Oral immunization with an anti-idiotypic antibody to the exoglycolipid antigen protects against experimental Chlamydia trachomatis infection.

Chlamydia trachomatis is the leading cause worldwide of preventable infectious blindness (trachoma) and sexually transmitted disease, including nongonoccocal urethritis and pelvic inflammatory disease. To date, no effective vaccine against C. trachomatis infection has been identified. A monoclonal anti-idiotypic antibody (anti-Id) to the chlamydial exoglycolipid antigen (GLXA) was tested in a murine model of ocular chlamydial infection for its ability to induce systemic immunity, which reduces microbiologic and clinical disease. The anti-Id to GLXA, delivered either systemically in soluble form or orally after encapsulation in poly(lactide) microspheres, induced significant protective immunity against ocular challenge of mice with a human biovar of C. trachomatis. Protection was associated with induction of anti-GLXA antibody and anti-chlamydial neutralizing antibody.

Intraocular tumor suppression of retinoblastoma gene-reconstituted retinoblastoma cells.

Human retinoblastoma is caused by mutational inactivation of the retinoblastoma suppressor gene (RB). We have examined intraocular tumorigenicity of retinoblastoma cells in which RB expression was achieved by retroviral transduction. Retinoblastoma cells were injected into the anterior chambers of severe combined immunodeficient mouse eyes, and tumorigenicity was assessed. RB-expressing retinoblastoma cells usually failed to form progressive tumors in the anterior chamber, whereas the parental, RB-negative line, WERI-Rb27, was rapidly tumorigenic. These results support the hypothesis that inactivation of the RB gene is critical for the growth of retinoblastoma tumors. The potential use of RB reconstitution for treating human retinoblastoma is suggested by our finding that intraocular tumor growth can be suppressed by RB expression.

Intracellular location of inapparently infecting Chlamydia in synovial tissue from patients with Reiter's syndrome.

Culture of Chlamydia trachomatis from synovial tissues/fluids from Reiter's syndrome (RS) patients frequently yields negative results. However, we have identified chlamydial RNA at that site in such patients, suggesting that viable organisms may be present. Here we define the cellular location of chlamydia within the synovium via in situ hybridization. Using a chlamydial ribosomal RNA-directed probe, we show that synovial tissue from culture-negative RS patients gives strong hybridization which is often localized to a subsynovial cell layer, rather than to the synovial lining; in some cases, hybridizing cells are dispersed through the synovium. All hybridization signal is located within host cells, indicating that infectious extracellular elementary bodies are rare or absent. These data confirm the extensive intracellular presence of inapparent chlamydia in the synovia of RS patients and provide some insight into the usual culture negativity of synovial tissues for the organism.

Modulation of immune-associated surface markers and cytokine production by murine retinal glial cells.

Murine retinal glia are normally negative for major histocompatibility complex (MHC) Class II antigens and express low levels of MHC Class I and intercellular adhesion molecule-1 (ICAM-1) as detected by avidin-biotin-peroxidase immunohistochemistry. These surface molecules associated with immune function were either induced (Class II) or upregulated (Class I and ICAM-1) on cultured retinal glial cells in a dose- and time-dependent manner following exposure to recombinant interferon-gamma (rIFN-gamma). MHC Class I and II expression by passaged and primary cells was maximal (> 90% positive) after incubation with 100 U/m1 of rIFN-gamma for 48 h. ICAM-1 expression by primary and passaged cells tripled between 48 and 72 h after exposure to 25 or 50 U/m1 of rIFN-gamma. By 72 h after exposure to 100 U/m1 of rIFN-gamma, 62% of the retinal glia were positive for ICAM-1, whereas under normal culture conditions these molecules were detected on < 3% of the retinal glia. Bacterial lipopolysaccharide (LPS), a known stimulator of central nervous system (CNS) astrocytes, increased ICAM-1 expression only 3-fold to 9% of cells staining positively, but neither MHC Class I nor Class II expression was altered from baseline levels. Surface expression of ICAM-1, MHC Class I, and MHC Class II was unaffected by exposure to either rTNF-alpha (1000 U/m1) or rIL-6 (100 U/m1) for 24 h. Under normal culture conditions, intracellular interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were detected immunohistochemically. Exposure to either rIFN-gamma or LPS induced more intense staining which correlated with increased secreted levels of both cytokines in culture supernatants. Levels of secreted TNF-alpha increased 6-fold after stimulation with LPS for 24 h, while secreted IL-6 increased over 9-fold. These results support the hypothesis that retinal glia may participate in intraretinal immune processes following stimulation during inflammatory and infections processes via either cell surface-or soluble mediator-dependent mechanisms or a combination of both.

