Ouabain Decreases Regulatory T Cell Number in Mice by Reducing IL-2 Secretion.

BACKGROUND: Ouabain (OUA) is a cardiotonic glycoside originally extracted from African plants. It has also been described as an endogenous component in mammals, being released in stress situations mainly by the adrenal gland. OUA has been reported to be capable of inhibiting mitogen-induced lymphocyte proliferation and also affects B and T lymphocytes. OBJECTIVES: The aim of this work is to show the effects of OUA in peripheral T lymphocytes. METHODS: In the in vivo experiments, mice were injected intraperitoneally for 3 consecutive days with RPMI medium (control group) or 0.56 mg/kg of OUA diluted in RPMI medium (OUA group). On the fourth day, spleen or mesenteric lymph nodes were removed. RESULTS: OUA significantly reduced the number of CD4+ T lymphocytes in the spleen, especially regulatory T cells (Tregs). In vitro OUA did not inhibit the proliferation of CD4+T lymphocytes stimulated with anti-CD3 neither was able to induce the apoptosis of CD4+ nor Tregs. There was no increase in the number or percentage of T lymphocytes in the mesenteric lymph nodes, suggesting that there was no preferential accumulation of these cells in this organ. Secretion of IL-2 by activated T lymphocytes was decreased by the OUA, explaining at least in part the reduction of Tregs, since this cytokine is involved in the peripheral conversion and maintenance of Tregs. CONCLUSION: The impact of this reduction in autoimmune diseases, allergy and cancer as well as the potential use of OUA as a therapeutic approach in tumor treatment still needs more investigation.

Coffee and herbal tea consumption is associated with lower liver stiffness in the general population: The Rotterdam study.

BACKGROUND & AIMS: Coffee and tea have been proposed to limit the progression of liver fibrosis in established liver disease, but it is unknown if this is also true for subclinical fibrosis. We therefore aimed to evaluate whether coffee and tea consumption are associated with liver stiffness in the general population. METHODS: The Rotterdam Study is an ongoing prospective population-based cohort. We included participants who underwent transient elastography, ultrasound and completed a food frequency questionnaire. Coffee and tea consumption were categorized into no, moderate (>0-3), or frequent (⩾3) intake (cups/day), and tea further into green, black and herbal tea (no/any). Significant fibrosis was defined as liver stiffness measurements (LSM) ⩾8.0kPa. We performed regression analyses relating coffee and tea intake with fibrosis, steatosis and log-transformed LSM and adjusted for energy, sugar and creamer intake, age, gender, BMI, steatosis/LSM, HOMA-IR, ALT, alcohol, smoking, soda, healthy diet index and physical activity. RESULTS: We included 2,424 participants (age 66.5±7.4; 43% male) of whom 5.2% had LSM ⩾8.0kPa and 34.6% steatosis. Proportion of LSM ⩾8.0kPa decreased with higher coffee consumption (7.8%, 6.9% and 4.1% for no, moderate and frequent respectively; Ptrend=0.006). This inverse association was confirmed in multivariable regression (ORmod 0.75, 95% CI 0.33-1.67; ORfreq 0.39, 95% CI 0.18-0.86; p=0.005). Amongst tea consumers, only herbal tea consumers (36.3%) had lower log-transformed LSM after adjustment (Beta-0.05, 95% CI-0.08;-0.02, p=0.001). Subtypes of tea were associated with steatosis in univariate but not multivariable analysis. CONCLUSIONS: In the general population, frequent coffee and herbal tea consumption were inversely related with liver stiffness but not steatosis. Longitudinal analyses, as well as studies validating and unravelling underlying mechanisms are needed. LAY SUMMARY: The Rotterdam Study is a large ongoing population study of suburban inhabitants of Rotterdam in whom data on liver stiffness, as proxy for liver fibrosis, presence of fatty liver on ultrasound and detailed information on coffee and tea consumption were obtained in 2,424 participants. The consumption of herbal tea and daily consumption of three or more cups of coffee was related to the presence of lower liver stiffness, independent of a great number of other lifestyle and environmental factors. Previous studies have found a protective effect of coffee on established liver disease and we now show for the first time that this effect is already measurable in the general population.

Age-standardized mortality rates related to viral hepatitis in Brazil.

