Effect of synthetic lipopeptides formulated in liposomes on the maturation of human dendritic cells

Diacylated (e.g. MALP-2) and triacylated (Pam(3)Cys derivatives) lipopeptides, deriving from the N-terminal moiety of respectively mycoplasmal and E. coli lipoproteins, are powerful adjuvants recognized by Toll-like receptors (TLR) which have been used successfully to trigger cell activation and immune responses. To design liposome-based vaccination constructs in which Th and CTL epitopes are conjugated to synthetic lipopeptide analogues anchored into the bilayers of the vesicles, the peptide moieties of the lipopeptides were functionalized with thiol-reactive groups, such as maleimide (Mal) or bromoacetyl, incorporated into liposomes and reacted with thiol carrying peptide epitopes. Because dendritic cells (DCs) play a key role as antigen-presenting cells in immune responses, in the present study we have evaluated the impact of the functionalization of lipopeptide analogues Pam(2)CAG, Pam(3)CAG and Ol(3)GAG on the phenotypic maturation of human monocyte-derived DCs. The intrinsic cellular activities of the lipopeptide analogues incorporated into liposomes were monitored, in vitro, by measuring the up-regulation of the cell-surface markers CD80, CD83, CD86 and HLA-DR. We found that in some cases their functionalization with thiol-reactive groups led to a loss of activity. The stimulatory potency can be ranked in the following order: Pam(3)CAG>/=Pam(2)CAG-Mal-Th approximately Pam(2)CAG-Mal>Pam(3)CAG-Mal-Th (where Th is a HS-peptide) and no appreciable activity was detected for Pam(3)CAG-Mal, Ol(3)CAG-Mal and Ol(3)CAG-Mal-Th. Our findings indicate that subtle modifications in the peptide moiety of lipopeptides have a great impact on the immunomodulatory properties of these molecules. For the engineering of liposome/lipopeptide-based vaccines, the maleimide derivative of Pam(2)CAG appears to be the best candidate.     

Immunoelectron microscopic investigation of surface coat of Wilms tumor cells. Dense lamina is composed of highly sialylated neural cell adhesion molecule

In previous studies we demonstrated the presence of polysialic acid in Wilms tumor by immunohistochemistry and immunoblotting. We now show by immunoelectron microscopy that the cell surface coat of Wilms tumor cells consists of a layer of amorphous material containing polysialic acid but not detectable amounts of laminin, laminin-nidogen complex, or low density proteoglycan. Therefore, Wilms tumor cells are covered by a highly developed and chemically specialized cell surface coat that does not represent a basement membrane, although it bears some structural similarities. Polysialic acid is present on neural cell adhesion molecule that exists in Wilms tumor as two isoforms of approximately 120 and 140 kDa. The cell surface coat exhibits variation in its thickness along the plasma membrane of a single tumor cell, and the variation is inversely related to the extent of cell-cell contact. It is therefore proposed that polysialic acid may modulate the behavior and invasive potential of Wilms tumor cells.     

Interpretation of skeletal muscle four-electrode impedance measurements using spatial and temporal frequency-dependent conductivities

Spatial and temporal frequency-dependent conductivities are used to interpret four-electrode conductivity measurements on skeletal muscle. The model qualitatively explains the observed dependence of the experimental data on the temporal frequency of the injected current, the angle between the electrode array and the fibre direction and the distance between the electrodes.     

The effect of tyrphostin AG-556 on intimal thickening in a mouse model of arterial injury

