RESUMO
Comparative study of the walls of the aorta, coronary artery, and a. basilaris detected for the first time intra- and extracellular depositions of Chlamydia pneumoniae in unstable atherosclerotic plaques. No chlamydia were detected in the intima of normal sites of the vascular wall and just negligible levels thereof in stable atherosclerotic plaques. An unstable plaque with intra- and extracellular colonies was characterized by infiltration of the cap and intima adjacent to the atheromatous core with mononuclear cells, primarily T cells. These data suggested that Chlamydia pneumoniae could play an important role in the development of immunoinflammatory processes in the vascular wall and promote destabilization and progressive development of atherosclerotic plaques in humans.
Assuntos
Aterosclerose/microbiologia , Aterosclerose/patologia , Chlamydophila pneumoniae/ultraestrutura , Idoso , Aorta/microbiologia , Aorta/patologia , Artéria Basilar/microbiologia , Artéria Basilar/patologia , Cadáver , Chlamydophila pneumoniae/imunologia , Vasos Coronários/microbiologia , Vasos Coronários/patologia , Técnicas Histológicas , Humanos , Masculino , Microscopia Eletrônica de Varredura , Pessoa de Meia-IdadeRESUMO
The sst1, "supersusceptibility to tuberculosis," locus has previously been shown to be a genetic determinant of host resistance to infection with the intracellular pathogen, Mycobacterium tuberculosis. Chlamydia pneumoniae is an obligate intracellular bacterium associated with community acquired pneumonia, and chronic infection with C. pneumoniae has been linked to asthma and atherosclerosis. C. pneumoniae is a highly adapted pathogen that can productively infect macrophages and inhibit host cell apoptosis. Here we examined the role of sst1 in regulating the host response to infection with C. pneumoniae. Although mice carrying the sst1 susceptible (sst1(S) ) locus were not impaired in their ability to clear the acute infection, they were dramatically less tolerant of the induced immune response, displaying higher clinical scores, more severe lung inflammation, exaggerated macrophage and neutrophil influx, and the development of fibrosis compared to wild type mice. This correlated with increased activated caspase-3 in the lungs of infected sst1(S) mice. Infection of sst1(S) macrophages with C. pneumoniae resulted in a shift in the secreted cytokine profile towards enhanced production of interferon-ß and interleukin-10, and induced apoptotic cell death, which was dependent on secretion of interferon-ß. Intriguingly macrophages from the sst1(S) mice failed to support normal chlamydial growth, resulting in arrested development and failure of the organism to complete its infectious cycle. We conclude that the sst1 locus regulates a shared macrophage-mediated innate defense mechanism against diverse intracellular bacterial pathogens. Its susceptibility allele leads to upregulation of type I interferon pathway, which, in the context of C. pneumoniae, results in decreased tolerance, but not resistance, to the infection. Further dissection of the relationship between type I interferons and host tolerance during infection with intracellular pathogens may provide identification of biomarkers and novel therapeutic targets.
Assuntos
Infecções por Chlamydophila/imunologia , Chlamydophila pneumoniae/imunologia , Loci Gênicos/imunologia , Imunidade Inata/fisiologia , Macrófagos Alveolares/imunologia , Pneumonia Bacteriana/imunologia , Animais , Caspase 3/genética , Caspase 3/imunologia , Infecções por Chlamydophila/genética , Infecções por Chlamydophila/patologia , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/ultraestrutura , Evasão da Resposta Imune/genética , Interferon beta/genética , Interferon beta/imunologia , Interleucina-10/genética , Interleucina-10/imunologia , Macrófagos Alveolares/ultraestrutura , Camundongos , Pneumonia Bacteriana/genética , Pneumonia Bacteriana/patologiaRESUMO
BACKGROUND: Limited body of evidence suggests that lipopolysaccharide of C. pneumoniae as well as C. pneumoniae-specific immune complexes can be detected and isolated from human serum. The aim of this study was to investigate the presence of viable elementary bodies of C.pneumoniae in serum samples of patients with acute coronary syndrome and healthy volunteers. MATERIAL AND METHODS: Serum specimens from 26 healthy volunteers and 56 patients with acute coronary syndrome were examined subsequently by serological (C.pneumoniae-specific IgA and IgG), PCR-based and bacteriological methods. Conventional, nested and TaqMan PCR were used to detect C.pneumoniae genetic markers (ompA and 16S rRNA) in DNA from serum specimens extracted with different methods. An alternative protocol which included culturing high-speed serum sediments in HL cells and further C.pneumoniae growth evaluation with immunofluorescence analysis and TaqMan PCR was established. Pellet fraction of PCR-positive serum specimens was also examined by immunoelectron microscopy. RESULTS: Best efficiency of final PCR product recovery from serum specimens has been shown with specific C. pneumoniae primers using phenol-chloroform DNA extraction protocol. TaqMan PCR analysis revealed that human serum of patients with acute coronary syndrome may contain genetic markers of C. pneumoniae with bacterial load range from 200 to 2000 copies/ml serum. However, reliability and reproducibility of TaqMan PCR were poor for serum specimens with low bacterial copy number (<200 /ml). Combination of bacteriological, immunofluorescence and PCR- based protocols applied for the evaluating HL cells infected with serum sediments revealed that 21.0 % of the patients with acute coronary syndrome have viable forms C.pneumoniae in serum. The detection rate of C.pneumoniae in healthy volunteers was much lower (7.7%). Immunological profile of the patients did not match accurately C.pneumoniae detection rate in serum specimens. Elementary bodies of C.pneumoniae with typical ultrastructural characteristics were also identified in serum sediments using immunoelectron microscopy. CONCLUSIONS: Viable forms C. pneumoniae with typical electron microscopic structure can be identified and isolated from serum specimens of the patients with acute coronary syndrome and some healthy volunteers. Increased detection rate of C. pneumoniae in serum among the patients with an acute coronary syndrome may contribute towards enhanced pro-inflammatory status in cardiovascular patients and development of secondary complications of atherosclerosis.
