Immunomonitoring followed by optimal dec therapy for successful management of clinical filariasis in an endemic area

Lymphatic filariasis continues to be the major cause of clinical morbidity in India and other developing tropical countries. One of the major lacunae in the effective management of clinical filarial cases is the non-availability of a suitable diagnostic test for confirming filaria aetiology in acute, chronic and occult clinical cases where microfilariae (mf) are not usually seen in peripheral circulation. Studies in our laboratory have shown the usefulness of filarial antibody and antigen assays using microfilarial excretory-secretory (mf ES) antigen in detecting microfilaraemic, acute and chronic filarial cases and in confirming filarial aetiology in occult infections. Diethylcarbamazine citrate (DEC) is the drug of choice for lymphatic filariasis. Different regimens of DEC have been explored in the treatment of microfilaraemic cases. Immunomonitoring has shown that the seroconversion of antigen and antibody positivity was found to be very helpful in determining appropriate period of DEC treatment for clinical relief and cure in clinical filarial patients and further they did not have recurrence in most of the cases. Optimal DEC (6mg/kg body wt/day for 21 days each month for 3–12 months) therapy was found to be very effective in acute and atypical clinical manifestations such as asthmatic bronchitis, pulmonary eosinophilia, monoarthritis, recurrent upper respiratory tract infections (URI), pneumonia (super imposed infections) in children and minimal hydrocele, epididymoorchitis, lymphangitis, lymphadenitis, acute abdomen, central serous retinopathy, tenosynovitis, pain and swelling in limbs and joints in adults living in filaria endemic areas.     

Host protective immunity and vaccine development studies in lymphatic filariasis

Lymphatic filariasis caused mainly by infection fromWuchereria bancrofti andBrugia malayi remains as the major cause of clinical morbidity in tropical and subtropical countries. Development of vaccine against filarial infection can act as additional measure to the existing therapeutic and vector control methods in the control of this disease. The main hurdles in the development of anti-filarial vaccine are the strict primate specificity ofWuchereria bancrofti, the paucity of parasite material, the diversity of clinical manifestations and their associated complex immune responses, lack of clear understanding on host-parasite interactions and the mechanisms involved in protective immunity. However in the past few years, the information generated in immuno-epidemiological studies, correlated with observations in experimental animals suggests that a filarial vaccine is feasible. Initially live irradiated infective larvae have been successfully used to induce high level of protective immunity in several animal models. Applying diverse strategies, variety of purified or recombinant filarial antigens have been explored for their ability to induce protection in different host-parasite systems. Some of these targeted filarial antigens induced high level of resistance in experimental animals against challenge infections. More focussed studies on thorough characterization of parasitological and immunological changes associated with resistance induced by such candidate protective antigens and on delivery mechanisms and safety aspects will be crucial in their selection for possible use in humans.     

Immunodiagnosis of human lymphatic filariasis: A review

A monoclonal antibody-based antigen detection system was used to detect the levels of circulating antigen in filarial patients before and after treatment with DEC and in normal individuals living in an area endemic forW. bancrofti infection in Chennai, India. The present study was to show the use of this assay as a means of efficient screening for filariásis in an endemic area where blood was absorbed onto the filter paper by finger prick during day time. The results of the antigen levels collected onto filter strips correlated with their corresponding plasma antigen levels (r=0.83). In microfilaraemics, DEC treatment did not alter the levels of circulating antigens upto a period of one month. We conclude that this monoclonal antibody based ELISA using filter strips may be used in daytime and can replace the existing routine night blood survey.     

Microscopic haematuria as an occult filarial infection in bhubaneshwar an endemic area for bancroftian filariasis

Sera samples of 7 microscopic haematuria cases collected before and after treatment with Diethylcarbamazine citrate, (DEC), 9 microfilaraemic cases and 19 endemic normal individuals were analysed for filarial antigen and IgG antibody levels. Filarial antigen was detected in 5 of the 7 microscopic haematuria cases, of which 3 turned negative for antigen after treatment with DEC. While none of the 7 haematuria cases were positive for filarial IgG antibodies, before the DEC treatment, all of them turned positive after DEC treatment. The sensitivity and specificity values(to detect mf +ve cases) were 89% and 90% respectively for the detection of filarial antigen and 78% and 95% respectively for the detection of filarial IgG antibodies.     

