Chemical Profiling of the Endoplasmic Reticulum Proteome Using Designer Labeling Reagents.

The endoplasmic reticulum (ER) is an organelle that performs a variety of essential cellular functions via interactions with other organelles. Despite its important role, chemical tools for profiling the composition and dynamics of ER proteins remain very limited because of the labile nature of these proteins. Here, we developed ER-localizable reactive molecules (called ERMs) as tools for ER-focused chemical proteomics. ERMs can spontaneously localize in the ER of living cells and selectively label ER-associated proteins with a combined affinity and imaging tag, enabling tag-mediated ER protein enrichment and identification with liquid chromatography tandem mass spectrometry (LC-MS/MS). Using this method, we performed proteomic analysis of the ER of HeLa cells and newly assigned three proteins, namely, PAICS, TXNL1, and PPIA, as ER-associated proteins. The ERM probes could be used simultaneously with the nucleus- and mitochondria-localizable reactive molecules previously developed by our group, which enabled orthogonal organellar chemoproteomics in a single biological sample. Moreover, quantitative analysis of the dynamic changes in ER-associated proteins in response to tunicamycin-induced ER stress was performed by combining ER-specific labeling with SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative MS technology. Our results demonstrated that ERM-based chemical proteomics provides a powerful tool for labeling and profiling ER-related proteins in living cells.

Cyclophilin A Controls Salmonella Internalization Levels and is Present at E. coli Actin-Rich Pedestals.

Salmonella enterica serovar Typhimurium (S. Typhimurium), enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) commandeer the actin cytoskeleton of their host cells as a crucial step in their infectious processes. These pathogens depend on the injection of their own effectors directly into target host cells in order to usurp cellular signaling pathways that lead to morphological actin rearrangements in those cells. Here we show that the PPIase Cyclophilin A (CypA) is a novel component of S. Typhimurium-induced membrane ruffles and functions to restrict bacterial invasion levels, as in cells depleted of CypA, bacterial loads increase. We also demonstrate that CypA requires the EPEC effector Tir as well as pedestal formation for its recruitment to bacterial attachment sites and that its presence at pedestals also holds during EHEC infections. Finally, we demonstrate that CypA is found at lamellipodia; actin-rich structures at the leading edge of motile cells. Our findings further establish CypA as a component of dynamic actin-rich structures formed during bacterial infections and within cells in general. Anat Rec, 301:2086-2094, 2018. © 2018 Wiley Periodicals, Inc.

Cyclophilin A expression and its prognostic significance in lung adenocarcinoma.

Cyclophilin A (CypA) has been reported to be upregulated in malignant tumors. CypA expression is thought to be associated with acquisition of tumor growth and anti-apoptotic function. Although upregulation of CypA has been reported in lung adenocarcinoma, its clinicopathological significance and roles in malignant progression remain unclear. Here we investigated the implications of CypA expression for outcome in patients with lung adenocarcinoma. Lung adenocarcinoma specimens from 198 cases were selected and reclassified according to the World Health Organization classification (4th edition) and the Noguchi classification. CypA expression was assessed by immunohistochemistry, and the H-score was calculated on the basis of intensity and proportion. The specificity of the antibody used was confirmed by Western blotting and the cut-off point was determined from the ROC curve. Sixty-seven cases (33.8%) had low CypA expression (CypA-L group) and 131 (66.2%) had high CypA expression (CypA-H group). Many cases of adenocarcinoma in situ were CypA-L, and advanced adenocarcinomas tended to be classified as CypA-H. Clinically, patients with CypA-H tumors showed a significantly poorer prognosis than those with CypA-L tumors. This is the first investigation of the implications of the CypA expression level in terms of the clinical characteristics of resected lung adenocarcinomas.

HIV-Enhancing Factors Are Secreted by Reproductive Epithelia upon Inoculation with Bacterial Vaginosis-Associated Bacteria.

