Assessment of tissue fibrosis in skin biopsies from patients with systemic sclerosis employing confocal laser scanning microscopy: an objective outcome measure for clinical trials?

OBJECTIVES: To obtain an objective, unbiased assessment of skin fibrosis in patients with SSc for use in clinical trials of SSc disease-modifying therapeutics. METHODS: Skin biopsies from the dorsal forearm of six patients with diffuse SSc and six healthy controls, and skin biopsies from the forearm of one patient with diffuse SSc before and following 1 year treatment with mycophenolate mofetil were analysed by confocal laser scanning microscopy (CLSM) with specific antibodies against collagen types I and III or fibronectin. The integrated density of fluorescence (IDF) was calculated employing National Institutes of Health-ImageJ software in at least four different fields per biopsy spanning the full dermal thickness. RESULTS: The intensities of collagen types I and III and fibronectin IDF were 174, 147 and 139% higher in SSc skin than in normal skin, respectively. All differences were statistically significant. The sum of the IDF values obtained for the three proteins yielded a comprehensive fibrosis score. The average fibrosis score for the six SSc samples was 28.3 x 10(6) compared with 18.6 x 10(6) for the six normal skin samples (P \u3c 0.0001). Comparison of skin biopsies obtained from the same SSc patient before treatment and after 12 months of treatment with mycophenolate mofetil showed a reduction of 39% in total fibrosis score after treatment. CONCLUSIONS: CLSM followed by quantitative image analysis provides an objective and unbiased assessment of skin fibrosis in SSc and could be a useful end-point for clinical trials with disease-modifying agents to monitor the response or progression of the disease

Bandwidth Selection and the Estimation of Treatment Effects with Unbalanced Data

This paper addresses the selection of smoothing parameters for estimating the average treatment effect on the treated using matching methods. Because precise estimation of the expected counterfactual is particularly important in regions containing the mass of the treated units, we define and implement weighted cross-validation approaches that improve over conventional methods by considering the location of the treated units in the selection of the smoothing parameters. We also implement a locally varying bandwidth method that uses larger bandwidths in areas where the mass of the treated units is located. A Monte Carlo study compares our proposed methods to the conventional unweighted method and to a related method inspired by Bergemann et al. (2005). The Monte Carlo analysis indicates efficiency gains from all methods that take account of the location of the treated units. We also apply all five methods to bandwidth selection in the context of the data from LaLonde\u27s (1986) study of the performance of non-experimental estimators using the experimental data from the National Supported Work (NSW) Demonstration program as a benchmark. Overall, both the Monte Carlo analysis and the empirical application show feasible precision gains for the weighted cross-validation and the locally varying bandwidth approaches

The role of allograft inflammatory factor 1 in systemic sclerosis

Purpose of review: The aim of this article is to review studies which support the hypothesis that allograft inflammatory factor-1, a protein initially identified in chronically rejected cardiac allografts, may be involved in the pathogenesis of the progressive fibroproliferative vasculopathy which is a hallmark of systemic sclerosis. Recent findings: Recent findings demonstrated elevated allograft inflammatory factor-1 expression both in systemic sclerosis affected tissues and peripheral blood mononuclear cells. A detailed immunohistopathologic study examined the tissue and cellular localization of the protein in affected systemic sclerosis tissues and demonstrated its expression in the endothelium of dermal and pulmonary vessels, in the pulmonary parenchyma, and in relevant inflammatory cells including T cells and macrophages. Furthermore, functional studies showed specific allograft inflammatory factor-1 isoform expression stimulation by transforming growth factor-[beta]. Summary: This review summarizes recent findings suggesting that allograft inflammatory factor-1 may play an important role in systemic sclerosis vasculopathy and provides supporting evidence to consider the molecule as a novel therapeutic target

Caveolin-1, TGF-β receptor internalization, and the pathogenesis of systemic sclerosis

PURPOSE OF REVIEW: To review the scientific literature supporting the participation of caveolin-1 in the pathogenesis of tissue fibrosis and the notion that modulation of the caveolin-1 pathway may represent a novel treatment for systemic sclerosis and other fibrotic diseases. RECENT FINDINGS: Caveolin-1 plays an important role in the regulation of transforming growth factor-beta (TGF-beta) signaling owing to its participation in TGF-beta receptor internalization. TGF-beta receptor internalized through caveolin-1 lipid rafts undergoes rapid degradation, effectively decreasing TGF-beta signaling. Studies have shown that caveolin-1 knockdown in vitro markedly increased collagen gene expression in normal human lung fibroblasts. Caveolin-1 was reduced in affected systemic sclerosis lungs and skin and in idiopathic pulmonary fibrosis lung tissues and fibroblasts. Increasing caveolin-1 expression markedly improved bleomycin-induced pulmonary fibrosis. Restoration of caveolin bioavailability employing penetratin, a cell-permeable peptide carrier for a bioactive caveolin-1 fragment, abrogated TGF-beta activation of cultured human dermal fibroblasts. Systemic administration of penetratin-caveolin-1 peptide to mice with bleomycin-induced lung fibrosis reduced fibrosis. SUMMARY: Caveolin-1 plays an important role in the regulation of TGF-beta signaling and participates in the pathogenesis of systemic sclerosis and idiopathic pulmonary fibrosis. Restoration of caveolin function employing active caveolin-1 fragments coupled to cell-permeable carrier peptides may represent a novel approach for their treatment

Decreased expression of caveolin 1 in patients with systemic sclerosis: crucial role in the pathogenesis of tissue fibrosis.

