Amplifying STING activation by cyclic dinucleotide-manganese particles for local and systemic cancer metalloimmunotherapy.

Nutritional metal ions play critical roles in many important immune processes. Hence, the effective modulation of metal ions may open up new forms of immunotherapy, termed as metalloimmunotherapy. Here, we demonstrate a prototype of cancer metalloimmunotherapy using cyclic dinucleotide (CDN) stimulator of interferon genes (STING) agonists and Mn2+. We screened various metal ions and discovered specific metal ions augmented STING agonist activity, wherein Mn2+ promoted a 12- to 77-fold potentiation effect across the prevalent human STING haplotypes. Notably, Mn2+ coordinated with CDN STING agonists to self-assemble into a nanoparticle (CDN-Mn2+ particle, CMP) that effectively delivered STING agonists to immune cells. The CMP, administered either by local intratumoural or systemic intravenous injection, initiated robust anti-tumour immunity, achieving remarkable therapeutic efficacy with minute doses of STING agonists in multiple murine tumour models. Overall, the CMP offers a new platform for local and systemic cancer treatments, and this work underscores the great potential of coordination nanomedicine for metalloimmunotherapy.

Haploidentical vs haplo-cord transplant in adults under 60 years receiving fludarabine and melphalan conditioning.

Haplo-identical transplant with posttransplant cyclophosphamide (haplo) and umbilical cord blood transplant supported by third-party CD34 cells (haplo-cord) are competing approaches to alternative donor transplant. We compared, in adults younger than age 60 years, the outcomes of 170 haplo at 1 institution with that of 137 haplo-cord at 2 other institutions. All received reduced intensity conditioning with fludarabine and melphalan ± total body irradiation. GVHD prophylaxis for haplo consisted of cyclophosphamide, tacrolimus, and mycophenolate, whereas haplo-cord received antithymocyte globulin, tacrolimus, and mycophenolate. Haplo transplant used mostly bone marrow, and peripheral blood stem cells were used in haplo-cord transplants. Haplo-cord were older and had more advanced disease. Haplo-cord hastened median time to neutrophil (11 vs 18 days, P = .001) and platelet recovery (22 vs 25 days, P = .03). At 4 years, overall survival (OS) was 50% for haplo-cord vs 49% for haplo. Progression-free survival (PFS) was 40% for haplo-cord vs 45% for haplo. In multivariate analysis, the disease risk index was significant for OS (hazard ratio, 1.8; 95% confidence interval, 1.48-2.17; P = .00) and PFS. Total body irradiation was associated with decreased recurrence and improved PFS, age >40 with increased nonrelapse mortality. The type of transplant had no effect on OS, PFS, relapse, or nonrelapse mortality. Cumulative incidence of grade 2-4 acute graft-versus-host disease (GVHD) by day 100 was 16% after haplo-cord vs 33% after haplo (P < .0001), but grade 3-4 GVHD was similar. Chronic GVHD at 1 year was 4% after haplo-cord vs 16% after haplo (P < .0001). Haplo or haplo-cord results in similar and encouraging outcomes. Haplo-cord is associated with more rapid neutrophil and platelet recovery and lower acute and chronic GVHD. Institutional review board authorization for this retrospective study was obtained at each institution. Some patients participated in trials registered at www.clinicaltrials.gov as #NCT01810588 and NCT01050946.

Brettanomyces bruxellensis SSU1 Haplotypes Confer Different Levels of Sulfite Tolerance When Expressed in a Saccharomyces cerevisiae SSU1 Null Mutant.