Immunologic modulation of virus-induced pathology in a murine model of acute herpetic retinal necrosis.

Unilateral inoculation of herpes simplex virus Type 1 (KOS strain) into the anterior chamber of BALB/c eyes produces an ocular disease with a distinctive differential pattern of retinal pathology. Specifically, the retina of the inoculated eye remains histologically intact, whereas the contralateral retina becomes necrotic. We demonstrate that retinal necrosis in opposite uninjected eyes directly correlates with the presence of herpes simplex viral antigens, whereas the intact retinas of virus-injected eyes are devoid of immunocytochemically detectable viral antigens. Immunosuppression or lack of a thymus results in bilateral retinal necrosis, with positive immunoperoxidase staining for viral antigens in both eyes. We have shown previously that retinal protection in both eyes can be restored to irradiated recipients by adoptive transfer of spleen cells from mice primed by AC injection of HSV. Our results with reconstituted and normal mice suggest that virus-mediated cytopathic effects underlie contralateral retinal necrosis since HSV antigens are localized to areas of retinal necrosis and their presence precedes the local inflammatory response; immunosuppression does not alter the development of contralateral retinal necrosis. They also indicate that ipsilateral retinal preservation reflects T cell-mediated inhibition of viral spread to retinas of injected eyes. Reconstitution of irradiated recipients with AC primed donor cells prevents immunohistochemically detectable virus and retinal necrosis in both eyes. In all experimental groups we failed to detect viral antigens in the absence of retinal pathology.

In vitro susceptibility of newborn murine retinal cells to herpes simplex virus type 1 infection.

We have investigated the in vitro susceptibility of murine neural retinal cells to infection by herpes simplex virus Type 1 (HSV-1). Retinal cells obtained from newborn C57B1/6 mice were cultured for 6 days and infected with varying doses of HSV-1. Infection was determined by ABC immunoperoxidase staining of fixed cultures for HSV-1 antigens. Retinal neurons, including amacrine cells, were highly susceptible to infection, with 100% of the multipolar neurons expressing viral antigens after 12 hr of infection. Glial cells and retinal pigment epithelial (RPE) cells also were 100% infected within 12-16 hr. Photoreceptor infection was not as fast, but all surviving photoreceptor cells and their precursors became infected by 24-48 hr postinoculation. Since embryonic chick photoreceptors are highly resistant to HSV-1, these results demonstrate that mammalian (murine) photoreceptor cells differ from avian photoreceptor cells in their susceptibility to in vitro HSV-1 infection. In addition, our current results suggest that the in vivo resistance of adult C57B1/6 mice to herpetic retinitis may not reside at the level of the individual retinal cell populations, although apparent differences in susceptibility exist among the various retinal cell subpopulations.

An immunogenetic analysis of resistance to herpes simplex virus retinitis in inbred strains of mice.

