Two Cases of Veno-occlusive Disease/Sinusoidal Obstruction Syndrome After Thioguanine Treatment for Acute Lymphoblastic Leukemia.

Veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) is a potentially life-threatening complication of hematopoietic cell transplantation conditioning or high-dose chemotherapy. The underlying pathogenesis involves toxic injury to hepatocytes and sinusoidal endothelial cells. Presenting symptoms include ascites, weight gain, hepatomegaly, and hyperbilirubinemia. Severe VOD/SOS with multiorgan failure has a mortality rate of >80% if left untreated. Thioguanine, a chemotherapy drug used to treat acute lymphoblastic leukemia, has been shown to cause VOD/SOS. Here, we describe cases of 2 patients who developed very severe VOD/SOS after starting thioguanine for acute lymphoblastic leukemia; both achieved complete remission with defibrotide and experienced no defibrotide-related adverse events.

Extended Treatment With Mesenchymal Stromal Cells-Frankfurt am Main in a Pediatric Patient With Steroid-refractory Acute Gastrointestinal Graft-Versus-Host Disease: Case Report and Review of the Literature.

In acute graft-versus-host disease (aGVHD) following allogeneic hematopoietic stem cell transplantation, there are various options available after the failure of initial steroid therapy. Since the publication of the first study in 2008, mesenchymal stromal cells (MSCs) have also been used with increasing frequency, including in pediatric patients with steroid-refractory aGVHD, and the manufacturing process has undergone further development. MSC-Frankfurt am Main (MSC-FFM, Obnitix), which is manufactured from pooled mononuclear bone marrow cells from 8 donors using a standardized process, resulted in a response rate of 84% in children with steroid-refractory aGVHD. We report on a 13-year-old female patient with acute myeloid leukemia who received Obnitix as a third-line treatment for gastrointestinal (GI) aGVHD in a life-threatening situation. The patient was initially given a total of 4 Obnitix infusions as per the regulatory approval, with her symptoms improving from day 9 after the first infusion. The second cycle of 4 Obnitix infusions followed due to persistent severe protein-losing enteropathy and resulted in complete remission. A systematic review of the literature on MSC in pediatric patients with steroid-refractory aGVHD confirms that MSC treatment beyond 4 weeks is employed in accordance with treatment protocols or on a case-by-case basis. To summarize, aGVHD activity can be checked endoscopically in patients with persistent GI symptoms and a second Obnitix cycle can then be administered if appropriate, with the goal of achieving complete remission. Future studies should also investigate the potential influence of tissue repair properties as an element in MSCs' efficacy in GI aGVHD.

Vitamin D supplementation does not prevent the testosterone decline in males with advanced heart failure: the EVITA trial.

PURPOSE: Observational studies indicate a positive association between circulating 25-hydroxyvitamin D (25OHD) and testosterone (T) concentrations. Because low 25OHD concentrations and T deficiency are considered to be a generalized phenomenon in patients with advanced heart failure (HF), we aimed to investigate whether vitamin D supplementation has beneficial effects on T indices in these patients. METHODS: In a pre-specified secondary analysis of the EVITA (effect of vitamin D on mortality in heart failure) randomized controlled trial, we analyzed in male subjects with 25OHD concentrations < 75 nmol/L the effect of a daily vitamin D3 supplement of 4000 IU for 3 years (n = 71) vs. placebo (n = 62) on total T (TT), sex hormone-binding globulin (SHBG), free T (fT), and bioactive T (BAT). We assessed changes from baseline until study termination and between-group differences at study termination. RESULTS: 25OHD increased in the placebo group from 36.6 nmol/L by 9.2 nmol/L (95% CI 3.2-15.1 nmol/L; P = 0.003) and in the vitamin D group from 36.5 nmol/L by 63.9 nmol/L (95% CI 52.6-75.3 nmol/L; P < 0.001), with a significant between-group difference at study termination (P < 0.001). TT and SHBG concentrations did not change significantly, neither in the placebo group nor in the vitamin D group (P = 0.845-0.082), but concentrations of fT and BAT declined significantly in both groups (P = 0.025-0.008). At study termination, there were no between-group differences in TT (P = 0.612), SHBG (P = 0.393), fT (P = 0.861), or BAT (P = 0.960). CONCLUSIONS: In male patients with advanced HF and low 25OHD concentrations, a daily vitamin D3 supplement of 4000 IU for 3 years did not prevent the decline in testosterone indices.

