Modulatory effects of tuberculosis vaccines on experimental autoimmune encephalomyelitis

Although BCG is the only accepted vaccine against tuberculosis (TB), its protective ability is very limited. Therefore, many new vaccines are being evaluated. Our group has been working on DNAhsp65 - a genetic construction containing the hsp65 gene from Mycobacterium leprae. In previous experimental works, we demonstrated that both DNAhsp65 alone or associated with BCG, in a prime-boost regimen, were effective to control TB. A possible deleterious effect related to autoimmunity needed to be tested because hsp65 is highly homologous to the correspondent mammalian protein. In this investigation we tested the effect of a previous immunization with DNAhsp65 alone or associated with BCG in a rat model of multiple sclerosis. Female Lewis rats were immunized with three doses of DNAhsp65 or primed with BCG followed by two DNAhsp65 boosters. The animals were, then, immunized with myelin associated with complete Freund's adjuvant to develop experimental autoimmune encephalomyelitis (EAE). The following parameters were evaluated: weight loss, clinical score, central nervous system (CNS) inflammation and anti-myelin immune response. No deleterious effect was associated with these immunizations schedules. Immunized animals equally lost weight, the clinical scores were similar and CNS inflammation did not increase. Interestingly, both procedures determined decreased inflammation in the brain and lumbar spinal cord. This was concurrent with a modulatory effect over cytokine production by peripheral lymphoid organs. Cell cultures from spleen and lymph nodes in vitro stimulated with myelin produced less IFN-gamma and IL-10, respectively. This phenomenon was more clear in rats immunized with the genetic vaccine alone than with the prime-boost strategy. Together the results suggest that these strategies for TB prophylaxis would not accelerate or aggravate multiple sclerosis, being therefore, safe in this aspect. In addition, they indicate that these vaccination regimens have a potential anti-inflammatory activity that could be better explored in the future.

Immunization with pVAXhsp65 Decreases Inflammation and Modulates Immune Response in Experimental Encephalomyelitis

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

Experimental autoimmune encephalomyelitis evolution was not modified by multiple infections with Strongyloides venezuelensis

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

Mucosa-associated but not luminal Escherichia coli is augmented in Crohn's disease and ulcerative colitis

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

Isolation of Shiga toxigenic Escherichia coli from butcheries in Taquaritinga city, State of São Paulo, Brazil

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

Resistance to experimental autoimmune encephalomyelitis development in Lewis rats from a conventional animal facility

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the brain and spinal cord that is mediated by CD4+ T lymphocytes specific to myelin components. In this study we compared development of EAE in Lewis rats from two colonies, one kept in pathogen-free conditions (CEMIB colony) and the other (Botucatu colony) kept in a conventional animal facility. Female Lewis rats were immunized with 100 µl of an emulsion containing 50 µg of myelin, associated with incomplete Freund's adjuvant plus Mycobacterium butyricum. Animals were daily evaluated for clinical score and weight. CEMIB colony presented high EAE incidence with clinical scores that varied from three to four along with significant weight losses. A variable disease incidence was observed in the Botucatu colony with clinical scores not higher than one and no weight loss. Immunological and histopathological characteristics were also compared after 20 days of immunization. Significant amounts of IFN-gamma, TNF-alpha and IL-10 were induced by myelin in cultures from CEMIB animals but not from the Botucatu colony. Significantly higher levels of anti-myelin IgG1 were detected in the CEMIB colony. Clear histopathological differences were also found. Cervical spinal cord sections from CEMIB animals showed typical perivascular inflammatory foci whereas samples from the Botucatu colony showed a scanty inflammatory infiltration. Helminths were found in animals from Botucatu colony but not, as expected, in the CEMIB pathogen-free animals. As the animals maintained in a conventional animal facility developed a very discrete clinical, and histopathological EAE in comparison to the rats kept in pathogen-free conditions, we believe that environmental factors such as intestinal parasites could underlie this resistance to EAE development, supporting the applicability of the hygiene hypothesis to EAE.

