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In the vast majority of patients with systemic mastocytosis (SM), the bone marrow is the primary extracutaneous site of disease. In addition to bone marrow involvement, other visceral organs such as the spleen, liver or the gastrointestinal tract, may also be affected. However, isolated involvement of a single extramedullary organ is rarely seen in SM. We report on two patients with SM with splenic involvement, lack of 'diagnostic' mast cell (MC) infiltrates in the bone marrow, and absence of skin lesions. In one patient, a myelodysplastic syndrome was diagnosed prior to the detection of SM. Both patients presented with massive splenomegaly and multifocal MC infiltrates in splenic tissues. These MCs also expressed CD25 as well as the C-KIT mutation D816V. In consecutive examinations, the mutation was also detected in the bone marrow in both patients suggesting diffuse infiltration with neoplastic cells. In summary, our data show that the spleen can be a primary site of disease in rare cases of SM. Mastocytosis should therefore be considered as a (rare) differential diagnosis in patients with splenomegaly of unknown etiology.

The European Competence Network on Mastocytosis (ECNM) is a Europe-wide, multinational cooperative approach attempting to improve recognition, diagnosis, and therapy of mastocytosis. The network is composed of local centers, physicians, and scientists who have dedicated their work to patients with mastocytosis. However, because of the rarity and complexity of the disease, each single component in the network alone would fail to meet important demands and to reach solid conclusions in this field of applied medicine. The ECNM represents an attempt to overcome this restriction as a cooperative multicenter platform that should serve as an important basis for the development of new therapeutic strategies and diagnostic concepts, and for the standardization of techniques used to determine diagnostic and prognostic parameters. Moreover, using future central databases and registries, a suitable infrastructure for the development of co-operative multicenter clinical trials will be established. In addition, the ECNM is dedicated to provide the best available information about the disease to patients and physicians.

We hypothesized that TGF-beta1 influences the metabolism of homocysteine (Hcy) and increases its cellular export, which may lead to hyperhomocysteinemia in patients with renal transplants. We exposed human renal proximal tubule epithelial cells (huRPTECs) to different concentrations of TGF-beta1, IL-1alpha, IL-10, or methionine and measured total Hcy (tHcy) in culture supernatants. We then examined the relationship between plasma levels of tHcy and TGF-beta1 in renal graft recipients. In multivariate analysis, the factors mediator (TGF-beta1, IL-1alpha, IL-10), mediator concentration, methionine concentration, and "mediator x concentration" interaction independently influenced tHcy concentrations in culture supernatants. A 31% increase in tHcy was observed after exposure of huRPTECs to TGF-beta1 compared to medium alone. However, TGF-beta1 plasma levels in kidney graft recipients showed no independent association with tHcy plasma concentrations. We demonstrated that the release of Hcy from huRPTECs is enhanced by TGF-beta1, but that TGF-beta1 plasma levels in renal graft recipients show no independent relationship with hyperhomocysteinemia.

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.

Background An increase in colony-forming progenitor cells (CFU) is typically seen in myeloproliferative disorders (MPD). Systemic mastocytosis (SM) is a haemopoietic neoplasm involving myeloid progenitors similar to MPD. In the present study, we measured the levels of peripheral blood (pb) and bone marrow (bm) CFU in patients with different categories of SM, and compared them with those obtained in MPD patients and healthy controls. Materials and methods Numbers of CFU (CFU-GM, BFU-E, CFU-GEMM) were measured in a colony assay in 25 patients with SM [indolent SM (ISM), n = 15; smouldering SM (SSM), n = 3; SM with an associated haematologic clonal non-mast cell lineage disease (SM-AHNMD), n = 5; aggressive SM (ASM), n = 1; mast cell leukaemia (MCL), n = 1] and 37 with MPD [chronic myeloid leukaemia (CML), n = 10; polycythemia vera (PV), n = 8; essential thrombocytosis (ET), n = 9; idiopathic myelofibrosis (IMF), n = 10]. Results In the patients with MPD, elevated numbers of pb CFU were detected in all groups when compared with healthy controls (P

Effects of the glutamate carboxypeptidase II ( GCP2 1561C>T) and reduced folate carrier ( RFC1 80G>A) allelic variants on folate and total homocysteine levels in kidney transplant patients. Background The effect of the glutamate carboxypeptidase II GCP2 1561C>T and the reduced folate carrier 1 RFC1 80G>A polymorphisms on folate and total homocysteine (tHcy) plasma levels of kidney transplant patients are unknown. Methods In a cross-sectional study of 730 kidney allograft recipients, GCP2 1561C>T, RFC1 80G>A, folate, and tHcy plasma levels were analyzed using linear regression models that allowed dependent covariates to follow a gamma distribution for univariate and multivariate analyses. Results The allele frequency for GCP2 1561C>T was 0.05, and 0.43 for RFC1 80G>A. Heterozygosity or homozygosity for GCP2 1561C>T was associated with higher folate plasma levels compared to patients without mutation ( P RFC1 80G>A showed no influence. Multiple testing, also including MTHFR 677C>T and MTHFR 1298A>C, revealed no interaction between the different genotypes and the folate plasma concentration. Neither GCP2 1561C>T nor RFC1 80G>A showed an association with tHcy plasma levels. Conclusion We conclude that GCP2 1561C>T is associated with elevated folate levels. GCP2 1561C>T and RFC1 80G>A are no major determinants of tHcy plasma levels in kidney transplant 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

OBJECTIVE The genetic background of hypertensive crisis is unknown. We examined the association of polymorphisms in genes involved in the renin-angiotensin-aldosterone-system with hypertensive crisis. DESIGN Population-based case-control study. SETTING Emergency department at a tertiary care university hospital. PATIENTS A total of 182 patients with essential hypertension who were admitted to an emergency department for treatment of hypertensive crisis and 182 age- and sex-matched healthy individuals. INTERVENTIONS None. MEASUREMENTS Analysis of polymorphisms in genes coding for angiotensinogen (AJT 704T-->C), angiotensin II receptor 1 (AGTR1 1166A-->C), renin (REN 2646G-->A), renin-binding protein (RENBP 61T-->C), alpha-adducin (ADD1 1378G-->T), beta-2-adrenergic receptor (ADRB2 46A-->G, 79C-->G), and angiotensin I converting enzyme (ACE I/D) was performed by polymerase chain reaction and restriction fragment length polymorphism analysis. MAIN RESULTS Among patients, the ACE I/D polymorphism showed a deviation from Hardy-Weinberg equilibrium (p =.01). In controls, all polymorphisms were in the Hardy-Weinberg equilibrium. The frequency of the DD genotype was increased in patients (n = 70, 38.5%) vs. controls (n = 51; 28.0%;p =.03; odds ratio, 1.61; 95% confidence interval, 1.03-2.50), which was due to the DD genotype in 40 male patients (44%) vs. 23 in male controls (25.3%;p =.004; odds ratio, 3.48; 95% confidence interval, 1.47-8.30). There were no differences in genotype distributions among other polymorphisms. CONCLUSION We demonstrate a possible association of the DD genotype with hypertensive crisis in men.

