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1.  Formulation of the Microbicide INP0341 for In Vivo Protection against a Vaginal Challenge by Chlamydia trachomatis 
PLoS ONE  2014;9(10):e110918.
The salicylidene acylhydrazide (SA) compounds have exhibited promising microbicidal properties. Previous reports have shown the SA compounds, using cell cultures, to exhibit activity against Chlamydia trachomatis, herpes simplex virus and HIV-1. In addition, using an animal model of a vaginal infection the SA compound INP0341, when dissolved in a liquid, was able to significantly protect mice from a vaginal infection with C. trachomatis. To expand upon this finding, in this report INP0341 was formulated as a vaginal gel, suitable for use in humans. Gelling agents (polymers) with inherent antimicrobial properties were chosen to maximize the total antimicrobial effect of the gel. In vitro formulation work generated a gel with suitable rheology and sustained drug release. A formulation containing 1 mM INP0341, 1.6 wt% Cremophor ELP (solubility enhancer) and 1.5 wt% poly(acrylic acid) (gelling and antimicrobial agent), was chosen for studies of efficacy and toxicity using a mouse model of a vaginal infection. The gel formulation was able to attenuate a vaginal challenge with C. trachomatis, serovar D. Formulations with and without INP0341 afforded protection, but the inclusion of INP0341 increased the protection. Mouse vaginal tissue treated with the formulation showed no indication of gel toxicity. The lack of toxicity was confirmed by in vitro assays using EpiVaginal tissues, which showed that a 24 h exposure to the gel formulation did not decrease the cell viability or the barrier function of the tissue. Therefore, the gel formulation described here appears to be a promising vaginal microbicide to prevent a C. trachomatis infection with the potential to be expanded to other sexually transmitted diseases.
doi:10.1371/journal.pone.0110918
PMCID: PMC4214720  PMID: 25356686
2.  Chlamydia pneumoniae Infection in Mice Induces Chronic Lung Inflammation, iBALT Formation, and Fibrosis 
PLoS ONE  2013;8(10):e77447.
Chlamydia pneumoniae (CP) lung infection can induce chronic lung inflammation and is associated with not only acute asthma but also COPD exacerbations. However, in mouse models of CP infection, most studies have investigated specifically the acute phase of the infection and not the longer-term chronic changes in the lungs. We infected C57BL/6 mice with 5×105 CP intratracheally and monitored inflammation, cellular infiltrates and cytokine levels over time to investigate the chronic inflammatory lung changes. While bacteria numbers declined by day 28, macrophage numbers remained high through day 35. Immune cell clusters were detected as early as day 14 and persisted through day 35, and stained positive for B, T, and follicular dendritic cells, indicating these clusters were inducible bronchus associated lymphoid tissues (iBALTs). Classically activated inflammatory M1 macrophages were the predominant subtype early on while alternatively activated M2 macrophages increased later during infection. Adoptive transfer of M1 but not M2 macrophages intratracheally 1 week after infection resulted in greater lung inflammation, severe fibrosis, and increased numbers of iBALTS 35 days after infection. In summary, we show that CP lung infection in mice induces chronic inflammatory changes including iBALT formations as well as fibrosis. These observations suggest that the M1 macrophages, which are part of the normal response to clear acute C. pneumoniae lung infection, result in an enhanced acute response when present in excess numbers, with greater inflammation, tissue injury, and severe fibrosis.
doi:10.1371/journal.pone.0077447
PMCID: PMC3808399  PMID: 24204830
3.  Protection of Mice From a Chlamydia trachomatis Vaginal Infection Using a Salicylidene Acylhydrazide, a Potential Microbicide 
The Journal of Infectious Diseases  2011;204(9):1313-1320.