Assuntos
Síndrome Coronariana Aguda/microbiologia , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/isolamento & purificação , Idoso , Proteínas da Membrana Bacteriana Externa/genética , Linhagem Celular , Chlamydophila pneumoniae/classificação , Chlamydophila pneumoniae/ultraestrutura , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genéticaRESUMO
Chlamydia pneumoniae is a common human and animal pathogen associated with upper and lower respiratory tract infections. Of the animal C. pneumoniae isolates, the koala nasal isolate (LPCoLN) is by far the best genetically characterised. This current study was designed to characterise the morphology and developmental events for the LPCoLN isolate, and our results showed several striking in vitro growth differences when compared to the human isolate, AR39. The LPCoLN inclusion size and morphology was distinct from AR39, and a much faster doubling time (3.4-4.9h versus 5.9-8.7h doubling time) was observed when grown in HEp-2 cell monolayers. Confocal and electron microscopy of LPCoLN confirmed large (9-30 microm in diameter) inclusions, that were heterogeneously shaped, compared to the small (5-9 microm in diameter), uniformly shaped inclusions of AR39. The morphology of the LPCoLN elementary body was round, and had a narrow or nonexistent periplasmic space, compared to the 'pear-shaped' morphology of AR39 EBs. While both isolates showed evidence of inclusion fusion, the level of fusion was much higher for LPCoLN (100%) compared to AR39 (30-40%). Our findings have provided new insights and identified key differences in the in vitro doubling time, size and morphology of an animal C. pneumoniae isolate.
Assuntos
Infecções por Chlamydophila/veterinária , Chlamydophila pneumoniae/fisiologia , Phascolarctidae/microbiologia , Infecções Respiratórias/veterinária , Animais , Infecções por Chlamydophila/microbiologia , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/crescimento & desenvolvimento , Chlamydophila pneumoniae/ultraestrutura , DNA Bacteriano/química , DNA Bacteriano/genética , Dosagem de Genes , Microscopia Confocal/veterinária , Microscopia Eletrônica de Transmissão/veterinária , Reação em Cadeia da Polimerase/veterinária , RNA Ribossômico 16S/química , RNA Ribossômico 16S/genética , Infecções Respiratórias/microbiologiaRESUMO
Since steroids are powerful anti-inflammatory agents and increase susceptibility to a variety of infections, including Chlamydia (Chlamydophila) pneumoniae respiratory tract infections, the effect of the steroid receptor antagonist RU486 (mifepristone) on C. pneumoniae growth in epithelial HEp-2 cells was examined. Treatment of HEp-2 cells with RU486 significantly inhibited the growth of C. pneumoniae in a dose-dependent manner. Electron microscopic studies also revealed that the treatment of infected cells with RU486 resulted in a marked destruction of infecting organisms. The addition of the host cell protein synthesis inhibitor cycloheximide to the infected cells did not alter the inhibition of C. pneumoniae growth by RU486. Pretreatment of C. pneumoniae organisms with RU486 before addition to culture also did not result in any modulation of bacterial growth in the cells. However, the binding of RU486 to C. pneumoniae organisms in cells at 24 h after infection was demonstrated by immune electron microscopy with anti-RU486 antibody. Incubation of cells with anti-RU486 antibody completely diminished the inhibition of C. pneumoniae growth by RU486. These results indicate that RU486 may directly bind to the bacteria within cells and cause the destruction of C. pneumoniae. This novel mode of regulation of C. pneumoniae growth in cells by RU486 might provide a new approach to understanding complicated aspects of C. pneumoniae infection.
