Cartilage-specific deletion of Alk5 gene results in a progressive osteoarthritis-like phenotype in mice.

OBJECTIVE: Previous studies have shown that Transforming growth factor-ß (TGF-ß)/TGFßRII-Smad3 signaling is involved in articular cartilage homeostasis. However, the role of TGF-ß/ALK5 signaling in articular cartilage homeostasis has not been fully defined. In this study, a combination of in vitro and in vivo approaches was used to elucidate the role of ALK5 signaling in articular cartilage homeostasis and the development of osteoarthritis (OA). DESIGN: Mice with inducible cartilage-specific deletion of Alk5 were generated to assess the role of ALK5 in OA development. Alterations in cartilage structure were evaluated histologically. The expressions of genes associated with articular cartilage homeostasis and TGF-ß signaling were analyzed by qRT-PCR, western blotting and immunohistochemistry. The chondrocyte apoptosis was detected by TUNEL staining and immunohistochemistry. In addition, the molecular mechanism underlying the effects of TGF-ß/ALK5 signaling on articular cartilage homeostasis was explored by analyzing the TGF-ß/ALK5 signaling-induced expression of proteoglycan 4 (PRG4) using specific inhibitors. RESULTS: Postnatal cartilage-specific deletion of Alk5 induced an OA-like phenotype with degradation of articular cartilage, synovial hyperplasia, osteophyte formation, subchondral sclerosis, as well as enhanced chondrocyte apoptosis, overproduction of catabolic factors, and decreased expressions of anabolic factors in chondrocytes. In addition, the expressions of PRG4 mRNA and protein were decreased in Alk5 conditional knockout mice. Furthermore, our results showed, for the first time, that TGF-ß/ALK5 signaling regulated PRG4 expression partially through the protein kinase A (PKA)-CREB signaling pathway. CONCLUSIONS: TGF-ß/ALK5 signaling maintains articular cartilage homeostasis, in part, by upregulating PRG4 expression through the PKA-CREB signaling pathway in articular chondrocytes.

[Health status and healthcare service utilization among children born to women with maternal syphilis in Shanghai].

Objective: To assess the health status and health service utilization of children born to syphilis infected mothers during pregnancy, in order to prevent mother-to-child transmission of syphilis to the newborns. Methods: Women with maternal syphilis were studied by trained researchers via phone calls, in Shanghai during 2014-2015. Data related to demographics, status of infection and health care, utilization by both mothers and their children were collected through specifically designed questionnaires. Non-parametric tests including chi-square were used to assess the health status and health service utilization of children born to mothers with different demographic and socioeconomic characteristics. Results: A total of 495 children born to mothers with maternal syphilis were recruited from 1 000 syphilis infected parturient women. A total of 61 out of the 495 children were diagnosed as having congenital syphilis (57 children were diagnosed at birth and another 4 were diagnosed during the follow-up period). Children born to women who received syphilis treatment during pregnancy were at lower risk on congenital syphilis (χ(2)=7.214, P=0.027). 37.8% of the children were reported to have had different illnesses in the past three months, mainly involving upper respiratory infections (32.3%) or diarrhea (3.6%). Children diagnosed with congenital syphilis showed a higher prevalence of different kinds of diseases, compared to those without congenital syphilis (47.5% vs. 36.6%). 81.6% of the children had received regular child health care services. Subjects with the following factors as: being immigrant, with lower education, unemployed, unmarried and multipara, were related to the less use of regular child healthcare services. Only 39.7% of the parents would inform the care-takers about the risk of congenital syphilis infection of their own children at the child health care centers. Mothers with residency of Shanghai, having higher education level and employed, were less willing to inform doctors about the risk of congenital syphilis infection of their children. Conclusions: Loss to follow-up among children born to syphilis infected pregnant women remained a serious problem. Few parents would be willing to inform the healthcare takers that their children are at risk of syphilis, when receiving child health care services at the centers. It was necessary to integrate the congenital syphilis follow-up programs into the routine child care services so as to timely diagnose and treat the patients with congenital syphilis.

Screening diabetes in tuberculosis patients in eastern rural China: a community-based cross-sectional study.

