Protective effects of Indigofera tinctoria L. against D-Galactosamine and carbon tetrachloride challenge on 'in situ' perfused rat liver.

The effect of pre-treatment with Indigofera tinctoria (IT) extract against the toxicity of D-Galactosamine (D-GalN) and carbon tetrachloride (CCl4) during 'in situ' perfusion of the liver for 2 hr was studied in rats. Release of LDH and levels of urea in the liver effluent perfusate, was studied and the rate of bile flow was monitored. Perfusion with D-Galactosamine (5 mM) or carbon tetrachloride (0.5 mM) resulted in increased LDH leakage, decreased urea levels in the liver effluent and reduction in bile flow. IT pretreatment (500 mg/kg body weight) in vivo ameliorated D-GalN and CCl4 induced adverse changes towards near normalcy and thereby indicates its hepatoprotective effects in rats.

Electrophysiological studies with repeated episodes of ischaemia on isolated rat heart.

In order to know the beneficial effect of preconditioning electrocardiography recording were used as tool to assess myocardial malfunction and for this perfusion apparatus was setup. Electrophysiological changes for each heart were recorded during perfusion at 1, 2, 3, 5, 10, 20, 30 and 60 min of global ischaemia and also during the equal period of reperfusion. Recordings dembnstrate that the normal rate was about 240 beats/min with an "R" amplitude of 4mV. During the first ischaemic episode of 1min the rate was 180 +/- 15 beats/min (counted as per 'R' wave deflection), at 2 mins it was 60 +/- 6 beats/min, at 3 min the rate was 40 +/- 2 beats/min, at 5 mins of ischaemia it was 90 +/- 6 beats/min, at 10 min 20 +/- 2 beats/min, at 20 min the rate was 60 +/- 4 beats/min, and at 30 mins there were nil beats/min. The recovery during all the periods of reperfusion was restored to between 120 and 180 beats/min in all episodes. Further after a 60 min of ischaemia the heart stopped to elicit any mechanical response. It is concluded that short term ischaemia can induce a resilient effect on the beating of the heart after a few episodes as seen subsequent to 1 and 2 min of ischaemia. Further, preconditioning was beneficial up to 30 min, beyond which the heart showed signs of fatigue and irreversible injury.

Mammalian coronary perfusion--an improved laboratory device.

An improvised device for coronary perfusion of isolated rat heart consisted of a reservoir of buffer pressurised with continuous bubbling of O2 connected to two double condensors in series. The outer jacket of each condenser was connected to a temperature controlled motorised water bath to ensure steady temperature of buffer. Isolated hearts could be tested with known agents directly through a syringe connected to the aortic cannula.

Effect of histamine and glucose on encystation of Entamoeba histolytica in vitro.

Isolates of Entamoeba histolytica grown and maintained in modified Boeck and Drbohlav's medium were used to examine the effect of histamine and glucose on encystation process in vitro. This revealed that 60-74% of the active and motile trophozoites of E. histolytica encysted within 72 hours in the presence of histamine and glucose, in contrast to 10-20% encystment in controls (with out histamine and glucose); a highly significant difference (P < 0.001). These observations also suggest the possibility of active role of histamine and glucose in encystation of E.histolytica in vivo.

Leucocyte migration inhibition index responses of patients of amoebic liver abscess to various antigens of Entamoeba histolytica.

The whole amoebic lysate (WAL), Sephadex G-200 eluted fraction-I. (F-I), detergent dissected membrane proteins (DDMP), amoebic membrane glycoprotein (AMG) and amoebal RNA were extracted from E. histolytica. Amoebal RNA recognized 98.21% where as F-I, AMG, DDMP and WAL recognised 92.85, 91.07, 89.28 and 75% of total 56 clinically proven and amoebic serology positive amoebic liver abscess cases respectively. Intensity of recognition of individual fraction i.e., leucocyte migration inhibition index of each fraction was found to be highly significant (P < 0.001) when compared to whole amoebic lysate. This indicate the leucocyte migration inhibition index responses of patients of amoebic liver abscess to various antigenic fractions of E. histolytica with variations.