Assuntos
Chlamydophila pneumoniae/efeitos dos fármacos , Chlamydophila pneumoniae/crescimento & desenvolvimento , Células Epiteliais/microbiologia , Mifepristona/farmacologia , Receptores de Glucocorticoides/antagonistas & inibidores , Animais , Linhagem Celular , Chlamydophila pneumoniae/metabolismo , Chlamydophila pneumoniae/ultraestrutura , Contagem de Colônia Microbiana , Células Epiteliais/ultraestrutura , Feminino , Humanos , Camundongos , Camundongos Endogâmicos ICR , Microscopia Eletrônica , Mifepristona/metabolismoRESUMO
To mimic in vivo conditions during chlamydial infections, Chlamydia trachomatis serovar D and Chlamydia pneumoniae CWL029 were cultured in low-oxygen atmospheres containing 4% O(2), with parallel controls cultured in atmospheric air. Both were enriched with 5% CO(2). The results showed a dramatic increase in the growth of C. pneumoniae but not of C. trachomatis.
Assuntos
Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydophila pneumoniae/crescimento & desenvolvimento , Corpos de Inclusão/ultraestrutura , Oxigênio/farmacologia , Ar , Chlamydia trachomatis/efeitos dos fármacos , Chlamydia trachomatis/genética , Chlamydia trachomatis/ultraestrutura , Chlamydophila pneumoniae/efeitos dos fármacos , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/ultraestrutura , Células HeLa , Humanos , Corpos de Inclusão/efeitos dos fármacos , Especificidade de Órgãos , Oxigênio/fisiologiaRESUMO
Chronic and acute infectious diseases have been implicated in modifying the risk of atherosclerosis independently or in collaboration with conventional risk factors. During the past two decades, the discussion on microbial agents and atherosclerosis has mainly been centred on Chlamydia pneumoniae. The strongest evidence linking Chlamydia pneumoniae and atherosclerotic disease comes from in vitro experimental studies. In this review, we summarize and critically evaluate the available data of human diagnostic and therapeutic studies on the association of Chlamydia pneumoniae with atherosclerosis. Taking into account the human in vivo data, there is currently insufficient proof linking Chlamydia pneumoniae to atherosclerosis. At present, there are no indications for antibiotic treatment targeted at Chlamydia pneumoniae in the management of atherosclerosis.
Assuntos
Aterosclerose/microbiologia , Infecções por Chlamydophila , Chlamydophila pneumoniae , Ensaios Clínicos Controlados Aleatórios como Assunto , Aterosclerose/tratamento farmacológico , Aterosclerose/imunologia , Infecções por Chlamydophila/diagnóstico , Infecções por Chlamydophila/tratamento farmacológico , Infecções por Chlamydophila/imunologia , Chlamydophila pneumoniae/imunologia , Chlamydophila pneumoniae/patogenicidade , Chlamydophila pneumoniae/ultraestrutura , Humanos , Projetos PilotoRESUMO
PURPOSE: Vulnerable plaques are characterized by a myxoid matrix, necrotic lipidic core, reactive oxygen species, and high levels of microorganisms. Aerobic microbes such as Chlamydophila pneumoniae and Mycoplasma pneumoniae usually do not survive in oxidative stress media. Archaea are anaerobic microbes with powerful anti-oxidative enzymes that allow detoxification of free radicals whose presence might favor the survival of aerobic microorganisms. We searched for archaeal organisms in vulnerable plaques, and possible associations with myxoid matrix, chlamydia, and mycoplasma bodies. METHODS: Twenty-nine tissue samples from 13 coronary artherectomies from large excentric ostial or bifurcational lesions were studied using optical and electron microscopy. Infectious agents compatible with archaea, chlamydia, and mycoplasma were semiquantified using electron micrographs and correlated with the amounts of fibromuscular tissue, myxoid matrix, and foam cells, as determined from semi-thin sections. Six of the cases were also submitted to polymerase chain reaction with archaeal primers. RESULTS: All 13 specimens showed archaeal-compatible structures and chlamydial and mycoplasmal bodies in at least 1 sample. There was a positive correlation between extent of the of myxoid matrix and archaeal bodies (r = 0.44, P = 0.02); between archaeal and mycoplasmal bodies (r = 0.41, P = 0.03), and between chlamydial bodies and foam cells (r = 0.42; P = 0.03). The PCR test was positive for archaeal DNA in 4 of the 6 fragments. DISCUSSION: DNA and forms suggestive of archaea are present in vulnerable plaques and may have a fundamental role in the proliferation of mycoplasma and chlamydia. This seems to be the first description of apparently pathogenic archaea in human internal organ lesions.
