Inhibition of polymorphonuclear leukocyte adherence suppresses no-reflow after focal cerebral ischemia in baboons.

BACKGROUND AND PURPOSE: While polymorphonuclear leukocytes may contribute to the "no-reflow" phenomenon after focal cardiac and skeletal muscle ischemia/reperfusion, their contribution to acute focal cerebral ischemia is unresolved. We have examined the role of polymorphonuclear leukocytes in microvascular perfusion defects after focal cerebral ischemia/reperfusion in a baboon model of reversible middle cerebral artery occlusion with the anti-CD18 monoclonal antibody IB4, which inhibits neutrophil adherence to endothelium. METHODS: Microvascular patency in the basal ganglia after 3-hour middle cerebral artery occlusion and 1-hour reperfusion (by india ink tracer perfusion) was quantified by computerized video imaging. Animals were randomized to receive intravenous IB4 infusion 15 minutes before reperfusion (n = 7) or to receive no treatment (n = 6). Binding of IB4 to baboon leukocytes was maximal within 5 minutes of infusion. RESULTS: In the untreated group, a significant reduction in patency was observed in microvessels less than 30 microns diameter: mean percent reflow was 51% in the capillary diameter class (4.0-7.5 microns) and 39% in the precapillary arteriole and postcapillary venule diameter class (7.5-30 microns). Infusion of IB4 before middle cerebral artery reperfusion increased reflow in microvessels of all size classes, most significantly in those 7.5-30 microns (p = 0.049) and 30-50 microns (p = 0.034) in diameter. CONCLUSIONS: These results suggest that CD18-mediated polymorphonuclear leukocyte-endothelium adherence contributes to no-reflow predominantly in noncapillary microvessels and at least partially to that in capillaries.     

Metabolism and pharmacokinetics of p-(3,3-dimethyl-1-triazeno) benzoic acid in M5076 sarcoma-bearing mice

Summary The pharmacokinetics of the anticancer agent p-(3,3-dimethyl-1-triazeno) benzoic acid (pCOOH-DMT), a drug now in phase I clinical trial in Europe, was investigated in C57 Bl female mice with M5076 reticulum-cell sarcoma that were treated i.v. with 200 mg/kg pCOOH-DMT. The drug disappeared from plasma with a terminal half-life of about 2.5 h. Plasma clearance was approximately 6 ml/min per kg. Distribution studies showed some differences in drug levels in different tissues. The highest levels were found in the tumor, liver, kidney and lung; lower levels were found in the spleen and gut, and the lowest, in the brain. The N-desmethyl derivative of pCOOH-DMT was not detectable in plasma or tissues of mice treated with the drug. Therefore, the previous evidence of low N-demethylation of pCOOH-DMT was confirmed. pCOOH-DMT glucuronide was identified by mass spectrometry and quantified by high-performance liquid chromatography (HPLC) in plasma, tissues and urine samples. pCOOH-DMT glucuronide appears to be the major urinary metabolite of pCOOH-DMT in mice. Another metabolite identified by mass spectrometry and quantified by HPLC in some tissues and urine was pCOOH-DMT glycinate.Abbreviations DTIlC 5-(3,3-dimethyl-l-triazeno)imidazole-4-carboxamide - pCOOH-DMT p-(3,3-dimethyl-l-triazeno)benzoic acid - pCOOH-MMT p-(3-methyl-l-triazeno)benzoic acid - pCONH₂-DMT p-(3,3-dimethyl-l-triazeno)carboxamide - BSTFA N,O-bis(trimethylsilyl)trifluoroacetamide - TMCS trimethylchlorosilane - TLC thin-layer chromatography - FAB fast atom bombardment - EI electron impact - M5 M5076 reticulum-cell sarcoma - t_1/2 beta-half-life - C₀ concentration time 0 - AUC area under the concentration vs time curve - Cl total clearance - V volume of distribution     

Exploring and embracing complexity in a distance-learning curriculum for physicians.

The recent pressures on clinical medicine such as the attention to medical error and the challenges of interdisciplinary care have also exerted pressure on health professions education. Educators must now gauge how to redesign education systems to adapt quickly to these disruptions. Sometimes disruptions can be self-inflicted, such as the VA National Quality Scholars Fellowship's decision to use interactive video (IV) as its primary medium for delivering the curriculum to its six sites around the nation. The authors describe how this disruption to their education system helped to fashion a learning environment that is adaptable. Along the journey from a classroom-based curriculum to an IV-based curriculum, the authors and others involved in the program learned the basic tenets of IV sessions, redefined the roles of the teachers and learners, and discovered an IV environment that functions as a complex adaptive learning system. This distance-learning curriculum can be a model for other health professions education, since it starts with simple rules, changes from within, has a tolerance for unpredictability, and continually moves forward and transforms itself despite tension.     

Phorbol esters enhance the cyclic GMP response of T84 cells to the heat-stable enterotoxin of Escherichia coli (STa).

