IntroductionHuman bone marrow stromal cells (MSCs), more recently referred to as mesenchymal stem cells, are capable of differentiating along multiple mesenchymal lineages in addition to supporting hematopoiesis. 1,2 Due to their potential for differentiation into osteocytes, chondrocytes, myocytes, and adipocytes, MSCs have emerged as a promising tool for clinical applications such as tissue engineering and cell and gene therapy. 3,4 MSCs are of inherently low immunogenicity and, more importantly, are capable of inhibiting allogeneic T-cell responses. 5-8 These intriguing observations have prompted clinical studies to investigate cotransplantation of MSCs in allogeneic hematopoietic stem cell transplantation (HSCT) in order to promote hematopoietic engraftment by preventing host-versusgraft reactivity and to suppress graft-versus-host reactions. 9,10 As of yet, the molecular mechanisms responsible for the immunosuppressive effects of MSCs have not been unequivocally identified. The reports describing a potential role of transforming growth factor-␤1 and hepatocyte growth factor as mediators of T-cell inhibition remain controversial, but most studies agree that soluble factors are involved. [6][7][8]11 In professional antigen-presenting cells (APCs), expression of indoleamine 2,3-dioxygenase (IDO) induced by interferon-␥ (IFN-␥) and other proinflammatory cytokines catalyzes conversion from tryptophan to kynurenine and has recently been identified as a major immunosuppressive effector pathway that inhibits T-cell responses to autoantigens and fetal alloantigens in vivo. [12][13][14][15][16] Based on these findings, we investigated whether MSCs exhibit IFN-␥-inducible IDO activity and whether this mechanism contributes to T-cell inhibition mediated by MSCs. Study design Culture of human bone marrow-derived MSCsBone marrow aspirates were harvested from volunteer donors who had provided informed consent; the study was approved by the institutional review board of the University Clinic, Düsseldorf, Germany. Primary human MSCs were generated as previously described 17 except that culture medium was supplemented with 3 ng/mL basic fibroblast growth factor (R&D Systems, Minneapolis, MN). Mixed lymphocyte reactions (MLRs)Standard 5-day MLR cultures were set up with 5 ϫ 10 4 mitomycin C-treated human peripheral blood mononuclear cells (PBMCs) as stimulators and 2 ϫ 10 5 human T cells purified using sheep red blood cell rosetting as responder cells. 5,11 In MSC/MLR coculture experiments, MLRs were performed on a layer of either 5 ϫ 10 3 or 2 ϫ 10 4 MSCs seeded one day before. IFN-␥ concentration was determined in MSC/MLR coculture supernatants using a commercially available enzyme-linked immunosorbent assay (ELISA; R&D Systems) according to manufacturer's instructions. Detection of IDO expression and activityMSCs were stimulated with IFN-␥ (R&D Systems) and assayed for IDO expression and function. Standard Western blot analysis for IDO protein expression was performed. 18 IDO enzyme activity following IFN-␥ stimulation of ...

Hyperhomocysteinemia, a proposed risk factor for cardiovascular disease, is also observed in other common disorders. The most frequent genetic cause of hyperhomocysteinemia is a mutated methylenetetrahydrofolate reductase (MTHFR), predominantly when folate status is impaired. MTHFR synthesizes a major methyl donor for homocysteine remethylation to methionine. We administered the alternate choline-derived methyl donor, betaine, to wild-type mice and to littermates with mild or severe hyperhomocysteinemia due to hetero- or homozygosity for a disruption of the Mthfr gene. On control diets, plasma homocysteine and liver choline metabolite levels were strongly dependent on the Mthfr genotype. Betaine supplementation decreased homocysteine in all three genotypes, restored liver betaine and phosphocholine pools, and prevented severe steatosis in Mthfr-deficient mice. Increasing betaine intake did not further decrease homocysteine. In humans with cardiovascular disease, we found a significant negative correlation between plasma betaine and homocysteine concentrations. Our results emphasize the strong interrelationship between homocysteine, folate, and choline metabolism. Hyperhomocysteinemic Mthfr-compromised mice appear to be much more sensitive to changes of choline/betaine intake than do wild-type animals. Hyperhomocysteinemia, in the range of that associated with folate deficiency or with homozygosity for the 677T MTHFR variant, may be associated with disturbed choline metabolism.