Assuntos
Archaea/isolamento & purificação , Chlamydophila pneumoniae/isolamento & purificação , Doença da Artéria Coronariana/microbiologia , Mycoplasma pneumoniae/isolamento & purificação , Idoso , Animais , Archaea/genética , Archaea/ultraestrutura , Chlamydophila pneumoniae/ultraestrutura , Doença da Artéria Coronariana/patologia , DNA Bacteriano , Feminino , Células Espumosas/ultraestrutura , Humanos , Lipídeos/análise , Masculino , Pessoa de Meia-Idade , Mycoplasma pneumoniae/ultraestrutura , Necrose/patologia , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/isolamento & purificação , Estatísticas não ParamétricasRESUMO
PURPOSE: Vulnerable plaques are characterized by a myxoid matrix, necrotic lipidic core, reactive oxygen species, and high levels of microorganisms. Aerobic microbes such as Chlamydophila pneumoniae and Mycoplasma pneumoniae usually do not survive in oxidative stress media. Archaea are anaerobic microbes with powerful anti-oxidative enzymes that allow detoxification of free radicals whose presence might favor the survival of aerobic microorganisms. We searched for archaeal organisms in vulnerable plaques, and possible associations with myxoid matrix, chlamydia, and mycoplasma bodies. METHODS: Twenty-nine tissue samples from 13 coronary artherectomies from large excentric ostial or bifurcational lesions were studied using optical and electron microscopy. Infectious agents compatible with archaea, chlamydia, and mycoplasma were semiquantified using electron micrographs and correlated with the amounts of fibromuscular tissue, myxoid matrix, and foam cells, as determined from semi-thin sections. Six of the cases were also submitted to polymerase chain reaction with archaeal primers. RESULTS: All 13 specimens showed archaeal-compatible structures and chlamydial and mycoplasmal bodies in at least 1 sample. There was a positive correlation between extent of the of myxoid matrix and archaeal bodies (r = 0.44, P = 0.02); between archaeal and mycoplasmal bodies (r = 0.41, P = 0.03), and between chlamydial bodies and foam cells (r = 0.42; P = 0.03). The PCR test was positive for archaeal DNA in 4 of the 6 fragments. DISCUSSION: DNA and forms suggestive of archaea are present in vulnerable plaques and may have a fundamental role in the proliferation of mycoplasma and chlamydia. This seems to be the first description of apparently pathogenic archaea in human internal organ lesions.
PROPOSTA: Placas vulneráveis são caracterizadas por matriz mixomatosa, centro lipídico necrótico, espécies reativas de oxigênio e alto níveis de microorganismos. Micróbios aeróbicos como Chlamydophila pneumoniae e Mycoplasma pneumoniae usualmente não sobrevivem em meio de estresse oxidativo. Arquéias são microorganismos anaeróbicos com poderosas enzimas anti-oxidantes que permitem detoxificação de radicais livres e a presença delas poderia favorecer a sobrevivência de micróbios aeróbicos. Pesquisamos por elementos de arquéia em placas vulneráveis e sua possível associação com degeneração mixomatosa da matriz e aumento do número de clamídias e micoplasmas. MÉTODOS: Vinte e nove amostras de 13 produtos de aterotomia de lesões grandes e excêntricas de óstio ou bifurcação de coronárias foram estudadas pela microscopia óptica e eletrônica. Agentes compatíveis com arquéia, clamídia e micoplasma foram semiquantificados pela microscopia eletrônica e correlacionados com quantidade de tecido fibromuscular, matriz mixomatosa e células xantomatosas. Seis casos foram também submetidos à reação em cadeia da polimerase com oligonucleotídeos de arquéia. RESULTADOS: Os 13 casos foram positivos para estruturas sugestivas de arquéia, micoplasma ou clamídia, em pelo menos uma amostra. Houve correlação positiva entre intensidade de matriz mixomatosa versus arquéia (r=0.44, p=0.02); arquéia versus micoplasma (r=0.41, p=0.03) e clamídia versus células xantomatosas r=0,42; 0.03). PCR foi positiva para DNA de arqueia em 4 dos 6 fragmentos. DISCUSSÃO: DNA e formas compatíveis com arquéia estão presentes em placas vulneráveis e podem ter papel fundamental na proliferação de micoplasma e clamídia. Este parece ser o primeiro relato de arquéia aparentemente patogênica em lesões de órgãos internos humanos.
