Human T-cell clones recognize a major M. leprae protein antigen expressed in E. coli

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. As with other intracellular parasites, protective immunity is dependent on T cells and cell-mediated immunity. In animal models, immunization with killed armadillo-derived M. leprae elicits strong T-cell responses, delayed-type hypersensitivity and protection against viable challenge. We have recently shown that killed M. leprae can induce delayed-type hypersensitivity in healthy human volunteers. Identification of the M. leprae antigens that are recognized by T cells and may be involved in protection has been hampered by the inability to cultivate the organism in vitro and by difficulties in antigen purification from limited quantities of armadillo-derived bacillus. Because genes for the major protein antigens of M. leprae as seen by mouse monoclonal antibodies have been isolated, it has become possible to test whether these individual antigens are recognized by T cells. We screened crude lambda gtll phage lysates of Escherichia coli containing individual M. leprae antigens using M. leprae-specific T-cell clones isolated from M. leprae-vaccinated volunteers. Using this method, we find that nearly half of the M. leprae-specific T-cell clones are stimulated to proliferate by lysates containing an epitope of a M. leprae protein of relative molecular mass 18,000 (18K).     

Genes for the major protein antigens of the leprosy parasite Mycobacterium leprae

Leprosy, a chronic infectious disease afflicting between 10 and 15 million people, is caused by the obligate intracellular parasite Mycobacterium leprae. Although M. leprae was the first identified bacterial pathogen of man, basic biochemical, immunological, diagnostic and therapeutic investigations have been severely limited because it remains one of the few human pathogens that have not been cultured in vitro. An M. leprae recombinant DNA expression library was constructed to provide a source of genes encoding proteins relevant for such studies. Monoclonal antibodies directed against M. leprae specific antigens have been used to isolate the genes encoding the five most immunogenic protein antigens of the leprosy bacillus. We report here that M. leprae specific epitopes recognized by all of 13 monoclonal antibodies tested were produced by recombinant phage in Escherichia coli.     

Cloned suppressor T cells from a lepromatous leprosy patient suppress Mycobacterium leprae reactive helper T cells

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. A characteristic feature of the disease is its remarkable spectrum of clinical symptoms correlating with the cellular immune responsiveness of the patient. At one pole of this spectrum are tuberculoid patients displaying both acquired cell-mediated immunity and delayed type hypersensitivity against the bacillus. At the other pole are lepromatous patients which show a specific T-cell unresponsiveness against M. leprae. In between those two poles variable degrees of tuberculoid and lepromatous features may be seen in borderline leprosy patients. Thus far, studies on the mechanism of the antigen specific unresponsiveness in lepromatous leprosy have been contradictory and difficult to interpret, probably because of the use of heterogeneous cell populations in those experiments. We have now succeeded in cloning M. leprae stimulated T-helper (TH) as well as T-suppressor (TS) cells from a borderline lepromatous patient. The TS-clones of this patient specifically suppress responses of peripheral TH cells as well as TH clones induced by both M. leprae and other mycobacteria, but not unrelated antigen or mitogen. These TS cells also completely suppress TH cell responses against a M. leprae specific protein with a relative molecular mass of 36,000 (36K), suggesting the presence of a suppression inducing determinant on this 36K M. leprae protein.     

The heparin-binding haemagglutinin of M. tuberculosis is required for extrapulmonary dissemination.

Tuberculosis remains the world's leading cause of death due to a single infectious agent, Mycobacterium tuberculosis, with 3 million deaths and 10 million new cases per year. The infection initiates in the lungs and can then spread rapidly to other tissues. The availability of the entire M. tuberculosis genome sequence and advances in gene disruption technologies have led to the identification of several mycobacterial determinants involved in virulence. However, no virulence factor specifically involved in the extrapulmonary dissemination of M. tuberculosis has been identified to date. Here we show that the disruption of the M. tuberculosis or Mycobacterium bovis Bacille Calmette-Guérin (BCG) hbhA gene encoding the heparin-binding haemagglutinin adhesin (HBHA) markedly affects mycobacterial interactions with epithelial cells, but not with macrophage-like cells. When nasally administered to mice, the mutant strains were severely impaired in spleen colonization, but not in lung colonization. Coating wild-type mycobacteria with anti-HBHA antibodies also impaired dissemination after intranasal infection. These results provide evidence that adhesins such as HBHA are required for extrapulmonary dissemination, and that interactions with non-phagocytic cells have an important role in the pathogenesis of tuberculosis. They also suggest that antibody responses to HBHA may add to immune protection against tuberculosis.     

