Elevated calprotectin levels are associated with mortality in patients with acute decompensation of liver cirrhosis.

BACKGROUND: Acute decompensation (AD) of cirrhosis is related to systemic inflammation and elevated circulating cytokines. In this context, biomarkers of inflammation, such as calprotectin, may be of prognostic value. AIM: To evaluate serum calprotectin levels in patients hospitalized for complications of cirrhosis. METHODS: This is a prospective cohort study that included 200 subjects hospitalized for complications of cirrhosis, 20 outpatients with stable cirrhosis, and 20 healthy controls. Serum calprotectin was measured by enzyme-linked immunosorbant assay. RESULTS: Calprotectin levels were higher among groups with cirrhosis when compared to healthy controls. Higher median calprotectin was related to Child-Pugh C, ascites, and hepatic encephalopathy. Higher calprotectin was related to acute-on-chronic liver failure (ACLF) and infection in the bivariate, but not in multivariate analysis. Calprotectin was not associated with survival among patients with ACLF; however, in patients with AD without ACLF, higher calprotectin was associated with a lower 30-d survival, even after adjustment for chronic liver failure-consortium (CLIF-C) AD score. A high-risk group (CLIF-C AD score ≥ 60 and calprotectin ≥ 580 ng/mL) was identified, which had a 30-d survival (27.3%) similar to that of patients with grade 3 ACLF (23.3%). CONCLUSION: Serum calprotectin is associated with prognosis in patients with AD without ACLF and may be useful in clinical practice to early identify patients with a very low short-term survival.

FACTORS ASSOCIATED WITH CIRCULATING ZONULIN IN INFLAMMATORY BOWEL DISEASE.

BACKGROUND: Inflammatory bowel disease (IBD) comprises the spectrum between Crohn's disease (CD) and ulcerative colitis (UC), a condition whose prevalence in countries such as Brazil has increased significantly in recent years. Changes in the intestinal epithelial barrier function and, consequently, an increase in intestinal permeability, have been suggested as important factors in the pathogenesis of different autoimmune conditions, including IBD. Therefore, there is a need for a practical tool to assess gut barrier integrity in these patients. OBJECTIVE: To study factors associated with serum zonulin levels, a marker of intestinal permeability, in patients with IBD. METHODS: This was a cross-sectional observational study that included 117 patients with IBD and 32 healthy controls. Disease activity was assessed by the Simple Clinical Colitis Activity Index (SCCAI) in UC and by the Harvey-Bradshaw Index (HBI) in CD subjects. Zonulin levels were measured by ELISA and inflammatory cytokines by Cytometric Bead Array, using commercially available kits. RESULTS: The mean age of IBD patients was 44.0±15.9 years, 66.7% were female, 57 subjects were diagnosed with CD and 60 with UC. At evaluation, clinical remission was observed in 56.7% of CD patients and in 59.2% of UC subjects. No differences were observed in zonulin levels when comparing IBD patients with the control group (95.28 ng/mL vs 96.61 ng/mL, P=0.573) and when comparing patients with CD to those with UC (79.68 ng/mL vs 106.10 ng/mL, P=0.887). Among IBD group, zonulin concentrations were higher among females, correlated positively with body mass index (BMI) and age; and negatively with hemoglobin and hematocrit. In patients with UC, zonulin correlated negatively with hemoglobin, hematocrit, and albumin; and positively with BMI and SCCAI. Among CD patients, zonulin was positively correlated with age and BMI, but not with HBI. No correlations were observed between zonulin and circulating cytokines in IBD patients. CONCLUSION: In this cohort mostly comprised of patients in clinical remission, serum zonulin levels were not higher in patients with IBD than healthy controls, and correlated with variables not linked to baseline disease, such as sex, age and BMI. However, zonulin correlated with clinical and laboratory parameters of disease severity and activity among subjects with UC, but not among patients with CD. These findings indicate a potential role for zonulin as a biomarker in IBD, particularly in UC.

