Transplanted adult human hepatic stem/progenitor cells prevent histogenesis of advanced hepatic fibrosis in mice induced by carbon tetrachloride

Transplantation of adult human hepatic stem/progenitor cells (hHSPCs) has been considered as an alternative therapy, replacing donor liver transplantation to treat liver cirrhosis. This study assessed the antifibrotic effects of hHSPCs in mice with fibrosis induced by carbon tetrachloride (CCl4) and examined the actions of hHSPCs on the fibrogenic activity of human hepatic stellate cells (HSCs) in a coculture system. Isolated hHSPCs expressed stem/progenitor cell phenotypic markers. Mice were given CCl4 (twice weekly for 7 weeks) and hHSPC transplantation weekly. CCl4 induced advanced fibrosis (bridging fibrosis and cirrhosis) in mice, which was prevented by hHSPC transplantation. The liver of hHSPC-transplanted mice showed only occasional short septa and focal parenchymal fibrosis, and a 50% reduction in hepatic collagen, assessed by Sirius red stain histomorphometry. Moreover, the proteins for α-smooth muscle actin (α-SMA) and collagen I were decreased. While α-SMA, collagen α1(I), and tissue inhibitor of metalloproproteinase-1 mRNAs were decreased, matrix metalloproteinase (MMP)-1 mRNA was increased, consistent with decreased fibrogenesis. MMP-2 and transforming growth factor-β were not affected. Alanine aminotransferase and aspartate aminotransferase were lower, suggesting improvement of liver function/damage. In coculture, hHSPCs elicited changes of α-SMA and fibrogenic molecules in HSCs similar to those observed in vivo, providing evidence for a functional link between hHSPCs and HSCs. A decreased HSC proliferation was noted. Thus, transplantation of hHSPCs prevents histogenesis of advanced liver fibrosis caused by CCl4. hHSPCs mediate downregulation of HSC activation coincident with modulation of fibrogenic molecule expression, leading to suppression of fibrogenesis both in vivo and in vitro

Transplanted adult human hepatic stem/progenitor cells prevent histogenesis of advanced hepatic fibrosis in mice induced by carbon tetrachloride

Transplantation of adult human hepatic stem/progenitor cells (hHSPCs) has been considered as an alternative therapy, replacing donor liver transplantation to treat liver cirrhosis. This study assessed the antifibrotic effects of hHSPCs in mice with fibrosis induced by carbon tetrachloride (CCI4) and examined the actions of hHSPCs on the fibrogenic activity of human hepatic stellate cells (HSCs) in a coculture system. Isolated hHSPCs expressed stem/progenitor cell phenotypic markers. Mice were given CCl4 (twice weekly for 7 weeks) and hHSPC transplantation weekly. CCl4 induced advanced fibrosis (bridging fibrosis and cirrhosis) in mice, which was prevented by hHSPC transplantation. The liver of hHSPC-transplanted mice showed only occasional short septa and focal parenchymal fibrosis, and a 50% reduction in hepatic collagen, assessed by Sirius red stain histomorphometry. Moreover, the proteins for a-smooth muscle actin (alpha-SMA) and collagen I were decreased. While alpha-SMA, collagen alpha 1(I), and tissue inhibitor of metalloproproteinase-1 mRNAs were decreased, matrix metalloproteinase (MMP)-1 mRNA was increased, consistent with decreased fibrogenesis. MMP-2 and transforming growth factor-beta were not affected. Alanine aminotransferase and aspartate aminotransferase were lower, suggesting improvement of liver function/damage. In coculture, hHSPCs elicited changes of alpha-SMA and fibrogenic molecules in HSCs similar to those observed in vivo, providing evidence for a functional link between hHSPCs and HSCs. A decreased HSC proliferation was noted. Thus, transplantation of hHSPCs prevents histogenesis of advanced liver fibrosis caused by CCl4. hHSPCs mediate down-regulation of HSC activation coincident with modulation of fibrogenic molecule expression, leading to suppression of fibrogenesis both in vivo and in vitro

X4 Human Immunodeficiency Virus Type 1 gp120 Promotes Human Hepatic Stellate Cell Activation and Collagen I Expression through Interactions with CXCR4

<div><h3>Background & Aims</h3><p>Patients coinfected with HIV-1 and HCV develop more rapid liver fibrosis than patients monoinfected with HCV. HIV RNA levels correlate with fibrosis progression implicating HIV directly in the fibrotic process. While activated hepatic stellate cells (HSCs) express the 2 major HIV chemokine coreceptors, CXCR4 and CCR5, little is known about the pro-fibrogenic effects of the HIV-1 envelope protein, gp120, on HSCs. We therefore examined the <em>in vitro</em> impact of X4 gp120 on HSC activation, collagen I expression, and underlying signaling pathways and examined the <em>in vivo</em> expression of gp120 in HIV/HCV coinfected livers.</p> <h3>Methods</h3><p>Primary human HSCs and LX-2 cells, a human HSC line, were challenged with X4 gp120 and expression of fibrogenic markers assessed by qRT-PCR and Western blot +/− either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Downstream intracellular signaling pathways were evaluated with Western blot and pre-treatment with specific pathway inhibitors. Gp120 immunostaining was performed on HIV/HCV coinfected liver biopsies.</p> <h3>Results</h3><p>X4 gp 120 significantly increased expression of alpha-smooth muscle actin (a-SMA) and collagen I in HSCs which was blocked by pre-incubation with either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Furthermore, X4 gp120 promoted Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and pretreatment with an ERK inhibitor attenuated HSC activation and collagen I expression. Sinusoidal staining for gp120 was evident in HIV/HCV coinfected livers.</p> <h3>Conclusions</h3><p>X4 HIV-1 gp120 is pro-fibrogenic through its interactions with CXCR4 on activated HSCs. The availability of small molecule inhibitors to CXCR4 make this a potential anti-fibrotic target in HIV/HCV coinfected patients.</p> </div

