Influence of coagulation factor, vitamin K epoxide reductase complex subunit 1, and cytochrome P450 2C9 gene polymorphisms on warfarin dose requirements

INTRODUCTION: The primary objective of this study was to determine whether variability in warfarin dose requirements is determined by common polymorphisms in genes whose products are involved in the pharmacodynamics and pharmacokinetics of warfarin, namely, the coagulation factors, vitamin K epoxide reductase complex subunit 1 (VKORC1), and cytochrome P450 (CYP) 2C9. METHODS: Patients (N = 350) receiving stable doses of warfarin at 3 consecutive visits were enrolled, and a deoxyribonucleic acid sample was collected. Samples were genotyped for polymorphisms in the factor II, factor VII, factor X, VKORC1, and CYP2C9 genes. A stepwise linear regression analysis was used to determine the independent effects of genetic and nongenetic factors on mean warfarin dose requirements. RESULTS: Variables associated with lower warfarin dose requirements were VKORC1 3673 AA genotype (P < .0001), VKORC1 3673 GA genotype (P < .0001), 1 variant CYP2C9 allele (P < .0001), 2 variant CYP2C9 alleles (P = .0004), increasing age (P = .0005), concomitant CYP2C9 inhibitors (P = .0005), and goal international normalized ratio (P = .01). Variables associated with higher warfarin dose requirements were weight (P < .0001), current smoker status (P = .0009), mean international normalized ratio (P = .001), concomitant CYP2C9 inducers (P = .006), factor X insertion/deletion genotype (P = .01), factor X insertion/insertion genotype (P = .04), factor VII deletion/deletion genotype (P = .04), and calculated vitamin K intake (P = .05). The linear regression model explained 51.4% of the variability in warfarin dose requirements. CONCLUSION: Polymorphisms in warfarin drug target and metabolizing enzyme genes, in addition to nongenetic factors, were important determinants of warfarin dose requirements.     

An ampD Gene in Pseudomonas aeruginosa Encodes a Negative Regulator of AmpC β-Lactamase Expression

The ampD and ampE genes of Pseudomonas aeruginosa PAO1 were cloned and characterized. These genes are transcribed in the same orientation and form an operon. The deduced polypeptide of P. aeruginosa ampD exhibited more than 60% similarity to the AmpD proteins of enterobacteria and Haemophilus influenzae. The ampD product transcomplemented Escherichia coli ampD mutants to wild-type β-lactamase expression.     

Functional activated carbon: from synthesis to groundwater fluoride removal

Developing green and functional adsorbents for the removal of inorganic pollutants from industrial wastewater is still a great challenge. Activated carbons (ACs) are promising eco-friendly materials for adsorption applications. This study reports on the preparation and functionalization of AC and its application for fluoride removal from water. Activated carbon was prepared from date stems, and the material was employed as a support for different modifications such as incorporation of Al(OH)₃, in situ dispersion of aluminum particles (Al⁰) and grafting of 3-(aminopropyl)triethoxysilane (APTES). The resulting functional adsorbents were fully characterized by Fourier transform infrared spectroscopy, scanning electronic microscopy, energy dispersive X-ray fluorescence, X-ray diffraction, differential scanning calorimetry and zeta potential analysis. The results evidenced successful surface modifications. All adsorbents had affinity for the removal of fluoride ions (F⁻). The highest F⁻ removal rate was up to 20 mg g⁻¹ for AC-Al(OH)₃. Removal of fluoride ions obeyed Langmuir isotherms and a second-order kinetic model, and reached 99% uptake. The AC-Al(OH)₃ adsorbent was successfully used to treat a groundwater solution contaminated by fluoride ions. These results open an interesting avenue for developing eco-friendly functionalized AC for adsorption applications.

Conversion and surface modification of date stems to obtain a relevant adsorbent to remove fluoride contamination.     

Hyperactivation of the hypothalamo‐pituitary‐adrenocortical axis in streptozotocin‐diabetic gerbils (Gerbillus gerbillus)