Assuntos
Humanos , Animais , Masculino , Feminino , Pessoa de Meia-Idade , Archaea/patogenicidade , Chlamydophila pneumoniae/isolamento & purificação , Doença da Artéria Coronariana/microbiologia , Mycoplasma pneumoniae/isolamento & purificação , Archaea/genética , Archaea/ultraestrutura , Chlamydophila pneumoniae/ultraestrutura , Doença da Artéria Coronariana/patologia , DNA Bacteriano , Células Espumosas/ultraestrutura , Lipídeos/análise , Mycoplasma pneumoniae/ultraestrutura , Necrose/patologia , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/isolamento & purificação , Estatísticas não ParamétricasRESUMO
Chlamydia pneumoniae is an important human respiratory pathogen that is responsible for an estimated 10% of community-acquired pneumonia and 5% of bronchitis and sinusitis cases. We examined changes in global protein expression profiles associated with the redifferentiation of reticulate body (RB) to elementary body (EB) as C. pneumoniae cells progressed from 24 to 48 h postinfection in HEp2 cells. Proteins corresponding to those showing the greatest changes in abundance in the beginning of the RB to EB transition were then identified from purified EBs. Among the 300 spots recognized, 35 proteins that were expressed at sufficiently high levels were identified by mass spectrometry. We identified C. pneumoniae proteins that showed more than 2-fold increases in abundance in the early stages of RB to EB transition, including several associated with amino acid and cofactor biosynthesis (Ndk, TrxA, Adk, PyrH, and BirA), maintenance of cytoplasmic protein function (GroEL/ES, DnaK, DksA, GrpE, HtrA, ClpP, ClpB, and Map), modification of the bacterial cell surface (CrpA, OmpA, and OmcB), energy metabolism (Tal and Pyk), and the putative transcriptional regulator TctD. This study identified C. pneumoniae proteins involved in the process of redifferentiation into mature, infective EBs and indicates bacterial metabolic pathways that may be involved in this transition. The proteins involved in RB to EB transition are key to C. pneumoniae infection and are perhaps suitable targets for therapeutic intervention.
Assuntos
Proteínas de Bactérias/análise , Chlamydophila pneumoniae/química , Proteínas da Membrana Bacteriana Externa/análise , Células Cultivadas , Infecções por Chlamydia/metabolismo , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/crescimento & desenvolvimento , Chlamydophila pneumoniae/ultraestrutura , Regulação Bacteriana da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , HumanosRESUMO
Chlamydia pneumoniae is an obligate intracellular pathogen that causes both acute and chronic human disease. Several in vitro models of chlamydial persistence have been established to mimic chlamydial persistence in vivo. We determined the expression patterns of 52 C. pneumoniae proteins, representing nine functional subgroups, from the gamma interferon (IFN-gamma) treatment (primarily tryptophan limitation) and iron limitation (IL) models of persistence compared to those following heat shock (HS) at 42 degrees C. Protein expression patterns of C. pneumoniae persistence indicates a strong stress component, as evidenced by the upregulation of proteins involved in protein folding, assembly, and modification. However, it is clearly more than just a stress response. In IFN persistence, but not IL or HS, amino acid and/or nucleotide biosynthesis proteins were found to be significantly upregulated. In contrast, proteins involved in the biosynthesis of cofactors, cellular processes, energy metabolism, transcription, and translation showed an increased in expression in only the IL model of persistence. These data represent the most extensive protein expression study of C. pneumoniae comparing the chlamydial heat shock stress response to two models of persistence and identifying the common and unique protein level responses during persistence.
Assuntos
Chlamydophila pneumoniae/fisiologia , Chlamydophila pneumoniae/patogenicidade , Proteínas de Choque Térmico/biossíntese , Modelos Biológicos , Proteoma/biossíntese , Proteômica , Linhagem Celular Tumoral , Chlamydophila pneumoniae/ultraestrutura , Temperatura Alta , Humanos , Interferon gama/fisiologia , Ferro/metabolismoRESUMO
Characteristic features of the persistent chlamydial developmental cycle, associated with chronic infections in both humans and animals, include the generation of non-replicative, morphologically aberrant bodies which are distinct from normal propagating reticulate bodies. Previous studies have correlated these morphological and metabolic changes with differential expression of diverse functional subsets of chlamydial genes. To further investigate these correlations, we compared mRNA expression of predicted chlamydial signal transduction genes between normal Chlamydophila pneumoniae A-03 infections in HEp-2 cells and those treated with gamma interferon (IFN-gamma) by using real-time RT-PCR. Inspection of the Cp. pneumoniae genome revealed at least 39 candidate signal transduction genes, of which 30 were differentially expressed in Cp. pneumoniae mediated persistence. Functional sub-groups of differentially expressed signal transduction genes include chlamydial GTPases (hflX, ychF, yhbZ and yphC), linked to bacterial cellular processes such as cell cycle control and ribosome assembly and stability. Other up-regulated signal transduction genes sharing similarity to bacterial stress response genes (htrA, surE, lytB and hrcA) were also detected. The transcriptional changes observed for the majority of signal transduction genes appear to be unique for Cp. pneumoniae, as similar changes were not observed in recent whole genomic analysis of C. trachomatis IFN-gamma mediated persistence. These results suggest that chlamydial signal transduction genes play potentially important roles in the establishment and maintenance of Cp. pneumoniae persistence, likely as part of the IFN-gamma response stimulon as described for C. trachomatis, but with considerable differences in the transcriptional profile.
