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Godwin O Ifere1, Fisseha Abebe2, Godwin A Ananaba1,31Department of Biological Sciences, 2Department of Mathematical Sciences, 3Center for Cancer Research and Therapeutic Development, Clark Atlanta University, Atlanta, GA, USABackground: To date there has not been any nationwide age-standardized incidence data reported for prostate cancer in Nigeria. We examined and integrated diverse trends in the age-specific incidence of prostate cancer into a comprehensive trend for Nigeria, and examined how best the existing data could generate a countrywide age-standardized incidence rate for the disease.Methods: Data were obtained from studies undertaken between 1970 and 2007 in referral hospital-based cancer registries. Records from at least one tertiary hospital in each of the six geopolitical zones of Nigeria were examined retrospectively. Data were also reported for the rural population in cross-sectional prospective studies. Age-standardized incidence rates and the annual incidence of disease were calculated.Results: Higher incidence rates for prostate cancer during this period were recorded for patients aged 60–69 years and 70–79 years, with a lower incidence rate for patients aged younger than 50 years. An exponential annual incidence rate of disease was observed in the 50–79 year age group and peaked at 70–79 years before dropping again at age 80 years. The results showed metastasis in more than half of these hospital-based prostate tumors.Conclusion: Our results suggest that prostate cancer occurs at a relatively young age in Nigerians and that hospital-based registry reports may not appropriately reflect the incidence of the disease in Nigeria. A countrywide screening program is urgently needed. Finally, the difference in reported stages of disease found in Nigerians and African-Americans versus Caucasians suggests biological differences in the prognosis. Nigeria may thus typify one of the ancestral populations that harbor inherited genes predisposing African-Americans to high-risk prostate cancer.Keywords: prostate cancer, annual age-standardized incidence rate, Nigeria cancer registry

Vitamin D hormone (1,25-dihydroxyvitamin D) is involved in innate immunity and induces host defense peptides in epithelial cells, suggesting its involvement in mucosal defense against infections. Chlamydia trachomatis is a major cause of bacterial sexually transmitted disease worldwide. We tested the hypothesis that the vitamin D endocrine system would attenuate chlamydial infection. Vitamin D receptor knock-out mice (VDR(-/-)) and wild-type mice (VDR(+/+)) were infected with 10(3) inclusion forming units of Chlamydia muridarum and cervical epithelial cells (HeLa cells) were infected with C. muridarum at multiplicity of infection 5:1 in the presence and absence of 1,25-dihydroxyvitamin D3. VDR(-/-) mice exhibited significantly higher bacterial loading than wild-type VDR(+/+) mice (P<0.01) and cleared the chlamydial infection in 39 days, compared with 18 days for VDR(+/+) mice. Monocytes and neutrophils were more numerous in the uterus and oviduct of VDR(-/-) mice than in VDR(+/+) mice (P<0.05) at d 45 after infection. Pre-treatment of HeLa cells with 10nM or 100nM 1,25-dihydroxyvitamin D3 decreased the infectivity of C. muridarum (P<0.001). Several differentially expressed protein spots were detected by proteomic analysis of chlamydial-infected HeLa cells pre-treated with 1,25-dihydroxyvitamin D3. Leukocyte elastase inhibitor (LEI), an anti-inflammatory protein, was up-regulated. Expression of LEI in the ovary and oviduct of infected VDR(+/+) mice was greater than that of infected VDR(-/-) mice. We conclude that the vitamin D endocrine system reduces the risk for prolonged chlamydial infections through regulation of several proteins and that LEI is involved in its anti-inflammatory activity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: The purpose of our study was to show the distinction between the apoptotic and anti-proliferative signaling of phytosterols and cholesterol-enrichment in prostate cancer cell lines, mediated by the differential transcription of caveolin-1, and N-myc downstream-regulated gene 1 (NDRG1), a pro-apoptotic androgen-regulated tumor suppressor., Methods: PC-3 and DU145 cells were treated with sterols (cholesterol and phytosterols) for 72h, followed by trypan blue dye-exclusion measurement of necrosis and cell growth measured with a Coulter counter. Sterol induction of cell growth-suppressor gene expression was evaluated by mRNA transcription using RT-PCR, while cell cycle analysis was performed by FACS analysis. Altered expression of Ndrg1 protein was confirmed by Western blot analysis. Apoptosis was evaluated by real time RT-PCR amplification of P53, Bcl-2 gene and its related pro- and anti-apoptotic family members., Results: Physiological doses (16microM) of cholesterol and phytosterols were not cytotoxic in these cells. Cholesterol-enrichment promoted cell growth (P<0.05), while phytosterols significantly induced growth-suppression (P<0.05) and apoptosis. Cell cycle analysis showed that contrary to cholesterol, phytosterols decreased mitotic subpopulations. We demonstrated for the first time that cholesterols concertedly attenuated the expression of caveolin-1 (cav-1) and NDRG1 genes in both prostate cancer cell lines. Phytosterols had the opposite effect by inducing overexpression of cav-1, a known mediator of androgen-dependent signals that presumably control cell growth or apoptosis., Conclusions: Cholesterol and phytosterol treatment differentially regulated the growth of prostate cancer cells and the expression of p53 and cav-1, a gene that regulates androgen-regulated signals. These sterols also differentially regulated cell cycle arrest, downstream pro-apoptotic androgen-regulated tumor suppressor, NDRG1 suggesting that cav-1 may mediate pro-apoptotic NDRG1 signals. Elucidation of the mechanism for sterol modulation of growth and apoptosis signaling may reveal potential targets for cancer prevention and/or chemotherapeutic intervention. Sterol regulation of NDRG1 transcription suggests its potential as biomarker for prediction of neoplasms that would be responsive to chemoprevention by phytosterols.

