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Background A proinflammatory environment characterized by the continuous activation of the innate immune system is thought to contribute to the markedly elevated mortality in haemodialysis (HD) patients with end-stage renal disease (ESRD). The presence of circulating cell-free DNA (cfDNA) has been demonstrated as biomarker in many pathologies. Methods We evaluated the occurrence of cfDNA in HD patients and its functional relevance for innate immunity and inflammation. Results Here, we found that cfDNA was enhanced in the plasma of ESRD patients after HD compared to healthy controls. Functionally, cfDNA selectively stimulated the production of the proinflammatory cytokine interleukin (IL)-6 by human monocytes, whereas tumour necrosis factor-α or IL-10 was not induced. Conversely, plasma from HD patients, but not from healthy controls or DNase I-treated HD plasma, induced IL-6 production from monocytes. Conclusion We provide the first evidence that cfDNA has selective immunostimulatory effects on human monocytes. This process may contribute to the proinflammatory milieu observed in HD patients.

Effects of TCN2 776C>G on vitamin B 12 , folate, and total homocysteine levels in kidney transplant patients. Background Controversy exists regarding the possible associations between a single nucleotide polymorphism of the transcobalamin II encoding gene ( TCN2 776C>G) and plasma levels of vitamin B 12 , folate, or total homocysteine. Methods In a cross-sectional study of 732 kidney allograft recipients, patients were categorized by TCN2 776C>G genotype. In univariate and multivariate linear regression models that allowed the outcome variables vitamin B 12 , folate, and total homocysteine plasma levels to follow a gamma distribution, we tested for possible associations of allelic variants of the TCN2 776C>G gene and these three dependent variables. Results The allele frequency for TCN2 776C>G was 0.46. Heterozygosity or homozygosity for TCN2 776C>G was not associated with plasma levels of vitamin B 12 (776CG, P = 0.22; 776GG, P = 0.89), folate (776CG, P = 0.91; 776GG, P = 0.84), or total homocysteine (776CG, P = 0.11; 776GG, P = 0.33) even after adjustment for several possible confounders. Conclusion We conclude from this largest study on the subject thus far that there are no associations between allelic variants of TCN2 776C>G and plasma vitamin B 12 , folate, or total homocysteine plasma levels in kidney transplant patients.

Background Oral ingestion of proteins or amino acids is associated with endothelial dysfunction. The effect of commercial amino acid peritoneal dialysis solutions on vascular function is unknown. Objective We compared the acute effect of intraperitoneal amino acid administration with that of intraperitoneal glucose administration on vascular function in peritoneal dialysis patients. Design In an open-label randomized, controlled, crossover and observer-blinded trial, we examined the acute effect of an intraperitoneal application of 2 L commercial 1.1% amino acid solution compared with that of a 2.27% glucose solution in 13 peritoneal dialysis patients. The primary endpoint was the change in forearm reactive hyperemia 6 h after instillation of either dialysis solution. Results After 6 h of dwell time, reactive hyperemia was substantially impaired after administration of the amino acid solution compared with the glucose solution (median difference: 202%; 95% CI: 57%, 368%; P = 0.007). In a comparison of differences between values at 6 h and those before treatment, reactive hyperemia significantly decreased during the dwell with the amino acid dialysis solution compared with that with the glucose dialysis solution (median difference: 242%; 95% CI: 53%, -457%; P = 0.013). In an analysis of smoking and nonsmoking patients separately, the difference in forearm blood flow between the 2 treatments was still statistically significant. Conclusions One 6-h dwell with a commercial amino acid dialysis solution acutely impairs forearm reactive hyperemia in smoking and nonsmoking peritoneal dialysis patients. Because endothelial dysfunction is associated with increased morbidity and mortality, the long-term use of these solutions may increase the risk of cardiovascular disease.

