Circulatory basophilia in guinea pigs with delayed-type hypersensitivity

Circulatory basophilia could be induced in inbred guinea pigs systematically immunized with ovalbumin and consequently provoked repeatedly with dissolved ovalbumin applied onto the mucosa of the nares or the outer eye. The degree of the increase in circulatory basophil granulocytes depended on the adjuvant used and was significantly more pronounced after immunization with Freund's complete adjuvant than with alhydrogel (A1(OH)3). The degree of basophilia was also dependent on the animal strain, but different in two strains selected for high-asthma trait.     

Sulfolipid deficiency does not affect the virulence of Mycobacterium tuberculosis H37Rv in mice and guinea pigs

Lipids that are found only in the cell envelope of pathogenic mycobacteria, such as those containing multiple methyl-branched fatty acids, have long been thought to play a role in pathogenesis. Among these complex lipids, sulfolipids have been the most extensively studied over the last 50 years. The numerous biological effects exhibited by purified sulfolipids on phagocytic cells led to the idea that these molecules are probably important virulence factors facilitating the intracellular survival of Mycobacterium tuberculosis. However, definitive evidence to support this concept has been lacking. The recent construction of an isogenic sulfolipid-deficient mutant of M. tuberculosis H37Rv (Sirakova et al., J. Biol. Chem. 276:16833-16839, 2001) has for the first time provided the opportunity to directly assess the contribution of these complex lipids to pathogenesis. In the present study, we show that against all expectations, sulfolipid deficiency does not significantly affect the replication, persistence, and pathogenicity of M. tuberculosis H37Rv in mice and guinea pigs or in cultured macrophages.     

Mononuclear cell function in Mycobacterium tuberculosis infected guinea pigs

In this study mononuclear cell function was studied in the lymph glands, spleen, and peripheral blood of Mycobacterium tuberculosis infected guinea pigs. Adherent cells from draining lymph nodes and spleens of infected animals spontaneously produced a factor which inhibited normal lymphocyte proliferative responses. As it has previously been shown that this factor activates a population of suppressor T cells, resident lymphocytes in the lymph nodes and spleen were examined and were shown to inhibit normal lymphocyte functions. It is suggested that adherent cells ingesting M. tuberculosis spontaneously release a suppressor cell activating factor (SCAF) which locally activates lymphocytes to become suppressor cells. Even at a time of overwhelming infection, peripheral blood adherent cells could not be shown to release SCAF and peripheral blood suppressor cells could not be identified. Although peripheral blood lymphocyte proliferative responses to PHA were normal in infected animals, their ability to produce the lymphokine macrophage inhibition factor was considerably reduced after the second week of infection. This dissociation between lymphocyte proliferation and lymphokine production is similar to that previously described in humans overwhelming tuberculosis.     

Antibody-mediated and delayed-type hypersensitivity reactions to Brucella skin test antigens in guinea pigs.

Cutaneous hypersensitivity responses to brucella antigens of different composition were studied in guinea pigs sensitized by infection with smooth brucella or immunization with killed rough brucella in adjuvant. These animals had circulating antibodies to smooth lipopolysaccharide or protein antigens, respectively. Intradermal skin tests, active cutaneous anaphylaxis, passive cutaneous anaphylaxis, and immunodiffusion tests were performed. Delayed-type hypersensitivity reactions uncomplicated by accompanying antibody-mediated reactions were seen only in infected guinea pigs with protein antigen that was entirely free of lipopolysaccharide. In the adjuvant-immunized animals, the protein antigen evoked overlapping antibody-mediated and delayed-type reactions. Lipopolysaccharide and polysaccharide preparations contained varying amounts of protein components. In infected animals, reactions of these antigens were clearly antibody mediated, but participation of delayed-type hypersensitivity could not be excluded. In adjuvant-immunized animals, the antibody-mediated reaction to the lipopolysaccharide preparation was caused by its protein component.     

Reactivity of neuroglia in guinea pigs sensitized by Mycobacterium tuberculosis

In guinea pigs with experimental allergic encephalomyelitis (EAE) induced by sensitization withMycobacterium tuberculosis cells mixed with petrolatum and Arlacel the reactivity of the glia was shown to change regularly depending on the stage and severity of the pathological process. Changes in reactivity determined by inhibition of glial migration in tissue culture were specific with respect to antigens from nerve tissue. The study of the mechanism of this phenomenon showed that similar changes in reactivity can be induced in a culture of glial cells from intact animals if blood serum or regional lymph gland cells taken from actively sensitized guinea pigs are added. It cannot yet be concluded from the available results that changes in reactivity of the glia to products of brain tissue destruction in guinea pigs with EAE are completely attributable to the action of humoral or cellular elements which penetrate into the CNS from the bloodstream or lymphatic tissue.     

