Effect of Giardia lamblia on duodenal disaccharidase levels in humans.

The study was conducted to detect the effect of giardiasis on human disaccharidase levels. Forty patients attending the medical outpatient department of PGIMER, Chandigarh were enrolled. Twenty patients, positive for Giardia lamblia comprised the study group while 20 patients negative for Giardia lamblia were taken as controls. Upper gastrointestinal endoscopy was performed in all patients. Estimation of lactase, sucrase, maltase and trehalase was done in biopsies. Histopathological investigation was carried out in all biopsy specimens after Haematoxylin and Eosin staining. Complaints of pain abdomen and bloating occurred commonly in giardiasis. Four biopsy samples in study group showed mild increase in lymphomononuclear infiltrate. Giardia lamblia was detected in 7 biopsies. Lactase levels were decreased significantly (p < 0.05) in giardiasis. Rest of the enzymes were comparable to the controls. No differences in the enzyme activities were observed between males and females in either group and with the duration of symptoms.

A monoclonal antibody-based test system for detection of Entamoeba histolytica-specific coproantigen.

BACKGROUND: Diagnosis of amebiasis based on stool microscopy or demonstration of anti-amebic antibodies has limitations. A diagnostic system based on demonstration of the parasite product in clinical specimens holds promise. METHODS: Murine monoclonal antibodies were developed against an Entamoeba histolytica-specific coproantigen. A monoclonal antibody (MoAb) 3D10 was employed in a double-antibody sandwich microELISA system for the detection of amebic coproantigen in fecal specimens. The system was evaluated in three groups of subjects: 63 patients with intestinal amebae, 27 with non-amebic parasitosis, and 57 apparently healthy controls. RESULTS: The MoAb 3D10 belonged to IgG1 isotype and recognized three antigens, with mol. wt. 36, 25 and 17 kDa in the crude extract of E. histolytica (HM1-IMSS), and an amebic coproantigen with MW 36 kDa in the stool supernatant from patients with intestinal amebae. The coproantigen was detected in the stool eluates of 56 (89%) patients with intestinal amebae and in none of the stool eluates from other subjects, thereby giving this system a sensitivity of 89% and specificity of 100% for the detection of intestinal amebae. CONCLUSIONS: This monoclonal antibody recognizes as intact epitope on the E. histolytica-specific coproantigen. The validity of the MoAb-based microELISA system needs to be established.

Anti-66 kDa antileishmanial antibodies as specific immunodiagnostic probe for visceral leishmaniasis.

A 66 kDa plasma membrane associated molecule of promastigotes of Leishmania donovani (MHOM/IN/1978/UR6) was affinity purified under acidic conditions. Employing purified 66 kDa antigen in micro ELISA, 36 (97.3%) of the 37 patients of visceral leishmaniasis (bone marrow aspirates positive for Leishman Donovan bodies) had detectable levels of anti 66 kDa anti leishmanial antibodies. The sera of the patients confirmed to have visceral leishmaniasis had significantly (P < 0.001) higher optical density values (0.636 +/- 0.230) as compared to sera (OD 0.185 +/- 0.131) from patients clinically suspected to have visceral leishmaniasis (bone marrow aspirates negative for Leishman Donovan bodies). None of the 35 sera from apparently healthy subjects from non endemic area had anti 66 kDa antibodies. However, sera from one (8.3%) of the 12 healthy subjects, who was a first degree relative of a patient of visceral leishmaniasis and residing in an area endemic for visceral leishmaniasis, had anti 66 kDa antibodies. It is felt that detection of anti 66 kDa antibodies in a micro ELISA assay provides a highly sensitive and specific tool for confirming ongoing visceral leishmaniasis.

Evaluation of commercially available antacid tablets.

Antacids are commonly used for the treatment of acid peptic disease. There is a need to study the relative merits of various brands of antacids available commercially. Twenty three brands of antacid tablet preparations were evaluated with regard to their composition, cost, dispersion time, pH, neutralising capacity and time taken for neutralisation. The cost of various tablets ranged from Rs 0.13 to Rs. 1.48 per tablet and the dispersion time from 20 to 90 minutes. The pH of the dispersed tablet solution ranged from 5.7 to 9.5. The neutralising capacity varied between 8 to 169 meq/tablet and the neutralizing time between 20 and 45 minutes. The cost: neutralizing ratio was calculated and ranged from 102 to 9867 x 10(-3) Rs/meq. A scoring system with a maximum score of 12 has been devised. The study provides a guide for choosing a more potent, quick neutralising and low cost antacid preparation.

