Utility of B-cell epitopes based peptides of RD1 and RD2 antigens for immunodiagnosis of pulmonary tuberculosis

Tuberculosis (TB) continues to be a major health problem due to lack of accurate, rapid, and cost-effective diagnostic tests. Serodiagnostic tests incorporating highly specific region of difference (RD) antigens (early secretory antigenic target 6 [ESAT-6], culture filtrate protein 10 [CFP-10], culture filtrate protein 21 [CFP-21], and mycobacterial protein from species tuberculosis 64 [MPT-64]) have recently been shown to be promising for specific diagnosis of TB in our lab. However, only few studies have reported the use of synthetic peptides of RD antigens, and none has used them to differentiate TB from sarcoidosis, a close mimic of smear-negative pulmonary TB (PTB) with entirely different management. The present study was conducted with an aim to study the utility of B-cell epitopes based peptides of RD1 (ESAT-6, CFP-10) and RD2 (CFP-21, MPT-64) antigens for immunodiagnosis of PTB for which sputum smear-positive PTB patients, sputum smear-negative PTB patients, sarcoidosis patients, and healthy controls (n = 24/group) were recruited. Bioinformatic software Bcepred was used to predict linear B-cell epitopes, using physico-chemical properties on a non-redundant dataset. Seven peptides as representative B-cell epitopes of ESAT-6, CFP-10, CFP-21, and MPT-64 were evaluated as targets of the antibody responses in TB patients and controls by enzyme-linked immunosorbent assay (ELISA). The current study showed sensitivity with individual peptides ranging from 37.5% to 83.3% for smear positive, 25% to 58.3% for smear negative as compared to 4.16% to 20.8% for sarcoidosis. Four out of 7 peptides that showed higher reactivity with TB patients and better discrimination from sarcoidosis patients representing ESAT-6, CFP-10, CFP-21, and MPT-64 were selected for multiepitope ELISA. The combination of peptides yielded 83.3% sensitivity for smear positive, 62.5% for smear negative, and only 4.16% for sarcoidosis. The specificity, however, for all the peptides/combination was 100%. Combination of peptides has proven to be better than individual peptides as per the latest criteria of the World Health Organization according to which a test that can replace smear microscopy with sensitivity of >90% for smear-positive patients and >65% for smear-negative TB patients with a specificity >95%, and thus, the present study suggests that a test based on combination of peptides selected from mycobacterial RD1 and RD2 antigens could be important for promoting an early diagnosis and management of otherwise difficult to diagnose smear-negative PTB patients. Moreover, it can also be used to discriminate sarcoidosis from PTB, thus preventing the misdiagnosis and mismanagement.     

Analysis of cellular response to isocyanate using N‐succinimidyl N‐methylcarbamate exposure in cultured mammalian cells

Isocyanates (R? N?C?O), one of the highly reactive industrial intermediates, possess the capability to modulate the bio‐molecules by forming toxic metabolites and adducts which may cause adverse health effects. Some of their toxic degradations have previously been unknown and overlooked; of which, molecular repercussions underlying their genetic hazards upon occupational/accidental exposures still remain as an intricate issue and are hitherto unknown. To assess the genotoxic potential of methyl isocyanate in cultured mammalian cells after in vitro exposure, we performed a study in three different normal cell lines MM55.K (mouse kidney epithelial), B/CMBA.Ov (mouse ovarian epithelial), and NIH/3T3 (primary mouse embryonic fibroblast). Cellular DNA damage response was studied for qualitative phosphorylation states of ATM, γH2AX proteins and quantitative state of p53 phosphorylation; DNA cell cycle analysis and measure of cellular apoptotic index before and after treatment were also investigated. Our results demonstrate that methyl isocyanate by negatively regulating the DNA damage response pathway, might promote cell cycle arrest, and apoptosis in cultured mammalian cells suggestive of causing genetic alterations. We anticipate that these data along with other studies reported in the literature would help to design better approaches in risk assessment of occupational and accidental exposure to isocyanates. We also predict that increasing knowledge on DNA damage‐triggered signaling leading to cell death could provide new strategies for investigating the effects of DNA repair disorders and decreased repair capacity on the toxicity and carcinogenic properties of environmental toxins. Environ. Mol. Mutagen., 2009. © 2009 Wiley‐Liss, Inc.     

Superficially invasive carcinoma of the vagina following treatment for cervical cancer: A report of six cases

Six patients with superficially invasive squamous carcinoma of the vagina are described. All patients meet recently proposed criteria for the diagnosis of microinvasive vaginal carcinoma. The depth of invasion measured from the surface was less than 2.5 mm. There was no lymph-vascular space involvement. The invasive foci arose within a field of carcinoma in situ. Five of these six patients had previously been treated for invasive cervical cancer with pelvic radiation from 82 to 246 months before the diagnosis of vaginal carcinoma. All but one patient had the carcinoma confined to the upper one-third of the vagina. All patients were treated with a single vaginal radium application following vaginectomy. One of these six patients expired from recurrent vaginal cancer 35 months following diagnosis. During the same 17-year period, 17 other cases of Stage I epidermoid cancer of the vagina were treated which did not meet the above criteria for microinvasion. There were no statistically significant differences between these two groups with regard to age at diagnosis, history of cervical cancer, hysterectomy, or pelvic radiation or in survival. Additional experience with early vaginal carcinoma is needed before microinvasive carcinoma of the vagina should be accepted as a distinct clinical entity.     

