Human Mannose-Binding Protein Inhibits Infection of HeLa Cells by Chlamydia trachomatis

The role that collectin (mannose-binding protein) may play in the host’s defense against chlamydial infection was investigated. Recombinant human mannose-binding protein was used in the inhibition of cell culture infection by Chlamydia trachomatis (C/TW-3/OT, E/UW-5/Cx, and L₂/434/Bu), Chlamydia pneumoniae (AR-39), and Chlamydia psittaci (6BC). Mannose-binding protein (MBP) inhibited infection of all chlamydial strains by at least 50% at 0.098 μg/ml for TW-3 and UW-5, and at 6.25 μg/ml for 434, AR-39, and 6BC. The ability of MBP to inhibit infection with strain L₂ was not affected by supplementation with complement or addition of an L₂-specific neutralizing monoclonal antibody. Enzyme-linked immunosorbent assay and dot blot analyses showed MBP bound to the surface of the organism to exert inhibition, which appeared to block the attachment of radiolabeled organisms to HeLa cells. Immunoblotting and affinity chromatography indicated that MBP binds to the 40-kDa glycoprotein (the major outer membrane protein) on the outer surface of the chlamydial elementary body. Hapten inhibition assays with monosaccharides and defined oligosaccharides showed that the inhibitory effects of MBP were abrogated by mannose or high-mannose type oligomannose-oligosaccharide. The latter carbohydrate is the ligand of the 40-kDa glycoprotein of C. trachomatis L₂, which is known to mediate attachment, suggesting that the MBP binds to high mannose moieties on the surface of chlamydial organisms. These results suggest that MBP plays a role in first-line host defense against chlamydial infection in humans.     

Systemic Chlamydia trachomatis infection in mice: a comparison of lymphogranuloma venereum and trachoma biovars.

We developed a murine model of systemic infection with Chlamydia trachomatis biovar lymphogranuloma venereum (LGV). The pathological features of this infection resemble those of human LGV infection since both are characterized by granuloma formation. Mice developed resistance to reinfection with LGV, and this resistance was based on cellular immune mechanisms since it was transferable with immune spleen cells but not with immune serum. Resistance required viable organisms for induction. We compared LGV biovar infection with trachoma biovar infection. Trachoma biovar produced similar but less marked microbiological and pathological features. Cross-immunity was less apparent between serovars from trachoma and LGV biovars than it was between serovars within the same biovar. This model of systemic C. trachomatis infection will be useful in exploring virulence features of LGV.     

Cytokine responses during mycobacterial and schistosomal antigen-induced pulmonary granuloma formation. Production of Th1 and Th2 cytokines and relative contribution of tumor necrosis factor.

Synchronized pulmonary granulomas (GRs) were induced in presensitized mice by intravenous embolization of polymer beads bound with purified protein derivative (PPD) of Mycobacteria tuberculosis or soluble antigens derived from Schistosoma mansoni eggs (SEA). Uncoated beads served as a foreign body control (CON). Antigen-coated beads elicited GRs with characteristic epithelioid macrophages and multinucleate giant cells by 4 days after embolization. Unlike PPD GR, SEA bead lesions contained eosinophils, whereas CON beads elicited only a limited mononuclear infiltrate. GRs and draining lymph nodes (LN) were assessed on days 2, 4, and 8 for Th1-(interleukin-2 [IL-2], interferon-gamma[IFN] and Th2-type (IL-4, IL-5, and IL-10) cytokines. CON GR produced only a small amount of IFN-gamma on day 2 and failed to induce a significant response in draining LN. In contrast, both PPD and SEA antigen-coated beads induced reactive lymphoid hyperplasia but differed greatly in local and regional cytokine profiles. PPD GR produced IFN-gamma on day 2 and the draining LN produced predominantly Th1 cytokines on days 2 and 4. In contrast, SEA beads GRs were dominated by Th2 cytokines. The corresponding LN produced IL-2 and IL-4 on day 2; IL-2, IL-4, IFN-gamma, and IL-10 on day 4; then IL-2, IFN-gamma, and IL-4 on day 8, probably reflecting maturational changes of T cells. Macrophages (MP) from bead GR also showed different patterns of IL-6 and tumor necrosis factor (TNF) production. Compared with CON GR, MPs from PPD GR were weak sources of IL-6, whereas those of SEA GR showed enhanced and accelerated production. In contrast, MP of PPD GR had augmented TNF-producing capacity, whereas those of SEA GR showed delayed TNF production. In vivo depletion of TNF, respectively, caused 40 and 10% decreases in PPD GR and SEA GR but had no effect on CON GR area, indicating that TNF contributed to a greater degree to the PPD response. These data show that depending on the inciting agent, GR can be mediated by different cytokines. Characterization of inflammatory lesions by cytokine profiles should allow design of more rational therapeutic interventions.     

