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Investigative Ophthalmology & Visual Science, Vol. 31, No. 10, October 1990
Copyright © Association for Research in Vision and Ophthalmology
Cell-Mediated Immunity Against Human Retinal
Extract, S-Antigen, and Interphotoreceptor Retinoid
Binding Protein in Onchocercal Chorioretinopathy
A. Von der Lelij,* A. Rorhovo,fi: J. 5. Stilmo,§ R. Hoekzema,t and A. Kijlstrat}:
Autoimmune mechanisms are thought to be involved in the pathogenesis of onchocercal chorioretinopathy. Cell-mediated immune responses to human retinal S-antigen, interphotoreceptor retinoid
binding protein (IRBP), and crude retinal extract were investigated in patients with onchocerciasis
from Sierra Leone, West Africa using a two-step migration-inhibition factor assay. Patients were
subdivided into three groups: (1) without ocular involvement (n = 10), (2) with ocular onchocerciasis
limited to the anterior segment (n = 19), and (3) with onchocercal chorioretinopathy (n = 21). A group
of endemic controls (n = 25) from Sierra Leone were also studied. The cellular immune response to
concanavalin A (Con A) was measured to assess the general capacity of lymphocytes to respond to a
mitogen. Four of 50 (8%) patients with onchocerciasis and four of 25 (16%) endemic controls reacted
with at least one retinal antigen. From the patients with onchocercal chorioretinopathy two of 21 (10%)
showed a positive cellular response. The general mitogen response tested with Con A was positive in
all these individuals. A role for an antiretinal autoimmune mechanism in the pathogenesis of onchocercal chorioretinopathy, as studied with human S-antigen, IRBP, or crude retinal extract, could not be
shown because the cellular response to these antigens did not differ in patients with or without
onchocercal chorioretinopathy or in endemic controls. Invest Ophthalmol Vis Sci 31:2031-2036,1990
Ocular onchocerciasis is a blinding disease. Visual
loss can be due to lesions of the anterior segment, eg,
sclerosing keratitis and/or lesions of the posterior
segment with chorioretinopathy and with optic nerve
atrophy. Pathologic changes are often found in both
eyes, although the lesions are usually not symmetric
and have progressed to different degrees.1
Autoimmunity may play a role in the pathogenesis
of this disease. Evidence for this was provided by
Vingtain et al,2 who found antibodies against bovine
retinal S-antigen and human retinal extract in patients with onchocerciasis from Mali; they claimed
there was a significant difference between patients
with and without chorioretinal involvement. Our
group also investigated the humoral immune re-
sponse against retinal antigens. Antibodies against
human S-antigen and interphotoreceptor retinoidbinding protein (IRBP) were detected in every patient
with onchocerciasis and in nearly every endemic
control from Sierra Leone, West Africa. No relationship could be found, however, between the level of
antibodies against the retinal proteins tested and the
occurrence of onchocercal chorioretinopathy.3
The role of cell-mediated immunity against retinal
antigens in ocular onchocerciasis is not yet known. In
experimental autoimmune uveoretinitis (EAU) induced by a retinal antigen such as S-antigen, it has
been shown that the uveoretinitis is primarily T-cell
mediated.4"6 This disease could not be elicited by
transfer of antiretinal antibodies, although some role
for these antibodies cannot be excluded in the disease
severity.7
An association of a cellular immune response to
human S-antigen with posterior uveitis and panuveitis of mixed etiology was reported by Doekes et al.8
Earlier studies by Nussenblatt et al 910 showed a proliferative response of lymphocytes to bovine S-antigen in patients with various uveitic entities, including
birdshot retinochoroidopathy and, recently, in patients with ocular toxoplasmosis.1'
We investigated whether a cell-mediated immune
From the *Department of Ophthalmology, Free University,
fThe Netherlands Ophthalmic Research Institute, and the ^Department of Ophthalmology, University of Amsterdam, Amsterdam, and the §F. C. Donders Institute for Ophthalmology, Utrecht,
The Netherlands.
Supported by a grant from the Algemene Nederlandse Vereniging ter Voorkoming van Blindheid and the Rotterdamse Vereniging Blindenbelangen.
