Search PubMed⌕ Search

Biomedical subjects

B R Bloom

Publications and source records attributed to B R Bloom.

At least 91 records · Page 5Linked to original sources
PubMed1995

Bacterial vaccine vectors and bacillus Calmette-Guérin.

Recent advances in biotechnology now allow a more modern approach to the development of vaccines, particularly that of recombinant vaccines. Bacterial vaccine vectors have the advantage over viral vectors in that the former have the ability to express a greater number of antigens in different forms. Although no recombinant bacterial vaccines are currently in use, bacillus Calmette-Guérin (BCG), Salmonella species, and Escherichia coli are being developed as vaccine vectors. We review plasmid systems and mutant strains developed for the expression of foreign antigens, with particular emphasis on those developed for BCG. We describe the development of antigen expression systems as well as the immune response elicited by recombinant BCG vaccine strains to bacterial and human immunodeficiency virus (HIV) antigens. A modified recombinant BCG carrier with selection for the stable maintenance of rDNA is proposed.

Administration, Oral↗
PubMed1995

Recombinant bacille Calmette-Guérin priming against measles.

Live attenuated measles virus (MV) vaccines are ineffective in young infants because of neutralization by maternal antibody. An immunization strategy that may permit priming for T cell memory for MV in infants at or shortly after birth uses recombinant bacille Calmette-Guérin expressing the full-length MV nucleocapsid (N) protein (rBCG::N). C3H/He mice immunized with rBCG::N developed T cell responses and ELISA antibodies to the N protein and low levels of neutralizing antibody after intracranial infection with MV strain CAM/R40. There was considerable reduction in the virus titer recovered from brain homogenates, a decrease in the incidence and severity of histologic encephalitis, and a decrease in mortality in rBCG::N-printed C3H/He mice compared with control mice. Given the limitations of existing live attenuated MV vaccines, these results encourage the further testing of rBCG::N vaccines in primate models.

Animals↗
PubMed1995

Effects of nitric oxide synthase inhibitors on murine infection with Mycobacterium tuberculosis.

We have recently demonstrated that the macrophage L-arginine-dependent cytotoxic pathway effectively kills the virulent Erdman strain of Mycobacterium tuberculosis in vitro via the generation of toxic reactive nitrogen intermediates by the enzyme nitric oxide synthase. This report demonstrates that two distinct inhibitors of nitric oxide synthase (aminoguanidine and NG-monomethyl-L-arginine) render similar deleterious effects on tuberculous infection in mice, as assessed by mortality, bacterial burden, and pathological tissue damage, thus confirming the importance of reactive nitrogen intermediates in resistance against M. tuberculosis.

Amino Acid Oxidoreductases↗
PubMed1995

In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis.

Insertional mutagenesis in Mycobacterium bovis BCG, a member of the slow-growing M. tuberculosis complex, was accomplished with transposons engineered from the Mycobacterium smegmatis insertion element IS1096. Transposons were created by placing a kanamycin resistance gene in several different positions in IS1096, and the resulting transposons were electroporated into BCG on nonreplicating plasmids. These analyses demonstrated that only one of the two open reading frames was necessary for transposition. A library of insertions was generated. Southern analysis of 23 kanamycin-resistant clones revealed that the transposons had inserted directly, with no evidence of cointegrate formation, into different restriction fragments in each clone. Sequence analysis of nine of the clones revealed junctional direct 8-bp repeats with only a slight similarity in target sites. These results suggest that IS1096-derived transposons transposed into the BCG genome in a relatively random fashion. Three auxotrophs, two for leucine and one for methionine, were isolated from the library of transposon insertions in BCG. They were characterized by sequencing and found to be homologous to the leuD gene of Escherichia coli and a sulfate-binding protein of cyanobacteria, respectively. When inoculated intravenously into C57BL/6 mice, the leucine auxotrophs, in contrast to the parent BCG strain or the methionine auxotroph, showed an inability to grow in vivo and were cleared within 7 weeks from the lungs and spleen.

Animals↗
PubMed1995

Safety and immunogenicity of a V3 loop synthetic peptide conjugated to purified protein derivative in HIV-seronegative volunteers.

