Genes associados à virulência e multirresistência de antimicrobianos em linhagens Trueperella Pyogenes isoladas de mastite e outras afecções em animais domésticos

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

Conformational polymorphism of the antidiabetic drug chlorpropamide

In this paper, the main features of Raman spectroscopy, one of the first choice methods in the study of polymorphism in pharmaceuticals, are presented taking chlorpropamide as a case of study. The antidiabetic drug chlorpropamide (1-[4-chlorobenzenesulphonyl]-3-propyl urea), which belongs to the sulfonylurea class, is known to exhibit, at least, six polymorphic phases. These forms are characterized not only by variations in their molecular packing but also in their molecular conformation. In this study, the polymorphism of chlorpropamide is discussed on the basis of Raman scattering measurements and quantum mechanical calculations. The main spectroscopic features that fingerprint the crystalline forms are correlated with the corresponding crystalline structures. Using a theoretical approach on the energy dependence of the conformers, simulated molecular torsion angles are plotted versus the formation energy, which provides a satisfactory agreement between the torsion angles at the energy minima and the experimental values observed in the different solid forms of chlorpropamide. Copyright (C) 2011 John Wiley & Sons, Ltd.

US Food and Drug Administration approval of generic versions of complex biologics: implications for the practicing physician using low molecular weight heparins

Low-molecular-weight heparins (LMWHs) have shown equivalent or superior efficacy and safety to unfractionated heparin as antithrombotic therapy for patients with acute coronary syndromes. Each approved LMWH is a pleotropic biological agent with a unique chemical, biochemical, biophysical and biological profile and displays different pharmacodynamic and pharmacokinetic profiles. As a result, LMWHs are neither equipotent in preclinical assays nor equivalent in terms of their clinical efficacy and safety. Previously, the US Food and Drug Administration (FDA) cautioned against using various LMWHs interchangeably, however recently, the FDA approved generic versions of LMWH that have not been tested in large clinical trials. This paper highlights the bio-chemical and pharmacological differences between the LMWH preparations that may result in different clinical outcomes, and also reviews the implications and challenges physicians face when generic versions of the original/innovator agents are approved for clinical use.

The role of myocardial scintigraphy in the assessment of cardiovascular risk in patients with end-stage chronic kidney disease on the waiting list for renal transplantation

The usefulness of stress myocardial perfusion scintigraphy for cardiovascular (CV) risk stratification in chronic kidney disease remains controversial. We tested the hypothesis that different clinical risk profiles influence the test. We assessed the prognostic value of myocardial scintigraphy in 892 consecutive renal transplant candidates classified into four risk groups: very high (aged epsilon 50 years, diabetes and CV disease), high (two factors), intermediate (one factor) and low (no factor). The incidence of CV events and death was 20 and 18, respectively (median follow-up 22 months). Altered stress testing was associated with an increased probability of cardiovascular events only in intermediate-risk (one risk factor) patients [30.3 versus 10, hazard ratio (HR) 2.37, confidence interval (CI) 1.693.33, P 0.0001]. Low-risk patients did well regardless of scan results. In patients with two or three risk factors, an altered stress test did not add to the already increased CV risk. Myocardial scintigraphy was related to overall mortality only in intermediate-risk patients (HR 2.8, CI 1.55.1, P 0.007). CV risk stratification based on myocardial stress testing is useful only in patients with just one risk factor. Screening may avoid unnecessary testing in 60 of patients, help stratifying for risk of events and provide an explanation for the inconsistent performance of myocardial scintigraphy.

Validation of the Subjective Handicap of Epilepsy (SHE) in Brazilian patients with epilepsy

The objectives of the study were to translate and adapt the Subjective Handicap of Epilepsy (SHE) instrument to Brazilian Portuguese and to determine its psychometric properties for the evaluation of quality of life in patients with epilepsy. A sample of 448 adult patients with epilepsy with different clinical profiles (investigation, preoperative period, postoperative period, and drug treatment follow-up) was evaluated with the SHE and the Epilepsy Surgery Inventory (ESI-55). Exploratory factorial analysis demonstrated that four factors explained 60.47% of the variance and were sensitive to discriminate the different clinical groups, with the preoperative group having the poorest quality of life. Internal consistency ranged from 0.92 to 0.96, and concurrent validity with the ESI-55 was moderate/strong (0.32-0.70). Test-retest reliability was confirmed, with an ICC value of 0.54 (2 days), 0.91 (7 days), and 0.97 (30 days). The SHE had satisfactory psychometric qualities for use in the Brazilian population, similar to those of the original version. The instrument seems to be more adequate in psychometric terms for the postoperative and drug treatment follow-up groups, and its use should be encouraged. (c) 2012 Elsevier Inc. All rights reserved.