Genetically restricted suppressor T-cell clones derived from lepromatous leprosy lesions

Leprosy is a spectral disease in which immune responses to Mycobacterium leprae correlate with the clinical, bacteriological and histopathological manifestations of disease, so study of its pathology provides insights into immunoregulatory mechanisms in man. At the tuberculoid pole, patients have few lesions in the skin which contain rare organisms and are able to mount strong cell-mediated immune responses to M. leprae antigens. In contrast, at the lepromatous pole, patients have disseminated skin lesions containing large numbers of acid-fast bacilli and are selectively unresponsive to antigens of M. leprae. M. leprae-induced suppressor cells derived from peripheral blood have been reported to be active in vitro, yet their in vivo significance has remained unclear. Because the focal point of the immune response to M. leprae is the skin lesion consisting of lymphocytes and macrophages, we have recently developed methods for isolating lymphocytes from skin biopsies of leprosy patients. We report here that two T8 clones derived from lepromatous leprosy skin biopsies, in the presence of lepromin, suppress concanavalin A (Con-A) responses both of peripheral blood mononuclear cells and of T4 clones in an HLA-D (HLA, histocompatibility locus antigen)-restricted manner. Moreover, these T8 clones suppressed responses of HLA-D-matched, but not HLA-D-mismatched antigen-responsive T4 clones to M. leprae antigens, indicating that T-cell suppression is major histocompatibility complex (MHC)-restricted at some level in man.     

Mycobacterium leprae-specific protein antigens defined by cloned human helper T cells

Leprosy displays a remarkable spectrum of symptoms correlating with the T-cell-mediated immune reactivity of the host against the causative organism, Mycobacterium leprae. At one pole of this spectrum are lepromatous leprosy patients showing a M. leprae-specific T-cell unresponsiveness; at the other are tuberculoid leprosy patients displaying both acquired immunity and delayed-type hypersensitivity against M. leprae which are thought to be conferred by helper T (Th) cells. Because well-defined M. leprae antigens are crucial for the prevention and control of leprosy, we have cloned M. leprae-reactive T cells (TLC) of the helper phenotype from a tuberculoid leprosy patient. As reported here, these TLC show an unexpected diversity in the recognition of M. leprae and related mycobacteria, which is different from that exhibited by monoclonal antibodies. Half of these TLC are completely or almost M. leprae-specific, whereas the other half are cross-reactive with most or all other mycobacteria. A M. leprae protein of relative molecular mass (Mr) 36,000 (36K) defined by a M. leprae-specific monoclonal antibody stimulates 4 out of 6 TLC tested. Each of these TLC recognizes a different antigenic determinant, one of which is M. leprae-specific. The previous paper describes other M. leprae-specific T-cell clones half of which recognize an epitope on a M. leprae protein of Mr 18 K.     

T-cell conditioned media reverse T-cell unresponsiveness in lepromatous leprosy

In some subjects the infective agent of leprosy, Mycobacterium leprae, causes disseminated (lepromatous) disease. Such subjects have a major role in the transmission of the disease and show deficient T-cell responses both in vivo and in vitro to M. leprae, but not to other antigens. Numerous studies have recently shown that T cells with functional capabilities after initial triggering with antigen can be maintained in a state of continuous proliferation in vitro when cultured in medium containing interleukin 2 (IL-2). Here we have studied the effect of IL-2 rich T-cell conditioned medium on lepromatous peripheral blood mononuclear cells. Our results show that although lepromatous T cells fail to produce IL-2 after exposure to M. leprae they can respond by proliferation to M. leprae in the presence of T-cell conditioned medium, suggesting that the unresponsiveness in lepromatous leprosy results from a deficiency in the production of IL-2 or related factors and not a lack of M. leprae-reactive T cells.     

