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Background: Despite their ubiquity across sub-Saharan Africa, private pharmacies are underutilized for HIV service delivery beyond the sale of HIV self-test kits. To understand what uptake of HIV prevention and treatment services might look like if private pharmacies offered clients free HIV self-testing and referral to clinic-based HIV services, we conducted a pilot study in Kenya., Methods: At 20 private pharmacies in Kisumu County, Kenya, pharmacy clients (≥ 18 years) purchasing sexual health-related products (e.g., contraception) were offered free HIV testing. Based on their test result and recent self-reported behaviors associated with HIV risk, clients were encouraged to consider pre-exposure prophylaxis (PrEP), post-exposure prophylaxis (PEP), or antiretroviral therapy (ART) initiation, informed where they could access free services, and issued a referral. We called clients three months after study completion to see if they had initiated the recommended service. Among clients who reported PrEP referral, we used Poisson regression models to examine characteristics associated with PrEP initiation and calculated adjusted prevalence ratios (aPRs)., Results: From March to June 2022, 1500 pharmacy clients completed HIV testing and were referred to clinic-based HIV services; in October 2022, 1178 (79%) were reached and meet our criteria for follow-up. Among those reached, the majority (63%, 742/1178) were women, the median age was 26 years (IQR 22-31), and few (4%, 51/1178) reported any prior PrEP use. At the pharmacy, most clients (96%, 1136/1178) tested HIV-negative and reported PrEP (95%, 1122/1178) or PEP (1%, 14/1178) referral; the remainder (4%, 42/1178) tested HIV-positive and reported ART referral. The uptake of ART (90%, 38/42) and PEP (86%, 12/14) among clients referred was high. The uptake of PrEP was only 9% (101/1122) among those referred and prior PrEP use was the only characteristic significantly associated with initiation (aPR 2.45, 95% confidence interval 1.19 to 5.07)., Conclusions: Although offering free HIV testing at private pharmacies led to the identification and referral of clients who could benefit from HIV services, additional interventions (e.g., incentives, patient navigators) may be needed to support PrEP referral follow-through. Alternatively, new delivery models that circumvent the need for referrals, such as same-day PrEP initiation at pharmacies, should be considered., Competing Interests: Declarations. Ethics approval and consent to participate: The protocol for this study was reviewed and approved by the Scientific Ethics Review Unit at the Kenya Medical Research Institute (Nairobi, Kenya) and the Institutional Review Board at the Fred Hutchinson Cancer Center (Seattle, United States). All participants provided written informed consent in English, Dholuo, or Kiswahili (the local languages) and received 500 Kenyan shillings during the study visit for their time and effort in completing study-related activities. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Objective: To determine the safety, tolerance, and adherence to self-administered intravaginal 5% fluorouracil (5FU) cream as adjuvant therapy following cervical intraepithelial neoplasia grade 2 or 3 (CIN2/3) treatment among women living with HIV (WLWH) in Kenya., Methods: A phase I pilot trial was performed among 12 WLWH in Kenya, aged 18-49 years between March 2023 and February 2024 (ClinicalTrial.gov NCT05362955). Participants self-administered 2 g of 5FU intravaginally every other week for eight applications. Safety was assessed using a standardized grading scale, and adherence was evaluated using self-report, inspection of used applicators, and weighing of the study drug., Results: The mean age and CD4 count were 43.9 years and 781 cells/mm 3 , respectively. Seven (58%) had an eighth-grade education or less. All 12 reported at least one grade I adverse event (AE), one (8%) reported a grade 2 AE, no grade 3 or 4 AEs were reported. Increased vaginal discharge (n = 9, 75%) and irritation (n = 5, 42%), with a mean duration of 3.2 and 2.8 days, respectively, were the most commonly reported AEs. Provider-observed AEs included grade 1 cervical erythema and superficial abrasions. All participants tolerated all eight 5FU doses, and 96% adherence was demonstrated., Conclusion: Self-administered 5FU following CIN2/3 treatment among WLWH in Kisumu, Kenya, was safe, tolerable, and associated with high adherence. Randomized trials are needed to investigate whether adjuvant 5FU can improve treatment outcomes or serve as primary cervical precancer treatment in sub-Saharan Africa. A self-administered therapy may be transformative in increasing access to treatment and, hence, secondary prevention of cervical cancer., (© 2024 International Federation of Gynecology and Obstetrics.)

