RESUMO
OBJECTIVE: To assess the presence of Chlamydia pneumoniae DNA in the joints of patients with reactive arthritis (ReA) and other arthritides. METHODS: DNA was prepared from synovial tissue (ST) and several synovial fluid (SF) samples from 188 patients with either ReA, undifferentiated oligoarthritis, or other forms of arthritis, and from 24 normal (non-arthritis) individuals. Preparations were screened using polymerase chain reaction (PCR) assays that independently targeted the C. pneumoniae 16S ribosomal RNA and major outer membrane protein genes. RESULTS: Twenty-seven of 212 ST samples (12.7%) were PCR positive for C. pneumoniae DNA; 10 SF samples from these 27 patients were similarly positive. Among the PCR-positive patients, 3 had ReA, 2 had Reiter's syndrome, 7 had undifferentiated oligoarthritis, 4 had undifferentiated monarthritis, 6 had rheumatoid arthritis, and 5 had other forms of arthritis. No samples from normal control individuals were PCR positive. CONCLUSION: DNA of C pneumoniae is present in synovial specimens from some arthritis patients. The prevalence of this organism in the joints was lower than that of C trachomatis, and synovial presence of the organism was not associated with any distinct clinical syndrome. Widely disseminated nucleic acids such as those of C. pneumoniae might have some role in the pathogenesis of several arthritides, since the organism was not found in the ST from normal control individuals.
Assuntos
Artrite Reumatoide/genética , Infecções por Chlamydia/genética , Chlamydia trachomatis/genética , Chlamydophila pneumoniae/genética , Líquido Sinovial/microbiologia , Membrana Sinovial/microbiologia , Artrite Reativa/etiologia , DNA Bacteriano/análise , Humanos , Articulações/química , Reação em Cadeia da Polimerase , Proibitinas , Líquido Sinovial/química , Membrana Sinovial/químicaRESUMO
Genetically determined differences in interleukin-10 (IL-10) and gamma interferon (IFN-gamma) responses in mice correlate with clearance of Chlamydia pneumonitis infection. We measured the synovial expression of IL-10 and IFN-gamma and additional cytokine genes in patients who had recent-onset Chlamydia-associated arthritis (Chl-AA). IL-10 and IFN-gamma mRNA were relatively abundant in recent-onset Chl-AA.
Assuntos
Artrite Infecciosa/genética , Artrite Infecciosa/imunologia , Infecções por Chlamydia/genética , Infecções por Chlamydia/imunologia , Chlamydia/patogenicidade , Interferon gama/genética , Interleucina-10/genética , Adulto , Animais , Artrite/genética , Artrite/imunologia , Artrite Infecciosa/etiologia , Estudos de Casos e Controles , Chlamydia/genética , Chlamydia/isolamento & purificação , Infecções por Chlamydia/etiologia , Citocinas/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Feminino , Expressão Gênica , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Membrana Sinovial/imunologiaRESUMO
The principal host cell for persistently infecting synovial Chlamydia trachomatis is the macrophage. During infection of human monocytes/macrophages in culture this bacterium displays aberrant morphology and produces no new elementary bodies, reflecting the situation in synovium. Here we investigate the metabolic status of C. trachomatis (serovar K) during an extended infection of human peripheral monocytes in vitro. Using reverse transcription-polymerase chain reaction assays, we have shown that primary transcripts from the chlamydial rRNA operons are present throughout a 10-day course of infection. Other assays targeting mRNAs from chlamydial genes encoding r-proteins S5 and L5, the glycyl-tRNA synthetase, the 60-kDa cysteine-rich outer membrane protein, and the KDO transferase indicate that these messengers are also present throughout the entire 10-day period. The gene encoding the 57-kDa heat-shock protein (hsp60) is expressed by the bacterium throughout the 10-day infection of cultured monocytes, but transcript levels from the gene encoding the major outer membrane protein (omp1) appear to be attenuated. Western analyses targeting these latter proteins confirm the presence of the hsp60 gene product, and the virtual absence of major outer membrane protein, in chlamydia-infected cultured human monocytes. Thus, during extended infection of human monocytes in vitro, chlamydia are non-productive but transcriptionally active; the pattern of transcriptional activity reflects that known for persistent C. trachomatis infection in vivo in synovial tissue.
