Accelerated membrane-assisted clean-up as a tool for the clean-up of extracts from biological tissues.

Accelerated membrane-assisted clean-up (AMAC) is a new technique to purify lipid-rich extracts of biota samples. It combines a separation of organic analytes and lipid-like matrix components using a semi-permeable membrane with the technical options provided by a Dionex ASE device which was developed for pressurized liquid extraction (PLE; Dionex trade name ASE for accelerated solvent extraction). The ASE device automates the procedure. It offers the possibility to increase the permeation of the analytes through the membrane via application of higher temperatures and automatic solvent exchange to ensure steep gradients across the membrane. The optimal operating parameters of AMAC were determined in this study. Satisfactory recoveries of over 70% for most target compounds, as well as excellent removal of lipid-like matrix components were achieved using 80 microm thick low-density polyethylene (LD-PE) membranes, at a temperature and pressure of 40 degrees C and 3.45 MPa, respectively, over 16 static cycles lasting 10 min each. The recoveries obtained were similar to those achieved using gel permeation chromatography (GPC), however AMAC showed a higher matrix removal capacity. AMAC removed up to 4.5 g of lipids in a single run, which was more than 98% of the original matrix content. Additionally, a 13-fold lower solvent consumption, compared with GPC, was achieved. AMAC separates analytes and matrix compounds mainly by size, making this method a beneficial tool for non-target or effect analysis. AMAC is especially applicable for processing large amounts of sample as a result of the high matrix removal capacity.

Cyclosporin A enhances elimination of intracellular L. major parasites by murine macrophages.

In this study we analyzed the influence of cyclosporin A (CyA) on the process of phagocytosis of L. major promastigotes and amastigotes by inflammatory peritoneal macrophages (MP) from BALB/c mice. Our data clearly demonstrate that CyA profoundly enhanced the degradation by peritoneal MP of both intracellular L. major promastigotes and amastigotes. This effect was T cell-independent and specifically associated with CyA, since the similarly structured cyclosporin F (CyF) was ineffective. CyA did not alter the replication and infectivity of extracellular parasites. From these results we conclude that the inhibition of intracellular parasite replication in the presence of CyA substantially contributes to the previously described suppressive effect of CyA on the development of L. major-induced lesions in BALB/c mice.

Studies on the release of cell-associated interleukin 1 by paraformaldehyde-treated murine macrophages.

Experiments are presented demonstrating the release of interleukin 1 (IL1) by lipopolysaccharide-stimulated murine macrophages (M phi) after treatment of the cells with paraformaldehyde (PFA). The IL1 activity set free was completely blocked by a rabbit anti-mouse IL1 alpha antiserum. Immunoprecipitation studies revealed that the IL1 released was of Mr 33 kDa, 23 kDa and 17 kDa. Incubation of PFA-treated M phi in the presence of leupeptin reduced the amount of detectable low molecular mass (i.e. 23 kDa and 17 kDa) IL1 alpha. Addition of leupeptin to PFA-treated M phi also reduced the amount of released IL1 activity, suggesting that proteolytic modification of the IL1 alpha precursor is involved in the generation of biological active IL1 alpha. It is proposed that the cell-associated 33-kDa IL1 is released from the M phi by vesiculation of parts of the M phi cell membrane, followed by a proteolytic cleavage step.

Kinetics of cell-mediated immunity developing during the course of Leishmania major infection in 'healer' and 'non-healer' mice: progressive impairment of response to and generation of interleukin-2.

Leishmania major (L. major)-infected mice of 'non-healer' (BALB/c) and 'healer' (C57BL/6) mouse-strain origin were studied with regard to the kinetics of cell-mediated immunity developing during the course of the disease. Cells obtained from lymph nodes draining L. major-infected footpads were comparatively analysed for their representation in the respective L3T4+, Lyt-2+ and sIg+ lymphocyte subsets; they were studied for their capacity to release interleukin-2 and to proliferate in response to L. major antigen and concanavalin A, including the determination of the frequencies of T cells proliferating antigen-specifically with or without an exogenous source of IL-2. The data obtained indicate L. major infection-induced long-lasting alterations in the cellular composition of the lymph node in both 'healer' and 'non-healer' mice. Moreover, they suggest that the inability of 'non-healer' mice to recover from L. major infection is associated with a progressive impairment of their lymph node T cells to release interleukin-2 in the culture supernatant and to respond to this lymphokine in vitro.

Murine cutaneous leishmaniasis: comparative study on the capacity of macrophages from "healer" and "non-healer" mouse strains to control L. tropica replication.

A comparison has been made between the capacity of macrophages of BALB/c "non-healer" mice and C57BL/6 "healer" mice to deal in vitro with Leishmania tropica (L. tropica) parasites in order to obtain a more detailed picture of the inherent contribution of macrophages to the susceptibility of BALB/c mice to infection with L. tropica and the resulting fatal course of the disease. In comparison to macrophages of C57BL/6 origin, BALB/c macrophages showed a higher parasite uptake and a higher infection rate; they allowed a more rapid transformation of L. tropica promastigotes into amastigotes and displayed less leishmanicidal activities. Lymphokine-rich culture supernatants induced activation of macrophages resulting in killing of L. tropica by macrophages of both, "non-healer" and "healer" mice. These supernatants also induced expression of Ia-antigens on infected "non-healer" and "healer" macrophages. The results of this study clearly point to the critical role of macrophage functions to either support a systemic leishmaniasis or to alternatively mount a protective immune response leading to a self-healing course of the disease.

Selection of bacterial mutants from Salmonella specifically recognizing determinants on the cell surface of activated T lymphocytes. A novel system to define cell surface structures.

The isolation of bacterial mutants from Salmonella is described with specific binding capacities to allogeneically or concanavalin A-activated T lymphocytes. The enrichment of these mutants was achieved by separation of T cell blasts with adhering mutants from nonresponsive lymphocytes and nonadhering bacteria through 1 x g sedimentation. Binding of the mutants was specific for T cells early after antigen or mitogen stimulation. No adherence was observed with unstimulated T or B lymphocytes and with B cell blasts. Further results suggested that the binding of Salmonella mutants was mediated by the heteropolysaccharide moiety of lipopolysaccharides with a specificity for protein receptor sites on activated T lymphocytes. Significantly, these heteropolysaccharides also inhibited the differentiation of prekiller to killer cells from allogeneic mixed lymphocyte cultures but did not depress the proliferative response or the activity of cytotoxic effector cells. Bacterial adherence, as well as polysaccharide activity in functional tests, showed strain specificity since reactivity could only be obtained with activated T cells from AKR, C57BL/6, C3H but not with BALB/c and A/J strain mice. It is discussed whether the heteropolysaccharides mimic the structure of naturally occurring molecules and thus compete for their receptor sites. Selection of bacterial mutants with adherence properties may become a general procedure for detecting cell surface molecules on lymphoid and nonlymphoid cells.