IgG subclass responses to proinflammatory fraction of Brugia malayi in human filariasis.

Background & objectives: Earlier we demonstrated that immunization with F6, a proinflammatory molecular fraction isolated from the human filarial parasite Brugia malayi, protected the host and eliminated the infection in Mastomys coucha by a Th1/Th2 response including IgG2a antibody response. Whether F6 molecules become accessible to human host during natural course of infection and elicit similar response is not known. The present study was undertaken to determine the profile of IgG subclasses specifically reactive to F6 in different categories of bancroftian filariasis cases to infer any relationship between the levels of a particular F6-specific IgG subclass and the infection or disease status. Methods: Serum samples of normal individuals from filariasis non-endemic regions of India like Jammu & Kashmir, Uttarakhand, and Chandigarh [(NEN-W; n=10), healthy subjects from USA (NEN-U; n=10) and three categories of bancroftian filariasis cases from endemic areas: endemic normals (EN; n=10) with no symptoms and no microfilariae, asymptomatic microfilaremics (ASM; n=10) and chronic symptomatic amicrofilaremics (CL; n=10) were assayed for F6-specific IgG1, IgG2, IgG3 and IgG4 by ELISA using SDS-PAGE-isolated F6 fraction of B. malayi adult worms. Results: Significantly high levels of F6-specific IgG1, IgG2 and IgG3 were found in CL (P<0.001) and EN (P<0.01-0.001) bancroftian filariasis cases compared to NEN-U. Significant levels of F6-specific IgG1 (P<0.01) and IgG2 (P<0.01) but not IgG3 were found in ASM cases compared to NEN-U. The most abundant was IgG2 which when compared to NEN-U, was significantly high in CL (P<0.001) and EN cases (P<0.001), followed by ASM (P<0.01). F6-specific IgG4 response in EN, ASM and CL subjects was not significantly different from the levels of NEN-U. Among the non-endemic normals, the NEN-W subjects showed significant reactivity with IgG2 (P<0.001) but not with IgG1, IgG3 and IgG4 as compared to NEN-U subjects. IgG subclass levels were different in different categories. Interpretation & conclusions: The high levels of F6 reactive IgG1, IgG2 and IgG3 in endemic normals and chronic symptomatic bancroftian patients, and IgG1 and IgG2 in asymptomatic microfilaraemics, suggest that F6 molecules of parasite are accessible in these subjects for IgG subclass-specific immune response and IgG2 may be related to pathogenesis. Studies using individual F6 molecules will be done to identify the molecule(s) involved in infection and protective immunity.

In vitro effect of four herbal plants on the motility of Brugia malayi microfilariae.

BACKGROUND & OBJECTIVE: Disease burden due to lymphatic filariasis is disproportionately high despite mass drug administration with conventional drugs. Usage of herbal drugs in traditional medicine is quite well known but largely empirical. Hence the present study was designed to screen the in vitro antifilarial effect of four herbal plants on Brugia malayi. METHODS: Motility of microfilariae of B. malayi after incubation for 48 h with aqueous/methanol extracts of Vitex negundo L. (roots), Butea monosperma L. (roots and leaves), Ricinus communis L. (leaves), and Aegle marmelos Corr. (leaves) was explored in the concentration range of 20 to 100 ng/ml for possible antifilarial effect by comparing with suitable solvent control. RESULTS: Butea monosperma leaves and roots, Vitex negundo root and Aegle marmelo leaves showed significant inhibition of motility of microfilariae as compared to controls whereas inhibitory activity demonstrated by Ricinus communis L. leaves was not significant. Antifilarial effects imparted by all these extracts were found to be a function of their relative concentrations. Inhibitory concentrations (IC(50)) for the plant extracts with significant antifilarial activity against Brugia malayi microfilariae in in vitro system have been derived to be 82, 83 and 70 ng/ml for Vitex negundo L., Butea monosperma L. and Aegle marmelos Corr. respectively. INTERPRETATION & CONCLUSION: The present study recorded significant antifilarial effect of all plant extracts studied except for Ricinus communis L. leaves and contributes to the development of database for novel drug candidates for human lymphatic filariasis.

Anti-microfilarial activity of methanolic extract of Vitex negundo and Aegle marmelos and their phytochemical analysis.

In the present study, methanolic extracts of roots of Vitex negundo L. and extracts of leaves of Vitex negundo L., Ricinus communis L. and Aegle marmelos Corr. were explored for possible antifilarial effect against Brugia malayi microfilariae. It was observed that among the herbal extracts, root extract of Vitex negundo L. and leaves extract of Aegle marmelos Corr. at 100 ng/ml concentration showed complete loss of motility of microfilariae after 48 hr of incubation. Thin layer chromatography of the extracts revealed the presence of alkaloids, saponin and flavonoids in the roots of Vitex negundo L. and coumarin in the leaves of Aegle marmelos Corr.