Specific inbred strains of mice have been shown to vary considerably in their resistance and susceptibility to herpes simplex virus (HSV) infection. We injected 2 X 10(5) plaque forming units (PFU) of the KOS strain of HSV-1 intracamerally into one eye of BALB/c, C57Bl/6, and F1 (BALB/c X C57Bl/6) mice. HSV-1 antigens were localized in frozen sections of enucleated eyes at 10 to 14 days post-inoculation. Injected eyes of BALB/c mice showed an anterior uveitis with HSV-1 antigens in the anterior segment and an intact retina free of HSV antigens. The retina of the contralateral uninjected eye was necrotic and contained HSV-1 antigens. In both C57Bl/6 and F1 mice, HSV antigens were limited to anterior segment structures in the injected eye, whereas, in contrast to BALB/c mice, the contralateral retina appeared histologically normal and contained no viral antigens. The C57Bl/6 and F1 strains remained relatively resistant to retinal infection even if pretreated with up to 800 Rads of irradiation. The retinas of normal or sublethally irradiated C57Bl/6 and F1, but not BALB/c strains, were also resistant to intravitreal injection of HSV. These results suggest that resistance to HSV retinitis is a dominantly inherited trait, which depends only partly upon immunologic factors and may be heavily influenced by the inherent ability of host cells from different murine strains to support a productive viral infection.

The dichotomy between herpes simplex virus type 1-induced ocular pathology and systemic immunity.

Herpes simplex virus Type 1 (HSV-1) was injected into the mouse eye by the intravitreal (into the vitreous chamber; VC) or translimbal (across the limbus into the anterior chamber; TxL) route. These routes were compared for ocular pathology and systemic immunity. After VC inoculation, the virus-injected eye developed a viral infection, with the majority of opposite, uninjected eyes remaining intact and free of virus over the 4 week period of observation. In contrast, following translimbal inoculation, the entire virus-injected eye developed infection and inflammation together with subsequent chorioretinitis in the opposite uninjected eye. Systemic immunity induced by VC or TxL virus inoculation was similar to the effects of anterior chamber (AC) inoculation of the same dose of HSV-1: T cell-mediated DTH responses were suppressed while levels of anti-HSV neutralizing antibody were enhanced, compared to subcutaneously primed positive control mice. These findings demonstrate that HSV-1-induced ocular pathology does not necessarily correlate directly with systemic immunity.

Herpes simplex virus type 1 alters transcript levels of tumor necrosis factor-alpha and interleukin-6 in retinal glial cells.

PURPOSE: Studies were performed to determine whether retinal Müller cells transcribe genes for the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF alpha). Isolated murine retinas were used to test whether these cytokines were upregulated in the retina in vivo after anterior chamber inoculation of herpes simplex virus type 1 (HSV-1). The effects of exposure to HSV-1 or interferon-gamma (IFN gamma) on transcript levels of these cytokines in cultured retinal glia also were examined. METHODS: In situ hybridization (ISH) using digoxigenin (DIG)-labeled RNA probes was used to localize mRNA for IL-6 and TNF alpha in cultured retinal glial cells. Changes in IL-6 and TNF alpha relative transcript levels were assessed in cultured retinal glial cells using a semiquantitative approach comprised of reverse transcription-polymerase chain reaction (RT-PCR) assay at low amplification cycle number followed by slot blotting and hybridization with DIG-labeled internal sequence probes. In the murine model of herpetic retinitis, the same methods were used to compare temporal changes in relative cytokine transcript levels in retinas isolated from eyes 1 to 7 days after anterior chamber injection of live HSV-1 (KOS strain; 2 x 10(4) pfu/eye) or buffer with levels in retinas isolated from normal, uninjected eyes. Densitometry was used to quantify relative signal changes obtained with serial diluted samples in slot blot assays. Cytokine signal was normalized to hypoxanthine phosphoribosyl transferase signal obtained from the same cDNA samples. RESULTS: Under baseline culture conditions, ISH and RT-PCR indicated that both IL-6 and TNF alpha were transcribed by cultured retinal glia. In vitro exposure to either viral (HSV-1) or inflammatory (IFN gamma) stimulants increased levels of these transcripts in a time-dependent manner. Peak TNF alpha mRNA levels were detected 4 hours after exposure to HSV, whereas IL-6 peaked 4 hours later (increases of 10.3 and 8.7 times over baseline, respectively). Differential increases in TNF alpha and IL-6 transcript levels were detected in retinas isolated from BALB/c mice that received anterior chamber injections of either HSV-1 or Hanks' balanced salt solution (HBSS). By day 3 after HSV-1 injection, increases of 4.5-fold in TNF alpha and 17-fold in IL-6 were detected, whereas substantially smaller changes in TNF alpha and IL-6 (1.5-fold and 6.3-fold, respectively) were observed in HBSS-injected eyes Virus-induced changes in TNF alpha mRNA levels occurred slightly earlier than for IL-6 because maximal levels of TNF alpha were detected 2 to 3 days after infection, but IL-6 peaked at day 3. CONCLUSIONS: Cultured retinal glial cells exhibit upregulated TNF alpha and IL-6 transcript levels after exposure to virus or inflammatory mediators. HSV-1 infection of the anterior segment of the mouse eye markedly upregulates TNF alpha and IL-6 mRNA levels compared to smaller responses to nonspecific inflammation. Taken together, these results identify retinal Müller cells as an intraretinal source of TNF alpha and IL-6 and support the potential of these resident cells to act as intraretinal modulators of immune and inflammatory responses.

