The generation of HepG2 transmitochondrial cybrids to reveal the role of mitochondrial genotype in idiosyncratic drug-induced liver injury.

Background: Evidence supports an important link between mitochondrial DNA (mtDNA) variation and adverse drug reactions such as idiosyncratic drug-induced liver injury (iDILI). Here, we describe the generation of HepG2-derived transmitochondrial cybrids, to investigate the impact of mtDNA variation on mitochondrial (dys)function and susceptibility to iDILI. This study created 10 cybrid cell lines, each containing distinct mitochondrial genotypes of haplogroup H or haplogroup J backgrounds. Methods: HepG2 cells were depleted of mtDNA to make rho zero cells, before the introduction of known mitochondrial genotypes using platelets from healthy volunteers (n=10), thus generating 10 transmitochondrial cybrid cell lines. The mitochondrial function of each was assessed at basal state and following treatment with compounds associated with iDILI; flutamide, 2-hydroxyflutamide, and tolcapone, and their less toxic counterparts bicalutamide and entacapone utilizing ATP assays and extracellular flux analysis. Results: Whilst only slight variations in basal mitochondrial function were observed between haplogroups H and J, haplogroup-specific responses were observed to the mitotoxic drugs. Haplogroup J showed increased susceptibility to inhibition by flutamide, 2-hydroxyflutamide, and tolcapone, via effects on selected mitochondrial complexes (I and II), and an uncoupling of the respiratory chain. Conclusions: This study demonstrates that HepG2 transmitochondrial cybrids can be created to contain the mitochondrial genotype of any individual of interest. This provides a practical and reproducible system to investigate the cellular consequences of variation in the mitochondrial genome, against a constant nuclear background. Additionally, the results show that inter-individual variation in mitochondrial haplogroup may be a factor in determining sensitivity to mitochondrial toxicants. Funding: This work was supported by the Centre for Drug Safety Science supported by the Medical Research Council, United Kingdom (Grant Number G0700654); and GlaxoSmithKline as part of an MRC-CASE studentship (grant number MR/L006758/1).

"Sex. Maybe We Should Do Things to be Healthy About It." Adolescent-Caregiver Discussions About Sex.

PURPOSE: Adolescents are disproportionately burdened with HIV. Numerous barriers limit adolescent pre-exposure prophylaxis (PrEP) use for HIV prevention. We explored adolescent-caregiver perspectives on discussing sexual health and PrEP to inform future caregiver interventions as a possible strategy to promote PrEP use. METHODS: We conducted separate in-depth interviews with adolescents aged 14-18 living in Alabama and their parent/guardian (caregiver). Interviews explored attitudes about sex, knowledge and attitudes about HIV prevention and PrEP, and attitudes about PrEP communication within adolescent-caregiver groups. Thematic analysis of adolescent and caregiver interviews was conducted independently and then triangulated to compare shared themes. RESULTS: Nine adolescents and seven caregivers contributed to five dyads and two triads. Adolescents had a median age of 16 years (range 14-18); five were girls (55%), and five were non-Hispanic Black (55%). Most caregivers were mothers (5, 71%), non-Hispanic Black (5, 71%), with a median age of 41 (36-56) years. All adolescents expressed willingness to involve their caregiver around PrEP use. Major themes included as follows: 1) caregiver efforts to overcome cultural taboos about sex and sexuality foster adolescents' willingness to talk with caregivers about sex; 2) evolving societal norms and reflections on their own upbringings motivate caregivers to discuss and support adolescents with sexual health; and 3) caregivers desire to engage in sexual health discussions with providers and support their teens with PrEP. DISCUSSION: As socio-cultural norms around sex evolve, adolescent-caregiver discussions about sexual health and PrEP may be an opportunity to increase PrEP use and reduce HIV infections among select adolescent sub-populations.

The Role of Mitochondrial DNA Variation in Drug Response: A Systematic Review.