The salicylidene acylhydrazide INP0341 inhibits growth of Chlamydia in HeLa cells, has negligible cell toxicity, and does not inhibit the growth of lactobacilli. The antichlamydial activity of INP0341 was retained when tested in vaginal and semen simulants. Vaginal tissue from INP0341-treated mice appeared similar to control sham-treated mice. To determine whether INP0341 can protect mice from a vaginal challenge, C3H/HeJ mice were either sham or INP0341 treated intravaginally pre- and postinoculation with 5 × 102 inclusion-forming units (IFUs) of Chlamydia trachomatis serovar D. Vaginal cultures taken over a month-long period showed a significant difference in the number of control mice that were culture positive versus the number in the INP0341-treated group, 100% (25/25) and 31% (8/26), respectively (P < .05). The quantity of IFUs shed and antibody titers to Chlamydia were significantly higher for the control group (P < .05). In summary, INP0341 is a promising compound to be considered for formulation as a vaginal microbicide.
doi:10.1093/infdis/jir552
PMCID: PMC3182314  PMID: 21933873
4.  Plasmacytoid Dendritic Cells Play a Role for Effective Innate Immune Responses during Chlamydia pneumoniae Infection in Mice 
PLoS ONE  2012;7(10):e48655.
Plasmacytoid dendritic cells (pDCs) are known for their robust antiviral response and their pro-tolerance effects towards allergic diseases and tissue engraftments. However, little is known about the role pDCs may play during a bacterial infection, including pulmonary Chlamydia pneumoniae (CP). In this study, we investigated the role of pDCs during pulmonary CP infection. Our results revealed that depletion of pDCs during acute CP infection in mice results in delayed and reduced lung inflammation, with an early delay in cellular recruitment and significant reduction in early cytokine production in the lungs. This was followed by impaired and delayed bacterial clearance from the lungs which then resulted in a severe and prolonged chronic inflammation and iBALT like structures containing large numbers of B and T cells in these animals. We also observed that increasing the pDC numbers in the lung by FLT3L treatment experimentally results in greater lung inflammation during acute CP infection. In contrast to these results, restimulation of T-cells in the draining lymph nodes of pDC-depleted mice induced greater amounts of proinflammatory cytokines than we observed in control mice. These results suggest that pDCs in the lung may provide critical proinflammatory innate immune responses in response to CP infection, but are suppressive towards adaptive immune responses in the lymph node. Thus pDCs in the lung and the draining lymph node appear to have different roles and phenotypes during acute CP infection and may play a role in host immune responses.
doi:10.1371/journal.pone.0048655
PMCID: PMC3485374  PMID: 23119083
5.  In vitro anti-HIV-1 activity of salicylidene acylhydrazide compounds 
Introduction
Salicylidene acylhydrazide compounds have been shown to inhibit bacterial pathogens, including Chlamydia and Neisseria gonorrhoeae. If such compounds could also target HIV-1, their potential use as topical microbicides to prevent sexually transmitted infections would be considerable. We determined the in vitro anti-HIV-1 activity, cytotoxicity and mechanism of action of several salicylidene acylhydrazides.
Methods
Inhibitory activity was assessed using TZMbl cells and primary peripheral blood mononuclear cells (PBMCs) as targets for HIV-1 infection. Anti-viral activity was measured against cell-free and cell-associated virus and in vaginal fluid and semen simulants. Since the anti-bacterial activity of salicylidene acylhydrazides is reversible by Fe2+, we determined whether Fe2+ and other cations could reverse the anti-HIV-1 activity of the compounds. We also employed real-time PCR to determine the stage affected in the HIV-1 replication cycle.
Results
We identified four compounds with 50% HIV-1 inhibitory concentrations of 1 to 7 μM. In vitro toxicity varied but was generally limited. Activity was similar against three R5 clade B primary isolates and whether targets for virus replication were TZMbl cells or PBMCs. Compounds inhibited cell-free and cell-associated virus and were active in vaginal fluid and semen simulants. Fe2+, but not other cations, reversed the anti-HIV-1 effect. Finally, inhibitory effect of the compounds occurred at a post-integration step.