Influence of mycophenolic acid and tacrolimus on homocysteine metabolism.BackgroundThe effect of mycophenolate mofetil (MMF) on homocysteine (Hcy) metabolism is unknown.MethodsThis in vitro study examined whether mycophenolic acid or tacrolimus influences the formation of Hcy as determined by measuring the total Hcy (tHcy) concentrations in supernatants of human renal proximal tubule epithelial cells. Cells were incubated with and without vitamins (folate, vitamin B6 and B12) in the presence of low or high methionine concentrations at different mycophenolic acid (0, or 5, or 20 μg/mL) or tacrolimus (0, or 10, or 25 ng/mL) concentrations for 24, 48 or 72 hours. The concentration of tHcy in culture supernatants was measured by a fluorescence polarization immunoassay. The effect of MMF on tHcy plasma levels was also examined in 454 kidney graft recipients.ResultsComparisons of tHcy levels in culture supernatants over time by four way ANOVA showed that methionine concentration (P < 0.00001), time (P < 0.00001), vitamins (P = 0.002728), and mycophenolic acid concentration (P = 0.000095) were all significant predictors of tHcy concentrations. This was due to significantly lower tHcy levels with using mycophenolic acid at a high concentration versus control at the 48- and 72-hour time points. By contrast, tacrolimus showed no effect in vitro. Among the kidney graft recipients, male patients on MMF therapy showed lower plasma tHcy concentrations as compared to those on azathioprine (P = 0.03).ConclusionOur study suggests a tHcy lowering effect of MMF in male transplant recipients, which improves the cardiovascular disease risk profile, whereas tacrolimus showed no effect.

Although systemic mastocytosis (SM) is a well-defined hematologic neoplasm, it is sometimes difficult to discriminate between SM and a reactive mast cell (MC) hyperplasia. We describe a patient with aplastic anemia who was treated with recombinant stem cell factor (SCF). In response to SCF, the patient showed transient hematologic improvement and developed a marked increase in MC as well as a transient increase in serum tryptase. Histologic and immunohistochemical examination revealed a huge increase in MC in the bone marrow with focal infiltrates similar to SM. However, most of the SM-criteria were not met: First, MC showed normal cytomorphological characteristics without significant atypias (no cytoplasmic extensions, no oval nuclei, no hypogranulated cytoplasm). Furthermore, bone marrow MC were CD2- and CD25-negative and did not exhibit the C-KIT 2468 A--T mutation (Asp-816-Val). After discontinuation of SCF the MC hyperplasia resolved confirming its reactive nature. Based on our case and similar cases mimicking mastocytosis, it seems of importance to apply recently established SM criteria in order to discriminate between reactive MC hyperplasia and true mastocytosis with certainty.

Diagnostic pathways are an essential subset of clinical pathways and a logical consequence of DRG-based reimbursement. They combine the principle of stepwise reflex and reflective testing with a management concept that helps to fulfill medical needs with organizational and economic efficacy. The two most common formats describing diagnostic pathways are graphical decision trees on paper and “if…then…else” rules on computers. From a laboratory point of view, diagnostic pathways represent “smart” test profiles, which – in contrast to conventional (inflexible) profiles – are not necessarily worked off completely, but just to a point, where a diagnostic decision can be made. This improves the cost-effectiveness of laboratory testing, while making sure that no essential tests are missed. The paper describes benefits and limitations of diagnostic pathways from a medical, organizational, and economic point of view. Their major advantage is also their major drawback, since they make the diagnostic process on the one hand extremely straight-forward and transparent, while on the other hand oversimplifying the underlying medical decision principles. This may provoke the abuse of their primarily medical intentions for mere economic purposes.

The proto-oncogene C-KIT encodes a tyrosine kinase receptor that is expressed on mast cells and haematopoietic stem cells and can show somatic mutations in patients with mastocytosis. Only scattered information is available about mutations in C-KIT in patients with other myeloid neoplasms. Moreover, the prevalence of mutations in C-KIT in bone marrow specimens of individuals with systemic mastocytosis is largely unknown. Using sequence analysis, we have screened cDNAs of the C-KIT domain encompassing codon 510-626 and codon 763-858 in bone marrow (BM) mononuclear cells (MNCs) of patients with myelodysplastic syndromes (n = 28) and patients with systemic mastocytosis (n = 12) for the presence of mutations. Furthermore, restriction fragment length polymorphism analysis was applied for identification of the C-KIT 2468A-->T and the C-KIT 1700T-->G mutation, as well as the C-KIT 1642A-->C polymorphism. All 11 patients with systemic indolent mastocytosis tested positive for C-KIT 2468A-->T. In contrast, no mutation was identified in the case of aggressive mastocytosis. Among patients with myelodysplastic syndromes, no patient showed a somatic mutation in C-KIT. The allele frequency for C-KIT 1642A-->C among the entire patient population was 0.038 and was 0.125 among age- and sex-matched healthy controls. Our data demonstrate that myelodysplastic syndromes without histological or cytological evidence of mastocytosis do not exhibit somatic mutations in exons 10, 11, 12, 16, 17 and 18 of C-KIT. In contrast, BM MNCs of patients with systemic indolent mastocytosis were all positive for C-KIT 2468A-->T and negative for additional mutations in these exons. The C-KIT 1642A-->C polymorphism is not associated with myelodysplastic syndrome or systemic mastocytosis.

