Red blood cells function as reservoirs of tumor DNA.

Novel screening techniques for early detection of lung cancer are urgently needed. Profiling circulating tumor cell-free DNA (ctDNA) has emerged as a promising tool for biopsy-free tumor genotyping. However, both the scarcity and short half-life of ctDNA substantially limit the sensitivity and clinical utility of ctDNA detection methodologies. Our discovery that red blood cells (RBCs) sequester mitochondrial DNA opens a new avenue for detecting circulating nucleic acids, as RBCs represent an unrecognized reservoir of circulating nucleic acid. Here, we show that RBCs acquire tumor DNA following coculture with lung cancer cell lines harboring Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR) mutations. RBC-bound tumor DNA is detectable in patients with early-stage non-small cell lung cancer (NSCLC) but not in healthy controls by qPCR. Our results collectively uncover a previously unrecognized yet easily accessible reservoir of tumor DNA, offering a promising foundation for future RBC-based tumor diagnostics.NEW & NOTEWORTHY We present a novel method for lung cancer detection by revealing RBCs as a reservoir for tumor DNA, overcoming the limitations of current circulating tumor ctDNA methodologies. By demonstrating that RBCs can capture tumor DNA, including critical mutations found in lung cancer, we provide a promising, biopsy-free avenue for early cancer diagnostics. This discovery opens up exciting possibilities for developing RBC-based diagnostic tools, significantly enhancing the sensitivity and clinical utility of noninvasive cancer detection.

Risk of venous thromboembolism for ovarian cancer patients during first-line therapy after implementation of an Enhanced Recovery After Surgery (ERAS) protocol.

OBJECTIVE: To determine incidence and risk factors for VTE for patients with advanced epithelial ovarian cancer undergoing first-line therapy, including cytoreductive surgery, on an Enhanced Recovery After Surgery (ERAS) protocol. METHODS: Medical records were reviewed for patients with FIGO stage IIIA-IVB epithelial ovarian, fallopian tube, or primary peritoneal cancer undergoing primary or interval cytoreductive surgery from March 2017 through September 2019. All patients were enrolled on an ERAS protocol, including 28-day postoperative VTE prophylaxis. Demographic information, medical history, perioperative characteristics, and ERAS compliance were evaluated using univariate and multivariate models. RESULTS: Of 230 patients undergoing cytoreductive surgery via laparotomy, 155 received neoadjuvant chemotherapy and 75 received primary cytoreduction. 38 patients had a VTE during the study period. 13 events (5.7%) were identified at time of diagnosis, 6 (3.9%) during neoadjuvant chemotherapy, 5 (2.2%) within 30 days after surgery, 5 (2.2%) between 30 days and 6 months after surgery, and 9 (3.9%) after the 6-month window. The cumulative incidence of VTE was 6.1% (95% CI, 4.3-8.8%) within 6 months after diagnosis and 8.5% (6.2-11.4%) within 1 year after diagnosis. Estimated blood loss (adjusted HR 1.22 [95% CI, 1.09-1.36], p = 0.001) and history of VTE (7.06 [2.34-21.29], p = 0.001) were independently associated with VTE. CONCLUSION: With implementation of an ERAS protocol, only 1 in 46 patients experienced a VTE within 30 days after surgery. However, overall VTE occurred in 1 in 16 patients during first-line therapy. Strategies to further reduce VTE risk, especially during neoadjuvant chemotherapy and surveillance, should be investigated.

Gynecologic oncology treatment modifications or delays in response to the COVID-19 pandemic in a publicly funded versus privately funded North American tertiary cancer center.

