A simple assay to quantify mycobacterial lipid antigen-specific T cell receptors in human tissues and blood.

T cell receptors (TCRs) encode the history of antigenic challenge within an individual and have the potential to serve as molecular markers of infection. In addition to peptide antigens bound to highly polymorphic MHC molecules, T cells have also evolved to recognize bacterial lipids when bound to non-polymorphic CD1 molecules. One such subset, germline-encoded, mycolyl lipid-reactive (GEM) T cells, recognizes mycobacterial cell wall lipids and expresses a conserved TCR-ɑ chain that is shared among genetically unrelated individuals. We developed a quantitative PCR assay to determine expression of the GEM TCR-ɑ nucleotide sequence in human tissues and blood. This assay was validated on plasmids and T cell lines. We tested blood samples from South African subjects with or without tuberculin reactivity or with active tuberculosis disease. We were able to detect GEM TCR-ɑ above the limit of detection in 92% of donors but found no difference in GEM TCR-ɑ expression among the three groups after normalizing for total TCR-ɑ expression. In a cohort of leprosy patients from Nepal, we successfully detected GEM TCR-ɑ in 100% of skin biopsies with histologically confirmed tuberculoid and lepromatous leprosy. Thus, GEM T cells constitute part of the T cell repertoire in the skin. However, GEM TCR-ɑ expression was not different between leprosy patients and control subjects after normalization. Further, these results reveal the feasibility of developing a simple, field deployable molecular diagnostic based on mycobacterial lipid antigen-specific TCR sequences that are readily detectable in human tissues and blood independent of genetic background.

Synthesis and immunogenicity of PG-tb1 monovalent glycoconjugate.

A PG-tb1 hapten from the West Beijing strains of Mycobacterium tuberculosis cell wall has been efficiently synthesized and conjugated to CRM197 in a simple way as linker-equipped carbohydrate by applying squaric acid chemistry for an original neoglycoprotein, creating a potent T-dependent conjugate vaccine. The intermediate monoester can be easily purified and the degree of incorporation can be monitored by MALDI-TOF mass spectrometry. After administered systemically in mice without any adjuvant, the conjugate induced high antigen-specific IgG levels in serum. Furthermore, following the third immunization, significant antibody titers frequently exceeding 0.8 million were observed in the sera of mice vaccinated with PG-CRM197 conjugate which showed the potential for preparation of TB vaccine.

Evaluation of diagnostic potential of Rv3803c and Rv2626c recombinant antigens in TB endemic country Pakistan.

To overcome and eliminate tuberculosis (TB), definitive, reliable, and rapid diagnosis is mandatory. Presently, the diagnostic potential of acute and latent stage TB specific antigens i.e., Rv3803c and Rv2626c was determined. Immunogenic recombinant genes of Rv3803c and Rv2626c antigens were cloned in bacterial expression vector pET23b and expressed product was purified. The homogeneity and structural integrity was confirmed by Western blot analysis. Diagnostic potential of Rv3803c and Rv2626c antigens was analyzed using the sera of 140 active TB patients (AFB smear positive) by indirect ELISA. Ten patients of leprosy and 94 healthy individuals were taken as disease and normal control respectively. The data was analyzed using R statistical package. The sensitivity and specificity of Rv3803c in active TB patients was of 69.3% and 76.4% respectively with an area under ROC curve of 0.77, whereas sensitivity and specificity of Rv2626c 77.1% and 85.1%, respectively. The area under ROC curve of Rv2626c was 0.89 which was significantly higher than Rv3803c (p < 0.0001). Recombinant antigens Rv3803c and Rv2626c have potential to be used as diagnostic markers for TB and need to evaluate with other antigens for differential diagnosis of TB.

Co-morbid infections in Hansen's disease patients in the United States: considerations for treatment.

120 patients attending a Hansen's disease public health satellite clinic were evaluated for selected latent co-morbidities, consisting of strongyloidiasis, Chagas disease, hepatitis B, HIV, and tuberculosis, and potential exacerbation by immunosuppressive therapy. Implications for treatment of Hansen's disease are discussed.

Active tuberculosis is characterized by an antigen specific and strictly localized expansion of effector T cells at the site of infection.

