Respuesta de las células HeLa y EA.hy926 al estrés por hipoxia y radiación ionizante a través de la vía JAK/STAT y la supervivencia celular

El comportamiento biológico de las células cancerosas es influenciado por el microambiente en el que se desarrollan y en este, factores como la angiogénesis o el estímulo de agentes estresores como la hipoxia, se han considerado críticos para su evolución y manejo terapéutico. Uno de los mecanismos moleculares implicados en la respuesta celular frente a estímulos estresores es la activación de vías de señalización intracelulares; en este estudio, se evaluó el estado de la vía JAK/STAT y en ella la expresión/activación de la proteína STAT3 en la línea tumoral (HeLa) y endotelial (EA.hy926), sometidas a hipoxia física y química con mesilato de deferoxamina durante 2, 6 y 24 horas. Adicionalmente, al considerar la importancia de la hipoxia como un agente modificador de la respuesta en el manejo del cáncer utilizando radiaciones ionizantes, se construyeron curvas de supervivencia celular que permitieron evaluar el comportamiento celular frente a estos estímulos. El presente estudio resalta la importancia de la hipoxia como un estímulo que modifica la activación de la proteína STAT3 y la supervivencia de células irradiadas en las dos líneas estudiadas.

Binding properties and adhesion-mediating regions of the major sheath protein of Treponema denticola ATCC 35405

There is growing evidence that a number of oral Treponema species, in particular Treponema denticola, are associated with the progression of human periodontal disease. The major sheath (or surface) protein (Msp) of T. denticola is implicated in adhesion of bacteria to host cells and tissue proteins and is likely to be an important virulence factor. However, the binding regions of the Msp are not known. We have purified from Escherichia coli recombinant Msp (rMsp) polypeptides corresponding to the following: full-length Msp (rMsp) minus 13 N-terminal amino acid (aa) residues, an amino-terminal fragment (rN-Msp, 189 aa residues), a 57-aa residue segment from the central region (rV-Msp), and a C-terminal fragment (rC-Msp, 272 aa residues). rMsp (530 aa residues) bound to immobilized fibronectin, keratin, laminin, collagen type 1, fibrinogen, hyaluronic acid, and heparin. The N- and V-region polypeptides, but not rC-Msp, also bound to these substrates. Binding of rMsp to fibronectin was targeted to the N-terminal heparin I/fibrin I domain. Antibodies to the N-region or V-region polypeptides, but not antibodies to the rC-Msp fragment, blocked adhesion of T. denticola ATCC 35405 cells to a range of host protein molecules. These results suggest that the N-terminal half of Msp carries epitopes that are surface exposed and that are involved in mediating adhesion. Binding of rMsp onto the cell surface of low-level fibronectin-binding Treponema isolates conferred a 10-fold increase in fibronectin binding. This confirms that Msp functions autonomously as an adhesin and raises the possibility that phenotypic complementation of virulence functions might occur within mixed populations of Treponema species.

Evidence of evolving extraintestinal enteroaggregative Escherichia coli ST38 clone

BACKGROUND: Several clones of extended-spectrum β-lactamase (ESBL)–producing extraintestinal pathogenic Escherichia coli (ExPEC) have globally expanded their distribution. ExPEC infections often originate from the patient’s own intestinal flora, although the degree of overlap between diarrheagenic E. coli and ExPEC pathotypes is unclear. Relatively little is known about antimicrobial drug resistance in the most common diarrheagenic E. coli groups, including enteroaggregative E. coli (EAEC), and bacterial gastroenteritis is generally managed without use of antimicrobial drugs. APPROACHES: We conducted this study to establish the presence and characteristics of ESBL-producing EAEC in a well-defined collection of ESBL-producing isolates. The isolates were from human and animal sources in Germany, the Netherlands, and the United Kingdom. DNA from 359 ESBL isolates was screened for the presence of the EAEC transport regulator gene (aggR), located on the EAEC plasmid, using a real-time PCR assay and the phylogroup was determined for each positive isolate. A microarray was used to detect ESBL genes, such as blaCTX-M, at the group level, as previously described. The antimicrobial drug susceptibilities of EAEC isolates were determined and virulence factors associated with intestinal and extraintestinal infection and with EAEC were investigated . RESULTS AND CONCLUSIONS: We assigned a virulence score (total number of virulence factor genes detected; maximum possible score 22) and a resistance score (total number of drug classes; maximum score 11) to each isolate. We isolated 11 EAEC from humans. Eight of the EAEC were isolated from urine specimens, and 1 was isolated from a blood culture; 63% belonged to phylogroup D (Table). EAEC ST38, the most common (55%) ST, was significantly associated with extraintestinal sites in the subset of 140 human isolates (Fisher exact test, p<0.0001)

