[Analysis and significance of HBV DNA below the lower detection limit of HBV RNA levels after long-term NAs antiviral therapy in patients with hepatitis B virus cirrhosis].

Objective: To analyze the significance of HBV DNA below the lower detection limit of HBV RNA levels after long-term nucleos(t)ide analogues (NAs) antiviral therapy in patients with hepatitis B virus cirrhosis. Methods: 97 cases with hepatitis B virus cirrhosis treated with NAs antiviral therapy for at least 3 years between May 2018 to July 2019 were selected. High-sensitivity HBV DNA (<20 IU/ml), alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (GGT), HBsAg, HBeAg and HBV RNA at least twice every 6 months were detected. According to Child-Pugh classification, HBeAg, HBsAg level, and HBV RNA level intergroup comparison was performed. Rank sum test, χ2 test and linear regression analysis were performed on the data. Results: Compared with the HBV RNA level of child-Pugh class A patients, the HBV RNA level of Child-Pugh class B+C patients were significantly higher [4.1 (0,4.9) log10 copies/ml and 2.0 (0,3.5) log10 copies/ml], and the difference was statistically significant (Z=2.370, P<0.05). According to different HBeAg levels, they were divided into HBeAg positive and negative group, and the quantitative comparison of HBV RNA levels between the two groups were 2.0 (0, 4.5) log10 copies/ml and 1.0 (1.0, 2.0) log10 copies/ml, respectively, and the difference was statistically significant (Z=3.233, P<0.05). According to different HBsAg levels, they were divided into three groups: HBsAg≤100 IU/ml, 100

Cancer-associated fibroblasts induce epithelial-mesenchymal transition of breast cancer cells through paracrine TGF-ß signalling.

BACKGROUND: Cancer-associated fibroblasts (CAFs) activated by tumour cells are the predominant type of stromal cells in breast cancer tissue. The reciprocal effect of CAFs on breast cancer cells and the underlying molecular mechanisms are not fully characterised. METHODS: Stromal fibroblasts were isolated from invasive breast cancer tissues and the conditioned medium of cultured CAFs (CAF-CM) was collected to culture the breast cancer cell lines MCF-7, T47D and MDA-MB-231. Neutralising antibody and small-molecule inhibitor were used to block the transforming growth factor-ß (TGF-ß) signalling derived from CAF-CM, which effect on breast cancer cells. RESULTS: The stromal fibroblasts isolated from breast cancer tissues showed CAF characteristics with high expression levels of α-smooth muscle actin and SDF1/CXCL12. The CAF-CM transformed breast cancer cell lines into more aggressive phenotypes, including enhanced cell-extracellular matrix adhesion, migration and invasion, and promoted epithelial-mesenchymal transition (EMT). Cancer-associated fibroblasts secreted more TGF-ß1 than TGF-ß2 and TGF-ß3, and activated the TGF-ß/Smad signalling pathway in breast cancer cells. The EMT phenotype of breast cancer cells induced by CAF-CM was reversed by blocking TGF-ß1 signalling. CONCLUSION: Cancer-associated fibroblasts promoted aggressive phenotypes of breast cancer cells through EMT induced by paracrine TGF-ß1. This might be a common mechanism for acquiring metastatic potential in breast cancer cells with different biological characteristics.

[The clinical observation of sirolimus combined with calcineurin inhibitors for steroid-resistant/steroid-dependent extensive cGVHD].

Objective: To observe the efficacy and safety of sirolimus combined with calcineurin inhibitor (CNI) in the treatment of glucocorticoid resistant/dependent extensive chronic graft-versus-host disease (cGVHD) . Methods: A total of 27 patients with steroid-resistant/steroid-dependent extensive cGVHD from November 2015 to January 2019 were enrolled and given sirolimus capsules combined with cyclosporine or tacrolimus to observe the clinical efficacy and adverse events. Results: The median duration of medication was 14.2 months and the mean duration was 16.7 months. The median follow-up time was 20.1 months (12.9-46.1 months) . Following the 6-month follow-up, 3 cases achieved complete response (CR) and 12 cases partial response (PR) . The overall response rate (ORR) was 55.6% ; for progression-free survival (PFS) , PFS-6 reached 88.9% (24/27) , and for overall survival (OS) , OS-6 was 100% . At the 1-year follow-up, there were 5 cases of CR and 11 cases of PR, ORR was 59.3% , PFS-12 reached 62.9% (17/27) , and OS-12 was 100% . The subgroup analysis found that the program was more effective for cGVHD in male donors and the target organ analysis had an advantage in the treatment of oral cavity, skin, and liver rejection. Adverse events were observed: hyperlipidemia 11.1% , oral ulcer 7.4% , fungal infection 11.1% , liver injury 3.7% , renal insufficiency 0, and no new CMV and EB viremia. Conclusion: Sirolimus combined with calcineurin inhibitors is effective in treating steroid-resistant/steroid-dependent extensive cGVHD, especially because adverse reactions (renal toxicity, CMV, EBV infection) are low in number, which is suitable for long-term treatment of cGVHD.