Objectives: We conducted an implementation science mental health treatment study in western Kenya, testing strategies for scale up of evidence-based mental health services for common adult disorders using a non-specialist workforce, integrated with existing primary care (Sequential Multiple, Assignment Randomized Trial of non-specialist-delivered psychotherapy (Interpersonal Psychotherapy) and/or medication (fluoxetine) for major depression and post-traumatic stress disorder (PTSD) (SMART DAPPER)). Because study launch coincided with the COVID-19 pandemic, participants were allowed to attend treatment visits via mHealth (audio-only mobile phone) or in-person. We conducted a secondary data analysis of the parent study to evaluate preference for mHealth or in-person treatment among our study participants, including rationale for choosing in-person or mHealth treatment modality, and comparison of baseline demographic and clinical characteristics., Design, Setting, Participants and Interventions: Participants were public sector primary care patients at Kisumu County Hospital in western Kenya with major depression and/or PTSD and were individually randomised to non-specialist delivery of evidence-based psychotherapy or medication (n=2162)., Outcomes: Treatment modality preference and rationale were ascertained before randomised assignment to treatment arm (psychotherapy or medication). The parent SMART DAPPER study baseline assessment included core demographic (age, gender, relationship status, income, clinic transport time and cost) and clinical data (eg, depression and PTSD symptoms, trauma exposures, medical comorbidities and history of mental healthcare). Given that this evaluation of mHealth treatment preference sought to identify the demographic and clinical characteristics of participants who chose in-person or mHealth treatment modality, we included most SMART DAPPER core measurement domains (not all subcategories)., Results: 649 (30.3%) SMART DAPPER participants preferred treatment via mHealth, rather than in person. The most cited rationales for choosing mHealth were affordability (18.5%) (eg, no transportation cost) and convenience (12.9%). On multivariate analysis, compared with those who preferred in-person treatment, participants who chose mHealth were younger and had higher constraints on receiving in-person treatment, including transport time 1.004 (1.00, 1.007) and finances 0.757 (0.612, 0.936). Higher PTSD symptoms 0.527 (0.395, 0.702) and higher disability 0.741 (0.559, 0.982) were associated with preference for in-person treatment., Conclusions: To our knowledge, this is the first study of public sector mental healthcare delivered by non-specialists via mHealth for major depression and/or PTSD in Sub-Saharan Africa. Our finding that mHealth treatment is preferred by approximately one-third of participants, particularly younger individuals with barriers to in-person care, may inform future mHealth research to (1) address knowledge gaps in mental health service implementation and (2) improve mental healthcare access to evidence-based treatment., Trial Registration Number: NCT03466346., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

SMART DAPPER is an implementation science study responding to mental health treatment gaps for depression and trauma-related disorders in Sub-Saharan Africa (SSA). We report on patient experiences in a study using a Sequential, Multiple Assignment Randomized Trial (SMART) design to test first and second line non-specialist treatment using psychotherapy (Interpersonal Psychotherapy [IPT] or medication (fluoxetine [FLX]), integrated within public sector primary care in western Kenya. An embedded qualitative study conducted in-depth interviews (n = 17) and three (n = 3) focus group discussions with participants (May to October 2021). Audio-recorded interviews were transcribed and translated into English; we deductively and inductively analyzed transcripts guided by grounded theoretical approaches and content analysis. We drew on the health belief model and socio-ecological framework to present