Neutralization of gamma interferon and tumor necrosis factor alpha blocks in vivo synthesis of nitrogen oxides from L-arginine and protection against Francisella tularensis infection in Mycobacterium bovis BCG-treated mice.

Peritoneal cells from Mycobacterium bovis BCG-infected C3H/HeN mice produced nitrite (NO2-, an oxidative end product of nitric oxide [NO] synthesis) and inhibited the growth of Francisella tularensis, a facultative intracellular bacterium. Both NO2- production and inhibition of bacterial growth were suppressed by NG-monomethyl-L-arginine, a substrate inhibitor of nitrogen oxidation of L-arginine, and monoclonal antibodies (MAbs) to gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). Intraperitoneal injection of mice with BCG increased urinary nitrate (NO3-) excretion coincident with development of activated macrophages capable of secreting nitrogen oxides and inhibiting F. tularensis growth in vitro. Eight days after BCG inoculation, mice survived a normally lethal intraperitoneal challenge with F. tularensis. Treatment of these BCG-infected mice with MAbs to IFN-gamma or TNF-alpha at the time of BCG inoculation reduced urinary NO3- levels to those found in normal uninfected mice for up to 14 days. The same anticytokine antibody treatment abolished BCG-mediated protection against F. tularensis: mice died within 4 to 6 days. Intraperitoneal administration of anti-IFN-gamma or anti-TNF-alpha antibody 8 days after BCG infection also reduced urinary NO3- and abolished protection against F. tularensis. Isotype control (immunoglobulin G) or anti-interleukin 4 MAbs had little effect on these parameters at any time of treatment. IFN-gamma and TNF-alpha were clearly involved in the regulation of macrophage activation by BCG in vivo. Protection against F. tularensis challenge by BCG depended upon the physiological generation of reactive nitrogen oxides induced by these cytokines.     

Pyrazinamide-monoresistant Mycobacterium tuberculosis in the United States

Mycobacterium bovis is naturally resistant to the antituberculosis drug pyrazinamide (PZA). To determine whether all Mycobacterium tuberculosis complex isolates demonstrating PZA monoresistance were truly M. bovis, we examined the phenotype and genotype of isolates reported as PZA monoresistant in five counties in California from January 1996 through June 1999. Isolates reported by local laboratories to be PZA monoresistant were sent to the state reference laboratory for repeat susceptibility testing using the BACTEC radiometric method and to the Centers for Disease Control and Prevention for pncA sequencing and PCR-restriction fragment length polymorphism (RFLP) analysis of the oxyR gene. Of 1,916 isolates, 14 were reported as PZA monoresistant and 11 were available for retesting. On repeat testing, 6 of the 11 isolates were identified as PZA-susceptible M. tuberculosis, 1 was identified as PZA-monoresistant M. bovis, and 1 was identified as M. bovis BCG. The three remaining isolates were identified as PZA-monoresistant M. tuberculosis. Sequencing of the pncA and oxyR genes genotypically confirmed the two M. bovis and the six susceptible M. tuberculosis species. Each of the three PZA-monoresistant M. tuberculosis isolates had different, previously unreported, pncA gene mutations: a 24-bp deletion in frame after codon 88, a base substitution at codon 104 (Ser104Cys), and a base substitution at codon 90 (Ile90Ser). This study demonstrates that PZA monoresistance is not an absolute marker of M. bovis species but may also occur in M. tuberculosis, associated with a number of different mutational events in the pncA gene. It is the first report of PZA-monoresistant M. tuberculosis in the United States.     

Association of ulcerative colitis with the inflammatory bowel disease susceptibility locus IBD2 in non-Jewish Caucasians and evidence of genetic heterogeneity among racial and ethnic populations with Crohn disease