The Vibrio cholerae ghost (rVCG) platform is an effective carrier and delivery system for designing efficacious Chlamydia vaccines. We investigated whether CTA2B, the nontoxic derivative of cholera toxin, can augment protective immunity conferred by an rVCG-based chlamydial vaccine and enhance cross-protection against heterologous chlamydial strains. An rVCG vaccine coexpressing chlamydial major outer membrane protein and CTA2B was genetically constructed and antigens were targeted to the inner membrane of V. cholerae before ghost production by gene E-mediated lysis. Effective immunomodulation by CTA2B was demonstrated by the ability of the vaccine construct to enhance the activation and maturation of dendritic cells in vitro. Also, C57BL/6 mice immunized via mucosal and systemic routes showed increased specific mucosal and systemic antibody and T-helper type-1 (Th1) responses, irrespective of the route. The enhanced production of IFN-gamma, but not IL-4 by genital mucosal and splenic T cells, indicated a predominantly Th1 response. Clearance of the Chlamydia muridarum vaginal infection was significantly enhanced by codelivery of the vaccine with CTA2B, with the intravaginal route showing a moderate advantage. These results indicate that the rVCG-based vaccine is capable of inducing cross-protection against heterologous chlamydial serovars and that incorporation of mucosal adjuvants, such as CTA2B in the rVCG delivery platform, may enhance protective immunity.

Background: The purpose of our study was to show the apoptotic and anti-proliferative effects of phytosterols as distinct from cholesterol effects on prostate cancer cell lines, and also their differential expression of caveolin-1, and a prostate specific gene, PCGEM1., Methods: PC-3 and DU145 cells were treated with sterols (cholesterol and phytosterols) for 48h, followed by trypan blue dye exclusion measurement of cytotoxicity and MTT cell proliferation assays, respectively. Cell cycle analysis was carried out microscopically, and by propidium iodide uptake using flow cytometry. Sterol induction of oncogenic gene expression was evaluated by RT-PCR. Apoptotic cells were identified by immunocytochemistry using DNA fragmentation method, and by annexin V adhesion using flow cytometry., Results: Physiological doses (16microM) of these sterols were not cytotoxic in these cells. Cholesterol-enrichment promoted mitosis (54 and 61% by microscopy; 40.8 and 34.08% by FACS analysis in PC-3 and DU145, respectively) and cell growth (P<0.05), while phytosterols suppressed mitosis (29 and 35% by microscopy; 27.71 and 17.37% by FACS analysis in PC-3 and DU145, respectively), and significantly induced tumor-suppression (P<0.05) and apoptosis. We demonstrated for the first time that cholesterols upregulated the expression of PCGEM1 even in androgen-insensitive prostate cancer cell lines. Phytosterols reversed this effect, while upregulating the expression of caveolin-1, a known mediator of androgen-dependent proto-oncogene signals that presumably control growth and anti-apoptosis., Conclusions: Phytosterol inhibition of PCGEM1 and cell growth and the overexpression of caveolin-1, suggests that poor disease prognosis anchors on the ability of caveolin-1 to regulate downstream oncogene(s) and apoptosis genes. Sterol intake may contribute to the disparity in incidence of prostate cancer, and elucidation of the mechanism for modulation of growth and apoptosis signaling may reveal potential targets for cancer prevention and/or chemotherapeutic intervention. Sterol regulation of PCGEM1 expression suggests its potential as biomarker for prediction of neoplasms that would be responsive to chemoprevention by phytosterols.

