the Beilinson
according
to
mm and neutropenia
of less
or exposure
to possibly
toxic
clinical
or laboratory
evidence
were made
as an essential
Fibroblasts
Mass
cultures
from human
fetal lung and extremities
were
made
from
an abortion
at
about
the seventh
to ninth
week of gestation.
The cells, suspended
by trypsinization,
were
seeded
in 50-mm
plastic
Petri
dishes
(Falcon
Co.) in 5 ml of Eagle’s
medium
with
a
fourfold
concentration
of amino
acids and vitamins
and 10%
fetal calf serum.
After
about
10 days
the cells reached
confluency,
and they were
then
subcultured
every
5 or 6 days
by seeding
5 X io
cells/50
mm Petri dish. At the seventh
subculture,
3 X 106 cells/iOO
mm
Petri
dish were
seeded
for the production
of conditioned
medium.
Four to 6 days
after
seeding,
when
the cells reached
confluency,
the medium
was removed
and the monolayers
were
washed
three
times
with
5-mI aliquots
of phosphate-buffered
saline
(pH 7.2).
Ten
milliliters
of medium
with 10%
fetal calf serum
were then added,
and conditioned
medium
was harvested
at 1-6 days
later.
The conditioned
medium
was filtered
through
a 0.45 s
Millipore
filter
and was stored
at -20#{176}C.
Assay
for
Colony-inducing
Activity
The
cells
used
for cloning
were
taken
from
human
bone
marrow
as described;9’2
2 x i#{248}nucleated
cells were seeded
for cloning
per 50 mm plastic
Petri dish (Falcon
Co.).
Metamyelocytes,
stab
forms,
and mature
granulocytes
were
not counted,
as they
were
presumably
no longer
able to multiply.
The cells were cloned
in soft agar (0.33%)
on a
harder
agar base
(0.5%)
in Eagle’s
medium
with a fourfold
concentration
of amino
acid
and vitamins
and 20%
fetal
calf serum
as described,6’-9
and conditioned
medium
was
added
to the lower
agar
layer.
Colonies
were
counted
microscopically
after
10-12
days
of incubation
with an inverted
or binocular
dissecting
microscope.
Only
colonies
containing more
than
50 cells
were counted;
two to six Petri dishes
were
seeded
per point.
The
results are given as the means
of the number
of colonies,
and the number
of colonies
per
Petri
dish
varied
up to about
25%
from
the mean.
Each
calculation
of the number
of
cells per colony
was based
on a count
of at least 30 pooled
colonies.
Colonies
were picked
from the agar with a capillary
tube,
and the cells
were
stained
with
May-GrunwaldGiemsa
and acetoorcein.
No colonies
were obtained
without
conditioned
medium.
RESULTS
Hematologic
eight
was
data
neutropenic
related.
All
Four
of these
and showed
appearance
gave
a
Yemenites
and
cloning
tested
with
the
peripheral
studied
Yemenite
were
no change
in
of leukoagglutinins.
normal
detected,
The
on
patients
the eight
pattern
the
myeloid
efficiency
conditioned
followed
their
in
blood
up
: erythroid
of bone
groups
for
from
3 yr
no
of
of
ratio
marrow
eight
in Table
had
blood
picture,
Examination
both
medium
of the
are shown
neutropenics
1.
an
of
absolute
in our
patients.
embryo
and
the
patients
neutropenia.
hematologic
cyclic
phenomena,
the bone
marrow
was within
cells
from
human
nonneutropenic
None
No
“left
clinic
and
(Table
no
2)
shift”
was
normal
limits.
the two
groups
was
fibroblasts.
Samples
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
GRANULOCYTE
COLONY-FORMING
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From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
748
MINTZ
AND
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From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
GRANULOCYTE
COLONY-FORMING
Fig. 1. Number
of cobnies/2
X 10 normal
human
bone marrow
cells
(patient
No. 1) with
human
embryo
fibroblast
conditioned
me-
fl
2
dium.
Bone
marrow
cells
counted
for seeding
were
nucleated
cells,
excluding
metamyelocytes,
stab forms,
and
mature
749
CELLS
Z
granulocytes.
Conditioned
medium
was
harvested
at daily intervals
(0-1 up to 0-6 days). There
were
no medium
changes
during
these 6 days.
of
conditioned
at
daily
medium
intervals
the
subsequent
16
of
cells
both
from
The
(Table
neutropenic
patients.
groups
The
harvested
6 days,
and
The
(Table
colonies
conditioned
from
the
the results
data
2,
obtained
2)
Fig.
patients
was
average
number
about
the
cultures
marrow
4-day
sample
was,
therefore,
with
that
medium
embryo
fibroblast
with normal
bone
No.
