Vaccination against tuberculosis by DNA injection.

There are 3 million deaths per annum worldwide due to tuberculosis, and AIDS is compounding the problem. A better vaccine than the live mycobacterium currently in use, bacillus Calmette-Guérin (BCG), is needed. When mice were injected with plasmid DNA encoding a single mycobacterial antigen (65-kDa heat shock protein, hsp65) they made specific cellular and humoral responses to the protein and became immune to subsequent challenge with Mycobacterium tuberculosis. Protection was equivalent to that obtained by vaccinating with live BCG, whereas immunizing with the protein was ineffective. Protection was also obtained with DNA encoding another mycobacterial antigen (36-kDa proline-rich antigen). These results suggest that DNA vaccination might yield improved vaccines to replace BCG.

A mycobacterial 65-kD heat shock protein induces antigen-specific suppression of adjuvant arthritis, but is not itself arthritogenic.

A recombinant (r)65-kD protein from Mycobacterium leprae, at levels far in excess of those present in whole mycobacteria, was unable to induce arthritis. Even when combined with a synthetic adjuvant, CP20961, to mimic the peptidoglycan adjuvant component of the mycobacterial cell wall, the r65-kD protein failed to induce arthritis. Pretreatment with as little as 1 microgram r65-kD protein protected rats against arthritis induced by M. tuberculosis, but this r65-kD protein was markedly less able to protect against arthritis induced by the synthetic adjuvant, CP20961, or type II collagen. The r65-kD protein appears, therefore, to produce an antigen-specific protection against arthritis induced by bacterial cell walls containing the 65-kD protein. Such protection can be overcome, however, by arthritogenic T lymphocytes, suggesting that protection occurs by preventing clonal proliferation of autoreactive T lymphocytes that are induced by the adjuvant properties of mycobacterial cell walls. How the r65-kD protein abrogates this particular adjuvant activity, and the nature of the arthritogenic self antigen(s), remain to be elucidated.

Tumor cells transfected with a bacterial heat-shock gene lose tumorigenicity and induce protection against tumors.

The gene encoding a highly immunogenic mycobacterial heat-shock protein (hsp65) was transfected into the murine macrophage tumor cell line J774. The resulting hsp65-expressing cells (J774-hsp65) were no longer able to produce tumors in syngeneic mice. This loss of tumorigenicity was not mediated through T cells since the transfected cells did not produce tumors in athymic mice. If mice are first immunized with the J774-hsp65 cells and then challenged with the parent J774 cells, the mice do not develop tumors, indicating that the presence of the mycobacterial hsp65 protein greatly enhances immunological recognition of unique structures expressed by the parent tumor cells. This is further confirmed by the demonstration in vitro of T cells derived from J774-hsp65-immunized mice that are cytotoxic for the parent J774 cells. The results provide the basis for a novel strategy for enhancing the immunological recognition and decreasing the tumorigenicity of transformed cells.

Recognition of a mycobacteria-specific epitope in the 65-kD heat-shock protein by synovial fluid-derived T cell clones.

Adjuvant arthritis in rats is induced by a T cell clone specific for amino acids 180-188 of the mycobacterial 65-kD heat-shock protein, and synovial T cell responses to this same Ag have been noted in human arthritis. We have isolated 65-kD Ag-specific T cell clones from synovial fluid mononuclear cells of a patient with acute arthritis, which, unlike the corresponding PBMC, showed a marked proliferative response to the 65-kD Ag. Using synthetic peptides corresponding to the whole sequence of the 65-kD Ag, all the clones were shown to recognize an epitope present in the first NH2-terminal peptide (amino acids 1-15), with no response to the adjacent peptide (amino acids 6-22) or to any other peptide. The complete dominance of this epitope in the response to the 65-kD Ag was shown by documenting responses to the peptide in PBMC obtained after recovery from the arthritis. This epitope, like that recognized by the rat arthritogenic T cell clone, is in a portion of the 65-kD sequence that is not conserved between bacteria and eukaryotes, so that in this case, joint inflammation could not be attributed to bacteria-induced T cell clones cross-reacting with the self 65-kD Ag.

