Urine levels of HMGB1 in Systemic Lupus Erythematosus patients with and without renal manifestations

Introduction

Lupus nephritis (LN) is a severe and frequent manifestation of systemic lupus erythematosus (SLE). Its pathogenesis has not been fully elucidated but immune complexes are considered to contribute to the inflammatory pathology in LN. High Mobility Group Box 1 (HMGB1) is a nuclear non-histone protein which is secreted from different types of cells during activation and/or cell death and may act as a pro-inflammatory mediator, alone or as part of DNA-containing immune complexes in SLE. Urinary excretion of HMGB1 might reflect renal inflammatory injury. To assess whether urinary HMGB1 reflects renal inflammation we determined serum levels of HMGB1 simultaneously with its urinary levels in SLE patients with and without LN in comparison to healthy controls (HC). We also analyzed urinary HMGB1 levels in relation with clinical and serological disease activity.

Methods

The study population consisted of 69 SLE patients and 17 HC. Twenty-one patients had biopsy proven active LN, 15 patients had a history of LN without current activity, and 33 patients had non-renal SLE. Serum and urine levels of HMGB1 were both measured by western blotting. Clinical and serological parameters were assessed according to routine procedures. In 17 patients with active LN a parallel analysis was performed on the expression of HMGB1 in renal biopsies.

Results

Serum and urinary levels of HMGB1 were significantly increased in patients with active LN compared to patients without active LN and HC. Similarly, renal tissue of active LN patients showed strong expression of HMGB1 at cytoplasmic and extracellular sites suggesting active release of HMGB1. Serum and urinary levels in patients without active LN were also significantly higher compared to HC. Urinary HMGB1 levels correlated with SLEDAI, and showed a negative correlation with complement C3 and C4.

Conclusion

Levels of HMGB1 in urine of SLE patients, in particular in those with active LN, are increased and correlate with SLEDAI scores. Renal tissue of LN patients shows increased release of nuclear HMGB1 compared to control renal tissue. HMGB1, although at lower levels, is, however, also present in the urine of patients without active LN. These data suggest that urinary HMGB1 might reflect both local renal inflammation as well as systemic inflammation.     

Effect of Statins on Venous Thromboembolic Events: A Meta-analysis of Published and Unpublished Evidence from Randomised Controlled Trials

Background

It has been suggested that statins substantially reduce the risk of venous thromboembolic events. We sought to test this hypothesis by performing a meta-analysis of both published and unpublished results from randomised trials of statins.

Methods and Findings

We searched MEDLINE, EMBASE, and Cochrane CENTRAL up to March 2012 for randomised controlled trials comparing statin with no statin, or comparing high dose versus standard dose statin, with 100 or more randomised participants and at least 6 months'' follow-up. Investigators were contacted for unpublished information about venous thromboembolic events during follow-up. Twenty-two trials of statin versus control (105,759 participants) and seven trials of an intensive versus a standard dose statin regimen (40,594 participants) were included. In trials of statin versus control, allocation to statin therapy did not significantly reduce the risk of venous thromboembolic events (465 [0.9%] statin versus 521 [1.0%] control, odds ratio [OR] = 0.89, 95% CI 0.78–1.01, p = 0.08) with no evidence of heterogeneity between effects on deep vein thrombosis (266 versus 311, OR 0.85, 95% CI 0.72–1.01) and effects on pulmonary embolism (205 versus 222, OR 0.92, 95% CI 0.76–1.12). Exclusion of the trial result that provided the motivation for our meta-analysis (JUPITER) had little impact on the findings for venous thromboembolic events (431 [0.9%] versus 461 [1.0%], OR = 0.93 [95% CI 0.82–1.07], p = 0.32 among the other 21 trials). There was no evidence that higher dose statin therapy reduced the risk of venous thromboembolic events compared with standard dose statin therapy (198 [1.0%] versus 202 [1.0%], OR = 0.98, 95% CI 0.80–1.20, p = 0.87). Risk of bias overall was small but a certain degree of effect underestimation due to random error cannot be ruled out. Please see later in the article for the Editors'' Summary.

Conclusions

The findings from this meta-analysis do not support the previous suggestion of a large protective effect of statins (or higher dose statins) on venous thromboembolic events. However, a more moderate reduction in risk up to about one-fifth cannot be ruled out.     