BACKGROUND: Liver-related mortality has been increasing worldwide. We aimed to estimate the age-standardized mortality rates from viral hepatitis in Brazil. METHODS: The Brazilian National Death Registry was analyzed from 2008 to 2014. Viral hepatitis deaths were defined by the following ICD-10 codes in the death certificate: hepatitis A [B15.0; B15.9]; hepatitis B [B16.2; B16.9; B18.1]; hepatitis C [B17.1; B18.2]; hepatitis Delta [B16.0; B16.1; B18.0; B17.0] and other viral hepatitis [B17.2; B17.8; B18.8; B18.9; B19.0; B19.9]. Crude mortality rates were calculated by the ratio between total number of deaths and estimated population. Mortality rates were age-standardized by the direct method using the WHO standard population. RESULTS: Thirty four thousand ,nine hundred seventy eight deaths had viral hepatitis mentioned in their death certificate [65% male, aged 58 years, 73% associated with hepatitis C]. Age-standardized mortality rate (95% CI) due to viral hepatitis was 2.695 (2.667-2.724) deaths per 100,000 inhabitants: South region had the higher rates [3.997 (3.911-4.085)]. Mortality rates associated with hepatitis A and Delta were 0.032 (0.029-0.035) and 0.028 (0.025-0.031), respectively. Hepatitis C mortality rates were 4-fold higher than those associated with hepatitis B [1.964 (1.940-1.989) vs 0.500 (0.488-0.512)]. South region had the higher rates for hepatitis C [3.163 (3.087-3.241)] and North had the higher rates for hepatitis A [0.066 (0.049-0.087)], B [0.986 (0.918-1.058)] and Delta [0.220 (0.190-0.253)]. CONCLUSION: Viral hepatitis remains a major public health issue in Brazil. Mortality rates were not homogeneous across the country, suggesting that health policies should be customized according to geographical location.

Learning curve and intra/interobserver agreement of transient elastography in chronic hepatitis C patients with or without HIV co-infection.

BACKGROUND AND OBJECTIVES: Liver stiffness measurement (LSM) by transient elastography has been validated as a noninvasive method to stage liver fibrosis. Few studies have evaluated the learning curve of this method and its reproducibility has led to controversy results. We aimed to evaluate the intra- and interobserver agreement of transient elastography as well as its learning curve for definition of an experimented operator. METHODS: We retrospectively analyzed 922 examinations performed in 544 patients during a training program of transient elastography. Patients with chronic hepatitis C with or without HIV co-infection that had two examinations by the training operator (intraobserver analysis; n=125) or examination by both training and experimented operators (interobserver analysis; n=151) in the same day were included. LSM was converted to METAVIR score: <7.1 as F0F1, 7.1-9.4 as F2, 9.5-12.4, as F3 and >12.4 kPa as F4. RESULTS: The overall intra- and interobserver intraclass correlation coefficient [ICC 95% CI] were 0.926 (0.901-0.951) and 0.912 (0.885-0.939), respectively. Measurements were correlated [Spearman's] in intra- [0.906, P<0.0001] and interobserver [0.907, P<0.0001] analysis. Reliability values [kappa (SE)] were k=0.74 (0.09) and k=0.85 (0.08) for fibrosis stages F ≥ 2 and k=0.77 (0.09) and k=0.75 (0.08) for cirrhosis in intra- and interobserver analysis, respectively. Agreement was improved when operator's experience was higher than 100 exams. However, it was observed discordance for fibrosis staging between examinations in a quarter of patients. CONCLUSION: Although there was a considerable discrepancy on fibrosis staging between examinations and a small power, transient elastography had an acceptable reproducibility in our population. Performance of at least 100 examinations should be used to define an experimented operator.

Dynamics of murine B lymphocytes is modulated by in vivo treatment with steroid ouabain.

Ouabain (OUA) is a steroid hormone capable of inhibiting the protein Na+K+ATPase present in the plasma membrane of cells. Ouabain was initially extracted from the roots of African trees such as Acocanthera ouabaio and Strophantus gratus seeds and later described as an endogenous component found in higher mammals. The adrenal gland is the main site of synthesis of ouabain and it is released in stressful situations, conditions similar to those where there is secretion of corticosteroids. Immunological functions have been shown to be regulated by ouabain. In order to understand the effects of ouabain on B lymphocyte populations in different lymphoid organs, mice received intraperitoneal injections of ouabain for 3 consecutive days. Twenty-four hours after the last injection, cells were analyzed by flow cytometry. In the spleen, ouabain modulated especially follicular B cells, inducing a significant decrease in the percentage and absolute numbers of those cells. Ouabain also reduced the absolute number of marginal zone B lymphocytes. No difference in the percentage or absolute number of B lymphocytes in the spleen forty-eight hours after the last injection was observed. An increase in the number of B cells was seen in mesenteric lymph nodes and this retention appears to be directly related to increased expression of CXCR5 chemokine receptor and reduction of CD62L, which also explains the observed reduction of B cells in the spleen. Our results indicate that ouabain regulates the dynamics of B lymphocytes in peripheral organs but production of total IgM and IgG in the serum of animals treated in vivo with ouabain was not affected.

Cytokines and neutrophils as important mediators of platelet-activating factor-induced kinin B1 receptor expression.

