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Cell
Antigens
H
and
Antigen
of
EDMOND
B antigens
have
human
fetal
The
cells.’
YUNI5
cytes
except
found
on the
AND
skin
of group
called
undertaken
0,
A and
on
J.
JORGE
has
Cells
adult
than
red
method.’
epidermal
found
and
the
cells
on HeLa
H antigen
can
Group
cells12
on all human
blood.4
Furthermore,
group
A2 individuals,2
“secretors,”5
to study
the
YUNI5
cells
other
agglutination
been
Bombay
0 and
On
Epidermal
human
antigen
erythrocytes
of
normoblasts
of group
individuals
research
was
This
shown
the
of the
tions
been
H group
III.
Human
HE ANTIGENIC
STRUCTURE
of body
be studied
by means
of the Coombs’
mixed
A and
the
Specialization.
Receptors
By
T
Cell
and
erythro-
it has been
in the secre-
cells.#{176}
on epidermal
cells
of
B persons.
MATERIALS
Epidermal
cell suspen.sion:
0, A1, A and B adult
Eighteen
placed
the
2
in
a sterile
specimens
were
jtg./nil.
sterile
and
dish
epidermal
cell
suspensions
the
specimens
was
by
obtained
were
and
by
suspensions
for
were
2 minutes.
and
capillary
to
Blood
then
The
approximately
gr&up
et
Fifty
antisera:
europeus)
nil.
of
saline at 4 C. The
suspension
after
this
0
storage
at 4
C.
by
37
C.
of
isotonic
mm.
removed
with
isotonic
was
The
anti-A
and
was
prepared
material
were
erythrocytes.
Serum
ml.
of
evaporation
this
in
a
material,
dialyzing
made
up
placed
in
was
removed
sheets
of
They
a
fine
the
of
from
epidermis
up
further
by
The
cell
spun
at
1500
The
and
rpm#{176}
cells
drawn
saline
a
The
.
pipette.
pipette.
supernatant
1 ml.
in
off
the
were
with
volume
ad-
ml.
sera
were
laboratory
obtained
commercially.
following
times
of
against
1:64
when
Bombay
bloodf
originally
bag
and
a capillary
and
3
broken
capillary
tubes
with
per
were
bore
in
)
saline
small
titer
from
Before
then
dermis
dialyzed
an anti-H
had
and
the
anti-B
our
4 days.
Medawar.7
The
saline
in
between
than
incubation
siliconized
cells
less
by
saline
dish
policeman.
resuspended
million
used,
( Fungizone
Petri
for
described
were
immediately
a
4 C.
trypsin
saline.
was
at
blood
specimens
antibiotics.
in
isotonic
cent
with
x 75
one
Fifty
After
a rubber
10
button
as
per
saline
final material
of group
concentrated
was
cell
prepared
( 0.5
not
placed
stored
from
The
containing
and
solution
obtained
death.
When
then
saline,
pipetting
in
once
solution
were
sterile
1 ml.
placed
contain
( [Jiex
in
supernatant
washed
saline
with
were
after
saline.
with
forceful
pipette.
anti-H
al.5
placed
isotonic
washed
at
free
specimens
6 hours
They
were
1 hour
it
repeated
The
resuspended
justed
for
of
isotonic
were
scraping
cc.
)
in
a trypsin
rndul)ated
epidermis
thus
with
than
isotonic
units/ml.
out
covered
scraping
with
200
skin
less
12-iS
wrung
trypsinization,
the
washed
squares,
Individual
mixture
containing
penicillin
gauze
Petri
jar
split
cadavers
group
the
2000
then
ml.
tested
with
was
separated
having
and
an
dialyzed
The
method
anti-H
of
of
Boyd
physiologic
a 4 per
titer
overnight
cent
hours
24
of
1:8,
against
From
the Department
of Laboratory
Medicine,
University
of Minnesota,
The
Medicat
School,
Minneapolis,
Minn.
