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Supplementary Figure 2 from Eradication of Tumor Colonization and Invasion by a B Cell–Specific Immunotoxin in a Murine Model for Human Primary Intraocular Lymphoma

Human primary intraocular lymphoma (PIOL) is predominantly a B cell–originated malignant disease with no appropriate animal models and effective therapies available. This study aimed to establish a mouse model to closely mimic human B-cell PIOL and to test the therapeutic potential of a recently developed immunotoxin targeting human B-cell lymphomas. Human B-cell lymphoma cells were intravitreally injected into severe combined immunodeficient mice. The resemblance of this tumor model to human PIOL was examined by fundoscopy, histopathology, immunohistochemistry, and evaluated for molecular markers. The therapeutic effectiveness of immunotoxin HA22 was tested by injecting the drug intravitreally. Results showed that the murine model resembles human PIOL closely. Pathologic examination revealed that the tumor cells initially colonized on the retinal surface, followed by infiltrating through the retinal layers, expanding preferentially in the subretinal space, and eventually penetrating through the retinal pigment epithelium into the choroid. Several putative molecular markers for human PIOL were expressed in vivo in this model. Tumor metastasis into the central nervous system was also observed. A single intravitreal injection of immunotoxin HA22 after the establishment of the PIOL resulted in complete regression of the tumor. This is the first report of a murine model that closely mimics human B-cell PIOL. This model may be a valuable tool in understanding the molecular pathogenesis of human PIOL and for the evaluation of new therapeutic approaches. The results of B cell–specific immunotoxin therapy may have clinical implications in treating human PIOL. (Cancer Res 2006; 66(21): 10586-93)

Supplementary Figure 1 from Eradication of Tumor Colonization and Invasion by a B Cell–Specific Immunotoxin in a Murine Model for Human Primary Intraocular Lymphoma

Immunological responses to pathogens are stringently regulated in the eye to prevent excessive inflammation that damage ocular tissues and compromise vision. Suppressors of cytokine signaling (SOCS) regulate intensity/duration of inflammatory responses. We have used SOCS1-deficient mice and retina-specific SOCS1 transgenic rats to investigate roles of SOCS1 in ocular herpes simplex virus (HSV-1) infection and non-infectious uveitis. We also genetically engineered cell-penetrating SOCS proteins (membrane-translocating sequence (MTS)-SOCS1, MTS-SOCS3) and examined whether they can be used to inhibit inflammatory cytokines. Overexpression of SOCS1 in transgenic rat eyes attenuated ocular HSV-1 infection while SOCS1-deficient mice developed severe non-infectious anterior uveitis, suggesting that SOCS1 may contribute to mechanism of ocular immune privilege by regulating trafficking of inflammatory cells into ocular tissues. Furthermore, MTS-SOCS1 inhibited IFN-γ-induced signal transducers and activators of transcription 1 (STAT1) activation by macrophages while MTS-SOCS3 suppressed expansion of pathogenic Th17 cells that mediate uveitis, indicating that MTS-SOCS proteins maybe used to treat ocular inflammatory diseases of infectious or autoimmune etiology.

Purpose To describe the clinical, morphologic, and immunohistochemical features of a case of paranasal natural killer/T-cell lymphoma (NKTL) with ocular involvement. Case report In March 2005 the patient presented with a maxillary sinusitis and upper nasal obstruction. In July she underwent a nasal computed tomography (CT) scan and multiple biopsies of the granulomatous tissue in the nasal fossae. The diagnosis was NK/T non-Hodgkin’s lymphoma nasal type, stage IV A. Afterwards she presented anterior uveitis. In September after the diagnosis of lymphoma the patient underwent a bone marrow biopsy and thoracic and abdominal CT scan. An ophthalmic examination including visual acuity assessment and fundoscopic examination was made. In October she started chemotherapy cycles. Maxillary CT scan and ophthalmic examinations were performed during the cycles. In January 2006 after severe recurrences of panuveitis a diagnostic vitrectomy was performed. Results A diagnosis of T-lymphoma cells in the vitreous was made; the tumor was most likely originating from her paranasal NKTL. The condition of the patient deteriorated rapidly and she expired on February 2006. Conclusions Nasal and paranasal sinus lymphomas are rare, but aggressive diseases with a tendency to invade tissues and spread to CNS, including the eye. Ocular manifestations prior to systemic ones may be useful to monitor the response to therapy.

