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Immunopathology and Electron Microscopy of Acanthamoeba Keratitis
Immunopathology and Electron Microscopy of Acanthamoeba Keratitis William Mathers, M . D . , Garth S t e v e n s , Jr., M . D . , Merlyn Rodrigues, M . D . , Chi Chao Chan, M . D . , Joseph G o l d , M . D . , G o v i n d a S. Visvesvara, P h . D . , Michael A. Lemp, M . D . , and Lorenz E. Zimmerman, M . D .
In order to evaluate the local cellular immune response to Acanthamoeba infection we performed immunohistochemical examinations of the corneal buttons of two patients with Acanthamoeba keratitis. We found that the corneal stroma was infiltrated with polymorphonuclear leukocytes and HLA-DR positive macrophages that appeared to be stromal keratocytes by light microscopy. Despite the presence of chronic inflammation in both patients, no stromal lymphocytes were seen in one patient and a sparse lymphocytic infiltrate was seen in the other patient. Electron microscopy confirmed the presence of macrophages, neutrophils, and Acanthamoeba organisms in these two patients. MORE THAN 50 cases of Acanthamoeba keratitis
have been described since 1974 including 24 cases reported to the Centers for Disease Con trol in 1986.1 The organism is difficult to culture from clinical specimens and in many cases the diagnosis is not made or even suspected before keratoplasty. Recent reports of rapid diagnosis
Accepted for publication Jan. 27, 1987. From the Cornea Service, Center For Sight, George town University (Drs. Mathers, Stevens, Gold, and Lemp); Laboratory of Pathology (Dr. Rodrigues) and Laboratory of Immunology (Drs. Stevens and Chan), National Eye Institute; Protozoal Diseases Branch, Cen ter for Infectious Diseases, Centers for Disease Control (Dr. Visvesvara); and Department of Pathology, George town University and Department of Ophthalmic Pa thology, Armed Forces Institute of Pathology (Dr. Zimmerman). This study was supported by an unrestricted grant from Research to Prevent Blindness, Inc. (Dr. Lemp). This study was presented in part at the annual meeting of the Ocular Microbiology and Immunology Group, Nov. 8, 1986, in New Orleans, Louisiana. Reprint requests to William Mathers, M.D., Universi ty of Arizona, Department of Ophthalmology, 1501 N. Campbell St., Tucson, AZ 85724.
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using immunofluorescent antibody 2 and calcofluor white 3 techniques suggest future im proved diagnostic accuracy. Until recently Acanthamoeba infections were most prominent in immunosuppressed indi viduals who developed fatal encephalitis. 4,5 The mechanism by which Acanthamoeba is able to cause progressive disease in normal hosts is unclear. Acanthamoeba keratitis is frequently present for several months before keratoplasty is performed, yet histologic examination of cor neal buttons has usually shown only acute inflammatory cells. 68 We recently examined two patients with Acanthamoeba keratitis in which we used immunohistochemical stains and transmission electron microscopy to examine the inflamma tory cells and organisms found in these infect ed corneas.
Case Reports Casel A 62-year-old man had a two-month history of pain and irritation with slight discharge in the right eye. He had worn extended wear contact lenses for the previous 4V2 years, but for two months had been unable to wear a contact lens in the right eye because of irrita tion. He admitted to wearing contact lenses under goggles while swimming in a public pool; however, he denied ocular pain until two months after stopping contact lens wear be cause of intolerance. He had recently been treated with topical vidarabine and prednisolone acetate 1% for a presumed herpes simplex keratitis. At our initial examination his best corrected visual acuity was R.E.: 20/200. There was a superficial punctate keratitis with irregu lar fluorescein staining in the central cornea
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Fig. 1 (Mathers and associates). Case 1. Top left, Paracentral ring infiltrate (arrow) of the right cornea. Top right, Corneal biopsy demonstrates cyst-like organisms in the stroma (hematoxylin and eosin, x250). Bottom left, Transplanted corneal tissue shows central crystalline opacities with a fern-like pattern.
and a ring of stromal opacification in the midcornea. His antiviral medication was slowly discontinued over the next several weeks. Two months later his visual acuity had de creased to counting fingers in the right eye, and there was an epithelial defect over the midcornea with a surrounding ring of stromal infil tration (Fig. 1, top left). Acanthamoeba keratitis was suspected, but no growth was obtained on scrapings of the stroma cultured on nonnutrient agar with Escherichia coli. Three days later his symptoms and stromal infiltrate had worsened. A 1.5 x 0.5 x 0.3-mm wedge corneal biopsy of the ring of infiltrate was performed. Light microscopy demonstrated numerous
Acanthamoeba cysts (Fig. 1, top right) and cul ture yielded motile trophozoites. The patient was treated with neomycin oph thalmic ointment hourly and propamidine isethionate eyedrops every four hours. The disease progressed despite therapy and a pene trating keratoplasty was performed one week later. Acanthamoeba cysts were identified in the corneal button as well as in culture. Three weeks after keratoplasty the eye devel oped increasing pain and redness. Crystalline stromal opacities with a fern-like pattern were present (Fig. 1, bottom left). Streptococcus viridans was cultured from scrapings. After four days of topical treatment with fortified ce-
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fazolin and gentamicin the transplant was re peated. Intrastromal colonies of gram-positive bacteria were present, but there was negligible infiltration by inflammatory cells. Rare Acanthamoeba cysts were found on light microscopic examination of material prepared for electron microscopy; cultures were negative for both Acanthamoeba and bacteria. The patient's cornea remained clear after discontinuing all medica tions except propamidine isethionate and topi cal corticosteroid daily. Ten weeks after the second transplant he developed a second pseudocrystalline keratopathy accompanied by fibrinous exudate in the anterior chamber. Cul tures were negative but the infiltrate cleared with the administration of topical cefazolin and gentamicin. A subsequent wound leak necessi tated repeat corneal grafting and the button demonstrated edema without bacteria or Acanthamoeba. The patient's graft remained clear for six weeks after the transplant while on a regi men of topical cefazolin until a deep infiltrate developed at the wound margin under intact epithelium. Cultures demonstrated Pseudomonas aeruginosa, which cleared with tobramycin therapy, and a subsequent wound leak led to his fourth corneal transplant. Case 2 A 35-year-old woman was examined because of a one-month history of pain, photophobia, and increased tearing in the right eye. She had worn soft contact lenses successfully for the previous 13 years. Visual acuity in the right eye was hand motion, and a central epithelial de fect was present with a paracentral ring of stromal infiltrate and a 1-mm hypopyon. Scrap ings of the corneal lesion and a paracentesis showed no organisms, and routine cultures were negative. The patient was treated with fortified cefazolin and gentamicin eyedrops as well as natamycin 5% applied topically. One week later the hypopyon had nearly resolved although the corneal haze persisted. The cornea was cultured again for Acanthamoeba, and although the plates initially showed no growth, they were subsequently reexamined and found to contain Acanthamoeba. Two weeks later when the lesion failed to improve, a corneal biopsy was performed. Although the small specimen proved inadequate for histologic examination, Acanthamoeba was cultured from the tissue. The patient was treated unsuc cessfully with topical propamidine isethionate every two hours as well as neomycin ointment. A penetrating keratoplasty was performed 12 days later. Histologic examination of the corne
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al button showed numerous cysts, primarily in the outer stromal layers beneath the areas of epithelial ulceration. Postoperatively the graft has remained clear for six months.