Role of intrauterine Rubella infection in the causation of congenital deafness.

Background : Rubella is a contagious viral infection associated with rash. 10-15% of women are susceptible to this infection in their childbearing years and the disease is usually transmitted to the developing fetus causing abortion or stillbirth or congenital Rubella syndrome (CRS). Sensorineural hearing loss is one of the most common abnormalities associated with CRS. Aims : The study was undertaken to determine the prevalence of Rubella induced congenital deafness in south India. Materials and Methods :The present study is carried out on 1076 hearing impaired children below 14 years of age to determine etiological factors for congenital hearing impairment. Various audiometric tests such as PTA, OAE and BERA were carried out to know the type and degree of hearing impairment. Development histories including the prenatal, perinatal and postnatal histories were collected using a standard questionnaire. Results :The results indicated very less percentage (1.57%) of cases with the history of maternal Rubella infection indicating a significant reduction of Rubella-induced deafness in the developing countries. Conclusions : The main reason behind the low percentage of children with rubella- induced hearing impairment may be due to appropriate immunization of mothers to the rubella infection during their child bearing years. The increasing awareness in the developing countries of this infection and its consequences is another important reason behind the low percentage of the affected population.

Immunological studies in children with hearing impairment.

An immunological study was carried out on 58 children below 14 years of age with sensorineural hearing loss of unknown aetiology. The observed elevated levels of IgE in 25.86% (n = 15) children and antinuclear antibodies in 10.34% (n = 6) children indicate that auto-immune activity has a role in the causation of hearing impairment.

Identification of 38kDa Brugia malayi microfilarial protease as a vaccine candidate for lymphatic filariasis.

A FPLC purified 38kDa protease (Bm mf S-7) isolated from B. malayi microfilarial soluble antigen was identified. It showed pronounced reactivity with sera collected from 'putatively immune' asymptomatic and amicrofilaraemic individuals residing in an endemic area for bancroftian filariasis. Further the immune protective activity of Bm mf S-7 antigen was evaluated in susceptible hosts, jirds (Meriones unguiculatus) against B. malayi filarial infection. The antigen showed 89% cytotoxicity against mf and 87-89% against infective (L3) larvae in in vitro antibody dependent cellular cytotoxicity Assay (ADCC) and in situ micropore chamber methods. Bm mf S-7 immunized jirds after challenge infection showed 81.5% reduction in the adult worm burden. The present study has shown that, the 38kDa microfilarial proteases (Bm mf S-7) could stimulate a strong protective immune response against microfilariae and infective larvae in jird model to block the transmission of filariasis. Analysis of IgG subclasses against Bm mf S-7 revealed a significant increase in IgG2 and IgG3 antibodies in endemic normals. Lymphocyte proliferation to Bm mf S-7 was significantly high in endemic normal group as compared to that in clinical and microfilarial carriers. Significantly enhanced levels of IFN-gamma in the culture supernatant of PBMC of endemic normals followed by stimulation with Bm mf S-7 suggest that the cellular response in this group is skewed towards Th 1 type.

Facio-auricular vertebral syndrome-a case report.

Facio Auricular Vertebral (FAV) or Goldenhar syndrome is a very rare kind of syndromic deafness and is inherited as autosomal dominant. A study was taken up to understand the prevalence of this syndrome in children below the age of 14 years with hearing loss. Out of 1073 children with hearing impairment, Goldenhar syndrome was observed only in 1 (0.09%) case. The child suffered severe hearing loss. Facial paralysis and hemifacial microsomia were prominent features observed in the child. Facio-Auricular-Vertebral syndrome is therefore synonymously used with Goldenhar syndrome.

Risk factors for acute myocardial infarction in a rural population of central India: a hospital-based case-control study.

