Interleukin‐6 initiates muscle‐ and adipose tissue wasting in a novel C57BL/6 model of cancer‐associated cachexia

BACKGROUND: Cancer‐associated cachexia (CAC) is a wasting syndrome drastically reducing efficacy of chemotherapy and life expectancy of patients. CAC affects up to 80% of cancer patients, yet the mechanisms underlying the disease are not well understood and no approved disease‐specific medication exists. As a multiorgan disorder, CAC can only be studied on an organismal level. To cover the diverse aetiologies of CAC, researchers rely on the availability of a multifaceted pool of cancer models with varying degrees of cachexia symptoms. So far, no tumour model syngeneic to C57BL/6 mice exists that allows direct comparison between cachexigenic‐ and non‐cachexigenic tumours. METHODS: MCA207 and CHX207 fibrosarcoma cells were intramuscularly implanted into male or female, 10–11‐week‐old C57BL/6J mice. Tumour tissues were subjected to magnetic resonance imaging, immunohistochemical‐, and transcriptomic analysis. Mice were analysed for tumour growth, body weight and ‐composition, food‐ and water intake, locomotor activity, O(2) consumption, CO(2) production, circulating blood cells, metabolites, and tumourkines. Mice were sacrificed with same tumour weights in all groups. Adipose tissues were examined using high‐resolution respirometry, lipolysis measurements in vitro and ex vivo, and radioactive tracer studies in vivo. Gene expression was determined in adipose‐ and muscle tissues by quantitative PCR and Western blotting analyses. Muscles and cultured myotubes were analysed histologically and by immunofluorescence microscopy for myofibre cross sectional area and myofibre diameter, respectively. Interleukin‐6 (Il‐6) was deleted from cancer cells using CRISPR/Cas9 mediated gene editing. RESULTS: CHX207, but not MCA207‐tumour‐bearing mice exhibited major clinical features of CAC, including systemic inflammation, increased plasma IL‐6 concentrations (190 pg/mL, P ≤ 0.0001), increased energy expenditure (+28%, P ≤ 0.01), adipose tissue loss (−47%, P ≤ 0.0001), skeletal muscle wasting (−18%, P ≤ 0.001), and body weight reduction (−13%, P ≤ 0.01) 13 days after cancer cell inoculation. Adipose tissue loss resulted from reduced lipid uptake and ‐synthesis combined with increased lipolysis but was not associated with elevated beta‐adrenergic signalling or adipose tissue browning. Muscle atrophy was evident by reduced myofibre cross sectional area (−21.8%, P ≤ 0.001), increased catabolic‐ and reduced anabolic signalling. Deletion of IL‐6 from CHX207 cancer cells completely protected CHX207(IL6KO)‐tumour‐bearing mice from CAC. CONCLUSIONS: In this study, we present CHX207 fibrosarcoma cells as a novel tool to investigate the mediators and metabolic consequences of CAC in C57BL/6 mice in comparison to non‐cachectic MCA207‐tumour‐bearing mice. IL‐6 represents an essential trigger for CAC development in CHX207‐tumour‐bearing mice

Applied immuno-epidemiological research: an approach for integrating existing knowledge into the statistical analysis of multiple immune markers.

BACKGROUND: Immunologists often measure several correlated immunological markers, such as concentrations of different cytokines produced by different immune cells and/or measured under different conditions, to draw insights from complex immunological mechanisms. Although there have been recent methodological efforts to improve the statistical analysis of immunological data, a framework is still needed for the simultaneous analysis of multiple, often correlated, immune markers. This framework would allow the immunologists' hypotheses about the underlying biological mechanisms to be integrated. RESULTS: We present an analytical approach for statistical analysis of correlated immune markers, such as those commonly collected in modern immuno-epidemiological studies. We demonstrate i) how to deal with interdependencies among multiple measurements of the same immune marker, ii) how to analyse association patterns among different markers, iii) how to aggregate different measures and/or markers to immunological summary scores, iv) how to model the inter-relationships among these scores, and v) how to use these scores in epidemiological association analyses. We illustrate the application of our approach to multiple cytokine measurements from 818 children enrolled in a large immuno-epidemiological study (SCAALA Salvador), which aimed to quantify the major immunological mechanisms underlying atopic diseases or asthma. We demonstrate how to aggregate systematically the information captured in multiple cytokine measurements to immunological summary scores aimed at reflecting the presumed underlying immunological mechanisms (Th1/Th2 balance and immune regulatory network). We show how these aggregated immune scores can be used as predictors in regression models with outcomes of immunological studies (e.g. specific IgE) and compare the results to those obtained by a traditional multivariate regression approach. CONCLUSION: The proposed analytical approach may be especially useful to quantify complex immune responses in immuno-epidemiological studies, where investigators examine the relationship among epidemiological patterns, immune response, and disease outcomes

