A long-term fulvestrant eluting implant is safe, non-toxic, and reduces the risk of breast cancer in in vivo models.

For individuals at high risk of developing breast cancer, interventions to mitigate this risk include surgical removal of their breasts and ovaries or five years treatment with the anti-estrogen tamoxifen or aromatase inhibitors. We hypothesized that a silicone based anti-estrogen-eluting implant placed within the breast would provide the risk reduction benefit of hormonal therapy, but without the adverse effects that limit compliance. To this end, we demonstrate that when placed adjacent to mammary tissue in the DMBA-induced rat breast cancer model a fulvestrant-eluting implant delays breast cancer with minimal systemic exposure. Using adult female sheep, fulvestrant-eluting implants were found to be safe and non-toxic when placed at the base of the udder for directed elution into the mammary tissue. At 30 days of elution, fulvestrant was found to penetrate mammary tissue forming a concentration gradient beyond 15 mm from the implant. Consistent with the small animal rat study, minimal systemic fulvestrant biodistribution was found. Together, these studies provide the proof of principle that a breast indwelling fulvestrant-eluting implant can reduce the risk of breast cancer and limit systemic exposure, while penetrating and distributing through breast tissue.

M. tuberculosis antigen-responsive IL17+ CD4 T cells are disproportionately spared in ART-suppressed HIV.

Background: Interleukin 17 producing CD4 T cells contribute to control of Mycobacterium tuberculosis (Mtb) infection in humans; whether infection with Human Immunodeficiency Virus (HIV) disproportionately affects distinct Th17 cell subsets that respond to Mtb are incompletely defined. Methods: We performed high-definition characterization of circulating Mtb-specific Th17 cells by spectral flow cytometry in people with latent TB and treated HIV (HIV-ART). We also measured kynurenine pathway activity in plasma by LC/MS and tested the hypothesis that tryptophan catabolism influences Th17 cell differentiation in this context. Results: We identified two categories of Th17 cells: TH17 (CD4+Vα7.2-CD161+CD26+) and T17 (CD4+Vα7.2-CCR6+CXCR3-) cells that were disproportionately reduced in LTBI with HIV-ART, yet Mtb-responsive IL17-producing CD4 T cells were preserved; we found that IL17-producing CD4 T cells dominate the response to Mtb antigen but not CMV antigen or staphylococcal enterotoxin B (SEB); and tryptophan catabolism negatively correlates with TH17 and T1T17 but not T17 cell frequencies. Conclusions: We found differential effects of ART-suppressed HIV on distinct categories of Th17 cells, that IL17-producing CD4 T cells dominate responses to Mtb but not CMV antigen or SEB, and that kynurenine pathway activity is associated with selective decreases of circulating Th17 cells that may contribute to tuberculosis immunity.

Female Genital Fibroblasts Diminish the In Vitro Efficacy of PrEP against HIV.

The efficacy of HIV pre-exposure prophylaxis (PrEP) is high in men who have sex with men, but much more variable in women, in a manner largely attributed to low adherence. This reduced efficacy, however, could also reflect biological factors. Transmission to women is typically via the female reproductive tract (FRT), and vaginal dysbiosis, genital inflammation, and other factors specific to the FRT mucosa can all increase transmission risk. We have demonstrated that mucosal fibroblasts from the lower and upper FRT can markedly enhance HIV infection of CD4+ T cells. Given the current testing of tenofovir disoproxil fumarate, cabotegravir, and dapivirine regimens as candidate PrEP agents for women, we set out to determine using in vitro assays whether endometrial stromal fibroblasts (eSF) isolated from the FRT can affect the anti-HIV activity of these PrEP drugs. We found that PrEP drugs exhibit significantly reduced antiviral efficacy in the presence of eSFs, not because of decreased PrEP drug availability, but rather of eSF-mediated enhancement of HIV infection. These findings suggest that drug combinations that target both the virus and infection-promoting factors in the FRT-such as mucosal fibroblasts-may be more effective than PrEP alone at preventing sexual transmission of HIV to women.