Growth and regression of human tumor cell lines in congenitally athymic (rnu/rnu) rats.

Athymic rats were inoculated with cells from nine human tumor tissue culture cell lines. Tumor growth was seen with seven of the cell lines; histologically, the tumors resembled the tumors from which the cell lines were originated. Only one cell line--a malignant melanoma--grew progressively. The remaining sex cell lines grew to a short period of time and then regressed.

1,25-dihydroxyvitamin D3 receptors in human epithelial cancer cell lines.

Specific, high-affinity cytosolic receptors for 1,25-dihydroxyvitamin D3 have been demonstrated in five human cancer cell lines. The cell lines were derived from tumors of breast, lung, cervix, and melanotic and amelanotic melanomas. Binding affinity (Kd) of the receptors for 1,25-dihydroxyvitamin D3 were all approximately 0.2 nM, and receptor content ranged from 21 to 174 fmol/mg cytosol protein. The receptors from all five cell lines sedimented at 3.2S on sucrose density gradients and exhibited preferential affinity for 1,25-dihydroxyvitamin D3 compared to other vitamin D metabolites.

Crystal structure of the transcription elongation/anti-termination factor NusA from Mycobacterium tuberculosis at 1.7 A resolution.

Mycobacterium tuberculosis is the cause of tuberculosis in humans, a disease that affects over a one-third of the world's population. This slow-growing pathogen has only one ribosomal RNA operon, thus making its transcriptional apparatus a fundamentally interesting target for drug discovery. NusA binds to RNA polymerase and modulates several of the ribosomal RNA transcriptional processes. Here, we report the crystal structure of NusA, and reveal that the molecule consists of four domains. They are organised as two distinct entities. The N-terminal domain (residues 1 to 99) that resembles the B chain of the Rad50cd ATP binding cassette-ATPase (ABC-ATPase) and a C-terminal module (residues 108 to 329) consisting of a ribosomal S1 protein domain followed by two K homology domains. The S1 and KH domains are tightly integrated together to form an extensive RNA-binding structure, but are flexibly tethered to the N-terminal domain. The molecule's surfaces and architecture provide insights into RNA and polymerase interactions and the mechanism of pause site discrimination. They also allow us to rationalize certain termination-defective and cold shock-sensitive mutations in the nusA gene that have been studied in Escherichia coli.

1,25-dihydroxyvitamin D3 and malignant melanoma: the presence of receptors and inhibition of cell growth in culture.

In this study we demonstrate the presence of specific, high-affinity receptors for 1,25-dihydroxyvitamin D3 in malignant melanoma. Receptors are present both in cultured melanoma cells and in melanoma tumor tissue produced by inoculation of cells into athymic rats. The receptor sediments at 3.25 on sucrose density gradients, possesses a preferential affinity for 1,25-(OH)2D3 and has an apparent Kd of 0.18 nM by Scatchard analysis. We also demonstrate that human melanoma cells are responsive to 1,25-(OH)2D3 in vitro. Inclusion of 1,25-(OH)2D3 in the culture medium produced a marked increase in cell doubling time. This inhibitory effect of the hormone on melanoma cell proliferation was dose-related and represents the first demonstration of a 1,25-(OH)2D3 mediated action on tumor cells.

Regulation of macrophage gene expression following invasion by Mycobacterium tuberculosis.