This study
has been supported
in part by a grant from the Minnesota
Heart
Association,
(10(1 lii part by a grant from the Graduate
School
of the University
of Minnesota.
Submitted
Apr. 16, 1.963; accepted
for publication
June
18, 1963.
0
International
fOrtho
$Confirmed
Clinical
Foundation,
by
Centrifuge,
Raritan,
Milwaukee
N.
Blood
Aloe.
J.
Center
and
Ruth
Sanger.
750
Biooo,
Vor..
22,
No.
(3 (DECESEBFR),
19(33
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
I-I ANTIGENS
2000
OF
ml.
of
E1’IDERMAL
physiologic
Erythrocyte
cytes
were
was
washed
was
before
pensions
of
kept
frozen
ml.
A1,
ml.
5
cent
in
and
the
group
B
A volume
).
per
cent
group
“detector
of
vials
0.5
-20
the
A,,
B,
titer
ml.
1:20.
erythro-
packed
cells
made.
C.
and
0
of
Bombay
Before
procedure
0
was
group
were
at
following
per
anti-H
and
suspensions
2 ml.
deglycerolized
labelled
18
group
per
and
0.5
and
was
of
saline,
and
and
experiment,
prepared
volume
samples
El)TA#{176} ( 1 mg.
ml.
thawed
the
were
15
final
ml.
glycerolized
material
mediately
in
in
were
this
The
Five
fresh
3 times
erythrocytes
of
saline.
suspensions:
collected
751
CELLS
use,
of
2 ml.
AABB.9
Im-
red
cell
sus-
with
serum.
)
Bombay
cells.”
METhoDS
Epidermal
anti-B
cells
and
two
experiment
All
supernatant
the
at
room
care
being
and
for
examined
as
and
drop
phase
As
+++
( weak )
+
can
he
absorption
ing
minute,
++++
reaction;
(very
strong),
in
the
of
six
A..
moved
with
to
last
the
the
cells by
ing
this,
with
sera
1 hour.
normal
and
of
of
the
was
that
per
at
of
least
cent;
cent
of
cells,
as well
reactions
cells
thoroughly
containing
then
centrifuged
a
forming
was
of
the
about
showed
in
classified
cells
1-10
the
detector
by
coverglass
erythrocytes
cent
represented
as the
each
tubes
reaction
(moderate),
the
incubated
under
of
per
to
and
resuspended
slide
positive
50
++
1 per
C. and
a
saline
added
were
completely
on
cell
the
were
deposited
isotonic
saline,
The
consisted
strength
cells
with
cells
20-25
at
placed
each
of
was
used
for
these
for
0.1
ml.
of
To
the
anti-A
at
in the
procedure
three
room
the
three
first
was
all
showed
per
cent;
reaction.
table
cells
of
1, the
of the
The
temperature
after
cell buttons
tubes
and
identical
with
epidermal
experiments.
cell
Each
specific
correspond-
suspension
experiment
epithelial
cell suspension.
the saline
supernatant
tubes,
anti-B.
completely
washing,
samples
and
first
or
removed
After
the
2 minutes
incubated
or anti-B
rest
suspension.
reaction
The
containing
were
saline.
(anti-A
The
B
pipette.
antisera
than
of
rpm
ml.
agitation
the
positive
10-50
epidermal
and
were
the
Each
epidermal
agitation
times
cell
deposit
was
agglutination
group
1500
0.15
cell
by
4
of
minutes
serum
the
2 minutes
the
of
incubation,
red
for
2
ml.
anti-A,
controlled.
and
a capillary
three,
cent
0.1
washed
Finally,
per
epidermal
tubes
at
and
meaning
One
different
After
minutes
A
mixed
to the
1 hour.
cells.
less
properly
group
ml.
antisera
treated
Bombay
for
the
suspension
about
experiments:
centrifuged
once
epidermal
rpm
1500
0.15
cells.