Quantification of N, N' N"-triethylenethiophosphoramide, N, N"-triethylenephosphoramide, cyclophosphamide, and 4-hydroxy-cyclophosphamide in microvolume human plasma to support neonatal and pediatric drug studies.

N, N' N"-triethylenethiophosphoramide (thiotepa) and cyclophosphamide (CP) are alkylating agents used for a variety of malignant and non-malignant disorders. Both drugs are metabolized by cytochrome P450 enzymes to form active metabolites. To support pharmacokinetic studies of thiotepa and CP in children, we sought to develop assays to determine parent drug and metabolite concentration in small volume plasma samples. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for assay development. CP metabolite 4-hydroxycyclophosphamide (4OHCP) was converted to the more stable semicarbazone derivative (4OHCP-SCZ) for quantitation. Samples (10 µL) were extracted by solid-phase extraction and injected onto the LC-MS/MS system equipped with a pentafluorophenyl reverse phase column (2.1 × 50 mm, 2.7 µm). Electrospray ionization in positive mode was used for detection. Multiple reaction monitoring of the precursor-to-product ion transitions m/z 190→147 for thiotepa, 174→131 for tepa, 261→233 for CP, and 334→221 for 4OHCP-SCZ was selected for quantification. The ion transitions m/z 202→155 for thiotepa-d12, 186→139 for tepa-d12, 267→237 for CP-d4, and 340→114 for 4OHCP-d4-SCZ were selected for the internal standard (IS) corresponding to each analyte. The less abundant IS ions from 37Cl were used for CP-d4 and 4OHCP-d4-SCZ to overcome the cross-talk interference from the analytes. Under optimized conditions, retention times were 0.67 min for tepa and its IS, 2.50 min for thiotepa and its IS, 2.52 min for 4OHCP-SCZ and its IS, and 2.86 min for CP and its IS. Total run time was 5 min per sample. The calibration ranges were 2.5-2,000ng/mL for thiotepa and tepa, 20-10,000ng/mL for CP and 20-5,000 ng/mL for 4OHCP; Dilution integrity for samples above the calibration range was validated with 10-fold dilution for thiotepa/tepa and 20-fold dilution for CP/4OHCP. Recoveries ranged from 86.3-93.4% for thiotepa, 86.3-89.0% for tepa, 90.2-107% for CP, and 99.3-115% for 4OHCP-SCZ. The IS normalized matrix effect was within (100±7) % for all 4 analytes. Plasma samples at room temperature were stable for at least 60 hours for thiotepa, 6 days for tepa, and 24 hours for CP and 4OHCP-SCZ. Plasma samples for thiotepa/tepa were stable after 4 freeze-thaw cycles, and for CP/4OHCP-SCZ were stable after 3 freeze-thaw cycles. The assays were validated and applied to clinical studies requiring small sample volumes.

Development and validation of an LC-MS/MS method for determination of hydroxychloroquine, its two metabolites, and azithromycin in EDTA-treated human plasma.

BACKGROUND: Hydroxychloroquine (HCQ) and azithromycin (AZM) are antimalarial drugs recently reported to be active against severe acute respiratory syndrome coronavirus- 2 (SARS-CoV-2), which is causing the global COVID-19 pandemic. In an emergency response to the pandemic, we aimed to develop a quantitation method for HCQ, its metabolites desethylhydroxychloroquine (DHCQ) and bisdesethylchloroquine (BDCQ), and AZM in human plasma. METHODS: Liquid chromatography tandem mass spectrometry was used to develop the method. Samples (20 µL) are extracted by solid-phase extraction and injected onto the LC-MS/MS system equipped with a PFP column (2.0 × 50 mm, 3 µm). ESI+ and MRM are used for detection. Ion pairs m/z 336.1→247.1 for HCQ, 308.1→179.1 for DHCQ, 264.1→179.1 for BDCQ, and 749.6→591.6 for AZM are selected for quantification. The ion pairs m/z 342.1→253.1, 314.1→181.1, 270.1→181.1, and 754.6→596.6 are selected for the corresponding deuterated internal standards (IS) HCQ-d4, DHCQ-d4, BDCQ-d4, and AZM-d5. The less abundant IS ions from 37Cl were used to overcome the interference from the analytes. RESULTS: Under optimized conditions, retention times are 0.78 min for BDCQ, 0.79 min for DHCQ, 0.92 min for HCQ and 1.87 min for AZM. Total run time is 3.5 min per sample. The calibration ranges are 2-1000 ng/mL for HCQ and AZM, 1-500 ng/mL for DHCQ and 0.5-250 ng/mL for BDCQ; samples above the range are validated for up to 10-fold dilution. Recoveries of the method ranged from 88.9-94.4% for HCQ, 88.6-92.9% for DHCQ, 88.7-90.9% for BDCQ, and 98.6%-102% for AZM. The IS normalized matrix effect were within (100±10) % for all 4 analytes. Blood samples are stable for at least 6 hr at room temperature. Plasma samples are stable for at least 66 hr at room temperature, 38 days at -70°C, and 4 freeze-thaw cycles. CONCLUSIONS: An LC-MS/MS method for simultaneous quantitation of HCQ, DHCQ, BDCQ, and AZM in human plasma was developed and validated for clinical studies requiring fast turnaround time and small samples volume.