Murine model of ocular infection by a human biovar of Chlamydia trachomatis.

PURPOSE: A human biovar of Chlamydia trachomatis was used to develop a murine model of ocular chlamydial infection. The inbred mouse model will allow detailed immunologic studies during ocular infection, and use of a human biovar for infection may aid in identification of appropriate vaccine strategies against chlamydial infections. METHODS: BALB/c, C3H/HeN, and C57B1/6J mice (n = 5 to 10 mice/group) were topically infected in the conjunctiva with C serovar of C. trachomatis. The effects were tested of single and repeated infection with 5000 inclusion-forming units (IFU) in 5 microliters and different inoculum doses. Conjunctival surfaces of both eyes were swabbed for microbiologic signs (isolation culture or direct fluorescent antibody staining) of infection over 4 to 6 weeks. Conjunctivae were removed for histopathologic study, and lymphocytes from draining cervical lymph nodes and spleens were tested for chlamydia-specific proliferative responses. Serum was obtained from all mice and tested for anti-chlamydial antibodies. RESULTS: BALB/c and C3H/HeN mice developed dose-dependent microbiologic, histopathologic, and immunologic evidence of ocular infection. Eyes of mice were culture-positive from day 7 through at least day 21, with the peak of infection at days 10 to 14 after infection. Histopathologically, the development of conjunctival subepithelial mononuclear infiltration, exudate, and loss of goblet cells occurred within 1 week. Dose-dependent lymphoproliferative responses to whole chlamydial elementary bodies were observed; anti-chlamydial antibody was detected by immunoblotting only in infected mice. CONCLUSIONS: Several strains of inbred mice are susceptible to human chlamydial biovars and may provide a useful alternative disease model in which to study the immunopathogenesis of ocular chlamydial infection and test of vaccine candidates derived from clinically relevant human biovars.

Immunohistochemical study of the local inflammatory response to chlamydial ocular infection.

Immunohistochemical staining of conjunctival biopsies from cynomolgus monkeys (Macaca fascicularis) was performed after they received a single primary ocular infection, a single secondary challenge infection, or repeated ocular inoculations with Chlamydia trachomatis. T cells of the suppressor/cytotoxic (OKT8F) phenotype predominated regardless of the infection protocol, and perifollicular T lymphocytes of both the suppressor/cytotoxic and helper (OKT4A) phenotypes appeared in large numbers during the peak inflammatory reaction. In repeatedly inoculated monkeys, T cells and follicles persisted until cessation of reinfection. IgM-bearing B lymphocytes comprised the majority of cells within follicles, with smaller numbers of IgG- or IgA-positive B cells. The major difference in the response to the various infection protocols was the increased number and persistence of follicles with repeated reinoculation. The finding of large numbers of T-suppressor/cytotoxic and T-helper cells in the infected conjunctiva supports a role for cell-mediated immunity in the local response to C. trachomatis ocular infection.