The addition of SO2 is practiced in the wine industry to mitigate the risk of microbial spoilage and to extend wine shelf-life. Generally, this strategy does not interfere with primary alcoholic fermentation, as wine strains of Saccharomyces cerevisiae exhibit significant SO2 tolerance, largely driven by the efflux pump Ssu1p. One of the key yeast species responsible for wine spoilage is Brettanomyces bruxellensis, which also exhibits strain-dependent SO2 tolerance, although this occurs via unknown mechanisms. To evaluate the factors responsible for the differential sulfite tolerance observed in B. bruxellensis strains, we employed a multifaceted approach to examine both expression and allelic differences in the BbSSU1 gene. Transcriptomic analysis following exposure to SO2 highlighted different inducible responses in two B. bruxellensis strains. It also revealed disproportionate transcription of one putative BbSSU1 haplotype in both genetic backgrounds. Here, we confirm the functionality of BbSSU1 by complementation of a null mutant in a S. cerevisiae wine strain. The expression of four distinct BbSSU1 haplotypes in the S. cerevisiae ΔSSU1 mutant revealed up to a 3-fold difference in conferred SO2 tolerance. Substitution of key amino acids distinguishing the encoded proteins was performed to evaluate their relative contribution to SO2 tolerance. Protein modeling of two haplotypes which differed in two amino acid residues suggested that these substitutions affect the binding of Ssu1p ligands near the channel opening. Taken together, preferential transcription of a BbSSU1 allele that encodes a more efficient Ssu1p transporter may represent one mechanism that contributes to differences in sulfite tolerances between B. bruxellensis strains.IMPORTANCEBrettanomyces bruxellensis is one of the most important wine spoilage microorganisms, with the use of sulfite being the major method to control spoilage. However, this species displays a wide intraspecies distribution in sulfite tolerance, with some strains capable of tolerating high concentrations of SO2, with relatively high concentrations of this antimicrobial needed for their control. Although SO2 tolerance has been studied in several organisms and particularly in S. cerevisiae, little is known about the mechanisms that confer SO2 tolerance in B. bruxellensis Here, we confirmed the functionality of the sulfite efflux pump encoded by BbSSU1 and determined the efficiencies of four different BbSSU1 haplotypes. Gene expression analysis showed greater expression of the haplotype conferring greater SO2 tolerance. Our results suggest that a combination of BbSSU1 haplotype efficiency, copy number, and haplotype expression levels likely contributes to the diverse SO2 tolerances observed for different B. bruxellensis strains.

Haplogroup J mitogenomes are the most sensitive to the pesticide rotenone: Relevance for human diseases.

There is growing evidence that the sequence variation of mitochondrial DNA (mtDNA), which clusters in population- and/or geographic-specific haplogroups, may result in functional effects that, in turn, become relevant in disease predisposition or protection, interaction with environmental factors and ultimately in modulating longevity. To unravel functional differences between mtDNA haplogroups we here employed transmitochondrial cytoplasmic hybrid cells (cybrids) grown in galactose medium, a culture condition that forces oxidative phosphorylation, and in the presence of rotenone, the classic inhibitor of respiratory Complex I. Under this experimental paradigm we assessed functional parameters such as cell viability and respiration, ATP synthesis, reactive oxygen species production and mtDNA copy number. Our analyses show that haplogroup J1, which is common in western Eurasian populations, is the most sensitive to rotenone, whereas K1 mitogenomes orchestrate the best compensation, possibly because of the haplogroup-specific missense variants impinging on Complex I function. Remarkably, haplogroups J1 and K1 fit the genetic associations previously established with Leber's hereditary optic neuropathy (LHON) for J1, as a penetrance enhancer, and with Parkinson's disease (PD) for K1, as a protective background. Our findings provide functional evidences supporting previous well-established genetic associations of specific haplogroups with two neurodegenerative pathologies, LHON and PD. Our experimental paradigm is instrumental to highlighting the subtle functional differences characterizing mtDNA haplogroups, which will be increasingly needed to dissect the role of mtDNA genetic variation in health, disease and longevity.

Retinoic acid and sodium butyrate suppress the cardiac expression of hypertrophic markers and proinflammatory mediators in Npr1 gene-disrupted haplotype mice.

