Deregulated expression of superoxide dismutase-2 correlates with different stages of cervical neoplasia

Objective: Superoxide dismutase-2 (SOD2) is considered one of the most important antioxidant enzymes that regulate cellular redox state in normal and tumorigenic cells. Overexpression of this enzyme may be involved in carcinogenesis, particularly in lung, gastric, colorectal and breast cancer. Methods: In the present study, we have evaluated SOD2 protein levels by immunohistochemistry (IHC) in 331 cervical histological samples including 31 low-grade cervical intraepithelial neoplasia (LSIL), 51 high-grade cervical intraepithelial neoplasia (HSIL), 197 squamous cervical carcinomas (SCC) and 52 cervical adenocarcinomas (ADENO). Results: We observed that SOD2 staining increases with cervical disease severity. Intense SOD2 staining was found in 13% of LSIL, 25.5% of HSIL and 40% of SCC. Moreover, 65.4% of ADENO exhibited intense SOD2 staining. Conclusions: Differences in the expression of SOD2 could potentially be used as a biomarker for the characterization of different stages of cervical disease.

High Prevalence of Clarithromycin Resistance and cagA, vacA, iceA2, and babA2 Genotypes of Helicobacter pylori in Brazilian Children

We isolated 45 Helicobacter pylori strains from 217 child patients. Resistance to clarithromycin, metronidazole, amoxicillin, and tetracycline was detected in 27%, 13%, 4%, and 0% of strains, respectively. The A2143G mutation was the most prevalent (67%) among clarithromycin-resistant strains. In addition, strain genotyping revealed a significant association between gastritis severity and the simultaneous presence of cagA, vacA s1m1, iceA2, and babA2 genes.

Immunohistochemical evaluation of macrophage activity and its relationship with apoptotic cell death in the polar forms of leprosy

The objective of the present study was to investigate the correlation between macrophage activity and apoptosis in the polar forms of leprosy because the immunopathological phenomena involved in these forms are still poorly understood For this purpose, 29 skin biopsy samples obtained from patients with the polar forms of leprosy were analyzed. Macrophage activity and apoptosis were evaluated by immunohistochemistry using lysozyme, CD68, iNOS and caspase 3 as markers The nonparametric Mann-Whitney test and Spearman`s linear correlation test were used for statistical analysis The results suggest that the apoptosis rate is under the direct influence of macrophage activity in lesions of patients with the tuberculoid form In contrast, in lepromatous lesions other factors seem to induce programmed cell death, possibly TGF-beta. Further studies are necessary to identify additional factors involved in the immunopathogenesis of leprosy. (C) 2010 Elsevier Ltd. All rights reserved

Characterization of leptospiral proteins that afford partial protection in hamsters against lethal challenge with Leptospira interrogans

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. The whole-genome sequence of Leptospira interrogans serovar Copenhageni together with bioinformatic tools allow us to search for novel antigen candidates suitable for improved vaccines against leptospirosis. This study focused on three genes encoding conserved hypothetical proteins predicted to be exported to the outer membrane. The genes were amplified by PCR from six predominant pathogenic serovars in Brazil. The genes were cloned and expressed in Escherichia coli strain BL21-SI using the expression vector pDEST17. The recombinant proteins tagged with N-terminal 6xHis were purified by metal-charged chromatography. The proteins were recognized by antibodies present in sera from hamsters that were experimentally infected. Immunization of hamsters followed by challenge with a lethal dose of a virulent strain of Leptospira showed that the recombinant protein rLIC12730 afforded statistically significant protection to animals (44 %), followed by rLIC10494 (40 %) and rLIC12922 (30 %). Immunization with these proteins produced an increase in antibody titres during subsequent boosters, suggesting the involvement of a T-helper 2 response. Although more studies are needed, these data suggest that rLIC12730 and rLIC10494 are promising candidates for a multivalent vaccine for the prevention of leptospirosis.

Therapeutic evaluation of free and liposome-loaded furazolidone in experimental visceral leishmaniasis

Drug delivery systems are promising pharmaceutical formulations used to improve the therapeutic index of drugs. In this study, we developed a liposomal formulation of furazolidone that targets Leishmania (Leishmania) chagasi amastigotes in a hamster model. Using laser scanning confocal microscopy, it was demonstrated that the liposomal drug co-localised with L. (L.) chagasi amastigotes within macrophages. Liposomal furazolidone administered intraperitoneally at 0.5 mg/kg for 12 consecutive days reduced spleen (74%) and liver (32%) parasite burden at a 100-fold lower dose than the free drug. Free furazolidone (50 mg/kg) also effectively reduced spleen (82.5%) and liver (85%) parasites; its in vitro activity against promastigotes and intracellular amastigotes demonstrated a high degree of parasite selectivity. Thus, furazolidone, both in the free and liposome-loaded formulation, is an effective inhibitor of L. (L.) chagasi, representing a possible cost-effective drug candidate for the treatment of visceral leishmaniasis. (C) 2010 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Application of Real-Time PCR Stool Assay for Helicobacter pylori Detection and Clarithromycin Susceptibility Testing in Brazilian Children