Leukadherin-1-Mediated Activation of CD11b Inhibits LPS-Induced Pro-inflammatory Response in Macrophages and Protects Mice Against Endotoxic Shock by Blocking LPS-TLR4 Interaction

Dysregulation of macrophage has been demonstrated to contribute to aberrant immune responses and inflammatory diseases. CD11b, expressed on macrophages, plays a critical role in regulating pathogen recognition, phagocytosis, and cell survival. In the present study, we explored the effect of leukadherin-1 (LA1), an agonist of CD11b, on regulating LPS-induced pro-inflammatory response in macrophages and endotoxic shock. Intriguingly, we found that LA1 could significantly reduce mortalities of mice and alleviated pathological injury of liver and lung in endotoxic shock. In vivo studies showed that LA1-induced activation of CD11b significantly inhibited the LPS-induced pro-inflammatory response in macrophages of mice. Moreover, LA1-induced activation of CD11b significantly inhibited LPS/IFN-γ-induced pro-inflammatory response in macrophages by inhibiting MAPKs and NF-κB signaling pathways in vitro. Furthermore, the mice injected with LA1-treated BMDMs showed fewer pathological lesions than those injected with vehicle-treated BMDMs in endotoxic shock. In addition, we found that activation of TLR4 by LPS could endocytose CD11b and activation of CD11b by LA1 could endocytose TLR4 in vitro and in vivo, subsequently blocking the binding of LPS with TLR4. Based on these findings, we concluded that LA1-induced activation of CD11b negatively regulates LPS-induced pro-inflammatory response in macrophages and subsequently protects mice from endotoxin shock by partially blocking LPS-TLR4 interaction. Our study provides a new insight into the role of CD11b in the pathogenesis of inflammatory diseases

Molecular cloning of human IL-16 gene and its expression in Escherichia Coli (34.17)

Abstract Objective: To clone human IL-16 gene, and construct its prokaryotic expression vector, and express it in E. coli DH5. Methods: The total RNA was extracted from peripheral blood mononuclear cells stimulated by histamine. And hIL-16 cDNA was amplified by using RT-PCR and cloned into pUC18 T-vector. After the sequence of hIL-16 cDNA was confirmed, the cDNA was inserted into prokaryotic expression vector pMAL-C2. The recombinant expression plasmid pMAL-IL-16 was identified by endonucleases digestion and PCR, and then transformed into E. coli DH5, human IL-16 expressed in DH5 was identified by SDS-PAGE and Western blotting method. Results: Obtained cDNA of hIL-16 was identical with that published on Genebank, and the prokaryotic expression vector pMAL-IL-16 was constructed correctly. Recombinant protein was expressed in E.coli when induced with IPTG and had a molecular weight of 60kD, wich is consistent with theoretical calculation. Conclusion: The hIL-16 cDNA was cloned and the prokaryotic expression plasmid pMAL-IL-16 was constructed successfully. The E. coli DH5 that stably expressed hIL-16 was obtained, which provide experimental basis for the further studying of functional activities and application of hIL-16. [Key words] Human interleukin-16, Molecular cloning, Prokaryotic expression</jats:p

Distinct roles of dendritic cell-derived iNOS in the control of effective and regulative dendritic cell differentiation.

Abstract The immune system exists in a delicate equilibrium between response and tolerance. Dendritic cells (DC) are a plastic lineage able to process and integrate signals from the microenvironment. But the regulation how DCs differentiate to effective or regulatory DC cells are incompletely understood. Inducible nitric oxide synthase (iNOS) derived NO plays critical roles in immune suppression of immune cells. But, it is still not clear what the function of DC cells-derived iNOS is in the regulation in inflammatory diseases. In this study, we demonstrated that DC-derived iNOS regulates balance of effective and regulatory DC cell differentiation. iNOS deficient mice displayed an increased effective DC phenotypes, whereas the percentage of regulatory DCs were comparable in wild-type and iNOS deficient mice in vivo and in vitro. The results were further supported by increased effective DCs from iNOS−/− BMDC cells. Activation of DCs by LPS/IFNg resulted in the expression of iNOS in WT mice. The iNOS inhibitor L-NIL enhanced effective DCs differentiation, mimicking the effect observed in iNOS deficient mice. NO donor SNAP suppressed effective DCs. iNOS−/− DCs result in more enhanced T cell activation. iNOS−/− mice infected with Citrobacter Rodentium led to more severe intestinal inflammation compared to WT mice and the results were correlated with more inflammatory cells infiltration in iNOS−/− in colon tissues. And iNOS−/− mice showed increased effective DCs in colon tissues than that in WT mice. Our results suggest that DC-derived iNOS negatively controls effective DC development and targeting DC-derived iNOS would lead to the new therapies for autoimmune/inflammatory diseases.</jats:p