Introduction of the AmpliChip CYP450 Test to a South African cohort: a platform comparative prospective cohort study.

BACKGROUND: Adverse drug reactions and lack of therapeutic efficacy associated with currently prescribed pharmacotherapeutics may be attributed, in part, to inter-individual variability in drug metabolism. Studies on the pharmacogenetics of Cytochrome P450 (CYP) enzymes offer insight into this variability. The objective of this study was to compare the AmpliChip CYP450 Test® (AmpliChip) to alternative genotyping platforms for phenotype prediction of CYP2C19 and CYP2D6 in a representative cohort of the South African population. METHODS: AmpliChip was used to screen for thirty-three CYP2D6 and three CYP2C19 alleles in two different cohorts. As a comparison cohort 2 was then genotyped using a CYP2D6 specific long range PCR with sequencing (CYP2D6 XL-PCR + Sequencing) platform and a PCR-RFLP platform for seven CYP2C19 alleles. RESULTS: Even though there was a low success rate for the AmpliChip, allele frequencies for both CYP2D6 and CYP2C19 were very similar between the two different cohorts. The CYP2D6 XL-PCR + Sequencing platform detected CYP2D6*5 more reliably and could correctly distinguish between CYP2D6*2 and *41 in the Black African individuals. Alleles not covered by the AmpliChip were identified and four novel CYP2D6 alleles were also detected. CYP2C19 PCR-RFLP identified CYP2C19*9,*15, *17 and *27 in the Black African individuals, with *2, *17 and *27 being relatively frequent in the cohort. Eliminating mismatches and identifying additional alleles will contribute to improving phenotype prediction for both enzymes. Phenotype prediction differed between platforms for both genes. CONCLUSION: Comprehensive genotyping of CYP2D6 and CYP2C19 with the platforms used in this study, would be more appropriate than AmpliChip for phenotypic prediction in the South African population. Pharmacogenetically important novel alleles may remain undiscovered when using assays that are designed according to Caucasian specific variation, unless alternate strategies are utilised.

HLA-DRB1 shared epitope genotyping using the revised classification and its association with circulating autoantibodies, acute phase reactants, cytokines and clinical indices of disease activity in a cohort of South African rheumatoid arthritis patients.

INTRODUCTION: The revised shared epitope (SE) concept in rheumatoid arthritis (RA) is based on the presence (S) or absence (X) of the SE RAA amino acid motif at positions 72 to 74 of the third hypervariable region of the various human leucocyte antigen (HLA)-DRB1 alleles. The purpose of this study was to investigate SE subtypes on the basis of the American College of Rheumatology 1987 revised criteria for the classification of RA in a cohort of South African RA patients (n = 143) and their association with clinical and circulating biomarkers of disease activity (autoantibodies, acute phase reactants and cytokines). METHODS: Genomic DNA was analysed using high-resolution recombinant sequence-specific oligonucleotide PCR typing of the HLA-DRB1 allele. Subtypes of the SE were classified according to the amino acids at positions 72 to 74 for the RAA sequence, and further sub-divided according to the amino acids at positions 70 and 71, which either contribute to (S2, S3P), or negate (S1, S3D) RA susceptibility. Disease activity was assessed on the basis of (1) Disease Activity Score in 28 joints using C-reactive protein (CRP), (2) rheumatoid factor (RF), (3) CRP and (4) serum amyloid A by nephelometry, anticyclic citrullinated peptide antibodies (aCCP) by an immunofluorometric procedure, and cytokines by multiplex bead array technology. RESULTS: Of the 143 RA patients, 81 (57%) were homozygous (SS) and 50 (35%) were heterozygous (SX) for the SE alleles with significant overexpression of S2 and S3P (respective odds ratios (ORs) 5.3 and 5.8; P < 0.0001), and 12 (8%) were classified as no SE allele (XX). Both the SS and SX groups showed a strong association with aCCP positivity (OR = 10.2 and P = 0.0010, OR = 9.2 and P = 0.0028, respectively) relative to the XX group. Clinical scores and concentrations of the other biomarkers of disease activity (RF, CRP and T helper cell type 1 (Th1), Th2, macrophage and fibroblast cytokines) were also generally higher in the SS group than in the SX and XX groups. CONCLUSIONS: RA susceptibility alleles investigated according to revised criteria for the classification of RA were significantly increased in South African RA patients and strongly associated with aCCP in particular as well as with circulating cytokines and disease severity.

Circulating cytokine profiles and their relationships with autoantibodies, acute phase reactants, and disease activity in patients with rheumatoid arthritis.

Our objective was to analyse the relationship between circulating cytokines, autoantibodies, acute phase reactants, and disease activity in DMARDs-naïve rheumatoid arthritis (RA) patients (n = 140). All cytokines were significantly higher in the RA cohort than in healthy controls. Moderate-to-strong positive intercorrelations were observed between Th1/Th2/macrophage/fibroblast-derived cytokines. RF correlated significantly with IL-1ß, IL-2, IL-4, IL-10, IL-12, G-CSF, GM-CSF, IFN-γ, and TNF (P < .0001), and aCCP and aMCV with IL-1ß, IL-2, IL-4, and IL-10 (P < .0002), while IL-6 correlated best with the acute phase reactants, CRP, and SAA (P < .0001). In patients with a DAS28 score of ≥5.1, IFN-γ, IL-1ß, IL-1Ra, TNF, GM-CSF, and VEGF were significantly correlated (P < .04-.001) with high disease activity (HDA). Circulating cytokines in RA reflect a multifaceted increase in immune reactivity encompassing Th1 and Th2 cells, monocytes/macrophages, and synovial fibroblasts, underscored by strong correlations between these cytokines, as well as their relationships with RF, aCCP, and aMCV, with some cytokines showing promise as biomarkers of HDA.

