Joseph M. Lyons

Vaccinations with Recombinant Variants of Aspergillus fumigatus Allergen Asp f 3 Protect Mice against Invasive Aspergillosis

ABSTRACT 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 predominately 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.

Do host genetic traits in the bacterial sensing system play a role in the development of Chlamydia trachomatis-associated tubal pathology in subfertile women?

BackgroundIn 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.MethodsOf 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.ResultsFollowing 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.ConclusionCarrying 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.

Vaccination of Corticosteroid Immunosuppressed Mice against Invasive Pulmonary Aspergillosis

Invasive pulmonary aspergillosis is an emerging devastating infection in the immunocompromised host that is treated with corticosteroids for neoplastic disease or for organ transplantation. By use of a model of invasive pulmonary aspergillosis in corticosteroid-treated CF-1 mice, prior infection and 2 Aspergillus fumigatus vaccine preparations (sonicate and filtrate) administered intranasally and subcutaneously were tested for efficacy in protecting against subsequent lethal A. fumigatus infection. The mortality rates were as follows: control subjects, 100%; prior infection, 12.5%; sonicate administered intranasally, 29%; sonicate given subcutaneously, 0%; filtrate given intranasally, 75%; and filtrate given subcutaneously, 50%. Prior infection and A. fumigatus sonicate vaccine administered by 2 routes protected corticosteroid-treated animals against subsequent lethal invasive pulmonary aspergillosis. The sonicate vaccine was more protective, but the subcutaneous route was more effective.

Challenges and directions for the pathogen hypothesis of Alzheimer’s disease

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 Alzheimers 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.

Differences in growth characteristics and elementary body associated cytotoxicity between Chlamydia trachomatis oculogenital serovars D and H and Chlamydia muridarum

Aim: In vitro growth and elementary body (EB) associated cytotoxicity of two Chlamydia trachomatis strains belonging to serovars D and H and C muridarum were compared to identify difference(s) that correlate with virulence variations between these strains in the mouse model of human female genital tract infection, and phenotypic characteristics that could explain human epidemiological data on serovar prevalence and levels of shedding during serovar D and H infection. Methods: Replication cycle kinetics, inclusion characteristics, and EB associated cytotoxicity were assessed in McCoy cell monolayers using culture, light microscopy, and lactate dehydrogenase release. Results: Over 72 hours, more rapid production and release of inclusion forming units (ifu) allowed C muridarum to initiate two replication rounds, resulting in 4–8 times more ifu/input unit of infection than with serovars D and H. Although C muridarum EBs were significantly more cytotoxic to McCoy cell monolayers than serovar D at moderate and high multiplicity of infection ratios (MOI), serovar H EBs were significantly more cytotoxic than C muridarum, even at the lowest MOI tested. Conclusions: These phenotypic differences are consistent with the more invasive course and severe pathological outcome of infection in mice infected with C muridarum, providing an objective basis for questioning the appropriateness of C muridarum as a surrogate for the human biovar of C trachomatis in the murine model of female genital tract infection. The differences seen between the human strains could help explain human epidemiological data relating to differences in prevalence and level of shedding that occurs during infection with oculogenital serovars D and H.

TLR9 KO MICE, HAPLOTYPES AND CPG INDICES IN CHLAMYDIA TRACHOMATIS INFECTION

Antiplatelet therapy is the cornerstone of treatment for patients with acute coronary syndrome undergoing percutaneous coronary intervention. Clopidogrel, in combination with aspirin, is associated with improvement in longterm vascular clinical outcomes in these patients and is currently the antiplatelet standard of care. However, a significant number of patients still experience secondary ischemic thrombotic events due to potential insufficient platelet inhibition or noncompliance. Therefore, the development of better and safer antiplatelet agents is of the utmost priority. Indeed, oral antiplatelet agents, such as aspirin in the ISIS-2 study and clopidogrel in the COMMIT mega trial, in moderate doses are among the very few classes of drugs that reduce absolute mortality in patients after acute vascular thrombotic events. Prasugrel (CS-747; LY-640315), an experimental third-generation oral thienopyridine, is a specific, irreversible antagonist of the platelet adenosine diphosphate P2Y(12) receptor. Preclinical and early phase clinical studies have shown that prasugrel has greater antiplatelet potency, lower variability in platelet response and faster onset of inhibition than clopidogrel. However, the doses of the drug chosen for further prasugrel developments are much higher (about 2.5-2.7 times higher) than those of conventional clopidogrel regimen(s). The recent TRITON trial assessed head-to-head prasugrel versus clopidogrel, both in addition to aspirin, and led to numerous controversies with regard to the fairness of the trial design, interpretation of its results, and the suitability of the high maintenance prasugrel dose for chronic preventive human use. We critically review various aspects of prasugrel development, focusing on the discrepancies between the official interpretation of the results and actual findings. We conclude that the benefits of prasugrel are exaggerated and that the risks are underestimated. Very careful maintenance dose selection and a flawless long-term safety profile for the new agents will become the keys to the success of future oral antiplatelet drug development.

Murine models of Chlamydia trachomatis genital tract infection: use of mouse pneumonitis strain versus human strains.

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

Acquired homotypic and heterotypic immunity against oculogenital Chlamydia trachomatis serovars following female genital tract infection in mice

BackgroundChlamydia 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.MethodsGroups 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.ResultsSerovar 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.ConclusionSerovar 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.

Efficacy of an Immune Modulator in Experimental Chlamydia trachomatis Infection of the Female Genital Tract

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.

The Influence of Vaginally Applied Imiquimod on the Course of Chlamydia trachomatis Serovar D Infection in a Murine Model

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.