BACKGROUND: Inflammation has been shown to play an important role in promoting the response to arterial injury and proinflammatory cytokines, such as tumor necrosis factor (TNF) alpha, are candidate mediators. AG-556 is a tyrosine kinase inhibitor proven to be effective in a model of multiple sclerosis-like syndrome in mice due to its immunomodulating effect. In the current study, we investigated the effect of the tyrphostin AG-556 on neointimal thickening and cytokine profile in a model of arterial injury in the mouse. METHODS: Injury was induced by external cuff placement on the left femoral artery of wild-type C57BL/6 mice. AG-556 dissolved in DMSO was injected intraperitoneally daily to the injured mice in a dosage of 2 mg/mouse. Control mice received DMSO injections. Histological analysis was carried out to assess neointimal formation. Splenocytes were cultured in the absence and presence of a mitogen for evaluation of thymidine incorporation and cytokine production. RESULTS: AG-556 treatment significantly attenuated intimal thickening (43,000+/-17,000 microm2; n=11) when compared to DMSO administration (286,000+/-127,000 microm2; n=10; P<0.05). Basal interferon-gamma production by splenocytes from AG-556-treated mice was increased by approximately 20-fold in comparison with levels in DMSO-treated animals, whereas Con-A induced secretion of the cytokine was similar between both groups. Levels of TNF-alpha, IL-4 and IL-10 in the culture supernatant from treated and non-treated animals did not differ significantly. CONCLUSION: The tyrosine kinase inhibitor AG-556 may have a role in the reduction of intimal thickening. The effect could be mediated via an immune modulating effect involving a significant increase in the smooth muscle cell inhibitory cytokine IFN-gamma.     

Protection of early phase hepatic ischemia-reperfusion injury by cholinergic agonists

Background  

Cytokine production is critical in ischemia/reperfusion (IR) injury. Acetylcholine binds to macrophages and inhibits cytokine synthesis, through the cholinergic anti-inflammatory pathway. This study examined the role of the cholinergic pathway in cytokine production and hepatic IR- injury.     

No evidence for transmission disequilibrium between a new marker at the myelin basic protein locus and multiple sclerosis in French patients

The myelin basic protein (MBP) gene is a candidate locus for susceptibility to multiple sclerosis. Several groups have tested a complex (TGGA)n repeat in the 5' region of this gene for association/linkage with multiple sclerosis, with divergent results. This region of tandem repetitive sequence has been subjected to complex rearrangements, and there is a possibility that alleles of the same size have different internal structures, which reduces the interest of this marker for linkage disequilibrium studies and may at least partly explain the conflicting results obtained so far. To overcome this problem, we isolated a new polymorphic (CA)n repeat within the Golli-MBP locus. The limited number of alleles identified makes this other marker suitable for transmission disequilibrium studies. We tested this marker for linkage with multiple sclerosis, using the transmission-disequilibrium test (TDT) on a sample of 196 nuclear families in which the genotypes of both parents could be unambiguously defined. We found no evidence of transmission disequilibrium between multiple sclerosis and any of the three alleles of this marker, even when the patients were subdivided according to their HLA-DRB1*1501 status. The present data thus provide no evidence for a contribution of the MBP gene to multiple sclerosis susceptibility in French patients.     

The possible role of protein degradation in altered membrane structure and function

Possible changes in membrane lipid assemetry may result in altered function with aging. Membrane proteolysis is an additional factor which must be considered, both with respect to modulation of membrane function and also as a methodological problem in analyses of membrane dynamics.     

Pneumotoxicity and thrombocytopenia after single injection of monocrotaline

Adult Sprague-Dawley rats were treated once with 105 mg/kg monocrotaline (MCT) subcutaneously or an equivalent volume of isotonic saline and examined 2, 5, 10, and 14 days later. The earliest changes observed were in the platelet count, which was decreased in the MCT animals at 2, 5, and 10 days postinjection. Clearance of perfused 5-hydroxytryptamine, a function of pulmonary vascular endothelium, was unaltered in isolated lungs of treated rats until 5 days after dosing but decreased progressively thereafter in the MCT animals and was 24% less than controls by 14 days. The magnitude of this effect was dose related. Inflow perfusion pressure was elevated in perfused lungs of MCT-treated animals at day 14. Right heart hypertrophy, measured as an increase in the ratio of right ventricle to left ventricle plus septum weights, was not evident until 14 days after treatment. A larger dose of MCT (130 mg/kg) resulted in significant mortality, whereas a lower dose (60 mg/kg) did not result in right ventricular hypertrophy 2 wk after treatment. The treatment regimen described has advantages over administration of MCT by ingestion and may prove suitable for investigations of the mechanism by which MCT results in pulmonary hypertension.