Fatores associados com os níveis séricos de zonulina na doença inflamatória intestinal / Factors associated with circulating zonulin in inflammatory bowel disease

ABSTRACT Background: Inflammatory bowel disease (IBD) comprises the spectrum between Crohn's disease (CD) and ulcerative colitis (UC), a condition whose prevalence in countries such as Brazil has increased significantly in recent years. Changes in the intestinal epithelial barrier function and, consequently, an increase in intestinal permeability, have been suggested as important factors in the pathogenesis of different autoimmune conditions, including IBD. Therefore, there is a need for a practical tool to assess gut barrier integrity in these patients. Objective: To study factors associated with serum zonulin levels, a marker of intestinal permeability, in patients with IBD. Methods: This was a cross-sectional observational study that included 117 patients with IBD and 32 healthy controls. Disease activity was assessed by the Simple Clinical Colitis Activity Index (SCCAI) in UC and by the Harvey-Bradshaw Index (HBI) in CD subjects. Zonulin levels were measured by ELISA and inflammatory cytokines by Cytometric Bead Array, using commercially available kits. Results: The mean age of IBD patients was 44.0±15.9 years, 66.7% were female, 57 subjects were diagnosed with CD and 60 with UC. At evaluation, clinical remission was observed in 56.7% of CD patients and in 59.2% of UC subjects. No differences were observed in zonulin levels when comparing IBD patients with the control group (95.28 ng/mL vs 96.61 ng/mL, P=0.573) and when comparing patients with CD to those with UC (79.68 ng/mL vs 106.10 ng/mL, P=0.887). Among IBD group, zonulin concentrations were higher among females, correlated positively with body mass index (BMI) and age; and negatively with hemoglobin and hematocrit. In patients with UC, zonulin correlated negatively with hemoglobin, hematocrit, and albumin; and positively with BMI and SCCAI. Among CD patients, zonulin was positively correlated with age and BMI, but not with HBI. No correlations were observed between zonulin and circulating cytokines in IBD patients. Conclusion: In this cohort mostly comprised of patients in clinical remission, serum zonulin levels were not higher in patients with IBD than healthy controls, and correlated with variables not linked to baseline disease, such as sex, age and BMI. However, zonulin correlated with clinical and laboratory parameters of disease severity and activity among subjects with UC, but not among patients with CD. These findings indicate a potential role for zonulin as a biomarker in IBD, particularly in UC.

RESUMO Contexto: A doença inflamatória intestinal (DII) compreende o espectro entre a doença de Crohn (DC) e a colite ulcerativa, condição esta cuja prevalência em países como o Brasil vem aumentando significativamente nos últimos anos. Alterações na função da barreira epitelial intestinal e, consequentemente, um aumento da permeabilidade intestinal, têm sido sugeridos como fatores importantes envolvidos na patogênese de diferentes condições autoimunes, dentre elas, a DII. Desta forma, existe a necessidade de uma ferramenta prática para avaliar a integridade da barreira epitelial intestinal nestes pacientes. Objetivo: Estudar os fatores associados com os níveis séricos de zonulina, um marcador da permeabilidade intestinal, em pacientes com DII. Métodos: Estudo observacional transversal que incluiu 117 pacientes com DII e 32 indivíduos que compuseram o grupo controle. A atividade da doença foi avaliada pelo Simple Cliniical Colitis Activity Index (SCCAI) na colite ulcerativa e pelo índice de Harvey-Bradshaw (IHB) em pacientes com DC. Os níveis de zonulina foram quantificados por ELISA e os níveis das citocinas inflamatórias pelo Cytometric Bead Array, utilizando kits comercialmente disponíveis. Resultados: A média de idade dos pacientes com DII foi de 44,0±15,9 anos, 66,7% eram do sexo feminino, 57 pacientes eram portadores de DC e 60 pacientes eram portadores de colite ulcerativa. No momento da avaliação clínico-laboratorial, 56,7% dos pacientes com DC encontravam-se em remissão clínica e, dentre os pacientes com colite ulcerativa, 59,2% deles assim se encontravam. Não foram observadas diferenças nos níveis séricos de zonulina entre pacientes com DII e grupo controle (95,28 ng/mL vs 96,61 ng/mL; P=0,573), assim como entre pacientes com DC e pacientes com colite ulcerativa (79,68 ng/mL vs 106,10 ng/mL, P=0,887). Dentre os pacientes com DII, as concentrações de zonulina foram mais elevadas no sexo feminino e correlacionaram-se positivamente com o índice de massa corporal (IMC) e com a idade, correlacionando-se negativamente com os níveis de hemoglobina e hematócrito. Nos pacientes com colite ulcerativa, as concentrações de zonulina correlacionaram-se negativamente com os parâmetros hemoglobina, hematócrito e albumina e, positivamente, com o IMC e com o SCCAI. Dentre os pacientes com DC, a zonulina sérica correlacionou-se positivamente com a idade e com o IMC, mas não com o IHB. Não foram observadas correlações entre os níveis de zonulina e as citocinas circulantes nos pacientes com DII. Conclusão: Nesta coorte constituída majoritariamente por pacientes em remissão clínica, os níveis séricos de zonulina não se mostraram aumentados em pacientes com DII em relação a indivíduos controles e correlacionaram-se com variáveis não relacionadas à doença de base, como com o sexo, com a idade e com o IMC. No entanto, os níveis séricos de zonulina correlacionaram-se com parâmetros clínicos e laboratoriais de gravidade e atividade da doença dentre os pacientes com colite ulcerativa, mas não dentre os pacientes com DC. Estes achados indicam um potencial papel da zonulina sérica como um biomarcador na DII, principalmente na colite ulcerativa.