Genetic diversity of heat-labile toxin expressed by enterotoxigenic Escherichia coli strains isolated from humans

The natural diversity of the eft operons, encoding the heat-labile toxin LT-I (LT), carried by enterotoxigenic Escherichia coli (ETEC) strains isolated from humans was investigated. For many years, LT was supposed to be represented by a rather conserved toxin, and one derivative, produced by the reference H10407 strain, was intensively studied either as a virulence factor or as a vaccine adjuvant. Amplicons encompassing the two LT-encoding genes (eltA and eltB) of 51 human-derived ETEC strains, either LT+ (25 strains) only or LT+/ST+ (26 strains), isolated from asymptomatic (24 strains) or diarrheic (27 strains) subjects, were subjected to restriction fragment length polymorphism (RFLP) analysis and DNA sequencing. Seven polymorphic RFLP types of the H10407 strain were detected with six (BsaI, DdeI, HhaI, HincII, HphI, and MspI) restriction enzymes. Additionally, the single-nucleotide polymorphic analysis revealed 50 base changes in the eft operon, including 21 polymorphic sites at eltA and 9 at eltB. Based on the deduced amino acid sequences, 16 LT types were identified, including LT1, expressed by the H10407 strain and 23 other strains belonging to seven different serotypes, and LT2, expressed by 11 strains of six different serotypes. In vitro experiments carried out with purified toxins indicated that no significant differences in GM1-binding affinity could be detected among LT1, LT2, and LT4. However, LT4, but not other toxin types, showed reduced toxic activities measured either in vitro with cultured cells (Y-1 cells) or in vivo in rabbit ligated ileal loops. Collectively, these results indicate that the natural diversity of LTs produced by wild-type ETEC strains isolated from human hosts is considerably larger than previously assumed and may impact the pathogeneses of the strains and the epidemiology of the disease.

Evaluation of laccases and melanization in clinical and environmental Cryptococcus neoformans samples by non-denaturing PAGE

The increased incidence of infections caused by the opportunistic pathogen Cryptococcus neoformans, which mainly affects immunocompromised patients but can also infect immunocompetent individuals, has needed additional studies on this micro-organism`s pathogenicity and factors related to virulence, such as enzyme production, for a better understanding of the aetiology of cryptococcosis. The aim of this study was to verify the applicability of non-denaturing PAGE for analysis of laccases by quantification of the amount of melanin pigment produced by clinical and environmental strains of C. neoformans. After incubation of the gel with the substrate L-dopa, strains produced melanin spots of a bright brown to black colour. Quantification of these spots was performed by densitometry analysis and the amount of melanin produced was calculated and compared among the strains. All strains showed laccase activity. Serotype B strains showed a higher melanin intensity than serotype A strains. Over half of the clinical strains (56.2%) showed the lowest melanin intensities, suggesting that melanin production may not be the main virulence factor against host defence. The clinical strain ICB 88 revealed two melanin spots on the gel, indicating the presence of two laccase isoforms. The environmental strains showed the highest values of melanin intensity, which may be related to previous exposure to environmental stress conditions.

Characterization of the alpha-haemolysin determinant from the human enteropathogenic Escherichia coli O26 plasmid pEO5

The 157-kb conjugative plasmid pEO5 encoding alpha-haemolysin in strains of human enteropathogenic Escherichia coli (EPEC) O26 was investigated for its relationship with EHEC-haemolysin-encoding plasmids of enterohaemorrhagic E. coli (EHEC) O26 and O157 strains. Plasmid pEO5 was found to be compatible with EHEC-virulence plasmids and did not hybridize in Southern blots with plasmid pO157 from the EHEC O157:H7 strain EDL933, indicating that both plasmids were unrelated. A 9227-bp stretch of pEO5 DNA encompassing the entire alpha-hlyCABD operon was sequenced and compared for similarity to plasmid and chromosomally inherited alpha-hly determinants. The alpha-hly determinant of pEO5 (7252 bp) and its upstream region was most similar to corresponding sequences of the murine E. coli alpha-hly plasmid pHly152, in particular, the structural alpha-hlyCABD genes (99.2% identity) and the regulatory hlyR regions (98.8% identity). pEO5 and alpha-hly plasmids of EPEC O26 strains from humans and cattle were very similar for the regions encompassing the structural alpha-hlyCABD genes. The major difference found between the hly regions of pHly152 and pEO5 is caused by the insertion of an IS2 element upstream of the hlyC gene in pHly152. The presence of transposon-like structures at both ends of the alpha-hly sequence indicates that this pEO5 virulence factor was probably acquired by horizontal gene transfer.