Mycobacterium tuberculosis (MTB)-specific cytokine responses in the peripheral blood and at the site of infection may differ significantly within the same individual, but the under-lying T-cell subset changes are largely unknown. Here, we measured effector and memory T-cell markers on CD4⁺ T cells (CD45RO, cysteine chemokine receptor (CCR)7, and CD27) in peripheral blood and at the site of active tuberculosis (TB). Additionally, T cells were stimulated overnight with purified protein derivative (PPD) and early secretory antigenic target (ESAT)-6 to determine which T-cell subset produces MTB-specific interferon (IFN)-γ. A striking decrease in CCR7 and CD27 expression on T cells was noted at the site of active TB. Likewise, IFN-γ expressing, ESAT-6 specific CD4⁺CD45RO⁺CD27⁻ T cells were dramatically increased at the site of infection but were not detectable in peripheral blood. An antigen-specific expansion of differentiated T cells at the site of active TB infection was poorly reflected in peripheral blood. Insight in these changes in MTB-specific effector T cells in different compartments of the body could lead to new approaches for immune-based diagnosis and interventions.

Prevalence and pattern of cross-reacting antibodies to HIV in patients with tuberculosis.

In many countries, HIV testing among tuberculosis (TB) patients is recommended so that both infections are appropriately treated. Cross-reacting antibodies to HIV antigens have been reported for several conditions, including TB, leprosy, malaria, and rheumatoid arthritis. To study the pattern and prevalence of cross-reacting antibodies to HIV antigens, we examined sera from 153 HIV-negative TB patients and 40 healthy individuals in Chennai, south India. We also studied the differences in cross-reactivity of various HIV antigens using two different Western blot kits. Of the 153 samples studied, 80 were tested using HIV Western blot and 73 were tested using INNOLIA. Most patients in the study had concordantly negative ELISA and rapid tests, and no subject had a positive Western blot. However, seven TB patients had antibodies that cross-reacted with HIV antigens, giving rise to an indeterminate result. While p51/55 was the most frequently recognized antigen in the Western blot assay, antibodies to sgp120 was most frequently identified in INNOLIA. Sequence similarities between the two organisms could be responsible for eliciting cross-reacting antibodies, since a few related epitopes were identified in HIV and Mycobacterium. These findings could have potential implications for the development of diagnostics and vaccines.

Alteration of the relative levels of iNKT cell subsets is associated with chronic mycobacterial infections.

CD1d-restricted invariant natural killer T cells (iNKT cells) have been identified as an important type of effector and regulatory T cell, but their roles in the chronic infectious diseases caused by Mycobacterium tuberculosis and Mycobacterium leprae remain poorly defined. Here, we studied circulating human iNKT cells in blood samples from tuberculosis (TB) and leprosy patients. We found that the percentages of iNKT cells among total circulating T cells in TB and leprosy patients were not significantly different from those in normal controls. However, both TB and leprosy patients showed a selective reduction of the proinflammatory CD4(-)CD8beta(-) (DN) iNKT cells with a proportionate increase in the CD4(+) iNKT cells. Similar phenotypic alterations in circulating iNKT cells were observed in a mouse model of M. tuberculosis infection. Taken together, these findings indicate that the selective reduction of circulating DN iNKT cells is associated with chronic infections caused by M. tuberculosis and M. leprae.

QuantiFERON TB Gold: a new method for latent tuberculosis infection.

QuantiFERON-TB Gold obtained approval in 2003 by the Food and Drug Administration as a valid tool for the diagnosis of latent tuberculosis. In this report, we evaluated its potential use in the immunological diagnosis of Mycobacterium tuberculosis infections in different groups of subjects. Our data indicate that QuantiFERON-TB Gold is specific for identifying subjects who have come into contact with M. tuberculosis and its use alongside traditional diagnostic techniques may be an important instrument for controlling tuberculosis.

[The estimation of protective efficacy of the fusion gene vaccine encoding tubercle antigen 85B and MPT64 in mice challenged with Mycobacterium tuberculosis].