were
containing
and
which
this
)
mm.
at
incubated
after
that
x 75
with
0.5
microscopy.
meaning
were
of
B cadavers
concentrated
mixed
2
of
of
(strong),
seen
antigens,
sisted
loss
ml.
with
of antibody
Blocking
group
A1,
were
,
for
and
and
pipette
were
were
1
A
centrifuged
C. ) for
0.1
One
by
( 10
tubes
rpm
avoid
agglutination
follows:
the
to
shaking.
mixed
( 20-25
1500
A,,
( ulex
a capillary
suspensions
rpm
of
cells
with
0,
sera
were
with
shaking
1500
gentle
a set
epidermal
taken
by
group
anti-H
tubes
at
composite
at
ing
the
temperature
centrifugation
the
of
The
by
mixed
of
removed
tubes.
from
kinds
consisted
suspension.
of
obtained
0.15
ml.
of
ulex
was
antisera
were
mixed
(20-25
C.) for
centrifugation
were
anti-H
that
and
further
in the
described
was
of
con-
The
tubes
then re-
added,
and
throughly
with
1 hour.
Followthe
cells
washed
treated
with sensitizlast three
tubes)
for
above.
RESULTS
Twelve
out of 18 preparations
of epidermal
cells
were
found
suitable
study.
Of them,
four belonged
to group
0, four to A1, two to A2 and two
Table
1 summarizes
the results
of the mixed
agglutination
reactions.
shows
that epidermal
cells contain
antigen
receptors
like those
contained
erythrocytes
Disodiun
of
Versenate.
the
same
ABO
blood
group:
Group
0
shows
no
antigen
for
to B.
It
on
re-
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752
YUNIS
Table
1.-Results
of the
Mixed
Erythrocyte.Epidermal
Agglutination
Epidermal
ABO
Bld
of Donor
Sensitization
Detector
( 1:64)
( 1:64 )
Bombay
( 1:20)
Bombay
( 1:20)
anti-A
( 1:256)
Ulex
Ulex
0
anti-B
0
Bombay
+++
negative
Ulex
A2
( 1:20)
( 1:20)
Bombay
Bombay
( 1:256)
( 1:256)
anti-A
anti-B
(1:64)
Ulex ( 1:64)
Ulex ( 1:64 )
Bombay
( 1:20 )
Bombay
( 1:20)
anti-A
( 1:256)
anti-B
( 1:256)
mixed
fA negative
A or
A1
B;
mixed
detector
group
A,
negative
++
negative
++
negative
hand,
dermal
cells
group
H
negative
0
Bombay
A1
B
+
negative
on group
B epidermal
ceptors
on
the
to
tors
demonstrated
were
set
cell
necessary
up
the
negative
Bombay
A1
negative
negative
were
on
the
In order
experiments.
group
on
A;
and
group
A2
group.
0 and
A, and
B the
epidermal
on
2.
on figure
group
cells. On
all human
1.
antigen
cells with
the
skin epi-
(fig. 2). However,
A2 epidermal
cells using
B. The presence of A and
cells
and
of
spatial
B and
B epidermal
H
position
to investigate
this
As shown
in table
A or group
figure
is seen
A, epidermal
blood
question
seen
reaction
demonstrated
A2 epidermal
raised
membrane.
blocking
+
is
antigen
of group
of their ABO
A, and
++
negative
agglutination
reactions
of group
than those of group
cells
negative
Bombay
0
that of group
than
tested regardless
receptors
group
A2 have
receptors
the mixed
agglutination
anti-H sera are stronger
++
negative
Bombay
cell agglutination
reactions
cells is stronger
other
H
and
+++
negative
0
B
cell
erythrocyte-epidermal
agglutination
0
A1
B
erythrocyte-epidermal
mixed
negative
0
Bombay
A1
B
anti-B(1:256)
0A positive
negative
B
0
A1
Ulex(1:64)
Ulex ( 1:64 )
Ulex ( 1:64 )
Bombay
( 1:20)
Bombay
( 1:20)
anti-A
( 1:256)
B
A1
Bombay
Ulex
A1
Results
negativef
negative
( 1:256)
(1:64)
Ulex ( 1:64)
Cells
0
A1
Bombay
Ulex(1:64)
The
Cell
Cells
Group
ceptors
YUNIS
Reaction
Ulex(1:64)
B.
