In vitro evaluation of growth reticence and anticancer potential of 5α,8α-epidioxy-24ᶓ-methylcholesta-6,22-dien-3ß-ol and ergosta-5,7,22-trien-3ß-ol bioactive isolated from an edible mushroom Lentinus tuberregium (fr.).

The focus point of this current work is to evaluate the anticancer and growth inhibitory efficacy of compounds 5α,8α-epidioxy-24ᶓ-methylcholesta-6,22-dien-3ß-ol (LT1), and Ergosta-5,7,22-trien-3ß-ol (LT2) of Lentinus tuberregium (Fr.) on three cell lines such as A673 (Rhabdomyosarcoma), MCF7 (breast cancer), and HCT116 (colorectal carcinoma) by MTT assay. LT1 and LT2 exerted maximal growth inhibition in the order as A673 > HCT116 > MCF7. Comparatively, LT1 was more potent in causing cell growth inhibition than LT2 in the A673 cancer cell line. Based on the MTT assay, A673 cells alone proceeded further as a model to evaluate the anticancer potential of LT1 and LT2 at three different semilogarithmic concentrations (3, 10, 30 µM). The cells exposed with compounds at 24 and 48 h were analyzed by flow cytometry. Exposure of LT1 at 3 and 10 µM concentrations for 24 h caused a G2-M arrest. At 10 µM concentration, cells also accumulated in the G0-G1 phase, indicating a G1 block. These effects were only transient as prolonged exposure (48 h) of LT1 treatment brought back the cell population to normalcy. Both the compounds only at 30 µM concentration have the potential to induce a hypodiploid peak (sub G0), indicating an induction of apoptosis which was explicit by nuclear condensation and fragmentation of nuclei in cells. The dose-dependent and compound-specific apoptotic induction was further confirmed by caspase activity higher in LT1 than LT2. The results highlight the significant growth inhibitory activity and anticancer potential of LT1 and LT2 which are recommended for further in-depth analysis.

GC-MS and Surface Characteristics of Polyvinyl Siloxane-An In Vitro Analysis.

OBJECTIVE: To assess the surface characteristics, composition and release of components of Polyvinyl siloxane (PVS) impression material and explore the use of the material other than for impressions. METHODS: Forty samples of 0.5-mm thickness and 8-mm diameter PVS discs, with few perforations, were prepared and divided into four groups. Group 1 in methanol was analyzed by GC-MS immediately and on 1st, 7th and 14th day. Group 2 and 3 placed in 10 mL of human saliva and in artificial nasal fluid, respectively, were analyzed by GC-MS at 24 hours. Group 4 was subjected to surface characteristics analysis by placing five PVS discs each in 1 mL of human saliva and 1 mL of artificial nasal fluid, respectively. RESULTS: On Day 1, Dodecanoic acid, methyl ester, Cyclononasiloxane, octadecamethyl, Cyclodecasiloxane and eicosamethyl (four peaks) were observed in Group 1. At 24 hours, Group 2 had additional compounds of 2-Decene,7-methyl-(Z)-, 2-Undecene,8-methyl-(Z)- and Lauryl acetate. Group 3 showed release of 32 compounds, but their retention time was less. Surface characteristics on Day 14 revealed no noticeable changes. CONCLUSIONS: The study revealed that there was a lack of any adverse component release from PVS for a period of 2 weeks based on GC-MS analysis.

Inhibition of MMP2-PEX by a novel ester of dihydroxy cinnamic and linoleic acid from the seagrass Cymodocea serrulata.