Occurrence of non-O157 Shiga toxin-producing Escherichia coli in dogs with diarrhea

Escherichia coli Shiga toxigênica (STEC) e E. coli Attaching- effacing (AEEC) têm sido associadas à doença diarréica em cachorros. Entre janeiro e dezembro de 2006, 92 cepas de E. coli isoladas de 25 cachorros diarréicos foram examinadas. As cepas foram analisadas para a detecção dos genes produtores de Shiga toxina (stx 1 e stx 2) e da intimina (eae). Por meio de PCR foi observado que sete cepas (7,6%) portavam o gene stx 1, cinco cepas (5,4%) carregavam o gene stx 2 e nenhum cepa apresentou ambos os genes associados. Nove cepas de E. coli (9,8%) apresentaram o gene eae isoladamente. Treze das cepas (62,0%) que apresentaram os genes stx ou eae também apresentaram a produção de a hemolisina. As cepas que apresentaram genes de virulência foram também examinadas em relação à resistência a 12 agentes antimicrobianos. As resistências mais comuns foram para cefalotina (85,7%), estreptomicina (81,0%), amoxicilina (71,4%) e gentamicina (71,4%).

Analysis of Escherichia coli isolated from bovine mastitic milk

Rangel P. & Marin J.M. 2009. Analysis of Escherichia coli isolated from bovine mastitic milk. Pesquisa Veterinaria Brasileira 29(5): 363-368. Departamento de Morfologia, Estomatologia e Fisiologia, Faculdade de Odontologia de Ribeirao Preto, Universidade de São Paulo, Avenida do Cafe s/n, Campus USP, Ribeirao Preto, SP 14040-904, Brazil. E-mail: jmmarin@forp.usp.brMastitis has been recognized for some time as the most costly disease in dairy herds. From February to November 2004, 670 samples of bovine mastitic milk from which 231 Escherichia coli strains were isolated, were collected from two Brazilian states. The strains were screened for the presence of Shiga toxin-producing (stx 1 and stx 2) and intimin (eae) genes. Twenty (8.6%) strains were detected by PCR to harbor the Shiga toxin genes (8 the stx 1 gene, 12 the stx 2 gene and none both of them). Two (0.8%) of the Escherichia coli strains studied were eae positive non Shiga toxin-producing. The strains were also examined for resistance to 12 antimicrobial agents. The predominantly observed resistance was to tetracycline (92.2%), streptomycin (90.4%), nalidixic acid (88.3%), amikacin (86.5%) and cephalothin (84.8%). Multidrug resistance was found among 152 isolates (65.8%).

CLINICAL STUDY of NATURAL INFECTION BY Trypanosoma cruzi IN DOGS RESIDING IN A RURAL AREA IN MATO GROSSO do SUL STATE, BRAZIL

This study was carried out to describe the clinical characteristics of natural infection caused by Trypanosoma cruzi in dogs that reside in a rural area of Mato Grosso do Sul State, Brazil. Conventional and nonconventional diagnostic methods were used for screening T. cruzi infection in 75 dogs that lived in the area. Cardiovascular tests and biochemical examination of sera were also performed in four confirmed positive dogs. The following techniques were employed: indirect immunofluorescence test (IFAT), enzyme-linked immunosorbent assay with T. cruzi epimastigote antigens (EAE-ELISA) and enzyme-linked immunosorbent assay with T. cruzi excreted-secreted trypomastigote antigens (TESA-ELISA) with antibodies detected in 45.33% (n = 34), 24.0% (n = 18) and 12.0% (n = 9) of the dogs, respectively. The current prevalence of the infection was confirmed as 10.7% (n = 8) by immunoblotting test with T. cruzi excreted-secreted antigens (TESA-blot). The test that showed the best concordance index (Kappa; 0.93), sensitivity (100%) and specificity (98.5%) was TESA-ELISA, that when associated with IFAT had the same results as those obtained by TESA-blot (10.7%). Three out of the four chagasic animals showed enlarged cardiac silhouette on X-ray and an increase of the P-wave duration and QRS complex in electrocardiogram. Two dogs presented conduction disturbances, right bundle branch block in one dog and first-degree atrioventricular block and sinus arrest in another. The ecodopplercardiography presented left-ventricular-wall thickness increased during diastole, decrease of the shortening fraction and inversion in the speed peaks of the E and A waves, indicating the presence of systolic and diastolic disorders. The four animals showed enzymatic activities of creatine kinase (221-404 U/L), MB fraction of creatine kinase (189-304 U/L), elevated total proteins (7.6-10.2 g/dL) and total globulins (4.6-7.7g/dL) and reduction of albumin/globulin ratio, which suggested a myocardial injury and continuous antigenic stimulus.