AND
receptors
on
of
re-
these
problem
it was
2, A or B recepcells
after
“block-
ing”
the H receptors
of the cells
by anti-H.
Likewise,
H receptors
were
demonstrated
on the cells after blocking
the A or B receptors by anti-A or
anti-B. From
these results, it is evident that A receptors and H antigen re-
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II ANTIGENS
OF
EPIDERMAL
753
CELLS
0%.
‘;
.t_
,
‘.‘.
‘01
,.!.
.0 .
,.#{149}.
..
.
‘---
-
4
--
Fig.
1.-Negative
sensitized
mixed
with
anti-H
agglutination
and
reaction
exposed
to
of group
Bombay
0 human
erythrocytes.
epidermal
Phase
ceUs
contrast
micro-
scopy.
ceptor
sites
receptor
on
sites
steric
A1 and
on
hindrance
A2
epidermal
B epidermal
cells
of the
two
sites
cells
are
is not
and
B receptors
spatially
and
separate
to
H
antigen
a degree
where
detectable.
DISCUSSION
By means
ble
to
of the
study
the
mixed
A,
B and
been
able
to corroborate
A and
B blood
group
were
able
to demonstrate
cells.
The
antigen
ABO
and
group
cells
with
to
the
present
antigens
The
are
anti-H
presence
of
in
on
donor.
A2 epidermal
sensitization
related
the
reaction
of Coombs,
human
it has
epidermal
cells.
this
paper
prove
beyond
all human
epidermal
mixed
agglutination
not
overlap
We
stronger
than
those
doubt
cells regardless
reactions
with
have
with
group
A,
that
H
of their
group
0
and
B after
addition
of group
0 erythrocytes.
This
may be
a greater
number
of H antigen
receptors
on group
of
on group
A and
B epidermal
cells
bodies
against
A or B receptors
(anti-A
or anti-B)
do not
on group
A or B epidermal
cells,
and also that antibodies
do
possi-
and
O and A2 epidermal
cells.
The blocking
experiments
(anti-H)
been
findings
of Coombs
et al.’ of the presence
of
on human
epidermal
cells.
In addition,
we
presence
of A antigen
on group
A2 epidermal
described
are
of
H
the
antigens
the
experiments
receptors
agglutination
the
cells.
These
results
are in
group
A2 erythrocytes.’#{176}
The finding
of H antigen
A
or
B
receptors
keeping
with
receptors
on
similar
human
on
group
prove
overlap
against
A or
experiments
epidermal
that
anti-
H receptors
H receptors
B epidermal
performed
cells
is interesting
on
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754
YUNIS
Fig.
2.-Specific
dermal
cell
positive
sensitized
mixed
with
anti-H
and
agglutination
group
0
reaction.
erythrocytes
Group
added.
ANI)
YUNIS
A, human
epiPhase
contrast
microscopy.
l)ecause
it
possible
is
be s)ecies-Specific
epiderrnal
cells.
be
will
The
results
of
of H receptors
Hogman12
primary
on
that
H substance
the experiments
using
has
cultivation
is
epidermal
demonstrated
of epidermal
persistance
of
on
might
than
the
the
11 substance
human
help
epidermal
to a better
H substance
on established
cancer
cells
understanding
in cancer
cells
and
loss
and
may
human
cell
lines
Kay
of
anti-
et al.’’
of A or B substances
of the urinary
tract.
human
cell lines and
the
investigated
at
being
of
for
the
cells,
are
cells
specific
established
an inverse
relationship
between
the content
epithelial
cells
and cancerous
epithelial
cells
H substance
present
and
other
presence
extent
elsewhere.”