Aims Large oral doses of betaine have proved effective in lowering plasma homocysteine in severe hyperhomocysteinaemia. The pharmacokinetic characteristics and metabolism of betaine in humans have not been assessed and drug monitoring for betaine therapy is not available. We studied the pharmacokinetics of betaine and its metabolite dimethylglycine (DMG) in healthy subjects and in three patients with homocystinuria. Methods Twelve male volunteers underwent an open-label study. After one single administration of 50 mg betaine kg -1 body weight and during continuous intake of twice daily 50 mg kg -1 body weight, serial blood samples and 24 h urines were collected to determine betaine and DMG plasma concentrations and urinary excretion, respectively. Patients were evaluated after one single dose of betaine. .70 h, respectively), whereas absorption remained unchanged. DMG concentrations increased significantly after betaine administration and accumulation occurred to the same extent as with betaine. Renal clearance was low and urinary excretion of betaine was equivalent to 4% of the ingested dose. Distribution and elimination kinetics in homocystinuric patients appeared to be accelerated. Conclusions Betaine plasma concentrations change rapidly after ingestion. Elimination half-life increased during continuous dosing over 5 days. Betaine is mainly eliminated by metabolism. More pharmacokinetic and pharmacodynamic studies in hyperhomocysteinaemic patients are needed to refine the current treatment with betaine.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (OMIM 201450) is the most common inherited disorder of fatty acid metabolism presenting with hypoglycaemia, hepatopathy and Reye-like symptoms during catabolism. In the past, the majority of patients carried the prevalent c.985A>G mutation in the ACADM gene. Since the introduction of newborn screening many other mutations with unknown clinical relevance have been identified in asymptomatic newborns. In order to identify functional effects of these mutant genotypes we correlated residual MCAD (OMIM 607008) activities as measured by octanoyl-CoA oxidation in lymphocytes with both genotype and relevant medical reports in 65 newborns harbouring mutant alleles. We identified true disease-causing mutations with residual activities of 0 to 20%. In individuals carrying the c.199T>C or c.127G>A mutation on one allele, residual activities were much higher and in the range of heterozygotes (31%–60%). Therefore, both mutations cannot clearly be associated with a clinical phenotype. This demonstrates a correlation between the octanoyl-CoA oxidation rate in lymphocytes and the clinical outcome. With newborn screening, the natural course of disease is difficult to assess. The octanoyl-CoA oxidation rate, therefore, allows a risk assessment at birth and the identification of new ACADM genotypes associated with asymptomatic disease variants.

A simple and convenient method using commercially available derivatization reagents is described for the measurement of betaine and N,N-dimethylglycine (DMG) in blood and urine. Precolumn derivatization of plasma or urine is performed directly in acetonitrile without extraction with p-bromophenacyl bromide and crown ether as catalyst. The p-bromophenacyl ester derivatives are then separated by high-performance liquid chromatography, using an isocratic system of acetonitrile and water containing choline. Effluent was monitored at 254 nm. The limit of detection was 5 μmol/L for betaine and 2 μmol/L for DMG. Analytical recovery was >97% for both analytes. Total and within-day CVs were 2.0–4.4% and 0.9–2.2% for DMG. For betaine, the total and within-day CVs were 1.3–5.3% and 0.4–3.8%, respectively. The method is precise and cost-effective and has been used successfully to determine the concentrations of DMG and betaine in human plasma and urine.

Kynurenine, the major degradation product of tryptophan has been shown to directly damage tissues, but its possible contribution to posttraumatic morbidity is unknown. Here, we studied the kinetics of kynurenine in patients after major trauma and whether this correlates with the development of posttraumatic sepsis. Kynurenine and tryptophan levels of 60 multiple-injured patients with Injury Severity Score of more than 16 were quantified prospectively by high-performance liquid chromatography. Blood samples were obtained daily from admission until day 10 after admission. Significantly increased kynurenine values were detectable already at day 1 after admission in blood from patients who later developed sepsis, regardless of injury pattern (P < 0.01). In contrast, kynurenine values of nonsepsis patients remained low throughout the observation period. However, all patients exhibited significantly decreased tryptophan values versus healthy controls (P < 0.01). Moreover, significantly increased kynurenine-tryptophan ratios rapidly predicted subsequent sepsis, multiple organ failure, and death (P < 0.01). Both increased kynurenine values and kynurenine-tryptophan ratios predicted posttraumatic development of sepsis and organ failure. This ought to be validated in subsequent studies.

The fatty acid (FA) composition of the main plasma lipids was analysed in eight well-nourished, generally healthy Nigerian children aged 14.1 +/- 7.2 months and in 17 malnourished children (8 marasmus, 9 kwashiorkor) aged 14.6 +/- 3.8 months within the first 2 days of admission at the Dept. of Child Health, University of Benin. In comparison to the control group, the malnourished children showed a marked decrease of polyunsaturated FA with low linoleic acid, mainly in sterol esters (STE), and severely reduced linoleic acid metabolites, including arachidonic acid, in all lipid fractions. omega-3-FA were not altered except for a reduction of docosapentaenoic and docosahexaenoic acids in phospholipids. Clearly increased values were found for saturated FA in STE and for the non-essential monoenoic FA in all lipid classes. This pattern indicates the presence of essential fatty acid deficiency in the malnourished children. There was no significant difference between marasmus and kwashiorkor. Eight malnourished children were followed up in the early phase of recovery during hospital treatment 14.0 +/- 3.1 days after obtaining the first sample. Linoleic acid had increased again in STE, but its metabolites were as low or even lower than before. An impaired activity of delta-6-desaturase, the rate limiting enzyme of linoleic acid metabolism, in suggested by elevated substrate-product-ratios of this enzyme in untreated children with protein energy malnutrition and in the early phase of recovery, which may be due to low insulin levels, protein and zinc deficiency. The trientetraen ratio (20:3 omega 9/20:4 omega 6) thus is not a reliable indicator of essential FA status in protein-energy malnutrition.

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