Valor prognóstico das expressões da proteína p53 e do fator de crescimento do endotélio vascular em pacientes com carcinoma epidermóide de esôfago submetidos a esofagectomia

As mutações no gene de supressão tumoral p53 estão entre as anormalidades genéticas mais comuns encontradas numa ampla variedade de tumores. Embora a função do gene p53 ainda não esteja completamente esclarecida, ele parece ser um fator de transcrição nuclear que controla a proliferação celular, a apoptose e a manutenção da estabilidade genética. A angiogênese é essencial para o crescimento e a metastatização de tumores sólidos. O Fator de Crescimento do Endotélio Vascular (VEGF, Vascular Endothelial Growth Factor), um fator de crescimento identificado recentemente com propriedades angiogênicas significativas, pode ser um importante regulador da angiogênese tumoral. A associação entre as expressões da proteína p53 e do VEGF e o prognóstico tem sido pouco estudada. Foram estudadas peças cirúrgicas de 47 pacientes com carcinoma epidermóide de esôfago (CEE) submetidos à esofagectomia em estágios II e III, utilizando-se coloração imuno-histoquímica. As expressões da proteína p53 e do VEGF foram observadas em 53% e 40% dos tumores, respectivamente. As expressões da proteína p53 e do VEGF coincidiram em somente 21% dos casos, e não foi encontrada correlação entre elas. Nenhum dos fatores clinicopatológicos se correlacionaram significativamente com as expressões da proteína p53 ou do VEGF. Em relação ao prognóstico, não havia associação significativa entre as expressões da proteína p53 e do VEGF e pior prognóstico. Os autores concluem que tanto a expressão da proteína p53 como a expressão do VEGF não se correlacionaram com o prognóstico em pacientes com CEE em estágios II e III.

Identificação e fatores de virulência de Candida spp isoladas da cavidade bucal de transplantados renais do hospital universitário Onofre Lopes em Natal-RN

Despite Candida species are often human commensals isolated from various oral sites such as: tongue, cheek and palatal mucosa plus subgingival region, there are some properties linked to the organism commonly known as virulence factors which confer them the ability to produce disease. Oral candidiasis is one of the main oral manifestations reported in literature related to kidney transplant patients. The objectives of the present study were to identify and investigate virulence factors of yeasts isolated from the oral cavity of kidney transplant recipients admitted at the Hospital Universitário Onofre Lopes, in Natal RN. Seventy Candida species isolated from 111 kidney transplant recipients were investigated in this study. Identification of the isolates was performed by using the evidence of germ tube formation, hypertonic broth, tolerance to grow at 42°C, micromorphology and biochemical profiles. We observed a high rate of isolation of yeasts from the oral cavity of kidney transplant recipients (63.1%) being C. albicans was the most prevalent species. Oral candidiasis was diagnosed in 14.4% of transplant recipients. We evaluated virulence properties of the isolates regarding to: biofilm formation on polystyrene microplates as well as XTT reduction, adherence to acrylic resin and human buccal epithelial cells and proteinase activity. Most isolates were able to form biofilm by the method of adhesion to polystyrene. All isolates of Candida spp. remained viable during biofilm formation when analyzed by the method of XTT reduction. The number of CFU attached to the acrylic resin suggested high adherence for C. parapsilosis. C. albicans isolates showed higher median adherence to human buccal epithelial cells than non-C. albicans Candida isolates. Nevertheless, this difference was not statistically significant. C. dubliniensis showed low ability to adhere to plastic and epithelial cells and biofilm formation. Proteolytic activity was observed for all the isolates investigated, including the unique isolate of C. dubliniensis. There was a statistically significant association between proteinase production and the presence of oral candidiasis. Studies related to oral candidiasis in renal transplant recipients are limited to clinical and epidemiological data, but investigations concerning Candida spp. virulence factor for this group of individuals are still scarce. We emphasize the importance of studies related to virulence factors of yeasts isolated from this population to contribute to the knowledge of microbiological aspects of oral candidiasis