Effect of MTHFR genotypes and hyperhomocysteinemia on patient and graft survival in kidney transplant recipients. Background The total homocysteine (tHcy) plasma level, which is partly determined by the MTHFR 677C→T genotype, may be associated with vascular disease. We prospectively examined the influence of MTHFR genotypes (677C→T, 1298A→C) and tHcy plasma concentration on all cause mortality and graft outcomes of renal transplant recipients. Methods Baseline tHcy plasma levels of 189 patients (three groups with either the MTHFR 677CC, CT or TT genotype, including 63 patients in each group, were matched for age, gender, body mass index and creatinine clearance at baseline), were obtained between September 1996 and May 1997. Follow-up data (time until return to dialysis therapy, time and cause of death) were collected from April to June 1999. Kaplan-Meier survival estimations were calculated and plotted, the groups (three MTHFR 677C→T genotype groups, or three MTHFR 1298A→C genotype groups, or two groups with tHcy plasma levels above/below 15 μmol/L) were compared by log-rank test. Age, gender, body mass index (BMI), time since transplantation, serum creatinine, creatinine clearance, combined MTHFR 677C→T/1298A→C genotypes, tHcy, folate and vitamin B 12 plasma levels were evaluated with regard to graft and patient survival in a multivariate Cox-proportional hazard regression model. Results During the follow-up period of 2.26 ± 0.66 years, 9 patients died (5 in the TT, 2 in the CT and 2 in the CC genotype group; P = 0.34) and 22 returned to dialysis treatment (7 in the TT, 9 in the CT and 6 in the CC genotype group; P = 0.65). There was also no influnce of MTHFR 1298A→C genotypes (AA genotype, 114 patients; AC genotype, 64 patients; CC genotype, 11 patients) on patient or graft survival ( P = 0.7087 and P = 0.1633, respectively). Two of 93 patients with a tHcy plasma level ≤15 μmol/L died, in contrast to 7 of 96 patients in the low tHcy > 15 μmol/L group, P = 0.0778. Two patients in the low tHcy group had to return to dialysis, in contrast to 20 patients in the high tHcy group ( P = 0.0001). In the multivariate model there was no significant predictor of patient survival, and the serum creatinine was the only predictor of graft survival ( P Conclusions In summary, our study shows that neither MTHFR 677C→T/1298A→C genotypes nor hyperhomocysteinemia are independently associated with patient or graft survival following kidney transplantation.

Objective The polymorphism 825C-->T in exon 10 of the gene GNB3 encoding the beta3 subunit of heterotrimeric guanine nucleotide binding regulatory proteins (G-proteins) results in a splicing variant (GNB3-s) in which the nucleotides 498-620 of exon 9 are deleted. The T allele has been shown to be overrepresented in patients with essential hypertension. Because GNB3-s may support the development of severe elevation of blood pressure, we hypothesized that GNB3 825C-->T may be present more frequently in patients with hypertensive crisis. Design Case control study. Setting Department of Emergency Medicine at the University Hospital of Vienna, Vienna, Austria. Patients A total of 174 patients admitted to an emergency department for treatment of hypertensive crisis diagnosed as suffering from essential hypertension. Interventions None. Measurements and main results Patients were genotyped for the 825C-->T transition in GNB3. An equal number of age- and gender-matched normotensive, healthy individuals served as the control population. The allele frequency of 825C-->T in the GNB3 gene was 0.310 in patients with hypertensive crisis and 0.342 in the control group. There was no difference in genotype distribution and allele frequency between the patients and the age- and gender-matched control group or between the observed prevalence and the occurrence rate expected from the Hardy-Weinberg principle within each group. Conclusions GNB3 825C-->T is not associated with the phenotype of hypertensive crisis in patients suffering from essential hypertension. Furthermore, our data do not support the concept that the 825C-->T transition in the GNB3 gene is associated with essential hypertension.

Homocysteine is associated with atherosclerosis and enhanced cardiovascular risk. In previous studies, treatment with folic acid up to 15 mg/d failed to correct hyperhomocysteinemia in the majority of end-stage renal disease patients. A dose of 30 or 60 mg of folic acid per day was compared with 15 mg/d in an attempt to normalize hyperhomocysteinemia in 150 hemodialysis patients. In a randomized, double-blind, multicenter study, 144 patients completed the 4-wk treatment period and 121 patients completed the 6-mo follow-up. Total homocysteine plasma levels were reduced by 32.1% (15 mg/d), 29. 9% (30 mg/d), or 37.8% (60 mg/d) with no significant differences found between the three treatment groups. Baseline total homocysteine plasma concentration was an independent predictor of the response to folic acid therapy (P = 0.0001), whereas the 5, 10-methylenetetrahydrofolate reductase polymorphisms (MTHFR 677C --> T and 1298A --> C) had no influence. Nevertheless, patients with the MTHFR 677TT genotype more frequently attained normal total homocysteine plasma levels than patients with the CC or CT genotype (P = 0.025). In response to 60 mg of folic acid per day, TT genotype patients had lower folate plasma levels compared to CC or CT genotype patients (P = 0.016). After completion of the 4-wk treatment period with 30 or 60 mg of folic acid per day, there was a marked rebound of total homocysteine plasma levels at the end of the follow-up in patients with the MTHFR 677TT genotype, which even exceeded baseline values in several patients (P = 0.0001). This study clearly demonstrates that doses of 30 or 60 mg of folic acid per day are not more effective than 15 mg/d in reducing hyperhomocysteinemia in regular hemodialysis patients. Patients with the MTHFR 677TT genotype are more likely to realize normal total homocysteine plasma levels. Folic acid at 30 or 60 mg/d but not 15 mg/d results in a rebound of total homocysteine plasma concentrations when treatment is stopped.

