An AC-5 cathepsin B-like protease purified from Haemonchus contortus excretory secretory products shows protective antigen potential for lambs

The immunogenic properties of cysteine proteases obtained from excretory/secretory products (ES) of Haemonchus contortus were investigated with a fraction purified with a recombinant H. contortus cystatin affinity column. The enrichment of H. contortus ES for cysteine protease was confirmed with substrate SDS-PAGE gels since the cystatin-binding fraction activity was three times higher than total ES, despite representing only 3% of total ES. This activity was inhibited by a specific cysteine protease inhibitor (E64) and by recombinant cystatin. The one-dimensional profile of the cystatin-binding fraction displayed a single band with a molecular mass of 43 kDa. Mass spectrometry showed this to be AC-5, a cathepsin B-like cysteine protease which had not been identified in ES products of H. contortus before. The cystatin binding fraction was tested as an immunogen in lambs which were vaccinated three times (week 0, 2.5 and 5), challenged with 10 000 L3 H. contortus (week 6) before necropsy and compared to unvaccinated challenge controls and another group given total ES (n = 10 per group). The group vaccinated with cystatin-binding proteins showed 36% and 32% mean worm burden and eggs per gram of faeces (EPG) reductions, respectively, compared to the controls but total ES was almost without effect. After challenge the cystatin-binding proteins induced significantly higher local and systemic ES specific IgA and IgG responses.

Leukotriene B(4)-loaded microspheres: a new therapeutic strategy to modulate cell activation

Background: Leukotriene B(4) (LTB(4)) is a potent inflammatory mediator that also stimulates the immune response. In addition, it promotes polymorphonuclear leukocyte phagocytosis, chemotaxis, chemokinesis and modulates cytokines release. Regarding chemical instability of the leukotriene molecule, in the present study we assessed the immunomodulatory activities conferred by LTB(4) released from microspheres (MS). A previous oil-in-water emulsion solvent extraction-evaporation method was chosen to prepare LTB(4)-loaded MS. Results: In the mice cremasteric microcirculation, intraescrotal injection of 0.1 ml of LTB(4)-loaded MS provoked significant increases in leukocyte rolling flux, adhesion and emigration besides significant decreases in the leukocyte rolling velocity. LTB(4)-loaded MS also increase peroxisome proliferator-activated receptor-alpha (PPAR alpha) expression by murine peritoneal macrophages and stimulate them to generate nitrite levels. Monocyte chemoattractant protein-I (MCP-I) and nitric oxide (NO) productions were also increased when human umbilical vein and artery endothelial cells (HUVECs and HUAECs, respectively) were stimulated with LTB(4)-loaded MS. Conclusion: LTB(4)-loaded MS preserve the biological activity of the encapsulated mediator indicating their use as a new strategy to modulate cell activation, especially in the innate immune response.

ECG scar quantification correlates with cardiac magnetic resonance scar size and prognostic factors in Chagas' disease

Objective To test the hypothesis that 12-lead ECG QRS scoring quantifies myocardial scar and correlates with disease severity in Chagas' heart disease. Design Patients underwent 12-lead ECG for QRS scoring and cardiac magnetic resonance with late gadolinium enhancement (CMR-LGE) to assess myocardial scar. Setting University of Sao Paulo Medical School, Sao Paulo, Brazil. Patients 44 Seropositive patients with Chagas' disease without a history of myocardial infarction and at low risk for coronary artery disease. Main outcome measures Correlation between QRS score, CMR-LGE scar size and left ventricular ejection fraction. Relation between QRS score, heart failure (HF) class and history of ventricular tachycardia (VT). Results QRS score correlated directly with CMR-LGE scar size (R=0.69, p<0.0001) and inversely with left ventricular ejection fraction (R=-0.54, p=0.0002), which remained significant in the subgroup with conduction defects. Patients with class II or III HF had significantly higher QRS scores than those with class I HF (5.1 +/- 3.4 vs 2.1 +/- 3.1 QRS points (p=0.002)) and patients with a history of VT had significantly higher QRS scores than those without a history of VT (5.3 +/- 3.2% vs 2.6 +/- 3.4 QRS points (p=0.02)). A QRS score >= 2 points had particularly good sensitivity and specificity (95% and 83%, respectively) for prediction of large CMR-LGE, and a QRS score >= 7 points had particularly high specificity (92% and 89%, respectively) for predicting significant left ventricular dysfunction and history of VT. Conclusions The wide availability of 12-lead ECG makes it an attractive screening tool and may enhance clinical risk stratification of patients at risk for more severe, symptomatic Chagas' heart disease.