Bacterial vaginosis is a common reproductive infection in which commensal vaginal lactobacilli are displaced by a mixed population of pathogenic bacteria. Bacterial vaginosis increases susceptibility to HIV, and it has been suggested that host innate immune responses to pathogenic bacteria contribute to enhanced infection, yet the cellular mechanisms mediating the increased HIV susceptibility remain uncharacterized. We evaluated the HIV-enhancing effects of bacterial vaginosis by inoculating endocervical epithelia with Atopobium vaginae, a bacterial vaginosis-associated bacteria, and assaying secreted factors for HIV-enhancing activity. When epithelia and A. vaginae were cocultured, we observed increased HIV-enhancing activity mediated by secreted low molecular weight factors. From this complex mixture we identified several upregulated host proteins, which functioned in combination to enhance HIV infection. These studies suggest that the host immune response to bacterial vaginosis-associated bacteria results in the release of HIV-enhancing factors. The combined activity of bacterial vaginosis-induced proteins likely mediates HIV enhancement.

Transcriptional expression study in the central nervous system of rats: what gene should be used as internal control?

OBJECTIVE: A growing number of published articles report the expression of specific genes with different behavior patterns in rats. The levels of messenger ribonucleic acid transcripts are usually analyzed by reverse transcription followed by polymerase chain reaction and quantified after normalization with an internal control or reference gene (housekeeping gene). Nevertheless, housekeeping genes exhibit different expression in the central nervous system, depending on the physiological conditions and the area of the brain to be studied. The choice of a good internal control gene is essential for obtaining reliable results. This study evaluated the expression of three housekeeping genes (beta-actin, cyclophilin A, and ubiquitin C) in different areas of the central nervous system in rats (olfactory bulb, hippocampus, striatum, and prefrontal cortex). METHODS: Wistar rats (virgin females, n=6) during the diestrum period were used. Total ribonucleic acid was extracted from each region of the brain; the complementary deoxyribonucleic acid was synthesized by reverse transcription and amplified by real-time quantitative polymerase chain reaction using SYBR™ Green and primers specific for each one of the reference genes. The stability of the expression was determined using NormFinder. RESULTS: Beta-actin was the most stable gene in the hippocampus and striatum, while cyclophilin A and ubiquitin C showed greater stability in the prefrontal cortex and the olfactory bulb, respectively. CONCLUSION: Based on our study, further studies of gene expression using rats as animal models should take into consideration these results when choosing a reliable internal control gene.

Transcriptional expression study in the central nervous system of rats: what gene should be used as internal control? / Estudo de expressão transcricional no sistema nervoso central de ratos: qual gene deve ser usado como controle interno?

Objective A growing number of published articles report the expression of specific genes with different behavior patterns in rats. The levels of messenger ribonucleic acid transcripts are usually analyzed by reverse transcription followed by polymerase chain reaction and quantified after normalization with an internal control or reference gene (housekeeping gene). Nevertheless, housekeeping genes exhibit different expression in the central nervous system, depending on the physiological conditions and the area of the brain to be studied. The choice of a good internal control gene is essential for obtaining reliable results. This study evaluated the expression of three housekeeping genes (beta-actin, cyclophilin A, and ubiquitin C) in different areas of the central nervous system in rats (olfactory bulb, hippocampus, striatum, and prefrontal cortex). Methods Wistar rats (virgin females, n=6) during the diestrum period were used. Total ribonucleic acid was extracted from each region of the brain; the complementary deoxyribonucleic acid was synthesized by reverse transcription and amplified by real-time quantitative polymerase chain reaction using SYBR™ Green and primers specific for each one of the reference genes. The stability of the expression was determined using NormFinder. Results Beta-actin was the most stable gene in the hippocampus and striatum, while cyclophilin A and ubiquitin C showed greater stability in the prefrontal cortex and the olfactory bulb, respectively. Conclusion Based on our study, further studies of gene expression using rats as animal models should take into consideration these results when choosing a reliable internal control gene. .