The objective of the present study was to examine the genetically determined differences in the natriuretic peptide receptor-A (NPRA) gene (Npr1) copies affecting the expression of cardiac hypertrophic markers, proinflammatory mediators, and matrix metalloproteinases (MMPs) in a gene-dose-dependent manner. We determined whether stimulation of Npr1 by all-trans retinoic acid (RA) and histone deacetylase (HDAC) inhibitor sodium butyric acid (SB) suppress the expression of cardiac disease markers. In the present study, we utilized Npr1 gene-disrupted heterozygous (Npr1(+/-), 1-copy), wild-type (Npr1(+/+), 2-copy), gene-duplicated (Npr1(++/+), 3-copy) mice, which were treated intraperitoneally with RA, SB, and a combination of RA/SB, a hybrid drug (HB) for 2 wk. Untreated 1-copy mice showed significantly increased heart weight-body weight (HW/BW) ratio, blood pressure, hypertrophic markers, including beta-myosin heavy chain (ß-MHC) and proto-oncogenes (c-fos and c-jun), proinflammatory mediator nuclear factor kappa B (NF-κB), and MMPs (MMP-2, MMP-9) compared with 2-copy and 3-copy mice. The heterozygous (haplotype) 1-copy mice treated with RA, SB, or HB, exhibited significant reduction in the expression of ß-MHC, c-fos, c-jun, NF-κB, MMP-2, and MMP-9. In drug-treated animals, the activity and expression levels of HDAC were significantly reduced and histone acetyltransferase activity and expression levels were increased. The drug treatments significantly increased the fractional shortening and reduced the systolic and diastolic parameters of the Npr1(+/-) mice hearts. Together, the present results demonstrate that a decreased Npr1 copy number enhanced the expression of hypertrophic markers, proinflammatory mediators, and MMPs, whereas an increased Npr1 repressed the cardiac disease markers in a gene-dose-dependent manner.

Novel point mutations in the ERG11 gene in clinical isolates of azole resistant Candida species.

The azoles are the class of medications most commonly used to fight infections caused by Candida sp. Typically, resistance can be attributed to mutations in ERG11 gene (CYP51) which encodes the cytochrome P450 14α-demethylase, the primary target for the activity of azoles. The objective of this study was to identify mutations in the coding region of theERG11 gene in clinical isolates of Candida species known to be resistant to azoles. We identified three new synonymous mutations in the ERG11 gene in the isolates of Candida glabrata (C108G, C423T and A1581G) and two new nonsynonymous mutations in the isolates of Candida krusei--A497C (Y166S) and G1570A (G524R). The functional consequence of these nonsynonymous mutations was predicted using evolutionary conservation scores. The G524R mutation did not have effect on 14α-demethylase functionality, while the Y166S mutation was found to affect the enzyme. This observation suggests a possible link between the mutation and dose-dependent sensitivity to voriconazole in the clinical isolate of C. krusei. Although the presence of the Y166S in phenotype of reduced azole sensitivity observed in isolate C. krusei demands investigation, it might contribute to the search of new therapeutic agents against resistant Candida isolates.

Novel point mutations in the ERG11 gene in clinical isolates of azole resistant Candida species

The azoles are the class of medications most commonly used to fight infections caused by Candida sp. Typically, resistance can be attributed to mutations in ERG11 gene (CYP51) which encodes the cytochrome P450 14α-demethylase, the primary target for the activity of azoles. The objective of this study was to identify mutations in the coding region of theERG11 gene in clinical isolates of Candidaspecies known to be resistant to azoles. We identified three new synonymous mutations in the ERG11 gene in the isolates of Candida glabrata (C108G, C423T and A1581G) and two new nonsynonymous mutations in the isolates of Candida krusei - A497C (Y166S) and G1570A (G524R). The functional consequence of these nonsynonymous mutations was predicted using evolutionary conservation scores. The G524R mutation did not have effect on 14α-demethylase functionality, while the Y166S mutation was found to affect the enzyme. This observation suggests a possible link between the mutation and dose-dependent sensitivity to voriconazole in the clinical isolate of C. krusei. Although the presence of the Y166S in phenotype of reduced azole sensitivity observed in isolate C. kruseidemands investigation, it might contribute to the search of new therapeutic agents against resistant Candida isolates.

Persistent HIV-1 replication maintains the tissue reservoir during therapy.

Lymphoid tissue is a key reservoir established by HIV-1 during acute infection. It is a site associated with viral production, storage of viral particles in immune complexes, and viral persistence. Although combinations of antiretroviral drugs usually suppress viral replication and reduce viral RNA to undetectable levels in blood, it is unclear whether treatment fully suppresses viral replication in lymphoid tissue reservoirs. Here we show that virus evolution and trafficking between tissue compartments continues in patients with undetectable levels of virus in their bloodstream. We present a spatial and dynamic model of persistent viral replication and spread that indicates why the development of drug resistance is not a foregone conclusion under conditions in which drug concentrations are insufficient to completely block virus replication. These data provide new insights into the evolutionary and infection dynamics of the virus population within the host, revealing that HIV-1 can continue to replicate and replenish the viral reservoir despite potent antiretroviral therapy.

