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T P Gillis

Publications and source records attributed to T P Gillis.

At least 19 recordsLinked to original sources
PubMed1997

Presence of F420-dependent glucose-6-phosphate dehydrogenase in Mycobacterium and Nocardia species, but absence from Streptomyces and Corynebacterium species and methanogenic Archaea.

A range of organisms known to contain F420 or to be relatives of mycobacteria were examined for F420-dependent glucose-6-phosphate dehydrogenase (FGD) and NADP-dependent glucose-6-phosphate dehydrogenase (NADP-G6PD) activities. All free-growing Mycobacterium species examined (including a virulent Mycobacterium tuberculosis strain) had FGD activities of 0.014-0.418 mumol min-1 mg protein-1, and NADP-G6PD activities of 0.013-0.636 mumol min-1 mg-1. Armadillo-grown Mycobacterium leprae had FGD activity of 0.008 mumol min-1 mg-1, but no detectable NADP-G6PD activity. Nocardia species also had FGD activity (0.088-0.154 mumol min-1 mg-1). Streptomyces and Corynebacterium species had no FGD, but had NADP-G6PD. Methanogenic Archaea had neither activity.

Corynebacterium↗
PubMed1997

Effects of essential fatty acid deficiency on prostaglandin E2 production and cell-mediated immunity in a mouse model of leprosy.

Results from animal and in vitro studies suggest that essential fatty acid (EFA) deficiency enhances cell-mediated immunity by reducing production of prostaglandins with immunosuppressive actions. However, direct experimental evidence that EFA deficiency enhances T-lymphocyte function in vivo has not been obtained. In this study, athymic (nu/nu) mice were infected in the footpads with Mycobacterium leprae and fed a linoleic acid-free diet. These mice, and infected nu/nu mice on control diets, were given an adoptive transfer of M. leprae-primed, T-cell-enriched lymphocytes. After 2 weeks, M. leprae bacilli were harvested from the recipient mice and bacterial viability was determined by the BACTEC system. M. leprae recovered from recipient mice fed control diets displayed little reduction in metabolic activity. In contrast, M. leprae from recipient mice fed the EFA-deficient (EFAD) diet exhibited markedly reduced viability. In vitro, donor cells from M. leprae-primed mice secreted elevated levels of gamma interferon upon exposure to the bacilli. These cells also exhibited an enhanced proliferative response, which was reduced by exogenous prostaglandin E2 (PGE2). In addition, M. leprae-infected granuloma macrophages (Mphi) from EFAD recipient nu/nu mice secreted significantly less PGE2 than granuloma Mphi from mice on control diets. These data suggest that enhanced levels of Mphi-generated PGE2, induced by M. leprae or its constituents, could act as an endogenous negative modulator of the immune response occurring in the microenvironment of the lepromatous granuloma.

Animals↗
PubMed1995

Ethanol fixation of sputum sediments for DNA-based detection of Mycobacterium tuberculosis.

The effect of ethanol fixation on PCR detection and viability of Mycobacterium tuberculosis in human sputum sediments was evaluated. M. tuberculosis seeded into sputum sediments was efficiently killed when treated for 1 h with 50, 70, or 95% ethanol. PCR amplification of a 123-bp fragment of the M. tuberculosis-specific IS6110 was not affected in ethanol-treated samples even when fixation was extended to 24 h. Ethanol fixation of sputum sediments did not affect the PCR detection of M. tuberculosis in clinical samples. PCR results from ethanol-treated clinical samples containing M. tuberculosis (smear positive and smear negative) or other respiratory pathogens correlated directly with the results by conventional detection methods for M. tuberculosis. Our results show that ethanol fixation of human sputum sediments containing M. tuberculosis significantly reduces the potential exposure of workers to viable M. tuberculosis without affecting DNA analysis by PCR. Also, ethanol fixation of sputum sediments provides a simple and inexpensive way to store and transport clinical specimens identified for DNA-based diagnostics without refrigeration.

Bacteriological Techniques↗
PubMed1994

Characterization of rifampin-resistance in pathogenic mycobacteria.