Conclusions
We identified salicylidene acylhydrazides with in vitro anti-HIV-1 activity in the μM range. The activity of these compounds against other sexually transmitted pathogens makes them potential candidates to formulate for use as a broad-spectrum topical genital microbicide.
doi:10.1016/j.ijantimicag.2012.05.023
PMCID: PMC3438335  PMID: 22819150
Salicylidene acylhydrazides; HIV; microbicide; iron chelation
6.  Candidate vaginal microbicides with activity against Chlamydia trachomatis and Neisseria gonorrhoeae 
Vaginal microbicides with activity towards organisms that cause sexually transmitted infections have been proposed as a strategy to reduce transmission. Small-molecule inhibitors of Chlamydia trachomatis serovar D belonging to the class of salicylidene acylhydrazides (INPs) have been shown to work through a mechanism that involves iron restriction. Expanding on this work, ten INPs were tested against a lymphogranuloma venereum strain of C. trachomatis serovar L2, Neisseria gonorrhoeae, and hydrogen peroxide-producing Lactobacillus crispatus and Lactobacillus jensenii. Seven INPs had minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations of <50 µM towards C. trachomatis L2. Three INPs had an MIC <12.5 µM against N. gonorrhoeae. Inhibition by was reversed by iron, holo-transferrin and holo-lactoferrin but not by the iron-poor forms of these compounds. The compounds exhibited no bactericidal activity toward Lactobacillus. The INPs were not cytotoxic to HeLa 229 cells. When INP 0341 was tested in a mouse model of a Chlamydia vaginal infection there was a significant reduction in the number of mice shedding C. trachomatis up to 4 days after infection (P < 0.01). In summary, select INPs are promising vaginal microbicide candidates as they inhibit the growth of two common sexually transmitted organisms in vitro, are active in a mouse model against C. trachomatis, are not cytotoxic and do not inhibit organisms that compose the normal vaginal flora.
doi:10.1016/j.ijantimicag.2010.03.018
PMCID: PMC2902681  PMID: 20605703
Vaginal microbicide; Sexually transmitted infections; Chlamydia trachomatis; LGV; Neisseria gonorrhoeae
7.  Chlamydia pneumoniae Infection Induced Allergic Airway Sensitization Is Controlled by Regulatory T-Cells and Plasmacytoid Dendritic Cells 
PLoS ONE  2011;6(6):e20784.
Chlamydia pneumoniae (CP) is associated with induction and exacerbation of asthma. CP infection can induce allergic airway sensitization in mice in a dose- and time-dependent manner. Allergen exposure 5 days after a low dose (mild-moderate), but not a high dose (severe) CP infection induces antigen sensitization in mice. Innate immune signals play a critical role in controlling CP infection induced allergic airway sensitization, however these mechanisms have not been fully elucidated. Wild-type, TLR2−/−, and TLR4−/− mice were infected intranasally (i.n.) with a low dose of CP, followed by i.n. exposure to human serum albumin (HSA) and challenged with HSA 2 weeks later. Airway inflammation, immunoglobulins, eosinophils, and goblet cells were measured. Low dose CP infection induced allergic sensitization in TLR2−/− mice, but not in TLR4−/− mice, due to differential Treg responses in these genotypes. TLR2−/− mice had reduced numbers of Tregs in the lung during CP infection while TLR4−/− mice had increased numbers. High dose CP infection resulted in an increase in Tregs and pDCs in lungs, which prevented antigen sensitization in WT mice. Depletion of Tregs or pDCs resulted in allergic airway sensitization. We conclude that Tregs and pDCs are critical determinants regulating CP infection-induced allergic sensitization. Furthermore, TLR2 and TLR4 signaling during CP infection may play a regulatory role through the modulation of Tregs.
doi:10.1371/journal.pone.0020784
PMCID: PMC3112152  PMID: 21695198
8.  Innate Immune Responses during Respiratory Infection with a Bacterial Pathogen Induce Allergic Airway Sensitization 
Background
The original hygiene hypothesis predicts that infections should protect against asthma, but does not account for increasing evidence that certain infections may also promote asthma development. A mechanistic reconciliation of these findings has not yet emerged. In particular, the role of innate immunity in this context is unclear.
Objective
To test whether bacterial respiratory infection causes airway sensitization towards an antigen encountered in parallel, and to elucidate the contribution of innate immune responses.