The dietary monounsaturated to saturated fatty acid ratio modulates the genetic effects of GCKR on serum lipid levels in children.

BACKGROUND: Glucokinase regulator (GCKR) plays important roles in the regulation of glucokinase (GK) activity and the metabolism of glucose and lipids. We investigated whether the association between GCKR genetic variants with serum lipids in Korean adults is replicated in children, and whether these genetic influences might be modulated by dietary monounsaturated fatty acid relative to saturated fatty acid (MUFA:SFA) ratio. METHODS: We genotyped 711 children for GCKR variants, used 7495 adults in KARE database, and analyzed anthropometric, biochemical, and dietary measurements. RESULTS: The major allele carriers of rs780094 and rs780092 in adults had significantly higher serum total cholesterol and triglycerides levels compared to noncarriers. Five variants in children, including rs780094 and rs780092, correlated similarly with high total and low-density lipoprotein (LDL) cholesterol. When the dietary MUFA:SFA ratio was dichotomized (MUFA:SFA≥1 or <1), the aggravating effects of the major allele on total cholesterol, LDL cholesterol, and triglycerides were only evident in the group in which MUFA:SFA ratio was <1. Additionally, we observed that the GCKR haplotype with a functional variant, rs1260326, influenced lower total and LDL cholesterol in children whose MUFA:SFA ratio was <1. CONCLUSION: We replicated the genetic association effect of GCKR on total cholesterol in children, and found that the interaction effects between GCKR genetic variants and the dietary MUFA:SFA ratio on lipid levels, were commonly observed in Korean adults and children.

Occurrence of multiple chloroquine-resistant Pfcrt haplotypes and emergence of the S(agt)VMNT type in Cameroonian Plasmodium falciparum.

OBJECTIVES: The main objective of this study was to unravel the distribution of different Pfcrt genotypes in the central, littoral, eastern and southern regions of Cameroon and also in locations bordering Gabon and Equatorial Guinea. This is because (i) the chloroquine-resistant malaria parasite Plasmodium falciparum shows a wide occurrence in Cameroon, (ii) mutations in the 72nd to 76th amino acid positions of the Pfcrt gene are known to confer resistance to chloroquine, and (iii) only a single chloroquine-resistant haplotype (C72V73I74E75T76) has so far been reported in Cameroon. METHODS: We followed a molecular approach with DNA sequencing of the second exon of the Pfcrt gene to identify single nucleotide polymorphisms in 180 P. falciparum field isolates sampled in five different locations in Cameroon. RESULTS: The chloroquine-resistant Pfcrt CVIET haplotype was most abundant, followed by the wild-type CVMNK haplotype. Five hitherto unreported chloroquine-resistant Pfcrt haplotypes were detected for the first time in Cameroonian P. falciparum, including the surprise appearance of the S(agt)VMNT haplotype. CONCLUSIONS: The high observed haplotype diversity of the chloroquine-resistant Pfcrt gene and the appearance of the S(agt)VMNT haplotype are daunting and can be attributed to drug pressure and/or the misuse of chloroquine and/or amodiaquine in Cameroon.

Human dopamine transporter gene: differential regulation of 18-kb haplotypes.

AIM: Since previous functional studies of short haplotypes and polymorphic sites of SLC6A3 have shown variant-dependent and drug-sensitive promoter activity, this study aimed to understand whether a large SLC6A3 regulatory region, containing these small haplotypes and polymorphic sites, can display haplotype-dependent promoter activity in a drug-sensitive and pathway-related manner. MATERIALS & METHODS: By creating and using a single copy number luciferase-reporter vector, we examined regulation of two different SLC6A3 haplotypes (A and B) of the 5´ 18-kb promoter and two known downstream regulatory variable number tandem repeats by 17 drugs in four different cellular models. RESULTS: The two regulatory haplotypes displayed up to 3.2-fold difference in promoter activity. The regulations were drug selective (37.5% of the drugs showed effects), and both haplotype and cell type dependent. Pathway analysis revealed at least 13 main signaling hubs targeting SLC6A3, including histone deacetylation, AKT, PKC and CK2 α-chains. CONCLUSION: SLC6A3 may be regulated via either its promoter or the variable number tandem repeats independently by specific signaling pathways and in a haplotype-dependent manner. Furthermore, we have developed the first pathway map for SLC6A3 regulation. These findings provide a framework for understanding complex and variant-dependent regulations of SLC6A3.

