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Investigative Ophthalmology & Visual Science, Vol. 32, No. 7, June 1991
Copyright © Association for Research in Vision and Ophthalmology
Class II Histocompatibility Antigen Expression by Cellular
Components of Vitreous and Subretinal Fluid
in Proliferative Vitreoretinopathy
C. Daudouin,* F. Brignole,t J. Doyle,j- D. Fredj-Reygrobeller,^ P. Lapolus4 ond P. Gasroud*
Proliferative vitreoretinopathy (PVR) is the major cause of failure in retinal detachment surgery. It is
characterized by the formation of membranes extending along both surfaces of the detached retina and
within the vitreous, but the nature of the growing cells has not yet been determined. Using cytologic and
immunocytologic procedures with 13 different monoclonal antibodies directed against Class II histocompatibility antigens and various markers of epithelial and immunocompetent cells, 30 specimens
were studied of vitreous or subretinal fluid removed from patients with PVR. Five main types of cells
could be identified: heavily pigmented cells, poorly pigmented ones, large totally unpigmented macrophage-resembling ones, smaller unpigmented cells, and lymphocytes. Analysis of intravitreal pigment
granules, using autofluorescence by epiillumination and cytologic procedures, showed two different
populations of pigmented cells: one with autofluorescent lipofuscin granules and the other with exclusively melanin pigment. Immunostaining procedures confirmed the epithelial nonmacrophage lineage
of the intravitreal and subretinal cells because most of these cells were positive for cytokeratin but
negative for macrophage markers. In addition, 40-100% of these epithelial-derived cells strongly
expressed Class II histocompatibility antigens HLA-DR and -DQ. Lymphocytes were found in 13
specimens; B-cells were seen, but no T-lymphocytes could be identified. These results confirm the
involvement of retinal pigment epithelial cells and the strong morphologic changes they undergo during
the course of PVR. Moreover, the expression of Class II histocompatibility antigens by the growing
cells may be related to inflammatory phenomena, but their eventual role in the development and the
extension of periretinal proliferation has not been determined. Invest Ophthalmol Vis Sci 32:20652072,1991
Proliferative vitreoretinopathy (PVR) is a common
complication of rhegmatogenous retinal detachment
characterized by migration and proliferation of
various types of cells on both surfaces of the detached
retina and within the vitreous body. Continuous synthesis of collagen and contractile elements by the
growing cells results in the formation of epiretinal or
subretinal membranes, causing extensive retinal retraction and failure in retinal reattachment surgery.
Several histologic studies1"8 were conducted on proliferative tissues in PVR and showed macrophages, fibroblasts, pigment epithelial cells, and glial cells as
components of the epiretinal membranes. Ultrastructural characteristics of fibroglial membranes are thus
well documented, but only a few studies examined the
cellular elements spreading throughout the vitreous
From the *Department of Ophthalmology, Saint-Roch Hospital,
and the Laboratories of fHematology and ^Pharmacology, Nice,
France.
Submitted for publication: October 2, 1990; accepted January
26, 1991.
Reprint requests: Dr. C. Baudouin, Department of Ophthalmology, 5 rue Pierre Devoluy, 06000, Nice, France.
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body and subretinal fluid during PVR. Moreover, in
previous immunohistologic studies, we found immunoglobulin (Ig) and complement deposits and abnormal Class II histocompatibility antigen expression in
the pars plana9 and epiretinal membranes10 at the pigment epithelial cell level, indicating eventual involvement of the immune system during the course of
PVR. We wished to characterize the cellular components of the different fluids surrounding the detached
retina and proliferative tissues better and to investigate Class II histocompatibility antigen expression by
intravitreal and subretinal cells in PVR.