Objetivo Um número crescente de artigos publicados relaciona a expressão de genes específicos com diferentes padrões de comportamento em ratos. Os níveis de transcritos de ácido ribonucleico mensageiro são geralmente analisados por transcrição reversa, seguida de reação em cadeia da polimerase, e quantificados após a normalização com um controle interno ou gene de referência (gene housekeeping). No entanto, os genes housekeeping exibem expressão diferencial no sistema nervoso central, dependendo das condições fisiológicas e da área do cérebro a ser estudada. A escolha de um bom gene de controle interno é essencial para a obtenção de resultados confiáveis. Este estudo avaliou a expressão de três genes housekeeping (beta-actina, ciclofilina A e ubiquitina C) em diferentes áreas do sistema nervoso central de ratos (bulbo olfatório, hipocampo, estriado e córtex pré-frontal). Métodos Foram usadas ratas Wistar (fêmeas virgens, n=6) durante o período de diestro. O ácido ribonucleico total foi extraído a partir de cada região do cérebro; o ácido desoxirribonucleico complementar foi sintetizado por transcrição reversa e amplificado por reação em cadeia da polimerase quantitativo em tempo real utilizando SYBR® Green e primers específicos para cada um dos genes de referência. A estabilidade de expressão foi determinada utilizando NormFinder. Resultados A beta-actina foi o gene mais estável no hipocampo e estriado, enquanto a ciclofilina A e a ubiquitina C apresentaram maior estabilidade no córtex pré-frontal e no bulbo olfatório, respectivamente. Conclusão Com base em nosso trabalho, estudos posteriores de expressão gênica utilizando ratos como modelos animais devem levar ...

Proteomic analysis and immune properties of exosomes released by macrophages infected with Mycobacterium avium.

The role of exosomes shed from Mycobacterium avium sp. paratuberculosis-infected macrophages in intercellular communication processes was examined. We compared the responses of resting macrophages infected with M. avium sp. paratuberculosis with those of resting macrophages treated with exosomes previously released from macrophages infected with M. avium sp. paratuberculosis. Some proteins components of exosomes released from resting macrophages infected with M. avium sp. paratuberculosis showed a significantly differential expression compared with exosomes from uninfected-macrophages. Both M. avium sp. paratuberculosis and exosomes from infected-cells enhanced the expression of CD80 and CD86 and the secretion of TNF-α and IFN-γ by macrophages. This suggests that exosomes from infected macrophages may be carriers of molecules, e.g. bacterial antigens and/or components from infected macrophages, that can elicit responses in resting cells. Two-dimensional analysis of the proteins present in exosomes from M. avium sp. paratuberculosis-infected macrophages compared with those from resting cells resulted in the identification by MALDI-TOF/TOF mass spectrometry of the following differentially expressed proteins: two actin isoforms, guanine nucleotide-binding protein ß-1, cofilin-1 and peptidyl-prolyl cis-trans isomerase A. The possible relevance of the changes observed and the biological functions of the proteins differentially present are discussed.

Decreased tissue levels of cyclophilin A, a cyclosporine a target and phospho-ERK1/2 in simvastatin patients with abdominal aortic aneurysm.

BACKGROUND: Cyclophilin A (CyPA), a cyclosporine A-binding protein, influences abdominal aortic aneurysm (AAA) formation and the ERK1/2 signalling pathway in animal and in vitro studies. Statins decrease CyPA in smooth muscle cells although their influence on CyPA in human AAA is unknown. MATERIAL AND METHODS: The study was performed on AAA wall-tissue samples obtained from 30 simvastatin-treated and 15 non-statin patients (2:1 case to control). The patients were matched by age, sex and AAA diameter. We investigated the gene expression of CyPA, its receptor extracellular matrix metalloproteinase inducer (EMMPRIN) by real-time RT-PCR. CyPA and EMMPRIN protein level and phosphorylated extracellular signal-regulated kinases 1 and 2 (ERK1/2) were measured by Western blot. RESULTS: The AAA wall tissue from simvastatin-treated patients had significantly lower CyPA gene expression and protein levels (P = 0.0018, P = 0.0083, respectively). Furthermore, phosphorylation of ERK1 and ERK2 was markedly suppressed in the simvastatin group (P = 0.0002, P = 0.0027, respectively). However, simvastatin did not influence EMMPRIN gene and protein expression. CONCLUSION: Simvastatin-treated patients with AAA exert lower CyPA messenger RNA (mRNA), as well as CyPA intracellular protein levels and a decreased amount of phospho-ERK1/2. Thus, the interference with signalling pathways leading to CyPA formation and ERK1/2 activation reveals a new anti-inflammatory role of statins in AAA.

Over-expression and potential role of cyclophilin A in human periodontitis.