Material and Methods Light microscopy—The corneal buttons were divided into three portions. Tissue for routine light microscopy was fixed in 10% formalin and paraffin-embedded sections were stained with hematoxylin and eosin and periodic acid Schiff. Immunohistochemical staining—Portions of recipient buttons were snap frozen in optimal cutting temperature compound, sectioned at 6 ixm, and stored at - 4 C for 48 hours before staining. Slides were air dried, fixed in acetone, and stained by indirect immunoperoxidase technique using the avidin-biotin-peroxidase complex method. 9 Slides for endogenous peroxidase were processed with light fixation and without addition of primary antibody. Slides were developed with diaminobenzidine and hydrogen peroxide. Counterstain was per formed with 1% methyl green. Mouse hybridoma monoclonal antibodies (Table) were used at a 1:40 dilution in Tris buffered saline. Antibodies to T lymphocytes include anti-Leu 2a (suppressor/cytotoxic sub set), 10 anti-Leu 3a (helper/inducer subset), 10 and anti-Leu 4 (pan T cell).11 The anti-Leu 14 anti body is specific for B lymphocytes 12 and antiLeu 7 for natural killer cells. 13 The OKT 614 is a thymocyte marker that also stains Langerhans cells of the epithelium. The anti-OKMl anti body stains cells of bone marrow derived monocyte lineage, and neutrophils. 15 Anti-Leu M-5 stains most macrophages and few neutro phils. 12 Anti-Mo-2 stains monocytes but no neutrophils. 16 Anti-HLA-DR stains cells that express a major histocompatibility complex Class II antigen. 17 Class II antigen expression has been noted on numerous cell types includ ing macrophages and stimulated keratocytes but does not occur on neutrophils. 16 Endoge nous peroxidase activity occurs in neutrophils and is weak in macrophages. Immunofluorescent staining—To determine the species of Acanthamoeba, we deparaffinized, dehydrated, and incubated paraffin sections with 1:20 and 1:100 dilutions of rabbit antiAcanthamoeba sera against A. castellanii, A. polyphaga, and A. culbertsoni. Transmission electron microscopy—For elec-
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TABLE IMMUNOHISTOCHEMICAL STAINS USED PERCENTAGE OF CORNEAL STROMAL CELLS STAINED, NEAR ULCER/DISTANT FROM ULCER* ANTIBODY
SPECIFICITY
PATIENT 1
PATIENT!
Mouse ascites None
Negative control Neutrophils (endogenous peroxldase) Monocytes/neutrophils15 (C3b receptor) Monocytes, neutrophils, Langerhans cells12 Class II histocompatibility antigen17 Monocytes16 T cell, suppressor/ cytotoxic subset10 T cell, helper subset10 Pan T cell marker11 Thymocyte marker14 Natural killer cells13 B lymphocytes12
2
2
2
2
80/60
100/20
40/10
80/30
98/98
100/60
NT/14 0
NT/5 0
0 0 0 0 0
8/1 10/3 0 0 0
OKM11 Leu M-5' HLA-DR* Mo-2* Leu 2a* Leu 3a* Leu 4* OKT6* Leu 7* Leu 14*
*NT, not tested. Ortho Diagnostic Systems. 'Becton Dickinson. f
tron microscopy, representative portions of corneal tissue were fixed in 2.5% glutaraldehyde, post fixed in 1% osmium tetroxide, dehy drated in ascending concentrations of alcohols, and embedded in epoxy resin. Sections 1 |xm thick were stained with toluidine blue. Thin sections were stained with uranyl acetate and lead citrate.
Results Routine light microscopy—In Patient 1, the minute biopsy specimen showed that the cor neal stroma was lightly infiltrated by polymor phonuclear leukocytes. Many Acanthamoeba cysts were present. Examination of the first keratoplasty specimen showed that the epithe lium was largely missing, and what remained was irregularly thinned and detached. Bow man's layer was generally intact but immedi ately beneath it the stroma was lightly but diffusely infiltrated by polymorphonuclear leu
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kocytes, as were the middle stromal layers. The deeper layers were more minimally infiltrated, but a layer of leukocytes also adhered to the endothelial surface. Amoebic cysts were irregu larly distributed throughout the corneal stroma but were more numerous in the superficial and middle layers. No vascularization or significant infiltration by mononuclear inflammatory cells was apparent. The second keratoplasty speci men showed superficial intrastromal colonies of gram-positive cocci with elongated tapered ends dissecting the adjacent stromal lamellae, but there were few inflammatory cells reacting to the bacteria. In Patient 2, examination of the keratoplasty specimen showed that the corneal epithelium was partially ulcerated, and Bowman's layer was largely absent. Where epithelium covered the button, it represented reepithelialization of previously ulcerated tissue. Acanthamoeba cysts were numerous in the superficial stromal layers in the denuded areas, where the infiltration by polymorphonuclear leukocytes was most in tense and where many of the inflammatory cells appeared degenerated. In this area the nuclei of the cells of the stroma appeared large, ovoid, and intensely basophilic. A light infil trate of mononuclear leukocytes was also pres ent in this area. Elsewhere, the stroma was lightly, but diffusely, infiltrated by polymor phonuclear leukocytes and a few mononuclear cells. No vascularization was observed. A few polymorphonuclear leukocytes were adherent to the degenerated endothelium. Immunopathology—By light microscopy nearly all stromal cells appeared as spindleshaped, keratocyte-like cells. In both Patients 1 and 2, 98% of the cells in the corneal stroma stained for the HLA-DR antigen (Fig. 2, top left and bottom left). OKM1 antibody stained 80% (Case 1) to 100% (Case 2) of the cells near the site of active ulceration but stained only 20% (Case 2) to 60% (Case 1) of stromal cells away from the active sites (Fig. 2, top middle and bottom middle) (Table). Anti-Leu M-5 antibody stained 40% (Case 1) to 80% (Case 2) of cells near the active sites and 10% (Case 1) to 30% (Case 2) elsewhere. Although endothelial kera tic precipitates in fixed sections showed numer ous neutrophils, in the frozen sections the keratic precipitates appeared as mononuclear cells and stained strongly with both antiOKM1, anti-Leu M-5, and anti-Mo-2. To distin guish further between macrophages and neu trophils, endogenous peroxidase activity in stromal cells was limited to 2% to 5% of stromal cells. Staining for the Mo-2 antigen indicated a
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Fig. 2 (Mathers and associates). Immunoperoxidase staining. Top left, Case 1. Anti-HLA-DR antibody (x 170). Top middle, Case 1. Anti-OKMl (x 220). Top right, Case 1. Anti-Leu 4 (x 220). Bottom left, Case 2. Anti-HLA-DR (x 220). Bottom middle, Case 2. Anti-OKMl (x 220). Bottom right, Case 2. Anti-Leu 4 (x330). In both Case 1 and Case 2 most cells of the corneal stroma express the HLA-DR antigen and the macrophage/monocyte marker OKM1. The cornea of Patient 2 demonstrates few T helper lymphocytes; the cornea of Patient 1 does not demonstrate any T helper lymphocytes.