Unexpected Diversity of Cellular Immune Responses against Nef and Vif in HIV-1-Infected Patients Who Spontaneously Control Viral Replication

Background: HIV-1-infected individuals who spontaneously control viral replication represent an example of successful containment of the AIDS virus. Understanding the anti-viral immune responses in these individuals may help in vaccine design. However, immune responses against HIV-1 are normally analyzed using HIV-1 consensus B 15-mers that overlap by 11 amino acids. Unfortunately, this method may underestimate the real breadth of the cellular immune responses against the autologous sequence of the infecting virus. Methodology and Principal Findings: Here we compared cellular immune responses against nef and vif-encoded consensus B 15-mer peptides to responses against HLA class I-predicted minimal optimal epitopes from consensus B and autologous sequences in six patients who have controlled HIV-1 replication. Interestingly, our analysis revealed that three of our patients had broader cellular immune responses against HLA class I-predicted minimal optimal epitopes from either autologous viruses or from the HIV-1 consensus B sequence, when compared to responses against the 15-mer HIV-1 type B consensus peptides. Conclusion and Significance: This suggests that the cellular immune responses against HIV-1 in controller patients may be broader than we had previously anticipated.

Factor (NF) kappa B polymorphism is associated with heart function in patients with heart failure

Background: Cardiac remodeling is generally an adverse sign and is associated with heart failure (HF) progression. NFkB, an important transcription factor involved in many cell survival pathways, has been implicated in the remodeling process, but its role in the heart is still controversial. Recently, a promoter polymorphism associated with a lesser activation of the NFKB1 gene was also associated with Dilated Cardiomyopathy. The purpose of this study was to evaluate the association of this polymorphism with clinical and functional characteristics of heart failure patients of different etiologies. Methods: A total of 493 patients with HF and 916 individuals from a cohort of individuals from the general population were investigated. The NFKB1-94 insertion/deletion ATTG polymorphism was genotyped by High Resolution Melt discrimination. Allele and genotype frequencies were compared between groups. In addition, frequencies or mean values of different phenotypes associated with cardiovascular disease were compared between genotype groups. Finally, patients were prospectively followed-up for death incidence and genotypes for the polymorphism were compared regarding disease onset and mortality incidence in HF patients. Results: We did not find differences in genotype and allelic frequencies between cases and controls. Interestingly, we found an association between the ATTG(1)/ATTG(1) genotype with right ventricle diameter (P = 0.001), left ventricle diastolic diameter (P = 0.04), and ejection fraction (EF) (P = 0.016), being the genotype ATTG(1)/ATTG(1) more frequent in patients with EF lower than 50% (P = 0.01). Finally, we observed a significantly earlier disease onset in ATTG(1)/ATTG(1) carriers. Conclusion: There is no genotype or allelic association between the studied polymorphism and the occurrence of HF in the tested population. However, our data suggest that a diminished activation of NFKB1, previously associated with the ATTG(1)/ATTG(1) genotype, may act modulating on the onset of disease and, once the individual has HF, the genotype may modulate disease severity by increasing cardiac remodeling and function deterioration.