Genomewide scanning has been used to identify chromosomal regions encoding susceptibility loci to inflammatory bowel disease (IBD). The greatest evidence for linkage to IBD has been reported for a region of chromosome 12q14 surrounding the microsatellite marker D12S83, with a logarithm of odds score of 5.47 and a positive transmission disequilibrium test, and which was subsequently named IBD2. We wished to confirm this locus by genotyping the highly polymorphic microsatellites D12S1022, D12S1056, and D12S83, spanning a continuous region on chromosome 12 of 342 kb, in a cohort of nonrelated individuals with ulcerative colitis (89 patients), Crohn disease (121 patients), and population-based control subjects (100 patients). In non-Jewish Caucasians, one D12S1022 allele, one D12S1056 genotype, and three D12S83 alleles were found to have statistically significant differences in distribution between the two disease groups and the control population. These data support a significant association of IBD with the IBD2 locus in close vicinity to the three markers studied. The replication of genetic risk loci in a case control association study may indicate susceptibility genes in this region and may facilitate identification of candidate genes for IBD. Subgroup analysis revealed a notable difference in genotype distribution among Jewish Caucasian and African American patients affected with Crohn disease when compared with similarly affected non-Jewish Caucasians. Using Fisher exact test, statistically significant distribution differences were observed for D12S1022 and D12S83. These data indicate that there may be significant genetic heterogeneity between different ethnic and racial IBD populations or may simply reflect differences in marker allele frequencies among populations.     

Incomplete rescue of cystic fibrosis transmembrane conductance regulator deficient mice by the human CFTR cDNA

We have used a mouse model to study the ability of human CFTR to correct the defect in mice deficient of the endogenous protein. In this model, expression of the endogenous Cftr gene was disrupted and replaced with a human CFTR cDNA by a gene targeted 'knock-in' event. Animals homozygous for the gene replacement failed to show neither improved intestinal pathology nor survival when compared to mice completely lacking CFTR. RNA analyses showed that the human CFTR sequence was transcribed from the targeted allele in the respiratory and intestinal epithelial cells. Furthermore, in vivo potential difference measurements showed that basal CFTR chloride channel activity was present in the apical membranes of both nasal and rectal epithelial cells in all homozygous knock-in animals examined. Ussing chamber studies showed, however, that the cAMP-mediated chloride channel function was impaired in the intestinal tract among the majority of homozygous knock-in animals. Hence, failure to correct the intestinal pathology associated with loss of endogenous CFTR was related to inefficient functional expression of the human protein in mice. These results emphasize the need to understand the tissue- specific expression and regulation of CFTR function when animal models are used in gene therapy studies.      

Ageing is associated with a decrease in the number of sarcolemmal ATP-sensitive K+ channels in a gender-dependent manner

The opening of sarcolemmal K(ATP) channels is considered to be an important endogenous cardioprotective mechanism. On the other hand, age-dependent changes in the myocardial susceptibility to ischemia and hypoxia have been observed in different species, including humans. Here, we have hypothesized that aging might be associated with the changes in sarcolemmal K(ATP) channels. Therefore, the main objective of the present study was to establish whether aging changes expression of cardiac sarcolemmal ATP-sensitive K+ (K(ATP)) channels. RT-PCR using primers specific for K(ATP) channel subunits, Kir6.2, Kir6.1 and SUR2A subunits was performed using total RNA from guinea-pig ventricular tissue. Whole cell electrophysiology was done on isolated guinea-pig ventricular cardiomyocytes. Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was performed on cardiac membrane fraction. Tissue and cells were harvested from young and old, male and female guinea-pigs. RT-PCR analysis did not reveal significant age-related changes in levels of Kir6.1 or Kir6.2 mRNAs. However, levels of SUR2A were significantly lower in old than in young females. Such age-differences were not observed with cardiac tissue from male animals. In both old and young males, pinacidil (100 microM) induced outward currents. The difference between current density of pinacidil-sensitive component in females, but not males, was statistically significant. Western blotting analysis revealed higher levels of Kir6.2 and SUR2A proteins in cardiac membrane fraction from young than old females. The present study demonstrates that in females, but not males, aging is associated with decrease in number of cardiac K(ATP) channels which is due to decrease in levels of the SUR2A subunit.     

Utilization of acidophil bodies in the diagnosis of recurrent hepatitis C infection after orthotopic liver transplantation.