Diagnostic utility of immunohistochemistry in distinguishing trichoepithelioma and basal cell carcinoma: evaluation using tissue microarray samples

Trichoepithelioma is a benign neoplasm that shares both clinical and histological features with basal cell carcinoma. It is important to distinguish these neoplasms because they require different clinical behavior and therapeutic planning. Many studies have addressed the use of immunohistochemistry to improve the differential diagnosis of these tumors. These studies present conflicting results when addressing the same markers, probably owing to the small number of basaloid tumors that comprised their studies, which generally did not exceed 50 cases. We built a tissue microarray with 162 trichoepithelioma and 328 basal cell carcinoma biopsies and tested a panel of immune markers composed of CD34, CD10, epithelial membrane antigen, Bcl-2, cytokeratins 15 and 20 and D2-40. The results were analyzed using multiple linear and logistic regression models. This analysis revealed a model that could differentiate trichoepithelioma from basal cell carcinoma in 36% of the cases. The panel of immunohistochemical markers required to differentiate between these tumors was composed of CD10, cytokeratin 15, cytokeratin 20 and D2-40. The results obtained in this work were generated from a large number of biopsies and resulted in the confirmation of overlapping epithelial and stromal immunohistochemical profiles from these basaloid tumors. The results also corroborate the point of view that trichoepithelioma and basal cell carcinoma tumors represent two different points in the differentiation of a single cell type. Despite the use of panels of immune markers, histopathological criteria associated with clinical data certainly remain the best guideline for the differential diagnosis of trichoepithelioma and basal cell carcinoma. Modern Pathology (2012) 25, 1345-1353; doi: 10.1038/modpathol.2012.96; published online 8 June 2012

Expression Analysis of Wound Healing Genes in Human Periapical Granulomas of Progressive and Stable Nature

Introduction: Wound healing process involves the activation of extracellular matrix components, remodeling enzymes, cellular adhesion molecules, growth factors, cytokines and chemokines genes. However, the molecular patterns underlying the healing process periapical environment remain unclear. Here we hypothesized that endodontic infection might result in an imbalance in the expression of wound healing genes involved in the pathogenesis of periapical lesions. Furthermore, we suggest that differential expression of wound healing markers in active and latent granulomas could account for different clinical outcomes for such lesions. Methods: Study samples consisted of 93 periapical granulomas collected after endodontic surgeries and 24 healthy periodontal ligament tissues collected from premolars extracted for orthodontic purposes as control samples. Of these, 10 periapical granulomas and 5 healthy periapical tissues were used for expression analysis of 84 wound healing genes by using a pathway-specific real-time polymerase chain reaction array. The remaining 83 granulomas and all 24 control specimens were used to validate the obtained array data by real-time polymerase chain reaction. Observed variations in expression of wound healing genes were analyzed according to the classification of periapical granulomas as active/progressive versus inactive/stable (as determined by receptor activator for nuclear factor kappa B ligand/osteoprotegerin expression ratio). Results: We observed a marked increase of 5-fold or greater in SERPINE1, TIMP1, COL1A1, COL5A1, VTN, CTGF, FGF7, TGFB1, TNF, CXCL11, ITGA4, and ITGA5 genes in the periapical granulomas when compared with control samples. SERPINE1, TIMP1, COL1A1, TGFB1, and ITGA4 mRNA expression was significantly higher in inactive compared with active periapical granulomas (P < .001), whereas TNF and CXCL11 mRNA expression was higher in active lesions (P < .001). Conclusions: The identification of novel gene targets that curb the progression status of periapical lesions might contribute to a more accurate diagnosis and lead to treatment modalities more conducive to endodontic success. (J Endod 2012;38:185-190)