different staining pattern; only 14% of cells in Case 1 and 5% in Case 2 stained positive for Mo-2. No sections that included the ulcer, how ever, were available for the Mo-2 antibody stain. T lymphocytes were not present in the stro ma of Patient 1 (Fig. 2, top right); in Patient 2 (Fig. 2, bottom right) the few T lymphocytes present were primarily of the Leu 3a (helper) subset. No cells of B lymphocyte lineage, natu
ral killer cells, or OKT 6 positive cells were seen. Immunofluorescent identification—Acanthamoeba organisms from Case 1 were identified as A. polyphaga. Organisms in Case 2 stained posi tive for A. castellani. Transmission electron microscopy—In the corneas of Patients 1 and 2 numerous cysts of Acanthamoeba were present, primarily in the superficial half of the stroma (Fig. 3). The outer
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Fig. 3 (Mathers and associates). Case 1. Electron micrograph showing an encysted Acanthamoeba organism with adjacent inflammatory cells. The outer ectocyst layer is focally disrupted (arrows). The organism shows mitochondria, lipid vacuoles, and lysosomes. A neutrophil is indicated (N). Inset shows well delineated cystic organisms in the anterior two thirds of the stroma (arrows) (x 630).
cyst wall of the organisms was often irregularly folded. Some cysts showed a nucleus and prominent nucleolus and cytoplasmic organelles including mitochondria, membranebound lipid vacuoles, and polyribosomes. The cyst wall consists of an inner endocyst layer and an outer ectocyst layer. Some organisms showed well-preserved organelles suggestive of a viable phase; others were disrupted with only cell wall remnants. The disrupted organ elles were primarily found adjacent to inflam matory cells, including neutrophils and macrophages (Fig. 4). In the regraft cornea (Case 1), there were large bacterial aggregates (cocci) in the superficial stroma (Fig. 5). The adjacent
stroma showed scant amoebic cysts and occa sional neutrophils (Fig. 6).
Discussion Acanthamoeba keratitis is a chronic infection that usually occurs in otherwise normal hosts and is associated with contact lens wear or other trauma. Despite the frequently noted vigorous corneal infiltration by neutrophils and a measurable antibody response, 5,18 Acanthamoeba infections frequently progress to corneal opacity or perforation.
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Fig. 4 (Mathers and associates). Case 2. Inset shows encysted amoebic organisms (arrows) in necrotic stroma (toluidine blue, x 500). Electron micrograph shows an encysted organism in corneal stroma. The protoplasm of the amoeba is partly shrunken and contains a nucleus (N), mitochondria, ribosomes, and occasional lysosomes (x 16,500).
Light microscopic examination of the two corneal buttons from our patients disclosed mild to moderate neutrophilic infiltrates. Hematoxylin and eosin stained frozen tissue sec tions adjacent to areas examined with immunohistochemical stains demonstrated fewer neu-
trophils, identifying a sampling difference be tween the routine light microscopic and immunopathologic findings. The immunohistochemical findings in both Cases 1 and 2 indicate an intense infiltration of cells staining for the macrophage markers OKM1 and Leu
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Fig. 5 (Mathers and associates). Case 2. Electron micrograph of stromal bacteria shows coccal forms with well-defined cell walls (x 58,500). M-5 and for the HLA-DR antigen. Most of these cells resembled keratocytes by light microsco py. These results might be explained by the alteration of resident keratocytes into cells ex pressing the macrophage markers OKM1 and Leu M-5 but not Mo-2. Keratocytes do express the HLA-DR antigen when stimulated by gamma interferon. 19 However, keratocyte or fibrocyte expression of OKM1, Leu M-5, or Mo-2 has not been reported, and our experi ence in staining numerous corneas with nonparasitic pathologic conditions with OKM1 does not support this concept (unpublished data). Neutrophils are strongly stained by the
OKM1 antibody; however, few are positive with anti-Leu M-5.12 Additionally, endogenous peroxidase activity was limited to 2% to 5% of the stromal cells. The presence of HLA-DR antigen on 98% of the stromal cells also argues against a heavy neutrophil infiltrate in these two patients, although it is conceivable that absorption of HLA-DR antigen into the cells of the stroma occurred. Few cells stained with the anti-Mo-2 antibody in this material, but sections available for this stain were not adjacent to the ulcer. Electron microscopy confirmed the presence of macrophages in the corneal stroma. We conclude that these immunohistochemical findings indicate a
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Fig. 6 (Mathers and associates). Case 1. Specimen from corneal transplant shows marked bacterial infection. Bacterial aggregates (curved arrow) are present in the superficial stroma. Bowman's layer (asterisk) is disrupted. The peripheral necrotic edge near the graft site shows, an encysted Acanthamoeba organism (arrow) with occasional mononuclear cells and neutrophils (toluidine blue, x550). significant macrophage infiltrate in these two patients. Sampling differences may explain the discrepancy between our routine histopathologic material that suggest a more conspicuous neutrophilic polymorphonuclear cell infiltrate than seen by immunopathologic evaluation. In general, Iymphocytic infiltration in the cornea is closely associated with vascularization. When vascularization is present, Iympho cytic and plasmacytic infiltrates are usually observed mainly in the immediate vicinity of blood vessels in the corneal stroma or in a vascular pannus. Granulomatous inflammation in Acanthamoeba keratitis has been previously described20,21; however, in most reports it has been observed that neutrophils are the predominant cellular infiltrate. Neutrophils are capable of killing the trophozoite form of the amoeba in vitro 22 when assisted by activated mononuclear cells, and they appear to be important in increasing sur vival after experimental intraperitoneal chal lenge. 23 However, an immune response to chronic inflammation can be expected to in volve macrophages, lymphocytes, and macrophage-derived epithelioid cells. Although the