Exposure to pesticides in utero impacts the fetal immune system and response to vaccination in infancy.

The use of pesticides to reduce mosquito vector populations is a cornerstone of global malaria control efforts, but the biological impact of most pesticides on human populations, including pregnant women and infants, is not known. Some pesticides, including carbamates, have been shown to perturb the human immune system. We measure the systemic absorption and immunologic effects of bendiocarb, a commonly used carbamate pesticide, following household spraying in a cohort of pregnant Ugandan women and their infants. We find that bendiocarb is present at high levels in maternal, umbilical cord, and infant plasma of individuals exposed during pregnancy, indicating that it is systemically absorbed and trans-placentally transferred to the fetus. Moreover, bendiocarb exposure is associated with numerous changes in fetal immune cell homeostasis and function, including a dose-dependent decrease in regulatory CD4 T cells, increased cytokine production, and inhibition of antigen-driven proliferation. Additionally, prenatal bendiocarb exposure is associated with higher post-vaccination measles titers at one year of age, suggesting that its impact on functional immunity may persist for many months after birth. These data indicate that in utero bendiocarb exposure has multiple previously unrecognized biological effects on the fetal immune system.

Methotrexate Decreases Tenofovir Exposure in Antiretroviral-Suppressed Individuals Living With HIV.

BACKGROUND: To mitigate increased risk of premature cardiovascular disease in antiretroviral therapy (ART) suppressed adults living with HIV (PWH), low-dose methotrexate (LDMTX) was evaluated in a multicenter randomized placebo controlled clinical trial of 176 PWH taking various ART regimens (ACTG A5314). Given shared methotrexate (MTX) and tenofovir (TFV) pharmacokinetic (PK) pathways, a substudy was conducted to investigate whether LDMTX alters TFV exposure. METHODS: Adults virally suppressed on ART for >24 weeks were randomized to LDMTX or placebo. The first 66 participants taking a tenofovir disoproxil fumarate-containing regimen underwent intensive PK sampling over 24 hours after the second dose of LDMTX 10 mg or placebo. TFV and MTX levels were quantified using validated mass spectrometry methods. TFV PK between LDMTX and placebo groups were compared and MTX PK was characterized. RESULTS: Forty-eight participants completed this substudy (n = 20 on LDMTX and 28 on placebo). Baseline characteristics were balanced except for protease inhibitor (PI)-use (25% in LDMTX and 43% in placebo groups). For TFV, AUC6 (primary endpoint), and AUC24,imputed, Cmax, and Cmin (secondary endpoints) were on average 22%, and 24%, 27%, and 31% less in the LDMTX versus placebo groups, with reductions in secondary endpoints reaching statistical significance. Additional analyses suggested a greater reduction in the absence of PI although not significant. CONCLUSION: Lower TFV AUC24,imputed and Cmax indicates that LDMTX reduces TFV exposure in PWH. However, this change was modest, not warranting a change in TFV dosing at this time. Further studies of TFV PK with LDMTX, especially without PI co-administration, are warranted.