Daily Supplementation with 4000 IU Vitamin D3 for Three Years Does Not Modify Cardiovascular Risk Markers in Patients with Advanced Heart Failure: The Effect of Vitamin D on Mortality in Heart Failure Trial.

BACKGROUND/AIMS: We aimed to investigate the effect of a moderately high vitamin D dose on lipid parameters and biochemical markers of vascular calcification (VC) in patients with established cardiovascular disease. METHODS: We included in this pre-specified secondary analysis of a randomized controlled trial 161 patients with advanced heart failure and 25-hydroxyvitamin D (25OHD) concentrations < 75 nmol/L (vitamin D group: n = 80; placebo group: n = 81), who received a daily vitamin D3 supplement of 4,000 IU for 3 years. We assessed between-group differences of the lipid parameters total-cholesterol, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, and triglycerides, and the VC markers fetuin-A and non-phosphorylated undercarboxylated matrix gla protein (MGP) at study termination, with adjustment for baseline values. RESULTS: Lipid parameters, the percentage of patients with dyslipoproteinemia, and VC markers did not differ significantly between groups at study termination (p values: 0.395-0.939). Likewise, vitamin D achieved no significant treatment effect on these markers in subgroup analyses in patients with 25OHD concentrations < 30 nmol/L, nonusers of lipid-lowering drugs, or diabetic patients (p values: 0.245-0.998). CONCLUSION: Our data indicate that vitamin D does not improve the lipid profile and does not influence the calcification inhibitors fetuin-A and non-phosphorylated undercarboxylated MGP in patients with advanced heart failure.

Effect of vitamin D on all-cause mortality in heart failure (EVITA): a 3-year randomized clinical trial with 4000 IU vitamin D daily.

AIMS: Circulating 25-hydroxyvitamin D (25OHD) levels <75 nmol/L are associated with a nonlinear increase in mortality risk. Such 25OHD levels are common in heart failure (HF). We therefore examined whether oral vitamin D supplementation reduces mortality in patients with advanced HF. METHODS AND RESULTS: Four hundred HF patients with 25OHD levels <75 nmol/L were randomized to receive 4000 IU vitamin D daily or matching placebo for 3 years. Primary endpoint was all-cause mortality. Key secondary outcome measures included hospitalization, resuscitation, mechanical circulatory support (MCS) implant, high urgent listing for heart transplantation, heart transplantation, and hypercalcaemia. Initial 25OHD levels were on average <40 nmol/L, remained around 40 nmol/L in patients assigned to placebo and plateaued around 100 nmol/L in patients assigned to vitamin D. Mortality was not different in patients receiving vitamin D (19.6%; n = 39) or placebo (17.9%; n = 36) with a hazard ratio (HR) of 1.09 [95% confidence interval (CI): 0.69-1.71; P = 0.726]. The need for MCS implant was however greater in patients assigned to vitamin D (15.4%, n = 28) vs. placebo [9.0%, n = 15; HR: 1.96 (95% CI: 1.04-3.66); P = 0.031]. Other secondary clinical endpoints were similar between groups. The incidence of hypercalcaemia was 6.2% (n = 10) and 3.1% (n = 5) in patients receiving vitamin D or placebo (P = 0.192). CONCLUSION: A daily vitamin D dose of 4000 IU did not reduce mortality in patients with advanced HF but was associated with a greater need for MCS implants. Data indicate caution regarding long-term supplementation with moderately high vitamin D doses. TRIAL REGISTRATION INFORMATION: clinicaltrials.gov Idenitfier: NCT01326650.