Objective: To test the hypothesis that the prevalence of hyperhomocysteinemia is increased in critically ill patients and correlates with disease severity and mortality in these patients. Design: A prospective study. Setting: Three medical intensive care units at the University of Vienna Medical School serving both medical and surgical patients. Patients: All consecutive admissions (n = 56) during a period of 4 wks. A total of 112 age- and gender-matched healthy individuals constituted the control group. Interventions: None. Measurements and Main Results: Blood samples were drawn within 24 hrs after admission for analysis of total homocysteine (tHcy), folate, vitamin B 6 levels, and vitamin B 12 levels as well as to identify the 677C→T polymorphism in the gene coding for the enzyme 5,10-methylenetetrahydrofolate reductase. Acute Physiology and Chronic Health Evaluation III scores at admission and 24 hrs after admission as well as 30-day survival were documented in all patients. Hyperhomocysteinemia was more prevalent in critically ill patients (16.1%; 95% confidence interval, 7.6% to 28.3%) compared with age- and gender-matched healthy individuals (5.4%; 95% confidence interval, 2.0% to 11.3%; chi-square test; p =.022). There was no difference in tHcy plasma concentrations in the first 24 hrs after admission to an intensive care unit between survivors and nonsurvivors. The 5,10-methylenetetrahydrofolate reductase 677C→T polymorphism had no influence on tHcy levels and survival of intensive care unit patients. Conclusions: The prevalence of hyperhomocysteinemia is increased in critically ill patients compared to age- and gender-matched healthy individuals. The clinical significance of this finding remains to be determined.

Effect of MTHFR 677C>T on plasma total homocysteine levels in renal graft recipients.BackgroundHyperhomocysteinemia is an established, independent risk factor for vascular disease morbidity and mortality. The 5,10-methylenetetrahydrofolate reductase (MTHFR) gene polymorphism C677T has been shown to result in increased total homocysteine concentrations on the basis of low folate levels caused by a decreased enzyme activity. The effect of this polymorphism on total homocysteine and folate plasma levels in renal transplant patients is unknown.MethodsWe screened 636 kidney graft recipients for the presence of the MTHFR C677T gene polymorphism. The major determinants of total homocysteine and folate plasma concentrations of 63 patients, who were identified to be homozygous for this gene polymorphism compared with heterozygotes (N = 63), and patients with wild-type alleles (N = 63), who were matched for sex, age, glomerular filtration rate (GFR), and body mass index, were identified by analysis of covariance. The variables included sex, age, GFR, body mass index, time since transplantation, folate and vitamin B12 levels, the use of azathioprine, and the MTHFR genotype. To investigate the impact of the kidney donor MTHFR genotype on total homocysteine and folate plasma concentrations, a similar model was applied in 111 kidney graft recipients with stable graft function, in whom the kidney donor C677T MTHFR gene polymorphism was determined.ResultsThe allele frequency of the C677T polymorphism in the MTHFR gene was 0.313 in the whole study population [wild-type (CC), 301; heterozygous (CT), 272; and homozygous mutant (TT), 63 patients, respectively] and showed no difference in the patient subgroups with various renal diseases. The MTHFR C677T gene polymorphism significantly influenced total homocysteine and folate plasma concentrations in renal transplant recipients (P = 0.0009 and P = 0.0002, respectively). Furthermore, a significant influence of the GFR (P = 0.0001), folate levels (P = 0.0001), age (P = 0.0001), body mass index (P = 0.0001), gender (P = 0.0005), and vitamin B12 levels (P = 0.004) on total homocysteine concentrations was observed. The donor MTHFR gene polymorphism had no influence on total homocysteine and folate levels. Geometric mean total homocysteine levels in patients homozygous for the mutant MTHFR allele were 18.6 μmol/liter compared with 14.6 μmol/liter and 14.9 μmol/liter in patients heterozygous for the MTHFR gene polymorphism and those with wild-type alleles (P < 0.05 for TT vs. CT and CC). Geometric mean folate levels were lower in CT and TT patients (11.2 and 10.2 nmol/liter) compared with CC patients (13.6 nmol/liter, P < 0.05 vs. CT and TT).ConclusionsThis study demonstrates that homozygosity for the C677T polymorphism in the MTHFR gene significantly increases total homocysteine concentrations and lowers folate levels in kidney graft recipients, even in patients with excellent renal function (GFR more than median). These findings have important implications for risk evaluation and vitamin intervention therapy in these patients who carry an increased risk for the development of cardiovascular disease.

A subset of patients with systemic mastocytosis (SM) develop acute myeloid leukaemia (AML). However, little is known about the biology of such leukaemias and their relationship to the mast cell (MC) lineage. We report on two female patients who suffered from SM and AML. According to FAB criteria, the leukaemias were classified as AML-M4 (patient 1) and AML-M0 (patient 2). The coexistence of the two distinct neoplasms (AML and SM) was demonstrable by immunostaining of serial bone marrow (BM) sections with monoclonal antibodies (mAb). In particular, the MC infiltrates were found to react with mAb against MC-tryptase and MC growth factor receptor c-kit (CD117), but not with mAb to CD15 or CD34. In contrast, the AML blasts were immunoreactive for CD15 (patient 1) or CD34 (patient 2), but did not express tryptase. The c-kit point mutation Asp Val at codon 816, considered to play a role in the transformation of MC progenitors, was detected in patient 1 in a BM cell fraction containing 4% MC. However, no c-kit mutation was found in pure AML blasts (

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.

The expression of the natural killer (NK) cell marker CD56 has been reported to occur in NK cell lymphomas/leukemias and a small group of peripheral T-cell lymphomas but has not been studied extensively in primary intestinal non-B-cell lymphomas. Normal human jejunal intraepithelial lymphocytes (IELs) are mainly T-cell receptor (TCR)-αβ + CD3 + CD8 + CD5 low and include an ∼15% fraction of CD56 + cells that could be the cells of origin for CD56 + intestinal T-cell lymphoma (ITL). To test this hypothesis, 70 cases diagnosed as ITL were immunophenotyped, and 15 CD56 + cases (21%) were identified. The majority of the CD56 + lymphomas was of monomorphic small to medium-sized histology, shared the common phenotype βF1 ± CD3e/cyt + CD8 + CD4 − CD5 − CD57 − TIA-1 + and had clonally rearranged TCR γ-chain genes. In contrast, the CD56 − lymphomas were mainly composed of pleomorphic medium and large cells or had a morphology most consistent with anaplastic large-cell lymphoma and were mostly CD8 − . These findings suggest that the majority of CD56 + intestinal lymphomas are morphologically and phenotypically distinct T-cell lymphomas most likely derived from activated cytotoxic CD56 + CD8 + IELs. Some overlapping histological and clinical features between CD56 + and CD56 − ITLs indicate that the former belong to the clinicopathological entity of ITL. The consistent expression of cytotoxic-granule-associated proteins introduces ITL (both CD56 + and CD56 − ) into the growing family of usually aggressive extranodal lymphomas of cytotoxic T-cell and NK-cell derivation. In contrast to putative NK-cell lymphoma of the sinonasal region, intestinal NK-cell lymphoma seems to be very rare.

