Soluble ST2 Levels Are Associated with Bleeding in Patients with Severe Leptospirosis

Background

Severe leptospirosis features bleeding and multi-organ failure, leading to shock and death. Currently it is assumed that both exaggerated inflammation and immune suppression contribute to mortality in sepsis. Indeed, several proinflammatory cytokines are reported to be induced during leptospirosis. Toll-like receptors, which play an important role in the initiation of an innate immune response, are inhibited by negative regulators including the membrane-bound ST2 (mST2) receptor. Soluble ST2 (sST2) has been implicated to inhibit signaling through mST2. The aim of this study was to determine the extent of sST2 and (pro-) inflammatory cytokine release in patients with severe leptospirosis.

Methodology and Principal Findings

In an observational study, 68 consecutive cases of severe leptospirosis were included. Soluble ST2 and cytokines (TNF-α, IL-1β, IL-6, IL-8, and IL-10) were repeatedly measured. To determine whether blood cells are a source of sST2 during infection, we undertook an in vitro experiment: human whole blood and peripheral blood mononuclear cells (PBMC) were stimulated with viable pathogenic Leptospira. All patients showed elevated sST2, IL-6, IL-8, and IL-10 levels on admission. Admission sST2 levels correlated with IL-6, IL-8, and IL-10. Thirty-four patients (50%) showed clinical bleeding. Soluble ST2 levels were significantly associated with bleeding overall (OR 2.0; 95%CI: 1.2–3.6) and severe bleeding (OR 5.1; 95%CI: 1.1–23.8). This association was unique, since none of the cytokines showed this correlation. Moreover, sST2 was associated with mortality (OR 2.4; 95%CI: 1.0–5.8). When either whole blood or isolated PBMCs were stimulated with Leptospira in vitro, no sST2 production could be detected.

Conclusions

Patients with severe leptospirosis demonstrated elevated plasma sST2 levels. Soluble ST2 levels were associated with bleeding and mortality. In vitro experiments showed that (white) blood cells are probably not the source. In this regard, sST2 could be an indicative marker for tissue damage in patients suffering from severe leptospirosis.     

A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]-hydrogenase

Hydrogenases are essential for H(2) cycling in microbial metabolism and serve as valuable blueprints for H(2)-based biotechnological applications. However, most hydrogenases are extremely oxygen sensitive and prone to inactivation by even traces of O(2). The O(2)-tolerant membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha H16 is one of the few examples that can perform H(2) uptake in the presence of ambient O(2). Here we show that O(2) tolerance is crucially related to a modification of the internal electron-transfer chain. The iron-sulfur cluster proximal to the active site is surrounded by six instead of four conserved coordinating cysteines. Removal of the two additional cysteines alters the electronic structure of the proximal iron-sulfur cluster and renders the catalytic activity sensitive to O(2) as shown by physiological, biochemical, spectroscopic and electrochemical studies. The data indicate that the mechanism of O(2) tolerance relies on the reductive removal of oxygenic species guided by the unique architecture of the electron relay rather than a restricted access of O(2) to the active site.     

Modulating mouse innate immunity to RNA viruses by expressing the Bos taurus Mx system

Mx proteins are interferon-induced members of the dynamin superfamily of large guanosine triphosphatases. These proteins have attracted much attention because some display antiviral activity against pathogenic RNA viruses, such as members of the orthomyxoviridae, bunyaviridae, and rhabdoviridae families. Among the diverse mammalian Mx proteins examined so far, we have recently demonstrated in vitro that the Bos taurus isoform 1 (boMx1) is endowed with exceptional anti-rabies-virus activity. This finding has prompted us to seek an appropriate in vivo model for confirming and evaluating gene therapy strategies. Using a BAC transgene, we have generated transgenic mouse lines expressing the antiviral boMx1 protein and boMx2 proteins under the control of their natural promoter and short- and long-range regulatory elements. Expressed boMx1 and boMx2 are correctly assembled, as deduced from mRNA sequencing and western blotting. Poly-I/C-subordinated expression of boMx1 was detected in various organs by immunohistochemistry, and transgenic lines were readily classified as high- or low-expression lines on the basis of tissue boMx1 concentrations measured by ELISA. Poly-I/C-induced Madin-Darby bovine kidney cells, bovine turbinate cells, and cultured cells from high-expression line of transgenic mice were found to contain about the same concentration of boMx1, suggesting that this protein is produced at near-physiological levels. Furthermore, insertion of the bovine Mx system rendered transgenic mice resistant to vesicular-stomatitis-virus-associated morbidity and mortality, and embryonic fibroblasts derived from high-expression transgenic mice were far less permissive to the virus. These results demonstrate that the Bos taurus Mx system is a powerful anti-VSV agent in vivo and suggest that the transgenic mouse lines generated here constitute a good model for studying in vivo the various antiviral functions—known and yet to be discovered—exerted by bovine Mx proteins, with priority emphasis on the antirabic function of boMx1. M.-M. Garigliany and K. Cloquette have contributed equally to the study.     

