B. C. Harinath

Role of oxidative stress and antioxidants in aetiopathogenesis and management of oral submucous fibrosis

Lipid peroxidation product, malonaldehyde (MDA) and antioxidants were estimated in plasma and erythrocytes of 34 cases of oral submucous fibrosis (OSMF) of different grades with equal number of healthy controls to evaluate the association of reactive oxygen species (ROS) and OSMF. While plasma MDA was found to be significantly higher in patients (3.3±0.4 nmole/ml, P<0.001) as compared to controls (2.4±0.5 nmole/ml), plasma beta carotene and vitamin E levels were found to be decreased significantly in patients (81.7±14.3 μg/100 ml, P<0.001; 9.3±0.9 mg/L, P<0.01 respectively) with respect to healthy controls (110±20.8 μg/100 ml and 10.1±1.2 mg/L). The decrease in beta-carotene and vitamin E was found to be more significant in OSMF grade II and III than in grade I. After 6 weeks of oral administration of beta-carotene and vitamin E, patients showed increase in plasma level of these two antioxidants along with decrease in MDA level associated with clinical improvement.

Detection of circulating antigen in bancroftian filariasis by sandwich ELISA using filarial serum IgG.

The utility of the IgG fraction of human filarial serum immunoglobulin in detecting circulating antigen by sandwich enzyme linked immunosorbent assay (ELISA) was studied. 27 of 33 sera from persons with microfilaraemia, 19 of 30 sera from clinical cases of filariasis, 4 of 30 sera from normal persons from a region endemic for filariasis showed the presence of circulating filarial antigen. All the 20 normal sera from the area where filariasis was not endemic gave negative reaction for filarial antigen. Those sera from persons with microfilaraemia that showed the presence of circulating antigen also showed an apparent positive correlation between the microfilarial density and the antigen titre.

Immunoprophylactic potential of a 120 kDa Brugia malayi adult antigen fraction, BmA-2 in lymphatic filariasis

A 120 kDa antigen containing SDS‐PAGE fraction BmA‐2 isolated from Brugia malayi adult parasite was highly reactive with normal sera from filarial endemic area. BmA‐2 was analysed for its propylactic potential in in vitro and in vivo. Sera collected from BmA‐2 immunized jirds induced a significant level (80 to 90%) of protection against infective larvae and microfilariae in in vitro ADCC assay as well as in in situ micropore chamber implantation studies. Mastomys natalensis immunized with BmA‐2 showed a significant level of protective response against circulating microfilariae by clearing 90% of them from circulation by fifth day after challenge infection. Immunization of jirds with BmA‐2 resulted in an enhanced level of antibody response against BmA‐2 and 88% reduction in the development of the parasites to the adult stage. Passive transfer of immunesera from jirds immunized with BmA‐2 to naive jirds resulted in 71% reduction in adult worm recovery as observed 90 days after challenge infection with B. malayi. On the other hand the passive transfer of nonadherent spleen cells from immune jirds did not show any significant effect on the development of parasite. Administration of jirds anti BmA‐2 serum to microfilaraemic jirds showed a temporary decrease in micrqfilarial count which was increased to pretherapeutic level within 100 days and there was no effect on the adult worms. This implies that the immune protective effect of BmA‐2 is mainly antibody dependent and active immunization with BmA‐2 is effective against filarial infection.,

Immunodiagnosis of tuberculosis: An update.