Reprint requests: A. Van der Lelij MD, The Netherlands Ophthalmic Research Institute, PO Box 12141 1100 AC Amsterdam
ZO, The Netherlands.
2031
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / October 1990
response against retinal antigens plays a role in the
pathogenesis of chorioretinal changes in onchocerciasis. The cellular immune response to retinal antigens was tested by measuring the production of migration inhibition factor (MIF) by peripheral mononuclear cells of patients with onchocerciasis and
endemic controls after incubation with human S-antigen, IRBP, or crude retinal extract.
Materials and Methods
Patients and Control Groups
Patients with onchocerciasis (n = 50) and endemic
controls (n = 25) tested in this study visited the Eye
Hospital Lunsar, which is situated in a hyperendemic
area for onchocerciasis in Sierra Leone, West Africa.
Onchocerciasis patients were selected on the basis of
a positive skin-snip test or a positive Mazzotti test.1
The mean age in this group was 40.3 yr (range, 12-71
yr), and the female:male ratio was 1:2.6. Individuals
with a negative skin-snip test and a negative Mazzotti
test were used as endemic controls. Many hospital
staff members were among this group. The mean age
of the control group was 30.1 yr (range, 13-67 yr) and
the female:male ratio, 1:1.1. The patients with onchocerciasis were subdivided in three subgroups: (1)
patients without ocular involvement (n = 10), (2)
patients with ocular onchocerciasis limited to the anterior segment (n = 19), and (3) patients with onchocercal chorioretinopathy (n = 21). The mean skinsnip count was five to 19 microfilariae for Group 1,
20-49 microfilariae for Group 2, and 50-100 microfilariae per skin-snip for Group 3. None of the patients were being treated with ivermectin, diethylcarbamazine, suramin, or immunosuppressive drugs at
that time or during previous 6 months. All subjects
were examined according to the Onchocerciasis Survey Form,1 recommended by the World Health Organization. Chorioretinopathy was scored in cases of
early disruption of retinal pigment alone or with exposure of the choroid and clumping of retinal pigment at any degree. Furthermore inflammatory activity in the posterior pole was noted.
Venous blood was drawn from patients and endemic controls using Venoject evacuated blood collecting tubes with heparin (Terumo Europe, Leuven,
Belgium). Peripheral blood mononuclear cells
(PBMC) were isolated from this heparinized blood by
centrifugation on Ficoll-Paque (Pharmacia, Uppsala,
Sweden), washed twice with phosphate-buffered saline (PBS), and used immediately in the first step of
the MIF assay.
Informed consent from patients and controls was
obtained after explanation of the study.
Vol. 31
Human S-antigen, IRBP, and Crude Retinal Extract
The retinal antigens were isolated from retinas of
white human donor eyes from which the cornea had
been removed for transplantation. These retinas were
stored at -20°C until use.
The isolation of human S-antigen was done as described previously.8 Briefly a 50% ammonium sulfate
precipitation of retinal extract was followed by DEAE
anion-exchange chromatography and gel filtration.
Human IRBP was purified using a modification of
the method described by Adler et al.1213 After thawing, the retinas were extracted with cold PBS containing 0.1 mM phenylmethyl-sulfonyl fluoride
(PMSF; Sigma, St. Louis, MO) and 0.02% NaN3, pH
7.4, for 1 hr at 4°C. After centrifugation for 30 min at
3000 X g, the pellet was resuspended in the same
solution, and the extraction procedure was repeated.
Both supernatants were collected and concentrated
by ultrafiltration on an Amicon XM-50 filter (Amicon, Danvers, MA). Further isolation was done by
30% ammonium sulfate precipitation and affinity
chromatography on a concanavalin A (Con A)-Sepharose column (Pharmacia) in PBS, containing 0.5 M
NaCl, 0.2 mM PMSF, and 0.02% (w/v) NaN3, (pH
7.4). Human IRBP was eluted with 2.0 M methyl a-D
glucopyranoside (Sigma) in the same buffer. Finally,
gel filtration on a Sephacryl-S300 column (Pharmacia) was done in the same buffer as that used for the
Con-A-Sepharose column, with the addition of 0.1%
(v/v) Tween 20. At every step of the isolation procedure, human IRBP was detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and an enzyme-linked immunosorbent assay
using a rabbit anti-bovine IRBP antiserum (kindly
provided by Dr. R. M. Broekhuyse, Nijmegen, The
Netherlands).