BACKGROUND: There is a paucity of data on the relative importance of various traditional risk factors for coronary artery disease among rural Indians. We conducted a prospective case-control study to determine the risk factors for acute myocardial infarction in a rural population of central India. METHODS: We recruited 111 consecutive patients admitted to our hospital with a first episode of acute myocardial infarction and 222 age- and sex-matched controls. Demographics, anthropometric measures, lipids, blood glucose, smoking and other lifestyle factors were compared among cases and controls. Multivariate analyses were used to identify the risk factors independently associated with acute myocardial infarction. RESULTS: Elevated fasting blood glucose (odds ratio [OR] 8.9; 95% confidence interval [CI] 4.5, 17.9), abnormal waist-hip ratio (OR 3.0; 95% CI 1.7, 5.4) and income (OR 4.0 and 5.9 for the high- and middle-income categories, compared to the lowest category) were independently associated with the first episode of acute myocardial infarction. Abnormal triglycerides (OR 1.7; 95% CI 0.9, 3.1) and current smoking (OR 1.9; 95% CI 0.9, 4.0) were risk factors but were not statistically significant. CONCLUSION: Reduction in blood glucose levels and truncal obesity may be important in controlling the burden of coronary artery disease in rural Indians.

Seroreactivity of 31 kDa and 41 kDa mycobacterial secretory proteins isolated from culture filtrate in extra pulmonary tuberculosis.

Despite rapid advances in molecular genetics for detection of mycobacteria, it is clear that interest in serodiagnosis remains high, especially for those situations in which a specimen may not contain the infecting agent in particular in extrapulmonary tuberculosis. Immune response to excretory-secretory (ES) proteins of Mycobacterium tuberculosis (M.tb) has been of diagnostic interest in tuberculosis. In earlier study from our laboratory, a secretory protein M.tb ES-31 has been shown to have diagnostic potential in pulmonary tuberculosis. Further, another M.tb H37Ra ES protein (ES-41) was isolated and purified by trichloroacetic acid solubilization followed by Fast Performance Liquid Chromatography (FPLC). These two protein fractions viz ES-31 and ES-41 secreted by M.tb H37 Ra bacilli were employed in stick indirect penicillinase ELISA to study seroreactivity in extra pulmonary tuberculosis namely tuberculous lymphadenopathy, tuberculous meningitis, abdominal tuberculosis and bone & joint tuberculosis. While using ES-31 antigen 88% (22/25) of tuberculous lymphadenopathy and 90% (9/10) of tuberculous meningitis cases showed positive reaction for tuberculous IgG antibody, ES-41 showed 80% positivity in both groups. In abdominal and bone & joint tuberculosis cases, ES-41 antigen showed better sensitivity of 81.5% (22/27) and 84.6% (22/26) respectively in IgG antibody detection compared to 70% (19/27) and 69.2% (18/26) shown by ES-31. This study is of interest that different antigen protein fractions of M.tb exhibit differential seroreactivity, as ES-31 protein showed good potential in detecting tuberculous IgG antibodies in tuberculous lymphadenopathy (TBLN) & tuberculous meningitis (TBM), while ES-41 in abdominal and bone & joint tuberculosis cases.

Analysis of IgG subclasses and IgE antibodies across the clinical spectrum of bancroftian filariasis in an endemic area.

Analysis of immune response in individuals with different clinical manifestations living in filaria endemic area will be of interest to understand the immunological events associated with the disease development in filaria infected endemic population. The levels of four IgG subclasses and IgE antibodies against Brugia malayi microfilarial excretory-secretory (Bm mf ES) antigen as well as circulating filarial antigen level were evaluated in 84 individuals belonging to different groups in an endemic area for bancroftian filariasis. Microfilaraemics showed significantly elevated levels of IgG4 and IgG3 antibodies compared to endemic normals (P < 0.02). As many as 70% of this group were positive for IgG4 & IgG3 antibodies. While Acute filarial cases had pronounced IgG1 antibodies(P < 0.001), the Grade I chronic cases showed higher levels of IgG3 and IgG4 antibodies (P < 0.02), Occult filarial cases had higher levels of IgG4 and IgG3 (P < 0.02) and also of IgG4 antibodies (P < 0.001). IgE antibodies were found to be elevated in microfilaraemics as well as other clinical filarial groups. Circulating filarial antigen was detected in 95% of microfilaraemics, 60% of acute cases, 75% to 90% of different grades of chronic filarial cases, 100% of occult cases and none of the endemic normals.

Pharmacotherapy for secondary prevention of stroke.

Stroke is a leading cause of mortality worldwide. It has well modifiable risk factors, which makes prevention an effective strategy. Antithrombotics and anticoagulants have been the main pharmacological options in secondary prevention. A number of new antiplatelet drugs have been introduced over the past decade. The more recent concepts in the understanding of stroke and atherosclerosis have paved the way for a number of newer pharmacological interventions like angiotensin enzyme inhibitors, statins and vitamins. The pharmacological armamentarium to treat stroke is expanding.

Sero-reactivity of two different antigenic protein fractions of M.tb H37Ra excretory-secretory antigen in pulmonary and extra-pulmonary tuberculosis.