Background: The triad of drug efficacy, toxicity and resistance underpins the risk-benefit balance of all therapeutics. The application of pharmacogenomics has the potential to improve the risk-benefit balance of a given therapeutic via the stratification of patient populations based on DNA variants. A growth in the understanding of the particulars of the mitochondrial genome, alongside the availability of techniques for its interrogation has resulted in a growing body of literature examining the impact of mitochondrial DNA (mtDNA) variation upon drug response. Objective: To critically evaluate and summarize the available literature, across a defined period, in a systematic fashion in order to map out the current landscape of the subject area and identify how the field may continue to advance. Methods: A systematic review of the literature published between January 2009 and December 2020 was conducted using the PubMed database with the following key inclusion criteria: reference to specific mtDNA polymorphisms or haplogroups, a core objective to examine associations between mtDNA variants and drug response, and research performed using human subjects or human in vitro models. Results: Review of the literature identified 24 articles reporting an investigation of the association between mtDNA variant(s) and drug efficacy, toxicity or resistance that met the key inclusion criteria. This included 10 articles examining mtDNA variations associated with antiretroviral therapy response, 4 articles examining mtDNA variants associated with anticancer agent response and 4 articles examining mtDNA variants associated with antimicrobial agent response. The remaining articles covered a wide breadth of medications and were therefore grouped together and referred to as "other." Conclusions: Investigation of the impact of mtDNA variation upon drug response has been sporadic to-date. Collective assessment of the associations identified in the articles was inconclusive due to heterogeneous methods and outcomes, limited racial/ethnic groups, lack of replication and inadequate statistical power. There remains a high degree of idiosyncrasy in drug response and this area has the potential to explain variation in drug response in a clinical setting, therefore further research is likely to be of clinical benefit.

Assessment of the impact of mitochondrial genotype upon drug-induced mitochondrial dysfunction in platelets derived from healthy volunteers.

Mitochondrial DNA (mtDNA) is highly polymorphic and encodes 13 proteins which are critical to the production of ATP via oxidative phosphorylation. As mtDNA is maternally inherited and undergoes negligible recombination, acquired mutations have subdivided the human population into several discrete haplogroups. Mitochondrial haplogroup has been found to significantly alter mitochondrial function and impact susceptibility to adverse drug reactions. Despite these findings, there are currently limited models to assess the effect of mtDNA variation upon susceptibility to adverse drug reactions. Platelets offer a potential personalised model of this variation, as their anucleate nature offers a source of mtDNA without interference from the nuclear genome. This study, therefore, aimed to determine the effect of mtDNA variation upon mitochondrial function and drug-induced mitochondrial dysfunction in a platelet model. The mtDNA haplogroup of 383 healthy volunteers was determined using next-generation mtDNA sequencing (Illumina MiSeq). Subsequently, 30 of these volunteers from mitochondrial haplogroups H, J, T and U were recalled to donate fresh, whole blood from which platelets were isolated. Platelet mitochondrial function was tested at basal state and upon treatment with compounds associated with both mitochondrial dysfunction and adverse drug reactions, flutamide, 2-hydroxyflutamide and tolcapone (10-250 µM) using extracellular flux analysis. This study has demonstrated that freshly-isolated platelets are a practical, primary cell model, which is amenable to the study of drug-induced mitochondrial dysfunction. Specifically, platelets from donors of haplogroup J have been found to have increased susceptibility to the inhibition of complex I-driven respiration by 2-hydroxyflutamide. At a time when individual susceptibility to adverse drug reactions is not fully understood, this study provides evidence that inter-individual variation in mitochondrial genotype could be a factor in determining sensitivity to mitochondrial toxicants associated with costly adverse drug reactions.

Efficacy of Lower Extremity Cycling Interventions for Youth with Intellectual Disabilities: A Systematic Review.

PURPOSE: To systematically examine the efficacy of lower extremity cycling interventions for youth with intellectual disability (ID). METHODS: Seven databases were searched from March 2000 to October 2019 for English-language articles on cycling interventions for youth with ID. The American Academy of Cerebral Palsy and Developmental Medicine guidelines were used for assigning levels of evidence and grading study quality. RESULTS: Eight articles met inclusion criteria. Children and young adults, 7-26 years (n = 229), with diagnoses of Down syndrome, autism spectrum disorder, or unspecified ID participated in the studies. Moderate evidence (one level II single subject design study) suggests that a specific cycling intervention can improve two-wheeled cycling skills in youth with ID. Weak evidence (level II group studies) supports stationary cycling for short-term improvements in cognitive skills and two-wheeled riding intervention for increasing physical activity one-year after intervention. CONCLUSIONS: Moderate to weak evidence exists to support two-wheeled cycling instructional programs or stationary cycling interventions for children and young adults with intellectual disabilities. Therapists can use this information when discussing cycling interventions for individuals with ID. Further research is needed to inform therapists in clinical decision-making related to dosing cycling interventions.