The homogeneity and the identity of human S-antigen and human IRBP were confirmed by SDSPAGE, using samples of approximately 10 ^g each,
with Coomassie Brillant Blue (Merck-Schuchardt,
Darmstadt, FRG) staining of the gels. Both proteins
showed one band on the gels. The molecular weight
was 50 kilodaltons (kD) and 135 kD, respectively
(data not shown).
The crude retinal extract was prepared by the same
extraction procedure as originally with human IRBP,
but instead of 1 hr, the extraction proceeded for 20 hr
at 4°C. After centrifugation for 30 min at 3000 X g,
the supernatant was collected.
Protein concentrations were determined with the
Bradford-method,14 using bovine serum albumin
(Sigma) as a standard.
The S-antigen, IRBP, and the crude retinal extract
were dialyzed and, if necessary diluted, in
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CELLULAR IMMUNITY TO RETINAL ANTIGENS IN ONCHOCERCIASIS / Von der Lelij er ol
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RPMI-1640 supplemented with 25 nM HEPES, 100
units/ml of penicillin, and 0.1 mg/ml of streptomycin
(Gibco, Grand Island, NY).
MI = mean area test supernatant/
MIF Assay
in which the control supernatant came from the tube
containing cells in culture medium without antigen.
A MI < 0.8 was taken as a positive reaction.
The cellular immune response against human Santigen, IRBP, or crude retinal extract was measured
in a MIF assay. The test consisted of two parts. In the
first step, done in the Eye Hospital Lunsar, Sierra
Leone, the PBMC were incubated with the test antigens. The cell supernatants were collected, frozen,
and subsequently tested in the second step for the
presence of MIF by using the human monocytoid
U937 cell-line as indicator cells. This second step was
done in our laboratory in Amsterdam, The Netherlands as described by Thurman et al.15
Briefly, the isolated PBMC were adjusted to 2.5
X 106 cells/ml in RPMI-1640 supplemented with 25
nM HEPES, 100 units/ml of penicillin, 0.1 mg/ml of
streptomycin (Gibco), and 10% heat-inactivated
pooled human serum from nontransfused male white
donors. One-ml aliquots of the cells were dispensed
into five separate 10-ml culture tubes (Nunc, Roskilde, Denmark). One tube served as a medium control for spontaneous MIF production of the cells; the
second tube was used to test the overall ability of the
cells to respond by adding Con A (in a final concentration of 25 Mg/ml). In three other tubes human Santigen (5 Mg/ml), IRBP (10 Mg/ml), and crude retinal
extract (103 Mg/ml) were added. A sixth tube contained culture medium alone without any cells. The
tubes were incubated for 18 hr at 37°C. Supernatants
were harvested by centrifugation (10 min at 1200 X g)
and stored at -20°C until the second step of the
assay.
For the second step of the assay, U937 cells were
harvested from permanent in vitro cultures, washed,
and adjusted to 500 X 106/ml in culture medium.
Seaplaque agarose (FMC Corporation, Rockland,
ME) was dissolved (22.5 mg/ml) in PBS at 100°C for
20 min and diluted 1:10 with warm (37°C) culture
medium. Equal volumes of this agarose solution and
U937 cells were mixed and kept at 37°C. One-microliter droplets were pipetted into the center of a flatbottom 96-well microtiter plate (Nunc) with a Hamilton syringe. After storage of the tray for 15 min at
4°C, 0.1 ml of the cell-culture supernatants were
added fourfold. The tray was incubated for 20 hr in a
humidified incubator at 37°C and 5% CO2 to allow
migration of the U937 cells out of the agarose droplets.
The migration areas were measured by planimetry
and the migration inhibition index (MI) was calculated as follows:
mean area control supernatant
Results
To investigate whether the cellular immune response against retinal antigens plays a possible role in
the pathogenesis of the chorioretinopathy in onchocerciasis, PBMC of onchocerciasis patients and endemic controls were challenged with human S-antigen, IRBP, or crude retinal extract.
