Preclinical Assessment of Adjunctive tPA and DNase for Peritoneal Dialysis Associated Peritonitis

A major complication of peritoneal dialysis is the development of peritonitis, which is associated with reduced technique and patient survival. The inflammatory response elicited by infection results in a fibrin and debris-rich environment within the peritoneal cavity, which may reduce the effectiveness of antimicrobial agents and predispose to recurrence or relapse of infection. Strategies to enhance responses to antimicrobial agents therefore have the potential to improve patient outcomes. This study presents pre-clinical data describing the compatibility of tPA and DNase in combination with antimicrobial agents used for the treatment of PD peritonitis. tPA and DNase were stable in standard dialysate solution and in the presence of antimicrobial agents, and were safe when given intraperitoneally in a mouse model with no evidence of local or systemic toxicity. Adjunctive tPA and DNase may have a role in the management of patients presenting with PD peritonitis.     

Characterization of Dahl salt-sensitive rats with genetic disruption of the A2B adenosine receptor gene: implications for A2B adenosine receptor signaling during hypertension

The A_2B adenosine receptor (AR) has emerged as a unique member of the AR family with contrasting roles during acute and chronic disease states. We utilized zinc-finger nuclease technology to create A_2BAR gene (Adora2b)-disrupted rats on the Dahl salt-sensitive (SS) genetic background. This strategy yielded a rat strain (SS-Adora2b mutant rats) with a 162-base pair in-frame deletion of Adora2b that included the start codon. Disruption of A_2BAR function in SS-Adora2b mutant rats was confirmed by loss of agonist (BAY 60-6583 or NECA)-induced cAMP accumulation and loss of interleukin-6 release from isolated fibroblasts. In addition, BAY 60-6583 produced a dose-dependent increase in glucose mobilization that was absent in SS-Adora2b mutants. Upon initial characterization, SS-Adora2b mutant rats were found to exhibit increased body weight, a transient delay in glucose clearance, and reduced proinflammatory cytokine production following challenge with lipopolysaccharide (LPS). In addition, blood pressure was elevated to a greater extent (∼15–20 mmHg) in SS-Adora2b mutants as they aged from 7 to 21 weeks. In contrast, hypertension augmented by Ang II infusion was attenuated in SS-Adora2b mutant rats. Despite differences in blood pressure, indices of renal and cardiac injury were similar in SS-Adora2b mutants during Ang II-augmented hypertension. We have successfully created and validated a new animal model that will be valuable for investigating the biology of the A_2BAR. Our data indicate varying roles for A_2BAR signaling in regulating blood pressure in SS rats, playing both anti- and prohypertensive roles depending on the pathogenic mechanisms that contribute to blood pressure elevation.     

MiR-101 and miR-144 Regulate the Expression of the CFTR Chloride Channel in the Lung

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that plays a critical role in the lung by maintaining fluid homeostasis. Absence or malfunction of CFTR leads to Cystic Fibrosis, a disease characterized by chronic infection and inflammation. We recently reported that air pollutants such as cigarette smoke and cadmium negatively regulate the expression of CFTR by affecting several steps in the biogenesis of CFTR protein. MicroRNAs (miRNAs) have recently received a great deal of attention as both biomarkers and therapeutics due to their ability to regulate multiple genes. Here, we show that cigarette smoke and cadmium up-regulate the expression of two miRNAs (miR-101 and miR-144) that are predicted to target CFTR in human bronchial epithelial cells. When premature miR-101 and miR-144 were transfected in human airway epithelial cells, they directly targeted the CFTR 3′UTR and suppressed the expression of the CFTR protein. Since miR-101 was highly up-regulated by cigarette smoke in vitro, we investigated whether such increase also occurred in vivo. Mice exposed to cigarette smoke for 4 weeks demonstrated an up-regulation of miR-101 and suppression of CFTR protein in their lungs. Finally, we show that miR-101 is highly expressed in lung samples from patients with severe chronic obstructive pulmonary disease (COPD) when compared to control patients. Taken together, these results suggest that chronic cigarette smoking up-regulates miR-101 and that this miRNA could contribute to suppression of CFTR in the lungs of COPD patients.     

Polypyrimidine Tract Binding Protein Prevents Activity of an Intronic Regulatory Element That Promotes Usage of a Composite 3��-Terminal Exon

Alternative splicing of 3′-terminal exons plays a critical role in gene expression by producing mRNA with distinct 3′-untranslated regions that regulate their fate and their expression. The Xenopus α-tropomyosin pre-mRNA possesses a composite internal/3′-terminal exon (exon 9A9′) that is differentially processed depending on the embryonic tissue. Exon 9A9′ is repressed in non-muscle tissue by the polypyrimidine tract binding protein, whereas it is selected as a 3′-terminal or internal exon in myotomal cells and adult striated muscles, respectively. We report here the identification of an intronic regulatory element, designated the upstream terminal exon enhancer (UTE), that is required for the specific usage of exon 9A9′ as a 3′-terminal exon in the myotome. We demonstrate that polypyrimidine tract binding protein prevents the activity of UTE in non-muscle cells, whereas a subclass of serine/arginine rich (SR) proteins promotes the selection of exon 9A9′ in a UTE-dependent way. Morpholino-targeted blocking of UTE in the embryo strongly reduced the inclusion of exon 9A9′ as a 3′-terminal exon in the endogenous mRNA, demonstrating the function of UTE under physiological circumstances. This strategy allowed us to reveal a splicing pathway that generates a mRNA with no in frame stop codon and whose steady-state level is translation-dependent. This result suggests that a non-stop decay mechanism participates in the strict control of the 3′-end processing of the α-tropomyosin pre-mRNA.     

Immunological Studies in Ugandan Patients with Hepatocellular Carcinoma

Immunological studies were performed on Ugandan patients with hepatocellular carcinoma to test the hypothesis that the high rate of persistence of hepatitis-associated antigen in these patients is the result of defects in host immune response. The responses to 1-chloro-2,4-dinitrobenzene sensitization and to a battery of recall skin test antigens were normal, as was the humoral antibody response to tularaemia antigen. Neither hypogammaglobulinaemia nor specific immunoglobulin deficiencies were found. Thus it appears unlikely that generalized defects in host immune responses can account for the high incidence of persistent hepatitis B virus infection found in Ugandan patients with hepatocellular carcinoma.