Systemic immunostimulation after retinal laser treatment in retinitis pigmentosa

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

Vol. 64, No. 1, July, pp. 76-83, 1992

Systemic Immunostimulation after Retinal Laser Treatment in Retinitis Pigmentosa LOWELL

*Department

L. WILLIAMS,*+$

BARRY T. SHANNON$§ ROBERT B. CHAMBERS,*? AND FREDERICK H. DAVIDORF*‘+

of Ophthalmology, fReti& Research Medicine, and Dthe Clinical Immunology

Laboratory, Laboratory

LAWRENCE

E. LEGUIRE,*+$

and ZDepartment of Pediatrics, the Ohio State University of Columbus Children’s Hospital, Columbus, Ohio 43205

Systemic immunostimulation followed an experimental treatment trial of scatter argon laser photocoagulation directed to the retina of one eye of 10 patients with heredodegenerative retinitis pigmentosa (RP). Significantly increased RP lymphocyte CD25, CD26, and CD4/CD26 activation epitope expressions over prelaser values and controls were found with a normalization of soluble interleukin-2 receptor secretion after laser treatment. Serum interferon-y was low both pre- and postlaser. Interestingly, when a panel of viral antibodies was tested, only those to rubella virus were elevated in the early postlaser period. The character of RP immunostimulation after laser-induced inflammation could be consistent with an antigenic stimulus from laserreleased retinal proteins which might be of autoimmune or latent infectious origin. Enhanced immune responses may be a common but unrecognized sequellae of retinal laser. 0 1992 AcademicPress,Inc.

College

of

showed similar function in the lasered and unlasered eyes (F. H. Davidorf, R. B. Chambers, L. E. Leguire, and L. L. Williams, unpublished data). However, the degree of systemic immunostimulation postlaser in RP patients was surprisingly large when considering the small foci of inflammation created in one eye by laser. In none of the patients was there a history of systemic illness, nor were there local or systemic toxic symptoms during the study period to explain the observed peripheral immune stimulus. Believing that this information may contribute to an understanding of the relationship of immunoregulation to RP pathogenesis (l-61, we have described RP patient immune system findings pre- and postlaser in detail. In addition, if these immune system sequellae commonly follow laser treatments, their characterization may assist in making therapeutic decisions. METHODS

INTRODUCTION

Patient Population and Study Plan

Immune alterations associated with T lymphocyte activation and regulation have been reported previously in the syndrome of retinal heredo-degeneration, retinitis pigrnentosa (RP) (1, 21, but the relevance of these changes to RP pathogenesis has not been well defined (3). In addition, although autoimmune responses to retinal proteins (4) and particularly S-antigens (5) have been identified, sufficient amounts of these antibodies that might lead to RP characteristic retinal lesions appear in only a small proportion of RP (4, 5). While there has been no effective treatment for RP (6), an ongoing experimental therapeutic trial of argon laser photocoagulation in RP offered an opportunity to examine RP lymphocyte expression and systemic cytokine secretion. It was anticipated that laserinduced local macrophage scavenger action in the retina (7) might be effective in clearing away the accumulation of unphagocytosed material and cellular debris seen by ultrastructure in RP retinas (81, possibly prolonging function in this progressive disease (6). Preliminary analysis of visual function after 2 years

Demographic and clinical characteristics of the 10 RP patients participating in the experimental laser treatment study are listed in Table 1. All patients fulfilled clinical requirements for the RP syndrome including progressive bilateral nyctalopia; reduced peripheral vision; abnormal to absent electroretinograms, with a retinal appearance of attenuated blood vessels; pallor of the epic nerve; and scattered peripheral pigmentary depositions (6). Approval of the Human Subjects Research Committee of the Ohio State University was obtained prior to the onset of the study and all requirements for the protection of patient rights were strictly observed. After informed consent phlebotomy was carried out before laser treatment and at planned intervals afterward. Controls for the prelaser laboratory tests were age-matched volunteers and family members without RP. Prelaser results were compared with postlaser as well as normal values. A coherent tuneable dye laser, Model 900, using the green only mode, provided the laser energy source. 78

0090-1229/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved

SYSTEMIC

TABLE

IMMUNOSTIMULATION

1

Demographic and Clinical Characteristics of Laser-Treated Retinitis Pigmentosa Patients Patient ID

Age

I II III IV V VI VII VIII IX

26 42 18 22 48 27 40 41 30

X

28

Visual acuity

Sex

RP type”

Years with RPb

Right

Left

Right

Left

M F M M F M M F F

Dom. Dom. Dom. Rec. Rec. Dom. Dom. Rec. Rec.