The search for a classic biomarker for prostate cancer has been on for a very long time, and it is still elusive. Despite the extensive and prolonged use of the prostate-specific antigen (PSA) as a screening tool for prostate cancer, many clinicians still identify its limitations in the grading of tumors, and monitoring of response to treatment. These limitations are based on the concealment of cancer by low levels of PSA, and over diagnosis and over treatment that are resultant of excessively high levels of antigens. To overcome these limitations, a plethora of candidate biomarkers have been investigated and identified. Regrettably, none of these putative biomarkers has shown ideal utility for prostate cancer detection and prognostication. In spite of these, various patents have been filed and granted for several of these biomarkers. The discovery of an exclusive marker for prostate cancer may be mired by the heterogeneity of the disease, since the molecular mediators of the disease are linked with its etiopathogenesis. Thus the search for biomarkers of prostate cancer must recognize its etiology and downstream mediators. In this review, PCGEM1, a patented prostatespecific non-coding RNA gene with an upregulated transcription in African American malignancy will be discussed in relationship to the etiology of prostate cancer.

We investigated the hypothesis that the enhanced Ag-presenting function of IL-10-deficient dendritic cells (DCs) is related to specific immunoregulatory cytoskeletal molecules expressed when exposed to Ags. We analyzed the role of a prominent cytoskeletal protein, LEK1, in the immunoregulation of DC functions; specifically cytokine secretion, costimulatory molecule expression, and T cell activation against Chlamydia. Targeted knockdown of LEK1 expression using specific antisense oligonucleotides resulted in the rapid maturation of Chlamydia-exposed DCs as measured by FACS analysis of key activation markers (i.e., CD14, CD40, CD54, CD80, CD86, CD197, CD205, and MHC class II). The secretion of mostly Th1 cytokines and chemokines (IL-1a, IL-9, IL-12, MIP-1a, and GM-CSF but not IL-4 and IL-10) was also enhanced by blocking of LEK1. The function of LEK1 in DC regulation involves cytoskeletal changes, since the dynamics of expression of vimentin and actin, key proteins of the cellular cytoskeleton, were altered after exposure of LEK1 knockdown DCs to Chlamydia. Furthermore, targeted inhibition of LEK1 expression resulted in the enhancement of the immunostimulatory capacity of DCs for T cell activation against Chlamydia. Thus, LEK1 knockdown DCs activated immune T cells at least 10-fold over untreated DCs. These results suggest that the effect of IL-10 deficiency is mediated through LEK1-related events that lead to rapid maturation of DCs and acquisition of the capacity to activate an elevated T cell response. Targeted modulation of LEK1 expression provides a novel strategy for augmenting the immunostimulatory function of DCs for inducing an effective immunity against pathogens.

Effective delivery systems are needed to design efficacious vaccines against the obligate intracellular bacterial pathogen, Chlamydia trachomatis. Potentially effective delivery vehicles should promote the induction of adequate levels of mucosal T-cell and antibody responses that mediate long-term protective immunity. Antigen targeting to the nasal-associated lymphoid tissue (NALT) is effective for inducing high levels of specific immune effectors in the genital mucosa, and therefore suitable for vaccine delivery against genital chlamydial infection. We tested the hypothesis that live attenuated influenza A viruses are effective viral vectors for intranasal delivery of subunit vaccines against genital chlamydial infection. Recombinant influenza A/PR8/34 (H1N1) viruses were generated by insertion of immunodominant T-cell epitopes from chlamydial major outer membrane protein into the stalk region of the neuraminidase gene. Intranasal immunization of mice with viral recombinants resulted in a strong T helper 1 (Th1) response against intact chlamydial elementary bodies. Also, immunized mice enjoyed a significant state of protective immunity (P > 0.002) by shedding less chlamydiae and rapidly clearing the infection. Furthermore, a high frequency of Chlamydia-specific Th1 was measured in the genital mucosal and systemic draining lymphoid tissues within 24 hr after challenge of vaccinated mice. Moreover, multiple epitope delivery provided a vaccine advantage over single recombinants. Besides, long-term protective immunity correlated with the preservation of a robustly high frequency of specific Th1 cells and elevated immunoglobulin G2a in genital secretions. Because live attenuated influenza virus vaccines are safe and acceptable for human use, they may provide a new and reliable approach to deliver efficacious vaccines against sexually transmitted diseases.