Effect of Chlamydia pneumoniae on cellular ATP content in mouse macrophages: role of Toll-like receptor 2

Chlamydiae are obligate intracellular gram-negative bacteria and are dependent on the host cell for ATP. Thus, chlamydial infection may alter the intracellular levels of ATP and affect all energy-dependent processes within the cell. We have shown that both live C. pneumoniae and inactivated C. pneumoniae induce markers of cell death prior to completion of the bacterial growth cycle. As depletion of ATP could account for the observed increase in cell death, the effects of C. pneumoniae on ATP concentrations within mouse macrophages were investigated. Live, heat-killed, and UV-inactivated C. pneumoniae cultures (at multiplicities of infection [MOIs] of 0.01, 0.1, and 1.0) were incubated with mouse bone marrow macrophages isolated from C57BL/6J mice and mice deficient in Toll-like receptors. Treatment of the macrophages with both live and inactivated C. pneumoniae increased the ATP content of the cells. In cells infected with live C. pneumoniae, the increase was inversely proportional to the MOI. In cells treated with inactivated C. pneumoniae, the increase in ATP content was smaller than that induced by infection with live organisms and was proportional to the MOI. The increase in ATP content early in the developmental cycle was independent of the growth of C. pneumoniae, while sustained induction required live organisms. The capacity of C. pneumoniae to increase the ATP content was ablated in macrophages deficient in expression of either Toll-like receptor 2 or the Toll-like receptor accessory protein MyD88. In contrast, no effect was observed in macrophages lacking expression of Toll-like receptor 4.     

Frequent presence of neuroendocrine small cells in thymic carcinoma: a light microscopic and immunohistochemical study

AIMS: Neuroendocrine differentiation has been described in conventional carcinomas of various organs. Small cells postulated to be neuroendocrine cells were observed previously in some thymic carcinomas. This study was conducted to confirm and characterize the presence of neuroendocrine small cells in thymic carcinomas by light microscopy and immunohistochemistry. METHODS AND RESULTS: Twenty-two thymic carcinomas were studied by light microscopy to detect the presence of small neuroendocrine-like cells. They were found in four of 10 squamous cell carcinomas (SCC) and seven of eight adenosquamous carcinomas (ASC). No small cells were observed in three lymphoepithelioma-like carcinomas (LELC) and one adenocarcinoma. The small cells were located within the tumour nests and constituted less than 1% of the entire tumour. In one case, small cells also extended outside the tumour nests. Rosette formation was seen in three cases. They were proved to be neuroendocrine cells by their immunoreactivity to neuron-specific enolase, chromogranin A, and/or synaptophysin. A few scattered neuroendocrine small cells were found only by immunohistochemistry in one case each of SCC, ASC, and LELC. The small cells were also strongly positive for cytokeratin (CK) 8 and CK18 but negative for CK19 and CK20. The predominant carcinoma cells other than the neuroendocrine small cells also displayed neuroendocrine markers in 68% of the cases studied. CONCLUSIONS: Neuroendocrine small cells can be recognized by light microscopic examination in approximately 61% of thymic SCC and ASC. Neuroendocrine markers, CK8 and CK18 can aid in confirming their presence. The neuroendocrine small cells present in thymic carcinomas are different from the main carcinoma cells displaying immunohistochemical neuroendocrine markers. The presence of neuroendocrine small cells could be an useful marker for the differentiation of thymic carcinomas from thymomas and carcinomas of other sites.     