Identification by mass spectrometry of CD8(+)-T-cell Mycobacterium tuberculosis epitopes within the Rv0341 gene product

Identification of Mycobacterium tuberculosis proteins that can provide immunological protection against tuberculosis is essential for the development of a more effective vaccine. To identify new vaccine targets, we have used immunoaffinity chromatography to isolate class I HLA-A*0201-peptide complexes from M. tuberculosis-infected cells and sequenced the isolated peptides by mass spectrometry. From this material, we have identified three peptides derived from a single M. tuberculosis protein that is encoded by the M. tuberculosis Rv0341 gene. Although no known protein encoded by the Rv0341 gene has been described, it is predicted to give rise to a 479-amino-acid protein with a molecular mass of 43.9 kDa. The three peptides identified are all nested and were found to be antigenic, in that they were capable of inducing peptide-specific, CD8(+) T cells from healthy blood donors in vitro and capable of recognizing and lysing M. tuberculosis-infected dendritic cells. This methodology provides a powerful tool for the identification of M. tuberculosis proteins that can be evaluated as potential vaccine candidates.     

Delayed hypersensitivity responses in mice and guinea pigs to Mycobacterium leprae, Mycobacterium vaccae, and Mycobacterium nonchromogenicum cytoplasmic proteins.

Antigenic relationships between Mycobacterium vaccae, M. nonchromogenicum, and M. leprae were examined in mice and guinea pigs injected with M. vaccae or M. nonchromogenicum suspensions. The growth of both organisms in outbred ICR and four inbred mouse strains was followed up to 30 days. M. nonchromogenicum persisted in the livers and spleens of the inbred mice substantially better than did the M. vaccae population in the same mouse strains. A translucent colony variant of M. vaccae isolated from the opossum survived in vivo better than the opaque colony isolated from opossums and cattle. Persistence of M. vaccae and M. nonchromogenicum was not markedly increased in T-cell-depleted (nude) mice. Normal mice infected with increasing numbers of M. vaccae did not develop delayed-type hypersensitivity to the homologous M. vaccae cytoplasmic protein antigen. When heat-killed M. vaccae were incorporated into Freund adjuvant, both mice and guinea pigs developed delayed hypersensitivity to cytoplasmic antigens prepared from M. vaccae, M. nonchromogenicum and M. vaccae vaccines cross-sensitized guinea pigs to the M. leprae cytoplasmic antigens.     

Three protein cocktails mediate delayed-type hypersensitivity responses indistinguishable from that elicited by purified protein derivative in the guinea pig model of Mycobacterium tuberculosis infection

Purified protein derivative (PPD) is a widely used reagent for the diagnosis of Mycobacterium tuberculosis infection. Recently, the molecular composition of PPD was defined, with hundreds of mycobacterial protein representatives making up PPD. Which, if any, of these specific products drive the potency of PPD remains in question. In this study, two proteins (DnaK and GroEL2) previously identified as dominant proteins in PPD were tested for the capacity to induce delayed-type hypersensitivity (DTH) responses in H37Rv-infected or BCG-vaccinated guinea pigs. These two proteins were used in pull-down assays to identify interacting PPD products. Six proteins were identified as interacting partners with DnaK and GroEL2, i.e., Rv0009, Rv0475, Rv0569, Rv0685, Rv2626c, and Rv2632c. These six proteins were tested alone and in combination with DnaK and GroEL2 for the capacity to induce a DTH response in the guinea pig model. From these studies, two cocktails, DnaK/GroEL2/Rv0009 and DnaK/GroEL2/Rv0685, were found to induce DTH responses in H37Rv-infected or BCG-vaccinated guinea pigs that were indistinguishable from DTH responses driven by a PPD injection. The mechanism by which DTH responses were induced was elucidated by histologic examination, analysis of activated CD4(+)/CD8(+) T cells, and cytokine mRNA expression at the site of the DTH response. PPD and the protein cocktails tested induced strong DTH responses in H37Rv-infected guinea pigs. Ex vivo phenotyping of T cells at the DTH site indicated that this response is mediated by activated CD4(+) and CD8(+) T cells, with increases in gamma interferon and tumor necrosis factor alpha, but not interleukin-10, at the site of the DTH response. Our results demonstrate for the first time that the PPD response can be mimicked at the molecular level with defined protein cocktails. The use of this defined product will allow a more thorough understanding of the DTH response and may provide a platform for more rapid and sensitive second-generation skin test reagents for the diagnosis of M. tuberculosis infection.     