Efficient arsenic metabolism--the AS3MT haplotype is associated with DNA methylation and expression of multiple genes around AS3MT.

Arsenic is a very potent toxicant. One major susceptibility factor for arsenic-related toxicity is the efficiency of arsenic metabolism. The efficiency, in turn, is associated with non-coding single nucleotide polymorphisms (SNPs) in the arsenic methyltransferase AS3MT on chromosome 10q24. However, the mechanism of action for these SNPs is not yet clarified. Here, we assessed the influence of genetic variation in AS3MT on DNA methylation and gene expression within 10q24, in people exposed to arsenic in drinking water. DNA was extracted from peripheral blood from women in the Argentinean Andes (N = 103) and from cord blood from new-borns in Bangladesh (N = 127). AS3MT SNPs were analyzed with Sequenom or Taqman assays. Whole genome epigenetic analysis with Infinium HumanMethylation450 BeadChip was performed on bisulphite-treated DNA. Whole genome gene expression analysis was performed with Illumina DirectHyb HumanHT-12 v4.0 on RNA from peripheral blood. Arsenic exposure was assessed by HPLC-ICPMS. In the Argentinean women, the major AS3MT haplotype, associated with more efficient arsenic metabolism, showed increased methylation of AS3MT (p = 10(-6)) and also differential methylation of several other genes within about 800 kilobasepairs: CNNM2 (p<10(-16)), NT5C2 (p<10(-16)), C10orf26 (p = 10(-8)), USMG5 (p = 10(-5)), TRIM8 (p = 10(-4)), and CALHM2 (p = 0.038) (adjusted for multiple comparisons). Similar, but weaker, associations between AS3MT haplotype and DNA methylation in 10q24 were observed in cord blood (Bangladesh). The haplotype-associated altered CpG methylation was correlated with reduced expression of AS3MT and CNNM2 (r(s) = -0.22 to -0.54), and with increased expression of NT5C2 and USMG5 (r(s) = 0.25 to 0.58). Taking other possibly influential variables into account in multivariable linear models did only to a minor extent alter the strength of the associations. In conclusion, the AS3MT haplotype status strongly predicted DNA methylation and gene expression of AS3MT as well as several genes in 10q24. This raises the possibility that several genes in this region are important for arsenic metabolism.

Genetic variation at the FADS1-FADS2 gene locus influences delta-5 desaturase activity and LC-PUFA proportions after fish oil supplement.

Delta-5 and delta-6 desaturases (D5D and D6D) are key enzymes in endogenous synthesis of long-chain PUFAs. In this sample of healthy subjects (n = 310), genotypes of single nucleotide polymorphisms (SNPs) rs174537, rs174561, and rs3834458 in the FADS1-FADS2 gene cluster were strongly associated with proportions of LC-PUFAs and desaturase activities estimated in plasma and erythrocytes. Minor allele carriage associated with decreased activities of D5D (FADS1) (5.84 × 10(-19) ≤ P ≤ 4.5 × 10(-18)) and D6D (FADS2) (6.05 × 10(-8) ≤ P ≤ 4.20 × 10(-7)) was accompanied by increased substrate and decreased product proportions (0.05 ≤ P ≤ 2.49 × 10(-16)). The significance of haplotype association with D5D activity (P = 2.19 × 10(-17)) was comparable to that of single SNPs, but haplotype association with D6D activity (P = 3.39 × 10(-28)) was much stronger. In a randomized controlled dietary intervention, increasing eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) intake significantly increased D5D (P = 4.0 × 10(-9)) and decreased D6D activity (P = 9.16 × 10(-6)) after doses of 0.45, 0.9, and 1.8 g/day for six months. Interaction of rs174537 genotype with treatment was a determinant of D5D activity estimated in plasma (P = 0.05). In conclusion, different sites at the FADS1-FADS2 locus appear to influence D5D and D6D activity, and rs174537 genotype interacts with dietary EPA+DHA to modulate D5D.