Higher risk of epilepsy in twins

The study explores the possibility of a higher risk of epilepsy in twins than in singletons. The concordance for epilepsy in MZ twins (66·7%) was higher than in DZ twins (12·5%), thus supporting the hypothesis of genetic predisposition. The incidence of epilepsy in twins is 46·7 per 1000 cotwins while in general population it varies from 3·6 to 10·38 per 1000. This shows that twins have appreciably higher risk of epilepsy than singletons. The present study suggests an association of the twinning phenomenon with a higher risk of epilepsy.     

Smokeless tobacco impairs the antioxidant defense in liver, lung, and kidney of rats.

The present study was designed to evaluate the effects of long-term use of aqueous extract of gutkha (a form of smokeless tobacco) on the antioxidant defense status and histopathological changes in liver, lung, and kidney of male Wistar rats. Animals were orally administered aqueous extract of smokeless tobacco (AEST) at a low dose (96 mg/kg body weight per day) for 2 and 32 weeks, and at a high dose (960 mg/kg body weight per day) for 2 weeks. High-dose AEST for 2 weeks decreased the hepatic glutathione (GSH) and glutathione peroxidase (GPx), and increased lipid peroxidation (Lpx) by 17%, 19%, and 20%, respectively. Low-dose AEST for 32 weeks significantly decreased (p < 0.05) the antioxidant status in these organs. In liver, AEST decreased GSH levels and the activities of superoxide dismutase (SOD), catalase (CAT), and GPx by 34.6%, 29%, 17.1%, and 17.4%, respectively, but it increased Lpx by 64%. In kidney, GSH, SOD, CAT, and GPx were decreased by 26.6%, 23%, 33%, and 18%, respectively, with an increase of Lpx by 65%. AEST decreased the lung GSH, SOD, CAT, and GPx, and increased lung Lpx by 43%, 28.5%, 37%, 40%, and 24%, respectively. However, no change in the plasma levels of vitamins A, C, and E were observed with AEST treatment. Histopathological findings suggest that administration of AEST at the high dose for 2 weeks or at the low dose for 32 weeks could cause mild to moderate inflammation in liver and lungs. In conclusion, a decrease in the antioxidant defense system and long-term inflammation caused by smokeless tobacco may be risk factors for gutkha-induced pathogenesis.     

Mutational analysis of the BRAF gene in human congenital and dysplastic melanocytic naevi

Eighteen congenital melanocytic naevi (CMN) from 17 patients and 18 dysplastic melanocytic naevi (DMN) from 18 patients were screened for mutations in the BRAF oncogene (present study) and the N-ras oncogene (in the course of two foregoing studies) by single-strand conformational polymorphism (SSCP)/sequencing analysis. BRAF mutations were demonstrated in both types of lesion. As a whole, 17 of 18 CMN (94.4%) and five of 18 DMN (27.7%) harboured either BRAF or N-ras mutations. As the BRAF oncogene is frequently found to be mutated in human cutaneous melanomas, it may constitute a risk factor for melanoma formation within CMN and DMN.     

Mutational analysis of 9 different tumour-associated genes in human malignant mesothelioma cell lines

Seven tumour suppressor genes (Chk1, Chk2, Apaf1, Rb1, p53, p16(INK4a) and p14(ARF)) and two oncogenes (N-ras and BRAF) were screened in nine human malignant melanoma (HMM) cell lines for point mutations or small deletions/insertions by DGGE, TGGE and SCCP analysis. For the first time in human mesothelioma, Chk1 gene mutations were detected in two of the nine investigated HMM cell lines. P53 gene mutations were found in three cell lines and p16(INK4a) mutations in 5. Mutation of the Chk1 gene implies a novel disruption mechanism of the p53 pathway in HMM, without affecting p53 itself. According to our knowledge, this is the first mutation screening of Chk1, Chk2, Apaf1 and Rb1 in human malignant mesothelioma.     

Coronary endarterectomy for diffuse extensive coronary artery disease

Introduction Atherosclerotic coronary artery disease is in an increasing trend in India. With the advancement of non-surgical methods of revascularisation, the patients coming for surgery are of less attractive anatomy. The role of coronary endarterectomy along with coronary artery bypass grafting for a selected group of these patients is quite promising. Materials and Methods From March 2000 to March 2005, out of 362 CABGs performed, 42 patients had undergone coronary endarterectomy. The age range being from 35 to 76 years, M: F is 38∶4 Hypertension was present in 26 (61%), diabetes mellitus in 20 (47.6%), smoking in 26 (61%) and dyslipidemia in 12 (28.5%) cases. Old myocardial infarction was present in 52.3% cases, unstable angina in 16.6%, stable angina in 23.8% and cardiogenic shock in 7.1% cases. All cases had undergone coronary artery bypass grafting with endarterectomy. Out of 18 LAD endarterectomies 17 cases LIMA was used as onlay patch. Result The average number of grafts anastomosed was 3.7. Single-vessel endarterectomy was done in 37, double vessel in 4 and four vessel in one case. LAD endarterectomy was done in 18, RCA in 12, diagonal in 10, intermediate in 1 and marginals in 8 cases. Postoperatively 3 patients had arrhythmia, two perioperative MI, one recurrent angina and one congestive cardiac failure (CCF). There was 2 (4.76%) mortality. Conclusion Hypertension and smoking are major risk factors. LAD is the most common artery requiring endarterectomy. Usage of LIMA following endarterectomy of LAD is quite satisfactory and short term results are encouraging.