Impact of Sirtuin 2 knockout on innate immune responses

Purpose/Objective: Histone deacetylases (HDACs) deacetylate histones and transcriptional regulators thereby affecting numerous biological functions. Seven mammalian sirtuins (SIRT1-7) constitute the NAD-dependent class III subfamily of HDACs. Sirtuins are the center of great interest due to their regulatory role in the control of metabolism, ageing and age-related diseases. Up to now, little is known about the influence of sirtuins on immune responses, and nothing about the role of SIRT2. The aim of the study was to analyze the influence of SIRT2 knockout on immune cell development and innate immune responses in vitro and in vivo. Materials and methods: SIRT2 germline knockout were produced on a C57BL/6J background. The cellularity of thymus and spleen was assessed by flow cytometry (n = 3). Bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs) and splenocytes were stimulated with LPS, Pam3CSK4 lipopeptide, CpG ODN, E. coli, S. aureus, TSST-1, SEB, anti-CD3+ CD28 and concanavalin A (n = 3_8). TNF, IL-2, IL-6, IL-12p40 and IFNc production, SIRT1_7 and CD40 expression, and proliferation were quantified by real time-PCR, ELISA, flow cytometry and H3-thymidine incorporation. Mice (n = 6_16) were challenged with LPS, TNF/D-galactosamine, E. coli and K. pneumonia titrated to cause either mild or severe infections or shock. Blood was collected to quantify cytokines and bacteria. Mortality was checked regularly. Results: SIRT2 is the most expressed sirtuin in macrophages and myeloid DCs. To test whether SIRT2 impacts on innate immune responses, we generated SIRT2 germline knockout mice. SIRT2-/- mice born at the expected Mendelian ratio and develop normally. The proportions and absolute numbers of DN1-4, DP and SP thymocytes, and of T-cells (DN and SP, naı¨ve and memory), B-cells (immature and mature), DCs (cDCs and pDCs) and granulocytes in the spleen are similar in SIRT2+/+ and SIRT2-/- mice. SIRT2+/+ and SIRT2-/- BMDMs, BMDCs and splenocytes produce cytokines (RNA and protein), upregulate CD40, and proliferate to the same extent. SIRT2+/+ and SIRT2-/- mice respond similarly (cytokine blood levels, bacterial counts and mortality) to non-severe and lethal endotoxemia, E. coli peritonitis, K. pneumonia pneumonia and TNF-induced shock. Conclusions: SIRT2 knockout has no dramatic impact on the development of immune cells and on innate immune responses in vitro and in vivo. Considering that SIRT2 may participate to control metabolic homeostasis, we are currently assessing the impact of SIRT2 deficiency on innate immune responses under metabolic stress.

High expression of co-stimulatory and adhesion molecules are observed on eosinophils during human Schistosoma mansoni infection

Herein we have focused attention on major phenotypic features of peripheral blood eosinophils from chronic Schistosoma mansoni-infected patients. For this purpose, detailed immunophenotypic profiles of a range of cell surface markers were performed, including activation markers (CD23/CD69/CD25/HLA-DR), co-stimulatory molecules (CD28/CD80/CD86), chemokine receptors (CXCR1/CXCR2/CCR3/CCR5) besides L-selectin-CD62L and adhesion molecules (CD18/CD54). Our major findings pointed out increased frequency of CD23+-cells, besides decreased percentages of CD69+-eosinophils, suggesting a chronic activation status with low frequency of early activated eosinophils in chronic S. mansoni-infected patients (INT) in comparison to non-infected individuals (NI). Moreover, a dichotomic expression of beta-chemokine receptors was observed during human schistosomiasis mansoni with higher CCR5 and lower levels of CCR3 observed between groups. Enhanced expression of co-stimulatory receptors (CD28/CD86) and adhesion molecules (CD54/CD18), besides striking lower frequency of L-selectin+ were reported for eosinophils from INT group as compared to NI. Interestingly, the frequency of CD62L+-eosinophils and a range of cell activation related molecules pointed out an opposite pattern of association in NI and INT, where only INT patients that display lower frequency of CD62L+-eosinophils (first CD62L tertile) kept the unusual relationship between the expression of L-selectin and the CD23 activation marker. These findings suggest that distinct dynamic of activation markers expressed by eosinophils may occur during chronic S. mansoni infection.

Reduction of alcohol consumption by brief alcohol intervention in primary care: systematic review and meta-analysis.