OBJECTIVE: To compare gynecologic oncology surgical treatment modifications and delays during the first wave of the COVID-19 pandemic between a publicly funded Canadian versus a privately funded American cancer center. METHODS: This is a retrospective cohort study of all planned gynecologic oncology surgeries at University Health Network (UHN) in Toronto, Canada and Brigham and Women's Hospital (BWH) in Boston, USA, between March 22,020 and July 302,020. Surgical treatment delays and modifications at both centers were compared to standard recommendations. Multivariable logistic regression was performed to adjust for confounders. RESULTS: A total of 450 surgical gynecologic oncology patients were included; 215 at UHN and 235 at BWH. There was a significant difference in median time from decision-to-treat to treatment (23 vs 15 days, p < 0.01) between UHN and BWH and a significant difference in treatment delays (32.56% vs 18.29%; p < 0.01) and modifications (8.37% vs 0.85%; p < 0.01), respectively. On multivariable analysis adjusting for age, race, treatment site and surgical priority status, treatment at UHN was an independent predictor of treatment modification (OR = 9.43,95% CI 1.81-49.05, p < 0.01). Treatment delays were higher at UHN (OR = 1.96,95% CI 1.14-3.36 p = 0.03) and for uterine disease (OR = 2.43, 95% CI 1.11-5.33, p = 0.03). CONCLUSION: During the first wave of COVID-19 pandemic, gynecologic oncology patients treated at a publicly funded Canadian center were 9.43 times more likely to have a surgical treatment modification and 1.96 times more likely to have a surgical delay compared to an equal volume privately funded center in the United States.

Acceptability of Long-Acting Injectable Cabotegravir (CAB LA) in HIV-Uninfected Individuals: HPTN 077.

Long-acting injectable PrEP could offer an alternative to daily oral PrEP, improve adherence and protection, if found acceptable, safe and effective. HPTN 077 evaluated injectable cabotegravir safety, tolerability and pharmacokinetics among HIV-uninfected males and females in sequentially-enrolled cohorts of two dosing strategies. We compared acceptability of product attributes, prevention preferences and future interest in injectable PrEP (FIIP) by region, sex-at-birth, arm and cohort and used multivariable analysis to identify FIIP determinants. Baseline injectable PrEP preferences were higher in non-U.S. sites and increased in both regions over time. In multivariable models, FIIP was most strongly associated with acceptability of product attributes, was higher in non-U.S. sites and more altruistic participants. Treatment arm and report of pain were not associated with FIIP. Injectable acceptability was highest in non-U.S. sites. Preferences for injectable versus other PrEP methods were higher among U.S. males than females, but higher among males and females in non-U.S. settings.

Management of pediatric plaque psoriasis using biologics.

BACKGROUND: Psoriasis is a chronic inflammatory disease with clinical manifestations of the skin that affect adults and children. In adults, biologics have revolutionized the treatment of moderate to severe plaque psoriasis where clear or almost clear is a tangible goal. Research on biologics has recently been extended to children. The introduction of these new therapeutic options has outpaced the limited guidelines in this population. OBJECTIVE: To provide a review of current data on biologics, with a proposal for a clinically relevant treatment algorithm on the management of moderate to severe plaque psoriasis in the pediatric population. METHODS: A Canadian panel with expertise in psoriasis, pediatric dermatology, and experience with consensus recommendation processes was selected to review the current landscape of pediatric psoriasis and clinical data on biologics plus identify special considerations for baseline workup and monitoring. Recommendations were reviewed and edited by each expert in an iterative process. CONCLUSION: A treatment algorithm for moderate to severe plaque psoriasis in pediatric patients is presented, incorporating approved biologics. Guidance on baseline screening and ongoing monitoring is also provided. Ultimately, treatment choice depends on the patient and his or her caregiver, with consideration of comorbidities, impact on quality of life, and relevant safety aspects.

Use of ablation and ultrasonic aspiration at primary debulking surgery in advanced stage ovarian, fallopian tube, and primary peritoneal cancer.

