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.

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.

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.

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.

Abstract. 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 nonvitamin supplemented patients maintained on continuous ambulatory peritoneal dialysis. Red blood cell availability of thiamine (␣-ETK) and of riboflavin (␣-EGR), along with other predictors of tHcy plasma levels, was considered in the analysis. There was a linear association of ␣-EGR with tHcy plasma concentrations (P ϭ 0.009), which was not observed for ␣-ETK. Among red blood cell vitamins, ␣-EGR was the only predictor of tHcy levels (P ϭ 0.035), whereas ␣-ETK, red blood cell pyridoxal-5-phosphate supply (␣-EGOT) and red blood cell folate levels had no effect. The risk for having a high tHcy plasma levels within the fourth quartile (plasma tHcy Ͼ38.3 mol/L) was increased by an ␣-EGR Ͼ median (odds ratio, 4.706; 95% confidence interval, 1.124 to 19.704; P ϭ 0.026). By way of contrast, ␣-ETK had no effect in these analyses. Independent predictors of tHcy plasma levels were serum albumin, ␣-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 ␣-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.The majority of patients with impaired renal function present elevated total homocysteine (tHcy) plasma levels (1). Established predictors of tHcy plasma levels in the renal failure population include serum albumin and serum creatinine levels, creatinine clearance, folate status, vitamin B 12 and vitamin B 6 levels, as well as genetic variants in enzymes involved in the folate cycle or in the remethylation of homocysteine (2-7). An elevated tHcy plasma level can indicate folate and/or vitamin B 12 deficiency (8) and is associated with a variety of pathologic conditions such as vascular disease (9 -11) or birth defects (12). Although genetic and nongenetic factors have been shown to determine tHcy concentrations of patients with renal insufficiency, the cause of hyperhomocysteinemia among these patients is not completely understood (13).The role of B-group vitamins other than vitamin B 6 or vitamin B 12 as determinants of hyperhomocysteinemia in the general population and in the setting of renal insufficiency is far from clear, although vitamin B 1 (thiamine pyrophosphate) and vitamin B 2 (riboflavin) are involved in the metabolism of methionine and homocysteine.We assumed that thiamine or riboflavin availability is a predictor of fasting tHcy plasma levels in end-stage renal disease (ESRD). To test this hypothesis, we performed a crosssectional study among a population of non-vitamin supplemented patients maintained on continuous ambulatory perito...

TCN2 776C>G does not influence holo-transcobalamin II or vitamin B12 levels, and has no major effect on tHcy concentrations of end-stage renal disease patients.

Abstract. This study was designed to examine the effect of two single nucleotide polymorphisms in the reduced folate carrier 1 (RFC1 80GϾA) and the glutamate carboxypeptidase 2 (GCP2 1561CϾT) 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 ϭ 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 1561CϾT genotype (P ϭ 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 Ͻ 0.001), creatinine (P Ͻ 0.001), and the 5,10-methylenetetrahydrofolate reductase (MTHFR) 677T allele (P ϭ 0.013) are independent predictors of tHcy plasma level explaining 49% of the variance of tHcy plasma concentration. GCP2 1561CϾT and RFC1 80GϾA showed no effect on tHcy and folate plasma level. In conclusion, GCP2 1561CϾT, but not RFC1 80GϾA, 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.In recent years, evidence accumulated indicating that the folate and homocysteine (Hcy) metabolism are partly under genetic control. In particular, the MTHFR 677CϾT polymorphism in the gene coding for 5,10-methylenetetrahydrofolate reductase was shown to influence total Hcy (tHcy) plasma and/or folate level in healthy adults (1) and in patients on hemodialysis treatment, peritoneal dialysis treatment, and in renal graft recipients (2-5). This effect can also be associated with riboflavin availability in end-stage renal disease (ESRD) (6). Interestingly, in renal failure patients, MTHFR 677CϾT modulates the response to folate therapy (7-9). In addition to MTHFR 677CϾT, MTHFR 1298AϾC, and methionine synthase (MTR) 2756AϾG have some effect on folate and/or Hcy metabolism in individuals with compromised renal function (10,11).Dietary folates are absorbed after hydrolysis of the terminal glutamate residues of folylpoly-␥-glutamates by the brushborder enzyme folylpoly-␥-glutamate carboxypeptidase (glutamate carboxypeptidase II). The reduced folate carrier then reabsorbs monoglutamylfolate from the enterohepatic cycle. Thus, both proteins regulate the availability of dietary and circulating folates.Recently, two polymorphisms located in the genes coding for glutamate carboxypeptidase II (GCP2 1561CϾT; H475Y) and for the reduced folate carrier (RFC1 80GϾA; R27H) have been shown to be associated with alterations in folate and Hcy metabolism in healthy individuals (12,13). Because folate and Hcy metabolism is markedly impaired in renal failure, these mutations could further contribute to hyperhomocysteinemia of ESRD. In the present study, we examined ...

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.