10 3 2 1 12 9 7 0.50 15

20130 20125 20140 20125 20120 20140 20125 20130 20120

20130 20125 20140 20125 20130 20125 20140 20140 20120

+I+ + + + -

+/+ + + + -

F

Rec.

0.25

20125

20140

+

+

ERG’

n The inheritance pattern of retinitis pigmentosa is recessive or dominant (Dam.). ’ Number of years of the diagnosis of retinitis pigmentosa. c Electroretinograms (ERG): recordable ( + ) or nonrecordable

(Rec.)

( - ).

With a three-mirror Goldman fundus lens, 200~pm size spots with a 0.05~set duration at a power setting of 100-200 mW created a gray-white reaction at the outer retina/RPE area. The laser photocoagulation used a total of 900 laser spots during four separate sessions 2 to 4 weeks apart to patients previously diagnosed with RP by accepted clinical standards (6). One eye was randomly chosen for treatment while the other eye remained as an internal control for comparison with the treated eye. Photographs, visual fields, visual acuity, and electroretinograms (6) were obtained at regular intervals. To correlate possible immune system changes with possible improvements in visual function after laser, peripheral blood counts and absolute numbers of peripheral lymphocyte epitope expression (9) were measured by flow cytometry before, during and following laser treatment. Serum soluble interleukin-2 receptor (IL-2R) (10) and interferon-y (11) contents and antibody responses to common viral agents (12) were also tested in RP patients before and after laser without laboratory knowledge of RP patient laser status. Immune

Function

Tests

Fluorescent flow cytometry for Lymphocyte epitope expression. Specific epitopes on peripheral blood lymphocytes were enumerated using monoclonal antibodies (MAb) conjugated with affinity-purified fluoresceinisothiocyanate (FITC) for CD2 (T-cell receptor), CD3 (pan T-cells), CD4 (helper-inducer T-cells), CD8 (cytotoxic-supressor T-cells), CD16 (natural killer cells), CD19 (B-cells), CD25 (interleukin-2 receptor cells), and CD26 (Tal activation antigen). Dual fluorescence employing FITC (green) and phycoerythrin (red) conjugated MAb detected CD4/CD29 (helper-inducer

AFTER

LASER

IN RP

79

memory T-cells), CD4/CD45RO (cytotoxic virgin T-cells), CD4/CD26 (antigen-activated CD4), and CD81 CD26 (antigen-activated CD8) cells (Coulter Immunology, Hialeah, FL; Becton-Dickinson, Mountainview CA; and Ortho Diagnostics, Raritan, NJ). Purified mouse myeloma proteins of the IgG classes (Coulter) served as negative control antigens. For staining, 100 p,l whole blood was added to 100 p,l PBS without Ca2+ and Mgz + with 10 p,l of the relevant MAb. After mixing, this suspension was incubated at 4°C for 30 min and washed twice by centrifugation at SOOg for 5 min at 4°C in 2 ml RPM1 1640 containing 5% fetal calf serum (Grand Island Biological Co., Grand Island, NY). After washing, erythrocyte lysing was accomplished with a lysing reagent at room temperature for 5 min. After centrifugation the cells were resuspended in 1.0 ml Isotol II (Coulter Diagnostics, Hialeah, FL) for analysis by flow cytometry (7, 8). Fluorescent-positive cells were counted with an automated flow cytometry system (EPICS-C, Coulter Electronics, Hialeah, FL) with the argon laser set at 488 nm light (500 mW) to excite both fluorochromes simultaneously. The forward light scatter and rightangle green fluorescence signals were collected as a two-parameter dot plot cytogram for analysis of fluorescent-positive and negative lymphocytes. The selected cytogram contained less than 1% monocytes demonstrated by positive staining with OKMl (CD14; Ortho Diagnostics). Data acquisition for detection of cells was governed by software written to collect lo4 cells (Coulter Electronics) with calculations made by log scale. The absolute numbers of positive cells, expressed per cubic milliliter of blood, were derived from the percentage of marker-positive lymphocytes x the white cell x the differential lymphocyte counts (Coulter counter S + IV, Coulter Electronics). Normal controls were included in daily assays. Reproductions from this assay showed less than 5% difference. Average values of RP patients and normals were compared by analysis of variance using an IBM software CRISP program. Soluble interleukin-2 receptor assay. From frozen (-20°C sera, the content of soluble interleukin-2 receptor (sIL-2R) was measured by an enzyme immunoassay (EIA) test kit (T-cell Sciences, Cambridge, MA). Briefly, an anti-IL-2R monoclonal Ab precoated onto polystryene microtiter wells was bound to sIL-2R in sera samples. An enzyme-conjugated anti-IL-2R monoclonal Ab which binds to a second distinct epitope on the IL-2R molecule was added to complete the sandwich. After washing away unreacted components, an added chromagen solution formed a colored end product proportional to the amount of IL-2R in the sample. The average absorbance of duplicate samples at 490 nm was compared to a standard curve prepared from six known IL-2R standards. Calculations were per-

