Correction to: Relationship between TRAF6 and deterioration of HCC: an immunohistochemical and in vitro study.

[This corrects the article DOI: 10.1186/s12935-016-0352-z.].

Expression of IL-22 in the Skin Causes Th2-Biased Immunity, Epidermal Barrier Dysfunction, and Pruritus via Stimulating Epithelial Th2 Cytokines and the GRP Pathway.

Increased expression of Th22 cytokine IL-22 is a characteristic finding in atopic dermatitis (AD). However, the specific role of IL-22 in the pathogenesis of AD in vivo has yet to be elucidated. Consistent with observations in human AD, IL-22 was significantly increased in the AD skin of mice after epicutaneous sensitization to house dust mite allergen. Utilizing a skin-specific inducible transgenic system, we show in the present study that expression of IL-22 in the skin of mice caused an AD-like phenotype characterized by chronic pruritic dermatitis associated with Th2-biased local and systemic immune responses, downregulation of epidermal differentiation complex genes, and enhanced dermatitis upon epicutaneous allergen exposure. IL-22 potently induced the expression of gastrin-releasing peptide (GRP), a neuropeptide pruritogen, in dermal immune cells and sensory afferents and in their skin-innervating sensory neurons. IL-22 also differentially upregulated the expression of GRP receptor (GRPR) on keratinocytes of AD skin. The number of GRP+ cells in the skin correlated with the AD severity and the intensity of pruritus. IL-22 directly upregulated the expression of epithelial-derived type 2 cytokines (thymic stromal lymphopoietin and IL-33) and GRP in primary keratinocytes. Furthermore, GRP not only strongly induced thymic stromal lymphopoietin but it also increased the expression of IL-33 and GRPR synergistically with IL-22. Importantly, we found that the expression of GRP was strikingly increased in the skin of patients with AD. These results indicate that IL-22 plays important pathogenic roles in the initiation and development of AD, in part through inducing keratinocyte production of type 2 cytokines and activation of the GRP/GRPR pathway.

Analysis of microarrays of miR-34a and its identification of prospective target gene signature in hepatocellular carcinoma.

BACKGROUND: Currently, some studies have demonstrated that miR-34a could serve as a suppressor of several cancers including hepatocellular carcinoma (HCC). Previously, we discovered that miR-34a was downregulated in HCC and involved in the tumorigenesis and progression of HCC; however, the mechanism remains unclear. The purpose of this study was to estimate the expression of miR-34a in HCC by applying the microarray profiles and analyzing the predicted targets of miR-34a and their related biological pathways of HCC. METHODS: Gene expression omnibus (GEO) datasets were conducted to identify the difference of miR-34a expression between HCC and corresponding normal tissues and to explore its relationship with HCC clinicopathologic features. The natural language processing (NLP), gene ontology (GO), pathway and network analyses were performed to analyze the genes associated with the carcinogenesis and progression of HCC and the targets of miR-34a predicted in silico. In addition, the integrative analysis was performed to explore the targets of miR-34a which were also relevant to HCC. RESULTS: The analysis of GEO datasets demonstrated that miR-34a was downregulated in HCC tissues, and no heterogeneity was observed (Std. Mean Difference(SMD) = 0.63, 95% confidence intervals(95%CI):[0.38, 0.88], P < 0.00001; Pheterogeneity = 0.08 I2 = 41%). However, no association was found between the expression value of miR-34a and any clinicopathologic characteristics. In the NLP analysis of HCC, we obtained 25 significant HCC-associated signaling pathways. Besides, we explored 1000 miR-34a-related genes and 5 significant signaling pathways in which CCND1 and Bcl-2 served as necessary hub genes. In the integrative analysis, we found 61 hub genes and 5 significant pathways, including cell cycle, cytokine-cytokine receptor interaction, notching pathway, p53 pathway and focal adhesion, which proposed the relevant functions of miR-34a in HCC. CONCLUSION: Our results may lead researchers to understand the molecular mechanism of miR-34a in the diagnosis, prognosis and therapy of HCC. Therefore, the interaction between miR-34a and its targets may promise better prediction and treatment for HCC. And the experiments in vivo and vitro will be conducted by our group to identify the specific mechanism of miR-34a in the progress and deterioration of HCC.

