Synovial Chlamydia trachomatis up regulates expression of a panel of genes similar to that transcribed by Mycobacterium tuberculosis during persistent infection.

BACKGROUND: Synovial tissues in patients with Chlamydia associated arthritis are persistently infected by C trachomatis, an organism for which genetic manipulation is not possible. M tuberculosis also engages in persistent infection, and because this bacterium is genetically tractable many groups have been able to define transcriptional characteristics of mycobacterial growth and persistence. OBJECTIVE: To investigate whether the pattern of gene expression underlying chlamydial persistence is similar to that underlying mycobacterial persistence. METHODS: 194 genes in M tuberculosis that are transcriptionally up regulated to support in vivo growth and persistence of that organism have previously been identified. Each of those genes was compared with the C trachomatis genome to identify orthologues. Expression of selected chlamydial orthologues so identified was assessed by real time RT-PCR in an in vitro model of chlamydial persistence and synovial tissues from patients who were PCR positive for C trachomatis at that site. RESULTS: 67 C trachomatis genes were identified as being orthologous to mycobacterial persistence related genes, representing 35% of the genes tested. The chlamydial orthologues fell into similar metabolic and other categories as those in M tuberculosis. Expression of a majority of selected chlamydial orthologues was strongly up regulated in an in vitro model of chlamydial persistence and in synovial tissues of relevant patients, compared with their expression during active infection. CONCLUSIONS: These observations provide new insight into the molecular genetic basis underlying chlamydial persistence, and indicate that this information can be obtained, in some instances, by extrapolating observations made in other biological systems and/or organisms.

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.

Expression of Chlamydia trachomatis genes encoding products required for DNA synthesis and cell division during active versus persistent infection.

During persistent infection, the intracellular bacterial pathogen Chlamydia trachomatis is viable but severely attenuates the production of new, infectious elementary bodies (EBs). To investigate the reasons for this lack of new EB output, we analysed the expression of chlamydial genes encoding products required for DNA replication and cell division, using in vitro models of active versus persistent infection and synovial tissue samples from patients with chronic Chlamydia-associated arthritis. Hep-2 cells were infected with K serovar C. trachomatis and harvested at t = 0-48 h post-infection (p.i; active). Human monocytes were infected similarly and harvested at t = 1-7 days p.i. (persistent). RNA preparations from infected/uninfected cells and patient samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) targeting polA, dnaA, mutS and parB mRNA, related to chlamydial DNA replication/segregation; these were expressed in infected Hep-2 cells from 11 to 48 h p.i; ftsK and ftsW, related to cell division, were expressed similarly. Real-time PCR analyses demonstrated that significant accumulation of chlamydial chromosome began at about 12 h p.i. in infected Hep-2 cells. In infected human monocytes, polA, dnaA, mutS and parB mRNA were produced from days 1-7 p.i. and were weakly expressed in patient samples. Real-time PCR indicated the continuing accumulation of chlamydial chromosome during the 7 day monocyte infection, although the rate of such accumulation was lower than that occurring during active growth. However, transcripts from ftsK and ftsW were detected only at 1 day p.i. in infected monocytes but not thereafter, and they were absent in all patient samples. Thus, genes whose products are required for chlamydial DNA replication are expressed during persistence, but transcription of genes whose products are required for cytokinesis is severely downregulated. These data explain, at least in part, the observed attenuation of new EB production during chlamydial persistence.

A Chlamydia pneumoniae-specific peptide induces experimental autoimmune encephalomyelitis in rats.

It has been reported recently that the bacterial respiratory pathogen Chlamydia pneumoniae is present in the cerebrospinal fluid of a subset of multiple sclerosis (MS) patients. However, it is not known whether this organism is a causative agent of MS, or merely an opportunistic pathogen that takes advantage of a disease process initiated by some other means. We report identification of a 20-mer peptide from a protein specific to C. pneumoniae which shares a 7-aa motif with a critical epitope of myelin basic protein, a major CNS Ag targeted by the autoimmune response in MS. This bacterial peptide induces a Th1 response accompanied by severe clinical and histological experimental autoimmune encephalomyelitis in Lewis rats, a condition closely reflective of many aspects of MS. Studies with peptide analogues suggest that different populations of encephalitogenic T cells are activated by the C. pneumoniae and myelin basic protein Ags. Mild experimental autoimmune encephalomyelitis was also observed when rats were immunized with sonicated C. pneumoniae in CFA.

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.

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.

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.

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.

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.

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.

T cells and trachoma. Their role in cicatricial disease.

Frozen sections of tarsoconjunctival biopsies with trachomatous scarring from 14 black adults undergoing corrective surgery for trichiasis, and "normal" tissue from three postmortem controls, were immunohistochemically stained for the major T- and B-cell subsets, and for macrophages and monocytes. T cells outnumbered B cells by 2 to 17 times, and macrophages and monocytes by approximately 20 times in all specimens. Biopsies were categorized as "inflamed" if a cumulative inflammatory score of cellular staining in the substantia propria with CD4, CD8, and OKM1 monoclonal antibodies was greater than that of control tissues. CD4+ lymphocytes predominated over CD8+ lymphocytes in 5 of 7 inflamed biopsies, whereas CD8+ lymphocytes predominated over CD4+ lymphocytes in 5 of 7 noninflamed biopsies. Lymphoid aggregates were present in five inflamed biopsies, but lacked germinal centers, centrally located B cells, or parafollicular T cells typical of the acute stage of trachoma. CD4+ and CD8+ lymphocytes also were observed in the epithelium and lumen of Meibomian glands. These observations indicate that the inflammatory infiltrate of the tarsoconjunctiva in the cicatricial stage of trachoma is comprised predominantly of T cells, and suggests that T cells may be involved in the genesis of tarsal thickening and conjunctival scarring seen in the later stages of trachoma.

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.

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.