findings, including perceived severity (motivations for taking part in the intervention), impacts of the intervention at the individual, interpersonal, and community and health systems levels as well as barriers and facilitators. Participants discussed family and marital conflict, loss of a child, loss of income or a job, and traumatic events such as a death or illness. Impacts at the individual level included reduced headaches, improved appetite and weight management, increased energy, improved sleep, better self-efficacy, and improved concentration, which was reported to lead to increased economic opportunities. At the interpersonal level, participants noted a reduction in conflict, better conflict management and resolution, increased harmony with family and community members, and improved relationships with their partners and children. Perceived challenges included balancing the intervention with livelihoods, preference for traditional medicines, actual or anticipated side effects with medication (FLX), mental health stigma, major life events, and perceived inadequate counseling and challenges with providers. The findings demonstrate the potential of the SMART DAPPER intervention for depression and trauma-related disorder treatments and underscore the challenges and barriers that must be addressed when scaling similar interventions. Trial registration: ClinicalTrials.gov identifier: NCT03466346., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Getahun et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: In Kenya, 65% of sexually active unmarried women use modern contraceptives, a population at increased risk of HIV acquisition compared to other populations. Anchoring HIV prevention services, including pre-exposure prophylaxis (PrEP), to trusted contraceptive delivery settings offers opportunities to efficiently reach this important population. In Kenya, almost half (40%) of women accessing contraception services do so outside traditional healthcare facilities, such as retail pharmacies. Thus, integrating PrEP services into retail pharmacies may increase options for reaching adolescent girls and young women (AGYW) who could benefit from PrEP. Efforts are underway to define care pathways for pharmacy-delivered PrEP services in Kenya, including unsupported and supported models with nurse navigators., Methods: The AGYW Pharmacy PrEP study is an unblinded 2-arm cluster-randomized controlled trial in Kisumu, Kenya. The objective is to determine the effect that unsupported versus supported pharmacy-delivered PrEP services has on PrEP initiation, persistence, and adherence among AGYW seeking contraception. Twenty retail pharmacies offering pharmacy provider-led PrEP delivery will be randomized 1:1 to either receive or not receive a nurse navigator to support PrEP delivery. Eligible AGYW (n = 1900 total, n = 950/arm) will be ≥ 15 years old, purchasing a method of contraception at the pharmacy. Trained pharmacy provider will offer eligible AGYW either daily oral PrEP or the monthly DPV vaginal ring. The primary trial outcomes are PrEP initiation (use of PrEP at 1 month), persistence (use of PrEP at 10 months), and adherence (quantified by levels of TFV or DPV in hair samples). Additionally, several secondary (STI incidence, PrEP method selection, predictors of PrEP adherence) and exploratory outcomes (HIV incidence, quality of care, contraceptive method mix) will be explored., Discussion: We hypothesize pharmacy-delivered PrEP services supported with nurse navigator, versus delivered by pharmacy providers alone, will improve PrEP outcomes among AGYW seeking contraception. Our results will help policy makers better understand how to potentially implement this novel differentiated service model for PrEP and prime pharmacies for the delivery of new PrEP agents in the pipeline (e.g., long-acting injectables and multi-purpose technologies). The study was initiated on May 13, 2023, and is expected to be completed by February 2025., Trial Registration: ClinicalTrials.gov (NCT05467306), with registration on July 20, 2022., (© 2024. The Author(s).)