Excretory-secretory proteins of Mycobacterium tuberculosis H37Ra, have been of diagnostic interest in pulmonary (PTB) and extrapulmonary tuberculosis (EPTB). Two different excretory-secretory antigenic proteins of M.tbH37Ra viz., EST-DE1 (a 6% TCA soluble and DEAE anion exchange purified antigen) and ESAS-7 (50% ammonium sulphate solubilized and SDS-PAGE fractionated antigen) were studied in stick-indirect penicillinase ELISA for detecting tuberculous IgG antibodies in serum samples of pulmonary as well as extrapulmonary tuberculosis (tuberculous lymphadenopathy (TBLN), tuberculous meningitis (TBM), bone & joint tuberculosis (B&J TB), abdominal tuberculosis (Abd. TB) patients. The ESAS-7 antigen has shown comparatively better seroreactivity (90%) than that of EST-DE1 antigen in pulmonary tuberculosis cases. The overall specificity of 93.2% using ESAS-7 antigen was also found better compared to 86.4% obtained using EST-DE1 antigen. Further, in extra pulmonary tuberculosis group, using ESAS-7 antigen 84% (21/25) of histopathologically confirmed TBLN cases and 90% (9/10) clinically diagnosed and ATT responded TBM cases showed positive reaction for tuberculous IgG antibody. The per cent positivity using EST-DE1 antigen was however comparatively low in TBLN and TBM cases, (76% and 80% respectively). In histopathologically proven bone and joint tuberculosis and abdominal tuberculosis cases EST-DE1 antigen showed better sensitivity of 75% and 83.3% respectively in IgG antibody detection compared to that of ESAS-7 antigen (50% and 66% respectively). From the present study, it can be envisaged that ESAS-7 antigenic fraction has a good potential in the diagnosis of pulmonary and certain extra-pulmonary tuberculosis infection (TBLN & TBM) whereas EST-DE1 was found to be better in detecting specific antibodies in bone & joint and abdominal tuberculosis.

Purification and characterization of a 31 kDa mycobacterial excretory-secretory antigenic protein with a diagnostic potential in pulmonary tuberculosis.

Proteins secreted into the culture medium by Mycobacterium tuberculosis are shown to be a source of antigens of immunodiagnostic importance. In our earlier study, we had reported a 31-37 kDa seroreactive gel-eluted antigenic fraction (ESAS-7), isolated from culture filtrate proteins of Mycobacterium tuberculosis H37Ra. In this report, we describe further purification of excretory-secretory ESAS-7 antigen fraction by fast protein liquid chromatography (FPLC) on Resource 'S' cation-exchange column and isolation of a more active and purified protein antigen fraction ESAS-7F. ESAS-7F antigen was characterized as a 31 kDa molecular weight glycoprotein containing a metallo-serine protease activity. N-terminal sequence analysis showed the first five amino acids as NTGQS (Asp-Thr-Gly-Glu-Ser). The present study helped in the isolation of a well characterized 31 kDa mycobacterial glycoprotein antigen with protease activity and diagnostic potential in detection of tuberculosis infection.

Seroreactivity of purified Brugia malayi microfilarial soluble and excretory-secretory antigens in different clinical presentations of bancroftian filariasis.

Brugia malayi microfilarial excretory-secretory (mf ES) and phosphate buffer saline soluble (mf S) antigens were fractionated by fast protein liquid chromatography (FPLC) on superdex 200 HR 10/30 gel filtration column. The active antigen fractions were identified and explored in comparison with whole mf ES and mf S antigens to detect filarial IgG antibodies in different groups viz microfilaraemics, acute, chronic and occult filarial cases of Wuchereria bancrofti infection and endemic and non-endemic normals. One of the fractions of mf ES antigen (ESF-6) and two fractions of mf S antigen (SF-2 & 3) were identified to be useful to detect filarial antibodies. A pooled preparation of these antigen fractions gave a sensitivity of 86.6% (for microfilaraemic cases) and a specificity of 95% to detect filarial IgG antibodies by indirect ELISA. The pooled FPLC purified mf antigens also showed 55-88% of cases of different grades of clinical filariasis and 65% of tropical pulmonary eosinophilia cases as positive for filarial antibodies. The pooled FPLC purified B. malayi mf antigens with higher specificity are preferable to whole mf ES and mf S antigens to detect active filarial infection in microfilaraemia and as well in different clinical entities of bancroftian filariasis.

Surgical filariasis: immunoscreening for filarial IgG antibodies using Wuchereria bancrofti microfilarial excretory-secretory antigen.