BACKGROUND AND OBJECTIVE: We previously demostrated that EMMPRIN participates in the periodontitis and its interaction with Cyclophilin A possibly exists in animal periodontitis models. This study is aimed to address the expression and potential role of cyclophilin A (CypA) in human periodontitis. MATERIAL AND METHODS: Gingival tissues and peripheral blood were collected from patients with moderate to severe periodontitis or from healthy donors. Western blotting and immunohistochemistry were performed to detect the expression and distribution of CypA in the gingival tissues. Peripheral blood mononuclear cells (PBMCs) and neutrophils were isolated from the peripheral blood by Ficoll-Paque density-gradient centrifugation. Chemotaxis assays were applied to evaluate the effects of different concentrations of CypA (100, 300 and 500 ng/mL) on the migration of PBMCs and neutrophils. Supernatants of human THP-1 cells were collected after treatment with 200 ng/mL of CypA for different periods of time (1, 3, 6, 12 and 24 h) to detect the levels of interleukin (IL)-1ß, IL-8 and tumor necrosis factor alpha (TNF-α) by ELISA. RESULTS: Western blot analyses revealed an increase of CypA expression in inflamed gingival tissues compared with healthy tissues. Immunohistochemistry identified that the over-expressed CypA was localized in the infiltrating cells and/or in the extracellular matrix in the inflamed gingival connective tissues. The positive infiltrating cells contained mononuclear cells and lobulated-nuclei neutrophils. Chemotactic assays showed that 300 ng/mL of CypA apparently facilitated the chemotaxis of PBMCs/neutrophils from healthy donors, compared with the no-treatment control (p < 0.01 for PBMCs, p < 0.05 for neutrophils), whereas 100 and 500 ng/mL of CypA only weakly enhanced the chemotaxis of PBMCs/neutrophils (p > 0.05 for PBMCs/neutrophils, not significant). The PBMCs/neutrophils from patients with periodontitis exhibited a stronger ability to migrate when stimulated with 300 ng/mL of CypA than did PBMCs/neutrophils from healthy donors (p < 0.05 for PBMCs, p < 0.01 for neutrophils). ELISA revealed that the level of TNF-α secreted by THP-1 cells was elevated after treatment with 200 ng/mL of CypA for 12 h compared with the no-treatment 0-h control (p < 0.05). The IL-8 level was sharply raised after 3 h of stimulation with 200 ng/mL of CypA (p < 0.01 compared with 0 h), but no significant change was observed at the other time points (p > 0.05). There was no statistical difference at any of the treatment time points for the secretion of IL-1ß (p > 0.05 for 1, 3, 6, 12 and 24 h compared with 0 h). CONCLUSIONS: CypA participates in the pathogenesis of human periodontitis. It may be involved in the inflammatory response of periodontal tissues through inducing the chemotaxis of PBMCs/neutrophils and the secretion of TNF-α/IL-8.

Cyclophilin A predicts clinical outcome in patients with congestive heart failure undergoing endomyocardial biopsy.

AIMS: Cyclophilin A (CyPA) represents a ubiquitous intracellular protein, which is secreted by inflammatory and by dying/necrotic cells. The aim of this study was to evaluate the prognostic relevance of CyPA expression in endomyocardial biopsies of consecutive patients with congestive heart failure. METHODS AND RESULTS: A total of 227 unselected patients (age 53.9 ± 15 years) with congestive heart failure undergoing endomyocardial biopsy for diagnostic reasons were enrolled. Biopsies were analysed using established histopathological and immunohistological criteria together with CyPA staining. Virus genome was studied by polymerase chain reaction. CyPA was significantly enhanced in patients with inflammatory cardiomyopathy (n = 127) as compared with patients with non-inflammatory cardiomyopathy (n = 100, P < 0.0001). During a mean follow-up of 16.3 months, 60 patients (26.4%) reached the primary endpoint, a composite of all-cause death, heart transplantation, malignant arrhythmia, and heart failure-related rehospitalization. Of all clinical (ejection fraction, New York Heart Association functional class), laboratory (brain natriuretic peptide), and immunohistological parameters (CyPA, extracellular matrix metalloproteinase inducer, CD68, CD3, major hisocompatibility complex II, and virus genome) tested, only CyPA was identified as an independent predictor for the composite endpoint [hazard ratio (HR) 2.4; 95% confidence interval (CI) 1.2-5.2; P = 0.019] as well as for all-cause death and heart transplantation alone (HR 4.7; 95% CI 1.1-19.8; P = 0.036). Subgroup analysis revealed CyPA as a predictor in patients with non-inflammatory cardiomyopathy for the composite endpoint (HR 3.0; 95% CI 1.3-6.6; P = 0.007) as well as all-cause death or heart transplantation alone (HR 6.4; 95% CI 1.4-28.1; P = 0.014). CONCLUSIONS: CyPA is an independent predictor of clinical outcome in patients with congestive heart failure undergoing endomyocardial biopsy.