Tuberculosis is still a major health problem in most developing countries and its incidence is rising in many developed countries. This resurgence has been attributed to the HIV epidemic and TB has been declared as a global health emergency by WHO in 1993. The diagnosis of tuberculosis mainly depends upon initial clinical suspicion and radiographic findings with subsequent bacteriological confirmation by sputum smear examination and culture. Lack of sensitivity in smear examination, non specificity of radiological findings, extended tum around time ofMycobacterium tuberculosis culture and difficulties in diagnosing paucibacillary, childhood and extrapulmonary tuberculosis has necessitated to explore the utility of immunodiagnosis of tuberculosis as a convenient and cost effective test to supplement clinical information for definite diagnosis. Many commercial tests are available in the market for diagnosis of TB. Most of these tests are based on the detection of IgG, IgA and IgM antibodies to specific mycobacterial antigen or mixture of antigens. Indigenous immunoassay systems have explored excretory-secretory ES-31 mycobacterial antigen for immunodiagnosis of TB. Many a time there is lack of consistent elevation in all the three Ig classes in active infection thus making it more important to determine the ideal antibody isotype assay for reliable diagnosis of tuberculosis and to save the costs of the patient for unnecessary investigations.

Isolation ofMycobacterium Tuberculosis 31 kDa antigen protein of diagnostic interest from culture filtrate using anti-ES-31 antibody by affinity chromatography.

Proteins secreted into the culture medium byMycobacterium tuberculosis (M. tb) are shown to be source of antigens of immunodiagnostic interest. Anin vitro released 31 kDa antigen ESAS-7F isolated fromM.tb H37Ra culture filtrate by salt precipitation, SDS-PAGE and cation exchange fast protein liquid chromatography (FPLC) was shown earlier to be a diagnostically important antigen fraction. In this report, we describe the isolation of ESAS-7F antigen using monospecific antibody coupled to sepharose CL-4B column. The percentage recovery of ESAS-7F antigen using affinity chromatography was approximately 8% of the total ES antigen proteins compared to 0.05% obtained by conventional purification steps using salt precipitation, SDS-PAGE and FPLC. Similar seroreactivity was observed by the antigen isolated by both the methods in indirect ELISA. Affinity chromatography helped in an increased recovery of ESAS-7F antigen and obviates the need for time consuming conventional purification steps.

Computational approach to understanding the mechanism of action of isoniazid, an anti-TB drug

Tuberculosis (TB) is an ancient disease caused by Mycobacterium tuberculosis (MTB), which remains a major cause for morbidity and mortality in several developing countries. Most drug-resistant MTB clinical strains are resistant to isoniazid (INH), a first-line anti-TB drug. Mutation in KatG, a catalase-peroxidase, of MTB is reported to be a major cause of INH resistance. Normally upon activation by KatG, INH is converted to an active intermediate which has antimycobacterial action in MTB. This INH intermediate in the presence of NADH forms INH-NAD adduct which inhibits inhA (2-trans-enoyl-acyl carrier protein reductase) of MTB, thus blocking the synthesis of mycolic acid, a major lipid of the mycobacterial cell wall. In this docking study, the high binding affinity of INH-NAD adduct towards InhA was observed in comparison with INH alone. In this study, two resistant mutants of KatG (S315T and S315N) were modeled using Modeller9v10 and docking analysis with INH was performed using AutoDock4.2 and the docking results of these mutants were compared with the wild type KatG. Docking results revealed the formation of a single hydrogen (H) bond between the secondary amine nitrogen (-NH) of INH with Thr or Asn residues in place of Serine at 315 position of KatG mutant strains respectively, whereas in the case of the wild type, there was no H-bond formation observed between INH and Ser315. The H-bond formation may prevent free radical formation by KatG in mutant strains thus the development of resistance to the drug. This in silico evidence may implicate the basis of INH resistance in KatG mutant strains.