B-cell compartment abnormalities are associated with ACLF and mortality in patients with liver cirrhosis.

BACKGROUND: Liver cirrhosis profoundly affects the immune system, leading to an immunological imbalance known as cirrhosis-associated immune dysfunction. AIMS: This study aimed to investigate B-cell disturbances in patients with acute decompensation (AD) of cirrhosis and assess relationships with prognosis and mortality. METHODS: The study included 39 patients with AD of cirrhosis, 29 patients with stable cirrhosis (SC), and 30 healthy controls (CTR). Circulating B-cell subsets and cytokine plasma levels were determined by flow cytometry. RESULTS: Cirrhotic groups showed higher percentages of naïve B cells, and lower percentages of CD27+ memory B cells (MBCs) than CTR. Further analysis comparing SC and AD revealed that the latter had higher frequencies of double-negative (DN) B cells and plasmablasts. Patients with more advanced liver disease exhibited a B-cell maturation shift toward MBCs and plasmablasts. Acute-on-chronic liver failure (ACLF) was associated with higher DN frequency. The Kaplan-Meier one-year survival probability was 92.9% in patients with >1.3% of transitional B cells and 27.3% in patients with <1.3%. CONCLUSIONS: B-cell subsets are markedly altered in cirrhotic patients, and cell profiles differ between stable and decompensated liver disease. Increased frequencies of DN B cells and reduced proportions of transitional B cells may be of great relevance in predicting ACLF and mortality, respectively.

Patterns of dendritic cell and monocyte subsets are associated with disease severity and mortality in liver cirrhosis patients.

Liver cirrhosis is often complicated by an immunological imbalance known as cirrhosis-associated immune dysfunction. This study aimed to investigate disturbances in circulating monocytes and dendritic cells in patients with acute decompensation (AD) of cirrhosis. The sample included 39 adult cirrhotic patients hospitalized for AD, 29 patients with stable cirrhosis (SC), and 30 healthy controls (CTR). Flow cytometry was used to analyze monocyte and dendritic cell subsets in whole blood and quantify cytokines in plasma samples. Cirrhotic groups showed higher frequencies of intermediate monocytes (iMo) than CTR. AD patients had lower percentages of nonclassical monocytes than CTR and SC. Cirrhotic patients had a profound reduction in absolute and relative dendritic cell numbers compared with CTR and showed higher plasmacytoid/classical dendritic cell ratios. Increased plasma levels of IL-6, IL-10, and IL-17A, elevated percentages of CD62L+ monocytes, and reduced HLA-DR expression on classical monocytes (cMo) were also observed in cirrhotic patients. Patients with more advanced liver disease showed increased cMo and reduced tissue macrophages (TiMas) frequencies. It was found that cMo percentages greater than 90.0% within the monocyte compartment and iMo and TiMas percentages lower than 5.7% and 8.6%, respectively, were associated with increased 90-day mortality. Monocytes and dendritic cells are deeply altered in cirrhotic patients, and subset profiles differ between stable and advanced liver disease. High cMo and low TiMas frequencies may be useful biomarkers of disease severity and mortality in liver cirrhosis.