Background and Purpose: Genital infections due to Chlamydia trachomatis pose a considerable public health challenge worldwide and a vaccine is urgently needed to protect against these infections. We examined whether a vaccine composed of a combination of the major outer membrane protein (MOMP) and porin B protein (PorB) of C. trachomatis would have a protective advantage over a single subunit construct., Methods: Single and multisubunit vaccines expressing MOMP and PorB were constructed and evaluated in the mouse model of genital infection. Thus, groups of female C57BL/6 mice were immunized intramuscularly with recombinant Vibrio cholerae ghosts (VCG) expressing the vaccine antigens or VCG alone and humoral and cell-mediated immune responses were evaluated., Results: Significant levels of Chlamydia-specific secretory immunoglobulin A and immunoglobulin G2a were detected in vaginal washes and serum of immunized mice. The multisubunit construct induced a significantly higher level of T-helper Type 1 response than the single subunits as measured by the amount of interferon-gamma produced by immune T cells in response to re-stimulation with ultraviolet-irradiated elementary bodies in vitro. Three weeks after the last immunization, animals were challenged intravaginally with 10(7) inclusion-forming units of C. trachomatis serovar D. There was a significant difference in the intensity and duration of vaginal shedding between the vaccine-immunized mice and controls. All the animals immunized with the multisubunit vaccine had completely resolved the infection 2 weeks post-challenge. Higher numbers of embryos were observed in vaccinated animals than in controls, indicating protection against infertility., Conclusion: These results underscore the potential, albeit moderate, vaccine advantage of the multisubunit formulation.

T cell immunity protects against diseases caused by the obligate intracellular bacterium Chlamydia trachomatis. Incidentally, host inflammatory response that includes T cells appears to also contribute to the pathogenesis of chlamydial diseases such as trachoma and tubal factor infertility (TFI). Therefore, designing effective prevention strategies requires a delineation of immune processes responsible for pathology and those mediating immunity, and identification of the immunogenetic factors predisposing to complication development. The chemokine receptor CCR5 is crucial for T cell activation and function since its deficiency causes suppression of T cell response. We investigated the hypothesis that the clearance of genital chlamydial infection in CCR5-deficient mice could be delayed in the short term; however, a beneficial effect could include protection against inflammation-related complications such as TFI. In a translational study in humans, we investigated the effect of a functional 32 bp deletion in the CCR5 gene on the risk of developing tubal pathology in Dutch Caucasian women with immunologic evidence [i.e., immunoglobulin G (IgG) responses] of chlamydial infection. When genitally-infected wild-type (WT) and CCR5 knockout (CCR5KO) mice were evaluated for microbiologic shedding of chlamydiae, there was a greater intensity of infection and delayed resolution in the knockout mice. However, compared to WT mice, the fertility of infected CCR5KO mice (measured by pregnancy rate) was only mildly affected in the short term and unaffected in the long term (70% vs 30% reduction in the short term, and 50 vs 0% in the long term, respectively). Immunobiologic analysis revealed that the diminished capacity of CCR5KO to control acute chlamydial infection correlated with the relatively low chemokine [interferon-inducible protein 10 (IP-10) and regulated upon activation normal cell expressed and secreted (RANTES)] and cytokine (mainly interferon-gamma and tumor necrosis factor-alpha) expression corresponding to a poor early T-helper I response. However, the reduced incidence of complications in the CCR5KO mice appears to correlate with the low activity of long term inflammatory mediators. Besides, the translational studies in humans revealed that among patients with positive anti-chlamydial IgG responses, tubal pathology correlated with a low incidence of CCR5delta32 deletion (7%), while women without tubal pathology had higher incidence of the CCR5delta32 deletion (31%) as compared to controls (19%). Thus, in mice and humans the inflammation associated with CCR5 function may predispose to development of complications of chlamydial infection, such as TFI.

Identification and targeting of novel immunobiological factors that regulate the induction of Th1 cells are crucial for designing effective vaccines against certain intracellular pathogens, including Chlamydia. IL-10-deficient dendritic cells (DC) are potent APCs and effective cellular vaccines that activate a high frequency of specific Th1 cells. To elucidate the molecular basis for the potency of the IL-10-deficient APC system, we tested the hypothesis that Chlamydia Ag-primed IL-10 knockout (IL-10KO) DC are quantitatively and qualitatively distinct in their metabolic characteristics relating to T cell activation. Using a combination of RT-PCR, two-dimensional gel electrophoresis, and MALDI-TOF-based proteomics analyses, the transcriptional and translational activities of Chlamydia-pulsed DC from wild-type and IL-10KO mice were assessed. IL-10 deficiency caused early maturation and activation of pulsed DC (i.e., high CD11c, CD40, CD80, CD83, CD86, IL-1, IL-12, and the T cell-attracting chemokine CCL27/CTACK) and consequently an enhanced ability to process and present Ags for a rapid and robust T cell activation. Supporting comparative proteomics revealed further that IL-10 deficient DC possess specific immunobiological properties, e.g., the T cell-attracting chemokine CCL27/CTACK, calcium-dependent protein kinase, and the IL-1/IL-12 inducer, NKR-P1A (CD161), which differentiated them immunologically from wild-type DC that express molecules relating to anti-inflammatory, differentiative, and metabolic processes, e.g., the anti-IL-12 molecule peroxisome proliferator-activated receptor-alpha and thymidine kinase. Collectively, these results provide a molecular basis for the high Th1-activating capacity of IL-10KO APC and may provide unique immunomodulation targets when designing vaccines against pathogens controlled by T cell immunity.

