Detection of the Tim-3 ligand, galectin-9, inside the allograft during a rejection episode

Introduction: Tim-3 is a Th1 lymphocytes membrane protein with inhibitory function. Its ligand, galectin-9, was recently identified and it is expressed in some lymphocyte subpopulation. In addition, endothelial cells and fibroblasts can also express galectin-9 according to the local cytokine milieu. Both molecules can act as important regulatory tools in the immune system. Aim: Evaluate the expression of these immunoregulatory molecules inside kidney allografts during acute rejection episodes. Methods: By using a quantitative polymerase chain reaction assay, we measured the levels of messenger RNA (mRNA) for galectin-9 and Tim-3 in 21 samples obtained at allograft nephrectomy. Five samples received the histological diagnosis of acute non-vascular rejection (ANVR), twelve of acute vascular rejection (AVR), and five of loss of non-immune cause (LNIC; as control). As cytolytic response markers we measured mRNA levels of granzyme B, interferon-gamma and perforin. The statistic analysis was performed using one way analysis of variance (ANOVA) and Pearson correlation. Results: The mean levels of Tim-3 mRNA expression were 13.99 +/- 6.99 for LNIC, 48.13 +/- 54.47 for RACNV and 238.63 +/- 333.14 for RAV (p = 0.004). For galectin-9, the mean values were 0.57 +/- 0.49 for LNIC, 0.66 +/- 0.36 for RACNV and 2.34 +/- 1.62 for RAV (p = 0.006). Furthermore, there was a positive correlation between both molecules (r = 0.526, p = 0.016). Also. granzyme B, perforin and interferon-gamma mRNA expression were different among the three groups. Conclusion: Messenger RNA level expressions of all the studied molecules were higher inside allografts with more severe rejection. Moreover, there was a positive correlation between galectin-9 and Tim-3 mRNA levels. The simultaneous expression of galectin-9 and Tim-3 may indicate an immunoregulatory function, during the ongoing cytotoxic response. (C) 2008 Elsevier B.V. All rights reserved.

Toll-like receptors-related genes in kidney transplant patients with chronic allograft nephropathy and acute rejection

Introduction: Toll-like receptors (TLR) comprehend an emerging family of receptors that recognize pathogen-associated molecular patterns and promote the activation of leukocytes. Surgical trauma and ischemia-reperfusion injury are likely to provide exposure to endogenous ligands for TLR in virtually all kidney transplant recipients. Methods: Macroarray (GEArray OHS-018.2 Series-Superarray) analyses of 128 genes involved in TLR signaling pathway were performed in nephrectomy samples of patients with chronic allograft nephropathy (CAN) and acute rejection (AR, vascular and non vascular). The analysis of each membrane was performed by GEArray Expression Analysis Suite 2.0. Results: Macroarray profile identified a gene expression signature that could discriminate CAN and AR. Three genes were significantly expressed between CAN and vascular AR: Pellino 2; IL 8 and UBE2V1. In relation to vascular and non-vascular AR, there were only two genes with statistical significance: IL-6 and IRAK-3. Conclusion: Vascular and non-vascular AR and CAN showed different expression of a few genes in TLR pathway. The analysis of nephrectomy showed that activation of TLR pathway is present in AR and CAN. (C) 2008 Elsevier B.V. All rights reserved.

Increased Activation of Stromal Interaction Molecule-1/Orai-1 in Aorta From Hypertensive Rats A Novel Insight Into Vascular Dysfunction

Disturbances in the regulation of cytosolic calcium (Ca(2+)) concentration play a key role in the vascular dysfunction associated with arterial hypertension. Stromal interaction molecules (STIMs) and Orai proteins represent a novel mechanism to control store-operated Ca(2+) entry. Although STIMs act as Ca(2+) sensors for the intracellular Ca(2+) stores, Orai is the putative pore-forming component of Ca(2+) release-activated Ca(2+) channels at the plasma membrane. We hypothesized that augmented activation of Ca(2+) release-activated Ca(2+)/Orai-1, through enhanced activity of STIM-1, plays a role in increased basal tonus and vascular reactivity in hypertensive animals. Endothelium-denuded aortic rings from Wistar-Kyoto and stroke-prone spontaneously hypertensive rats were used to evaluate contractions because of Ca(2+) influx. Depletion of intracellular Ca(2+) stores, which induces Ca(2+) release-activated Ca(2+) activation, was performed by placing arteries in Ca(2+) free-EGTA buffer. The addition of the Ca(2+) regular buffer produced greater contractions in aortas from stroke-prone spontaneously hypertensive rats versus Wistar-Kyoto rats. Thapsigargin (10 mu mol/L), an inhibitor of the sarcoplasmic reticulum Ca(2+) ATPase, further increased these contractions, especially in stroke-prone spontaneously hypertensive rat aorta. Addition of the Ca(2+) release-activated Ca(2+) channel inhibitors 2-aminoethoxydiphenyl borate (100 mu mol/L) or gadolinium (100 mu mol/L), as well as neutralizing antibodies to STIM-1 or Orai-1, abolished thapsigargin-increased contraction and the differences in spontaneous tone between the groups. Expression of Orai-1 and STIM-1 proteins was increased in aorta from stroke-prone spontaneously hypertensive rats when compared with Wistar-Kyoto rats. These results support the hypothesis that both Orai-1 and STIM-1 contribute to abnormal vascular function in hypertension. Augmented activation of STIM-1/Orai-1 may represent the mechanism that leads to impaired control of intracellular Ca(2+) levels in hypertension. (Hypertension. 2009; 53[part 2]: 409-416.)