Effect of Vitamin D or Activated Vitamin D on Circulating 1,25-Dihydroxyvitamin D Concentrations: A Systematic Review and Metaanalysis of Randomized Controlled Trials.

BACKGROUND: Evidence is accumulating that circulating 1,25-dihydroxyvitamin D [1,25(OH)2D] concentrations are inversely related to overall mortality. METHODS: We searched PubMed, Embase and ISI Web of Science for randomized controlled trials with a control group receiving a placebo instead of vitamin D/activated vitamin D and performed a metaanalysis to evaluate the effect of oral vitamin D/activated vitamin D on circulating 1,25(OH)2D concentrations using a random effects model. RESULTS: We included 52 vitamin D intervention groups (4796 individuals) and 14 intervention groups with activated vitamin D (668 individuals). Vitamin D supplements increased circulating 1,25(OH)2D by 12.2 pmol/L (95% CI, 7.8-16.5 pmol/L) and 18.8 pmol/L (95% CI, 9.2-28.4 pmol/L) if only studies with a low risk of bias in study design and reporting were considered (n = 18). There was significant heterogeneity among studies (Cohran's Q P < 0.001, I(2) = 91%). The incremental effect was larger in studies using vitamin D alone compared with coadministration of calcium supplements (18.6 pmol/L; 95% CI, 12.7-24.4 pmol/L vs 4.9 pmol/L; 95% CI, -0.4 to 10.2 pmol/L; P = 0.001), and if quantification was performed with RIA vs other methods (17.1 pmol/L; 95% CI, 11.1-23.1 pmol/L vs 6.9 pmol/L; 95% CI, 1.0-12.8 pmol/L; P = 0.02). Activated vitamin D increased the mean circulating 1,25(OH)2D by 20.5 pmol/L (95% CI, 8.3-32.7 pmol/L; P = 0.04). Again, there was evidence for significant heterogeneity among studies (Cochran Q = 85.4; P < 0.001; I(2) = 87%), but subgroup analysis did not identify parameters significantly influencing the increment in 1,25(OH)2D concentrations. CONCLUSIONS: Both vitamin D and activated vitamin D significantly increase circulating 1,25(OH)2D concentrations, but in vitamin D users this increase is suppressed by calcium coadministration.

Effects of the environmental contaminants DEHP and TCDD on estradiol synthesis and aryl hydrocarbon receptor and peroxisome proliferator-activated receptor signalling in the human granulosa cell line KGN.

Environmental contaminants binding to transcription factors, such as the aryl hydrocarbon receptor (AhR) and the alpha and gamma peroxisome proliferator-activated receptors (PPARs), contribute to adverse effects on the reproductive system. Expressing both the AhR and PPARs, the human granulosa cell line KGN offers the opportunity to investigate the regulatory mechanisms involved in receptor crosstalk, independent of overriding hormonal control. The aim of the present study was to investigate the impact of two environmental contaminants, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, an AhR ligand) and di-(2-ethylhexyl) phthalate (DEHP, a PPAR ligand), on gonadotrophin sensitivity and estrogen synthesis in KGN cells. Accumulation of the DEHP metabolite mono-(2-ethylhexyl) phthalate (MEHP) in DEHP-exposed cells was measured by high-performance liquid chromatography mass spectrometry, thereby demonstrating DEHP metabolism to MEHP by KGN cells. By employing TCDD ( an AhR agonist), rosiglitazone (a PPARgamma agonist) or bezafibrate (a PPARalpha agonist), the presence of a functional AhR and PPAR cascade was confirmed in KGN cells. Cytotoxicity testing revealed no effect on KGN cell proliferation for the concentrations of TCDD and DEHP used in the current study. FSH-stimulated cells were exposed to TCDD, DEHP or a mix of both and estradiol synthesis was measured by enzyme-linked immunosorbent assay and gene expression by quantitative RT-PCR. Exposure decreased estradiol synthesis (TCDD, DEHP, mix) and reduced the mRNA expression of CYP19 aromatase (DEHP, mix) and FSHR (DEHP). DEHP induced the expression of the alpha and gamma PPARs and AhR, an effect which was inhibited by selective PPAR antagonists. Studies in the human granulosa cell line KGN show that the action of endocrine-disrupting chemicals may be due to a direct activation of AhR, for example by TCDD, and by a transactivation via PPARs, for example by DEHP, inducing subsequent transcriptional changes with a broad range of effects on granulosa cell function.