Pharmacogenetic interactions between antiretroviral drugs and vaginally administered hormonal contraceptives.

OBJECTIVE: In AIDS Clinical Trials Group study A5316, efavirenz lowered plasma concentrations of etonogestrel and ethinyl estradiol, given as a vaginal ring, while atazanavir/ritonavir increased etonogestrel and lowered ethinyl estradiol concentrations. We characterized the pharmacogenetics of these interactions. METHODS: In A5316, women with HIV enrolled into control (no antiretrovirals), efavirenz [600 mg daily with nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs)], and atazanavir/ritonavir (300/100 mg daily with NRTIs) groups. On day 0, a vaginal ring was inserted, releasing etonogestrel/ethinyl estradiol 120/15 µg/day. Intensive plasma sampling for antiretrovirals was obtained on days 0 and 21, and single samples for etonogestrel and ethinyl estradiol on days 7, 14, and 21. Seventeen genetic polymorphisms were analyzed. RESULTS: The 72 participants in this analysis included 25, 24 and 23 in the control, efavirenz, and atazanavir/ritonavir groups, respectively. At day 21 in the efavirenz group, CYP2B6 genotype was associated with increased plasma efavirenz exposure (P = 3.2 × 10), decreased plasma concentrations of etonogestrel (P = 1.7 × 10), and decreased ethinyl estradiol (P = 6.7 × 10). Compared to controls, efavirenz reduced median etonogestrel concentrations by at least 93% in CYP2B6 slow metabolizers versus approximately 75% in normal and intermediate metabolizers. Efavirenz reduced median ethinyl estradiol concentrations by 75% in CYP2B6 slow metabolizers versus approximately 41% in normal and intermediate metabolizers. CONCLUSION: CYP2B6 slow metabolizer genotype worsens the pharmacokinetic interaction of efavirenz with hormonal contraceptives administered by vaginal ring. Efavirenz dose reduction in CYP2B6 slow metabolizers may reduce, but will likely not eliminate, this interaction.

Antiretroviral therapy and vaginally administered contraceptive hormones: a three-arm, pharmacokinetic study.

BACKGROUND: Drug-drug interactions between orally administered antiretroviral therapy (ART) and hormones released from an intravaginal ring are not known. We hypothesised that ART containing either efavirenz or ritonavir-boosted atazanavir would alter plasma concentrations of vaginally administered etonogestrel and ethinylestradiol but that ART concentrations would be unchanged during use of an intravaginal ring. METHODS: We did a parallel, three-group, pharmacokinetic evaluation at HIV clinics in Asia (two sites), South America (five), sub-Saharan Africa (three), and the USA (11) between Dec 30, 2014, and Sept 12, 2016. We enrolled women with HIV who were either ART-naive (control group; n=25), receiving efavirenz-based ART (n=25), or receiving atazanavir-ritonavir-based ART (n=24). Women receiving ART were required to be on the same regimen for at least 30 days, with 400 copies or less per mL of plasma HIV-1 RNA; women not receiving ART had CD4 counts of 350 cells per µL or less. We excluded participants who had a bilateral oophorectomy or conditions that were contraindicated in the intravaginal ring product labelling. An intravaginal ring releasing etonogestrel and ethinylestradiol was inserted at entry (day 0). Single plasma samples for hormone concentrations were collected on days 7, 14, and 21 after intravaginal ring insertion. The primary outcome was the plasma concentration of etonogestrel and ethinylestradiol on day 21. Etonogestrel and ethinylestradiol concentrations were compared between each ART group and the control group by geometric mean ratio (GMR) with 90% CIs and Wilcoxon rank-sum test. As secondary outcomes, efavirenz or ritonavir-boosted atazanavir concentrations were assessed by 8-h intensive pharmacokinetic sampling at entry before intravaginal ring insertion and before intravaginal ring removal on day 21. Antiretroviral areas under the concentration-time curve (AUC0-8 h) were compared before and after intravaginal ring insertion by GMR (90% CI) and Wilcoxon signed-rank test. This study is registered with ClinicalTrials.gov, number NCT01903031. FINDINGS: Between Dec 30, 2014, and Sept 12, 2016, we enrolled 84 participants in the study; ten participants were excluded from the primary hormone analysis. 74 participants met the primary endpoint: 25 in the control group, 25 in the efavirenz group, and 24 in the atazanavir group. On day 21 of intravaginal ring use, participants receiving efavirenz had 79% lower etonogestrel (GMR 0·21, 90% CI 0·16-0·28; p<0·0001) and 59% lower ethinylestradiol (0·41, 0·32-0·52; p<0·0001) concentrations compared with the control group. By contrast, participants receiving ritonavir-boosted atazanavir had 71% higher etonogestrel (1·71, 1·37-2·14; p<0·0001), yet 38% lower ethinylestradiol (0·62, 0·49-0·79; p=0·0037) compared with the control group. The AUC0-8 h of efavirenz or atazanavir did not differ between the groups. INTERPRETATION: Hormone exposure was significantly lower when an intravaginal ring contraceptive was combined with efavirenz-based ART. Further studies designed to examine pharmacodynamic endpoints, such as ovulation, when intravaginal ring hormones are combined with efavirenz are warranted. FUNDING: National Institutes of Health, through the AIDS Clinical Trials Group and the International Maternal Pediatric Adolescent AIDS Clinical Trials Network, National Institute of Allergy and Infectious Diseases, Eunice Kennedy Shriver National Institute of Child Health and Human Development, and the National Institute of Mental Health.