The effectiveness of biofeedback therapy in managing Bladder Bowel Dysfunction in children: A systematic review.

PURPOSE: The purpose of this study was to examine the effects of biofeedback therapy as a non-invasive intervention to treat Bladder Bowel Dysfunction in pediatrics. METHODS: Six databases were searched between February 2016 and September 2016. Biofeedback studies for children aged 4-16 with idiopathic urinary or fecal incontinence were included. Articles were excluded on subjects' medical histories, study design, timeline of study, and lacking expert review. Quality was determined using Sackett's Levels of Evidence and the PEDro scale. RESULTS: Twelve articles were included in the review. Quality of evidence was moderate, as the average PEDro score of the selected articles was 5.3. The participants' ages ranged from 4-16 years old. Studies demonstrated that a multifactorial approach consisting of biofeedback therapy and behavioral modification can be successful in resolving Bladder Bowel Dysfunction. CONCLUSION: Biofeedback is a beneficial treatment for children with dysfunctional voiding and functional fecal incontinence. More conclusive research needs to be completed to explore the effects of biofeedback therapy treatment to make more concrete conclusions. Healthcare professionals should consider biofeedback as an alternative approach in conjunction with traditional treatments. A multidisciplinary approach is best when treating dysfunctional voiding and functional fecal incontinence in the pediatric population.

Identification of the Additional Mitochondrial Liabilities of 2-Hydroxyflutamide When Compared With its Parent Compound, Flutamide in HepG2 Cells.

The androgen receptor antagonist, flutamide, is strongly associated with idiosyncratic drug-induced liver injury (DILI). Following administration, flutamide undergoes extensive first-pass metabolism to its primary metabolite, 2-hydroxyflutamide. Flutamide is a known mitochondrial toxicant; however there has been limited investigation into the potential mitochondrial toxicity of 2-hydroxyflutamide and its contribution to flutamide-induced liver injury. In this study we have used the acute glucose or galactose-conditioning of HepG2 cells to compare the mitochondrial toxicity of flutamide, 2-hydroxyflutamide and the structurally-related, non-hepatotoxic androgen receptor antagonist, bicalutamide. Compound-induced changes in mitochondrial oxygen consumption rate were assessed using Seahorse technology. Permeabilization of cells and delivery of specific substrates and inhibitors of the various respiratory complexes provided more detailed information on the origin of mitochondrial perturbations. These analyses were supported by assessment of downstream impacts including changes in cellular NAD(+)/NADH ratio. Bicalutamide was not found to be a mitochondrial toxicant, yet flutamide and 2-hydroxyflutamide significantly reduced basal and maximal respiration. Both flutamide and 2-hydroxyflutamide significantly reduced respiratory complex I-linked respiration, though 2-hydroxyflutamide also significantly decreased complex II and V-linked respiration; liabilities not demonstrated by the parent compound. This study has identified for the first time, the additional mitochondrial liabilities of the major metabolite, 2-hydroxyflutamide compared with its parent drug, flutamide. Given the rapid production of this metabolite upon administration of flutamide, but not bicalutamide, we propose that the additional mitochondrial toxicity of 2-hydroxyflutamide may fundamentally contribute to the idiosyncratic DILI seen in flutamide-treated, but not bicalutamide-treated patients.

Postpartum healthcare after gestational diabetes and hypertension.

