Effects of bile acids on biliary epithelial cell proliferation and portal fibroblast activation using rat liver slices

During cholestasis, bile acids accumulate in the liver, and induce cellular alterations. Cholestasis is a major cause of liver fibrosis. We have used precision-cut liver slices (PCLS) in culture to investigate the effects of bile acids on hepatic cells. Rat PCLS were placed on an insert in a vial containing culture medium, and gently agitated on a roller platform. PCLS were treated with 100 microM taurolithocholate (TLC), taurodeoxycholate (TDC) or taurocholate (TC) for 24 or 48 h. PCLS viability was measured, and immunohistochemistry was performed with antibodies against active caspase 3, platelet-derived growth factor (PDGF) receptor-beta and ED-A fibronectin. TDC and TLC, two hydrophobic bile acids, induced hepatocyte necrosis and apoptosis, whereas TC, an hydrophilic bile acid, improved slice viability as compared with controls. Both TDC and TC induced biliary epithelial cell proliferation, together with portal fibroblast proliferation and activation, as shown by PDGF receptor-beta and ED-A fibronectin expression. TLC induced biliary epithelial cell apoptosis. Our results indicate that individual bile acids induce cell type-specific effects in a complex liver microenvironment. The fact that PCLS support biliary epithelial cell and portal fibroblast proliferation will make this model very useful for the study of the mechanisms involved in portal fibrosis.     

Toxoplasma gondii, HCV, and HBV seroprevalence and co-infection among HIV-positive and -negative pregnant women in Burkina Faso

Toxoplasma gondii (T. gondii) infections can cause serious complications in HIV-infected pregnant women, leading to miscarriage, stillbirth, birth defects (e.g., mental retardation, blindness, epilepsy etc.) and could favor or enhance the mother-to-child transmission of HCV, HBV, and HIV vertical transmission. From May 20, 2004 to August 3, 2005, 336 18-45 years aged pregnant women, were enrolled for an investigation of the prevalence of serum antibodies against T. gondii, HCV, HBV, and HIV using ELISA. The prevalence of T. gondii, HCV, and HBV in pregnant women was 25.3%, 5.4%, and 9.8%, respectively and the HIV serostatus (61.6%) seems to be associated with greater prevalence rates of both T. gondii (28.5% vs. 20.2%) and HBV (11.6% vs. 7.0%). Without taking into account HIV, only 65.5% (220 of 336) of the women were not infected with these agents. The co-infection rate between HIV-infected and -negative women was different statistically: T. gondii/HBV 0.048 versus 0.015, T. gondii/HCV 0.014 versus 0.008, and HCV/HBV 0.005 versus 0.008, respectively. The elevated co-infection rate in HIV-positive women demonstrated that they are exposed to T. gondii, HCV, and HBV infections prevalently by sexual contact.     

The chemokine CXCL3 is responsible for the constitutive chemotactic activity of bovine milk for neutrophils

Bovine milk is known to exert a potent chemotactic activity on neutrophils, but the responsible agent has not been identified. The objective of the study was to characterize the main biochemical component responsible for this chemotactic activity. A neutrophil shape change assay was used to locate active milk fractions separated by chromatography. A single protein was isolated and identified by amino acid sequencing and mass spectrometry as CXCL3. Recombinant bovine chemokines and specific antibodies were used to show that normal milk contains active concentrations of CXCL1 (1-5ng/ml) and CXCL3 (100-500ng/ml), whereas CXCL2 and CXCL8/IL-8 were not detected. Depletion experiments with antibodies showed that CXCL3 was the main chemotaxin for neutrophils in normal (non-mastitic) milk. The chemokine CXCL3 was located by immunohistochemistry in mammary epithelial cells, and abundant mRNA was found in uninflamed mammary tissue, suggesting constitutive secretion by the lactating mammary epithelium. These results indicate that CXCL3/GRO-gamma is the major chemotactic factor for neutrophils in bovine milk in the absence of inflammation, and that it is secreted constitutively in milk by mammary epithelial cells. This finding prompts the question of the biological significance of permanent high concentrations of a CXC chemokine in milk.     