80

WILLIAMS

formed on a Biokinetics Microplate Reader EL 312 (BioTek Instruments, Highland Park, WI). Comparisons between RP patients and normals were made by ANOVA using the CRISP IBM Program. assay. To detect the content of human immune interferon-y (hIFN-y) (111, an enzymelinked immunosorbent assay (ELISA) was used (Intertest-y, Genzyme, Boston MA). Specific for hIFN-y, the monoclonal Ab coated on the microtiter plate demonstrates no detectable cross-reactivity with human interleukin-l-a, interleukin-6, or tumor necrosis factor-o and is capable of detecting 100 pg/ml (2.5 units/ ml) of IFN-y. In the procedure, after the serum addition in duplicate, a second goat polyclonal Ab is bound to multiple epitopes on the IFN-y. A third Ab, biotinlabeled donkey anti-goat Ig, was then recognized by streptavidin-peroxidase reaction with OPD chromagen at 492 nm. The absorbance, proportional to the concentration of IFN-r, was quantified by comparison with known IFN-y standards and calculated as above. Human interferon-y

Antiviral antibody detection by ELISA. Antiviral immunity of serum of RP patients was tested to common viral agents: herpes simplex-I and -11 (HSV-I, -II), cytomegalovirus (CMV), rubella, rubeola, and varicella-zoster (VZV). Specific viral antigen enzymelinked immunosorbent assays (ELISA) (12) (MA Bioproducts, Walkersville, MD) were used in masked fashion by the State of Ohio Health Department Laboratory. Values of RP patients were determined by a linear regression analysis of the test absorbance values with known kit standards and categorized as negative (no response), low (comparable to the lower 15% range of the normal Poisson distribution), medium (average response as in 70% of normal range), or high (comparable to the upper 15% of normal range). Actual values were compared to age-matched controls from central Ohio by analysis of variance using IBM CRISP software program. RESULTS

ET AL.

patients, although the total WBC count fell to the lower ranges of normal in 5 of the 10 patients just after laser (data not shown). Lymphocyte epitope expression prelaser. As found by others (l-3), RP prelaser values were different from those of normal controls in several lymphocyte expressions (Table 2). Activation of T and B cells, demonstrated by elevated average absolute numbers of the T-cell activation marker CD25 (interleukin-2 receptor) and elevated numbers of CD26 (Tall positive cells, was consistent with an antigen-activated stimulus. Dually labeled CD4 subsets, CD4XD26 (activated helpers) and CDB/CD26 (activated cytolyticl cells, supported the observation of an expanded CD4 compartment (Table 2). Additionly, natural killer cells (CD16) were increased prior to laser treatment, while other peripheral lymphocyte expressions were similar to normal ranges (Table 21. Lymphocyte expressionpostlaser. The postlaser pattern of RP lymphocyte expressions was consistent with a further stimulation of systemic immunoregulation (19) (Table 2). Average numbers of CD25 lymphocytes. above normal before laser treatment, rose afterward significantly from prelaser levels (P < 0.005). CD26positive cells (antigen-activated cells) also rose significantly from pre- to postlaser times (P < 0.005). While both CD4/CD26 and CD8/CD26 subsets increased after laser treatment, only antigen-activated helper cells (CD4CD26) showed a significant enhancement postla-

TABLE 2 Pre- and Postlaser Lymphocyte Epitope Expression of 10 Retinitis Pigmentosa Patients Compared to Normals Epitope” CD4 CD8 CD16

Ocular Symptoms Postlaser

Ocular symptoms following laser were minimal. There had been no subjective symptoms of pain, fever, or unusual discomfort after the laser to one eye other than occasional slight blurring of vision for 24 hr. There was no evidence of infectious foci at any time in any patient. At the termination of the laser treatments there was a peripheral distribution of laser scars in the retina.