Análise do perfil de expressão dos genes da cana-de-açúcar envolvidos na interação com Leifsonia xyli subsp: xyli

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Adherence and intracellular parasitism of Paracoccidioides brasiliensis in Vero cells

Paracoccidioides brasiliensis is a dimorphic fungus known to produce invasive systemic disease in humans. The 43-kDa glycoprotein of P, brasiliensis is the major diagnostic antigen of paracoccidioidomycosis and may act as a virulence factor, since it is a receptor for laminin. Very little is known about early interact-ions between this fungus and the host cells, so we developed in vitro a model system employing cultured mammalian cells (Vero cells), in order to investigate the factors and virulence mechanisms of P. brasiliensis related to the adhesion and invasion process. We found that there is a permanent interaction after 30 min of contact between the fungus and the cells. The yeasts multiply in the cells for between 5 and 24 h. Different strains of P, brasiliensis were compared, and strain 18 thigh virulence) was the most strongly adherent, followed by strain 113 (virulent), 265 (considered of low virulence) and 113M(mutant obtained by ultraviolet radiation, deficient in gp43). P. brasiliensis adhered to the epithelial cells by a narrow tube, while depressions were noticed in the cell surface, suggesting an active cavitation process. An inhibition assay was performed and it was verified that anti-gp43 serum and a pool of sera from individuals with paracoccidioidomycosis were able to inhibit the adhesion of P. brasiliensis to the Vero cells. Glycoprotein 43 (gp43) antiserum abolished 85 % of the binding activity of P. brasiliensis. This fungus can also invade the Vero cells, and intraepithelial parasitism could be an escape mechanism in paracoccidioidomycosis. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

In vitro adherence of Candida albicans isolated from patients with chronic periodontitis

Adherence is considered an extremely important virulence factor in yeast. Objective: The aim of this study was to analyze the adherence to epithelial cells of C. albicans isolated from patients with chronic periodontitis in comparison to healthy patients. Material and methods: Candida albicans cells isolated from individuals with chronic periodontitis (n=25) and healthy controls (n=25) were included in this study. Suspensions of C. albicans (10(6) cells/rnL) and epithelial cells (10(5) cells/mL) were mixed and incubated at 37 degrees C for 1 h. The number of yeasts adhered to 25 epithelial cells was counted. Results: The number of C. albicans cells adhered to epithelial cells was statistically higher in the chronic periodontitis group than in the control group (Student's t-test, p=0.000). Conclusion: The results of the present study suggest a higher Candida adherence of samples isolated from patients with chronic periodontitis.

Caracterização fenotípica de leveduras isoladas da mucosa vaginal em mulheres adultas

OBJETIVO: caracterizar fenotipicamente leveduras isoladas do conteúdo vaginal de 223 mulheres adultas, sintomáticas (S) e assintomáticas (A) para vulvovaginite, e determinar os indicadores clínicos que possivelmente levam ao surgimento de sinais e sintomas relacionados ao acometimento da mucosa por essa patologia. MÉTODOS: inicialmente foi aplicado um questionário, com questões abertas e fechadas, sobre dados clínicos epidemiológicos. Logo, ocorreu o diagnóstico micológico com semeadura em meio Chrom Agar Candida, identificação micromorfológica e bioquímica. Métodos específicos para detecção de fatores de virulência, proteinase e fosfolipase foram empregados. A análise estatística das variáveis foi estabelecida utilizando os testes χ2 e χ2 de Pearson. RESULTADOS: Candida albicans foi a espécie mais prevalente (87%, S e 67%, A), seguida de Candida glabrata (4%, S e 17%, A). O número de mulheres que referiram adoção de anticoncepcionais foi mais alto entre as sintomáticas, 77%. Nos dois grupos estudados, em torno de 87% apresentaram ciclos menstruais regulares, 57% das mulheres eram casadas com idade entre 30 a 40 anos. em relação a práticas sexuais, houve para parte das pacientes, concomitância entre os hábitos, anal, oral e vaginal. em relação à fosfolipase, apenas Candida albicans produziu este fator de virulência em 37,5%. A proteinase foi detectada em Candida albicans, Candida glabrata e Candida parapsilosis. Esse último fator de virulência esteve associado, principalmente, a isolados de pacientes sintomáticas. CONCLUSÕES: a colonização e infecção da mucosa vaginal por levedura é real com diversas espécies de Candida presentes. No entanto, Candida albicans se destaca como espécie prevalente em mucosa vaginal de mulheres adultas. Fica evidente a emergência de espécies de Candida não albicans, algumas com resistência intrínseca aos azólicos, tais como Candida glabrata, Candida parapsilosis, Candida tropicalis, e Candida guillermondii, o que pode ser explicado pelo uso inadequado de medicamentos e tratamento empírico.