Humoral and cell-mediated immune responses to live recombinant BCG-HIV vaccines

Several viral and bacterial live recombinant vaccine vehicles are being developed to produce a new generation of vaccines against a broad spectrum of infectious diseases. The human tuberculosis vaccine Mycobacterium bovis bacillus Calmette-Guerin (BCG) has features that make it a particularly attractive live recombinant vaccine vehicle. BCG and other mycobacteria are highly effective adjuvants, and the immune response to mycobacteria has been studied extensively. With nearly two billion immunizations, BCG has a long record of safe use in man. It is one of the few vaccines that can be given at birth, it engenders long-lived immune responses with only a single dose, and there is a worldwide distribution network with experience in BCG vaccination. Recently developed molecular genetic tools and methods for mycobacteria have provided the means to introduce foreign genes into BCG. Here we report that a variety of human immunodeficiency virus type 1 polypeptides can be expressed in BCG recombinants under the control of the mycobacterial hsp70 promoter and that the foreign polypeptides produced in BCG can induce antibody and T-cell responses. These results demonstrate that BCG can be used as a live recombinant vaccine vehicle to induce immune responses to pathogen proteins produced by the bacillus.     

Therapy of tuberculosis in mice by DNA vaccination.

Mycobacterium tuberculosis continues to kill about 3 million people every year, more than any other single infectious agent. This is attributed primarily to an inadequate immune response towards infecting bacteria, which suffer growth inhibition rather than death and subsequently multiply catastrophically. Although the bacillus Calmette-Guerin (BCG) vaccine is widely used, it has major limitations as a preventative measure. In addition, effective treatment requires that patients take large doses of antibacterial drug combinations for at least 6 months after diagnosis, which is difficult to achieve in many parts of the world and is further restricted by the emergence of multidrug-resistant strains of M. tuberculosis. In these circumstances, immunotherapy to boost the efficiency of the immune system in infected patients could be a valuable adjunct to antibacterial chemotherapy. Here we show in mice that DNA vaccines, initially designed to prevent infection, can also have a pronounced therapeutic action. In heavily infected mice, DNA vaccinations can switch the immune response from one that is relatively inefficient and gives bacterial stasis to one that kills bacteria. Application of such immunotherapy in conjunction with conventional chemotherapeutic antibacterial drugs might result in faster or more certain cure of the disease in humans.     

结核分枝杆菌Rv2450基因真核表达质粒的构建及表达

构建结核分枝杆菌Rv2450基因真核表达载体.PCR扩增Rv2450基因,测序正确后克隆入真核表达载体pCDNA3.1（-）,重组质粒酶切鉴定正确后以阳离子聚合物转染P815细胞,分别以RT-PCR方法检测mRNA表达和间接免疫荧光技术检测目的蛋白的表达.结果构建了重组质粒pCDNA-Rv2450,RT-PCR结果证明Rv2450可在P815细胞中转录,用间接免疫荧光检测,表达有Rv2450蛋白的细胞着染.成功构建了结核分枝杆菌Rv2450基因的真核表达载体pCDNA-Rv2450,Rv2450基因可以在P815细胞中表达.     

麻风杆菌多途径接种裸鼠的研究

用麻风杆菌经静脉内、后足垫和耳皮下多途径接种裸鼠。在裸鼠接种菌后307、334、497、和625天分别剖杀1—2只裸鼠,取接种足垫和耳部皮损、胭窝淋巴结、鼻、坐骨神经、尾部皮肤损害、舌、肝、脾、肺、肾和心脏称重后制成匀浆,作菌计数。同时各取部分组织作组织病理学检查。实验结果表明,裸鼠经过多途径接种麻风杆菌后,其菌量增殖明显高于以往报告的单途径接种方法所取得的结果。尤其在接种部位的后足垫及耳部,最高菌量分别达到2.85×10~11/g(497天)和6.28×10~12/g(625天)。虽然麻风杆菌的世代时间近似,但对数生长期时间明显延长。结果也表明,多途径接种方法能使裸鼠发生更严重的病变和系统性播散。组织病理学检查表明,经抗酸染色的足垫组织切片中,在横纹肌、血管壁内皮细胞和血管中均可见到大量抗酸杆菌(AFB)及菌团,皮神经束膜内外和雪旺氏细胞中也找到了许多AFB。最终在整个足垫组织中充满了AFB。HE染色呈瘤型麻风进行性病变,肉芽肿浸润由含大量AFB的泡沫细胞和巨噬细胞构成,直至泡沫细胞组成的麻风瘤病变完全代替了正常结构。各个部位相比较,低体温部位如足垫、耳、鼻及尾部仍然是麻风菌繁殖的优势部位。横纹肌及外周神经是麻风菌好侵犯之处,也显示了麻风菌在裸鼠体内感染的特点。     