Methods
Mice were infected with different doses of Chlamydia pneumoniae (CP) followed by exposure to human serum albumin (HSA) and challenge with HSA two weeks later. Airway inflammation, immunoglobulins and lymph node cytokines were assessed. Furthermore, adoptive transfer of dendritic cells (DCs) and depletion of regulatory T-cells (Tregs) was employed.
Results
CP-pneumonia induced sensitization towards HSA resulting in eosinophilic airway inflammation after HSA-challenge. Airway sensitization depended upon severity and timing of infection: low-dose infection and antigen exposure within 5 days of infection induced allergic sensitization, while high-dose infection or antigen exposure 10 days after infection did not. Temporal and dose-related effects reflected DC activation, and could be reproduced by adoptive transfer of HSA-pulsed lung DCs from infected mice. MyD88 deficiency in DCs abolished antigen sensitization, and depletion of Tregs prolonged the time window in which sensitization could occur.
Conclusions
We conclude that moderate but not severe pulmonary bacterial infection can induce allergic sensitization to inert inhaled antigens by a mechanism that requires MyD88-dependent DC activation and is controlled by Tregs.
doi:10.1016/j.jaci.2008.06.038
PMCID: PMC3052793  PMID: 18774395
Asthma; allergic sensitization; adjuvant; dendritic cell; Chlamydia pneumoniae; regulatory T cells; bacterial pneumonia; allergen
9.  TLR/MyD88 and LXRα Signaling Pathways Reciprocally Control Chlamydia Pneumoniae-Induced Acceleration of Atherosclerosis 
Experimental and clinical studies link Chlamydia pneumoniae infection to atherogenesis and athero-thrombotic events, but the underlying mechanisms are unclear. We tested the hypothesis that C. pneumoniae-induced acceleration of atherosclerosis in ApoE−/− mice is reciprocally modulated by activation of TLR-mediated innate immune or LXRα signaling pathways. We infected ApoE−/− mice and ApoE−/− mice that also lacked TLR2 or TLR4 or MyD88 or LXRα intranasally with C. pneumoniae followed by high-fat diet feeding for 4 months. Mock infected littermates served as controls. Atherosclerosis was assessed in aortic sinuses and in en face preparation of whole aorta. The numbers of activated dendritic cells (DCs) within plaques, and serum levels of cholesterol and proinflammatory cytokines were also measured. C. pneumoniae infection markedly accelerated atherosclerosis in ApoE deficient mice that was associated with increased numbers of activated DCs in aortic sinus plaques and higher circulating levels of MCP-1, IL-12p40, IL-6 and TNF-α. In contrast, C. pneumoniae infection had only a minimal effect on atherosclerosis, accumulation of activated DCs in the sinus plaques, or circulating cytokine increases in ApoE−/− mice that were also deficient in either TLR2, TLR4, or MyD88. However, C. pneumoniae-induced acceleration of atherosclerosis in ApoE−/− mice was further enhanced in ApoE−/−/LXRα−/− double knockout mice, and was accompanied by higher serum levels of IL-6 and TNF-α. We conclude that C. pneumoniae-infection accelerates atherosclerosis in hypercholesterolemic mice predominantly through a TLR/MyD88-dependent mechanism, and that LXRα appears to reciprocally modulate and reduce the pro-atherogenic effects of C. pneumoniae infection.
PMCID: PMC2683843  PMID: 18981139
13.  The NOD/RIP2 Pathway Is Essential for Host Defenses Against Chlamydophila pneumoniae Lung Infection 
PLoS Pathogens  2009;5(4):e1000379.