Background: Scalable PTSD screening strategies must be brief, accurate and capable of administration by a non-specialized workforce., Methods: We used PTSD as determined by the structured clinical interview as our gold standard and considered predictors sets of (a) Posttraumatic Stress Checklist-5 (PCL-5), (b) Primary Care PTSD Screen for the DSM-5 (PC-PTSD) and, (c) PCL-5 and PC-PTSD questions to identify the optimal items for PTSD screening for public sector settings in Kenya. A logistic regression model using LASSO was fit by minimizing the average squared error in the validation data. Area under the receiver operating characteristic curve (AUROC) measured discrimination performance., Results: Penalized regression analysis suggested a screening tool that sums the Likert scale values of two PCL-5 questions-intrusive thoughts of the stressful experience (#1) and insomnia (#21). This had an AUROC of 0.85 (using hold-out test data) for predicting PTSD as evaluated by the MINI, which outperformed the PC-PTSD. The AUROC was similar in subgroups defined by age, sex, and number of categories of trauma experienced (all AUROCs>0.83) except those with no trauma history- AUROC was 0.78., Conclusion: In some East African settings, a 2-item PTSD screening tool may outperform longer screeners and is easily scaled by a non-specialist workforce., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Meffert, Mathai, Ongeri, Neylan, Mwai, Onyango, Akena, Rota, Otieno, Obura, Wangia, Opiyo, Muchembre, Oluoch, Wambura, Mbwayo, Kahn, Cohen, Bukusi, Aarons, Burger, Jin, McCulloch and Njuguna Kahonge.)

Cells respond to lysosomal membrane permeabilization by membrane repair or selective macroautophagy of damaged lysosomes, termed lysophagy, but it is not fully understood how this decision is made. Here, we uncover a pathway in human cells that detects lipid bilayer perturbations in the limiting membrane of compromised lysosomes, which fail to be repaired, and then initiates ubiquitin-triggered lysophagy. We find that SPG20 binds the repair factor IST1 on damaged lysosomes and, importantly, integrates that with the detection of damage-associated lipid-packing defects of the lysosomal membrane. Detection occurs via sensory amphipathic helices in SPG20 before rupture of the membrane. If lipid-packing defects are extensive, such as during lipid peroxidation, SPG20 recruits and activates ITCH, which marks the damaged lysosome with lysine-63-linked ubiquitin chains to initiate lysophagy and thus triages the lysosome for destruction. With SPG20 being linked to neurodegeneration, these findings highlight the relevance of a coordinated lysosomal damage response for cellular homeostasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) make up a group of anthropogenic chemicals with a myriad of applications. However, some PFAS have been shown to negatively impact human health and the environment, leading to increased regulation, with some countries making efforts to phase out their use. PFAS fate in the environment is driven by physical, chemical, and biological processes, with microbial communities in matrices such as soil and sewage sludge being known to generate a range of low-molecular-weight PFAS metabolites. Proposed metabolic intermediates for both mixed and pure microbial cultures include fluorinated carboxylates that may be activated by CoA prior to β-oxidation and defluorination, although thus far, no PFAS-CoA adducts have been reported. Herein, we expressed and purified acyl-CoA synthetase (ACS) from the soil bacterium Gordonia sp. strain NB4-1Y and performed an analysis of substrate scope and enzyme kinetics using fluorinated and nonfluorinated carboxylates. We determined that ACS was able to catalyze the formation of CoA adducts of 3,3,3-trifluoropropionic acid, 5,5,5-trifluoropentanoic acid, 4,5,5-trifluoropent-4-enoic acid, and 4,4,5,5,5-pentafluoropentanoic acid. Kinetic analysis revealed a 90-98% decrease in k cat between nonfluorinated carboxylates and their fluorinated analogues. This provides evidence to validate proposed enzymatic pathways for microbial PFAS metabolism that proceed via an activation step involving the formation of