In situ hybridization for the detection and localization of swine Chlamydia trachomatis.
Gnotobiotic piglets were inoculated intralaryngeally with swine Chlamydia trachomatis strain R33 or orally with swine C. trachmatis strain R27. Archived formalin-fixed, paraffin-embedded tissues from piglets euthanatized 4-7 days postinoculation were examined by in situ hybridization for C. trachomatis nucleic acid using a nonradioactive digoxigenin-labeled DNA probes that targeted specific ribosomal RNA or omp1 mRNA molecules of the swine C. trachomatis strains. Positive hybridization signals were detected in bronchial epithelial cells, bronchiolar epithelial cells, pneumocytes, alveolar and interstitial macrophages, and jejunal and ileal enterocytes. Chlamydia-infected cells had a strong signal that was confined to the intracytoplasmic inclusions. Positive hybridization signals were not detected in tissue sections from an uninfected control piglet or in C. psittaci-infected sheep placenta. The morphology of host cells was preserved despite the relatively high temperature required in parts of the incubation procedure. The data indicate that in situ hybridization can be used to detect swine C. trachomatis in formalin-fixed, paraffin-embedded tissue specimens. (+info)
Analysis of hepatitis G virus (HGV) RNA, antibody to HGV envelope protein, and risk factors for blood donors coinfected with HGV and hepatitis C virus.
Serologic, biochemical, and molecular analyses were used to study hepatitis G virus (HGV), antibody to the HGV envelope protein (anti-E2), risk factors, clinical significance, and the impact of HGV on coexistent hepatitis C virus (HCV). Among 329 donors with confirmed HCV infection, 12% were HGV RNA-positive and 44% were anti-E2-positive (total exposure, 56%). HGV RNA and anti-E2 were mutually exclusive except in 9 donors (1.5%); 8 of 9 subsequently lost HGV RNA but anti-E2 persisted. HGV had little impact on alanine aminotransferase, aspartate aminotransferase, or gamma-glutamyl transpeptidase in donors with HGV infection alone or those coinfected with HCV. A multivariate analysis showed that intravenous drug abuse was the leading risk factor for HGV transmission, followed by blood transfusion, snorting cocaine, imprisonment, and a history of sexually transmitted diseases. In summary, HGV and HCV infections were frequently associated and shared common parenteral risk factors; HGV did not appear to cause hepatitis or to worsen the course of coexistent hepatitis C. (+info)
In vitro scanning saturation mutagenesis of all the specificity determining residues in an antibody binding site.
For the first time, each specificity determining residue (SDR) in the binding site of an antibody has been replaced with every other possible single amino acid substitution, and the resulting mutants analyzed for binding affinity and specificity. The studies were conducted on a variant of the 26-10 antidigoxin single chain Fv (scFv) using in vitro scanning saturation mutagenesis, a new process that allows the high throughput production and characterization of antibody mutants [Burks,E.A., Chen,G., Georgiou,G. and Iverson,B.L. (1997) Proc. Natl Acad. Sci. USA, 94, 412-417]. Single amino acid mutants of 26-10 scFv were identified that modulated specificity in dramatic fashion. The overall plasticity of the antibody binding site with respect to amino acid replacement was also evaluated, revealing that 86% of all mutants retained measurable binding activity. Finally, by analyzing the physical properties of amino acid substitutions with respect to their effect on hapten binding, conclusions were drawn regarding the functional role played by the wild-type residue at each SDR position. The reported results highlight the value of in vitro scanning saturation mutagenesis for engineering antibody binding specificity, for evaluating the plasticity of proteins, and for comprehensive structure-function studies and analysis. (+info)
Identification by PCR of Helicobacter pylori in subgingival plaque of adult periodontitis patients.
The PCR was used to detect the presence of Helicobacter pylori in subgingival plaque samples from patients with adult periodontitis. Primers based upon the 16S ribosomal RNA (rRNA) gene sequence of H. pylori were used in a single round of PCR to amplify a 295-bp DNA fragment and the identity of the amplified products was confirmed by Southern blot hybridisation to a digoxigenin-labelled H. pylori probe. Further confirmation of product identity was obtained by DNA sequencing of a proportion of the amplified products. The assay was demonstrated to be specific for H. pylori with a lower limit of detection of 100 fg of bacterial genomic DNA. In all, 73 samples from 29 patients were analysed, of which 24 (33%) were H. pylori-positive by PCR; the proportion of patients carrying H. pylori in at least one sampled site was 38% (11 of 29). This is the first study to demonstrate the presence of H. pylori in the subgingival plaque of patients with adult periodontitis and indicates that, in this patient group at least, subgingival plaque may be a reservoir for H. pylori infection. (+info)
Evidence for ovarian granulosa stem cells: telomerase activity and localization of the telomerase ribonucleic acid component in bovine ovarian follicles.