Detection of Entamoeba histolytica antigens in stool in amebiasis.

BACKGROUND: Stool microscopy, the conventional method of diagnosing intestinal amebiasis, fails to detect Entamoeba histolytica in more than 30-40% of clinically suspected cases. Demonstration of parasite products in clinical specimens has been suggested as an alternative. However, the usefulness of demonstrating amebic antigen in the stools of clinical cases needs to be assessed. METHODS: A double-antibody sandwich enzyme linked immunosorbent assay (ELISA) using anti-trophozoite antibodies to capture E histolytica specific coproantigen(s) was carried out on stools obtained from 31 patients with microscopically confirmed non-dysenteric amebic colitis, 18 patients with intestinal parasites other than E histolytica and 41 apparently healthy subjects. RESULTS: The assay detected E histolytica specific coproantigen(s) in stools of 23 (74.2%) of 31 subjects with non-dysenteric amebic colitis, none of 18 with other parasitic infections and 1 (2.4%) of 41 apparently healthy subjects. CONCLUSION: Our results provide evidence for the presence of E histolytica specific coproantigen(s) in stool eluates from patients with amebic infection; this finding can be exploited for confirming ongoing amebic infection. However, the sensitivity of the assay needs to be improved by the use of relevant monospecific/monoclonal antibodies.

Isolation & immunochemical characterization of diagnostically relevant antigens of Echinococcus granulosus.

Two hydatid specific polypeptides with molecular masses of 8 kDa and 116 kDa have been successfully isolated from E. granulosus hydatid cyst fluid using affinity chromatography. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and western immunoblot analysis under reducing and denaturing conditions indicated the 116 kDa purified antigen to be a hetero-tetramer consisting of 45 kDa, 66 kDa, 75 kDa and 116 kDa subunits linked by disulphide bonds while the 8 kDa purified antigen was found to be a monomer polypeptide. Affinity purified 116 kDa molecule was heat-labile, sensitive to treatment with pronase, trypsin and pepsin and its immunoreactivity as assessed in enzyme linked immunosorbent assay remained unaltered on treatment with sodium metaperiodate. The affinity purified 8 kDa molecule was heat-stable, sensitive to proteolytic enzymes and also sodium metaperiodate oxidation. Lectin binding studies revealed that the 8 kDa molecule specifically bound Concanavalin A and Triticum vulgaris, and thus had varies; is directly proportional to-D-glucose and N-acetyl D-glucosamine sugar moieties. The immunoreactivity of both the antigens remained unaltered on treatment with lipase. However, biochemical estimation of total lipid content revealed the affinity purified 116 kDa antigen to contain 6.25 per cent total lipids suggesting it to be lipoproteinic in nature. The 8 kDa antigen had no detectable total lipids biochemically. All sera from patients confirmed to have hydatidosis recognised the 8 kDa and 116 kDa polypeptides. However, sera from seven subjects with other parasitic infections also recognised the 116 kDa antigen though not the 8 kDa antigen. The data suggested that the recognition of 8 kDa antigen of E. granulosus has potential for specific immunodiagnosis of hydatidosis.

Evaluation of antacid gel preparations available commercially in the Indian market.

A scoring system based on the neutralising capacity, cost efficiency and time of buffering of twenty four commercially available antacid gels was analysed. A gel scoring eight out of the ten points was considered as the best antacid. The study provides a practical guide in choosing a quick neutralizing and low cost antacid gel.

Variations in isoenzymes of cloned & uncloned axenic Entamoeba histolytica without bacterial association.

Five clones of axenic E. histolytica (HMI) grown as discrete colonies in semisolid agar medium were adapted in liquid medium and labelled as HMI-C121, HMI-C131, HMI-C143, HMI-C144 and HMI-C145. Isoenzymes of these 5 clones of E. histolytica (HMI) were investigated in starch gel electrophoresis. There were no differences in the electromobility of maleate NADP oxidoreductase and glucosephosphoisomerase amongst the five clones and uncloned cultures of axenic E. histolytica. The relative electromobility (rf) of a single phosphoglucomutase (PGM) band of uncloned Mexican E. histolytica (HMI) and Indian axenic E. histolytica (KCG: 0986: 11) cultures and cloned E. histolytica HMI-C121, HMI-C145 was 0.087 while a single PGM band of uncloned E. histolytica (NIH: 200) and cloned E. histolytica HMI-C131, HMI-C143 and HMI-C144 cultures had rf of 0.075. Isoenzyme characterization of four cloned HMI-C121, HMI-C131, HMI-C143, HMI-C144 cultures of axenic E. histolytica (HMI) revealed existence of three bands of hexokinase (HK). The additional third band of HK was located close to the place of application of lysate and had rf ranging from 0.11-0.14. The data indicated that parent axenic E. histolytica (HMI) consisted of several populations and each population expressed different isoenzyme pattern without an association of amoebic cultures with any bacterial species.