Assuntos
Proteínas de Bactérias/biossíntese , Chlamydia trachomatis/metabolismo , Monócitos/microbiologia , Anticorpos Monoclonais , Especificidade de Anticorpos , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Western Blotting , Chaperonina 60/biossíntese , Chaperonina 60/genética , Chlamydia trachomatis/genética , Eletroforese em Gel de Ágar , Humanos , Reação em Cadeia da Polimerase , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição GênicaRESUMO
OBJECTIVE: To use standard molecular methods to define the prevalence and metabolic characteristics of Chlamydia trachomatis during infection of fallopian tubes in women with ectopic pregnancies. DESIGN: Polymerase chain reaction (PCR)- and reverse transcription-PCR (RT-PCR)-based assessment of presence of chlamydial DNA and various RNA species in fallopian tube biopsy samples. SETTING: Hospital and molecular genetics laboratory. PATIENTS: Ten women of varying ages, each presenting with ectopic pregnancy. MAIN OUTCOME MEASURE(S): Positive signal in specific chlamydia-directed PCR and RT-PCR assays. RESULT(S): Nucleic acid preparations from 7 of the 10 fallopian tube patient samples were PCR-positive for C. trachomatis DNA. Each of the 7 PCR-positive samples also showed the presence of several transcripts from the bacterium, including primary transcripts from the ribosomal RNA operons. CONCLUSION(S): A higher proportion of ectopic pregnancies than was believed previously may be attributable to infection of the fallopian tubes by C. trachomatis. The presence of various chlamydial RNA molecules suggests that viable, metabolically active bacteria were present in fallopian tubes of the patients studied.
Assuntos
Chlamydia trachomatis/crescimento & desenvolvimento , Tubas Uterinas/microbiologia , Gravidez Ectópica/microbiologia , Adulto , Feminino , Células HeLa , Humanos , Reação em Cadeia da Polimerase/métodos , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We assessed whether the intracellular bacterium Chlamydia pneumoniae was present in post-mortem brain samples from patients with and without late-onset Alzheimer's disease (AD), since some indirect evidence seems to suggest that infection with the organism might be associated with the disease. Nucleic acids prepared from those samples were screened by polymerase chain reaction (PCR) assay for DNA sequences from the bacterium, and such analyses showed that brain areas with typical AD-related neuropathology were positive for the organism in 17/19 AD patients. Similar analyses of identical brain areas of 18/19 control patients were PCR-negative. Electron- and immunoelectron-microscopic studies of tissues from affected AD brain regions identified chlamydial elementary and reticulate bodies, but similar examinations of non-AD brains were negative for the bacterium. Culture studies of a subset of affected AD brain tissues for C. pneumoniae were strongly positive, while identically performed analyses of non-AD brain tissues were negative. Reverse transcription (RT)-PCR assays using RNA from affected areas of AD brains confirmed that transcripts from two important C. pneumoniae genes were present in those samples but not in controls. Immunohistochemical examination of AD brains, but not those of controls, identified C. pneumoniae within pericytes, microglia, and astroglia. Further immunolabelling studies confirmed the organisms' intracellular presence primarily in areas of neuropathology in the AD brain. Thus, C. pneumoniae is present, viable, and transcriptionally active in areas of neuropathology in the AD brain, possibly suggesting that infection with the organism is a risk factor for late-onset AD.