Matrix metalloproteinases (MMPs) are pivotal for cancer cell migration and metastasis which are generally over-expressed in such cell types. Many drugs targeting MMPs do so by binding to the conserved catalytic domains and thus exhibit poor selectivity due to domain-similarities with other proteases. We report herein the binding of a novel compound [3-(E-3,4-dihydroxycinnamaoyloxyl)-2-hydroxypropyl 9Z, 12Z-octadeca-9, 12-dienoate; Mol. wt: 516.67 Da], (C1), isolated from a seagrass, Cymodocea serrulata to the unconserved hemopexin-like (PEX) domain of MMP2 (- 9.258 kcal/mol). MD simulations for 25 ns, suggest stable ligand-target binding. In addition, C1 killed an ovarian cancer cell line, PA1 at IC50: 5.8 µM (lesser than Doxorubicin: 8.6 µM) and formed micronuclei, apoptotic bodies and nucleoplasmic bridges whilst causing DNA laddering, S and G2/M phase dual arrests and MMP disturbance, suggesting intrinsic apoptosis. The molecule increased mRNA transcripts of BAX and BAD and down-regulated cell survival genes, Bcl-xL, Bcl-2, MMP2 and MMP9. The chemical and structural details of C1 were deduced through FT-IR, GC-MS, ESI-MS, 1H and 13C NMR [both 1D and 2D] spectra.

De novo transcriptome analysis unravels tissue-specific expression of candidate genes involved in major secondary metabolite biosynthetic pathways of Plumbago zeylanica: implication for pharmacological potential.

KEY MESSAGE: The present study provides comparative transcriptome analysis, besides identifying functional secondary metabolite genes of Plumbago zeylanica with pharmacological potential for future functional genomics, and metabolomic engineering of secondary metabolites from this plant towards diversified biomedical applications. ABSTRACT: Plumbago zeylanica is a widely used medicinal plant of the traditional Indian system of medicine with wide pharmacological potential to treat several disorders. The present study aimed to carry out comparative transcriptome analysis in leaf and root tissue of P. zeylanica using Illumina paired end sequencing to identify tissue-specific functional genes involved in the biosynthesis of secondary metabolites, contributing to its therapeutic efficacy. De novo sequencing assembly resulted in the identification of 62,321 "Unigenes" transcripts with an average size of 1325 bp. Functional annotation using BLAST2GO resulted in the identification of 50,301 annotated transcripts (80.71%) and GO assigned to 18,814 transcripts. KEGG pathway annotation of the "Unigenes" revealed that 2465 transcripts could be assigned to 242 KEGG pathway maps wherein the number of transcripts involved in secondary metabolism was distinct in root and leaf transcriptome. Among the secondary metabolite biosynthesis pathways, the cluster of "Unigenes" encoding enzymes of 'Phenylpropanoid biosynthesis pathway' represents the largest group (84 transcripts) followed by 'Terpenoid Backbone biosynthesis' (48 transcripts). The transcript levels of the candidate unigenes encoding key enzymes of phenylpropanoid (PAL, TAL) and flavanoid biosynthesis (CHS, ANS, FLS) pathways were up-regulated in root, while the expression levels of candidate "Unigenes" transcript for monoterpenoid (DXS, ISPF), diterpenoid biosynthesis (SPS, SDS) and indole alkaloid pathways (STR) were significantly higher in leaf of P. zeylanica. Interestingly, validation of differential gene expression profile by qRT-PCR also confirmed that candidate "Unigenes" enzymes of phenylpropanoid and flavonoid biosynthesis were highly expressed in the root, while the key regulatory enzymes of terpenoid and indole alkaloid compounds were up-regulated in the leaf, suggesting that (differences in) the levels of these functional genes could be attributed to the (differential) pharmacological activity (between root and leaf) in tissues of P. zeylanica.

Establishment of T-lymphocyte subset reference intervals in a healthy adult population in Chennai, India.

Estimation of CD4+ T-lymphocytes continues to be an important aspect for monitoring HIV disease progression and response to antiretroviral therapy. Most of the diagnostic laboratories often rely on western text books for CD4+ T-lymphocyte reference values, which could, often be unreliable for usage in local settings. Therefore, we attempted to establish the reference values for T-lymphocyte subsets among healthy adults in a cross-sectional study carried out at the YRG Centre for AIDS Research and Education (YRG CARE) in Chennai, south India, in 213 (84 female and 129 male) healthy, HIV-1/2 seronegative adults as volunteers. Whole blood specimens were processed for CD4+, CD8+ T-lymphocyte estimation and haematological parameters. The established range of CD4+ T-lymphocyte counts for men and women were 383-1347 cells/microl (mean 865 and median 845 cells/microl) and 448-1593 cells/microl (mean 1021 and median 954 cells/microl), respectively. Women had significantly higher absolute CD4+ Tlymphocyte counts (P<0.001) and CD4+:CD8+ T-lymphocyte ratio as compared to men. The established normal range of CD4+ T-lymphocyte % was 21-59 (mean 40.2 and median 40.1). The influence of age was not observed in any of the parameters except CD4+/CD8+ T-lymphocyte ratio with the >45 yr age group. Further studies with greater sample size may be required to define the staging of HIV disease in relation to the normal CD4 T-lymphocyte count in the general population.