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.

Background The pathophysiology of hypertensive crises is poorly understood. To date, no information is available about genetic determinants underlying the individual risk for development of hypertensive urgencies or emergencies. Recently, mutations in the beta subunit (h beta ENaC) and the gamma subunit (h gamma ENaC) of the human epithelial sodium channel (hENaC) have been shown to result in excessive elevation of blood pressure in patients with Liddle's syndrome. Methods Using polymerase chain reaction and direct sequencing of amplification products we have screened 90 consecutive out-patients with hypertensive urgency or hypertensive emergency for the presence of mutations in the carboxy terminus of these genes. Furthermore, serum potassium concentrations were determined in all 90 patients, and serum aldosterone levels and plasma renin activity were measured in a subset of 34 patients. Results Among 71 patients with hypertensive urgency (78.9%) and 19 patients with hypertensive emergency (21.1%) not one individual showed a mutation in genomic DNA extending from codon 532 to codon 637 of h beta ENaC and from codon 525 to codon 651 of h gamma ENaC. Twelve of 90 patients showed mild hypokalaemia (13.3%), 16 of 34 patients had a plasma renin activity below the lower normal range (47.1%) and one of 34 patients had a low serum aldosterone concentration (2.9%). Conclusions The present study clearly demonstrates the absence of mutations in the carboxy terminus of the h beta ENaC and h gamma ENaC gene of hENaC in an Austrian cohort of 90 patients suffering from hypertensive crisis.

Major determinants of hyperhomocysteinemia in peritoneal dialysis patients. The mechanisms leading to elevated total homocysteine concentrations in peritoneal dialysis patients are only partially understood. We show that a common polymorphism in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene (C677T transition) results in increased total homocysteine levels in peritoneal dialysis patients compared to age- and sex-matched healthy individuals. The allelic frequency of the C677T transition in the MTHFR gene in peritoneal dialysis patients (0.29) was comparable to the frequency in healthy individuals (0.34). Separate comparison of the total homocysteine plasma levels between non-carriers of the MTHFR polymorphism (C/C), heterozygous (C/T) and homozygous (T/T) subjects was performed by analysis of covariance in the patient and the control group. In the patient group the mean total homocysteine level was 61.7 ± 40.1 μmol/liter in individuals with the (T/T) genotype, which was significantly higher than the total homocysteine concentration of 23.1 ± 15.8 μmol/liter in (C/T) patients and 22.2 ± 11.1 μmol/liter for non-carriers ( P = 0.0001). Vitamin B 12 ( P = 0.0001), folate ( P = 0.0005), serum creatinine ( P = 0.016), albumin ( P = 0.0157) and dialysis center ( P = 0.0173) significantly influenced total homocysteine plasma levels in peritoneal dialysis patients, whereas this was not the case for age, gender, weekly Kt/V, weekly creatinine clearance, residual renal function, duration of dialysis, mode of peritoneal dialysis and vitamin intake. Folate levels in peritoneal dialysis patients were significantly affected by the MTHFR genotype ( P = 0.016). Elevated total homocysteine levels in diabetic patients with cardiovascular disease were associated with increased cardiovascular morbidity. In summary, the present study provides evidence that homozygosity for the C677T transition in the MTHFR gene, low vitamin B 12 and low folate levels result in elevated total homocysteine levels in peritoneal dialysis patients.

interest for the management of allograft recipients in whom monoclonal and polyclonal gammaglobulinaeBackground. C-reactive protein (CRP) and ferritin serum levels represent routine laboratory parameters mia are frequently observed. We therefore recommend reanalysis of the respective plasma proteins by latexin the monitoring of renal failure patients. Analysis of CRP, ferritin and other serum proteins can be per- free assays in patients with hypergammaglobinaemia showing no clinical signs of an acute infectious disease formed using latex-enhanced or non-latex-enhanced immunoassays. We report on a renal transplant patient or malignant disorder. with polyclonal IgM hypergammaglobulinaemia having markedly elevated serum CRP and ferritin Key words: latex immunoassays; hypergammagloblevels (as detected by latex-enhanced immunoassays) ulinaemia; C-reactive protein; ferritin in the absence of clinical signs of an infectious or malignant disorder. Methods. CRP and ferritin serum levels were determined with various immunoassays with and without Introduction latex enhancement. To characterize the causative agent for the elevated CRP and ferritin values, the patient’s C-reactive protein (CRP) and ferritin are routine laborand a control serum were fractionated by gel filtration atory parameters in the monitoring of renal failure on a Sephacryl S-300 column. Serum fractions were patients. For quantification of CRP and ferritin a subjected to further analysis for reactivity in CRP and variety of methods such as turbidimetry or nepheloferritin assays. In addition, patient’s serum samples metry have been developed. In principle all these were investigated for reactivity with various other methods use a heterologous antibody directed against latex-based immunoassays (rheumatoid factor, antis- the target protein. In order to improve sensitivity of treptolysin O, antistreptococcal DNase B). immunoassays, enhancement using latex particles is Results. Using latex-enhanced CRP and ferritin frequently applied [1 ]. Falsely high levels of CRP have immunoassays, markedly elevated serum levels were been reported in patients with high titres of rheumatoid obtained (CRP 726 mg/l determined by turbidimetry, factor [2‐4] or monoclonal gammopathies, predomi398 mg/l determined by nephelometry; ferritin, nantly of the IgM type [5‐8]. In the latter case, the 20 000 mg/l determined by turbidimetry). In contrast, mechanism resulting in the falsely high CRP determinaassays without latex enhancement revealed levels tion has been shown to be due to a non-specific within the normal range for both serum proteins (CRP reaction between the patient’s monoclonal immuno

For a long time the biology of mast cells has remained an enigma. During the past few decades, molecular research has filled many of the gaps in our understanding of this multifunctional cell type. Several concepts are now established about the origin and development of mast cells. However, the mechanisms leading to autonomous proliferation of mast cells in patients with indolent or aggressive mastocytosis still require further investigation. This article focuses on the recent advances in the field of mast cell development and abnormal mast cell proliferation, and reviews data from the most significant mouse models, which have provided the basis for many of these new insights.

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.