The administration of an efficacious vaccine is the most effective long-term measure to control the oculogenital infections caused by Chlamydia trachomatis in humans. Chlamydia genome sequencing has identified a number of potential vaccine candidates, and the current challenge is to develop an effective delivery vehicle for induction of a high level of mucosal T and complementary B cell responses. Vibrio cholerae ghosts (VCG) are nontoxic, effective delivery vehicles with potent adjuvant properties, and are capable of inducing both T cell and Ab responses in mucosal tissues. We investigated the hypothesis that rVCG could serve as effective delivery vehicles for single or multiple subunit chlamydial vaccines to induce a high level of protective immunity. rVCG-expressing chlamydial outer membrane proteins were produced by a two-step genetic process, involving cloning of Omp genes in V. cholerae, followed by gene E-mediated lysis of the cells. The immunogenicity and vaccine efficacy of rVCG-expressing single and multiple subunits were compared. Immunologic analysis indicated that i.m. immunization of mice with either vaccine construct induced a strong mucosal and systemic specific Th1 response against the whole chlamydial organism. However, there was an immunogenic advantage associated with the multiple subunit vaccine that induced a higher frequency of Th1 cells and a relatively greater ability to confer protective immunity, compared with the single subunit construct. These results support the operational theory that the ability of a vaccine to confer protective immunity against Chlamydia is a function of the level of Th1 response elicited.

Immunity to intracellular microbial pathogens, including Chlamydia species, is controlled primarily by cell-mediated effector mechanisms, yet, the absence of antibodies results in inefficient microbial clearance. We investigated the hypothesis that certain Fc receptor functions promote the rapid induction of elevated T helper type 1 (Th1) response, which effectively clears chlamydiae. FcR(-/-) mice exhibited a delayed and reduced frequency of Chlamydia-specific Th1 cells, compared to FcR(+/+) mice. In vitro, antichlamydial antibodies increased the rate of Th1 activation by FcR(+/+) but not FcR(-/-) antigen-presenting cells. FcR(-/-) dendritic cells and the T cell-associated IgG2A and IgA mediate enhanced Th1 activation by antibodies. Immunization with chlamydia-antibody complexes induced elevated and protective Th1 response. These results provide a mechanistic basis for requiring both T cell and humoral immune responses in protective immunity and vaccine evaluation. Findings offer a paradigm in host defense wherein different effector components function indirectly to maximize the principal effector mechanism.

An efficacious vaccine is needed to control the morbidity and burden of rising healthcare costs associated with genital Chlamydia trachomatis infection. Despite considerable efforts, the development of reliable chlamydial vaccines using conventional strategies has proven to be elusive. The 40kDa major outer membrane protein (MOMP) of C. trachomatis is so far the most promising candidate for a subunit vaccine. The lack of satisfactory protective immunity with MOMP-based vaccine regimens to date would suggest that either MOMP alone is inadequate as a vaccine candidate or better delivery systems are needed to optimize the effect of MOMP. Recombinant Vibrio cholerae ghosts (rVCG) are attractive for use as non-living vaccines because they possess strong adjuvant properties and are excellent vehicles for delivery of antigens of vaccine relevance to mucosal sites. The suitability of the ghost technology for designing an anti-chlamydial vaccine was evaluated by constructing a rVCG vector-based candidate vaccine expressing MOMP (rVCG-MOMP) and assessing vaccine efficacy in a murine model of C. trachomatis genital infection. Intramuscular delivery of the rVCG-MOMP vaccine induced elevated local genital mucosal as well as systemic Th1 responses. In addition, immune T cells from immunized mice could transfer partial protection against a C. trachomatis genital challenge to nai;ve mice. These results suggest that rVCG expressing chlamydial proteins may constitute a suitable subunit vaccine for inducing an efficient mucosal T cell response that protects against C. trachomatis infection. Altogether, the potency and relatively low production cost of rVCG offer a significant technical advantage as a chlamydial vaccine.