Human primary intraocular lymphoma (PIOL) is predominantly a B cell–originated malignant disease with no appropriate animal models and effective therapies available. This study aimed to establish a mouse model to closely mimic human B-cell PIOL and to test the therapeutic potential of a recently developed immunotoxin targeting human B-cell lymphomas. Human B-cell lymphoma cells were intravitreally injected into severe combined immunodeficient mice. The resemblance of this tumor model to human PIOL was examined by fundoscopy, histopathology, immunohistochemistry, and evaluated for molecular markers. The therapeutic effectiveness of immunotoxin HA22 was tested by injecting the drug intravitreally. Results showed that the murine model resembles human PIOL closely. Pathologic examination revealed that the tumor cells initially colonized on the retinal surface, followed by infiltrating through the retinal layers, expanding preferentially in the subretinal space, and eventually penetrating through the retinal pigment epithelium into the choroid. Several putative molecular markers for human PIOL were expressed in vivo in this model. Tumor metastasis into the central nervous system was also observed. A single intravitreal injection of immunotoxin HA22 after the establishment of the PIOL resulted in complete regression of the tumor. This is the first report of a murine model that closely mimics human B-cell PIOL. This model may be a valuable tool in understanding the molecular pathogenesis of human PIOL and for the evaluation of new therapeutic approaches. The results of B cell–specific immunotoxin therapy may have clinical implications in treating human PIOL. (Cancer Res 2006; 66(21): 10586-93)

Testicular lymphoma is a rare neoplasm of the testis that is most commonly seen in older patients. It metastasizes preferentially to extranodal sites, including the skin, central nervous system, Waldeyer ring, contralateral testis, and lung. Two case reports of patients with a history of testicular lymphoma who developed involvement of the vitreous and retina are presented. These are interesting cases as the testis, central nervous system, and eye are all immune privileged organs, which may account for occurrence of disease in these sites. Histopathologic examination of diagnostic vitrectomy specimens from both cases showed atypical lymphoid cells with immunoglobulin heavy chain (IgH) gene rearrangements, consistent with the diagnosis of intraocular B-cell lymphoma. The results of a literature review of all reports of ocular involvement with testicular lymphoma are discussed. Patients with testicular lymphoma are at risk for relapse, particularly in the central nervous system. Clinicians should be suspicious for intraocular lymphoma in patients with a history of testicular lymphoma who present with vitritis or retinal lesions.

To determine whether a definite diagnosis of malignant lymphoma can be made from paraffin-embedded archived orbital specimens by gene rearrangement analysis using microdissection and polymerase chain reaction (PCR).Specimens from four patients with histopathologically diagnosed orbital malignant lymphoma were examined. The malignant cells were microdissected off the paraffin-embedded specimens. DNA was extracted from the cells, and the immunoglobulin heavy chain ( IgH) gene was amplified by PCR. Gene rearrangements were detected by using primers for the third framework (FR3A), the second framework (FR2A), and the complementary determining region 3 (CDR3). Translocation of the B-cell lymphoma/leukemia-2 ( bcl-2) gene was also examined.Malignant cells were present on the slides of the paraffin-embedded specimens of three of four cases. The specimens from these three cases showed IgH rearrangements for FR3A, FR2A, and CDR3. A bcl-2-associated translocation was also detected in one case.Gene rearrangement analysis is applicable to paraffin-embedded archived orbital specimens to confirm a diagnosis of malignant lymphoma. The advantage of this method is that only a small specimen is needed because the detection sensitivity is high.

Sex hormones have been associated with the prevalence, susceptibility, and severity of autoimmune disease. Although the exact mechanism is unknown, sex hormones are reported to influence cytokine production, specifically by affecting the balance of Th1 and Th2 effector cells. We evaluated the effect of estrogen, progesterone, and testosterone in autoimmune uveoretinitis (EAU), a rodent model of human ocular autoimmune disease.Lewis rats implanted with either beta-estradiol (estrogen), 5-dihydrotestosterone (5-DHT), norgestrel (progesterone), or estrogen plus progesterone were immunized with the retinal antigen interphotoreceptor retinoid binding protein (IRBP) peptide. Evaluation of EAU was based on histology of the eyes and measurement of peripheral immunological responses of DTH and lymphocyte proliferation to S-antigen. Quantitative RT-PCR was used to measure IFN-gamma and IL-10 mRNA in the eyes.In female rats 5-DHT significantly decreased, estrogen slightly enhanced, but progesterone or estrogen + progesterone did not affect EAU. In contrast, in male rats 5-DHT slightly decreased, estrogen moderately decreased, progesterone did not effect, but, estrogen + progesterone slightly decreased EAU. The results correlated with the ocular levels of Th1 (IFN-gamma) and Th2 (IL-10) cytokine messengers.The data support the hypothesis that sex hormones may affect autoimmune diseases by inducing changes in the cytokine balance. This suggests that sex hormone therapy could be considered as an adjunct to anti-inflammatory agents to treat ocular autoimmune diseases in humans.