CD19 CD25 CD26 CD4/CD29 CD4/CD26

CD8/CD26

Immune Function Studies Prelaser and Postlaser

Prior to and after laser treatment, white blood count (WBC) and differential cell counts remained within the control range in all Peripheral

blood counts.

RP prelaser 1620’ (6061 778 (283) 385 (200) 361 (155) 561 (2821 592 1151) 512 (277) 104 (70) 117 (86)

P value c.01 ns c.01

ns c.001 <.Ol ns <.OOl <.OOl

RP postlaser 1534 1417) 862 (3451 505 1197)

415 (182) 1034 1348) a44 (182) 616 (166) 244 (74) 206 (‘78)

P’

value c.01 ns c.001

ne c.001 <.Ol <.Ol <.OOl <.OOl

Normals 1093 13571 734 (317) 168 1132) 485 12091 194 (49) 356 1120: 308 (158) 4i 1181 45 (22)

a Surface epitope nomenclature (see Methods for identification). * Absolute lymphocyte number (SD) per mm3 (see Methods). ’ P values = significant difference between pre- or postlaser numbers and normal

(last

column);

ns, not significant.

SYSTEMIC IMMUNOSTIMULATlON

ser (P < 0.001). In addition, the CD16 subset, identifying a portion of the natural killer cell population, and CD4/CD29, memory helper-inducer cells, showed slight increases from pre- to postlaser times while the CD4 helper cell compartment was elevated over the normal range throughout. These combined data suggested the possibility of an antigen stimulation associated with retinal laser treatment (Table 2) (17, 20). There were no changes from normal expression in other cellular epitopes measured including CD2, CD3, CD8, CD19, CD4XD45R0, and HLA-DR (data not shown). Viral antibody values pre- and postlaser. Prelaser average antiviral antibodies (Ab) of RP patients to the common viral agents, rubella, CMV, rubeola, HSV-I and -11, and VZV were within normal range (Table 3). In contrast after laser treatment, only the average RP group Ab to rubella virus rose significantly. Moreover, an increase in anti-rubella Ab occurred in every RP patient’s serum. While slight Ab elevations to other viruses occurred in individual serum post laser, all Ab values returned to prelaser levels by 6 months after laser. Importantly, there were no retinal lesions suggestive of infectious foci in postlaser RP fundus examinations.

AFTER LASER IN RP

81

above the normal range. Also in contrast to the prelaser treatment values, correlation of sIL-2R content to cellular IL-2R expression in RP (1034 + 348 cells/mm3) postlaser showed a more normal positive value (r = +0.486; P < 0.01) (22). Interferon-y content pre- and postlaser. Since elevated levels of serum interleukin-2 are usually associated with an increased activated cell production of IFN-y (18), we measured RP IFN-y prelaser treatment (187 2 107 pg/ml; mean + SD) and postlaser treatment (168 + 122 pglml). There was no difference between these values and no rise in any individual patient’s serum, although 3 of the 10 RP had detectable IFN-y (350-545 pg/ml) before and after laser treatment. Approximately 100 pg/ml IFN-?/ are normally found in supernatant fluids of unactivated cells (18). Since there was marked lymphocyte activation in both pre- and especially postlaser RP cells, the lack of IFN-1, in RP sera at both times is unusual (16). DISCUSSION