No difference could be shown between patients
with or without onchocercal chorioretinopathy and
endemic controls with regard to their ability to react
in vitro to human S-antigen, IRBP, or crude retinal
extract (Fig. 1). The general mitogenic responsiveness
of West Africans when tested with Con A was positive.
Lymphocytes from four of 50 (8%) patients with
onchocerciasis showed MIF production (MI < 0.80)
after overnight incubation with at least one of the
retinal preparations (Table 1). One patient without
ocular involvement had a positive response to human
S-antigen (one often [10%]) and did not react with
human IRBP or crude retinal extract. A second patient with ocular onchocerciasis, but without chorioretinal changes, showed a MI < 0.80 in response to
both human S-antigen and IRBP (one of 19 [5%]) but
not to crude retinal extract. Of the remaining two
patients with onchocercal chorioretinopathy, one patient responded to human S-antigen, IRBP, and
crude retinal extract. The other patient's PBMC
reacted only to human S-antigen and IRBP (two of 21
[10%] and one of 21 [5%], respectively) (Fig. 1; Table 2).
In the group of endemic controls four of 25 (16%)
positive reactions were observed after challenge of
their cells with at least two of the three retinal proteins (Table 1). Two of them showed MIF production
with human S-antigen, IRBP, and crude retinal extract, and the other two only reacted to S-antigen and
IRBP (Fig. 1; Table 2).
The general capacity of the PBMC of the patients
and controls to respond in vitro was tested by incubating the cells with the mitogen Con A. All individuals showed a strong positive response, when tested
with Con A (Fig. 1). The mean MI in the four tested
groups varied between 0.24-0.31 (with standard deviations varying between 0.08-0.14).
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Vol. 31
INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Ocrober 1990
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RETINAL
EXTRACT
Fig. 1. Migration inhibition index of endemic control subjects (—) (n = 25); onchocerciasis patients without ocular imvolvement (•) (n
= 10); patients with ocular onchocerciasis, but limited to the anterior segment (•) (n = 19); and patients with onchocercal chorioretinopathy
(A)(n = 2l).
Discussion
For several decades autoimmune mechanisms
have been thought to play a role in the pathogenesis
of the chorioretinal changes in ocular onchocerciasis.
In our study no relationship could be established between a cellular immune responsiveness to human
S-antigen, IRBP, or crude retinal extract and the occurrence of chorioretinopathy in onchocerciasis.
Only two of 21 patients (10%) with onchocercal chorioretinal lesions showed a reaction to at least two of
these retinal proteins, whereas four of 25 individuals
(16%) of the endemic control group reacted positively.
There is evidence implicating inflammation, mediated primarily by T-cells, in EAU,4"6 although a
role has been claimed for a humoral immune response against retinal antigens in this model.1617 In
addition to this, several investigators confirmed the
observation that an association exists between some
form of clinical uveitis and cell-mediated immunity
against retinal antigens.8""
There are no reports to our knowledge on cell-mediated autoimmunity against retinal antigens in ocular onchocerciasis. Only defects in cellular immunity
have been described with preservation of reactivity to
mitogens in onchocerciasis. Diminished cell-mediated immunity to parasite-related and nonparasite-
Table 1. Cellular immunity against retinal antigens in Sierra Leone (West Africa)
No. (percentage) of individuals* with a migration inhibition index < 0.80
Onchocerciasis patients
Without ocular involvement
With ocular involvement
Limited to the anterior segment
Chorioretinopathy
Endemic control subjects
n
Con A
S-aniigen
IRBP
Retinal extract
50
10
50(100)
10(100)
4(8)
1(10)
3(6)
0(0)
1(2)
0(0)
19
21
25
19(100)
21(100)
25(100)
1(5)
2(10)
4(16)
1(5)
2(10)
4(16)
0(0)
1(5)
2(8)
* See table 2 for specific response to each antigen in separate individuals.
