868 Reports
2. Van Horn, D. L., Edelhauser, H. F., and
Aaberg, T. M.: In vivo effects of air and
sulfur hexafluoride gas on rabbit corneal
endothelium, INVEST. OPHTHALMOL. 11: 1028,
1972.
3. Fineberg, E., Machemer, R., Sullivan, P.: The
use of gas in the management of retinal detachment. II. Experimental evaluation of sulfur
hexafluoride, Am. J. Ophthalmol. In press.
4. Tenney, S. M., Carpenter, F. G., and Rahn, H.:
Gas transfers in a sulfur hexafluoride pneumoperitoneum, J. Appl. Physiol. 6: 201, 1953.
5. Pieper, J., Canfield, R. E., and Rahn, H.:
Absorption of various inert gases from subcutaneous gas pockets in rats, J. Appl. Physiol.
17: 268, 1962.
6. Tucker, R. W., and Tenney, S. M.: Inert gas
exchange in subcutaneous gas pockets in airbreathing animals, Resp. Physiol. 1: 151, 1966.
7. Khodadoust, A. A.: Penetrating keratoplasty in
the rabbit, Am. J. Ophthalmol. 66: 899, 1968.
8. Hamilton, L. H., and Kory, R. C.: Application
of gas chromatography to respiratory gas analysis, J. Appl. Physiol. 15: 829, 1960.
9. Sendroy, J., Jr., and Collison, H. A.: Solubility of SF(1 in biologic fluids, Fed. Proc. 14:
279, 1955.
Growth factors in aqueous humor of normal and inflamed eyes of rabbits. D.
BENEZRA AND U. SACHS.
A factor partially inhibiting the blast transformation phenomenon is demonstrated in aqueous
humor drawn from normal eyes. When the anterior segment of the eye is inflamed, this factor
is overwhelmed by others and an enhancing stimulus is obtained.
The repair mechanism of the iris has been a
puzzle since the early surgical attempts on this
tissue. Iridectomy perfomed under aseptic conditions remains, for years, exactly as it was immediately after the operation.1 Experimental studies
on iris healing showed no tendency to form scar
tissue.'- The role of aqueous humor in modulating
the iris reaction has been postulated and contradictory evidence has been reported.!> ' We report
herein the influence of aqueous humors, from normal and inflamed rabbit eyes, on the transformation of lymphocytes in vitro.
Methods. Animals. Mongrel adult rabbits weighing 2 to 3 kilograms were used. Calf corneas
for xenograft transplantation were obtained immediately after death.
Aqueous humor. Three different pooled primary
rabbit aqueous humors were used: (1) aqueous
humor drawn from normal eyes will be referred
to as "normal h"; (2) aqueous humor drawn
from eyes after diathermy applications on cornea
Investigative Ophthalmology
November 1974
during the most active phase"' will be referred
to as "diathermy h"; and (3) aqueous humor
drawn from eyes during severe graft rejection of
a pocket lamellar xenograft" will be referred to
as "graft h."
The paracentesis was carried out under aseptic
conditions with a 30-gauge needle; 0.15 ml. of
aqueous was drawn from each eye and pooled
separately for the three different primary humors.
All humors were spun down, before use, at 1,000
g for 10 minutes in order to remove any cellular
debris.
Lymphocyte culture. Rabbit spleen cell cultures
were prepared by aseptic removal of the spleen,
trimming of the fat tissue, and washing with
Tc-199 medium (Gibco E-12) containing antibiotics.7 After teasing with forceps and a bundle
of needles, the spleen cell suspension was allowed
to stand for setting of large cell aggregrates and
the homogenous supernatant suspension was centrifuged at 250 g and washed once with the medium. Aliquots of 2 x 10" lymphocytes were transferred into 17 by 100 mm. plastic tubes (Falcon
2001) with the addition of 15 per cent rabbit serum
and Tc-199 medium, or aqueous humor, to the
volume of 2.0 ml. The final volume of all cultures
was kept at 2.0 ml. Therefore, there is a smaller
volume of medium M199 in cultures in which a
bigger volume of aqueous humor is added. However, for a certain volume of aqueous humor,
the same amount of medium is added for the
three different sets of cultures along the whole
study. The cultures were nonspecifically stimulated
by 50 Mg of phytohemagglutinin P (PHA, Difco)
or 0.1 ml. of staphylococcal filtrate (SF) prepared in our laboratory.8 Specific stimulation of
lymphocytes was carried out by addition of 0.1
ml. of 20 per cent sheep red blood cells (SRBC)
to cultures of previously sensitized cells.9 The
cultures were incubated for different intervals
(three days for the experiments with PHA and
five days for cultures stimulated by SF or SRBC)
at 37° C. with 5 per cent CO2 atmosphere. The
degree of response was determined by the extent
of tritiated thymidine uptakes- 9 when 0.5 /iCi
of :!H-thymidine (Swartz Bio-Research Inc.) is
added 24 hours before harvesting. All cultures
were run in triplicates and the results are represented as the mean value for each set.