Vitamin D supplementation, body weight and human serum 25-hydroxyvitamin D response: a systematic review.

PURPOSE: There is considerable variation in incremental circulating 25-hydroxyvitamin D (25OHD) levels on vitamin D supplements, even when similar age groups and identical vitamin D doses are compared. We therefore aimed to investigate the importance of body weight for the dose-response relation in circulating 25OHD. METHODS: We performed a systematic review of randomized placebo-controlled vitamin D supplementation trials in all age groups ≥10 years to clarify the influence of body weight and other parameters on incremental circulating 25OHD levels (difference between baseline and in-study values) in vitamin D-deficient and non-deficient individuals. RESULTS: We included 144 cohorts from 94 independent studies, published from 1990 to November 2012, in our systematic review. There was a logarithmic association between vitamin D dose per kg body weight per day and increment in circulating 25OHD. In multivariable regression analysis, vitamin D dose per kg body weight per day could explain 34.5% of variation in circulating 25OHD. Additional significant predictors were type of supplement (vitamin D2 or vitamin D3), age, concomitant intake of calcium supplements and baseline 25OHD, explaining 9.8, 3.7, 2.4 and 1.9%, respectively, of the variation in circulating 25OHD. CONCLUSIONS: This systematic review demonstrates that body weight is an important predictor of variation in circulating 25OHD in cohorts on vitamin D supplements. Our model provides an estimate of the daily vitamin D dose that is necessary for achieving adequate circulating 25OHD levels in vitamin D-insufficient or vitamin D-deficient individuals/cohorts with different body weights and ages.

Analysis of risk factors for hepatic sinusoidal obstruction syndrome following allogeneic hematopoietic stem cell transplantation in pediatric patients.

PURPOSE: Hepatic sinusoidal obstruction syndrome (SOS) represents a serious complication following hematopoietic stem cell transplantation (HSCT). Our study aimed to investigate important risk factors of SOS in a pediatric population. METHODS: This retrospective study analyzed 105 children, adolescents and young adults who underwent allogeneic HSCT at our pediatric HSCT center in Jena. The observation period was 12 years and SOS was defined by the pediatric criteria of the European Society for Blood and Marrow Transplantation (EBMT). RESULTS: 15 out of all 105 patients developed SOS (14.3%). The median time from HSCT to SOS diagnosis was 12 days. The mortality rate of SOS was 20.0%. In univariate analyses, we identified the significant risk factors of patient age < 1 year [odds ratio (OR) = 7.25, p = 0.037], prior treatment with gemtuzumab ozogamicin (OR = 11.00, p = 0.020), high pretransplant ferritin levels above 1500 ng/mL (OR = 4.00, p = 0.033), 2000 ng/mL (OR = 4.69, p = 0.016), and 2400 ng/mL (OR = 5.29, p = 0.005) as well as international normalized ratio (INR) ≥ 1.3 (OR = 5.91, p = 0.009). The following risk factors could be confirmed in multivariate analysis: treatment with gemtuzumab ozogamicin (OR = 9.24, p = 0.048), ferritin > 2400 ng/mL (OR = 5.74, p = 0.023), and INR ≥ 1.3 (OR = 8.02, p = 0.007). CONCLUSION: Our study confirms several risk factors from the current literature. Additionally, this is the first report on the risk factor of high pretransplant INR.