Diagnostic use of Polyclonal Antibodies Raised in Mouse Ascitic Fluid in Bancroftian Filariasis

Polyclonal antibodies were produced against Brugia malayi adult antigens (BmA (PBS) SAg and BmA (SDS) SAg) in mouse ascitic fluid by immunising Balb/c mice intraperitoneally with high ratio of adjuvant to immunogen. The diagnostic use of these antibodies in detecting circulating filarial antigen in bancroftian filariasis was studied by sandwich enzyme-linked immunosorbent assay (sandwich ELISA) using stick assay system. Both antibodies raised against PBS and SDS soluble antigens were found to be equally sensitive and relatively specific in detection of circulating filarial antigen. When anti BmA (PBS) SAg antibody was used in sandwich ELISA, 90% of microfilaraemic sera, 30-40% of acute and sub acute filarial sera, 20% of chronic filarial sera, 7% of endemic normal sera and none of 15 non-endemic normal sera were positive for filarial antigen. Using anti BmA (SDS) Sag antibody, 93% of microfilarial sera, 40% of acute and sub acute filarial sera, 20% of chronic filarial sera and none of 15 endemic and non-endemic normal sera showed the presence of filarial antigen. The filarial antigen detection using anti BmA S Ag antibodies produced in mouse ascitic fluid in sandwich ELISA may be useful in detection of active stage (microfilaraemia) of infection.

The utility of human filarial serum in the detection of circulating antigen.

The utility of human filarial serum immunoglobulin (FSI) in detecting circulating antigen in filarial sera was studied by counter immunoelectrophoresis (CIEP) and the indirect haemagglutination test (IHAT). CIEP was found to be better than IHAT. 23 out of 30 sera from persons with microfilaeremia and one of 30 clinical cases of filariasis, but none of the normal sera or sera from those with helminths other than filariae, showed the presence of circulating filarial antigen in CIEP. FSI was fractionated by DEAE- Sephadex A-50 column chromatography and the antibody active in CIEP was found to be IgG in nature.

Detection of filarial infection usingWuchereria bancrofti microfilariae culture antigen and filter paper blood samples in enzyme linked immunosorbent assay

Blood collected on filter paper by finger-prick gave results comparable to intravenous serum samples when analysed by enzyme-linked immunosorbent assay (ELISA). All the 100 microfilaraemia, 5 out of 100 endemic normals and none of the 10 nonendemic normal filter paper blood samples showed the presence of filarial antibody when tested by this method,using culture antigen and anti-immunoglubulins, class G, M and A — penicillinase conjugate. When the same samples were screened for the presence of IgM antibody, 91 out of 100 microfilaraemia, 13 out of 100 endemic normal and none of the 10 nonendemic normal samples showed a positive reaction. Enzyme linked immunosorbent assay, using culture antigen and filter paper blood samples, appears to work in large field studies for detection of filarial infection.

Serodiagnosis of childhood tuberculosis by ELISA.

Objective: Diagnosis of childhood tuberculosis remains an enigma despite many recent technological developments. The present study has been taken up with the aim to assess the diagnostic potential of mycobacterium tuberculosis excretory-secretory ES-31 antigen and affinity purified anti ES-31 antibodies in the serodiagnosis of different spectrum of childhood tuberculosis.Methods: Mycobacterium tuberculosis H37 Ra excretory-secretory antigen (ES-31) and affinity purified goat anti ES-31 antibodies were used in stick penicillinase ELISA for IgG antibody detection and stick Sandwich penicillinase ELISA for detection of circulating free and immune complexed antigen in the sera of 230 children.Results: Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in both pulmonary and extrapulmonary form of childhood tuberculosis and overall sensitivity of 81.4% with a specificity of 93% was achieved for detection of antitubercular IgG antibodies. Of the five cases of pulmonary tuberculosis showing absence of IgG antibody, 3 showed the presence of CIC-Ag and one was found positive for both free and CIC-Ag. Similarly out of 8 cases of extrapulmonary childhood tuberculosis missed by IgG detection 5 were found to be positive for CIC-Ag and 1 showed the positive reaction for both free and immune complexed antigens.Conclusion: IgG antibody to excretory-secretory antigen ES-31 is found to be having good specificity with acceptable sensitivity in detecting different forms of childhood tuberculosis. Further detection of circulating free and / or immunecomplexed antigen can be used as an adjunct tool in the diagnosis of childhood tuberculosis.