Low sodium to potassium ratio in spot urine sample is associated with progression to acute kidney injury and mortality in hospitalized patients with cirrhosis.

BACKGROUND: sodium to potassium ratio in spot urine sample (Na/Kur) is a surrogate marker of sodium excretion that is recommended for the management of patients with ascites due to cirrhosis. AIMS: to investigate Na/Kur ratio and fractional excretion of sodium (FENa) in patients admitted with decompensated cirrhosis, evaluating its relationship with acute kidney injury (AKI) and prognosis. METHODS: prospective cohort study included 225 adult subjects. Urine samples were obtained within 48 h of hospitalization. RESULTS: AKI at admission was observed in 32.9% of patients and was associated with lower Na/Kur ratio, but not FENa. Among 151 subjects initially without kidney dysfunction, AKI at some point during hospitalization occurred in 26.2% and was independently associated with low Na/Kur ratio at admission. AKI was observed in 44% of the patients with Na/Kur ratio < 1 and only in 8% when values ≥ 2. Na/Kur ratio at admission was independently associated with 30-day mortality, with Kaplan-Meier survival probability of 78.8% for Na/Kur ratio < 1 and 93.6% for values ≥ 1. CONCLUSIONS: low Na/Kur ratio in spot urine sample is associated with progression to AKI and lower short-term survival in patients hospitalized for decompensated cirrhosis.

Prevalence of Factor V Leiden in a healthy population in Santa Catarina, Southern Brazil.

Thrombophilic disorders are found in 50% of patients with venous thromboembolism, and factor V Leiden (FVL) is the most common genetic risk factor for the development of these conditions. FVL prevalence varies according to population group. In Europe, many countries have a high prevalence of the mutation, including Portugal, Germany, and Italy. Santa Catarina State, southern Brazil, was colonized by different European nations; most inhabitants are descendants of Portuguese, Italian, and German immigrants. There are, however, no data on the prevalence of FVL in the state. This study aimed to determine FVL prevalence in a healthy population in Santa Catarina and assess whether there is an association between the mutation and demographic characteristics, thereby contributing to the understanding of the heterogeneity of prevalence of this important VTE risk factor and racial or geographical differences in the incidence of thrombotic diseases. Analysis of the FVL mutation was performed on 400 blood donors using the PCR technique followed by enzymatic digestion. The findings show that 2.5% of the participants were heterozygous for FVL, and none were homozygous. No association was found between the presence of FVL in heterozygosis and individual characteristics. In conclusion, this study found a prevalence of FVL in heterozygosis of 2.5% among healthy individuals in Santa Catarina, Brazil. Further studies are needed to assess the prevalence of FVL in other regions of the country, determine the distribution of the mutation among population groups, and evaluate how these factors affect the incidence of thrombotic diseases.

Elevated neopterin levels are associated with acute-on-chronic liver failure and mortality in patients with liver cirrhosis.

BACKGROUND: Macrophage activation plays a central role in hepatic and systemic inflammation and is involved in the pathogenesis of acute-on-chronic liver failure (ACLF). AIMS: This study aimed to investigate neopterin levels in patients admitted for acute decompensation (AD) of cirrhosis, evaluating its relationship with ACLF and prognosis. METHODS: This prospective cohort study included 205 adult subjects hospitalized for AD of cirrhosis. Twenty-one healthy subjects and 89 patients with stable cirrhosis were evaluated as controls. RESULTS: Circulating neopterin was higher in AD as compared to stable cirrhosis and healthy controls (p<0.001). ACLF was independently associated with higher neopterin levels (OR 1.015, 95% CI 1.002-1.028, p = 0.025). In the multivariate Cox regression analysis, neopterin levels (HR = 1.002, IC 95% 1.000-1.004, p = 0.041), Child-Pugh class C, and ACLF were predictors of 30-day survival. Among patients with ACLF, the Kaplan-Meier survival probability was 71.4% in those with neopterin levels < 25 nmol/L and 31.0% if neopterin ≥ 25 nmol/L (p<0.001). CONCLUSIONS: Higher circulating neopterin was associated with ACLF in patients hospitalized for AD of cirrhosis. Neopterin levels were also independently predictors of high short-term mortality, especially among patients with ACLF, and could represent a useful biomarker of macrophage activation in clinical practice.