Materials and Methods
This study was done on 30 specimens, 16 samples
of vitreous and 14 of subretinal fluid, obtained surgically in 23 patients (age range, 16-78 yr) with retinal
detachment and PVR as described by the Retina Society Terminology Committee.11 Retinal detachments
were 1 week to 2 months in duration. In eight cases,
unsuccessful retinal detachment surgery, including
cryotherapy and scleral buckling, was done
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / June 1991
previously. Six patients underwent cataract surgery 4
months to 5 years before retinal detachment. In the
other nine patients, no ocular surgery was done. In all
cases, pigmented dots and clumps could be seen biomicroscopically in the vitreous, and the detached retina was retracted partially or totally by fibroglial
membranes spreading along the inner and/or outer
surfaces of the retina.
Vitreous was removed by posterior vitrectomy
through the pars plana from 16 patients. The vitreous
gel was removed completely except for the peripheral
vitreous adjacent to the vitreous base and epiretinal
membranes excised separately with vitreous forceps.
Thus the whole vitreous was collected for cytologic
examination. Subretinal fluid was sampled from 14
patients, either by intraocular aspiration through the
retinal hole after posterior vitrectomy (6 cases) or
after a cut down through the sclera in the other 8
patients who had a recent retinal detachment with
beginning periretinal retraction but who did not undergo vitrectomy. Subretinal fluids were aspirated
from the most highly elevated retinal area, after heat
coagulation of the sclera and choroid to prevent
bleeding. Contaminated samples were discarded. In
seven patients, both vitreous and subretinal fluid
could be collected.
First all these specimens were centrifuged at 1600
rpm for 10 min. Cytocentrifuge smears were then prepared (1200 rpm, 10 min) and fixed for 10 min in
methanol at 4°C. Cytologic examination was conducted using May Griinwald Giemsa, periodic acidSchiff, and alpha-naphthyl acetate esterase staining
procedures. Toluidine blue was used to identify the
nature of intracellular pigment granules; melanin
granules appear brownish yellow and lipofuscin
greenish-blue when stained with this dye.12 Specimens
also were examined immunocytologically with 13
different monoclonal or polyclonal antibodies, directed against histocompatibility antigens HLA-DR
(OKDR and 12; Ortho and Coulter, respectively),
HLA-DQ (IOT2d; Immunotech), epithelial markers,
epithelial membrane antigen (EMA; Dakopatts), 56kilodalton cytokeratin (clone KL1; Immunotech),
glial fibrillary acidic protein (GFAP; Dakopatts), fibronectin (Immunotech), macrophage (OKM5;
Ortho, and M02; Coulter), and lymphocyte markers
(T3, T4, T8, and B4; Coulter). Antilymphocyte
markers were tested only when standard cytologic examination showed the presence of lymphocytes or
lymphocyte-resembling cells.
Immunofluorescence and immunoperoxidase procedures were done with previously described methods.9 Briefly, the primary antibodies in a 1:50 dilution
were layered on slides for 1 hr, before washing in
phosphate-buffered saline at pH 7.4, and incubation
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Vol. 32
for 30 min with the secondary antibody, fluorescein
isothiocyanate-labeled anti-rabbit (for anti-GFAP antiserum) or anti-mouse IgG antiserum (Dakopatts).
For immunoperoxidase procedures, biotin-conjugated anti-mouse IgG antiserum was used as secondary antibody. Then avidin-biotin peroxidase complexes (Dakopatts) were reacted for 30 min before developing in aminoethyl carbazole hydrogen peroxide
solution. The percentages of positive cells were obtained by counting at least 100 cells in each specimen.
Damaged cells or cells without nuclei were not
counted.
Negative control reactions were prepared by omission of the primary antibody or substitution by serum
from the same species. In each patient, positive controls for markers of immunocompetent cells were obtained by the same technique on peripheral blood
lymphocytes. To assess the nature of pigment granules in ciliary and retinal pigment epithelial cells, 5ixm thick whole-eye frozen sections were prepared
from six eyes obtained by autopsy in six subjects without any known ocular disease. Autofluorescence analysis and toluidine blue staining were done on these
sections.
Results
The results of cytologic examination of vitreous
specimens was independent of the length or importance of the retinal detachment and proliferative processes. There was no difference between cellular patterns from patients who previously underwent cataract or retinal detachment surgery and those without
any previous surgical intervention.
Five major cellular components were found as follows.