OBJECTIVE: To evaluate the protective efficacy of the fusion DNA vaccine (AM) encoding tubercle Ag85B and MPT64 in mice infected with Mycobacterium tuberculosis. METHODS: C57BL/6 mice were intramuscularly immunized with the DNA vaccines. The mice were challenged with 10(6) CFU H37Rv via lateral tail vein 35 days later after the third immunization for DNA vaccine groups and 100 days later for BCG vaccinated group. The mice in vaccinated groups and control groups were sacrificed 42 days later following challenge. The lungs and spleens were removed respectively, and the number of CFU in organs and histopathologic changes was determined. The antibody level, IFN-gamma, IL-4 and the survival time in all of the mice were evaluated. RESULTS: Antibody titer of pcDNA/Ag85B + pcDNA/MPT64 group and pcDNA/AM group was higher than that of other groups (P < 0.05). The level of IFN-gamma produced by spleen lymphocytes and spleen lymphocyte proliferation from BCG group, pcDNA/Ag85B, pcDNA/Ag85B + pcDNA/MPT64 group and pcDNA/AM group was higher than that of other groups (P < 0.05). No IL-4 was found in all groups. The number of bacterial colonies in the lungs and spleens was significantly decreased at 6th week postchallenge in all the vaccinated groups (P < 0.05), especially in BCG group (P < 0.01). The pulmonary histopathological changes were observed 6 weeks later following challenge with M. tuberculosis H37Rv. In PBS and pcDNA3.1 groups, the lesion was characterized by seroplastic inflammatory infiltration and lung tissue necrosis, in BCG group by granulomas and numerous macrophages, lymphocytes and a few epithelioid cells. The lesion in pcDNA/Ag85B groups was characterized by seroplastic inflammatory infiltration and a few macrophages, in pcDNA/Ag85B + pcDNA/MPT64 group and pcDNA/AM group, by granulomas, numerous macrophages and lymphocytes. The lesion in spleen was different from the lung and characterized by proliferative lymphocytes and inflammatory infiltration. The results in spleen were similar to those in lung. The survival time of BCG vaccinated mice after challenge with M. tuberculosis H37Rv was longer than that of other groups. The survival time of AM group was longer than that of other DNA vaccine groups. CONCLUSION: The pcDNA/AM can improve the protective efficacy in immunized mice against M. tuberculosis.

Presence of a 88 kDa Eales protein in uveitis, tuberculosis, leprosy and rheumatoid arthritis.

BACKGROUND: Eales disease (ED) is an idiopathic retinal vasculitis affecting young adult males. We have earlier reported the identification, purification and partial characterization of a novel 88 kDa protein found in the serum of patients with ED. The aim of the present study was to look for the 88 kDa protein in serum samples obtained from cases of retinal vasculitis mimicking ED and in other systemic inflammatory diseases. MATERIAL/METHODS: Serum samples from healthy volunteers and from patients with ED, uveitis, parsplanitis ocular sarcoidosis, toxoplasmosis, leprosy, diabetic retinopathy, viral hepatitis, and rheumatoid arthritis were analyzed for the presence of the 88 kDa protein by polyacralymide gel electrophoresis (PAGE). The immunological identity of the 88 kDa protein found in ED and in other diseases was investigated by Western blot. Immunohistochemistry was performed on epiretinal membranes (ERM) obtained from ED patients to localize the 88 kDa protein. RESULTS: 88 kDa protein were detected in serum samples obtained from patients with posterior uveitis, tuberculosis, leprosy and rheumatoid arthritis. The 88 kDa protein found in serum from patients with ED is immunologically identical to that found in other systemic inflammatory conditions. 88 kDa protein was localized in inflammatory cells and in nonvascular endothelium in ERMs obtained from patients with ED. CONCLUSIONS: We have identified a novel acute phase reactant, which is elaborated in ocular and systemic inflammatory conditions other than Eales disease. Further work is necessary to decipher the precise role of the 88 kDa protein in the pathophysiology of these inflammatory diseases.