INTRODUCTION: Mycobacteria are intracellular pathogens which survive and grow in host macrophages. M. tuberculosis bacilli enter the macrophage via binding to several distinct cell surface molecules. Following phagocytosis, sustained intracellular bacterial growth depends on the ability to avoid destruction by macrophage-mediated host defences such as lysosomal enzymes, reactive oxygen and the reactive nitrogen intermediates. We used differential display reverse transcription polymerase chain reaction (DD RT-PCR) to identify host genes which are regulated during infection and hence which might be involved in the host-parasite cross talk. RESULTS: Live M. tuberculosis (strain H37Rv) was used to infect Balb/c peritoneal murine macrophages. mRNA from infected and uninfected macrophages was isolated at different time intervals after phagocytosis and subjected to DD RT-PCR. Oligo dT12NV and random 10mer primers were used for PCR amplification of cDNA. Macrophage genes which appeared to be differently regulated during infection were subjected to further reamplification by PCR in order to clone and sequence them. The differential expression of the selected bands was further analysed by an RNA protection assay and a Northern blot. RESULTS: Several differentially regulated bands were identified. One band, of 158 bp, was down regulated after infection. Sequencing of this band revealed a high level of homology (95% identity) to mouse cytochrome c oxidase subunit VIIc. The downregulation was specific for live virulent Mtb, while live BCG, heat killed Mtb and latex beads-mediated phagocytosis did not affect the transcriptional level of this enzyme. CONCLUSIONS: The cytochrome oxidase enzyme complex of the inner mytochondrial membrane catalyzes the reaction between ferrocytochrome c and oxygen. The reaction is the terminal event in the electron transport scheme. Downregulation of cytochrome c oxidase subunit VIIc could interfere with: (1) the host apoptotic programme; or (2) the host respiratory burst.

The effect of antigen presenting cells on the cytokine profiles of stable and reactional lepromatous leprosy patients.

In view of varied reports on the Th1/Th2 paradigm in leprosy, we used a novel real time (RT) fluorogenic reverse transcriptase based PCR (RT-PCR) to measure cytokine expression in peripheral blood cells from lepromatous leprosy patients with stable disease and those suffering from erythema nodosum leprosum (ENL/Type II) reactions. To evaluate the role of accessory cells in Th cell differentiation, co-expression of Th cytokines interferon gamma (IFNgamma) and interleukin (IL) 4 and regulatory cytokines IL 10 and IL 12 was compared in antigen stimulated peripheral blood mononuclear cells (PBMC), cultures containing T cells reconstituted with autologous monocytes (MO) and cultures containing T cells reconstituted with autologous dendritic cells (DC). 7/8 stable lepromatous leprosy patients showed co-expression of both IFNgamma and IL 4, suggesting a Th0 or a combination of Th1 + Th2 subsets in PBMC. The RT-PCR demonstrated that stable lepromatous patients and patients in ENL had significantly higher levels of IFNgamma mRNA molecules compared to IL 4. In fact, 5/8 ENL patients had undetectable levels of IL 4 mRNA, with a skewing of the cytokine response towards a Th1-like profile. Consistent with this. IL 12p40 mRNA molecules were significantly higher in the PBMC of ENL patients compared to stable lepromatous patients (P < 0.01). Reconstitution of purified T cells with autologous DC and MO from the stable lepromatous group resulted in down regulation of IL 4 (P < 0.03 for DC and P < 0.02 for MO) and IL 10 (P < 0. 01 for DC and P < 0.02 for MO), and a consequent skewing towards a Th1 profile similar to that seen in ENL patients. The fact that accessory cells could alter the cytokine profile in the reconstituted cultures suggests that they may play a role in determining Th subset differentiation in chronic diseases, and may influence the immunological stability of such diseases.

Intermittent chemotherapy of experimental leprosy in mice.

In this study we assess the degree of prolonged bacteriostasis of Mycobacterium leprae after temporary exposure to ehtionamide or thiacetazone, and relate this to their efficacy when administered intermittently to mice with experimental leprosy infections. The results show that temporary exposure of M. leprae to either of these drugs results in a prolonged bacteriostatic effect, but that efficacy is rapidly lost as the interval between doses is increased. Using the mouse foot pad system, growth of M. leprae is not inhibited by thiacetazone when the frequency of administration is less than three times weekly. When ethionamide is administered once weekly, growth of M. leprae is inhibited but bactericidal activity is lost. When ethionamide is administered in combination with continuous dapsone therapy, either continuously or three times weekly, the bactericidal activity of the drug combination is greater than when either drug is administered alone. However, when ethionamide is administered once weekly in combination with continuous dapsone treatment, the bactericidal effect is identical to that when dapsone is given alone: that is, ethionamide makes no contribution to the combination.

