[Show abstract][Hide abstract] ABSTRACT: Rapid and accurate diagnosis is important for preventing transmission of Mycobacterium tuberculosis. Currently available tuberculosis (TB) diagnostic methods lack desired sensitivity and specificity, and require sophisticated equipment and skilled workforce including weeks' long duration to yield results. In this study, extracellular proteins or secretory protein antigens of M. tuberculosis H37Rv have been isolated using ion exchange chromatography, immunocharacterized and exploited for the development of efficient enzyme-linked immunosorbent assay (ELISA) for diagnosis of active TB with enhanced specificity and sensitivity. Apparent molecular masses for purified proteins were found to be 6, 27, 30, 38 and 64 kDa. Out of five purified proteins, one (64 kDa) was found to be novel. Of the five proteins, four (6, 27, 30 and 38 kDa) were found significant to be used in the development of ELISA for pulmonary and extra-pulmonary TB. The immune responses of serum samples of TB patients and other healthy subjects against the above-mentioned antigens' cocktail were evaluated. Critical parameters of newly developed ELISA were optimized and it was observed that the cocktail antigens have a greater specificity (98.06 %) and sensitivity (98.67 %) as compared to other commercially available diagnostic tests. The present findings suggest that the developed ELISA is an effective tool for routine screening and early-stage diagnosis of TB.
[Show abstract][Hide abstract] ABSTRACT: Acute myocardial infarction (AMI) is the leading cause of death worldwide, with early diagnosis still being difficult. Promising new cardiac biomarkers such as troponins and creatine kinase (CK) isoforms are being studied and integrated into clinical practice for early diagnosis of AMI. The cardiac-specific troponins I and T (cTnI and cTnT) have good sensitivity and specificity as indicators of myocardial necrosis and are superior to CK and its MB isoenzyme (CK-MB) in this regard. Besides being potential biologic markers, cardiac troponins also provide significant prognostic information. The introduction of novel high-sensitivity troponin assays has enabled more sensitive and timely diagnosis or exclusion of acute coronary syndromes. This review summarizes the available information on the potential of troponins and other cardiac markers in early diagnosis and prognosis of AMI, and provides perspectives on future diagnostic approaches to AMI.
[Show abstract][Hide abstract] ABSTRACT: HIV (human immunodeficiency virus) infection has now become endemic worldwide and AIDS ranks fourth among the world's top killers of mankind. A rapid and accurate HIV testing assay is pre-requisites for practical applicability of diagnostic tests. The aim of present study was to design peptides cocktail as an antigen and development of ELISA test for HIV-1/2 antibody detection, with enhanced sensitivity and specificity. A novel peptide stretch V3-I, covering immunodominant epitope corresponding to V3 hypervariable loop of gp120 antigens of selected Indian isolates, has been studied and incorporated in an antigenic cocktail of gp36, gp41, and rp24 of HIV-1/2. Peptides from these antigens were chemically synthesized and an additional cysteine residue was added at both amino- and carboxyl- terminal sequences of each peptide in order to form inter and intramolecular disulfide bond for the folding of peptides. This generated conformational epitopes with increased oligomericity and stability of peptide sequences; and attachment of antigen to the solid support of ELISA plates. The use of antigenic cocktail of folded peptides and recombinant p24 enhanced sensitivity and specificity of ELISA test. Evaluation of the test using 1123 serum samples in comparison with Boston Biomedical Incorporation (BBI) panels showed 100% sensitivity and 99.3% specificity with no cross reactivity tribulation. In conclusion, "HIV Screen test" detects HIV 1/2 antibodies with a high degree of sensitivity and specificity and could be a promising tool for seroscreening of blood during transfusion, counseling and diagnosis of HIV-1/2.
Journal of immunological methods 10/2012; · 2.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Survivin is known to be highly-expressed in various carcinomas; and is associated with their biologically aggressive characteristics and drug resistance. We have previously reported survivin as an important anti-apototic protein involved in head and neck squamous cell carcinoma (HNSCC) tumorigenesis and providing resistance to conventional cancer therapies. The purpose of present study was to investigate the potential of survivin as a therapeutic target in HNSCC. This study was designed to explore the effect(s) of survivin-siRNA therapy on the apoptosis in HNSCC cells, and its influence on cisplatin-sensitivity. Lentivirus vector was developed to deliver survivin specific siRNA into cancer cells. The data indicates that silencing of survivin-mediated by Lentivirus-siRNA therapy effectively suppressed cancer cell proliferation and induced caspase-dependent apoptosis in HNSCC cells. The study also shows that the response of HNSCC cells to cisplatin drug treatment at clinically relevant level was limited. We observed survivin to be a key factor involved in this cisplatin-resistance mechanism, and down-regulation of survivin significantly increases sensitivity of cancer cells to cisplatin-mediated apoptosis. Thus, this combination therapy acts as a multimodality regimen with significant potential to improve clinical outcomes in head and neck cancers.