The emergence of rifampin-resistant strains of pathogenic mycobacteria has threatened the usefulness of this drug in treating mycobacterial diseases. Critical to the treatment of individuals infected with resistant strains is the rapid identification of these strains directly from clinical specimens. It has been shown that resistance to rifampin in Mycobacterium tuberculosis and Mycobacterium leprae apparently involves mutations in the rpoB gene encoding the beta-subunit of the RNA polymerases of these species. DNA sequences were obtained from a 305-bp fragment of the rpoB gene from 110 rifampin-resistant and 10 rifampin-susceptible strains of M. tuberculosis from diverse geographical regions throughout the world. In 102 of 110 rifampin-resistant strains 16 mutations affecting 13 amino acids were observed. No mutations were observed in rifampin-susceptible strains. No association was found between particular mutations in the rpoB gene and drug susceptibility patterns of multidrug-resistant M. tuberculosis strains. Drug-resistant M. tuberculosis strains from the same outbreak and exhibiting the same IS6110 DNA fingerprint and drug susceptibility pattern contained the same mutation in the rpoB gene. However, mutations are not correlated with IS6110 profiling outside of epidemics. The evolution of rifampin resistance as a consequence of mutations in the rpoB gene was documented in a patient who developed rifampin resistance during the course of treatment. Rifampin-resistant strains of M. leprae, Mycobacterium avium, and Mycobacterium africanum contained mutations in the rpoB gene similar to that documented for M. tuberculosis. This information served as the basis for developing a rapid DNA diagnostic assay (PCR-heteroduplex formation) for the detection of rifampin susceptibility of M. tuberculosis.

Amino Acid Sequence↗
PubMed1994

Clinical trial of fusidic acid for lepromatous leprosy.

Fusidic acid was assessed for antileprosy activity in nine lepromatous leprosy patients. Patients received fusidic acid at either 500 mg/day for 12 weeks or 750 mg/day for 4 weeks followed by 500 mg/day for 8 weeks. All patients showed time-dependent clinical improvement and decreases in bacillary morphological index, radiorespirometric activity and PCR signal, and in serum phenolic glycolipid I. Fusidic acid appears to be a weakly bactericidal antileprosy agent which may have a role in the multidrug treatment of leprosy pending an evaluation of lepra-reaction-suppressive activity.

Adolescent↗
PubMed1992

Detection of Mycobacterium leprae and the potential for monitoring antileprosy drug therapy directly from skin biopsies by PCR.

An improved protocol for PCR analysis of Mycobacterium leprae-infected tissues, based on enzymatic lysis, has been developed and used to demonstrate the feasibility of using PCR for detecting M. leprae in routine skin biopsies taken from leprosy patients throughout the clinical spectrum. Of 92 multibacillary patients tested, 99% were PCR-positive using gel electrophoresis or DNA hybridization to detect the amplified product. Similar analysis of paucibacillary patients, in which only one of 27 biopsies had demonstrable AFB microscopically, gave a positivity rate of 74%. No PCR signals were demonstrated from skin biopsies from seven patients with non-leprosy dermatoses and one AIDS patient with a disseminated atypical mycobacteriosis. Evaluation of leprosy patients with antileprosy drug therapy prior to biopsy demonstrated that PCR signals were either greatly diminished or absent after 2 months of continuous antibiotic therapy. PCR was also able to detect the presence of M. leprae in tissues of patients receiving antibacterial therapy when patients were suspected of harbouring drug-resistant M. leprae.

Anti-Bacterial Agents↗
PubMed1992

DNA hybridization analysis of mycobacterial DNA using the 18-kDa protein gene of Mycobacterium leprae.

DNA hybridization studies using a 611-base pair (bp) probe, encoding the entire 18-kDa protein of Mycobacterium leprae, demonstrated that M. simiae, M. intracellulare, M. kansasii, M. terrae, ADM-2, M. avium, M. scrofulaceum, M. gordonae and M. chelonei appear to possess DNA sequences homologous to the 18-kDa protein gene of M. leprae. RFLP analysis revealed that the restriction sites in the M. leprae 18-kDa gene were not conserved in the putative gene homologs of M. simiae and M. intracellulare. The restriction patterns observed with the 611-bp probe were useful in differentiating M. intracellulare, M. simiae, and M. leprae from each other, as well as in distinguishing strains of M. simiae serovar 1. Finally, the presence of homologous sequences in various mycobacteria did not affect the specificity of a previously described PCR test for detection of M. leprae, based on the M. leprae 18-kDa protein gene.