[The clinical observation of serum specific biomarkers in patients with chronic graft-versus-host disease].

Objective: Chronic graft-versus-host disease (cGVHD) is a major long-term complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) . It is important to study the changes of serum biomarkers expression in patients for early diagnosis and treatment. Methods: The expression levels of five serum protein markers (IL-1b, IL-16, CXCL9, CCL19, CCL17) in patients with or without cGVHD after allo-HSCT were detected by liquid suspension microarray. Results: Compared with the control group without cGVHD, the expression levels of CXCL9 and CCL17 in serum of patients with cGVHD were significantly increased (P<0.05) . CCL17 was correlated with the severity of cGVHD (P<0.001) . CXCL9 was significantly increased in the serum of patients with skin lesion (P<0.01) , and CCL17 was significantly expressed in cGVHD patients with liver as the target organ (P<0.01) . Conclusion: The combination of CXCL9 and CCL17 can be used as serum biomarkers of cGVHD, which has certain reference value in assisting the diagnosis and evaluation of cGVHD severity.

Role of GSPE in improving early cerebral vascular damage by inhibition of Profilin-1 expression in a ouabain-induced hypertension model.

OBJECTIVE: Grape seed proanthocyanidin extract (GSPE), as one of the most popular natural drug extracted from the grape, has been reported to improve endothelial function and arteriosclerosis. However, little is known about the influence of GSPE on hypertension and vascular remodeling. Profilin-1, an Actin-binding protein, is closely involved in the remodeling of large vessels in ouabain-induced hypertension. To date, there is no effective prevention or treatment in place for the high incidence of ischemic stroke associated with hypertension. In this study, we aimed to determine the role of GSPE via inhibition Profilin-1 in ischemic cerebral cortices of ouabain-hypertension rats and potentially provide a new target to prevent stroke associated with hypertension. MATERIALS AND METHODS: The blood pressure of male Sprague-Dawley (SD) rats was measured during a period of ouabain-induced hypertension. The expression of Profilin-1, vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in the cerebral cortex were determined by quantitative Real Time-PCR (qRT-PCR) and Western blot. Histopathological and behavioral tests were also conducted. RESULTS: Blood pressure elevation started at week 5 and reached clinical standards for hypertension at week 8. GSPE was proved to suppress Profilin-1 and VEGF levels through inhibition of Profilin-1-protein kinase B (AKT)-hypoxia inducible factor-1α (HIF-1α) signal pathway and promote eNOS expression. Moreover, the histopathological and ethiological improvement was observed in GSPE over-expression and Profilin-1 inhibition groups. CONCLUSIONS: We detected that GSPE could improve cerebral vascular damage through inhibiting Profilin-1 in an ouabain-induced hypertension model.

Overexpression of ezrin and galectin-3 as predictors of poor prognosis of cervical cancer.

The aim of this study was to explore the correlation of ezrin and galectin-3 expressions with prognosis in cervical cancer. The immunohistochemical method was applied to detect ezrin and galectin-3 expressions in normal cervix tissues (n=30), cervicitis tissues (n=28), cervical intraepithelial neoplasia (CIN) tissues (classified as I-III, n=89), and cervical carcinoma tissues (n=84). Follow-up was conducted for 5 to 78 months to analyze the correlation of protein expressions with prognosis. Ezrin and galectin-3 expressions in cervical cancer were significantly higher than in normal cervix, cervicitis and CIN (all P<0.05), and expressions in CIN were significantly higher than in normal cervix and cervicitis (both P<0.05). The expressions of ezrin and galectin-3 were both related with histological grade, deep myometrial invasion and lymph node metastasis (all P<0.05). Spearman analysis showed that ezrin expression was positively correlated with galectin-3 expression in cervical cancer (r=0.355, P<0.05). The survival rate of patients with high expressions of ezrin and galectin-3 was significantly lower than those with low expressions of proteins (both P<0.05). The expressions of ezrin and galectin-3, histological grade, depth of stromal invasion, and lymph node metastasis are risk factors affecting the survival rate of patients with cervical cancer. The expressions of ezrin and galectin-3 were correlated with the development of cervical cancer, and overexpressions of those proteins were indicative of poor prognosis in patients with cervical cancer.