Current Gene Therapy 09/2012; · 5.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lactoglobulin (β-LG) genetic polymorphisms are important and well known due to their effects on quantitative traits and technological properties of milk. At the DNA level, polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) allows for the detection of unknown polymorphisms at β-LG loci. Here we describe the usefulness of the PCR-SSCP technique for β-LG typing. In the present study, we amplified and sequenced the part of promoter region and all the seven exons containing the entire coding and untranslated region for the β-lactoglobulin gene in best dairy goat breeds of India namely: Jamunapari and Jakhrana. Nine polymorphisms were detected, one in the l promoter region, four in the introns and four in the exons of the β-lactoglobulin gene. All polymorphisms were single nucleotide substitutions. The polymorphisms in the coding region did not produce any amino acid change. Key words: β-Lactoglobulin gene, dairy goats, polymorphism, single nucleotide polymorphism (SNP), single strand conformational polymorphism (SSCP).
AFRICAN JOURNAL OF BIOTECHNOLOGY 07/2012; 11(50-DOI:10.5897/AJB12.884):11057-11064. · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cyclooxygenase-2 (Cox-2) is an inducible enzyme involved in the conversion of arachidonic acid to prostaglandin and other eicosanoids. Molecular pathology studies have revealed that Cox-2 is over-expressed in cancer and stroma cells during tumor progression, and anti-cancer chemo-radiotherapies induce expression of Cox-2 in cancer cells. Elevated tumor Cox-2 is associated with increased angiogenesis, tumor invasion and promotion of tumor cell resistance to apoptosis. Several experimental and clinical studies have established potent anti-cancer activity of NSAID (Non-steroidal anti-inflammatory drugs) and other Cox-2 inhibitors such as celecoxib. Much attention is being focused on Cox-2 inhibitors as beneficial target for cancer chemotherapy. The mode of action of Cox-2 and its inhibitors remains unclear. Further clinical application needs to be investigated for comprehending Cox-2 biological functions and establishing it as an effective target in cancer therapy.
Current drug targets 03/2011; 12(7):1082-93. · 3.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The present study deals with the evaluation of the efficacy of oxaliplatin and paclitaxel combination as a potential strategy in controlling HNSCC cell proliferation and the assessment of correlation between occurrence of apoptosis and changes in expression of survivin (IAP). The panel cell lines included two HNSCC cell lines (Cal27 and NT8e) and one normal cell line (293) with differential level of survivin expression in accordance with chemosensitivity. The cytotoxicity and effect of drugs on apoptosis was determined, separately and in combination. Combined treatment of cells with paclitaxel and oxaliplatin resulted in significantly higher cytotoxicity as compared to individual single drug treatment. Cytotoxicity was prominent in paclitaxel to oxaliplatin (pacl-oxal) sequence treatment with an approximate two-fold increase in apoptosis as compared to oxaliplatin to paclitaxel (oxal-pacl) sequence treatment. Paclitaxel treatment also caused increased survivin expression showing reduced apoptosis at low concentration. Oxaliplatin, when combined with paclitaxel, decreased the survivin level with increased cell death. Inhibition of survivin by a small interfering RNA (siRNA) method also increased the sensitivity of the cancer cell lines to paclitaxel whereas over-expression of survivin in the transfected 293-cell line provided resistance. In conclusion, the interaction between drugs was synergistic and schedule-dependent. Survivin played a critical role in paclitaxel resistance through the suppression of apoptosis, and a significant induction of apoptosis was observed when oxaliplatin was combined with paclitaxel at least in part by the down-regulation of survivin.
Current cancer drug targets 11/2010; 10(7):660-9. · 5.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oxaliplatin is integrated in treatment strategies against a variety of cancers including radiation protocols. Herein, as a new strategy we tested feasibility and rationale of oxaliplatin in combination with radiation to control proliferation of head and neck squamous cell carcinoma (HNSCC) cells and discussed survivin-related signaling and apoptosis induction.
Cytotoxicity and apoptosis induced by radiation and/or oxaliplatin were examined in relation to survivin status using two HNSCC cell lines viz., Cal27 and NT8e, and one normal 293-cell line. Survivin gene knockdown by siRNA was also tested in relevance to oxaliplatin-mediated radiosensitization effects.