Effect of TCN2 776C>G on vitamin B 12 cellular availability in end-stage renal disease patients. Background Transcobalamin II is a serum protein that transports vitamin B 12 from the intestine to the tissues. This complex, holo-transcobalamin II, may reflect vitamin B 12 availability in the body. Conflicting data exist with regard to the effect of a polymorphism in the gene coding for transcobalamin II, TCN2 776C>G, on transcobalamin II levels in the general population, which in turn may affect holo-transcobalamin II, vitamin B 12 , as well as total homocysteine (tHcy) plasma levels. The effect of TCN2 776C>G on vitamin B 12 cellular availability in dialysis patients is unknown. Methods We examined the effect of TCN2 776C>G on holo-transcobalamin II, vitamin B 12 , and tHcy plasma concentrations in 120 dialysis patients. Results Holo-transcobalamin II levels were normal or supranormal in all patients and showed a linear association with albumin ( r = 0.205, P = 0.025) and with vitamin B 12 ( r = 0.778, P = 0.001), but not with age, creatinine, body mass index, tHcy, ln-tHcy, vitamin B 6 , plasma folate, and red blood cell folate concentration. TCN2 776C>G showed no effect on holo-transcobalamin II, vitamin B 12 , and tHcy concentration [one-way analysis of variance (ANOVA), post-hoc Scheffe test]. Multiple linear regression analyses showed that albumin and B 12 are independently associated with holo-transcobalamin II, whereas TCN2 776C>G and MTHFR 677C>T had no effect. Independent predictors of ln-tHcy included creatinine, red blood cell folate, and the MTHFR 677TT genotype. There was also an effect of the TCN2 776CC genotype on ln-tHcy levels in this multivariate analysis, however, that deserves cautious interpretation because there was no effect of TCN2 genotypes by ANOVA and Scheffe test [median ln-tHcy concentrations according to TCN2 genotypes (μmol/L): CC, 3.22; CG, 3.30; GG, 3.23]. Conclusion TCN2 776C>G does not influence holo-transcobalamin II or vitamin B 12 levels, and has no major effect on tHcy concentrations of end-stage renal disease patients.

This study was designed to examine the effect of two single nucleotide polymorphisms in the reduced folate carrier 1 ( RFC1 80G>A) and the glutamate carboxypeptidase 2 ( GCP2 1561C>T) gene on total homocysteine (tHcy) plasma level and folate status in 120 chronic dialysis patients. Red blood cell folate concentration was higher in patients with the GCP2 CT or TT genotype (ANOVA, P = 0.04). Among patient groups with different RFC1 genotypes, red blood cell folate level was not significantly different. A multivariate analysis confirmed that the GCP2 1561C>T genotype ( P = 0.011) had a significant influence on the red blood cell folate concentration. Overall, serum folate, creatinine, and the GCP2 polymorphism explained nearly 50% of the variance of red blood cell folate. A linear multivariate regression analysis showed that red blood cell folate ( P P MTHFR ) 677T allele ( P = 0.013) are independent predictors of tHcy plasma level explaining 49% of the variance of tHcy plasma concentration. GCP2 1561C>T and RFC1 80G>A showed no effect on tHcy and folate plasma level. In conclusion, GCP2 1561C>T, but not RFC1 80G>A, is a predictor of red blood cell folate level in chronic dialysis patients. Both polymorphisms have no major effect on tHcy plasma concentration in end-stage renal disease patients. E-mail: Gere.Sunder-Plassmann@univie.ac.at

Endothelial cell adhesion molecule and PMNL response to inflammatory stimuli and AGE-modified fibronectin. Background Atherosclerotic vascular disease is the leading cause of death in patients with diabetes mellitus and end-stage renal disease. Advanced glycation end products (AGEs) are strongly suggested to be involved in the pathogenesis of atherosclerosis in these patients who also frequently experience infectious complications. We hypothesized that the interaction of AGEs and inflammatory mediators contributes to the up-regulation of endothelial cell activation. Methods We investigated the effect of advanced glycated fibronectin in the presence or absence of inflammatory stimuli on the endothelial cell surface and mRNA expression of cell adhesion molecules. Furthermore, the influence of advanced glycated fibronectin on the transendothelial migration pattern of polymorphonuclear cells was analyzed. Results Exposure to advanced glycated fibronectin together with inflammatory stimuli such as interleukin (IL)-1α, tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS) led to a significant increase in the surface expression of the cell adhesion molecules E-selectin, ICAM-1, VCAM-1 and PECAM-1 on endothelial cells. Soluble AGEs in combination with advanced glycated fibronectin significantly enhanced the endothelial cell surface expression of ICAM-1, VCAM-1 and PECAM-1, whereas this was not the case for E-selectin. At the transcriptional level short-time exposure of endothelial cells to advanced glycated fibronectin and inflammatory mediators resulted in an increased expression of E-selectin, ICAM-1 and VCAM-1 mRNA levels, whereas PECAM-1 repeatedly showed a significant decrease of gene transcript levels. An increase of mRNA levels was also observed for E-selectin, ICAM-1, VCAM-1 and PECAM-1 following incubation with a combination of advanced glycated fibronectin and soluble advanced glycation end-products. Furthermore, polymorphonuclear cells responded with a sevenfold increase in transendothelial migration following exposure of endothelial cells to advanced glycated fibronectin and inflammatory mediators. Conclusions These results suggest that the combination of matrix glycation and inflammation up-regulates the activation of the endothelial cell adhesion cascade, a mechanism that might contribute to the increased burden of atherosclerotic morbidity and mortality in patients suffering from diabetes mellitus or chronic renal failure.

