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L K Riley

Publications and source records attributed to L K Riley.

At least 73 records · Page 4Linked to original sources
PubMed1994

Development of a monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay for detection of Bacillus piliformis isolate-specific antibodies in laboratory animals.

A competitive inhibition enzyme-linked immunosorbent assay (ELISA) was developed to detect Bacillus piliformis isolate-specific antibodies in serum specimens from rats and gerbils experimentally infected with B. piliformis isolates R1, R2, or M. Detection was based on the ability of serum antibodies to block binding of B. piliformis isolate-specific monoclonal antibodies to purified B. piliformis flagella. Application of this assay to serum specimens collected from sham-infected or experimentally infected rats and gerbils demonstrated that the serum specimens were capable of specifically inhibiting the binding of B. piliformis isolate-specific monoclonal antibodies to homologous flagella preparations (> 70% inhibition) only when the serum specimens were from animals infected with the homologous B. piliformis isolate. Only one false-negative and false-positive result were obtained when 80 serum specimens were tested by this competitive inhibition ELISA. In addition, we demonstrated that little nonspecific inhibition of monoclonal antibody binding occurred (< 30% inhibition) in this immunoassay specific inhibition of monoclonal antibody binding by serum was due to serum antibody and a serum's ability to inhibit binding of monoclonal antibodies to purified B. piliformis flagella was correlated with antibody reactivity with B. piliformis flagella but not with serum antibody reactivity to whole B. piliformis organisms. These results suggest that this monoclonal antibody-based competitive inhibition assay could be successfully applied to the serologic identification of isolates involved in naturally occurring B. piliformis infections in laboratory animals.

Animals↗
PubMed1994

Experimentally induced intravaginal Tritrichomonas foetus infection in the estrogenized mouse.

Studies were initiated to establish and maintain intravaginal Tritrichomonas foetus infections in female BALB/c mice as a model for elucidation of parasite and host factors that affect the course of vaginal protozoan infections. Results of these studies indicated that T. foetus infections could only be established in mice in which estrus was induced and maintained. Over a period of several weeks, mice induced to estrus by weekly administration of estradiol cypionate exhibited purulent vaginal discharge and perivulvar abscesses. Implantation of silastic tubing containing 15 micrograms of estradiol-17 beta proved effective in induction and maintenance of estrus and avoided the animal health problems associated with estradiol cypionate treatment. Results of quantitative experiments indicated that the duration of trichomonad infection was influenced by initial colonization of the vagina, i.e., mice with high numbers of vaginal trichomonads at 7 days after infection maintained infections longer than did mice with lower numbers of vaginal parasites. Weekly administration of either 2 or 4 mg of methylprednisolone acetate to estrogenized mice did not extend the duration of T. foetus infections, thereby suggesting that the immune response did not limit the establishment and maintenance of primary vaginal trichomonad infections. Study of estrogenized BALB/c nu/nu mice supported these observations in that establishment of T. foetus infections was difficult in nu/nu mice and that, in most nu/nu mice (76%), the course of infection was not lengthened (mean, 1.9 weeks). When examined by electron microscopy, the earliest lesions were characterized by degeneration and necrosis of chondrocytes, along with degradation of cartilage matrix. These findings confirm that quinolone arthropathy develops in juvenile rabbits and is similar to quinolone arthropathy in other laboratory animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Tyzzer's infection: host specificity of Clostridium piliforme isolates.

Tyzzer's disease, a well-recognized syndrome in numerous laboratory animal species, is caused by the obligate intracellular bacterium, Clostridium piliforme. Distinct isolates of C. piliforme from various laboratory animal species have been identified based on protein and antigenic heterogeneity. The goal of this study was to examine the host specificity of three well-characterized isolates of C. piliforme. Groups of mice, rats, and hamsters were experimentally infected with isolates obtained from a naturally infected mouse (M1), a naturally infected rat (R1), and a naturally infected hamster (H2). To assess infection status, animals were monitored serologically for antibody to C. piliforme over a 12-week period. Evaluation of results indicated that the M1 isolate infected rats and mice but not hamsters, whereas the R1 and H2 isolates infected only the host species from which the isolates were originally obtained. These findings suggest that C. piliforme isolates can be categorized into two types: 1) cross-infective isolates, such as M1, which can infect more than one laboratory animal species, and 2) isolates, such as R1 and H2, which have a more limited host range within laboratory animal species. These results emphasize the need to consider the host specificity of C. piliforme isolates when investigating outbreaks of Tyzzer's disease.

