Nitrosative stress parameters and the level of oxidized DNA bases in patients with multiple sclerosis

Multiple sclerosis (MS) is a neurodegenerative disease with various factors affecting its etiology. Overproduction of nitric oxide and subsequent lesions of biopolymers are some of the possible causes of the disease. This study aimed to measure the most relevant nitrosative and oxidative stress biomarkers and the level of modified DNA bases in patients with MS. Each parameter was assayed in 25 patients with MS and 25 healthy controls. This study involved detecting blood plasma and serum nitric oxide metabolites by chemiluminescence detector Sievers NOA-280i, malondialdehyde (MDA) measurements with thiobarbituric acid reactive substance (TBARS) assay, detection of oxidized purines and pyrimidines with the enzyme-modified comet assay. Statistical analysis of the results was performed by one-way analysis of variance (ANOVA) and unpaired t test for the comparison of less than three data sets. DNA single-strand breaks, levels of modified purines and pyrimidines, as well as nitrite and nitrate levels in plasma and serum samples, were significantly higher in patients with MS than in healthy controls. On the contrary, MDA levels appeared to be lower in patients with MS.

     

CTLA-4 controls follicular helper T-cell differentiation by regulating the strength of CD28 engagement

Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) is an essential regulator of T-cell responses, and its absence precipitates lethal T-cell hyperactivity. However, whether CTLA-4 acts simply to veto the activation of certain clones or plays a more nuanced role in shaping the quality of T-cell responses is not clear. Here we report that T cells in CTLA-4–deficient mice show spontaneous T-follicular helper (T_FH) differentiation in vivo, and this is accompanied by the appearance of large germinal centers (GCs). Remarkably, short-term blockade with anti–CTLA-4 antibody in wild-type mice is sufficient to elicit T_FH generation and GC development. The latter occurs in a CD28-dependent manner, consistent with the known role of CTLA-4 in regulating the CD28 pathway. CTLA-4 can act by down-regulating CD80 and CD86 on antigen presenting cells (APCs), thereby altering the level of CD28 engagement. To mimic reduced CD28 ligation, we used mice heterozygous for CD28, revealing that the magnitude of CD28 engagement is tightly linked to the propensity for T_FH differentiation. In contrast, other parameters of T-cell activation, including CD62L down-regulation and Ki67 expression, were relatively insensitive to altered CD28 level. Altered T_FH generation as a result of graded reduction in CD28 was associated with decreased numbers of GC B cells and a reduction in overall GC size. These data support a model in which CTLA-4 control of immunity goes beyond vetoing T-cell priming and encompasses the regulation of T_FH differentiation by graded control of CD28 engagement.Control of the magnitude and nature of adaptive immune responses is critical for health. The cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4)/CD28 axis has long been known to control the magnitude of T-cell responses, however whether it also influences their nature has not been clear. Early studies suggested that CD28 may be particularly important for Th2 differentiation (1, 2), although others identified roles for CD28 in both Th1 and Th2 responses (3, 4). It is known that CD28 is an absolute requirement for the differentiation of follicular helper T cells (T_FHs) that support germinal center (GC) formation (5, 6). However, these studies generally make use of CD28-deficient T cells, and therefore, results may reflect a failure of the cells to properly activate, proliferate, or survive, particularly given the known contribution of CD28 to these processes.A key outstanding question is whether CD28 costimulation in vivo is more complex than a binary checkpoint for T-cell priming. It is clear that expression of costimulatory ligands on antigen presenting cells (APCs) fluctuates in response to environmental stimuli, being up-regulated by inflammatory cytokines and TLR agonists and down-regulated by Treg-expressed CTLA-4 (7–11). Thus, variable levels of costimulatory ligands will be available for CD28 binding depending on the microenvironmental context. However, whether this simply alters the number of T cells that achieve the required threshold to commit to a response or whether it influences the nature of the response is not clear. Effective immune homeostasis appears to be reliant on maintaining an appropriate level of CD28 engagement. For example, basal expression of CD28 ligands, in particular dendritic cell-expressed CD86 (12), is critical to Treg homeostasis (13, 14), whereas excessive CD28 engagement in the absence of CTLA-4 results in lethal autoimmunity (15, 16). Nonetheless, distinguishing whether these effects are simply quantitative is not straightforward. Thus, although levels of CD28 ligands are clearly variable in vivo, our understanding of the impact of altering the level of CD28 engagement is still incomplete.To explore the impact of varying levels of CD28 ligation, we have used the CTLA-4–deficient mouse as a model of excessive CD28 stimulation. In these mice, we observed a striking skewing toward T_FH differentiation, with induction of IL-21 and spontaneous formation of GCs. In a complementary approach, we used CD28 heterozygosity to decrease T cell CD28 expression: This revealed that the level of CD28 engagement is tightly coupled to the level of inducible T-cell costimulator (ICOS) induction, T_FH generation, and GC formation, whereas other parameters of T-cell activation were less affected. Finally, we demonstrate that induction of the microRNA cluster miR17-92, recently linked with T_FH differentiation (17, 18), varies proportionally with APC costimulatory ligand expression and is modulated by CTLA-4 deficiency or blockade. Collectively, these data suggest that the CTLA-4/CD28 axis provides quantitative and qualitative control of T-cell help for humoral immunity.     

