The pharmacogenetics of tacrolimus in renal transplant patients: association with tremors, new-onset diabetes and other clinical events.

Our study is the first study to investigate the effect of SNPs in CYP3A5, CYP3A4, ABCB1 and POR genes on the incidence of tremors, nephrotoxicity, and diabetes mellitus. A total of 223 renal transplant patients receiving tacrolimus and mycophenolate mofetil (MMF) were recruited. Both adults and children patients participated in the study. Genotyping was performed using PROFLEX-PCR followed by RFLP. MPA and tacrolimus plasma concentrations were measured by immunoassay. The AUC0-12h of MMF was estimated by a Bayesian method. We found a statistically significant association between the CYP3A5*3 and CYP3A4*1B genotypes and the tacrolimus exposure. We found a lower occurrence of nephrotoxicity (p = 0.03), tremor (p = 0.01), and new-onset diabetes (p = 0.002) associated with CYP3A5*1 allele. The CYP3A4*1B allele was significantly associated with a lower occurrence of new-onset diabetes (p = 0.026). The CYP3A5*1 allele was significantly associated with an increased risk of acute and chronic rejection (p = 0.03 and p < 0.001, respectively). Our results support the usefulness of tacrolimus pharmacokinetics in pre-kidney transplant assessments.

Modulation of macrophage phagocytosis in vitro-A role for cholinergic stimulation?

Acetylcholine is synthetized and released from neural cells, but also by non-neuronal cells such as epithelial cells or keratinocytes. Cholinergic agonists enhance the phagocytosis of zymosan particles in primary peritoneal macrophages. The aim of this study was to investigate the effect of carbachol stimulation on phagocytosis in a macrophage cell line using microspheres. The murine cell line MH-S was used in a phagocytosis assay with fluorescent latex beads. The amount of the ingested beads was determined using flow cytometry. Gene expression was investigated using polymerase chain reaction. Gene expression of the muscarinic receptors M1, M3, M4 and M5 but not M2 was found. Carbachol slightly increased the phagocytosis of microspheres in the macrophages. A co-stimulation using lipopolysaccharide and carbachol did not increase the effect of lipopolysaccharide alone. In conclusion, cholinergic stimulation in vitro only moderately modulates the phagocytosis of microspheres. M2 might have a role in stimulation of macrophage phagocytosis.

MDR-1 and CYP3A5 Polymorphisms in Pediatric Idiopathic Nephrotic Syndrome: Impact on Susceptibility and Response to Steroids (Preliminary Results).

BACKGROUND: Oral steroid treatment is the first line of therapy for childhood nephrotic syndrome (NS). However, resistance to this treatment has been observed in some patients. Here, we investigated the association of two steroid metabolism-related genes with susceptibility to childhood NS and the steroid response. METHODS: We genotyped the single nucleotide polymorphisms (SNP) of MDR-1 [C1236T (rs1128503), G2677T/A (rs2032582), and C3435T (rs1045642)] and the CYP3A5 gene (A6986G) in 63 NS patients and 110 age and gender matched controls by PCR-RFLP. RESULTS: Based on multivariate logistic regression analysis carrying the G2677A A allele seemed to multiply both the risk of NS and the risk of developing glucocorticoid (GC) resistance by three-fold (OR = 3.50, [1.37 - 7.06] , p < 0.001, OR = 3.07, [1.06 - 26.10], p = 0.048, respectively). When combined into haplotype, the TAT (1236_T, 2677_A, and 3435_T) haplotype conferred a two-fold NS risk (OR = 2.26, [1.11 - 4.58], p = 0.023) and almost three-fold risk to develop resistance to GC (OR = 2.69, [1.12 - 8.79], p = 0.044). However, TAT carriers seemed to have less risk to develop NS at late age (OR = 0.34, [0.12 - 0.92], p = 0.037). The C1236T (MDR-1) and the A6986G (CYP3A5) polymorphisms showed a trend of association to GC resistance but these associations did not reach the statistical significance (OR = 2.83, [0.54 - 14.67], p = 0.294), (OR = 2.11, [0.53 - 8.38], p = 0.28), respectively. CONCLUSIONS: Here we report that only the G2677A polymorphism was associated to NS susceptibility and steroid resistance. The TAT haplotype was associated with NS susceptibility especially at an early age and with steroid resistance.