Deficient Regulatory T Cell Activity and Low Frequency of IL-17-Producing T Cells Correlate with the Extent of Cardiomyopathy in Human Chagas' Disease

Background: Myocardium damage during Chagas' disease results from the immunological imbalance between pro-and production of anti-inflammatory cytokines and has been explained based on the Th1-Th2 dichotomy and regulatory T cell activity. Recently, we demonstrated that IL-17 produced during experimental T. cruzi infection regulates Th1 cells differentiation and parasite induced myocarditis. Here, we investigated the role of IL-17 and regulatory T cell during human Chagas' disease. Methodology/Principal Findings: First, we observed CD4(+)IL-17(+) T cells in culture of peripheral blood mononuclear cells (PBMC) from Chagas' disease patients and we evaluated Th1, Th2, Th17 cytokine profile production in the PBMC cells from Chagas' disease patients (cardiomyopathy-free, and with mild, moderate or severe cardiomyopathy) cultured with T. cruzi antigen. Cultures of PBMC from patients with moderate and severe cardiomyopathy produced high levels of TNF-alpha, IFN-gamma and low levels of IL-10, when compared to mild cardiomyopathy or cardiomyopathy-free patients. Flow cytometry analysis showed higher CD4(+)IL-17(+) cells in PBMC cultured from patients without or with mild cardiomyopathy, in comparison to patients with moderate or severe cardiomyopathy. We then analyzed the presence and function of regulatory T cells in all patients. All groups of Chagas' disease patients presented the same frequency of CD4(+)CD25(+) regulatory T cells. However, CD4(+)CD25(+) T cells from patients with mild cardiomyopathy or cardiomyopathy-free showed higher suppressive activity than those with moderate and severe cardiomyopathy. IFN-gamma levels during chronic Chagas' disease are inversely correlated to the LVEF (P = 0.007, r = -0.614), while regulatory T cell activity is directly correlated with LVEF (P = 0.022, r = 0.500). Conclusion/Significance: These results indicate that reduced production of the cytokines IL-10 and IL-17 in association with high levels of IFN-gamma and TNF-alpha is correlated with the severity of the Chagas' disease cardiomyopathy, and the immunological imbalance observed may be causally related with deficient suppressor activity of regulatory T cells that controls myocardial inflammation.

Genetic diversity of Leishmania amazonensis strains isolated in northeastern Brazil as revealed by DNA sequencing, PCR-based analyses and molecular karyotyping

Abstract Background Leishmania (Leishmania) amazonensis infection in man results in a clinical spectrum of disease manifestations ranging from cutaneous to mucosal or visceral involvement. In the present study, we have investigated the genetic variability of 18 L. amazonensis strains isolated in northeastern Brazil from patients with different clinical manifestations of leishmaniasis. Parasite DNA was analyzed by sequencing of the ITS flanking the 5.8 S subunit of the ribosomal RNA genes, by RAPD and SSR-PCR and by PFGE followed by hybridization with gene-specific probes. Results ITS sequencing and PCR-based methods revealed genetic heterogeneity among the L. amazonensis isolates examined and molecular karyotyping also showed variation in the chromosome size of different isolates. Unrooted genetic trees separated strains into different groups. Conclusion These results indicate that L. amazonensis strains isolated from leishmaniasis patients from northeastern Brazil are genetically diverse, however, no correlation between genetic polymorphism and phenotype were found.