Disrupting the EMMPRIN (CD147)-cyclophilin A interaction reduces infarct size and preserves systolic function after myocardial ischemia and reperfusion.

OBJECTIVE: Inflammation and proteolysis crucially contribute to myocardial ischemia and reperfusion injury. The extracellular matrix metalloproteinase inducer EMMPRIN (CD147) and its ligand cyclophilin A (CyPA) may be involved in both processes. The aim of the study was to characterize the role of the CD147 and CyPA interplay in myocardial ischemia/reperfusion (I/R) injury. METHODS AND RESULTS: Immunohistochemistry showed enhanced expression of CD147 and CyPA in myocardial sections from human autopsies of patients who had died from acute myocardial infarction and from mice at 24 hours after I/R. At 24 hours and 7 days after I/R, the infarct size was reduced in CD147(+/-) mice vs CD147(+/+) mice (C57Bl/6), in mice (C57Bl/6) treated with monoclonal antibody anti-CD147 vs control monoclonal antibody, and in CyPA(-/-) mice vs CyPA(+/+) mice (129S6/SvEv), all of which are associated with reduced monocyte and neutrophil recruitment at 24 hours and with a preserved systolic function at 7 days. The combination of CyPA(-/-) mice with anti-CD147 treatment did not yield further protection compared with either inhibition strategy alone. In vitro, treatment with CyPA induced monocyte chemotaxis in a CD147- and phosphatidylinositol 3-kinase-dependent manner and induced monocyte rolling and adhesion to endothelium (human umbilical vein endothelial cells) under flow in a CD147-dependent manner. CONCLUSION: CD147 and its ligand CyPA are inflammatory mediators after myocardial ischemia and reperfusion and represent potential targets to prevent myocardial I/R injury.

Increased expression of peroxiredoxin 6 and cyclophilin A in squamous cell carcinoma of the tongue.

OBJECTIVES: The aim of this study was to assess the expression levels of two proteins, such as PRDX6 and cyclophilin A (CypA), and to evaluate their relationship with clinicopathologic features and survival in tongue squamous cell carcinomas (TSCCs). MATERIAL AND METHODS: An immunohistochemical study was performed comprising a total of 42 tissue samples of patients suffering from TSCCs as well as 10 corresponding adjacent normal tissues. After detection of PRDX6 and CypA, their expression levels were semiquantitatively evaluated and correlated with clinicopathologic variables. RESULTS: Both PRDX6 and CypA expressions were significantly higher in tissue samples of TSCCs compared with the 10 corresponding adjacent normal tissues (P < 0.01). A statistically significant correlation in TSCCs regarding the expression of PRDX6 and CypA was revealed (P = 0.005), and the lymphadenectasis was correlated with PRDX6 (P < 0.05). Results of a multivariate analysis revealed age, CypA expression, cervical lymph node metastases, and tongue cancer differentiation to be independent prognostic variables in respect of the overall survival rate (P < 0.05). CONCLUSIONS: It could be detected that PRDX6 and CypA are associated with tumorigenesis in TSCCs. High levels of CypA expression may predict reduced survival time.

Strain-specific differences in the impact of human TRIM5alpha, different TRIM5alpha alleles, and the inhibition of capsid-cyclophilin A interactions on the infectivity of HIV-1.

HIV-1 infectivity is strongly restricted by TRIM5α from certain primate species but has been described as being only marginally susceptible to human TRIM5α. In this study, we evaluated the effects of the modulation of human TRIM5α activity (pretreatment of target cells with alpha interferon, expression of a pre-miRNA targeting TRIM5α, and/or overexpression of TRIM5γ), the inhibition of cyclophilin A (CypA)-CA interactions, and the expression of different allelic variants of human TRIM5α on the infectivity of a series of recombinant viruses carrying different patient-derived Gag-protease sequences. We show that HIV-1 displays virus-specific differences in its sensitivity to human TRIM5α and in its sensitivity to different TRIM5α alleles. The effect of inhibiting CypA-CA interactions is also strain specific, and blocking these interactions can either inhibit or improve viral infectivity, depending on the isolate studied. The inhibition of CypA-CA interactions also modulates viral sensitivity to human TRIM5α. In the absence of CypA-CA interactions, most viruses displayed increased sensitivity to the inhibitory effects of TRIM5α on viral replication, but one isolate showed a paradoxical decrease in sensitivity to TRIM5α. Taken together, these findings support a model in which three interlinked factors--capsid sequence, CypA levels, and TRIM5α--interact to determine capsid stability and therefore viral infectivity.