CoA adducts., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Introduction: HIV self-testing (HIVST) has the potential to support daily oral pre-exposure prophylaxis (PrEP) delivery in private pharmacies, but many national guidelines have not approved HIVST for PrEP dispensing. In Kenya, pharmacy providers are permitted to deliver HIVST, but often do not have the required certification to deliver rapid diagnostic testing (RDT). We estimated the performance of provider-delivered HIVST compared to RDT, the standard of care for PrEP delivery, at private pharmacies in Kenya to inform decisions on the use of HIVST for PrEP scale-up., Methods: At 20 pharmacies in Kisumu County, we trained pharmacy providers (pharmacists and pharmaceutical technologists) on blood-based HIVST use and client assistance (if requested). We recruited pharmacy clients purchasing sexual and reproductive health-related products (e.g. condoms) and enrolled those ≥18 years with self-reported behaviours associated with HIV risk. Enrolled clients received HIVST with associated provider counselling, followed by RDT by a certified HIV testing services (HTS) counsellor. Pharmacy providers and clients independently interpreted HIVST results prior to RDT (results interpreted only by the HTS counsellor). We calculated the sensitivity and specificity of pharmacy provider-delivered HIVST compared to HTS counsellor-administered RDT., Results: Between March and June 2022, we screened 1691 clients and enrolled 1500; 64% (954/1500) were female and the median age was 26 years (IQR 22-31). We additionally enrolled 40 providers; 42% (17/40) were pharmacy owners and their median years of experience was 6 (IQR 4-10). The majority (79%, 1190/1500) of clients requested provider assistance with HIVST and providers spent a median of 20 minutes (IQR 15-43) with each HIVST client. The sensitivity of provider-delivered HIVST at the pharmacy was high when interpreted by providers (98.5%, 95% CI 97.8%, 99.1%) and clients (98.8%, 95% CI 98.0%, 99.3%), as was the specificity of HIVST in this setting (provider-interpretation: 96.9%, 95% CI 89.2%, 99.6%; client-interpretation: 93.8%, 95% CI 84.8%, 98.3%)., Conclusions: When compared to the national HIV testing algorithm, provider-delivered blood-based HIVST at private pharmacies in Kenya performed well. These findings suggest that blood-based HIVST may be a useful tool to support PrEP initiation and continuation at private pharmacies and potentially other community-based delivery settings., (© 2023 The Authors. Journal of the International AIDS Society published by John Wiley & Sons Ltd on behalf of International AIDS Society.)

Natural killer (NK) cells recognize virally infected cells and tumors. NK cell function depends on balanced signaling from activating receptors, recognizing products from tumors or viruses, and inhibitory receptors (such as KIR/Ly49), which recognize major histocompatibility complex class I (MHC-I) molecules. KIR/Ly49 signaling preserves tolerance to self but also conveys reactivity toward MHC-I-low target cells in a process known as NK cell education. Here, we found that NK cell tolerance and education were determined by the subcellular localization of the tyrosine phosphatase SHP-1. In mice lacking MHC-I molecules, uneducated, self-tolerant Ly49A + NK cells showed accumulation of SHP-1 in the activating immune synapse, where it colocalized with F-actin and the signaling adaptor protein SLP-76. Education of Ly49A + NK cells by the MHC-I molecule H2D d led to reduced synaptic accumulation of SHP-1, accompanied by augmented signaling from activating receptors. Education was also linked to reduced transcription of Ptpn6 , which encodes SHP-1. Moreover, synaptic SHP-1 accumulation was reduced in NK cells carrying the H2D d -educated receptor Ly49G2 but not in those carrying the noneducating receptor Ly49I. Colocalization of Ly49A and SHP-1 outside of the synapse was more frequent in educated compared with uneducated NK cells, suggesting a role for Ly49A in preventing synaptic SHP-1 accumulation in NK cell education. Thus, distinct patterning of SHP-1 in the activating NK cell synapse may determine NK cell tolerance.