Microbial metabolic exchange--the chemotype-to-phenotype link

The function of microbial interactions is to enable microorganisms to survive by establishing a homeostasis between microbial neighbors and local environments. A microorganism can respond to environmental stimuli using metabolic exchange-the transfer of molecular factors, including small molecules and proteins. Microbial interactions not only influence the survival of the microbes but also have roles in morphological and developmental processes of the organisms themselves and their neighbors. This, in turn, shapes the entire habitat of these organisms. Here we highlight our current understanding of metabolic exchange as well as the emergence of new technologies that are allowing us to eavesdrop on microbial conversations comprising dozens to hundreds of secreted metabolites that control the behavior, survival and differentiation of members of the community. The goal of the rapidly advancing field studying multifactorial metabolic exchange is to devise a microbial 'Rosetta stone' in order to understand the language by which microbial interactions are negotiated and, ultimately, to control the outcome of these conversations.     

Metabolic effects of the iodothyronine functional analogue TRC150094 on the liver and skeletal muscle of high-fat diet fed overweight rats: an integrated proteomic study

A novel functional iodothyronine analogue, TRC150094, which has a much lower potency toward thyroid hormone receptor (α1/β1) activation than triiodothyronine, has been shown to be effective at reducing adiposity in rats simultaneously receiving a high-fat diet (HFD). Here, by combining metabolic, functional and proteomic analysis, we studied how the hepatic and skeletal muscle phenotypes might respond to TRC150094 treatment in HFD-fed overweight rats. Drug treatment increased both the liver and skeletal muscle mitochondrial oxidative capacities without altering mitochondrial efficiency. Coherently, in terms of individual respiratory in-gel activity, blue-native analysis revealed an increased activity of complex V in the liver and of complexes II and V in tibialis muscle in TCR150094-treated animals. Subsequently, the identification of differentially expressed proteins and the analysis of their interrelations gave an integrated view of the phenotypic/metabolic adaptations occurring in the liver and muscle proteomes during drug treatment. TRC150094 significantly altered the expression of several proteins involved in key liver metabolic pathways, including amino acid and nitrogen metabolism, and fructose and mannose metabolism. The canonical pathways most strongly influenced by TRC150094 in tibialis muscle included glycolysis and gluconeogenesis, amino acid, fructose and mannose metabolism, and cell signaling. The phenotypic/metabolic influence of TRC150094 on the liver and skeletal muscle of HFD-fed overweight rats suggests the potential clinical application of this iodothyronine analogue in ameliorating metabolic risk parameters altered by diet regimens.     

Treatment-associated polymorphisms in protease are significantly associated with higher viral load and lower CD4 count in newly diagnosed drug-naive HIV-1 infected patients

Background

Bone marrow stromal cell antigen 2 (BST-2) is a cellular factor that restricts the egress of viruses such as human immunodeficiency virus (HIV-1) from the surface of infected cells, preventing infection of new cells. BST-2 is variably expressed in most cell types, and its expression is enhanced by cytokines such as type I interferon alpha (IFN-??). In this present study, we used the beta-retrovirus, mouse mammary tumor virus (MMTV) as a model to examine the role of mouse BST-2 in host infection in vivo.

Results

By using RNA interference, we show that loss of BST-2 enhances MMTV replication in cultured mammary tumor cells and in vivo. In cultured cells, BST-2 inhibits virus accumulation in the culture medium, and co-localizes at the cell surface with virus structural proteins. Furthermore, both scanning electron micrograph (SEM) and transmission electron micrograph (TEM) show that MMTV accumulates on the surface of IFN??-stimulated cells.

Conclusions

Our data provide evidence that BST-2 restricts MMTV release from naturally infected cells and that BST-2 is an antiviral factor in vivo.     

Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function

Background

The Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF) region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region.

Results

The dysfunctional HIV group O8 nef allele was restored to the consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2) and non-canonical (B2 and C1422) HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF??AAA) and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef??s association with the cellular p21-activated kinase 2 (PAK2), the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN) were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck.

Conclusion

Based on these findings, we propose that this highly conserved three amino acid VGF motif together with the acidic cluster and the proline-rich motif form a previously unrecognized amphipathic surface on Nef. This surface appears to be essential for the majority of Nef functions and thus represents a prime target for the pharmacological inhibition of Nef.     

