Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes

<p>Abstract</p> <p>Background</p> <p>MMP-13 and IGFBP-5 are important factors involved in osteoarthritis (OA). We investigated whether two highly predicted microRNAs (miRNAs), miR-140 and miR-27a, regulate these two genes in human OA chondrocytes.</p> <p>Methods</p> <p>Gene expression was determined by real-time PCR. The effect of each miRNA on IGFBP-5 and MMP-13 expression/production was evaluated by transiently transfecting their precursors (pre-miRNAs) and inhibitors (anti-miRNAs) into human OA chondrocytes. Modulation of IGFBP-5, miR-140 and miR-27a expression was determined upon treatment of OA chondrocytes with cytokines and growth factors.</p> <p>Results</p> <p>IGFBP-5 was expressed in human chondrocytes with its level significantly lower (p < 0.04) in OA. Five computational algorithms identified miR-140 and miR-27a as possible regulators of MMP-13 and IGFBP-5 expression. Data showed that both miRNAs were expressed in chondrocytes. There was a significant reduction (77%, p < 0.01) in miR-140 expression in OA compared to the normal chondrocytes, whereas miR-27a expression was only slightly decreased (23%). Transfection with pre-miR-140 significantly decreased (p = 0.0002) and with anti-miR-140 significantly increased (p = 0.05) IGFBP-5 expression at 24 hours, while pre-miR-27a did not affect either MMP-13 or IGFBP-5. Treatment with anti-miR-27a, but not with anti-miR-140, significantly increased the expression of both MMP-13 (p < 0.05) and IGFBP-5 (p < 0.01) after 72 hours of incubation. MMP-13 and IGFBP-5 protein production followed the same pattern as their expression profile. These data suggest that IGFBP-5 is a direct target of miR-140, whereas miR-27a down-regulates, likely indirectly, both MMP-13 and IGFBP-5.</p> <p>Conclusion</p> <p>This study is the first to show the regulation of these miRNAs in human OA chondrocytes. Their effect on two genes involved in OA pathophysiology adds another level of complexity to gene regulation, which could open up novel avenues in OA therapeutic strategies.</p

Phase I study of IMGN901, a CD56-targeting antibody-drug conjugate, in patients with CD56-positive solid tumors.

Background IMGN901 is a CD56-targeting antibody-drug conjugate designed for tumor-selective delivery of the cytotoxic maytansinoid DM1. This phase 1 study investigated the safety, tolerability, pharmacokinetics, and preliminary activity of IMGN901 in patients with CD56-expressing solid tumors. Methods Patients were enrolled in cohorts of escalating IMGN901 doses, administered intravenously, on 3 consecutive days every 21 days. A dose-expansion phase accrued patients with small cell lung cancer (SCLC), Merkel cell carcinoma (MCC), or ovarian cancer. Results Fifty-two patients were treated at doses escalating from 4 to 94 mg/m(2)/day. The maximum tolerated dose (MTD) was determined to be 75 mg/m(2). Dose-limiting toxicities included fatigue, neuropathy, headache or meningitis-like symptoms, chest pain, dyspnea, and myalgias. In the dose-expansion phase (n = 45), seven patients received 75 mg/m(2) and 38 received 60 mg/m(2) for up to 21 cycles. The recommended phase 2 dose (RP2D) was established at 60 mg/m(2) during dose expansion. Overall, treatment-emergent adverse events (TEAEs) were experienced by 96.9 % of all patients, the majority of which were Grade 1 or 2. The most commonly reported Grade 3 or 4 TEAEs were hyponatremia and dyspnea (each 8.2 %). Responses included 1 complete response (CR), 1 clinical CR, and 1 unconfirmed partial response (PR) in MCC; and 1 unconfirmed PR in SCLC. Stable disease was seen for 25 % of all evaluable patients who received doses ≥60 mg/m(2). Conclusions The RP2D for IMGN901 of 60 mg/m(2) administered for 3 consecutive days every 3 weeks was associated with an acceptable tolerability profile. Objective responses were observed in patients with advanced CD56+ cancers

Selective Hyper-responsiveness of the Interferon System in Major Depressive Disorders and Depression Induced by Interferon Therapy