Modulation of cellular immune response by cytokines in bancroftian filariasis

Human lymphatic filariasis is a chronic helminth infection caused byWuchereria bancrofti andBrugia malayi. Wide spectrum of clinical manifestations are seen in different clinical groups of filariasis patients which comprises asymptomatic patients with circulating microfilaria (MF), individuals with chronic lymphatic obstruction (CP), Endemic normals (EN) who are asymptomatic and amicrofilaraemic or the relatively rare tropical pulmonary eosinophilia (TPE). The cellular immune response to this infection varies in different clinical groups of filarial patients. ranging from normal lymphocyte proliferative response in EN individuals to lymphocyte hyporesponsiveness in MF to total parasite antigen [Brugia malayi antigen (BMA)]. But in response to recombinant filarial antigen (pRJ51) the lymphocyte proliferation is restored in MF patients. Interestingly the lymphocytes from MFs responded normally to parasite antigen when EN serum was added in the culture whereas sera from MFs failed to revert the lymphocyte hyporesponsiveness. In order to study the molecular mechanisms responsible for parasite antigen specific lymphocyte hyporesponsiveness, we analysed both the Th1 and Th2 type cytokine gene expression profile in different clinical groups of filarasis patients. MF individuals expressed elevated Th2 type cytokines like IL-4, IL-5 and IL-10 and decreased levels of IL-2 and IFN-γ in response to parasite antigen. Chronic patients have elevated levels of both Th1 and Th2 type cytokines in response to parasite antigen. The EN individuals had a purely Th1 type pattern with absence of IL-4 and IL-5 expression. These studies clearly demonstrate the role of Th2 cytokine like IL-10 in antigen specific hyporesponsiveness seen in MF patients. Any methods to arrest the progression of this disease should concentrate on the means to revert the Th2 type into Th1 type response in the MF patients either by Th1 type cytokine therapy or by using recombinant filarial antigen which stimulates the Th1 response. Further the recombinant filarial antigen which induces Th1 type cytokine response could be used for immunoprophylactic studies.     

Detection of tubercular antibody and antigen in sera of bone and joint tuberculosis

Trichloroacetic acid (TCA) solubilized and DEAE fractionatedMycobacterium tuberculosis H₃₇Ra excretory-secretory (ES) antigen viz., Mtb EST DE1 and affinity purified goat antibodies to the TCA solubilized ES antigen (Mtb EST) were explored in detecting tubercular antibody and antigen respectively in sera of bone and joint tuberculosis by indirect and sandwich ELISA. Out of total 36 bone & joint tuberculosis cases, tubercular antibody was detected by indirect ELISA in 30 patients (sensitivity 83%), while circulating tubercular antigen was detected by sandwich ELISA in 27 patients (sensitivity 75%). Out of 34 non tubercular disease control cases, 10 patients showed positive reaction for antibody while only 4 patients showed positive reaction for antigen. In another group of 34 healthy subjects who were screened, 4 individuals showed positive reaction for tubercular antibody and 2 cases for antigen. This study shows that antigen detection assay using affinity purified anti Mtb EST antigen antibody is superior with overall specificity of 91% as compared to antibody detection assay with 75% specificity in bone & joint tuberculosis.     

Filarial antibody and antigen detection in different clinical conditions in an endemic area

The study was conducted in filarial endemic area for various clinical presentations and diagnosis of occult filariasis. A total of 157 cases of various clinical presentations namely tropical pulmonary eosinophilia, monoarthritis, polyarthritis, glomerulonephropathy, tenosynovitis, inguinal lymphadenopathy, generalised lymphadenopathy, retroperitonial lymphadenopathy, endomyocardial fibrosis and acute conjunctivitis were screened for filarial antigen and antibody by enzyme linked immunosorbent assay (ELISA). Out of 157 cases, 107 cases were positive for antigen or antibody, suggesting the role of filarial infection in these clinical presentations. All the 107 cases were treated with diethylcarbamazine citrate (DEC) and some of the patients who were followed showed relief in signs and symptoms. Hence assay of filarial antigen and/or antibody may be useful for diagnosing occult filarial syndromes for better management and further appropriate treatment.     

