Enhanced T cell responsiveness to Mycobacterium bovis BCG r32-kDa Ag correlates with successful anti-tuberculosis treatment in humans.

Th1 and Th2 cytokines play key role in protection from and pathogenesis of mycobacterial infection and their dynamic changes may predict clinical outcome of the patient. Patients with tuberculosis (TB) have a poorer cellular immune response to recombinant 32-kDa antigen (Ag) of Mycobacterium bovis (r32-kDa M. bovis) than do healthy volunteers. The basis for this observation was studied by evaluating the Th1 (gamma interferon [IFN-γ]) produced in response to the r32-kDa Ag M. bovis by peripheral blood mononuclear cells (PBMC) from patients with pulmonary TB (n=20), extra-pulmonary TB (n=13) and from healthy volunteers (n=9). Recombinant 32-kDa M. bovis stimulated PBMC from TB patients produced significantly lower levels of IFN-γ at 0 month, and increased at 2-4, and 6 months of treatment and were highly significant (p<0.000) compared to the responses in controls. The ratios of IFN-γ to IL-10 were low in patients newly diagnosed and improved both during and after treatment. The present study concludes that the levels of in vitro response to M. bovis BCG r32-kDa Ag leading to the specific release of IFN-γ increased after anti-tuberculosis treatment and seems to reflect the clinical status of the patient, thus reiterating the utility of this antigen in T cell based assays as a surrogate marker of cell mediated responses.

In vitro levels of interleukin 10 (IL-10) and IL-12 in response to a recombinant 32-kilodalton antigen of Mycobacterium bovis BCG after treatment for tuberculosis.

Cell-mediated immunity plays a major role in conferring protection against tuberculosis (TB) on an individual. It is not known whether the immune status correlates with the bacterial load or whether the immunity improves after treatment. Also, it may be important to monitor treatment by being able to discriminate between active disease and successfully treated TB. The main aim of this study was to investigate the usefulness of a recombinant 32-kDa antigen (r32-kDa Ag) of Mycobacterium bovis BCG (Ag85A-BCG) as a diagnostic marker in patients being treated for TB. Specifically, the in vitro T-cell assays and the release of interleukin-12 (IL-12) (Th1-type cytokine) and IL-10 (Th2-type cytokine) in response to the r32-kDa Ag of BCG were assayed in patients with either pulmonary (sputum positive/negative, n = 74) or extrapulmonary TB (n = 49) and healthy controls. The proliferative responses of stimulated cells at 0, 2 to 4, and 6 months of treatment increased and were highly significant (P < 0.000) compared to the responses in controls. The increase in IL-12 and decrease in IL-10 release suggest that there is cytokine expression modification during different stages of TB, and treatment seems to have an influence on the levels of these cytokines, suggesting an augmentation in the protective responses. The in vitro response to the M. bovis BCG r32-kDa Ag may be useful in monitoring treatment of TB.

The co-operonic PE25/PPE41 protein complex of Mycobacterium tuberculosis elicits increased humoral and cell mediated immune response.

BACKGROUND: Many of the PE/PPE proteins are either surface localized or secreted outside and are thought to be a source of antigenic variation in the host. The exact role of these proteins are still elusive. We previously reported that the PPE41 protein induces high B cell response in TB patients. The PE/PPE genes are not randomly distributed in the genome but are organized as operons and the operon containing PE25 and PPE41 genes co-transcribe and their products interact with each other. METHODOLOGY/PRINCIPAL FINDING: We now describe the antigenic properties of the PE25, PPE41 and PE25/PPE41 protein complex coded by a single operon. The PPE41 and PE25/PPE41 protein complex induces significant (p<0.0001) B cell response in sera derived from TB patients and in mouse model as compared to the PE25 protein. Further, mice immunized with the PE25/PPE41 complex and PPE41 proteins showed significant (p<0.00001) proliferation of splenocyte as compared to the mice immunized with the PE25 protein and saline. Flow cytometric analysis showed 15-22% enhancement of CD8+ and CD4+ T cell populations when immunized with the PPE41 or PE25/PPE41 complex as compared to a marginal increase (8-10%) in the mice immunized with the PE25 protein. The PPE41 and PE25/PPE41 complex can also induce higher levels of IFN-gamma, TNF-alpha and IL-2 cytokines. CONCLUSION: While this study documents the differential immunological response to the complex of PE and PPE vis-à-vis the individual proteins, it also highlights their potential as a candidate vaccine against tuberculosis.