OBJECTIVES: To develop a peptide-based model for a preventive vaccine for HIV-1 infection. DESIGN: Phase I trial in HIV-1-seronegative volunteers. PARTICIPANTS: Adult healthy subjects HIV-1-antibody-seronegative in an enzyme-linked immunosorbent assay, screened for tuberculin [purified protein derivative (PPD)] reactivity with 2 tuberculin units PPD-administered intradermally. INTERVENTIONS: Submicrogram doses of a PPD conjugate with a peptide of the primary neutralizing domain (PND) of HIV-1MN (PPD-MN-PND) were administered intradermally to tuberculin skin-test-positive and -negative volunteers. RESULTS: Antibodies to the MN-PND were measured after two immunizations in 10 out of 11 PPD skin-test-positive volunteers. After the fourth immunization high-affinity antibodies were detected, which persisted for over 1 year. High titers of MN-PND-specific immunoglobulin (Ig) G and IgA were detected in the serum and saliva of all volunteers tested. Serum antibodies were cross-reactive with PND peptide from some other HIV-1 strains but neutralized only the HIV-1MN prototype. Human leukocyte antigen (HLA)-B7-restricted MN-PND-specific cytotoxic T lymphocytes (CTL) were also detected. CONCLUSIONS: The PPD-MN-PND vaccine at submicrogram doses is safe and immunogenic in PPD skin-test-positive healthy adult volunteers. Long lasting humoral immune responses in the serum and saliva were possibly accompanied by HLA-B7-restricted CTL responses. This is a vaccine prototype that can be rapidly and inexpensively modified to include other peptide epitopes. It is especially suitable for use in a worldwide multibillion Bacillus Calmette-Guérin (BCG)-primed or tuberculosis-exposed population at risk for HIV-1 infection.

AIDS Vaccines↗
PubMed1994

Nonpeptide ligands for human gamma delta T cells.

gamma delta T cells respond to a variety of microbial pathogens and transformed cells. Their limited receptor repertoire and activation by mycobacterial antigens resistant to proteases suggest that they may recognize nonpeptide antigens. We have tested a variety of nonpeptide molecules for stimulation of human gamma delta T cells. Synthetic alkyl phosphates, particularly monoethyl phosphate (MEP), selectively activated gamma delta T cells and stimulated their proliferation in vitro. All gamma delta T cells stimulated by MEP expressed V gamma 2/V delta 2 receptors. The purified natural ligand of mycobacteria is chemically similar to, though distinct from, MEP and contains a phosphate residue that is critical for biological activity. Recognition and expansion of a specific T-cell receptor-bearing population to non-peptide ligands is unprecedented among T cells. We suggest that MEP mimics small natural ligands capable of expanding one subset of gamma delta T cells and that this recognition of nonpeptide antigens may play an important role in human immunity to pathogens.

Antibodies↗
PubMed1994

Transmission of tuberculosis in New York City. An analysis by DNA fingerprinting and conventional epidemiologic methods.

BACKGROUND: The incidence of tuberculosis and drug resistance is increasing in the United States, but it is not clear how much of the increase is due to reactivation of latent infection and how much to recent transmission. METHODS: We performed DNA fingerprinting using restriction-fragment-length polymorphism (RFLP) analysis of at least one isolate from every patient with confirmed tuberculosis at a major hospital in the Bronx, New York, from December 1, 1989, through December 31, 1992. Medical records and census-tract data were reviewed for relevant clinical, social, and demographic data. RESULTS: Of 130 patients with tuberculosis, 104 adults (80 percent) had complete medical records and isolates whose DNA fingerprints could be evaluated. Isolates from 65 patients (62.5 percent) had unique RFLP patterns, whereas isolates from 39 patients (37.5 percent) had RFLP patterns that were identical to those of an isolate from at least 1 other study patient; the isolates in the latter group were classified into 12 clusters. Patients whose isolates were included in one of the clusters were inferred to have recently transmitted disease. Independent risk factors for having a clustered isolate included seropositivity for the human immunodeficiency virus (HIV) (odds ratio for Hispanic patients, 4.31; P = 0.02; for non-Hispanic patients, 3.12; P = 0.07), Hispanic ethnicity combined with HIV seronegativity (odds ratio, 5.13; P = 0.05), infection with drug-resistant tuberculosis (odds ratio, 4.52; P = 0.005), and younger age (odds ratio, 1.59; P = 0.02). Residence in sections of the Bronx with a median household income below $20,000 was also associated with having a clustered isolate (odds ratio, 3.22; P = 0.04). CONCLUSIONS: In the inner-city community we studied, recently transmitted tuberculosis accounts for approximately 40 percent of the incident cases and almost two thirds of drug-resistant cases. Recent transmission of tuberculosis, and not only reactivation of latent disease, contributes substantially to the increase in tuberculosis.