Measuring Free‐Living Energy Expenditure and Physical Activity with Triaxial Accelerometry

Objective: To investigate the ability of a newly developed triaxial accelerometer to predict total energy expenditure (EE) (TEE) and activity‐related EE (AEE) in free‐living conditions. Research Methods and Procedures: Subjects were 29 healthy subjects between the ages of 18 and 40. The Triaxial Accelerometer for Movement Registration (Tracmor) was worn for 15 consecutive days. Tracmor output was defined as activity counts per day (ACD) for the sum of all three axes or each axis separately (ACD‐X, ACD‐Y, ACD‐Z). TEE was measured with the doubly labeled water technique. Sleeping metabolic rate (SMR) was measured during an overnight stay in a respiration chamber. The physical activity level was calculated as TEE × SMR^?1, and AEE was calculated as [(0.9 × TEE) ? SMR]. Body composition was calculated from body weight, body volume, and total body water using Siri's three‐compartment model. Results: Age, height, body mass, and ACD explained 83% of the variation in TEE [standard error of estimate (SEE) = 1.00 MJ/d] and 81% of the variation in AEE (SEE = 0.70 MJ/d). The partial correlations for ACD were 0.73 (p < 0.001) and 0.79 (p < 0.001) with TEE and AEE, respectively. When data on SMR or body composition were used with ACD, the explained variation in TEE was 90% (SEE = 0.74 and 0.77 MJ/d, respectively). The increase in the explained variation using three axes instead of one axis (vertical) was 5% (p < 0.05). Discussion: The correlations between Tracmor output and EE measures are the highest reported so far. To measure daily life activities, the use of triaxial accelerometry seems beneficial to uniaxial.     

The Immunogenetic Architecture of Autoimmune Disease

The development of most autoimmune diseases includes a strong heritable component. This genetic contribution to disease ranges from simple Mendelian inheritance of causative alleles to the complex interactions of multiple weak loci influencing risk. The genetic variants responsible for disease are being discovered through a range of strategies from linkage studies to genome-wide association studies. Despite the rapid advances in genetic analysis, substantial components of the heritable risk remain unexplained, either owing to the contribution of an as-yet unidentified, “hidden,” component of risk, or through the underappreciated effects of known risk loci. Surprisingly, despite the variation in genetic control, a great deal of conservation appears in the biological processes influenced by risk alleles, with several key immunological pathways being modified in autoimmune diseases covering a broad spectrum of clinical manifestations. The primary translational potential of this knowledge is in the rational design of new therapeutics to exploit the role of these key pathways in influencing disease. With significant further advances in understanding the genetic risk factors and their biological mechanisms, the possibility of genetically tailored (or “personalized”) therapy may be realized.Autoimmune diseases affect a significant proportion of the population, with >4% of the European population suffering from one or more of these disorders (Vyse and Todd 1996; Cooper et al. 2009; Eaton et al. 2010). Although all autoimmune diseases share similarities in the basic immunological mechanisms, in other aspects, such as clinical manifestation and age of onset, individual diseases vary widely. A few rare autoimmune diseases with Mendelian inheritance patterns within families occur including APS-1 (autoimmune polyendocrine syndrome type 1), IPEX (immunodysregulation, polyendocrinopathy, and enteropathy X-linked) syndrome, and ALPS (autoimmune lymphoproliferative syndrome). Most autoimmune diseases are, however, multifactorial in nature, with susceptibility controlled by multiple genetic and environmental factors.The genetic component of more common autoimmune diseases can be calculated in several different manners, including the sibling recurrence risk (λ_s) and the twin concordance rate. The sibling recurrence risk is the ratio of the lifetime risk in siblings of patients to the lifetime population risk, whereas the twin concordance rate measures the proportion of the siblings of affected twins that are also affected. Most common autoimmune diseases, such as multiple sclerosis (MS), type 1 diabetes (T1D), rheumatoid arthritis (RA), and inflammatory bowel disease (IBD) are characterized by a sibling recurrence risk between 6 and 20 (Vyse and Todd 1996), and concordance rates of 25%–50% in monozygotic twins and 2%–12% in dizygotic twins (Cooper et al. 1999). A substantial proportion of relatives may also have subclinical evidence of autoimmunity without developing clinically overt disease. For example, 19% of healthy siblings of MS patients show antibody production in the cerebrospinal fluid, compared to 4% of unrelated healthy controls (Haghighi et al. 2000), whereas 4% of healthy first-degree relatives display lesions that are indistinguishable from those seen in patients and are not seen in unrelated healthy controls (De Stefano et al. 2006). Furthermore, comorbidity with the development of several autoimmune diseases in the same patient and clustering of several autoimmune diseases within families above what is expected by chance appear common (Cooper et al. 2009; Zhernakova et al. 2009). Together these data show a strong genetic component to autoimmune disease development.     