Genetic affinity of two south Indian ethnic groups with other populations.

Humans appear to have spread to several parts of the Indian subcontinent by the middle Paleolithic period. It also provided a major passage for the distribution of modern humans. For the first time generic human leucocyte antigen (HLA) class II (DRB1) genotyping was performed using polymerase chain reaction-sequence-specific primer (PCR-SSP) (low-resolution) technique in two endogamous groups (Hindu-Brahmins and Sunni-Muslims) from the southern state of Andhra Pradesh in order to decipher the genetic affinity between them and with other populations. Calculation of genetic distances, construction of neighbour-joining dendograms and principal-component (P-C) maps were executed using HLA allelic frequencies. The present study reveals the genetic affinity of the Brahmin and Muslim populations from the state of Andhra Pradesh. The dendograms demonstrated that Indian populations form a separate cluster with oriental populations on one side and the eastern and the western Mediterranean populations on the other side. The principal component maps showed the clustering of Indian populations in the lower-right quadrant, where in the Sunni-Muslims occupied a position more closely to the north Indian Brahmins and the Bhargavas than to the geographically related south Hindu-Brahmin group. The study hypothesizes that unlike Brahmins who can be considered as an immediate stock of the Caucasians, the ancestors of Muslims might have been the result of an admixture between the Indo-Europeans and proto-Dravidians.

Interferon-gamma Low producer genotype +874 overrepresented in Bacillus Calmette-Guerin nonresponding children.

BACKGROUND: There are limited reports on the influence of the immune regulatory genotypes on the efficacy of Bacillus Calmette-Guerin (BCG) in man. This study was designed to evaluate the influence of the cytokine genotype interferon (IFN)-gamma +874T/A on T cell in vitro assays in BCG nonresponders (negative to either in vivo or in vitro test with purified protein derivative or both). METHODS: Ninety healthy children who were without any clinical evidence of the disease, 45 with a BCG-scar and the remaining 45 without scar were assessed for in vitro T cell responses. CD4+ and CD8+ cell counts were measured by flow cytometry. r32kDaBCG (Ag85A-BCG) protein was used to stimulate T cells and IFN-gamma cytokine concentration in the cultures were measured by enzyme-linked immunosorbent assay. Polymorphism in IFN-gamma (+874T/A) region was detected by amplification refractory mutation system-polymerase chain reaction. RESULTS: T cell subsets were within the normal range in all subjects. Children with TT genotype showed significantly higher antigen-induced IFN-gamma (P < 0.001) as compared with those with AT/AA genotype. The highest values were observed in children with TT genotype combined with positive antigen-specific peripheral blood mononuclear cells proliferation. Seventy-five percent of the vaccinated children with TT genotype showed high amounts of stimulated IFN-gamma compared with 66% of scar negative and 16% of scar positive but with AA genotype. CONCLUSIONS: IFN-gamma (+874T/A) polymorphism seemed to be a strong and independent predictor for clinical outcome of both scar-positive and scar-negative children. These results may help in planning future vaccination strategies. The ability to mount in vitro lymphoproliferation did not distinguish the success or failure of BCG vaccination nor predict susceptibility to the disease.

Age-related waning of in vitro Interferon-gamma levels against r32kDaBCG in BCG vaccinated children.

