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Meropenem is a broad-spectrum carbapenem antibiotic with mostly renal excretion. Conflicting data are available regarding meropenem pharmacokinetics in critically ill neonates on concomitant continuous renal replacement therapy (CRRT) and/or extracorporeal membrane oxygenation (ECMO). Our objectives were to assess meropenem clearance in a neonate on CRRT and ECMO, compare it to previously published data and assess whether dose recommendations can be generalized in this population.A 2.5 kg male infant with a large diaphragmatic hernia was delivered by cesarean section at week 35 and immediately mechanically ventilated due to shock and respiratory insufficiency. He underwent surgical correction of the hernia, but developed recurrent sepsis, multiorgan failure and pulmonary hypertension. He remained mechanically ventilated and required ECMO and continuous venovenous hemodiafiltration. He was started on meropenem 40 mg/kg/dose, every 8 hs for Enterobacter cloacae bacteremia and sepsis, but due to lack of clinical and microbiologic response despite in vitro susceptibility, he was started on a continuous meropenem infusion of 240 mg/kg/d, based on dose recommendations in a similar case. We measured steady state meropenem plasma concentrations on 2 occasions, during ECMO and continuous venovenous hemodiafiltration (CVVHDF) and then on CVVHDF only.Meropenem serum concentrations were 90 and 64 mg/L on the first and second occasion (therapeutic target concentration, 10 mg/L) corresponding to a clearance of 1.9 and 2.6 mL/kg/min. This clearance estimate was substantially lower than that reported in toddlers on CRRT and ECMO in some studies.In neonates and infants, meropenem clearance is difficult to predict because of dynamic ontogenetic changes in renal function. This problem is further aggravated in acutely ill infants with decreased renal function, renal replacement therapy and/or ECMO. Therefore, Target Concentration Intervention based on meropenem plasma concentrations is indispensable to ensure therapeutic exposure in this population.

BACKGROUND Therapeutic drug monitoring (TDM) for busulfan supports dose adjustment during conditioning for stem cell transplantation. The authors aimed to develop and validate limited sampling strategies (LSS) of 4-5 samples for a precise estimation of the area under concentration (AUC)-time curve of busulfan, in plasma as an alternative to an intensive sampling strategy (ISS) requiring 9-10 samples. METHODS ISS TDM data from 297 patients (≤18 years of age) were used. AUCLSS was calculated using the trapezoidal rule and multiple linear regression (MLR). Unlike more complex modeling methods, MLR does not require sophisticated software or advanced training of personnel. MLR coefficients were estimated in the development subset containing randomly selected 50% of the records and were then used to calculate the AUCLSS of the remaining records (the validation subset). The agreement between dose adjustment recommendations (DAR) based on ISS and LSS, in the validation subset, was evaluated by a Bland-Altman analysis. A DAR deviating from an ISS-based reference by

BACKGROUND Thiopurines are efficacious in the treatment of Crohn's disease and were recently shown to induce T-cell apoptosis by modulation of Rac1 activation. To assess whether polymorphisms in Rac1 and other apoptosis-related genes, combined with clinical parameters, can predict response to thiopurines. METHODS A retrospective cohort of 156 thiopurine-treated patients with Crohn's disease was genotyped for 11 single-nucleotide polymorphisms (SNPs): 9 SNPs in Rac1, 1 SNP in the Fas ligand -843 T>C, and 1 SNP in the Caspase-9 93 C>T. Clinical data were extracted from the medical charts. Odds ratios (ORs) and 95% confidence intervals (CIs) of the association between demographic, clinical, and genetic variables and thiopurine response rates were calculated. RESULTS The overall response rate to thiopurines was 74% (115/156). The Rac1 SNP rs34932801 heterozygote genotype GC was associated with a lower response rate compared with the wild-type GG genotype (46% versus 76%; OR = 0.26; 95% CI, 0.08-0.91; P = 0.036). Only wild-type homozygotes were found for 5 Rac1 SNPs. None of the other 3 Rac1 SNPs were associated with response to thiopurines. Patients with Montreal B3 behavior pattern responded worse than those with a B1 behavior pattern (59%, versus 80%; OR = 0.37; 95% CI, 0.17-0.83; P = 0.016). Sephardic Jews had a lower response rate to thiopurines compared with Jews of Ashkenazi or mixed ancestry (60% versus 82%; OR = 0.32; 95% CI, 0.15-0.69, P = 0.003). CONCLUSIONS Rac1 SNP rs34932801carriage, Montreal B3 disease behavior, and a Sephardic Jewish origin were associated with unfavorable response to thiopurines. Corroboration of these associations in larger cohorts is warranted.

