Detection of histidine rich protein & lactate dehydrogenase of Plasmodium falciparum in malaria patients by sandwich ELISA using in-house reagents.

Background & objectives: Despite major control efforts, malaria remains a major public health problem that still causes high mortality rate worldwide especially in Africa and Asia. Accurate and confirmatory diagnosis before treatment initiation is the only way to control the disease. The present study was undertaken to develop reagents using sandwich ELISA for simultaneous detection of PfHRP2 (Plasmodium falciparum histidine rich protein) and PfLDH (P. falciparum lactate dehydrogenase) antigens in the proven malaria cases. Methods: The antibodies were raised against two epitopes of PfHRP2 protein and three unique and unexplored epitopes of PfLDH protein. These antibodies were able to detect PfHRP2 and PfLDH antigens in culture supernatant and parasitized RBC lysate of P. falciparum, respectively up to 50 parasites/μl. The in-house reagents were tested in 200 P. falciparum positive patients residing in Baghpat district of Uttar Pradesh in northern India. Results: Microsphere (PLGA) with CpG ODN were used to generate high titre and high affinity antibodies against selected peptides of PfHRP-2 and pLDH antigen in mice and rabbit. The peptide specific peak titre varied from 12,800 - 102,400 with an affinity ranging 0.73 - 3.0 mM. The indigenously developed reagents are able to detect PfHRP2 and PfLDH antigens as low as 75 parasites/μl of blood with a very high sensitivity (96-100%) and specificity (100%). Interpretation & conclusions: The study highlight the identification of unique epitopes of PfHRP2 and PfLDH, and the generated antibodies against these antigens were used for quantitative estimation of these two antigens using sandwich ELISA. No corresreactivity with P. vivax infected patients was observed with the sera.

Protective effect of resveratrol and vitamin E against ethanol-induced oxidative damage in mice: Biochemical and immunological basis.

The metabolism of ethanol gives rise to the generation of excess amounts of reactive oxygen species and is also associated with immune dysfunction. We examined the efficacy of resveratrol and vitamin E on the immunomodulatory activity and vascular function in mice with liver abnormalities induced by chronic ethanol consumption by measuring the protein, liver-specific transaminase enzymes, antioxidant enzymes and non-enzymes such as reduced glutathione (GSH) content, thiobarbituric acid reactive substance (TBARS) level, nitrite level, and activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GPx) and glutathione-S-transferase (GST), and cytokines such as interleukin (IL)-2, IL-4, IL-10, tumor necrosis factor (TNF)-, gamma interferon (IFN-), vascular endothelial growth factor (VEGF)-A and transforming growth factor (TGF)-1 in mice blood. Ethanol (1.6 g/kg body wt/day) exposure for 12 wks significantly increased TBARS and nitrite levels and GST activity, and significantly decreased GSH content and the activities of SOD, CAT, GR and GPx in whole blood hemolyzate of 8-10 wks-old male BALB/c mice (weighing 20-30 g). Ethanol exposure also elevated the activities of transaminase enzymes (AST and ALT), IL-10, TNF-, IFN-, VEGF-A and TGF-1, while decreasing the albumin concentration and IL-4 activity in the serum. Both resveratrol (5 mg kg-1 day-1) and vitamin E (80 mg kg-1 day-1) treatment significantly reduced AST, ALT, GST, IL-10, TNF-, IFN-, VEGF-A and TGF-1 activities and levels of TBARS and nitrite, and elevated albumin content, GSH level and activities of SOD, CAT, GR and GPx, compared to ethanol-treated group. Thus, results from the study demonstrated that both resveratrol (5 mg kg-1 day-1) and vitamin E (80 mg kg-1 day-1) can effectively ameliorate ethanol (1.6 g kg-1 day-1)-induced oxidative challenges, immunomodulatory activity and angiogenesis processes.

Time-dependent effects of ethanol on blood oxidative stress parameters and cytokines.