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder''s nociceptive innervation and initiate voiding contractions that expel the bladder''s contents. Interestingly, the bladder''s sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity¹. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models². Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions³.Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value⁴. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.     

A resource for the conditional ablation of microRNAs in the mouse

The importance of miRNAs during development and disease processes is well established. However, most studies have been done in cells or with patient tissues, and therefore the physiological roles of miRNAs are not well understood. To unravel in vivo functions of miRNAs, we have generated conditional, reporter-tagged knockout-first mice for numerous evolutionarily conserved miRNAs. Here, we report the generation of 162 miRNA targeting vectors, 64 targeted ES cell lines, and 46 germline-transmitted miRNA knockout mice. In vivo lacZ reporter analysis in 18 lines revealed highly tissue-specific expression patterns and their miRNA expression profiling matched closely with published expression data. Most miRNA knockout mice tested were viable, supporting a mechanism by which miRNAs act redundantly with other miRNAs or other pathways. These data and collection of resources will be of value for the in vivo dissection of miRNA functions in mouse models.     

The calm mouse: an animal model of stress reduction

Chronic stress is associated with negative health outcomes and is linked with neuroendocrine changes, deleterious effects on innate and adaptive immunity, and central nervous system neuropathology. Although stress management is commonly advocated clinically, there is insufficient mechanistic understanding of how decreasing stress affects disease pathogenesis. Therefore, we have developed a "calm mouse model" with caging enhancements designed to reduce murine stress. Male BALB/c mice were divided into four groups: control (Cntl), standard caging; calm (Calm), large caging to reduce animal density, a cardboard nest box for shelter, paper nesting material to promote innate nesting behavior, and a polycarbonate tube to mimic tunneling; control exercise (Cntl Ex), standard caging with a running wheel, known to reduce stress; and calm exercise (Calm Ex), calm caging with a running wheel. Calm, Cntl Ex and Calm Ex animals exhibited significantly less corticosterone production than Cntl animals. We also observed changes in spleen mass, and in vitro splenocyte studies demonstrated that Calm Ex animals had innate and adaptive immune responses that were more sensitive to acute handling stress than those in Cntl. Calm animals gained greater body mass than Cntl, although they had similar food intake, and we also observed changes in body composition, using magnetic resonance imaging. Together, our results suggest that the Calm mouse model represents a promising approach to studying the biological effects of stress reduction in the context of health and in conjunction with existing disease models.     

Gene expression in flow sorted mouse teratocarcinoma X human fibroblast heterokaryons

Abstract. Mouse teratocarcinoma cells and primary human fibroblasts were fluorescently labelled with fluorescein isothiocyanate (F1TC)- and trimethylrhodamine isothiocyanate (TR1TC)-stearylamine respectively. After fusion populations highly enriched for red-green heterokaryons (around 80%) were isolated from the fusion mixture using a FACS II cell sorter.
To study gene expression in the early hybrids [³⁵S] methionine-labelled proteins synthesized by the sorted cells at two and three days after fusion were analysed by two-dimensional gel electrophoresis. Three spots were denser in gels of the fused cells than in those of 1:1 mixtures of parental cells. For one of these proteins it could be demonstrated that this reflects the enhanced synthesis of a mouse-specific protein present only in small amounts in teratocarcinoma cells. All three proteins were synthesized in relatively large amounts by differentiated mouse cells.
Collagen (type I) synthesis by the sorted hybrid cells was studied by analysing the [³H] proline-labelled material secreted into the medium. Analysis by sodium dodecyl sulphate (SDS)-gel electrophoresis and two-dimensional non-equilibrium pH gradient electrophoresis showed that the material secreted by the fused cells five days after fusion was the same as that secreted by the human fibroblasts. No evidence was obtained for synthesis of mouse α2(I) collagen. The amount of collagen produced by the sorted cells five days after fusion was about half the amount produced by the human fibroblasts. Immunofluorescence studies also showed that collagen synthesis was not suppressed after fusion both in heterokaryons and synkaryons.
In conclusion, we did not find evidence for activation of a previously completely silent mouse gene in the fused cells. The results show, however, that the fused cells do resemble the differentiated fibroblasts rather than the undifferentiated teratocarcinoma cells.