Cecilia Cuffini

Susceptibility of Prostate Epithelial Cells to Chlamydia muridarum Infection and Their Role in Innate Immunity by Recruitment of Intracellular Toll-Like Receptors 4 and 2 and MyD88 to the Inclusion

ABSTRACT Although Chlamydia infections are widespread throughout the world, data about immunopathogenesis of genitourinary tract infections in males are very limited. In the present work we present an in vitro model of male genital tract-derived epithelial cells, more precisely prostate epithelial cells (PEC), to analyze if they are susceptible and able to respond to Chlamydia muridarum infection. Our results demonstrate that rat PEC are susceptible to C. muridarum infection and respond to this pathogen by up-regulating different proinflammatory cytokine and chemokine genes that could participate in the recruitment and local activation of immune cells, therefore influencing innate and adaptive immune responses during Chlamydia infection. Moreover, we analyzed the expression of Toll-like receptor 4 (TLR4), TLR2, and related molecules on PEC and the effect of C. muridarum infection on their expression. Our results demonstrate that PEC express significant levels of TLR4, CD14, TLR2, and the adaptor molecule MyD88 and up-regulate these proteins in response to C. muridarum infection. Indeed, TLR4, CD14, TLR2, and the adaptor MyD88 are specifically recruited to the vicinity of the bacterial inclusion, suggesting that these TLRs are actively engaged in signaling from this intracellular location in these cells. This is, to our knowledge, the first time that an in vitro model of infection with Chlamydia of male tract-derived epithelial cells has been achieved, and it provides the opportunity to determine how these cells respond and participate in modulating innate and adaptive immune response during Chlamydia infections.

Male Rodent Genital Tract Infection With Chlamydia Muridarum: Persistence in the Prostate Gland That Triggers Self-Immune Reactions in Genetically Susceptible Hosts

PURPOSE We investigated Chlamydia trachomatis infection and its pathogenic consequences in the male rodent genital tract. MATERIALS AND METHODS Male rats were inoculated in the meatal urethra with Chlamydia muridarum. We sought bacterial DNA at early and late times after inoculation in different parts of the male genital tract. Histological alterations and the immune response against prostate antigens were analyzed. RESULTS Male rats showed ascending infection with wide dissemination of bacteria in the genital tract at an early time point after inoculation. At later stages bacteria persisted only in some parts of the genital tract and in the prostate gland. C. muridarum was also detected in semen in a high proportion of rats irrespective of an acute or chronic stage of infection. Histological alterations that accompanied C. muridarum were especially observed in the prostate and mainly composed of CD3+ cell infiltration. Positive humoral and cellular responses against prostate antigens were noted in a considerable number of infected rats. NOD mice, an autoimmune, prostatitis prone strain, showed a similar pattern with C. muridarum in the prostate of 100% of infected mice, which was again accompanied by mononuclear cell infiltration and antibodies against prostate antigens at early and late times after inoculation. CONCLUSIONS Results reveal that C. muridarum infects the male rodent genitourinary tract with special persistence in the prostate gland, where it causes chronic inflammation that in turn may act as a trigger factor for self-immune reactions in susceptible hosts.

Molecular evidence of Chlamydophila pneumoniae infection in reptiles in Argentina

In the central area of Argentina, the epidemiological and molecular characteristics of Chlamydophila pneumoniae infections in reptiles are still unknown. A nested polymerase chain reaction of the rpoB gene was used to detect C. pneumoniae in cloacal swab samples from 19 reptiles at a recreational area. Eleven (57.89%) reptiles were positive; the sequencing and phylogenetic analysis confirmed the presence of this bacterium. Neither C. pneumoniae DNA in the caregivers pharynges nor IgM antibodies anti-C. pneumoniae in their serum samples were detected; however, caregivers presented very high titers of IgG anti-C. pneumoniae. The detection of C. pneumoniae DNA in reptiles demonstrated the circulation of this agent in the recreational area and could be responsible for the exacerbated immune response of the personnel handling the reptiles, which suggests a potential zoonotic cycle. This is the first report of the detection of C. pneumoniae in reptiles in Argentina.