Cytokines in autoimmune lacrimal gland disease in MRL/MpJ mice.

PURPOSE: MRL/MpJ-+/+ (MRL/+) and MRL/MpJ-lpr/lpr (MRL/lpr) mice show spontaneous development of a T-cell-driven lacrimal gland inflammation that is a model for Sjögren syndrome. The lacrimal gland lesions in these mice were evaluated by quantitative RT-PCR for selected cytokine mRNA for the relative contributions of T-helper (Th)1 versus Th2 immune responses and by RT-PCR and immunohistochemistry for the contribution of the interleukin (IL)-2/IL-2 receptor (IL-2R) autocrine pathway. METHODS: RNA was isolated from lacrimal glands of MRL/+ mice ages 1 to 9 months and from MRL/lpr mice ages 1 through 5 months, and competitive RT-PCR was used to quantify mRNA for the cytokines IL-2, -4, -10, and -12 and interferon (IFN)-gamma. Frozen sections of lacrimal glands from MRL/+ and MRL/lpr mice ages 2 through 5 months were stained for the IL-2R. RESULTS: IL-2 and -12 mRNA transcripts were below the limit of detection (<10(-3) fg/pg hypoxanthine phosphoribosyl transferase gene; HPRT) in both MRL/+ and MRL/lpr mice of all ages. When detectable, IFN-gamma transcripts were present in low amounts and were below the limit of detection in most samples. IL-4 transcripts were present in 100- to 1000-fold greater amounts than IFN-gamma transcripts. IL-10 transcripts were detectable in both MRL/+ and MRL/lpr mice. IL-2R typically was detected on less than 10% of lymphocytes infiltrating lacrimal gland lesions in both substrains. CONCLUSIONS: On the basis of RT-PCR for cytokine mRNA, autoimmune lacrimal gland lesions in MRL/+ and MRL/lpr mice appear to be largely Th2-mediated. There does not appear to be a direct role for the IL-2/IL-2R autocrine pathway within the microenvironment of the lacrimal gland.

Th1 versus Th2 immune responses in autoimmune lacrimal gland disease in MRL/Mp mice.

PURPOSE: In MRL/Mp-lpr/lpr (MRL/lpr) and MRL/Mp-+/+ (MRL/+) mice, a T-cell-driven lacrimal gland inflammation spontaneously develops that is a model for Sjögren's syndrome. The lacrimal gland lesions in these mice were evaluated by immunohistochemistry for the relative contributions of T-helper (Th)1 versus Th2 immune responses. METHODS: Frozen sections of lacrimal glands from MRL/lpr and MRL/+ mice ages 1 through 5 months were stained with monoclonal antibodies to the cytokines interferon (IFN)-gamma and interleukin (IL)4 and to the cell surface costimulatory molecules B7-1 and B7-2, which are associated with Th1 and Th2 responses, respectively. RESULTS: The median proportion of cells staining for IL-4 ranged from 30% to 67% over time for MRL/lpr mice and from 30% to 55% for MRL/+ mice. The median proportion of cells staining for IFN-gamma ranged from 1% to 5% for MRL/lpr mice and from 0% to 3% for MRL/+ mice. The proportion of cells staining positively for IL-4 was significantly greater than for IFN-gamma in both MRL/lpr (mean difference, 33%; P = 0.0001) and MRL/+ mice (mean difference, 42%; P = 0.0002). The median proportion of cells staining positively for B7-2 ranged from 20% to 38% for MRL/lpr mice and from 16% to 34% for MRL/+ mice. The median proportion of cells staining for B7-1 ranged from 2% to 10% for MRL/lpr mice and from 2% to 5% for MRL/+ mice. The proportion of cells staining positively for B7-2 was significantly greater than for B7-1 for both MRL/lpr mice (mean difference, 15%; P = 0.001) and for MRL/+ mice (mean difference, 19%; P = 0.006). CONCLUSIONS: On the basis of immunohistochemistry for cytokines and costimulatory molecules, inflammatory lacrimal gland lesions in MRL/lpr and MRL/+ mice appear to be a largely Th2 phenomenon.