Lack of Galectin-3 Disturbs Mesenteric Lymph Node Homeostasis and B Cell Niches in the Course of Schistosoma mansoni Infection

Galectin-3 is a beta-galactoside-binding protein that has been shown to regulate pathophysiological processes, including cellular activation, differentiation and apoptosis. Recently, we showed that galectin-3 acts as a potent inhibitor of B cell differentiation into plasma cells. Here, we have investigated whether galectin-3 interferes with the lymphoid organization of B cell compartments in mesenteric lymph nodes (MLNs) during chronic schistosomiasis, using WT and galectin-3(-/-) mice. Schistosoma mansoni synthesizes GalNAc beta 1-4(Fuc alpha 1-3) GlcNAc(Lac-DiNAc) structures (N-acetylgalactosamine beta 1-4 N-acetylglucosamine), which are known to interact with galectin-3 and elicit an intense humoral response. Antigens derived from the eggs and adult worms are continuously drained to MLNs and induce a polyclonal B cell activation. In the present work, we observed that chronically-infected galectin-3(-/-) mice exhibited a significant reduced amount of macrophages and B lymphocytes followed by drastic histological changes in B lymphocyte and plasma cell niches in the MLNs. The lack of galectin-3 favored an increase in the lymphoid follicle number, but made follicular cells more susceptible to apoptotic stimuli. There were an excessive quantity of apoptotic bodies, higher number of annexin V(+)/PI(-) cells, and reduced clearance of follicular apoptotic cells in the course of schistosomiasis. Here, we observed that galectin-3 was expressed in nonlymphoid follicular cells and its absence was associated with severe damage to tissue architecture. Thus, we convey new information on the role of galectin-3 in regulation of histological events associated with B lymphocyte and plasma cell niches, apoptosis, phagocytosis and cell cycle properties in the MLNs of mice challenged with S. mansoni.

Characterization of Hepatitis B virus (HBV) genotypes in patients from Rondonia, Brazil

Background: Hepatitis B virus (HBV) can be classified into nine genotypes (A-I) defined by sequence divergence of more than 8% based on the complete genome. This study aims to identify the genotypic distribution of HBV in 40 HBsAg-positive patients from Rondonia, Brazil. A fragment of 1306 bp partially comprising surface and polymerase overlapping genes was amplified by PCR. Amplified DNA was purified and sequenced. Amplified DNA was purified and sequenced on an ABI PRISM (R) 377 Automatic Sequencer (Applied Biosystems, Foster City, CA, USA). The obtained sequences were aligned with reference sequences obtained from the GenBank using Clustal X software and then edited with Se-Al software. Phylogenetic analyses were conducted by the Markov Chain Monte Carlo (MCMC) approach using BEAST v.1.5.3. Results: The subgenotypes distribution was A1 (37.1%), D3 (22.8%), F2a (20.0%), D4 (17.1%) and D2 (2.8%). Conclusions: These results for the first HBV genotypic characterization in Rondonia state are consistent with other studies in Brazil, showing the presence of several HBV genotypes that reflects the mixed origin of the population, involving descendants from Native Americans, Europeans, and Africans.

Detection of Hepatitis B virus subgenotype A1 in a Quilombo community from Maranhao, Brazil

Background: The Brazilian population is mainly descendant from European colonizers, Africans and Native Americans. Some Afro-descendants lived in small isolated communities since the slavery period. The epidemiological status of HBV infection in Quilombos communities from northeast of Brazil remains unknown. The aim of this study was to characterize the HBV genotypes circulating inside a Quilombo isolated community from Maranhao State, Brazil. Methods: Seventy-two samples from Frechal Quilombo community at Maranhao were collected. All serum samples were screened by enzyme-linked immunosorbent assays for the presence of hepatitis B surface antigen ( HBsAg). HBsAg positive samples were submitted to DNA extraction and a fragment of 1306 bp partially comprising HBsAg and polymerase coding regions (S/POL) was amplified by nested PCR and its nucleotide sequence was determined. Viral isolates were genotyped by phylogenetic analysis using reference sequences from each genotype obtained from GenBank (n = 320). Sequences were aligned using Muscle software and edited in the SE-AL software. Bayesian phylogenetic analyses were conducted using Markov Chain Monte Carlo (MCMC) method to obtain the MCC tree using BEAST v.1.5.3. Results: Of the 72 individuals, 9 (12.5%) were HBsAg-positive and 4 of them were successfully sequenced for the 1306 bp fragment. All these samples were genotype A1 and grouped together with other sequences reported from Brazil. Conclusions: The present study represents the first report on the HBV genotypes characterization of this community in the Maranhao state in Brazil where a high HBsAg frequency was found. In this study, we reported a high frequency of HBV infection and the exclusive presence of subgenotype A1 in an Afro-descendent community in the Maranhao State, Brazil.