1. Rounded typical pigment epithelial cells (Fig. 1),
with a large heavily pigmented cytoplasm masking
the nucleus (5-30% of total vitreous cells), were
seen in 13 specimens. Under epiillumination, two
different kinds of pigmented cells were found: 3070% contained yellow autofluorescent pigment
granules, identified as lipofuscin by toluidine blue
staining. In contrast, the other heavily pigmented
cells did not contain lipofuscin but only nonautofluorescent pigment, identified as melanin by toluidine blue.
2. Large partially pigmented cells (30-50%), with
rare granules of melanin and/or lipofuscin and apparently empty vacuoles, were found (Fig. 1). Nuclear anomalies often were seen, such as notches,
duplication, and even triplication.
3. Large rounded totally unpigmented cells (Fig. 1),
with similar nuclear abnormalities and intracytoplasmic vacuoles, were found (10-30%). Although
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VITREOUS AND 5UBRETINAL FLUID IN PVR / Doudouin er ol
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Fig. 1. Cytologic staining
of intravitreal cells collected
in a case of proliferative vitreoretinopathy, showing a
heavily pigmented cell (arrowhead) and four macrophage-resembling cells containing various amounts of
pigmented dots (arrows).
These different kinds of cells
were identified by immunocytologic procedures as epithelial-derived cells (May
Griinwald Giemsa, original
magnification X55O).
resembling macrophages, these cells were negative
to specific macrophage staining with alphanaphthyl acetate esterase.
4. Smaller unpigmented cells (10-50%), with a condensed rounded or lightly elongated cytoplasm
surrounding a large nucleus, were seen (Fig. 2). In
some specimens, occasional spindle-shaped cells,
with morphologic features of fibroblasts, were
found.
5. Rare well-characterized lymphocytes were seen in
six vitreous specimens. The absence of red blood
cells confirmed the lack of hematogenic contamination of the specimens.
Identification of these different kinds of cells was
achieved with immunocytologic procedures (Table
1). In 40-80% of intravitreal cells, either heavily pigmented or totally unpigmented ones, large or smaller,
were positively stained for EMA and cytokeratin (Fig.
2), confirming their epithelial origin. There was no
reaction to antimacrophage monoclonal antibodies.
Cells with different morphologies had a similar pattern of reactivity and comparable percentages of positivity to cytokeratin. Fibronectin was found in four
specimens on a minority of intravitreal cells (5-30%),
mainly small unpigmented ones. In contrast, GFAP
was negative. The fifth type of cells, lymphocytes, appeared to be mostly B-cells (B4 positive), with T3, T4,
and T8 monoclonal antibodies negative in all specimens.
Moreover, immunofluorescence and immunoperoxidase procedures on vitreous fluid showed numerous Class II histocompatibility antigen-expressing
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cells (Figs. 3, 4). A strong reactivity was found in 40100% of intravitreal cells. Both pigmented and nonpigmented, large and smaller cells widely expressed
HLA-DR and -DQ determinants (Table 1). There was
no difference in these percentages or the pattern reac-
Fig. 2. Immunofluorescence reactivity of anti-cytokeratin monoclonal antibodies in a small, partially elongated intravitreal cell.
Nucleus is counterstained with propidium iodide (X800).
2068.
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / June 1991
Table 1. Immunostaining reactivity of cellular components of vitreous gel (V) and subretinal fluid (SRF) in
proliferative vitreoretinopathy
Mean percentages of positive
cells (SD)
No. of positive
specimens
Range (%)
Antibodies
V
SRF
V
SRF
V
SRF
Cytokeratin
EMA*
anti-HLA DR.
anti-HLA DQ
GFAP
Fibronectin
0KM5 and M02
T-cell markersf
B-cell markersf
74(18.3)
58(15.7)
67 (28.9)
59 (23.6)
68(14.1)
66(17.3)
82 (20.6)
76(18.2)
50-80
40-75
40-100
40-90
—
0-30
—
—
0-5
50-90
40-80
50-100
40-95
—.,
16
13
16
16
0
14
11
14
14
0
0
—
6(5.3)
—
2(1.5)
—
3 (2.7)
•
•
,
0-10
4
#
0
6
0
0
7
* Not performed in six specimens.
f Only performed when lymphocytes could be seen on cytologic examination.
tivity between the different kinds of cells and between
melanin- or lipofuscin-containing cells. There was no
relationship between the expression of Class II antigens and the importance of pigmentation. Both intravitreal cells from patients with and without previous
ocular surgery similarly expressed these antigens.