A comparative histologic study of the fibrillin microfibrillar system in the lens capsule of normal subjects and subjects with Marfan syndrome.

PURPOSE: To gain a better understanding of the pathogenesis of ectopia lentis and myopia in Marfan syndrome, studies were performed to determine the distribution and structure of fibrillin microfibrils in the lens capsule of normal subjects and of subjects with Marfan syndrome. METHODS: Frozen sections and/or flat mounts of lens capsules were prepared from six autopsy eyes, nine surgical capsulotomy specimens obtained at the time of cataract extraction, and five capsules from patients with Marfan syndrome obtained at intracapsular lens extraction. Avidin-biotin-peroxidase complex (ABC) immunoperoxidase or immunofluorescence staining with monoclonal antifibrillin antibody was used to localize fibrillin in lens capsules. Image analysis was also performed to compare the amount of fibrillin expression in normal and Marfan syndrome capsules. RESULTS: Based on fibrillin staining patterns, we identified three distinct zones in the equatorial and periequatorial regions of the normal lens capsule. Zone I, a 0.75-mm-wide peripheral ring of the anterior capsule, contained radial bundles of fibrillin fibers. In Zone II, a 1-mm-wide meshwork of fibrillin-rich fibers encircled the equator and served as an insertion platform for zonular fibers. Zone III was composed of radial, 0.1-mm-wide bands arranged in a periodic fashion in the most peripheral part of the posterior capsule. Fibrillin fibers were abnormal and disrupted in all three zones in patients with Marfan syndrome. The amount of fibrillin staining per unit area was significantly reduced in Marfan capsules compared with normal capsules (16-26% versus 49-56% per unit area, respectively; P < 0.001). CONCLUSIONS: Fibrillin was a major constituent of the peripheral and equatorial areas of the lens capsule. Zonular fibers, also rich in fibrillin, insert into the equatorial region, primarily in Zone II. Possibly, fibrillin played a role in the ability of the lens to change its configuration during accommodation. The observed qualitative and quantitative abnormalities in fibrillin expression in the lens capsule of patients with Marfan syndrome supported a causal relationship to lens abnormalities in these patients.

A chlamydial major outer membrane protein extract as a trachoma vaccine candidate.

PURPOSE: As shown in infected humans and in animal models of chlamydial infection, the major outer membrane protein (MOMP) of Chlamydia trachomatis is immunogenically potent. The purpose of this investigation was to test in the cynomolgus monkey model of trachoma a new extract of MOMP as a candidate vaccine against ocular chlamydial infection. METHOD: The nonionic detergent octyl-beta-D glucopyranoside (OGP) was used to extract MOMP from purified C. trachomatis (serovar C) elementary bodies. Protective immunization with OGP-MOMP by mucosal and systemic routes was compared in the cynomolgus monkey model of trachoma. All control and immunized monkeys were challenged by topical application of infectious C. trachomatis to the conjunctivae 35 days after the initiation of immunization. RESULTS: Immunization with OGP-extracted MOMP successfully induced chlamydia-specific local and systemic immunity to MOMP and to whole organism before challenge and early clearance of infection by systemically immunized monkeys. Although ocular disease was not significantly reduced in either immunized group compared to control animals, the lowest clinical and microbiologic disease scores developed in two animals in the mucosal group with the highest immunoglobulin A tear antibody titers at day 0 to 14, whereas higher tear and serum immunoglobulin G correlated with reduced disease in the systemically immunized group. CONCLUSIONS: These data demonstrate that despite evidence of vigorous MOMP-specific and other chlamydia-specific serologic and cell-mediated immunity, as well as anamnestic serologic responses to chlamydia, vaccination with OGP-MOMP was only partially protective against chlamydial ocular disease. The partial protection correlated best with tear immunoglobulin A responses after mucosal immunization and with local and systemic immunoglobulin G responses after peripheral immunization, suggesting that alternative chlamydial antigens may have to be considered in future vaccine development to induce more effective protective immunity and that evaluation of efficacy must be appropriate to route of immunization.