A randomized, placebo-controlled, double-blind, prospective trial to evaluate the effect of vildagliptin in new-onset diabetes mellitus after kidney transplantation

<p>Abstract</p> <p>Background</p> <p>New-onset diabetes mellitus after transplantation (NODAT), a frequent and serious complication after transplantation, is associated with decreased graft and patient survival. Currently, it is diagnosed and treated primarily according to existing guidelines for type II diabetes. To date, only a few trials have studied antidiabetic drugs in patients with NODAT. Vildagliptin is a novel dipeptidyl peptidase-4 (DPP-4) inhibitor that improves pancreatic islet function by enhancing both α- and β-cell responsiveness to increased blood glucose. Experimental data show potential protective effects of DPP-4 inhibitors on islet function after exogenous stress stimuli including immunosuppressants. Therefore, the therapy of NODAT with this class of compounds seems attractive. At present, vildagliptin is used to treat type II diabetes as monotherapy or in combination with other antidiabetic drugs, since that it efficiently decreases glycated hemoglobin (HbA1c) values. Additionally, vildagliptin has been shown to be safe in patients with moderately impaired kidney function. This study will evaluate the safety and efficacy of vildagliptin monotherapy in renal transplant recipients with recently diagnosed NODAT.</p> <p>Methods/Design</p> <p>This study is a randomized, placebo-controlled, double-blind, prospective phase II trial. Using the results of routinely performed oral glucose tolerance tests (OGTT) in stable renal transplant patients at our center, we will recruit patients without a history of diabetes and a 2 h glucose value surpassing 200 mg/dl (11.1 mmol/l). They are randomized to receive either 50 mg vildagliptin or placebo once daily. A total of 32 patients with newly diagnosed NODAT will be included. The primary endpoint is the difference in the 2 h glucose value between baseline and the repeated OGTT performed 3 months after treatment start, compared between the vildagliptin- and the placebo-group. Secondary endpoints include changes in HbA1c and fasting plasma glucose (FPG). The safety of vildagliptin in renal transplant patients will be assessed by the number of symptomatic hypoglycemic episodes (glucose <72 mg/dl or 4 mmol/l), the number of adverse events, and possible medication-associated side-effects.</p> <p>Discussion</p> <p>NODAT is a severe complication after kidney transplantation. Few trials have assessed the safety and efficacy of antidiabetic drugs for these patients. The purpose of this study is to assess the safety and efficacy of vildagliptin in renal transplant patients with NODAT.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov NCT00980356</p

mTORC1 is essential for early steps during Schwann cell differentiation of amniotic fluid stem cells and regulates lipogenic gene expression.

Schwann cell development is hallmarked by the induction of a lipogenic profile. Here we used amniotic fluid stem (AFS) cells and focused on the mechanisms occurring during early steps of differentiation along the Schwann cell lineage. Therefore, we initiated Schwann cell differentiation in AFS cells and monitored as well as modulated the activity of the mechanistic target of rapamycin (mTOR) pathway, the major regulator of anabolic processes. Our results show that mTOR complex 1 (mTORC1) activity is essential for glial marker expression and expression of Sterol Regulatory Element-Binding Protein (SREBP) target genes. Moreover, SREBP target gene activation by statin treatment promoted lipogenic gene expression, induced mTORC1 activation and stimulated Schwann cell differentiation. To investigate mTORC1 downstream signaling we expressed a mutant S6K1, which subsequently induced the expression of the Schwann cell marker S100b, but did not affect lipogenic gene expression. This suggests that S6K1 dependent and independent pathways downstream of mTORC1 drive AFS cells to early Schwann cell differentiation and lipogenic gene expression. In conclusion our results propose that future strategies for peripheral nervous system regeneration will depend on ways to efficiently induce the mTORC1 pathway