BACKGROUND: Numerous trials of the efficacy of brief alcohol intervention have been conducted in various settings among individuals with a wide range of alcohol disorders. Nevertheless, the efficacy of the intervention is likely to be influenced by the context. We evaluated the evidence of efficacy of brief alcohol interventions aimed at reducing long-term alcohol use and related harm in individuals attending primary care facilities but not seeking help for alcohol-related problems. METHODS: We selected randomized trials reporting at least 1 outcome related to alcohol consumption conducted in outpatients who were actively attending primary care centers or seeing providers. Data sources were the Cochrane Central Register of Controlled Trials, MEDLINE, PsycINFO, ISI Web of Science, ETOH database, and bibliographies of retrieved references and previous reviews. Study selection and data abstraction were performed independently and in duplicate. We assessed the validity of the studies and performed a meta-analysis of studies reporting alcohol consumption at 6 or 12 months of follow-up. RESULTS: We examined 19 trials that included 5639 individuals. Seventeen trials reported a measure of alcohol consumption, of which 8 reported a significant effect of intervention. The adjusted intention-to-treat analysis showed a mean pooled difference of -38 g of ethanol (approximately 4 drinks) per week (95% confidence interval, -51 to -24 g/wk) in favor of the brief alcohol intervention group. Evidence of other outcome measures was inconclusive. CONCLUSION: Focusing on patients in primary care, our systematic review and meta-analysis indicated that brief alcohol intervention is effective in reducing alcohol consumption at 6 and 12 months.

Patterns of co-association of C-reactive protein and nitric oxide in malaria in endemic areas of Iran

In addition to numerous immune factors, C-reactive protein (CRP) and nitric oxide (NO) are believed to be molecules of malaria immunopathology. The objective of this study was to detect CRP and NO inductions by agglutination latex test and Griess microassay respectively in both control and malaria groups from endemic areas of Iran, including Southeastern (SE) (Sistan & Balouchestan, Hormozgan, Kerman) and Northwestern (NW) provinces (Ardabil). The results indicated that CRP and NO are produced in all malaria endemic areas of Iran. In addition, more CRP and NO positive cases were observed amongst malaria patients in comparison with those in control group. A variable co-association of CRP/NO production were detected between control and malaria groups, which depended upon the malaria endemic areas and the type of plasmodia infection. The percentage of CRP/NO positive cases was observed to be lower in NW compare to SE region, which may be due to the different type of plasmodium in the NW (Plasmodium vivax) with SE area (P. vivax, Plasmodium falciparum, mixed infection). The fluctuations in CRP/NO induction may be consistent with genetic background of patients. Although, CRP/NO may play important role in malaria, their actual function and interaction in clinical forms of disease remains unclear.

In vitro activity of thienyl-2-nitropropene compounds against Trypanosoma cruzi

The in vitro activity of four 2-nitropropene derivatives, 1-(3-benzothienyl)-2-nitropropene (N1), 1-(3-thienyl)-2-nitropropene (N2), 1-(5-bromo-2-thienyl)-2-nitropropene (N3) and 1-(4-bromo-2-thienyl)-2-nitropropene (N4), were tested against cultures of the parasite Trypanosoma cruzi. Cytotoxicity studies were performed using Vero cells. The blood trypomastigotes, amastigotes and epimastigotes showed differential degrees of sensitivity towards the four tested compounds; the highest activity against the epimastigotes and blood tripomastigotes was exhibited by N1, followed by N3, N4 and finally N2. In contrast, whereas the compounds N1, N3 and N4 exerted similar magnitudes of activity against amastigotes, N2 was found to be a much less potent compound. According to our results, the compound N1 had the highest level of activity (IC50: 0.6 μM) against epimastigotes.