Serodiagnosis of tuberculosis using two ELISA systems

Serodiagnosis by ELISA has been widely explored over the years, in the diagnosis of tuberculosis. Two ELISA systems were evaluated for detection of mycobacterial antibodies in pulmonary and extra pulmonary tuberculosis. The two test assays explored were ERBA LISA (TB IgG) test (Anda Biologicals) which uses A60 antigen complex found in the cytosol of typical and atypical mycobacteria, and SEVA TB (IgG) ELISA, which uses a 31 kDa, glycoprotein antigen purified fromM. tb H₃₇Ra culture filtrate. Sera from 98 proven tuberculosis [pulmonary TB (48), tuberculous lymphadenopathy (30), tuberculous meningitis (15) & genitourinary TB (5)] were studied along with 32 healthy controls. The overall positivity obtained using ERBA LISA (TB IgG) test and SEVA TB (IgG) ELISA test was 72.9% and 91.6% in pulmonary tuberculosis, 43.3% and 76.6% in tuberculous lymphadenopathy respectively. The sensitivity of ERBA LISA test in tuberculous meningitis and genito-urinary TB was significantly low (26.6% & 40% respectively) compared to sensitivity obtained using SEVA TB ELISA (86.6% & 60% respectively) with overall specificity of 60% and 87.5%. Thus SEVA TB IgG ELISA test was found to be more sensitive than ERBA LISA in detecting IgG antibodies in tuberculous sera, in particular in extra pulmonary tuberculosis cases.     

Assessment of Hematological Parameters in Pulmonary Tuberculosis Patients

The aim of the study was the assessment of hematological parameters in pulmonary tuberculosis patients. Forty patients diagnosed with tuberculosis were recruited from the Institute of Thoracic Medicine on the basis of history, clinical examination, chest radiography, sputum examination and related laboratory parameters and were compared with age and sex matched healthy volunteers (n = 40). Hematological parameters and CRP in tuberculosis patients were determined. The mean values for serum hemoglobin level, RBC count and platelet count in PTB was found to be less (p < 0.001). Erythrocyte sedimentation rate (ESR), plasma C-reactive protein, WBC count in PTB subjects was increased (p < 0.001 for ESR & CRP, p < 0.05 for WBCs) and all were statistically significant. This study demonstrated that serum hemoglobin level, RBC count and platelet count was decreased in tuberculosis patients whereas ESR, CRP and WBC count was increased when compared with healthy controls.     

Analysis of SEVA TB ES-31 antigen specific immunoglobulins IgM, IgA and IgG in sera of sputum and culture positive pulmonary tuberculosis

Tuberculosis remains major health problem in India and developing countries Immunodiagnosis has important role in screening, diagnosis and management of tuberculosis. SEVA TB ES-31 antigen has shown potential in detecting tuberculous IgG antibody in earlier studies from our laboratory. In the present study we have analysedSEVA TB ES-31 antigen specific immunoglobulinsIgM, IgA and IgG in clinically and bacteriologically confirmed pulmonary tuberculosis cases to determine the usefulness of specific immunoglobulin class in the diagnosis of patients attending the hospital. Of the 30 cases of pulmonary tuberculosis 25 (83.3%) were positive for IgG, 19 (63.3%) for IgM and 16 (53.3%) for IgA. On combining IgG and IgM positivity, sensitivity was increased to 93.3%. While combining IgG and IgA positivity, sensitivity increased to 90%. However specificity was decreased to 66.6% and 70% for both of these combinations respectively. It could be envisaged from this study that IgG antibody detection against ES-31 antigen showed acceptable sensitivity (83.3%) and specificity (86.6%) compared to IgM or IgA alone or in combination. When immune responses were analysed according to degree of sputum positivity, IgG response was observed to be predominant in all grades, compared to IgM or IgA antibody. The addition of IgM or IgA as an adjunct test increases the sensitivity but at the cost of specificity. Hence the detection of IgG alone is more useful compared to IgM or IgA assay, in detecting tuberculosis disease cases coming to the hospital.     

Diagnostic potential of fractionatedMycobacterium tuberculosis H37Ra excretory-secretory (EST-DE1) antigen in pulmonary tuberculosis

Tuberculosis is emerging as a major public health problem in developing and developed world. Early and precise diagnosis is of prime importance in successful control of infection. Indirect ELISA with penicillinase as marker was developed using purifiedM. tuberculosis excretory-secretory (EST-DE₁) antigen for detecting IgG antibodies in pulmonary tuberculosis. The assay System gave a overall sensitivity of 82% for both smear positive and smear negative pulmonary tuberculosis cases with a specificity of 84%. The positive and negative predictive values were 75% and 88% respectivaly. Further studies with EST-DE₁ antigen revealed that, it contains two of the active antigen fractions of Mtb EST antigen i.e. Mtb EST-4 (56–68 KDa) and Mtb EST-6 (37–45 KDa), as demonstrated by inhibition ELISA. Reactivity with monoclonal antibodies HGT 3a showed the presence of 38 KDa molecule in EST-DE₁ antigen.     