AIDS-Related Opportunistic Infections↗
PubMed1994

Leprosy vaccine.

Explore the source record for details and available documents.

Bacterial Proteins↗
PubMed1994

Evidence for a superantigen in human tuberculosis.

T cells are not only required for resistance to tuberculosis, but they likely contribute to the tissue damage characteristic of the disease. To define better the T cell populations that contribute to the immunopathogenesis of human tuberculosis, we investigated the T cell receptor (TCR) beta chain repertoire expressed in patients with tuberculous pleuritis. Analysis by polymerase chain reaction and flow cytometry indicated an expansion of V beta 8+ T cells at the site of disease in some donors, suggesting the possibility that Mycobacterium tuberculosis contains a superantigen. M. tuberculosis induced strong T cell proliferative responses in tuberculin-negative healthy donors in vitro, with preferential expansion of V beta 8+ T cells, independent of the CDR3 region. T cell stimulation was MHC class II-dependent and did not require antigen processing by the antigen-presenting cells. These findings are consistent with the presence of a superantigen in M. tuberculosis, aspects of which may contribute to the immunopathology of tuberculosis and to the adjuvant properties of M. tuberculosis.

Amino Acid Sequence↗
PubMed1994

Experimental approaches to mechanisms of protection and pathogenesis in M. tuberculosis infection.

It has, for many years, been widely assumed that the fundamental mechanism of protection in tuberculosis infection is a CD4 T cell response producing lymphokines that activate macrophages to kill or restrict the intracellular growth of M. tuberculosis. Just as certain cytokines, e.g. IFN-gamma, are currently perceived to be important for protection, others, particularly tumor necrosis factor (TNF), are thought to be responsible for much of the tissue destruction associated with the disease. Yet there are remarkably few critical experimental or clinical data that have defined the immunological requirements for protection and pathogenesis. One of the initial stimuli to the work we have undertaken has been careful reflection on the results of the many prospective trials of BCG against tuberculosis. Two aspects have impelled us to reconsider conventional wisdom. The first, of course, is the wide discrepancy in the degree of protection imparted, ranging from 0% in South India to 77% in the British MRC trial (1, 2). The second is that, in all trials that examined them, skin test conversions to tuberculin positivity were 85% or greater, indicating a disparity between the presence of delayed hypersensitivity to tuberculin and protection. We and others have argued (1, 2) that there are multiple possible explanations for this discrepancy, the principal one being protection caused by infection with environmental mycobacteria. But, the general point raised is whether cell mediated immunity as manifested by CD4+ cell production of lymphokines and macrophage activation is a sufficient mechanism for protection against M. tuberculosis infection.

Animals↗
PubMed1994

Isolation and characterization of the aspartokinase and aspartate semialdehyde dehydrogenase operon from mycobacteria.

Diaminopimelic acid (DAP) is a major component of the peptidoglycan layer of the mycobacterial cell wall. The mycobacterial cell wall has been implicated as a potential virulence factor and is highly immunogenic. The pathway for biosynthesis of DAP may serve as a target in the design of antimycobacterial agents and construction of in vivo selection systems. Despite its significance, this biosynthetic pathway is poorly understood in mycobacteria. In order to develop a better understanding of mycobacterial DAP biosynthesis, the aspartate semialdehyde dehydrogenase (asd) genes of Mycobacterium smegmatis, bacille Calmette-Guerin (BCG), Mycobacterium avium, Mycobacterium leprae, and Mycobacterium tuberculosis were isolated. The M. smegmatis asd gene was isolated by complementation in Escherichia coli. This gene was then used to isolate the asd genes from other mycobacteria. The asd-complementing fragments from BCG and M. smegmatis were sequenced. An open reading frame upstream of the mycobacterial asd gene was identified as the mycobacterial aspartokinase gene (ask). Primer extension analysis revealed that the only transcriptional start in this region is found 5' of the ask gene. This observation indicates that the mycobacterial ask and asd genes are in an operon.