Increased oxidants and reduced antioxidants in irradiated parenteral nutrition solutions may contribute to the inflammatory response.

BACKGROUND/OBJECTIVES: To measure reactive oxidant production and the decline in antioxidant potential in commercially available, irradiated parenteral nutrition (PN) solutions and the effect that these have on oxidant production in patients in the intensive care unit. SUBJECTS AND METHODS: Vitamin E and malondialdehyde in irradiated and nonirradiated commercially available, PN solutions were measured. The PBN (alpha-phenyl-n-test-butylnitrone (PBN) spin trap was used to measure free radicals and TEMPOL (2,2,6,6-tetramethyl-4-hydroxy-piperidine-oxyl) was used to assess antioxidant capacity. The irradiated PN was administered (as per unit protocol) to 10 patients with gut failure and plasma and urinary isoprostanes and interleukin-6 (IL-6) were measured 1 hour preadministration, at the time of, and 1 and 2 hours postadministration of PN. RESULTS: Irradiation reduced vitamin E significantly (P < .0025). Malondialdehyde products were present in both samples, but more so in irradiated samples (P < .0001), as were free radicals measured by PBN spin trapping. Irradiated samples had a higher scavenging capacity of TEMPOL free radical due to depletion of antioxidants in irradiated samples. Urinary isoprostanes increased at time 2 by 6.3 units relative to time 0 and by 5.23 units relative to time 1(Friedman ANOVA: P < .01413). CONCLUSIONS: Lipid hydroperoxides are formed in PN solutions and increase further following irradiation. This is associated with a significant reduction in vitamin E and antioxidant potential. The increase in urinary isoprostanes indicates a potentially proinflammatory effect of irradiated PN.

Palladium attenuates the pro-inflammatory interactions of C5a, interleukin-8 and pneumolysin with human neutrophils.

The primary objective of this study was to investigate the effects of cobalt (Co(2 +)), palladium (Pd(2 +)), platinum (Pt(4 +)) and vanadium (V(2 +), V(3 +), V(4 +) and V(5 +)) on the ability of the neutrophil chemoattractants C5a and IL-8, as well as the pneumococcal toxin, pneumolysin, to activate human neutrophils in vitro. Neutrophil activation was determined according to the magnitude of the increase in cytosolic Ca(2 +) concentrations using a fura-2/AM-based, spectrofluorimetric procedure, as well as by a chemotaxis assay using modified Boyden chambers. In initial screening studies, in which the metals were used at a fixed concentration of 25 mu M, the Ca(2 +)-mobilizing interactions of C5a, IL-8, and pneumolysin were unaffected by exposure to Co(2 +), Pt(4 +) and V(2 + - 5 +). However, exposure of C5a, IL-8, and pneumolysin to Pd(2 +) resulted in either partial (IL-8) or complete (C5a and pneumolysin) loss of Ca(2 +) -mobilizing and chemotactic activities. In dose-response experiments, these effects of Pd(2 +) were detectable at a threshold concentration of 6.5 mu M. These observations demonstrate that exposure to Pd(2 +) may compromise innate host defenses, a previously unrecognized potential health threat of environmental and/or occupational exposure to a ubiquitous heavy metal.

Vanadium promotes hydroxyl radical formation by activated human neutrophils.

This study was undertaken to investigate the effects of vanadium in the +2, +3, +4, and +5 valence states on superoxide generation, myeloperoxidase (MPO) activity, and hydroxyl radical formation by activated human neutrophils in vitro, using lucigenin-enhanced chemiluminescence (LECL), autoiodination, and electron spin resonance with 5,5-dimethyl-l-pyrroline N-oxide as the spin trap, respectively. At concentrations of up to 25 microM, vanadium, in the four different valence states used, did not affect the LECL responses of neutrophils activated with either the chemoattractant, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (1 microM), or the phorbol ester, phorbol 12-myristate 12-acetate (25 ng/ml). However, exposure to vanadium in the +2, +3, and +4, but not the +5, valence states was accompanied by significant augmentation of hydroxyl radical formation by activated neutrophils and attenuation of MPO-mediated iodination. With respect to hydroxyl radical formation, similar effects were observed using cell-free systems containing either hydrogen peroxide (100 microM) or xanthine/xanthine oxidase together with vanadium (+2, +3, +4), while the activity of purified MPO was inhibited by the metal in these valence states. These results demonstrate that vanadium in the +2, +3, and +4 valence states interacts prooxidatively with human neutrophils, competing effectively with MPO for hydrogen peroxide to promote formation of the highly toxic hydroxyl radical.