Optimized assay and storage conditions for enzyme activity profiling of ectomycorrhizae.

The aim of a joint effort by different research teams was to provide an improved procedure for enzyme activity profiling of field-sampled ectomycorrhizae, including recommendations on the best conditions and maximum duration for storage of ectomycorrhizal samples. A more simplified and efficient protocol compared to formerly published procedures was achieved by using manufactured 96-filter plates in combination with a vacuum manifold and by optimizing incubation times. Major improvements were achieved by performing the series of eight enzyme assays with a single series of root samples instead of two series, reducing the time needed for sample preparation, minimizing error-prone steps such as pipetting and morphotyping, and facilitating subsequent DNA analyses due to the reduced sequencing effort. The best preservation of samples proved to be storage in soil at 4-6 °C in the form of undisturbed soil cores containing roots. Enzyme activities were maintained for up to 4 weeks under these conditions. Short-term storage of washed roots and ectomycorrhizal tips overnight in water did not cause substantial changes in enzyme activity profiles. No optimal means for longer-term storage by freezing at -20 °C or storage in 100% ethanol were recommended.

Pro-inflammatory effects of DEHP in SGBS-derived adipocytes and THP-1 macrophages.

In the member countries of the Organization for Economic Co-operation and Development (OECD), overweight and obesity affect the majority of the population. The use of environmental chemicals, such as the plasticizer DEHP, has largely increased simultaneously with this development. DEHP is an "obesogen" that interferes with normal adipocyte differentiation and energy homeostasis. Obesity in turn is accompanied by chronic low-grade adipose tissue inflammation, leading to metabolic disorders such as type II diabetes. The main actors in adipose tissue inflammation are adipocytes and macrophages. However, the impact of DEHP on adipose tissue inflammation and the crosstalk between adipocytes and macrophages are unknown and the subjects of the current study. The influence of DEHP on inflammation was investigated in human Simpson-Golabi-Behmel syndrome (SGBS)-derived adipocytes and human THP-1 macrophages. The proinflammatory markers IL8, MCP1, IL1ß, TNFα and others were measured (qRT-PCR, ELISA) in SGBS-derived adipocytes treated with DEHP [day 0 (d0)-d4; 50 µg/ml] and THP-1 macrophages cultured with conditioned medium (CM) from DEHP-treated adipocytes (SGBS-CM) (from d4 and d8). DEHP exposure led to a proinflammatory state in SGBS-derived adipocytes (e.g., increased secretion of IL8 and MCP1). Surprisingly, exposure of THP-1 macrophages to SGBS-CM did not show DEHP-induced effects. However, we demonstrated that medium containing (pre)adipocyte-secreted factors had a significant impact on the expression and secretion of macrophage and inflammatory markers in THP-1 macrophages in general and led to the significantly increased accumulation of intracellular lipid droplets.

The endocrine disruptor DEHP and the ECS: analysis of a possible crosstalk.

Studies of the last decade associated the environmental contamination by di-(2-ethylhexyl)-phthalate (DEHP) with obesity and endocrine malfunction. DEHP was found to interact with several receptors - among them are receptors of the endocannabinoid system (ECS) with high expression levels in adipose tissue. Furthermore, the correlation for BMI and body fat to the serum endocannabinoid level raises the question if the obesogenic and endocrine-disrupting DEHP effects are mediated via the ECS. We therefore characterized the ECS in a human cell model of adipogenesis using the SGBS preadipocytes to subsequently investigate if DEHP exposure affects the intrinsic ECS. The receptors of the ECS and the endocannabinoid-metabolizing enzymes were upregulated during normal adipogenesis, accompanied by an increasing secretion of the adipokines adiponectin and leptin. DEHP affected the secretion of both adipokines but not the ECS, suggesting DEHP to alter the endocrine function of adipocytes without the involvement of the intrinsic ECS.

