Lipid transfer inhibitor protein (apolipoprotein F) concentration in normolipidemic and hyperlipidemic subjects.

Lipid transfer inhibitor protein (LTIP) is an important regulator of cholesteryl ester transfer protein function. We report the development of an immunoassay for LTIP and its use to quantify LTIP in plasma of varying lipid contents. A rabbit antibody against bacterially produced recombinant LTIP detected two LTIP isoforms in plasma differing in carbohydrate content. This antibody was used in a competitive, enzyme-linked immunoassay that uses partially purified LTIP bound to microtiter plates. To optimize LTIP immunoreactivity, plasma samples required preincubation in 1% Tween-20 and 0.5% Nonidet P-40. In normolipidemic plasma, LTIP averaged 83.5 microg/ml. LTIP was 31% higher in males than in females. LTIP was positively associated with HDL cholesterol in normolipidemic males but not in females. In hypertriglyceridemic males, LTIP was only 56% of control values, whereas in hypertriglyceridemic females, LTIP tended to increase. Additionally, in males with normal cholesterol and triglyceride (TG) < or = 200 mg/dl, LTIP varied inversely with plasma TG. Overall, we have confirmed the negative association between plasma TG levels and LTIP previously suggested by a small data set, but now we demonstrate that this effect is seen only in males. The mechanisms underlying this gender-specific response to TG, and why LTIP and HDL levels correlate in males but not in females, remain to be determined.

Inhibition of inducible Nitric Oxide Synthase by a mustard gas analog in murine macrophages.

BACKGROUND: 2-Chloroethyl ethyl sulphide (CEES) is a sulphur vesicating agent and an analogue of the chemical warfare agent 2,2'-dichlorodiethyl sulphide, or sulphur mustard gas (HD). Both CEES and HD are alkylating agents that influence cellular thiols and are highly toxic. In a previous publication, we reported that lipopolysaccharide (LPS) enhances the cytotoxicity of CEES in murine RAW264.7 macrophages. In the present investigation, we studied the influence of CEES on nitric oxide (NO) production in LPS stimulated RAW264.7 cells since NO signalling affects inflammation, cell death, and wound healing. Murine macrophages stimulated with LPS produce NO almost exclusively via inducible nitric oxide synthase (iNOS) activity. We suggest that the influence of CEES or HD on the cellular production of NO could play an important role in the pathophysiological responses of tissues to these toxicants. In particular, it is known that macrophage generated NO synthesised by iNOS plays a critical role in wound healing. RESULTS: We initially confirmed that in LPS stimulated RAW264.7 macrophages NO is exclusively generated by the iNOS form of nitric oxide synthase. CEES treatment inhibited the synthesis of NO (after 24 hours) in viable LPS-stimulated RAW264.7 macrophages as measured by either nitrite secretion into the culture medium or the intracellular conversion of 4,5-diaminofluorescein diacetate (DAF-2DA) or dichlorofluorescin diacetate (DCFH-DA). Western blots showed that CEES transiently decreased the expression of iNOS protein; however, treatment of active iNOS with CEES in vitro did not inhibit its enzymatic activity CONCLUSION: CEES inhibits NO production in LPS stimulated macrophages by decreasing iNOS protein expression. Decreased iNOS expression is likely the result of CEES induced alteration in the nuclear factor kappa B (NF-kappaB) signalling pathway. Since NO can act as an antioxidant, the CEES induced down-regulation of iNOS in LPS-stimulated macrophages could elevate oxidative stress. Since macrophage generated NO is known to play a key role in cutaneous wound healing, it is possible that this work has physiological relevance with respect to the healing of HD induced skin blisters.

Ricin at Subpicomole Concentrations Induce Cytolytic Activity and Stimulate Lymphokine Secretion by Human Peripheral Blood Mononuclear Cells.

Human PBMCs were stimulated by ricin at very low concentrations (10(-14) M and lower) for 48 h at 37 degrees C. Cytolytic activity to K562 cell line and lymphokine secretion were been measured. PBMCs possessed the highest cytotoxicity (63 +/- 2.8% and 64 +/- 3.1%, respectively) to K562 cell line in the presence of 10(-15) M or 10(-16) M ricin. The high secretion of IL-1beta (2000 pg/ml), IL-2 (1300 pg/ml) and TNF-alpha (2200 pg/ml) was detected in the presence of 10(-15) M ricin. The mechanisms of the target cell death induced by PBMCs after co-incubation with various lectins: 10(-8) M phytohemagglutinin, 10(-8) M concanavalin A and ricin at 10(-15) M or 10(-16) M concentrations were compared. Internucleosomal DNA fragmentation characteristic of apoptotic mechanisms of the target cell death in all cases were observed, and the maximum of DNA fragmentation was registered in the presence of 10(-15) M ricin.

Purification and Partial Characterization of a 14 kDa Protein Enhancing Mitogen-Stimulated Proliferation of Human Peripheral Blood Mononuclear Cells.

Cytotoxicity of plasma free platelets or peripheral blood mononuclear cells (PBMCs) upon stimulation with Ca-ionophore A23187 at various (10(-9) - 10(-6) M) concentrations has been studied on human erythroid leukaemia cell line (K562). A 14 kDa protein, which did not display any cytotoxic activity to K562 cells, but stimulate PBMC proliferation has been isolated from the supernatant of plasma free platelets stimulated by 10(-7) M Ca-ionophore. The protein displayed a very slight mitogenic effect on PBMCs, but it enhanced proliferative response of PBMC stimulated by concanavalin A at suboptimal concentrations. The p14 N-terminal sequence ((1)VLERTXA(7)-) is identical to the region 99-103 residues of the human MHC class II antigen DQ-beta chain. Also, we identified this 14 kDa protein in the supernatant of PBMCs stimulated by 10(-7) M Ca-ionophore. Its N-terminal sequence (VLERTXA-) is identical to the one of p14 from the stimulated plasma-free platelet supernatant.

Stimulation of Human Platelet Cytotoxicity by Various Activators in the Absence of Antibodies: Release of Thromboxane A2 and Soluble Cytotoxic Factors. Purification and Partial Characterisation of 14 and 28 kDa Cytotoxic Proteins.

The cytolytic activity of human plasma free platelets treated with various stimulators (Ca-ionophore, PAF (platelet activating factor), PHA, and ricin) has been studied on human erythroid leukemia cell line (K562). The 14 kDa and 28 kDa platelet cytotoxic factors (PCF) have been purified from the supernatant obtained from platelets stimulated by 10(-7) M Ca-ionophore. The PCF-14 N-terminal sequence ((1)YAPQXQFGP(9)-) appeared to be homologous to the region 241-249 residues of the human C1s complement component. The PCF-28 N-terminal sequence (DVGLT-) did not display any homology to known human proteins. The PCF-14 and the PCF-28 have been detected both in the supernatants of platelets treated with various stimulators (10(-7) M Ca-ionophore A23187, or 10(-8) M PAF, or 10(-9) M PHA, or 10(-13) M ricin) and in the extracts of disrupted platelets treated by these stimulators. All of these stimulators enhanced the production of thromboxane A(2) by platelets, as evidenced from the accumulation of thromboxane B(2), a spontaneous hydrolysis product of thromboxane A(2). These results indicate that platelet cytotoxicity to the K562 cells and the release of PCF-14 and PCF-28 might be mediated by the stimulation of thromboxane A(2) synthesis, when platelets have been activated through the cycloxygenase pathway.