Rubella Virus Does Not Induce Apoptosis in Primary Human Embryo Fibroblast Cultures: A Possible Way of Viral Persistence in Congenital Infection

Congenital rubella is a persistent infection that contrasts with acute postnatal infection. Basis of the Rubella virus (RV) persistence still remain unknown, though several hypotheses have been postulated. RV induces apoptosis in cell lines, maybe as a way of cell-autonomous defense mechanism against virus. Considering the pattern of c-oncogenes expression during embryogenesis, which promotes proliferation while it inhibits apoptosis in specific cells, at certain times, it can be proposed that when RV infection establishes early in gestation, embryo cells that are proliferating have their apoptotic pathways shut down; then infected proliferating embryo cells cannot execute their apoptotic death program. We here report that RV induces apoptosis in human normal-term placenta chorionic villi explants (CVE) and in monolayers of cytotrophoblasts (CTB), but does not induce apoptosis in primary human embryo fibroblasts (HEF) cultures. These results suggest distinct responses to RV infection when comparing differentiated cells, as CTB, to cells with high proliferating potential, as HEF. RV shoots apoptosis in the former, whereas in fibroblastic dividing cells derived from embryo, RV appears not to be enough stimulus to activate the genetic program of cell death.

Mutation Detection of E6 and LCR Genes from HPV 16 Associated with Carcinogenesis

Human papillomavirus (HPV) is responsible for one of the most frequent sexually transmitted infections. The first phylogenetic analysis was based on a LCR region fragment. Nowadays, 4 variants are known: African (Af-1, Af-2), Asian-American (AA) and European (E). However the existence of sub-lineages of the European variant havs been proposed, specific mutations in the E6 and LCR sequences being possibly related to persistent viral infections. The aim of this study was a phylogenetic study of HPV16 sequences of endocervical samples from Cordoba, in order to detect the circulating lineages and analyze the presence of mutations that could be correlated with malignant disease. The phylogenetic analysis determined that 86% of the samples belonged to the E variant, 7% to AF-1 and the remaining 7% to AF-2. The most frequent mutation in LCR sequences was G7521A, in 80% of the analyzed samples; it affects the binding site of a transcription factor that could contribute to carcinogenesis. In the E6 sequences, the most common mutation was T350G (L83V), detected in 67% of the samples, associated with increased risk of persistent infection. The high detection rate of the European lineage correlated with patterns of human migration. This study emphasizes the importance of recognizing circulating lineages, as well as the detection of mutations associated with high-grade neoplastic lesions that could be correlated to the development of carcinogenic lesions.

Genetic diversity of Chlamydia among captive birds from central Argentina

To study the occurrence of Chlamydia spp. and their genetic diversity, we analysed 793 cloacal swabs from 12 avian orders, including 76 genera, obtained from 80 species of asymptomatic wild and captive birds that were examined with conventional nested polymerase chain reaction and quantitative polymerase chain reaction. Chlamydia spp. were not detected in wild birds; however, four species (Chlamydia psittaci, Chlamydia pecorum, Chlamydia pneumoniae and Chlamydia gallinacea) were identified among captive birds (Passeriformes, n = 20; Psittaciformes, n = 15; Rheiformes, n = 8; Falconiformes n = 2; Piciformes n = 2; Anseriformes n = 1; Galliformes n = 1; Strigiformes n = 1). Two pathogens (C. pneumoniae and C. pecorum) were identified simultaneously in samples obtained from captive birds. Based on nucleotide-sequence variations of the ompA gene, three C. psittaci-positive samples detected were grouped into a cluster with the genotype WC derived from mammalian hosts. A single positive sample was phylogenetically related to a new strain of C. gallinacea. This report contributes to our increasing understanding of the abundance of Chlamydia in the animal kingdom.

