Thomas J. Velenosi

Silencing IDO in dendritic cells: A novel approach to enhance cancer immunotherapy in a murine breast cancer model

Cancer immunotherapeutic agents (vaccines) in the form of antigen‐loaded dendritic cells (DCs) reached an important milestone with the recent approval of Provenge, the first DC vaccine for treatment of prostate cancer. Although this heralds a new era of tumor immunotherapy, it also highlights the compelling need to optimize such DC‐based therapies as they are increasingly tested and used to treat human patients. In this study we sought to augment and enhance the antitumor activity of a DC‐based vaccine using siRNA to silence expression of immunosuppressive enzyme indoleamine 2,3‐dioxygenase (IDO) in DCs. We report here that DCs loaded with tumor antigens, but with siRNA‐silenced IDO expression, were introduced into 4T1 breast tumor‐bearing mice, the treatment: (i) lengthened the time required for tumor onset, (ii) decreased tumor size compared to tumors grown for equal lengths of time in mice treated with antigen‐loaded DCs without IDO silencing and (iii) reduced CD4+ and CD8+ T cell apoptosis. Furthermore, immunization with IDO‐silenced DCs enhanced tumor antigen‐specific T cell proliferation and CTL activity, and decreased numbers of CD4+CD25+Foxp3+ Treg. This study provides evidence to support silencing of immunosuppressive genes (IDO) as an effective strategy to enhance the efficacy of DC‐based cancer immunotherapeutic.

Down-regulation of hepatic CYP3A and CYP2C mediated metabolism in rats with moderate chronic kidney disease.

Expression and activity of drug-metabolizing enzymes are decreased in severe kidney disease; however, only a small percentage of patients with chronic kidney disease (CKD) are at the final stage of the disease. This study aimed to determine the changes in drug-metabolizing enzyme function and expression in rats with varying degrees of kidney disease. Sprague-Dawley rats were subjected to surgical procedures that allowed the generation of three distinct models of kidney function: normal kidney function, moderate kidney function, and severe kidney disease. Forty-two days after surgery, rats were sacrificed and hepatic CYP3A and CYP2C expression was determined. In addition, enzymatic activity was determined in liver microsomes by evaluating midazolam (CYP3A), testosterone (CYP3A and CYP2C), and tolbutamide (CYP2C) enzyme kinetics. Both moderate and severe kidney disease were associated with a reduction in CYP3A2 and CYP2C11 expression (p < 0.05). Likewise, moderate kidney disease resulted in more than a 60% decrease in enzyme activity (Vmax) for CYP2C11 and CYP3A, compared with controls (p < 0.05). When the degree of kidney disease was correlated with metabolic activity, an exponential decline in CYP2C- and CYP3A-mediated metabolism was observed. Our results demonstrate that CYP3A and CYP2C expression and activity are decreased in both moderate and severe CKD. Our data suggest that drug metabolism is significantly decreased in the earlier stages of CKD and imply that patients with moderate CKD may be subject to unpredictable pharmacokinetics.

N-Acetylcysteine prevents congenital heart defects induced by pregestational diabetes

BackgroundPregestational diabetes is a major risk factor of congenital heart defects (CHDs). Glutathione is depleted and reactive oxygen species (ROS) production is elevated in diabetes. In the present study, we aimed to examine whether treatment with N-acetylcysteine (NAC), which increases glutathione synthesis and inhibits ROS production, prevents CHDs induced by pregestational diabetes.MethodsFemale mice were treated with streptozotocin (STZ) to induce pregestational diabetes prior to breeding with normal males to produce offspring. Some diabetic mice were treated with N-acetylcysteine (NAC) in drinking water from E0.5 to the end of gestation or harvesting of the embryos. CHDs were identified by histology. ROS levels, cell proliferation and gene expression in the fetal heart were analyzed.ResultsOur data show that pregestational diabetes resulted in CHDs in 58% of the offspring, including ventricular septal defect (VSD), atrial septal defect (ASD), atrioventricular septal defects (AVSD), transposition of great arteries (TGA), double outlet right ventricle (DORV) and tetralogy of Fallot (TOF). Treatment with NAC in drinking water in pregestational diabetic mice completely eliminated the incidence of AVSD, TGA, TOF and significantly diminished the incidence of ASD and VSD. Furthermore, pregestational diabetes increased ROS, impaired cell proliferation, and altered Gata4, Gata5 and Vegf-a expression in the fetal heart of diabetic offspring, which were all prevented by NAC treatment.ConclusionsTreatment with NAC increases GSH levels, decreases ROS levels in the fetal heart and prevents the development of CHDs in the offspring of pregestational diabetes. Our study suggests that NAC may have therapeutic potential in the prevention of CHDs induced by pregestational diabetes.