结核分枝杆菌mpt64基因真核表达载体的构建及表达

构建结核分枝杆菌分泌蛋白mpt64基因真核表达载体。通过PCR法从MTBH37Rv株基因组中扩增mpt64基因,插入pGEM-T-easy载体中,序列测定正确后,将其亚克隆到真核表达载体pcDNA3．1（-）,重组质粒酶切鉴定正确后,以Lipo-fectamine2000转染COS-7细胞后,分别以RT-PCR方法检测mRNA表达和间接免疫荧光技术检测目的蛋白的表达。构建了真核表达载体pcDNA-mpt64,RT-PCR结果证明mpt64基因可在COS-7细胞中转录,用间接免疫荧光检测,有表达mpt64蛋白的细胞着染。结果表明,构建结核分枝杆菌mpt64基因的真核表达载体pcDNA-mpt64成功,mpt64基因可以在COS-7细胞中表达。     

基于生物信息学的结核杆菌候选药靶基因的分析筛选

结核分支杆菌(Mycobacterium tuberculosis,MTB)是结核病的致病菌,MTB基因组和蛋白质组的研究为筛选新的抗MTB药物提供了可能．鉴于前人所做的工作,目前比较一致的意见是：与MTB细胞壁的合成相关的酶类,尤其是与脂肪酸合成和降解相关的酶类,是筛选抗结核杆菌药物的理想靶标．选取与脂肪酸合成、分解、转运相关的187个基因,分析blast结果,剔除与人和其他生物(如大肠杆菌等)蛋白序列相似性大于60％的基因．利用相关生物软件挑选出至少有一个糖基位点且具有溶剂可及性的抗原基因9个．本研究为以后分析药靶基因编码蛋白质的三维结构、利用现有的生物技术和有关的化合物对候选药靶进行功能研究、在细胞水平加以验证打下基础．     

结核分枝杆菌的抗逆性与致病性研究进展

结核病是由结核分枝杆菌(Mycobacterium tuberculosis,MTB)复合群感染导致的人畜共患传染病,在新型冠状病毒感染暴发前是全球死亡人数最多的单一传染病。结核病致死率高,主要原因是其致病菌结核分枝杆菌具有极强的毒力且可以抵御多种外界环境胁迫因子。本文主要介绍结核分枝杆菌对理化胁迫的耐受性,以及结核分枝杆菌的耐药性、致病性与免疫性,为深入研究结核病的发病机制和研发新型的抗结核药物提供理论指导。     

The DNA sequence and analysis of human chromosome 13

Chromosome 13 is the largest acrocentric human chromosome. It carries genes involved in cancer including the breast cancer type 2 (BRCA2) and retinoblastoma (RB1) genes, is frequently rearranged in B-cell chronic lymphocytic leukaemia, and contains the DAOA locus associated with bipolar disorder and schizophrenia. We describe completion and analysis of 95.5 megabases (Mb) of sequence from chromosome 13, which contains 633 genes and 296 pseudogenes. We estimate that more than 95.4% of the protein-coding genes of this chromosome have been identified, on the basis of comparison with other vertebrate genome sequences. Additionally, 105 putative non-coding RNA genes were found. Chromosome 13 has one of the lowest gene densities (6.5 genes per Mb) among human chromosomes, and contains a central region of 38 Mb where the gene density drops to only 3.1 genes per Mb.     

Sequence and analysis of chromosome 2 of the plant Arabidopsis thaliana

Arabidopsis thaliana (Arabidopsis) is unique among plant model organisms in having a small genome (130-140 Mb), excellent physical and genetic maps, and little repetitive DNA. Here we report the sequence of chromosome 2 from the Columbia ecotype in two gap-free assemblies (contigs) of 3.6 and 16 megabases (Mb). The latter represents the longest published stretch of uninterrupted DNA sequence assembled from any organism to date. Chromosome 2 represents 15% of the genome and encodes 4,037 genes, 49% of which have no predicted function. Roughly 250 tandem gene duplications were found in addition to large-scale duplications of about 0.5 and 4.5 Mb between chromosomes 2 and 1 and between chromosomes 2 and 4, respectively. Sequencing of nearly 2 Mb within the genetically defined centromere revealed a low density of recognizable genes, and a high density and diverse range of vestigial and presumably inactive mobile elements. More unexpected is what appears to be a recent insertion of a continuous stretch of 75% of the mitochondrial genome into chromosome 2.     