A clinical study and immunoscreening was conducted on 363 suspected filarial patients attending the surgery out patient division of the MGIMS, Sevegram. The disease was significantly higher in males (86%) than in females (14%). Majority (52.9%) of the cases were in the age group of 11-30 years. The distribution of cases into three different grades of infection showed, 52.6%, 33.3% and 14.1% of the cases having acute (grade I), sub-acute (grade II) and chronic (grade III) stages of infection respectively. While 73% of the cases had genital manifestations, 23% were with lymphatic obstruction in limbs and the rest of the 4% suffered from manifestations like cellulitis, abscesses, haematuria and chyluria. Filarial IgG antibodies against microfilarial excretory-secretory (mf ES) antigen were detected in 89% of cases with genital manifestations, 87% of lymphoedema cases, 67% of lymphadenitis cases and 60% of cases with other clinical manifestations and 3% of endemic normals.

Immunodiagnosis of pulmonary tuberculosis by concomitant detection of antigen and antibodies of excretory-secretory protein of Mycobacterium tuberculosis H37Ra.

With a view to diagnosing tuberculosis in populations in endemic areas, excretory-secretory antigen fraction (Mtb EST -6) of purified Mycobacterium tuberculosis H37Ra and affinity purified polyclonal antibodies against Mtb EST were used to detect both antibodies and circulating antigen in the sera of patients and disease-free individuals. Indirect stick penicillinase ELISA system using Mtb EST-6 detected antigen-specific IgG antibody in 84% of sputum positive, 77% of sputum negative pulmonary tuberculosis patients and 7% of healthy and 11% of subjects with nontub~rculosis diseases. Similarly, a sandwich penicillinase ELISA system using affinity purified anti Mtb EST antibodies detected circulating antigen in 83% and 61 % of sputum positive and negative pulmonary tuberculosis subjects. In contrast only 24% of healthy and 18% of disease controls showed seropositivity. Antibody assay showed higher sensitivity and specificity (83% and 91 % respectively) compared to antigen detection (sensitivity of 79% and specificity of 79%). However, by concomitant use of both assays it was possible to enhance the specificity of detection to 98%, though sensitivity was reduced marginally to 70%. The present study confirms the presence of both antigen and specific antibodies in the circulation during clinical disease and draws attention to the utility of Mtb EST -6 as a diagnostic marker of pulmonary tuberculosis

Immunoprophylactic studies with a 43 kDa human circulating filarial antigen and a cross reactive 120 kDa Brugia malayi sodium dodecyl sulphate soluble antigen in filariasis.

Bancroftian filariasis is a major public health problem affecting about 120 million people all over the world. Immunoprophylaxis may serve as an additional adjunct along with chemotherapy and anti larval measures for successful filaria control. Circulating filarial antigen fraction (CFA2-6) containing 43 kDa antigen and adult Brugia malayi sodium dodecyl sulphate (S DS) soluble antigen fraction BmA-2 with a 120 kDa molecule were earlier shown to be reactive with endemic normal sera by immunoblotting and indirect ELISA techniques. BmA-2 was found to be highly cross reactive with CFA2-6. Sera raised against both the antigen fractions showed about 9 0 % cytotoxicity to the parasites in the presence of jird peritoneal cells in in vitro as well as by in situ micropore chamber implantation technique. Further in in vivo studies using animal model, jirds CFA2-6 and BmA-2 could induce about 90% protection to infection in immunized animals. In passive transfer studies of immunity it has been observed that BmA-2 induced protection is mainly antibody mediated.

Detection of filarial antigen by inhibition enzyme linked immunosorbent assay using fractionated Brugia malayi microfilarial excretory secretory antigen.

Detection of filarial antigen in blood or urine samples would provide an accurate indication of active infection. The absence of a simple, well established animal model and limitations in getting the required amount of parasite material from human sources have been the main obstacles for the diagnosis ofWuchereria bancrofti infection. An inhibition ELISA has been developed for detection of filarial antigen using a partially purified Brugia malayi mf ES antigen (BmE DE1) and its affinity purified antibodies. Filarial antigen was detected in the sera of 88% of microfilaraemic, 60% of chronic filarial, 17% of endemic normal and none of the non-endemic normal subjects. The sensitivity and specificity of the assay were 88% and 89% respectively. Moreover, undiluted urine samples from 82% of microfilaraemic and 17% of endemic normal, but none of the non-endemic normal samples showed the presence of filarial antigen.With the limitations on the availability of sufficient homologous parasite materials, the inhibition ELISA using BmE DE1 and anti BmE DE1 antibodies shows promise for the detection of active infection in bancroftian filariasis in man.Moreover, its detection in urine makes it more possible to test patients in field areas.