AKT activation was not essential for hepatocellular carcinoma cell survival under glucose deprivation.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide, with a dismal 5-year survival rate less than 15%. The present study aimed to investigate whether AKT inhibition and glucose deprivation could synergistically kill HCC cells and the molecular mechanisms involved. HCC cells were starved in glucose deprivation, and then the resultant cell death was determined by flow cytometry and mitochondrial oxygen consumption rates using a Seahorse XF-24 Extracellular Flux Analyzer. Glucose deprivation reduced mitochondrial oxygen consumption rates for ATP production, enhanced mitochondrial proton leaks, reduced Mcl-1 expression, and subsequently caused significant cell death in the sensitive HepG2 and HCC-M cells. In the resistant Hep3B and Huh7 cells that survived, glucose starvation induced time-dependent AKT activation. However, blockage of AKT activation using chemical inhibitors (ZSTK474 and LY290042) or specific AKT1-targeting siRNAs could not markedly sensitize glucose deprivation-induced cell death. In contrast, AKT inhibitors or AKT1-targeting siRNAs significantly protected the sensitive HepG2 cells from glucose deprivation-induced cell death. More importantly, AKT inhibition mechanically suppressed mTOR activity and induced the prosurvival autophagy pathway in the sensitive HCC cells. Taken together, these data demonstrated that AKT activity was not essential for HCC cell survival during glucose deprivation. The reduction of mTOR activity and induction of the autophagy pathway may hinder the potential application of AKT inhibitors in the cancer therapy of solid tumors such as HCC.

Relationship between TRAF6 and deterioration of HCC: an immunohistochemical and in vitro study.

OBJECTIVE: To explore the relationship between tumor necrosis factor receptor-associated factor 6 (TRAF6) and the clinicopathological features in HCC as well as its biological function. METHODS: Totally, 412 liver tissues were collected, including 171 hepatocellular carcinoma (HCC) and their corresponding non-tumor tissues, 37 cirrhosis and 33 normal liver tissues. The expression of TRAF6 was assessed by immunohistochemistry. Then, analysis of the correlations between TRAF6 expression and clinicopathological parameters in HCC was conducted. Furtherer, in vitro experiments on HepG2 and Hep3B cells were performed to validate the biological function of TRAF6 on HCC cells. TRAF6 siRNA was transfected into HepG2 and Hep3B cell lines and TRAF6 expression was evaluated with RT-qPCR and western blot. The assays of cell viability, proliferation, apoptosis and caspase-3/7 activity were carried out to investigate the effects of TRAF6 on HCC cells with RNA interference. Cell viability was assessed with Cell Titer-Blue kit. Cell proliferation was tested with MTS kit. Cell apoptosis was checked through morphologic detection with fluorescence microscope, as well as caspase-3/7 activity was measured with fluorogenic substrate detection. RESULTS: The positive expression rate of TRAF6 protein was 49.7 % in HCC, significantly higher than that of normal liver (12.1 %), cirrhosis (21.6 %) and adjacent non-cancerous tissues (36.3 %, all P < 0.05). Upregulated TRAF6 was detected in groups with metastasis (Z = -2.058, P = 0.04) and with low micro-vessel density (MVD) expression (Z = -2.813, P = 0.005). Spearman correlation analysis further showed that the expression of TRAF6 was positively correlated with distant metastasis (r = 0.158, P = 0.039) and negatively associated with MVD (r = -0.249, P = 0.004). Besides, knock-down of TRAF6 mRNA in HCC cell lines HepG2 and Hep3B both resulted in cell viability and proliferation inhibition, also cell apoptosis induction and caspase-3/7 activity activation. CONCLUSIONS: TRAF6 may contribute to metastasis and deterioration of the HCC via influencing cell growth and apoptosis. Thus, TRAF6 might become a predictive and therapeutic biomarker for HCC.

TRPA1-dependent pruritus in IL-13-induced chronic atopic dermatitis.