The effect of COX-2 inhibitors on the formation of heterotopic bone in a rat model : O1323

Aims: 1) to create a new and reproducible animal model to produce heterotopic ossification (HO) 2) to be able to exactly quantify the amount of HO using a microCT scan and 3) to prove the hypothesis that COX-2 inhibitors are efficacious in the prevention of HO. Methods: We developed a IACUC-approved Lewis rat model, in which the ventral side of the right femur was scraped to mechanically disrupt the periosteum. By clamping the vastus intermedius ischemic injury to the muscle was produced to enhance HO. Finally homologous bone marrow from a donor rat was placed on the anterior surface of the femur. Half of the study group (8 rats) received chow mixed with a COX-2 inhibitor, while the other half received normal chow. After 6 weeks the animals were sacrificed, the femurs removed and imaged by microCT. Grading of HO was based on the thickness of ectopic bone as evaluated in a blinded fashion by 3 independent observers. Results: All animals developed bilateral HO. Rats treated with COX-2 inhibitors developed significantly less ectopic bone than the control group rats. Conclusions: The results suggest that we have created a very reliable, reproducible model to form ectopic bone in rats. Using the microCT we can precisely quantify the amount of HO. We have been able to show that COX-2 inhibitors significantly decrease the amount of HO formation and are thus a good alternative to non-specific NSAIDs with their potential serious side effects on the gastrointestinal tract and on hemo-stastis.

Tea consumption and incidence of type 2 diabetes in Europe: the EPIC-InterAct case-cohort study.

BACKGROUND In previous meta-analyses, tea consumption has been associated with lower incidence of type 2 diabetes. It is unclear, however, if tea is associated inversely over the entire range of intake. Therefore, we investigated the association between tea consumption and incidence of type 2 diabetes in a European population. METHODOLOGY/PRINCIPAL FINDINGS The EPIC-InterAct case-cohort study was conducted in 26 centers in 8 European countries and consists of a total of 12,403 incident type 2 diabetes cases and a stratified subcohort of 16,835 individuals from a total cohort of 340,234 participants with 3.99 million person-years of follow-up. Country-specific Hazard Ratios (HR) for incidence of type 2 diabetes were obtained after adjustment for lifestyle and dietary factors using a Cox regression adapted for a case-cohort design. Subsequently, country-specific HR were combined using a random effects meta-analysis. Tea consumption was studied as categorical variable (0, >0-<1, 1-<4, ≥ 4 cups/day). The dose-response of the association was further explored by restricted cubic spline regression. Country specific medians of tea consumption ranged from 0 cups/day in Spain to 4 cups/day in United Kingdom. Tea consumption was associated inversely with incidence of type 2 diabetes; the HR was 0.84 [95%CI 0.71, 1.00] when participants who drank ≥ 4 cups of tea per day were compared with non-drinkers (p(linear trend) = 0.04). Incidence of type 2 diabetes already tended to be lower with tea consumption of 1-<4 cups/day (HR = 0.93 [95%CI 0.81, 1.05]). Spline regression did not suggest a non-linear association (p(non-linearity) = 0.20). CONCLUSIONS/SIGNIFICANCE A linear inverse association was observed between tea consumption and incidence of type 2 diabetes. People who drink at least 4 cups of tea per day may have a 16% lower risk of developing type 2 diabetes than non-tea drinkers.

Common variants of the liver fatty acid binding protein gene influence the risk of type 2 diabetes and insulin resistance in Spanish population

SUMMARY The main objective was to evaluate the association between SNPs and haplotypes of the FABP1-4 genes and type 2 diabetes, as well as its interaction with fat intake, in one general Spanish population. The association was replicated in a second population in which HOMA index was also evaluated. METHODS 1217 unrelated individuals were selected from a population-based study [Hortega study: 605 women; mean age 54 y; 7.8% with type 2 diabetes]. The replication population included 805 subjects from Segovia, a neighboring region of Spain (446 females; mean age 52 y; 10.3% with type 2 diabetes). DM2 mellitus was defined in a similar way in both studies. Fifteen SNPs previously associated with metabolic traits or with potential influence in the gene expression within the FABP1-4 genes were genotyped with SNPlex and tested. Age, sex and BMI were used as covariates in the logistic regression model. RESULTS One polymorphism (rs2197076) and two haplotypes of the FABP-1 showed a strong association with the risk of DM2 in the original population. This association was further confirmed in the second population as well as in the pooled sample. None of the other analyzed variants in FABP2, FABP3 and FABP4 genes were associated. There was not a formal interaction between rs2197076 and fat intake. A significant association between the rs2197076 and the haplotypes of the FABP1 and HOMA-IR was also present in the replication population. CONCLUSIONS The study supports the role of common variants of the FABP-1 gene in the development of type 2 diabetes in Caucasians.

Risk of type 2 diabetes according to traditional and emerging anthropometric indices in Spain, a Mediterranean country with high prevalence of obesity: results from a large-scale prospective cohort study.