Unusual clinical outcome of primary Hyperoxaluria type 1 in Tunisian patients carrying 33_34InsC mutation.

BACKGROUND: Primary hyperoxaluria type 1 (PH1), is a rare and heterogeneous disease and one of major causes of renal insufficiency in Tunisia, caused by mutations in the AGXT gene. 33-34InsC mutation, was mainly described in children with a severe clinical feature leading to early death, but it was uncommonly reported in adult patients. METHODS: Common mutations in AGXT were tested using PCR/RFLP technique in 111 patients (68 adult, 43 children) with suspected PH1. RESULTS: We described 16 cases (eight adult and eight children) with a 33-34InsC mutation with a median age of 24 years [6 months - 73 years]. All children were in end stage renal disease (ESRD) at the median age of 3 years due to lithiasis and/or nephrocalcinosis. Unfortunately, 75% of them died with a median age of 2.5 years. For the majority of adults only spontaneous elimination of urolithiasis were noted, 37.5% preserved until now a normal renal function and 62.5% of them reached ESRD at the median age of 55.8 ± 12.31 years old. CONCLUSION: In this study 33-34InsC mutation gives a controversial clinical effect in children and adults. The implication of other genetic and/or environmental factors can play a crucial role in determining the ultimate phenotype.

Genetic Variation in the Renin-Angiotensin System and Diabetic Nephropathy in the Tunisian Population.

BACKGROUND: The aim of this study was to evaluate the association of ACE, angiotensinogen (AGT) and angiotensin II receptor type I (AGTR1) polymorphisms with diabetic nephropathy (DN) in Tunisians. METHODS: The study population comprised 236 type 2 diabetic patients: with nephropathy (DN = 47) and without nephropathy (DM = 189). Genotyping of ACE-I/D-rs1799752, ACE-rs4343G>A, AGT-rs5050A>C, AGT-rs 4762C>T, AGT-rs699A>G, and AGTR1-rs5186A>C was performed by PCR-RFLP. Haplotype and statistical analysis were realized using SNP Analyzer2.0 and SPSS20, respectively. RESULTS: Genotype frequencies were in Hardy-Weinberg equilibrium. After adjustment for potential confounding factors (age, gender, diabetes duration, hypertension…), an increased risk for DN was associated with mutated alleles of rs4762 (OR = 10.25, p = 0.001), rs699 (OR = 22.21, p < 0.001), and rs5186 (OR = 11.25, p < 0.001). However, mutated alleles of rs1799752 seemed to be protector (OR = 0.41, p = 0.011). Adjusted ORs of DN associated with the ACE haplotype (DA) was (OR = 9.56, p = 0.047) and with the ACE-AGT haplotype (ATADAA) was (OR = 5.38, p = 0.032). CONCLUSIONS: This study indicates that common variants in ACE, AGT, and AGTR1 seem to play a role in genetic susceptibility to DN in Tunisian population and provides evidence for a disease haplotype: ATADAA.

Mutational Analysis of Agxt in Tunisian Population with Primary Hyperoxaluria Type 1.

BACKGROUND: Primary hyperoxaluria type 1 (PH1) is an autosomal recessive metabolic disorder caused by inherited mutations in the AGXT gene encoding liver peroxisomal alanine:glyoxylate aminotransferase (AGT). PH1 is a clinically and genetically heterogeneous disorder. The aim of our study was to analyze and characterize the mutational spectrum of PH1 in Tunisian patients. MATERIALS AND METHODS: Molecular studies of 146 Tunisian patients suspected with PH were performed by PCR/Restriction fragment length polymorphism (RFLP) to detect seven mutations described as the most common. Direct sequencing for the 11 exons was performed in patients in whom any mutation was not identified. RESULTS: The genetic diagnosis of PH1 was confirmed in 62.3% of patients. The first molecular approach based on PCR/restriction enzyme test was positive in 37.6% of patients, whereas the second molecular approach based on whole gene sequencing was successful in 24% of cases. Twelve pathogenic mutations were detected in our cohort. Two mutations were novel, and five were detected for the first time in Tunisians. The three most frequent mutations were p.Ile244Thr, p.Gly190Arg, and c.33dupC, with a frequency of 43.4%, 21.4%, and 13.1%, respectively. CONCLUSION: The two novel mutations detected in our study extend the spectrum of known AGXT gene mutations. The screen for the mutations identified in this study can provide a useful, cost-effective, and first-line investigation in Tunisian PH1 patients.