We describe a multiplex polymerase chain reaction (PCR) method suitable for the detection of all T-cell receptor (TCR) gamma-chain gene rearrangements in patients with lymphoproliferative diseases. 40 patients with various lymphoproliferative disorders and 40 healthy individuals were tested. Clonal TCR gamma rearrangements were identified in all patients with malignant disorders, and in one of 10 cases with established reactive lymphocytosis but not in normal controls. In all individuals testing positive, the patient's specific V and J segment involved in the rearrangement could be determined by simply splitting the multiplex primer mix. Our data show that the multiplex PCR technique enables rapid, simple and sensitive screening for clonal TCR gamma chain gene rearrangements.

Purpose: Anderson-Fabry disease is an inherited lysosomal storage disease with a broad and unspecific range of symptoms, a painful course of disease and early death. The recent development of new enzyme therapy emphasises the need for early diagnosis and treatment of undiagnosed patients. One of the affected organs of Anderson-Fabry disease is the eye. Cornea verticillata – corneal opacities and corneal dystrophy – as well as tortuositas vasorum can occur in an early stage of the disease affecting almost all hemizygous men and more than 70% of heterozygous women. In order to identify unknown cases with Anderson-Fabry disease, we carried out a screening programme contacting Austrian ophthalmologists. Methods: All 658 Austrian ophthalmologists were asked to record patients with cornea verticillata as well as tortuositas vasorum – twice at an interval of 3 months. Results: 33% of the contacted ophthalmologists replied, identifying 5 patients suspected of having Anderson-Fabry disease. After additional examinations including tests for enzyme activities Anderson-Fabry disease was confirmed in 1 man. Conclusion: We have identified 1 case with Anderson-Fabry disease through our ophthalmology screening programme among a population of approximately of 8 million. Ophthalmologic screening programmes for ocular manifestations typical of Anderson-Fabry disease are limited because of the moderate visual affection in these patients. Nevertheless, considering the limited options to detect such cases otherwise, ophthalmologists have a major responsibility to identify patients with Anderson-Fabry disease on routine examinations.

Although mast cells are hematopoietic cells, little is known about the origin of their precursors in vivo. In this study, the origin (donor v recipient genotype) of human mast cells (MCs) was analyzed in a patient who underwent allogeneic bone marrow transplantation (BMT). The patient presented with secondary acute myeloid leukemia (French-American- British classification, M2) arising from refractory anemia with excess of blast cells and bone marrow (BM) mastocytosis. Transplantation was performed in chemotherapy-induced complete remission. On days 88, 126, 198, and 494 after BMT, mast cells were enriched to homogeneity from bone marrow mononuclear cells (BM MNCs) by cell sorting for CD117+/CD34- cells. Purified mast cell populations were CD117(c-kit)+ (> 95%), CD34- (< 1%), CD3- (< 1%), CD14- (< 1%), and virtually free of contaminating cells as assessed by Giemsa staining. The genotype of MCs was analyzed after amplification by polymerase chain reaction (PCR) of a variable number tandem repeat (VNTR) region within intron 40 of the von Willebrand factor (vWF) gene. Unexpectedly, on days 88 and 126 after BMT, sorted MCs displayed recipient genotype as shown by vWF.VNTR-PCR. However, on days 198 and 494, PCR analysis showed a switch to donor genotype in isolated mast cells. Peripheral blood (PB) and BM MNC as well as highly enriched (sorted) CD3+ T cells (PB, BM), CD4+ helper T cells (PB), CD8+ T cells (PB), CD19+ B cells (PB), CD14+ monocytes (PB, BM), and CD34+ precursor cells (BM) showed donor genotype throughout the observation period. Together, these results provide evidence that human MCs developed in vivo from transplanted hematopoietic stem cells. Engraftment and in vivo differentiation of MCs from early hematopoietic progenitor cells may be a prolonged process.

Entamoeba histolytica has an unusual nuclear structure characterized by a low degree of chromatin condensation and the absence of stainable metaphase chromosomes. Although nucleosome-like particles were observed, no information about histones was available so far. In this paper we describe a cDNA clone with significant homology to H3 histones that was isolated from a library of pathogenic E. histolytica. The complete cDNA encodes a 15-kDa polypeptide, which like the histone sequence from Volvox carteri is shorter by one residue than the human homologue. The amino acid sequence has only 69% identity with human H3.3 histone and 67% identity with the human H3.1 histone. This is the highest degree of sequence divergence observed for any eukaryote H3 histone sequence. Our results indicate that this divergence may contribute to the unusual chromatin structure of E. histolytica.

Because Fabry disease is medically important and effective interventions exist, an early diagnosis in symptomatic individuals would be desirable. However, the diagnosis is often delayed for several years after first symptoms have developed. One approach to overcome this clinical deficit is case finding studies among people with medical problems typically found in Fabry disease patients. During the last decade such studies revealed a surprising high prevalence of Fabry disease among patients with chronic kidney disease, heart disease, or stroke. Because specific treatment is available, several experts endorsed case finding studies in high risk populations within an organized frame or even routine testing of individuals with stroke, kidney- or heart disease. The methodologies recommended for case finding strategies include testing for enzyme activity in dried blood spots or examination of urine specimen for increased excretion of globotriaosylceramide. Together with newborn screening the results of large case finding studies among patients with stroke, left ventricular hypertrophy, or chronic kidney disease will allow for a better approximation of the true prevalence of the disease and will probably result in an improvement of clinical care of affected individuals.

The poor general state of health of the Austrian youth - which is possibly representative for the western industrial world - will have dramatic effects on our health care system in years to come. Health risks among adolescents, including smoking, alcohol, obesity, lack of physical activity and an unhealthy diet, will lead to an increase in chronic diseases. A preventive measure against such a development could be to reinforce health awareness through the use of web and mobile applications supporting self observation and behavior change. In this paper, we present an overview of the latest developments in the area of mobile wellness and take a look at the features of applications that constitutes the current state of the art, as well as their shortcomings and ways of overcoming these. Finally, we discuss the possibilities offered by new technological developments in the area of mobile devices and by incorporating the characteristics that make up the Web 2.0.