Objective To report a case of adult T-cell leukemia/lymphoma (ATL) presenting as a bilateral retinal vasculitis and diagnosed by molecular detection of a rearrangement in the T-cell receptor (TCR) and the presence of the human T-cell lymphotropic virus type 1 (HTLV-1) pol gene in the malignant lymphoid cells. Design Case report. Methods Routine histologic and immunohistochemical analyses were performed on the retinal biopsy specimen before referral to the National Eye Institute. Lymphoid cells associated with granulomatous inflammation infiltrating the retina and surrounding retinal blood vessels were microdissected from the paraffin sections of the retinal biopsy specimen. The polymerase chain reaction (PCR) was performed using primers for the TCR gene and HTLV-1 pol and gag genes. Results Microscopic examination showed a necrotizing granulomatous retinal vasculitis with a predominant T-cell infiltrate detected by immunohistochemistry. Molecular analysis demonstrated a clonal rearrangement of the TCR and the presence of the HTLV-1 pol gene in the microdissected lymphoid cells diagnostic of ATL. Conclusions Necrotizing retinitis and retinal vasculitis are rare manifestations of ATL. Human T-cell lymphotropic virus type 1 infection should be considered in the differential diagnosis of patients from endemic areas who have retinal vasculitis at presentation. This case further demonstrates the usefulness of microdissection and PCR for the diagnosis of ocular disease, including HTLV-1 infection.

Ocular inflammation leads to vision loss through the destruction and scarring of delicate tissues along the visual axis. To identify inflammatory mediators involved in this process, we used real time RT-PCR to quantify the expression of mRNA transcripts of 34 cytokines, 26 chemokines, and 14 chemokine receptors at certain time points during T cell-mediated ocular inflammation. We induced disease by adoptive transfer of Ag-specific Th1 or Th2 cells into recipients expressing the target Ag in their eyes. We also compared the mediator expression patterns seen in adoptive transfer-induced inflammation with that seen in mouse eyes developing experimental autoimmune uveoretinitis. In addition, we used laser capture microdissection to examine chemokine mRNA production by both retinal pigment epithelium cells and infiltrating leukocytes in inflamed eyes. Major findings included the following: 1) Three patterns of expression of the inflammation-related molecules were seen in recipients of adoptively transferred Th cells: preferential expression in Th1 recipients, or in Th2 recipients, or similar expression in both recipient groups. 2) In experimental autoimmune uveoretinitis, the inflammatory mediator expression pattern largely paralleled that seen in Th1-induced disease. 3) Both retinal pigment epithelium and infiltrating leukocytes expressed chemokine transcripts in distinct, but overlapping patterns in inflamed eyes. 4) Interestingly, trancripts of multiple cytokines, chemokines, and chemokine receptors were constitutively expressed in high levels in mouse eyes. Seven of these molecules have not been previously associated with the eye. These data underscore the multiplicity of mediators that participate in the pathogenesis of eye inflammation and point to upstream cytokines as potential therapeutic targets.

Primary intraocular lymphoma, a variant of primary central nervous system lymphoma with ocular involvement, is a large B-cell non-Hodgkin's lymphoma. Some cases of primary intraocular lymphoma have been reported to be associated with microorganisms including Epstein-Barr virus (EBV) and human herpes virus-8 (HHV-8), but not parasites. We analyzed 10 cases of primary intraocular lymphoma using microdissection and PCR. Tumor and normal cells were microdissected from ocular tissue on slides and subjected to PCR for genes from Toxoplasma gondii, EBV, and HHV-8. We detected Toxoplasma gondii, not HHV-8 or EBV, DNA in the lymphoma but not in normal cells of two cases that resembled ocular toxoplasmosis clinically. We speculate that Toxoplasma gondii may play a role in some forms of primary intraocular B-cell lymphoma.

Objective Primary intraocular lymphoma, a non-Hodgkin's lymphoma, is a primary central nervous system lymphoma (PCNSL). Diagnosis is usually made by identifying malignant, large B lymphocytes in the vitreous, eye, brain, and cerebral spinal fluid; however, these cells are few, friable, and difficult to recognize. Recently, clonal heavy chain immunoglobulin (IgH) gene rearrangement and bcl-2 gene translocation have been reported in systemic B-cell lymphoma and are used for the detection of malignant cells and in making a diagnosis. The authors investigated the molecular changes in three eyes and a chorioretinal biopsy specimen of four patients with PCNSL. Design Human tissue study. Materials Five ocular specimens of PCNSL were collected. Intervention The first patient had a diagnostic enucleation of the left eye. The second patient underwent diagnostic chorioretinal biopsy. In the third case, a pair of autopsied eyes with reactive lymphoplasmacytic infiltrates of a patient with acquired immune deficiency syndrome (AIDS) were studied. In the fourth case, an enucleated eye of a patient with AIDS-associated lymphoma was sampled. Main outcome measures The bcl-2 and IgH genes of the lymphoma cells from routine, paraffin-embedded, formaldehyde-fixed, or frozen histologic tissue sections were analyzed using microdissection and polymerase chain reaction (PCR) technique. Results Lymphoma cells obtained from the above four cases showed IgH rearrangement gene in the third framework of the V H region. Bcl-2-associated translocation also was detected in three cases (cases 1, 2, and 4). Conclusion Rearrangement of the IgH gene can serve as a molecular marker for PCNSL. Microdissection allows for procurement and analysis of specific, selected, minute cell populations that are obtained from histologic sections of the complex, heterogeneous tissue. Translocation of IgH and bcl-2, the apoptotic "survival" signal and proto-oncogene, could contribute to the pathogenesis of PCNSL. The combination of microdissection and PCR is a powerful tool for studies of small lesions and cell populations and for understanding disease mechanisms.