Background: Helicobacter pylori ClariRes assay is a novel commercially available real-time PCR assay allowing H. pylori detection and clarithromycin susceptibility testing in either gastric biopsy or stool specimens. Objective: The aim of this study was to validate the novel biprobe real-time assay in stool specimens from 217 dyspeptic children. Methods: DNA from gastric biopsies and stool specimens were obtained and submitted to the biprobe real time assay for H. pylori detection and clarithromycin susceptibility testing. Results: The sensitivity, specificity, and test accuracy were 69, 100 and 93.9% for the detection of H. pylori infection and 83.3, 100 and 95.6%, for detection of clarithromycin resistance. Conclusion: This assay proved to be appropriate for H. pylori clarithromycin susceptibility testing, particularly in children populations where a high prevalence of clarithromycin-resistant strains is suspected.

Global Analysis of Protein Palmitoylation in African Trypanosomes

Many eukaryotic proteins are posttranslationally modified by the esterification of cysteine thiols to long-chain fatty acids. This modification, protein palmitoylation, is catalyzed by a large family of palmitoyl acyltransferases that share an Asp-His-His-Cys Cys-rich domain but differ in their subcellular localizations and substrate specificities. In Trypanosoma brucei, the flagellated protozoan parasite that causes African sleeping sickness, protein palmitoylation has been observed for a few proteins, but the extent and consequences of this modification are largely unknown. We undertook the present study to investigate T. brucei protein palmitoylation at both the enzyme and substrate levels. Treatment of parasites with an inhibitor of total protein palmitoylation caused potent growth inhibition, yet there was no effect on growth by the separate, selective inhibition of each of the 12 individual T. brucei palmitoyl acyltransferases. This suggested either that T. brucei evolved functional redundancy for the palmitoylation of essential palmitoyl proteins or that palmitoylation of some proteins is catalyzed by a noncanonical transferase. To identify the palmitoylated proteins in T. brucei, we performed acyl biotin exchange chemistry on parasite lysates, followed by streptavidin chromatography, two-dimensional liquid chromatography-tandem mass spectrometry protein identification, and QSpec statistical analysis. A total of 124 palmitoylated proteins were identified, with an estimated false discovery rate of 1.0%. This palmitoyl proteome includes all of the known palmitoyl proteins in procyclic-stage T. brucei as well as several proteins whose homologues are palmitoylated in other organisms. Their sequences demonstrate the variety of substrate motifs that support palmitoylation, and their identities illustrate the range of cellular processes affected by palmitoylation in these important pathogens.

Exacerbation of Leishmania (Viannia) shawi infection in BALB/c mice after immunization with soluble antigen from amastigote forms

The present study aimed to evaluate the effects of immunization with soluble amastigote (AmaAg) and promastigote (ProAg) antigens from Leishmania (Viannia) shawi on the course of infection in BALB/c mice. After immunization with AmaAg, the challenged group showed greater lesion size and parasite load in the skin and lymph nodes, associated with diminished interleukin (IL)-2, IL-4, IL-10, interferon (IFN)-gamma and nitrate levels in the supernatant of lymph node cell cultures, together with increases in transforming growth factor (TGF)-beta concentrations and humoral immune response. In contrast, immunization with ProAg led to smaller lesion size with reduced numbers of viable parasites in the skin. Protection was associated with increases in IL-12, IFN-gamma, TGF-beta and nitrates and decreases in IL-4 and IL-10 levels. Concerning humoral immune response, a significant reduction in anti-leishmania immunoglobulin G was verified in the ProAg-challenged group. Analysis of these results suggests that AmaAg induced a suppressive cellular immune response in mice, favouring the spread of infection, whereas ProAg induced partial protection associated with increased cellular immune response.

Whole-Genome Characterization of Human and Simian Immunodeficiency Virus Intrahost Diversity by Ultradeep Pyrosequencing

Rapid evolution and high intrahost sequence diversity are hallmarks of human and simian immunodeficiency virus (HIV/SIV) infection. Minor viral variants have important implications for drug resistance, receptor tropism, and immune evasion. Here, we used ultradeep pyrosequencing to sequence complete HIV/SIV genomes, detecting variants present at a frequency as low as 1%. This approach provides a more complete characterization of the viral population than is possible with conventional methods, revealing low-level drug resistance and detecting previously hidden changes in the viral population. While this work applies pyrosequencing to immunodeficiency viruses, this approach could be applied to virtually any viral pathogen.