Results. Table I represents the effect of various
concentrations of aqueous humor on the specific
blast transformation of rabbit lymphocytes previously sensitized to SRBC. In all experiments,
normal h at high concentrations of 0.2 to 0.3
ml. per culture (10 to 15 per cent) inhibited
significantly the thymidine uptake while graft h
enhanced the transforming ability. Diathermy h
did not show marked influence. Similar results
were obtained when the nonspecific blast formation induced by SF or PHA was tested: high
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Volume 13
Nunilicr 11
Reports 869
Table I. Effect of various aqueous humors on the specific blast transformation induced by
SRBC
Experiment
No.
1
2
3
Origin of
aqueous
Normal h
Diathermy h
Graft h
Normal h
Diathermy h
Graft h
Normal h
Diathermy h
Graft h
Control
4,602f
4,205
4,457
7,309
7,514
7,256
3,209
3,201
3,154
0.05 ml."
4,150
4,391
4,875
7,592
7,503
7,844
3,502
3,098
3,365
0.2 ml.
2,788
4,297
6,984
6,241
8,044
9,068
3,005
4,187
4,351
0.1 ml.
3,824
4,144
5,077
7,145
7,656
8,112
3,419
3,508
3,717
0.3 ml.
2,651
4,495
6,598
5,846
7,965
10,463
2,641
4,006
4,803
"Volume of aqueous added per culture.
fNumber representing the mean counts per minute of three cultures.
Table II. Effect of various aqueous humors on blast transformation induced by SF or PHA
Stimulant
SF
PHA
Origin of
Control
aqueous
Normal h
22,435t
Diathermy h 22,197
Graft h
22,308
Normal h
10,329
Diathermy h 9,874
Graft h
8,955
0.05 ml.0
20,814
25,425
26,543
11,025
10,128
9,018
0.1 ml.
20,205
26,721
27,928
9,203
9,531
10,888
0.15 ml.
21,961
24,110
31,226
7,851
10,415
11,308
0.2 ml.
20,973
26,721
33,887
4,311
9,087
13,467
0.25 ml.
16,493
27,613
31,196
3,854
8,974
15,872
0.3 ml.
15,498
28,315
30,812
2,093
10,509
17,045
°Volume of aqueous added per culture.
{Number representing the mean counts per minute of three cultures.
concentrations of normal h inhibited markedly
the thymidine uptake while graft h enhanced the
transforming ability by 30 to 85 per cent compared to control cultures. Most significant results
were obtained when the transformation was induced by PHA (Table II). Diathermy h had a
slight enhancing effect on cultures stimulated
by SF while cultures stimulated by PHA were
not significantly affected by this aqueous.
Although in most experiments, normal h tended
to inhibit the transformation ability and graft
h to enhance it, unexplained differences between
experiments were recorded (Table I).
Discussion. The presence of an "inhibitor" of
lymphocyte proliferation in the normal aqueous
humor has been demonstrated. These results are
in correlation with previous findings showing the
inhibitory effect of aqueous humor on the proliferation of cells in tissue culture.4
This inhibitor is either destroyed or overwhelmed by enhancing factors when inflammatory
processes are induced in the anterior segment.
Aqueous humor drawn from eyes after xenograft
rejection reaction had the most marked enhancing effect on all cultures. Aqueous humor drawn
after induction of vascularization of cornea by
controlled traumatic stimulus had only a slight
effect on the extent of blast transformation.
Whether this difference is due to the basic dissimilarity between a mechanical and an immuno-
logical stimulus or to the magnitude of inflammation (or vascularization) remains to be elucidated.
We are thankful for the expert assistance of
Mrs. R. Edelstein and the technical help of Miss
H. Gunessin.
From the Department of Ophthalmology, Immunology Research Laboratory, Hadassah Hebrew
University Hospital and Medical School, Jerusalem,
Israel. This work was partly supported by the
Swiss Fund for Ophthalmologic Research. Submitted for publication June 17, 1974. Reprint requests: Dr. D. Benezra.
Key words: lymphocyte cultures, blast transformation, phytohemagglutinin, staphylococcal filtrate,
aqueous humor, diathermy, xenograft.
REFERENCES
1. Henderson, T.: The histology of iridectomy,
Ophthalmol. Review 26: 191, 1967.
2. Daniel, R. K.: Healing of the iris in rabbits
following experimental iridectomy, Arch. Ophthalmol. 31: 293, 1944.
3. Snell, A. C., Jr., and Favata, B. Y.: The
development of resistance to reinoculation and
of circulating cytotoxins in response to heterologous ocular tumor transplantation in the
Guinea pig, Cancer Res. 11: 335, 1951.