Genetic Variation in <Emphasis Type="Italic">CD166</Emphasis> Gene and Its Association with Bladder Cancer Risk in North Indian Population

Adhesion molecules play a key role in cancer progression and tumorigenesis. Genetic polymorphism of adhesion molecules may alter the normal functioning thereby leading to bladder cancer susceptibility. Hence we aimed to evaluate three SNPs of CD166 gene (CD166rs6437585 C/T, CD166rs10511244 C/T, and CD166rs1157 A/G) in bladder cancer patients and normal controls of North Indian population. A total of 270 healthy controls and 240 confirmed bladder cancer patients were recruited for this study. Three SNPs of CD166 gene viz. CD166rs6437585 C/T, CD166rs10511244 C/T, and CD166rs1157 A/G were selected for this study. CD166rs6437585 C/T and CD166rs10511244 C/T were genotyped by Taqman allelic discrimination assay and CD166rs1157 A/G was genotyped by PCR–RFLP. The statistical analysis was done using the SPSS software, version 16.0 (SPSS, Chicago, IL), and p < 0.05 was considered statistically significant. Haplotypic analysis was done by using SNP analyzer version 1.2A. CD166rs6437585 C/T and CD166rs10511244 C/T showed significant association with reduced risk in bladder cancer while CD166rs1157 A/G showed significant high risk along with association at genotypic and allelic levels. Haplotypic analysis showed 1.8-folds risk in CCG combination, whereas CTA and TCG showed significant association with reduced risk. Further stratification on the basis of smoking, tumor grade/stage and BGC therapy revealed no association of these three polymorphic sites of CD166. Our study suggests that CD166rs6437585 C/T and CD166rs10511244 C/T are predictive for the reduced risk of bladder cancer, whereas CD166rs1157 A/G had shown significant association with high risk of bladder cancer in North Indians. This somehow suggests that CD166rs1157 A/G can be used as a marker for risk prediction of bladder cancer.     

Effect of Tulsi (<Emphasis Type="Italic">Ocimum sanctum</Emphasis> Linn.) Supplementation on Metabolic Parameters and Liver Enzymes in Young Overweight and Obese Subjects

Ocimum sanctum Linn. (also known as Tulsi) is a sacred Indian plant, the beneficial role of which, in obesity and diabetes is described traditionally. This is a randomized, parallel group, open label pilot study to investigate the effect of O. sanctum on metabolic and biochemical parameters in thirty overweight/obese subjects, divided into two groups A and B. Group A (n = 16) received one 250 mg capsule of Tulsi (O. sanctum) extract twice daily in empty stomach for 8 weeks and group B (n = 14) received no intervention. Statistically significant improvements in the values of serum triglycerides (p = 0.019); low density lipoprotein (p = 0.001); high density lipoprotein (p = 0.001); very low density lipoprotein (p = 0.019); Body Mass Index, BMI (p = 0.005); plasma insulin (p = 0.021) and insulin resistance (p = 0.049) were observed after 8 weeks in the O. sanctum intervention group. The improvement in HDL-C in the intervention group when compared to the control group was also statistically significant (p = 0.037). There was no significant alteration of the liver enzymes SGOT and SGPT in both the intervention (p = 0.141; p = 0.074) and control arms (p = 0.102; p = 0.055) respectively. These observations clearly indicate the beneficial effects of O. sanctum on various biochemical parameters in young overweight/obese subjects.     

Immunization with <Emphasis Type="Italic">Wuchereria bancrofti</Emphasis> Glutathione-S-transferase Elicits a Mixed Th1/Th2 Type of Protective Immune Response Against Filarial Infection in Mastomys

Lymphatic filariasis is a mosquito borne parasitic infection and can severely affect the normal working ability of an individual. Currently there is no vaccine available to prevent this infection and the development of a potential vaccine could effectively support the on-going mass drug administration program by World Health Organization (WHO). Filarial parasites have complex mechanisms to modulate the host immune responses against them. The glutathione-S-transferases (GST) are the important enzymes effectively involved to counteract the oxidative free radicals produced by the host. In the present study, we have shown that the mastomys which are fully permissible rodents for Brugia malayi when immunized with Wuchereria bancrofti recombinant GST (rWbGST) could induce 65.5 % in situ cytotoxicity against B. malayi infective (L₃) larvae. There was a balanced Th1/Th2 immune response in the vaccinated animals, characterized by higher levels of WbGST-specific IgG1 and IgG2a antibodies and pronounced IFN-γ, IL-10 and IL-4 cytokines production by the spleen cells.     