Effects of anti-TNF therapy on glucose metabolism in patients with ankylosing spondylitis, psoriatic arthritis or juvenile idiopathic arthritis

The objective of this study was to evaluate the influence of anti-tumor necrosis factor (anti-TNF) in juvenile idiopathic arthritis (DA), ankylosing spondylitis (AS) or psoriatic arthritis (PsA). Sixty-two patients were investigated: 7 DA; 37 AS; and 18 PsA. Caucasian race accounted for 79% and 29% were female. Mean age was 40.4 +/- 12.6years. None of the patients had a history of diabetes, and none had used oral hypoglycemic agents or insulin. Treatment was with adalimumab, infliximab and etanercept. Glucose, inflammatory markers and prednisone dose were assessed at baseline, as well as after three and six months of treatment. The mean erythrocyte sedimentation rate was significantly lower at three months and six months than at baseline (13.7 +/- 18.0 and 18 +/- 22.5 vs. 27.9 +/- 23.4 mm; p = 0.001). At baseline, three months and six months, we found the following: mean C-reactive protein levels were comparable (22.1 +/- 22.7, 14.5 +/- 30.7 and 16.0 +/- 23.8 mg/L, respectively; p = 0.26); mean glucose levels remained unchanged (90.8 +/- 22.2 mg/dl, 89.5 +/- 14.6 mg/dl and 89.8 +/- 13.6 mg/dl, respectively; p = 0.91); and mean prednisone doses were low and stable (3.9 +/- 4.9 mg/day, 3.7 +/- 4.8 mg/day and 2.6 +/- 4.0 mg/day, respectively; p = 0.23). During the first six months of treatment, anti-TNF therapy does not seem to influence glucose metabolism in JIA, AS or PsA. (C) 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

Real-time quantitative PCR in cerebral toxoplasmosis diagnosis of Brazilian human immunodeficiency virus-infected patients

Cerebral toxoplasmosis is the most common cerebral mass lesion in AIDS patients in Brazil, and results in high mortality and morbidity, despite free access to HAART (highly active antiretroviral treatment). Molecular diagnosis based on conventional PCR (cnPCR) or real-time quantitative PCR (qrtPCR) has been indispensable for definitive diagnosis. We report here the evaluation of qrtPCR with blood and cerebrospinal fluid (CSF) samples from AIDS patients in Brazil. This prospective study was conducted for 2 years, analysing DNA samples extracted from 149 AIDS patients (98 blood and 51 CSF samples) with confirmed clinical and radiological diagnosis The laboratory diagnosis included cnPCR (with the B22/B23 primer set) and indirect immunofluorescence (IF). For qrtPCR, two primer sets were simultaneously designed based on described genes and using a 6-carboxyfluorescein dye-labelled TaqMan MGB (minor groove binder) probe One was Bug, which amplified a sequence from the B1 gene The other was the RETg, which amplified a PCR product of the 529 bp sequence. The overall cnPCR and qrtPCR results were positive results were observed in 33.6% (50) patients The sensitivities were 98% for cnPCR (B22/B23), and 86 and 98% for qrtPCR (B1Tg and RETg, respectively). Negative reactions were observed in 66 4% patients. The specificities were 97% for cnPCR and qrtPCR (B1Tg). and 88.8% for RETg These data show that RETg PCR is highly sensitive as it amplifies a repeat region with many copies; however, its specificity is lower than the other markers However, B1Tg PCR had good specificity, but lower sensitivity Among the patients, 20 had blood and CSF collected simultaneously Thus, their results permitted us to analyse and compare molecular, serological and clinical diagnosis for a better understanding of the different scenarios of laboratorial and clinical diagnosis. For nine patients with confirmed cerebral toxoplasmosis diagnosis, four scenarios were observed: (i) and (ii) negative molecular diagnosis for CSF and positive for blood with variable IF titres for the sera and CSF (negative or positive), (iii) positive molecular diagnosis with CSF and negative with blood, and (iv) positive molecular diagnosis in both samples. In the latter two situations, normally the IF titres in sera and CSF are variable. Other opportunistic infections were shown in 11 patients Despite the IF titres in sera and CSF being variable, all of them had negative molecular diagnosis for both samples qrtPCR allows for a rapid identification of Toxoplasma gondii DNA in patient samples; in a minority of cases discrepancies occur with the cnPCR.