macrophages in our patients were seen to phagocytize Acanthamoeba cysts, they were not effective in recruiting lymphocytes into the cornea. The lack of Iymphocytic infiltration in Case 1 and the sparse Iymphocytic infiltration in Case 2 may indicate ineffective lymphocyte recruitment by the macrophages. Feedback stimulation of macrophages from activated lymphocytes through lymphokines, such as gamma interferon, is important for optimal macrophage/monocyte stimulation, which in turn can stimulate neutrophil function. 2223 The lack of vigorous Iymphocytic infiltration, whether because of lack of recruitment by mac rophages or secondary to a lack of vasculariza tion, may contribute to the failure of the im mune system to eliminate the encysted organisms. Unlike other forms of chronic kera titis that usually develop vascularization with a Iymphocytic and plasma cell infiltrate, no vas cularization was seen in our cases. Blackman and associates 24 suggested that the keratitis in these patients (a neutrophil and macrophage response) results from a secon dary inflammatory response, a result of necro sis of the amoebic organisms and not a pri-
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mary immune response to the organisms. Our finding of ineffective lymphocyte recruitment supports the concept that the organism has the ability to either mask its antigens from the cellular immune system or to suppress the function of infiltrating macrophages. References 1. Centers for Disease Control: Acanthamoeba kera titis associated with contact lenses—United States. M.M.W.R. 35:405, 1986. 2. Epstein, R. J., Wilson, L. A, Visvesvara, G. S., and Plourde, E. G.: Rapid diagnosis of Acanthamoeba keratitis from corneal scrapings using indirect fluorescent antibody staining. Arch. Ophthalmol. 104:1318, 1986. 3. Wilhelmus, K. R., Osato, M. S., Font, R. L., Robinson, N. M., and Jones, D. B.: Rapid diagnosis of Acanthamoeba keratitis using calcofluor white. Arch. Ophthalmol. 104:1309, 1986. 4. Visvesvara, G. S.: Free-living pathogenic amoe bae. In Lennette, E. H., Bellows, A., Hausler, W. J., Jr., and Truant, J. P. (eds.): Manual of Clinical Micro biology, 3rd ed. Washington, D.C., American Socie ty for Microbiology, 1980, pp. 704-708. 5. Jones, D. B., Visvesvara, G. S., and Robinson, H. M.: Acanthamoeba polyphaga keratitis and Acanthamoeba uveitis associated with fatal meningoencephalitis. Trans. Ophthalmol. Soc. U.K. 95:221, 1975. 6. Hirst, L. W., Green, W. R., Merz, W., Kaufmann, C , Visvesvara, G. S., Jensen, A., and Howard, M.: Management of Acanthamoeba kerati tis. A case report and review of the literature. Oph thalmology 91:1105, 1984. 7. Samples, J. R., Binder, P. S., Luibel, F. J., Font, R. L., Visvesvara, G. S., and Peter, C. R.: Acanthamoeba keratitis possibly acquired from a hot tub. Arch. Ophthalmol. 102:707, 1984. 8. Theodore, F. H., Jakobiec, F. A., Juechter, K. B., Pearl, M. A., Troutman, R. C , Pang, P. M., and Iwamoto, T.: The diagnostic value of a ring infiltrate in canthamoebic keratitis. Ophthalmology 92:1471, 1985. 9. Hsu, S. M., Raine, L., and Fanger, H.: The use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. A comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29:577, 1981. 10. Engleman, E. G., Benike, C. J., Glickman, E., and Evans, R. L.: Antibodies to membrane struc tures that distinguish suppressor/cytotoxic and help er T lymphocyte subpopulations block the mixed leukocyte reaction in man. J. Exp. Med. 153:193, 1981. 11. Ledbetter, J. A., Evans, R. L., Lipinski, M.,
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Cunningham-Rundles, C , Good, R. A., and Henzenberg, L. A.: Evolutional conservation of sur face molecules that distinguish T lymphocyte helper/ inducer and cytotoxic/suppressor subpopulations in mouse and man. J. Exp. Med. 153:310, 1981. 12. Schwarting, R., Stein, H., and Wang, C. Y.: The monoclonal antibodies anti-S-HCI-1 (anti Leu-14) and anti-S-HCl-3 (anti Leu M-5) allow the diagnosis of hairy cell leukemia. Blood 65:974, 1985. 13. Abo, T., and Bach, C M . : A differentiation antigen of human NK and K cells identified by a monoclonal antibody (HNK-1). J. Immunol. 127: 1024, 1981. 14. Janossy, G., Tidman, N., Papageriou, E. S., Kung, P. C , and Goldstein, G.: Distribution of T lymphocyte subsets in the human bone marrow and thymus. An analysis with monoclonal antibodies. J. Immunol. 126:1608, 1981. 15. Breard, J., Reinherz, E. L., Kung, P. C , Goldstein, G., and Schlossman, S. F.: A monoclonal antibody reactive with human peripheral blood monocytes. J. Immunol. 124:1943, 1980. 16. Todd, R. F., Ill, Biondi, A., and Roach, J. A.: Human macrophage antigens. In van Furth, R. (ed.): Mononuclear Phagocytes. Boston, Martinus Nijhoff, 1985, p. 31. 17. Reinherz, E. L., Kung, P. C , Pesando, J. M., Ritz, J., Goldstein, G., and Schlossman, S. F.: la determinants on human T cell subsets defined by monoclonal antibody. Activation stimuli required for expression. J. Exp. Med. 150:1472, 1979. 18. Wright, P., Warhurst, D., and Jones, B. R.: Acanthamoeba keratitis successfully treated medical ly. Br. J. Ophthalmol. 69:778, 1985. 19. Young, E., Stark, W., and Prendergast, R. A.: Immunology of corneal allograft rejection. HLA-Dr antigens on human corneal cells. Invest. Ophthal mol. Vis. Sci. 26:571, 1985. 20. Hanssens, M., de Jonckheere, J. F., and de Meunynck, C : Acanthamoeba keratitis. A clinicopathological case report. Int. Ophthalmol. 7:203, 1985. 21. Witschel, H., Sundmacher, R., and Seitz, H. M.: Amoeben-keratitis, Ein kinisch-histopathologischer Fallbericht. Klin. Monatsbl. Augenheilkd. 185:46, 1984. 22. Ferrante, A., and Abell, T. J.: Conditioned medium from stimulated mononuclear leukocytes augments human neutrophil-mediated killing of a virulent Acanthamoeba sp. Infect. Immun. 51:607, 1986. 23. Markowitz, S. M., Sobieski, T., Martinez, A. J., and Duma, R. J.: Experimental Acanthamoeba infections in mice pretreated with methylprednisolone or tetracycline. Am. J. Pathol. 92:733, 1978. 24. Blackman, H. J., Rao, N. A., Lemp, M. A., and Visvesvara, G. S.: Acanthamoeba keratitis suc cessfully treated with penetrating keratoplasty. Sug gested immunogenic mechanisms of action. Cornea 3:125, 1984.