Unexpected increased cell-mediated and humoral immune responses followed experimental argon laser photocoagulation in a group of 10 patients with the heredo-degenerative retinal disease, retinitis pigmentosa (6). Enhanced immunoregulation may be a comSoluble IL-2R content/cellular IL-2R expression ramon, but unrecognized, sequella of retinal laser which tio. The soluble 55-kDa component (sIL-2R) of the ILis considered to produce only small local inflammatory 2R (10) was increased in RP patient sera (758 + 233 U/ml) before laser treatment compared to normals (365 responses and local macrophage scavenger activity in ? 147 U/ml; P < O.OOl), but only one half of the RP the laser-treated eye (7). To clarify this issue we analyzed the systemic immunostimulation that followed patients had values above the first SD of normal range. laser in these RP patients. Clearly, their immunoregHowever, when sIL-2R content was compared to celluulation had not been normal before laser treatment, as lar IL-2R expression (561 + 282 cells/mm31 prelaser, there was an unusual negative correlation between the was previously reported (l-5). An increased expression of two activation epitopes, CD25 (IL-2R) (14) and CD26 values (r = -0.434). (Tal, antigen-activated T-cells), was accompanied by a Evidence of immunostimulation was found in the slight enhancement of the entire CD4 compartment postlaser sIL-2R response. The average RP sIL-2R conwith increased expressions of CD4/CD26- and CD8/ tent had increased to 927 + 353 U/ml (P < 0.001 comCD26-activated subsets and some expansion of the pared to normal) and now all 10 RP patients had levels CDl6-positive (natural killer) compartment. However, after laser treatment, significant increases in RP-activated epitopes developed. An average douTABLE 3 IL-2R expression suggested a new Comparison of Antiviral Antibodies of 10 RP Patients Pre- bling of cellular stimulation of immune cells by the cytokine cascade and Postlaser to 25 Normals associated with macrophage interleukin-1 secretion Rubella” CMV Rubeola HSV-1 HSV-2 VZV during the normal processing of foreign antigen (19). 0.46b RP prelaser 0.42 0.28 0.56 0.42 0.43 Consistent with laser-related induction of this cascade (0.22) (0.39) (0.15) (0.30) (0.25) (0.22) were slightly increased numbers of CD16 cells, an acRP postlaser 0.72* 0.51 0.32 0.62 0.48 0.44 tivation response associated with viral or tumor pres(0.22) (0.32) (0.23) (0.42) (0.45) (0.37) ence (20). Significantly greater expression of the antiNormals 0.28 0.33 0.29 0.85 0.45 0.35 gen-activated CD26 molecule, also reflected in marked (0.21) (0.37) (0.17) (0.53) (0.28) (0.24) enhancement of CD41CD26 subset numbers, implied a ELISA determinations by specific antiviral kits for rubella, cy- an additional stimulus directed toward an antigentomegalovirus (CMV), rubeola (measles), herpes simplex-I and -11 related event. There was a slight postlaser treatment (HSV I, II), and varicella-zoster virus (VZV). increase in CD4/CD29, antigen memory helper cells, a b Average (SD) antibody values determined by kit standards. * Significantly different from normal; P < 0.001. compartment characterized as antigen-responsive,

82

WILLIAMS

which increases after exposure to infectious agents with age (20). In RP prelaser lymphocytes, we found an unusual negative correlation between IL-2R lymphocyte surface expression and the serum content of its 55kDa released soluble IL-2R protein (22). Normally, increased synthesis of surface IL-2R during infections leads to high levels of its released component, sIL-2R in sera with an equivalent ratio between them (10,22). A reverse of the serum sIL-2R/IL-2R cellular expression ratio suggests that despite elevated IL-2R expression on activated cells, the soluble form is either not being formed or not being released (23). However, in these RP patients since increased amounts of RP sIL-2R during postlaser systemic immunostimulation restored a normal positive sIL-2R/IL-2R ratio, elimination of a block of sIL-2R secretion was suggested (22, 23). Although the function of sIL-2R is still under debate, an inverse (negative) ratio of sIL-2R to IL-2R, present during certain phases of persistent infection with HIV-l, is considered secondary to HIV-l interference with effective cellular production of interleukin-2 (23). Prelaser RP sera showed an impaired production of immune IFN-r also described in RP by Detrick et al. (2). However, laser treatment and systemic immunostimulation did not initiate production of this cytokine as expected (18). Low IFN-r secretion has been also described in certain phases of HIV-l infections (23). Therefore, abnormal immunoregulation in RP is supported by finding both increased and defective production of cytokines normally secreted in an immune activation response (19). It is surprising that this abnormal pattern is also causally linked to virally mediated interference and suppression (22-24). Systemic immunostimulation of postlaser treatment RP sera might lead to slightly elevated antiviral antibodies toward common viral agents. However, a significant elevation of only the anti-rubella antibody, occurring in all RP patients in the immediate postlaser period without fever or rash, suggests the possibility of latent viral reactivation during laser-induced retinal inflammation (25). While rubella virus is known to target the retina in the congenital rubella syndrome and can establish invisible persistent infections in human cells (25), rubella infections have rarely been recognized in the retina after birth (6). Increased antirubella antibodies have been reported in RP patients, but their relevance to RP syndrome is unknown (26). In summary, although both increased activation expressions (1,27) and inhibited immune responses (l-3) were found previously in RP patients, these immune alterations were not clearly linked to distinct elements of RP retinal pathology or to genetic patterns of RP (6). We suggest instead that these findings could represent an activated immune response to an endogenous antigenie stimulus that cycles on and off under the control of normal immunoregulatory feedback mechanisms