OBJECTIVES: To understand the prevalence of diabetes mellitus (DM) and tuberculosis (TB) comorbidity in rural China and to identify factors associated with TB-DM comorbidity and screening efficacy. METHODS: A community-based cross-sectional study was carried out in four counties in eastern rural China. All TB patients newly registered from April 2013 to March 2014 were screened for DM using fasting blood glucose (FBG). Screening-positive patients were further examined using glycosylated haemoglobin A1C (HbA1c). RESULTS: Ninety-seven (7.7%) of the 1252 recruited TB patients had DM, 44 (45.4%) of whom were newly diagnosed. The DM-TB patients were significantly older than non-diabetics (mean age 57 ± 13 years vs. 49 ± 19 years, P < 0.001). The risk of DM-TB was higher in patients aged >40 years (OR 3.039) and in overweight patients (OR 2.595). The number needed to screen (NNS) among TB patients to identify one case of DM was 12.97. The NNS to identify one new DM patient (27.4) was lower in participants aged >40 years (20.5), those who were illiterate (19.9), those with a family history of DM (9.3), those with missing bacille Calmette-Guérin vaccination (11.3), current smokers (14.2) and those with body mass index >24 (11.4). CONCLUSION: Regular DM screening in TB patients is practical in rural China. Better efficacy of DM-TB detection could be obtained by screening high-risk populations, such as overweight TB patients or those with a family history of DM.

Protective roles of pulmonary rehabilitation mixture in experimental pulmonary fibrosis in vitro and in vivo.

Abnormal high mobility group protein B1 (HMGB1) activation is involved in the pathogenesis of pulmonary fibrosis. Pulmonary rehabilitation mixture (PRM), which combines extracts from eight traditional Chinese medicines, has very good lung protection in clinical use. However, it is not known if PRM has anti-fibrotic activity. In this study, we investigated the effects of PRM on transforming growth factor-ß1 (TGF-ß1)-mediated and bleomycin (BLM)-induced pulmonary fibrosis in vitro and in vivo. The effects of PRM on TGF-ß1-mediated epithelial-mesenchymal transition (EMT) in A549 cells, on the proliferation of human lung fibroblasts (HLF-1) in vitro, and on BLM-induced pulmonary fibrosis in vivo were investigated. PRM treatment resulted in a reduction of EMT in A549 cells that was associated with attenuating an increase of vimentin and a decrease of E-cadherin. PRM inhibited the proliferation of HLF-1 at an IC50 of 0.51 µg/mL. PRM ameliorated BLM-induced pulmonary fibrosis in rats, with reduction of histopathological scores and collagen deposition, and a decrease in α-smooth muscle actin (α-SMA) and HMGB1 expression. An increase in receptor for advanced glycation end-product (RAGE) expression was found in BLM-instilled lungs. PRM significantly decreased EMT and prevented pulmonary fibrosis through decreasing HMGB1 and regulating RAGE in vitro and in vivo. PRM inhibited TGF-ß1-induced EMT via decreased HMGB1 and vimentin and increased RAGE and E-cadherin levels. In summary, PRM prevented experimental pulmonary fibrosis by modulating the HMGB1/RAGE pathway.

Deaths of tuberculosis patients in urban China: a retrospective cohort study.

BACKGROUND: The case-fatality rate for tuberculosis (TB) remains high in China, whereas risk factors for deaths among TB cases are still unclear. DESIGN: This was a retrospective cohort study of all pulmonary TB patients registered in four districts of Shanghai from 2004 to 2008. Data were derived from the China National TB Surveillance System. A total of 4271 patients were followed up in communities. Data were analysed using Cox regression. RESULTS: The percentage of all-cause deaths in the study population was 15% 2-6 years after the most recent TB diagnosis. TB was responsible for only 17% of all deaths; male sex was significantly associated with all-cause deaths. After adjustment for sex, age and treatment history, four factors were significantly and independently associated with increased risk of death: psychopathy, chronic bronchitis, cancer and the presence of multiple diseases. TB was responsible for 7.2 potential years of life lost (PYLL); PYLL was higher in females (8.2) than males (5.3). CONCLUSIONS: TB remains a significant problem in urban China. Implementation of improved clinical management, prevention strategies and other public health programmes should target TB patients with chronic conditions, particularly those with multiple diseases.

Preclinical pharmacology of TP1, a novel potent triple reuptake inhibitor with antidepressant properties.