Assuntos
Infecções por Chlamydophila/imunologia , Infecções por Chlamydophila/microbiologia , Chlamydophila pneumoniae/genética , Regulação Bacteriana da Expressão Gênica , Interferon gama/imunologia , Transdução de Sinais/genética , Linhagem Celular , Infecções por Chlamydophila/induzido quimicamente , Chlamydophila pneumoniae/efeitos dos fármacos , Chlamydophila pneumoniae/ultraestrutura , Perfilação da Expressão Gênica , Humanos , Interferon gama/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
In this study the effects of 2-amino-phenoxazine-3-one (phenoxazine derivate, Phx-3) on Chlamydia (Chlamydophila) pneumoniae growth in human monocytic THP-1 cells as well as human epithelial HEp-2 cells were examined. Cells were infected with bacteria at an m.o.i. of 10 by centrifugation. After washing to remove any remaining bacteria, the cells were incubated with or without Phx-3 in the presence or absence of tryptophan for 72 h. The bacteria in cells were assessed by staining of chlamydial inclusions with FITC-labelled anti-chlamydial antibody, electron microscopic analysis, real-time RT-PCR specific for C. pneumoniae 16S rRNA and propagation on HEp-2 cells. Treatment with Phx-3 significantly inhibited growth of C. pneumoniae in THP-1 and HEp-2 cells. A decrease in the number of bacterial 16S rRNA transcripts was also confirmed in both cell lines by real-time RT-PCR. Electron microscopic studies revealed that treatment with Phx-3 induces bacterial destruction in most of the inclusion bodies in these cells. Addition of tryptophan to the culture slightly blocked the growth inhibition of C. pneumoniae by Phx-3. The reagents did not show any cytotoxicity to the cells at the concentrations used. The results suggest that Phx-3 inhibits C. pneumoniae replication in human monocytic cells as well as epithelial cells, partially depending on the tryptophan-metabolic pathway of host cells. Thus, Phx-3 might be a useful compound for controlling C. pneumoniae growth in cells and may be an alternative conventional therapy.
Assuntos
Chlamydophila pneumoniae/crescimento & desenvolvimento , Oxazinas/farmacologia , Inibidores da Aromatase/farmacologia , Linhagem Celular , Chlamydophila pneumoniae/efeitos dos fármacos , Chlamydophila pneumoniae/ultraestrutura , Células Epiteliais/microbiologia , Humanos , Microscopia Eletrônica , Monócitos/microbiologia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Triptofano/farmacologiaRESUMO
We investigated the extent to which tobacco smoke could induce persistence of Chlamydia pneumoniae in human endothelial cells. Aortic and coronary artery endothelia were infected in the absence or presence of non-cytotoxic concentrations of tobacco smoke medium. Following exposure to smoke medium, chlamydial inclusions were smaller and demonstrated fewer genome copies as determined by real-time PCR. Enumeration of inclusion-forming units (IFU) established a significant smoke-mediated, dose-dependent inhibition of elementary bodies (EB). Host cell apoptosis did not contribute to the observed restriction of productive infection. Ultrastructure analysis demonstrated an arrest in chlamydial development following smoke-exposure, with a predominance of reticulate bodies (RB) observed inside inclusions. Recovery of viable IFU was achieved with removal of smoke-medium and addition of L-tryptophan. In the presence of smoke, C. pneumoniae infection demonstrated all the characteristics of persistence in human endothelia cells. This is the first time that primary human arterial endothelial cells have been shown to support chlamydial persistence. Tobacco smoke is a well-characterized risk factor for progression of atherosclerosis, but a novel means of inducing chlamydial persistence in vascular cells. Thus, smoking may additionally contribute to atherosclerotic disease by inducing a persistent chlamydial infection in arterial endothelium.
Assuntos
Chlamydophila pneumoniae/crescimento & desenvolvimento , Células Endoteliais/microbiologia , Nicotiana , Fumaça , Aorta , Apoptose , Aterosclerose/etiologia , Linhagem Celular , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/ultraestrutura , Contagem de Colônia Microbiana , Vasos Coronários , Células Endoteliais/ultraestrutura , Dosagem de Genes , Humanos , Corpos de Inclusão , Reação em Cadeia da PolimeraseRESUMO
To examine a possible relationship between Chlamydia pneumoniae infection and multiple sclerosis (MS), we undertook an immunohistochemical (IHC), molecular, and ultrastructural comparison of central nervous system (CNS) tissue and cerebrospinal fluid (CSF) sediment from patients with MS and control individuals with other neurological diseases (ONDs). In 7 of 20 MS cases, IHC staining was seen in association with ependymal surfaces and periventricular regions of formalin-fixed brain tissue, by use of 3 different antichlamydial antibodies. There was no staining with any of the 3 antichlamydial antibodies in formalin-fixed brain tissue from OND controls (n=17). With available frozen CNS tissue, polymerase chain reaction (PCR) studies for the presence of C. pneumoniae genes were performed. The presence of a PCR signal was confirmed in 5 of 8 MS cases and in 3 of 18 OND controls. In an examination of CSF sediment by electron microscopy, we observed electron-dense structures resembling chlamydial organisms in CSF sediments from 11 of 20 MS cases and 2 of 12 OND controls. The presence of immunogold-labeled electron-dense bodies was correlated with the presence of a PCR signal in 10 of 11 MS cases. Results of studies using these different approaches support our suspicion of the presence of chlamydial organisms in the CNS, in a subset of patients with MS.