Microinjection of histamine into the cerebellar vermis impairs emotional memory consolidation in mice

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

Atitudes em relação à Bioestatística de discentes e docentes da Faculdade de Ciências Farmacêuticas de Araraquara-UNESP

In this study, the attitudes of students and teachers at the Faculty of Pharmaceutical Sciences of Araraquara (UNESP University) towards Biostatistics were assessed. The Survey of Attitudes Toward Statistics (SATS) scale was used as the measuring instrument. The reproducibility of the scale was estimated by Cohen's Kappa (κ) coefficient with linear weighting and its internal consistency by Cronbach's alpha coefficient (α). The individuals were first placed in two groups, according to their positive or negative attitude toward Statistics; then, the association of their attitude with the variables of interest was tested by the chi-squared (χ 2) test at a significance level of 5%. The sample consisted of 272 undergraduate students, 83 graduate students and 24 teachers, predominantly female (78.2%). Among the students, 67.5% participated in the scientific research initial training program. Reproducibility and internal consistency of the scale were adequate (κ=0.7093; α=0.9334). Most of the subjects (74.4%) had a positive attitude toward Statistics. Significant association was found between attitude and functional activity (p=0.0204), the course taken (p=0.0316) and effort (p=0.0002). Thus, it was concluded that the great majority of the participants had a positive attitude towards Biostatistics and that undergraduate students and those who reported good performance in Biostatistics showed a significantly higher proportion of positive attitude than the other students.

Guía de evaluación ambiental estratégica

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Contamination of cattle carcasses by Escherichia coli shiga like toxin with high antimicrobials resistence

During processing of cattle carcasses, contamination may occurs with the transfer of microbiota of animals feaces to carcasses. This contamination many times may be by Escherichia coli carriers of virulence factor as stx and eae genes being classified as Shiga like toxin. Shiga toxin-producing Escherichia coli (STEC) is recognized wordwide as human pathogen. A survey was performed to determine the sensibility profile to several antimicrobial drugs of STEC in carcasses obtained from an abattoir in Brazil between March 2008 and August at 2009. A total of 120 STEC were isolated. All isolates were confirmed as being E. coli by their biochemical analysis and submitted to polymerase chain reaction (PCR) for detection of stx, eae and ehly genes. No strains was isolated being carriers of ehly gene. The number of isolates carriers of eae gene were 48/120. The most frequent resistance was seen against cephalothin (84.0%), streptomycin (45.0%), nalidixic acid (42.0%) and tetracycline (20.0%). Multidrug resistance (MDR) to three or more antimicrobial agents was observed in 46 (38.3%) E. coli isolates. The findings of STEC and MRD show that cattle carcasses may be a reservoir of pathogenic bacterial for the consumer public. © 2011 Academic Journals.

Dissection of the role of pili and type 2 and 3 secretion systems in adherence and biofilm formation of an atypical enteropathogenic escherichia coli strain

Atypical enteropathogenic Escherichia coli (aEPEC) strains are diarrheal pathogens that lack bundle-forming pilus production but possess the virulence-associated locus of enterocyte effacement. aEPEC strain 1551-2 produces localized adherence (LA) on HeLa cells; however, its isogenic intimin (eae) mutant produces a diffuse-adherence (DA) pattern. In this study, we aimed to identify the DA-associated adhesin of the 1551-2 eae mutant. Electron microscopy of 1551-2 identified rigid rod-like pili composed of an 18-kDa protein, which was identified as the major pilin subunit of type 1 pilus (T1P) by mass spectrometry analysis. Deletion of fimA in 1551-2 affected biofilm formation but had no effect on adherence properties. Analysis of secreted proteins in supernatants of this strain identified a 150-kDa protein corresponding to SslE, a type 2 secreted protein that was recently reported to be involved in biofilm formation of rabbit and human EPEC strains. However, neither adherence nor biofilm formation was affected in a 1551-2 sslE mutant. We then investigated the role of the EspA filament associated with the type 3 secretion system (T3SS) in DA by generating a double eae espA mutant. This strain was no longer adherent, strongly suggesting that the T3SS translocon is the DA adhesin. In agreement with these results, specific anti-EspA antibodies blocked adherence of the 1551-2 eae mutant. Our data support a role for intimin in LA, for the T3SS translocon in DA, and for T1P in biofilm formation, all of which may act in concert to facilitate host intestinal colonization by aEPEC strains. ©2013, American Society for Microbiology.

Efeito da vacina gênica para tuberculose(pVAXhsp65) na encefalite autoimune experiemtal

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