ET AL.

(28, 29). An autoimmune component has often been considered in RP pathogenesis (l-6) but autoimmune responses to specific retinal proteins are not consistently identified in RP (4, 5). However, such a mild antigenic stimulus present in RP patients (l-3) might be intensified by laser-induced inflammation. A systemically released antigen from the inflamed retina could possibly be either a RP genetically-altered or even a normal retinal protein leading to an enhanced autoimmune response (4, 5), or alternately, a laserinduced activation of an indolent, latent viral infection in the retina (25, 26). Although our data cannot distinguish between these two alternatives, they implicate immune component participation in the pathogenesis of the underlying genetic disease process in RP (5). Moreover, marked systemic immunostimulation after a single eye laser treatment questions the use of the nonlasered eye for a suitable control to judge benefit to the lasered eye. In addition, if similar immunostimulation occurs in other conditions treated routinely by retinal laser, recognition and characterization of their immune system responses may introduce a new understanding as well as suggest new therapeutic modalities. ACKNOWLEDGMENTS The authors appreciate the support of the Children’s Hospital Research Foundation, the Lehenbauer Fund, and the Ohio Lion’s Research Fund, and the technical expertise of Christy Steffen, Cindy Taylor, and Deborah Jacobs. REFERENCES 1. Williams, L. L., Shannon, B. T., and Leguire, L. E., Immune alterations associated with T lymphocyte activation and regulation in retinitis pigmentosa patients. Clin. Immunol. Immunopathol. 43, 380-389, 1988. 2. Detrick, B., Newsome, D. A., Percopo. C. M., and Hooks, J. .I.. Class II antigen expression and gamma interferon modulation oi monocytes and retinal pigment epithelial cells from patients with retinitis pigmentosa. Clin. Immunol. Immunopathol. 36. 210-221, 1985. 3. Newsome, D. A., Quinn, T. C., Hess, A. I)., and Pitha-Rowe, P. M.. Cellular immune status in retinitis pigmentosa. Ophthulmology 95, 1696-1703, 1988. 4. Heckenlively, J. R., Solish, A. M., Chant, S. M., and MeyersElliott, R. H., Autoimmunity in hereditary retinal degenerations. II. Clinical studies: Anti-retinal antibodies and fluorescein angiogram findings. Br. J. Ophthalmol. 69, 758-764. 1985. 5. Thirkill, C. E., Roth, A. M., Takemoto, D. J., Tyler, N. K.. and Keltner, J. L., Antibody indications of secondary and superimposed retinal hypersensitivity in retinitis pigmentosa. Am. .I. Ophthalmol. 112, 132-137, 1991. 6. Heckenlively, J. R. (Ed.), In “Retinitis Pigmentosa,” Chaps. l-4 and 8, Lippincott, Philadelphia, 1988. 7. Peyman, G. A., Ocular effects of various laser wavelengths. Sure. Ophthalmol. 28, 391-404, 1984. 8. Flannery, J. G., Farber, D. B., Bird, A. C., and Bok, D., Degenerative changes in a retina affected with autosomal dominant retinitis pigmentosa. Invest. Ophthnlmol. Visual Sci. 30, 191211,1989.

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IMMUNOSTIMUI

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Received January 16, 1992; accepted with revision April 6, 1992

,ATION AFTER LASER IN RP

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