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No. 10
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CELLULAR IMMUNITY TO RETINAL ANTIGENS IN ONCHOCERCIASIS / Von der Lelij er ol
Table 2. Patients and control subjects* with a positive cell-mediated immune response to retinal proteins
Onchocerciasis patients
Without ocular involvement
With ocular involvement
Limited to the anterior segment
Chorioretinopathy
Endemic control subjects
Patient
no.
Age
55
22
20
46
53
22
28
70
71
40
45
35
19
32
40
20
Sex
MF per
skin snip
S-antigen
IRBP
Retinal
extract
1-4
M
F
M
F
F
F
F
1-4
>100
1-4
0
0
0
0
* All individuals, including patients and control subjects who did not
)t react
to human S-antigen, IRBP, or retinal extract, had a positive cellular response
SDonse
to Con A.
MF = microfilariae.
related antigens was reported.18 2I It is possible, that
this results in a protective mechanism avoiding tissue
damage due to reaction against dead micronlariae in
this parasitic infection.
We measured the cellular immune response to
human S-antigen, IRBP, and crude retinal extract in
patients with onchocerciasis with and without chorioretinal involvement and in endemic controls from
Sierra Leone, using a two-step MIF-assay. Type IV
(Gell and Coombs) allergic reactions, presumed to
play a role in cellular autoimmunity, are often investigated using the lymphocyte transformation test. A
better and more consistent correlation exists, however, between the MIF assay and the delayed-type
hypersensitivity skin reaction.22
Neither cell-mediated nor humoral autoimmunity
against retinal antigens such as human S-antigen,
IRBP, and crude retinal extract could specifically be
shown in patients with ocular onchocerciasis and, in
particular, in patients with onchocercal chorioretinopathy compared with the endemic controls. We
are inclined to return to an earlier concept in which
microfilariae of Onchocerca volvulus or their toxins
are directly involved in the pathogenesis of the chorioretinal changes in onchocerciasis. Histologic studies report microfilariae in the retina and choroid,23"25
and Murphy et al26 described microfilariae in the living retina examined with a three-mirror contact lens
at the biomicroscope. There is also the observation
that posterior-segment lesions can freshly develop or
progress after treatment with diethylcarbamazine,
probably by killing the microfilariae in situ, which
gives further support to this hypothesis.27
2. Vingtain P, Thillaye B, Karpouzas I, and Faure JP: Longitudinal study of microfilarial infestation and humoral immune
response to filarial and retinal antigens in onchocerciasis patients treated with ivermectin. Ophthalmic Res 20:951, 1988.
3. Van der Lelij A, Doekes G, Hwan BS, Vetter JCM, Rietveld E,
Stilma JS, and Kijlstra A: Humoral autoimmune response
against S-antigen and IRBP in ocular onchocerciasis. Invest
Ophthalmol Vis Sci 31:1374, 1990.
4. Mochizuki M, Kuwabara T, McAllister CG, Nussenblatt RB,
and Gery I: Adoptive transfer of experimental autoimmune
uveoretinitis in rats: Immunopathogenic mechanisms and histologic features. Invest Ophthalmol Vis Sci 26:1, 1985.
5. Caspi RR, Roberge FG, McAllister CG, El-Saied M, Kuwabara T, Gery I, Hanna E, and Nussenblatt RB: T cell lines
mediating experimental autoimmune uveoretinitis (EAU) in
the rat. J Immunol 136:928, 1986.
6. Merryman CF, Donoso LA, Sery TW, Sciutto E, Bauer A, and
Shinohara T: S-antigen: Adoptive transfer of experimental autoimmune uveitis following immunization with a small synthetic peptide. Arch Ophthalmol 105:841, 1987.
7. Gery I, Mochizuki M, and Nussenblatt RB: Retinal specific
antigens and immunopathogenic processes they provoke. In
Progress in Retinal Research, Osborne N and Chader J, editors. New York, Pergamon Press, 1986, pp. 75-109.
8. Doekes G, Van der Gaag R, Rothova A, Van Kooyk Y,
Broersma L, Zaal MJM, Dijkman G, Fortuin ME, Baarsma
GS, and Kijlstra A: Humoral and cellular immune responsiveness to human S-antigen in uveitis. Curr Eye Res 6:909, 1987.
