Effects of Hypoxia on the Growth and Development of the Fetal Ovine Hepatocytes in Primary Culture.

OBJECTIVE: To investigate the development and characterizations of the hepatocytes isolated from fetal ovine and to determine the effect of hypoxia on their growth and metabolism. METHODS: Fresh hepatocytes were isolated from the liver of fetal ovine at late gestation, cultured in specific media, and exposed to normoxia (21% O2) or hypoxia (2% O2). The cellular characteristics and population purity were identified by immunocytochemistry and flow cytometry (FCM). The effects of hypoxia on cell cycle and apoptosis of the hepatocytes were evaluated by FCM, whereas the cellular ultrastructure changes were examined with a transmission electron microscope. RESULTS: The cell purity of hepatocytes was over 95%. Under hypoxia exposure, the hepatocytes showed a gradual increase in proportion at the S phase and in proliferative index, followed with a compatible increase in apoptosis and progressively decreased cell viability. Additionally, the organelles of the hepatocytes demonstrated dramatic changes, including swelling of mitochondria, disorder in cristae arrangement, expansion of endoplasmic reticulum, and a large number of circular lipid droplets emerging in the cytoplasm. CONCLUSION: Fetal ovine hepatocytes could be primarily cultured in a short-term culture system with a high purity of over 95% and with their preserved original characteristics. Hypoxia could induce changes in ultrastructural and inhibit the proliferation of cultured fetal ovine hepatocytes through apoptotic mechanisms.

Hypothetical protein Cpn0423 triggers NOD2 activation and contributes to Chlamydia pneumoniae-mediated inflammation.

BACKGROUND: Chlamydia pneumoniae (C. pneumoniae) is pathogenic to humans, by causing pulmonary inflammation or bronchitis in both adolescents and young adults. However, the molecular signals linking C. pneumoniae components to inflammation remain elusive. This study was to investigate the effect of Chlamydia-specific Cpn0423 of C. pneumoniae on C. pneumoniae-mediated inflammation. RESULTS: Cpn0423 was detected outside of C. pneumoniae inclusions, which induced production of several cytokines including macrophage inflammatory protein-2 (MIP-2) and interleukins (ILs). Production of the Cpn0423-induced cytokines was markedly reduced in cells pretreated with NOD2-siRNA, but not with negative control oligonucleotides. Mice treated with Cpn0423 through intranasal administration exhibited pulmonary inflammation as evidenced by infiltration of inflammatory cells, increased inflammatory scores in the lung histology, recruitment of neutrophils and increased cytokines levels in the BALF. CONCLUSION: Cpn0423 could be sensed by NOD2, which was identified as an essential element in a pathway contributing to the development of C. pneumoniae -mediated inflammation.

Development and evaluation of a MAb-based ELISA for detection of Chlamydophila pneumoniae infection with variable domain 2 and 3 of the major outer membrane protein.

OBJECTIVE: This paper aims to develop a monoclonal antibodies (MAbs)- based ELISA for detecting Chlamydophila pneumoniae (C. pneumoniae) antigens in humans with the variable domains (VD) 2 and 3 of the major outer membrane protein (MOMPVD2-VD3) and to assess its sensitivity and specificity by comparing with a widely used MAb that is able to recognize the elementary bodies of C. pneumoniae. METHODS: MOMPVD2-VD3 were overexpressed in Escherichia coli and purified by affinity chromatography. Mice were immunized with the recombinant antigen, and hybridomas secreting MAbs were screened. Three stable hybridomas clones were selected and named 5D6, 7G3, and 8C9. The MAbs-based ELISA was scrutinized for species-specific recognition with a number of human throat swab samples from Group I (156 patients with typical respiratory illness clinically confirmed before) and Group II (57 healthy donors). RESULTS: In Group I, 55 positive cases were detected by anti-EB MAb-based ELISA, 51 cases were positive by MAbs 5D6-based ELISA, and 33 and 38 cases were positive by MAb 8C9 and 7G3-based ELISA respectively. Of the 57 samples from Group II "healthy donors", 5 were positive and 52 were negative with both anti-EB and 5D6-based tests, while 2 and 3 positive cases were identified by the other two MAb-based ELISAs respectively. CONCLUSION: The novel MOMPVD2-VD3 MAb-based assay may have higher specificity than the anti-EB MAb, which may possibly be used as an alternative tool for the diagnosis of C. pneumoniae infection.