The diagnosis of Anderson-Fabry disease is often delayed or even missed. As severe renal manifestations are a hallmark of alfa-galactosidase A (AGAL) deficiency, we tested the hypothesis that Anderson-Fabry disease is under-recognized among male kidney transplant recipients. This nation-wide study in Austria enrolled 1306 patients (ca 65% of all kidney transplanted males) from 30 kidney centers. AGAL activity was determined from filter paper dried blood spots by a fluorescence assay. A positive screening test was defined by an AGAL activity below 1.5 nmol/h/ml. In patients with a positive blood spot-screening test, AGAL activity was re-examined in peripheral blood leukocytes. Genetic testing for mutations in the GLA gene was performed by sequencing to confirm the diagnosis of Anderson-Fabry disease. Two previously not recognized cases with Anderson-Fabry disease were identified. Our study is the first showing that a diagnosis of Anderson-Fabry disease can be missed even in patients who undergo kidney transplantation. Case-finding strategies may be considered a useful tool for diagnosis of this rare disease that may be somewhat more prevalent among kidney transplant recipients compared with dialysis populations.

Background Basophilic crisis and eosinophilia are well recognized features of advanced chronic myeloid leukaemia. In other myeloid neoplasms, however, transformation with marked basophilia and eosinophilia is considered unusual. Design We examined the long-term follow-up of 322 patients with de novo myelodysplastic syndromes (MDS) to define the frequency of basophilic, eosinophilic and mixed lineage (basophilic and eosinophilic) transformation. Results Of all patients, only one developed mixed lineage crisis (≥ 20% basophils and ≥ 20% eosinophils). In this patient, who initially suffered from chronic myelomonocytic leukaemia, basophils increased to 48% and eosinophils up to 31% at the time of progression. Mixed lineage crisis was not accompanied by an increase in blast cells or organomegaly. The presence of BCR/ABL and other relevant fusion gene products (FIP1L1/PDGFRA, AML1/ETO, PML/RARα, CBFβ/MYH11) were excluded by PCR. Myelomastocytic transformation/myelomastocytic leukaemia and primary mast cell disease were excluded by histology, KIT mutation analysis, electron microscopy and immunophenotyping. Basophils were thus found to be CD123+, CD203c+, BB1+, KIT- cells, and to express a functional IgE-receptor. Among the other patients with MDS examined, 4(1·2%) were found to have marked basophilia (≥ 20%) and 7(2·1%) were found to have massive eosinophilia ( ≥ 20%), whereas mixed-lineage crisis was detected in none of them. Conclusions Mixed basophil/eosinophil crisis may develop in patients with MDS but is an extremely rare event.

Background Systemic mastocytosis (SM) is a clonal myeloid disorder characterized by abnormal accumulation and growth of mast cells (MC) in internal organs. In most cases, the bone marrow is involved. Expression of CD25 in bone marrow MC, with or without coexpression of CD2, is an important minor SM criterion. So far, most studies have examined CD25-expression on MC by flow cytometry. Materials and methods We examined the expression of CD25 in MC in patients with SM (n = 25) by immunohistochemistry (IHC) and compared these data with results obtained by flow cytometric assessment of CD25-expression. In addition, we compared CD25-staining results with that obtained with an antibody against CD2. Results In a majority of all patients (> 80%), CD25 was detectable by both staining techniques. However, in one patient, CD25 was only detectable on MC by IHC, but not by flow cytometry, and in two patients in whom IHC could not be applied because of lack of compact MC infiltrates, flow cytometry revealed aberrant expression of CD25. The antibody against CD2 produced diagnostic staining results in a smaller group of patients (flow cytometry: 65%; IHC: 28% of SM cases) compared to CD25 (> 80%). Conclusions CD25-IHC is equally diagnostic and sensitive in SM compared to flow cytometry and thus can be recommended as a diagnostic test. Our data also suggest that the diagnostic value of CD25 exceeds that of CD2, and that optimal assessment of CD25-expression in neoplastic MC in all patients requires the application of both techniques, flow cytometry and IHC.

Hypercalcemia in persistent secondary hyperparathyroidism after kidney transplantation is considered to result from increased bone resorption. Bone biopsies' studies, however, have never been performed in these patients. Bone biopsies after double tetracycline labeling were obtained from 17 patients with hypercalcemic hyperparathyroidism and an estimated glomerular filtration rate > 30 mL/min/1.73 m2. Serologic bone markers, calcitriol, intact fibroblast growth factor-23 (iFGF-23), and serum and 24h urine concentration of calcium and phosphate were measured in all patients. Tubular maximum for phosphate corrected for GFR (TmP/GFR), and the fractional excretion of calcium (FeCa) were calculated. High-turnover renal osteodystrophy (ROD) was present in nine and low-turnover ROD in eight patients. The bone formation rate was significantly associated with bone alkaline phosphatase, c-telopeptide and osteocalcin. In patients with high turnover ROD, osteocalcin was also significantly higher than in patients with decreased bone formation. The FeCa was normal or below normal in 14/17 patients. TmP/GFR was below normal in all patients. Neither intact PTH nor iFGF-23 was associated with TmP/GFR, FeCa or any histomorphometric bone parameter. We conclude that hypercalcemia of posttransplant hyperparathyroidism can be associated with high or low turnover bone disease. Decreased calcium excretion suggests an additive tubular effect on hypercalcemia.

In kidney transplant recipients (KTR), C-reactive protein (CRP) has been shown to be associated with increased mortality, but data on this association within the high-sensitivity (hs) range of CRP (5 mg/l) are lacking. We prospectively studied 710 prevalent and stable KTR over6 years. We thawed frozen plasma and measured baseline hs-CRP using an ultrasensitive assay. Detailed clinical and demographic baseline characteristics were available for study. We stratified patients by quartile of hs-CRP within the hs range (5 mg/l), and also included KTRs whose hs-CRP was above the hs range (5-10 and10 mg/l). We used multivariate proportional hazards models to test for independent associations. After careful multivariate adjustment, we found a J-shaped association between hs-CRP and mortality. Compared with KTR whose hs-CRP was in the second lowest quartile of hs-CRP (0.06-1.26 mg/l), patients in the lowest quartile (0.06 mg/l) had more than twice their mortality risk (HR = 2.07; 95% CI: 1.05-4.07), as did patients whose hs-CRP wasor =2.44 mg/l (all HRs2.27). No association was found between hs-CRP and death-censored allograft loss. In contrast to the general population, the association between hs-CRP and mortality in KTRs is not linear, but J-shaped, suggesting that KTRs with very low hs-CRP may also be at increased risk of death.