Here we investigated the role of the Nod/Rip2 pathway in host responses to Chlamydophila pneumoniae–induced pneumonia in mice. Rip2−/− mice infected with C. pneumoniae exhibited impaired iNOS expression and NO production, and delayed neutrophil recruitment to the lungs. Levels of IL-6 and IFN-γ levels as well as KC and MIP-2 levels in bronchoalveolar lavage fluid (BALF) were significantly decreased in Rip2−/− mice compared to wild-type (WT) mice at day 3. Rip2−/− mice showed significant delay in bacterial clearance from the lungs and developed more severe and chronic lung inflammation that continued even on day 35 and led to increased mortality, whereas WT mice cleared the bacterial load, recovered from acute pneumonia, and survived. Both Nod1−/− and Nod2−/− mice also showed delayed bacterial clearance, suggesting that C. pneumoniae is recognized by both of these intracellular receptors. Bone marrow chimera experiments demonstrated that Rip2 in BM-derived cells rather than non-hematopoietic stromal cells played a key role in host responses in the lungs and clearance of C. pneumoniae. Furthermore, adoptive transfer of WT macrophages intratracheally was able to rescue the bacterial clearance defect in Rip2−/− mice. These results demonstrate that in addition to the TLR/MyD88 pathway, the Nod/Rip2 signaling pathway also plays a significant role in intracellular recognition, innate immune host responses, and ultimately has a decisive impact on clearance of C. pneumoniae from the lungs and survival of the infectious challenge.
Author Summary
Chlamydophila pneumoniae (C. pneumoniae) is a common intracellular parasite that causes lung infections and contributes to several diseases characterized by chronic inflammation. Toll-like receptors expressed on the cell surface detect C. pneumoniae and mount a vigorous defense, but it is not known how the cell defends itself once the pathogen has taken up residence as a parasite. We reasoned that cytosolic pattern recognition receptors called Nods (nucleotide oligomerization domain) that detect microbes that gain entry into the cell might be involved. Using mice genetically deficient in Nod1 and Nod2 or their common downstream adaptor (Rip2), we show that in lung infection, Nod proteins are indeed essential in directing a defense against C. pneumoniae. Mice with defective Nod/Rip2-dependent signaling exhibited delayed recruitment of neutrophils, blunted production of pro-inflammatory cytokines and chemokines, and evidence of defective iNOS expression and NO production. These impaired responses led to delayed clearance of bacteria, intense persistent lung inflammation, and increased mortality. By performing bone marrow transplantation experiments and direct transfer of cells into the lungs of mice, we demonstrated that intact Nod-dependent signaling in bone marrow–derived cells was critical in the defense against C. pneumoniae. Our results indicate that Nod proteins also play an important role in host defense against C. pneumoniae. Coordinated and sequential activation of TLR and Nod signaling pathways may be necessary for an efficient immune response and host defense against C. pneumoniae.
doi:10.1371/journal.ppat.1000379
PMCID: PMC2660273  PMID: 19360122
14.  Reversal of the Antichlamydial Activity of Putative Type III Secretion Inhibitors by Iron▿  
Infection and Immunity  2007;75(7):3478-3489.
INPs, which are chemically synthesized compounds belonging to a class of acylated hydrazones of salicylaldehydes, can inhibit the growth of Chlamydiaceae. Evidence has been presented that in Yersinia and Chlamydia INPs may affect the type III secretion (T3S) system. In the present study 25 INPs were screened for antichlamydial activity at a concentration of 50 μM, and 14 were able to completely inhibit the growth of Chlamydia trachomatis serovar D in McCoy and HeLa 229 cells. The antichlamydial activities of two of these INPs, INPs 0341 and 0400, were further characterized due to their low cytotoxicity. These compounds were found to inhibit C. trachomatis in a dose-dependent manner; were not toxic to elementary bodies; were cidal at a concentration of ≥20 μM; inhibited all Chlamydiaceae tested; and could inhibit the development of C. trachomatis as determined by the yield of progeny when they were added up to 24 h postinfection. INP 0341 was able to affect the expression of several T3S genes. Compared to the expression in control cultures, lcrH-1, copB, and incA, all middle- to late-expressed T3S genes, were not expressed in the INP 0341-treated cultures 24 to 36 h postinfection. Iron, supplied as ferrous sulfate, as ferric chloride, or as holo-transferrin, was able to negate the antichlamydial properties of the INPs. In contrast, apo-transferrin and other divalent metal ions tested were not able to reverse the inhibitory effect of the INPs. In conclusion, the potent antichlamydial activity of INPs is directly or indirectly linked with iron, and this inhibition of Chlamydia has an effect on the T3S system of this intracellular pathogen.
doi:10.1128/IAI.00023-07
PMCID: PMC1932962  PMID: 17470544
15.  Interaction between Components of the Type III Secretion System of Chlamydiaceae 
Journal of Bacteriology  2005;187(2):473-479.
