Mycobacterium bovis, the causative agent of bovine tuberculosis, has no known geographical boundaries. M. bovis infection occurs in diverse groups of animals, which include farm animals of economic importance, wildlife, and humans (1,16,17,31). Despite these ominous features of M. bovis, to date there have been only projected global estimates of the disease burden. There has been no international effort to determine the actual disease burden, owing to the nonavailability of a reliable user-friendly technology for early detection of M. bovis in clinical samples. Bovine tuberculosis has been on the increase in developed countries and continues to occur in developing countries (6, 14, 16). In Africa, approximately 85% of cattle and 82% of the human population live in areas where the disease is prevalent (6, 26). There are limited reports from India (24, 27, 38, 40) and from underdeveloped countries (6, 16) relating to the prevalence of and infection with M. bovis in cattle. Detection of M. bovis in bovine samples has become necessary, as infected animals are potentially capable of infecting humans (zoonotic tuberculosis) (6). Besides M. bovis, transmission of M. tuberculosis from infected humans to animals and back has been reported (reverse zoonosis) (10,12,41). Hence, M. bovis and M. tuberculosis pose a potential health hazard to both animals and humans (1,15,43).The identification of the closely related members of the Mycobacterium tuberculosis complex (MTC) has remained a challenging task in diagnostic laboratories (7,30,44). MTC includes a variety of closely related mycobacteria, namely, M. tuberculosis, M. bovis, M. canetti, M. africanum, and M. microti. A panel of classical tests based on microbiological features such as growth rate and phenotypic and biochemical characteristics has conventionally been utilized to distinguish members of MTC (30). However, these tests are slow, cumbersome, unreliable, and time-consuming. The high degree of variability among these tests warrants the development of molecular biological tools for identification of MTC members. In this regard, multiple gene targets have been used to date to detect and differentiate genetically identical species, such as M. tuberculosis and M. bovis. The gene targets include pncA (2,29,36), gyrB (5), oxyR (42), and katG (19). Huard et al. (21) and Richter et al. (33) have targeted multiple loci and genes to differentiate M. tuberculosis from M. bovis. However, to date no single accepted protocol(s) that can unambiguously differentiate all members of the MTC is available. Identification of the etiological agent belonging to MTC is important for determination of the origin and reservoirs of infection and also for implementation of appropriate public health measures.Our laboratory earlier described a PCR-restriction fragment length polymorphism (RFLP) method utilizing the hupB gene,

Mycobacterium tuberculosis inhibits gamma interferon (IFN-␥Mycobacterium tuberculosis is a facultative intracellular pathogen that resides and multiplies within human macrophages. Gamma interferon (IFN-␥), the predominant inducer of macrophage-mediated microbicidal functions (36), has been shown to be required for the prevention of progressive M. tuberculosis infection (13,19). In tuberculosis patients, IFN-␥ has been detected in CD4ϩ T cells and culture supernatants (8) as well as in infectious foci (7). Despite the availability of IFN-␥, the immune system is unable to eradicate the infection, indicating that M. tuberculosis selectively inhibits macrophage responsiveness to 61). This reduced response results in the inefficient induction of IFN-␥-inducible genes, such as major histocompatibility complex class II and others (24, 61).IFN-␥ binds to its cell surface receptor, IFN-␥R, which consists of two heterodimeric subunits, IFN-␥R1 (␣, ligand binding) and IFN-␥R2 (␤, signaling subunit) (6). The IFN-␥R is MATERIALS AND METHODSSubjects. Informed consent obtained from a cohort of 13 pulmonary tuberculosis (PTB) patients of the outpatient department of LRS Hospital of Tuberculosis and Respiratory Diseases and 16 laboratory personnel of the Biotechnology Department, AIIMS, New Delhi, India, were included in the study. All patients were administered standard antitubercular therapy (ATT). Six of them were available for the assessment of the level of expression of IFN-␥R1 at various time points prior to and after therapy. Scrutinizing patient clinical histories, physical examinations, and laboratory investigations ruled out the occurrence of concom-* Corresponding author. Mailing address:

No abstract

No abstract

Pooled polyvalent sera from lepromatous leprosy patients were used to screen a Agtll recombinant DNA expression library of Mycobacterium leprae in order to identify the relevant antigens recognized by the human immune response. Of the 300,000 phages screened, 4 clones were identified that coded for fusion proteins of the same molecular mass. The fusion protein from clone LSR2 was tested for immunoreactivity in assays using peripheral blood cells and sera from 11 laboratory personnel and 105 patients across the leprosy spectrum. LSR2 protein appears to be predominantly a T-cell antigen. It evokes similar lymphoproliferative responses as the native bacillus both at the individual level and in the leprosy spectrum as a whole. Though only 50% of patient sera with anti-M. leprae antibodies reacted with the fusion protein, the pattern of reactivity in the antibody responses was also similar for the various clinical types. The coding regions of clones LSR1 and LSR2 are identical. They show no homology with sequences stored in data banks and encode a protein of 89 amino acids with a calculated molecular mass of -10 kDa.Leprosy is a chronic infectious disease caused by noncultivable Mycobacterium leprae. The clinicopathological spectrum observed in this disease reflects the variability in the host immune responses to the pathogen (1). Protective immunity is mainly effected by cellular responses as evidenced by the presence of optimal T-cell functions in the localized paucibacillary form of tuberculoid (TT) leprosy. In contrast, the generalized multibacillary lepromatous (LL) leprosy shows antigen-specific T-cell anergy concomitant with the presence of high levels of specific and crossreactive mycobacterial antibodies (2, 3). Young et al. (4) constructed a genomic library of M. leprae in the Agtll expression vector. By using monoclonal antibodies (mAbs) as screening reagents, genes coding for 12-, 18-, 28-, 36-, and 65-kDa proteins of M. leprae were identified. Whereas some of these have been shown to share homology with the heat shock proteins of various species (5-8), the 18-kDa protein has been found to be stimulatory for human T helper clones (9) and for peripheral blood cells from healthy contacts (10).With a view to identifying genes expressing proteins recognized by the human immune response to natural M. leprae infection, we have used polyclonal antibodies obtained from pooled sera of lepromatous patients to screen the Agtll DNA expression library. We have identified four clones coding for a fusion protein of the same molecular mass. § It appears to be a dominant T-cell antigen and mimics the native bacillus in lymphoproliferative responses of all clinical types of leprosy patients. It is also recognized by the sera of 50-70o of the patients having anti-M. leprae antibodies. MATERIALS AND METHODSSubjects. The study included 105 leprosy patients attending the Hansen disease clinic of Safdarjung Hospital (New Delhi) and 11 healthy laboratory personnel with >3 years of constant contact with patients. The type...