With subretinal fluid analysis, similar results were
found. Cytologic examination also showed heavily
pigmented, lightly pigmented, and the two different
types, small and large, of unpigmented cells. In seven
specimens, a few lymphocytes could also be seen.
However, in contrast with vitreous samples, autofluorescent lipofuscin granules could be observed
under epiillumination in all pigment-containing cells.
Subretinal cells (Fig. 5) also strongly expressed HLA-
DR and -DQ determinants on their membranes.
There was no difference in the percentages of positivity between the four kinds of cells, pigmented or unpigmented, large or small. Both pigmented and unpigmented cells widely reacted with EMA and cytokeratin. No cell was positive for antimacrophage
antibodies, and macrophage-specific staining was negative on all cells. A few B-lymphocytes were found in
seven specimens, but the other tested antibodies (fibronectin, GFAP, T3, T4, and T8) were negative.
Pigment examination on normal eye sections
showed, either by autofluorescence or toluidine blue
staining, a sharp delineation between retinal pigment
epithelium (continuously and strongly autofluorescent [Fig. 6A] or greenish-blue when stained by tolu-
Fig. 3. Immunofluorescence reactivity of two intravitreal cells with anti-HLA
DR monoclonal antibodies:
the bright white dots are
autofluorescent lipofuscin
granules. Note nuclear duplication in these two cells
(propidium iodide counterstain, original magnification
X800).
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VITREOUS AND SUDRETINAL FLUID IN PVR / Baudouin er al
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Fig. 4. HLA DR antigens
in two partially pigmented intravitreal cells. In contrast to
Figure 3, the intracytoplasmic pigment is nonautofluorescent melanin (arrows).
Note also the duplication of
the nuclei (immunofluorescence, propidium iodide
counterstain, original magnification X55O).
idine blue) and ciliary or iris pigment epithelial cells
totally devoid of lipofuscin. The latter were not autofluorescent (Fig. 6B) and were stained brownish by
toluidine blue. Only a few granules of lipofuscin could
be seen in pigment epithelial cells of pars plana adjacent to the peripheral retina.
Discussion
Several reports show that epiretinal membranes result from proliferation of various types of cells, in-
Fig. 5. Various types of
subretinal cells strongly expressing HLA DQ antigens:
large lipofuscin-containing
(arrow) and smaller unpigmented cells can be seen
which similarly express class
II antigens (immunofluorescence, propidium iodide
counterstain, original magnification X550).
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cluding pigment epithelial ones (probably migrating
through retinal tears) and glial cells growing through
disruptions of the inner retinal membrane.1"3 Epiretinal membranes also were found to be composed of
fibroblast-like and macrophage-Iike cells, but the origin of these cells is controversial, even though immunohistologic studies suggest that they could originate from metaplastic retinal pigment epithelial
cells.48 Animal models of PVR show that retinal pigment epithelial cells rapidly change their morphology
when implanted into the vitreous body or when they
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INVE5TIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / June 1991
Fig. 6. Normal eye sections showing the bright continuous autofluorescence of retinal pigment epithelium (A), partially masking
Bruch's membrane (arrows) and the melanin-containing pigment
epithelium of pars plana (B), underlined by the autofluorescent
Bruch's membrane (arrows). Ch = choroid, PE = pigment epithelium, R = retina, RPE = retinal pigment epithelium, S = sclera
(original magnification X300).
proliferate on the retinal surface,13 lose their pigment
granules, and no longer resemble typical pigment epithelial cells. This may result in confusion in identification of the cell types involved in epiretinal membranes.