PAF injection into the rat paw is accompanied by the concomitant activation of NF-kappaB and neutrophil influx, which appears to be relevant to the up-regulation of kinin B1 receptors. Herein, we analyse the role of TNF-alpha and IL-1beta production for PAF-induced B1 receptor upregulation in the rat paw. Additionally, we evaluate how cytokine production and neutrophil migration fit into the temporal sequence of events leading to PAF-induced B1 receptor upregulation. In our experiments, treatment with PAF resulted in a marked increase of B1 receptor-mediated paw oedema and in situ production of TNF-alpha at 1 h and IL-1beta at 3 and 6 h later. B1 receptor-mediated paw oedema was significantly inhibited by anti-TNF-alpha antibody and by interleukin-1 receptor antagonist (IRA). TNF-alpha was necessary for the local PAF-induced IL-1beta production. NF-kappaB blocker PDTC prevented the production of both TNF-alpha and IL-1beta, indicating that cytokine production is NF-kappaB dependent. Depletion of neutrophils with an anti-PMN antibody prevented IL-1beta, but not TNF-alpha, production. Although both TNF-alpha and IL-1beta are relevant to functional B1 receptor upregulation, PAF-induced increase in B1 receptor mRNA was markedly suppressed by anti-TNF-alpha and, to a lesser extent, by IRA. B1 receptor mRNA expression was also prevented by the anti-PMN antibody. In conclusion, the activation of the TNF-alpha/neutrophil axis by PAF seems to be sufficient for B1 receptor mRNA production. However, the TNF-alpha/neutrophil axis is also necessary for IL-1beta production. These two processes might lead to the appearance of functional kinin B1 upregulation receptors in vivo after PAF treatment.

Association of Schistosoma mansoni-specific IgG and IgE antibody production and clinical schistosomiasis status in a rural area of Minas Gerais, Brazil.

BACKGROUND: Studies in murine models and human populations have indicated that the collagen-rich granulomatous response against parasite eggs trapped in the liver is associated with the development of severe hepatosplenic schistosomiasis, characterized by periportal fibrosis and portal hypertension. The role of the humoral response in parasite susceptibility has been well established, but its participation in disease severity remains poorly understood. In this work, we evaluated the relationship between parasite-reactive IgE and IgG levels and schistosomiasis morbidity in infected patients with similar parasite burdens. METHODOLOGY/PRINCIPAL FINDINGS: Ninety-seven Schistosoma mansoni-infected individuals were subjected to clinical examination and abdominal ultrasound analysis. IgG reactivity and IgE concentration against Schistosoma mansoni soluble egg antigens (SEA) and adult worm antigen preparation (SWAP) were evaluated by ELISA assay. Multivariable linear regression models were used to evaluate the relationship between parasite-reactive antibodies and the co-variables investigated. The study population showed low parasite burden (median 30 eggs/g feces), constant re-infection, and signs of fibrosis was detected in more than 30% of individuals. Most infected individuals showed IgG reactivity, and the median concentrations of IgE anti-SEA and anti-SWAP antibodies were 1,870 and 1,375 ng/mL, respectively. There was no association between parasite burden and antibody response or any parameter of disease severity. However, IgG anti-SWAP level was positively associated with morbidity parameters, such as spleen size and thickness of portal vein at the entrance and secondary branch. In contrast, the data also revealed independent inverse correlations between concentration of parasite-reactive IgE and gallbladder wall thickness, a marker of fibrosis in schistosomiasis. CONCLUSIONS/SIGNIFICANCE: The data indicate that IgG anti-SWAP is positively associated with severe schistosomiasis, independently of parasite burden, while high production of parasite-specific IgE is associated with mild disease in the human population. Antibody profiles are good correlates for schistosomiasis severity and could be tested as biomarkers of disease severity.

Age-Standardized Mortality Rates Related to Cirrhosis in Brazil from 2000 to 2012: A Nationwide Analysis

ABSTRACT Background. Cirrhosis remains the most frequent liver-related cause of death worldwide and we aimed to evaluate its burden in Brazil from 2000 to 2012. Material and methods. The Brazilian National Death Registry was analyzed from 2000 to 2012. Death by cirrhosis was defined by the presence of I85, K73 and/or K74 ICD 10 codes in contributing or underlying causes of death on the death certificate (DC). Crude mortality rates were calculated as the ratio of the absolute number of deaths and the estimated population. Mortality rates were age-adjusted by the direct standardization method using the WHO standard population. Results. A total of 265,180 deaths where cirrhosis was mentioned on the DC [77% male, aged 56 years] occurred from 2000 to 2012. Cirrhosis codes were present in 46% of liver-related deaths and 2% of all deaths in this period. Despite an increase in the absolute number of deaths (n = 18,245 to 22,340), the age-standardized mortality rates (95%CI) decreased from 13.32 (13.16-13.48) to 11.71 (11.59-11.83) per 100,000 inhabitants from 2000 to 2012 (p < 0.001). This trend was not uniform across the country, with decreases in death rates in the South [14.46 (14.07-14.87) to 10.89 (10.59-11.19)] and Southeast [15.85 (15.6-16.09) to 12.52 (12.34-12.70)] and increases in the North [8.84 (8.24-9.43) to 11.53 (11.08-11.99)] and Northeast [9.41 (9.13-9.69) to 10.93 (10.68-11.17)] (p < 0.001 for all). Conclusion. Cirrhosis remains a major public health issue, despite the reduction in mortality rates in the last decade.