The International Collaboration on Air Pollution and Pregnancy Outcomes: Initial Results

BACKGROUND: The findings of prior studies of air pollution effects on adverse birth outcomes are difficult to synthesize because of differences in study design. OBJECTIVES: The International Collaboration on Air Pollution and Pregnancy Outcomes was formed to understand how differences in research methods contribute to variations in findings. We initiated a feasibility study to a) assess the ability of geographically diverse research groups to analyze their data sets using a common protocol and b) perform location-specific analyses of air pollution effects on birth weight using a standardized statistical approach. METHODS: Fourteen research groups from nine countries participated. We developed a protocol to estimate odds ratios (ORs) for the association between particulate matter <= 10 mu m in aerodynamic diameter (PM(10)) and low birth weight (LBW) among term births, adjusted first for socioeconomic status (SES) and second for additional location-specific variables. RESULTS: Among locations with data for the PM(10) analysis, ORs estimating the relative risk of term LBW associated with a 10-mu g/m(3) increase in average PM(10) concentration during pregnancy, adjusted for SES, ranged from 0.63 [95% confidence interval (CI), 0.30-1.35] for the Netherlands to 1.15 (95% CI, 0.61-2.18) for Vancouver, with six research groups reporting statistically significant adverse associations. We found evidence of statistically significant heterogeneity in estimated effects among locations. CONCLUSIONS: Variability in PM(10)-LBW relationships among study locations remained despite use of a common statistical approach. A more detailed meta-analysis and use of more complex protocols for future analysis may uncover reasons for heterogeneity across locations. However, our findings confirm the potential for a diverse group of researchers to analyze their data in a standardized way to improve understanding of air pollution effects on birth outcomes.

Frequency and genotypic distribution of GB virus C (GBV-C) among Colombian population with Hepatitis B (HBV) or Hepatitis C (HCV) infection

Background: GB virus C (GBV-C) is an enveloped positive-sense ssRNA virus belonging to the Flaviviridae family. Studies on the genetic variability of the GBV-C reveals the existence of six genotypes: genotype 1 predominates in West Africa, genotype 2 in Europe and America, genotype 3 in Asia, genotype 4 in Southwest Asia, genotype 5 in South Africa and genotype 6 in Indonesia. The aim of this study was to determine the frequency and genotypic distribution of GBV-C in the Colombian population. Methods: Two groups were analyzed: i) 408 Colombian blood donors infected with HCV (n = 250) and HBV (n = 158) from Bogota and ii) 99 indigenous people with HBV infection from Leticia, Amazonas. A fragment of 344 bp from the 5' untranslated region (5' UTR) was amplified by nested RT PCR. Viral sequences were genotyped by phylogenetic analysis using reference sequences from each genotype obtained from GenBank (n = 160). Bayesian phylogenetic analyses were conducted using Markov chain Monte Carlo (MCMC) approach to obtain the MCC tree using BEAST v. 1.5.3. Results: Among blood donors, from 158 HBsAg positive samples, eight 5.06% (n = 8) were positive for GBV-C and from 250 anti-HCV positive samples, 3.2%(n = 8) were positive for GBV-C. Also, 7.7% (n = 7) GBV-C positive samples were found among indigenous people from Leticia. A phylogenetic analysis revealed the presence of the following GBV-C genotypes among blood donors: 2a (41.6%), 1 (33.3%), 3 (16.6%) and 2b (8.3%). All genotype 1 sequences were found in co-infection with HBV and 4/5 sequences genotype 2a were found in co-infection with HCV. All sequences from indigenous people from Leticia were classified as genotype 3. The presence of GBV-C infection was not correlated with the sex (p = 0.43), age (p = 0.38) or origin (p = 0.17). Conclusions: It was found a high frequency of GBV-C genotype 1 and 2 in blood donors. The presence of genotype 3 in indigenous population was previously reported from Santa Marta region in Colombia and in native people from Venezuela and Bolivia. This fact may be correlated to the ancient movements of Asian people to South America a long time ago.