This study was designed to investigate the HPA‐axis impairment in the streptozotocin (STZ)‐diabetic gerbils (Gerbillus gerbillus). Twenty‐six male gerbils (body weight ~27 g) were divided into 3 groups: vehicle control (n = 10), 2 days of diabetes (n = 09) and 30 days of diabetes (n = 07). The latter 2 groups received an intraperitoneal injection of STZ (150 mg/kg of body weight). At 2 and 30 days of diabetes, streptozotocin‐diabetic gerbils underwent a retro‐orbital puncture for assessment of biochemical and hormonal parameters. Subsequently the animals were decapitated and the adrenal glands were removed, weighed and processed for light microscopy and stereology. Nondiabetic control gerbils that had been injected with citrate buffer were examined as a comparison. At 2 days of diabetes, STZ gerbils exhibited symptoms that are characteristic of human diabetes type 1. The adrenal gland showed significant increase in weight, associated with a larger cortex layer, hypertrophy of the fasciculate cells and a significant decrease in the nucleocytoplasmic index. These changes were associated with higher plasma ACTH and cortisol concentrations compared to nondiabetic controls. At 30 days postdiabetes, ACTH levels remained elevated, whereas cortisol levels decreased compared to the early stage of diabetes. Histological analysis revealed the existence of a band of connective tissue (collagen) that separates the cortical and medullary zones and is not present in humans or laboratory rodents, which represents a striking change seen throughout the disease. STZ‐induced diabetes mellitus in Gerbillus gerbillus resulted in hyperactivation of the HPA axis in the early stages of diabetes mellitus which did not persist into the final stages of the disease, suggesting a possible reduction in adrenocortical sensitivity over time.     

Implications of Polymorphisms in the BCKDK and GATA‐4 Gene Regions on Stable Warfarin Dose in African Americans

VKORC1 and CYP2C9 genotypes explain less variability in warfarin dose requirements in African Americans compared with Europeans. Variants in BCKDK and GATA‐4 gene regions, purported to regulate VKORC1 and CYP2C9 expression, have been shown to play an important role in warfarin dose requirements in Europeans and Asians, respectively. We sought to determine whether rs56314408 near BCKDK or GATA‐4 rs2645400 influence warfarin dose requirements in 200 African Americans. Unlike the strong linkage disequilibrium (LD) between rs56314408 and VKORC1 rs9923231 in Europeans, they were not in LD in African Americans. No associations were found on univariate analysis. On multivariable analysis, rs56314408 was associated (P = 0.027) with dose in a regression model excluding VKORC1 rs9923231, and GATA‐4 rs2645400 was associated (P = 0.032) with dose in a model excluding CYP2C (CYP2C9*2, *3, *5, *6, *8, and *11, CYP2C rs12777823) variants. Neither variant contributed to dose in the model that included both VKORC1 rs9923231 and CYP2C variants. Our results do not support contributions of the studied variants to warfarin dose requirements in African Americans. However, they illustrate the value of studies in African descent populations, who have low LD in their genome, in teasing out genetic variation underlying drug response associations. They also emphasize the importance of confirming associations in persons of African ancestry.

Study Highlights

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

The literature is limited in warfarin pharmacogenomic studies in African Americans. VKORC1 and CYP2C9 genotypes explain less variability in warfarin dose requirements in African Americans compared with those of European ancestry. Variants in BCKDK and GATA‐4 gene regions, purported to regulate VKORC1 and CYP2C9 expression, have been studied in Europeans and Asians, respectively, but not in African Americans.

• WHAT QUESTION DID THIS STUDY ADDRESS?

We sought to determine whether rs56314408 near BCKDK or GATA‐4 rs2645400 influence warfarin dose requirements in African Americans.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

Our results do not support major contributions of studied variants to warfarin dose requirements in African Americans. However, our findings illustrate the value of studies in African descent populations, who have low linkage disequilibrium in their genome, in teasing out genetic variation underlying drug response associations. They also highlight the importance of confirming associations in persons of African ancestry.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

Results do not support the inclusion of rs56314408 near BCKDK or GATA‐4 rs2645400 in pharmacogenomic dosing algorithms for African Americans.

Warfarin remains commonly prescribed despite the availability of direct‐acting oral anticoagulants. ¹ It is, however, a challenging drug to dose because of its narrow therapeutic index and pronounced interpatient variability in dose requirements. Warfarin dose requirements are influenced by VKORC1 and CYP 2C9 genotypes and clinical factors. ² VKORC1‐1639G>A (rs9923231) and CYP2C9*2 and *3 are the variants most commonly included in pharmacogenetic dosing algorithms and explain more than 30% of dose variability in Europeans, but only about 10% of the variability in African Americans. ³ , ⁴ , ⁵ , ⁶ The CYP2C9*5, *6, *8, and *11 and the rs12777823 variant in the CYP2C cluster explain an additional 11% of the dose variability in African Americans. ⁴ , ⁷ , ⁸ Pharmacogenomic studies on warfarin are mostly done in Europeans and Asians. There is, therefore, a gap in the literature on warfarin pharmacogenomic studies in minorities like African Americans who show differences in minor allele frequencies (MAF) and have a lower linkage disequilibrium (LD) pattern. ⁹ A recent study in a European population suggested that the rs56314408 C>T single nucleotide polymorphism (SNP), located on chromosome 16 in an enhancer upstream of the branched chain ketoacid dehydrogenase kinase (BCKDK) gene, could be a functional variant regulating VKORC1 gene expression. ¹⁰ However, rs56314408 is in high LD with VKORC1 rs9923231 in Europeans, and thus, whether this SNP influences warfarin dose independent of the VKORC1 rs9923231 variant could not be determined in Europeans. Therefore, Cavalli et al. mentioned that it is warranted to genotype rs56314408 in warfarin‐treated African Americans to see whether it improves warfarin dose predictions. ¹⁰ In another study, the GATA binding protein 4 (GATA‐4) rs2645400 T>G variant was significantly associated with stable warfarin dose in Asians with prosthetic cardiac valves having the homozygous wild‐type (i.e., *1/*1) CYP2C9 genotype. On multivariable analysis, rs2645400/rs4841588 combination increased contribution to the overall warfarin dose variability. This study, therefore, suggested that GATA‐4 plays a role in the regulation of CYP2C9 gene expression and can be predictive of stable warfarin dose. ¹¹ Neither rs56314408 nor rs2645400 has been studied in African Americans. Therefore, this study aimed to determine whether the rs56314408 variant near BCKDK, which is in low LD with VKORC1 rs9923231 in African Americans, and/or the GATA‐4 rs2645400 variant are associated with warfarin dose requirements in African Americans and explain additional variability in dose beyond that of the VKORC1 and CYP2C (CYP2C9*2, *3, *5, *6, *8, and *11 and CYP2C rs12777823) polymorphisms. In our study, we are using these two SNPs as cases to illustrate the importance of carrying out pharmacogenomic studies in African Americans.     