The pendrin/SLC26A4 Cl−/HCO3− exchanger, encoded by the PDS gene, is expressed in cortical collecting duct (CCD) non-A intercalated cells. Pendrin is essential for CCD bicarbonate secretion and is also involved in NaCl balance and blood pressure regulation. The intestinal peptide uroguanylin (UGN) is produced in response to oral salt load and can function as an “intestinal natriuretic hormone.” We aimed to investigate whether UGN modulates pendrin activity and to explore the molecular mechanisms responsible for this modulation. Injection of UGN into mice resulted in decreased pendrin mRNA and protein expression in the kidney. UGN decreased endogenous pendrin mRNA levels in HEK293 cells. A 4.2-kb human PDS (h PDS) promoter sequence and consecutive 5′ deletion products were cloned into luciferase reporter vectors and transiently transfected into HEK293 cells. Exposure of transfected cells to UGN decreased h PDS promoter activity. This UGN-induced effect on the h PDS promoter occurred within a 52-bp region encompassing a single heat shock element (HSE). The effect of UGN on the promoter was abolished when the HSE located between nt −1119 and −1115 was absent or was mutated. Furthermore, treatment of HEK293 cells with heat shock factor 1 (HSF1) small interfering RNA (siRNA) reversed the UGN-induced decrease in endogenous PDS mRNA level. In conclusion, pendrin-mediated Cl−/HCO3− exchange in the renal tubule may be regulated transcriptionally by the peptide hormone UGN. UGN exerts its inhibitory activity on the h PDS promoter likely via HSF1 action at a defined HSE site. These data define a novel signaling pathway involved in the enterorenal axis controlling electrolyte and water homeostasis.

Hematopoietic SCT (HSCT) from HLA-matched donors is sometimes complicated by GVHD or graft rejection, because of mismatched mHA. This study presents data suggesting the involvement of glutathione S-transferase theta-1 (GSTT1), a phase II detoxifying enzyme encoded by GSTT1, in Ab-mediated rejection of HSCT in children with congenital hemoglobinopathies (CHs). Mismatch of GSTT1, which often features a deletion polymorphism variant, can have major consequences in solid organ transplantation outcome. In liver transplantation, it has been shown to lead to de novo hepatitis, whereas in kidney transplantation, chronic allograft rejection has been documented. In this study on 18 children with CH who underwent HSCT, five cases of graft rejection occurred, all in GSTT1-null patients, four of which featured anti-GSTT1 antibodies. The data suggest that when GSTT1-null patients are transplanted with a GSTT1-positive graft, rejection due to an Ab-mediated immune response against GSTT1 displayed on transplanted stem cells may take place. Thus, it seems that detection of anti-GSTT1 antibodies in patients with a GSTT1-null genotype before transplantation may be predictive of graft rejection in the event of a GSTT1-positive donor.

Thiopurines are effective for maintenance of remission in inflammatory bowel disease (IBD) in only about half of patients. Predictors of response may assist in selecting the most appropriate patients for thiopurine therapy. Thiopurines inhibit Rac1, a GTPase that exerts an antiapoptotic effect on T-lymphocytes. A genetic association was recently demonstrated between a Rac1 single nucleotide polymorphism (SNP) and poorer response to thiopurines in adult patients with Crohn disease. We aimed to determine whether Rac1 SNPs are associated with response to thiopurines in children with IBD.Children with IBD treated with thiopurines were prospectively followed for 1 year and were genotyped for 3 Rac1 SNPs previously found to be relevant to IBD: rs10951982, rs4720672, and rs34932801. The rate of sustained steroid-free remission (SSFR) without treatment escalation by 12 months was compared between wild types (WTs) and heterozygotes.A total of 59 patients were studied (63% boys, 80% having Crohn disease, mean age 13 ± 4.1). Nineteen of the 41 WT (46%) and 9 of the 15 (60%) heterozygotes for rs10951982 were in SSFR (P = 0.55). Similarly, 21 of the 45 (47%) WT and 8 of the 12 (67%) heterozygotes for rs4720672 were in remission (P = 0.33). Finally, 21 of the 45 (47%) WT and 3 of the 5 (60%) heterozygotes for rs34932801 were in remission (P = 0.66). All of the 3 comparisons remained nonsignificant in a sensitivity analysis of only the patients with Crohn disease.We did not find an association between 3 Rac1 SNPs and thiopurine effectiveness by 12 months in a prospective study of children with IBD. Other predictors of response should be sought to optimize patient selection for thiopurine therapy.