Lymphocyte suppression in leprosy induced by unique M. leprae glycolipid

Leprosy remains a significant medical and social problem in many developing countries. The varied forms of the disease form a spectrum. At one pole, tuberculoid leprosy, patients develop high levels of cell-mediated immunity which results in the killing and clearing of bacilli in the tissues. At the lepromatous pole, patients exhibit a selective immunological unresponsiveness to antigens of Mycobacterium leprae so that the organisms inexorably multiply in the skin. We have suggested that in lepromatous leprosy one or a small number of unique antigenic determinants present on M. leprae might induce specific suppressor cells that inhibit the reactivity of helper T-cell clones capable of recognizing other specific or cross reactive determinants. Although unique epitopes have been identified by monoclonal antibodies on a small number of M. leprae proteins, the only unique species of antigen present in M. leprae, and not on any other species of mycobacteria so far examined, is a phenolic glycolipid (gly-I). We show here that this unique antigen of M. leprae is capable of inducing suppression of mitogenic responses of lepromatous patients' lymphocytes in vitro and provide evidence that the suppressor T cells recognize the specific terminal trisaccharide moiety.     

Cloning of the mycobacterial epitope recognized by T lymphocytes in adjuvant arthritis

Adjuvant arthritis (AA) is a chronic disease inducible in rats by immunization with an antigen of Mycobacterium tuberculosis. After the isolation of arthritogenic T-cell lines and clones, it became possible to demonstrate that the critical M. tuberculosis antigen contained an epitope cross-reactive with a self-antigen in joint cartilage. Like AA rats, patients suffering from rheumatoid arthritis demonstrated specific T-lymphocyte reactivity to the M. tuberculosis fraction containing the cross-reactive epitope. To characterize the critical M. tuberculosis epitope we used AA T-cell clones to screen mycobacterial antigens expressed in Escherichia coli and genetically engineered truncated proteins and synthetic peptides. The AA T-cell clones recognized an epitope formed by the amino acids at positions 180-188 in the sequence of a Mycobacterium bovis BCG antigen. Administration of this antigen to rats induced resistance to subsequent attempts to produce AA.     

Transposition of an antibiotic resistance element in mycobacteria

Bacterial resistance to antibiotics is often plasmid-mediated and the associated resistance genes encoded by transposable elements. Mycobacteria, including the human pathogens Mycobacterium tuberculosis and M. leprae, are resistant to many antibiotics, and their cell-surface structure is believed to be largely responsible for the wide range of resistance phenotypes. Antibiotic-resistance plasmids have so far not been implicated in resistance of mycobacteria to antibiotics. Nevertheless, antibiotic-modifying activities such as aminoglycoside acetyltransferases and phosphotransferases have been detected in fast-growing species. beta-lactamases have also been found in most fast- and slow-growing mycobacteria. To date no mycobacterial antibiotic-resistance genes have been isolated and characterized. We now report the isolation, cloning and sequencing of a genetic region responsible for resistance to sulphonamides in M. fortuitum. This region also contains an open reading frame homologous to one present in Tn1696 (member of the Tn21 family) which encodes a site-specific integrase. The mycobacterial resistance element is flanked by repeated sequences of 880 base pairs similar to the insertion elements of the IS6 family found in Gram+ and Gram- bacteria. The insertion element is shown to transpose to different sites in the chromosome of a related fast-growing species, M. smegmatis. The characterization of this element should permit transposon mutagenesis in the analysis of mycobacterial virulence and related problems.     

Evolutionary genomics: codon volatility does not detect selection

Plotkin et al. introduce a method to detect selection that is based on an index called codon volatility and that uses only the sequence of a single genome, claiming that this method is applicable to a large range of sequenced organisms. Volatility for a given codon is the ratio of non-synonymous codons to all sense codons accessible by one point mutation. The significance of each gene's volatility is assessed by comparison with a simulated distribution of 10(6) synonymous versions of each gene, with synonymous codons drawn randomly from average genome frequencies. Here we re-examine their method and data and find that codon volatility does not detect selection, and that, even if it did, the genomes of Mycobacterium tuberculosis and Plasmodium falciparum, as well as those of most sequenced organisms, do not meet the assumptions necessary for application of their method.