Presence of a 88 kDa Eales protein in uveitis, tuberculosis, leprosy and rheumatoid arthritis

BACKGROUND: Eales disease (ED) is an idiopathic retinal vasculitis affecting young adult males. We have earlier reported the identification, purification and partial characterization of a novel 88 kDa protein found in the serum of patients with ED. The aim of the present study was to look for the 88 kDa protein in serum samples obtained from cases of retinal vasculitis mimicking ED and in other systemic inflammatory diseases. MATERIAL/METHODS: Serum samples from healthy volunteers and from patients with ED, uveitis, parsplanitis ocular sarcoidosis, toxoplasmosis, leprosy, diabetic retinopathy, viral hepatitis, and rheumatoid arthritis were analyzed for the presence of the 88 kDa protein by polyacralymide gel electrophoresis (PAGE). The immunological identity of the 88 kDa protein found in ED and in other diseases was investigated by Western blot. Immunohistochemistry was performed on epiretinal membranes (ERM) obtained from ED patients to localize the 88 kDa protein. RESULTS: 88 kDa protein were detected in serum samples obtained from patients with posterior uveitis, tuberculosis, leprosy and rheumatoid arthritis. The 88 kDa protein found in serum from patients with ED is immunologically identical to that found in other systemic inflammatory conditions. 88 kDa protein was localized in inflammatory cells and in nonvascular endothelium in ERMs obtained from patients with ED. CONCLUSIONS: We have identified a novel acute phase reactant, which is elaborated in ocular and systemic inflammatory conditions other than Eales disease. Further work is necessary to decipher the precise role of the 88 kDa protein in the pathophysiology of these inflammatory diseases.

Onchocerciasis modulates the immune response to mycobacterial antigens.

Chronic helminth infection induces a type-2 cellular immune response. In contrast to this, mycobacterial infections commonly induce a type-1 immune response which is considered protective. Type-2 responses and diminished type-1 responses to mycobacteria have been previously correlated with active infection states such as pulmonary tuberculosis and lepromatous leprosy. The present study examines the immune responses of children exposed to both the helminth parasite Onchocerca volvulus and the mycobacterial infections, Mycobacterium tuberculosis and M. leprae. Proliferation of peripheral blood mononuclear cells (PBMC) and production of IL-4 in response to both helminth and mycobacterial antigen (PPD) decreased dramatically with increasing microfilarial (MF) density. Although interferon-gamma (IFN-gamma) production strongly correlated with cellular proliferation, it was surprisingly not related to MF density for either antigen. IL-4 production in response to helminth antigen and PPD increased with ascending children's age. IFN-gamma and cellular proliferation to PPD were not related to age, but in response to helminth antigen were significantly higher in children of age 9-12 years than children of either the younger age group (5-8 years) or the older group (13-16 years). Thus, there was a MF density-related down-regulation of cellular responsiveness and age-related skewing toward type 2 which was paralleled in response to both the helminth antigen and PPD. This parasite-induced immunomodulation of the response to mycobacteria correlates with a previous report of doubled incidence of lepromatous leprosy in onchocerciasis hyperendemic regions. Moreover, this demonstration that helminth infection in humans can modulate the immune response to a concurrent infection or immunological challenge is of critical importance to future vaccination strategies.

Diagnostic and prognostic potential of a competitive enzyme-linked immunosorbent assay for leishmaniasis in India.

A Leishmania donovani species-specific monoclonal antibody (monoclonal antibody D2) was evaluated for its diagnostic and prognostic potential by a competitive enzyme-linked immunosorbent assay (C-ELISA) in sera from Indian patients with visceral leishmaniasis (VL) and seven patients with post-kala-azar dermal leishmaniasis (PKDL). These results were compared with those obtained by microscopy with Giemsa-stained tissue smears and a direct enzyme-linked immunosorbent assay (direct ELISA) with crude parasite antigen. Of 121 patients with clinically diagnosed VL examined, 103 (85.1%) were positive and 11 (9.1%) were negative by all three methods. An additional 7 (5.8%) who were negative by microscopy were positive by both C-ELISA and direct ELISA. Seven PKDL patients were also examined and were found to be positive by all three methods. Analysis of the chemotherapeutic response to sodium antimony gluconate of these 110 serologically positive VL patients showed that 57 (51.8%) were drug responsive and 53 (48.2%) were drug resistant. The C-ELISA with sera from 20 longitudinally monitored VL patients before and after chemotherapy showed a significant decrease in percent inhibition of monoclonal antibody D2 in drug-responsive patients. However, in drug-unresponsive patients, the percent inhibition of D2 was unchanged or was slightly increased. Our results therefore indicate (i) the applicability of L. donovani species-specific monoclonal antibody D2 for sensitive and specific serodiagnosis by C-ELISA, (ii) that the C-ELISA is more sensitive than microscopy, especially for early diagnosis, (iii) that L. donovani is still the main causative agent of VL, irrespective of the chemotherapeutic response, and (iv) that the C-ELISA can be used to evaluate the success of drug treatment.

