Evaluation of heat shock proteins for discriminating between latent tuberculosis infection and active tuberculosis: a preliminary report

The diagnosis of a latent tuberculosis infection [LTBI] is of the utmost concern. The available tests, the tuberculin skin test [TST] and the Quantiferon-TB Gold test [QFT-G] cannot discriminate between active TB and LTBI. Therefore, the aim of the study is to identify new biomarkers that can discriminate between active TB and LTBI and can also assess the risk of the individual developing active TB. In total, 55 blood samples were collected, of which 10 samples were from the active TB infection group, 10 were from the high-risk exposure group, 23 were from the low-risk exposure group, and 12 were from healthy controls living in a non-TB endemic area. A panel of heat shock proteins [Hsps], including host Hsp25, Hsp60, Hsp70, and Hsp90 and Mycobacterium tuberculosis [MTB] Hsp16, were evaluated in all of the collected samples using ELISA. The levels of the host Hsp[s] [Hsp25, Hsp60, Hsp70 and Hsp90] and MTB Hsp16 were significantly [p=0.05] elevated in the active TB group compared to the high-risk exposure group, the low-risk exposure group and the control group. Notably, the levels of the same panel of Hsp[s] were elevated in the high-risk exposure group compared to the low-risk exposure group. On follow-up, out of the 10 high-risk exposure participants, 3 converted into active TB, indicating that this group has the highest risk of developing TB. Thus, the evaluated panel of Hsp[s] can discriminate between LTBI and active TB. They can also identify individuals who are at the highest risk of developing active TB. Because they can be rapidly detected, Hsp[s] have an edge over the existing diagnostic tools for LTBI. The evaluation of these proteins will be useful in designing better diagnostic methods for LTBI

assessment of cytokines in quantiferon supernatants for the diagnosis of latent tb infection in a tribal population of Melghat, India

The tuberculin skin test [TST] and interferon-gamma release assays [IGRA], namely, the QuantiFERON-TB Gold test [QFT], remain the standard immuno-logical diagnostic tools for latent tuberculosis [TB] infection [LTBI]. However, the sub-optimal detection rates of both of these tests are major impediments in recognizing the population at risk. This study was aimed at evaluating additional cytokines besides interferon-gamma [IFN-gamma] as biomarkers for improving LTBI diagnosis in the tribal population of Melghat, India. Seventy-four close TB contacts were stratified by QFT and TST results into: [i] QFT+/TST+ [n = 26], [ii] QFT+/TST- [n = 12], [iii] QFT-/TST- [n = 35] and [iv] QFT-/TST+ [n = 1] groups. A panel of cytokines [IL-6, IL-10, TNF-alpha and IL-2R] was then evaluated in antigen-stimulated QFT cell-free culture supernatants using IMMULITE-1000, an automated immunoassay analyzer. Cytokine estimation showed significantly higher levels of IL-6 in the QFT+/TST+ group, while significantly higher levels of IL-10 were found in the QFT-/TST- group. Correlation analysis identified a positive correlation between IL-6 and the QFT response [r = 0.6723, P< 0.0001], while a negative correlation was seen between QFT and IL-10 expression [r=-0.3271, P = 0.0044]. Similarly, IL-6 was positively correlated with TST levels [r = 0.6631, P< 0.0001], and conversely, a negative correlation was found between TST and IL-10 expression [r=-0.5698, P<0.0001]. The positive and negative predictive values of IL-6 were found to be 92.59 and 93.33%, respectively, and the positive and negative predictive values of IL-10 were 96.55 and 91.18%, respectively. No significant impact of the demographic characteristics on cytokine positivity was observed. Our preliminary results suggest that the evaluation of additional cytokines in QFT cell-free culture supernatants may be valuable for the identification of LTBI. Combining IL-6 and IL-10 with QFT and/or TST could markedly improve the detection accuracy of LTBI. Our observations require investigation in larger well-characterized cohorts along with follow-up studies to further confirm the study outcome

Investigation of Immune Biomarkers Using Subcutaneous Model of M. tuberculosis Infection in BALB/c Mice: A Preliminary Report