Correlation between membrane glycoprotein and detergent dissected membrane protein in the assessment of LMIF: a report of 51 ALA cases.

Assessment of 51 amoebic liver abscess cases for leukocyte migration inhibition factor released using membrane glycoprotein and detergent dissected membrane protein (DDMP) of axenic Entamoeba histolytica (NIH:200). Lymphokines release by T lymphocytes in response to purified amoebic membrane glycoprotein (PAMG) against whole amoebic lysate (WAL), dissect out protein against whole amoebic lysate and membrane glycoprotein against dissected protein was tested by leukocyte migration inhibition test on blood samples from proved amoebic liver abscess cases. A significant increase was noted in the release of lymphokines and 100% positivity was observed with both PAMG and DDMP compared to 78% with whole amoebic lysate. The difference between means leukocyte migration indices of the membrane glycoprotein and whole amoebic lysate, detergent dissected protein and whole amoebic lysate with regards to release LMIF were found to be highly significant (P < 0.001), (P < 0.005) respectively. But insignificant difference and very much similarity was noted between the means of membrane glycoprotein and dissect out protein sensitized T lymphocytes with regards to lymphokine release in vitro. This shows the patients had high degree of leukocyte sensitized to pure amoebic membrane glycoprotein and detergent dissected membrane protein compared to whole amoebic lysate. These findings indicate that detergent dissected protein has similar antigenicity with membrane glycoprotein in elicitation cell mediated immune response in amoebic liver abscess cases.

Detection of sialic acid in trophozoites of Entamoeba histolytica (NIH:200).

In two successive experiments detection of sialic acid was achieved from trophozoites of axenically grown E. histolytica (NIH:200) using silicic acid column chromatography for the separation of sialosylated lipids and asialosylated lipids from total lipids. Sialic acid of the sialosylated lipids was detected through thiobarbituric acid assay followed by acid hydrolysis. These findings indicate that presence of sialic acid is not only a character of the cystic stage but also that of trophozoites of E. histolytica. Detection of sialic acid confirmed the electronegative charge of the surface membrane (glycocalyx) of E. histolytica trophozoites.

Correlation between antiamoebic IgG and autoreactive anti-IgG in amoebic liver abscess cases.

IgG isolated and purified from a healthy human serum through Sephadex G-200 and protein A CL 4B sepharose chromatography was used for detection of its own antibodies and correlated with the antiamoebic antibody titres in amoebic liver abscess cases. The mean titres with standard deviation of the self reactive antibodies to serum IgG both ALA cases and healthy controls show a highly significant difference, and antiamoebic antibody titres (IgG) are very much correlated with the autoreactive anti IgG titres in amoebic liver abscess cases. This correlation suggests that as antiamoebic IgG levels reach to its maximum, autoreactive anti IgG are produced to switch off antiamoebic anti IgG production in amoebic liver abscess cases.

Role of pure and biologically active amoebal RNA in assessment of lymphokines: a report of 55 ALA cases.

Amoebic liver abscess cases (55) were assessed for release of lymphokines (LMIF) using pure and biologically active amoebal RNA of axenic Entamoeba histolytica (NIH: 200) obtained with cesium chloride centrifugation. Lymphokines released by T lymphocytes in response to both amoebal RNA and whole amoebic lysate (WAL) were tested by leukocyte migration inhibition test (LMIT) on blood samples from amoebic liver abscess cases. A significant increase was observed in the release of lymphokine and 100% positivity was observed with amoebal RNA compared to whole amoebic extract with a positivity of only 78%. The difference between means leukocyte migration inhibition of the above two with regards to release of lymphokine was highly significant (P less than 0.001). This shows that patients had high degree of leukocyte sensitization to amoebal RNA of E. histolytica compared to whole amoebic lysate. These findings suggest that the amoebal RNA plays an important role as a potent antigen in the elicitation of cell mediated immune responses in amoebic liver abscess cases.

Autoimmunity in amoebiasis.