Alcohol consumption is implicated in the genesis of a spectrum of liver abnormalities, which are associated with a number of factors. In the present study, time-dependent effects of ethanol on cytokines (TNF-alpha, IL-2, IL-4, IL-10, IFN-gamma, VEGF-A and TGF-beta1) in serum, and blood oxidative stress parameters such as reduced glutathione content, TBARS level and activities of GPx, GR, GST, catalase and SOD in 8-10 weeks-old male BALB/c mice have been investigated. Ethanol administered @ 1.6 g/kg body wt/day significantly increased the activities of liver marker enzymes AST, ALT and ALP. Serum nitrite levels and haemolysate TBARS level also increased, while total antioxidant status in serum and GSH content in whole blood hemolysate decreased from 4th week onwards of exposure. In spite of the increased serum nitrite level and GST activity in the haemolysate, albumin level in serum, GPx and GR activities in haemolysate decreased after 12 weeks of exposure. Chronic ethanol treatment did not show any effect on IL-2, but IL-4 level was reduced and other cytokines such as IL-10, TNF-alpha, IFN-gamma, TGF-beta1 and VEGF-A levels were increased significantly after 12 weeks. The study indicates a relationship between free radical generation and immune response, and suggests that ethanol-induced liver damage is associated with oxidative stress and immunological alterations in a time-dependent manner.

Suppressive effect of strychnos nux-vomica on induction of ovalbumin-specific IgE antibody response in mice.

Strychnos nux-vomica Linn. (SNV; Loganiaceae), a medicinal plant has been used as folk medicine for alleviating inflammation, joint pains and allergic symptoms. In the present study, we examined its possible immunomodulatory effect on induction of ovalbumin (OVA)-specific IgE antibody response in a murine model, as evaluated by passive cutaneous anaphylaxis (PCA). The OVA-specific IgE antibody response was significantly suppressed in BALB/c mice (H-2d), following intraperitoneal administration of aqueous stem extract of the plant along with OVA. Furthermore, the different doses of SNV extract were found to significantly suppress the induction of OVA-specific IgE antibody response. The anti-OVA IgE antibody response was suppressed in different haplotypes of mice viz., C57BL/6 (H-2b) and SWR/J (H-29). However, preliminary findings revealed no significant change in the total IgG antibody response against OVA, as evaluated by ELISA. These results confirm the suppressive activity of S. nux-vomica on allergen-specific IgE antibody response and suggest its possible application in allergic conditions. Keywords: Strychnlos nux-vomica, Immunomodulation, Immunosuppression, IgE antibody response, Passive cutaneous anaphylaxis, ELISA

Liposomal delivery of Mycobacterium leprae antigen(s) with murabutide and Trat peptide inhibits Fas-mediated apoptosis of peripheral blood mononuclear cells derived from leprosy patients.

Protective immunity against intracellular pathogen Mycobacterium leprae is dependent on the activation of T cells. Repeated stimulation of T cells by M. leprae antigens MLCwA (M. leprae total cell wall antigen) and ManLAM (mannose capped lipoarabinomannan) may lead to apoptosis in leprosy patients. In the present study, inhibition of the Fas-induced apoptosis of peripheral blood mononuclear cells of leprosy patients was investigated using above M. leprae antigen(s), in combination with immunomodulators murabutide (MB) and a Trat peptide in particulate form (liposome). Incubation of the cells with particulate mode of antigen presentation led to both decreased percentage of propidium iodide (PI) positive cells and T cells expressing Fas-FasL, as well as decreased caspase-8/-3 activities in the lepromatous patients, thereby inhibiting apoptosis, while converse was true with stimulation with soluble antigen. Concurrently, there was an upregulation of antiapoptotic protein Bcl-X(L) in the lepromatous patients, thereby inhibiting apoptosis. Thus, the liposomal formulation of antigen promoted proliferation of anergized T cell by inhibiting apoptosis through decreased expression of death receptors and caspase activities and increased expression of anti-apoptotic protein Bcl-X(L) in these patients.

Effect of lecithin with vitamin-B complex and tocopheryl acetate on long-term effect of ethanol induced immunomodulatory activities.