We examined the effect of protein kinase C (PKC) activation on the cyclic GMP response to heat-stable enterotoxin (STa) in a colonic carcinoma intestinal epithelial cell line, T84 cells. Our results demonstrate that the active phorbol ester analog, phorbol dibutyrate, but not the inactive alpha-phorbol dibutyrate, acts synergistically with STa to elevate cyclic GMP in intact T84 cells. The effect is observed in the absence or presence of the phosphodiesterase inhibitor, isobutylmethylxanthine, which suggests that phorbol dibutyrate modifies cyclic GMP synthesis rather than cyclic GMP degradation. In contrast to several systems in which prolonged treatment with phorbol ester desensitizes PKC-mediated responses, the cyclic GMP response in T84 cells is not diminished by prolonged treatment of T84 cells with phorbol dibutyrate. Also, transient treatment of T84 cells with phorbol dibutyrate enhances subsequent STa-stimulated cyclic GMP accumulation. These observations suggest that PKC activation produces a long-lived signal in T84 cells which enhances cyclic GMP accumulation in response to STa. Second messenger "cross talk" [T. Yoshimasa, D. R. Sibley, M. Bouvier, R. J. Lefkowitz, and M. G. Caron, Nature (London) 327:67-70, 1987] may be important in the pathogenesis of diarrheal disease.     

Diverged evolution of recent equine-2 influenza (H3N8) viruses in the Western Hemisphere

Summary.  We reported previously that equine-2 influenza A virus (H3N8) had evolved into two genetically and antigenically distinct “Eurasian” and “American” lineages. Phylogenetic analysis, using the HA1 gene of more recent American isolates, indicated a further divergence of these viruses into three evolution lineages: A South American lineage, a Kentucky lineage, and a Florida lineage. These multiple evolution pathways were not due to geographic barriers, as viruses from different lineages co-circulated. For the Kentucky lineage, the evolution rate was estimated to be 0.89 amino acid substitutions per year, which agreed with the previously estimated rate of 0.8. For the South American lineage, the evolution rate was estimated to be only 0.27 amino acid substitutions per year. This low evolution rate was probably due to a unique alternating Ser138 to Ala138 substitutions at antigenic site A. For the Kentucky lineage, there was a preference for sequential nonsynonymous substitutions at antigenic site B, which was also a “hot spot” for amino acid substitutions. Convalescent sera had minimal cross-reactivity to viruses of different lineages, indicating antigenic distinctions among these viruses. In contrast to human H3N2 viruses, our results suggested that the evolution of equine-2 influenza virus resembled the multiple evolution pathways of influenza B virus. Accepted December 14, 2000 Received September 19, 2000     

Scaffold protein harmonin (USH1C) provides molecular links between Usher syndrome type 1 and type 2

Usher syndrome (USH) is the most frequent cause of combined deaf-blindness in man. USH is clinically and genetically heterogeneous with at least 11 chromosomal loci assigned to the three USH types (USH1A-G, USH2A-C, USH3A). Although the different USH types exhibit almost the same phenotype in human, the identified USH genes encode for proteins which belong to very different protein classes and families. We and others recently reported that the scaffold protein harmonin (USH1C-gene product) integrates all identified USH1 molecules in a USH1-protein network. Here, we investigated the relationship between the USH2 molecules and this USH1-protein network. We show a molecular interaction between the scaffold protein harmonin (USH1C) and the USH2A protein, VLGR1 (USH2C) and the candidate for USH2B, NBC3. We pinpoint these interactions to interactions between the PDZ1 domain of harmonin and the PDZ-binding motifs at the C-termini of the USH2 proteins and NBC3. We demonstrate that USH2A, VLGR1 and NBC3 are co-expressed with the USH1-protein harmonin in the synaptic terminals of both retinal photoreceptors and inner ear hair cells. In hair cells, these USH proteins are also localized in the signal uptaking stereocilia. Our data indicate that the USH2 proteins and NBC3 are further partners in the supramolecular USH-protein network in the retina and inner ear which shed new light on the function of USH2 proteins and the entire USH-protein network. These findings provide first evidence for a molecular linkage between the pathophysiology in USH1 and USH2. The organization of USH molecules in a mutual 'interactome' related to the disease can explain the common phenotype in USH.     

Absence of proteinase-activated receptor-1 signaling affords protection from bleomycin-induced lung inflammation and fibrosis

Activation of the coagulation cascade is commonly observed in the lungs of patients with both acute and chronic inflammatory and fibrotic lung disorders, as well as in animal models of these disorders. The aim of this study was to examine the contribution of the major thrombin receptor, proteinase-activated receptor-1 (PAR-1), during the acute inflammatory and chronic fibrotic phases of lung injury induced by intratracheal instillation of bleomycin in mice. Inflammatory cell recruitment and increases in bronchoalveolar lavage fluid (BALF) protein were attenuated by 56 +/- 10% (P < 0.05) and 53 +/- 12% (P < 0.05), respectively, in PAR-1-deficient (PAR-1-/-) mice compared with wild-type (WT) mice. PAR-1-/- mice were also protected from bleomycin-induced pulmonary fibrosis with total lung collagen accumulation reduced by 59 +/- 5% (P < 0.05). The protection afforded by PAR-1 deficiency was accompanied by significant reductions in pulmonary levels of the potent PAR-1-inducible proinflammatory and profibrotic mediators, monocyte chemoattractant protein-1 (MCP-1), transforming growth factor-beta-1 (TGF-beta1), and connective tissue growth factor/fibroblast-inducible secreted protein-12 (CTGF/FISP12). In addition, PAR-1 was highly expressed in inflammatory and fibroproliferative lesions in lung sections obtained from patients with fibrotic lung disease. These data show for the first time that PAR-1 signaling plays a key role in experimentally induced lung injury, and they further identify PAR-1 as one of the critical receptors involved in orchestrating the interplay between coagulation, inflammation, and remodeling in response to tissue injury.