A Rapid Subtractive Immunization Method to Prepare Discriminatory Monoclonal Antibodies for Food E. coli O157:H7 Contamination

To detect food E. coli O157:H7 contamination rapidly and accurately, it is essential to prepare high specific monoclonal antibodies (mAbs) against the pathogen. Cyclophosphamide (Cy)-mediated subtractive immunization strategy was performed in mice to generate mAbs that react with E. coli O157:H7, but not with other affiliated bacteria. Specificity of 19 mAbs was evaluated by ELISA and/or dot-immunogold filtration assay (DIGFA). Immunogloubin typing, affinity and binding antigens of 5 selected mAbs were also analysed. MAbs 1D8, 4A7, 5A2 were found to have high reactivity with E. coli O157:H7 and no cross-reactivity with 80 other strains of bacteria including Salmonella sp., Shigella sp., Proteus sp., Yersinia enterocolitica, Staphylococcus aureus, Klebsiella pneumoniae, Citrobacter freundii and other non-E. coli O157:H7 enteric bacteria. Their ascetic titers reached 1∶106 with E. coli O157:H7 and affinity constants ranged from 1.57×1010 to 2.79×1010 L/mol. The antigens recognized by them were different localized proteins. Furthermore, immune-colloidal gold probe coated with mAb 5A2 could specifically distinguish minced beef contaminated by E. coli O157:H7 from 84 other bacterial contaminations. The Cy-mediated subtractive immunization procedure coupled with hybridoma technology is a rapid and efficient approach to prepare discriminatory mAbs for detection of E. coli O157:H7 contamination in food

Integrin-mediated type II TGF-beta receptor tyrosine dephosphorylation controls SWIAD-dependent profibrotic signaling

Tubulointerstitial fibrosis underlies all forms of end-stage kidney disease. TGF-beta mediates both the development and the progression of kidney fibrosis through binding and activation of the serine/threonine kinase type II TGF-beta receptor (T beta RII), which in turn promotes a T beta RI-mediated SMAD-dependent fibrotic signaling cascade. Autophosphorylation of serine residues within T beta RII is considered the principal regulatory mechanism of T beta RII-induced signaling; however, there are 5 tyrosine residues within the cytoplasmic tail that could potentially mediate T beta RII-dependent SMAD activation. Here, we determined that phosphorylation of tyrosines within the T beta RII tail was essential for SMAD-dependent fibrotic signaling within cells of the kidney collecting duct. Conversely, the T cell protein tyrosine phosphatase (TCPTP) dephosphorylated T beta RII tail tyrosine residues, resulting in inhibition of T beta R-dependent fibrotic signaling. The collagen-binding receptor integrin OM was required for recruitment of TCPTP to the T beta RII tail, as mice lacking this integrin exhibited impaired TCPTP-mediated tyrosine dephosphorylation of T beta RII that led to severe fibrosis in a unilateral ureteral obstruction model of renal fibrosis. Together, these findings uncover a crosstalk between integrin alpha 1 beta 1 and T beta RII that is essential for T beta RII-mediated SMAD activation and fibrotic signaling pathways

Oxidative injury is a common consequence of BMPR2 mutations

Hereditary pulmonary arterial hypertension (PAH) is usually caused by mutations in BMPR2. Mutations are found throughout the gene, and common molecular consequences of different types of mutation are not known. Knowledge of common molecular consequences would provide insight into the molecular etiology of the disease. The objective of this study was to determine the common molecular consequences across classes of BMPR2 mutation. Increased superoxide and peroxide production and alterations in genes associated with oxidative stress were a common consequence of stable transfection of the vascular smooth muscle cells, with three distinct classes of BMPR2 mutation, in the ligand binding domain, the kinase domain and the cytoplasmic tail domain. Measurement of oxidized lipids in whole lung from transgenic mice expressing a mutation in the BMPR2 cytoplasmic tail showed a 50% increase in isoprostanes and a two-fold increase in isofurans, suggesting increased reactive oxygen species (ROS) of mitochondrial origin. Immunohistochemistry on BMPR2 transgenic mouse lung showed that oxidative stress was vascular-specific. Electron microscopy showed decreased mitochondrial size and variability in the pulmonary vessels from BMPR2-mutant mice. Measurement of oxidized lipids in urine from humans with BMPR2 mutations demonstrated increased ROS, regardless of disease status. Immunohistochemistry on hereditary PAH patient lung confirmed oxidative stress specific to the vasculature. Increased oxidative stress, likely of mitochondrial origin, is a common consequence of BMPR2 mutation across mutation types in cell culture, mice and humans

Dendritic Cell Amiloride-Sensitive Channels Mediate Sodium-Induced Inflammation and Hypertension

Summary: Sodium accumulates in the interstitium and promotes inflammation through poorly defined mechanisms. We describe a pathway by which sodium enters dendritic cells (DCs) through amiloride-sensitive channels including the alpha and gamma subunits of the epithelial sodium channel and the sodium hydrogen exchanger 1. This leads to calcium influx via the sodium calcium exchanger, activation of protein kinase C (PKC), phosphorylation of p47phox, and association of p47phox with gp91phox. The assembled NADPH oxidase produces superoxide with subsequent formation of immunogenic isolevuglandin (IsoLG)-protein adducts. DCs activated by excess sodium produce increased interleukin-1β (IL-1β) and promote T cell production of cytokines IL-17A and interferon gamma (IFN-γ). When adoptively transferred into naive mice, these DCs prime hypertension in response to a sub-pressor dose of angiotensin II. These findings provide a mechanistic link between salt, inflammation, and hypertension involving increased oxidative stress and IsoLG production in DCs. : Barbaro et al. describe a pathway by which increased extracellular sodium activates dendritic cells. This pathway potentially explains the link between excessive salt intake, inflammation, and high blood pressure. Keywords: hypertension, sodium chloride, dendritic cells, isolevuglandins, oxidative stress, NADPH oxidase, amiloride, ENaC, calciu