Cytokine production in thromboangiitis obliterans patients: new evidence for an immune-mediated inflammatory disorder.

OBJECTIVE: To study IL-6, IL-10, IL-12 levels and circulating immune complexes (CIC) containing IgG, IgM or IgA in sera of 14 TAO patients and 12 healthy blood donors. To evaluates the ability of TAO PBMC to produce IL-6, IL-10, IL-12, as well as to detect PBMC apoptosis after stimulation with different stimuli. METHODS: In vitro stimulation of PBMC with lypopolysaccharide (LPS), phytohemagglutinin (PHA), C3 binding glycoprotein from uscuta europea (C3bgp), pokeweed mitogen (PWM), and dexamethasone (DM) were performed. The quantities of the secreted cytokines in sera and in culture supernatants, as well as CIC were detected by ELISA. The apoptosis was assessed according to nuclear morphology, after acridine orange staining, by fluorescence microscopy. RESULTS: Significantly higher IL-6 levels in the patients', than in the controls' sera was found. An increased production of IL-6 and IL-12 in TAO PBMC supernatants was detected, regardless of the stimuli used. A hyporeactivity of TAO PBMC toward IL-10 production was found after C3bgp, LPS, PHA and PWM stimulation, compared to the controls' PBMC. The spontaneous and induced apoptosis was significantly higher in TAO compared to the control group. Increased CIC quantities were detected in 75% of the patients tested. According to the CIC isotype, the IgG CIC positives (75%) prevailed over IgA CIC positives (50 %). CONCLUSION: The altered production of IL-6, IL-12 and IL-10, the increased apoptosis as well as the elevated levels of CIC could be a reason for the persisting immune inflammation in TAO.

Changes in serum levels of cytokines in mice injected with an immunostimulator C3bgp isolated from Cuscuta europea.

The effect of C3 binding glycoprotein (C3bgp), isolated from Cuscuta europea seeds on induction of in vivo cytokine synthesis was investigated. Different groups of mice were stimulated with 30 microg C3bgp per mouse, injected intraperitoneally. The quantitative determination of IL-1alpha, IL-2, IL-4, IL-6, IL-10, TNF-alpha and IFN-gamma was performed in mouse sera by ELISA. The quantities of these cytokines were measured at different hours: 1, 2, 3, 4, 5, 6, 7, and 24 h after injection. No significant changes in serum level of IL-2, IL-4 and TNF-alpha in experimental animal groups were found. A little increase of IL-1alpha, moderate elevation of IL-10 and IFN-gamma (5- to 6-fold more) and strong release, more than 10-fold of IL-6 in sera of C3bgp-treated mice were detected. The results obtained from C3bgp stimulated cultures of mouse peritoneal macrophages and mouse splenocytes suggest that C3bgp binds to mouse peritoneal macrophages and induces production mainly of IL-6, followed by IFN-gamma and in a very low degree of IL-1alpha and IL-10. Based on the results presented, we conclude that the increased level of IL-6 was the basic after injection of C3bgp and that the mouse macrophages were the major cell targets for the C3bgp effect.

Two glycine containing 2-chloroethylnitrosoureas--a comparative study on some physicochemical properties, in vivo antimelanomic effects and immunomodulatory properties.

Physicochemical properties such as alkylating and carbamoylating activity and in vivo antimelanomic effects against B16 melanoma of the spin labeled (nitroxyl free radical containing) glycine nitrosourea (SLCNUgly) and its nonlabeled analogue (ChCNUgly), synthesized in our laboratory are studied and compared to those of antitumour drug 3-cyclohexyl-1-(2-chloroethyl)-1-nitrosourea (CCNU). We have demonstrated that introducing of glycine moiety in the nitrosourea structure in practice does not affect either alkylating or carbamoylating activity. On the other hand replacement of cyclohexyl moiety in ChCNUgly structure with nitroxyl free radical leads to a decrease in carbamoylating activity and an increase in alkylating activity. Compound ChCNUgly showed in vivo a higher antimelanomic activity against B16 melanoma in comparison with CCNU and SLCNUgly. It completely inhibited B16 melanoma growth (TGI=100%) at a dose 64.0 mg/kg. Moreover, we established that joint i.p. application in normal mice of SLCNUgly plus a new immunostimulator (C3bgp) formerly isolated in our laboratory led to a 75% restoration in immune function with respect to antibody production measured by Jerne hemolytic plaque assay. In contrast, no immunostimulation was found after joint application of C3bgp plus ChCNUgly or CCNU at the same experimental conditions. Based on these preliminary results, a possibility for developing of new combination immunochemotherapy schemes for treatment of human cancers is discussed.

Preliminary studies on the immunomodulatory effect of the C3 binding glycoprotein isolated from Cuscuta europea.

This study investigates the immunomodulatory effect of a C3 binding glycoprotein (C3bgp), isolated from the parasitic plant Cuscuta europea. When BALB/c mice, immunized with sheep red blood cells (SRBC), were given a single intraperitoneal injection of C3bgp a dose-dependent immunostimulation was observed. The stimulation was assessed by an increase in the number of haemolytic plaque forming cells (PFC) and haemaglutination titres. The induction was time dependent in respect to the administration of both the C3bgp and SRBC. When C3bgp was applied 24 h before SRBC at a dose of 30 microg per mouse (1.2 mg/kg), a well expressed immunostimulation was found. It was also found that giving C3bgp to mice, which had previously been treated with the immunosuppressive drug cyclophosphamide (CY), produced an increase in PFC. The immune response was also restored in vitro experiments were performed using human whole blood cultures stimulated with 30 microg/ml C3bgp in the presence or absence of egg albumin (OVA) as antigen for 72 to 168 h. In C3bgp stimulated cultures it was found that after 120 h there was a high expression of the CD 19+ subset of the activation antigen CD25 (IL-2R) as assessed by flow cytometric phenotype analysis. Supernatants from cultures with different stimuli were assayed by a solid phase ELISA for the determination of OVA-specific IgM at 120, 144 and 168 h. It was found that C3bgp application alone, failed to enhance OVA specific IgM, but significantly high levels of IgM in cultures containing C3bgp and OVA, were detected. Overall it has been shown that the C3 binding glycoprotein, as obtained from the parasitic plant Cuscuta europea, has strong immunostimulatory properties both in vivo and in vitro.