The accurate measurement of T-cell receptor (TCR) repertoire changes requires the analysis of a representative sampling of complex T-cell populations. The number and frequency of clonally expanded TCR beta-chain transcripts bearing distinct CDR3 sequences were accurately determined using a simple DNA heteroduplex tracking assay. This method allowed major and minor clonal expansions (> or = 1% of a Vbeta subfamily's transcripts) to be rapidly and reproducibly quantified. Oligoclonal CD8 + cell expansions were detected in all young adults tested, while CD4 + cells generally expressed more polyclonal beta-chain repertoires. The same pattern of CD8 + cells oligoclonality and CD4 + cells polyclonality was observed in asymptomatic HIV-1 infected individuals with high CD4 + cell counts. CD8 + CD45RA + and CD8 + CD45RO + cell fractions both displayed oligoclonal, although distinct, TCR beta chain repertoires while CD8 + cells from umbilical cord blood were generally polyclonal. Oligoclonal CD8 + cell repertoires from young adults were generally stable over a period of weeks, although minor, transient, clonal expansions could also be detected in the absence of symptomatic infections. DNA heteroduplex tracking analysis provided a higher level of sensitivity for the detection of TCR beta chain transcript expansions than CDR3 length (spectrotyping/immunoscope) analysis. DNA heteroduplex tracking of TCR beta-chain transcripts is therefore a simple and sensitive method for assessing the level of clonality and for measuring changes in the TCR beta chain repertoire of different T-cell populations.

Experimental melanin-protein induced uveitis (EMIU) is a T-cell mediated autoimmune uveitis induced by immunization with bovine uveal melanin protein. Gp100, a melanocyte lineage-specific protein, is identified as a human melanoma antigen. A recombinant adenovirus construct encoding gp100 (Ad2CMV-gp100) has been used as a vaccine for cancer therapy. This study examines the effect of Ad2CMV-gp100 on EMIU. To induce EMIU, rats were injected intraperitoneally on day 7 before immunization with ad2CMV-gp100, control adenovirus encoding LacZ (Ad2CMV-LacZ), or no virus. On day 21 after immunization, the right eye was processed for histology and the left eye was analysed for cytokines by quantitative reverse transcriptase-polymerase chain reaction. Western blot analysis showed that uveal melanin-protein contains gp100. In three independent experiments, ocular inflammation was significantly suppressed, and expression of ocular IL-12p40 mRNA was much lower in the rats which received Ad2CMV-gp100 before immunization than in those that received Ad2CMV-LacZ or no virus. No abnormalities developed in rats which received Ad2CMV-gp100 or Ad2CMV-LacZ alone. Therefore, Ad2CMV-gp100 injection prevents the development of EMIU, at least in part, through cytokine regulation.

Eyelid nodules were investigated in a patient with Carney complex who was heterozygous for the most commonly known PRKAR1A-inactivating mutation, c.578delTG. Immunohistochemical studies confirmed the diagnosis of myxoma. Loss of heterozygosity was not present, suggesting that haploinsufficiency alone was responsible for tumorigenesis of this eyelid lesion.