Fibroglial membrane formation along the retina,
however, appears as the result of complex proliferative phenomena which occur very early in the course
of PVR and involve other structures besides the retina. The vitreous gel of patients with PVR usually
contains brown pigmented dots and clumps, even
when epiretinal membranes cannot be seen, which
indicates strong extraretinal cellular processes before
epiretinal membrane formation. Moreover, because
retinal pigment epithelial cells are supposed to proliferate actively and participate in sub- and epiretinal
membrane development, studies on the subretinal
fluid that accumulates in the space between the neural
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Vol. 32
retina and pigment epithelium may be important in
understanding the pathophysiologic events leading to
the formation of this periretinal tissue. Biochemical
analysis of subretinal fluid has been well documented, 1415 but cytologic studies on cellular components of subretinal fluid and vitreous gel are few.
An ultrastructural work of Feeney and associates12
showed two major cell types in subretinal fluid: typical pigment epithelial cells and macrophage-like pigment-containing ones. In addition to classic cytologic
procedures, we used immunocytologic methods to
identify cellular components present in the vitreous
and subretinal fluid of patients with PVR. We therefore found different cellular patterns such as typical
pigment epithelial cells and lightly pigmented or totally unpigmented cells, including macrophage-resembling ones. Immunostaining for epithelial and
macrophage markers showed evidence that these cells
were of epithelial origin and not macrophage derived.
They widely expressed epithelial markers, mainly cytokeratin, a characteristic determinant of retinal pigment epithelial cells.4 In addition, some of the observed cells, the small unpigmented ones, although
morphologically different from typical pigment epithelial cells, were also identified as epithelial-derived
cells. In contrast was the absence of GFAP-positive
glial cells, even though they have been found in epiretinal membranes.Ii5 We suggest that glial cells probably spread along the detached retina but do not reach
the vitreous cavity or subretinal space. A minority of
cells in each different morphologic pattern, however,
did not react with our markers and could not be precisely identified. In all cases, these cells were negative
to all the tested antibodies, and the absence of any
kind of reactivity could be related to cellular alterations during specimen collection.
These patterns could thus be the result of strong
ultrastructural changes occurring in proliferating pigment epithelial cells. They are consistent with those of
animal models of PVR, in which cultured retinal pigment epithelial cells are injected into the vitreous
body. 13 Similarly, retinal pigment epithelial cells
overlaid with vitreous, 16 collagen, or fibrin17 have
been shown to lose their normal epithelial characteristics and, gaining fibroblast-like features, migrate into
the surrounding extracellular matrix. Vitreous stimulation of pigment epithelial cells through retinal tears
may thus play a role in the development of PVR.
Moreover, characterization of lipofuscin and melanin content, either by autofluorescence evaluation or
histochemical staining, showed that about 50% of the
intravitreal strongly pigmented cells contained only
melanin and not lipofuscin granules. The large
amounts of pigment granules in these cells could not
result from phagocytosis of intravitreal debris. Lipofuscin pigments result from a deficient degradation of
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VITREOUS AND SUBRETINAL FLUID IN PVR / Boudouin er ol
phagocytized shed outer segments in intracytoplasmic phagolysosomes.18 They begin to accumulate in
retinal pigment epithelial cells as early as the 16th
postnatal month.19 The presence of intravitreal cells
containing large amounts of melanin pigment without any lipofuscin granules suggests that retinal pigment epithelial cells are not the only ones involved in
proliferative phenomena during PVR and that ciliary
process or iris pigment epithelial cells, which are devoid of lipofuscin pigment as assessed on normal eye
sections, could also migrate and proliferate in the vitreous body. This is consistent with our previous immunohistologic study done on pars plana specimens
from patients with PVR. We showed the involvement
of ciliary pigment epithelial cells, on which Ig and
complement deposits and abnormal expression of
Class II major histocompatibility complex antigens
could be seen.9 These antigens also were found at the
surface of numerous pigmented cells in epiretinal
membranes in PVR.710 In the current study, most
intravitreal and subretinal cells, whether heavily or
lightly pigmented or unpigmented, strongly expressed
HLA-DR and -DQ antigens.