1 (Fig.
1) showed
that
the
of colony
formation.
This
sample
showed
nonneutropenic
in
1 to
experiments.
patients
the
were
from
cells
from
patient
highest
induction
the
25
Percent
bone
average
twice
that
of cells
per
gave
used
marrow
cells
cloning
of
from
efficiency
the
colony
the
for
cells
was
from
similar
2).
in all the
assays
were
granulocytic
with
cells
in all states
from
CM
2.
Fig.
of
Average
cobonies/2
X
cells
nonneutropenic
des) and eight
marrow
(open
circles)
conditioned
days)
human
fibroblasts.
Bone
cells
counted
were
nucleated
stab
10
bone
from
eight
(black
cirneutropenic
patients
with
medium
from
cluding
number
for
forms,
0
(0-4
marrow
seeding
cells,
and
S
‘6
embryo
ex-
metamyelocytes,
granulocytes.
.
mature
0
25
12.5
Percent
25
conditioned
50
medium
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
MINTZ
750
myeloblast
were
in
to
mature
observed
vitro.
The
induced
human
formation
and
granulocytes.
granulocytes
same
the
colonies,
neutrophil
in the
conditioned
of
a small
No
in bone
about
number
of
granulocyte
mixed
smears
or
rat
bone
with
SACHS
abnormalities
morphologic
marrow
medium
70%
AND
colonies,
in
the
colonies
marrow
30%
cells
macrophage
colonies.
DISCUSSION
Among
the
ethnic
form
Jewish
various
families
penia
familial
genetic
dominant
benign
contrast
transmission
to
neutropenia
in
kathexis”
with
series
in
there
than
the
normal
The
cells.
the
a
cells,’#{176}”82#{176}
and
in the
The
blood
this
present
fibroblasts
can
of human
granulocyte
the
cannot
can
be
explain
bone
marrow
rapid
destruction
the
The
average
to
agranuloand
the
the
of
in
as
in
in the
the
release
peripheral
mature
absolute
blood.
granulocytes
neutropenia
of
number
was
same
colony-forming
a defect
control
higher
in
colonies
interest.
neutropenia
to
feedback
of
gfanulocytes
absolute
due
difference
genetic
of granulocyte
was
excluded.
the
in
the
about
the
granulocyte
may
colony-forming
of colony-forming
patients.
results
and
to a deficiency
a more
deficiency
the
“myeloIn
of the
infantile
was
a
as
or
the number
of particular
in mature
that
shift
findings,
efficiency
neutropenia
from
neutropenic
cells,2’
due
of
blood
in
deficient
cloning
showed
of
granulocytes,
cells
twice
with
indicate
the
possibility
peripheral
result
was
the
not
that
granulocytes
the
obtained
marrow
was
these
patients
granulocytes.’7
in young
of
mode
right
nonneutropenics-is
therefore,
suggested
mature
However,
the
no
an
with
in
recessive
mature
mature
by
to infection,’3
Yemenite
of about
dis-
be transmitted
American
family
tendency
of
neutro-
hematologic
autosomal
lack
genetic
in certain
absolute
other
an
and
view
average
and
or
to
to
in the
or
no increase
results
and
patients
It is
cells
bone
an
shift
In
in
the
controls.9’5
present
results,
Yemenite
also
blood.
promyelocytes,
a minor
neutropenia,’4”6
was
to
with
where
cells
left
an
contrast
or
the
origin,3
a relative
hypersegmented
peripheral
where
in
also
of
degenerating
the
in
cytosis
in
forms
no
is
infection
marrow
was
neutropenics
is
of
bone
efficiency-namely
the
rich
other
patients,
cloning
in
the
There
agranulocytosis,
has been
reported
is the case
for
as
neutropenia
suggested.’4
described
This
neutropenia
gene,45
of
or
of African
there
to
chronic
pattern.
Yemenite
origin,4’5
tendency
congenital
has been
Examination
normal
neutropenia
In people
Yemenite
a special
This
of
outstanding.
of
without
orders.
autosomal
forms
is quite
have
embryo
serve
also
shown
kidney,22
as a good
that,
in addition
conditioned
source
of the
to spleen,9
medium
inducer
from
required
peripheral
human
for
the
embryo
formation
colonies.
REFERENCES
Wintrobe
MM:
Clinical
Hematology
(ed 6). Philadelphia,
Lea & Febiger,
1967
2, Editorial:
Chronic
idiopathic
neutropenia.
Lancet
2:1282,
1968
1.
3.
penia
2:1023,
4.