BACKGROUND: Gestational diabetes and hypertensive disorders of pregnancy identify women with an elevated lifetime risk of diabetes and cardiovascular disease. METHODS: Prospective cohort of women recruited from the postpartum service of a large community-based academic obstetrical hospital after delivery of a pregnancy complicated by gestational diabetes (GDM) or a hypertensive disorder of pregnancy (HDP). Interviews were conducted, and validated surveys completed, before hospital discharge and again 3 months postpartum. RESULTS: The study sample included 249 women: 111 with GDM, 127 with HDP, and 11 with both. Most, 230 (92.4%) had a PCP prior to pregnancy and 97 (39.0%) reported an office visit with their PCP during the prenatal period. Of the 176 (70.7%) participants who attended the 3-month study visit, 169 (96.0%) women with either diagnosis reported they had attended their 6-week postpartum visit. By the 3-month study visit, 51 (57.9%) women with GDM had completed follow-up glucose testing; 93 (97.9%) with HDP had follow-up blood pressure testing; and 101 (57.4%) with either diagnosis recalled ever having completed lipid screening. Women least likely to complete screening tests were those who had no college education, less than a high school level of health literacy, and who were not privately insured. CONCLUSION: There are important opportunities to improve postpartum testing for diabetes and CVD risk factor assessment. Most women were connected to primary care suggesting a "hand-off" to a primary care physician after pregnancy is feasible. More robust strategies may be needed to improve follow-up care for women with less education, lower health literacy, and those without private health insurance.

Impact of Celecoxib restrictions in medicare beneficiaries with arthritis.

OBJECTIVE: To compare the incidence of serious gastrointestinal (GI) complications and associated medical costs in a population with either osteoarthritis (OA) or rheumatoid arthritis (RA) enrolled in Medicare plans with celecoxib formulary restrictions versus plans without such restrictions. METHODS: This study was a retrospective cohort analysis of Medicare members in plans with and without celecoxib restrictions. Members diagnosed with OA or RA were identified and followed for 1 year. RESULTS: The restricted group had higher levels of nonselective nonsteroidal anti-inflammatory drug use (51% vs 40%, p <.001), and celecoxib use was double in the unrestricted group (16% vs 8%, p <.001). The incidence of a serious GI complication was slightly higher in the restricted group (5.4% vs 4.6%, P <.001). The adjusted mean serious GI complication-related cost for the restricted group was more than 15 times higher than that for the nonrestricted group ($1559 [95% confidence interval (CI) $1341-$1811] vs $101 [95% CI $87-$117]), adjusted mean arthritis-related medical costs were $5733 per year (95% CI $5097-$6448) for the restricted group and $3170 (95% CI $2816-$3569) for the unrestricted group. CONCLUSIONS: The restricted group had significantly less use of celecoxib, indicating that restriction was effective at reducing celecoxib utilization. Although limitations exist when comparing populations from different health plans, and the underlying causes of serious GI complications are multifactorial, the restricted group had a higher incidence of serious GI complications and higher costs related to serious GI complications and arthritis.

Nonadherence to oral linezolid after hospitalization: a retrospective claims analysis of the incidence and consequence of claim reversals.

BACKGROUND: Linezolid is available in an oral as well as an intravenous formulation. It is an oxazolidinone antibiotic and is effective in treating resistant gram-positive organisms such as methicillin-resistant Staphylococcus aureus and multidrug-resistant Streptococcus pneumoniae. OBJECTIVES: The goals of this study were to identify the incidence of claim reversals for oral linezolid in members who were recently discharged from a hospital and to study the subsequent pattern of health care utilization to quantify the consequences for members who have a reversed linezolid claim. METHODS: This study was a retrospective claims analysis of Humana Medicare Advantage Prescription Drug patients who had a claim for oral linezolid after an inpatient discharge between April 1, 2006, and June 30, 2008. The incidence of reversed claims among those with a linezolid prescription was measured as a proxy for medication adherence. Propensity scores were calculated to account for differences in patients' propensity to have a reversed claim. The association of the claim reversal with subsequent expenditures was assessed through 3 multivariate regression models wherein the dependent variables were drug, medical, and total costs for the 60-day period after discharge. The key independent variable was the occurrence of a reversed linezolid claim, and control variables included the propensity score quartiles and other clinical and demographic characteristics. All costs were provided in US dollars and from the year in which they occurred. RESULTS: Of 1046 patients identified (mean [SD] age, 69 [12] years; 51% male), 252 patients (24.1%) had a claim reversal for linezolid. Among these, 125 patients (49.6%) received linezolid within 10 days of the initial reversal, 39 patients (15.5%) received other antibiotics, and 88 patients (34.9%) did not receive any antibiotics. The unadjusted, mean outpatient drug costs were $696 and $2265 for patients with and without a reversal, respectively, whereas mean medical costs were $13,567 and $9355. Multivariable analyses revealed that members who did not receive linezolid after the claim reversal had significantly higher medical expenditures (Wald χ(2), 8.370; P = 0.004) and lower drug expenditures (Wald χ(2), 122.630; P < 0.01). The total costs did not differ significantly between the 2 groups (Wald χ(2), 1.540; P = 0.215), however, as the medical savings were partially negated by the higher drug costs. CONCLUSION: These patients with a reversed outpatient claim for linezolid had lower outpatient drug costs and higher medical costs in the 60-day period after the reversal.