BACKGROUND: Mycobacterium bovis BCG vaccine has displayed inconsistent efficacy in different trials conducted in various geographical regions. Nevertheless, it significantly reduces the risk of severe childhood tuberculosis and continues to be used to prevent tuberculosis in many countries. Many studies revealed that efficacy of vaccine wanes with age. Most of the studies were based on in vivo and in vitro responses to tuberculin. With the advent of newer tests such as in vitro interferon-gamma assays and identification of potent immunogenic mycobacterial proteins there is a need to corroborate the observations. This study aims at ascertaining the need for a booster at a later age as indicated by in vitro release of IFN-gamma while evaluating Ag85A as an antigen. METHODS: Ninety healthy children who were without any clinical evidence of the disease, 45 with a BCG-scar and the remaining 45 without scar and 25 with tuberculosis were included in the study. The incidence of TB was analyzed in 216 children attending a DOTS clinic during 1996-2005. CD3+, CD4+ and CD8+ cell counts were measured by Flow cytometry. r32kDaBCG (Ag85A-BCG) protein was used to stimulate T cells in in vitro T cell responses and interferon-gamma (IFN-gamma) cytokine levels in the supernatants were measured by ELISA. RESULTS: High incidence of TB was observed in age group 13-14 years followed by children in the age group 10-12 years (Chi-square 242.22; p < 0.000). T cell subsets were within the normal range in all subjects. 79% of vaccinated children showed positive proliferative responses with a mean SI value of 4.98 +/- 1.99 while only 39% of the unvaccinated and 58% of the tuberculosis children showed positive responses with mean values of 2.9 +/- 1.6 (p < 0.001) and 2.9 +/- 1.7(p < 0.057), respectively. The stimulation indices in vaccinated children decreased in the older children concurring with an increase in the incidence of TB. CONCLUSION: Significantly high levels of in vitro IFN-gamma demonstrated in BCG vaccinated children in our study substantiate the observation that BCG is effective in children, but the effect may wane with age. The immunity could be boosted using modified r32kDa (Ag85A) of BCG.

Cost and cost-effectiveness of PPM-DOTS for tuberculosis control: evidence from India.

OBJECTIVE: To assess the cost and cost-effectiveness of the Public-Private Mix DOTS (PPM-DOTS) strategy for tuberculosis (TB) control in India. METHODS: We collected data on the costs and effects of pilot PPM-DOTS projects in Delhi and Hyderabad using documentary data and interviews. The cost of PPM-DOTS was compared with public sector DOTS (i.e. DOTS delivered through public sector facilities only) and non-DOTS treatment in the private sector. Costs for 2002 in US$ were assessed for the public sector, private practitioners, and patients/attendants. Effectiveness was measured as the number of cases successfully treated. FINDINGS: The average cost per patient treated was US$ 111-123 for PPM-DOTS and public sector DOTS, and US$ 111-172 for non-DOTS treatment in the private sector. From the public sector's perspective, the cost per patient treated was lower in PPM-DOTS projects than in public sector DOTS programmes (US$ 24-33 versus US$ 63). DOTS implementation in either the public or private sectors improved treatment outcomes and substantially lowered costs incurred by patients and their attendants, compared to non-DOTS treatment in the private sector (US$ 50-60 for DOTS compared to over US$ 100 for non-DOTS). The average cost-effectiveness of PPM-DOTS and public sector DOTS was similar, at US$ 120-140 per patient successfully treated, compared to US$ 218-338 for non-DOTS private sector treatment. Incremental cost-effectiveness analysis showed that PPM-DOTS can improve effectiveness while also lowering costs. CONCLUSION: PPM-DOTS can be an affordable and cost-effective approach to improving TB control in India, and can substantially lower the economic burden of TB for patients.

Role of HLA-B51 and HLA-B52 in susceptibility to pulmonary tuberculosis.