Image-based personalized Finite Element Models (pFEM) could detect alterations in physiological deformation of human vertebral bodies, but their accuracy has been seldom reported. Meaningful validation experiments should allow vertebral endplate deformability and ensure well-controlled boundary conditions. This study aimed to (i) validate a new loading system to apply a homogeneous pressure on the vertebral endplate during vertebral body compression regardless of endplate deformation; (ii) perform a pilot study on human vertebral bodies measuring surface displacements and strains with Digital Image Correlation (DIC); (iii) determine the accuracy of pFEM of the vertebral bodies. Homogeneous pressure application was achieved by pressurizing a fluid silicone encased in a rubber silicone film acting on the cranial endplate. The loading system was validated by comparing DIC-measured longitudinal strains and lower-end contact pressures, measured on three homogeneous pseudovertebrae of constant transversal section at 2.0 kN, against theoretically calculated values. Longitudinal strains and contact pressures were rather homogeneous, and their mean values close to theoretical calculations (5% underestimation). DIC measurements of surface longitudinal and circumferential displacements and strains were obtained on three human vertebral bodies at 2.0 kN. Complete displacement and strain maps were achieved for anterolateral aspects with random errors ≤0.2 μm and ≤30 μstrain, respectively. Venous plexus and double curvatures limited the completeness and accuracy of DIC data in posterior aspects. pFEM of vertebral bodies, including cortical bone mapping, were built from computed tomography images. In anterolateral aspects, pFEM accuracy of the three vertebrae was: (i) comparable to literature in terms of longitudinal displacements (R2>0.8); (ii) extended to circumferential displacements (pooled data: R2>0.9) and longitudinal strains (zero median error, 95% error: <27%). Circumferential strains were overestimated (median error: 39%). The new methods presented may permit to study how physiological and pathologic conditions influence the ability of vertebral endplates/bodies to sustain loads., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Viscous dissipation causes significant energy losses in fluid flows; in ducts, laminar flows provide the minimum resistance to the motion, whereas turbulence substantially increases the friction at the wall and the consequent energy requirements for pumping. Great effort is currently being devoted to find new strategies to reduce the energy losses induced by turbulence. Here we propose a simple and novel drag-reduction technique which achieves substantial energy savings in internal flows. Our approach consists in driving the flow with a temporally intermittent pumping, unlike the common practice of a constant pumping. We alternate "pump on" phases where the flow accelerates, and "pump off" phases where the flow decays freely. The flow cyclically enters a quasi-laminar state during the acceleration, and transitions to a more classic turbulent state during the deceleration. Our numerical results demonstrate that important energy savings can be achieved by simply modulating the power injection into the system over time. The physical understanding of this process can help the industry in reducing the waste of energy, creating economical benefits and preserving the environment by reducing harmful emissions., (© 2023. The Author(s).)

Anticancer T cells acquire a dysfunctional state characterized by poor effector function and expression of inhibitory receptors, such as PD-1. Blockade of PD-1 leads to T cell reinvigoration and is increasingly applied as an effective anticancer treatment. Recent work challenged the commonly held view that the phosphatase PTPN11 (known as SHP-2) is essential for PD-1 signaling in T cells, suggesting functional redundancy with the homologous phosphatase PTPN6 (SHP-1). Therefore, we investigated the effect of concomitant Ptpn6 and Ptpn11 deletion in T cells on their ability to mount antitumour responses. In vivo data show that neither sustained nor acute Ptpn6/11 deletion improves T cell-mediated tumor control. Sustained loss of Ptpn6/11 also impairs the therapeutic effects of anti-PD1 treatment. In vitro results show that Ptpn6/11-deleted CD8 + T cells exhibit impaired expansion due to a survival defect and proteomics analyses reveal substantial alterations, including in apoptosis-related pathways. These data indicate that concomitant ablation of Ptpn6/11 in polyclonal T cells fails to improve their anticancer properties, implying that caution shall be taken when considering their inhibition for immunotherapeutic approaches., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: The adoptive transfer of chimeric antigen receptor (CAR)-T cells has emerged as a potent immunotherapy against some hematological malignancies but not yet for epithelial-derived