OBJECTIVES: Ovarian cancer patients with miliary disease have the lowest rates of complete surgical resection and poorest survival. Adjunct surgical techniques may potentially increase rates of complete surgical resection. No studies have evaluated the use of these techniques in primary debulking surgery for ovarian cancer patients with miliary disease. The aim of this study was to examine the use of adjunct surgical techniques during primary debulking surgery for patients with advanced epithelial ovarian, fallopian tube, and primary peritoneal cancer with miliary disease. METHODS: Medical records of patients with International Federation of Gynecology and Obstetrics (FIGO) stages IIIC-IVB epithelial ovarian, fallopian tube, or primary peritoneal cancer with miliary disease undergoing primary debulking surgery from January 2010 to December 2014 were reviewed. Adjunct surgical techniques were defined as ultrasonic surgical aspiration, argon enhanced electrocautery, thermal plasma energy, and traditional electrocautery ablation. Patients undergoing surgery with and without these devices were compared with respect to demographics, operative characteristics, postoperative complications, residual disease, progression free survival and overall survival. RESULTS: A total of 135 patients with miliary disease underwent primary debulking surgery, of which 30 (22.2%) patients used adjunct surgical techniques. The most common devices were ultrasonic surgical aspiration (40%) and argon enhanced electrocautery (36.7%). The most common sites of use were diaphragm (63.3%), pelvic peritoneum (30%), bowel mesentery (20%), and large bowel serosa (20%). There were no differences in age, stage, primary site, histology, operative time, surgical complexity, or postoperative complications for patients operated on with or without these devices. Volume of residual disease was similar (0.1-1 cm: 60% with adjunct techniques versus 68.6% without; complete surgical resection: 16.7% with adjunct techniques versus 13.3% without; p=0.67). For patients with ≤1 cm residual disease, median progression free survival (15 versus 15 months, p=0.65) and median overall survival (40 versus 55 months, p=0.38) were also similar. CONCLUSION: Adjunct surgical techniques may be incorporated during primary debulking surgery for patients with advanced epithelial ovarian, fallopian tube, or primary peritoneal cancer with miliary disease; however, these do not improve the rate of optimal cytoreduction.

The Longitudinal Effects of Non-injection Substance Use on Sustained HIV Viral Load Undetectability Among MSM and Heterosexual Men in Brazil and Thailand: The Role of ART Adherence and Depressive Symptoms (HPTN 063).

The effect of non-injection substance use on HIV viral load (VL) is understudied in international settings. Data are from HPTN063, a longitudinal observational study of HIV-infected individuals in Brazil, Thailand, and Zambia, with focus on men with VL data (Brazil = 146; Thailand = 159). Generalized linear mixed models (GLMM) assessed whether non-injection substance use (stimulants, cannabis, alcohol, polysubstance) was associated with VL undetectability. ART adherence and depressive symptoms were examined as mediators of the association. In Thailand, substance use was not significantly associated with VL undetectability or ART adherence, but alcohol misuse among MSM was associated with increased odds of depression (AOR = 2.75; 95% CI 1.20, 6.32, p = 0.02). In Brazil, alcohol misuse by MSM was associated with decreased odds of undetectable VL (AOR = 0.34; 95% CI 0.13, 0.92, p = 0.03). Polysubstance use by heterosexual men in Brazil was associated with decreased odds of ART adherence (AOR = 0.25; 95% CI 0.08, 0.78, p = 0.02). VL suppression appears attainable among non-injection substance users. Substance use interventions among HIV-positive men should address depression, adherence, and VL undetectability.

Safety, tolerability, and pharmacokinetics of long-acting injectable cabotegravir in low-risk HIV-uninfected individuals: HPTN 077, a phase 2a randomized controlled trial.