MHC class I-restricted CD8+ T cells are important for the generation of protective immune responses in MTB infection. CD8+ CTL (cytotoxic T-lymphocyte)-derived IFN-g may be especially important both for cells lacking MHC class II molecules, e.g. in the lung and for macrophages where mycobacteria can evade recognition during chronic infection by sequestering their antigens away from sensitized CD4+ T cells. This study was designed to detect any association of MHC class I (HLA-B) molecules with pulmonary tuberculosis. A total of 75 individuals, comprising of 33 patients with pulmonary tuberculosis; 12 HIV patients who developed tuberculosis and 30 healthy controls, were included in the study. They were typed for HLA-B by the PCR-SSP method. The results of only HLA-B alleles, which are highly significant, are presented here. The number of healthy individuals with HLA-B52 was significantly high when compared to the patient groups (healthy versus TB: 21.2% versus 0.0%, OR=0.0, P<0.0001, P(c)=0.003; healthy versus HIV-TB: 21.2% versus 16.7%; OR=0.74; P<0.001; P(c)=0.003). In contrast, the number of patients, both TB- and HIV-TB-positive, with HLA-B51 was significantly high when compared to the healthy group of individuals (TB versus healthy: 36.7% versus 3%; OR=18.53; P<0.0001; P(c)=0.001; HIV-TB versus healthy: 41.7% versus 3%; OR=22.86; P<0.0001; P(c)=0.001). Only one healthy control was positive to HLA-B51; however this individual also had HLA-B52. The results of this study suggest that HLA-B52(5) has a negative, i.e. a protective association and HLA-B51(5) has a positive (susceptible) association, for pulmonary tuberculosis. Studies on HLA-B51 and HLA-B52 in a larger population to assess their role in tuberculosis may be useful for TB-vaccination strategies, since HLA profiles are likely to be related to vaccine efficacy.

Prevalence of drug resistance under the DOTS strategy in Hyderabad, South India, 2001-2003.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB), defined as resistance to at least isoniazid (INH) and rifampicin (RMP), is considered a threat to TB control. Implementation of DOTS ensures high cure rates and prevents MDR. OBJECTIVE: To study the prevalence of MDR-TB from a retrospective analysis of the data in a tuberculosis unit where DOTS was implemented over a period of 6 years through public private mix (PPM). METHODS: Drug susceptibility testing of Mycobacterium tuberculosis samples isolated from the cultures of newly registered and retreatment sputum smear-positive cases during 2001-2003. RESULTS: During the study, 909 sputum-positive cases were registered and analysed. Of these, 714 were new and 195 were retreatment sputum-positive cases. INH resistance was found in 3.2% (23) of new and 9.2% (18) of retreatment cases. RMP resistance was present in 1.5% (11) of new and 7.2% (14) of retreatment cases. MDR was present only in 0.14% (1) of new and 2% (4) of retreatment cases. New cases had cure rates of 96% compared to 85% in retreatment cases. CONCLUSION: The prevalence of MDR-TB is low where success rates are high.

Fluorescent amplified fragment length polymorphism (FAFLP) based molecular epidemiology of hospital infections in a tertiary care setting in Hyderabad, India.

Bacterial isolates from respiratory and urinary tract infections in an Indian hospital setting were genotyped using FAFLP analysis. The 77 different isolates analyzed belonged to five genera namely Escherichia, Staphylococcus, Pseudomonas, Enterobacter and Pantoea. Before carrying out FAFLP analysis all the isolates were subjected to16S-23S ribosomal RNA-based species identification. Cluster analysis of FAFLP profiles of 77 isolates generated five groups corresponding to five bacterial genera that are used in the study. Further analyses of the dendrograms revealed efficient species and strain differentiation. Cluster analysis identified genetically distant clones among the clinical isolates of Staphylococcus aureus, two distinct genetic lineages among the Escherichia coli strains and a single cluster of closely related Pseudomonas aeruginosa isolates. Ribosomal spacer region amplification identified different species accurately but intraspecies discrimination could not be accomplished completely. Comparison of FAFLP profiles of our isolates, with a pilot database of validated strains, was very useful in identification and worked better in conjunction with dendrogram analysis.

Influence of socioeconomic status on lung function and prediction equations in Indian children.

