Chlamydia trachomatis infections demonstrate remarkable differences in clinical course that are approximately 40% based on host genetic variation. Here, we study the single nucleotide polymorphisms (SNPs) and their haplotypes in TLR2, TLR4 and TLR9 (TLR2+2477G > A; TLR2 -16934T > A; TLR4+896A > G; TLR9-1237T > C and TLR9 +2848G > A) in relation to the susceptibility to, and severity of C. trachomatis infections. We analysed the five SNPs in a cohort of 770 Dutch Caucasian women either attending a sexually transmitted diseases outpatient clinic (n = 731) or having complaints of subfertility (n = 39). Haplotype analyses showed a trend for TLR2 haplotype I (-16934T/+2477G) to protect against the development of symptoms and tubal pathology (P-trend = 0.03) after Chlamydia infection. In the susceptibility cohort, TLR9 haplotype III (-1237C/+2848A) showed a significant decreasing trend in the development of symptoms after C. trachomatis infection (P = 0.02, OR: 0.55, 95% CI: 0.33-0.91). Logistic regression of the TLR2 haplotypes, TLR4+896A > G, and TLR9 haplotypes showed that the TLR2 haplotype combinations AG-TA and AG-TG confer risk (OR 3.4 (P = 0.01) and 1.6 (P = 0.03)), while the TLR9 haplotype combination TG-TA protects against C. trachomatis infections (OR: 0.4, P = 0.004). Our study shows that both TLR2 and TLR9 genes and SNP combinations do influence the clinical course of Chlamydia infections.

Chlamydia trachomatis infections demonstrate remarkable differences in clinical course that are approximately 40% based on host genetic variation. Here, we study the single nucleotide polymorphisms (SNPs) and their haplotypes in TLR2, TLR4 and TLR9 (TLR2 +2477G>A; TLR2 –16934T>A; TLR4+896A>G; TLR9 –1237T>C and TLR9 +2848G>A) in relation to the susceptibility to, and severity of C. trachomatis infections. We analysed the five SNPs in a cohort of 770 Dutch Caucasian women either attending a sexually transmitted diseases outpatient clinic (n = 731) or having complaints of subfertility (n = 39). Haplotype analyses showed a trend for TLR2 haplotype I (–16934T/+2477G) to protect against the development of symptoms and tubal pathology (Ptrend = 0.03) after Chlamydia infection. In the susceptibility cohort, TLR9 haplotype III (–1237C/+2848A) showed a significant decreasing trend in the development of symptoms after C. trachomatis infection (P = 0.02, OR: 0.55, 95%CI: 0.33–0.91). Logistic regression of the TLR2 haplotypes, TLR4+896A>G, and TLR9 haplotypes showed that the TLR2 haplotype combinations AG-TA and AG-TG confer risk (OR 3.4 (P = 0.01) and 1.6 (P = 0.03)), while the TLR9 haplotype combination TG-TA protects against C. trachomatis infections (OR: 0.4, P = 0.004). Our study shows that both TLR2 and TLR9 genes and SNP combinations do influence the clinical course of Chlamydia infections.

Despite the recent development of new anti-mould agents, there remains a significant incidence of invasive aspergillosis in the most immunocompromised hosts and the response to these agents is still dismal. There is a need for a different approach: prevention by vaccination. We have demonstrated that a hyphal sonicate of Aspergillus fumigatus was capable of conferring protection against subsequent invasive pulmonary aspergillosis in corticosteroid immunosuppressed mice. Subcutaneous vaccination was superior to nasal vaccination. Mice exposed intranasally to viable conidia were noted to respond serologically to a 19 kDa protein. This protein was identified as the allergen Asp f 3 by mass spectrometry. Vaccination with recombinant Asp f 3 was protective. Truncated forms of Asp f 3 that lacked either one of the two known IgE binding sites were cloned and also demonstrated protection against aspergillosis. Although all of these recombinant proteins required an adjuvant (TiterMax) for efficacy, a particulate preparation of rAsp f 3 was also found to be protective without requiring adjuvant. At least two T-cell epitopes (11-mer and 13-mer) have been identified in Asp f 3. There are homologues of Asp f 3 in other Aspergillus species as well as in other moulds (Coccidioides posadasii, Penicillium citrinum) and yeasts (Candida albicans, C. boidinii, Saccharomyces cerevisiae). Asp f 3, truncated non-allergenic versions of Asp f 3, and T-cells epitopes of Asp f 3 are potential candidates for vaccines potentially capable of protecting immunocompromised hosts against invasive aspergillosis.

