Simon C. Watkins

The direct effects of pro-inflammatory cytokines on the contractility of mammalian heart were studied. Tumor necrosis factor alpha, interleukin-6, and interleukin-2 inhibited contractility of isolated hamster papillary muscles in a concentration-dependent, reversible manner. The nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) blocked these negative inotropic effects. L-Arginine reversed the inhibition by L-NMMA. Removal of the endocardial endothelium did not alter these responses. These findings demonstrate that the direct negative inotropic effect of cytokines is mediated through a myocardial nitric oxide synthase. The regulation of pro-inflammatory cytokines and myocardial nitric oxide synthase may provide new therapeutic strategies for the treatment of cardiac disease.

Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses to induce/maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs.

We examined the hypothesis that an excess accumulation of intramuscular lipid (IMCL) is associated with insulin resistance and that this may be mediated by the oxidative capacity of muscle. Nine sedentary lean (L) and 11 obese (O) subjects, 8 obese subjects with type 2 diabetes mellitus (D), and 9 lean, exercise-trained (T) subjects volunteered for this study. Insulin sensitivity (M) determined during a hyperinsulinemic (40 mU x m(-2)min(-1)) euglycemic clamp was greater (P < 0.01) in L and T, compared with O and D (9.45 +/- 0.59 and 10.26 +/- 0.78 vs. 5.51 +/- 0.61 and 1.15 +/- 0.83 mg x min(-1)kg fat free mass(-1), respectively). IMCL in percutaneous vastus lateralis biopsy specimens by quantitative image analysis of Oil Red O staining was approximately 2-fold higher in D than in L (3.04 +/- 0.39 vs. 1.40 +/- 0.28% area as lipid; P < 0.01). IMCL was also higher in T (2.36 +/- 0.37), compared with L (P < 0.01). The oxidative capacity of muscle determined with succinate dehydrogenase staining of muscle fibers was higher in T, compared with L, O, and D (50.0 +/- 4.4, 36.1 +/- 4.4, 29.7 +/- 3.8, and 33.4 +/- 4.7 optical density units, respectively; P < 0.01). IMCL was negatively associated with M (r = -0.57, P < 0.05) when endurance-trained subjects were excluded from the analysis, and this association was independent of body mass index. However, the relationship between IMCL and M was not significant when trained individuals were included. There was a positive association between the oxidative capacity and M among nondiabetics (r = 0.37, P < 0.05). In summary, skeletal muscle of trained endurance athletes is markedly insulin sensitive and has a high oxidative capacity, despite having an elevated lipid content. In conclusion, the capacity for lipid oxidation may be an important mediator of the association between excess muscle lipid accumulation and insulin resistance.

Exosomes are nanovesicles released by leukocytes and epithelial cells. Although their function remains enigmatic, exosomes are a source of antigen and transfer functional major histocompatibility complex (MHC)-I/peptide complexes to dendritic cells (DCs) for CD8(+) T-cell activation. Here we demonstrate that exosomes also are internalized and processed by immature DCs for presentation to CD4(+) T cells. Endocytosed exosomes are sorted into the endocytic compartment of DCs for processing, followed by loading of exosome-derived peptides in MHC-II molecules for presentation to CD4(+) T cells. Targeting of exosomes to DCs is mediated via milk fat globule (MFG)-E8/lactadherin, CD11a, CD54, phosphatidylserine, and the tetraspanins CD9 and CD81 on the exosome and alpha(v)/beta(3) integrin, and CD11a and CD54 on the DCs. Circulating exosomes are internalized by DCs and specialized phagocytes of the spleen and by hepatic Kupffer cells. Internalization of blood-borne allogeneic exosomes by splenic DCs does not affect DC maturation and is followed by loading of the exosome-derived allopeptide IEalpha(52-68) in IA(b) by host CD8alpha(+) DCs for presentation to CD4(+) T cells. These data imply that exosomes present in circulation or extracellular fluids constitute an alternative source of self- or allopeptides for DCs during maintenance of peripheral tolerance or initiation of the indirect pathway of allorecognition in transplantation.