结核分枝杆菌Rv0073基因在大肠杆菌中的表达及纯化

目的 构建结核分枝杆菌(MTB) Rv0073基因原核表达载体并进行表达和纯化.方法 以MTB H37Rv基因组DNA为模板,采用聚合酶链反应(PCR)扩增目的基因片段,构建原核表达载体pET26b-Rv0073,经测序确定无误后转化至大肠杆菌(E.coli)感受态细胞BL21中.用聚丙烯酰氨凝胶电泳(SDS-PAGE)方法检测重组蛋白表达,检测异丙基-β-D-硫代半乳糖苷(IPTG)诱导不同时间、不同温度条件下重组蛋白表达量.采用His镍磁珠进行外源蛋白小量纯化.结果 成功构建重组表达质粒,重组蛋白经IPTG诱导后,2h开始明显表达且表达量无时间依赖性,在不同温度诱导下,重组蛋白的表达量随温度的增高而减少.重组蛋白以包涵体形式存在,经His镍磁珠纯化后获得重组蛋白.结论 成功构建并表达Rv0073蛋白,为后续Rv0073的大量纯化及其功能研究奠定了基础.     

Deletion of the Mycobacterium tuberculosis resuscitation-promoting factor Rv1009 gene results in delayed reactivation from chronic tuberculosis

Approximately one-third of the human population is latently infected with Mycobacterium tuberculosis, comprising a critical reservoir for disease reactivation. Despite the importance of latency in maintaining M. tuberculosis in the human population, little is known about the mycobacterial factors that regulate persistence and reactivation. Previous in vitro studies have implicated a family of five related M. tuberculosis proteins, called resuscitation promoting factors (Rpfs), in regulating mycobacterial growth. We studied the in vivo role of M. tuberculosis rpf genes in an established mouse model of M. tuberculosis persistence and reactivation. After an aerosol infection with the M. tuberculosis Erdman wild type (Erdman) or single-deletion rpf mutants to establish chronic infections in mice, reactivation was induced by administration of the nitric oxide (NO) synthase inhibitor aminoguanidine. Of the five rpf deletion mutants tested, one (deltaRv1009) exhibited a delayed reactivation phenotype, manifested by delayed postreactivation growth kinetics and prolonged median survival times among infected animals. Immunophenotypic analysis suggested differences in pulmonary B-cell responses between Erdman- and deltaRv1009-infected mice at advanced stages of reactivation. Analysis of rpf gene expression in the lungs of Erdman-infected mice revealed that relative expression of four of the five rpf-like genes was diminished at late times following reactivation, when bacterial numbers had increased substantially, suggesting that rpf gene expression may be regulated in a growth phase-dependent manner. To our knowledge, deltaRv1009 is the first M. tuberculosis mutant to have a specific defect in reactivation without accompanying growth defects in vitro or during acute infection in vivo.     

Adjuvant activity of carbohydrate analogs of N-acetylmuramyl-L-alanyl-D-isoglutamine on the induction of delayed-type hypersensitivity to azobenzenearsonate-N-acetyl-L-tyrosine in guinea pigs.

The adjuvant activity of 29 kinds of carbohydrate analogs of N-acetylmuramyl-L-alanyl-D-isoglutamine on the induction of delayed-type hypersensitivity to azobenzenearsonate-N-acetyl-L-tyrosine was examined in guinea pigs. It was found that the D-manno- and D-galacto-type muramyldipeptides were as active as the D-gluco-type muramyldipeptide (MDP). The structural requirement of the carbohydrate moiety of analogs of N-acetylmuramyl-L-alanyl-D-isoglutamine is discussed.     

Magnetic resonance imaging of pulmonary lesions in guinea pigs infected with Mycobacterium tuberculosis

We utilized magnetic resonance imaging to visualize lesions in the lungs of guinea pigs infected by low-dose aerosol exposure to Mycobacterium tuberculosis. Lesions were prominent in such images, and colorized three-dimensional reconstructions of images revealed a very uniform distribution in the lungs. Lesion numbers after 1 month were approximately similar to the aerosol exposure algorithm, suggesting that each was established by a single bacterium. Numbers of lesions in unprotected and vaccinated animals were similar over the first month but increased thereafter in the control animals, indicating secondary lesion development. Whereas lesion sizes increased progressively in control guinea pigs, lesions remained small in BCG-vaccinated animals. A prominent feature of the disease pathology in unprotected animals was rapid and severe lymphadenopathy of the mediastinal lymph node cluster, which is paradoxical given the strong state of cellular immunity at this time. Further development of this technical approach could be very useful in tracking lesion size, number, and progression in the search for new tuberculosis vaccines.     