Though an important percentage of patients with chronic hepatitis C virus (HCV) undergoing interferon (IFN) therapy develop depressive symptoms, the role of the IFN system in the pathogenesis of depressive disorders is not well understood.50 patients with HCV infection were treated with standard combination therapy (pegylated IFN-α2a/ribavirin). IFN-induced gene expression was analyzed to identify genes which are differentially regulated in patients with or without IFN-induced depression. For validation, PBMC from 22 psychiatric patients with a severe depressive episode (SDE) and 11 controls were cultivated in vitro with pegylated IFN-α2a and gene expression was analyzed.IFN-induced depression in HCV patients was associated with selective upregulation of 15 genes, including 6 genes that were previously described to be relevant for major depressive disorders or neuronal development. In addition, increased endogenous IFN-production and selective hyper-responsiveness of these genes to IFN stimulation were observed in SDE patients.Our data suggest that selective hyper-responsiveness to exogenous (IFN therapy) or endogenous (depressive disorders) type I IFNs may lead to the development of depressive symptoms. These data could lead to the discovery of novel therapeutic approaches to treat IFN-induced and major depressive disorders

All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells

BACKGROUND & AIMS: Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen. METHODS: Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity. RESULTS: Cell preparation yielded the following cell counts per gram of liver tissue: 2.0+/-0.4x107 hepatocytes, 1.8+/-0.5x106 Kupffer cells, 4.3+/-1.9x105 liver sinusoidal endothelial cells, and 3.2+/-0.5x105 stellate cells. Hepatocytes were identified by albumin (95.5+/-1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5+/-1.2%) and exhibited phagocytic activity, as determined with 1mum latex beads. Endothelial cells were CD146+ (97.8+/-1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of alpha-smooth muscle actin (97.1+/-1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence. CONCLUSIONS: Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease

Correlation of histopathologic characteristics to protein expression and function in malignant melanoma

BACKGROUND: Metastatic melanoma is still one of the most prevalent skin cancers, which upon progression has neither a prognostic marker nor a specific and lasting treatment. Proteomic analysis is a versatile approach with high throughput data and results that can be used for characterizing tissue samples. However, such analysis is hampered by the complexity of the disease, heterogeneity of patients, tumors, and samples themselves. With the long term aim of quest for better diagnostics biomarkers, as well as predictive and prognostic markers, we focused on relating high resolution proteomics data to careful histopathological evaluation of the tumor samples and patient survival information. PATIENTS AND METHODS: Regional lymph node metastases obtained from ten patients with metastatic melanoma (stage III) were analyzed by histopathology and proteomics using mass spectrometry. Out of the ten patients, six had clinical follow-up data. The protein deep mining mass spectrometry data was related to the histopathology tumor tissue sections adjacent to the area used for deep-mining. Clinical follow-up data provided information on disease progression which could be linked to protein expression aiming to identify tissue-based specific protein markers for metastatic melanoma and prognostic factors for prediction of progression of stage III disease. RESULTS: In this feasibility study, several proteins were identified that positively correlated to tumor tissue content including IF6, ARF4, MUC18, UBC12, CSPG4, PCNA, PMEL and MAGD2. The study also identified MYC, HNF4A and TGFB1 as top upstream regulators correlating to tumor tissue content. Other proteins were inversely correlated to tumor tissue content, the most significant being; TENX, EHD2, ZA2G, AOC3, FETUA and THRB. A number of proteins were significantly related to clinical outcome, among these, HEXB, PKM and GPNMB stood out, as hallmarks of processes involved in progression from stage III to stage IV disease and poor survival. CONCLUSION: In this feasibility study, promising results show the feasibility of relating proteomics to histopathology and clinical outcome, and insight thus can be gained into the molecular processes driving the disease. The combined analysis of histological features including the sample cellular composition with protein expression of each metastasis enabled the identification of novel, differentially expressed proteins. Further studies are necessary to determine whether these putative biomarkers can be utilized in diagnostics and prognostic prediction of metastatic melanoma

The Naturally Occurring YMDD Mutation among Patients Chronically Infected HBV and Untreated with Lamivudine: A Systematic Review and Meta-Analysis