Evaluation and screening of vaccines against tuberculosis depends on development of proper cost effective disease models along with identification of different immune markers that can be used as surrogate endpoints of protection in preclinical and clinical studies. The objective of the present study was therefore evaluation of subcutaneous model of M.tuberculosis infection along with investigation of different immune biomarkers of tuberculosis infection in BALB/c mice. Groups of mice were infected subcutaneously with two different doses : high (2x10(6) CFU) and low doses (2x10(2) CFU) of M.tuberculosis and immune markers including humoral and cellular markers were evaluated 30 days post M.tuberculosis infections. Based on results, we found that high dose of subcutaneous infection produced chronic disease with significant (p<0.001) production of immune markers of infection like IFNgamma, heat shock antigens (65, 71) and antibody titres against panel of M.tuberculosis antigens (ESAT-6, CFP-10, Ag85B, 45kDa, GroES, Hsp-16) all of which correlated with high bacterial burden in lungs and spleen. To conclude high dose of subcutaneous infection produces chronic TB infection in mice and can be used as convenient alternative to aerosol models in resource limited settings. Moreover assessment of immune markers namely mycobacterial antigens and antibodies can provide us valuable insights on modulation of immune response post infection. However further investigations along with optimization of study protocols are needed to justify the outcome of present study and establish such markers as surrogate endpoints of vaccine protection in preclinical and clinical studies in future.

Comparative evaluation of booster efficacies of BCG, Ag85B, and Ag85B peptides based vaccines to boost BCG induced immunity in BALB/c mice: a pilot study

PURPOSE: In the present study booster efficacies of Ag85 B, Bacillus Calmette-Guerin (BCG), and Ag85B peptides were evaluated using prime boost regimes in BALB/c mice. MATERIALS AND METHODS: Mice were primed with BCG vaccine and subsequently boosted with Ag85B, BCG and cocktail of Ag85B peptides. RESULTS: Based on analysis of immune response it was observed mice boosted with Ag85B peptides showed significant (p < 0.001) cytokines levels (interferon gamma, interleukin 12) and BCG specific antibodies (anti-BCG and anti-purified protein derivative titre) compared to booster dose of BCG, Ag85B and BCG alone. CONCLUSION: Our pilot results suggest that prime boost regimes with Ag85B peptides can boost waning BCG induced immunity and may improve immunogenicity of BCG vaccine. However, lot of work is further needed using experimental model of tuberculosis infection to justify the result.

Evaluation of Mycobacterium tuberculosis 16 and 71 Heat Shock Proteins in the Cerebrospinal Fluid of Tuberculous Meningitis Patients.

Aims: To evaluate the efficiency of two potential Mycobacterium tuberculosis (M. tb) heat shock proteins (Hsps) towards the improvement of tuberculous meningitis (TBM) diagnosis. Study Design: The patients were divided into TBM (confirmed and suspected) and non TBM group. The cerebrospinal fluid (CSF) was collected and evaluated for M. tb Hsp 16 and 71.The Indirect ELISA results of M. tb Hsp 71 were compared with polymerase chain reaction (PCR). Place and Duration of Study: Biochemistry Research Laboratory, Central India Institute of Medical Sciences, Nagpur between June 2009 and July 2010. Methodology: 29 TBM and 22 non TBM CSF samples were collected. Indirect ELISA was performed for evaluating the, M. tb Hsp16 and Hsp71 in the collected samples, individually as well as in combination. The ELISA method for detection of M. tb Hsp 71 was also compared with in house PCR technique for TBM diagnosis. Results: The data analysis was done with MedCalc® Software. M. tb Hsp16 showed positivity of 58.62% and negativity of 68.18%. Similarly for M. tb Hsp71, positivity is 89.65% and negativity is 68.18%.The results of ELISA for M. tb Hsp71 was compared with PCR technique and concordance was also calculated. Of the Hsp ELISA positive group for M. tb Hsp71, 24 were PCR positive and 2 were PCR negative with the 92.30 % concordance in TBM patients and in non TBM patients the concordance was observed to be 93.30%. Use of the monoclonal antibody Hsp 71 appear preferable over individual use of M. tb Hsp 16 and combined use of both Hsp and yield optimum results. Conclusion: Our data suggest that the detection of M. tb Hsp 71 in the CSF sample of TBM patients can be useful for the diagnosis of TBM patients. These predictors, however, need further work to validate reliability.