The D1790G mutation was found in all 24 patients of an extended long QT family but not in 200 chromosomes carried by healthy individuals. We describe a 37-year-old man presenting with a typical spontaneous type 1 Brugada pattern who in electrophysiological testing had easily inducible ventricular fibrillation. At the age of 47 years he had an atrial ventricular type 2 block documented by an exercise test and a Holter monitor. Genetic analysis revealed a known D1790G mutation in the gene encoding of the sodium channel (SCN5A) that until now has been associated only with the long QT phenotype. Although this mutation has not been associated with a reduction of sodium channel expression, we hypothesize that sodium currents are further diminished due to the 20-mV shift of the steady-state inactivation curve, and this could contribute to the Brugada phenotype. This case is important as it allows a better understanding of the underlying molecular mechanisms of Brugada syndrome. Moreover, this observation raises concern about the safety of class IC drug therapy in long QT type 3 patients and quinidine therapy in Brugada patients, and emphasizes the importance of a thorough clinical and genetic evaluation.

What is the central question of this study? In the kidney, the bulk of the filtered Mg(2+) is reabsorbed in the thick ascending limb by paracellular conductance, mediated by the tight junction protein, claudin-16, which is encoded by the gene CLDN16. The role of 1,25-dihydroxyvitamin D [1,25(OH)2 VitD] in renal Mg(2+) handling is unclear. We aimed to explore the molecular mechanisms underlying the effect of 1,25(OH)2 VitD on claudin-16-mediated Mg(2+) transport. What is the main finding and its importance? Paracellular, claudin-16-mediated Mg(2+) transport is transcriptionally repressed by 1,25(OH)2 VitD, probably via a Ca(2+)-sensing receptor-dependent mechanism. This renal effect of 1,25(OH)2 VitD may serve as an adaptive mechanism to the 1,25(OH)2 VitD-induced enteric hyperabsorption of dietary Mg(2+). Magnesium is reabsorbed in the thick ascending limb by paracellular conductance, mediated by the CLDN16-encoded tight junction protein, claudin-16. However, the role of 1,25-dihydroxyvitamin D [1,25(OH)2 VitD] in renal Mg(2+) handling is unclear. We have shown that Mg(2+) depletion increases and 1,25(OH)2 VitD inhibits CLDN16 transcription. We have now explored further the molecular mechanisms underlying the effect of 1,25(OH)2 VitD on claudin-16-mediated Mg(2+) transport. Adult mice received parenteral 1,25(OH)2 VitD or 1,25(OH)2 VitD combined with either high-Mg(2+) or low-Mg(2+) diets. Administration of 1,25(OH)2 VitD enhanced urinary excretion of Mg(2+) and Ca(2+). The 1,25(OH)2 VitD also increased renal Ca(2+)-sensing receptor (CaSR) mRNA and decreased renal claudin-16 and claudin-19 mRNA and claudin-16 protein, but did not affect renal claudin-2 mRNA. The 1,25(OH)2 VitD reversed the expected increase in claudin-16 mRNA in Mg(2+)-depleted animals. Comparably treated HEK 293 cells showed similar changes in claudin-16 mRNA, but 1,25(OH)2 VitD did not alter mRNA of the TRPM6 Mg(2+) channel. A luciferase reporter vector containing 2.5 kb of 5'-flanking DNA sequence from human CLDN16 (hCLDN16) was transfected into HEK 293 and OK cells. The hCLDN16 promoter activity was modestly decreased by 1,25(OH)2 VitD, but markedly inhibited in HEK 293 cells coexpressing CaSR. Coexpression in OK cells of dominant-negative CaSR completely abolished inhibition of hCLDN16 promoter activity by 1,25(OH)2 VitD. The 1,25(OH)2 VitD-induced decrease in hCLDN16 promoter activity was attenuated in Mg(2+)-depleted HEK 293 cells. In conclusion, 1,25(OH)2 VitD transcriptionally inhibits claudin-16 expression by a mechanism sensitive to CaSR and Mg(2+). This renal effect of 1,25(OH)2 VitD may serve as an adaptive response to the 1,25(OH)2 VitD-induced increase in intestinal Mg(2+) absorption.