In polycythemia vera (PV) erythroid colonies that grow in vitro in the absence of exogenous erythropoietin (EPO) arise from the abnormal clone that is responsible for overproduction of red blood cells. Although the mechanism of autonomous formation of burst-forming units-erythroid (BFU-E) is not fully understood, a spontaneous release of growth regulatory molecules by PV cells and/or by accessory cells is likely to be involved. Because of its cytokine synthesis inhibiting action, interleukin-10 (IL-10) could be a potentially useful molecule to modulate abnormal erythropoiesis in PV. We studied the effect of recombinant human IL-10 on the EPO-independent growth of erythroid bursts derived from peripheral blood mononuclear cells (PBMNCs) of patients with PV. IL-10 showed a profound, dose-dependent, and specific inhibitory effect on autonomous BFU-E formation. Ten nanograms per milliliter of IL-10 significantly suppressed spontaneous growth of erythroid colonies in methylcellulose in five of five PV patients tested with a mean inhibition by 81% (range, 72-94). To elucidate the possible mechanism of the inhibitory action of IL-10 we further studied the effect of anticytokine antibodies on autonomous BFU-E growth and the ability of exogenous cytokines to restore IL-10–induced suppression of erythroid colony growth. Among a panel of growth regulatory factors tested (granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-3, granulocyte colony-stimulating factor, stem cell factor, and insulin-like growth factor-1) GM-CSF was the only molecule for which both an inhibition of spontaneous BFU-E formation by its respective antibody as well as a significant restimulation of erythroid colonies in IL-10-treated cultures by exogenous addition was found. Moreover, inhibition of GM-CSF production by IL-10 was shown in PV PBMNCs at the mRNA level. Our data indicate that autonomous BFU-E growth in PV can be profoundly inhibited by IL-10 and that this inhibitory effect seems to be at least in part secondary to suppression of endogenous GM-CSF production. © 1998 by The American Society of Hematology.

Spontaneous growth of myeloid colonies (colony-forming unit–granulocyte-macrophage [CFU-GM]) can be observed in methylcellulose cultures containing peripheral blood mononuclear cells (PB-MNCs) and is supposedly caused by the release of colony-stimulating factors (CSF ) by accessory cells. Because of its cytokine synthesis-inhibiting effects on T lymphocytes and monocytes, interleukin-10 (IL-10) may be a potential candidate for indirect modulation of hematopoiesis. We studied the effect of recombinant human IL-10 (rhIL-10) on spontaneous growth of myeloid colonies derived from human PB-MNCs. A total of 10 ng/mL of IL-10 almost completely inhibited spontaneous CFU-GM proliferation (by 95.1%; P < .001, n = 7) in unseparated PB-MNCs. This effect was dose-dependent and specific, because a neutralizing anti–IL-10 antibody was able to prevent IL-10–induced suppression of CFU-GM growth. Spontaneous CFU-GM growth, which required the presence of both monocytes (CD14+ cells) and T lymphocytes (CD3+ cells), was also greatly suppressed by a neutralizing anti–granulocyte-macrophage CSF (GM-CSF ) antibody but was only slightly or not at all inhibited by antibodies against G-CSF or IL-3. Moreover, IL-10–suppressed colony growth could be completely restored by the addition of exogenous GM-CSF. Using semiquantitative polymerase chain reaction, we were able to show that GM-CSF transcripts that spontaneously increased in PB-MNCs within 48 hours of culture were markedly reduced by the addition of IL-10. Inhibiton of GM-CSF production in PB-MNCs by IL-10 was also confirmed at the protein level by measuring GM-CSF levels in suspension cultures. Our findings suggest that autonomous CFU-GM growth, resulting from an interaction of monocytes and T lymphocytes, is mainly caused by endogenous GM-CSF release and can be profoundly suppressed by the addition of exogenous IL-10. Considering the strong inhibitory action of IL-10 on GM-CSF production and spontaneous cell growth in vitro, this cytokine may be useful in myeloid malignancies in which autocrine and/or paracrine mechanisms involving GM-CSF are likely to play a pathogenetic role.