The prevailing paradigm for designing potentially efficacious vaccines against the obligate intracellular bacterium, Chlamydia trachomatis, advocates regimens capable of inducing a mucosal antigen-specific T helper type 1 (Th1) response. However, recent reports indicate that rapid and efficient clearance of a secondary infection also requires certain B-cell functions. We investigated the hypothesis that Fc receptor (FcR)-mediated antibody effector mechanisms are important B-cell-related functions involved in controlling a chlamydial genital reinfection. Microbiological analysis of genital chlamydial infection in FcR knockout (FcRKO) mice lacking the activatory FcgammaRI (CD64) and FcRgammaIII (CD16), as well as the inhibitory FcgammaRIIB1 (CD32), revealed a greater intensity of secondary infection (i.e. bacterial shedding) in FcRminus sign/minus sign as compared to FcR+/+ mice; however, the course of the primary infection was indistinguishable in both animals. Pathologically, FcRKO mice suffered greater ascending infection than immunocompetent wild-type (WT) mice after a secondary infection. Immunological evaluation indicated that the presence of specific anti-chlamydial antibodies enhanced chlamydial antigen presentation for induction of a Th1 response by FcR+/+, but not FcRminus sign/minus sign, antigen-presenting cells. In addition, specific anti-chlamydial antibodies augmented both macrophage killing of infected epithelial cells by antibody-dependent cellular cytotoxicity (ADCC) and macrophage inhibition of productive growth of chlamydiae in co-cultures. These results indicate that B cells participate in anti-chlamydial immunity via FcR-mediated effector functions of antibodies, which are operative during reinfections. Such effector functions include ADCC, and possibly enhanced uptake, processing and presentation of chlamydial antigens for rapid induction of a Th1 response, all facilitating the early clearance of an infection. These findings suggest that a future anti-chlamydial vaccine should elicit both humoral and T-cell-mediated immune responses for optimal memory response and vaccine efficacy.

Current design strategies for vaccines against certain microbial pathogens, including Chlamydia trachomatis, require the induction and targeting of specific immune effectors to the local sites of infection known as the mucosal effector sites. Chemokines and their receptors are important mediators of leukocyte trafficking and of the controlled recruitment of specific leukocyte clonotypes during host defense against infections and during inflammation. We analyzed the dynamics of chemokine and chemokine receptor expression in genital mucosae during genital chlamydial infection in a murine model to determine how these molecular entities influence the development of immunity and the clearance of infection. A time course study revealed an increase of up to threefold in the levels of expression of RANTES, monocyte chemotactic protein 1 (MCP-1), gamma-interferon-inducible protein 10 (IP-10), macrophage inflammatory protein 1alpha (MIP-1alpha), and intercellular adhesion molecule type 1 (ICAM-1) after genital infection with the C. trachomatis agent of mouse pneumonitis. Peak levels of expression of RANTES, MCP-1, and MIP-1alpha occurred by day 7 after primary infection, while those of IP-10 and ICAM-1 peaked by day 21. Expression levels of these molecules decreased by day 42 after primary infection, by which time all animals had resolved the infection, suggesting an infection-driven regulation of expression. A rapid upregulation of expression of these molecules was observed after secondary infection. The presence of cells bearing the chemokine receptors CCR5 and CXCR3, known to be preferentially expressed on Th1 and dendritic cells, was also synchronous with the kinetics of immune induction in the genital tract and clearance of infection. Results demonstrated that genital chlamydial infection is associated with a significant induction of chemokines and chemokine receptors that are involved in the recruitment of Th1 cells into the site of infection. Future studies will focus on how selective modulation of chemokines and their receptors can be used to optimize long-term immunity against CHLAMYDIA:

A new paradigm for designing vaccines against certain microbial pathogens, including Chlamydia trachomatis, is based on the induction of local mucosal Th1 response. IL-10 is an anti-inflammatory cytokine that exerts negative immunoregulatory influence on Th1 response. This study investigated whether biochemical modulation of endogenous IL-10 expression at the level of APCs is a practical strategy for enhancing the specific Th1 response against pathogens controlled by Th1 immunity. The results revealed that the high resistance of genetically engineered IL-10-/- (IL-10KO) mice to genital chlamydial infection is a function of the predilection of their APCs to rapidly and preferentially activate a high Th1 response. Thus, in microbiological analysis, IL-10KO mice suffered a shorter duration of infection, less microbial burden, and limited ascending infection than immunocompetent wild-type mice. Also, IL-10KO were resistant to reinfection after 8 wk of the primary infection. Cellular and molecular immunologic evaluation indicated that IL-10KO mice induced greater frequency of chlamydial-specific Th1 response following C. trachomatis infection. Moreover, IL-10KO APCs or antisense IL-10 oligonucleotide-treated wild-type APCs were potent activators of Th1 response from naive or immune T cells. Furthermore, both Ag-pulsed dendritic cells from IL-10KO mice and IL-10 antisense-treated dendritic cells from wild-type mice were efficient cellular vaccines in adoptive immunotherapeutic vaccination against genital chlamydial infection. These findings may furnish a novel immunotherapeutic strategy for boosting the Th1 response against T cell-controlled pathogens and tumors, using IL-10-deficient APCs as vaccine delivery agents.