Amino Acid Sequence↗
PubMed1994

Use of in vivo complementation in Mycobacterium tuberculosis to identify a genomic fragment associated with virulence.

Novel molecular tools and genetic methods were developed to isolate genomic fragments of Mycobacterium tuberculosis that may be associated with virulence. We sought to restore virulence, a characteristic of M. tuberculosis that is correlated with growth rate in mouse spleen and lung tissue, to the avirulent strain H37Ra by complementation. A representative library of the virulent M. tuberculosis strain H37Rv was constructed and transformed into H37Ra. Enrichment for individual faster-growing recombinants was achieved by passage of pools of H37Ra transformants harboring the H37Rv library through mice. A molecular strategy was devised to isolate and clone the H37Rv genomic DNA fragment ivg, which conferred a more rapid in vivo growth rate to H37Ra.

Animals↗
PubMed1994

Genetic determination of the meso-diaminopimelate biosynthetic pathway of mycobacteria.

The increasing incidence of multiple-drug-resistant mycobacterial infections indicates that the development of new methods for treatment of mycobacterial diseases should be a high priority. meso-Diaminopimelic acid (DAP), a key component of a highly immunogenic subunit of the mycobacterial peptidoglycan layer, has been implicated as a potential virulence factor. The mycobacterial DAP biosynthetic pathway could serve as a target for design of new antimycobacterial agents as well as the construction of in vivo selection systems. We have isolated the asd, dapA, dapB, dapD, and dapE genes involved in the DAP biosynthetic pathway of Mycobacterium bovis BCG. These genes were isolated by complementation of Escherichia coli mutations with an expression library of BCG DNA. Our analysis of these genes suggests that BCG may use more than one pathway for biosynthesis of DAP. The nucleotide sequence of the BCG dapB gene was determined. The activity of the product of this gene in Escherichia coli provided evidence that the gene may encode a novel bifunctional dihydrodipicolinate reductase and DAP dehydrogenase.

Amino Acid Sequence↗
PubMed1994

Diagnosis of cutaneous leishmaniasis and species discrimination of parasites by PCR and hybridization.

The aim of this study was to assess the efficacy of PCR methodology in establishing the diagnosis of cutaneous leishmaniasis in patients from areas of endemicity in Venezuela. Biopsies from 233 patients with cutaneous ulcers suggestive of leishmaniasis were analyzed by PCR, employing oligonucleotides directed against conserved regions of kinetoplast DNA (kDNA), and the PCR products were then hybridized to nonradioactively labeled, species-specific, cloned kDNA fragments. The ability of PCR to detect Leishmania cells was compared with those of the conventional methodologies: skin testing with killed promastigotes (Montenegro test), examination of Giemsa-stained biopsy smears, and in vitro culture of biopsy tissue. The PCR-hybridization technique detected the presence of Leishmania cells in 98% of patients clinically diagnosed as having leishmaniasis and also positive by the Montenegro skin test. In comparison, leishmania positivity was found in only 42% of cultures and 64% of biopsy smears. By hybridizing the PCR product to new kDNA probes specific for either Leishmania mexicana or Leishmania braziliensis, we found that both species are major causes of cutaneous leishmaniasis in Venezuela, and the species identification was confirmed by restriction enzyme analysis of kDNA from biopsy cultures. This work demonstrates that PCR coupled with hybridization is useful not only for the diagnosis of cutaneous leishmaniasis but also for the taxonomic discrimination essential for both epidemiology and therapy. This technique can be used to diagnose leishmaniasis in a country in which the disease is endemic and can perhaps be adapted for use in a rural clinic.

Animals↗
PubMed1994

Role of macrophage oxidative burst in the action of anthrax lethal toxin.