Chlamydia trachomatis species-specific epitope detected on mouse biovar outer membrane protein.

A common antigenic determinant on the chlamydial major outer membrane protein was detected on each of the three Chlamydia trachomatis biovars (trachoma, lymphogranuloma venereum, and mouse). This determinant was prominently displayed on the surface of chlamydial strains from both the trachoma and lymphogranuloma venereum biovars. However, detection of this determinant on a mouse biovar strain required denaturation by sodium dodecyl sulfate or periodate oxidation. This determinant provides a definable taxonomic link between the three biovars of C. trachomatis.     

Reticulate bodies as single antigen in Chlamydia trachomatis serology with microimmunofluorescence.

Formalin-fixed, purified reticulate bodies (RB) of Chlamydia trachomatis immunotype C/TW-3/OT were used as a serological test antigen in the microimmunofluorescence test. The sensitivity and specificity of the RB antigen were compared to elementary bodies (EB) used as antigens in the detection of C. trachomatis antibodies in human sera by microimmunofluorescence. RB reacted with all known C. trachomatis immunotypes with the same sensitivity as the homotypic EB. In routine serology with sera and endocervical secretions, the RB antigen had a sensitivity similar to that of the EB in detecting serum antibodies, endocervical secretion antibodies, and antibodies of immunoglobulin M and G classes. No false-positive reactions were detected with control sera. All positive reactions showed type-specific antibodies against an EB immunotype. RB seemed to demonstrate chlamydial group reactivity; sera from 10 psittacosis patients diagnosed clinically and serologically by complement fixation showed five positive, three equivocal, and two negative reactions. By immunofluorescence, RB appeared as distinct rings demonstrating uniform peripheral surface fluorescence at their rims. The EB appeared as pinpoint-sized dots. C/TW-3/OT RB used as a single test anitgen should provide a simple and sensitive serological assay for the detection of C. trachomatis antibody.     

Duke Surgery Research Central: an open-source Web application for the improvement of compliance with research regulation

Background  

Although regulatory compliance in academic research is enforced by law to ensure high quality and safety to participants, its implementation is frequently hindered by cost and logistical barriers. In order to decrease these barriers, we have developed a Web-based application, Duke Surgery Research Central (DSRC), to monitor and streamline the regulatory research process.     

Extended epidemic of nosocomial urinary tract infections caused by Serratia marcescens

In recent years a significant increase in the incidence of Serratia marcescens infections was noted at the Chang Gung Memorial Hospital, Taoyuan, Taiwan. A review of laboratory (1991 to 2002) and infection control (1995 to 2002) records showed the possibility of an extended epidemic of nosocomial urinary tract infections (UTIs) caused by S. marcescens. Therefore, in 1998 and 1999, 87 isolates were collected from patients with such infections and examined and another 51 isolates were collected in 2001 and 2002. The patients were mostly elderly or the infections were associated with the use of several invasive devices. S. marcescens was usually the only pathogen found in urine cultures in our study. Neither prior infections nor disseminated infections with the organism were observed in these patients. Resistance to most antibiotics except imipenem was noted. Two genotyping methods, pulsed-field gel electrophoresis and infrequent-restriction-site PCR, were used to examine the isolates. A total of 12 genotypes were identified, and 2 predominant genotypes were found in 72 (82.8%) of the 87 isolates derived from all over the hospital. However, 63.9% of the isolates of the two genotypes were from neurology wards. A subsequent intervention by infection control personnel reduced the infection rate greatly. The number and proportion of the two predominant genotypes were significantly reduced among the 51 isolates collected in 2001 and 2002. Thus, a chronic and long-lasting epidemic of nosocomial UTIs caused by S. marcescens was identified and a successful intervention was carried out. Both a cautious review of laboratory and infection control data and an efficient genotyping system are necessary to identify such a cryptic epidemic and further contribute to the quality of patient care.