Recent studies in animal models of genital chlamydial disease revealed that early recruitment of dendritic cells and specific T helper type-1 (Th1) cells into the genital mucosae is crucial for reducing the severity of the acute phase of a cervico-vaginal infection and arresting ascending disease. These immune effectors are therefore important for preventing major complications of genital chlamydial infection. Other in vitro studies showed that intercellular adhesion molecule-1 (ICAM-1) plays a role in the antichlamydial action of specific CD4+ and CD8+ T cells. In the present study, we investigated the clinicopathological consequences of ICAM-1 deficiency during chlamydial genital infection in ICAM-1 knockout (ICAM-1KO) mice, and analysed the cellular and molecular immunological bases for any observed pathology or complication. Following a primary genital infection of female ICAM-l-/- and ICAM-1+/+ mice, the intensity of the disease during the first 3 weeks (as assessed by shedding of chlamydiae in the genital tract) was significantly greater in ICAM-1KO mice than in ICAM-1+/+ mice (P < 0.0001), although both ICAM-l-/- and ICAM-1+/+ mice subsequently cleared the primary infection. There was greater ascending disease during the initial stage of the infection, and a higher incidence of tubal disease (hydrosalpinx formation) after multiple infections in ICAM-l-/- mice. Analysis of the cellular and molecular bases for the increased acute and ascending disease in ICAM-l-/- mice revealed that the high affinity of ICAM-1 for leucocyte function antigen type-1 is a property that promotes rapid activation of specific Th1 cells, as well as their early recruitment into the genital mucosa. Moreover, ICAM-1 was more important for naive T-cell activation than primed Th1 cells, although its absence delayed or suppressed immune T-cell activation by at least 50%. Taken together, these results indicated that ICAM-1 is crucial for rapid T-cell activation, early recruitment and control of genitally acquired Chlamydia trachomatis.

The induction of local T helper type 1 (Th1)-mediated cellular immunity is crucial for resistance of mice to genital infection by the obligate intracellular bacterium Chlamydia trachomatis. We tested the hypothesis that the route of immunization that elicits relatively high numbers of chlamydia-specific, gamma interferon (IFN-gamma)-secreting T lymphocytes (ISTLs) in the genital tract would induce optimal protective immunity against reinfection. Female BALB/c mice were infected intravaginally (i.v.), intranasally (i.n.), orally (p.o.), or subcutaneously (s.c.) with C. trachomatis. At days 7, 14, 21, and 28 postinfection, T cells isolated from the genital tract tissues were restimulated with chlamydial antigen in vitro, and the amounts of IFN-gamma induced were measured by a sandwiched enzyme-linked immunosorbent assay method. At day 7 postinfection, i.n.- and i.v.-immunized mice had high levels of chlamydia-specific ISTLs in their genital tracts (203.58 +/- 68.1 and 225.5 +/- 12.1 pg/ml, respectively). However, there were no detectable ISTLs in the genital tracts of p.o.- or s.c.-infected mice. When preinfected mice were challenged i.v. 70 days later, animals preexposed by the i.n. route were highly resistant to reinfection, with greatly reduced chlamydial burden, and suffered an attenuated infection that resolved by day 6 postchallenge. Animals preexposed by the i.v. route were modestly protected, whereas p.o. and s.c. groups were indistinguishable in this regard from control mice. The resistance of i.n.-immunized mice (and to some extent the i.v.-exposed mice) to reinfection was associated with early appearance (within 24 h) of high levels of genital ISTLs compared with mice preinfected by other routes. Furthermore, although i.n. and i.v.-immunized mice had comparable levels of chlamydia-specific immunoglobulin A (IgA) antibodies in their vaginal washes, the levels of IgG2a were four- sixfold higher in i.n.-immunized mice than in any of the other groups. The results suggested that immunization routes that foster rapid induction of vigorous genital mucosal cell-mediated immune (CMI) effectors (e.g., IFN-gamma), the CMI-associated humoral effector, IgG2a, and to some extent secretory IgA produce protective immunity against chlamydial genital infection. Therefore, i.n. immunization is a potential delivery route of choice in the development of a vaccine against Chlamydia.