BACKGROUND: Major symptoms and death from systemic Bacillus anthracis infections are mediated by the action of the pathogen's lethal toxin on host macrophages. High levels of the toxin are cytolytic to macrophages, whereas low levels stimulate these cells to produce cytokines (interleukin-1 beta and tumor necrosis factor-alpha), which induce systemic shock and death. MATERIALS AND METHODS: Experiments were performed to assess the possibility that the oxidative burst may be involved in one or both of lethal toxin's effects on macrophages. Toximediated cell lysis, superoxide anion and cytokine production were measured. Effects of antioxidants and macrophage mutations were examined. RESULTS: RAW264.7 murine macrophages treated with high levels of toxin released large amounts of superoxide anion, beginning at about 1 hr, which correlates with the onset of cytolysis. Cytolysis could be blocked with various exogenous antioxidants or with N-acetyl-L-cysteine and methionine, which promote production of the endogenous antioxidant, glutathione. Mutant murine macrophage lines deficient in production of reactive oxygen intermediates (ROIs) were relatively insensitive to the lytic effects of the toxin, whereas a line with increased oxidative burst potential showed elevated sensitivity. Also, cultured blood monocyte-derived macrophages from a patient with Chronic Granulomatous Disease, a disorder in which the phagocyte's oxidative burst is disabled, were totally resistant to toxin, in contrast to control monocytes. CONCLUSIONS: These results imply that the cytolytic effect of the toxin is mediated by ROIs. Additionally, cytokine production and consequent pathologies showed partial dependence on macrophage ROIs. Antioxidants moderately inhibited toxin-induced cytokine production in vitro, and BALB/c mice pretreated with N-acetyl-L-cysteine or mepacrine showed partial protection against lethal toxin. Thus ROIs are involved in both the cytolytic action of anthrax lethal toxin and the overall pathologic process in vivo.

Animals↗
PubMed1993

Effective immunization against cutaneous leishmaniasis with recombinant bacille Calmette-Guérin expressing the Leishmania surface proteinase gp63.

Leishmania parasites cause a spectrum of diseases that afflict the populations of 86 countries in the world. The parasites can survive within the lysosomal compartments of the host's macrophages, unless those macrophages are appropriately activated. Despite the fact that protective immunity can be induced by vaccination with crude parasite preparations, little progress has been made toward a defined vaccine for humans. In this study the gene encoding the Leishmania surface proteinase gp63 was cloned and expressed as a cytoplasmic protein in a bacille Calmette-Guérin (BCG) vaccine strain. BALB/c and CBA/J mice were inoculated with a single dose of recombinant BCG and challenged with infective Leishmania major or Leishmania mexicana promastigotes. Significant protection was observed in both mouse strains against L. mexicana and in CBA/J against L. major, whereas only a delay in L. major growth was seen in BALB/c mice. Recombinant BCG also engendered a strong protective response against challenge with amastigotes of L. mexicana, demonstrating that the induced immune response recognized the intracellular form of the parasite. The results support the view that recombinant BCG expressing gp63 may prove a useful vaccine for inducing protective cell-mediated immune responses to Leishmania species causing American cutaneous leishmaniasis.

Animals↗
PubMed1993

An essential role for interferon gamma in resistance to Mycobacterium tuberculosis infection.

Tuberculosis, a major health problem in developing countries, has reemerged in recent years in many industrialized countries. The increased susceptibility of immunocompromised individuals to tuberculosis, and many experimental studies indicate that T cell-mediated immunity plays an important role in resistance. The lymphokine interferon gamma (IFN-gamma) is thought to be a principal mediator of macrophage activation and resistance to intracellular pathogens. Mice have been developed which fail to produce IFN-gamma (gko), because of a targeted disruption of the gene for IFN-gamma. Upon infection with Mycobacterium tuberculosis, although they develop granulomas, gko mice fail to produce reactive nitrogen intermediates and are unable to restrict the growth of the bacilli. In contrast to control mice, gko mice exhibit heightened tissue necrosis and succumb to a rapid and fatal course of tuberculosis that could be delayed, but not prevented, by treatment with exogenous recombinant IFN-gamma.

Amino Acid Oxidoreductases↗