BACKGROUND: Cabotegravir (CAB) is a novel strand-transfer integrase inhibitor being developed for HIV treatment and prevention. CAB is formulated both as an immediate-release oral tablet for daily administration and as a long-acting injectable suspension (long-acting CAB [CAB LA]) for intramuscular (IM) administration, which delivers prolonged plasma exposure to the drug after IM injection. HIV Prevention Trials Network study 077 (HPTN 077) evaluated the safety, tolerability, and pharmacokinetics of CAB LA in HIV-uninfected males and females at 8 sites in Brazil, Malawi, South Africa, and the United States. METHODS AND FINDINGS: HPTN 077 was a double-blind, placebo-controlled phase 2a trial. Healthy individuals age 18-65 years at low HIV risk were randomized (3:1) to receive CAB or placebo (PBO). In the initial oral phase, participants received 1 daily oral tablet (CAB or PBO) for 4 weeks. Those without safety concerns in the oral phase continued and received injections in the injection phase (Cohort 1: 3 injections of CAB LA 800 mg or 0.9% saline as PBO IM every 12 weeks for 3 injection cycles; Cohort 2: CAB LA 600 mg or PBO IM for 5 injection cycles; the first 2 injections in Cohort 2 were separated by 4 weeks, the rest by 8 weeks). The primary analysis included weeks 5 to 41 of study participation, encompassing the injection phase. The cohorts were enrolled sequentially. Primary outcomes were safety and tolerability. Secondary outcomes included pharmacokinetics and events occurring during the oral and injection phases. Between February 9, 2015, and May 27, 2016, the study screened 443 individuals and enrolled 110 participants in Cohort 1 and 89 eligible participants in Cohort 2. Participant population characteristics were as follows: 66% female at birth; median age 31 years; 27% non-Hispanic white, 41% non-Hispanic black, 24% Hispanic/Latino, 3% Asian, and 6% mixed/other; and 6 transgender men and 1 transgender woman. Twenty-two (11%) participants discontinued the oral study product; 6 of these were for clinical or laboratory adverse events (AEs). Of those who received at least 1 CAB LA injection, 80% of Cohort 1 and 92% of Cohort 2 participants completed all injections; injection course completion rates were not different from those in the PBO arm. Injection site reactions (ISRs) were common (92% of Cohort 1 and 88% of Cohort 2 participants who received CAB LA reported any ISR). ISRs were mostly Grade 1 (mild) to Grade 2 (moderate), and 1 ISR event (Cohort 1) led to product discontinuation. Grade 2 or higher ISRs were the only AEs reported more commonly among CAB LA recipients than PBO recipients. Two Grade 3 (severe) ISRs occurred in CAB recipients, 1 in each cohort, but did not lead to product discontinuation in either case. Seven incident sexually transmitted infections were diagnosed in 6 participants. One HIV infection occurred in a participant 48 weeks after last injection of CAB LA: CAB was not detectable in plasma both at the time of first reactive HIV test and at the study visit 12 weeks prior to the first reactive test. Participants in Cohort 2 (unlike Cohort 1) consistently met prespecified pharmacokinetic targets of at least 95% of participants maintaining CAB trough concentrations above PA-IC90, and 80% maintaining trough concentrations above 4× PA-IC90. Study limitations include a modest sample size, a short course of injections, and a low-risk study population. CONCLUSIONS: In this study, CAB LA was well tolerated at the doses and dosing intervals used. ISRs were common, but infrequently led to product discontinuation. CAB LA 600 mg every 8 weeks met pharmacokinetic targets for both male and female study participants. The safety and pharmacokinetic results observed support the further development of CAB LA, and efficacy studies of CAB LA for HIV treatment and prevention are in progress. TRIAL REGISTRATION: ClinicalTrials.gov Registry: ClinicalTrials.gov Trial number: NCT02178800.

Analysis of HLA A*02 association with vaccine efficacy in the RV144 HIV-1 vaccine trial.

UNLABELLED: The RV144 HIV-1 vaccine trial demonstrated partial efficacy of 31% against HIV-1 infection. Studies into possible correlates of protection found that antibodies specific to the V1 and V2 (V1/V2) region of envelope correlated inversely with infection risk and that viruses isolated from trial participants contained genetic signatures of vaccine-induced pressure in the V1/V2 region. We explored the hypothesis that the genetic signatures in V1 and V2 could be partly attributed to selection by vaccine-primed T cells. We performed a T-cell-based sieve analysis of breakthrough viruses in the RV144 trial and found evidence of predicted HLA binding escape that was greater in vaccine versus placebo recipients. The predicted escape depended on class I HLA A*02- and A*11-restricted epitopes in the MN strain rgp120 vaccine immunogen. Though we hypothesized that this was indicative of postacquisition selection pressure, we also found that vaccine efficacy (VE) was greater in A*02-positive (A*02(+)) participants than in A*02(-) participants (VE = 54% versus 3%, P = 0.05). Vaccine efficacy against viruses with a lysine residue at site 169, important to antibody binding and implicated in vaccine-induced immune pressure, was also greater in A*02(+) participants (VE = 74% versus 15%, P = 0.02). Additionally, a reanalysis of vaccine-induced immune responses that focused on those that were shown to correlate with infection risk suggested that the humoral responses may have differed in A*02(+) participants. These exploratory and hypothesis-generating analyses indicate there may be an association between a class I HLA allele and vaccine efficacy, highlighting the importance of considering HLA alleles and host immune genetics in HIV vaccine trials. IMPORTANCE: The RV144 trial was the first to show efficacy against HIV-1 infection. Subsequently, much effort has been directed toward understanding the mechanisms of protection. Here, we conducted a T-cell-based sieve analysis, which compared the genetic sequences of viruses isolated from infected vaccine and placebo recipients. Though we hypothesized that the observed sieve effect indicated postacquisition T-cell selection, we also found that vaccine efficacy was greater for participants who expressed HLA A*02, an allele implicated in the sieve analysis. Though HLA alleles have been associated with disease progression and viral load in HIV-1 infection, these data are the first to suggest the association of a class I HLA allele and vaccine efficacy. While these statistical analyses do not provide mechanistic evidence of protection in RV144, they generate testable hypotheses for the HIV vaccine community and they highlight the importance of assessing the impact of host immune genetics in vaccine-induced immunity and protection. (This study has been registered at ClinicalTrials.gov under registration no. NCT00223080.).