A new way to use transit-time flow measurement for coronary artery bypass grafting

 Open in a separate windowOBJECTIVESTransit-time flow measurement is a recognized method for graft evaluation in coronary surgery. However, single flow measurement has been associated with a low specificity for detecting graft dysfunction. The goal of this study was to assess the value of transit-time flow measurement for assessing in situ internal mammary artery grafts during non-existent native coronary circulation and the relevance of collateral blood flow in target vessels.METHODSBetween 2014 and 2018, a total of 134 patients undergoing on-pump coronary artery bypass grafting were evaluated using transit-time flow measurement. We analysed 111 single left internal mammary artery and 57 single right internal mammary artery bypasses. Correlations between coronary relevant parameters were calculated using Spearman’s ρ coefficient. Risk factors for decreased flow with an arrested heart (FAH) <30 ml/min and an increased pulsatility index (PI) >3.0 as well as flow reduction >30% were calculated.RESULTSFAH correlated with the diameter of the target vessel (Spearman’s ρ = 0.32; P < 0.001), the amount of blood distribution (Spearman’s ρ = 0.34; P < 0.001), the PI (Spearman’s ρ = 0.19; P = 0.019) and the degree of stenosis (Spearman’s ρ = −0.17; P = 0.042). The percentage of flow change was found to correlate with the PI (Spearman’s ρ = −0.47; P < 0.0001), the degree of stenosis (Spearman’s ρ = 0.42; P < 0.001), the diameter of the target vessel (Spearman’s ρ = −0.22; P = 0.008) and the area of blood distribution (Spearman’s ρ = −0.19; P = 0.018). A small blood distribution area was the only risk factor for decreased FAH [odds ratio (OR) 8.43, confidence interval (CI) 95% (3.04–23.41); P < 0.001]. Binary logistic regression identified PI [OR 2.05, CI 95% (1.36–3.10); P = 0.001], FAH [OR 0.98, CI 95% (0.97–0.99); P = 0.005] and degree of stenosis [OR 0.95, CI 95% (0.92–0.99); P = 0.011] as risk factors for decreased flow after cardiopulmonary bypass (<30 ml/min). An increased PI (>3) was mainly influenced by percentage of flow change [OR 0.99, CI 95% (0.98–1.00); P = 0.031].CONCLUSIONSFAH and percentage of flow change are related to the dimensions of the target vessel and the degree of stenosis. The addition of flow measurements with the heart arrested provides additional information about the bypass graft, the quality of the anastomosis and the physiology of the coronary circulation.