9. Nussenblatt RB, Mittal KK, Ryan S, Green WR, and Maumenee AE: Birdshot retinochoroidopathy associated with
HLA-29 antigen and immune responsiveness to retinal S-antigen. Am J Ophthalmol 94:147, 1982.
10. Nussenblatt RB, Gery I, Ballintine EJ, and Wacker WB: Cellular immune responsiveness of uveitis patients to retinal Santigen. Am J Ophthalmol 89:173, 1980.
11. Nussenblatt RB, Mittal KK, Fuhrman S, Sharma SD, and
Palestine AG: Lymphocyte proliferative responses of patients
with ocular toxoplasmosis to parasite and retinal antigens. Am
J Ophthalmol 107:632, 1989.
12. Adler JA and Evans CD: Some functional characteristics of
purified bovine interphotoreceptor retinol-binding protein.
Invest Ophthalmol Vis Sci 26:273, 1985.
13. Adler JA and Klucznik KM: Proteins and glycoproteins of the
bovine interphotoreceptor matrix: Composition and fractionation. Exp Eye Res 34:423, 1982.
14. Bradford MM: A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248, 1976.
Key words: onchocerciasis, retinal autoimmunity, cell-mediated immunity, S-antigen, IRBP
References
1. Buck AA: Onchocerciasis: Symptomatology, Pathology, Diagnosis. Geneva, World Health Organization, 1974.
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / October 1990
15. Thurman GB, Stull HB, Miller PJ, Stevenson HC, and Oldham RK: Utilization of purified human monocytes in the agarose droplet assay for measuring migration inhibitory factors. J
Immunol Methods 65:41, 1983.
16. Marak GE Jr, Wacker WB, Rao NA, Jack R, and Ward PA:
Effects of complement depletion on experimental allergic
uveitis. Ophthalmic Res 11:97, 1979.
17. De Kozak Y, Sainte-Laudy J, Benveniste J, and Faure JP:
Evidence for immediate hypersensitivity phenomena in experimental autoimmune uveoretinitis. Eur J Immunol 11:612,
1981.
18. Ward DJ, Nutman TB, Zea-Flores G, Portocarrero C, Lujan
A, and Ottesen EA: Onchocerciasis and immunity in humans:
Enhanced T-cell responsiveness to parasite antigen in putatively immune individuals. J Infect Dis 157:536, 1988.
19. Greene BM, Fanning MM, and Ellner JJ: Non-specific suppression of antigen-induced lymphocyte blastogenesis in Onchocerca volvulus infection in man. Clin Exp Immunol 52:259,
1983.
20. Greene BM, Gbakima AA, Albiez EJ, and Taylor HR: Humoral and cellular responses to Onchocerca volvulus infection
in humans. Rev Infect Dis 7:789, 1985.
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21. Gallin M, Edmonds K, Ellner JJ, Erttmann KD, White AT,
Newland HS, Taylor HR, and Greene BM: Cell-mediated immune responses in human infection with Onchocerca volvulus.
J Immunol 140:1999, 1988.
22. Van de Plassche-Boers EM, Drexhage HA, Kokje-Kleingeld
M, and Leezenberg HA: Parameters of T-cell mediated immunity to commensal micro-organisms in patients with chronic
purulent rhinosinusitis: A comparison between delayed type
hypersensitivity skin test, lymphocyte transformation test and
macrophage migration inhibition factor assay. Clin Exp Immunol 66:516, 1986.
23. Rodger FC and Chir M: The pathogenesis and pathology of
ocular onchocerciasis. Am J Ophthalmol 49:327, 1960.
24. Paul EV and Zimmerman LE: Some observations on the ocular pathology of onchocerciasis. Hum Pathol 1:581, 1970.
25. Neumann E and Gunders E: Pathogenesis of the posterior
segment lesion of ocular onchocerciasis. Am J Ophthalmol
75:82, 1973.
26. Murphy RP, Taylor HR, and Greene BM: Chorioretinal damage in onchocerciasis. Am J Ophthalmol 98:519, 1984.
27. Anderson J and Fuglsang H: Effects of diethylcarbamazine on
ocular onchocerciasis. Trop Med Parasitol 27:263, 1976.
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