Indicators for prediction of Mycobacterium tuberculosis positivity detected with bronchoalveolar lavage fluid.

BACKGROUND: The diagnosis of active pulmonary tuberculosis (TB) remains a challenge in clinic, especially for sputum negative pulmonary TB. Bronchoalveolar lavage fluid (BALF) has higher sensitivity than sputum for detection of Mycobacterium tuberculosis (Mtb). However, bronchoscopy is invasive and costly, and not suitable for all patients. In order to make TB patients get more benefit from BALF for diagnosis, we explore which indicator might be used to optimize the choice of bronchoscopy. METHODS: A total of 1539 sputum-smear-negative pulmonary TB suspects who underwent bronchoscopy were recruited for evaluation. The sensitivity, specificity and accuracy of Mtb detection in sputum and BALF were compared. Odds ratios and 95% confidence intervals were used to assess variables that associated with positive acid-fast bacilli (AFB) smear, Mtb culture and nucleic acid amplification test (NAAT) of BALF in sputum-negative and non-sputum-producing pulmonary TB suspects. RESULTS: BALF has significantly higher sensitivity (63.4%) than sputum (43.5%) for Mtb detection by culture and NAAT. 19.7% (122/620) sputum-negative and 40.0% (163/408) non-sputum-producing suspects had positive bacteriological results in BALF. Among sputum-negative and non-sputum-producing pulmonary TB suspects, the positivity of Mtb detection in BALF is associated with a younger age, the presence of pulmonary cavities and a positive result of interferon-gamma release assay (IGRA). Sputum-negative patients under 35 years old with positive IGRA and pulmonary cavity had 84.8% positivity of Mtb in BALF. CONCLUSIONS: Our study indicated that combination of age, the presence of pulmonary cavity, and the result of IGRA is useful to predict the positivity of Mtb detection in BALF among sputum-negative and non-sputum producing pulmonary TB suspects. Those who are under 35 years old, positive for the presence of pulmonary cavity and IGRA, should undergo bronchoscopy to collect BAFL for Mtb tests, as they have the highest possibility to get bacteriologically confirmation of TB.

Delta-like ligand 4 correlates with endothelial proliferation and vessel maturation in human malignant glioma.

AIM: To investigate the role of delta-like ligand 4 (DLL4) in the angiogenesis of high-grade malignant glioma. MATERIALS AND METHODS: DLL4 expression and microvessel density (MVD) were detected by immunohistochemistry in 51 human high-grade malignant glioma tissue samples. The vessel maturation index (VMI) was calculated as the percentage of a-smooth muscle actin (a-SMA)-positive vessels in relation to the amount of CD31-positive vessels. Double fluorescent immunostaining for CD31 and EphrinB2 or EphB4 was performed to identify the arterial (EphrinB2) or venous (EphB4) origins of glioma microvessels. RESULTS: Strong immunostaining of DLL4 and a positive correlation of DLL4 with the MVD were observed in high-grade malignant gliomas. The VMI of the DLL4-positive group was significantly higher than that of the DLL4-negative group. However, no significant association was found between DLL4 and EphrinB2 or EphB4 in high-grade gliomas. CONCLUSION: DLL4 may be an important regulator for vessel proliferation and maturation in human high-grade malignant gliomas.

[Effect of metformin on apoptosis of renal cell carcinoma cells in vitro and its mechanisms].

OBJECTIVE: To evaluate the effect of metformin on the apoptosis of renal cell carcinoma (RCC) cells in vitro and its mechanisms. METHODS: Fluorescent microscopy and flow cytometry were used to examine the changes in the apoptosis of 786-O cells after metformin treatment. The possible signaling molecules involved in this process were analyzed by immunoblot analysis of AMP-activated protein kinase (AMPK) signaling and caspase 9. RESULTS: Metformin induced apoptosis and caspase 9 activation in 786-O cells in low-serum medium but not in normal-serum medium. Metformin also induced AMPK activation in 786-O cells, but this activation was not associated with the cell proliferation inhibition or apoptosis-inducing effect of metformin. CONCLUSION: Metformin can induce apoptosis of RCC cells in vitro, suggesting its potential as a therapeutic agent for RCC.

Metformin induces G1 cell cycle arrest and inhibits cell proliferation in nasopharyngeal carcinoma cells.

It has been reported that metformin, a biguanide derivative widely used in type II diabetic patients, has antitumor activities in some cancers by activation of AMP-activated protein kinase (AMPK). But its role in nasopharyngeal carcinoma (NPC) is not known. Here, we reported for the first time that 1-50 mM of metformin in a dose- and time-dependent manner suppressed cell proliferation and colony formation in NPC cell line, C666-1. Further studies revealed that the protein level of cyclin D1 decreased and the percentage of the cells in G0/G1 phase increased by 5 mM metformin treatment. Metformin also induced the phosphorylation of AMPK (T172) in a time-dependent manner. Mammalian target of rapamycin complex 1 (mTORC1), which is negatively regulated by AMPK and plays a central role in cell growth and proliferation, was inhibited by metformin, as manifested by dephosphorylation of its downstream targets 40S ribosomal S6 kinase 1 (S6K1) (T389), the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) (T37/46) and S6 (S235/236) in C666-1 cells. In a summary, metformin prevents proliferation of C666-1 cells by down-regulating cyclin D1 level and inducing G1 cell cycle arrest. AMPK-mediated inhibition of mTORC1 signaling may be involved in this process.