Pharmacokinetic considerations in chronic kidney disease and patients requiring dialysis

Introduction: Chronic kidney disease (CKD) is the progressive decline in renal function over time. Patients with end-stage renal disease require renal replacement therapy such as hemodialysis to support life. Hemodialysis patients require several medications to treat a variety of comorbid conditions. Polypharmacy accompanied by alterations in the pharmacokinetics of medications places hemodialysis patients at increased risk of drug accumulation and adverse events. Areas covered: We review alterations in the pharmacokinetics of drugs in hemodialysis patients. The major areas of pharmacokinetics, absorption, distribution, metabolism and excretion, are covered and, where appropriate, differences between dialysis patients and non-dialysis CKD patients are compared. In addition, we review the importance of drug dialyzability and its potential impact on drug efficacy. Finally, we describe important clinical examples demonstrating nonrenal drug clearance is significantly altered in CKD. Expert opinion: Decreases in renal drug excretion experienced by hemodialysis patients have been known for years. Recent animal and human clinical pharmacokinetic studies have highlighted that nonrenal clearance of drugs is also substantially decreased in CKD. Clinical pharmacokinetic studies are required to determine the optimal dosage of drugs in CKD and hemodialysis patients in order to decrease the incidence of adverse medication events in these patient populations.

In vitro and in vivo assessment of renal drug transporters in the disposition of mesna and dimesna.

Mesna and its dimer, dimesna, are coadministered for mitigation of ifosfamide‐ and cisplatin‐induced toxicities, respectively. Dimesna is selectively reduced to mesna in the kidney, producing its protective effects. In vitro screens of uptake and efflux transporters revealed saturable uptake by renal organic anion transporters OAT1, OAT3, and OAT4. Efflux transporters breast cancer resistance protein; multidrug and toxin extrusion 1 (MATE1); multidrug resistance proteins MRP1, MRP2, MRP4, and MRP5; and P‐glycoprotein (Pgp) significantly reduced dimesna accumulation. Further investigation demonstrated that renal apical efflux transporters MATE1, MRP2, and Pgp were also capable of mesna efflux. Administration of OAT inhibitor probenecid to healthy subjects significantly increased combined mesna and dimesna plasma exposure (91% ± 34%) while decreasing the renal clearance due to net secretion (67.0% ± 12.7%) and steady‐state volume of distribution (45.2% ± 13.4%). Thus, the kidney represents a significant sink of total mesna, whereas function of renal drug transporters facilitates clearance in excess of glomerular filtration rate and likely the presence of active mesna in the urine. Loss of renal transporter function due to genetic variability or drug—drug interactions may decrease the efficacy of chemoprotectants, increasing the risk of ifosfamide‐ and cisplatin‐induced toxicities.

Untargeted plasma and tissue metabolomics in rats with chronic kidney disease given AST-120

Chronic kidney disease (CKD) results in the accumulation of metabolic waste products that are normally cleared by the kidney, known as uremia. Many of these waste products are from bacteria metabolites in the gut. Accumulation of uremic toxins in plasma and tissue, as well as the gut-plasma-tissue metabolic axis are important for understanding pathophysiological mechanisms of comorbidities in CKD. In this study, an untargeted metabolomics approach was used to determine uremic toxin accumulation in plasma, liver, heart and kidney tissue in rats with adenine-induced CKD. Rats with CKD were also given AST-120, a spherical carbon adsorbent, to assess metabolic changes in plasma and tissues with the removal of gut-derived uremic toxins. AST-120 decreased >55% of metabolites that were increased in plasma, liver and heart tissue of rats with CKD. CKD was primarily defined by 8 gut-derived uremic toxins, which were significantly increased in plasma and all tissues. These metabolites were derived from aromatic amino acids and soy protein including: indoxyl sulfate, p-cresyl sulfate, hippuric acid, phenyl sulfate, pyrocatechol sulfate, 4-ethylphenyl sulfate, p-cresol glucuronide and equol 7-glucuronide. Our results highlight the importance of diet and gut-derived metabolites in the accumulation of uremic toxins and define the gut-plasma-tissue metabolic axis in CKD.