Concomitant nevirapine impacts pharmacokinetic exposure to the antimalarial artemether-lumefantrine in African children.

BACKGROUND: The antiretroviral drug nevirapine and the antimalarial artemisinin-based combination therapy artemether-lumefantrine are commonly co-administered to treat malaria in the context of HIV. Nevirapine is a known inhibitor of cytochrome P450 3A4, which metabolizes artemether and lumefantrine. To address the concern that the antiretroviral nevirapine impacts the antimalarial artemether-lumefantrine pharmacokinetics, a prospective non-randomized controlled study in children presenting with uncomplicated malaria and HIV in sub-Saharan Africa was carried out. METHODS: Participants received artemether-lumefantrine (20/120 mg weight-based BID) for 3 days during nevirapine-based antiretroviral therapy (ART) co-administration (158-266 mg/m2 QD). HIV positive participants who were not yet on ART drugs were also enrolled as the control group. The target enrollment was children aged 3-12 years (n = 24 in each group). Intensive pharmacokinetics after the last artemether-lumefantrine dose was assessed for artemether, its active metabolite dihydroartemisinin, and lumefantrine. Pharmacokinetic parameters (area under the plasma concentration vs. time curve (AUC), maximum concentration and day 7 lumefantrine concentrations) were estimated using non-compartmental methods and compared to controls. RESULTS: Nineteen children (16 on nevirapine and three not on ART) enrolled. Fifteen of the 16 (aged 4 to 11 years) on nevirapine-based ART were included in the pharmacokinetic analysis. Due to evolving WHO HIV treatment guidelines, insufficient children were enrolled in the control group (n = 3), so the pharmacokinetic data were compared to a historical control group of 20 HIV-uninfected children 5-12 years of age who also presented with malaria and underwent identical study procedures. Decreases of pharmacokinetic exposure [as estimated by AUC (AUC0-8hr)] were marginally significant for artemether (by -46%, p = 0.08) and dihydroartemisinin (-22%, p = 0.06) in the children on nevirapine-based ART, compared to when artemether-lumefantrine was administered alone. Similarly, peak concentration was decreased by 50% (p = 0.07) for artemether and 36% (p = 0.01) for dihydroartemisinin. In contrast, exposure to lumefantrine increased significantly in the context of nevirapine [AUC0-120hr:123% (p<0.001); Cday7:116% (p<0.001), Cmax: 95% (p<0.001)]. CONCLUSIONS: Nevirapine-based ART increases the exposure to lumefantrine in pre-pubescent children with a trend toward diminished artemether and dihydroartemisinin exposure. These findings contrast with other studies indicating NVP reduces or results in no change in exposure of antimalarial drugs, and may be specific to this age group (4-12 years). Considering the excellent safety profile of artemether-lumefantrine, the increase in lumefantrine is not of concern. However, the reduction in artemisinin exposure may warrant further study, and suggests that dosage adjustment of artemether-lumefantrine with nevirapine-based ART in children is likely warranted.