MicroRNA profiles in serum samples from Acute-On-Chronic Liver Failure patients and miR-25-3p as a potential biomarker for survival prediction.

Acute-on-chronic liver failure (ACLF) is a condition characterized by acute decompensation of cirrhosis, associated with organ failure(s), and high short-term mortality. The microRNAs or miRNAs are small non-coding RNA molecules, stable in circulating samples such as biological fluids, and the difference in expression levels may indicate the presence, absence and/or stage of the disease. We analyzed here the miRNA profiling to identify potential diagnostic or prognostic biomarkers for ACLF. The major miRNAs discovered were validated in a cohort of patients with acute decompensation of cirrhosis grouped in no ACLF or ACLF according to EASL-CLIF definition. Relationship between serum miRNAs and variables associated with liver-damage and survival outcomes were verified to identify possible prognostic markers. Our results showed twenty altered miRNAs between no ACLF and ACLF patients, and twenty-seven in patients who died in 30 days compared with who survived. In validation phase, miR-223-3p and miR-25-3p were significantly altered in ACLF patients and in those who died in 30 days. miR-223-3p and miR-25-3p expression were associated with the lowest survival in 30 days. The decrease in miR-223-3p and miR-25-3p expression was associated with the presence of ACLF and poor prognosis. Of these, miR-25-3p was independently related to ACLF and 30-day mortality.

Applicability of Sepsis-3 criteria and quick Sequential Organ Failure Assessment in patients with cirrhosis hospitalised for bacterial infections.

BACKGROUND & AIMS: An algorithm including Sepsis-3 criteria and quick Sequential Organ Failure Assessment (qSOFA) was recently proposed to predict severity of infection in cirrhosis. However, its applicability among patients without a baseline SOFA available for Sepsis-3 definition is unknown. We sought to investigate the applicability and prognostic value of qSOFA and Sepsis-3 criteria in patients with cirrhosis hospitalised for bacterial infections, without pre-hospitalisation SOFA. METHODS: In this cohort study, 164 patients were followed up to 30 days. Data collection, including the prognostic models, was performed at admission and at day-3. RESULTS: All patients fulfilled Sepsis-3 criteria (admission SOFA ≥ 2) and, therefore, admission Sepsis-3 was not included in further analysis. Admission qSOFA was an independent predictor of survival (HR = 2.271, P = 0.015). For patients initially classified as high risk by qSOFA, Chronic Liver Failure - Sequential Organ Failure Assessment (CLIF-SOFA) was the only prognostic predictor. Among patients initially classified as low risk by qSOFA, the following parameters evaluated at day-3 were independent predictors of survival: qSOFA, acute-on-chronic liver failure, and Child-Pugh classification. Although not independently related to survival, Sepsis-3 criteria at day-3 was associated with lower 30-day survival in Kaplan-Meier analysis (66% vs 85%, P = 0.008). However, prognosis was better predicted by day-3 qSOFA, with 30-day Kaplan-Meier survival probability of 88% when qSOFA < 2 and 24% among those with qSOFA ≥ 2. CONCLUSION: Sepsis-3 criteria evaluated at admission are very limited in infected patients with cirrhosis without baseline SOFA. qSOFA was independently related to survival and appears to be a valuable tool for determining severity of infection and to follow patients initially classified as low risk.

MicroRNA profiles in serum samples from patients with stable cirrhosis and miRNA-21 as a predictor of transplant-free survival.