Members of the family Chlamydiaceae possess at least 13 genes, distributed throughout the chromosome, that are homologous with genes of known type III secretion systems (TTS). The aim of this study was to use putative TTS proteins of Chlamydophila pneumoniae, whose equivalents in other bacterial TTS function as chaperones, to identify interactions between chlamydial proteins. Using the BacterioMatch Two-Hybrid Vector system (Stratagene, La Jolla, Calif.), lcrH-2 and sycE, positions 1021 and 0325, respectively, from C. pneumoniae CM-1 were used as “bait” to identify target genes (positions 0324, 0705, 0708, 0808 to 0810, 1016 to 1020, and 1022) in close proximity on the chromosome. Interaction between the products of the lcrH-2 (1021) and lcrE (copN) (0324) genes was detected and confirmed by pull-down experiments and enzyme immunoassays using recombinant LcrH-2 and LcrE. As further confirmation of this interaction, the homologous genes from Chlamydia trachomatis, serovar E, and Chlamydophila psittaci, Texas turkey, were also cloned in the two-hybrid system to determine if LcrH-2 and LcrE would interact with their orthologs in other species. Consistent with their genetic relatedness, LcrH-2 from C. pneumoniae interacted with LcrE produced from the three species of Chlamydiaceae; LcrH-2 from C. psittaci reacted with LcrE from C. pneumoniae but not from C. trachomatis; and C. trachomatis LcrH-2 did not react with LcrE from the other two species. Deletions from the N and C termini of LcrE from C. pneumoniae identified the 50 C-terminal amino acids as essential for the interaction with LcrH-2. Thus, it appears that in the Chlamydiaceae TTS, LcrH-2 interacts with LcrE, and therefore it may serve as a chaperone for this protein.
doi:10.1128/JB.187.2.473-479.2005
PMCID: PMC543553  PMID: 15629918
16.  Temporal Expression of Type III Secretion Genes of Chlamydia pneumoniae  
Infection and Immunity  2003;71(5):2555-2562.
Chlamydia pneumoniae has been shown to possess at least 13 genes that are homologous with other known type III secretion (TTS) systems. Upon infection of HEp-2 cells with C. pneumoniae, the expression of these genes was followed by reverse transcriptase PCR throughout the developmental cycle of this obligate intracellular pathogen. In addition, expression was analyzed when C. pneumoniae was grown in the presence of human gamma interferon (IFN-γ). The groEL-1, ompA, and omcB genes were used as markers for the early, middle, and late stages of the developmental cycle, respectively, and the inhibition of expression of the fstK gene was used as a marker for the effect of IFN-γ on the maturation of C. pneumoniae. In the absence of IFN-γ, the TTS genes were expressed as follows: early stage (1.5 to 8 h), yscC, yscS, yscL, yscJ and lcrH-2; middle stage (by 12 to 18 h), lcrD, yscN, and yscR; and late stage (by 24 h), lcrE, sycE, lcrH-1, and yscT. Of the genes expressed early, the lcrH-2 gene was detected the earliest, at 1.5 h. Expression of the yscU gene was not detected at any of the time points examined. Under the influence of IFN-γ, the cluster of TTS genes that were normally not expressed until the middle to late stages of the developmental cycle, namely, lcrD, lcrE, and sycE, as well as lcrH-1, were down-regulated, and expression could not be detected up to 48 h. In contrast, the expression of the other TTS genes appeared to be unchanged in the presence of IFN-γ. The lcrH-1 and lcrH-2 genes differed from one another in both their temporal expression and response to IFN-γ. In other TTS systems, these genes code for proteins that function in regulation of effector protein synthesis as well as serve as chaperones for proteins that provide for the translocation of the effector proteins into the host cell. In summary, the expression pattern of the TTS genes of C. pneumoniae examined suggests that they are temporally regulated throughout the developmental cycle. Furthermore, paralleling the inhibition of the maturation of the reticulate body to the elementary body, TTS genes expressed in the later stages of the cycle appear to be down-regulated when the organism is grown in the presence of IFN-γ.