Parasite Clearance and Artemether Pharmacokinetics Parameters Over the Course of Artemether-Lumefantrine Treatment for Malaria in Human Immunodeficiency Virus (HIV)-Infected and HIV-Uninfected Ugandan Children.

BACKGROUND: Artemisinins are primarily responsible for initial parasite clearance. Antimalarial pharmacokinetics (PK), human immunodeficiency virus (HIV) infection, and antiretroviral therapy have been shown to impact treatment outcomes, although their impact on early parasite clearance in children has not been well characterized. METHODS: Parasite clearance parameters were generated from twice-daily blood smears in HIV-infected and HIV-uninfected Ugandan children treated with artemether-lumefantrine (AL). Artemether and dihydroartemisinin (DHA) area-under-the-curve from 0-8 hours (AUC0-8hr) after the 1st AL dose was compared with AUC0-8hr after the last (6th) dose in a concurrently enrolled cohort. The association between post-1st dose artemisinin AUC0-8hr and parasite clearance was assessed. RESULTS: Parasite clearance was longer in HIV-infected versus HIV-uninfected children (median, 3.5 vs 2.8 hours; P = .003). Artemether AUC0-8hr was 3- to 4-fold lower after the 6th dose versus the 1st dose of AL in HIV-infected children on nevirapine- or lopinavir/ritionavir-based regimens and in HIV-uninfected children (P ≤ .002, 1st vs 6th-dose comparisons). Children on efavirenz exhibited combined post-1st dose artemether/DHA exposure that was significantly lower than those on lopinavir/ritonavir and HIV-uninfected children. Multiple regression analysis supported that the effect of artemether/DHA exposure on parasite clearance was significantly moderated by HIV status. CONCLUSIONS: Parasite clearance rates remain rapid in Uganda and were not found to associate with PK exposure. However, significant decreases in artemisinin PK with repeated dosing in nearly all children, coupled with small, but significant increase in parasite clearance half-life in those with HIV, may have important implications for AL efficacy, particularly because reports of artemisinin resistance are increasing.

The effect of malnutrition on the pharmacokinetics and virologic outcomes of lopinavir, efavirenz and nevirapine in food insecure HIV-infected children in Tororo, Uganda.

BACKGROUND: Malnutrition may impact the pharmacokinetics (PKs) of antiretroviral medications and virologic responses in HIV-infected children. The authors therefore evaluated the PK of nevirapine (NVP), efavirenz (EFV) and lopinavir (LPV) in associations with nutritional status in a cohort of HIV-infected Ugandan children. METHODS: Sparse dried blood spot samples from Ugandan children were used to estimate plasma concentrations. Historical PK data from children from 3 resource-rich countries (RRC) were utilized to develop the PK models. RESULTS: Concentrations in 330 dried blood spot from 163 Ugandan children aged 0.7-7 years were analyzed in reference to plasma PK data (1189 samples) from 204 children from RRC aged 0.5-12 years. Among Ugandan children, 48% was malnourished (underweight, thin or stunted). Compared to RRC, Ugandan children exhibited reduced bioavailability of EFV and LPV; 11% (P=0.045) and 18% (P=0.008), respectively. In contrast, NVP bioavailability was 46% higher in Ugandan children (P<0.001) with a trend toward greater bioavailability when malnourished. Children receiving LPV, EFV or NVP had comparable risk of virologic failure. Among children on NVP, low height and weight for age Z scores were associated with reduced risk of virologic failure (P=0.034, P=0.068, respectively). CONCLUSIONS: Ugandan children demonstrated lower EFV and LPV and higher NVP exposure compared to children in RRC, perhaps reflecting the consequence of malnutrition on bioavailability. In children receiving NVP, the relation between exposure, malnutrition and outcome turned out to be marginally significant. Further investigations are warranted using more intensive PK measurements and adequate adherence assessments, to further assess causes of virologic failure in Ugandan children.