A vaccine that effectively protects immunocompromised patients against invasive aspergillosis is a novel approach to a universally fatal disease. Here we present a rationale for selection and in vivo testing of potential protein vaccine candidates, based on the modification of an immunodominant fungal allergen for which we demonstrate immunoprotective properties. Pulmonary exposure to viable Aspergillus fumigatus conidia as well as vaccination with crude hyphal extracts protects corticosteroid-immunosuppressed mice against invasive aspergillosis (J. I. Ito and J. M. Lyons, J. Infect. Dis. 186:869-871, 2002). Sera from the latter animals contain antibodies with numerous and diverse antigen specificities, whereas sera from conidium-exposed mice contain antibodies predominantly against allergen Asp f 3 (and some against Asp f 1), as identified by mass spectrometry. Subcutaneous immunization with recombinant Asp f 3 (rAsp f 3) but not with Asp f 1 was protective. The lungs of Asp f 3-vaccinated survivors were free of hyphae and showed only a patchy low-density infiltrate of mononuclear cells. In contrast, the nonimmunized animals died with invasive hyphal elements and a compact peribronchial infiltrate of predominantly polymorphonuclear leukocytes. Three truncated versions of rAsp f 3, spanning amino acid residues 15 to 168 [rAsp f 3(15-168)], 1 to 142, and 15 to 142 and lacking the known bipartite sequence required for IgE binding, were also shown to be protective. Remarkably, vaccination with either rAsp f 3(1-142) or rAsp f 3(15-168) drastically diminished the production of antigen-specific antibodies compared to vaccination with the full-length rAsp f 3(1-168) or the double-truncated rAsp f 3(15-142) version. Our findings point to a possible mechanism in which Asp f 3 vaccination induces a cellular immune response that upon infection results in the activation of lymphocytes that in turn enhances and/or restores the function of corticosteroid-suppressed macrophages to clear fungal elements in the lungs.

Objective. The aim of this study was to determine if vaginal application of the immune response modifier imiquimod (Aldara cream, 3M Pharmaceuticals, St Paul, Minn) would alter the course and/or outcome of female genital tract infection with a human isolate of Chlamydia trachomatis in a murine model. Methods. Groups of CF-1 mice were treated with Aldara on three different schedules: (1) ongoing beginning 5 days prior to and continuing through day 5 of infection; (2) a single prophylactic dose 2 hours prior to infection; and (3) therapeutic from day 4 to day 14 of infection. Mice were infected vaginally with a serovar D strain of C trachomatis, and monitored by culture to determine the level of shedding and duration of infection. Results. We observed a significant reduction in both duration of infection and the level of shedding during the acute phase in mice treated on an ongoing basis commencing 5 days prior to infection. There was no effect with respect to the other regimens. Conclusion. These results demonstrate that ongoing Aldara treatment has efficacy and may enhance local innate immunity which reduces the duration of subsequent infection with human isolates of C trachomatis in a murine model of female genital tract infection.

Background: In women, Chlamydia (C.) trachomatis upper genital tract infection can cause distal tubal damage and occlusion, increasing the risk of tubal factor subfertility and ectopic pregnancy. Variations, like single nucleotide polymorphisms (SNPs), in immunologically important host genes are assumed to play a role in the course and outcome of a C. trachomatis infection. We studied whether genetic traits (carrying multiple SNPs in different genes) in the bacterial sensing system are associated with an aberrant immune response and subsequently with tubal pathology following a C. trachomatis infection. The genes studied all encode for pattern recognition receptors (PRRs) involved in sensing bacterial components.Methods: Of 227 subfertile women, serum was available for C. trachomatis IgG antibody testing and genotyping (common versus rare allele) of the PRR genes TLR9, TLR4, CD14 and CARD15/NOD2. In all women, a laparoscopy was performed to assess the grade of tubal pathology. Tubal pathology was defined as extensive peri-adnexal adhesions and/or distal occlusion of at least one tube.Results: Following a C. trachomatis infection (i.e. C. trachomatis IgG positive), subfertile women carrying two or more SNPs in C. trachomatis PRR genes were at increased risk of tubal pathology compared to women carrying less than two SNPs (73% vs 33% risk). The differences were not statistically significant (P=0.15), but a trend was observed.Conclusion: Carrying multiple SNPs in C. trachomatis PRR genes tends to result in an aberrant immune response and a higher risk of tubal pathology following a C. trachomatis infection. Larger studies are needed to confirm our preliminary findings.