Assuntos
Doença de Alzheimer/microbiologia , Encéfalo/microbiologia , Chlamydophila pneumoniae/isolamento & purificação , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Anticorpos Monoclonais , Encéfalo/ultraestrutura , Chlamydophila pneumoniae/genética , DNA Bacteriano/análise , Feminino , Genes Bacterianos , Humanos , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Bacteriano/análiseRESUMO
OBJECTIVE: We used reverse transcription-polymerase chain reaction (RT-PCR) assays to assess expression of genes from Chlamydia trachomatis in synovial tissues of patients with reactive arthritis (ReA)/Reiter's syndrome (RS) to determine viability/metabolic activity of the bacterium in joints of infected patients. METHODS: Synovial biopsies were obtained from 18 patients with ReA, RS, or other arthritides; nucleic acids from 16 samples were PCR positive for chlamydial chromosomal DNA. RT-PCR assays targeting primary transcripts from C. trachomatis rRNA operons, and mRNA from the bacterial omp1, hsp60, glyQS, and r-protein S5 and L5 genes, were used to characterize viability/metabolic activity. Host actin mRNA was assessed as control in each sample preparation. RESULTS: RT-PCR of host cell actin mRNA in the 18 patient samples confirmed the quality of all RNA preparations. RNA from 14/16 PCR positive samples was positive by RT-PCR for chlamydial rRNA primary transcripts. Each of these same 14 samples was also RT-PCR positive in assays targeting glyQS, r-protein S5 and L5, and hsp60 mRNA. However, none of the 14 samples showing chlamydial rRNA and mRNA was positive for omp1 transcripts. CONCLUSION: Synovial chlamydia are viable/metabolically active, since primary rRNA transcripts and mRNA from chlamydial genes specifying components of the bacterial protein synthetic system were present in most patient samples assayed. Expression of omp1, encoding the major outer membrane protein, is strongly attenuated in persistently infecting synovial chlamydia, while that of hsp60, specifying a highly immunogenic heat shock protein of the organism, is not downregulated.
Assuntos
Artrite Reativa/microbiologia , Infecções por Chlamydia/genética , Chlamydia trachomatis/genética , Regulação Bacteriana da Expressão Gênica , Porinas , Membrana Sinovial/microbiologia , Adulto , Idoso , Proteínas da Membrana Bacteriana Externa/genética , Chaperonina 60/genética , Infecções por Chlamydia/diagnóstico , Chlamydia trachomatis/isolamento & purificação , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Proibitinas , RNA Bacteriano/análise , Transcrição GênicaRESUMO
Previous studies have suggested that monocytes may play a role in the dissemination of Chlamydia trachomatis, and in establishment of persistent infection with this bacterium. Infection of cultured human peripheral blood monocytes with C. trachomatis serovar K produced persistent, nonproductive infection. Transmission electron microscopy of such infected cultures revealed single or multiple Chlamydia in monocyte inclusions over a culture period of 10 days. Those inclusions were aberrant, and normal reticulate bodies within the inclusions were not observed. Immunoelectron microscopy showed the chlamydial major outer membrane protein and lipopolysaccharide to be associated with the bacterial plasma membrane. Lipopolysaccharide was also identified in the monocyte cytoplasm. Molecular analyses of primary chlamydial rRNA transcripts demonstrated that the organism is viable and metabolically active within monocyte inclusions. However, attempts to overcome chlamydial growth arrest by incubation of Chlamydia-infected monocytes with tryptophan, and antibodies against alpha interferon, gamma interferon, or tumor necrosis factor, were all ineffective, suggesting that known mechanisms of growth inhibition do not hold in human monocytes. These observations indicate that infection of human peripheral blood monocytes with C. trachomatis may be involved in the genesis/maintenance of extra-urogenital inflammation, since non-culturable, metabolically active bacteria persist in those cells.