Novel MesoPorous BioGlass/silk scaffold containing adPDGF-B and adBMP7 for the repair of periodontal defects in beagle dogs.

AIM: The local delivery of growth factors via gene therapy has gained tremendous awareness in recent years due to their sustained growth factor delivery to target tissues. The aim of this study was to fabricate and investigate a scaffold able to release growth factors via gene therapy for the repair of periodontal tissues. MATERIALS AND METHODS: Novel mesoporous bioglass (MBG)/silk fibrin scaffold combined with BMP7 and/or PDGF-B adenovirus was fabricated and tested in vitro for cell migration, proliferation and differentiation. Furthermore, acute-type buccal dehiscence periodontal defects (mesiodistal width × depth: 5 × 5 mm) were created on the buccal portion of the maxillary premolars in five normal male beagle dogs (12 months old, 15.0 ± 2.0 kg) and histologically examined for periodontal regeneration following implantation of the following five groups: (1) no scaffold, (2) MBG/silk scaffold alone, (3) scaffold + adPDGF-B, (4) scaffold + adBMP7, (5) scaffold + adPDGF-b + adBMP7. RESULTS: In vitro findings demonstrated that adPDGF-B was able to rapidly recruit periodontal ligament (PDL) cells over sixfold more effectively than adBMP7, whereas adBMP7 was more able to induce osteoblast differentiation of PDL cells. In vivo findings demonstrate that scaffolds loaded with adPDGF-B were able to partially regenerate the periodontal ligament while adBMP7 scaffolds primarily improved new bone formation. The combination of both adPDGF-B and adBMP7 synergistically promoted periodontal regeneration by allowing up to two times greater regeneration of the periodontal ligament, alveolar bone and cementum when compared to each adenovirus used alone. CONCLUSIONS: Although both PDGF-B and BMP7 are individually capable of promoting periodontal regeneration to some degree, their combination synergistically promotes wound healing in acute-type buccal dehiscence periodontal defects when delivered simultaneously. This study demonstrates the promise for successful delivery of low-cost, effective growth factor delivery via gene therapy for the treatment of periodontal defects.

Interleukin-6 mediates the platelet abnormalities and thrombogenesis associated with experimental colitis.

Clinical studies and animal experimentation have shown that colonic inflammation is associated with an increased number and reactivity of platelets, coagulation abnormalities, and enhanced thrombus formation. The objective of this study was to define the contribution of IL-6 to the thrombocytosis, exaggerated agonist-induced platelet aggregation, and enhanced extra-intestinal thrombosis that occur during experimental colitis. The number of mature and immature platelets, platelet life span, thrombin-induced platelet aggregation response, and light/dye-induced thrombus formation in cremaster muscle arterioles were measured in wild-type (WT) and IL-6-deficient (IL-6(-/-)) mice with dextran sodium sulfate (DSS)-induced colitis. DSS colitis in WT mice was associated with thrombocytosis with an elevated number of both mature and immature platelets and no change in platelet life span. The thrombocytosis response was absent in IL-6(-/-) mice. DSS treatment also enhanced the platelet aggregation response to thrombin and accelerated thrombus development in WT mice, but not in IL-6(-/-) mice. Exogenous IL-6 administered to WT mice elicited a dose-dependent enhancement of thrombus formation. These findings indicate that IL-6 mediates the thrombocytosis, platelet hyperreactivity, and accelerated thrombus development associated with experimental colitis. The IL-6-dependent colitis-induced thrombocytosis appears to result from an enhancement of thrombopoiesis because platelet life span is unchanged.