Methotrexate (MTX), a folate antagonist employed for treating a wide range of malignancies, has an extensive interpatient variability, resulting in unpredictable toxicity. The present study evaluated the impact of single gene polymorphisms (SNPs: rs1801133 and rs1801131 in the MTHFR gene; rs4149056 and rs11045879 in the SLC01B1 gene; and rs2032582 and rs1045642 in the ABCB1 transporter gene) on MTX blood levels and toxicity in samples from 69 patients with diffuse large-B-cell lymphoma (DLBCL) treated with high dose intravenous (HD IV) MTX,2 g/m(2). None of the studied genotypes was found to be associated with a statistically significant risk for elevated MTX levels at 24-48 h after completing therapy with MTX. Ancestral alleles (T) for SLC01B1 rs4149056 (T521C) and SLC01B1 rs11045879 (intron C21273886T) were found to be associated with an increased risk for MTX-related toxicity (p0.05 and p = 0.07, respectively), emphasizing the potential importance of employing pharmacogenetic assessment for personalized medicine.

Thiopurines are effective in attaining and maintaining remission in patients with inflammatory bowel diseases (IBD). The major drawback of these drugs are their serious adverse effects (SAE), highlighting the importance of preemptive identification of patients at risk. We aimed to examine whether gene polymorphisms in GSTM1, GSTT1 and TPMT, combined with various clinical parameters, can predict thiopurine induced SAE.A retrospective cohort of 176 Crohn's Disease (CD) patients treated with thiopurines (131 with 6MP and 45 with azathioprine) was genotyped for common polymorphisms in GSTM1, GSTT1 and TPMT. Clinical data including SAE, age, ethnicity, gender and smoking status were extracted from patient charts. SAEs evaluated were myelosuppression, hepatotoxicity and pancreatitis. Associations between demographic, clinical, and genetic variables and thiopurine induced SAE were assessed.Twenty-four patients (14%) developed SAE, revealing a significant association between thiopurine induced SAE and GSTM1-null genotype (P=0.05), older age (P=0.016) and active smoking status (P=0.043) and SAE. On multi-variant analysis, past or current smokers were at increased risk for developing thiopurine related SAE (OR 2.915, CI 95%: 1.199- 7.084), specifically pancreatitis (p0.001). No association was found between TPMT or GSTT1 polymorphisms and the development of SAE.Active smoking and GSTM1-null genotype appear to be risk factors for thiopurine induced SAEs (i.e. myelosuppression, hepatotoxicity and pancreatitis) in patients with CD. Corroboration of these associations in larger cohorts is warranted.

MTHFR C677T is a common gene polymorphism that has been shown to be associated with hyperhomocysteinemia. Studies on the role of MTHFR in inflammatory bowel diseases (IBD) have yielded conflicting results, perhaps due in part to genetic heterogeneity. The prevalence of the MTHFR C677T variant allele varies according to Jewish subpopulations: Ashkenazi vs non-Ashkenazi. The aim of this study was to examine the association between MTHFR C677T genotype and IBD in the different Jewish populations. DNA samples were assessed for the presence of the MTHFR C677T variant allele in 445 Jewish Israeli IBD patients: 338 with Crohn's disease [CD] (214 Ashkenazi and 124 non-Ashkenazi Jews) and 107 with ulcerative colitis [UC] (73 Ashkenazi and 34 non-Ashkenazi Jews), and in 347 healthy controls: 173 Ashkenazi and 174 Non-Ashkenazi Jews. Possible genotype–phenotype associations were investigated. We showed a significantly higher frequency of MTHFR 677T variant genotypes in non-Ashkenazi CD patients: Odds ratio of 1.86 for heterozygotes (CT) and 2.89 for homozygotes (TT) compared to non-Ashkenazi healthy controls. No significant association was found for UC in non-Ashkenazi patients or for CD or UC in Ashkenazi patients. Our findings suggest that the MTHFR 677T variant may contribute to the risk of CD in non-Ashkenazi but not Ashkenazi Jews. This may result from genetic heterogeneity and highlights the complexity of the genetic etiology of IBD.