We have previously postulated that granulosa cells of developing follicles arise from a population of stem cells. Stem cells and cancer cells can divide indefinitely partly because they express telomerase. Telomerase is a ribonucleoprotein enzyme that repairs the ends of telomeres that otherwise shorten progressively upon each successive cell division. In this study we carried out cell cycle analyses and examined telomerase expression to examine our hypothesis. Preantral (60-100 microm) and small (1 mm) follicles, as well as granulosa cells from medium-sized (3 mm) and large (6-8 mm) follicles, were isolated. Cell cycle analyses and expression of Ki-67, a cell cycle-related protein, were undertaken on follicles of each size (n = 3) by flow cytometry; 12% to 16% of granulosa cells in all follicles were in the S phase, and less than 2% were in the G(2)/M phase. Telomerase activity (n = 3) was highest in the small preantral follicles, declining at the 1-mm stage and even further at the 3-mm stage. In situ hybridization histochemistry was carried out on bovine ovaries, and telomerase RNA was detected in the granulosa cells of growing follicles but not primordial follicles. Two major patterns of staining were observed in the membrana granulosa of antral follicles: staining in the middle and antral layers, and staining in the middle and basal layers. No staining was detected in oocytes. Our results strongly support our hypothesis that granulosa cells arise from a population of stem cells. (+info)
Detection and localization of Mycoplasma hyopneumoniae DNA in lungs from naturally infected pigs by in situ hybridization using a digoxigenin-labeled probe.
Mycoplasma hyopneumoniae DNA was detected in 20 naturally infected pigs by in situ hybridization using a nonradioactive digoxigenin-labeled DNA probe. A 520-base-pair DNA probe targeting a reiterative sequence of the M. hyopneumoniae genome was generated by the polymerase chain reaction. All 20 pigs infected with M. hyopneumoniae had distinct and positive hybridization signals without background staining. A strong hybridization signal was detected mainly in the luminal surface of bronchial and bronchiolar lining epithelial cells, whereas no hybridization signal was seen in the cytoplasm of bronchial and bronchiolar lining epithelial cells. When hybridization signal was detected in the luminal surface of bronchial and bronchiolar lining epithelial cells, a given bronchus or bronchiole had peribronchiolar lymphoid hyperplastic tissues. Hybridization signals were not seen in the peribronchiolar lymphoid hyperplastic tissues. A less intense signal was detected in the interstitial and alveolar macrophages randomly scattered in the thickened alveolar septa and spaces. Hybridization signal was rarely detected in the type I pneumocytes. The in situ hybridization technique developed in this study was useful for detection of M. hyopneumoniae nucleic acids in tissues taken from naturally infected piglets and may be a valuable technique for studying the pathogenesis of M. hyopneumoniae infection. (+info)
Nonisotopic detection of Loma salmonae (microspora) in rainbow trout (Oncorhynchus mykiss) gills by in situ hybridization.
Loma salmonae, a microsporidian parasite of salmonids of the genus Oncorhynchus, is a significant cause of economic loss in pen-reared chinook salmon (O. tschawytscha). Final stages of L. salmonae infections are easily recognized by the xenomas that form in the gills during sporogony. However, early prexenoma stages of infection (3 weeks or less after infection) are difficult to detect on histologic slides. An L. salmonae-specific single-stranded DNA probe labeled with digoxigenin was used to detect these prexenoma stages of L salmonae by in situ hybridization in experimentally infected rainbow trout. This method allows detection of the parasite in the gills only 2 weeks after infection, providing a sensitive and specific way of detecting L. salmonae during the early stages of infection. (+info)
In situ distribution of hepatitis C virus replicative-intermediate RNA in hepatic tissue and its correlation with liver disease.
Liver failure from chronic hepatitis C is the leading indication for liver transplantation in the United States. However, the pathogenesis of liver injury resulting from chronic hepatitis C virus (HCV) infection is not well understood. To examine the relationship between HCV replication in liver tissue and hepatocellular injury, a strand-specific in situ hybridization procedure was developed. The sensitivity and specificity of digoxigenin-labeled riboprobes were optimized by analyzing Northern blots and cell lines expressing HCV RNAs. For the current study, both genomic (sense) and replicative-intermediate (antisense) HCV RNAs were detected and quantified in 8 of 8 liver tissue specimens from infected patients versus 0 of 11 liver tissue specimens from noninfected controls. The distribution pattern for HCV replicative-intermediate RNA in liver was different from that for HCV genomic RNA. HCV genomic RNA was variably distributed throughout infected livers and was located primarily in the cytoplasm of hepatocytes, with some signal in fibroblasts and/or macrophages in the surrounding fibroconnective tissue. However, HCV replicative-intermediate RNA showed a more focal pattern of distribution and was exclusively localized in the cytoplasm of hepatocytes. There was no significant relationship between the distribution pattern for HCV genomic RNA and any indices of hepatocellular injury. However, a highly significant correlation was observed between the percentage of cells staining positive for replicative-intermediate RNA and the degree of hepatic inflammatory activity (P, < 0.0001). Furthermore, the ratio of cells staining positive for HCV replicative-intermediate versus genomic RNA correlated with the histological severity of liver injury (P, 0. 0065), supporting the hypothesis that active replication of HCV in liver tissue may be a significant determinant of hepatocellular injury. (+info)