Niche-specific association of Aeromonas ribotypes from human and environmental origin

A total of 88 Aeromonas isolates from distinct locations and sources (39 from extraintestinal infections, 31 from diarrhoeic, ten from non-diarrhoeic faeces, all human, and eight from fresh water) were subjected to phenospecies identification, serotyping, ribotyping and detection of some virulence markers. The strains belonged to four different phenospecies marked by 19 O serogroups and 38 ribotypes. No strong correlation between these parameters was found, and no group, as defined by the typing methods, could be characterized with a particular set of virulence markers. There was a clear association of ribotypes with the source of the strains. Cluster analysis allowed the identification of a complex of ribotypes belonging to distinct but related sources, including clinical and environmental isolates. These results suggest that ribotyping may be an epidemiological tool suitable for the study of Aeromonas infections.

Importância de Yersinia enterocolitica em microbiologia médica

Y. enterocolitica is a human invasive enteropathogen which causes a number of intestinal and extraintestinal clinical symptoms of various degrees of severity, ranging from mild gastroenteritis to mesenteric lymphadenitis, which mimics appendicitis and in rare cases can evolve to septicemia. Infection by Y. enterocolitica can also lead to post-infection immunological sequelae including arthritis, erythema nodosum and glomerulonephritis. Pathogenic Y. enterocolitica strains have traditionally been linked to specific biotypes and serogroups and associated to a variety of phenotypic characteristics related to virulence. Molecular genetics studies have pointed to the importance of the pYV virulence plasmid, which encodes various virulence genes, as well that of specific chromosomal virulence genes, in determining the pathogenesis of this bacterium. Intestinal infections by Y. enterocolitica are mostly self-limiting and usually do not need an antibiotic treatment. The occurrence of this microorganism is not as frequently described in Brazil as it is in other countries, such as Japan, USA and many European countries. This review focuses on the general characteristics, pathogenesis, clinical symptoms, virulence characteristics, treatment and antibiotic susceptibility of Yersinia enterocolitica strains isolated in Brazil and around the world.

Detecção da produção de slime por estafilococos coagulase-negativa isolados de cateter venoso central

Slime production is an important virulence factor of coagulase-negative Staphylococcus spp., allowing them to attach to smooth surfaces of biomaterials, and it has been associated with infections of implanted medical devices. In the present study the production of slime capsules in 27 strains of coagulase-negative Staphylococcus was investigated by culture in Congo Red agar (77.7% positivity), spectrophotometric or microplate method (81.4% positivity) and scanning electron microscopy (88.9% positivity). The resistance of coagulase-negative strains of Staphylococcus to various antimicrobial agents was also determined by agar disk diffusion. The proportion of strains resistant to penicillin G, oxacillin, erythromycin, clindamycin and gentamicin among the slime-producing staphylococci was 88.9%, 70.4%, 81.5%, 66.7% and 59.2%, respectively; all of the coagulase-negative staphylococci were susceptible to vancomycin. The strains isolated from central venous catheters were identified by a conventional method and the API Staph system. The 27 coagulase-negative Staphylococcus strains were identified as: S. saprophyticus (3.7%), S. xylosus (7.4%), S. haemolyticus (14.8%), S. epidermidis (37.0%), S. warneri (14.8%), S. lugdunensis (7.4%), S. hominis (7.4%), S. schleiferi (3.7%) and S. chromogenes (3.7%). It can be concluded that in the most of the coagulase-negative Staphylococcus species there was an association between slime production, the nosocomial origin of the strains and reduced sensitivity to the antibiotics, suggesting a pathogenic potential in the hospital environment.