The present study was carried out to assess the influence of socioeconomic status on lung functions and to suggest prediction equations for Indian children. For this purpose, 2,616 normal, healthy schoolchildren aged between 5-15 years were recruited. Boys were classified into three groups, i.e., high-income (HIG), middle-income (MIG), and low-income (LIG), while girls were classified into HIG and LIG groups, based on socioeconomic status (SES). Height, weight, chest circumference, body surface area (BSA), fat-free mass (FFM), and body fat were assessed. Forced expiratory volume in 1 sec (FEV1), forced vital capacity (FVC), FEV1/FVC ratio, and peak expiratory flow rate (PEFR) were measured. The results, before and after adjustment of physical characteristics, showed that anthropometry, body composition, and lung functions were significantly higher in HIG compared to MIG and LIG children, while in girls, no differences were observed in physical characteristics after adjustments. Multiple linear regression equations were developed to predict FEV1, FVC, and PEFR, using independent variables like age, height, fat-free mass, and SES. It is opined that these equations could be used as Indian reference equations for healthy children based on the SES.

Tuberculin specific T cell responses in BCG vaccinated children.

The effector mechanisms of BCG protection were examined 5-7 years after vaccination. The in vitro lymphoproliferation, following stimulation with tuberculin, in normal, (A) vaccinated and (B) unvaccinated children and children with tuberculosis (C), were assayed. The mean stimulation index (SI) of lymphocyte transformation in normal subjects were significantly (P < 0.05) higher than those with tuberculosis. The ratio of tuberculin-specific CD4 to CD8 cells in short-term cultures were significantly (P less than 0.05) higher in the vaccinees. In group (A), 70 % had positive ratios as against 20 %and 0 %in groups (B) and (C), respectively. Secretion of IL-2 by the cells was significantly (P < 0.05) high in the vaccinated. None of the unvaccinated children had positive levels of IL-2. The vaccinees also had highly significant (P < 0.01) levels of IFN-)in the supernatants of cell-cultures, following tuberculin stimulation. In majority of the BCG vaccinated children, the stimulation of specific TH1 cells seem to be considerably high, in short-term in vitro cultures. While these responses were not so marked in the unvaccinated, they were almost totally absent in the patients.

Cell-mediated immune responses in children towards secreted proteins of Mycobacterium bovis BCG.

BACKGROUND: There is a need to develop an improved anti-TB vaccine for adequate control and elimination of tuberculosis, to control the spread of MDR-TB and TB/HIV co-infection. Studies in children have indicated that BCG vaccination has certain beneficial effects, especially against miliary TB and TB meningitis, but needs to be improved for protection against pulmonary tuberculosis. OBJECTIVE: The aim of the present study was to identify the immunogenic proteins in the culture filtrate (CF) of Mycobacterium bovis BCG by studying the effector mechanism of protection in children, which may help in the formulation of an effective vaccine against tuberculosis. MATERIALS AND METHODS: Lymphoproliferative responses (LTT) and the levels of IL-2 and IFN-gamma (Th1 cytokines) released into the culture supernatants were estimated (by ELISA) in short-term cultures of PBMC of BCG vaccinated (n=25) and unvaccinated (n=15) children and children with tuberculosis (n=15) against 10 different fractions of CF (mol.wt > or = 90-<14 kDa). RESULTS: The mean stimulation indices (SI) in LTT against all the (10) fractions in vaccinated children were high when compared to the unvaccinated and diseased groups; however, of the 10 fractions, F3, F4, F6, F8 and F9 elicited significantly higher SIs than the other fractions (p<0.05). In the vaccinated children, the SI (5.58+/-1.57), levels of IL-2 (381.66+/-16.40 pg/ml) and IFN-gamma (732+/-62.36 pg/ml) in the cultures stimulated by fraction 8 (34-30k Da) were significantly higher (p<0.001), than responses to the other fractions and also to those of other groups of children. CONCLUSION: Thus, the peptides within the cluster 34-30 kDa appear to be promising vaccine candidates by virtue of their immunodominant nature specifically priming Th1 cells in normal, BCG-vaccinated children.

Use of specific IgE to fractions of gynandropsis gynandra pollen inin vitro diagnostic test.