Immunohistochemical localization of fibrillin in human ocular tissues. Relevance to the Marfan syndrome.

OBJECTIVE: To better understand the ocular manifestations of the Marfan syndrome, we investigated the distribution of fibrillin in normal human ocular tissues. Fibrillin, a microfibrillar glycoprotein component of the extracellular matrix, has been found to be the defective gene product in the Marfan syndrome. METHODS: Frozen sections from seven pairs of normal eyes were stained with mouse anti-human fibrillin antibodies using the avidin-biotin immunoperoxidase technique. RESULTS: In the anterior segment, the following exhibited positive staining for fibrillin: the lens capsule and zonules; connective tissues of the iris, ciliary body, ciliary processes, and conjunctiva; and the basement membrane regions of the corneal epithelium and endothelium of Schlemm's canal. Posteriorly, fibrillin localized to the lamina cribrosa, sclera, choroid, and Bruch's membrane. CONCLUSIONS: Fibrillin is widely distributed in ocular connective tissues. The implications of defects in these tissues and the resultant ocular abnormalities in the Marfan syndrome such as ectopia lentis and glaucoma are discussed.

Vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGFbeta), and interleukin-6 (IL-6) in experimental herpesvirus retinopathy: association with inflammation and viral infection.

Experimental herpesvirus retinopathy presents a unique model of a transient inflammatory response in the virus-injected eye and subsequent acute retinal necrosis and chronic inflammation in the contralateral eye. For 6 days after infection, VEGF, TGFbeta1, and TGFbeta2 were associated only with inflammatory cells in the injected eye. By 6 days (after viral antigens were no longer detected), VEGF and TGFbeta2 were upregulated in retinas of injected eyes until 8-10 days. In contralateral eyes, VEGF was first demonstrated in the retina at 6-7 days (prior to the appearance of viral antigens) and TGFbeta2 at 7-8 days. Staining for these factors was also evident around areas of necrosis. The VEGF receptor, flt-1, was associated with ganglion cells and the inner nuclear layer of normal and experimental mice and it was also demonstrated around areas of necrosis. Another VEGF receptor, flk-1, was localized to Muller cell processes and the outer plexiform layer in normal and experimental mice. Coincident with VEGF upregulation in the retinas of herpesvirus-1 injected mice, there was increased flk-1 in ganglion cells and the inner and outer nuclear layers. IL-6 was associated with Muller cell endfeet in normal mice. Following unilateral intraocular inoculation, IL-6 spread along the MUller cell processes and some astrocytes demonstrated IL-6 in both eyes at 6-8 days. The present study demonstrates that intraocular inoculation of herpesvirus is sufficient to induce VEGF, flk-1, TGFbeta2, and IL-6 in the retinas of injected and contralateral eyes. Further investigation of common signaling pathways for these factors during responses to viral infection and the development of acute retinal necrosis could provide information useful for therapeutic intervention in human herpesvirus retinopathy.

Microfibril abnormalities of the lens capsule in patients with Marfan syndrome and ectopia lentis.