Background: Several recent reports have demonstrated that tyrosine (Y)-methionine (M)-aspartic acid (D)-aspartic acid (D) (YMDD) motif mutations can naturally occur in chronic HBV patients without antiviral treatment such as lamivudine therapy. This paper aims to assess the overall spontaneous incidence and related risk factors of YMDD-motif mutations among lamivudine-naïve chronic HBV carriers, so as to provide some clue for clinical treatment of hepatitis B. Methodology/Principal Findings: Chinese and English literatures were searched for studies reporting natural YMDD mutations among untreated chronic HBV patients from 2001 to 2010. The incidence estimates were summarized and analyzed by meta-analyses. Forty-seven eligible articles from eight countries were selected in this review (13 in English and 34 in Chinese). The pooled incidence of YMDD-motif mutation among untreated chronic HBV patients from eight countries was 12.21 % (95 % CI: 9.69%–14.95%). China had an incidence of 13.38 % (95 % CI: 10.90%–16.07%) and seven other countries had an incidence of 9.90 % (95 % CI: 3.28%–19.55%), respectively. Lamivudine therapy would increase the risk of mutations 5.23 times higher than the untreated patients. A higher HBV DNA copy number was associated with increased incidence of natural YMDD mutation. No significant difference was found in YMDD mutation incidence between groups of different gender, age, HBeAg status, patients ’ ALT (alanine aminotransferase) level, and between the groups of HBV genotype B and C. Conclusions: The YMDD-motif mutations can occur spontaneously with a relatively high incidence in CHB patient

Downregulation of the AU-Rich RNA-Binding Protein ZFP36 in Chronic HBV Patients: Implications for Anti-Inflammatory Therapy

Inflammation caused by chronic hepatitis B virus (HBV) infection is associated with the development of cirrhosis and hepatocellular carcinoma; however, the mechanisms by which HBV infection induces inflammation and inflammatory cytokine production remain largely unknown. We analyzed the gene expression patterns of lymphocytes from chronic HBV-infected patients and found that the expression of ZFP36, an AU-rich element (ARE)-binding protein, was dramatically reduced in CD4+ and CD8+ T lymphocytes from chronic HBV patients. ZFP36 expression was also reduced in CD14+ monocytes and in total PBMCs from chronic HBV patients. To investigate the functional consequences of reduced ZFP36 expression, we knocked down ZFP36 in PBMCs from healthy donors using siRNA. siRNA-mediated silencing of ZFP36 resulted in dramatically increased expression of multiple inflammatory cytokines, most of which were also increased in the plasma of chronic HBV patients. Furthermore, we found that IL-8 and RANTES induced ZFP36 downregulation, and this effect was mediated through protein kinase C. Importantly, we found that HBsAg stimulated PBMCs to express IL-8 and RANTES, resulting in decreased ZFP36 expression. Our results suggest that an inflammatory feedback loop involving HBsAg, ZFP36, and inflammatory cytokines may play a critical role in the pathogenesis of chronic HBV and further indicate that ZFP36 may be an important target for anti-inflammatory therapy during chronic HBV infection

Cationic Amino Acid Transporter-2 Regulates Immunity by Modulating Arginase Activity

Cationic amino acid transporters (CAT) are important regulators of NOS2 and ARG1 activity because they regulate L-arginine availability. However, their role in the development of Th1/Th2 effector functions following infection has not been investigated. Here we dissect the function of CAT2 by studying two infectious disease models characterized by the development of polarized Th1 or Th2-type responses. We show that CAT2−/− mice are significantly more susceptible to the Th1-inducing pathogen Toxoplasma gondii. Although T. gondii infected CAT2−/− mice developed stronger IFN-γ responses, nitric oxide (NO) production was significantly impaired, which contributed to their enhanced susceptibility. In contrast, CAT2−/− mice infected with the Th2-inducing pathogen Schistosoma mansoni displayed no change in susceptibility to infection, although they succumbed to schistosomiasis at an accelerated rate. Granuloma formation and fibrosis, pathological features regulated by Th2 cytokines, were also exacerbated even though their Th2 response was reduced. Finally, while IL-13 blockade was highly efficacious in wild-type mice, the development of fibrosis in CAT2−/− mice was largely IL-13-independent. Instead, the exacerbated pathology was associated with increased arginase activity in fibroblasts and alternatively activated macrophages, both in vitro and in vivo. Thus, by controlling NOS2 and arginase activity, CAT2 functions as a potent regulator of immunity