The study was aimed at presence of specific IgE antibody levelsinvitro to the identified antigen. Based on positive skin test with Gynandropsis gynandra and elevated levels of total IgE (>325 IU/ml) 104 patients were selected. Healthy, asymptomatic individuals (25) with low total IgE (<325 IU/ml) were included as controls. The mean OD values by ELISA for specific IgE were 0.67±0.21, 0.57±0.18 and 0.56±0.18 with whole pollen antigen, 46-37 kD fraction and 36-32 kD fraction, respectively. The specificity and sensitivity between skin test positivity with whole pollen antigen verses fraction with mol.wt 46-37 kD was 90% and 90% and for fraction with mol.wt 36-32 kD was found to be 81.1% and 89.4%. The clusters with molecular weights 46-37 kD and 36-32 kD may be useful inin vitro diagnostic test. Fractions within these clusters need to be identified for a higher specificity.

Pulmonary function tests in Indian girls--prediction equations.

OBJECTIVE: The present study was conducted to evaluate pulmonary functions and develop prediction equations in Indian girls. METHODS: 1038 normal healthy schoolgirls in the age group of 5 to 15 years were selected for the present study. The anthropometrical parameters like height, weight, sitting height and chest circumference were measured and body surface area (BSA) and fat free mass (FFM) were derived using equations. The pulmonary functions such as FEV1, FVC, FEV1/FVC% and PEFR were studied. RESULTS: The height, sitting height, weight, BSA, chest circumference, body fat as well as FEV1, FVC, FEV1/FVC% and PEFR were comparable with Indian standards. Multiple regression equations were developed to predict FEV1, FVC and PEFR using anthropometrical indices like height, fat free mass and age or chest circumference in view of significantly high correlation of these parameters with lung function variables [height and FEV1 (r-0.90), height and FVC (r-0.899), height and PEFR (r-0.891), chest and FEV1 (r-0.868), chest and FVC (r-0.867), chest and PEFR (r-0.83)]. CONCLUSION: The regression equations to predict the pulmonary functions were presented using the independent variables like height, fat free mass and chest circumference or age, since these variables have shown very strong predictability for FEV1, FVC and PEFR. The equations presented in this study can be considered as referral standards for Indian girls.

Mycobacterium tuberculosis (Mtb) isocitrate dehydrogenases show strong B cell response and distinguish vaccinated controls from TB patients.

Proteins released from Mycobacterium tuberculosis (Mtb) during late logarithmic growth phase are often considered candidate components of immunogenic or autolysis markers. One such protein is isocitrate dehydrogenase (ICD), a key regulatory enzyme in the citric acid cycle. We have evaluated the immunogenic properties of two isoforms of Mtb ICD and compared them with the control antigens heat-shock protein 60 and purified protein derivative (PPD). PPD lacks the sensitivity to distinguish between bacillus Calmette-Guérin (BCG)-vaccinated and tuberculosis (TB)-infected populations, and, therefore, epidemiological relevance of PPD in BCG-vaccinated regions is debatable. We show that Mtb ICDs elicit a strong B cell response in TB-infected populations and can differentiate between healthy BCG-vaccinated populations and those with TB. The study population (n = 215) was categorized into different groups, namely, patients with fresh infection (n = 42), relapsed TB cases (n = 32), patients with extrapulmonary TB (n = 35), clinically healthy donors (n = 44), nontuberculous mycobacteria patients (n = 30), and non-TB patients (culture negative for acid-fast bacteria but carrying other infections, n = 32). The Mtb ICDs showed statistically significant antigenic distinction between healthy BCG-vaccinated controls and TB patients (P < 0.0001) and those with other infections. Although extrapulmonary infections could not be discriminated from healthy controls by heat-shock protein 60 (P = 0.2177), interestingly, the Mtb ICDs could significantly (P < 0.0001) do so. Our results highlight the immunodominant, immunosensitive, and immunospecific nature of Mtb ICDs and point to an unusual property of this tricarboxylic acid energy cycle enzyme.

Molecular genotyping of a large, multicentric collection of tubercle bacilli indicates geographical partitioning of strain variation and has implications for global epidemiology of Mycobacterium tuberculosis.