Most primary central nervous system lymphomas (PCNSLs) and primary vitreoretinal lymphomas (PVRLs) are B-cell lymphomas that produce high levels of interleukin (IL)-10, which is linked to rapid disease progression. The IL-10 (-1082) G → A polymorphism (IL-10 SNP) is associated with improved survival in certain non-CNS lymphoma patients. PDCD4 is a tumor suppressor gene and upstream regulator of IL-10. This study examined the correlation between the IL-10 SNP, PDCD4 mRNA expression, and IL-10 expression (at transcript and protein levels) in these lymphoma cells.Single-nucleotide polymorphism (SNP)-typing at IL-10 (-1082) was performed after microdissecting cytospun PVRL cells from 26 specimens. Vitreal IL-10 and IL-6 levels were measured by ELISA. PCNSL cells from 52 paraffin-embedded sections were microdissected and SNP typed on genomic DNA. RT-PCR was performed to analyze expression of IL-10 and PDCD4 mRNA. IL-10 (-1082) SNP typing was performed on blood samples of 96 healthy controls. We measured IL-10 (-1082) SNP expression in 26 PVRLs and 52 PCNSLs and examined its relationship with IL-10 protein and gene expression, respectively.More PVRL patients expressed one copy of the IL-10 ( -1082 ) G → A SNP with the GA genotype compared to controls. The frequencies of the three genotypes (AA, AG, GG) significantly differed in PVRL versus controls and in PCNSL versus controls. In PVRLs, the vitreal IL-10/IL-6 ratio was higher in IL-10 (-1082) AG and IL-10 (-1082) AA patients, compared to IL-10 (-1082) GG patients. IL-10 mRNA expression was higher in IL-10 (-1082) AG and IL-10 (-1082) AA PCNSLs, compared to IL-10 (-1082) GG PCNSLs. No correlation was found between IL-10 and PDCD4 expression levels in 37 PCNSL samples.PVRL and PCNSL patients had similar IL-10 (-1082) A allele frequencies, but genotype distributions differed from healthy controls. The findings suggest that the IL-10 (-1082) A allele is a risk factor for higher IL-10 levels in PVRLs and PCNSLs. Higher IL-10 levels have been correlated with more aggressive disease in both PVRLs and PCNSLs, making this finding an important and potentially clinically significant observation.

To the Editor: Two herpesviruses, human herpesvirus-8 (HHV-8) and Epstein-Barr virus (EBV), are able to contribute to lymphomagenesis in humans.[1][1] HHV-8 is documented not only in a strong association with Kaposi’s sarcoma, but also in a high percentage of primary effusion lymphoma, Castleman

1. Masi AT, Hunder GG, Lie JT, et al. The American College of Rheumatology 1990 criteria for the classification of ChurgStrauss syndrome (allergic granulomatosis and angiitis). Arthritis Rheum 1990;33:1094–1100. 2. Takanashi T, Uchida S, Arita M, et al. Orbital inflammatory pseudotumor and ischemic vasculitis in Churg-Strauss syndrome: report of two cases and review of the literature. Ophthalmology 2001;108:1129–1133. 3. Penfold PL, Wen L, Madigan MC, et al. Triamcinolone acetonide modulates permeability and intercellular adhesion molecule-1 (ICAM-1) expression of the ECV304 cell line: implications for macular degeneration. Clin Exp Immunol 2000;121:458–465. 4. Jonas JB, Kreissig I, Degenring RF. Treatment of oedematous, proliferative and neovascular diseases by intravitreal triamcinolone acetonide. Klin Monatsbl Augenheilkd 2003;220:384–390. 5. Jonas JB, Kreissig I, Degenring R. Intraocular pressure after intravitreal injection of triamcinolone acetonide. Br J Ophthalmol 2003;87:24–27.

PURPOSE. The glucocorticoid-induced TNF-related receptor (GITR) plays a pivotal role in regulating the suppressive function of CD4CD25 regulatory T cells. GITR is also involved in the inhibition of T-cell receptor–induced apoptosis and the upregulation of inducible nitric oxide synthase (iNOS). GITR expression on CD4 T cells has been shown to correlate with the disease course of noninfectious uveitis. The current study was conducted to examine the expression of glucocorticoidinduced TNF-related receptor ligand (GITRL) and its regulation by ocular tissue. METHODS. Immunohistochemistry and confocal immunofluorescence microscopy were performed on human ocular tissue to examine the in vivo protein expression of GITRL. The regulation of GITRL was investigated by culturing retinal pigment epithelium (RPE) with proinflammatory cytokines and performing immunocytochemistry and reverse transcription– polymerase chain reaction (RT-PCR). The in vivo mRNA expression of GITRL was studied by RT-PCR on RNA from microdissected tissue sections. RESULTS. Both immunohistochemistry and confocal immunofluorescence microscopy demonstrated that GITRL was expressed constitutively on RPE and photoreceptor inner segments. Immunocytochemistry demonstrated that in vitro stimulated RPE cells expressed GITRL at the protein level, and RT-PCR showed that GITRL was upregulated at 24 hours by proinflammatory cytokines. Constitutive GITRL mRNA expression in vivo was confirmed by RT-PCR analysis of microdissected tissue. CONCLUSIONS. GITRL is expressed constitutively on RPE and in high levels on photoreceptor inner segments. The upregulation of GITRL by proinflammatory cytokines suggests that GITRL may play an important role in ocular immunity. The high level of constitutive GITRL expression on photoreceptor inner segments suggests that photoreceptors participate in the