Assuntos
Antígenos de Bactérias/análise , Sistema Nervoso Central/microbiologia , Chlamydophila pneumoniae/isolamento & purificação , DNA Bacteriano/análise , Esclerose Múltipla/microbiologia , Encéfalo/microbiologia , Sistema Nervoso Central/ultraestrutura , Infecções por Chlamydia/complicações , Infecções por Chlamydia/microbiologia , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/imunologia , Chlamydophila pneumoniae/ultraestrutura , DNA Bacteriano/líquido cefalorraquidiano , Humanos , Imuno-Histoquímica , Microscopia Eletrônica , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Chlamydiales are familiar causes of acute and chronic infections in humans and animals. Human pulmonary emphysema is a component of chronic obstructive pulmonary disease (COPD) and a condition in which chronic inflammation manifested as bronchiolitis and intra-alveolar accumulation of macrophages is common. It is generally presumed to be of infectious origin. Previous investigations based on serology and immunohistochemistry indicated Chlamydophila pneumoniae infection in cases of COPD. Furthermore, immunofluorescence with genus-specific antibodies and electron microscopy suggested involvement of chlamydial infection in most cases of pulmonary emphysema, but these findings could not be verified by PCR. Therefore, we examined the possibility of other chlamydial species being present in these patients. METHODS: Tissue samples from patients having undergone lung volume reduction surgery for advanced alpha-1 antitrypsin deficiency (AATD, n = 6) or non-alpha-1 antitrypsin deficiency emphysema (n = 34) or wedge resection for hamartochondroma (n = 14) were examined by transmission electron microscopy and PCR. RESULTS: In all cases of AATD and 79.4% of non-AATD, persistent chlamydial infection was detected by ultrastructural examination. Intra-alveolar accumulation of macrophages and acute as well as chronic bronchiolitis were seen in all positive cases. The presence of Chlamydia psittaci was demonstrated by PCR in lung tissue of 66.7% AATD vs. 29.0% non-AATD emphysema patients. Partial DNA sequencing of four positive samples confirmed the identity of the agent as Chlamydophila psittaci. In contrast, Chlamydophila pneumoniae was detected only in one AATD patient. Lung tissue of the control group of non-smokers with hamartochondroma was completely negative for chlamydial bodies by TEM or chlamydial DNA by PCR. CONCLUSIONS: These data indicate a role of Chlamydophila psittaci in pulmonary emphysema by linking this chronic inflammatory process to a chronic infectious condition. This raises interesting questions on pathogenesis and source of infection.
Assuntos
Chlamydophila psittaci/isolamento & purificação , Pulmão/microbiologia , Psitacose/complicações , Enfisema Pulmonar/microbiologia , Deficiência de alfa 1-Antitripsina/complicações , Sequência de Bases , Estudos de Casos e Controles , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/isolamento & purificação , Chlamydophila pneumoniae/ultraestrutura , Chlamydophila psittaci/genética , Chlamydophila psittaci/ultraestrutura , DNA Bacteriano/análise , DNA Bacteriano/química , DNA Ribossômico/química , Feminino , Hamartoma/microbiologia , Humanos , Pulmão/patologia , Pulmão/ultraestrutura , Pneumopatias/microbiologia , Masculino , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Psitacose/microbiologia , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/patologia , RNA Ribossômico 16S/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Fumar/efeitos adversosRESUMO
The literature data and the results of the authors' experiments allow one to consider Chlamydia pneumoniae as one of the leading risk factor of development of atherosclerosis and ischemic heart disease. Features of atherosclerosis morphogenesis in the presence of Chlamydia are described. Data, showing similarity of cell reactions, characterizing immune inflammation in mLDL deposit in arterial wall and obligate parasites are described. Synergism in the action of mLDL and chlamydial infection may induce a "malignant" course of atherogenesis promoting thrombosis and ischemic heart disease excerbation.