Shaper
AG,
in people
Lewis
P:
of African
Genetic
origin.
neutroLancet
1971
Djaldetti
M,
Joshua
H,
Kalderon
M:
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
GRANULOCYTE
Familial
COLONY-FORMING
CELLS
leukopenia-neutropenia
enite
Jews.
Bull
Res
in
Counc
Yem-
Israel
Cutting
chronic
13.
E9 :24,
1961
nign
61 :876,
5. Feinaro
penia
M,
in
of
of
the
gress
of
Basel,
New-York,
6.
Med,
Tel
Aviv,
1968,
Sachs
L:
in
tissue
cells
clones
of
stance
from
normal
Y,
control
phage
of
and
Acad
M,
L,
In
in
vol
Century-Crofts,
induced
E,
by
a
12.
Mintz
activity
T,
Sachs
differentiation
[New
U,
Sachs
for
form
of
L:
Sci
colonies
in normal
serum
and serum
patients
with
leukemia
and Hodgkin’s
ease. Blood,
in press
idio-
recognized
1968
“Myelokathexis”-a
N
and
granulocytopenia,
Engl
J
of
Med
270:699,
58:1480,
1967
M,
Ichikawa
Paran
USA
20.
Normal
37:6,
pro-
in
marrow
21.
from
dis-
iol
Pike
growth
1971
L:
Feed-
development
of
colonies.
Proc
NatI
Howard
Morley
A,
The
in
colony-forming
vitro
to neutropenia.
II.
Acad
KA,
response
BL, Robinson
colony
76:77,
22.
bone
1969
the
Sachs
I.
Acad
D,
Blood
1971
marrow
1972
granulocytes.
F Jr:
Natl
granulocyte
by
Rickard
and
cell
cells
the
62:81,
L:
of
colonies.
Proc
Y,
of
and
Inhibition
DH,
Sachs
development
granulocyte
inhibition
Sci
Pluznik
of the
macrophages.
macrophage
growth
Differences
agranu-
279:1015,
chronic
case.
by
USA
19.
Appleton-
bone
of
L:
Chronic
newly
WW:
a
Sachs
genetic
JW:
J Med
18. Ichikawa
Y,
Feedback
inhibition
leu-
237:276,
human
new
5,
proliferation
1971
A
Engi
report
Natl
L:
Linman
Zuelzer
from
inducing
Bioll
N
17.
Proc
leukemia
RA,
1970
Levin
infantile
38:74,
AM:
raeu-
283:1072,
meyloid
neutropenia.
Stohlman
myeloid
in
Mauer
congenital
M,
of
Blood
Kyle
pathic
Inhibition
cell
York,
Paran
maturation
macrophage
Regulation
J Med
Y,
locytosis.
dif-
p 217
Landau
of
Nature
inducing
I. New
1970,
Fibach
NatI
hematopoietic
(ed):
beMed
1964
Resnitzky
of
E,
in
induction
back
control
AS
Hematopoiesis,
macro-
1970
of
Gordon
differentiation
Y,
cells
vitro
and
Kauder
Engi
Barak
entity.
In
granulocyte
patients.
67:1542,
L:
L:
Proc
Barak
of
development
11.
Cell
of
hematopoietic
USA
Sachs
and
sub-
Sachs
colonies.
non-leukemic
Sci
clones,
a
Exp
1966
Sachs
in
and
10.
D,
induction
ferentiation
Acad
by
development
56:488,
vitro
kemic
the
USA
Paran
In
Pluznik
granulocyte
Sci
9.
cells
medium.
of
N
vitro
16.
induction
1966
Ichikawa
vitro
of
J Cell
JE: Familial
Ann Intern
myelopoiesis
15.
In
cloning
D,
Defective
1967
culture.
Wriedt
tropenia.
p 172
The
“mast”
conditioned
45:553,
8.
Con-
HO,
Land
neutropenia.
1964
14.
in Pro-
International
Karger
DH,
neutro-
origin,
Physiol
66:319,
1965
Pluznik
DH, Sachs
L: The
7.
tein.
Familial
Yemenite
Ass
“mast”
Comp
WJ:
Ninth
Life
Pluznik
normal
Res
Alkan
Jews
ceedings
P:
751
WA:
growth
in
Human
vitro.
bone
J Cell
Phys-
1970
Brown
of
marrow.
CH,
Carbone
normal
J NatI
and
PP:
leukemic
Cancer
In
vitro
human
Inst
46:989,
From www.bloodjournal.org by guest on July 12, 2017. For personal use only.
1973 41: 745-751
Normal Granulocyte Colony-forming Cells in the Bone Marrow of Yemenite
Jews With Genetic Neutropenia
Uri Mintz and Leo Sachs
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