Coronary reserve impairment prevents the improvement of left ventricular dysfunction and adversely affects the long-term outcome of patients with hypertensive dilated cardiomyopathy

In hypertension, left ventricular (LV) hypertrophy develops as an adaptive mechanism to compensate for increased afterload and thus preserve systolic function. Associated structural changes such as microvascular disease might potentially interfere with this mechanism, producing pathological hypertrophy. A poorer outcome is expected to occur when LV function is put in jeopardy by impaired coronary reserve. The aim of this study was to evaluate the role of coronary reserve in the long-term outcome of patients with hypertensive dilated cardiomyopathy. Between 1996 and 2000, 45 patients, 30 of them male, with 52 +/- 11 years and LV fractional shortening <30% were enrolled and followed until 2006. Coronary flow velocity reserve was assessed by transesophageal Doppler of the left anterior descending coronary artery. Sixteen patients showed >= 10% improvement in LV fractional shortening after 17 +/- 6 months. Coronary reserve was the only variable independently related to this improvement. Total mortality was 38% in 10 years. The Cox model identified coronary reserve (hazard ratio = 0.814; 95% CI = 0.72-0.92), LV mass, low diastolic blood pressure, and male gender as independent predictors of mortality. In hypertensive dilated cardiomyopathy, coronary reserve impairment adversely affects survival, possibly by interfering with the improvement of LV dysfunction. J Am Soc Hypertens 2010;4(1):14-21. (C) 2010 American Society of Hypertension. All rights reserved.

trans, trans-2,4-decadienal induces mitochondrial dysfunction and oxidative stress

Lipid peroxidation produces a large number of reactive aldehydes as secondary products. We have previously shown that the reaction of cytochrome c with trans,trans-2, 4-decadienal (DDE), an aldehyde generated as a product of lipid peroxidation in cell membranes, results in the formation of adducts. Mass spectrometry analysis indicated that His-33, Lys-39, Lys-72 and Lys-100 in cytochrome c were modified by DDE. In the present work, we investigated the effect of DDE on isolated rat liver mitochondria. DDE (162 mu M) treatment increases the rate of mitochondrial oxygen consumption. Extensive mitochondrial swelling upon treatment with DDE (900 nM-162 mu M) was observed by light scattering and transmission electron microscopy experiments. DDE-induced loss of inner mitochondrial membrane potentials, monitored by safranin O fluorescence, was also observed. Furthermore, DDE-treated mitochondria showed an increase in lipid peroxidation, as monitored by MDA formation. These results suggest that reactive aldehydes promote mitochondrial dysfunction.

cis-4-decenoic acid provokes mitochondrial bioenergetic dysfunction in rat brain

Aims: In the present work we investigated the in vitro effect of cis-4-decenoic acid, the pathognomonic metabolite of medium-chain acyl-CoA dehydrogenase deficiency, on various parameters of bioenergetic homeostasis in rat brain mitochondria. Main methods: Respiratory parameters determined by oxygen consumption were evaluated, as well as membrane potential, NAD(P)H content, swelling and cytochrome c release in mitochondrial preparations from rat brain, using glutamate plus malate or succinate as substrates. The activities of citric acid cycle enzymes were also assessed. Key findings: cis-4-decenoic acid markedly increased state 4 respiration, whereas state 3 respiration and the respiratory control ratio were decreased. The ADP/O ratio, the mitochondrial membrane potential, the matrix NAD(P)H levels and aconitase activity were also diminished by cis-4-decenoic acid. These data indicate that this fatty acid acts as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor. cis-4-decenoic acid also provoked a marked mitochondrial swelling when either KCl or sucrose was used in the incubation medium and also induced cytochrome c release from mitochondria, suggesting a non-selective permeabilization of the inner mitochondria! membrane. Significance: It is therefore presumed that impairment of mitochondrial homeostasis provoked by cis-4-decenoic acid may be involved in the brain dysfunction observed in medium-chain acyl-CoA dehydrogenase deficient patients. (C) 2010 Elsevier Inc. All rights reserved.