Survivin plays a critical role in mediating radiation-resistance in part through suppression of apoptosis via a caspase-dependent mechanism. Oxaliplatin treatment significantly decreased expression of survivin in cancer cells within 24-72 h. Apoptotic cells and caspase-3 activity were increased parallely with decrease in cell viability, if irradiated during this sensitive period. The cytotoxicity of oxaliplatin and radiation combination was greater than additive. Survivin gene knockdown experiments have demonstrated the role of survivin in radiosensitization of cancer cells mediated by oxaliplatin.
Higher expression of survivin is a critical factor for radioresistance in HNSCC cell lines. Pre-treatment of cancer cells with oxaliplatin significantly increased the radiosensitivity through induction of apoptosis by potently inhibiting survivin.
Radiotherapy and Oncology 08/2010; 96(2):267-73. · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium tuberculosis is one of the most dangerous human pathogens
evolved on the Planet that poses big challenge to the scientists working on diagnostics and therapeutics. There is an urgent requirement for the development of a novel candidate as an immunoprophylactic agent (vaccine). The complete genome sequencing of M. tuberculosis
H37 Rv had paved way for the identification of candidate antigens of prophylactic significance. Numerous proteins especially membrane proteins acting as immunogens or subunit vaccines are profiled and investigated for immunoprophylactic activity. The protein of 71 kDa, which elicits the response of both B cell/T cell has been used for the development of candidate vaccine. However the use of proteinaceious antigens in
immunoprophylaxis is not encouraging for various reasons. The lipid based immunogenic antigens find to be more promising in this regard, since lipids escape the coventional antigen processing and are routed through CD1 system by a different pathway. The lipid antigens are endocytosed through a pathway involving protease resistant proteins such as saposin.
Saposin molecules help CD1 molecules to present the antigen to the T Cell and NKT cells. The NKT cells play a larger regulatory role and have immunomodulatory properties and hence NKT cells are attractive targets for the development of immunotherapies. Another advantage of lipid Ag induced immune response is its specificity, which is attributed by δ,γ region of the variable chain of the TCR. This region of TCR are not diverse in it function. Hence the rate of elimination of mycobacterium will be rapid if the lipid Ag is challenged. The other advantages of priming with lipid moieties include, i. rate of mutation of CD1 molecules is not rapid, limited polymorphism of CD1 etc. The use of liposome technology in enhancing
the immune response is under active invesitgation. Mycobacterial mannophosphoinositides (PIMs) encapsulated in liposomes made of egg phosphatidylcholine (EPC) and cholesterol (CH) (2:1.5 molar ratio) were able to induce humoral and delayed type hypersensitivity (DTH, foot-pad swelling reaction) responses in mice without the help of any carrier protein.
Animals immunized with this liposome glycophospholipid antigen demonstrated enhanced percent survival on intravenous challenge with virulent M. tuberculosis. The paper deals with the immunoprophylactic studies carried out with M. tuberculosis derived lipid based antigens.
ADVANCES IN BIOMEDICAL RESEARCH. 2010. 01/2010; 407- 416.
[Show abstract][Hide abstract] ABSTRACT: Tuberculosis is the second most infectious disease and is the leading cause of death in the tropical World. The Mycobacterium tuberculosis infects about 1.7 billion people every year and is responsible for more than 2 million deaths globally each year. Overall, one-third of the world’s population is currently infected with the M. tb bacillus without demonstrable symptoms.
A delayed or missed diagnosis of active infection and prevalence of drug resistant strains of M. tuberculosis are major causes of transmission and mortality. With the threat of such an epidemic looming, and despite an enormous amount of research since the time of Koch, we still have no
simple, rapid, sensitive, and specific test to detect and differentiate most or all patients with active tuberculosis from those with quiescent lesions, previous vaccination, or other diseases, or even from those who are completely healthy. Tuberculosis co-infected with HIV has further
complicated the diagnosis since. HIV-positive patients with active TB are more likely to have negative sputum smears and atypical chest X-rays than their HIV-negative counterparts. Timely and accurate identification of subjects infected with M. tuberculosis and rapid laboratory confirmation of tuberculosis are two effective public health measures that can be taken to combat the tuberculosis epidemic across the globe. The focus of this paper is to review the diagnostic potential of proteinaceous and non proteinaceous antigens in various diagnostic assays and promises of liposome based antibody or antigen detection methods for diagnosis of tuberculosis
[Show abstract][Hide abstract] ABSTRACT: Survivin, an inhibitor of apoptosis, is overexpressed in cancer. It has been implicated in both prevention of apoptosis and cell cycle regulation. We investigated the distribution of antiapoptotic protein survivin in 29 oral squamous cell carcinoma (OSCC) and 16 oral premalignant lesions. It has been suggested that wild-type p53 represses survivin expression. Therefore, we investigated the status of p53 in relation to survivin to determine the potential involvement in oral tumorigenesis.