Monoclonal antibodies which inhibit in vitro cytotoxicity of axenic Entamoeba histolytica.

A panel of 12 independent hybridoma cell lines secreting monoclonal antibodies to axenic E. histolytica (HM1) have been developed. A hybridoma cell line P4 C4 P2 F8 C8 (clone C8) produced monoclonal antibodies (MoAb C8) of IgG1 isotype which recognised a 29 KD surface associated antigen of amoebic trophozoites in Western immunoblot. Immunofluorescent probing with MoAb C8 employing live and acetone fixed amoebic trophozoites indicated 29 KD molecule on the surface plasma membrane of E. histolytica trophozoites. The MoAb C8 also agglutinated the live amoebic trophozoites. Pretreatment of amoebic trophozoites with anti 29 KD monoclonal antibody significantly (P less than 0.01) inhibited in vitro cytotoxicity of amoebic trophozoites to the cultured baby hamster kidney (BHK-21) cells. MoAb recognised a 29 KD molecule of E. histolytica trophozoites which mediated cytotoxic potentials of the parasite. The absence or variable degree of expression of cytotoxic 29 KD molecule may possibly serve as a marker to differentiate virulent/avirulent populations or strains of E. histolytica.

Characterization of surface associated antigens of axenic Giardia lamblia trophozoites & their recognition by human sera.

Two surface associated antigens (GLSA-82 and GLSA-56) of axenically grown G. lamblia trophozoites (PI strain) were affinity purified from its sonic extract. Both GLSA-82 and GLSA-56 were heat labile, sensitive to treatment with pronase, trypsin and were also sodium metaperiodate modifiable as assessed by micro ELISA. Lectin binding studies revealed that GLSA-82 specifically bound concanavalin A and pokeweed mitogen, and had alpha-methyl mannoside and n-acetyl-B-d-glucosamine sugar moieties. However, GLSA-56 selectively bound Ricinus communis agglutinin and phytohaemagglutinin, and had B-d-galactose and n-acetyl-B-d-galastosamine as sugar moieties. Human sera obtained during acute G. lamblia infection recognised GLSA-82 and GLSA-56 antigens. However, the antibody levels to GLSA-82 were significantly lower (P less than 0.05) during active giardiasis infection. Such surface associated antigens may be target of antiparasitic immune responses and thus, may modulate disease processes.

Prevention of malaria-induced foetal abnormalities following immunization of mice with Plasmodium berghei merozoite antigen.

Pre-pregnancy immunization of Swiss albino mice with merozoite antigen of P. berghei entrapped in multilamellar phosphatidyl choline liposomes resulted in (i) increased prepatent period, (ii) either no or low parasitaemic levels, (iii) reduced mortality, and (iv) normal foetal and placental development, upon challenge with P. berghei on 13th gestational day. The unimmunized animals which received either phosphate buffered saline or empty multilamellar phosphatidyl choline liposomes before pregnancy developed high parasitaemic and 30-40 per cent animals died before parturition while 60-70 per cent unimmunized animals revealed foetal abnormalities such as low body weight and larger spleen size. Placentae of unprotected animals had hyperplasia of trophoblastic membrane and plugging of placental sinusoids with parasitized erythrocytes and malarial pigments. The data suggest that prior immunization of animals with merozoite antigen entrapped in multilamellar phosphatidyl choline liposomes could abrogate the ill effects induced by malaria infection under the stress of pregnancy.

Immunobiological studies with mycobacterial ribonucleic acid-protein complex: Part II--Mechanism of protective immunity against experimental tuberculosis.

Passive transfer of protective antituberculous immunity against LD50 dose of M. tuberculosis H37Rv was found to be mainly mediated by immune T-cells harvested from spleens of donor mice immunized with Myc. RNA-P-FIA complexes as monitored by indices of percent survival, root specific lung weight, lung density and by bacterial enumeration from different organs. Treatment of immune T-cells with anti-Thy 1.2. monoclonal antibodies plus complement prior to passive transfer, completely abrogated its protective effect thereby confirming their protective nature. Passive transfer of immune sera as well as immune T + B cells did not induce any enhancement in protective immunity.