The detection of Mycobacterium leprae protein and carbohydrate antigens in skin and nerve from leprosy patients with type 1 (reversal) reactions.

Type 1 (reversal) reactions are the most common immunological complications of leprosy. These episodes of delayed hypersensitivity produce severe local immunopathology and ultimately nerve damage. To date, the Mycobacterium leprae antigens associated with type 1 reactions have not been identified. Using monoclonal antibodies to defined protein and carbohydrate M. leprae epitopes (65, 35 and 28 kd and lipoarabinomannan [LAM]) in a two-step immunoperoxidase staining technique, M. leprae antigens were demonstrated in skin and nerve biopsies from patients in reversal reaction. Antigen presence and staining patterns were similar in skin and nerve lesions, implying that the pathological processes are similar in the two sites. Antigens were present both in macrophages and Schwann cells but also as a diffuse extracellular infiltrate associated with the inflammatory infiltrate. The 28-kd antigen was present most strongly and may be a potential candidate antigen for initiating type 1 reactions. LAM also stained strongly and persisted after treatment. The possible roles of LAM and 65 kd in the cellular events of type 1 reactions are discussed.

Determination and evolutionary significance of nucleotide sequences near to the 3'-end of 16S ribosomal RNA of mycobacteria.

The nucleotide sequence at the 3'-end of 16Sr-RNA (nucleotides 1305-1508) was determined, by the primer extension method, for Mycobacterium smegmatis, Mycobacterium tuberculosis and Mycobacterium vaccae, in addition to Mycobacterium leprae. No differences in nucleotide sequence were detected, indicating that this region of 16SrRNA is highly conserved among mycobacteria. The nucleotide sequence common to the four above-mentioned mycobacteria differs from that reported for species of other genera. For example, for helix 39 (nucleotides 1408-1491) the mycobacterial sequence has 58% similarity with the Escherichia coli sequence, 74% similarity with the Bacillus subtilis sequence and 93% similarity with the Streptomyces sequence. The observations support the assignment of M. leprae to the genus Mycobacterium.

A novel method for the isolation of mycobacterial DNA.

DNA was isolated from mycobacteria by a simplified procedure. Cells were suspended in 6 M guanidinium chloride, the suspension was cooled to -70 degrees C, then incubated at 65 degrees C for 10 min, cooled in ice, deproteinized by chloroform and DNA was recovered from the supernatant. The procedure was used to obtain DNA from several mycobacteria (1 x 10(9) or more cells) including Mycobacterium neoaurum, M. fortuitum, M. phlei and M. smegmatis. Each of the species was shown to have two ribosomal RNA operons per genome, and preliminary evidence was obtained which suggests that one of these operons is homologous with one of the operons of M. smegmatis.

The effect of freezing and storage in liquid nitrogen on the viability and growth of Mycobacterium leprae.

The effects of rapid and slow rates of freezing in liquid nitrogen, storage in liquid nitrogen for 12 months, and the rate of subsequent thawing on the viability and growth of M. leprae in the mouse footpad were studied. Some loss of viability of M. leprae was detected, and this was found to be associated with the freezing process, rather than with storage or thawing. Slow freezing was less deleterious than quick freezing, with a loss of viability of 90% compared with 98%. The growth pattern of M. leprae was unaffected except for a delay in the appearance of growth caused by the loss of viability, though there was some evidence of an increased lag phase of one strain, possibly due to the repair of sub-lethally damaged organisms.

The 16S ribosomal RNA of Mycobacterium leprae contains a unique sequence which can be used for identification by the polymerase chain reaction.