High proportion of isosporiasis among HIV-infected patients with diarrhea in southern India.

We investigated 245 diarrheal stool specimens from HIV-positive subjects between January 2003 and December 2006 to determine the etiological role of coproparasites. Parasitic etiology was observed in 91 (37.1%) cases. Isospora belli (26.1%) was the most common parasite followed by Entameba histolytica/dispar (3.3%), Cryptosporidium spp. (2.9%), Giardia intestinalis (1.6%), and Strongyloides stercoralis (1.2%). Interesting trends of significant increase in the number of cases of I. belli and decline in Cryptosporidium spp. were observed during the study period.

Prevalence of aflatoxin B1 in liver biopsies of proven hepatocellular carcinoma in India determined by an in-house immunoperoxidase test.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death in the world. The incidence of HCC in India is reportedly low and varies from 0.2 to 1.9 %. Aflatoxins, secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus, are potent human carcinogens implicated in HCC. The prevalence of aflatoxin B1 (AFB1) as co-carcinogen was analysed using an in-house immunoperoxidase test in 31 liver biopsies and 7 liver-resection specimens from histopathologically proven HCC, and in 15 liver biopsies from cirrhosis patients (control group). Serum was tested for hepatitis B and C serological markers using commercial assays, and for AFB1 using an in-house ELISA with a sensitivity of approximately 1 ng ml(-1) for AFB1. In spite of positive AFB1 immunostaining in HCC cases, all serum specimens, from both HCC and the control groups, were AFB1-negative. There were 18 (58.1 %) HCC cases that revealed AFB1 in liver biopsies; 68.8 % (n=11) of non-B non-C hepatitis cases with HCC and 46.1 % (n=6) of the hepatitis B surface-antigen-positive subjects were positive for AFB1. Out of the two hepatitis B/hepatitis C virus co-infected cases, one was positive for AFB1. Of seven tumour-resection samples, six were positive for AFB1. Only one case revealed AFB1 in the non-tumour area of the resected material. Thus AFB1 staining was significantly associated with tumour tissue (P=0.03). Aflatoxins proved to have a significant association with HCC in this peninsular part of the subcontinent. The impact seems to be a cumulative process, as revealed by the AFB1 deposits in HCC liver tissue, even though the serum levels were undetectable.

Diagnostic utility of serologic markers for genital chlamydial infection in STD patients in Chennai, India.

OBJECTIVES: To evaluate the diagnostic utility of serological markers for C. trachomatis in different clinical groups of STD patients. METHODS: Blood and genital swab specimens were collected from symptomatic STD patients (n=143) attending the STD out patient clinic at the Institute of STDs, Government General hospital, Chennai who enrolled for the study. Serological determination for IgM, IgA and IgG antibodies to C. trachomatis was done using commercial kits. PCR analysis was performed on genital swab samples by using plasmid and major outer membrane protein (MOMP) based primers and patients who were positive by both PCR assays were considered as proven cases of C. trachomatis infection. The serological marker positivity was analysed with PCR positivity. RESULTS: Serologic positivity by IgM, IgA and IgG was 22.4%, 28.7% and 58.7% respectively. The PCR analysis showed 44 (30.8%) cases with confirmed C. trachomatis infection. Seropositivity for IgM (34.1% (15/44) vs. 17.2% (17/99); P<0.05) as well as for IgA (40.9% (18/44) vs. 23.2% (23/99); P<0.05) significantly correlated to PCR positivity, while significant correlation was not seen with IgG positivity. The overall seropositivity (IgM/IgA/IgG) in the study population was 68.5%. CONCLUSIONS: The observations of the present study indicate a high exposure rate to chlamydial infection in STD clinic patients in India. The study also suggests the usefulness of serology instead of PCR to trace chlamydial etiology, especially in deep-seated upper genital tract diseases and to facilitate better clinical management as there was good correlation between serology and PCR positivity.