Isolation of Chlamydophila pneumoniae from atheromas of the carotid artery and their antibiotics susceptibility profile

BACKGROUND Atherosclerosis is pathogenically similar to a chronic inflammatory response. Peripheral arterial disease (PAD) is a common manifestation of atherosclerosis. Chlamydophila pneumoniae has been suggested to play a role in the origin of PAD. OBJECTIVE To determine whether C. pneumoniae is present in atherosclerosis lesions of the carotid artery wall in patients with PAD through several diagnostic methods and to characterize C. pneumoniae susceptibility profiles. METHODS The presence of C. pneumoniae in 9 tissue samples from atherosclerotic lesions obtained by carotid endarterectomy was investigated by 3 methods. Karnofsky-fixed specimens were examined by transmission electron microscopy (TEM), isolation of C. pneumoniae was attempted in LLCMK2 cell structure (ICC), and the presence of chlamydial DNA was investigated by polymerase chain reaction (PCR). The in vitro activities of azithromycin, roxithromycin and penicillin were tested in 4 isolations and the reference strain of C. pneumoniae (AR39). RESULTS C. pneumoniae was detected in atherosclerotic plaques from 4 patients with PAD. The pathogen was identified by TEM, PCR and ICC. We report data of the in vitro susceptibility of 4 strains. These strains did not differ from respiratory AR39 strain in their susceptibility patterns to azithromycin, roxithromycin and penicillin. CONCLUSIONS C. pneumoniae is frequently found in the advanced carotid atherosclerotic lesions of patients undergoing endarterectomy. Although these findings do not establish causality in carotid artery atherosclerosis, they should stimulate investigation of the possible causal or pathogenic role of C. pneumoniae. Notably, the profiles of antibiotic susceptibility of C. pneumoniae isolated from 4 of the patients did not differ from those of the reference strain.

Genotyping of C. psittaci in central area of Argentina

In central area of Argentina, the epidemiologic and molecular characteristics of Chlamydophila psittaci infections are still unknown. Nested polymerase chain reaction of domains II, III, and IV of the omp A gene was used to detect Chlamydophila in 43 pharyngeal swab samples from patients with suspected human psittacosis (2010-2011); 9 (21%) of them yielded positive results. Molecular typing was performed by direct sequencing demonstrating the presence of C. psittaci genotypes A, E/B, and WC.

Circulation of human papillomavirus (HPV) genotypes in women from Córdoba, Argentina, with squamous intraepithelial lesions

Human papillomavirus (HPV) can induce a wide spectrum of squamous intraepithelial lesions (SIL) of varying severity. The aim of the present study was to establish the frequency of HPV infection and identify the genotypes circulating in women from Córdoba, Argentina, in relation to age and cytology. A total of 186 women, between 18 and 65 years old, with antecedents of SIL, underwent a pelvic examination and had cervical cells collected for cytology and HPV DNA detection. Ninety-six samples (51.6%) were positive for HPV detection, and sixty-three (65.6%) of them showed the presence of at least one HR-HPV. Low- and high-grade SIL showed significant association in patients younger than 35 years of age. We found 18 different genotypes, with a greater presence of HR-HPV. Genotypes 16 and 6 were the most frequent. Seven (7.3%) multiple infections, 85.7% of which had at least one HR-HPV, were detected. The detection of a large number of different HPV genotypes is a warning sign. It is thus necessary to strengthen the monitoring of the circulation of high-risk genotypes, currently less prevalent in intraepithelial lesions, as a control measure for the possible impact of the implementation of vaccines against genotypes 16 and 18.

Cell-fusion assay for the detection of rubella virus in Vero cells

BACKGROUND AND OBJECTIVES Rubella virus (RV) produces a subtle and slow-developing cytopathic effect in Vero cells that is difficult to recognize, especially at low multiplicities of infection. In order to facilitate the detection of RV in cell culture, we standardized a low-pH virus-mediated cell-fusion assay. STUDY DESIGN The incubation periods, temperatures, pH and multiplicity of infection were established. The specificity of the method was tested by immunofluorescence assay and cell-fusion inhibition by specific sera. RESULTS Six days post infection, Vero cells were treated for 5 min with fusion medium. After that, monolayers were incubated with medium at neutral pH for 16 h and then stained. Gigantic cells with multiple nuclei were observed. CONCLUSIONS The method allowed the observation of unequivocal images that are easier to recognize than the cytopathic effect caused by RV in the same cell line. At the same time, the method is simple, accessible and shown to be specific to demonstrate the replication of several strains and isolates of RV in Vero cells.