Type 1 CD4+-T-cell-mediated immunity is crucial for the resolution of chlamydial infection of the murine female genital tract. Previous studies demonstrating a correlation between CD4+-T-cell-mediated inhibition of chlamydial growth and gamma interferon (IFN-gamma)-mediated induction of nitric oxide synthase suggested a potential role for the nitric oxide (NO) effector pathway in the clearance of Chlamydia from genital epithelial cells by the immune system. To clarify the role of this pathway, the growth levels of Chlamydia trachomatis organisms in normal (iNOS+/+) mice and in genetically engineered mice lacking the inducible nitric oxide synthase (iNOS) gene (iNOS-/- mice) were compared. There was no significant difference in the course of genital chlamydial infections in iNOS+/+ and iNOS-/- mice as determined by recovery of Chlamydia organisms shed from genital epithelial cells. Dissemination of Chlamydia to the spleen and lungs occurred to a greater extent in iNOS-/- than in iNOS+/+ mice, which correlated with a marginal increase in the susceptibility of macrophages from iNOS-/- mice to chlamydial infection in vitro. However, infections were rapidly cleared from all affected tissues, with no clinical signs of disease. The finding of minimal dissemination in iNOS-/- mice suggested that activation of the iNOS effector pathway was not the primary target of IFN-gamma during CD4+-T-cell-mediated control of chlamydial growth in macrophages because previous reports demonstrated extensive and often fatal dissemination of Chlamydia in mice lacking IFN-gamma. In summary, these results indicate that the iNOS effector pathway is not required for elimination of Chlamydia from epithelial cells lining the female genital tract of mice although it may contribute to the control of dissemination of C. trachomatis by infected macrophages.

The antimicrobial activity of T cell-derived cytokines, especially interferon (IFN)-gamma, against intracellular pathogens, such as Chlamydia trachomatis, involves the induction of 3 major biochemical processes: tryptophan catabolism, nitric oxide (NO) induction and intracellular iron (Fe) deprivation. Since the epithelial cell is the natural target of chlamydial infection, the presence of these antimicrobial systems in the cell would suggest that they may be involved in T cell control of intracellular multiplication of Chlamydia. However, the controversy over whether these 3 antimicrobial processes are present in both mice and humans has precluded the assessment of the relative contribution of each of the 3 mechanisms to chlamydial inhibition in the same epithelial cell from either mice or humans. In the present study, we identified a Chlamydia-susceptible human epithelial cell line, RT4, that possesses the 3 antimicrobial systems, and we examined the role of nitric oxide (NO) induction, and deprivation of tryptophan or Fe in cytokine-induced inhibition of chlamydiae. It was found that the 3 antimicrobial systems contributed to cytokine-mediated inhibition of the intracellular growth of Chlamydia. NO induction accounted for approximately 20% of the growth inhibition; tryptophan catabolism contributed approximately 30%; iron deprivation was least effective; but the combination of the 3 systems accounted for greater than 60% of the inhibition observed. These results indicate that immune control of chlamydial growth in human epithelial cells may involve multiple mechanisms that include NO induction, tryptophan catabolism and Fe deprivation.

The clinical and epidemiologic features of respiratory syncytial virus (RSV) infections suggest that RSV-specific antibody may sometimes contribute to the disease process. Recently, it has been demonstrated that virus-specific antibody can enhance RSV infection of macrophagelike cells in vitro. We evaluated the possibility that antibody might also enhance RSV stimulation of the bronchoactive mediator of inflammation leukotriene C-4 (LTC4)in a macrophagelike cell line, U937. The addition of RSV led to little increase in LTC4 production, but addition of RSV plus anti-RSV antibody increased production to a level similar to that achieved with calcium ionophore, a known stimulator of LTC4 production. The antibody-enhanced increase in LTC4 production occurred rapidly (within 15 min), peaked at 60 min, and achieved levels 1.5- to 3.0-fold above that for cells or cells plus virus. RSV plus anti-RSV antibodies in the form of polyclonal serum, monoclonal antibodies, or F(ab')2 fragments and parainfluenza virus types 1 and 3 plus their respective antibodies all increased LTC4 levels over that for the virus alone. These results demonstrate that antibody plus the corresponding virus or protein can increase leukotriene production. This phenomenon could contribute to diseases, such as RSV bronchiolitis, that appear to be caused by an interaction between the virus (or antigen) and host immunity.

Phenotypically typical Staphylococcus aureus was isolated frequently from the necrotic bone and liver of poultry suffering from femoral head necrosis. Occasionally strains were isolated that differed from typical Staph. aureus in one or more of the major diagnostic tests, i.e. coagulase production, anaerobic fermentation of mannitol and production of a heat-stable deoxyribonuclease. Such atypical strains were also isolated from nasal swabs of healthy birds. Tests for enterotoxin production demonstrated that some atypical strains from both sick and healthy birds are capable of producing staphylococcal enterotoxins.