IgG response to some mycobacterial antigens in selected leprosy patients.

Serum samples from selected leprosy patients with putative tuberculosis were tested by indirect ELISA to determine the level of IgG antibody against six mycobacterial antigen preparations. PCR-positive leprosy patients were confirmed with PGL-I ELISA. A ratio of antibodies to antigens of tuberculosis and leprosy was found to be a valuable serological marker for tuberculosis in long-treated leprosy patients.

Immunoreactive antigens of a candidate leprosy vaccine: Mycobacterium habana.

Mycobacterium habana (M. simiae serovar-1) is a candidate vaccine for mycobacterial infections on the basis of the protection shown by this strain. We prepared 3 fractions of M. habana, i.e. the cell wall (CW), the cell membrane (CM) and the cytosol (CS). Protein antigens of these fractions were resolved by SDS-PAGE and subsequently probed with the sera of leprosy and tuberculosis patients and also antiBCG antibodies. We saw 3 major protein bands at congruent to 33 kD in the CW, congruent to 38 kD in the CM and congruent to 22 kD in the cytosol (CS) after coomassie blue staining of the gels. Pool leprosy patients' serum had identified proteins of congruent to 26 kD in CW, congruent to 35 and congruent to 18 kD in CM and congruent to 24 kD in the CS which have not been seen by the TB patient's serum pool. Pool serum of tuberculosis patients has identified 1 protein at congruent to 10 kD in the CW and a broad band between 20 and 24 kD and 1 at congruent to 4 kD in the CM which have not been visualized in the pool leprosy patient's serum lane. The proteins of M. habana which are recognized only by leprosy antisera or only by tuberculosis antisera could be exploited for developing diagnostic agents against these infections.

ELISA test for tuberculosis.

The Elisa test for diagnosis of tuberculosis using highly purified A 60 antigen extracted from mycobacteria was developed by Anda Biologicals, France, during the late 1980s. It is claimed to have about 95% sensitivity and specificity. Both IgG and IgM antibodies can be separately tested. IgM antibodies appear early in the disease and IgG appear later. The test is negative in healthy, normal subjects and is not related to tuberculin test or BCG vaccination status. The A 60 antigen is common to many mycobacteria including M. tuberculosis, M. leprae, M. bovis, and M. avium. Hence, clinical, radiological and other laboratory data must be considered along with the results of Elisa test for final diagnosis. Tuberculosis is still the most important bacterial infection in India. Because of its high prevalence, normal subjects and proved tuberculosis patients were first surveyed to determine the cut off values for IgG and IgM antibodies among local population. Subsequently the test was offered in India for general use. This paper describes our experience of the test since 1990, on samples referred by various practitioners and laboratories in Bombay and other cities. Result on 5840 IgG tests and 2101 IgM tests shows that the A 60 Elisa test is well accepted and is a useful laboratory aid in the diagnosis of pulmonary and extrapulmonary tuberculosis.

Synthesis of the trisaccharide-protein conjugate of the phenolic glycolipid of Mycobacterium tuberculosis for the serodiagnosis of tuberculosis.

The trisaccharide segment of the phenolic glycolipid (PGL) of Mycobacterium tuberculosis, 2-O-methyl-3-O-[3-O-(2,3,4-tri-O-methyl-alpha-L-fucopyranosyl)-alpha-L- rhamnopyranosyl]-alpha-L-rhamnopyranose, was synthesized in the form of the p-(2-methoxycarbonylethyl)phenyl glycoside by a stepwise condensation. 2,4-Di-O-benzyl-3-O-acetyl-alpha-L-rhamnopyranosyl chloride was coupled to p-(2-methoxycarbonylethyl)phenyl 4-O-benzyl-2-O-methyl-alpha-L-rhamnopyranoside in the presence of silver triflate, and 2,3,4-tri-O-methyl-alpha-L-rhamnopyranosyl chloride was then coupled to the deacetylated disaccharide by the same procedure. The trisaccharide was deblocked and coupled to BSA, giving the neoglycoconjugate TB-NT-P-BSA. TB-NT-P-BSA showed its possibility as a useful tool for the serodiagnosis of tuberculosis.