Targeting the non-neuronal cholinergic system in macrophages for the management of infectious diseases and cancer: challenge and promise.

Macrophages represent key players of the immune system exerting highly effective defense mechanisms against microbial infections and cancer that include phagocytosis and programmed cell removal. Recent findings highlight the relevance of the non-neuronal cholinergic system for the regulation of macrophage function that opens promising new concepts for the treatment of infectious diseases and cancer. This mini review summarizes our present knowledge on this topic and gives an outlook on future developments.

The C677T MTHFR genotypes influence the efficacy of B9 and B12 vitamins supplementation to lowering plasma total homocysteine in hemodialysis.

BACKGROUND: Hyperhomocysteinaemia, an independent risk factor for cardiovascular diseases, is common in hemodialysis patients (HD) and particularly in those homozygous for polymorphism of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene. B vitamins supplementation has been shown to lower plasma total homocysteine (tHcy), but this has been contreversed in several groups. The aim of our study was to explore the response of tHcy in hemodialysis (HD) patients to individual supplementation with folic acid (B9) and/or vitamin B12, based on carrier status for the (MTHFR) polymorphism. METHODS: 132HD were randomized according to C677TMTHFR genotypes into 2 groups (AandB). The group (A) was treated initially with B9 (10mg/day orally) for 2 months (t1) and then with B12 vitamin (cyanocobalamin ampoule of 1000 µg) for the following 2 months (t2), then association of B9 and B12 for 2 months (t3). The group (B) was supplemented initially with vitamin B12 (t1), then with folic acid (t2) and then B9 + B12 for 2 months (t3). A wash-out period of 2 months followed the treatment in both groups (t4). We determined tHcy, B9 and B12 concentrations at each time. RESULTS: In group A, we noted that the decrease in tHcy becomes significant for CC when patients were supplemented with vit B12 only (p = 0.009). While, B9 + vit B12 supplementation did not seem to improve a significant effect compared with B12 alone. For genotypes (CT) and (TT) we noticed a significant decrease in tHcy at t1 (p = 0.038; 0.005 respectively) and at (t3; CT p = 0.024; TT p = 0.017). In group B, for genotypes CC, the decrease in tHcy became significant at t3 (vit B12 + B9; p = 0.031). For genotypes (CT) and (TT), at the replacement of vit B12 by B9, tHcy was significantly decreased (p = 0.036; 0.012, respectively). The combination of the 2 vitamins (t3) showed no difference compared to folate alone. In the 2 groups (t4), there was an significant increase of tHcy again for 3 genotypes. CONCLUSION: Supplementation with B vitamins correlated to the MTHFR genotypes has been shown to lower significantly tHcy in HD patients.

Immunohistochemical expression of nuclear factor erythroid-2-related factor 2 and heme oxygenase 1 in normal bovine lung and bovine lung infected with Mannheimia haemolytica.

Mannheimia haemolytica is an important cause of pneumonia in feedlot cattle. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a redox-sensitive transcription factor responsible for the induction of antioxidant enzymes, such as heme oxygenase 1 (HO-1), within the lung. The expression of Nrf2 and HO-1 was immunohistochemically evaluated in 4 calves 24 h after experimental infection with M. haemolytica. Calves receiving normal saline served as controls. In the infected lungs, cytoplasmic Nrf2 expression was high in macrophages and bronchioles and low in alveolar epithelium, whereas nuclear expression was high in endothelial cells, macrophages, and bronchioles and lowest in alveolar epithelium. Normal lung samples displayed only faint Nrf2 cytoplasmic staining within bronchiolar epithelium. Expression of HO-1 was detected within the cytoplasm of macrophages and bronchiolar epithelial cells in all infected lung samples, whereas normal lungs displayed only weak cytoplasmic staining in bronchiolar epithelial cells. These findings suggest that bronchiolar epithelial cells and macrophages up-regulate Nrf2 expression early in the course of infection, which results in increased expression of HO-1 within these cells.