Animals↗
PubMed1994

Characterization of cilia-associated respiratory bacillus isolates from rats and rabbits.

Isolates of the cilia-associated respiratory (CAR) bacillus were harvested from the trachea of three naturally infected rats and five naturally infected rabbits and were grown on 3T3 mouse fibroblast cells. Isolates were compared by growth characteristics in mammalian cell culture, by use of protein and DNA analyses, and by experimental infections of BALB/c mice. Examination of CAR bacillus isolates by transmission electron microscopy indicated that organisms from rats and rabbits were similar in appearance and had an acidic mucopolysaccharide layer. In culture, the isolates from rats appeared larger than the rabbit isolates and formed large multiorganism aggregates, whereas isolates obtained from rabbits did not. Protein and antigenic analyses and DNA ribotyping revealed minor differences between isolates but could not be used to distinguish the rat from the rabbit isolates. Mice experimentally inoculated with CAR bacillus of rat origin developed interciliary colonization, seroconverted, and developed microscopic pulmonary lesions. Mice inoculated with isolates of rabbit origin did not display intraciliary colonization, seroconvert, or develop pulmonary disease. The findings of this study indicate that CAR bacillus isolates of rat and rabbit origins may be distinct strains and suggest that, in mice, isolates of rat origin may be more virulent than those of rabbit origin.

3T3 Cells↗
PubMed1993

In vitro proliferation of a canine granulocytic Ehrlichia.

Canine granulocytic Ehrlichia sp., an agent which parasitizes the neutrophilic leukocytes in dogs, was transiently propagated in vitro. Dogs were experimentally inoculated with blood containing canine granulocytic Ehrlichia. Bacteremias in experimentally infected dogs varied from 1.2 to 9.3% granulocytes infected. Granulocytes from experimentally infected dogs were harvested and cultured in the presence of RPMI 1640 medium supplemented with fetal bovine serum, conditioned medium, and HEPES buffer. The percentages of granulocytes containing ehrlichial morulae increased significantly with time for 2 to 4 days, with at least one culture from each dog achieving 20% of granulocytes infected. Granulocytes taken from infected dogs early in bacteremia yielded cultures with the greatest percentage of infected cells. By 5 days post-infection the percentage of infected granulocytes decreased as did leukocyte viability. Attempts to maintain the in vitro cultures for prolonged periods by addition of uninfected granulocytes failed to increase the number of infected host cells, suggesting that no new infections were initiated and that observed increases in the percentage of infected cells in in vitro cultures were due to growth of the organism in granulocytes that were infected in vivo.

Animals↗
PubMed1993

Antigenic diversity in flagellar epitopes among Bacillus piliformis isolates.

Monoclonal antibodies (MAbs) were developed to Bacillus piliformis isolate-specific flagellar epitopes and used to group B. piliformis isolates on the basis of epitope expression. BALB/c mice immunised with flagella purified from various B. piliformis isolates served as the source of immune spleen cells for fusion with SP2/0Ag14 myeloma cells. Evaluation of hybridoma culture medium by ELISA against various bacterial species and B. piliformis isolates indicated that 482 of 2127 hybridomas secreted antibodies specific for B. piliformis. Specificity of MAbs for flagellar epitopes was demonstrated by indirect fluorescent antibody assays and Western blot analyses. Probing of 10 B. piliformis isolates with MAbs indicated that four B. piliformis isolates each possessed a distinct and isolate-specific flagellar epitope; five other isolates shared a common flagellar epitope. One isolate did not react with any of the MAbs specific for flagellar epitopes. Thus, B. piliformis isolates could be grouped into six antigenically distinct groups based upon flagellar epitope expression. Additionally, a MAb reactive with a cell-associated component recognised all but one isolate. This serological grouping of B. piliformis isolates agrees with the grouping of isolates based upon genetic and physiological characteristics, and supports the assertion that there are different strains among B. piliformis isolates.