BACKGROUND: The distinction between acute rejection and early recurrent hepatitis C infection (RHCV) in the setting of orthotopic liver transplantation is often difficult. In liver biopsies acidophil bodies and lobular hepatitis are used to suggest a diagnosis of RHCV over rejection, however, the reliability of this practice has not been established. Because portal tract changes in RHCV and rejection often overlap, we sought to determine whether the degree of hepatocyte acidophil body formation seen on liver biopsies could be used to distinguish between these two conditions. METHODS: Quantification of acidophil bodies was performed on liver biopsies in orthotopic liver transplant patients with RHCV (n = 10), non-hepatitis C orthotopic liver transplant patients with uncomplicated rejection episodes (n = 10) and non-transplant patients with chronic hepatitis C infection (n = 10). Hematoxylin and Eosin stained slides from all three groups were randomized and tissue segments 1.0 cm in length and of variable width (0.04-0.13 cm) were examined at 200x magnification in a blinded fashion by two pathologists in order to quantify the number of acidophil bodies/cm(2). Lobular chronic inflammation was also graded on a 0-3+ scale. RESULTS: Liver biopsies taken at the onset of RHCV exhibited 606 +/- 101 acidophil bodies/cm(2) (mean +/- standard error of mean, range 200-1390). These counts were significantly greater (P =.0061, paired 2-tailed t-test) than the 241 +/- 53 acidophil bodies/cm(2) (range 80-514) for acute rejection, and the 194 +/- 21 acidophil bodies/cm(2) (range 100-333) for non-liver transplant chronic hepatitis C infection (P =.0013). No difference in lobular inflammation between index RHCV and rejection biopsies was detected. CONCLUSIONS: Although there is overlap, on average there are twice as many acidophil bodies in the initial stage of RHCV when compared with acute rejection (average of 55 per linear cm in RHCV versus 21 per linear cm for rejection). Lobular inflammation was not a reliable indicator of the initial onset of RHCV.     

Human MSH2 binds to trinucleotide repeat DNA structures associated with neurodegenerative diseases

The expansion of trinucleotide repeat sequences is associated with several neurodegenerative diseases. The mechanism of this expansion is unknown but may involve slipped-strand structures where adjacent rather than perfect complementary sequences of a trinucleotide repeat become paired. Here, we have studied the interaction of the human mismatch repair protein MSH2 with slipped-strand structures formed from a triplet repeat sequence in order to address the possible role of MSH2 in trinucleotide expansion. Genomic clones of the myotonic dystrophy locus containing disease-relevant lengths of (CTG)n x (CAG)n triplet repeats were examined. We have constructed two types of slipped-strand structures by annealing complementary strands of DNA containing: (i) equal numbers of trinucleotide repeats (homoduplex slipped structures or S-DNA) or (ii) different numbers of repeats (heteroduplex slipped intermediates or SI-DNA). SI-DNAs having an excess of either CTG or CAG repeats were structurally distinct and could be separated electrophoretically and studied individually. Using a band-shift assay, the MSH2 was shown to bind to both S-DNA and SI-DNA in a structure- specific manner. The affinity of MSH2 increased with the length of the repeat sequence. Furthermore, MSH2 bound preferentially to looped-out CAG repeat sequences, implicating a strand asymmetry in MSH2 recognition. Our results are consistent with the idea that MSH2 may participate in trinucleotide repeat expansion via its role in repair and/or recombination.      

Megalencephaly in the epileptic chicken: a morphometric study of the adult brain

The epileptic chicken is a genetic model of generalized epilepsy in which epilepsy is combined with megalencephaly. We have performed a morphometric study of the brains of adult epileptic hens, using heterozygous carrier hens as controls. There is no obvious disorder of cell form or of architectural arrangement in the megalencephalic brains. We have found that the enlargement of the epileptic brain is not uniform: it is most marked in the telencephalon, and is present to a lesser degree in the cerebellum, but neither the optic tectum nor the diencephalic nucleus rotundus shows a significant increase in size. The enlarged regions are characterized by a decrease in the packing density of neurons. There is an increase in the total neuron population in some of the enlarged areas (archistriatum), despite the lower density per unit volume, but in other enlarged areas (hippocampus) there is no difference in total neuron numbers. The glial cells, by contrast, show no significant alteration in packing density. These findings suggest that the megalencephaly of the epileptic chicken is due to an increase in neuron size, with a contribution from increased numbers of neurons and glial cells. The epileptic chicken may provide a valuable model for further dynamic studies of aberrant neuronal development, and of structural-functional relationships in epilepsy.