In order to evaluate the local cellular immune response to Acanthamoeba infection we performed immunohistochemical examinations of the corneal buttons of two patients with Acanthamoeba keratitis. We found that the corneal stroma was infiltrated with polymorphonuclear leukocytes and HLA-DR positive macrophages that appeared to be stromal keratocytes by light microscopy. Despite the presence of chronic inflammation in both patients, no stromal lymphocytes were seen in one patient and a sparse lymphocytic infiltrate was seen in the other patient. Electron microscopy confirmed the presence of macrophages, neutrophils, and Acanthamoeba organisms in these two patients. MORE THAN 50 cases of Acanthamoeba keratitis
have been described since 1974 including 24 cases reported to the Centers for Disease Con trol in 1986.1 The organism is difficult to culture from clinical specimens and in many cases the diagnosis is not made or even suspected before keratoplasty. Recent reports of rapid diagnosis
Accepted for publication Jan. 27, 1987. From the Cornea Service, Center For Sight, George town University (Drs. Mathers, Stevens, Gold, and Lemp); Laboratory of Pathology (Dr. Rodrigues) and Laboratory of Immunology (Drs. Stevens and Chan), National Eye Institute; Protozoal Diseases Branch, Cen ter for Infectious Diseases, Centers for Disease Control (Dr. Visvesvara); and Department of Pathology, George town University and Department of Ophthalmic Pa thology, Armed Forces Institute of Pathology (Dr. Zimmerman). This study was supported by an unrestricted grant from Research to Prevent Blindness, Inc. (Dr. Lemp). This study was presented in part at the annual meeting of the Ocular Microbiology and Immunology Group, Nov. 8, 1986, in New Orleans, Louisiana. Reprint requests to William Mathers, M.D., Universi ty of Arizona, Department of Ophthalmology, 1501 N. Campbell St., Tucson, AZ 85724.
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using immunofluorescent antibody 2 and calcofluor white 3 techniques suggest future im proved diagnostic accuracy. Until recently Acanthamoeba infections were most prominent in immunosuppressed indi viduals who developed fatal encephalitis. 4,5 The mechanism by which Acanthamoeba is able to cause progressive disease in normal hosts is unclear. Acanthamoeba keratitis is frequently present for several months before keratoplasty is performed, yet histologic examination of cor neal buttons has usually shown only acute inflammatory cells. 68 We recently examined two patients with Acanthamoeba keratitis in which we used immunohistochemical stains and transmission electron microscopy to examine the inflamma tory cells and organisms found in these infect ed corneas.
Case Reports Casel A 62-year-old man had a two-month history of pain and irritation with slight discharge in the right eye. He had worn extended wear contact lenses for the previous 4V2 years, but for two months had been unable to wear a contact lens in the right eye because of irrita tion. He admitted to wearing contact lenses under goggles while swimming in a public pool; however, he denied ocular pain until two months after stopping contact lens wear be cause of intolerance. He had recently been treated with topical vidarabine and prednisolone acetate 1% for a presumed herpes simplex keratitis. At our initial examination his best corrected visual acuity was R.E.: 20/200. There was a superficial punctate keratitis with irregu lar fluorescein staining in the central cornea
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Fig. 1 (Mathers and associates). Case 1. Top left, Paracentral ring infiltrate (arrow) of the right cornea. Top right, Corneal biopsy demonstrates cyst-like organisms in the stroma (hematoxylin and eosin, x250). Bottom left, Transplanted corneal tissue shows central crystalline opacities with a fern-like pattern.
and a ring of stromal opacification in the midcornea. His antiviral medication was slowly discontinued over the next several weeks. Two months later his visual acuity had de creased to counting fingers in the right eye, and there was an epithelial defect over the midcornea with a surrounding ring of stromal infil tration (Fig. 1, top left). Acanthamoeba keratitis was suspected, but no growth was obtained on scrapings of the stroma cultured on nonnutrient agar with Escherichia coli. Three days later his symptoms and stromal infiltrate had worsened. A 1.5 x 0.5 x 0.3-mm wedge corneal biopsy of the ring of infiltrate was performed. Light microscopy demonstrated numerous
Acanthamoeba cysts (Fig. 1, top right) and cul ture yielded motile trophozoites. The patient was treated with neomycin oph thalmic ointment hourly and propamidine isethionate eyedrops every four hours. The disease progressed despite therapy and a pene trating keratoplasty was performed one week later. Acanthamoeba cysts were identified in the corneal button as well as in culture. Three weeks after keratoplasty the eye devel oped increasing pain and redness. Crystalline stromal opacities with a fern-like pattern were present (Fig. 1, bottom left). Streptococcus viridans was cultured from scrapings. After four days of topical treatment with fortified ce-
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fazolin and gentamicin the transplant was re peated. Intrastromal colonies of gram-positive bacteria were present, but there was negligible infiltration by inflammatory cells. Rare Acanthamoeba cysts were found on light microscopic examination of material prepared for electron microscopy; cultures were negative for both Acanthamoeba and bacteria. The patient's cornea remained clear after discontinuing all medica tions except propamidine isethionate and topi cal corticosteroid daily. Ten weeks after the second transplant he developed a second pseudocrystalline keratopathy accompanied by fibrinous exudate in the anterior chamber. Cul tures were negative but the infiltrate cleared with the administration of topical cefazolin and gentamicin. A subsequent wound leak necessi tated repeat corneal grafting and the button demonstrated edema without bacteria or Acanthamoeba. The patient's graft remained clear for six weeks after the transplant while on a regi men of topical cefazolin until a deep infiltrate developed at the wound margin under intact epithelium. Cultures demonstrated Pseudomonas aeruginosa, which cleared with tobramycin therapy, and a subsequent wound leak led to his fourth corneal transplant. Case 2 A 35-year-old woman was examined because of a one-month history of pain, photophobia, and increased tearing in the right eye. She had worn soft contact lenses successfully for the previous 13 years. Visual acuity in the right eye was hand motion, and a central epithelial de fect was present with a paracentral ring of stromal infiltrate and a 1-mm hypopyon. Scrap ings of the corneal lesion and a paracentesis showed no organisms, and routine cultures were negative. The patient was treated with fortified cefazolin and gentamicin eyedrops as well as natamycin 5% applied topically. One week later the hypopyon had nearly resolved although the corneal haze persisted. The cornea was cultured again for Acanthamoeba, and although the plates initially showed no growth, they were subsequently reexamined and found to contain Acanthamoeba. Two weeks later when the lesion failed to improve, a corneal biopsy was performed. Although the small specimen proved inadequate for histologic examination, Acanthamoeba was cultured from the tissue. The patient was treated unsuc cessfully with topical propamidine isethionate every two hours as well as neomycin ointment. A penetrating keratoplasty was performed 12 days later. Histologic examination of the corne
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al button showed numerous cysts, primarily in the outer stromal layers beneath the areas of epithelial ulceration. Postoperatively the graft has remained clear for six months.