Oral cancer tissues were freshly obtained at the time of surgery and classified as per general rules of head and neck cancer (TNM classification). Immunohistochemistry and reverse transcriptase-polymerase chain reaction were conducted to study the expression of survivin and p53. The Fisher's exact test was employed to determine the association of survivin and p53 with clinicopathologic parameters of the subjects being studied.
Positive staining for survivin was found in 72% OSCC and 44% oral premalignant lesions with no immunoreactions in the corresponding normal tissues. For p53, 59% OSCC, 38% premalignant lesions, and 14% normal tissues were positive. Importantly, about half of the p53-positive OSCC and premalignant tissues also showed survivin positivity (28% OSCC and 18% premalignant lesions). Further, it is observed that the number of survivin positive cells was significantly higher in the p53-positive group. Survivin is expressed in a varying proportion of cells, and in majority of patients it was localized in cytoplasm, whereas p53 is strictly restricted to the nucleus. The survivin expression levels in both primary OSCC and premalignant lesions were significantly higher than in normal oral tissues (OSCC, p < .0008; premalignant lesions, p < .04). No significant correlations between survivin and p53 expression with clinicopathologic parameters were found.
Frequent overexpression of apoptosis regulators, survivin and p53, in OSCC as well as in oral premalignant lesions were found. Overexpression of these 2 markers in premalignant lesions suggest a role in early stages of oral carcinogenesis.
Head & Neck 04/2009; 31(8):1039-48. · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A total of 1360 subjects with clinically confirmed pulmonary and extra-pulmonary tuberculosis (TB) and other non-tuberculous conditions.
To develop a rapid, sensitive and specific diagnostic test for the detection of the glycolipid antigen of Mycobacterium tuberculosis in a variety of clinical samples.
Affinity-purified rabbit anti-glycolipid antibodies (IgG) were coupled to liposome particles (0.2-0.4 microm) in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinamide to prepare the working reagent of the TB/M card test.
Antibody-conjugated liposomes, when determined with the glycolipid antigens present in the specimens, formed a dark blue agglutination within 4 min. No clumping was observed in samples from normal healthy subjects or patients with other diseases. The test was shown to be effective in detecting glycolipid antigens of M. tuberculosis in clinical samples from patients with active TB with as low as 1 ng/ml analytical sensitivity, 97.4% clinical sensitivity and 96.9% specificity.
The TB/M card test was found to be comparatively economical (4 Indian Rupees or US$ 0.09/test), rapid (4 min) and seems fairly useful for mass testing of a variety of biological specimens (cerebrospinal, pleural and synovial fluids, serum, tissue biopsy extract) from patients with tuberculous meningitis, pulmonary TB and other extra-pulmonary TB in endemic countries.
The international journal of tuberculosis and lung disease: the official journal of the International Union against Tuberculosis and Lung Disease 11/2007; 11(10):1143-51. · 2.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In June 1981, the Centers for Disease Control and Prevention reported the first evidence of a new disease that would later become known as Acquired Immunodeficiency Syndrome (AIDS). HIV and Aids: Basic Elements and Priorities is a concise collection of all aspects of this disease and a source of readily available knowledge. It examines all currently advocated preventive measures such as health education, condom use, safer sex practices, and treatment of sexually transmitted infections. Coverage includes: up-to-date information on multiple dimensions of the HIV/AIDS epidemic; a discussion on new anti–retroviral therapy/drugs, new drugs under clinical trials and preventive HIV vaccine; coverage of current ethical, legal and social issues related to HIV/AIDS; an evaluation of general public awareness about HIV/AIDS; a global perspective and information about HIV/AIDS. • Recommended by the World Health Organization Eastern Mediterranean Region Office (WHO-EMRO) as a basic resource for medical faculty libraries.
First edited by Kartikeyan S, Bharmal RN, Tiwari RP, Bisen PS, 08/2007; Springer., ISBN: 978-9048174454
[Show abstract][Hide abstract] ABSTRACT: A simple and cost-effective diagnostic tool (TB Screen Test) for the screening of patients with pulmonary and extrapulmonary tuberculosis and for differentiation of those individuals from individuals without tuberculosis, other common infections, and healthy controls has been developed. The serological responses of purified mycobacterial glycolipid antigens were examined by a liposome agglutination assay. The assay was able to detect very low antiglycolipid antibody concentrations in the infected individuals. The sera from the tuberculosis patient group had significantly higher concentrations of antiglycolipid antibody than the sera from uninfected control subjects, with 94% sensitivity and 98.3% specificity. Glycolipids of Mycobacterium tuberculosis H37Rv antigens were isolated, purified, and characterized. After interchelation with liposome particles, these purified antigens specifically bound to the antiglycolipid antibodies present in the sera of patients with tuberculosis, resulting in the formation of a blue agglutination. This protocol clearly differentiates healthy controls and M. bovis BCG-vaccinated subjects from those with active tuberculosis. The resultant diagnostic tool, the TB Screen Test, is more economical and rapid (4 min) than other currently available products and can be used for the mass screening of a heavily afflicted population.