Immunobiological studies with mycobacterial ribonucleic acid-protein complex: Part I--Immune responses and protective role against experimental tuberculosis.

Ribonucleic acid (RNA) isolated from M. tuberculosis H37Ra was found to be native in nature as determined by hyperchromicity studies using ribonuclease. Mycobacterial RNA-protein (Myc. RNA-P) when injected as RNA-P-FIA complexes induced weak humoral immune responses and strong cell-mediated immune (CMI) responses which were directed against Myc. RNA. Protection comparable to BCG was induced in mice immunized with RNA-FIA complexes against LD50 dose of M. tuberculosis as monitored by increased survival rates, decreased lung density, root specific lung weight (RSLW) and by decreased viable counts of M. tuberculosis in lung, liver and spleen of immunized mice. Enzymatic degradation studies revealed Myc. RNA component to specifically mediate protection while the protein component was found ineffective.

Gut associated immune responses in clinical and experimental giardiasis: an overview.

Giardia lamblia, the protozoan parasite, first described by Von Leewenhoek in 1681, has come into prominence in last quarter century because of mounting awareness that it may cause significant morbidity and loss of man power. Earlier thought to be a commensal organism, it has been recognised as a true intestinal pathogen in the past three decades. Nevertheless, the mechanism of the disease caused by this protozoan parasite (in the human host's small intestine) continues to remain unexplained. The infection with G. lamblia is worldwide with an average prevalence of 12.5 per cent and is especially common in children and may cause failure of child to thrive. The G. lamblia infection has been implicated in a number of water borne epidemics and is important cause of traveller's diarrhoea all over the world. Infection with G. lamblia may be entirely asymptomatic, may produce a mild, self limiting illness or chronic diarrhoea with or without malabsorption. The reasons for such variations in severity are not clearly understood. However, interplay of virulence of parasite, nutritional status and type of the host immune responses and its effect on intestinal mucosa appear to modulate the infection.

Interactions of macrophages from in vivo stimulated guineapigs & the trophozoites of Entamoeba histolytica.

The interactions between peritoneal macrophages obtained from guineapigs immunized with a Sephadex chromatographed fraction (FI) of crude amoebic extract proteins and trophozoites of axenic E. histolytica (NIH:200) were studied in vitro in the presence or absence of anti FI antiamoebic antibodies. Unstimulated macrophages killed trophozoites to some extent. Incorporation of anti FI antiamoebic antibodies or IgG of FI antibodies in the amoebae macrophages mixture significantly enhanced the cytotoxic potentials of both unstimulated and in vivo stimulated peritoneal macrophages. Transmission electron microscopy revealed a close contact between macrophages and E. histolytica trophozoites. A large number of lysosomal vacuoles were seen accumulating near the contact areas. The contact between peritoneal macrophages and amoebic trophozoites in the presence of anti FI antibodies led to extensive degeneration of amoebic trophozoites. The antiamoebic antibodies induced cytotoxicity by macrophages in an opsonic manner. The data suggest that antiamoebic IgG binding to the trophozoites by its Fab portion and to the macrophages by its Fc portion initiated the cytotoxic mechanism(s) of macrophages.

Functional characterization of intestinal intraepithelium & lamina propria lymphocytes from Giardia lamblia infected mice.

Quantitation of T cell subsets in intraepithelium and lamina propria during the course of experimental G. lamblia infection in the inbred mice revealed increased influx of Thy 1.2+ (T cells) and Lyt 2.2+ (suppressor/cytotoxic T cells) during the establishment (3-5 day post-inoculation) and acute (9-11 day post-inoculation) phases of infection. The influx of these cells reduced as the parasite load declined. In contrast, no significant changes were noticed in lamina propria and intraepithelium Lyt 1.1+ (helper T cell) cells during the establishment or acute phase but such cells increased significantly in the decline (17-21 day post-inoculation) phase of infection. Further, both intraepithelium and lamina propria lymphocytes isolated from uninfected or infected animals failed to kill G. lamblia trophozoites in vitro in the absence or presence of antigiardial antibodies. Our data suggest that the clearance of G. lamblia trophozoites was not mediated by cytotoxic T cells. However, the induction of helper T cells during the declining phase of infection might be an important mechanism for the induction of parasite specific antibody response leading to the immune elimination of G. lamblia trophozoites from the gut.