Depletion of T regulatory cells through selection of CD127-positive cells results in a population enriched in memory T cells: implications for anti-tumor cell therapy

Background

Donor lymphocyte infusions can induce remissions in patients with relapse after allogeneic hematopoietic stem cell transplantation. Nevertheless, some grafted patients never display any signs of alloreactivity, either following allogeneic hematopoietic stem cell transplantation or after donor lymphocyte infusions. Consequently, they do not develop graft-versus-host disease and frequently do not respond to donor lymphocyte infusions. In a recently published clinical trial, we observed that elimination of CD4⁺CD25⁺Foxp3⁺ natural regulatory T cells from the donor lymphocyte product could improve alloreactivity and the associated anti-tumor effect in a small proportion of patients with relapsed hematologic malignancies. Here, we aimed to improve the effect of donor lymphocyte infusion by modifying the procedure for depletion of T regulatory cells.

Design and Methods

We directly compared depletion of regulatory T cells from human peripheral blood mononuclear cells achieved by selection of CD127-positive cells or by selection of CD25-negative cells. We tested the manipulated products (i) in vitro in mixed lymphocyte reactions and against pathogen-derived recall antigens and (ii) in vivo in experimental graft-versus-host disease.

Results

In vitro, we found that depletion of regulatory T cells through CD127 positive selection improved both alloreactive and pathogen-specific immune responses. In vivo, we observed accelerated donor T-cell division and enhanced graft-versus-host disease due to efficient regulatory T-cell depletion accompanied by enrichment in memory T cells.

Conclusions

Our results show that the strategy of CD127 positive selection is an efficient way of eliminating regulatory T cells from donor lymphocyte infusions and improves alloreactivity. This supports the investigation of CD127 positive selection in place of elimination of CD25-positive cells for clinical applications.Key words: CD127 positive selection, Treg, alloreactivity, donor lymphocyte infusion     

Decreased heart rate variability may predict the progression of carotid atherosclerosis in type 2 diabetes

Heart rate variability (HRV), a measure of autonomic function, can predict survival outcomes. Cardiovascular disease is a known complication of diabetes, and we aimed to determine if autonomic dysfunction was associated with carotid artery atherosclerotic plaques in type 2 diabetic patients. We assessed frequency domain HRV from power spectral analysis of 24 h Holter ECG recordings, expiration/inspiration (E/I) ratio during deep breathing, acceleration index (AI) of R–R interval in response to head-up tilt, and the degree of carotid artery atherosclerosis in 61 type-2 diabetic patients (39 males, 45–69 years). Studies were carried out 5–6 years after diagnosis (baseline) and repeated 8 years after diagnosis (follow-up). At baseline, patients diagnosed with autonomic neuropathy, with abnormal E/I ratio and abnormal AI measurements, had decreased low frequency (LF) HRV. Baseline E/I ratio correlated with day (r = 0.34; P < 0.001) and night-time (r = 0.44; P < 0.001) LF power. Night-time HRV did not differ in patient with and without autonomic neuropathy. Reduced common carotid artery diameter and atherosclerotic intima-media thickness (IMT) both correlated with HRV at baseline. At follow-up, all HRV variables decreased significantly. Furthermore, patients with lower LF power at baseline, had a larger increase in the thickness of the carotid bulb intima-media at follow-up. Our results show that LF HRV power is associated with the extent and progression of carotid atherosclerosis in type 2 diabetes. A low LF HRV may predict the progression of atherosclerosis in these patients.