High dose busulfan (BU) has become a mainstay in conditioning regimens for hematopoietic stem cell transplantation (HSCT), despite its unpredictable response, narrow therapeutic index and severe toxicity. The present study provides an integration of pharmacokinetic and genetic data of 63 adults with acute myeloid leukemia (AML) preconditioned for HSCT with high dose oral BU, with the aim of defining biomarkers predictive of poor BU metabolism. BU area under the concentration time curve (AUC) demonstrated that 76% of the patients achieved target AUC; 24% required dose modification. The main findings of this study were: (1) AML patients carrying the GSTP1 rs1695 variant allele were at risk of developing supra-therapeutic BU-AUC due to reduced BU clearance. (2) Combined polymorphisms in GSTM1 and ABCB1 were associated with BU clearance and AUC rates. In conclusion, GST and ABCB1 genotyping may assist care-givers in personalizing BU dosage with less trial-and-error and may enable preemptive identification of patients at risk for BU toxicity.

The involvement of oxidant/antioxidant imbalance in the development of inflammatory bowel disease (IBD) is well documented. Two members of the glutathione S-transferase (GST) family of enzymes, GSTM1 and GSTT1, known to take part in cellular protection against electrophiles, demonstrate common deletion variants (termed null) associated with impaired enzyme function. To evaluate the effect of GSTM1/GSTT1 genotype on IBD susceptibility in a Israeli cohort and to study the correlation between GSTM1/GSTT1 genotype, smoking status, and a variety of clinical characteristics of IBD. A cohort of 606 Israeli IBD patients (453 with Crohn’s disease [CD] and 153 with ulcerative colitis [UC]) and 528 ethnically matched healthy controls were genotyped for the null variants of GSTM1 and GSTT1. In patients, phenotype–genotype correlations were examined. Ethnic stratification of healthy controls revealed a higher frequency of GSTT1-null in Jewish and Arab Moslem individuals compared to Druze individuals (P

Busulfan (BU), often used in high dose for myeloablation before hematopoietic stem cell transplantation (HSCT), has been implicated in certain HSCT toxicities, including the occurrence of hepatic veno-occlusive disease (HVOD). In addition to weight and age, gene polymorphisms in specific members of the glutathione-transferase (GST) gene family (A1, P1, M1, and T1), involved in BU metabolism, may play a role in the wide inter-patient variability in systemic BU concentrations.The present study integrated clinical data regarding the occurrence of HVOD, graft versus host disease (GVHD), BU pharmacokinetics and GSTA1, GSTP1, GSTM1, and GSTT1 genotypes of 18 children who received BU in their pre-HSCT conditioning regimen. The children were all treated for congenital hemoglobinopathies and were all of Arab Moslem descent.The data demonstrate an association between GSTA1 and GSTP1 genotypes and BU-maximal concentration (C(max)) (P = 0.01, P = 0.02, respectively), area under the concentration-time curve (AUC) (P = 0.02, P = 0.01, respectively) and oral BU clearance/kg body weight (P0.02, P = 0.08, respectively). GSTM1-null individuals demonstrated lower BU-AUC/Kg compared to GSTM1-positive individuals. In addition, an association between GVHD and GSTM1-null genotype was found.GSTA1, GSTP1, and GSTM1 genotyping prior to HSCT in children with congenital hemoglobinopathies may allow better prediction of oral BU kinetics and the need for BU dose adjustment, as well as prediction of transplant related toxicity such as GVHD, thereby improving clinical outcome.