Metformin stimulates osteoprotegerin and reduces RANKL expression in osteoblasts and ovariectomized rats.

Anti-diabetic drug metformin has been shown to enhance osteoblasts differentiation and inhibit osteoclast differentiation in vitro and prevent bone loss in ovariectomized (OVX) rats. But the mechanisms through which metformin regulates osteoclastogensis are not known. Osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) are cytokines predominantly secreted by osteoblasts and play critical roles in the differentiation and function of osteoclasts. In this study, we demonstrated that metformin dose-dependently stimulated OPG and reduced RANKL mRNA and protein expression in mouse calvarial osteoblasts and osteoblastic cell line MC3T3-E1. Inhibition of AMP-activated protein kinase (AMPK) and CaM kinase kinase (CaMKK), two targets of metformin, suppressed endogenous and metformin-induced OPG secretion in osteoblasts. Moreover, supernatant of osteoblasts treated with metformin reduced formation of tartrate resistant acid phosphatase (TRAP)-positive multi-nucleated cells in Raw264.7 cells. Most importantly, metformin significantly increased total body bone mineral density, prevented bone loss and decreased TRAP-positive cells in OVX rats proximal tibiae, accompanied with an increase of OPG and decrease of RANKL expression. These in vivo and in vitro studies suggest that metformin reduces RANKL and stimulates OPG expression in osteoblasts, further inhibits osteoclast differentiation and prevents bone loss in OVX rats.

[Expression and antibody preparation of FKBP38 and its application].

OBJECTIVE: To obtain recombinant N-and C-terminal of FKBP38 and prepare anti-FKBP38 polyclonal antibody for Western blotting (WB), immunohistochemical (IHC) and immunofluorescence (IF) analyses. METHODS: The N-terminal (1-207 aa) and C-terminal (209-387 aa) cDNA of FKBP38 were sub-cloned from the full-length cDNA of FKBP38 and ligated to prokaryotic expression plasmid pGEX-6P-1 for construction of the recombinant vectors pGEX-6P-1-FKBP38-N and pGEX-6P-1-FKBP38-C. After sequencing, the recombinant vectors were transformed into E.coli BL21 and GST-tagged FKBP38-NT and FKBP38-CT were induced by IPTG. The proteins were purified by Glutathione affinity chromatography column and characterized by SDS-PAGE. Rabbits were immunized with the purified recombinant protein to prepare the antiserum, which were analyzed by WB, IHC and IF. RESULTS: The recombinant vectors pGEX-6P-1-FKBP38-N and pGEX-6P-1-FKBP38-C were successfully constructed. After IPTG induction, the E.coli transformed with these plasmids expressed GST-tagged protein, which was successfully purified. Western blotting demonstrated that the purified antibody could specifically bind to FKBP38 in various cell lines. Immunofluorescence assay showed that FKBP38 was located mainly on the mitochondria. Immunohistochemical analysis revealed cytoplasmic location of FKBP38 in breast cells. CONCLUSION: We successfully expressed and purified N- and C-terminal of FKBP38, and FKBP38 polyclonal antibody we prepared can specifically recognize FKBP38 in SB, IF and IHC assays, which facilitates further functional investigation of FKBP38.

[The expression of hepatocyte growth factor and its receptor in brain astrocytomas].

OBJECTIVE: To investigate the expression of hepatocyte growth factor (HGF) mRNA and its receptor (c-Met) mRNA in brain astrocytomas and their relationships with tumor proliferation, angiogenesis, clinical pathology and prognosis. METHODS: The expression of HGF mRNA, c-Met mRNA in the resected tumor tissues of 76 patients with brain astrocytomas, 43 males and 33 females, aged 20 - 71, were detected by in situ hybridization. Immunohistochemistry technique was used to test the expression of proliferating cell nuclear antigen (PCNA) protein and the microvessel density (MVD) was determined by immunohistochemistry with monoclonal antibody against CD34. RESULTS: The positive rates of expression of HGF, c-Met and PCNA in low pathologic grades of brain astrocytoma were 34.5%, 44.8% and 15% +/- 9% respectively, and in high pathologic grades of brain astrocytoma were 34.5%, 44.8% and 48% +/- 12% respectively (P < 0.05). MVD in low and high pathologic grades of brain astrocytoma were 17 +/- 7 and 31 +/- 13 respectively (P < 0.05). The expression of HGF, c-Met, PCNA and CD34 was not related to sex, age, position of tumor and diameter of tumor. The expression of c-Met was related to the expression of HGF, PCNA and the MVD in the tumor tissues of these patients. The pathological grade, position of tumor, HGF, c-Met, PCNA, MVD had a significant influence on the survival time. CONCLUSION: HGF/c-Met plays an important role in the formation and progression of the brain astrocytoma and can promote tumor proliferation and intratumoral microvascular formation, and is closely related to the prognosis of the patients.