Background Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen causing female genital tract infection throughout the world. Reinfection with the same serovar, as well as multiple infections with different serovars, occurs in humans. Using a murine model of female C. trachomatis genital tract infection, we determined if homotypic and/or heterotypic protection against reinfection was induced following infection with human oculogenital strains of C. trachomatis belonging to two serovars (D and H) that have been shown to vary significantly in the course of infection in the murine model. Methods Groups of outbred CF-1 mice were reinfected intravaginally with a strain of either serovar D or H, two months after initial infection with these strains. Cellular immune and serologic status, both quantitative and qualitative, was assessed following initial infection, and the course of infection was monitored by culturing vaginal samples collected every 2–7 days following reinfection. Results Serovar D was both more virulent (longer duration of infection) and immunogenic (higher level of circulating and vaginal IgG and higher incidence of IgA in vaginal secretions) in the mouse genital tract. Although both serovars induced cross-reacting antibodies during the course of primary infection, prior infection with serovar H resulted in only a slight reduction in the median duration of infection against homotypic reinfection (p ~ 0.10), while prior infection with serovar D resulted in significant reduction in the median duration of infection against both homotypic (p < 0.01) and heterotypic reinfection (p < 0.01) when compared to primary infection in age and conditions matched controls. Conclusion Serovar D infection resulted in significant homotypic and heterotypic protection against reinfection, while primary infection with serovar H resulted in only slight homotypic protection. In addition to being the first demonstration of acquired heterotypic immunity between human oculogenital serovars, the differences in the level and extent of this immunity could in part explain the stable difference in serovar prevalence among human isolates.

Sir—We read with great interest the article by Ramsey and colleagues [1]. In their study, they concluded that imiquimod had no efficacy in controlling Chlamydia trachomatis MoPn (mouse pneumonitis agent) infection in a murine model of female genital tract infection. Imiquimod is an immune response modifier that induces the production of an array of essentially Th1 response promoting cytokines, most notably tumor necrosis factor-alpha and interferon-gamma, the pluripotent cytokine that is considered essential in both the innate and the acquired immune response to C. trachomatis genital tract infection. We, as well as others, have shown that the course of female genital tract infection with MoPn in IFNgamma knockout mice is only minimally different when compared with genetically intact mice, whereas infection with a human isolate of C. trachomatis serovar D is uncontrolled during the acute phase and of significantly longer duration in interferon-gamma deficient mice [2, 3]. Therefore, we investigated the effect of various regimens of vaginally applied imiquimod on the course of infection in essentially the same murine model of C. trachomatis female genital tract infection as used by Ramsey and colleagues, but employing a strain belonging to the human oculogenital biovar of C. trachomatis. In brief, CF-1 mice treated with progesterone were inoculated intravaginally by direct instillation of 10 ml of bacterial suspension containing 1 X 10 inclusion-forming units (ifu) of C. trachomatis serovar D [4]. On the days before (7) and after ( + ) infection as indicated in the results table, 10 ml of imiquimod (Aldara), diluted 1:4 in saline or a placebo similar in composition to the inactive base used in the imiquimod preparation, was administered intravaginally [5]. The presence of Chlamydia in the lower genital tract was determined by culturing the vaginal contents as previously described, and the duration of infection between groups was analyzed using the Wilcoxon rank sum test [2, 4, 5]. As displayed below, we observed a statistically significant reduction in the median duration of infection in mice treated prophylactically with imiquimod several times before and during the acute phase of C. trachomatis infection, i.e. 4 days for treated animals compared with 19 days for the group receiving placebo. No effect on the course of infection was seen with other regimens, a single application 2 h before or multiple applications every other day beginning 4 days after chlamydial inoculation and ending on day 14 (data not shown). Remarkably, in the prophylactically treated group on day 2, no effect was observed on the incidence of infection when compared with the placebo group. However, by day 4 differences between the groups were apparent, implying that treated mice were primed to respond more rapidly to infection, while apparently unaltered in their susceptibility to initial infection. In mice, imiquimod is reported to act through the Toll-like receptor 7 (TLR7) MyD88dependent signalling pathway This suggests that, in contrast to the speculation of Ramsey and colleagues, sufficient quantities of the TLR7 are expressed in the mouse genital epithelium or in tissues adjacent to the genital epithelium to allow a response to imiquimod.