Effect of nutritional supplements and protease inhibitor on the yield of es antigens ofBrugia malayi microfilariaein vitro

Brugia malayi microfilarial excretory-secretory (mf ES) antigens obtained byin vitro maintenance of mf are important tools in the immunodiagnosis of bancroftian filariasis. To increase the yield of mf ES products, the effect of nutritional supplements on the culture medium (RPMI 1640) and the maintenance temperature were studied. Supplementation of RPMI 1640 medium with organic acids and sugars of Grace's insect culture medium forin vitro maintenance of 10 lakhs of mf in 40 ml medium increased the yield of mf ES antigen from 152 μg to 364 μg of mean protein, content and the mean antigen titre from 200 to 400. Supplementation with phenyl methyl sulphonyl fluoride (PMSF) and shift in the culture temperature resulted in a further increase in the yield of mf ES antigen to 502 μg of mean total protein with an antigen titre of 800. The modification resulted in a net increase of 3 fold in the protein content and 4 fold in the antigen titre of ES products. The above modifications in thein vitro maintenance of mf did not affect the diagnostic quality of mf ES antigen, which gave a sensitivity and specificity of 80% and 90% respectively in detection of filarial IgG antibodies inWuchereria bancrofti infected cases.     

Assay of tubercular antibody, circulating free and immune complexed antigen in the diagnosis of pulmonary tuberculosis

Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in confimed pulmonary tuberculosis sera by ELISA, using ES-31 antigen and affinity purified anti ES-31 antibody. Twenty three of 25 (92%) tuberculosis sera were positive for IgG antibody to ES-31 antigen. Using anti ES-31 antibody, free tubercular antigen could be detected in 20 of 25 (80%) cases whereas circulating immune complexed antigen (CIC-Ag) in 18 of 25 (72%) cases by sandwich ELISA. Of the two sera showing absence of antibody, one showed presence of free and CIC-Ag whereas the other showed the presence of free antigen. Thus antigen assay may be used as an adjunct tool for confirmation of pulmonary tuberculosis.     

<Emphasis Type="Italic">DRD4</Emphasis> and <Emphasis Type="Italic">DAT1</Emphasis> VNTR Genotyping in Children with Attention Deficit Hyperactivity Disorder

The dopamine receptor-D4 and the dopamine transporter have been investigated for their role in attention deficit hyperactivity disorder (ADHD) in children. Reports of their genetic association with ADHD have shown mixed results. The aim of the study was to evaluate the association of variable number tandem repeats (VNTRs) of the DRD4 and DAT1 genes with ADHD in children. A pilot 1:1 case control study, with 44 clinically confirmed ADHD cases and 44 age/gender matched healthy controls, was conducted at a tertiary care centre in Mumbai. Variable number tandem repeats of DRD4 exon 3, DAT1 intron 8 and 3′UTR were genotyped by PCR-AGE. Several allele repeats of the genes were observed in the screened subjects. Statistical significance was observed for the 10R/10R genotype of the DAT1 3′UTR VNTR between cases and controls.     

R&amp;D collaboration: role of <Emphasis Type="Italic">B</Emphasis>a in knowledge-creating networks

Research and development (R&D) collaboration between universities and business is a vital form of new knowledge creation in knowledge-intensive high-technology business environments. Increasingly, collaboration occurs in networks. A key element in forming these collaborate networks is shared knowledge creation, which is dependent on the Ba, the SECI process, and knowledge assets. This paper argues that Ba plays a major role for successful knowledge creation through R&D collaboration between university and business. A Ba is a perception of a place – which can be virtual – and a shared purpose. The absence of a Ba is a significant barrier to success, but building a Ba takes collaborative time and effort. This paper addresses the problems in the successful formation of such networks based on insights from biotechnology, an area where this type of collaboration has been and still is common, but not always successful or unproblematic.