Background: Obesity is a major risk factor for type 2 diabetes mellitus (T2DM). A proper anthropometric characterisation of T2DM risk is essential for disease prevention and clinical risk assessement. Methods: Longitudinal study in 37 733 participants (63% women) of the Spanish EPIC (European Prospective Investigation into Cancer and Nutrition) cohort without prevalent diabetes. Detailed questionnaire information was collected at baseline and anthropometric data gathered following standard procedures. A total of 2513 verified incident T2DM cases occurred after 12.1 years of mean follow-up. Multivariable Cox regression was used to calculate hazard ratios of T2DM by levels of anthropometric variables. Results: Overall and central obesity were independently associated with T2DM risk. BMI showed the strongest association with T2DM in men whereas waist-related indices were stronger independent predictors in women. Waist-to-height ratio revealed the largest area under the ROC curve in men and women, with optimal cut-offs at 0.60 and 0.58, respectively. The most discriminative waist circumference (WC) cut-off values were 99.4 cm in men and 90.4 cm in women. Absolute risk of T2DM was higher in men than women for any combination of age, BMI and WC categories, and remained low in normal-waist women. The population risk of T2DM attributable to obesity was 17% in men and 31% in women. Conclusions: Diabetes risk was associated with higher overall and central obesity indices even at normal BMI and WC values. The measurement of waist circumference in the clinical setting is strongly recommended for the evaluation of future T2DM risk in women.

Negative control of keratinocyte differentiation by Rho/CRIK signaling coupled with up-regulation of KyoT1/2 (FHL1) expression.

Rho GTPases integrate control of cell structure and adhesion with downstream signaling events. In keratinocytes, RhoA is activated at early times of differentiation and plays an essential function in establishment of cell-cell adhesion. We report here that, surprisingly, Rho signaling suppresses downstream gene expression events associated with differentiation. Similar inhibitory effects are exerted by a specific Rho effector, CRIK (Citron kinase), which is selectively down-modulated with differentiation, thereby allowing the normal process to occur. The suppressing function of Rho/CRIK on differentiation is associated with induction of KyoT1/2, a LIM domain protein gene implicated in integrin-mediated processes and/or Notch signaling. Like activated Rho and CRIK, elevated KyoT1/2 expression suppresses differentiation. Thus, Rho signaling exerts an unexpectedly complex role in keratinocyte differentiation, which is coupled with induction of KyoT1/2, a LIM domain protein gene with a potentially important role in control of cell self renewal.

High-frequency linkage of co-expressing T-DNA in transgenic Arabidopsis thaliana transformed by vacuum-infiltration of Agrobacterium tumefaciens

The efficiency of co-expression and linkage of distinct T-DNAs present in separate Agrobacterium tumefaciens was analysed in Arabidopsis thaliana transformed by the vacuum infiltration method. Co-expression was monitored by the synthesis of three bacterial proteins involved in the production of polyhydroxybutyrate (PHB) in the plastids. Out of 80 kanamycin-resistant transgenic plants analysed, 13 plants were co-transformed with the two distinct T-DNAs and produced PHB. Of those, 7 lines had a kanamycin-resistance segregation ratio consistent with the presence of a single functional insert. Genetic linkage between the distinct T-DNAs was demonstrated for all 13 PHB-producing lines, while physical linkage between the distinct T-DNAs was shown for 12 out of 13 lines. T-DNAs were frequently linked in an inverted orientation about the left borders. Transformation of A. thaliana by the co-infiltration of two A. tumefaciens containing distinct T-DNAs is, thus, an efficient approach for the integration and expression of several transgenes at a single locus. This approach will facilitate the creation and study of novel metabolic pathways requiring the expression of numerous transgenes.

Role of cyclooxygenase-2 in Trypanosoma cruzisurvival in the early stages of parasite host-cell interaction

Chagas disease, caused by the intracellular protozoan Trypanosoma cruzi, is a serious health problem in Latin America. During this parasitic infection, the heart is one of the major organs affected. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. When cells are infected with T. cruzi, they develop an inflammatory response, in which cyclooxygenase-2 (COX-2) catalyses rate-limiting steps in the arachidonic acid pathway. However, how the parasite interaction modulates COX-2 activity is poorly understood. In this study, the H9c2 cell line was used as our model and we investigated cellular and biochemical aspects during the initial 48 h of parasitic infection. Oscillatory activity of COX-2 was observed, which correlated with the control of the pro-inflammatory environment in infected cells. Interestingly, subcellular trafficking was also verified, correlated with the control of Cox-2 mRNA or the activated COX-2 protein in cells, which is directly connected with the assemble of stress granules structures. Our collective findings suggest that in the very early stage of the T. cruzi-host cell interaction, the parasite is able to modulate the cellular metabolism in order to survives.