Determination of artemether and dihydroartemisinin in human plasma with a new hydrogen peroxide stabilization method.

BACKGROUND: Numerous methods have been reported for the determination of artemether (ARM) and its metabolite dihydroartemisinin (DHA) in plasma. However, stability issues in patient plasma have not received enough attention. RESULTS: An LC-MS/MS method for simultaneous determination of ARM and DHA in human plasma (K3EDTA) turned out to be problematic: ARM and DHA were degraded partially or completely in some patient plasma samples as indicated by the stable isotope-labeled internal standards. We postulated iron II (Fe(2+)) in hemoglobin or its derived products from malaria patients causes degradation of the drugs, and found that hydrogen peroxide (H2O2) protected the drugs from degradation. Acidifying plasma increased recovery of ARM significantly. Using only 50 µl of plasma sample, the method has a LLOQ at 0.5 ng/ml for both ARM and DHA. CONCLUSION: H2O2 is a stabilizing agent for artemisinin derivatives. The modified method is reliable and sensitive.

Distinct genes related to drug response identified in ER positive and ER negative breast cancer cell lines.

Breast cancer patients have different responses to chemotherapeutic treatments. Genes associated with drug response can provide insight to understand the mechanisms of drug resistance, identify promising therapeutic opportunities, and facilitate personalized treatment. Estrogen receptor (ER) positive and ER negative breast cancer have distinct clinical behavior and molecular properties. However, to date, few studies have rigorously assessed drug response genes in them. In this study, our goal was to systematically identify genes associated with multidrug response in ER positive and ER negative breast cancer cell lines. We tested 27 human breast cell lines for response to seven chemotherapeutic agents (cyclophosphamide, docetaxel, doxorubicin, epirubicin, fluorouracil, gemcitabine, and paclitaxel). We integrated publicly available gene expression profiles of these cell lines with their in vitro drug response patterns, then applied meta-analysis to identify genes related to multidrug response in ER positive and ER negative cells separately. One hundred eighty-eight genes were identified as related to multidrug response in ER positive and 32 genes in ER negative breast cell lines. Of these, only three genes (DBI, TOP2A, and PMVK) were common to both cell types. TOP2A was positively associated with drug response, and DBI was negatively associated with drug response. Interestingly, PMVK was positively associated with drug response in ER positive cells and negatively in ER negative cells. Functional analysis showed that while cell cycle affects drug response in both ER positive and negative cells, most biological processes that are involved in drug response are distinct. A number of signaling pathways that are uniquely enriched in ER positive cells have complex cross talk with ER signaling, while in ER negative cells, enriched pathways are related to metabolic functions. Taken together, our analysis indicates that distinct mechanisms are involved in multidrug response in ER positive and ER negative breast cells.

Rapid variable-number tandem-repeat genotyping for Mycobacterium leprae clinical specimens.

Mycobacterium leprae is the noncultivable pathogen of leprosy. Since the genome sequence of an isolate of M. leprae has become available, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been explored as a tool for strain typing and identification of chains of transmission of leprosy. In order to discover VNTRs and develop methods transferable to clinical samples, MLVA was applied to a global collection of M. leprae isolates derived from leprosy patients and propagated in armadillo hosts. PCR amplification, agarose gel electrophoresis, and sequencing methods were applied to DNA extracts from these infected armadillo tissues (n = 21). We identified polymorphisms in 15 out of 25 short-tandem-repeat (STR) loci previously selected by in silico analyses of the M. leprae genome. We then developed multiplex PCR for amplification of these 15 loci in four separate PCRs suitable for fluorescent fragment length analysis and demonstrated STR profiles highly concordant with those from the sequencing methods. Subsequently, we extended this method to DNA extracts from human clinical specimens, such as skin biopsy specimens (n = 30). With these techniques, mapping of multiple loci and differentiation of genotypes have been possible using total DNA extracts from limited amounts of clinical samples at a reduced cost and with less time. These practical methods are therefore available and applicable to answer focused epidemiological questions and to allow monitoring of the transmission of M. leprae in different countries where leprosy is endemic.