Periodontal disease and diabetes mellitus

Periodontal disease (PD) is one of the most commonly known human chronic disorders. The relationship between PD and several systemic diseases such as diabetes mellitus (DM) has been increasingly recognized over the past decades. Objective The purpose of this review is to provide the reader with knowledge concerning the relationship between PD and DM. Many articles have been published in the English and Portuguese literature over the last 50 years examining the relationship between these two chronic diseases. Data interpretation is often confounded by varying definitions of DM, PD and different clinical criteria were applied to determine the prevalence, extent and severity of PD, levels of glycemic control and diabetes-related complications. Methods This paper provides a broad overview of the predominant findings from research conducted using the BBO (Bibliografia Brasileira de Odontologia), MEDLINE, LILACS and PubMed for Controlled Trials databases, in English and Portuguese languages published from 1960 to October 2012. Primary research reports on investigations of relationships between DM/DM control, PD/periodontal treatment and PD/DM/diabetes-related complications identified relevant papers and meta-analyses published in this period. Results 7This paper describes the relationship between PD and DM and answers the following questions: 1- The effect of DM on PD, 2- The effects of glycemic control on PD and 3- The effects of PD on glycemic control and on diabetes-related complications. Conclusions The scientific evidence reviewed supports diabetes having an adverse effect on periodontal health and PD having an adverse effect on glycemic control and on diabetes-related complications. Further research is needed to clarify these relationships and larger, prospective, controlled trials with ethnically diverse populations are warranted to establish that treating PD can positively influence glycemic control and possibly reduce the burden of diabetes-related complications.

Airway disease: similarities and differences between asthma, COPD and bronchiectasis

Airway diseases are highly prevalent worldwide; however, the prevalence of these diseases is underestimated. Although these diseases present several common characteristics, they have different clinical outcomes. The differentiation between asthma, chronic obstructive pulmonary disease and bronchiectasis in the early stage of disease is extremely important for the adoption of appropriate therapeutic measures. However, because of the high prevalence of these diseases and the common pathophysiological pathways, some patients with different diseases may present with similar symptoms. The objective of this review is to highlight the similarities and differences between these diseases in terms of the risk factors, pathophysiology, symptoms, diagnosis and treatment.

Identification of Urban Leprosy Clusters

Overpopulation of urban areas results from constant migrations that cause disordered urban growth, constituting clusters defined as sets of people or activities concentrated in relatively small physical spaces that often involve precarious conditions. Aim. Using residential grouping, the aim was to identify possible clusters of individuals in São José do Rio Preto, Sao Paulo, Brazil, who have or have had leprosy. Methods. A population-based, descriptive, ecological study using the MapInfo and CrimeStat techniques, geoprocessing, and space-time analysis evaluated the location of 425 people treated for leprosy between 1998 and 2010. Clusters were defined as concentrations of at least 8 people with leprosy; a distance of up to 300 meters between residences was adopted. Additionally, the year of starting treatment and the clinical forms of the disease were analyzed. Results. Ninety-eight (23.1%) of 425 geocoded cases were located within one of ten clusters identified in this study, and 129 cases (30.3%) were in the region of a second-order cluster, an area considered of high risk for the disease. Conclusion.This study identified ten clusters of leprosy cases in the city and identified an area of high risk for the appearance of new cases of the disease.

Le figure femminili all'ombra di Cosa Nostra: la rappresentazione della donna mafiosa siciliana sulla stampa italiana del secondo dopoguerra

This thesis investigates the representation of Sicilian mafia women on the Italian press after the second postwar, in particular by examining three Italian newspapers, Il Corriere della Sera, L’Ora and Il Giornale di Sicilia. The focus is on the 1963 – 1983 twenty-year period, after which there has been a big change in the world of Sicilian crime organization with the phenomenon of pentitismo that changes a lot of thing in the whole universe of mafia. In this research there are two aspects very different but very central at the same time. On one hand the careful counting of quoted newspapers and the filing of the articles about general mafia and mafia women, which rendered a whole database about the interest for these woman figures founded in print, in a historical period never analyzed from this point of view; on the other hand the interpretation of these articles and the different representation forms. The founded material was compared with the cultural Italian history of that period to understand if there was a difference between general woman perception and that of mafia woman and if there was also an iconographic prototype of southern woman which it is possible to apply on photographs and descriptions. In fact the most important result of the thesis is to underline that, in front of a woman who belongs to mafia context, the Italia press represented the female gender as first and the mafia gender as the second one.