Transcriptional Errors in Human Immunodeficiency Virus Type 1 Generate Targets for T-Cell Responses

We measured T-cell responses to human immunodeficiency virus type 1 (HIV-1) cryptic epitopes encoded by regions of the viral genome not normally translated into viral proteins. T-cell responses to cryptic epitopes and to regions normally spliced out of the HIV-1 viral proteins Rev and Tat were detected in HIV-1-infected subjects.

Comparative Study of the Standard Fluorescent Antibody to Membrane Antigen (FAMA) Assay and a Flow Cytometry-Adapted FAMA Assay To Assess Immunity to Varicella-Zoster Virus

A flow cytometry-adapted fluorescent antibody to membrane antigen (FAMA) assay to detect IgG antibodies against varicella-zoster virus (VZV) was developed and tested in 62 serum samples, showing 90.32% accuracy obtained from a receiver operating characteristic (ROC) curve with a 0.9125 (95% confidence interval [CI], 0.829 to 1.00) area below the curve compared to the result with standard FAMA.

HLA-DR in Brazilian patients with polyarteritis nodosa (PAN) and microscopic polyangiitis (MPA)

The aim of this study was to evaluate the frequency and clinical associations of HLA-DR alleles in Brazilian Caucasian patients with polyarteritis nodosa (PAN) or microscopic polyangiitis (MPA). We evaluated 29 Caucasian patients with vasculitis classified as PAN or MPA according to the American College of Rheumatology (ACR) 1990 Criteria, Chapel Hill Consensus Conference (CHCC) nomenclature for vasculitis and EULAR recommendations for conducting clinical studies in systemic vasculitis. HLA-DR alleles were typed using polymerase chain reaction-amplified DNA, hybridized with sequence-specific low resolution primers. DNA obtained from 59 Caucasian healthy blood donors were used as control. In order to evaluate if a specific HLA may have influence on the clinical profile of those diseases, we also divided the patients according to Birmingham vasculitis score (BVAS) and Five-Factors Score (FFS) at the time of diagnosis. Increased frequency of HLA-DRB1*16 (p = 0.023) and DRB4*01 (p = 0.048) was found in patients with higher disease activity at the time of diagnosis (BVAS >= 22). Patients with less severe disease (FFS = 0) had a higher frequency of HLA-DRB1*03 (p = 0.011). Patients with gastrointestinal tract involvement had significantly increased frequency of HLA-DRB1*11 or B1*12 (p = 0.046), B1*13 (p = 0.021) and B3 (p = 0.008). In contrast, patients with renal disease, had higher frequency of DRB1*15 or DRB1*16 (p = 0.035) and B5 (p = 0.035). In the subgroup of patients with MPA, increased frequency of HLA-DRB1*15 was found in patients with BVAS >= 22 (p = 0.038) and FFS >= 1 (p = 0.039) suggesting that this allele is associated with more aggressive disease. Antineutrophil cytoplasmic antibodies (ANCA) negative MPA patients had significantly increased frequency of HLA-DRB1*11 or DRB1*12 when compared to ANCA positive patients (p = 0.023). Our results suggest that HLA-DR alleles may influence PAN and MPA clinical expression and outcome and that in MPA they participate in the mechanisms involved in the development to ANCA.

Targeted Mutagenesis in Pathogenic Leptospira Species: Disruption of the LigB Gene Does Not Affect Virulence in Animal Models of Leptospirosis

The pathogenic mechanisms of Leptospira interrogans, the causal agent of leptospirosis, remain largely unknown. This is mainly due to the lack of tools for genetically manipulating pathogenic Leptospira species. Thus, homologous recombination between introduced DNA and the corresponding chromosomal locus has never been demonstrated for this pathogen. Leptospiral immunoglobulin-like repeat (Lig) proteins were previously identified as putative Leptospira virulence factors. In this study, a ligB mutant was constructed by allelic exchange in L. interrogans; in this mutant a spectinomycin resistance (Spc(r)) gene replaced a portion of the ligB coding sequence. Gene disruption was confirmed by PCR, immunoblot analysis, and immunofluorescence studies. The ligB mutant did not show decrease virulence compared to the wild-type strain in the hamster model of leptospirosis. In addition, inoculation of rats with the ligB mutant induced persistent colonization of the kidneys. Finally, LigB was not required to mediate bacterial adherence to cultured cells. Taken together, our data provide the first evidence of site-directed homologous recombination in pathogenic Leptospira species. Furthermore, our data suggest that LigB does not play a major role in dissemination of the pathogen in the host and in the development of acute disease manifestations or persistent renal colonization.