Chronic debilitating pruritus is a cardinal feature of atopic dermatitis (AD). Little is known about the underlying mechanisms. Antihistamines lack efficacy in treating itch in AD, suggesting the existence of histamine-independent itch pathways in AD. Transient receptor potential ankyrin 1 (TRPA1) is essential in the signaling pathways that promote histamine-independent itch. In this study, we tested the hypothesis that TRPA1-dependent neural pathways play a key role in chronic itch in AD using an IL-13-transgenic mouse model of AD. In these mice, IL-13 causes chronic AD characterized by intensive chronic itch associated with markedly enhanced growth of dermal neuropeptide-secreting afferent nerve fibers and enhanced expression of TRPA1 in dermal sensory nerve fibers, their dorsal root ganglia, and mast cells. Inhibition of TRPA1 with a specific antagonist in these mice selectively attenuated itch-evoked scratching. Genetic deletion of mast cells in these mice led to significantly diminished itch-scratching behaviors and reduced TRPA1 expression in dermal neuropeptide containing afferents in the AD skin. Interestingly, IL-13 strongly stimulates TRPA1 expression, which is functional in calcium mobilization in mast cells. In accordance with these observations in the AD mice, TRPA1 expression was highly enhanced in the dermal afferent nerves, mast cells, and the epidermis in the lesional skin biopsies from patients with AD, but not in the skin from healthy subjects. These studies demonstrate a novel neural mechanism underlying chronic itch in AD and highlight the complex interactions among TRPA1(+) dermal afferent nerves and TRPA1(+) mast cells in a Th2-dominated inflammatory environment.

Up-regulation of heparan sulfate 6-O-sulfation in idiopathic pulmonary fibrosis.

Heparan sulfate proteoglycans (HSPGs) are integral components of the lung. Changes in HSPGs have been documented in idiopathic pulmonary fibrosis (IPF). Many of the biological functions of HSPGs are mediated by heparan sulfate (HS) side chains, and little is understood about these side chains in the pathogenesis of IPF. The aims of this study were to compare HS structure between normal and IPF lungs and to examine how changes in HS regulate the fibrotic process. HS disaccharide analysis revealed that HS 6-O-sulfation was significantly increased in IPF lungs compared with normal lungs, concomitant with overexpression of HS 6-O-sulfotransferases 1 and 2 (HS6ST1/2) mRNA. Immunohistochemistry revealed that HS6ST2 was specifically expressed in bronchial epithelial cells, including those lining the honeycomb cysts in IPF lungs, whereas HS6ST1 had a broad expression pattern. Lung fibroblasts in the fibroblastic foci of IPF lungs expressed HS6ST1, and overexpression of HS6ST1 mRNA was observed in primary lung fibroblasts isolated from IPF lungs compared with those from normal lungs. In vitro, small interference RNA-mediated silencing of HS6ST1 in primary normal lung fibroblasts resulted in reduced Smad2 expression and activation and in reduced expression of collagen I and α-smooth muscle actin after TGF-ß1 stimulation. Similar results were obtained in primary IPF lung fibroblasts. Furthermore, silencing of HS6ST1 in normal and IPF lung fibroblasts resulted in significant down-regulation of TßRIII (betaglycan). In summary, HS 6-O-sulfation is up-regulated in IPF with overexpression of HS6ST1 and HS6ST2, and overexpression of HS6ST1 in lung fibroblasts may regulate their fibrotic responses to TGF-ß1.

Overexpression of Sulf2 in idiopathic pulmonary fibrosis.

