Liposome entrapped allergen reduces plasma histamine in sensitized mice.

Immunotherapy of allergic diseases is associated with problems of adverse systemic reactions. We have shown earlier that liposome entrapped allergen (LEA) is effective in inducing IgG response and restricting IgE response in immunized mice. This mode of treatment may be more effective and safer if it can prevent anaphylaxis. To determine this feature, mice were administered allergen preparations repeatedly and later challenged with the same allergen. Mice given liposomal preparation showed lower specific IgE response as compared to the mice given free allergen or alum adsorbed allergen of Artemisia scoparia. Specific IgG response was higher in mice immunized with LEA. The mice immunized with liposomal preparation survived whereas others injected with free allergen or alum adsorbed allergen died probably due to anaphylaxis. High levels of histamine were observed in mice injected with free allergen as compared to the mice injected LEA. The increase in plasma histamine level may be the cause of anaphylaxis during allergen challenge. In conclusion, LEA could be used as a safe and effective mode of immunotherapy for allergy diseases, since it reduces plasma histamine levels considerably thereby reducing the chances of anaphylaxis.

Umbilical cord blood as a source of CD34 positive haematopoietic stem cells.

In this paper we have used a monoclonal antibody to CD34 an antigen expressed solely on stem cells, and stem cell colony assays to show that umbilical cord blood has nearly the same number of functional stem cells as compared to normal bone-marrow. The number of CD34+ve cells in cord blood being 2 to 2.7 per cent, whereas bone-marrow had 3 to 3.5 per cent. The multi-potent colony forming cells (CFU-GEMM) were 60 +/- 18 in cord blood per 2 x 10(5) mononuclear cells (MNCs), whereas normal bone-marrow had 70 +/- 10 per 2 x 10(5) MNCs. Enrichment of these stem cells on Percoll gradients was successful for normal bone-marrow but not for cord blood.

T cell responses in Hodgkin's disease: frequency distribution of IL-2 producing cells and quantitation of IL-2 produced per cell.

T cell dysfunction in Hodgkin's disease (HD) is well documented. Since interleukin-2 (IL-2) plays a pivotal role in T cell proliferation, we have investigated frequency distribution of IL-2 producing phytohaemagglutinin (PHA)-stimulated lymphocytes from HD patients compared to that of healthy donors using two limiting dilution (LD) culture systems in which autologous peripheral blood lymphocytes (PBL) and Epstein Barr Virus transformed allogeneic B lymphoblastoid cell lines (EBV-LCL) have been used as feeders. The latter provided better conditions for IL-2 production by single cells, as evident from the enhanced frequencies obtained (For healthy donors: 1/67 +/- 1545.5 using EBV-LCL and 1/1123 +/- 1.7438 using autologous PBL as feeders). The data showed significantly reduced frequency of IL-2 producing cells as well as reduced quantity of IL-2 produced per cell in HD even after using/EBV-LCL as feeders, the amount of IL-2 produced per activated responder cell in HD patients being 0.825-1.3 pg/well (p < 0.001) as compared to 1.48-2.43 pg/well in healthy donors. Thus, the EBV-LCL feeders did provide better culture conditions for estimating frequencies of functional T cells. However these cell lines were unable to restore in vitro the abnormalities in functional properties of T cells in HD.

Impairment in cellular protein phosphorylation by mitogen activated lymphocytes from patients with Hodgkin's disease.

T cell activation process in patients of Hodgkin's disease was studied in terms of cellular protein phosphorylation following interaction of T lymphocytes with mitogen PHA. Peripheral blood lymphocytes from Hodgkin's disease patients and healthy donors, labelled with [32P] were activated with PHA. The cell lysates were subjected to SDS-PAGE, 2-dimensional gel analysis and were autoradiographed. It was observed that lymphocytes from both Hodgkin's disease patients and healthy donors followed similar time kinetics of phosphorylation. Nine of the eleven major protein bands, resolved on SDS-PAGE in the molecular weight range of 15.7-98 kD showed reduced phosphorylation (ratios of densitometric readings taken after and before stimulation) compared to that of healthy donors. Isoelectric focusing of these major protein bands in 2-dimensional gels further resolved them into about 27 proteins. Most of these showed increased phosphorylation in lysates of activated lymphocytes from healthy donors compared to that of Hodgkin's disease patients. The results showed a defect even at an early stage in terms of inadequate cellular protein phosphorylation.

A study on antigenic and allergenic changes during storage in three different biological extracts.

The stability of three allergens common in tropical countries was evaluated under different storage conditions. Prosopis juliflora (PJ), Rhizopus nigricans (RN), and wheat dust (WD), were taken as representatives of various groups of allergens viz, pollen, fungi and dust. The extracts were stored in buffer containing phenol (0.4%) or glycerol (50%) at temperatures ranging from 4-55 degrees C for 15 to 60 days. Protein content of PJ extract was reduced remarkably when it was stored at 40 degrees C for 45 days. Thin layer isoelectric focusing and rocket immunoelectrophoresis of PJ showed that certain antigenic proteins degrade rapidly even at 25 degrees C as early as day 15. However, two to three proteins of PJ remain stable at a higher temperature (40 degrees C) for two months. Relative radioallergosorbent test (RAST) inhibition showed substantial loss of allergenic activity in all the three extracts, when stored at higher temperatures (25-55 degrees C) even for short durations, i.e., 15 days. Extracts (PJ and RN) containing 50% glycerol were found to be stable, retaining more than 50% activity, even when stored at 55 degrees C for 40 days, while extracts without glycerol lost more than 75% of their allergenic activity. However, addition of glycerol did not change the stability of wheat dust allergenic extract. The present findings indicate that allergenic extracts behave differently when stored. Hence, the stability of each extract should be determined individually.

Development, characterization & potential clinical use of monoclonal antibodies against human alphafoetoprotein.

Four anti-alphafoetoprotein (AFP) monoclonal antibodies (MAb) were raised in the laboratory and characterized using ELISA and immunodot assays. The affinity constants of the MAbs, analysed by scatchard plots, ranged from 3.1 X 10(8) to 2.15 X 10(9) M/l. Epitope analysis using competition RIA indicated that MAb 5E2D7 and 5E2E3 recognize different epitopes on AFP. This combination was used to set up a two site sandwich ELISA with HRPO conjugated 5E2D7. AFP values in sera of hepatocellular carcinoma patients and pregnant women were quantitated using sandwich ELISA. The anti-AFP MAbs showed strong reactivity when tested on hepatoma tissue sections using immunoperoxidase technique.