Material and Methods Light microscopy—The corneal buttons were divided into three portions. Tissue for routine light microscopy was fixed in 10% formalin and paraffin-embedded sections were stained with hematoxylin and eosin and periodic acid Schiff. Immunohistochemical staining—Portions of recipient buttons were snap frozen in optimal cutting temperature compound, sectioned at 6 ixm, and stored at - 4 C for 48 hours before staining. Slides were air dried, fixed in acetone, and stained by indirect immunoperoxidase technique using the avidin-biotin-peroxidase complex method. 9 Slides for endogenous peroxidase were processed with light fixation and without addition of primary antibody. Slides were developed with diaminobenzidine and hydrogen peroxide. Counterstain was per formed with 1% methyl green. Mouse hybridoma monoclonal antibodies (Table) were used at a 1:40 dilution in Tris buffered saline. Antibodies to T lymphocytes include anti-Leu 2a (suppressor/cytotoxic sub set), 10 anti-Leu 3a (helper/inducer subset), 10 and anti-Leu 4 (pan T cell).11 The anti-Leu 14 anti body is specific for B lymphocytes 12 and antiLeu 7 for natural killer cells. 13 The OKT 614 is a thymocyte marker that also stains Langerhans cells of the epithelium. The anti-OKMl anti body stains cells of bone marrow derived monocyte lineage, and neutrophils. 15 Anti-Leu M-5 stains most macrophages and few neutro phils. 12 Anti-Mo-2 stains monocytes but no neutrophils. 16 Anti-HLA-DR stains cells that express a major histocompatibility complex Class II antigen. 17 Class II antigen expression has been noted on numerous cell types includ ing macrophages and stimulated keratocytes but does not occur on neutrophils. 16 Endoge nous peroxidase activity occurs in neutrophils and is weak in macrophages. Immunofluorescent staining—To determine the species of Acanthamoeba, we deparaffinized, dehydrated, and incubated paraffin sections with 1:20 and 1:100 dilutions of rabbit antiAcanthamoeba sera against A. castellanii, A. polyphaga, and A. culbertsoni. Transmission electron microscopy—For elec-
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TABLE IMMUNOHISTOCHEMICAL STAINS USED PERCENTAGE OF CORNEAL STROMAL CELLS STAINED, NEAR ULCER/DISTANT FROM ULCER* ANTIBODY
SPECIFICITY
PATIENT 1
PATIENT!
Mouse ascites None
Negative control Neutrophils (endogenous peroxldase) Monocytes/neutrophils15 (C3b receptor) Monocytes, neutrophils, Langerhans cells12 Class II histocompatibility antigen17 Monocytes16 T cell, suppressor/ cytotoxic subset10 T cell, helper subset10 Pan T cell marker11 Thymocyte marker14 Natural killer cells13 B lymphocytes12
2
2
2
2
80/60
100/20
40/10
80/30
98/98
100/60
NT/14 0
NT/5 0
0 0 0 0 0
8/1 10/3 0 0 0
OKM11 Leu M-5' HLA-DR* Mo-2* Leu 2a* Leu 3a* Leu 4* OKT6* Leu 7* Leu 14*
*NT, not tested. Ortho Diagnostic Systems. 'Becton Dickinson. f
tron microscopy, representative portions of corneal tissue were fixed in 2.5% glutaraldehyde, post fixed in 1% osmium tetroxide, dehy drated in ascending concentrations of alcohols, and embedded in epoxy resin. Sections 1 |xm thick were stained with toluidine blue. Thin sections were stained with uranyl acetate and lead citrate.
Results Routine light microscopy—In Patient 1, the minute biopsy specimen showed that the cor neal stroma was lightly infiltrated by polymor phonuclear leukocytes. Many Acanthamoeba cysts were present. Examination of the first keratoplasty specimen showed that the epithe lium was largely missing, and what remained was irregularly thinned and detached. Bow man's layer was generally intact but immedi ately beneath it the stroma was lightly but diffusely infiltrated by polymorphonuclear leu
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kocytes, as were the middle stromal layers. The deeper layers were more minimally infiltrated, but a layer of leukocytes also adhered to the endothelial surface. Amoebic cysts were irregu larly distributed throughout the corneal stroma but were more numerous in the superficial and middle layers. No vascularization or significant infiltration by mononuclear inflammatory cells was apparent. The second keratoplasty speci men showed superficial intrastromal colonies of gram-positive cocci with elongated tapered ends dissecting the adjacent stromal lamellae, but there were few inflammatory cells reacting to the bacteria. In Patient 2, examination of the keratoplasty specimen showed that the corneal epithelium was partially ulcerated, and Bowman's layer was largely absent. Where epithelium covered the button, it represented reepithelialization of previously ulcerated tissue. Acanthamoeba cysts were numerous in the superficial stromal layers in the denuded areas, where the infiltration by polymorphonuclear leukocytes was most in tense and where many of the inflammatory cells appeared degenerated. In this area the nuclei of the cells of the stroma appeared large, ovoid, and intensely basophilic. A light infil trate of mononuclear leukocytes was also pres ent in this area. Elsewhere, the stroma was lightly, but diffusely, infiltrated by polymor phonuclear leukocytes and a few mononuclear cells. No vascularization was observed. A few polymorphonuclear leukocytes were adherent to the degenerated endothelium. Immunopathology—By light microscopy nearly all stromal cells appeared as spindleshaped, keratocyte-like cells. In both Patients 1 and 2, 98% of the cells in the corneal stroma stained for the HLA-DR antigen (Fig. 2, top left and bottom left). OKM1 antibody stained 80% (Case 1) to 100% (Case 2) of the cells near the site of active ulceration but stained only 20% (Case 2) to 60% (Case 1) of stromal cells away from the active sites (Fig. 2, top middle and bottom middle) (Table). Anti-Leu M-5 antibody stained 40% (Case 1) to 80% (Case 2) of cells near the active sites and 10% (Case 1) to 30% (Case 2) elsewhere. Although endothelial kera tic precipitates in fixed sections showed numer ous neutrophils, in the frozen sections the keratic precipitates appeared as mononuclear cells and stained strongly with both antiOKM1, anti-Leu M-5, and anti-Mo-2. To distin guish further between macrophages and neu trophils, endogenous peroxidase activity in stromal cells was limited to 2% to 5% of stromal cells. Staining for the Mo-2 antigen indicated a
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Fig. 2 (Mathers and associates). Immunoperoxidase staining. Top left, Case 1. Anti-HLA-DR antibody (x 170). Top middle, Case 1. Anti-OKMl (x 220). Top right, Case 1. Anti-Leu 4 (x 220). Bottom left, Case 2. Anti-HLA-DR (x 220). Bottom middle, Case 2. Anti-OKMl (x 220). Bottom right, Case 2. Anti-Leu 4 (x330). In both Case 1 and Case 2 most cells of the corneal stroma express the HLA-DR antigen and the macrophage/monocyte marker OKM1. The cornea of Patient 2 demonstrates few T helper lymphocytes; the cornea of Patient 1 does not demonstrate any T helper lymphocytes.