Long-term creatine intake is beneficial to muscle performance during resistance training

Vandenberghe, K., M. Goris, P. Van Hecke, M. Van Leemputte,L. Vangerven, and P. Hespel. Long-term creatine intake isbeneficial to muscle performance during resistance training. J. Appl. Physiol. 83(6):2055-2063, 1997.The effects of oral creatine supplementation onmuscle phosphocreatine (PCr) concentration, muscle strength, and bodycomposition were investigated in young female volunteers(n = 19) during 10 wk ofresistance training (3 h/wk). Compared with placebo, 4 days ofhigh-dose creatine intake (20 g/day) increased(P < 0.05) muscle PCr concentration by 6%. Thereafter, this increase was maintained during 10 wk of training associated with low-dose creatine intake (5 g/day).Compared with placebo, maximal strength of the muscle groups trained,maximal intermittent exercise capacity of the arm flexors, and fat-free mass were increased 20-25, 10-25, and 60% more(P < 0.05), respectively, duringcreatine supplementation. Muscle PCr and strength, intermittent exercise capacity, and fat-free mass subsequently remained at a higherlevel in the creatine group than in the placebo group during 10 wk ofdetraining while low-dose creatine was continued. Finally, on cessationof creatine intake, muscle PCr in the creatine group returned to normalwithin 4 wk. It is concluded that long-term creatine supplementationenhances the progress of muscle strength during resistance training insedentary females.

     

ATP x Mg-dependent protein phosphatase from rabbit skeletal muscle. I. Purification of the enzyme and its regulation by the interaction with an activating protein factor

An ATP x Mg-dependent protein phosphatase (FC) was purified to near homogeneity from rabbit muscle. The enzyme was completely devoid of any spontaneous activity but could be activated by a protein activator (FA) in the presence of ATP and Mg ions. The inactive phosphatase migrated as a single protein band on sodium dodecyl sulfate-gel electrophoresis, and in discontinuous gel electrophoresis, where the potential phosphatase activity was located in the main protein band. The molecular weight determined by sodium dodecyl sulfate electrophoresis or by sucrose density centrifugation was found to be 70,000. FC migrated on gel filtration as a 140,000 molecular weight species. The activation by FA was not paralleled by an incorporation of [32P]-phosphate into the ATP x Mg-dependent phosphatase, and from the kinetics of activation a protein-protein interaction with ATP x Mg as a necessary factor, can be inferred as the mechanism of activation. After activation by FA and ATP X Mg, the purified enzyme had a specific activity of 10,000 units/mg of protein, and a Km for rabbit muscle phosphorylase a of approximately 1.0 mg/ml. The activated enzyme did not release [32P]phosphate from 32[-labeled rabbit muscle synthase b, prepared from glucagon-treated dogs. It did, however, remove all the 32P label from phosphorylase b kinase, autophosphorylated to the level of 2.0 mol/mol of 1.3 X 10(6) molecular weight.