Tuberculosis continues to be a major killer disease, despite an all-out effort launched against it in the postgenomic era. We describe here the population structure of Mycobacterium tuberculosis strains, as revealed by a chromosome-wide scan of fluorescent amplified fragment length polymorphisms (FAFLPs), for more than 1,100 independent isolates from 11 different countries. The bacterial strains were genotyped based on a total of 136 +/- 1 different FAFLP markers at the genome sequence interface, with details on IS6110 profiles, drug resistance status, clinicopathological observations, and host status integrated into the analysis process. The strains were found to cluster with possible geographic affinities, including the parameters of host species type, IS6110 profile, and drug susceptibility status. Of the five most commonly amplified fragment sets (or amplitypes), type A predominated in strains of mixed origin, deposited in The Netherlands; type B was exclusively observed for Indian isolates; type C was found mainly in strains from Peru and Australia; and types D and E predominated in European strains from France and Italy. The amplitypes were independent of certain large sequence polymorphisms representing two important deletions, TbD1 and Rd9. It appears that M. tuberculosis has a high genomic diversity with a possible geographic evolution. This may have occurred due to specific genomic deletions and synonymous substitutions selected rigorously against host defenses and environmental stresses on an evolutionary timescale. The genotypic data reported here are additionally significant for genotype-phenotype correlations and for determining whether pathogen diversity is a reflection f the host population diversity.

PPE antigen Rv2430c of Mycobacterium tuberculosis induces a strong B-cell response.

The variation in sequence and length in the C-terminal region among members of the unique PE (Pro-Glu) and PPE (Pro-Pro-Glu) protein families of Mycobacterium tuberculosis is a likely source of antigenic variation, giving rise to the speculation that these protein families could be immunologically important. Based on in silico analysis, we selected a hypothetical open reading frame (ORF) encoding a protein belonging to the PPE family and having epitopes with predictably higher antigenic indexes. Reverse transcriptase PCR using total RNA extracted from in vitro-cultured M. tuberculosis H37Rv generated an mRNA product corresponding to this gene, indicating the expression of this ORF (Rv2430c) at the mRNA level. Recombinant protein expressed in Escherichia coli was used to screen the sera of M. tuberculosis-infected patients, as well as those of clinically healthy controls (n = 10), by enzyme-linked immunosorbent assay. The panel of patient sera comprised sera from fresh infection cases (category 1; n = 32), patients with relapsed tuberculosis (category 2; n = 30), and extrapulmonary cases (category 3; n = 30). Category 2 and 3 sera had strong antibody responses to the PPE antigen, equal to or higher than those to other well-known antigens, such as Hsp10 or purified protein derivative (PPD). However, a higher percentage of patients belonging to category 1, as opposed to clinically healthy controls, showed stronger antibody response against the PPE protein when probed with anti-immunoglobulin M (IgM) (71 versus 37.5%) or anti-IgG (62.5 versus 28.12%). Our results reveal that this PPE ORF induces a strong B-cell response compared to that generated by M. tuberculosis Hsp10 or PPD, pointing to the immunodominant nature of the protein.

Study on lung function tests and prediction equations in Indian male children.

OBJECTIVE: The present study was carried out to evaluate lung functions and develop prediction equations in Indian boys. SUBJECTS: 1555 normal healthy schoolboys from Hyderabad city who were in the age group of 5 to 15 years were selected for the present study. DESIGN: The anthropometric parameters such as height, sitting height, weight, and chest circumference were measured and body surface area (BSA) and percent body fat (% Fat) were derived. The lung functions studied were FEV1, FVC, FEV1% and PEFR. RESULTS: The height, sitting height, weight, BSA, chest circumference, body fat as well as FEV1, FVC, FEV1 % and PEFR were comparable with Indian boys. The height for age, weight for age and weight for height were found to be lower than 50th percentile of NCHS standards in the subjects studied. Similarly the lung function values of the study population were found to be lower than the values of corresponding western population. CONCLUSION: Regression equations were derived to predict FEV1, FVC and PEFR using physical characteristics. Height, chest circumference and fat free mass were the best predictors for FEV1, FVC, and PEFR. Age, height, sitting height, weight, chest circumference and fat free mass showed significant association with lung functions.