HIV-1 western blot assay: What determines an indeterminate status?

BACKGROUND: The Western blot assay is the gold standard for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1). However, indeterminate Western blot reactivity to HIV-1 proteins may occur in individuals, who may not be infected with HIV. AIM: This retrospective study was aimed to determine the diagnostic value of the interpretation criteria in relation to commercial kits for HIV-1 diagnosis. METHODS AND MATERIALS: A total of 556 serum/plasma specimens collected from high-risk population attending our HIV clinic from 2000-2004 were tested by three different western blot kits: NEW LAV BLOT I (n=244), HIV BLOT 2.2; (n=112), Genetic Systems HIV-1 (n=237). And the results of western blot strips were analyzed using the various interpretation criteria: WHO/NACO, CDC/ ASTPHLD, ARC, FDA, CRSS and JHU. Some specimens were run on more than one kit. RT-PCR assay was performed on 5 specimens, which were indeterminate with LAV BLOT I. RESULTS: The discrepancy in LAV BLOT I positive results were between 157(64)-176(72), and indeterminate results were between 44(18) to 63(25). No such variations were observed in genetic systems. There are some HIV negative (by PCR) specimens were indeterminate in LAV BLOT I revealing the kit more sensitive and less effective for diagnostic purpose. CONCLUSION: The genetic systems kit is superior to other kits we analyzed and its results are concordant with HIV-1 PCR results. To report, the choice of western blot commercial kit is paramount important than the use of particular interpretation criteria for the diagnosis of HIV-1.

Genital chlamydial infection in STD patients: its relation to HIV infection.

In the present report, we have analysed C.trachomatis infection and HIV positivity among patients (n-143) who attended the STD clinic at the Institute of STDs, Government General Hospital, Chennai. HIV positivity rate was significantly high among those with chlamydial infection than in those without chlamydial infection (29.5% (13/44) vs. 11.1% (11/99); p<0.05). The results of the present study suggest the association between C.trachomatis and HIV infections and reinforce the need for routine screening for C.trachomatis as a necessary intervention to reduce the burden of chlamydial diseases and to reduce the risk of HIV and its spread in India.

Community prevalence of hepatitis B infection and modes of transmission in Tamil Nadu, India.

BACKGROUND AND OBJECTIVE: There have been very few community based studies on prevalence of hepatitis B virus (HBV) infection in India. We undertook this study to determine the prevalence of HBV infection in a southern State of India, Tamil Nadu and to describe the important factors related to transmission of the virus in the community. METHODS: Analysis of stored blood samples from a representative population of Tamil Nadu from an earlier community cluster survey on sexually transmitted diseases (STD) prevalence using proportionate to population size (PPS) technique was done. Serum markers of HBV viz., hapatitis B surface antigen (HBsAg), hepatitis B e antigen (HBe Ag) and antibody to surface antigen (anti-HBs) were performed. RESULTS: 1981 subjects were screened in the study. HBsAg prevalence was 5.7 per cent (CI 4.6- 6.8) with 23.5 per cent (25/106) of these having positive HBe-antigen. Community seroprevalence (HbsAg + anti-HBs) of hepatitis B infection was 27.4 per cent (CI: 25.3-29.5) with the highest prevalence of 32.7 per cent (CI: 30.2-35.2) noted in the 15-20 yr age group. Significant independent association (OR 1.4; P=0.006) was detected with family history of exposure to HBV infection by logistic modeling. Other risk factors noted to have significant association were use of disposable needles during injection (OR 0.5; P=0.02) in men, smoking (OR 3; P=0.04) and use of condom (OR 0.6; P=0.08) in women. INTERPRETATION AND CONCLUSION: This community based study shows a high prevalence of hepatitis B infection in the state of Tamil Nadu with the highest prevalence being in the younger (15-20 yr) age group. High prevalence rate in childhood with e-antigenemia seen in 23.5 per cent of HBsAg positive subjects suggest childhood transmission. Poor injection practices and high-risk sexual behavior were found to be additional risk factors for transmission of the disease in the community.