MicroRNAs (miRNAs) have remarkable potential as diagnostic and prognostic markers because of their roles in disease pathogenesis. miRNAs can be released into the bloodstream, where they are sufficiently stable to be detected noninvasively. Here, we prospectively evaluated serum levels of miR-21, miR-34a, miR-122, miR-181b, and miR-885-5p in patients with stable cirrhosis. Total RNA was extracted from the sera of patients with cirrhosis and healthy individuals, and the expression levels of the target miRNAs were analyzed by reverse transcription-quantitative polymerase chain reaction. Serum miRNAs levels were correlated with liver function parameters, etiology, and complications of cirrhosis. Circulating miR-34a, miR-122, and miR-885-5p levels were higher in patients with cirrhosis than in healthy individuals. These miRNAs were positively correlated with alanine aminotransferase and aspartate aminotransferase levels, and the relative expression levels were higher in hepatitis C virus-infected patients and lower in patients with Child-Pugh C cirrhosis. miR-122 and miR-885-5p levels were also positively correlated with γ-glutamyl transpeptidase concentrations. miR-21 was associated with transplant-free survival in univariate Cox regression analysis and remained independently associated with survival after adjustment for age, Child-Pugh classification, Model for End-stage Liver Disease score, and history of previous decompensation in multivariate Cox regression analysis. These data suggested that miR-34a, miR-122, and miR-885-5p levels may be more related to the inflammatory process and ongoing hepatocyte damage in patients with cirrhosis. Moreover, miR-21 levels were independently associated with shorter transplant-free survival and may be used as a prognostic tool in outpatients with stable cirrhosis.

A composição química dos cálculos da vesícula biliar - um estudo em pacientes de Santa Catarina / The chemical composition of gallstones: a study in patients from Santa Catarina

Objetivo: investigar a composição química dos cálculos da vesícula biliar em pacientes colecistectomizados no Hospital Universitário (HU) da Universidade Federal de Santa Catarina (UFSC) naturais e procedentes do estado de Santa Catarina e relacionar com parâmetros epidemiológicos e fatores de risco. Método: os cálculos de 117 pacientes naturais e procedentes do estado de Santa Catarina operados consecutivamente em 2015 no HU - UFSC foram analisados por método bioquímico para determinação da sua composição sendo classificados em cálculos de colesterol, mistos e pigmentados. Os pacientes doadores foram submetidos à entrevista para coleta de dados demográficos e epidemiológicos. Dosagens séricas de colesterol e triglicerídeos também foram realizadas. Resultados: a análise dos dados revelou que os cálculos de colesterol foram predominantes seguidos dos do tipo misto e por último dos pigmentados. Os fatores de risco tradicionais para litíase mostraram significância estatística. Conclusão: este estudo confirma a maior prevalência de cálculos de colesterol em uma população geral. Os fatores de risco clássicos não se associam a tipos específicos de cálculos biliares. Os cálculos tipo pigmentados não foram significativamente associados com a presença dos fatores de risco clássicos para colelitíase.

Objective: to determine the chemical composition of gallstones in patients submitted to cholecistectomy in the University Hospital (HU) at the Federal University of Santa Catarina ( UFSC) and correlate with epidemiological features and risk factors. Methods: Gallstones of 117 patients born and living in Santa Catarina state who undergone cholecystectomy at the UFSC / HU in 2015 were analyzed to determine their chemical composition and classified in: cholesterol, mixed and pigmented types. The patients were interviewed to obtain data about demographics, epidemiological features and risk factors for lithiasis. Blood samples for cholesterol and triglycerides levels were also taken. Results: Data analysis revealed that cholesterol stones were predominant followed by mixed and pigmented ones. Conclusion: This study confirms greater prevalence of cholesterol stones in a general population. Classical risk factors are not associated with specific types of gallstones. Pigmented stones were not significantly associated with classical risk factors for cholelithiasis.

Evaluation of CD307a expression patterns during normal B-cell maturation and in B-cell malignancies by flow cytometry.