4. Kornblueth, W., and Tenenbaum, E.: The
inhibitory effect of aqueous humor on the
growth of cells in tissue cultures, Am. J.
Ophthalmol. 42: 70, 1956.
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870 Reports
5. Campbell, F. W., and Michaelson, I. C :
Blood-vessel formation in the cornea, Br. J.
Ophthalmol. 33: 248, 1949.
6. Benezra, D., and Sachs, U.: Growth and
transplantation of organ cultured corneas, in
preparation.
7. Benezra, D., Gery, I., and Davies, A. M.:
Transformation of rabbit lymphocytes by specific antigens, Proc. Soc. Exp. Biol. Med. 125:
1305, 1967.
8. Benezra, D., and Hochman, A.: In vitro
activation of lymphocytes from patients with
malignant diseases. I. Kinetics and differences
in magnitude of response, Isr. J. Med. Sci.
7: 553, 1971.
9. Benezra, D., Gery, I., and Davies, A. M.:
Specificity of blast transformation. II. Studies
with erythrocyte antigens, Int. Arch. Allergy
39: 488, 1970.
Cytological changes in the conjunctiva in
the megaloblastic anemias. J. D. BRODRICK, I. M. STRACHAN, AND T. SPEED.
An attempt has been made to demonstrate increased nuclear size of the conjunctival cells in
the viegaloblastic anemias. Conjunctival scrapings
were taken, wet fixation employed, and nuclear
diameters measured under the microscope. The
resulting data was subjected to statistical analysis.
Two approximate tests of the null hypothesis that
the affected group has the same nuclear size as
the control group have been performed. Both tests
reject this hypothesis at the 5 to 10 per cent level.
Cytologic changes in the mucous membrane of
the mouth and vagina are well recognized in patients suffering from megaloblastic anemia. The
nature of these changes is similar irrespective of
whether folic acid or vitamin Bi2 deficiency is the
underlying cause and it would be somewhat
surprising if similar cytologic changes did not
occur in the conjunctiva. As far as we are able to
ascertain, the only attempt made to find these
expected changes led to the conclusion that there
was no alteration of the conjunctival cytology in
megaloblastic anemia.1
This communication deals with the results of
our investigations into the conjunctiva] cytology
of proved cases of megaloblastic anemia.
Material and methods. Patients were obtained
from the Department of Hematology, having been
referred there for sternal marrow examination on
the basis of symptoms, clinical examination, and
demonstration of the characteristic changes in the
peripheral blood film. Before treatment was commenced, conjunctival scrapings were taken by the
same observer following instillation of 0.5 per
cent Ophthaine. The specimens were spread onto
Investigative Ophthalmology
November 1974
Table I. Table showing high and low classification
in control and affected specimens
High
Control
Affected
Total
4
9
13
Low
Total
10
11
21
the center of a slide, immersed immediately in
50:50 absolute alcohol and ether, and fixed for
24 hours. They were then air dried, taken down
through 70 per cent and 50 per cent alcohol to
distilled water, treated with hot (60° C.) N HC1
for 10 minutes, washed in distilled water, and
stained with 1 per cent Cresyl fast violet acetate.
The stained sections were then taken through 95
per cent absolute alcohol and xylol and mounted
in Canada Balsam. Scrapings were taken from a
series of clinically normal control specimens and
the whole series was randomized so that the subsequent microscopic observations were carried out
"blind."
When the affected patients were ultimately
identified, the hematology record was consulted
for details of the blood picture, sternal marrow,
hemoglobin, serum vitamin Bi2, and serum folate.
The smears were viewed monocularly with a
microscope using the xlOO oil immersion objective and an eyepiece graticule. Certain criteria
were adopted regarding the nuclei to be measured.
In general, the largest, most regular nuclei in the
field were selected and those with overlapping or
contiguous borders and shrunken, irregular, or
abnormal nuclei due to artifacts of preparation
were excluded.
One-hundred nuclei were selected on each slide
and two measurements were made on each nucleus
at 90° to each other. The measurements were made
in arbitrary scale division to the nearest half-unit,
so that the effect of magnification and eyepiece
distance can be discounted.
The results obtained were submitted for statistical analysis.
Results. Data. The data available is in the form
of measurement of areas (in arbitrary units) of
nuclear cross-sections.
A Mann-Whitney Test was performed with the
null hypothesis being that the control and affected
groups were the same (as far as the nuclear area
was concerned) and (one-sided) alternative that
the affected group had larger nuclear areas than
the control group. The test statistic was significant
at the 2.5 per cent level, indicating an upward
shift in the area of nuclei of affected individuals
in comparison with the control group (Fig. 1).
Another more crude analysis was performed
as follows: The averages were classified as high
(H) or low (L) according to whether they were
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