PURPOSE: To determine the distribution and structure of fibrillin microfibrils in the three fibrillin-rich lens capsule zones of subjects with the Marfan syndrome. METHODS: Capsules were dissected from nine lenses extracted intracapsularly from Marfan syndrome patients. The capsules were divided and mounted flat on gelatin-coated glass slides. ABC immunoperoxidase staining with monoclonal anti-fibrillin antibody was used to visualize and localize fibrillin in these specimens. The staining patterns and microscopic structure of microfibrils were compared to those of normal controls. RESULTS: There were no bundles of fibrillin fibers in Zone I - a 0.75-mm wide peripheral ring of the anterior capsule that normally contains radial bunches of fibrillin fibers; instead, fine disorganized fibrillin-positive fragments were dispersed in this region. The size and shape of the fragments varied among patients. In contrast to normal lenses, there was only light staining for fibrillin in Zone II - a 1-mm wide meshwork of normally fibrillin-rich fibers that encircles the equator and serves as an insertion platform for most zonular fibers. The radial periodic bands of Zone III - a 0.1-mm wide ring on the most peripheral part of the normal posterior capsule - were identifiable in some samples, but stained only faintly for fibrillin. CONCLUSION: Fibrillin microfibrils are disrupted and fragmented in the lens capsule of patients with the Marfan syndrome. The qualitative, quantitative, and structural abnormalities of fibrillin deposition in the lens capsule of these patients support a causal relationship to lens abnormalities in this disease.

Case report: in situ hybridization for detection of inapparent infection with Chlamydia trachomatis in synovial tissue of a patient with Reiter's syndrome.

The authors have shown that protein antigens, RNA, and DNA from Chlamydia trachomatis are present in synovial tissues of patients with Reiter's syndrome (RS). However, those studies gave no insight into the host cell type involved or the precise tissue location of the bacteria. To address such issues, the authors developed an in situ hybridization system to detect chlamydia, and they used that system to examine synovial biopsies from a patient with RS and a patient without RS. The in situ system uses a previously described digoxigenin-labeled DNA probe that hybridizes with chlamydial 16S rRNA sequences in paraformaldehyde-fixed samples. Control studies with chlamydia-infected and uninfected HeLa cells confirmed that the in situ system is as sensitive as is direct fluorescence cytology for detection of the organism. Morphology of host and chlamydia cells is preserved after hybridization. Studies using synovial tissue from an osteoarthritis patient produced no in situ hybridization signal, but similar hybridization to tissue from a culture-/direct fluorescence cytology- negative RS patient had a strong intracellular signal for chlamydia within a subsynovial cell layer. These in situ hybridization results confirm the extensive presence of chlamydia in synovia and extend the authors' earlier observation that chlamydia RNA is present in the synovia of patients with RS. The data also confirm their electron microscopy studies, indicating that chlamydia are intracellular in synovial tissue, and they further show that infected host cells are located beneath the synovial lining.

Rapid communication: Cervical Chlamydia trachomatis in women at low risk for infection.

BACKGROUND: Evidence suggests that the bacterium Chlamydia trachomatis can cause asymptomatic genital infection in persons at risk for acquisition of the organism. We employed 2 independent molecular screening systems to assess such inapparent cervical chlamydial infections in low-risk female patients attending general (non-STD) clinics in 2 locations. METHODS: Three hundred seventy-five cervical swab samples were obtained in duplicate from patients attending a general women's clinic (278 samples) and a colposcopy clinic (97 samples). One set of samples from the general clinic was screened by a highly-specific molecular hybridization system, using a probe targeting the chlamydial 16S ribosomal RNA; the other set was screened with the use of the Chlamydiazyme test. Samples from the colposcopy clinic were screened using a sensitive and specific polymerase chain reaction (PCR) assay system targeting chlamydia; the duplicates were assayed by direct fluorescent antibody assay (DFA). RESULTS: Of the 278 patients screened by RNA-directed hybridization, 6.5% were positive for C. trachomatis, in contrast to screening of duplicate samples via Chlamydiazyme, which indicated that 3.6% were infected. PCR-based screening of the additional 97 patients gave a positivity rate of 17.5% for the organism, whereas DFA on duplicate samples from this group showed only 7.5% positive. CONCLUSIONS: These observations suggest that the level of asymptomatic cervical C. trachomatis infection is significant even in women who are at low risk for such infections; the data also indicate that results from standard laboratory screening for chlamydia should be viewed with caution.