OBJECTIVE: Recent studies have implicated caveolin 1 in the regulation of transforming growth factor beta (TGFbeta) downstream signaling. Given the crucial role of TGFbeta in the pathogenesis of systemic sclerosis (SSc), we sought to determine whether caveolin 1 is also involved in the pathogenesis of tissue fibrosis in SSc. We analyzed the expression of CAV1 in affected SSc tissues, studied the effects of lack of expression of CAV1 in vitro and in vivo, and analyzed the effects of restoration of caveolin 1 function on the fibrotic phenotype of SSc fibroblasts in vitro. METHODS: CAV1 expression in tissues was analyzed by immunofluorescence and confocal microscopy. The extent of tissue fibrosis in Cav1-knockout mice was assessed by histologic/histochemical analyses and quantified by hydroxyproline assays. Cav1-null and SSc fibroblast phenotypes and protein production were analyzed by real-time polymerase chain reaction, immunofluorescence, Western blot, and multiplexed enzyme-linked immunosorbent assay techniques. The effects of restoration of caveolin 1 function in SSc fibroblasts in vitro were also examined using a cell-permeable recombinant CAV1 peptide. RESULTS: CAV1 was markedly decreased in the affected lungs and skin of SSc patients. Cav1-knockout mice developed pulmonary and skin fibrosis. Down-regulation of caveolin 1 was maintained in cultured SSc fibroblasts, and restoration of caveolin 1 function in vitro normalized their phenotype and abrogated TGFbeta stimulation through inhibition of Smad3 activation. CONCLUSION: Caveolin 1 appears to participate in the pathogenesis of tissue fibrosis in SSc. Restoration of caveolin 1 function by treatment with a cell-permeable peptide corresponding to the CAV1 scaffolding domain may be a novel therapeutic approach in SSc

Coupling of Smoothened to inhibitory G proteins reduces voltage-gated K

SMO (Smoothened), the central transducer of Hedgehog signaling, is coupled to heterotrimeric Gi proteins in many cell types, including cardiomyocytes. In this study, we report that activation of SMO with SHH (Sonic Hedgehog) or a small agonist, purmorphamine, rapidly causes a prolongation of the action potential duration that is sensitive to a SMO inhibitor. In contrast, neither of the SMO agonists prolonged the action potential in cardiomyocytes from transgenic GiCT/TTA mice, in which Gi signaling is impaired, suggesting that the effect of SMO is mediated by Gi proteins. Investigation of the mechanism underlying the change in action potential kinetics revealed that activation of SMO selectively reduces outward voltage-gated K+ repolarizing (Kv) currents in isolated cardiomyocytes and that it induces a down-regulation of membrane levels of Kv4.3 in cardiomyocytes and intact hearts from WT but not from GiCT/TTA mice. Moreover, perfusion of intact hearts with Shh or purmorphamine increased the ventricular repolarization time (QT interval) and induced ventricular arrhythmias. Our data constitute the first report that acute, noncanonical Hh signaling mediated by Gi proteins regulates K+ currents density in cardiomyocytes and sensitizes the heart to the development of ventricular arrhythmias. © 2018 Cheng et al

Expression of allograft inflammatory factor 1 in tissues from patients with systemic sclerosis and in vitro differential expression of its isoforms in response to transforming growth factor beta

OBJECTIVE: Allograft inflammatory factor 1 (AIF-1), a protein initially identified in chronically rejected rat cardiac allografts, is involved in the immune response and proliferative vasculopathy that occurs during allograft rejection. Three well-characterized isoforms of AIF-1 result from alternative messenger RNA (mRNA) splicing. We previously identified a strong association of systemic sclerosis (SSc) with a polymorphism in AIF-1 isoform 2. The purpose of this study was to investigate AIF-1 expression in affected tissues from patients with SSc and to examine the regulation of its isoforms by transforming growth factor beta (TGFbeta). METHODS: AIF-1 in the skin and lung tissues of patients with SSc was analyzed by immunochemistry. AIF-1 isoform expression in response to TGFbeta and interferon-gamma stimulation was examined by quantitative polymerase chain reaction (PCR). RESULTS: AIF-1 protein was present in affected vessels of the lung and skin lesions of patients with SSc. Quantitative PCR showed an average of 14-fold higher mRNA levels in affected SSc skin than in normal skin. Double-label immunofluorescence staining demonstrated that T cells, macrophages, and endothelial cells in affected tissues expressed AIF-1. Stimulation of peripheral blood mononuclear cells with TGFbeta caused a specific and significant increase in the expression of AIF-1 isoform 2 transcripts (P \u3c 0.005), which was due to stabilization of AIF-1 isoform 2 mRNA. CONCLUSION: These data suggest that AIF-1 plays an important role in the pathogenesis of SSc owing to its increased expression in affected tissues and to the specific stimulation of AIF-1 isoform 2 by TGFbeta