Murine alveolar macrophages rapidly accumulate intranasally administered SARS-CoV-2 Spike protein leading to neutrophil recruitment and damage.

The trimeric SARS-CoV-2 Spike protein mediates viral attachment facilitating cell entry. Most COVID-19 vaccines direct mammalian cells to express the Spike protein or deliver it directly via inoculation to engender a protective immune response. The trafficking and cellular tropism of the Spike protein in vivo and its impact on immune cells remains incompletely elucidated. In this study, we inoculated mice intranasally, intravenously, and subcutaneously with fluorescently labeled recombinant SARS-CoV-2 Spike protein. Using flow cytometry and imaging techniques, we analyzed its localization, immune cell tropism, and acute functional impact. Intranasal administration led to rapid lung alveolar macrophage uptake, pulmonary vascular leakage, and neutrophil recruitment and damage. When injected near the inguinal lymph node medullary, but not subcapsular macrophages, captured the protein, while scrotal injection recruited and fragmented neutrophils. Widespread endothelial and liver Kupffer cell uptake followed intravenous administration. Human peripheral blood cells B cells, neutrophils, monocytes, and myeloid dendritic cells all efficiently bound Spike protein. Exposure to the Spike protein enhanced neutrophil NETosis and augmented human macrophage TNF-α (tumor necrosis factor-α) and IL-6 production. Human and murine immune cells employed C-type lectin receptors and Siglecs to help capture the Spike protein. This study highlights the potential toxicity of the SARS-CoV-2 Spike protein for mammalian cells and illustrates the central role for alveolar macrophage in pathogenic protein uptake.

Allergic and infectious complications of Etonogestrel implant placements: A case series.

Patients rarely experience complications at the time of Etonogestrel subdermal contraceptive implant placement. Few case reports describe infection or allergy as a complication at the time of implant insertion. In this case series, we discuss three infections and one allergic reaction following Etonogestrel implant placement, review six previous case reports of eight cases of infection or allergy, and discuss management of these complications. We highlight differential diagnosis when encountering a placement complication, considerations of dermatologic conditions when placing Etonogestrel implants, and discuss when to consider removal of the implant when a complication occurs.

Murine Alveolar Macrophages Rapidly Accumulate Intranasally Administered SARS-CoV-2 Spike Protein leading to Neutrophil Recruitment and Damage.

The trimeric SARS-CoV-2 Spike protein mediates viral attachment facilitating cell entry. Most COVID-19 vaccines direct mammalian cells to express the Spike protein or deliver it directly via inoculation to engender a protective immune response. The trafficking and cellular tropism of the Spike protein in vivo and its impact on immune cells remains incompletely elucidated. In this study we inoculated mice intranasally, intravenously, and subcutaneously with fluorescently labeled recombinant SARS-CoV-2 Spike protein. Using flow cytometry and imaging techniques we analyzed its localization, immune cell tropism, and acute functional impact. Intranasal administration led to rapid lung alveolar macrophage uptake, pulmonary vascular leakage, and neutrophil recruitment and damage. When injected near the inguinal lymph node medullary, but not subcapsular macrophages, captured the protein, while scrotal injection recruited and fragmented neutrophils. Wide-spread endothelial and liver Kupffer cell uptake followed intravenous administration. Human peripheral blood cells B cells, neutrophils, monocytes, and myeloid dendritic cells all efficiently bound Spike protein. Exposure to the Spike protein enhanced neutrophil NETosis and augmented human macrophage TNF-α and IL-6 production. Human and murine immune cells employed C-type lectin receptors and Siglecs to help capture the Spike protein. This study highlights the potential toxicity of the SARS-CoV-2 Spike protein for mammalian cells and illustrates the central role for alveolar macrophage in pathogenic protein uptake.

Comparing and combining data across multiple sources via integration of paired-sample data to correct for measurement error.