Population genetic analysis of white shrimp, Litopenaeus setiferus, using microsatellite genetic markers.

The white shrimp (Litopenaeus setiferus) is a commercially and recreationally valuable species, yet little is known of its population structure or genetic diversity. White shrimp are distributed along the Atlantic coast of the United States and from the west coast of Florida to the Bay of Campeche, Mexico. In this study, shrimp were collected from North Carolina, South Carolina (four separate collections were taken from 1995 to 1999), Georgia, the Atlantic and Gulf coasts of Florida, Louisiana, Texas and Mexico. DNA was isolated from these individuals, and genetic variation was assessed at six microsatellite loci. These loci were, for the most part, highly polymorphic with an average expected heterozygosity of 0.68. Deviations from Hardy-Weinberg proportions were observed over all samples, but experimental results suggested the presence of null alleles, which confounded a biological interpretation of this result. Pairwise tests of the similarity of allele frequency distributions and distance measure analyses showed broad-scale genetic homogeneity superimposed over occasional indications of random geographical and temporal differentiation. FST and RST estimates over all loci and samples were 0.002 or less and indicated little population structure. Weak but significant genetic differentiation was evident only between pooled western Atlantic and pooled Gulf of Mexico samples. Within the Gulf of Mexico or within the western Atlantic, the large-scale genetic homogeneity observed may be a consequence of genetic mixing resulting from pelagic larvae and adult migrations, while the random local genetic differentiation may be a result of genetic sampling or experimental sampling error. The weak differentiation between shrimp from the Gulf of Mexico and the western Atlantic can be explained by a relatively recent separation of these two populations and/or small amounts of ongoing gene flow.

Formulary management of colony-stimulating factors.

The outcome of a formulary interchange from filgrastim to sargramostim for the amelioration of neutropenia for outpatients receiving myelosuppressive chemotherapy was evaluated. The pharmacy department at the James Graham Brown Cancer Center of the University of Louisville Hospital implemented a therapeutic interchange program by following the Joint Commission on Accreditation of Healthcare Organizations performance methodology, incorporating four key elements: plan, do, check, and act. After the pharmacy and therapeutics committee agreed that filgrastim and sargramostim are therapeutically equivalent, the pharmacy initiated the interchange, with a commitment to collect outcomes data to analyze the impact of the program on patient outcomes. Inclusion criteria included patient age of > or = 18 years, the presence of solid tumors or lymphoma, and current treatment with traditional chemotherapy. Patient demographics and cycle-specific data were collected for 31 patients receiving sargramostim and 20 patients receiving filgrastim from August 2000 to July 2001. Absolute neutrophil counts (ANCs) were measured before initiating and after discontinuing colony-stimulating factors. The majority (70%) of all growth factor use was initiated within one to four days of the last chemotherapy dose. No appreciable difference was found between agents for median ANC at any measured time point. The majority of patients exceeded the target ANC of 1500 cells/mm3 at the time of growth factor discontinuation. There were no significant differences in the number of patients that had adverse effects or in the number of cycles resulting in an adverse event between groups. Sargramostim demonstrated a 21% cost savings over filgrastim ($1036 versus $1318, respectively). The formulary switch from filgrastim to sargramostim resulted in a significant cost savings for the institution without increasing incidence of adverse effects and negative outcomes associated with growth factor use.