Neutral Ceramidase Secreted Via Exosome Protects Against Palmitate-Induced Apoptosis in INS-1 Cells.

Aims: To investigate the effects of neutral ceramidase (NCDase) packaged in exosomes that are secreted from ß-cells on free fatty acid (FFA)-induced ß-cells apoptosis and its role in regulation of sphingolipid-mediated signaling pathway. Methods: HPLC and Western blotting were performed to determine NCDase activity and expression. Annexin V-fluorescein-isothiocyanate/propidium iodide flow cytometry was used to assess apoptosis. Electrospray ionization tandem mass spectrometry was used for ceramide (Cer), sphingosine-1-phosphate (S1P), and sphingosine (SPH) determination. Results: INS-1 cells over-expressed NCDase secreted active NCDase via exosomes. Exosomes isolated from the cultured medium of INS-1 cells that oxpressed NCDase could ameliorate palmitate-induced apoptosis. Furthermore, the results showed that exosome-derived NCDase treatment reduced intracellular Cer/S1P ratio. Conclusions: ß-cell secreted active NCDase via exosome, the exosome-packaged-NCDase protected ß-cells from FFA-induced apoptosis through regulating sphingolipid metabolites and it might be a potential treatment for ß-cell lipotoxicity and type 2 diabetes.

Roles of PECAM-1 in cell function and disease progression.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) belongs to immunoglobulin superfamily, which is key factor for adhesion and accumulation of platelets. It is proved that PECAM-1 is closely correlative with cell migration, proliferation, apoptosis, signal transduction and cellular immunity. Meanwhile, PECAM-1 involves in multiple clinical diseases, such as atherosclerosis, thrombosis and leukemia. This paper reviewed the structure and function of PECAM-1, and its roles in cell function and disease generation and progression.

Hydrophilic Thr can replace the hydrophobic and absolutely conservative A3Val in insulin.

A mutant insulin, [A3Thr]human insulin, was obtained by means of site-directed mutagenesis. The [A3Thr]human insulin retains 50% receptor-binding potency and nearly total in vivo biological activity compared with native insulin, and can be crystallized using the same condition of native insulin. The results demonstrate that the absolutely conservative and hydrophobic valine at A3 can be substituted by hydrophilic threonine.

Conformational stability of porcine serum transferrin.

The conformation of porcine serum ferric transferrin (Tf) and its stability against denaturation were studied by circular dichroism. Tf was estimated to have 19-24% alpha-helix and 50-55% beta-sheet based on the methods of Chang et al. (Chang, C.T., Wu, C.-S.C., & Yang, J.T., 1978, Anal. Biochem. 91, 13-31) and Provencher and Glöckner (Provencher, S.W. & Glöckner, J., 1981, Biochemistry 20, 33-37). Removal of the bound ferric ions (apo-Tf) did not alter the overall conformation, but there were subtle changes in local conformation based on its near-UV CD spectrum. The Tfs were stable between pH 3.5 and 11. Denaturation by guanidine hydrochloride (Gu-HCl) showed two transitions at 1.6 and 3.4 M denaturant. The process of denaturation by acid and base was reversible, whereas that by Gu-HCl was partially reversible. The irreversible thermal unfolding of Tfs began at temperatures above 60 degrees C and was not complete even at 80 degrees C. The bound irons (based on absorbance at 460 nm) were completely released at pH < 4 or in Gu-HCl solution above 1.7 M, when the protein began to unfold, but they remained intact in neutral solution even at 85 degrees C. The NH2- and COOH-terminal halves of the Tf molecule obtained by limited trypsin digestion had CD spectra similar to the spectrum of native Tf, and the COOH-terminal fragment had more stable secondary structure than the NH2-terminal fragment.

Bioavailability of selenium to residents in a low-selenium area of China.