Alterazioni dell'immunità umorale nei pazienti con sarcoma di Kaposi classico: caratterizzazione dei linfociti B circolanti e delle loro sottopopolazioni

Objectives: Human Herpesvirus 8 (HHV-8) is the etiological agent of Kaposi’s Sarcoma (KS) and it is also associated with two B cell lymphoproliferative diseases: primary effusion lymphoma (PEL), and the plasmablastic form of multicentric Castelman’s disease (MCD). HHV-8 establishes persistent infection in the host with tropism for multiple cell types. In KS patients, the virus is found in tumor-spindle cells, peripheral blood monocytes, endothelial progenitor circulating cells, T and B lymphocytes. Peripheral B cells represent one of the major virus reservoir, but the consequences of HHV-8 infection of these cells have been poorly characterized. Therefore, in this study the frequency, the immunophenotypic profile and the functional activity of different peripheral B cell subsets in patients with classic KS (cKS) was analysed in order to identify potential alterations of these cells. The classic variant of KS is ideal to perform such studies, as it lacks confounding factors such as HIV or EBV infection and immunosuppression. Methods: Whole-blood samples from patients with the classical form of KS (cKS) (n=62) and healthy age and sex-matched seronegative controls (HSN) (n=43) were analyzed by multiparametric flow-cytometry to determine the frequency of B cells and their subpopulations, as well as their surface expression of immunoglobulins and activation markers. Results: The frequency of circulating B cells was significantly higher in cKS patients than in controls. In particular, the analysis of the B cell subsets revealed a higher frequency of naïve B cells (CD19+CD27-), among which transitional CD19+CD38highCD5+ and pre-naïve (CD27-CD38intCD5+ ) B cells demonstrated an expansion. Memory B cells (CD19+CD27+) did not differ between the two study groups, except from a higher frequency of CD19+CD27+IgM+IgD+ B cells, the typical phenotype of marginal zone (MZ) B cells, in cKS patients. The characterization of membrane surface activation markers showed lower levels of the activation marker HLA-DR only on CD27- B cells, while CD80 and CD86 were less represented in all the the B cells from cKS patients. Moreover, B cells from cKS patients were smaller and with less granules than the ones from controls. Conclusion: Taken together, these results clearly indicate that circulating B cells are altered in patients with cKS, showing an expansion of the immature phenotypes. These B cell alterations may be due to an indirect viral effect rather than to a direct one: the cytokines expressed in the microenvironment typical of cKS may cause a faster release of immature cells from the bone marrow and a lower grade of peripheral differentiation, as already suggested for other chronic viral infections such as HIV and HCV. Further studies will be necessary to understand how these alterations contribute to the pathogenesis of KS and, eventually, to the different clinical evolution of the disease.

Reverse genetic studies of Benyvirus - Polymyxa betae molecular interaction: Role of the RNA4-encoded protein in virus transmission