The alcoholic liver disease usually causes overall immunological alterations which might be attributed to hepatic disease, to ethanol action, and/or to malnourishment. In the present study, efficacy of lecithin with vitamin-B complex to treat ethanol induced immunomodulatory activity was compared with the effect of lecithin alone and tocopheryl acetate (vitamin E). Ethanol (1.6 g/kg body wt/day for 12 weeks) exposure increased thiobarbituric acid reactive substance (TBARS) level, while decreased superoxide dismutase (SOD) activity and reduced glutathione (GSH) content in whole blood hemolysate of 8-10 week-old male BALB/c mice (weighing 20-30 g). The activities of transaminase (AST and ALT) enzymes, interleukin (IL)-10 and gamma interferon (IFN-gamma) elevated, while IL-2 and IL-4 reduced in mice serum due to ethanol exposure. These suggested that oxidative stress and immunomodulatory activities were interdependent and associated with ethanol induced liver damage. Lecithin treatment significantly reduced AST (32.44%), ALT (32.09%), IL-10 (25.63%) activities and TBARS content (12.76%) compared to ethanol treated group. However, lecithin with vitamin-B complex treatment, significantly reduced AST (62.83%); ALT (61.96%); IL-10 (35.88%); IFN-gamma (22.55%) activities and TBARS content (31.58%), while significantly elevated GSH content (36.49%) and SOD activity (61.21%). Tocopheryl acetate treatment significantly reduced AST (62.83%); ALT (61.54%); IL-10 (36.35%): IFN-gamma (23.28%) activities and TBARS content (35.84%). while significantly elevated GSH content (28.76%) and SOD activity (62.42%) compared to ethanol treated group. These findings persuasively argued that lecithin with vitamin-B complex was a new promising therapeutic approach in controlling ethanol induced immunomodulatory activities involving liver damage processes. Prevention of oxidative stress with correction of nutritional deficiency caused alteration in the ethanol-induced immunomodulatory activities and associated liver diseases.

Isolation and characterization of allergens from the seeds of Vigna sinensis.

Allergenic components of cowpea vegetable green seeds (Vigna sinensis) were isolated based on solubility, isoelectric precipitation and molecular mass. The allergenicity of the cowpea fractions was monitored by enzyme-linked immunosorbent assay (ELISA) and skin-prick test. The allergenic albumin fraction was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and IgE-specific immunoblotting. The 41 and 55 kDa protein components were found to be major allergens and the allergenicity was resistant to heat and proteolytic enzyme digestion. This study confirms the presence of potent allergens in cowpea seeds.

Estimation & significance of serum & synovial fluid malondialdehyde levels in rheumatoid arthritis.

Serum and synovial fluid (SF) levels of malondialdehyde (MDA), a marker of free radical induced lipid peroxidation, were estimated in patients of rheumatoid arthritis (RA) and compared with healthy controls and patients of osteoarthritis (OA). While serum MDA levels were similar in healthy controls (0.24 +/- 0.10 nmol/ml) and OA (0.28 +/- 0.11 nmol/ml), the serum levels in RA (0.47 +/- 0.19 nmol/ml) were significantly higher as compared to both healthy controls and OA patients; and correlated with synovial fluid (SF) MDA levels. No difference was observed in SF-MDA levels in RA (0.17 +/- 0.07 nmol/ml) and OA (0.16 +/- 0.09). MDA levels did not correlate with markers of disease activity in RA like joint counts, duration of morning stiffness, erythrocyte sedimentation rate etc. Increased serum MDA levels in RA suggest the role of free radicals in the pathogenesis of this inflammatory arthropathy and support the need for further studies assessing the therapeutic role of free radical scavengers in RA.

Risk assessment of tobacco, alcohol and diet in cancers of base tongue and oral tongue--a case control study.

This is a retrospective case-control study of male tongue cancer patients seen at Tata memorial Hospital, Bombay, during the years 1980-84. The purpose of the study was to identify the association of tobacco, alcohol, diet and literacy status with respect to cancers of two sub sites of tongue namely anterior portion of the tongue (AT) (ICD 1411-1414) and base of the tongue (BT) (ICD 1410). There were 142 male AT patients and 495 BT patients interviewed during the period. 635 interviewed male patients who were free of any disease were considered as control. Bidi smoking was found to be a significant risk factor for BT patients and tobacco chewing for AT patients respectively. Alcohol drinkers showed about 45% to 79% excess risk for both sites of tongue cancer. Illiteracy and non vegetarian diet proved to be a significant factor for AT patients only. The study brings out that the location of cancer has got a direct bearing with the type of tobacco use and other related habits and this inturn may provide meaningful interpretation of variations observed in the incidence of tongue cancer around the world.