Triple reuptake inhibitors (TRIs) that block the dopamine transporter (DAT), norepinephrine transporter (NET), and serotonin transporter (SERT) are being developed as a new class of antidepressant that may have better efficacy and fewer side effects compared with traditional antidepressants. The purpose of this study was to characterize a new chemical entity, 4-[2-(dimethylamino)-1-(1-hydroxycyclohexyl)ethyl] phenyl 4-methoxybenzoate hydrochloride (TP1). TP1 was designed as a prodrug of desvenlafaxine. Competitive radioligand binding assays were performed using cells expressing the human dopamine (DA) transporter (hDAT), the human serotonin (5-HT) transporter (hSERT), and the human norepinephrine (NE) transporter (hNET) with K(i) values for TP1 of 190 nM, 2076 nM, and 1023 nM, respectively. Uptake assays were performed with IC(50) values for TP1 of 712 nM, 521 nM, and 628 nM, respectively. TP1 (0.06 mmol/kg, orally) rapidly penetrated rat brain and hypothalamus, translated into desvenlafaxine within 1 h, and demonstrated higher bioavailability and better pharmacokinetic properties than desvenlafaxine succinate (DVS). TP1 (0.06 mmol/kg, orally) significantly increased extracellular levels of DA, NE, and 5-HT compared with baseline in the rat hypothalamus by microdialysis assay. In dose-response assays, oral administration of TP1 reduced the time of immobility in a dose-dependent manner during tail suspension test and forced swimming test (FST). This antidepressant-like effect manifests in the absence of significant increases in motor activity even at doses of up to 32 mg/kg. The ability of TP1 to inhibit the reuptake of three biogenic amines closely linked to the etiology of depression may result in a therapeutic profile different from antidepressants that inhibit the reuptake of serotonin and/or NE.

Protective roles of pulmonary rehabilitation mixture in experimental pulmonary fibrosis in vitro and in vivo

Abnormal high mobility group protein B1 (HMGB1) activation is involved in the pathogenesis of pulmonary fibrosis. Pulmonary rehabilitation mixture (PRM), which combines extracts from eight traditional Chinese medicines, has very good lung protection in clinical use. However, it is not known if PRM has anti-fibrotic activity. In this study, we investigated the effects of PRM on transforming growth factor-β1 (TGF-β1)-mediated and bleomycin (BLM)-induced pulmonary fibrosis in vitro and in vivo. The effects of PRM on TGF-β1-mediated epithelial-mesenchymal transition (EMT) in A549 cells, on the proliferation of human lung fibroblasts (HLF-1) in vitro, and on BLM-induced pulmonary fibrosis in vivo were investigated. PRM treatment resulted in a reduction of EMT in A549 cells that was associated with attenuating an increase of vimentin and a decrease of E-cadherin. PRM inhibited the proliferation of HLF-1 at an IC50 of 0.51 µg/mL. PRM ameliorated BLM-induced pulmonary fibrosis in rats, with reduction of histopathological scores and collagen deposition, and a decrease in α-smooth muscle actin (α-SMA) and HMGB1 expression. An increase in receptor for advanced glycation end-product (RAGE) expression was found in BLM-instilled lungs. PRM significantly decreased EMT and prevented pulmonary fibrosis through decreasing HMGB1 and regulating RAGE in vitro and in vivo. PRM inhibited TGF-β1-induced EMT via decreased HMGB1 and vimentin and increased RAGE and E-cadherin levels. In summary, PRM prevented experimental pulmonary fibrosis by modulating the HMGB1/RAGE pathway.

Cardioprotection with forsythoside B in rat myocardial ischemia-reperfusion injury: relation to inflammation response.

The present study was undertaken to examine the effect of forsythoside B (FB) on rat myocardial ischemia-reperfusion (I/R) model and elucidate the potential mechanism. Left ventricular systolic pressure (LVSP) and +/-dp/dt(max) were detected. Blood samples were collected to determine serum levels of troponin T (Tn-T), TNF-alpha and IL-6. Hearts were harvested to assess histopathological change and infarct size, determine content of MDA, myeloperoxidase (MPO), SOD and GPx activities, analyze expression of high-mobility group box 1 (HMGB1), phosphor-I kappaB-alpha and phosphor-nuclear factor kappaB (NF-kappaB) in ischemic myocardial tissue by Western blot. Compared with control group, rats treatment with FB showed a significant recovery in myocardial function with improvement of LVSP and +/-dp/dt(max). The myocardial infarct volume, serum levels of Tn-T, TNF-alpha and IL-6, content of MDA and MPO activity in myocardial tissue were all reduced, protein expression of HMGB1, phosphor-I kappaB-alpha and phosphor-NF-kappaB were down-regulated, while attenuated the decrease of SOD and GPx activities. Besides, the infiltration of polymorph nuclear leukocytes (PMNs) and histopathological damages in myocardium were decreased in FB treated groups. These findings suggested that FB rescued cardiac function from I/R injury by limiting inflammation response and its antioxidant properties.