Mannheimia haemolytica est une cause importante de pneumonie chez les bovins en parc d'engraissement. Le facteur érythroïde-2 nucléaire apparenté au facteur 2 (Nrf2) est un facteur transcriptionnel sensible au potentiel redox responsable de l'induction d'enzymes antioxidants, tel que l'hème oxygénase 1 (HO-1), dans le poumon. L'expression de Nrf2 et HO-1 fut évaluée par épreuve immunohistochimique chez quatre veaux 24 h après une infection expérimentale avec M. haemolytica. Les veaux témoins ont reçu de la saline. Dans les poumons infectés, l'expression cytoplasmique de Nrf2 était élevée dans les macrophages et les bronchioles et faible dans l'épithélium alvéolaire, alors que l'expression nucléaire était élevée dans les cellules endothéliales, macrophages et bronchioles, et à son plus faible dans l'épithélium alvéolaire. Les échantillons de poumons normaux montraient seulement une faible coloration cytoplasmique pour Nrf2 dans l'épithélium des bronchioles. L'expression de HO-1 fut détectée dans le cytoplasme des macrophages et des cellules épithéliales des bronchioles de tous les échantillons de poumons infectés, alors que les échantillons de poumons normaux ne montraient qu'une faible coloration cytoplasmique dans les cellules épithéliales des bronchioles. Ces données suggèrent que les cellules épithéliales des bronchioles et les macrophages régulent à la hausse l'expression de Nrf2 tôt lors de l'infection, ce qui résulte en une expression augmentée d'HO-1 à l'intérieur de ces cellules.(Traduit par Docteur Serge Messier).

Association between haptoglobin 2-2 genotype and coronary artery disease and its severity in a tunisian population.

Haptoglobin (Hp) polymorphism generates three common human genotypes (Hp1-1, Hp2-1, and Hp2-2), having functional differences, related to the risk of development of cardiovascular diseases. These functions are a consequence of hemoglobin binding that leads to the synthesis of an antioxidant like ferritin. We explored the association of Hp polymorphism with significant coronary stenosis (SCS) and its severity within 400 Tunisian patients, using genotyping, biochemical parameters, and the Gensini score. After adjustments for age and gender, Hp2-2 was associated with the highest ferritin but the lowest Hp concentrations. After adjustments for confounding parameters, the OR of SCS associated with Hp2-2 was 1.74 (95% CI 1.18-2.58; p = 0.005). This effect was enhanced within diabetics (OR 1.90, 95% CI 1.11-3.24; p = 0.018), obese subjects (OR 1.98, 95% CI 1.10-4.86; p = 0.034), and smokers (OR 4.17, 95% CI 1.54-1.29; p = 0.005). The Hp2-2 genotype is associated with an increase in SCS especially in diabetics, the obese, and smokers.

Six lipoprotein lipase gene polymorphisms, lipid profile and coronary stenosis in a Tunisian population.

Lipoprotein lipase (LPL) is the rate-limiting enzyme in the hydrolysis of triglyceride-rich lipoprotein particles (Chylomicrons and very-low-density lipoprotein). LPL polymorphisms' effects on lipids and coronary artery disease are controversial among studies and populations. Our aim was to study the association between six polymorphisms, haplotypes and significant coronary stenosis (SCS), disease severity and lipid parameters in Tunisian patients. LPL PvuII, 93 T/G, 188 G/E, HindIII, N291S and D9N polymorphisms were analyzed in 316 patients who underwent coronary angiography. Assessment of coronary angiograms identified SCS as the presence of stenosis >50 % in at least one major coronary artery. The stenosis severity was determined by using Gensini score and vessels number. A significant association of SCS with TT of the HindIII polymorphism was showed (odds ratio (OR): 2.84, 95 % CI, 1.19-7.40, p = 0.017) and TG (OR: 1.77, 95 % CI, 1.99-2.82, p = 0.033). The mutated HindIII genotype was significantly associated with increased TG and ApoB/ApoA-I ratio and with decreased HDL-C. Haplotype analysis showed that OR of SCS associated with the CTGTAG haplotype was 2.12 (95 % CI 1.05-4.25, p = 0.032) and with CGGGAA was 0.71 (95 % CI 0.26-1.95, p = 0.022) compared to the CTGTAA. Significant difference in Gensini score was observed among HindIII genotype and haplotypes. A significant association between the mutated genotype of HindIII polymorphism and decreased HDL-C level and increased ApoB/ApoA-I ratio and TG level was showed. Our results suggest that HindIII and D9N polymorphisms and CTGTAG haplotype seem to be considered as marker of predisposition to coronary stenosis. In another hand, HindIII and haplotypes were related to stenosis severity.