Estimate of cancer incidence in India in 1991.

Cancer incidence and eighteen site-specific age standardised rates in India were estimated for the year 1991. With the establishment of National Cancer Registry Programme, incidence rates per 100,000 are available from six metropolitan registries and one rural registry. Using population census data for India in 1991, about 609,000 new cancer cases were estimated to have been diagnosed in the country in 1991. The estimated age standardised rates per 100,000 were 96.4 for males and 88.2 for females. The five most common cancers were lung (10.6%) pharynx (9.1%), oesophagus (6.7%), tongue (6.6%) and stomach (5.7%) among males and cervix (23.5%), breast (19.3%), ovary (5.5%) oesophagus (4.4%), and mouth (3.9%) among females. A comparison of estimated ASRs for two two largest countries in Asia (China and India) showed differences in the pattern of cancer.

Preparation & standardization of the goat lung surfactant extract.

Exogenous goat lung surfactant extract (GLSE) was obtained by chloroform-methanol extraction of the centrifuged lung lavage. Four lungs provided around 250 mg of surfactant. GLSE has a phospholipid content of 50 percent, cholesterol of 1.5 percent and protein content under 1 percent. The preparation was bacteriologically sterile and had a shelf life of at least one month at 4 degrees C. Endotracheal administration of surfactant (50 mg/kg) in a rat lung model rendered surfactant deficient by saline lavage, restored the compliance characteristics to normal, thus documenting biological activity of the preparation. The cost of a course of 2 doses of surfactant replacement therapy was estimated to be as low as Rs 500/-. The study opens up the possibility of surfactant replacement therapy for the management of neonatal respiratory distress syndrome in India by an indigenous product at an affordable cost.

Immune response against the non-repeat region (293-310) of the circumsporozoite protein of Plasmodium vivax.

Immunization by peptides based on the repeat sequences of Plasmodium falciparum or P. vivax antigen(s) have shown inconsistent results during clinical trials in humans. This could be attributed to the lack of T-cell help or antigenic polymorphism. Thus, attention has been focused towards the more conserved non-repeat regions. The present study was undertaken to map the antigenic determinant in the vicinity of region II (outside the repeat) of CS protein of P. vivax. The immunogenicity of the peptide was studied alone and after linking with polytuftsin (PT), using alum and Freund's adjuvant, in inbred strains of mice with different genetic backgrounds. The humoral response and antigen induced T-cell proliferation assays clearly demonstrated the immunomodulatory activity of PT. Comparable results were observed with antigen(s) administered either in alum or Freund's adjuvant. The induction of IgG2a and IgG2b antibody isotypes by both, peptide as well as the conjugate, may indicate that the T-helper response involved is of Th1 type. Further the immunofluorescence studies have shown that antibodies recognized the air dried sporozoites of P. cynomolgi. The results thus show that the above sequence has overlapping B and T-cell determinants and that alum can be substituted for Freund's adjuvant in generating an effective immune response.

Leukocyte glucose 6 phosphate dehydrogenase as prognostic indicator in ANLL and CML.

Glucose 6 phosphate dehydrogenase (G6PD) was estimated in the leucocytes of 35 patients with acute non-lymphocytic leukemia (ANLL) and 10 patients with chronic myeloid leukemia (CML). G6PD levels were found to be significantly decreased in majority of the patients with ANLL while it was increased in all CML patients. Variation in G6PD was found to be dependent on the percentage of myelocytes inANLL. Cytogenetic analysis was also carried out in these patients. Correlation analysis of leucocyte G6PD activity and karyotype with prognostic assessment clearly indicated the association of (s) high percentage of chromosomal abnormalities especially translocations, (b) low survival and remission rates, with patients having decreased G6PD activity when compared to patients with normal activity in ANLL. The studies indicate that leucocyte G6PD may be useful as a diagnostic and prognostic tool.

Red cell enzymopathies in leukemias.

