New frontiers in fibrotic disease therapies: The focus of the Joan and Joel Rosenbloom Center for Fibrotic Diseases at Thomas Jefferson University.

Fibrotic diseases constitute a world-wide major health problem, but research support remains inadequate in comparison to the need. Although considerable understanding of the pathogenesis of fibrotic reactions has been attained, no completely effective therapies exist. Although fibrotic disorders are diverse, it is universally appreciated that a particular cell type with unique characteristics, the myofibroblast, is responsible for replacement of functioning tissue with non-functional scar tissue. Understanding the cellular and molecular mechanisms responsible for the creation of myofibroblasts and their activities is central to the development of therapies. Critical signaling cascades, initiated primarily by TGF-ß, but also involving other cytokines which stimulate pro-fibrotic reactions in the myofibroblast, offer potential therapeutic targets. However, because of the multiplicity and complex interactions of these signaling pathways, it is very unlikely that any single drug will be successful in modifying a major fibrotic disease. Therefore, we have chosen to examine the effectiveness of administration of several drug combinations in a mouse pneumoconiosis model. Such treatment proved to be effective. Because fibrotic diseases that tend to be chronic, are difficult to monitor, and are patient variable, implementation of clinical trials is difficult and expensive. Therefore, we have made efforts to identify and validate non-invasive biomarkers found in urine and blood. We describe the potential utility of five such markers: (i) the EDA form of fibronectin (Fn(EDA)), (ii) lysyl oxidase (LOX), (iii) lysyl oxidase-like protein 2 (LoxL2), (iv) connective tissue growth factor (CTGF, CCNII), and (v) the N-terminal propeptide of type III procollagen (PIIINP).

Nitric oxide synthase: an endogenous source of elevated nitrite in infected urine.

To further define the endogenous sources of urine nitrite in urinary tract infections, we measured urinary nitrite levels by the Griess method and assayed urinary nitric oxide (NO) synthase activity by the conversion of 14C-arginine to 14C-citrulline. Endogenous production of 14C-citrulline was confirmed by thin layer chromatography. Exogenous L-arginine increased nitrite production in whole infected urine, but not in bacteria isolated from infected urine. Urinary tract infections significantly increased NO synthase activity in soluble urine fractions, although soluble activity was less than 10% of particulate activity. Urine particulate fractions from women with non-infected urine had greater NO synthase activity than particulate fractions from men with non-infected urine, 11 +/- 2 and 0.2 +/- 0.1 picomol/min/mg protein, respectively. Urinary tract infections increased NO synthase activity in urine particulate fractions from women and men, 99 +/- 20 and 48 +/- 9 picomol/min/mg protein, respectively. The conversion of 14C-arginine to 14C-citrulline required NADPH, was calcium independent, and was inhibited to a greater extent by L-canavanine than by NG-monomethyl-L-arginine or NG-nitro-L-arginine. Human infected urine contains an isoform of NO synthase which is an endogenous source of urine nitrite.

Rapid detection of urinary tract infection caused by Escherichia coli or Proteeae species.

Beta-glucuronidase and phenylalanine deaminase tests for screening urine specimens to provide rapid reporting (2 hours) of Escherichia coli and Proteeae species were evaluated. A total of 2,318 urine specimens were processed for these two tests. For the detection of Escherichia coli in urine, the sensitivity and specificity of the beta-glucuronidase test were 0.96 and 0.99 respectively; predictive positive and negative values were 0.97 and 0.99. For the detection of Proteeae in urine, the sensitivity and specificity of the phenylalanine test were 0.92 and 0.99. Predictive values were 0.99 for a negative test and 0.95 for a positive test. The data suggest that beta-glucuronidase and phenylalanine deaminase tests performed directly in urine sediment have potential usefulness as rapid and reliable tests that are easy to perform and interpret for the diagnosis of urinary tract infections caused by Escherichia coli or Proteeae species.