Animals↗
PubMed1993

In vitro model of adhesion and invasion by Bacillus piliformis.

An in vitro model of Bacillus piliformis infection was developed to investigate the mechanisms of adhesion and internalization of this obligate intracellular bacterium. Adhesion and internalization events were examined by electron microscopic evaluation of infected Caco-2 cell monolayers. A few bacteria were identified in apical surface invaginations and in vacuoles subjacent to the apical surface, whereas the majority of bacteria were observed free within the cytoplasm, suggesting that B. piliformis entered epithelial cells via a phagocytic process and rapidly escaped the phagosome. To confirm that host cell phagocytosis was involved in entry of B. piliformis into mammalian cells, Intestine 407 cells were treated with the phagocytic inhibitor cytochalasin D, infected with B. piliformis, and evaluated for bacterial internalization by double-fluorescence labeling. The results showed decreased intracellular bacteria, suggesting that internalization was dependent on host cell microfilament function. To examine the role of B. piliformis in internalization, growth of live and Formalin-killed bacteria was compared. Dead bacteria were not internalized, suggesting that B. piliformis actively participates in internalization. B. piliformis appears to enter host cells by a bacterially directed phagocytic process. The in vitro system described should prove invaluable in further investigations of B. piliformis pathogenic mechanisms.

Actin Cytoskeleton↗
PubMed1992

Cytotoxicity of Bacillus piliformis.

Seven isolates of B. piliformis, the agent of Tyzzer's disease, obtained from various host species, were examined for cytotoxic activity by incubating culture filtrates on BRL 3A rat-hepatocyte and 3T3 mouse-fibroblast cell lines. One isolate exhibited cytopathic effects on BRL 3A cells, but not on 3T3 cells. Three other isolates were strongly cytotoxic for 3T3 cells but only slightly so for BRL 3A cells. The remaining three isolates showed no cytotoxicity for either cell line. The cytotoxic products were greater than 100 kDa in mol. wt, thermolabile, and partly destroyed by trypsin treatment. The data show that some B. piliformis isolates produce cytotoxic proteins, which may contribute to the pathogenesis of Tyzzer's disease.

Animals↗
PubMed1992

Evaluation of a subcutaneously implanted chamber for antibody production in rabbits.

Polyclonal antibody production in subcutaneous chambers was compared to traditional antibody production methods in rabbits. The chamber, a sterilized plastic wiffle golf ball that had been surgically implanted in the subcutis of the thoracic region, was immunized via a percutaneous injection of antigen into the core of the ball through one of the perforations in the chamber wall. Rabbits bearing chambers were immunized on the same schedule and with the same concentrations of antigens as were provided the adjuvant injected rabbits. Fluid volumes of 12 to 22 ml could be removed from each chamber at weekly intervals. Chamber antibody to specific microbial antigens was equal to or better than serum antibody produced to the same antigens with Freund's or acrylamide adjuvants. The comfort of the rabbit, the ease in chamber immunization, and the recovery of high titer antibody in large volumes make the subcutaneous chamber an attractive method for polyclonal antibody production.

Animals↗
PubMed1992

Subclinical infection and transmission of Tyzzer's disease in rats.