In biomedical research such as the development of vaccines for infectious diseases or cancer, study outcomes measured by an assay or device are often collected from multiple sources or laboratories. Measurement error that may vary between laboratories needs to be adjusted for when combining samples across data sources. We incorporate such adjustment in the main study by comparing and combining independent samples from different laboratories via integration of external data, collected on paired samples from the same two laboratories. We propose the following: (i) normalization of individual-level data from two laboratories to the same scale via the expectation of true measurements conditioning on the observed; (ii) comparison of mean assay values between two independent samples in the main study accounting for inter-source measurement error; and (iii) sample size calculations of the paired-sample study so that hypothesis testing error rates are appropriately controlled in the main study comparison. Because the goal is not to estimate the true underlying measurements but to combine data on the same scale, our proposed methods do not require that the true values for the error-prone measurements are known in the external data. Simulation results under a variety of scenarios demonstrate satisfactory finite sample performance of our proposed methods when measurement errors vary. We illustrate our methods using real enzyme-linked immunosorbent spot assay data generated by two HIV vaccine laboratories.

Metabolism of Long-Acting Rilpivirine After Intramuscular Injection: HIV Prevention Trials Network Study 076 (HPTN 076).

A long-acting injectable formulation of rilpivirine (RPV), a non-nucleoside reverse transcriptase inhibitor, is currently under investigation for use in human immunodeficiency virus (HIV) maintenance therapy. We previously characterized RPV metabolism after oral dosing and identified seven metabolites: four metabolites resulting from mono- or dioxygenation of the 2,6-dimethylphenyl ring itself or either of the two methyl groups located on that ring, one N-linked RPV glucuronide conjugate, and two O-linked RPV glucuronides produced via glucuronidation of mono- and dihydroxymethyl metabolites. However, as is true for most drugs, the metabolism of RPV after injection has yet to be reported. The phase II clinical trial HPTN 076 enrolled 136 HIV-uninfected women and investigated the safety and acceptability of long-acting injectable RPV for use in HIV pre-exposure prophylaxis. Through the analysis of plasma samples from 80 of these participants in the active product arm of the study, we were able to detect 2 metabolites after intramuscular injection of long-acting RPV, 2-hydroxymethyl-RPV, and RPV N-glucuronide. Of the total of 80 individuals, 72 participants exhibited detectable levels of 2-hydroxymethyl-RPV in plasma samples whereas RPV N-glucuronide was detectable in plasma samples of 78 participants. In addition, RPV N-glucuronide was detectable in rectal fluid, cervicovaginal fluid, and vaginal tissue. To investigate potential genetic variation in genes encoding enzymes relevant to RPV metabolism, we isolated genomic DNA and performed next-generation sequencing of CYP3A4, CYP3A5, UGT1A1 and UGT1A4. From these analyses, four missense variants were detected for CYP3A4 whereas one missense variant and one frameshift variant were detected for CYP3A5. A total of eight missense variants of UGT1A4 were detected, whereas two variants were detected for UGT1A1; however, these variants did not appear to account for the observed interindividual variability in metabolite levels. These findings provide insight into the metabolism of long-acting RPV and contribute to an overall understanding of metabolism after oral dosing versus injection. ClinicalTrials.gov Identifier: NCT02165202.

Unrestrained Gαi2 Signaling Disrupts Neutrophil Trafficking, Aging, and Clearance.

Neutrophil trafficking, homeostatic and pathogen elicited, depends upon chemoattractant receptors triggering heterotrimeric G-protein Gαißγ signaling, whose magnitude and kinetics are governed by RGS protein/Gαi interactions. RGS proteins typically limit Gαi signaling by reducing the duration that Gαi subunits remain GTP bound and able to activate downstream effectors. Yet how in totality RGS proteins shape neutrophil chemoattractant receptor activated responses remains unclear. Here, we show that C57Bl/6 mouse neutrophils containing a genomic knock-in of a mutation that disables all RGS protein-Gαi2 interactions (G184S) cannot properly balance chemoattractant receptor signaling, nor appropriately respond to inflammatory insults. Mutant neutrophils accumulate in mouse bone marrow, spleen, lung, and liver; despite neutropenia and an intrinsic inability to properly mobilize from the bone marrow. In vitro they rapidly adhere to ICAM-1 coated plates, but in vivo they poorly adhere to blood vessel endothelium. Those few neutrophils that cross blood vessels and enter tissues migrate haphazardly. Following Concanavalin-A administration fragmented G184S neutrophils accumulate in liver sinusoids leading to thrombo-inflammation and perivasculitis. Thus, neutrophil Gαi2/RGS protein interactions both limit and facilitate Gαi2 signaling thereby promoting normal neutrophil trafficking, aging, and clearance.