Paradoxical Effects of Rapamycin on Experimental House Dust Mite-Induced Asthma

The mammalian target of rapamycin (mTOR) modulates immune responses and cellular proliferation. The objective of this study was to assess whether inhibition of mTOR with rapamycin modifies disease severity in two experimental murine models of house dust mite (HDM)-induced asthma. In an induction model, rapamycin was administered to BALB/c mice coincident with nasal HDM challenges for 3 weeks. In a treatment model, nasal HDM challenges were performed for 6 weeks and rapamycin treatment was administered during weeks 4 through 6. In the induction model, rapamycin significantly attenuated airway inflammation, airway hyperreactivity (AHR) and goblet cell hyperplasia. In contrast, treatment of established HDM-induced asthma with rapamycin exacerbated AHR and airway inflammation, whereas goblet cell hyperplasia was not modified. Phosphorylation of the S6 ribosomal protein, which is downstream of mTORC1, was increased after 3 weeks, but not 6 weeks of HDM-challenge. Rapamycin reduced S6 phosphorylation in HDM-challenged mice in both the induction and treatment models. Thus, the paradoxical effects of rapamycin on asthma severity paralleled the activation of mTOR signaling. Lastly, mediastinal lymph node re-stimulation experiments showed that treatment of rapamycin-naive T cells with ex vivo rapamycin decreased antigen-specific Th2 cytokine production, whereas prior exposure to in vivo rapamycin rendered T cells refractory to the suppressive effects of ex vivo rapamycin. We conclude that rapamycin had paradoxical effects on the pathogenesis of experimental HDM-induced asthma. Thus, consistent with the context-dependent effects of rapamycin on inflammation, the timing of mTOR inhibition may be an important determinant of efficacy and toxicity in HDM-induced asthma

Lipopolysaccharide Renders Transgenic Mice Expressing Human Serum Amyloid P Component Sensitive to Shiga Toxin 2

Transgenic C57BL/6 mice expressing human serum amyloid P component (HuSAP) are resistant to Shiga toxin 2 (Stx2) at dosages that are lethal in HuSAP-negative wild-type mice. However, it is well established that Stx2 initiates extra-intestinal complications such as the haemolytic-uremic syndrome despite the presence of HuSAP in human sera. We now demonstrate that co-administering purified Escherichia coli O55 lipopolysaccharide (LPS), at a dosage of 300 ng/g body weight, to HuSAP-transgenic mice increases their susceptibility to the lethal effects of Stx2. The enhanced susceptibility to Stx2 correlated with an increased expression of genes encoding the pro-inflammatory cytokine TNFα and chemokines of the CXC and CC families in the kidneys of LPS-treated mice, 48 hours after the Stx2/LPS challenge. Co-administering the glucocorticoid dexamethasone, but not the LPS neutralizing cationic peptide LL-37, protected LPS-sensitized HuSAP-transgenic mice from lethal doses of Stx2. Dexamethasone protection was specifically associated with decreased expression of the same inflammatory mediators (CXC and CC-type chemokines and TNFα) linked to enhanced susceptibility caused by LPS. The studies reveal further details about the complex cascade of host-related events that are initiated by Stx2 as well as establish a new animal model system in which to investigate strategies for diminishing serious Stx2-mediated complications in humans infected with enterohemorrhagic E. coli strains

Pathogen Proteins Eliciting Antibodies Do Not Share Epitopes with Host Proteins: A Bioinformatics Approach

The best way to prevent diseases caused by pathogens is by the use of vaccines. The advent of genomics enables genome-wide searches of new vaccine candidates, called reverse vaccinology. The most common strategy to apply reverse vaccinology is by designing subunit recombinant vaccines, which usually generate an humoral immune response due to B-cell epitopes in proteins. A major problem for this strategy is the identification of protective immunogenic proteins from the surfome of the pathogen. Epitope mimicry may lead to auto-immune phenomena related to several human diseases. A sequence-based computational analysis has been carried out applying the BLASTP algorithm. Therefore, two huge databases have been created, one with the most complete and current linear B-cell epitopes, and the other one with the surface-protein sequences of the main human respiratory bacterial pathogens. We found that none of the 7353 linear B-cell epitopes analysed shares any sequence identity region with human proteins capable of generating antibodies, and that only 1% of the 2175 exposed proteins analysed contain a stretch of shared sequence with the human proteome. These findings suggest the existence of a mechanism to avoid autoimmunity. We also propose a strategy for corroborating or warning about the viability of a protein linear B-cell epitope as a putative vaccine candidate in a reverse vaccinology study; so, epitopes without any sequence identity with human proteins should be very good vaccine candidates, and the other way around