Background: In the presence of several coexisting infections, superimposed tissue necrosis or tissue metaplasia, it may be difficult to recognize standard histologic morphology on hematoxylin-eosin slides. Tissue microdissection combined with the polymerase chain reaction (PCR-MD) offers the advantages of high specificity and relative speed. The objective of this study was to describe the use of PCR-MD in identifying potentiating viral co-infection in patients with HIV/AIDS with retinitis and choroiditis. Methods: Eyes from two patients with HIV/AIDS with several ocular infections were studied by a variety of techniques, including standard histologic examination, immunochemistry, electron microscopy and in situ hybridization. PCR-MD was used to identify coexisting viral infections. Results: Histologic examination showed cytomegalovirus retinitis in both cases. Use of PCR-MD allowed the identification of Epstein-Barr virus within a zone of fulminant varicella-zoster virus retinitis in one patient. PCR-MD confirmed the presence of human herpesvirus 8 in the second patient, who had ocular lymphoma. Interpretation: PCR-MD can be used to demonstrate coexisting viral infection in ocular specimens from patients with unusually fulminant courses. Co-infections may contribute to the observed clinical course and should be considered in patients with rapid progression or unusual presentation.

Monocyte chemoattractant protein (MCP)-1 (CCL-2) is a chemokine with chemoattractant properties for monocytes, memory T cells, natural killer cells, mast cells, and basophils. To delineate the role played by MCP-1 in acute anterior uveitis, a common ocular inflammation, MCP-1(-/-) mice and wild-type matched control mice were analyzed for the development of endotoxin-induced uveitis (EIU) in response to subcutaneous injection of a sublethal dose of lipopolysaccharide (LPS).EIU was induced in MCP-1(-/-) and wild-type control mice by a single subcutaneous injection of Salmonella typhimurium LPS endotoxin at day 0. Alternatively, MCP-1(-/-) mice were injected subcutaneously with LPS plus recombinant MCP-1 at day 0 and with recombinant MCP-1 6 hours later. Mice were killed at day 1 or 3 after injection. Serum levels of IL-1alpha, IL-1beta, IL-6, IFN-gamma, TNF-alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein (MIP)-1alpha, MIP-2, regulated on activation normal T-cell expressed and secreted (RANTES), and MCP-1 were determined by ELISA. Eyes were collected and analyzed histologically and by RT-PCR for MCP-1, IFN-gamma, IL-6, TNF-alpha, beta-actin, MCP-5, RANTES, KC, inflammatory protein (IP)-10, and toll-like receptor (TLR)-4.EIU was strongly reduced in MCP-1(-/-) mice compared with wild-type control mice. The number of ocular inflammatory cells was significantly reduced. Moreover, intraocular IFN-gamma transcription was increased. EIU was induced in MCP-1(-/-) mice by co-administration of recombinant rat MCP-1 and LPS.Data indicate that MCP-1 plays a crucial role in the induction of EIU. MCP-1 may be a new therapeutic strategy for acute anterior uveitis.

Sight-damaging ocular inflammation is often mediated by T-helper (Th) lymphocytes. The population of Th cells is divided into two major subsets, designated Th1 and Th2, that differ by their cytokine production and biological activities. In the present study, the capacity of Th1 and Th2 cells to induce ocular inflammation was examined.Ocular inflammation was induced in transgenic (Tg) mice that express hen egg lysozyme (HEL) in their lens, by adoptively transferring Th cells that transgenically express HEL-specific receptor. Th1 and Th2 populations were polarized in vitro, and their selective cytokine production was determined by conventional methods. Levels of ocular inflammation were monitored by conventional histologic methods. Infiltrating cells were collected from sections of inflamed eyes by microdissection, and their cytokine production was examined by RT-PCR.Th1 cells were highly immunopathogenic, producing disease in naive recipients at numbers as low as 0.12 x 10(6), whereas Th2 cells were inactive in these recipients, even at 30 x 10(6). Th2 cells, however, produced inflammation when transferred into sublethally irradiated recipients. Distinctive histopathologic changes characterized ocular inflammation induced by the two types of Th cells. Cytokine analysis of infiltrating cells in recipient mouse eyes, as well as of splenocytes of these mice demonstrated that the transferred cells retained their type specificity. Coinjecting Th2 and Th1 cells did not alleviate the ocular disease in naive recipients and even exacerbated the immunopathogenic process in irradiated recipients.Th2 cells are capable of inducing ocular inflammation, but only in immunodeficient mice, and are profoundly inferior to Th1 cells in their immunopathogenic capacity.