BACKGROUND: Flow cytometric immunophenotyping is deemed a fundamental tool for the diagnosis of B-cell neoplasms. Currently, the investigation of novel immunophenotypic markers has gained importance, as they can assist in the precise subclassification of B-cell malignancies by flow cytometry. Therefore, the purpose of the present study was to evaluate the expression of CD307a during normal B-cell maturation and in B-cell malignancies as well as to investigate its potential role in the differential diagnosis of these entities. METHODS: CD307a expression was assessed by flow cytometry in normal precursor and mature B cells and in 115 samples collected from patients diagnosed with precursor and mature B-cell neoplasms. CD307a expression was compared between neoplastic and normal B cells. RESULTS: B-acute lymphoblastic leukemia cases exhibited minimal expression of CD307a, displaying a similar expression pattern to that of normal B-cell precursors. Mantle cell lymphoma (MCL) cases showed the lowest levels of CD307a among mature B-cell neoplasms. CD307a expression was statistically lower in MCL cases than in chronic B lymphocytic leukemia (CLL) and marginal zone lymphoma (MZL) cases. No statistical differences were observed between CD307a expression in neoplastic and normal plasma cells. CONCLUSION: These results indicate that the assessment of CD307a expression by flow cytometry could be helpful to distinguish CLL from MCL, and the latter from MZL. Although these results are not entirely conclusive, they provide a basis for further studies in a larger cohort of patients. © 2018 International Clinical Cytometry Society.

S-nitrosylation of Mycobacterium tuberculosis tyrosine phosphatase A (PtpA) induces its structural instability.

S-nitrosylation is associated with signal transduction and microbicidal activity of nitric oxide (NO). We have recently described the S-nitrosylation of Mycobacterium tuberculosis protein tyrosine phosphatase A, PtpA, an enzyme that plays an important role in mycobacteria survival inside macrophages. This post-translational modification decreases the activity of the enzyme upon modification of a single Cys residue, C53. The aim of the present work was the investigation of the effect of S-nitrosylation in PtpA kinetic parameters, thermal stability and structure. It was observed that the K(M) of nitrosylated PtpA was similar to its unmodified form, but the V(max) was significantly reduced. In contrast, treatment of PtpA C53A with GSNO, did not alter either K(M) or V(max). These results confirmed that PtpA S-nitrosylation occurs specifically in the non-catalytic C53 and that this modification does not affect substrate affinity. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy techniques it was shown that PtpA S-nitrosylation decreased protein thermal stability and promoted a local effect in the surroundings of the C53 residue, which interfered in both protein stability and function.

Mycobacterium tuberculosis tyrosine phosphatase A (PtpA) activity is modulated by S-nitrosylation.

M. tuberculosis PtpA and PtpB, the only two phosphotyrosine phosphatases (Ptps) present in this pathogen, play an important role in mycobacteria survival inside macrophages. The aim of the present work was to investigate M. tuberculosis PtpA and PtpB susceptibility to S-nitrosylation, a reversible covalent bond between nitric oxide (NO) and specific cysteine (sulfur) residues in proteins. PtpB was not modified by NO, in contrast, PtpA Cys53 was identified by site directed mutagenesis as the target of S-nitrosylation.

A transthyretin-related protein is functionally expressed in Herbaspirillum seropedicae.

Transthyretin-related proteins (TRPs) constitute a family of proteins structurally related to transthyretin (TTR) and are found in a large range of bacterial, fungal, plant, invertebrate, and vertebrate species. However, it was recently recognized that both prokaryotic and eukaryotic members of this family are not functionally related to transthyretins. TRPs are in fact involved in the purine catabolic pathway and function as hydroxyisourate hydrolases. An open reading frame encoding a protein similar to the Escherichia coli TRP was identified in Herbaspirillum seropedicae genome (Hs_TRP). It was cloned, overexpressed in E. coli, and purified to homogeneity. Mass spectrometry data confirmed the identity of this protein, and circular dichroism spectrum indicated a predominance of beta-sheet structure, as expected for a TRP. We have demonstrated that Hs_TRP is a 5-hydroxyisourate hydrolase and by site-directed mutagenesis the importance of three conserved catalytic residues for Hs_TRP activity was further confirmed. The production of large quantities of this recombinant protein opens up the possibility of obtaining its 3D-structure and will help further investigations into purine catabolism.