Bacterial Proteins↗
PubMed1992

Effects of fixation on polymerase chain reaction detection of Mycobacterium leprae.

The effects of standard fixatives (10% neutral buffered formalin, ethanol and mercury based) on the detection of Mycobacterium leprae DNA by the polymerase chain reaction (PCR) were studied. Mercury-based fixatives (Zenker's and Carnoy-Lebrun's fluids) strongly inhibited PCR amplification of M. leprae DNA. Ten percent neutral buffered formalin was inhibitory, but significant inhibition was observed only when fixation times exceeded 24 h. Ethanol-based fixatives provided the best medium for holding specimens for subsequent PCR with both free bacilli and skin biopsy specimens containing M. leprae. The M. leprae-specific, 360-bp region of the 18-kDa protein gene could be amplified from paraffin-embedded sections of formalin-fixed skin biopsy specimens from patients with either multibacillary or paucibacillary infections when proper fixation conditions were used. Results of the study demonstrate that tissues properly fixed with two standard fixatives (10% neutral buffered formalin and 50 or 70% ethanol) can be analyzed by PCR for the presence of M. leprae with no loss in specificity and only minimal diminution in sensitivity compared with the specificities and sensitivities obtained by use of freshly prepared, unfixed specimens.

Animals↗
PubMed1991

Comparison of polymerase chain reaction technique with other methods for detection of Mycobacterium leprae in tissues of wild nine-banded armadillos.

Thirty, nine-banded armadillos weighing between 3 and 5 kilograms trapped from an area endemic for armadillo leprosy were collected at random; killed, autopsied and examined histopathologically. Also, one of the right inguinal lymph nodes was removed under sterile precautions and examined using PCR, direct smear examination, mouse footpad study, culture in laboratory media and histopathology with a view to detecting Mycobacterium leprae. Blood was collected at death and tested for IgM antibodies to PGL-1. According to the PCR study of the inguinal lymph nodes 16 of 30 armadillos (53.3%) had evidence of M. leprae. Significant levels of IgM antibodies to PGL-1 and identifiable lepromatous granuloma in inguinal lymph nodes were found in 2 animals (6.7%) with advanced disseminated disease. The prevalence of generalized leprosy according to autopsy study was 13.3% and according to histopathological examination of ear tissue 3.3%. The presence of M. leprae in the tissues evoked no special tissue reaction in the early stages. The pattern of spread of the disease in 2 animals closely resembled that found in experimental animals infected intracutaneously. Initiation of infection by inoculation of M. leprae through thorn pricks remains a distinct possibility.

Animals↗
PubMed1990

Atopy and IgE in patients with leprosy.

The atopic status of patients with leprosy was assessed by medical history, physical examination, serum total IgE, and specific IgE antibodies to common allergens (by skin testing and RAST). Tests for specific IgE antibody to Mycobacterium leprae were performed by RAST and immunoblotting technique. We studied 28 patients with leprosy and 49 control subjects. The two groups did not differ significantly in the prevalence of atopic disease. The IgE level was significantly higher in the patients, however, than in the control subjects, whether there was atopy (296.1 versus 96.3 IU/ml) or not atopy (72.9 versus 18.9 IU/ml). Neither RAST nor immunoblotting technique detected significant levels of IgE antibodies to M. leprae. Our data indicate that leprosy was associated with increased total IgE level, but clinical atopy in patients with leprosy was similar to that in control subjects. The observed IgE increase in patients with leprosy appears to be generally nonspecific.

Adolescent↗
PubMed1990

The use of a specific DNA probe and polymerase chain reaction for the detection of Mycobacterium leprae.

A DNA probe encoding approximately 80% of the 18-kDa protein gene of Mycobacterium leprae was isolated and tested for specificity by assessing hybridization of the probe to genomic DNA from taxonomically related and unrelated DNA samples. The 360-base-pair (bp) probe was specific for M. leprae DNA and did not hybridize with genomic DNA from 18 species of bacteria nor with DNA from human, murine, and armadillo sources. Oligonucleotide primers were synthesized corresponding to the 5' and 3' ends of the 360-bp fragment to yield a fragment of similar size on amplification of M. leprae DNA by the polymerase chain reaction (PCR). A simple procedure for DNA extraction from M. leprae-infected tissues was developed that provided suitable template DNA for amplification. The PCR test was specific for M. leprae DNA from human and murine sources and detected M. leprae DNA in biopsies from leprosy patients and from control and uninfected human skin biopsy preparations seeded with as few as 100 M. leprae.