The kinetics of various cytokines in the eye plays a critical role in endotoxin-induced uveitis (EIU). This study examined the cytokine kinetics and susceptibility of EIU in four mice strains.Four strains of TLR-4 or Toll-like receptor-4 (Lps, lipopolysaccharide-susceptible) gene-positive mice (C3H/HeN of H-2(k), C57/B6 of H-2(b), Balb/C of H-2(d), and 129/J of H-2(b)) were injected subcutaneously with either lipopolysaccharide (LPS) in phosphate-buffered saline (PBS) or PBS alone in two repeated experiments. Mice were sacrificed 1, 3, 6, 24 (1 d), 72 (3 d), 120 (5 d), or 168 (7 d) hours after LPS injection. Ocular histology and reverse transcriptase-polymerase chain reaction (RT-PCR) to detect ocular interleukin-1 alpha (IL-1 alpha), IL-6, tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA were performed. Serum IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha levels were measured using enzyme-linked immunosorbent assay (ELISA).No ocular inflammation was present in any mice within six hours after LPS injection. Only the C3H/HeN mice developed a biphasic ocular inflammatory response (1 d and 5 d), during which all proinflammatory cytokine messages were expressed. In the other three strains with minimal (129/J and Balb/C) to mild (C57/B6) EIU that peaked at 1 d, IL-6 mRNA was barely detectable in C57/B6 and Balb/C; GM-CSF mRNA was also present in C57/B6. Serum IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha were high in all EIU mice within six hours after LPS injection. Control mice did not develop uveitis or measurable cytokine messages.In the most susceptible strain, C3H/HeN, EIU was biphasic and correlated to multiple proinflammatory cytokines released in the eye. The less susceptible mice strains exhibited a monophasic response to LPS that may result from no cytokine cascade.

Objective To examine the kinetics and mechanisms of endotoxin-induced uveitis in the mouse. Methods C3H/HeN mice were injected subcutaneously with 0.3 mg of Salmonella typhimurium lipopolysaccharide (LPS) in 0.1 mL of phosphate-buffered saline solution or phosphate-buffered saline solution alone in 3 separate experiments; mice were killed after 1, 3, 5, and 7 days. In 2 other separate experiments, mice were killed 1, 3, 6, and 24 hours after LPS injection. All eyes were collected for histological examination, immunohistochemical analyses, aqueous protein level determination, and reverse transcriptase–polymerase chain reaction for ocular interleukin (IL)1α, IL-6, tumor necrosis factor α, and granulocyte-macrophage colony-stimulating factor messenger RNA (mRNA). Enzyme-linked immunosorbent assay was used to measure tumor necrosis factor α and IL-6 levels in aqueous and serum samples. Results Results were consistent for all experiments. Numbers of ocular inflammatory cells and levels of aqueous protein peaked 1 and 5 days after LPS injection. Control mice did not develop inflammation. Serum and aqueous IL-6 and ocular IL-6 mRNA levels peaked at 1 day and subsided at 3 days. However, ocular IL-1α, tumor necrosis factor α, and granulocyte-macrophage colony-stimulating factor mRNA appeared, peaked, and subsided at 3, 5, and 7 days, respectively. Predominant infiltrating cells were neutrophils at 1 day and macrophages at 5 days. Although no ocular inflammatory cells were detected before 24 hours after LPS injection, tumor necrosis factor α mRNA was noticed at 1 hour, peaked at 3 hours, and disappeared at 6 hours and granulocyte-macrophage colony-stimulating factor mRNA was spotted only at 3 hours after LPS injection. Conclusions The ocular inflammatory response to C3H/HeN mouse endotoxin-induced uveitis is biphasic for 7 days. The first wave appears at day 1 and subsides by day 3. A second, higher peak appears at day 5. The 2 inflammatory waves are related to the kinetics of the different cytokines released in the eye. This is in contrast to the rat monophasic endotoxin-induced uveitis model, which has only one peak of intense inflammation associated with cytokine release. Clinical Relevance A biphasic inflammatory response associated with cytokine release lasting several days is observed in C3H/HeN mice with endotoxin-induced uveitis. Because human anterior uveitis has a tendency to be recurrent in nature, this might be a better experimental model.

PURPOSE: To report metastatic Waldenstrom macroglobulinemia cells with immunoglobulin heavy chain gene rearrangement in the vitreous and the blood. METHODS: A 58-year-old man with Waldenstrom macroglobulinemia developed bilateral vitreitis. Diagnostic vitrectomy was performed on the left eye. The vitreous cells and the peripheral blood lymphocytes were analyzed using microdissection and polymerase chain reaction amplification. RESULTS: Vitrectomy specimen of the left eye contained a few degenerated cells. Molecular analysis showed immunoglobulin heavy chain gene rearrangement at the third complementary determining region of the vitreal infiltrating cells and peripheral blood lymphocytes. CONCLUSIONS: Waldenstrom macroglobulinemia rarely metastasizes to the vitreous. Molecular detection of the immunoglobulin heavy chain gene third complementary determining region rearrangement is helpful in the diagnosis of the malignancy. Microdissection combined with polymerase chain reaction is a useful and innovative tool for molecular pathological investigation.