doi:10.1128/IAI.71.5.2555-2562.2003
PMCID: PMC153279  PMID: 12704128
17.  Inclusion Fluorescent-Antibody Test as a Screening Assay for Detection of Antibodies to Chlamydia pneumoniae 
A study was conducted to determine the ability of the inclusion immunofluorescence assay (inclusion IFA) to act as a screening test to detect samples with antibodies to Chlamydia pneumoniae; microimmunofluorescence (MIF) was used as the “gold standard.” In addition, the inclusion IFA was compared using HEp-2 cells infected with either C. pneumoniae CM-1 or Chlamydia trachomatis serovar E. A total of 331 serum samples representing a range of MIF titers were evaluated. The sensitivities of the inclusion IFA for detecting samples with C. pneumoniae MIF titers of ≥16 were 96.9 and 74.8% with C. pneumoniae- and C. trachomatis-infected cells, respectively. For samples with an elevated C. pneumoniae MIF titer of ≥512, the sensitivities of the C. pneumoniae- and C. trachomatis-based inclusion IFA were 97.0 and 8.8%, respectively. These results suggest that the inclusion IFA is not a genus-specific test, as evidenced by the failure of the C. trachomatis-infected cells to detect a significant number of samples with C. pneumoniae antibodies. Samples that had elevated C. pneumoniae inclusion IFA and MIF titers but that were found negative (titer, <16) by the C. trachomatis inclusion IFA were further tested by an in vitro neutralization assay for functional antibodies that might not have been detected by the serological assays. The in vitro neutralization results corroborated the serological results in that all seven sera tested had a neutralization titer for C. pneumoniae (range, 20 to 225), while all but one failed to have any effect on the infectivity of C. trachomatis serovar E. While the C. pneumoniae inclusion IFA had a high sensitivity for detecting chlamydial antibodies, depending on whether it was used as a screening test for detecting samples with low (≥16) or elevated (≥512) MIF titers, its specificity ranged from 53.4 to 77.1%. In conclusion, the inclusion IFA with C. pneumoniae-infected cells was best suited as a sensitive screening test for identifying specimens with elevated MIF titers (those associated with a possible acute infection with C. pneumoniae).
doi:10.1128/CDLI.9.3.562-567.2002
PMCID: PMC119991  PMID: 11986260
18.  Pre-clinical pharmacokinetics and anti-chlamydial activity of salicylidene acylhydrazide inhibitors of bacterial type III secretion 
The Journal of Antibiotics  2012;65(8):397-404.
Salicylidene acylhydrazides belong to a class of compounds shown to inhibit bacterial type III secretion (T3S) in pathogenic Gram-negative bacteria. This class of compounds also inhibits growth and replication of Chlamydiae, strict intracellular bacteria that possess a T3S system. In this study a library of 58 salicylidene acylhydrazides was screened to identify inhibitors of Chlamydia growth. Compounds inhibiting growth of both Chlamydia trachomatis and Chlamydophila pneumoniae were tested for cell toxicity and seven compounds were selected for preliminary pharmacokinetic analysis in mice using cassette dosing. Two compounds, ME0177 and ME0192, were further investigated by individual pharmacokinetic analysis. Compound ME0177 had a relatively high peak plasma concentration (Cmax) and area under curve and therefore may be considered for systemic treatment of Chlamydia infections. The other compound, ME0192, had poor pharmacokinetic properties but the highest anti-chlamydial activity in vitro and therefore was tested for topical treatment in a mouse vaginal infection model. ME0192 administered vaginally significantly reduced the infectious burden of C. trachomatis and the number of infected mice.
doi:10.1038/ja.2012.43
PMCID: PMC3428607  PMID: 22669447
Chlamydophila pneumoniae; Chlamydia trachomatis; pre-clinical pharmacokinetics; type III secretion inhibitor; vaginal microbicide; virulence inhibitor

Results 1-18 (18)