[EGFR expression and EGF stimulation of proliferation in human liver carcinoma cells].

Epidermal growth factor receptor (EGFR) gene expression and growth stimulation of EGF on human hepatoma cells of cell lines BEL-7404 and SMMC-7721 were studied. 125I-EGF binding assay was used to measure the binding characteristics and the amounts of EGFR on these cells. The binding time course and the binding competition assay showed that the binding of 125I-EGF to 7404 cells was saturable and specific. Scatchard analysis of EGF binding curve indicated that 7404 and 7721 cells expressed approximately 1.1 x 10(5) and 0.7 x 10(5) EGFRs per cell with binding affinity (Kd) 2.1 nM and 1.8 nM respectively. Northern hybridization and immunoblotting analysis showed the EGFR gene expression products in 7404 and 7721 cells were 5.6 Kb mRNA and 170 Kilo-dalton glycoprotein. Anchorage-dependent growth of 7404 and 7721 cells was stimulated in the presence of nanogram quantities of EGF in medium containing 10% calf serum or 0.5% calf serum. The factors in serum appeared to act synergitically in stimulating of cell proliferation. EGF also stimulated the anchorage-independent growth of 7404 and 7721 cells in soft agar. The results suggest that EGFR is actively expressed in human hepatoma 7404 and 7721 cells and EGF may be one of the mitogens needed for the growth of hepatoma cells.

[Determination of sotalol hydrochloride by reversed-phase high performance liquid chromatography].

An RP-HPLC method for determination and detection of sotalol hydrochloride is described. The baseline separation was achieved on ODS column within 15 minutes by using 0.1% HAc-acetonitrile (80:20, V/V) as mobile phase at a flow rate 1.5 mL/min, and the wavelength was set at 227 nm. The linear range was 5-45 mg/L (r = 0.9991) and the limit of detection was 1 mg/L(S/N > 3). The intra-day and inter-day RSDs were 0.20% and 0.93% respectively.

[Gene cloning, expression of fusion protein TGF alpha-PE 40 and its inhibition activity on cancer cell growth].

Recombinant plasmid pYX382 and pYX3825 were constructed by fusing the cDNA encoding transforming growth factor type alpha (TGF alpha) to Pseudomonas exotoxin gene (PE) in which the cell recognition domain was deleted. The chimeric proteins produced by host E. Coli cells BL21 transformed by plasmid pYX382 and pYX3825 are termed TGF alpha-PE 40 which reacts with antibody against TGF alpha or antibody against PE in immunoblotting to show a 46 kd protein band reflecting the fusion of 56 kD TGF alpha peptide and 40 kD truncated Pseudomonas exotoxin molecule. An additional signal sequence OmpA was inserted into upstream region of TGF alpha cDNA in plasmid pYX3825 resulting in the partly secreting of expression product into medium and periplasm of the cells. TGF alpha-PE 40 was purified from medium by MONO Q ion exchange column and TSK 250 gel filtration column attached to Pharmacia EPLC system. The TGF alpha-PE 40 molecules showed a very strong activities inhibiting the protein synthesis and killing the cancer cells overexpressing EGF receptor on the cell surface.

State of hepatitis B virus DNA in leucocytes of hepatitis B patients.

Hepatitis B virus (HBV) DNA in leucocytes from 50 hepatitis patients with various patterns of HBV serological markers and serum HBV DNA and 13 normal controls were examined by Southern blot hybridization with 32P-labeled 3.2 Kb HBV DNA. A free form of HBV DNA was observed in leucocytes of 8 patients, 7 of whom were positive for serum HBeAg, and in 6 patients an integrated form of HBV DNA was identified. HBV DNA was not identified in leucocytes from 13 normal controls. The free form of HBV DNA in leucocytes existed as a heterogeneous smear from 2.0 to 3.2 Kb, similar to the pattern in liver and hepatocellular carcinoma cells but different from serum HBV DNA in which the 3.2 Kb band was absent. The banding pattern of the integrated form of HBV DNA in leucocytes varied among different patients. During preparation of white blood cells and purification of HBV DNA probes, it was important to remove plasma contamination and traces of pBR322, respectively. The presence of extrachromosomal DNA sequences partially homologous to pBR322 could cause false results. The presence of a free and integrated form of HBV DNA in leucocytes is important for explaining the biology of HBV, the harbouring and replication sites of extrahepatic origin, the mechanism of recurrent infection, and the rationale of the treatment of hepatitis B.