A 3 year post-intervention follow-up on mortality in advanced heart failure (EVITA vitamin D supplementation trial).

AIMS: Vitamin D supplementation is widely used in the clinical setting, but its effects on mortality and cardiovascular outcomes in patients with heart failure are unclear. This paper reports outcome data that were collected during follow-up of 3 years after closure of the EVITA trial (a 3 year randomized, placebo-controlled, intervention study with 4000 IU vitamin D daily in patients with advanced heart failure), to capture potential latency effects of vitamin D supplementation on clinical outcomes. METHODS AND RESULTS: The prespecified primary endpoint was overall mortality. Secondary endpoints included hospitalization, mechanical circulatory support implantation, high urgent listing for heart transplantation, and heart transplantation. For group comparisons, we used Cox regression models with a time-dependent categorical covariate. The calculated net difference in circulating 25-hydroxyvitamin D between the vitamin D and placebo groups dropped from 60.9 nmol/L at the end of the active study period to 3.2 nmol/L at the end of the post-intervention period. During the entire 6 year period, 73 patients (36.5%) died in the placebo group and 76 (38.8%) in the vitamin D group. Out of these 149 patients, 36 and 39 died during the first 3 years, and 37 and 37 during the second 3 years, respectively. The hazard ratio (HR) for mortality in the vitamin D versus the placebo group was 1.06 [95% confidence interval (CI): 0.68-1.66] for the first 3 years and 1.07 (95% CI: 0.68-1.70) for the 3 year post-intervention follow-up. Compared with the placebo group, the HRs for hospitalization and for mechanical circulatory support implant were significantly higher in the vitamin D group during vitamin D supplementation (HR = 1.31, 95% CI: 1.01-1.68 and HR = 2.01, 95% CI: 1.08-3.76, respectively) but not after vitamin D discontinuation (HR = 1.10, 95% CI: 0.62-1.94 and HR = 0.99, 95% CI: 0.38-2.56, respectively). There was no significant time-dependent effect on the risk of high urgent listing for heart transplantation and heart transplantation. CONCLUSIONS: No beneficial latency effects of vitamin D supplementation on overall mortality could be demonstrated. Instead, the disappearance of unfavourable findings in the vitamin D group (higher HRs for hospitalization and for mechanical circulatory support implant) after vitamin D discontinuation supports the assumption of adverse vitamin D effects on the cardiovascular system at doses of 4000 IU daily.

Androstenedione changes steroidogenic activity of SGBS cells.

The rapid increase of obesity during the last decades and its future prospects are alarming. Besides the general discussed causes of obesity, the 'Developmental Origins of Health and Disease' (DOHaD) hypothesis received more attention in recent years. This hypothesis postulates an adverse influence during early development that programs the unborn child for metabolic dysfunctions later in life. Childhood obesity - an as much increasing problem - can be predisposed by maternal overweight and diabetes. Both, obesity and hyperinsulinemia are major causes of female hyperandrogenemia. As predicted by the DOHaD hypothesis and shown in animal models, developmental androgen excess can lead to metabolic abnormalities in offspring. In this study, we investigated, if androgen exposure adversely affects the adipogenic differentiation of preadipocytes and the endocrine function of adult adipocytes. The human SGBS preadipocyte model was used to affirm the de novo biosynthesis of steroid hormones under normal adipogenesis conditions. Normal adipogenesis was paralleled by an increase of corticosteroids and androgens, whereas estrogen remained at a steady level. Treatment with androstenedione had no effect on SGBS proliferation and differentiation, but adult adipocytes exhibited a significant higher accumulation of triglycerides. Progesterone (up to 2-fold), testosterone (up to 38-fold) and cortisone (up to 1.4-fold) - but not cortisol - were elevated by androstenedione administration in adult adipocytes. Estrogen was not altered. Data suggest that androgen does not negatively influence adipogenic differentiation, but steroidogenic function of SGBS adipocytes.