Experimental melanin protein-induced uveitis (EMIU) is an autoimmune uveitis induced by immunization with uveal melanin protein. Fas and FasL enhancement is reported in rats with EMIU. Tricyclodecan-9-yl-xanthogenate (D609), a specific inhibitor of phosphatidylcholine-specific phospholipase C, inhibits inducible nitric oxide synthase (iNOS) induction. In two independent experiments, 35 Lewis rats with EMIU received either D609 or PBS daily. The eyes and draining lymph nodes were collected for histology, analyses of nitrite, peroxide, and superoxide dismutase, Fas and FasL immunochemistry, in situ hybridization for iNOS mRNA and in situ apoptosis detection at the peak of the disease. Both experiments showed significant inhibition of EMIU by D609. Decreases in nitrite and peroxide, increase of superoxide dismutase and lower expressions of iNOS mRNA were found in D609-treated, as compared to PBS-treated eyes. There was mild enhancement of Fas and FasL in the eyes and lymph nodes of D609-injected animals. DNA fragmentation was increased in the lymph nodes of D609-treated rats. We conclude that iNOS activation is responsible for NO production in eyes with EMIU. The suppressive effect of D609 on EMIU may result from scavenging NO and activating apoptosis previously inhibited by NO along with other anti-inflammatory effects.

Background The diagnosis of primary intraocular lymphoma is difficult in many cases even with conventional cytological tests using vitreous samples. Recently new diagnostic tests, such as microdissection and polymerase chain reaction (PCR) and measurement of cytokines using intraocular samples, have been applied to the diagnosis of the disease. We report here a case where we used the new diagnostic tests and the results aided us to make a diagnosis of intraocular lymphoma. Case A 68-year-old woman with an initial diagnosis of bilateral idiopathic uveitis with steroid-resistant vitreous opacities underwent a vitreous biopsy. The cytological examinations of the vitreous samples revealed class III. The microdissection and PCR using the vitreous samples detected IgH rearrangement gene in the third framework (FR3A), the complementary determining region 3(CDR3) of the V H region and Bcl-2-associated translocation. The interleukin (IL)-10 to IL-6 ratio in the vitreous fluid was greater than 100. Because the results of the examinations strongly suggested intraocular lymphoma, the patient was treated with radiation and chemotherapy. One month after the therapy, however, the patient developed multiple metastatic lesions in the brain. The clinical course of the patient together with the new diagnostic results of examinations led to a diagnosis of intraocular lymphoma. Conclusion A combination of tests, such as conventional cytology, microdissection, and PCR, and cytokine assay using intraocular biopsy samples, is useful to make a diagnosis of intraocular lymphoma.

Objective To evaluate choroidal lesions in histological sections from the enucleated eye of a patient with chronic ocular histoplasmosis syndrome for the presence of Histoplasma capsulatum DNA. Methods Laser-capture microdissection was used to procure cells from macular and midperipheral choroidal lesions in a deparaffinized hematoxylin-eosin–stained section prepared from the enucleated left eye of a patient with an ipsilateral choroidal melanoma and bilateral chronic histoplasmosis syndrome. The captured cells were initially subjected to polymerase chain reaction (PCR) amplification using a pair of primers unique to each end of the nucleotide sequences that are complementary to the DNA known to flank the internal transcribed spacer regions of the ribosomal RNA genes of H capsulatum. This product was then reamplified using a second set of internally situated nested primers. The results were compared with a positive control sample of H capsulatum DNA and with a negative microdissected sample from noninflamed choroid in the same slide. Results Products of H capsulatum DNA were identified in both samples of microdissected tissue and the positive control. They were absent in the negative control. Conclusion The observations provide molecular biological evidence linking the chronic choroidal lesions to earlier infection by H capsulatum .

A rapid and simple method is described for the incorporation of monoclonal antibody coupled with palmitic acid into liposomes prepared by the reverse-phase evaporation method (Szoka, F. and Papahadjopoulos, D. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4194-4198). Palmitoyl antibody in 0.15% deoxycholate is added to a liposome suspension after the majority of the organic solvent has been removed by evaporation. Efficient incorporation (over 80%) of palmitoyl antibody occurred without leakage of the encapsulated drug. Native, unmodified antibody did not incorporate under identical conditions. About 50% of the incorporated antibodies could be readily digested by protease, while most of an internal protein marker was not, suggesting that about half of the antibodies were exposed on the outer surfaces of liposomes. Target-specific binding of antibody-liposomes has also been demonstrated in vitro with the RDM-4 lymphoma cells. This method offers a rapid and highly efficient attachment of functional antibody molecules to liposomes with high capture efficiency of drugs, and therefore should be useful in target-specific delivery of drugs mediated by liposomes.