Blends of wood chips from oak and cherry: impact on the general phenolic parameters and sensory profile of a white wine during the aging process

Today, there is a restricted knowledge about the potential impact of the use of different wood chip species on the white wine aging process. This lack of knowledge is even greater when wood species are used in blends of different species. Thus, the aim was to carry out a comparative analysis of the impact of different blends of wood chip species, involving oak and cherry wood, on different phenolic and color parameters, browning potential index and sensory profile of a white wine during the aging process. During the aging time studied, the use of wood chips induced an increase of wine phenolic content and color intensity, particularly in wines aged in contact with cherry wood chips alone or in blends with oak wood chips. A similar tendency was also detected for browning potential index. Regarding the sensorial results, the use of different wood chip species has an impact on the increase of "woody aroma" for the wines aged with oak wood chip species and also an increase of "body" and "astringency" descriptors for the wines aged with oak chips alone or blended with cherry chips. This work advances our understanding of the impact of different wood chip species separately or by the use of blends on white wine quality

NFκB activation and stimulation of chemokine production in normal human macrophages by the gadolinium-based magnetic resonance contrast agent Omniscan: possible role in the pathogenesis of nephrogenic systemic fibrosis.

OBJECTIVE: Nephrogenic systemic fibrosis (NSF) is a generalised fibrotic disorder occurring in certain individuals with renal insufficiency exposed to gadolinium-based contrast agents (GdBCA) for MRI. Histopathological examination of affected tissues shows increased numbers of activated macrophages. To elucidate the mechanisms responsible for macrophage activation, the effects of the GdBCA Omniscan on normal human macrophage global gene expression, chemokine production and nuclear factor κB (NFκB) activation was examined. METHODS: Normal human monocyte-derived macrophages were incubated with Omniscan (50 mM) and their gene expression analysed by microarrays and real-time PCR. Macrophage chemokine production was assayed by multiplex ELISA. NFκB activation was assessed by NFκB nuclear localisation and quantitation of intracellular levels of inducible nitric oxide synthase (iNOS) protein. A specific cell-permeable NFκB peptide inhibitor was used to abrogate NFκB stimulation of chemokine and iNOS protein levels. CCL8/MCP-2 in affected skin of patients with NSF was examined by indirect immunofluorescence. RESULTS: Omniscan caused a profound change in the transcriptome of differentiated human normal macrophages in vitro, including a large increase in the expression of genes encoding CC and CXC chemokines. It induced rapid nuclear localisation of NFκB and stimulation of iNOS protein levels and chemokine production which were blocked by an NFκB inhibitory peptide. CCL8/MCP-2, the most upregulated chemokine following in vitro macrophage exposure to Omniscan, was strongly increased in NSF-affected skin. CONCLUSION: The GdBCA Omniscan induces potent stimulation of macrophage gene expression, NFκB activation and increased NFκB-mediated production of CC and CXC chemokines and iNOS. These alterations may play a crucial role in the pathogenesis of NSF

Calcificaciones heterotópicas en cirugía protésica de cadera: Prótesis bicénctricas vs híbridas

Realizamos el seguimiento clínico y radiológico de 202 pacientes a los que se implantó una Prótesis Total de Cadera cementada (PTC). Analizamos la presencia de calcificaciones heterotópicas (CH) y su graduación, estudiando separadamente prótesis híbridas y bicéntricas. Valoramos los factores de riesgo relacionados con la aparición de CH, tamaño de implantes, alineación final, lado intervenido y relación con la presencia de complicaciones locales y sistemáticas. La prevalencia de CH fue de 35.5% (grado I, 19%; grado II, 10%; grado III, 5% y grado IV 1,5%); 9,9 % sintomáticas. Una mayor proporción de CH fue observada en varones (p=0,005); vástagos de mayor tamaño (p=0,001). No existen diferencias significativas (p=0,059) entre pacientes con prótesis híbridas frente a aquellos con prótesis bicéntricas. La valoración de Harris mostró diferencias en el seguimiento del parámetro dolor a los 24 meses entre los grupos con y sin CH.The clinical and radiological follow-up of 202 patients with a cemented Hip Artrhoplasty (HA) was reviewed. The presence of periarticular heterotopic ossifications (PHO) and its gradation was compared i9n hybrid and bipolar arthroplasties. Risk factors related to the appearance of PHO, implants size, final alignment, operated side and relationship to the postoperative local and systemic complications were several of the parameter analyzed. The presence of PHO was 35,5% (degree I, 19%; degree II, 10%; degree III, 5% and degree IV, 1,5%); 9,9% symptomatic. A greater proportion of PHO was observed in males (p=0,005) greater zie stems (p=0,001) and patients with postoperative local complications (p=0,001). There were no statistical differences between patients with hybrid hip arthoplasties as compared to those with bipolar his arthroplasties. The Harris score showed differences as regards pain at 24 months follow-up between the groups whit an without PHO