Performance of two commercial enzyme-linked immunosorbent assay kits using recombinant glycoprotein G2 antigen for detection of herpes simplex virus type 2 specific antibodies.

For 93 stored serum samples tested by HerpeSelect2 and the Euroimmun enzyme-linked immunosorbent assay for detection of herpes simplex virus type 2-specific immunoglobulin G antibodies, the concordance of positive and negative results was 100%. Moreover, all the results that were equivocal by HerpeSelect2 (negative by Euroimmun) were confirmed as being negative by a Western blot assay.

Genital syndromes and syndromic management of vaginal discharge in a community setting.

The objective of the study was to determine the community prevalence of genital syndromes in women and evaluate the syndromic management of vaginal discharge in this setting. A representative sample for the state of Tamilnadu was chosen using probability proportional to size cluster technique. Thirty clusters were selected from three districts. Demographic, sexual behaviour, risk factors, clinical and laboratory data were collected from the selected population using a structured questionnaire. Direct smear examination for Trichomonas vaginalis, culture for Neisseria gonorrhoeae and Haemophilus ducreyi, serological tests for syphilis (RPR and TPHA), hepatitis B (Hbs Ag ELISA), IgM and IgG antibodies to HSV2 (Novum diagnostics, Germany) and PCR test for detection of C. trachomatis from urine were done. There were 1157 women in the selected population. On examination, vaginal discharge was the most common genital syndrome (38.4%). The sensitivity, specificity, positive and negative predictive value of vaginal discharge as a marker for STD in women was found to be 43.3%, 61.6%, 10.7% and 91.1%, respectively. We concluded that treatment on the basis of syndromic management would result in over-treatment of 90% of women with vaginal discharge.

Chlamydia trachomatis genital infection in apparently healthy adult population of Tamil Nadu, India: a population-based study.

Since the epidemiology of Chlamydia trachomatis infection in apparently healthy population has not been studied in India, a population-based study was conducted in the state of Tamil Nadu, India in order to analyse the prevalence of genital chlamydial infections in the community and to implement control programmes. A representative sample was taken from three randomly selected districts by using the 'probability proportional to size' cluster survey method. Households were the basic units of clusters. Adults aged 15-45 years, pre-identified from the selected households were enrolled during the medical camps conducted for a major study on community prevalence of sexually transmitted diseases in Tamil Nadu. Blood and urine samples collected from the study subjects were tested by enzyme-linked immunosorbent assay (ELISA) for anti-chlamydial IgM antibodies and by the commercial Amplicor polymerase chain reaction (PCR) test for chlamydial DNA. The prevalence of anti-C. trachomatis antibodies determined by IgM-ELISA was 2.4% (95% CI 1.6%-3.2%). The prevalence of genital chlamydial infection determined by PCR was 1.1% (95% CI 0.5%-1.7%). Majority of the detected infections (68.8%) were asymptomatic. This is the first Indian report on the prevalence of genital chlamydial infections in the general population. It is concluded that this study provides evidence for a substantial burden of approximately 10 million asymptomatic genital chlamydial infection cases in the sexually active age groups in the general population of India.

Evaluation of immunogenicity and safety of Genevac B: a new recombinant hepatitis B vaccine in comparison with Engerix B and Shanvac B in healthy adults.