Correction to "Identification of Novel UGT1A1 Variants Including UGT1A1 454C>A through the Genotyping of Healthy Participants of the HPTN 077 Study".

[This corrects the article DOI: 10.1021/acsptsci.0c00181.].

Identification of Novel UGT1A1 Variants Including UGT1A1 454C>A through the Genotyping of Healthy Participants of the HPTN 077 Study.

Cabotegravir (CAB) is an integrase strand-transfer inhibitor of HIV that has proven effective for HIV treatment and prevention in a long-acting injectable formulation, typically preceded by an oral formulation lead-in phase. Previous in vitro studies have demonstrated that CAB is primarily metabolized via glucuronidation by uridine diphosphate glucuronosyltransferase (UGT) 1A1 and 1A9. In this study, we performed next-generation sequencing of genomic DNA isolated from the HPTN 077 participants to explore the variants within UGT1A1 and UGT1A9. Additionally, to enable correlation of UGT1A1 and UGT1A9 genotypes with plasma CAB-glucuronide levels, we quantified glucuronidated CAB following both oral administration of CAB and intramuscular injection of long-acting CAB. From these studies, 48 previously unreported variants of UGT1A1 and UGT1A9 were detected. Notably, 5/68 individuals carried a UGT1A1 454C>A variant that resulted in amino acid substitution P152T, and the use of in silico tools predicted a deleterious effect of the P152T substitution. Thus, the impact of this mutant on a range of UGT1A1 substrates was tested using a COS-7 cell-based assay. The glucuronide conjugates of CAB, dolutegravir, and raltegravir, were not formed in the COS-7 cells expressing the UGT1A1 P152T mutant. Further, formation of glucuronides of raloxifene and 7-ethyl-10-hydroxycamptothecin were reduced in the cells expressing the UGT1A1 P152T mutant. Using the same approach, we tested the activities of two UGT1A9 mutants, UGT1A9 H217Y and UGT1A9 R464G, and found that these mutations were tolerated and decreased function, respectively. These data provide insight into previously unreported genetic variants of UGT1A1 and UGT1A9.

Family history and risk of recurrent cystitis and pyelonephritis in women.

PURPOSE: Recurrent urinary tract infections and pyelonephritis have risk factors suggesting genetic sources. Family history variables indicative of genetic risk merit further investigation. We evaluated the risk of recurrent cystitis and pyelonephritis in women with and those without a family history of urinary tract infection. MATERIALS AND METHODS: We conducted a population based case-control study of 1,261 women 18 to 49 years old enrolled in a Northwest health plan. Participants were cases identified from plan databases with documented recurrent cystitis (431) or pyelonephritis (400). Shared controls (430) were similar age women with no urinary tract infection history. We evaluated the history of urinary tract infection and pyelonephritis in first-degree female relatives (mother, sister[s], daughter[s]) and other covariates, ascertained through questionnaires and computerized databases. RESULTS: Of the cases 70.9% with recurrent cystitis and 75.2% with pyelonephritis, and of the controls 42.4% reported a urinary tract infection history in 1 or more female relative (p <0.001 for each case group vs controls). In both case groups odds ratios were significantly increased for women reporting a urinary tract infection history in their mother, sister(s) or daughter(s). Risk increased with a greater number of affected relatives. In women with 1 vs 2 or more relatives the ORs for recurrent cystitis were 3.1 (95% CI 2.1, 4.7) and 5.0 (3.1, 8.1), and the ORs for pyelonephritis were 3.3 (2.2, 5.0) and 5.5 (3.4, 9.0), respectively. CONCLUSIONS: In these community dwelling women a urinary tract infection history in female relatives was strongly and consistently associated with urinary tract infection recurrence and pyelonephritis. Risk estimates increased with stronger family history indices, suggesting a genetic component for increased susceptibility to these infections.