Evidence for an Independent and Cumulative Effect of Postprandial Hypertriglyceridemia and Hyperglycemia on Endothelial Dysfunction and Oxidative Stress Generation

Background— Postprandial hypertriglyceridemia and hyperglycemia are considered risk factors for cardiovascular disease. Evidence suggests that postprandial hypertriglyceridemia and hyperglycemia induce endothelial dysfunction through oxidative stress; however, the distinct role of these two factors is a matter of debate. Methods and Results— Thirty type 2 diabetic patients and 20 normal subjects ate 3 different meals: a high-fat meal; 75 g glucose alone; and high-fat meal plus glucose. Glycemia, triglyceridemia, nitrotyrosine, and endothelial function were assayed during the tests. Subsequently, diabetics took 40 mg/d simvastatin or placebo for 12 weeks. The 3 tests were performed again at baseline, between 3 to 6 days after the start, and at the end of each study. High-fat load and glucose alone produced a decrease of endothelial function and an increase of nitrotyrosine in normal and diabetic subjects. These effects were more pronounced when high fat and glucose were combined. Short-term simvastatin treatment had no effect on lipid parameters but reduced the effect on endothelial function and nitrotyrosine observed during each different test. Long-term simvastatin treatment was accompanied by a lower increase in postprandial triglycerides, which was followed by smaller variations of endothelial function and nitrotyrosine during the tests. Conclusions— This study shows an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial function, suggesting oxidative stress as common mediator of such effect. Simvastatin shows a beneficial effect on oxidative stress and endothelial dysfunction, which may be ascribed to a direct effect as well as the lipid-lowering action of the drug

Costimulatory Effects of an Immunodominant Parasite Antigen Paradoxically Prevent Induction of Optimal CD8 T Cell Protective Immunity

<div><p><i>Trypanosoma cruzi</i> infection is controlled but not eliminated by host immunity. The <i>T</i>. <i>cruzi</i> trans-sialidase (TS) gene superfamily encodes immunodominant protective antigens, but expression of altered peptide ligands by different TS genes has been hypothesized to promote immunoevasion. We molecularly defined TS epitopes to determine their importance for protection versus parasite persistence. Peptide-pulsed dendritic cell vaccination experiments demonstrated that one pair of immunodominant CD4⁺ and CD8⁺ TS peptides alone can induce protective immunity (100% survival post-lethal parasite challenge). TS DNA vaccines have been shown by us (and others) to protect BALB/c mice against <i>T</i>. <i>cruzi</i> challenge. We generated a new TS vaccine in which the immunodominant TS CD8⁺ epitope MHC anchoring positions were mutated, rendering the mutant TS vaccine incapable of inducing immunity to the immunodominant CD8 epitope. Immunization of mice with wild type (WT) and mutant TS vaccines demonstrated that vaccines encoding enzymatically active protein and the immunodominant CD8⁺ T cell epitope enhance subdominant pathogen-specific CD8⁺ T cell responses. More specifically, CD8⁺ T cells from WT TS DNA vaccinated mice were responsive to 14 predicted CD8⁺ TS epitopes, while T cells from mutant TS DNA vaccinated mice were responsive to just one of these 14 predicted TS epitopes. Molecular and structural biology studies revealed that this novel costimulatory mechanism involves CD45 signaling triggered by enzymatically active TS. This enhancing effect on subdominant T cells negatively regulates protective immunity. Using peptide-pulsed DC vaccination experiments, we have shown that vaccines inducing both immunodominant and subdominant epitope responses were significantly less protective than vaccines inducing only immunodominant-specific responses. These results have important implications for <i>T</i>. <i>cruzi</i> vaccine development. Of broader significance, we demonstrate that increasing breadth of T cell epitope responses induced by vaccination is not always advantageous for host immunity.</p></div

CD4⁺ TSaa57-74 (p7)/IA^d- and CD8⁺ TSaa359-367 (TSKd1)/K^d-specific T cell responses are minimally sufficient for induction of protective <i>T</i>. <i>cruzi</i> immunity.

<p>Panel A shows a schematic of the TS consensus protein (all 12–15 active TS subfamily members have at least 90% homology within their catalytic domains), and immunodominant TS CD4 and CD8 epitopes. In panels B and C, BALB/c mice were vaccinated with dendritic cells (DC) pulsed (or not) with this pair of CD4 and CD8 epitopes and later challenged with <i>T</i>. <i>cruzi</i>. BALB/c CD11c⁺ splenic DC were purified from BALB/c mice 2 weeks after injection of 5x10⁶ B16-Flt3L-producing cells and 1 day after i.v. injection of 1μg LPS. 1x10⁶ DC pulsed (or left unpulsed) with 50 μg/ml of the indicated peptides were injected i.v. into groups of naïve BALB/c mice 3 times, 2 weeks apart. Mice were challenged 1 month later with <i>T</i>. <i>cruzi</i> (N = 5 in each of the two control groups and N = 10 in DC+TS peptide group). Both parasitemia (B; 2 weeks post-infection) and mortality (C) were significantly reduced in mice given DC pulsed with both TSaa57-74 (p7) and TSaa359-367 (TSKd1) [*p<0.001 by Mann-Whitney U test (B) and *p<0.01 by 2-tailed Fisher exact and Log-Rank [Mantel-Cox] tests(C)]. Survival results are representative of 3 independent experiments.</p

The WT DNA vaccine induces CD8⁺ T cell responses directed against both immunodominant and subdominant T cell epitopes.

<p>BALB/c mice were vaccinated i.m. twice, two weeks apart with 100μg of WT TS DNA or TSKd1 null TS DNA. Four weeks later, CD8⁺ splenic T cells from these vaccinated mice were stimulated overnight in IFN-γ ELISPOT assays with APC (A20 cells) pulsed with TS peptides predicted to bind BALB/c MHC [H2-K^d (A), H2- D^d (B) and H2-L^d(C). Results are representative of 3 experiments. As expected, vaccination of mice with WT but not TSKd1 null TS DNA constructs elicited T cell responses to TSKd1. CD8⁺ T cells from WT TS DNA vaccinated mice also responded to TS peptides Kd2, Kd5, Kd6, Kd7, Kd8, Kd9, Dd2, Dd6, Ld1 and Ld2, while cells from TSKd1 null vaccinate mice responded to only TS Kd8. Mutation of the 2 binding residues of TSKd1 to H2-Kd1 resulted in a marked immunofocusing of CD8⁺ T cell responses. Panel D further shows that WT TS DNA vaccination induced higher levels of TS-specific antibody, compared with TSKd1 null DNA vaccination.</p