The entry and survival of Mycobacterium tuberculosis (Mtb) within host cells is orchestrated partly by an essential histone-like protein HupB (Rv2986c). Despite being an essential drug target, the lack of structural information has impeded the development of inhibitors targeting the indispensable and multifunctional C-terminal domain (CTD) of HupB. To bypass the requirement for structural information in the classical drug discovery route, we generated a panel of DNA aptamers against HupB protein through systemic evolution of ligands by exponential (SELEX) enrichment. Two G-quadruplex-forming high-affinity aptamers (HupB-4T and HupB-13T) were identified, each of which bound two distinct sites on full-length HupB, with an estimated KD of ∼1.72 μM and ∼0.17 μM, respectively, for the high-affinity sites. While HupB-4T robustly inhibited DNA-binding activity of HupB in vitro, both the aptamers recognized surface-located HupB and significantly blocked Mtb entry into THP-1 monocytic cells (p < 0.0001). In summary, DNA aptamers generated in this study block DNA-binding activity of HupB, inhibit virulent Mtb infection in host cells, and demonstrate aptamers to be inhibitors of HupB functions. This study also illustrates the utility of SELEX in developing inhibitors against essential targets for whom structural information is not available.

No abstract

The epidermal changes that occur in human cutaneous immune responses have been investigated in the tuberculin reaction and in the lesions of tuberculoid and lepromatous leprosy and cutaneous leishmaniasis. In each situation, there was a dermal accumulation of monocytes and T cells, and the epidermis exhibited thickening. In the tuberculin response, the thickness of the epidermis sometimes doubled in 48-72 hr, and this was attributed to increases in both size and number of keratinocytes. In addition, the phenotype of the keratinocytes changed from la-to Ia'.Similar changes in keratinocyte Ia-antigen expression occurred in the epidermis overlying untreated tuberculoid leprosy and cutaneous leishmaniasis lesions, but not in lepromatous leprosy. We suggest that one or more epidermal growth factors may be generated in the course of a delayed immune reaction in the dermis.The cutaneous lesions of lepromatous leprosy are characterized by a sparse lymphocytic infiltrate and foamy macrophages laden with Mycobacteria leprae. In contrast, tuberculoid leprosy lesions exhibit large numbers of lymphocytes, granuloma formation, and the absence of appreciable numbers of intracellular bacteria (1, 2). Monoclonal antibodies to human leukocytes and their subsets have been used to determine the phenotype of the cells in dermal infiltrates (3-6). These studies showed that in lepromatous lesions there is not only a marked reduction in the numbers of T cells (compared to tuberculoid lesions) but also a selective absence of T lymphocytes of the OKT4+ subset.We wanted to establish whether this represented a specific unresponsiveness to M. leprae antigens or a more general defect in the emigration and accumulation of OKT4+ T cells. For this purpose we generated tuberculin reactions in the dermis of lepromatous patients by use of the purified protein derivative of tuberculin (PPD). During the course of these studies, we observed striking changes in the thickness and Ia-antigen expression of the epidermis overlying the PPDinduced delayed-type hypersensitivity (DTH) lesions. Similar changes also occur in the epidermis overlying the lesions of patients with tuberculoid leprosy and cutaneous leishmaniasis. MATERIALS AND METHODSThe Generation of DTH Response to PPD. After informed consent was obtained, we evaluated the DTH response to 5 units of PPD in 90 Indian leprosy patients from New Delhi (a highly endemic area for tuberculosis). The study group included 25 lepromatous (LL) (see diagnosis below), 10 borderline lepromatous (BL), and 55 tuberculoid (BT and TT) patients and 50 non-leprosy control individuals. Sex and age distribution were as follows: BL and LL patients, 3 female and 32 male, ages 16-68 (median 30) years; BT and TT patients, 12 female and 43 male, ages 11-68 (median 30) years; non-leprosy controls, 15 female and 35 male, ages 15-65 (median 28) years. The leprosy patients were examined in collaboration with A. K. Sharma and R. S. Mishra (Department of Dermatology, Safdarjung Hospital, New Delhi). Antigen was injected i...