Expression of mitofusin 2(R94Q) in a transgenic mouse leads to Charcot-Marie-Tooth neuropathy type 2A.

Charcot-Marie-Tooth disease type 2A is an autosomal dominant axonal form of peripheral neuropathy caused by mutations in the mitofusin 2 gene. Mitofusin 2 encodes a mitochondrial outer membrane protein that participates in mitochondrial fusion in mammalian cells. How mutations in this protein lead to Charcot-Marie-Tooth disease type 2A pathophysiology remains unclear. We have generated a transgenic mouse expressing either a mutated (R94Q) or wild-type form of human mitofusin 2 in neurons to evaluate whether the R94Q mutation was sufficient for inducing a Charcot-Marie-Tooth disease type 2A phenotype. Only mice expressing mitofusin 2(R94Q) developed locomotor impairments and gait defects thus mimicking the Charcot-Marie-Tooth disease type 2A neuropathy. In these animals, the number of mitochondria per axon was significantly increased in the distal part of the sciatic nerve axons with a diameter smaller than 3.5 microm. Importantly, the analysis of R94Q transgenic animals also revealed an age-related shift in the size of myelinated axons leading to an over-representation of axons smaller than 3.5 microm. Together these data suggest a link between an increased number of mitochondria in axons and a shift in axonal size distribution in mitofusin 2(R94Q) transgenic animals that may contribute to their neurological phenotype.

An autoregulatory loop controlling CYP1A1 gene expression: role of H(2)O(2) and NFI.

Cytochrome P450 1A1 (CYP1A1), like many monooxygenases, can produce reactive oxygen species during its catalytic cycle. Apart from the well-characterized xenobiotic-elicited induction, the regulatory mechanisms involved in the control of the steady-state activity of CYP1A1 have not been elucidated. We show here that reactive oxygen species generated from the activity of CYP1A1 limit the levels of induced CYP1A1 mRNAs. The mechanism involves the repression of the CYP1A1 gene promoter activity in a negative-feedback autoregulatory loop. Indeed, increasing the CYP1A1 activity by transfecting CYP1A1 expression vectors into hepatoma cells elicited an oxidative stress and led to the repression of a reporter gene driven by the CYP1A1 gene promoter. This negative autoregulation is abolished by ellipticine (an inhibitor of CYP1A1) and by catalase (which catalyzes H(2)O(2) catabolism), thus implying that H(2)O(2) is an intermediate. Down-regulation is also abolished by the mutation of the proximal nuclear factor I (NFI) site in the promoter. The transactivating domain of NFI/CTF was found to act in synergy with the arylhydrocarbon receptor pathway during the induction of CYP1A1 by 2,3,7,8-tetrachloro-p-dibenzodioxin. Using an NFI/CTF-Gal4 fusion, we show that NFI/CTF transactivating function is decreased by a high activity of CYP1A1. This regulation is also abolished by catalase or ellipticine. Consistently, the transactivating function of NFI/CTF is repressed in cells treated with H(2)O(2), a novel finding indicating that the transactivating domain of a transcription factor can be targeted by oxidative stress. In conclusion, an autoregulatory loop leads to the fine tuning of the CYP1A1 gene expression through the down-regulation of NFI activity by CYP1A1-based H(2)O(2) production. This mechanism allows a limitation of the potentially toxic CYP1A1 activity within the cell.

The required beta cell research for improving treatment of type 2 diabetes.

In healthy individuals, insulin resistance is associated with physiological conditions such as pregnancy or body weight gain and triggers an increase in beta cell number and insulin secretion capacity to preserve normoglycaemia. Failure of this beta cell compensation capacity is a fundamental cause of diabetic hyperglycaemia. Incomplete understanding of the molecular mechanisms controlling the plasticity of adult beta cells mechanisms and how these cells fail during the pathogenesis of diabetes strongly limits the ability to develop new beta cell-specific therapies. Here, current knowledge of the signalling pathways controlling beta cell plasticity is reviewed, and possible directions for future research are discussed.