Animals↗
PubMed1990

Confirmation of a false-positive result associated with a competition inhibition assay used for detecting antibodies to a protein epitope of Mycobacterium leprae.

A competitive antibody-binding assay (CABA) was developed to detect antibodies in infected armadillos and leprosy patients which compete with an M. leprae-specific 125I monoclonal antibody IIIE9 for the species-specific M. leprae-IIIE9 epitope on the 65-kD protein. The results suggest armadillos and leprosy patients produce antibodies that inhibit the binding of 125I-IIIE9 monoclonal antibody to the IIIE9 epitope on crude, native 65-kD protein preparations. When purified, recombinant 65-kD protein was substituted for crude antigen, there was no evidence in the CABA of antibody to the IIIE9 epitope. False-positive results, possibly induced by steric hindrance, are likely to be associated with CABA which incorporate crude cell wall extracts as solid-phase antigen.

Animals↗
PubMed1990

Geographically distinct isolates of Mycobacterium leprae exhibit no genotypic diversity by restriction fragment-length polymorphism analysis.

Differentiation of microorganisms for taxonomic purposes is based primarily on phenotypic characteristics, which are the direct or cumulative result of gene expression. Since expression of phenotypic characteristics usually relies on in vitro growth of a microorganism, non-cultivable organisms, such as Mycobacterium leprae, present major problems for the identification of potential variants based on phenotypic similarities or differences between individual isolates. We have employed the use of restriction fragment-length polymorphism (RFLP) analysis of chromosomal DNA of M. leprae isolates, including human isolates from geographically distinct regions of the world and isolates from a Sooty Mangabey monkey and an armadillo, to assess the relatedness among these isolates. Restriction endonuclease (EcoRI, BstEII, PstI, and PvuI) digests of chromosomal DNA were analysed using DNA probes encoding all or part of the 12 kD, 18 kD, 28 kD, 65 kD and 70 kD proteins of M. leprae as well as a probe containing an M. leprae-specific sequence repeated up to 20 times in the M. leprae chromosome. Comparison of the resulting autoradiographs showed that the RFLP patterns were all identical, indicating that these isolates contained no polymorphism with respect to the restriction endonuclease sites analysed. In addition, RFLP patterns of two separate human M. leprae isolates remained unchanged after three cycles of experimental infection in the armadillo model. These results indicated that the M. leprae isolates tested in this study were indistinguishable at the genotypic level, strongly suggesting homogeneity among members of this species.

Animals↗
PubMed1989

Analysis of human antibody epitopes on the 65-kilodalton protein of Mycobacterium leprae by using synthetic peptides.

In order to study antibody reactivity to the Mycobacterium leprae 65-kilodalton (kDa) antigen, peptides representing overlapping sequences of the 65-kDa protein were synthesized, and a recombinant protein expression system for r65-kDa was constructed. Mouse monoclonal antibodies and leprosy patient seroreactivity to peptides and r65-kDa were tested by an enzyme-linked immunosorbent assay. All seven of the monoclonal antibodies used in this study reacted with their previously defined epitopes when tested against peptides. All monoclonal antibodies also reacted with r65-kDa. Leprosy patient seroreactivity to peptides and r65-kDa was seen in about one-third of active multibacillary cases. Specimens from patients positive for antibodies to peptides were seen to recognize different epitopes than did mouse monoclonal antibodies used in this study. It is concluded that substantial differences exist between mouse monoclonal antibodies and human leprosy patient reactivity to the 65-kDa antigen and that human seroreactivity to the 65-kDa antigen is indicative of a highly elevated bacillary load.

Amino Acid Sequence↗
PubMed1989

A study of the relatedness of Mycobacterium leprae isolates using restriction fragment length polymorphism analysis.