Two isolates of Bacillus piliformis originally obtained from rats from Japan and Indiana were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Protein and antigen profiles revealed heterogeneity between the two isolates, demonstrating that more than one isolate of B. piliformis is capable of infecting rats. Results of parallel infection and transmission studies with the two isolates were almost identical. Orally inoculated rats remained asymptomatic; however, enzyme-linked immunosorbent assay results revealed a significant increase in serum antibodies to B. piliformis. Formalin-killed B. piliformis elicited no serum antibody response among rats inoculated orally, indicating that viable organisms, capable of replicating within the host, are needed to induce a systemic humoral response. Naive rats and weanling gerbils were housed on soiled bedding from the experimentally infected, asymptomatic, seropositive rats. Although gerbils showed no clinical signs or histopathologic evidence of Tyzzer's disease, rats housed on bedding collected 1 or 2 weeks postinoculation seroconverted and remained seropositive but asymptomatic throughout the study. These results demonstrate that subclinically infected rats are capable of transmitting B. piliformis to naive rats and suggest that the histopathologic evaluation of sentinel gerbils may not be an effective method for detecting all strains of B. piliformis.

Animals↗
PubMed1991

Bacillus piliformis flagellar antigens for serodiagnosis of Tyzzer's disease.

Purified flagella from multiple isolates of Bacillus piliformis were obtained and examined by electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot (immunoblot) analyses were used to assess the purity, antigenicity, and cross-reactivity of purified flagellar preparations. SDS-PAGE demonstrated a single, major protein band evident at approximately 53 to 56 kDa in all isolates tested. Results of Western blot analyses indicated a lack of cross-reactivity between flagellar antigens and heterologous isolates. Enzyme-linked immunosorbent assays (ELISAs) were used to compare the efficacies of flagellar preparations from the various isolates as antigens in detecting B. piliformis serum antibodies from several host species. ELISA results indicated that no single flagellar preparation could be relied on to consistently identify serum antibodies in all the host species tested; however, ELISAs that utilized a trivalent flagellar antigen preparation were shown to be specific and sensitive for the detection of antibodies to B. piliformis.

Animals↗
PubMed1991

Detection of serum antibodies to Bacillus piliformis in mice and rats using an enzyme-linked immunosorbent assay.

Rats and mice were infected with Bacillus piliformis organisms at a dosage which resulted in clinical signs of Tyzzer's disease in gerbils. Although rats and mice did not show clinical signs of disease, rising antibody titers to B. piliformis were detected by enzyme-linked immunosorbent assay (ELISA) 2 to 6 weeks post-inoculation and remained at positive levels 11 weeks post-inoculation. Western blot analyses of sera from experimentally infected animals revealed banding patterns nearly identical to those obtained using hyperimmune serum. Results indicated that elevated ELISA titers reflected production of specific antibodies directed against antigens of B. piliformis. ELISA and Western blot analyses of naturally infected animals yielded similar results. These findings suggest that immunoassays such as ELISA can be used to detect subclinically infected rats and mice in the absence of clinical signs or histopathologic evidence of Tyzzer's disease.

Animals↗
PubMed1990

Protein and antigenic heterogeneity among isolates of Bacillus piliformis.

Protein and antigenic heterogeneity among isolates of Bacillus piliformis, the etiologic agent of Tyzzer's disease, were investigated. The seven isolates utilized in this study were originally isolated from naturally infected animals of different animal species and diverse geographical locations. Isolates were propagated in mammalian cell lines, and bacterial extracts were prepared. Protein and antigenic profiles were compared among isolates, using Coomassie blue-stained polyacrylamide gels and Western blot (immunoblot) analyses, respectively. Results showed differences in protein and antigen banding patterns, indicating diversity among isolates. Western blots probed with serum preabsorbed with a heterologous bacterial extract revealed that numerous antigens have different electrophoretic mobilities among isolates but apparently share common epitopes. Immunodominant cross-reactive antigens may be candidate proteins useful for development of improved serologic diagnostic tests, allowing identification of animals infected with a wide range of B. piliformis isolates.

Animals↗
PubMed1990

Identification of enterotoxigenic Escherichia coli by colony hybridization with nonradioactive digoxigenin-labeled DNA probes.