Hepatitis B (HBV), Hepatitis C (HCV) and Hepatitis Delta (HDV) Viruses in the Colombian Population-How Is the Epidemiological Situation?

Background: Viral hepatitis B, C and delta still remain a serious problem worldwide. In Colombia, data from 1980s described that HBV and HDV infection are important causes of hepatitis, but little is known about HCV infection. The aim of this study was to determine the currently frequency of HBV, HCV and HDV in four different Colombian regions. Methodology/Principal Findings: This study was conducted in 697 habitants from 4 Colombian departments: Amazonas, Choco, Magdalena and San Andres Islands. Epidemiological data were obtained from an interview applied to each individual aiming to evaluate risk factors related to HBV, HCV or HDV infections. All samples were tested for HBsAg, anti-HBc, anti-HBs and anti-HCV markers. Samples that were positive to HBsAg and/or anti-HBc were tested to anti-HDV. Concerning the geographical origin of the samples, the three HBV markers showed a statistically significant difference: HBsAg (p = 0.033) and anti-HBc (p < 0.001) were more frequent in Amazonas and Magdalena departments. Isolated anti-HBs (a marker of previous vaccination) frequencies were: Choco (53.26%), Amazonas (32.88%), Magdalena (17.0%) and San Andres (15.33%) p < 0.001. Prevalence of anti-HBc increased with age; HBsAg varied from 1.97 to 8.39% (p = 0.033). Amazonas department showed the highest frequency for anti-HCV marker (5.68%), while the lowest frequency was found in San Andres Island (0.66%). Anti-HDV was found in 9 (5.20%) out of 173 anti-HBc and/or HBsAg positive samples, 8 of them from the Amazonas region and 1 from them Magdalena department. Conclusions/Significance: In conclusion, HBV, HCV and HDV infections are detected throughout Colombia in frequency levels that would place some areas as hyperendemic for HBV, especially those found in Amazonas and Magdalena departments. Novel strategies to increase HBV immunization in the rural population and to strengthen HCV surveillance are reinforced by these results.

Maternal immunization with ovalbumin prevents neonatal allergy development and up-regulates inhibitory receptor Fc gamma RIIB expression on B cells

Background: Preconception allergen immunization prevents neonatal allergen sensitization in mice by a complex interaction between regulatory cells/factors and antibodies. The present study assessed the influence of maternal immunization with ovalbumin (OVA) on the immune response of 3 day-old and 3 week-old offspring immunized or non-immunized with OVA and evaluated the effect of IgG treatment during fetal development or neonatal period. Results: Maternal immunization with OVA showed increased levels of Fc gamma RIIb expression in splenic B cells of neonates, which were maintained for up to 3 weeks and not affected by additional postnatal OVA immunization. Maternal immunization also exerted a down-modulatory effect on both IL-4 and IFN-gamma-secreting T cells and IL-4 and IL-12-secreting B cells. Furthermore, immunized neonates from immunized mothers showed a marked inhibition of antigen-specifc IgE Ab production and lowered Th2/Th1 cytokine levels, whereas displaying enhanced Fc gamma RIIb expression on B cells. These offspring also showed reduced antigen-specific proliferative response and lowered B cell responsiveness. Moreover, in vitro evaluation revealed an impairment of B cell activation upon engagement of B cell antigen receptor by IgG from OVA-immunized mice. Finally, in vivo IgG transference during pregnancy or breastfeeding revealed that maternal Ab transference was able to increase regulatory cytokines, such as IL-10, in the prenatal stage; yet only the postnatal treatment prevented neonatal sensitization. None of the IgG treatments induced immunological changes in the offspring, as it was observed for those from OVA-immunized mothers. Conclusion: Maternal immunization upregulates the inhibitory Fc gamma RIIb expression on offspring B cells, avoiding skewed Th2 response and development of allergy. These findings contribute to the advancement of prophylactic strategies to prevent allergic diseases in early life.

Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections

Interference by autofluorescence is one of the major concerns of immunofluorescence analysis of in situ hybridization-based diagnostic assays. We present a useful technique that reduces autofluorescent background without affecting the tissue integrity or direct immunofluorescence signals in brain sections. Using six different protocols, such as ammonia/ethanol, Sudan Black B (SBB) in 70% ethanol, photobleaching with UV light and different combinations of them in both formalin-fixed paraffin-embedded and frozen human brain tissue sections, we have found that tissue treatment of SBB in a concentration of 0.1% in 70% ethanol is the best approach to reduce/eliminate tissue autofluorescence and background, while preserving the specific fluorescence hybridization signals. This strategy is a feasible, non-time consuming method that provides a reasonable compromise between total reduction of the tissue autofluorescence and maintenance of specific fluorescent labels.

Increased Levels of NOTCH1, NF-kappa B, and Other Interconnected Transcription Factors Characterize Primitive Sets of Hematopoietic Stem Cells

As previously shown, higher levels of NOTCH1 and increased NF-kappa B signaling is a distinctive feature of the more primitive umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSCs), as compared to bone marrow ( BM). Differences between BM and UCB cell composition also account for this finding. The CD133 marker defines a more primitive cell subset among CD34+ HSC with a proposed hemangioblast potential. To further evaluate the molecular basis related to the more primitive characteristics of UCB and CD133+ HSC, immunomagnetically purified human CD34+ and CD133+ cells from BM and UCB were used on gene expression microarrays studies. UCB CD34+ cells contained a significantly higher proportion of CD133+ cells than BM (70% and 40%, respectively). Cluster analysis showed that BM CD133+ cells grouped with the UCB cells ( CD133+ and CD34+) rather than to BM CD34+ cells. Compared with CD34+ cells, CD133+ had a higher expression of many transcription factors (TFs). Promoter analysis on all these TF genes revealed a significantly higher frequency ( than expected by chance) of NF-kappa B-binding sites (BS), including potentially novel NF-kappa B targets such as RUNX1, GATA3, and USF1. Selected transcripts of TF related to primitive hematopoiesis and self-renewal, such as RUNX1, GATA3, USF1, TAL1, HOXA9, HOXB4, NOTCH1, RELB, and NFKB2 were evaluated by real-time PCR and were all significantly positively correlated. Taken together, our data indicate the existence of an interconnected transcriptional network characterized by higher levels of NOTCH1, NF-kappa B, and other important TFs on more primitive HSC sets.

Expression of eight genes of nuclear factor-kappa B pathway in multiple myeloma using bone marrow aspirates obtained at diagnosis

Purpose: To evaluate the expression of NF-kappa B pathway genes in total bone marrow samples obtained from MM at diagnosis using real-time quantitative PCR and to evaluate its possible correlation with disease clinical features and survival. Material and methods: Expression of eight genes related to NF-kappa B pathway (NFKB1, IKB, RANK, RANKL, OPG, IL6, VCAM1 and ICAM1) were studied in 53 bone marrow samples from newly diagnosed MM patients and in seven normal controls, using the Taqman system. Genes were considered overexpressed when tumor expression level was at least four times higher than that observed in normal samples. Results: The percentages of overexpression of the eight genes were: NFKB1 0%, IKB 22.6%, RANK 15.1%, RANKL 31.3%, OPG 7.5%, IL6 39.6%, VCAM1 10% and ICAM1 26%. We found association between IL6 expression level and International Staging System (ISS) (p = 0.01), meaning that MM patients with high ISS scores have more chance of overexpression of IL6. The mean value of ICAM1 relative expression was also associated with the ISS score (p = 0.02). Regarding OS, cases with IL6 overexpression present worse evolution than cases with IL6 normal expression (p = 0.04). Conclusion: We demonstrated that total bone marrow aspirates can be used as a source of material for gene expression studies in MM. In this context, we confirmed that IL6 overexpression was significantly associated with worse survival and we described that it is associated with high ISS scores. Also, ICAM1 was overexpressed in 26% of cases and its level was associated with ISS scores.