Assuntos
Infecções por Chlamydia/microbiologia , Chlamydia trachomatis/patogenicidade , Anticorpos Bloqueadores/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Membrana Celular/química , Membrana Celular/imunologia , Membrana Celular/ultraestrutura , Células Cultivadas , Chlamydia trachomatis/crescimento & desenvolvimento , Chlamydia trachomatis/ultraestrutura , Doença Crônica , Citoplasma/metabolismo , DNA Bacteriano/genética , Humanos , Interferon-alfa/imunologia , Interferon gama/imunologia , Lipopolissacarídeos/imunologia , Microscopia Eletrônica , Microscopia Imunoeletrônica , Monócitos/microbiologia , Monócitos/ultraestrutura , Reação em Cadeia da Polimerase , RNA Bacteriano/genética , RNA de Transferência/análise , Transcrição Gênica , Triptofano/farmacologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Considerable evidence suggests that viable Chlamydia trachomatis are present in joint tissues of patients with Reiter's syndrome/reactive arthritis (RS/ReA), but the use of antibiotics to treat such patients remains controversial. We investigated the continued presence of chlamydia in synovial tissues of patients with RS/ReA; these patients had been treated with antibiotics for relatively extended periods, had shown clinical improvement, but had persistent active disease. Knee synovial tissue was obtained from two patients with RS/ReA and two controls with osteoarthritis (OA). Each sample was screened for chlamydia by culture, direct fluorescent antibody assay (DFA), in situ hybridization (ISH), and polymerase chain reaction (PCR).Synovial tissues from antibiotic-treated RS/ReA patients were negative for chlamydia when analyzed by culture and DFA, but positive when analyzed by ISH for chlamydial RNA and by PCR for chlamydial DNA. Samples from OA patients were negative by all screening methods. Thus, antibiotic treatment does not appear to easily eradicate chlamydia from the joints of RS/ReA patients. Rather, the organism can persist in synovial tissue in a form not detectable by routine laboratory screening methods. Further studies are needed to determine whether antibiotic regimens other than those used here can eradicate synovial chlamydia and to determine how this relates to disease activity. Optimal therapy for patients with RS/ ReA is therefore not yet clear.
RESUMO
OBJECTIVE: Difficulties in detecting Chlamydia trachomatis in human joints by polymerase chain reaction (PCR) may be related to whether synovial tissue or synovial fluid (SF) is used as the source of DNA in PCR amplification. In this study, a new PCR assay was developed and used to compare chlamydial DNA in paired samples of SF and synovial tissue from patients with arthritis. METHODS: The PCR assay targeted the ribosomal RNA operons, which are present in 2 copies on the C trachomatis chromosome. DNA from several relevant bacteria and chlamydial serovars was used for testing this screening system. The detection of chlamydial DNA in nucleic acid preparations from matched samples of SF and synovial tissue was compared by PCR assay. Samples were obtained from 55 patients, including patients with reactive arthritis, Reiter's syndrome, and other arthropathies. RESULTS: Testing of the PCR screening system confirmed it to be highly specific and sensitive. Use of this assay to screen DNA from SF and synovial tissue samples showed that 29 (53%) of 55 synovial tissue preparations were positive for chlamydial DNA, but only 16 (29%) of the matched SF samples from these 29 patients were similarly positive. Five (9%) of 55 SF samples, but not their tissue counterparts, were positive for chlamydial DNA by PCR. CONCLUSION: Detection of chlamydial DNA in the joints of patients by PCR gives positive results more often when synovial tissue rather than SF is the source of target nucleic acids. Although synovial tissue is the source of choice for the most reliable determination of chlamydia in the joint, both synovial tissue and SF should be assayed if possible.
Assuntos
Infecções por Chlamydia/diagnóstico , Chlamydia trachomatis/genética , Líquido Sinovial/microbiologia , Membrana Sinovial/microbiologia , Adulto , Idoso , Chlamydia trachomatis/isolamento & purificação , Primers do DNA , Feminino , Testes Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Bacteriano/análise , RNA Bacteriano/isolamento & purificação , RNA Ribossômico/análise , RNA Ribossômico/isolamento & purificaçãoRESUMO
The objective of this study was to determine the effects of orally administered misoprostol and diclofenac on inflammation caused by particulates in the rat subcutaneous air-pouch model. Subcutaneous air pouches were formed in male Sprague-Dawley rats. The animals were premedicated by gavage with either saline, diclofenac, misoprostol, or both diclofenac and misoprostol. The air pouches were injected with either polymethyl methacrylate particles or monosodium urate crystals, and the pouch fluids were obtained at 1, 6, 24, 48, and 72 h. Leukocyte influx, tumor necrosis factor, neutral metalloprotease activity, and prostaglandin E(2) levels were measured. It was determined that leukocyte influx was inhibited by diclofenac in the acute inflammation caused by monosodium urate crystals only. In all animals receiving diclofenac, prostaglandin E(2) (PGE(2)) levels were reduced, whereas tumor necrosis factor levels were elevated. The elevation of tumor necrosis factor was prevented by the addition of misoprostol. It is concluded that the oral administration of diclofenac or misoprostol can affect levels of specific mediators involved in particulate-induced inflammation in the subcutaneous air pouch.