Class II antigens are membrane glycoproteins directly involved in the presentation of an antigen to
helper T-lymphocytes. They are normally restricted
to immunocompetent cells, such as macrophages, Blymphocytes, or activated T-cells, but aberrant expression of Class II antigens by resident epithelial cells
has been documented in various autoimmune diseases.20'21 The hypothesis that expression of Class II
antigens by target cells might enable them to present
autoantigens to helper T-lymphocytes which could
initiate an autoimmune reaction was proposed by
Bottazzo and associates.22 In the eye, HLA-DR was
expressed by retinal pigment epithelial cells in retinitis pigmentosa,23 uveitis,24 and PVR.7>9>1° Moreover,
experimental studies stressed the prominent role of
Class II expression in the initiation and perpetuation
of intraocular immune responses. Class II antigen expression by retinal pigment epithelial cells thus is one
of the earliest events in the development of experimental autoimmune uveoretinitis and precedes induction
of local inflammation.25 In addition, immunotherapy
by monoclonal antibodies to Class II determinants
has been shown to reduce the course of various experimentally induced autoimmune diseases. Anti-Class II
antigen treatment was efficient in inhibiting or suppressing ocular inflammation in experimental autoimmune uveoretinitis,26'27 even when the antibodies were administered 5-7 days after antigen injection.
Our studies indicate that pigment epithelial cells
during the course of PVR express HLA-DR and -DQ
determinants in the ciliary body,9 epiretinal membranes,10 vitreous gel, and subretinal fluid. Normal
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ciliary and retinal pigment epithelial cells do not.9
However Class II antigen expression by retinal pigment epithelial cells in PVR is not the consequence of
inflammation induced by previous surgical interventions. Nine of our 23 patients had not undergone any
ocular surgery before specimen collection.
Even if Class II antigen induction on proliferative
cells is only an epiphenomenon resulting from a nonspecific inflammatory reaction, we can hypothesize
that the expression of HLA-DR and -DQ antigens by
retinal pigment epithelial cells or pigment epithelialderived ones during the development of PVR may
allow them to function as effective antigen-presenting
cells and induce an immune reaction. Some reports
cite the possibility of autoimmune phenomena in retinal detachment. Using assays of cellular immunity,
patients with long-standing retinal detachment28-29 or
epiretinal proliferation30 showed autoimmune responses to retinal or uveal antigens. Serum antibodies
to retina also were detected in 60% of patients with
retinal detachment.29 More recently, experimental
studies in animal models of retinal detachment
showed cellular immunity to three purified retinal antigens: interphotoreceptor retinoid-binding protein,
S-antigen, and opsin.31 Our findings of lymphocytes
in vitreous and subretinal fluid, together with
previous reports on the presence of lymphoid cells in
experimentally induced epiretinal membranes,32 provide additional indications on the intervention of the
immune system in PVR.
Mechanisms of such immune involvement are speculative, but Class II antigen expression by the proliferating retinal pigment epithelial cells and the eventual
subsequent immune response could be dependent on
the liberation of some of the growth factors which lead
to periretinal cell proliferation after retinal detachment. Various growth factors, known to stimulate proliferation of retinal pigment epithelial cells,33'34 such
as platelet-derived growth factor, epidermal growth
factor, and fibroblast growth factor, enhance the production of gamma interferon,35 a potent inducer of
HLA-DR expression by retinal pigment epithelial
cells both in vitro3637 and in animal models.38 Some
of these growth promoting factors, possibly liberated
from their storage sites after retinal detachment, may
induce the target cells to proliferate and, at the same
time, to express Class II antigens, resulting in additional inflammatory phenomena.
However it remains to be determined whether or
not the Class II antigen-mediated immune phenomena play a role in the development of PVR and the
extension of epiretinal membranes after retinal detachment. Further studies are needed to identify the
respective roles of growth factors, class II antigen expression, and immune mediators in proliferative diseases.
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / June 1991
Key words: Class II antigens, retinal detachment, proliferative vitreoretinopathy, vitreous, retinal pigment epithelium
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