Previously, we have shown that heparan sulfate (HS) 6-O-endosulfatase 1 (Sulf1) is a transforming growth factor-ß1 (TGF-ß1)-responsive gene in normal human lung fibroblasts and functions as a negative feedback regulator of TGF-ß1 and that TGF-ß1 induces the expression of Sulf1 as well as that of the closely related Sulf2 in a murine model of pulmonary fibrosis. In this study, we focused on the role of Sulf2 in modulating TGF-ß1 function and the development of pulmonary fibrosis. We found that Sulf2 mRNA was overexpressed in lung samples from human patients with idiopathic pulmonary fibrosis (IPF), and Sulf2 protein was specifically localized to the hyperplastic type II alveolar epithelial cells (AECs). In vitro, TGF-ß1 induced the expression of Sulf2 with accompanied HS 6-O-desulfation in A549 cells, adenocarcinoma cells derived from the type II alveolar epithelium. Using small interference RNA to block Sulf2 expression, we observed a biphasic TGF-ß1 response with early enhanced Smad activation, but eventually reduced TGF-ß1 target gene expression in Sulf2 knockdown A549 cells compared with the control cells. To study the role of Sulf2 in normal type II AECs, we isolated primary type II cells from wild-type and Sulf2 knockout mice. We observed enhanced Smad activation as well as enhanced TGF-ß1 target gene expression in Sulf2 knockout type II AECs compared with wild-type type II AECs. In conclusion, Sulf2 is overexpressed in IPF and may play a role in regulating TGF-ß1 signaling in type II AECs.

In vitro and in vivo activities of a new lead compound I2906 against Mycobacterium tuberculosis.

BACKGROUND: Due to the long duration of treatment and the emergence of multidrug-resistant strains, new antitubercular agents are urgently needed. I2906, as a novel lead, was screened and tested for efficacy in vitro and in vivo. METHODS: To determine the efficacy of I2906,the minimum inhibitory concentrations against Mycobacterium tuberculosis and cytotoxicity were tested, and its in vivo activities were assessed by administering it to mice infected with M. tuberculosis H37Rv or multidrug-resistant strain. RESULTS: Under in vitro conditions, I2906 showed excellent antimycobacterial activities and low cytotoxicity. In a murine model infected with M. tuberculosis H37Rv, the reductions on bacterial loads of both lungs and spleen were statistically significant (p < 0.05) between I2906-treated mice and untreated controls after 4 weeks. Further, the colony-forming unit counts in the lungs were dramatically lower (p < 0.05) than that of isoniazid-treated mice by the addition of I2906 after 8 weeks. Moreover, survival rate was increased by I2906 treatment. For multidrug-resistant strain infection, bacterial counts were reduced significantly in the lungs and spleen due to I2906 treatment in comparison with data from untreated controls (p < 0.05). CONCLUSIONS: I2906 displayed potential antimicrobial activities against M. tuberculosis H37Rv and drug-resistant strains in vitro and in vivo, and could improve efficacy of isoniazid in vivo.

Quantitative Analysis of Hepatic Microcirculation in Rabbits After Liver Ischemia-Reperfusion Injury Using Contrast-Enhanced Ultrasound.

Previous studies have shown that contrast-enhanced ultrasound (CEUS) can be used quantitatively to analyze microcirculation blood perfusion in hepatocellular carcinoma patients. However, limited data have described the application of CEUS in hepatic microcirculation after liver ischemic-reperfusion injury (IRI). The purpose of this study was to explore the use of CEUS quantitatively to assess liver microcirculation after liver IRI. We randomly sorted 45 New Zealand rabbits into 3 groups (15 in each). Group A was a control group in which the rabbits underwent laparotomy alone. In groups B and C, hepatic blood was blocked for 30 min. Simultaneously, rabbits in group C underwent left lateral lobe resection. After 30 min of ischemia, CEUS was conducted after 0 h, 1 h, 6 h and 24 h of reperfusion in the 3 groups. Time-intensity curves (TICs) for CEUS were constructed and quantitative parameters (maximum intensity [IMAX], rise time [RT], time to peak [TTP] and mean transit time [mTT]) were obtained. In addition, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were computed to estimate liver function before the operation and at 0 h, 1 h, 6 h and 24 h after reperfusion, respectively. Pathologic changes in the liver after reperfusion were also observed. Simultaneously, the correlations between serum transaminase and a variety of quantitative analysis parameters were analyzed. In groups B and C, the IMAX value decreased; whereas RT, TTP, mTT and serum ALT and AST levels increased significantly in comparison with those in group A after 0 h and 1 h of reperfusion. The pathology revealed that erythrocytes were destroyed and microcirculation was disturbed. Then, at 6 h of reperfusion, the IMAX continued to decrease. Additionally, the levels of RT, TTP, mTT and serum ALT and AST increased in comparison with those at 1 h of reperfusion. The pathologic analysis revealed inflammatory cell aggregation and leukocyte infiltration. After 24 h of reperfusion, the IMAX was reduced in comparison with that of the 6-h group. The levels of RT, TTP, mTT and serum ALT and serum AST were increased in comparison with that of the 6-h group. These findings were in accordance with the pathologic analysis. In addition, serum transaminase had a negative correlation with IMAX (p < 0.001) and a positive correlation with RT, TTP and mTT (all p < 0.001). So, in conclusion, the quantitative analysis of CEUS can be used to assess hepatic microcirculation after liver IRI.