Induction of delayed-type hypersensitivity to Mycobacterium leprae in healthy individuals.

Delayed-type hypersensitivity (DTH) to a soluble Mycobacterium leprae skin test antigen (SML) was successfully induced in healthy volunteers following immunization with 2 X 10(8) killed armadillo-derived M. leprae. No better sensitization was obtained by a mixture of live BCG and killed M. leprae. The relative specificity of the DTH reaction to SML has been demonstrated in this study, since little cross-reactivity was observed to PPD, after immunization with BCG ro M, leprae alone, or combined. Moreover, armadillo-derived M. leprae readily induced a specific hypersensitivity with the time course DTH response associated with protective immunity suggesting that this bacterial preparation may be a candidate for an effective anti-leprosy vaccine.     

Development of delayed-type hypersensitivity during Mycobacterium lepraemurium infection in mice.

Various preparations of Mycobacterium lepraemurium were used to elicit delayed-type hypersensitivity in the footpad of mice infected with this organism. With a sonicated preparation of the mycobacterium, a significant increase in footpad swelling was elicited in mice infected with M. lepraemurium 5 weeks previously, but not in BCG-infected animals or uninfected controls. This footpad reaction was shown to peak at 24 h and to be associated with an infiltration of mononuclear cells. The kinetics of footpad swelling, its association with lymphoproliferation, and its dependence on T lymphocytes were each examined. The results support the hypothesis that this is a delayed-type hypersensitivity reaction. The ability to transfer this reactivity to normal mice with cells but not serum offers further confirmation that this hypersensitivity is dependent on cell-mediated immunological mechanisms rather than humoral antibody. The relevance of this to the study of the immunological response of mice to murine leprosy is discussed.     

Boosting with poxviruses enhances Mycobacterium bovis BCG efficacy against tuberculosis in guinea pigs

Tuberculosis is rising in the developing world due to poor health care, human immunodeficiency virus type 1 infection, and the low protective efficacy of the Mycobacterium bovis BCG vaccine. A new vaccination strategy that could protect adults in the developing world from tuberculosis could have a huge impact on public health. We show that BCG boosted by poxviruses expressing antigen 85A induced unprecedented 100% protection of guinea pigs from high-dose aerosol challenge with Mycobacterium tuberculosis, suggesting a strategy for enhancing and prolonging the efficacy of BCG.     

Rapid accumulation of eosinophils in lung lesions in guinea pigs infected with Mycobacterium tuberculosis

Guinea pig eosinophils were positively identified in bronchoalveolar lavage populations and in the lung granulomas of Mycobacterium tuberculosis-infected guinea pigs. It is possible that the rapid influx of these cells, and their subsequent degranulation during acute pulmonary tuberculosis, may play a key role in the susceptibility of this animal model.     

T-cell responses to the Mycobacterium tuberculosis-specific antigen ESAT-6 in Brazilian tuberculosis patients

The Mycobacterium tuberculosis-specific ESAT-6 antigen induces highly potent T-cell responses and production of gamma interferon (IFN-gamma), which play a critical role in protective cell-mediated immunity against tuberculosis (TB). In the present study, IFN-gamma secretion by peripheral blood mononuclear cells (PBMCs) in response to M. tuberculosis ESAT-6 in Brazilian TB patients was investigated in relation to clinical disease types, such as pleurisy and cavitary pulmonary TB. Leprosy patients, patients with pulmonary diseases other than TB, and healthy donors were assayed as control groups. Sixty percent of the TB patients indeed recognized M. tuberculosis ESAT-6, as did 50% of the leprosy patients and 60% of the non-TB controls. Nevertheless, the levels of IFN-gamma in response to the antigen ESAT, but not to antigen 85B (Ag85B) and purified protein derivative (PPD), were significantly lower in controls than in patients with treated TB or pleural or cavitary TB. Moreover, according to Mycobacterium bovis BCG vaccination status, only 59% of the vaccinated TB patients responded to ESAT in vitro, whereas 100% of them responded to PPD. Both CD4 and CD8 T cells were able to release IFN-gamma in response to ESAT. The present data demonstrate the specificity of ESAT-6 of M. tuberculosis and its ability to discriminate TB patients from controls, including leprosy patients. However, to obtain specificity, it is necessary to include quantitative IFN-gamma production in response to the antigen as well, and this might limit the use of ESAT-6-based immunodiagnosis of M. tuberculosis infection in an area of TB endemicity.