The inability to cultivate Mycobacterium leprae in vitro has severely hampered comprehensive phenotypic analysis of individual isolates, leaving unanswered the question of the relatedness of these isolates. Since the nucleotide sequence of a bacterial chromosome is its "genetic fingerprint", we have employed the use of restriction fragment length polymorphism (RFLP) analysis of chromosomal DNA of M. leprae isolates to assess the relatedness among these isolates. DNA of M. leprae was harvested from infected armadillo tissue originally inoculated with bacilli from lepromatous lesions of human patients from geographically distinct regions of the world. Restriction endonuclease (EcoRI, PstI, and PvuI) digests of chromosomal DNA were analysed using DNA probes encoding all or part of the 28-kDa, 65-kDa and 70-kDa proteins of M. leprae. Comparison of the resultant autoradiographs showed that the RFLP patterns were all identical indicating that these isolates contained no polymorphism with respect to the restriction endonuclease sites analysed. These results indicated that the M. leprae isolates tested in this study were indistinguishable at the genotypic level, suggesting the possibility of homogeneity among members of the species, M. leprae.

Animals↗
PubMed1988

Genetic control of murine T cell proliferative responses to Mycobacterium leprae. V. Evidence for cross-reactivity between host antigens and Mycobacterium leprae.

T cell proliferative responses to Mycobacterium leprae were measured by immunization of mice at the base of the tail with Ag and challenging lymphocytes from draining lymph nodes in culture with M. leprae. C57BL/10J and B10.BR mice were identified as low responder mice and the congenic strains B10.M, B10.Q, and B10.AKM as high responders whereas F1 (high x low) hybrid mice were found to be low responders. The cellular basis of low responsiveness did not appear to result from a defect in Ag-presenting cells or the activation of suppressor T cells by M. leprae. The influence of the environment in which T cells developed on responsiveness to M. leprae was analyzed in chimeric mice prepared by irradiating F1(C57BL/10J x B10.M) mice and reconstituting with bone marrow from C57BL/10J, B10.M, or F1 donors. Six weeks later, chimeric mice were immunized with M. leprae, lymph node cells were subsequently prepared, and H-2 phenotyped and challenged in culture with M. leprae Ag. T cell proliferative responses were found to be low in all cases, similar to those observed using lymph node cells from F1 hybrid mice. These results suggested that high responder B10.M lymphocytes developing in the irradiated F1 mice became tolerized to antigenic determinants found on M. leprae. This implied cross-reactive epitopes existed between some mouse strains and M. leprae. Low responsiveness to M. leprae in low responder and F1 hybrid mice may result from tolerance to H-2-encoded Ag that show cross-reactivity with M. leprae.

Animals↗
PubMed1988

Leprosy.

Leprosy affects over 10 million people in the world. The disease is a model of graded cell-mediated immunity, in this case to the causative organism, Mycobacterium leprae. The clinical manifestations are due to (i) bacterial progression, (ii) immunologic responses of the host, (iii) peripheral nerve damage due to either or both bacterial progression and immunologic responses of the host, and (iv) preventable secondary deformities following nerve damage, which account for most of the stigma of the disease. Treatment modalities are now available to control or minimize the effects of bacterial progression, harmful immunologic responses of the host, peripheral nerve damage, and secondary deformities. Unique biochemical characteristics of M. leprae reside in the cell wall and associated macromolecules. Some of these molecules are potent immunogens in humans, while others constitute the structural integrity of the bacillus. Proteins of M. leprae are currently under intensive investigation as a result of deoxyribonucleic acid cloning of M. leprae genes. Structure-function and antigenic relationships of M. leprae proteins should become available by using recombinant deoxyribonucleic acid procedures coupled with T- and B-cell cloning to advance our understanding of the immunologic reactions encountered in Hansen's disease. Until recently, the study of the immunology of leprosy has been stymied by the lack of immunologically specific M. leprae antigens. The definition of specific antigens and production of recombinant and synthetic immunologic reagents have fostered state-of-the-art research efforts into new immunodiagnostic procedures and development of a leprosy vaccine. Also discussed is progress in understanding of the mechanism(s) underlying the M. leprae-specific immunodeficiency associated with lepromatous leprosy, including the role of suppressor T cells and defective macrophage function. Metabolic studies of M. leprae suggest intact catabolic pathways and energy generation with purine bases and catalase as possible growth factors. Special attention may also need to be given to biophysical parameters for eventual in vitro cultivation. Rapid in vitro systems, using quantitation of bacillary metabolic activity, may soon replace the lengthy mouse footpad test for determining the viability and drug susceptibility of the leprosy bacillus.

Animals↗