Amoebic liver abscess (ALA) and symptomatic intestinal amoebiasis cases were assessed by indirect haemagglutination assay for auto-reactive IgG and IgA class of antibodies in response to healthy human serum IgG and IgA. The present results indicated the presence of autoreactive IgG and IgA class of antibodies in ALA and intestinal amoebiasis respectively.

Detection of autoreactive antibodies to serum IgG and IgA in amoebic liver abscess cases.

Assessment of autoreactive antibodies in response to healthy human serum IgA and IgG was performed by indirect haemagglutination assay on serum samples from 81 amoebic liver abscess cases for IgA and 70 for IgG. Appropriate controls were taken simultaneously. IgA, IgG were isolated and purified from a healthy human serum through Sephadex G-200 and protein A CL 4B sepharose chromatography. These immunoglobulins were used for the detection of its own antibodies in amoebic liver abscess cases. This revealed that 43.20% and 48.50% of the cases were positive for IgA and IgG respectively, where as only 19.35% and 28.30% of the controls were in positive category (IgA and IgG respectively). The mean titres with standard deviation of the autoreactive antibodies to serum IgA both in ALA cases and controls shows a highly significant difference between tests and controls (P less than 0.001). Similarly the mean titres with standard deviation both in ALA and controls for the serum IgG differed significantly (P less than 0.001). This suggests the presence of autoreactive antibodies against serum IgA and IgG in amoebic liver abscess cases.

Detergent dissection of membrane proteins of Entamoeba histolytica and its effect on lymphokine release in in vitro.

Fifty-two amoebic liver abscess cases were assessed for the release of lymphokines (LMIF) using detergent dissected membrane proteins (DDMP) of axenic Entamoeba histolytica (NIH:200) obtained with sodium deoxycholate treatment. Lymphokines release by T lymphocytes in response to both DDMP and whole amoebic lysate (WAL) was tested by leukocyte migration inhibition test on blood samples from amoebic liver abscess cases. A significant increase was noted in the release of LMIF and 100% positivity was observed with DDMP compared to whole amoebic extract with a positivity of 73%. The difference between means of the above two with regards to release of LMIF was found to be highly significant (P less than 0.005). This shows the patients had high degree of leukocyte sensitization to surface antigens of E. histolytica compared to the whole amoebic lysate. These findings suggest that the antigens shed might have important role as a potent antigen in elicitation of CMI response in amoebic liver abscess cases.

Immunogenicity of detergent membrane proteins of Entamoeba histolytica.

The lymphokine release and antibody production were assessed in the peripheral blood of 52 and 48 cases of amoebic liver abscess respectively, by employing detergent dissected membrane proteins (DDMP) of axenic Entamoeba histolytica (NIH:200). Lymphokine release by T lymphocytes in response to both DDMP and whole amoebic lysate (WAL) was performed by leukocyte migration inhibition test. A highly increased release of LMIF and 100 per cent positivity was observed with DDMP where as the same for whole amoebic extract, was only 73 per cent. The difference between the means of the above two values with regards to release of LMIF, was found to be highly significant (P less than 0.005). Antibodies production in response to both DDMP and whole amoebic lysate was performed by indirect haemagglutination assay on blood samples from amoebic liver abscess cases. A 53 folds increased titres of IHA and cent percent positivity was observed with DDMP compared to WAL. The difference between mean titres of the above two with regards to detection of antibodies, was found to be highly significant (P less than 0.001). This shows that the patients, had high degree of leukocyte sensitization and production of antibodies which will not be assessed simply with WAL. These findings suggest that the shed material might have important role as a potent antigen in elicitation cell mediated and humoral immune response in amoebic liver abscess cases.

Penicillic acid as Na+,K+ and Ca2+ channel blocker in isolated frog's heart at toxic levels.

The effect of penicillic acid on isolated frog's heart has been studied along with ions of Na+,K+ and Ca2+. Penicillic acid has been found to inhibit the entry of these ions into cardiac tissue thereby arresting the action of the heart. The blockage can be washed away by perfusion with Ringer's solution.