Summary Disturbances in calcium and phosphate metabolism have been linked to increased mortality in hemodialysis patients, but not in kidney transplant recipients (KTR). We enrolled 733 KTR from the Vienna General Hospital into this study. Detailed demographic, clinical, laboratory, and transplant-related information was collected at baseline. We used the Austrian Dialysis and Transplantation Registry for follow-up. Using multivariate proportional hazard regression, we examined the independent associations between serum calcium, serum phosphate, and calcium phosphate (CaPO4) product with the outcomes of death from any cause and kidney allograft loss. Over a median follow-up of >6 years, 154 patients died and 259 kidney allografts were lost. Associations with serum calcium, phosphate concentrations, and CaPO4 product concentrations were found for allograft loss, but not for patient mortality. Patients in the highest quintile of phosphate concentration and CaPO4 product had an increased risk for allograft loss compared with patients in the lowest quintile of these parameters (hazards ratio, HR = 2.15; 95% confidence interval, CI: 1.36–3.40 and HR = 1.72; 95% CI: 1.10–2.71, respectively). High calcium levels were associated with a reduced risk for allograft loss. Results were even more pronounced for death-censored allograft loss. High concentrations of serum phosphate and CaPO4 product were associated with an increased risk for allograft loss in these KTR, whereas high serum calcium concentrations seemed to lower the risk.

Patients with chronic kidney disease who are on dialysis or with a kidney transplant have higher total plasma homocysteine concentrations than individuals who are free from kidney disease. Several single-nucleotide polymorphisms of genes encoding enzymes that are involved in homocysteine metabolism have been studied in these patients. These polymorphisms are located in genes encoding of 5,10-methylenetetrahydrofolate reductase (MTHFR), methionine synthase reductase, methionine synthase, cystathionine beta-synthase, glutamate carboxy peptidase II, reduced folate carrier 1, and transcobalamin II. Among the single-nucleotide polymorphisms studied, only MTHFR 677CT was associated consistently with total plasma homocysteine levels, but there currently is no evidence of any association between MTHFR 677CT genotype and long-term outcomes.

Currently, no evidence is available on the putative associations between a novel single nucleotide polymorphism of the 5,10-methylenetetrahydrofolate reductase gene MTHFR 1793GA and plasma levels of vitamin B(12), folate, or total homocysteine (tHcy).In a cross-sectional study of 730 kidney allograft recipients, patients were categorized by MTHFR 1793GA genotype. In univariate and multivariate linear regression models that allowed the outcome variables vitamin B(12), folate, and tHcy plasma levels to follow a gamma distribution, we tested for possible associations of allelic variants of MTHFR 1793GA and these three dependent variables. As hypothesized in previous work, we specifically evaluated possible effect modification between the MTHFR 1793GA and 1298AC mutations on these outcomes.The allele frequency for MTHFR 1793GA was 0.052. Heterozygosity (N= 72) or homozygosity (N= 2) for MTHFR 1793GA was not independently associated with plasma levels of vitamin B(12) (P= 0.33) or tHcy (P= 0.70), but a borderline association with higher folate concentrations was detected (Deltafolate = 1.91 nmol/L) (95% CI -0.03 to 3.86 nmol/L) (P= 0.05). Further, we found strong and significant positive interactions between the MTHFR 1793GA and 1298AC mutations on vitamin B(12) concentrations.Higher folate concentrations in kidney transplant recipients with MTHFR 1793GA or 1793AA and markedly higher concentrations of vitamin B(12) in patients with combined MTHFR 1793GA and 1298AC mutations may contribute to the survival advantage that has been postulated for such patients showing these genotypes.

Elevated plasma total homocysteine (tHcy) concentrations are associated with premature cardiovascular disease. We assessed tHcy, folate, vitamin B12 (Vit B12), vitamin B6 (Vit B6), and genetic polymorphisms potentially enhancing tHcy in patients with juvenile idiopathic arthritis (JIA) and healthy controls.Open study of 56 consecutive patients with JIA and 62 controls.tHcy concentrations were normal in JIA patients (mean 6.5 +/- 2 micromol/l) and controls (mean 7.5 +/- 2.2 micromol/l). Folate concentrations were significantly higher in JIA patients (40.2 +/- 67.9 ng/ml) compared to controls (13.6 +/- 8.2 ng/ml). The prevalence of genetic polymorphisms coding for key enzymes in the homocysteine pathway did not differ between patients and controls. Erythrocyte sedimentation rate (ESR) showed significant inverse correlations with circulating Vit B6 and tHcy concentrations.No evidence for hyperhomocysteinemia or evidence for a specific genetic predisposition for hyperhomocysteinemia was present in patients with JIA. Elevated ESR is not associated with hyperhomocysteinemia.

Mast cell disorders are defined by an abnormal accumulation of tissue mast cells (MCs) in one or more organ systems. Symptoms in mastocytosis result from MC-derived mediators and, less frequently, from destructive infiltration of MCs. Cutaneous mastocytosis (CM) is a benign disease of the skin and may regress spontaneously. Systemic mastocytosis (SM) is a persistent disease in which a somatic c-kit mutation at codon 816 is usually detectable in MCs and their progenitors. The clinical course in these patients is variable ranging from asymptomatic for years to highly aggressive and rapidly devastating. The WHO discriminates five categories of SM: indolent SM (ISM), aggressive SM (ASM), SM with associated clonal hematological non-MC-lineage disease (AHNMD), and mast cell leukemia (MCL). The c-kit mutation D816V is quite common and may be found in all SM-categories. In SM-AHNMD, additional genetic abnormalities have been reported, whereas no additional defects are yet known for ASM or MCL. Patients with ISM and CM are treated with "mediator-targeting" drugs, whereas patients with ASM or MCL are candidates for cytoreductive therapy. The use of "Kit-targeting" tyrosine kinase inhibitors such as STI571 (Imatinib, Gleevec), has also been suggested. However, the D816V mutation of c-kit is associated with relative resistance against STI571. Therefore, these patients require alternative targeted drugs or new drug-combinations. In patients with SM-AHNMD, separate treatment plans for the SM-component and the AHNMD should be established. Examples include the use of STI571 in patients with SM plus hypereosinophilic syndrome (SM-HES) and the FIPL1/PDGFRA fusion gene target, or chemotherapy for eradication of AML in patients with SM-AML.