Inhibitory effect of recombinant TGFalpha-PE40 on neointimal proliferation after arterial balloon injury.

AIM: To investigate inhibitory effects of recombinant transforming growth factor alpha-Pseudomonas exotoxin 40 fusion protein (TGFalpha-PE40; TP40) on neointimal proliferation after arterial balloon injury. METHODS: Forty male rabbits fed a cholesterol rich diet were randomly divided into TP40 15 microg, 30 microg, 60 microg, physiologic saline control, and normal artery groups (n=8). Rabbits in the treatment groups were treated by local administration of TP40 (15 microg, 30 microg, and 60 microg per rabbit) 24 h postinjury, and those in the control group were treated by physiologic saline 24 h postinjury. Remained 8 rabbits in normal artery group were treated by TP40 (60 microg). Optical microscope, electron microscope, and computer image analysis were used to study arterial segments 2 weeks after treatment. RESULTS: Irregular thickening of the arterial intima, large amounts of smooth muscle cells (SMC) within the neointima, and stenosis of the arterial cavity were observed in the physiologic saline control group. Great inhibition of intimal proliferation and prevention of stenosis of the arterial cavity were observed in the TP40 treated groups that were examined 2 weeks postinjury by optical microscope. The uninjured carotids were histologically normal. Lots of destructural and necrotic SMC were observed in media in TP40 60 microg group by electron microscope. Computer image analysis showed that the neointimal area of the TP40-treated groups was markedly smaller than that of the saline control group (P < 0.01). CONCLUSION: Recombinant TP40 greatly inhibited neointimal proliferation after arterial balloon injury.

Proteome alterations in human hepatoma cells transfected with antisense epidermal growth factor receptor sequence.

The epidermal growth factor (EGF) is a member of the growth factor superfamily that can stimulate the proliferation of many types of cells. Overexpression of EGF receptor (EGFR) was observed in many types of cancer cells. Anti-EGFR antibodies or antisense nucleic acid sequences of EGFR can suppress the growth of hepatoma cells. In order to further investigate the proteome alterations associated with malignant growth of the human hepatoma cells and the influence of EGFR signal pathway on the cellular proteome, we have comparatively analyzed the proteomes of human hepatoma cells transfected with antisense EGFR sequence (cell strain JX-1) and its control cells (cell strain JX-0) by two-dimensional (2-D) gel electrophoresis and mass spectrometry. Image analysis of silver-stained 2-D gels revealed that 40 protein spots showed significant expression changes in JX-1 cells compared to JX-0 cells. Three of them, including the tumor suppressor protein maspin, changed with tendency to the normal levels. Two protein spots were identified as HSP27 in the same gel, and one of them had a reduced level in JX-1 cells. The apparent alterations of HSP27 in expression level might be the results from their differential chemical modifications, suggesting the effect of dynamic post-translational modifications of proteins on the growth of hepatoma cells. Other proteins such as glutathione peroxidase (GPX-1) and 14-3-3-sigma also exhibited altered expression in JX-1 cells, and their functional implications are discussed.

Inhibitory effect of recombinant TGF alpha-PE40 on vascular smooth muscle cell proliferation.

AIM: To study inhibitory effect of recombinant transforming growth factor alpha-Pseudomonas exotoxin fusion protein (TP40) on proliferation of the cultured vascular smooth muscle cells (SMC). METHODS: Expression of epidermal growth factor receptor (EGFR) mRNA and EGFR in cultured proliferating and quiescent SMC was analyzed with Northern blot and immunohistochemistry. Inhibitory effects of TP40 on SMC proliferation and protein synthesis were analyzed with crystal violet staining and [3H]leucine incorporation. Competition assays were performed by the addition of 100-fold excess of EGF. RESULTS: Expression of EGFR mRNA and EGFR in rapidly proliferating SMC increased than that in quiescent SMC. When the concentration of TP40 was 10 or 100 micrograms.L-1, inhibitory effects of TP40 on rapidly proliferating SMC proliferation and protein synthesis were much higher than that on quiescent SMC (P < 0.01), and the IC50 of [3H]leucine incorporation against rapidly proliferating and quiescent SMC were 8.01 (5.05-12.69) and 121.95 (90.98-163.47) micrograms.L-1. Excess EGF completely blocked inhibitory effects of TP40. CONCLUSION: The rapidly proliferating SMC express EGFR at a high level. TP40 selectively inhibited the proliferation of rapidly proliferating SMC. The cytotoxic effects of TP40 were specifically mediated by EGFR.