PURPOSE: Genevac B, a new indigenous recombinant hepatitis B vaccine was evaluated for its immunogenicity and safety in comparison with Engerix B (Smithkline Beecham Biologicals, Belgium) and Shanvac B (Shantha Biotechnics, India) in healthy adult volunteers. METHODS: While 240 study subjects were included in the Genevac B group, 80 each were the subjects for Engerix B and Shanvac B. A three dose regimen of 0,1,2 months was adopted with 20 gm dosage uniformly in all the three groups. Vaccinees were assessed during prevaccination, followup and post vaccination periods for clinical, haematological, biochemical and immunological parameters for safety and immunogenicity. RESULTS: Successful follow-up in all parameters for four months could be achieved in 92.5% (222/240) for Genevac B study subjects and the same was 85% (68/80) and 80% (64/80) for Engerix B and Shanvac B respectively. While 100% seroconversion was observed in all the three groups, the rate of seroprotectivity was 99.5% by Genevac B, 98.5% by Engerix B and 98.4% for Shanvac B. However the difference was not statistically significant (p>0.05). The GMT values of anti HBs after one month of completion of the vaccination were 735.50, 718.23 and 662.20 mIU/mL respectively. No systemic reaction was either seen or reported by the volunteers during the vaccination process of Genevac B and other two vaccines. Clinical, haematological and biochemical safety parameters remained within normal limits throughout the study period. CONCLUSION: The study confirms that Genevac B, the new recombinant Hepatitis B vaccine has the acceptable international standards of safety and immunogenicity.

Geographical difference in antimicrobial resistance pattern of Helicobacter pylori clinical isolates from Indian patients: Multicentric study.

AIM: To assess the pattern of antimicrobial resistance of Helicobacter pylori isolates from peptic ulcer disease patients of Chandigarh, Delhi, Lucknow, Hyderabad and Chennai in India, and to recommend an updated anti-H. pylori treatment regimen to be used in these areas. METHODS: Two hundred and fifty-nine H. pylori isolates from patients with peptic ulcer disease reporting for clinical management to the Post Graduate Institute of Medical Education and Research, Chandigarh; All India Institute of Medical Sciences, New Delhi; Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow; Deccan College of Medical Sciences and Allied Hospitals, Hyderabad; and hospitals in Chennai in collaboration with the Dr ALM Post Graduate Institute of Basic Medical Sciences were analyzed for their levels of antibiotic susceptibility to metronidazole, clarithromycin, amoxycillin, ciprofloxacin and tetracycline. The Epsilometer test (E-test), a quantitative disc diffusion antibiotic susceptibility testing method, was adopted in all the centers. The pattern of single and multiple resistance at the respective centers and at the national level were analyzed. RESULTS: Overall H. pylori resistance rate was 77.9% to metronidazole, 44.7% to clarithromycin and 32.8% to amoxycillin. Multiple resistance was seen in 112/259 isolates (43.2%) and these were two/three and four drug resistance pattern to metronidazole, clarithromycin, amoxycillin observed (13.2, 32 and 2.56%, respectively). Metronidazole resistance was high in Lucknow, Chennai and Hyderabad (68, 88.2 and 100%, respectively) and moderate in Delhi (37.5%) and Chandigarh (38.2%). Ciprofloxacin and tetracycline resistance was the least, ranging from 1.0 to 4%. CONCLUSION: In the Indian population, the prevalence of resistance of H. pylori is very high to metronidazole, moderate to clarithromycin and amoxycillin and low to ciprofloxacin and tetracycline. The rate of resistance was higher in southern India than in northern India. The E-test emerges as a reliable quantitative antibiotic susceptibility test. A change in antibiotic policy to provide scope for rotation of antibiotics in the treatment of H. pylori in India is a public health emergency.

Prevalence of antibodies to hepatitis A and hepatitis E virus in urban school children in Chennai.

With similar feco-oral mode of transmission of Hepatitis A and E viruses, and improving levels of personal hygiene among higher socioeconomic population, periodic surveillance on HAV/HEV exposure pattern may be of immense public health value. One such attempt was made in Tamilnadu, India by analysing the presence of antibodies to HAV and HEV in 185 healthy children of 6 months to 12 years of age. While anti HAV positivity was 96.9% by 12 years of age, anti HEV positivity fluctuated between 5.3-16.7%. The study suggests the necessity for developing a vaccine for HEV to prevent the frequent occurrence of HEV outbreaks in India, since natural HEV exposure does not bestow significant protection as observed in HAV.