different staining pattern; only 14% of cells in Case 1 and 5% in Case 2 stained positive for Mo-2. No sections that included the ulcer, how ever, were available for the Mo-2 antibody stain. T lymphocytes were not present in the stro ma of Patient 1 (Fig. 2, top right); in Patient 2 (Fig. 2, bottom right) the few T lymphocytes present were primarily of the Leu 3a (helper) subset. No cells of B lymphocyte lineage, natu
ral killer cells, or OKT 6 positive cells were seen. Immunofluorescent identification—Acanthamoeba organisms from Case 1 were identified as A. polyphaga. Organisms in Case 2 stained posi tive for A. castellani. Transmission electron microscopy—In the corneas of Patients 1 and 2 numerous cysts of Acanthamoeba were present, primarily in the superficial half of the stroma (Fig. 3). The outer
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Fig. 3 (Mathers and associates). Case 1. Electron micrograph showing an encysted Acanthamoeba organism with adjacent inflammatory cells. The outer ectocyst layer is focally disrupted (arrows). The organism shows mitochondria, lipid vacuoles, and lysosomes. A neutrophil is indicated (N). Inset shows well delineated cystic organisms in the anterior two thirds of the stroma (arrows) (x 630).
cyst wall of the organisms was often irregularly folded. Some cysts showed a nucleus and prominent nucleolus and cytoplasmic organelles including mitochondria, membranebound lipid vacuoles, and polyribosomes. The cyst wall consists of an inner endocyst layer and an outer ectocyst layer. Some organisms showed well-preserved organelles suggestive of a viable phase; others were disrupted with only cell wall remnants. The disrupted organ elles were primarily found adjacent to inflam matory cells, including neutrophils and macrophages (Fig. 4). In the regraft cornea (Case 1), there were large bacterial aggregates (cocci) in the superficial stroma (Fig. 5). The adjacent
stroma showed scant amoebic cysts and occa sional neutrophils (Fig. 6).
Discussion Acanthamoeba keratitis is a chronic infection that usually occurs in otherwise normal hosts and is associated with contact lens wear or other trauma. Despite the frequently noted vigorous corneal infiltration by neutrophils and a measurable antibody response, 5,18 Acanthamoeba infections frequently progress to corneal opacity or perforation.
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Fig. 4 (Mathers and associates). Case 2. Inset shows encysted amoebic organisms (arrows) in necrotic stroma (toluidine blue, x 500). Electron micrograph shows an encysted organism in corneal stroma. The protoplasm of the amoeba is partly shrunken and contains a nucleus (N), mitochondria, ribosomes, and occasional lysosomes (x 16,500).
Light microscopic examination of the two corneal buttons from our patients disclosed mild to moderate neutrophilic infiltrates. Hematoxylin and eosin stained frozen tissue sec tions adjacent to areas examined with immunohistochemical stains demonstrated fewer neu-
trophils, identifying a sampling difference be tween the routine light microscopic and immunopathologic findings. The immunohistochemical findings in both Cases 1 and 2 indicate an intense infiltration of cells staining for the macrophage markers OKM1 and Leu
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Fig. 5 (Mathers and associates). Case 2. Electron micrograph of stromal bacteria shows coccal forms with well-defined cell walls (x 58,500). M-5 and for the HLA-DR antigen. Most of these cells resembled keratocytes by light microsco py. These results might be explained by the alteration of resident keratocytes into cells ex pressing the macrophage markers OKM1 and Leu M-5 but not Mo-2. Keratocytes do express the HLA-DR antigen when stimulated by gamma interferon. 19 However, keratocyte or fibrocyte expression of OKM1, Leu M-5, or Mo-2 has not been reported, and our experi ence in staining numerous corneas with nonparasitic pathologic conditions with OKM1 does not support this concept (unpublished data). Neutrophils are strongly stained by the
OKM1 antibody; however, few are positive with anti-Leu M-5.12 Additionally, endogenous peroxidase activity was limited to 2% to 5% of the stromal cells. The presence of HLA-DR antigen on 98% of the stromal cells also argues against a heavy neutrophil infiltrate in these two patients, although it is conceivable that absorption of HLA-DR antigen into the cells of the stroma occurred. Few cells stained with the anti-Mo-2 antibody in this material, but sections available for this stain were not adjacent to the ulcer. Electron microscopy confirmed the presence of macrophages in the corneal stroma. We conclude that these immunohistochemical findings indicate a
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Fig. 6 (Mathers and associates). Case 1. Specimen from corneal transplant shows marked bacterial infection. Bacterial aggregates (curved arrow) are present in the superficial stroma. Bowman's layer (asterisk) is disrupted. The peripheral necrotic edge near the graft site shows, an encysted Acanthamoeba organism (arrow) with occasional mononuclear cells and neutrophils (toluidine blue, x550). significant macrophage infiltrate in these two patients. Sampling differences may explain the discrepancy between our routine histopathologic material that suggest a more conspicuous neutrophilic polymorphonuclear cell infiltrate than seen by immunopathologic evaluation. In general, Iymphocytic infiltration in the cornea is closely associated with vascularization. When vascularization is present, Iympho cytic and plasmacytic infiltrates are usually observed mainly in the immediate vicinity of blood vessels in the corneal stroma or in a vascular pannus. Granulomatous inflammation in Acanthamoeba keratitis has been previously described20,21; however, in most reports it has been observed that neutrophils are the predominant cellular infiltrate. Neutrophils are capable of killing the trophozoite form of the amoeba in vitro 22 when assisted by activated mononuclear cells, and they appear to be important in increasing sur vival after experimental intraperitoneal chal lenge. 23 However, an immune response to chronic inflammation can be expected to in volve macrophages, lymphocytes, and macrophage-derived epithelioid cells. Although the