A population pharmacokinetic-pharmacogenetic analysis of atazanavir.

Atazanavir is a first-line HIV protease inhibitor commonly co-dosed with ritonavir. Ritonavir inhibits atazanavir metabolism, decreasing variability and increasing plasma concentrations. However, ritonavir use results in higher costs and increased drug-related adverse events. Elucidating atazanavir pharmacokinetics might allow for individualized ritonavir boosting. We previously demonstrated that genetically determined CYP3A5 nonexpression was associated with slower atazanavir clearance CL/F and higher trough concentrations. This effect was prominent in non-African-American men but absent in African-Americans. The present study considers additional genetic predictors of atazanavir CL/F with a focus on race differences. Nine polymorphisms in CYP3A4, ABCG2, NR1I2 (PXR), and SLCO1B1 were evaluated; 330 plasma samples from 30 HIV-negative volunteers, balanced by sex, race, and CYP3A5 expressor status, were available. Analyses were performed using nonlinear mixed-effects modeling (NONMEM). The following factors were univariately associated with atazanavir CL/F (% effect) : African-American race (decreased 35%), female sex (decreased 25%), older age (decreased 1.7%/year), CYP3A5 nonexpressors (decreased 26%), ABCB1 CGC haplotype carriers (1236C/2677G/3435C) (decreased 33%), and CYP3A4*1B carriers (decreased 31%). However, an independent genetic explanation for the differential race effect could not be identified. An interaction was observed with PXR 63396 C>T and CYP3A5 expressor status (p=0.0002). CYP3A5 nonexpressors with a PXR 63396 CC genotype had 37% slower CL/F versus those with CT or TT genotypes. For CYP3A5 expressors, those with a PXR 63396 CC genotype had 63% faster CL/F versus those with CT or TT genotypes. Although this study has as its main limitation a small overall sample size, these results nonetheless provide new leads and impetus to evaluate ways to individualize the need for ritonavir boosting using demographic and genetic predictors of atazanavir pharmacokinetics.

Influence of ABCB1 (P-glycoprotein) haplotypes on nortriptyline pharmacokinetics and nortriptyline-induced postural hypotension in healthy volunteers.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: A single nucleotide polymorphism in ABCB1, which encodes P-glycoprotein, has retrospectively been associated with symptoms of nortriptyline-induced postural hypotension in depressed patients. This finding needs to be replicated in independent studies before recommendations regarding pharmacogenetic testing can be made. WHAT THIS STUDY ADDS: In a prospective study of healthy volunteers homozygous for ABCB1 (1236-2677-3435, TTT/TTT or CGC/CGC), a single dose of nortriptyline was administered, plasma exposure was determined and blood pressure and heart rate were monitored during posture change. No differences between ABCB1 haplotype groups were found in plasma exposure of nortriptyline and its active metabolites, E- and Z-10-hydroxynortriptyline. The heart rate response to posture change was increased with nortriptyline, whereas there was no difference in blood pressure response. However, no differences between haplotype groups were observed except that the pre dose heart rate response to standing was greater in the TTT than CGC homozygotes. The association between ABCB1 polymorphisms and nortriptyline-induced postural hypotension found in a previous study could not be confirmed. The results raise the possibility of a predisposition in heart rate response in the TTT homozygotes rather than an effect of nortriptyline. AIMS To investigate the influence of ABCB1 (1236-2677-3435) polymorphisms on nortriptyline pharmacokinetics and nortriptyline-induced postural hypotension in healthy volunteers. METHODS: Genetic screening of 67 healthy volunteers identified eight CGC homozygotes and nine TTT homozygotes of ABCB1 (1236-2677-3435), who were administered a single dose of nortriptyline 25 mg. Plasma exposure of nortriptyline and its active metabolites, E- and Z-10-hydroxynortriptyline, was determined over 72 h. Heart rate and blood pressure responses to posture change (active standing and passive head-up tilt) were measured continuously using finger plethysmography. RESULTS: There were no differences in plasma exposure between ABCB1 haplotype groups, as the geometric mean (95% CI) AUC(0,72 h) ratios were 0.98 (0.94, 1.03), 1.02 (0.96, 1.09) and 0.95 (0.80, 1.10) for nortriptyline, E- and Z-10-hydroxynortriptyline, respectively. The pre dose heart rate response to standing was greater in the TTT than CGC homozygotes (mean (95% CI) difference 7.4 (1.5, 13.4) beats min(-1) , P = 0.02). At t(max) at 8 h post dose, nortriptyline increased the heart rate response to posture change in all subjects with mean (95% CI) Δ heart rate values of 7.4 (3.6, 11.3) beats min(-1) on active standing (P = 0.0009) and 4.8 (2.0, 7.6) beats min(-1) on head-up tilt (P = 0.002), but no difference was observed between haplotype groups. There was no difference in blood pressure response to posture change in either group. CONCLUSION: The association between ABCB1 polymorphisms and nortriptyline-induced postural hypotension found in the previous study could not be confirmed. The results raise the possibility of a predisposition in heart rate response in the TTT homozygotes rather than an effect of nortriptyline.