Analysis of microRNA expression profiling identifies miR-155 and miR-155* as potential diagnostic markers for active tuberculosis: a preliminary study.

To explore biologic behaviors and disease relevance of microRNAs (miRNAs) in the development of active tuberculosis (ATB), we investigated the expression profile of Mycobacterium tuberculosis (MTB) purified protein derivative (PPD)-induced miRNAs to determine the specific miRNAs involved in the pathogenesis of ATB. The expression profile of miRNA under PPD challenge was first measured using microarray analysis in peripheral blood mononuclear cells isolated from ATB patients and healthy controls (HC). The remarkably reactive miRNAs were then validated in a larger cohort by quantitative real-time polymerase chain reaction (qRT-PCR). The receiver operating characteristic (ROC) curve was plotted to evaluate the diagnostic value of the determined PPD-responsive miRNAs. The potential targets for those miRNAs were also predicted by computational programs. Fourteen of 866 human miRNAs exhibited at least 1.8-fold difference in the ratio of expression level before and after stimulation with PPD between the ATB and HC groups. The qRT-PCR study validated the findings from microarray-based screening, in which miR-155 exhibited a fold change of 1.4 in the HC group and 3.7 in the ATB group upon PPD stimulation (p < 0.0001); miR-155* exhibited a fold change of 1.9 in the HC and 4.6 in the ATB group (p < 0.005). In ROC plots, the area under the curve was 0.8972 for miR-155 and 0.7945 for miR-155*. The background expression of these 2 microRNAs exhibited no differences between the ATB and HC groups. miR-155 and miR-155* exhibited characteristic expression by TB-specific antigen, suggesting that they can be potential diagnostic markers under the challenge of specific MTB antigens.

Nasal administration of a novel recombinant human parathyroid hormone (1-34) analog for the treatment of osteoporosis of ovariectomized rats.

Synthetic human parathyroid (1-34) (hPTH (1-34)) is known to have the full biological activity of the holohormone for osteoporosis. This study is about designing a novel analog of hPTH (1-34) which is more suitable for intranasal administration. We likewise evaluate effectiveness of the nasal drops against osteoroporosis. Through fusion expression of combining gene, cell disruption, inclusion body washing, ethanol fraction precipitation, acid hydrolysis, and CM-52 ion exchange column chromatography Pro-Pro-[Arg¹¹] hPTH (1-34)-Pro-Pro was designed and produced. Nasal drops of Pro-Pro-[Arg¹¹] hPTH (1-34)-Pro-Pro were prepared and administrated to ovariectomized rats. After 12 weeks of raising, Bone Material Densities (BMD) of vertebrae were examined by Dual Energy X-Ray Absorptiometry (DEXA). The average BMD of these groups treated with nasal drops of the peptide were 28.0%-47.2% (P<0.01) higher than that of the group treated with normal saline (NS). The subchondral bone plates of the femoral heads were examined by scanning electron microscopy and a defined planar section was photographed. Percentage of the area of the cancellous bone was calculated. Percentages of the groups treated with nasal drops of the peptide increased; values were significantly different to that of the group treated with NS (P<0.001) and were even equivalent to that of normal groups. These results show that nasal drops of Pro-Pro-[Arg¹¹] hPTH (1-34)-Pro-Pro are effective against osteoporosis.