Autonomous release of hematopoietic growth factors may play a crucial role in the pathogenesis of certain hematological malignancies. Because of its cytokine synthesis-inhibiting action, interleukin 10 (IL-10) could be a potentially useful molecule to affect leukemic cell growth in such disorders. Chronic myelomonocytic leukemia (CMML) cells spontaneously form myeloid colonies (colony-forming units-granulocyte/macrophage) in methylcellulose, suggesting an autocrine growth factor-mediated mechanism. We studied the effect of recombinant human IL-10 (rhIL-10) on the in vitro growth of mononuclear cells obtained from peripheral blood or bone marrow of patients with CMML. IL-10 specifically binding to leukemic cells had a profound and dose-dependent inhibitory effect on autonomous in vitro growth of CMML cells. IL-10 significantly inhibited the spontaneous growth of myeloid colonies in methylcellulose in 10/11 patients, and autonomous CMML cell growth in suspension in 5/5 patients tested. Spontaneous colony growth from CMML cells was also markedly reduced by addition of antigranulocyte/macrophage colony-stimulating factor (GM-CSF) antibodies, but not by addition of antibodies against G-CSF, IL-3, or IL-6, IL-10-induced suppression of CMML cell growth was reversed by the addition of exogenous GM-CSF and correlated with a substantial decrease in GM-CSF production by leukemic cells, both at the mRNA and protein levels. Our data indicate that IL-10 profoundly inhibits the autonomous growth of CMML cells in vitro most likely through suppression of endogenous GM-CSF release. This observation suggests therapeutic evaluation of rhIL-10 in patients with CMML.

Controversy exists regarding the possible associations between a single nucleotide polymorphism of the transcobalamin II encoding gene (TCN2 776CG) and plasma levels of vitamin B(12), folate, or total homocysteine.In a cross-sectional study of 732 kidney allograft recipients, patients were categorized by TCN2 776CG genotype. In univariate and multivariate linear regression models that allowed the outcome variables vitamin B(12), folate, and total homocysteine plasma levels to follow a gamma distribution, we tested for possible associations of allelic variants of the TCN2 776CG gene and these three dependent variables.The allele frequency for TCN2 776CG was 0.46. Heterozygosity or homozygosity for TCN2 776CG was not associated with plasma levels of vitamin B(12) (776CG, P= 0.22; 776GG, P= 0.89), folate (776CG, P= 0.91; 776GG, P= 0.84), or total homocysteine (776CG, P= 0.11; 776GG, P= 0.33) even after adjustment for several possible confounders.We conclude from this largest study on the subject thus far that there are no associations between allelic variants of TCN2 776CG and plasma vitamin B(12), folate, or total homocysteine plasma levels in kidney transplant patients.

The effect of thiamine (vitamin B(1)) or riboflavin (vitamin B(2)) availability on fasting total homocysteine (tHcy) plasma levels in end-stage renal disease patients is unknown. A cross-sectional study was performed in a population of non-vitamin supplemented patients maintained on continuous ambulatory peritoneal dialysis. Red blood cell availability of thiamine (alpha-ETK) and of riboflavin (alpha-EGR), along with other predictors of tHcy plasma levels, was considered in the analysis. There was a linear association of alpha-EGR with tHcy plasma concentrations (P = 0.009), which was not observed for alpha-ETK. Among red blood cell vitamins, alpha-EGR was the only predictor of tHcy levels (P = 0.035), whereas alpha-ETK, red blood cell pyridoxal-5-phosphate supply (alpha-EGOT) and red blood cell folate levels had no effect. The risk for having a high tHcy plasma levels within the fourth quartile (plasma tHcy38.3 micromol/L) was increased by an alpha-EGRmedian (odds ratio, 4.706; 95% confidence interval, 1.124 to 19.704; P = 0.026). By way of contrast, alpha-ETK had no effect in these analyses. Independent predictors of tHcy plasma levels were serum albumin, alpha-EGR, red blood cell folate, and certain MTHFR genotypes. A logistic regression analysis showed that the MTHFR genotype is a predictor for having a tHcy plasma concentration within the fourth quartile. In summary, riboflavin availability, as measured by alpha-EGR, is a determinant of fasting tHcy plasma levels in peritoneal dialysis patients. This finding may have implications for tHcy lowering therapy in individuals with end-stage renal disease.