We have previously shown that female outbred CF-1 mice are susceptible to prolonged genital tract infection with the oculogenital serovars (D-K) of Chlamydia trachomatis, and that partial homotypic and heterotypic protection against reinfection is induced. To understand the possible role of inherent T-helper 1 (Th1)/Th2 polarity bias on both the course of infection and the level of acquired immunity induced by infection, 2 immunologically different and well-characterized inbred strains of mice, BALB/c and C57BL/6, were studied in this model. Groups of mice were inoculated intravaginally with C. trachomatis serovar D (Ct D) and monitored by culture to determine the duration of initial infection. Two months later, mice were reinfected, and monitored along with age- and condition-matched control groups. Plasma and vaginal secretions were collected for serologic analysis and specific delayed-type hypersensitivity was assessed by footpad swelling. Initial infection in C57BL/6 mice was comparable in duration to outbred CF-1 mice (median duration 42 versus 43.5 days), while BALB/c mice had a shorter median duration of initial infection (12 days). All strains had significantly shorter durations of infection following reinfection. BALB/c mice shed 4-10 times more inclusion-forming units (IFU) than both C57BL/6 and CF-1 mice on sample days during the first week of infection and all strains shed less IFU during reinfection. C57BL/6 and BALB/c mice had significantly lower anti-Ct D immunoglobulin G titers in both plasma and vaginal secretions than CF-1 mice following resolution of infection; the frequency of immunoglobulin A seropositive vaginal secretions was less in both inbred strains, being significantly less in the case of C57BL/6 mice. Qualitative analysis of the antigen specificity and isotype composition revealed differences among the mouse strains. All 3 strains had detectable levels of specific footpad swelling on day 14 of infection, whereas only BALB/c mice showed a significant response at 70 days post-infection. Significant differences between 2 strains of mice that differ in Th1/Th2 polarity bias were observed in: 1) the duration of infection; 2) the level of bacterial shedding during infection; and 3) the quantitative and qualitative cellular and humoral responses made in response to female genital tract infection with a human oculogenital isolate of C. trachomatis. In addition, a similar and significant level of partial acquired immunity to reinfection was observed in both strains, suggesting that inherent Th1/Th2 polarity bias present upon initial infection does not prevent the development of a protective immune response within the genital tract during infection with an oculogenital isolate of C. trachomatis.

This paper critically reviews the possibility that infiltration of the brain by pathogens (e.g. Herpes simplex virus type 1 (HSV1) or Chlamydophila pneumoniae (Cp)) acts as a trigger or co-factor for Alzheimer’s disease (AD). The evidence currently available is limited and in some cases inconsistent, but it does justify the need for more vigorous investigation of this hypothesis. An issue of particular concern is the paucity of experimental evidence showing that pathogens can elicit the neuropathological changes and cognitive deficits that characterise AD. Other weaknesses include a failure to obtain independent confirmation of Cp in AD brains, and a lack of evidence for HSV1 proteins or intact virions in AD brain tissue. Future avenues of investigation that might prove fruitful include epidemiological investigations of the incidence of AD in individuals who are either immunosuppressed or have received chronic antiviral or antibiotic therapy. There is also a need to consider systemic infections as potential contributors to the pathogenesis of AD.

Morrison and Morrison ([11][1]) analyzed by in situ immunohistochemistry the progression of the inflammatory and cytokine responses in the genital tracts of mice. They provided an important foundation from which we can study the effects of experimentally induced perturbations in the systemic immune

Epidemiological studies have yet to identify a single cause for the most common late-onset form of Alzheimer's disease. The common respiratory pathogen Chlamydia pneumoniae recently has been implicated as a risk factor for this form of Alzheimer's disease. Were this true, there would be a dramatic shift in current paradigms of Alzheimer's disease research and treatment. In the absence of published confirmation, we obtained postmortem brain tissue from late-onset Alzheimer's disease patients (n = 15) and representative controls (n = 5) and extracted DNA from up to six separate brain regions in each instance, including those areas particularly relevant to Alzheimer's disease neuropathology. Each sample of DNA (n = 101) was assayed five times or more for the presence of C. pneumoniae DNA using a nested-PCR protocol targeting a species-specific gene sequence coding for the major outer membrane protein of this organism. We were unable unequivocally to detect C. pneumoniae in any of the 101 samples tested by PCR and failed to culture the organism from tissue samples. We conclude that C. pneumoniae is neither strongly nor uniquely associated with the neuropathology seen in late-onset Alzheimer's disease.

An enzyme-linked immunosorbent assay for antibody to the lipopolysaccharides (LPSs) of a rough strain of Escherichia coli (J5) and Neisseria gonorrhoeae was developed. When standard methods of this assay were applied to these LPSs, no antibody was measured. By radiolabeling smooth and rough endotoxins with 51Cr, it was discovered that the rough LPS was not adhering to the polystyrene tubes. When Mg++ was added, however, all endotoxins absorbed to the solid phase. With this assay antiserum to core glycolipid had higher titers against its smooth parent strain than did homologous glycolipid had higher titers against its smooth parent strain that did homologous antiserum, and antisera to gonococci had high homologous but low heterologous titers. Thus, these data demonstrate that bulky O side chains do not hinder the penetration of antibody to core glycolipid and that LPSs from gonococci are heterogeneous but do cross-react.