Role of Vitamin D in Preventing and Treating Selected Extraskeletal Diseases-An Umbrella Review.

Evidence is accumulating that vitamin D may have beneficial effects on respiratory tract, autoimmune, neuro-degenerative, and mental diseases. The present umbrella review of systematic reviews (SRs) of cohort studies and randomised controlled trials (RCTs), plus single Mendelian randomisation studies aims to update current knowledge on the potential role of vitamin D in preventing and treating these extraskeletal diseases. Altogether, 73 SRs were identified. Observational data on primary prevention suggest an inverse association between vitamin D status and the risk of acute respiratory tract infections (ARI), dementia and cognitive decline, and depression, whereas studies regarding asthma, multiple sclerosis (MS), and type 1 diabetes mellitus (T1DM) are scarce. SRs of RCTs support observational data only for the risk of ARI. No respective RCTs are available for the prevention of chronic obstructive pulmonary disease (COPD), MS, and T1DM. SRs of RCTs indicate beneficial therapeutic effects in vitamin D-deficient patients with asthma and COPD, while effects on major depression and T1DM need to be further elucidated. Mendelian randomisation studies do not consistently support the results of SRs. Since several limitations of the included SRs and existing RCTs do not permit definitive conclusions regarding vitamin D and the selected diseases, further high-quality RCTs are warranted.

Prevalence and dynamics of clonal hematopoiesis caused by leukemia-associated mutations in elderly individuals without hematologic disorders.

Clonal hematopoiesis is frequently observed in elderly people. To investigate the prevalence and dynamics of genetic alterations among healthy elderly individuals, a cohort of 50 people >80 years was genotyped for commonly mutated leukemia-associated genes by targeted deep next-generation sequencing. A total of 16 somatic mutations were identified in 13/50 (26%) individuals. Mutations occurred at low variant allele frequencies (median 11.7%) and remained virtually stable over 3 years without development of hematologic malignancies in affected individuals. With DNMT3A mutations most frequently detected, another cohort of 160 healthy people spanning all age groups was sequenced specifically for DNMT3A revealing an overall mutation rate of 6.2% (13/210) and an age-dependent increase of mutation prevalence. A significant difference (p = 0.017) in the DNMT3A expression pattern was detected between younger and healthy elderly people as determined by qRT-PCR. To evaluate the selection of clonal hematopoietic stem cells (HSCs), bone marrow of two healthy individuals with mutant DNMT3A was transplanted in a humanized mouse model. Xenografts displayed stable kinetics of DNMT3A mutations over 8 months. These findings indicate that the appearance of low-level clones with leukemia-associated mutations is a common age-associated phenomenon, but insufficient to initiate clonal selection and expansion without the additional influence of other factors.

Regulation of arginase-1 expression in macrophages by a protein kinase A type I and histone deacetylase dependent pathway.

The aim of the current study was to investigate the cAMP-dependent regulation of arginase-1 (ARG1) expression in RAW-macrophages. Basal ARG1 mRNA expression was low and increased upon incubation with the cAMP analogue Br-cAMP. We used selective agonists of protein kinase A type I (PKAI), type II (PKAII) and exchange protein directly activated by cAMP (EPAC) to determine the pathway responsible for ARG1 expression. Activation of PKAI led to a significant up-regulation of ARG1 mRNA expression and arginase enzyme activity. In contrast, neither activation of PKAII nor activation of EPAC affected ARG1 expression. In addition, it has been shown that histone deacetylase (HDAC) activity plays a critical role in cAMP-dependent transcriptional regulation. Incubation with Br-cAMP and the HDAC inhibitor trichostatin A (TSA) led to a concentration-dependent suppression of ARG1 expression. These data indicate that cAMP-dependent activation of ARG1 expression is mediated by PKAI and requires histone deacetylation.