Nucleotide sequence data for bacterial 16S ribosomal RNA was used to identify oligodeoxyribonucleotide primers suitable for probing the rRNA gene of mycobacteria and related organisms, with the polymerase chain reaction. The method enabled us to distinguish mycobacteria from other closely related genera, and to differentiate between slow- and fast-growing mycobacteria. Mycobacterium leprae fell within the slow-growing group of mycobacteria but there are significant differences between the sequence of the M. leprae 16S rRNA gene and that of other slow-growing mycobacteria. These differences were used to devise a rapid, non-radioactive method for detecting M. leprae in infected tissue.

Susceptibility of Mycobacterium leprae to the bactericidal activity of mouse peritoneal macrophages and to hydrogen peroxide.

Macrophages from athymic nude mice were infected in vitro with Mycobacterium leprae to study the intracellular fate of this organism. Using the proportional bactericidal test, we have shown that the viability of M. leprae declines rapidly within these macrophages, although results of clearance experiments demonstrate that live and killed organisms are cleared at comparable rates. We have also shown that M. leprae is susceptible to the bactericidal effects of hydrogen peroxide and we suggest that hydrogen peroxide generated by macrophages is responsible for the killing of intracellular M. leprae.

The effect of body temperature and cell-mediated immunity on the growth of Mycobacterium marinum and Mycobacterium leprae in mice.

Evidence is presented that the high susceptibility of armadillos to infection with Mycobacterium leprae cannot be explained solely in terms of body temperature because mutant mice maintained with a body temperature similar to that of armadillos do not become heavily infected with M. leprae. The depression of cell-mediated immunity accompanying the low body temperature is not sufficient to produce an overwhelming infection. The results obtained with M. marinum suggest that whereas lack of cell-mediated immunity or a low body temperature result in a moderately enhanced infection in the mouse a combination of both of these factors is required to produce an overwhelming infection involving the internal organs.

The cytokine response to bacille Calmette Guérin vaccination in South India.

SETTING: Evaluation of immune response after BCG vaccination in a population from South India where BCG vaccination was a failure. OBJECTIVE: To study the cell-mediated immune responses (CMI) in vitro by assessing skin test conversion, lymphocyte proliferation and cytokine patterns before and after BCG vaccination in 20 Mantoux negative subjects, and to compare this with that of 20 naturally Mantoux positive subjects. DESIGN: In vitro lymphocyte proliferation and cytokine responses to various mycobacterial antigens were studied in 12 subjects from each group. The cytokines interferon gamma (IFN-gamma), interleukin (IL)-4 and IL-10 were measured by both reverse transcriptase polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). RESULTS: All of those who were initially Mantoux negative but one subject converted to positivity following vaccination, confirming that BCG vaccination does cause skin test conversion. However, in vitro proliferative responses to phytohaemagglutinin (PHA), purified protein derivative (PPD) and Mycobacterium tuberculosis remained largely unaltered by vaccination. The production of IFN-gamma was significantly higher in PPD-positive individuals compared to the PPD-negative group, and BCG vaccination of the latter did not change the levels of IFN-gamma, IL-4 or IL-10. CONCLUSIONS: The finding that PPD-negative individuals did not produce IFN-gamma even following vaccination and skin test conversion suggests that BCG had little effect in driving the immune response towards a protective Th1 type.

The microbiology of Mycobacterium leprae; progress in the last 30 years.

Over the last 30 years, there have been dramatic changes in the way Mycobacterium leprae is studied in the microbiology laboratory. The organism still has not been grown in vitro but, starting with demonstration of growth in the footpads of mice and culminating in the application of molecular biological and genetic techniques, we are now in a position to circumvent some of the difficulties arising from lack of cultivability. Such studies are providing us with new insights into the basic biology of the organism and are likely to provide new tools which will be of value in the clinical laboratory. In this article, I briefly outline the progress which has been made, and the potential applications of molecular techniques in such areas as bacterial identification and drug-resistance testing.