Assuntos
Arteriosclerose/complicações , Arteriosclerose/microbiologia , Infecções por Chlamydophila/complicações , Infecções por Chlamydophila/microbiologia , Chlamydophila pneumoniae/patogenicidade , Animais , Arteriosclerose/patologia , Infecções por Chlamydophila/patologia , Chlamydophila pneumoniae/isolamento & purificação , Chlamydophila pneumoniae/ultraestrutura , Humanos , Modelos Biológicos , Fatores de RiscoRESUMO
OBJECTIVE: Research results showed that Chlamydia pneumoniae infection is related to atherosclerosis. C. pneumoniae infection may exacerbate atherogenesis. We investigated the presence of this microorganism for patients who underwent carotid endarterectomy and evaluated clinical values of C. pneumoniae infection on carotid stenosis. METHODS: Twenty patients with carotid stenosis were enrolled in this prospective study between 1997 and 1999. The patients were observed on whether they were positive or negative in four C. pneumoniae measures, namely; IgA titers, IgG titers, presence of electron microscopy, and immunocytochemistry in the endarterectomy specimens. Possible clinical findings for atherosclerosis were also observed of Chlamydial measures such as the percentage of carotid stenosis, cholesterol and triglyceride levels, smoking status, symptomatic or non-transient ischaemic attack or stroke, previous ischaemic event, calcification at surgery, ulceration on angiographies, ulceration at surgery and hypertension were included in this evaluation. RESULTS: Specific C. pneumoniae IgG were detected as positive in 9 (45%) of 20 patient samples. These patients were regarded as having chronic Chlamydia pneumoniae infection. None of the patients were positive for IgA antibody. This result demonstrated no evidence of reinfection. Immunocytochemistry and electron microscopy were positive in 7 (35%) of the 20 patients and correlated with positive serological results. The proportion of previous ischaemic events, calcification at surgery, ulceration on angiography, and ulceration at surgery were found significantly higher ( p < 0.05 ) for patients who are positive for chlamydial measures than those who are negative. CONCLUSION: The results of this study demonstrated an association between C. pneumoniae to atherosclerosis. The proportion of patients who are positive for Chlamydia measures (IgG titers, electron microscopy, and immunocytochemistry) is significantly higher for those who were positive for each of these clinical variables (PIE, CALCI, U1, and U2) than who were negative. We emphasise, the higher incidence in clinical variables of PIE, CALCI, U1, and U2 in Chlamydia measures positive group may support the association of C. pneumoniae with atherosclerotic events.
Assuntos
Aterosclerose/etiologia , Estenose das Carótidas/etiologia , Infecções por Chlamydophila/complicações , Chlamydophila pneumoniae , Adulto , Idoso , Anticorpos/metabolismo , Anticorpos Antibacterianos/imunologia , Aterosclerose/microbiologia , Aterosclerose/patologia , Estenose das Carótidas/microbiologia , Infecções por Chlamydophila/patologia , Chlamydophila pneumoniae/imunologia , Chlamydophila pneumoniae/ultraestrutura , Angiografia Coronária/métodos , Feminino , Humanos , Masculino , Microscopia Eletrônica de Transmissão/métodos , Pessoa de Meia-Idade , Estudos Retrospectivos , Testes Sorológicos/métodos , Tomografia Computadorizada por Raios X/métodosRESUMO
Persistent growth forms of Chlamydia pneumoniae have been associated with chronic infections in vivo. We investigated the effects of first-line therapeutics on the induction of persistence by monitoring recoverable organisms, gene expression of membrane proteins, and morphology. We found that all of the antibiotics tested have distinct and subinhibitory concentrations at which they induce persistence.
Assuntos
Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Infecções por Chlamydia/tratamento farmacológico , Infecções por Chlamydia/microbiologia , Chlamydophila pneumoniae/efeitos dos fármacos , Antibacterianos/farmacologia , Chlamydophila pneumoniae/crescimento & desenvolvimento , Chlamydophila pneumoniae/ultraestrutura , Imunofluorescência , Células HeLa , Humanos , Microscopia EletrônicaRESUMO
OBJECTIVE: To study atheromas, Mycoplasma pneumoniae (M. pneumoniae), and Chlamydia pneumoniae (C. pneumoniae). METHODS: C. pneumoniae was studied with immunohistochemistry and M. pneumoniae with in situ hybridization (ISH), in segments of coronary arteries (SCA) as follows: group A - thrombosed ruptured plaques (TRP) of 23 patients who died due to acute myocardial infarction (AMI); group B - 23 nonruptured plaques (NRP) of group A patients; group C - NRP of 11 coronary patients who did not die due to AMI; and group D - 11 SCA from patients with dilated cardiomyopathy or Chagas' disease without atherosclerosis. RESULTS: The mean number of C. pneumoniae+ cells/400x in groups A, B, C, and D was, respectively, 3.3 +/- 3.6; 1.0 +/- 1.3; 1.2 +/- 2.4; and 0.4 +/- 0.3; and the percentage of M. pneumoniae area was, respectively, 3.9 +/- 3.5; 1.5 +/- 1.6; 0.9 +/- 0.9; and 0.4 +/- 0.2. More M. pneumoniae and C. pneumoniae were found in of group A than in group B (P<0.01). Good correlation was seen between the area of the vessel and the M. pneumoniae area in the plaque (r = 0.46; P=0.001) and between C. pneumoniae+ cells and CD4+ T lymphocytes (r = 0.42; P<0.01). The number of C. pneumoniae+ cells correlated with CD20+ B cells (r=0.48; P<0.01). CONCLUSION: M. pneumoniae and C. pneumoniae are more frequently found in TRP correlate with the intensity of the inflammation and diameter of the vessel (positive remodeling).