Decreased nuclear receptor activity and epigenetic modulation associates with down-regulation of hepatic drug-metabolizing enzymes in chronic kidney disease

Patients with chronic kidney disease (CKD) require many medications. CYP2C and CYP3A drug‐metabolizing enzymes play a critical role in determining the pharmacokinetics of the majority of prescribed medications. These enzymes are transcriptionally regulated by the nuclear receptors pregnane X receptor (PXR) and hepatic nuclear factor 4α (HNF4α). Expression of CYP2C and CYP3A is decreased in CKD; however, the mechanisms by which this occurs is unknown. We induced CKD in rats by 5/6 nephrectomy and used chromatin immunoprecipitation (ChIP) to determine nuclear receptor‐ and epigenetic alteration‐mediated differences in the promoter region of the CYP2C and CYP3A genes. RNA polymerase II and HNF‐4α binding was decreased 76 and 57% in the CYP2C11 promotor and 71 and 77% in the CYP3A2 promoter, respectively (P<0.05). ChIP also revealed a 57% decrease in PXR binding to the CYP3A2 promoter in CKD rats (P<0.05). The decrease in PXR and HNF‐4α binding was accompanied by diminished histone 4 acetylation in the CYP3A2 promoter (48%) and histone 3 acetylation in the CYP2C11 (77%) and CYP3A2 (77%) promoter loci for nuclear receptor activation (P<0.05). This study suggests that decreased nuclear receptor binding and histone acetylation may contribute to the mechanism of drug‐metabolizing enzyme down‐regulation and altered pharmacokinetics in CKD.–Velenosi, T. J., Feere, D. A., Sohi, G., Hardy, D. B., Urquhart, B. L. Decreased nuclear receptor activity and epigenetic modulation associates with down‐regulation of hepatic drug‐metabolizing enzymes in chronic kidney disease. FASEB J. 28, 5388–5397 (2014). www.fasebj.org

Metabolomic Response of Skeletal Muscle to Aerobic Exercise Training in Insulin Resistant Type 1 Diabetic Rats

The etiology of insulin resistance in Type 1 Diabetes (T1D) is unknown, however it affects approximately 20% of T1D patients. Intramyocellular lipids (IMCL) have been identified as a mechanism of insulin resistance. We examined skeletal muscle of T1D rats to determine if alterations in lipid metabolism were evident and whether aerobic exercise training improves IMCL and insulin resistance. To do so, 48 male Sprague-Dawley rats were divided into control (C), sedentary diabetes (D) and diabetes exercise (DX) groups. Following multiple low-dose Streptozotocin (STZ) injections (20 mg/kg), glycemia (9–15 mM) was maintained using insulin treatment. DX were treadmill trained at high intensity (~75% V02max; 5days/week) for 10 weeks. The results demonstrate that D exhibited insulin resistance compared with C and DX, indicated by decreased glucose infusion rate during a hyperinsulinemic-euglycemic clamp (p < 0.05). There were no differences between C and DX, suggesting that exercise improved insulin resistance (p < 0.05). Metabolomics analysis revealed a significant shift in lipid metabolism whereby notable fatty acid metabolites (arachidonic acid, palmitic acid and several polyunsaturated fatty acids) were significantly elevated in D compared to C and DX. Based on the intermediates observed, insulin resistance in T1D is characterized by an insulin-desensitizing intramyocellular fatty acid metabolite profile that is ameliorated with exercise training.

Effect of erythropoietin on hepatic cytochrome P450 expression and function in an adenine-fed rat model of chronic kidney disease

Erythropoietin (EPO) is used to treat anaemia associated with chronic kidney disease (CKD). Hypoxia is associated with anaemia and is known to cause a decrease in cytochrome P450 (P450) expression. As EPO production is regulated by hypoxia, we investigated the role of EPO on P450 expression and function.

Metabolic products of the intestinal microbiome and extremes of atherosclerosis

BACKGROUND AND AIMS There is increasing awareness that the intestinal microbiome plays an important role in human health. We investigated its role in the burden of carotid atherosclerosis, measured by ultrasound as total plaque area. METHODS Multiple regression with traditional risk factors was used to identify three phenotypes among 316/3056 patients attending vascular prevention clinics. Residual score (RES; i.e. the distance off the regression line, similar to standard deviation) was used to identify the 5% of patients with much less plaque than predicted by their risk factors (Protected, RES <-2), the 90% with about as much plaque as predicted (Explained, RES -2 to 2), and the 5% with much more plaque than predicted (Unexplained RES >2). Metabolic products of the intestinal microbiome that accumulate in renal failure - gut-derived uremic toxins (GDUT) - were assayed in plasma by ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. RESULTS Plasma levels of trimethylamine n-oxide (TMAO), p-cresyl sulfate, p-cresyl glucuronide, and phenylacetylglutamine were significantly lower among patients with the Protected phenotype, and higher in those with the Unexplained phenotype, despite no significant differences in renal function or in dietary intake of nutrient precursors of GDUT. In linear multiple regression with a broad panel of risk factors, TMAO (p = 0.011) and p-cresyl sulfate (p = 0.011) were significant independent predictors of carotid plaque burden. CONCLUSIONS The intestinal microbiome appears to play an important role in atherosclerosis. These findings raise the possibility of novel approaches to treatment of atherosclerosis such as fecal transplantation and probiotics.