solid tumors. One critical issue is the paucity of broadly expressed solid tumor antigens (TAs), and another is the presence of suppressive mechanisms in the tumor microenvironment (TME) that can impair CAR-T cell homing, extravasation and effector functions. TAs expressed by endothelial cells of the tumor vasculature are of clinical interest for CAR therapy because of their genomic stability and accessibility to circulating T cells, as well as their expression across multiple tumor types. In this study, we sought to explore limitations to the efficacy of second-generation (2G) murine CAR-T cells redirected against the vascular endothelial growth factor receptor-2 (VEGFR-2) with the well-characterized single-chain variable fragment DC101., Methods: Primary murine T cells were retrovirally transduced to express a 2G anti-VEGFR-2-CAR, and the in vitro binding to VEGFR-2, as well as reactivity against TA-expressing cells, was evaluated in the absence versus presence of exogenous VEGF-A. The CAR-T cells were further tested in vivo for tumor control alone and in combination with anti-VEGF-A antibody. Finally, we performed ex vivo phenotypic analyses of tumor-infiltrating CAR-T cells for the two treatment groups., Results: In line with previous reports, we observed poor control of B16 melanoma by the 2G anti-VEGFR-2 CAR-T cells as a monotherapy. We further showed that VEGFR-2 is not downregulated by B16 melanoma tumors post treatment, but that its soluble ligand VEGF-A is upregulated and furthermore competes in vitro with the CAR-T cells for binding to VEGFR-2. This competition resulted in impaired CAR-T cell adhesion and effector function in vitro that could be restored in the presence of anti-VEGF-A antibody. Finally, we demonstrated that coadministration of anti-VEGF-A antibody in vivo promoted CAR-T cell persistence and tumor control and was associated with reduced frequencies of PD-1 + Ki67 - and LAG-3 + Ki67 - CAR-T cells in the TME., Conclusions: This study represents the first example of impaired function of a vasculature-targeted CAR by an angiogenic ligand and rationalizes the use of combinatorial therapies that target the tumor vasculature and augment CAR-T cell effector function., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The mechanisms maintaining adult lymphatic vascular specialization throughout life and their role in coordinating inter-organ communication to sustain homeostasis remain elusive. We report that inactivation of the mechanosensitive transcription factor Foxc2 in adult lymphatic endothelium leads to a stepwise intestine-to-lung systemic failure. Foxc2 loss compromised the gut epithelial barrier, promoted dysbiosis and bacterial translocation to peripheral lymph nodes, and increased circulating levels of purine metabolites and angiopoietin-2. Commensal microbiota depletion dampened systemic pro-inflammatory cytokine levels, corrected intestinal lymphatic dysfunction, and improved survival. Foxc2 loss skewed the specialization of lymphatic endothelial subsets, leading to populations with mixed, pro-fibrotic identities and to emergence of lymph node-like endothelial cells. Our study uncovers a cross-talk between lymphatic vascular function and commensal microbiota, provides single-cell atlas of lymphatic endothelial subtypes, and reveals organ-specific and systemic effects of dysfunctional lymphatics. These effects potentially contribute to the pathogenesis of diseases, such as inflammatory bowel disease, cancer, or lymphedema., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Limited clinical benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, but coengineering strategies to generate so-called fourth-generation (4G) CAR-T cells are advancing toward overcoming barriers in the tumor microenvironment (TME) for improved responses. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we describe optimized methods for the efficient retroviral transduction and expansion of murine T lymphocytes of a predominantly central memory T cell (TCM cell) phenotype. We present a bicistronic retroviral vector encoding both a tumor vasculature-targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages. The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells in immunocompetent mice, an important step toward the acceleration of effective therapies reaching the clinic., Competing Interests: Disclosures: G. Coukos reported grants from Celgene, Boehringer-Ingelheim, Roche, BMS, Iovance Therapeutics, and Kite Pharma; personal fees from Genentech, Roche, BMS, AstraZeneca, NextCure, Geneos Tx, and Sanofi/Aventis outside the submitted work; and had patents in the domain of antibodies and vaccines targeting the tumor vasculature as well as technologies related to T cell expansion and engineering for T cell therapy. G. Coukos holds patents around TEM1 antibodies and receives royalties from the University of Pennsylvania. No other disclosures were reported., (© 2020 Lanitis et al.)