[Show abstract][Hide abstract] ABSTRACT: A recombinant DNA strategy was applied to analyze and screen the shotgun expression library from a clinically confirmed local virulent isolate of Mycobacterium tuberculosis with sera from tuberculosis patients, which led to expression and purification of highly immunoreactive and specific mycobacterial antigens expressed during the course of active disease which could be of diagnostic significance. An enzyme-linked immunoassay for diagnosis of tuberculosis was devised by using a shotgun immunoexpression library in the lambdagt11 vector. DNA from a virulent M. tuberculosis patient isolate (TBW-33) confirmed with the BACTEC 460 system was sheared and expressed to generate shotgun polypeptides. beta-Galactosidase fusion proteins capable of demarcating active tuberculosis infections from Mycobacterium bovis BCG-vaccinated healthy subjects or people harboring environmental mycobacteria were selected by comparative immunoreactivity studies. Promising mycobacterial DNA cassettes were subcloned and expressed into the glutathione S-transferase (GST) fusion vector pGEX-5X-1 with a strong tac promoter and were expressed in Escherichia coli BL21. These fusion proteins were severed at a built-in factor Xa recognition site to separate the GST tags and were utilized in an indirect enzyme-linked immunoassay for serodiagnosis of patients with active tuberculosis. The system offered a clear demarcation between BCG-vaccinated healthy subjects and patients with active tuberculosis and proved to be effective in detecting pulmonary as well as extrapulmonary tuberculosis, with an overall sensitivity of 84.33% and an overall specificity of 93.62%.
[Show abstract][Hide abstract] ABSTRACT: Rapid diagnosis and treatment are important for preventing transmission of Mycobacterium tuberculosis. However, the diagnosis of tuberculosis continues to pose serious problems, mainly because of difficulties in differentiating between patients with active tuberculosis and those with healed lesions, normal mycobacterium boris BCG (Bacillus Calmette Guerin) vaccinated individuals, and unvaccinated Manteux positives. Physicians still rely on conventional methods such as Ziehl-Neelsen (ZN) staining, fluorochrome staining, sputum culture, gastric lavage, and other non-traditional methods. Although the tuberculin test has aided in the diagnosis of tuberculosis for more than 85 years, its interpretation is difficult because sensitization with nontuberculous mycobacteria leads to false-positive tests. There have been numerous unsuccessful attempts to develop clinically useful serodiagnostic kits for tuberculosis. A number of proteinaceous and nonprotein antigens (such as acyltrehaloses and phenolglycolipids) have been explored from time to time for the development of such assays but they have not proved to be clinically useful. It has been difficult to develop an ELISA utilizing a suitable antigen because M. tuberculosis shares a large number of antigenic proteins with other microorganisms that may or may not be pathogenic. With the advent of molecular biology techniques, there have been significant advances in nucleic acid-based amplification and hybridization, which are helping to rectify existing flaws in the diagnosis of tuberculosis. The detection of mycobacterial DNA in clinical samples by polymerase chain reaction (PCR) is a promising approach for the rapid diagnosis of tuberculous infection. However, the PCR results must be corrected for the presence of inhibitors as well as for DNA contamination. In the modern era of genetics, marked by proteomics and genomics, the day is not far off when DNA chip-based hybridization assays will instantly reveal mycobacterial infections.
[Show abstract][Hide abstract] ABSTRACT: A rapid test for detecting HIV infection in blood, based on the principle of RBC agglutination was assessed and commercialized. The test is based on the principle of using recombinant anti-RBC monoclonal antibody (Mab) Fab molecules fused to immunodominant portions of the HIV epitopes of HIV-1 gp41, HIV-2 gp36 and HIV-1 p24 as reagents. The test reaction is visible to the naked eye and detects anti-HIV antibodies in 5 minutes or less. This kit was used to test a significant number of samples, its performance was compared to a popular ELISA based kit and the positives were confirmed with a popular Western Blot assay. From these studies, the kit was found to be highly sensitive and specific.