Amino acid alterations in Gag that confer the ability to grow in simian cells on HIV-1 are located at a narrow CA region.

We previously generated a prototype monkey-tropic human immunodeficiency virus type 1 (HIV-1) designated NL-DT5R. This viral clone has a small region of simian immunodeficiency virus (SIV) within Gag capsid (CA) protein and also SIV Vif protein, but displays a poor growth phenotype in simian cells. To improve the growth potential of NL-DT5R, we have constructed a series of its gag variant viruses. Out of fourteen viral clones generated, five were infectious for simian HSC-F cells, and two of the infectious variants grew similarly with NL-DT5R. Taking their genome structures into consideration, our data here clearly show that a narrow CA region within the Gag protein, i.e., the domain around cyclophilin A (CypA)-binding loop, is critical for the growth ability of HIV-1 in simian cells.

Throughput and efficiency of a mass spectrometry-based screening assay for protein-ligand binding detection.

An H/D exchange- and MALDI mass spectrometry-based screening assay was applied to search for novel ligands that bind to cyclophilin A, a potential therapeutic and diagnostic target in lung cancer. The assay is based on stability of unpurified proteins from rates of H/D exchange (SUPREX), which exploits the H/D exchange properties of amide protons to measure the increase in a protein's thermodynamic stability upon ligand binding in solution. The current study evaluates the throughput and efficiency with which 880 potential ligands from the Prestwick Chemical Library (Illkirch, France) could be screened for binding to cyclophilin A. Screening was performed at a rate of 3 min/ligand using a conventional MALDI mass spectrometer. False positive and false negative rates, based on a set of control data, were as low as 0% and 9%, respectively. Based on the 880-member library screening, a false positive rate of 0% was observed when a two-tier selection strategy was implemented. Although novel ligands for cyclophilin A were not discovered, cyclosporin A, a known ligand to CypA and a blind control in the library, was identified as a hit. We also describe a new strategy to eliminate some of the complications related to back exchange that can arise in screening applications of SUPREX.

A proteomic study of the effects of ramipril on post-infarction left ventricular remodelling in the rabbit.

OBJECTIVES: In this study, we used a proteomic approach to investigate the potential proteins regulated by ramipril in post-infarction left ventricular remodelling in the rabbit. METHODS AND RESULTS: Myocardial infarction (MI) was induced in male New Zealand White rabbits (2.5-3 kg) by ligation of the left anterior descending coronary artery. Two months later, the rabbits were either left untreated (MI group) or were treated daily for one month with 0.1 mg/kg wt of ramipril (ramipril group), then sacrificed. One month of ramipril treatment resulted in a significant improvement in the LV ejection fraction (LVEF) and a decrease in hydroxyproline content. The protein profiles of LV tissue showed that ramipril caused upregulation of glutathione peroxidase, superoxide dismutase (SOD), and heart-type fatty acid binding-protein (h-FABP) and downregulation of HSP27 and cyclophilin A. Ramipril treatment caused an increase in catalase, glutathione peroxidase, and SOD activity in the LV tissue. Oxidized glutathione levels and the GSSG/GSH ratio in the heart tissue were lower in the ramipril group than in the MI group. CONCLUSIONS: Ramipril increased antioxidative protein expression and enzyme activity, which could partly explain the role of ramipril in attenuating LV remodelling. In addition, the present study identifies several potential protein targets which may help to explain the mechanism by which ramipril exerts its effect in post-infarction LV remodelling in the rabbit.

Proteomic analysis of Trypanosoma cruzi resistance to Benznidazole.