Beet necrotic yellow vein virus (BNYVV), the leading infectious agent that affects sugar beet, is included within viruses transmitted through the soil from plasmodiophorid as Polymyxa betae. BNYVV is the causal agent of Rhizomania, which induces abnormal rootlet proliferation and is widespread in the sugar beet growing areas in Europe, Asia and America; for review see (Peltier et al., 2008). In this latter continent, Beet soil-borne mosaic virus (BSBMV) has been identified (Lee et al., 2001) and belongs to the benyvirus genus together with BNYVV, both vectored by P. betae. BSBMV is widely distributed only in the United States and it has not been reported yet in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs evidenced similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSBMV and BNYVV in two different species (Rush et al., 2003). Benyviruses field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs -1 contains a single long ORF encoding polypeptide that shares amino acid homology with known viral RNA-dependent RNA polymerases (RdRp) and helicases. RNAs -2 contains six ORFs: capsid protein (CP), one readthrough protein, triple gene block proteins (TGB) that are required for cell-to-cell virus movement and the sixth 14 kDa ORF is a post-translation gene silencing suppressor. RNAs -3 is involved on disease symptoms and is essential for virus systemic movement. BSBMV RNA-3 can be trans-replicated, trans-encapsidated by the BNYVV helper strain (RNA-1 and -2) (Ratti et al., 2009). BNYVV RNA-4 encoded one 31 kDa protein and is essential for vector interactions and virus transmission by P. betae (Rahim et al., 2007). BNYVV RNA-5 encoded 26 kDa protein that improve virus infections and accumulation in the hosts. We are interest on BSBMV effect on Rhizomania studies using powerful tools as full-length infectious cDNA clones. B-type full-length infectious cDNA clones are available (Quillet et al., 1989) as well as A/P-type RNA-3, -4 and -5 from BNYVV (unpublished). A-type BNYVV full-length clones are also available, but RNA-1 cDNA clone still need to be modified. During the PhD program, we start production of BSBMV full-length cDNA clones and we investigate molecular interactions between plant and Benyviruses exploiting biological, epidemiological and molecular similarities/divergences between BSBMV and BNYVV. During my PhD researchrs we obtained full length infectious cDNA clones of BSBMV RNA-1 and -2 and we demonstrate that they transcripts are replicated and packaged in planta and able to substitute BNYVV RNA-1 or RNA-2 in a chimeric viral progeny (BSBMV RNA-1 + BNYVV RNA-2 or BNYVV RNA-1 + BSBMV RNA-2). During BSBMV full-length cDNA clones production, unexpected 1,730 nts long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil. Sequence analysis of the new BSBMV RNA-4 form revealed high identity (~100%) with published version of BSBMV RNA-4 sequence (NC_003508) between nucleotides 1-608 and 1,138-1,730, however the new form shows 528 additionally nucleotides between positions 608-1,138 (FJ424610). Two putative ORFs has been identified, the first one (nucleotides 383 to 1,234), encode a protein with predicted mass of 32 kDa (p32) and the second one (nucleotides 885 to 1,244) express an expected product of 13 kDa (p13). As for BSBMV RNA-3 (Ratti et al., 2009), full-length BSBMV RNA-4 cDNA clone permitted to obtain infectious transcripts that BNYVV viral machinery (Stras12) is able to replicate and to encapsidate in planta. Moreover, we demonstrated that BSBMV RNA-4 can substitute BNYVV RNA-4 for an efficient transmission through the vector P. betae in Beta vulgaris plants, demonstrating a very high correlation between BNYVV and BSBMV. At the same time, using BNYVV helper strain, we studied BSBMV RNA-4’s protein expression in planta. We associated a local necrotic lesions phenotype to the p32 protein expression onto mechanically inoculated C. quinoa. Flag or GFP-tagged sequences of p32 and p13 have been expressed in viral context, using Rep3 replicons, based on BNYVV RNA-3. Western blot analyses of local lesions contents, using FLAG-specific antibody, revealed a high molecular weight protein, which suggest either a strong interaction of BSBMV RNA4’s protein with host protein(s) or post translational modifications. GFP-fusion sequences permitted the subcellular localization of BSBMV RNA4’s proteins. Moreover we demonstrated the absence of self-activation domains on p32 by yeast two hybrid system approaches. We also confirmed that p32 protein is essential for virus transmission by P. betae using BNYVV helper strain and BNYVV RNA-3 and we investigated its role by the use of different deleted forms of p32 protein. Serial mechanical inoculation of wild-type BSBMV on C. quinoa plants were performed every 7 days. Deleted form of BSBMV RNA-4 (1298 bp) appeared after 14 passages and its sequence analysis shows deletion of 433 nucleotides between positions 611 and 1044 of RNA-4 new form. We demonstrated that this deleted form can’t support transmission by P. betae using BNYVV helper strain and BNYVV RNA-3, moreover we confirmed our hypothesis that BSBMV RNA-4 described by Lee et al. (2001) is a deleted form. Interesting after 21 passages we identifed one chimeric form of BSBMV RNA-4 and BSBMV RNA-3 (1146 bp). Two putative ORFs has been identified on its sequence, the first one (nucleotides 383 to 562), encode a protein with predicted mass of 7 kDa (p7), corresponding to the N-terminal of p32 protein encoded by BSBMV RNA-4; the second one (nucleotides 562 to 789) express an expected product of 9 kDa (p9) corresponding to the C-terminal of p29 encoded by BSBMV RNA-3. Results obtained by our research in this topic opened new research lines that our laboratories will develop in a closely future. In particular BSBMV p32 and its mutated forms will be used to identify factors, as host or vector protein(s), involved in the virus transmission through P. betae. The new results could allow selection or production of sugar beet plants able to prevent virus transmission then able to reduce viral inoculum in the soil.