Enterotoxigenic Escherichia coli (ETEC) strains were readily identified in pure and mixed cultures with nonradioactive, digoxigenin-labeled DNA probes coding for heat-labile (LTI) and heat-stable (STaI, STaII, and STb) enterotoxins. Digoxigenin-labeled ETEC fragments were more sensitive than and exhibited less nonspecific background contamination than biotinylated ETEC probes.

Animals↗
PubMed1989

Effects of theophylline on human natural killer cells.

Theophylline has been shown previously to inhibit a number of cellular immune functions of granulocytes and T-lymphocytes. In the present report, we demonstrate that theophylline, in a dose-dependent fashion, suppresses human natural killer (NK) cell activity in vitro. To determine if theophylline produces quantitative or qualitative alterations in NK cells in vivo we quantitated peripheral blood NK cells with three monoclonal antibodies and FACS analysis and measured NK cytolytic activity in eight normal volunteers who took theophylline for eight days. No change was noted in the number of cytolytic activity of NK cells over the eight days of monitoring. We conclude that theophylline does not alter NK cells in vivo when given in therapeutic doses.

Adult↗
PubMed1989

Effects of coumarin (1,2-benzopyrone) and cimetidine on peripheral blood lymphocytes, natural killer cells, and monocytes in patients with advanced malignancies.

The combination of coumarin and cimetidine has yielded objective tumor regressions in patients with metastatic renal cell carcinoma and malignant melanoma. While the mechanism of action of cimetidine appears to be immunomodulatory, coumarin appears to have direct effects on tumor cells as well as immunomodulatory activity. We utilized monoclonal antibody labeling techniques to monitor peripheral blood lymphocyte, natural killer (NK) cell, and monocyte phenotypes in patients treated with coumarin and cimetidine. Patients received coumarin 100 mg orally daily for 14 days; on day 15 cimetidine 300 mg four times daily was added and both drugs were continued until disease progression. Studies were performed pre treatment and at 2, 4, and 8 weeks on therapy. There were no alterations in T-cells, helper/inducer T-cells, cytotoxic/suppressor T-cells, B-cells, Ia + lymphocytes, or NK cells. However, an increase was noted by 2 weeks on therapy in the percentage of monocytes and the percentage of DR+ monocytes. This change in the monocyte population occurred in the presence of coumarin alone, before the institution of cimetidine. While this treatment appears to increase DR expression by monocytes, further studies with larger numbers of patients are needed to determine if this observed change is related to antitumor response.

Adult↗
PubMed1988

Human terminal deoxyribonucleotidyltransferase: molecular cloning and structural analysis of the gene and 5' flanking region.

Human terminal deoxyribonucleotidyltransferase (nucleoside-triphosphate:DNA deoxynucleotidylexotransferase, EC 2.7.7.31) cDNA contains an open reading frame of 1530 base pairs (bp) corresponding to a protein containing 510 amino acids. The encoded protein is a template-independent DNA polymerase found only in a restricted population of normal and malignant prelymphocytes. To begin to investigate the genetic elements responsible for the tissue-specific expression of terminal deoxyribonucleotidyltransferase, genomic clones containing the entire human gene were isolated and characterized. Initially, cDNA clones were isolated from a library generated from the human lymphoblastoid cell line, MOLT-4R. A cDNA clone containing the entire coding region of the protein was used to isolate a series of overlapping clones from two human genomic libraries. The gene comprises 11 exons and 10 introns and spans 49.4 kilobases. The 5' flanking region (709 bp) including exon 1 was sequenced. Several putative transcription initiation sites were mapped. Within 500 nucleotides of the translation start site, a series of promoter elements was detected. "TATA" and "CAAT" sequences, respectively, were found to start at nucleotides -185 and -204, -328, and 465 and -505. Start sites were found for a cyclic AMP-dependent promoter analog at nucleotide -121, an eight-base sequence corresponding to the IgG promoter enhancer (cd) at nucleotide -455, and an analog of the IgG promoter (pd) at nucleotide -159. These findings suggest that transcripts coding for terminal deoxyribonucleotidyltransferase may be variable in length and that transcription may be influenced by a variety of genetic elements.

Base Sequence↗