Red cell glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PK) levels were estimated in patients with leukemias, to see if there was a consistent enzyme defect and correlation of enzyme levels with stage of the disease with prognostic assessment. G6PD levels were found to be significantly increased or decreased in majority of the patients with acute leukemias. Increased activity was seen in majority of the patients with L1 and decreased activity in L2 subtype of acute lymphocytic leukemia (ALL). In chronic myeloid leukemia (CML) G6PD activity was consistently increased, with levels being highly elevated in chronic phase and moderately in blast phase. Variation in G6PD activity was found to be related to the stage of disease and was of prognostic significance. PK was found to be normal or decreased. Fetal haemoglobin levels were also estimated in 30 patients with leukemias and were found to be elevated in most patients. Red cell G6PD may be useful for staging of the disease, as prognostic indicator and predictor of relapse.

Epidemiological observations on cancer of the oesophagus--a review of Indian studies.

This is an epidemiological review on cancer of the oesophagus. In this attempt, all aspects of epidemiological factors based on national and international studies on oesophageal cancer have been brought out. The problem of this cancer in Indian context has been documented. The association of tobacco and alcohol habits with oesophageal cancer has been confirmed from the studies conducted in India. There is an urgent need to educate the common people about the harmful effect of these two habits and governments and voluntary organisation should take effective steps for its prevention.

Leukocyte glucose 6 phosphate dehydrogenase (G6PD) as prognostic indicator in children with acute lymphocytic leukemia.

G6PD activity, estimated in 37 patients with acute lymphocytic leukemia (ALL) prior to therapy was found to be significantly decreased in 78.37% of the patients with ALL while it was normal in other patients. Variation in G6PD was found to be dependent on the percentage of myelocytes. Correlation analysis of leukocyte G6PD activity with karyotype indicated that patients with normal karyotype with normal G6PD activity had good prognosis while those with abnormal G6PD with abnormal karyotype had poor prognosis. Subjects with normal karyotype and abnormal G6PD and vice versa had intermediate prognosis. Thus the results clearly indicate that leukocyte G6PD may be used as a diagnostic and prognostic tool.

Humoral immune responses to the Plasmodium falciparum antigen Pf155 RESA in adults with differential clinical conditions from an Indian zone where malaria is endemic.

Ring infected erythrocyte surface antigen of Plasmodium falciparum (Pf155/RESA) has been considered as a vaccine candidate. However, the relative immunogenicity of this antigen has not been studied in Indian populations. Pf155/RESA was investigated for its immunogenicity by studying humoral immune responses against Pf155/RESA and Pf155/RESA derived peptides (P1, P2 representing immunodominant epitopes from the 3' and P3 from the 5' repeat regions) by erythrocyte membrane immunofluorescence (EMIF) assay and by enzyme linked immunosorbent assay (ELISA) in P. falciparum primed donors living in hyperendemic malarious areas (Orissa State, India) where P. falciparum infections are highly prevalent. Subjects of different clinical status namely acute (A), clinically immune (CI) and acute with history of repeated P. falciparum infections (R) were included in this study. All the donors were seropositive against the crude antigen. There was considerable variation in the responses among the donors. While humoral responses in the plasmas against the P2 peptide (EENV)4 were significantly higher in magnitude and in frequency in the CI donors than in the A donors, no positive response was seen in the R donors. The responses to the peptides P1 (EENVEHDA)2 and P3 (DDEHVEEPTVA)2 were poor both in the A and in the CI groups. Whereas, most of the R donors were seropositive against the P3. The present results indicate that Pf155/RESA contains B cell epitopes which were recognized differently by the immune system of individuals living in malaria-hyperendemic areas of India who have been primed by natural infection. Our studies also suggest that in order to investigate the possible functional role of a given antigen, study of immune responses against the antigen in donors of different clinical status may be useful.

Comparative assessment of pedal pressing rates of self-stimulation of hypothalamus and midbrain with both square wave and sine wave stimulus parameters.

In Wistar rats, the regional differences of pedal pressing rates of self-stimulation (SS) of lateral hypothalamus (LH) and substantia nigra-ventral tegmental area (SN-VTA) were assessed with electrodes implanted in both regions in each subject. Average of SS rates of SN-VTA sites was significantly higher than that of LH sites, tested with both sine wave and square wave types of stimuli. There was no significant difference in SS rates between males and females, and also in the females between different days of oestrus cycles. The high rates of robust SS observed in this study relative to SS rates reported in past literature were probably due not only to the placements of electrodes in the main substrates of SS, but also to the parameters of stimulus used (0.25 sec trains of sine waves through bipolar electrodes).