Functional proteomics mapping of a human signaling pathway

Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.     

Human lung myofibroblasts as effectors of the inflammatory process: the common receptor gamma chain is induced by Th2 cytokines,and CD40 ligand is induced by lipopolysaccharide,thrombin and TNF-alpha

The common gamma (gamma c) chain, shared by Th1 and Th2 cytokines, is fundamental for the activation of hematopoietic cells, but its role in non-hematopoietic tissues has not been explored. Here we show that in normal lung fibroblasts IL-4 and IL-13 induce the expression of the gamma c chain and its association with Janus kinase (JAK) 3, while lung myofibroblasts constitutively express a gamma c chain displaying a limited association with JAK3. In the latter cells, without exogenous cytokines, gamma c/JAK3 controls, through autocrine loops, tyrosine kinase (TYK) 2 phosphorylation and the balance between functional (IL-4Ralpha, IL-13Ralpha 1) and decoy (IL-13Ralpha 2) high-affinity receptors. Moreover, JAK3 is also associated with a pre-phosphorylated IL-4Ralpha and CD40. This novel "heterotrimer" (p-IL-4Ralpha, CD40/JAK3) is functional and controls STAT3 phosphorylation and CD40 expression, as shown by use of the specific JAK3 inhibitor WHI-P31. In basal culture conditions, CD40 signaling could be induced by the transient establishment of inter-fibroblastic CD40/CD40 ligand (CD40L) functional bridges. Indeed, powerful pro-inflammatory stimuli such as lipopolysaccharide and thrombin can rapidly mobilize CD40L at the surface of lung myofibroblasts. These interactions are modified by IL-13, which triggers the formation of a new type of functional receptor (p-IL-4Ralpha /IL-13Ralpha 1/gamma c) and also the recruitment and the phosphorylation of JAK3. Treatment with JAK3 inhibitors blocks IL-13-induced phosphorylation of JAK2, TYK2 and STAT3, but not of JAK1 and STAT6. These data underline (1) the pivotal role of the gamma c chain, CD40/CD40L, JAK3 and IL-13 in the inflammatory-like activation of lung myofibroblasts, (2) the cell-type restraint effects of IL-13 on these cells, and (3) the potential usefulness of JAK3 inhibitors in the treatment of asthma.     

Congenital posterior cervical spine malformation due to biallelic c.240‐4T>G RIPPLY2 variant: A discrete entity

The clinical and radiological spectrum of spondylocostal dysostosis syndromes encompasses distinctive costo‐vertebral anomalies. RIPPLY2 biallelic pathogenic variants were described in two distinct cervical spine malformation syndromes: Klippel–Feil syndrome and posterior cervical spine malformation. RIPPLY2 is involved in the determination of rostro‐caudal polarity and somite patterning during development. To date, only four cases have been reported. The current report aims at further delineating the posterior malformation in three new patients. Three patients from two unrelated families underwent clinical and radiological examination through X‐ray, 3D computed tomography and brain magnetic resonance imaging. After informed consent was obtained, family‐based whole exome sequencing (WES) was performed. Complex vertebral segmentation defects in the cervico‐thoracic spine were observed in all patients. WES led to the identification of the homozygous splicing variant c.240‐4T>G in all subjects. This variant is predicted to result in aberrant splicing of Exon 4. The current report highlights a subtype of cervical spine malformation with major atlo‐axoidal malformation compromising spinal cord integrity. This distinctive mutation‐specific pattern of malformation differs from Klippel–Feil syndrome and broadens the current classification, defining a sub‐type of RIPPLY2‐related skeletal disorder. Of note, the phenotype of one patient overlaps with oculo‐auriculo‐vertebral spectrum disorder.