The first proteomic analysis of Trypanosoma cruzi resistance to Benznidazole (BZ) is presented. The differential proteome of T. cruzi with selected in vivo resistance to Benznidazole (BZR and Clone27R), its susceptible pairs (BZS and Clone9S), and a pair from a population with Benznidazole- in vitro-induced resistance (17LER) and the susceptible pair 17WTS were analyzed by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS) for protein identification. Out of 137 spots analyzed through MS, 110 were identified as 56 distinct proteins. Out of the 56 distinct proteins, 36 were present in resistant, 9 in susceptible, and 11 in both phenotypes. Among the proteins identified in resistant samples, 5 were found in Cl 27R and in BZR (calpain-like cysteine peptidase, hypothetical protein conserved 26 kDa, putative peptidase, peroxiredoxin and tyrosine amino transferase) and 4 in Cl 27R and 17LER (cyclophilin A, glutamate dehydrogenase, iron superoxide dismutase and nucleoside diphosphate kinase). As for the proteins identified in Benznidazole-susceptible samples, PGF-2a was found in BZS and 17WTS. A functional category analysis showed that the proteins involved with transcription and protein destination were overexpressed for the Benznidazole-resistant phenotype. Thus, the present study provides large-scale, protein-related information for investigation of the mechanism of T. cruzi resistance to Benznidazole.

The cyclophilin inhibitor Debio-025 shows potent anti-hepatitis C effect in patients coinfected with hepatitis C and human immunodeficiency virus.

UNLABELLED: Debio-025 is an oral cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus activity in vitro. Its effect on viral load as well as its influence on intracellular Cyp levels was investigated in a randomized, double-blind, placebo-controlled study. Mean hepatitis C viral load decreased significantly by 3.6 log(10) after a 14-day oral treatment with 1200 mg twice daily (P < 0.0001) with an effect against the 3 genotypes (1, 3, and 4) represented in the study. In addition, the absence of viral rebound during treatment indicates that Debio-025 has a high barrier for the selection of resistance. In Debio-025-treated patients, cyclophilin B (CypB) levels in peripheral blood mononuclear cells decreased from 67 +/- 6 (standard error) ng/mg protein (baseline) to 5 +/- 1 ng/mg protein at day 15 (P < 0.01). CONCLUSION: Debio-025 induced a strong drop in CypB levels, coinciding with the decrease in hepatitis C viral load. These are the first preliminary human data supporting the hypothesis that CypB may play an important role in hepatitis C virus replication and that Cyp inhibition is a valid target for the development of anti-hepatitis C drugs.

Muscle contraction accelerates IL-6 mRNA expression in the rat masseter muscle.

OBJECTIVE: This study was conducted to determine if interleukin-6 (IL-6) and interleukin-1beta (IL-1beta) mRNA expression increase in response to muscle contraction caused by repetitive electrical stimulation of the rat masseter muscle. METHODS: Male Wistar rats weighing 140-160 g were divided randomly into the following three groups: electrical stimulation (ES) group (n=21), carrageenan injection (CI) group (n=24), and ES under dantrolene sodium (muscle relaxant) injection (ESDI) group (n=7). ES or CI was done to the left masseter; and mock ES or mock CI to the right. Muscle tissues on both sides were sampled for total RNA isolation. Real-time RT-PCR was performed, with the cyclophilin A (CypA) mRNA level in each sample as an internal control. Mean relative IL-6 (il-6/cypA) and IL-1beta (il-1beta/cypA) mRNA levels were compared between the experimental and mock-treated sides within each group. RESULTS: Mean IL-6/CypA levels in the ES- or CI-treated muscle significantly increased, without any significant incremental change observed in either mock-treated muscle. Interestingly, the increase in the il-6/cypA level caused by the ES was suppressed by the injection of dantrolene sodium in the ESDI group. Furthermore, the mean il-1beta/cypA level in the CI-treated masseter also significantly increased without any significant incremental change observed in the mock-treated muscle. However, there was no significant difference in the mean il-1beta/cypA levels in the masseter between the ES- and the mock-treated sides. CONCLUSIONS: These results show that IL-6 mRNA expression in the rat masseter muscle was accelerated by the CI or by repetitive muscle contraction induced by ES. Since the mRNA level of IL-1beta, a well-known proinflammatory cytokine, was not altered by the contraction, the accelerated IL-6 mRNA expression elicited by the muscle contraction does not seem to be related to local inflammation.