Impact of ABCG2 polymorphisms on the clinical outcome and toxicity of gefitinib in non-small-cell lung cancer patients.

AIMS: The current study investigates whether or not functional polymorphisms in the ATP-binding cassette transporter gene ABCG2 might affect gefitinib activity and/or toxicity in non-small-cell lung cancer (NSCLC) patients. MATERIALS & METHODS: Towards this end, ABCG2 polymorphisms and expression were assessed in DNA and tumors from 94 NSCLC patients treated with gefitinib, whereas their associations with toxicity/response and time-to-progression/overall survival were evaluated using Pearson-χ(2) and log-rank-test, respectively. RESULTS: Patients carrying an ABCG2 -15622T/T genotype or harboring at least one TT copy in the ABCG2 (1143C/T, -15622C/T) haplotype developed significantly more grade 2/3 diarrhea (p < 0.01). No associations were found between polymorphisms and outcome. Consistently, ABCG2 protein levels in tumors were not significantly different between patients harboring different ABCG2 variants. CONCLUSION: The ABCG2 -15622C/T polymorphism and ABCG2 (1143C/T, -15622C/T) haplotype resulted in a gefitinib-dependent, moderate-to-severe diarrhea suggesting that these pharmacogenetic markers should be considered to optimize NSCLC treatment.

Temporal and geographical genetic variation in the amphipod Melita plumulosa (Crustacea: Melitidae): Link of a localized change in haplotype frequencies to a chemical spill.

Anthropogenic effects such as contamination affect the genetic structure of populations. This study examined the temporal and geographical patterns of genetic diversity among populations of the benthic crustacean amphipod Melita plumulosa in the Parramatta River (Sydney, Australia), following an industrial chemical spill. The spill of an acrylate/methacrylate co-polymer in naphtha solvent occurred in July 2006. M. plumulosa were sampled temporally between December 2006 and November 2009 and spatially in November 2009. Genetic variation was examined at the mitochondrial cytochrome c oxidase subunit I locus. Notably, nucleotide diversity was low and Tajima's D was significantly negative amongst amphipods collected immediately downstream from the spill for 10 months. We hypothesize that the spill had a significant localized effect on the genetic diversity of M. plumulosa. Alternate explanations include an alternate and unknown toxicant or a localized sampling bias. Future proposed studies will dissect these alternatives.

Cytochrome P450 1B1 gene polymorphisms as predictors of anticancer drug activity: studies with in vitro models.