For 8 wk 5 groups of 10 men each were given 0.5 g/day DL-methionine, 150 micrograms Se/day as sodium selenite with or without methionine or 150 micrograms Se/day as selenomethionine with or without methionine. Twenty subjects received placebo as controls. Initially plasma Se rose more rapidly than RBC Se. Increases in Se levels were significantly greater with selenomethionine than with the selenite supplement. In the placebo and methionine supplemented groups neither plasma nor RBC Se varied significantly over the course of the study. Supplementation with selenium resulted in marked increases in plasma and RBC GSH-Px within 2 and 4 wk, respectively. Plasma and RBC GSH-Px activity did not differ significantly between Se-supplemented groups. These studies suggest that selenomethionine-Se was more effective in raising plasma and RBC Se than was selenite-Se. Methionine supplements may increase the bioavailability of selenium in severely deficient subjects.

Selenium intake and metabolic balance of 10 men from a low selenium area of China.

Selenium intake and urinary and fecal Se excretion of 10 healthy men from a low Se area in China were determined for three consecutive days, in summer, fall, and winter of 1983, and the spring of 1984 while self-selected diets were being consumed. Mean daily Se intake was 8.8 micrograms/day with a range of 2.3-35.5 micrograms/day, and was far below the recommended range of safe and adequate Se intake of 50-200 micrograms Se/day (National Academy of Sciences/National Research Council). Mean urinary and fecal Se outputs were 3.7 and 3.4 micrograms Se/day, respectively. Mean Se balance during this time was +1.8 micrograms Se/day. Apparent absorption of Se approximated 57%. The low Se intake in this area is a cause for concern since the residents of Molimo may be at risk for Se deficiency diseases.

Cerebellar granule cells acquire transferrin-free iron by a carrier-mediated process.

In this study, the mechanism of transferrin-free iron uptake by brain neuronal cells was investigated using the cultured cerebellar granule cells. Effects of incubation time, iron concentration, temperature and other divalent metals on the cellular uptake were determined. After five days of plating, the cells were incubated with different concentrations of transferrin-free iron in isotonic sucrose solution at different temperatures for a certain time. The cellular transferrin-free iron uptake was analysed by measuring the cellular radioactivity with a gamma-counter. The result showed that the cultured cerebellar granule cells had the capacity to acquire transferrin-free iron at pH 6.5, at which it was demonstrated that transferrin binds iron very poorly and only very little transferrin can be internalized by reticulocytes and HeLa cells. The iron uptake by cells increased with incubation time in a linear manner at a rate of 0.1076 pmol/microg protein/min within the first 10 min. The uptake was time- and temperature-dependent, iron concentration saturable, and inhibited by several divalent metal ions, such as Co2+, Zn2+, Mn2+ and Ni2+. These characteristics of transferrin-free iron uptake by the cultured cerebellar granule cells observed in this study, similar to those obtained from cells outside of the brain, implied that a carrier-mediated iron transport system might be present on the membrane of this type of brain neuronal cells. In addition, no significant difference in malondialdehyde measurement was found when the cells were incubated without or with the lower concentrations of iron (< 4 microM) for 20 min at 37 degrees C, demonstrating that this system was valid for studying membrane iron transport in this type of brain neuronal cell.

Inhibition of the transforming ability of cigarette smoking condensate-treated human fetal lung DNA induced by oltipraz.

Rat-1 cells transfected by genomic DNA of human fetal lung explants treated with 100 micrograms/ml of oltipraz (5-(2-pyrazimyl)-4-methyl-1, 2-dithiol-3-thione) for 14 hr or 100 micrograms/ml of cigarette smoking condensate for 6 hr formed 0 to 8 transformation foci, respectively. If 100 micrograms/ml of oltipraz was added to culture of human fetal lung explants 8 hr prior to the treatment of cigarette smoking condensate, the Rat-1 cells transfected by genomic DNA of human fetal lung explants formed only two foci. In addition, the growth speed of Rat-1 cells transfected by genomic DNA of human fetal lung treated with both oltipraz and cigarette smoking condensate was lower than that transfected by cigarette smoking condensate-treated human fetal lung DNA. Our results indicate that oltipraz can block the irreversible change of human fetal lung DNA caused by cigarette smoking condensate, and the results suggest the possibility of using oltipraz as control in the experimental initiation of human lung carcinogenesis.