B during
have
found
in normal
the
the
The
pIll)lished
A and
geils
that
to
the
loss
established
cell
of
isoantigens
culture
lines.
SUMMARY
This
article
demonstrate
cells,
and
locations
describes
the
also
than
H
the
the
the
antigen
presence
H
receptors
application
Le
presente
in A and
B epidermal
communication
mixte
epidermic
human
IN
A2, B, e 0 e etiam
le
de
agglutination
method
to
A,, A2, B and
0 epidermal
receptors in different spatial
cells.
INTERLINGUA
describe
in le demonstration
A,,
mixed
on human
B antigen
SUMMARLO
glutination
of the
receptors
of A and
application
receptores
del
presentia
del
de
methodo
antigenos
de receptores
de
ag-
H in cellulas
de antigeno
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
11 ANTIGENS
Table
OF
EPIDERMAL
2.-Results
Will Block
of
Epidermal
Blood
Group
Blocking
Experiments
to Determine
whether
anti.A
or
Cells
of Donor
Blocking
Serum
A,
Sensitizing
Serum
titiII
Detector
anti-A
tiiti-A
Cell
Results
0
++
A,
+++
Bombay
0
.iiiti-H
negative
A,
++
Bombay
A.,
anti-Il
n(gative
0
anti-A
+++
A,
++
Bombay
anti-A
negative
0
A1
anti-H
______
+++
++
negative
Bombay
B
anti-H
anti-B
anti-B
0
++
B
+++
Bombay
0
anti-H
negative
++
B
++
Bombay
A e B in locos spatial altere que
epidermic
antiB
the Site of Reaction
of anti.H
on Group A and B Epidermal
and Also to Determine
whether
anti.H
Will Block the
Site of Reaction
of anti-A or anti-B or Group A
and B Epidermal
Cells
Cells,
AB()
755
CELLS
illos del receptores
negative
de antigenos
H
in cellulas
A e B.
ACKNOWLEDGMENT
The
authors
grateful
are
to
Dr.
K.
Gerhard
Brand
for
his
valuable
criticism
\V.
M.:
A
acter
related
to the
1:903,
1952.
parts
in
of
this work.
REFERENCES
1.
Coombs,
R.
Rouillard,
group
R.
A.,
L.
M.:
on
antigens
Bedford,
cells demonstrated
nation.
2.
Yunis,
Lancet
J. J., and
and
cell
I)Iood
A and
human
by
mixed
11:461,
1956.
Yunis,
E.:
specialization.
group
D.,
antigens
Cell
and
B blood
epidermal
agglutiantigens
R.,
H.
Morgan,
M.,
W.
Sanger,
T.
J., and
W.
The
Race,
Watkins,
6.
of
blood
J.,
T.
detection
group
ship
norinoblasts.
R.,
Morgan,
blood
of
Blood
22:53,
1963.
:3. Hogman,
C. F.: Blood
group
antigens
A and
B determined
by means
of
mixed
agglutination
on cultured
cells
of human
fetal
kidney,
liver,
spleen,
lung,
heart
and skin.
Vox Sang.
4:
319, 1959.
4. Bhende,
Y. M.,
Deshpande,
C.
K.,
Bhatia,
5.
M.:
I. A study
on
cet
“new”
0
the
char-
Lan-
gene
and
the
0
relation-
substance
the agglutinogens
A and B.
Exper.
Path.
29:159,
1948.
Coombs,
R.
R. A.:
Identification
to
of
analysis
logical
mixed
with
Culture.
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1963 22: 750-756
Cell Antigens and Cell Specialization. III. On the H Antigen Receptors of
Human Epidermal Cells
EDMOND YUNIS and JORGE J. YUNIS
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