macrophages in our patients were seen to phagocytize Acanthamoeba cysts, they were not effective in recruiting lymphocytes into the cornea. The lack of Iymphocytic infiltration in Case 1 and the sparse Iymphocytic infiltration in Case 2 may indicate ineffective lymphocyte recruitment by the macrophages. Feedback stimulation of macrophages from activated lymphocytes through lymphokines, such as gamma interferon, is important for optimal macrophage/monocyte stimulation, which in turn can stimulate neutrophil function. 2223 The lack of vigorous Iymphocytic infiltration, whether because of lack of recruitment by mac rophages or secondary to a lack of vasculariza tion, may contribute to the failure of the im mune system to eliminate the encysted organisms. Unlike other forms of chronic kera titis that usually develop vascularization with a Iymphocytic and plasma cell infiltrate, no vas cularization was seen in our cases. Blackman and associates 24 suggested that the keratitis in these patients (a neutrophil and macrophage response) results from a secon dary inflammatory response, a result of necro sis of the amoebic organisms and not a pri-
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mary immune response to the organisms. Our finding of ineffective lymphocyte recruitment supports the concept that the organism has the ability to either mask its antigens from the cellular immune system or to suppress the function of infiltrating macrophages. References 1. Centers for Disease Control: Acanthamoeba kera titis associated with contact lenses—United States. M.M.W.R. 35:405, 1986. 2. Epstein, R. J., Wilson, L. A, Visvesvara, G. S., and Plourde, E. G.: Rapid diagnosis of Acanthamoeba keratitis from corneal scrapings using indirect fluorescent antibody staining. Arch. Ophthalmol. 104:1318, 1986. 3. Wilhelmus, K. R., Osato, M. S., Font, R. L., Robinson, N. M., and Jones, D. B.: Rapid diagnosis of Acanthamoeba keratitis using calcofluor white. Arch. Ophthalmol. 104:1309, 1986. 4. Visvesvara, G. S.: Free-living pathogenic amoe bae. In Lennette, E. H., Bellows, A., Hausler, W. J., Jr., and Truant, J. P. (eds.): Manual of Clinical Micro biology, 3rd ed. Washington, D.C., American Socie ty for Microbiology, 1980, pp. 704-708. 5. Jones, D. B., Visvesvara, G. S., and Robinson, H. M.: Acanthamoeba polyphaga keratitis and Acanthamoeba uveitis associated with fatal meningoencephalitis. Trans. Ophthalmol. Soc. U.K. 95:221, 1975. 6. Hirst, L. W., Green, W. R., Merz, W., Kaufmann, C , Visvesvara, G. S., Jensen, A., and Howard, M.: Management of Acanthamoeba kerati tis. A case report and review of the literature. Oph thalmology 91:1105, 1984. 7. Samples, J. R., Binder, P. S., Luibel, F. J., Font, R. L., Visvesvara, G. S., and Peter, C. R.: Acanthamoeba keratitis possibly acquired from a hot tub. Arch. Ophthalmol. 102:707, 1984. 8. Theodore, F. H., Jakobiec, F. A., Juechter, K. B., Pearl, M. A., Troutman, R. C , Pang, P. M., and Iwamoto, T.: The diagnostic value of a ring infiltrate in canthamoebic keratitis. Ophthalmology 92:1471, 1985. 9. Hsu, S. M., Raine, L., and Fanger, H.: The use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques. A comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 29:577, 1981. 10. Engleman, E. G., Benike, C. J., Glickman, E., and Evans, R. L.: Antibodies to membrane struc tures that distinguish suppressor/cytotoxic and help er T lymphocyte subpopulations block the mixed leukocyte reaction in man. J. Exp. Med. 153:193, 1981. 11. Ledbetter, J. A., Evans, R. L., Lipinski, M.,
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Cunningham-Rundles, C , Good, R. A., and Henzenberg, L. A.: Evolutional conservation of sur face molecules that distinguish T lymphocyte helper/ inducer and cytotoxic/suppressor subpopulations in mouse and man. J. Exp. Med. 153:310, 1981. 12. Schwarting, R., Stein, H., and Wang, C. Y.: The monoclonal antibodies anti-S-HCI-1 (anti Leu-14) and anti-S-HCl-3 (anti Leu M-5) allow the diagnosis of hairy cell leukemia. Blood 65:974, 1985. 13. Abo, T., and Bach, C M . : A differentiation antigen of human NK and K cells identified by a monoclonal antibody (HNK-1). J. Immunol. 127: 1024, 1981. 14. Janossy, G., Tidman, N., Papageriou, E. S., Kung, P. C , and Goldstein, G.: Distribution of T lymphocyte subsets in the human bone marrow and thymus. An analysis with monoclonal antibodies. J. Immunol. 126:1608, 1981. 15. Breard, J., Reinherz, E. L., Kung, P. C , Goldstein, G., and Schlossman, S. F.: A monoclonal antibody reactive with human peripheral blood monocytes. J. Immunol. 124:1943, 1980. 16. Todd, R. F., Ill, Biondi, A., and Roach, J. A.: Human macrophage antigens. In van Furth, R. (ed.): Mononuclear Phagocytes. Boston, Martinus Nijhoff, 1985, p. 31. 17. Reinherz, E. L., Kung, P. C , Pesando, J. M., Ritz, J., Goldstein, G., and Schlossman, S. F.: la determinants on human T cell subsets defined by monoclonal antibody. Activation stimuli required for expression. J. Exp. Med. 150:1472, 1979. 18. Wright, P., Warhurst, D., and Jones, B. R.: Acanthamoeba keratitis successfully treated medical ly. Br. J. Ophthalmol. 69:778, 1985. 19. Young, E., Stark, W., and Prendergast, R. A.: Immunology of corneal allograft rejection. HLA-Dr antigens on human corneal cells. Invest. Ophthal mol. Vis. Sci. 26:571, 1985. 20. Hanssens, M., de Jonckheere, J. F., and de Meunynck, C : Acanthamoeba keratitis. A clinicopathological case report. Int. Ophthalmol. 7:203, 1985. 21. Witschel, H., Sundmacher, R., and Seitz, H. M.: Amoeben-keratitis, Ein kinisch-histopathologischer Fallbericht. Klin. Monatsbl. Augenheilkd. 185:46, 1984. 22. Ferrante, A., and Abell, T. J.: Conditioned medium from stimulated mononuclear leukocytes augments human neutrophil-mediated killing of a virulent Acanthamoeba sp. Infect. Immun. 51:607, 1986. 23. Markowitz, S. M., Sobieski, T., Martinez, A. J., and Duma, R. J.: Experimental Acanthamoeba infections in mice pretreated with methylprednisolone or tetracycline. Am. J. Pathol. 92:733, 1978. 24. Blackman, H. J., Rao, N. A., Lemp, M. A., and Visvesvara, G. S.: Acanthamoeba keratitis suc cessfully treated with penetrating keratoplasty. Sug gested immunogenic mechanisms of action. Cornea 3:125, 1984.