Antioxidant potential of fagonia arabica against the chemical ischemia-induced in PC12 cells

The imbalance between pro-oxidants and anti-oxidants leads to generation of oxygen/nitrogen free radicals which are implicated in several neurodegenerative diseases. Fagonia arabica is an ethno-pharmacologically important Ayurvedic herb known to have many medicinal properties like anti-inflammatory, analgesic and antipyretic effects. However, its antioxidant potential has not been investigated so far. The present study was designed to investigate the antioxidant potential of F. arabica and its neuroprotective effect on chemical ischemia induced in PC12 cells. Chemical ischemia was induced through exposing the cells to uncoupler of oxidative phosphorylation sodium azide [5.0 mM] and competitive inhibitor of glycolysis 2-deoxy-glucose [2.0 mM] for 2 h followed by 24 h reperfusion with normal culture medium. Total polyphenolic content [TPC] and antioxidant potential of the herb was measured using DPPH and ABTS + scavenging and ferric ion reducing antioxidant potential [FRAP] assays; its effect on neuroprotection and energy metabolism was also studied. The ischemic injury was characterized by impaired energy status as indicated by decreased ATP levels in the cells, accompanied by increased lactic acid content. Both the changes favourably responded to F. arabica and offered considerable neuroprotection from ischemia and helped to maintain the cellular viability and mitochondrial integrity of the cells. F. arabica showed considerable amount of TPC and antioxidant activity. This study reveals the antioxidant potential of F. arabica and its protective efficacy against ischemia/reperfusion mediated cell death. F. arabica thus can be considered for further studies for the development of the prophylactic or therapeutic agent for the treatment of ischemic stroke

Effect of repeat dose of BCG vaccination on humoral response in mice model.

BCG is the only vaccine presently available against tuberculosis but it is estimated to prevent only 5% of the all potentially vaccine-preventable deaths due to Tuberculosis. Keeping these in view the present study has been undertaken to evaluate the efficacy of BCG and the effect of repeat dose of BCG on antimycobacterial humoral response in mouse model. To improve BCG immunogenicity, specific anti-mycobacterial immune responses (anti-BCG titre and total IgG level) were evaluated in mouse model using boost immunization protocols with the BCG vaccine. Mice induced with a repeat dose of BCG showed an increased anti mycobacterial humoral response, which gradually declined few weeks after single dose of BCG administration. The results suggest improved efficacy of BCG vaccine by giving repeat dose of BCG that can enhance the level of immunoprotection against tuberculosis as opposed to a single BCG dose.

Secretory factors of human neuroblastoma (IMR-32) and human glioblastoma (U87MG) cell lines induce neurite outgrowths in PC12 cells.

Neurite outgrowth is essential for the communication of the nervous system. The rat Pheochromocytoma (PC12) cells are commonly used in the neuronal cell study. It is well known that exogenous stimuli such as Nerve Growth Factor (NGF) induce neurite outgrowth. In the present study it has been investigated whether or not the conditioned medium from human neuroblastoma cell line (IMR-32) and human glioblastoma cell line (U87MG) may augment neurite outgrowth in PC12 cells. PC12 were cultured with and without conditioned media of IMR-32 and U87MG. The result showed that both the conditioned media induce neurite outgrowth within 48 hr and stops further proliferation of PC12 cells. However no outgrowth was noted in PC12 cells incubated without conditioned medium. In conclusion, it is shown that both the conditioned media (IMR-32 and U87MG) have the potential to induce the neurite outgrowth in the PC12 cells.

Pattern of leprosy disabilities in Gorakhpur (Uttar Pradesh).

Out of 514 leprosy cases studied, 229(44.56%) had disability. Disability was most commonly seen in lepromatous leprosy. There was an increasing trend in disability with increasing age of patient and duration of disease. Disability rate was higher in males as compared to females. Nerve thickening and reactional states were more common in disabled cases. Dapsone treated group showed a disability rate of 63.8% as compared to 30.0% in untreated group. Hand was the most commonly affected site and mobile claw hand was the single most common disability. The overall disability index-D.I. (2) of Bachelli was 1.25 and lepromatous cases had highest D.I. (1.89). Disability index was higher in males and was found to increase with increasing age of patient and duration of disease.

A study of cutaneous lesions of leprosy on male genitalia.

The cutaneous lesions of leprosy on male genitalia were studied. They were found in 2.9% of cases examined in this series. They were seen most commonly in borderline group. It is emphasised that it is not uncommon to find lesions on male genitalia in leprosy.

A study of palmo-plantar lesions in leprosy: a preliminary report.

Out of 500 leprosy patients screened for palmar and/or plantar lesions, eighteen cases were detected. They were classified according to Ridley-Jopling classification. In majority of cases it was macular lesion. Cases were from TT, BT and BB group. In 50% cases, palmo-plantar involvement was associated with type I reaction. In 66.7% cases, it was extension of patch from surrounding area, 11.1% cases isolated lesions were seen and in 22.2% cases both extension as well as isolated lesions were present.

Calcification of ulnar nerve in leprosy--a case report.

A case of Tuberculoid leprosy who had taken irregular treatment for five years and had thickened ulnar nerve which on radiological examination showed calcification, is reported.