Renal tubular Mg(2+) reabsorption is mediated predominantly by the tight junction channel protein claudin-16 which is encoded by the gene CLDN16. Hypermagnesemia decreases, whereas hypomagnesemia increases Mg(2+) reabsorption. This study examines the role of claudin-16 in the adaptive response of the kidney to Mg(2+) availability.Mice received a low-, normal- or high Mg(2+) diet for up to 3 days. Mg(2+)-loaded animals displayed hypermagnesemia with increasing urine Mg(2+)/Ca(2+) levels paralleled by a decrease in claudin-16 protein and mRNA in the kidney. Mg(2+)- deprived animals developed hypomagnesemia with decreasing urine Mg(2+)/Ca(2+) levels associated with an increase in claudin-16 protein and mRNA abundance. Mg(2+) depletion markedly increased and Mg(2+) load decreased endogenous claudin-16 mRNA levels in calcium-sensing receptor-transfected HEK293 cells compared with native HEK293 cells. The effect of Mg(2+) availability on the human CLDN16 (hCLDN16) gene promoter was examined. Using a 2.5kb hCLDN16 5'-flanking DNA sequence, we show that magnesium depletion increases and Mg(2+) load decreases hCLDN16 promoter activity in transfected HEK293 cells.Changes in Mg(2+) availability may influence claudin-16 mediated Mg(2+) transport at the transcriptional level. The possible involvement of the cell membrane bound Ca(2+)/Mg(2+) sensing receptor or the potential role of a hypothetical Mg(2+) response element on the CLDN16 promoter in the Mg(2+)-induced response remains to be explored.

Individuals with intermediate or no thiopurine S-methyltransferase (TPMT) activity are at risk of hematotoxicity when treated with standard doses of thiopurines, thus, pretreatment identification of these individuals is of major importance. The purpose of this study was to determine the frequency and distribution of TPMT polymorphic variants, known to functionally impair TPMT activity, in the highly heterogeneous Israeli population.TPMT genotyping of individuals representing three major demographic groups in Israel was carried out by PCR restriction fragment length polymorphism and high-resolution melting.Frequencies of TPMT risk alleles differed significantly among the screened Israeli subpopulations: Druze showed fivefold and twofold higher frequencies than Jews and Moslems, respectively. Specifically, allelic frequencies of TPMT*3A were 0.73% (95% CI 0.34-1.45%), 0.79% (95% CI 0.16-2.39%), and 3.19% (95% CI 1.78-5.58%) in Jews, Moslems, and Druze, respectively. Although not found in Jews, TPMT*3C was found at an allelic frequency of 1.05% (95% CI 0.31-2.76%) and 0.75% (95% CI 0.02-2.84%) in Moslems and Druze. TPMT*2 and TPMT*3B were not detected in any of the Israeli subpopulations studied.These data indicate that the Israeli population displays a distinct TPMT genetic variability that is comprised of a mix of three major genetically diverse subpopulations, each with its unique TPMT allelic frequency distribution pattern and likelihood of developing an adverse reaction to thiopurine drugs.

Background: The best described primary inherited proximal tubulopathies include X-linked hypercalciuric nephrolithiasis (XLHN), caused by a mutation in the chloride channel gene CLCN5, and classic Fanconi's syndrome, the genetic basis of which is unknown. The aim of this study is to examine the clinical, biochemical, and genetic characteristics of a highly consanguineous Druze family with autosomal recessive proximal tubulopathy and hypercalciuria (ARPTH), a syndrome not reported previously. Methods: Three children (2 girls, 1 boy) of the family referred for evaluation of renal glycosuria and hypercalciuria and 10 of their close relatives were evaluated clinically and biochemically. All study participants underwent genetic analysis to exclude involvement of the CLCN5 gene. Results: Evaluation of the 3 affected children showed glycosuria, generalized aminoaciduria, hypouricemia, uricosuria, low molecular weight (LMW) proteinuria, and hypercalciuria in all 3 children and phosphaturia in 2 children. They had no metabolic acidosis or renal insufficiency. One affected girl had nephrocalcinosis. Two children had a history of growth retardation and radiological findings of metabolic bone disease. Parathyroid hormone and 1,25-dihydroxyvitamin D [1,25(OH) 2 Vit D] blood levels in affected children were normal. Unaffected family members examined had no renal tubular defects or LMW proteinuria. Genetic linkage analysis excluded cosegregation of the ARPTH phenotype with the CLCN5 locus. Conclusion: ARPTH is a new syndrome characterized by nonacidotic proximal tubulopathy, hypercalciuria, metabolic bone disease, and growth retardation. It can be distinguished from XLHN by its autosomal recessive mode of inheritance and normal serum levels of calciotropic hormones, as well as the absence of LMW proteinuria in obligate carriers. The gene mutated in ARPTH remains to be identified.