Cytochrome P450 1B1 (CYP1B1) is found in tumor tissue and is suspected to play a role in oncogenesis and drug resistance. CYP1B1 gene polymorphisms have been associated with the risk of developing lung and other cancers. They may be associated with tumor response to anticancer drugs. We have determined 4 frequent nonsynonymous gene polymorphisms of CYP1B1 in the human tumor cell lines panels of the National Cancer Institute (NCI) and the Japanese Foundation for Cancer Research (JFCR): rs10012 (R48G), rs1056827 (A119S), rs1056836 (L432V), and rs1800440 (N453S). Numerous anticancer drugs have been tested against these panels that offer the opportunity to detect associations between gene polymorphisms and drug sensitivity. CYP1B1 single nucleotide polymorphisms were in marked linkage disequilibrium. The L432V allelic variants were significantly associated with reduced sensitivity to DNA-interacting anticancer agents, alkylators, camptothecins, topoisomerase II inhibitors, and some antimetabolites. For instance, in the NCI panel, cell lines homozygous for the V432 allele were globally 2-fold resistant to alkylating agents (P = 5 × 10(-10)) and 4.5-fold to camptothecins (P = 6.6 × 10(-9)) than cell lines homozygous for the L432 allele. Similar features were exhibited by the JFCR panel. Cell lines homozygous for the V432 allele were globally less sensitive to DNA-interfering drugs than cell lines having at least 1 common allele. There was no significant association between mRNA expression of CYP1B1 and CYP1B1 genotype, and no significant association between CYP1B1 mRNA expression and drug cytotoxicity. These observations open the way to clinical studies exploring the role of CYP1B1 gene polymorphisms for predicting tumor sensitivity to chemotherapy.

No significant effect of ABCB1 haplotypes on the pharmacokinetics of fluvastatin, pravastatin, lovastatin, and rosuvastatin.

AIMS: This study aimed to investigate possible effects of ABCB1 genotype on fluvastatin, pravastatin, lovastatin, and rosuvastatin pharmacokinetics. METHODS: In a fixed-order crossover study, 10 healthy volunteers with the ABCB1 c.1236C/C-c.2677G/G-c.3435C/C (CGC/CGC) genotype and 10 with the c.1236T/T-c.2677T/T-c.3435T/T (TTT/TTT) genotype ingested a single 20-mg dose of fluvastatin, pravastatin, lovastatin, and rosuvastatin. Plasma fluvastatin, pravastatin, and lovastatin concentrations were measured up to 12 h and plasma and urine rosuvastatin concentrations up to 48 and 24 h, respectively. RESULTS: The ABCB1 genotype had no significant effect on the pharmacokinetics of any of the investigated statins. The geometric mean ratio (95% confidence interval) of the area under the plasma concentration-time curve from 0 h to infinity (AUC(0-infinity)) in participants with the TTT/TTT genotype to that in those with the CGC/CGC genotype was 0.96 (0.77, 1.20; P= 0.737) for fluvastatin, 0.92 (0.53, 1.62; P= 0.772) for pravastatin, 0.83 (0.36, 1.90; P= 0.644) for lovastatin, 1.25 (0.72, 2.17; P= 0.400) for lovastatin acid, and 1.10 (0.73, 1.65; P= 0.626) for rosuvastatin. The AUC(0-infinity) of lovastatin acid correlated significantly with that of rosuvastatin (r= 0.570, P= 0.009), but none of the other AUC(0-infinity) pairs showed a significant correlation. CONCLUSIONS: These data suggest that the ABCB1 c.1236C-c.2677G-c.3435C and c.1236T-c.2677T-c.3435T haplotypes play no significant role in the interindividual variability in the pharmacokinetics of fluvastatin, pravastatin, lovastatin, and rosuvastatin.

Pharmacodynamic genes do not influence risk of neutropenia in cancer patients treated with moderately high-dose irinotecan.

AIMS: A recent study found that variation in camptothecin pharmacodynamic genes (TOP1, PARP1, TDP1 and XRCC1) correlated with efficacy and risk of neutropenia in irinotecan-treated cancer patients (median dose: 180 mg/m2), which suggests that these genes might predict outcomes to irinotecan-based therapies. The present study was conducted to evaluate previous gene associations using an independent sample of patients receiving irinotecan. MATERIALS & METHODS: DNA was isolated from 85 advanced cancer patients treated with 300 or 350 mg/m2 irinotecan and genotyped for haplotype-tag polymorphisms across TOP1, PARP1, TDP1 and XRCC1. Associations between genotypes and haplotypes and log(absolute neutrophil count nadirs) were assessed by linear regression. RESULTS: No associations were observed. CONCLUSION: Our findings suggest that the genes we tested do not influence toxicity of irinotecan when adminstered at 300-350 mg/m2.