Lentiviral expression of wild-type LAMA3A restores cell adhesion in airway basal cells from children with epidermolysis bullosa.

The hallmark of epidermolysis bullosa (EB) is fragile attachment of epithelia due to genetic variants in cell adhesion genes. We describe 16 EB patients treated in the ear, nose, and throat department of a tertiary pediatric hospital linked to the United Kingdom's national EB unit between 1992 and 2023. Patients suffered a high degree of morbidity and mortality from laryngotracheal stenosis. Variants in laminin subunit alpha-3 (LAMA3) were found in 10/15 patients where genotype was available. LAMA3 encodes a subunit of the laminin-332 heterotrimeric extracellular matrix protein complex and is expressed by airway epithelial basal stem cells. We investigated the benefit of restoring wild-type LAMA3 expression in primary EB patient-derived basal cell cultures. EB basal cells demonstrated weak adhesion to cell culture substrates, but could otherwise be expanded similarly to non-EB basal cells. In vitro lentiviral overexpression of LAMA3A in EB basal cells enabled them to differentiate in air-liquid interface cultures, producing cilia with normal ciliary beat frequency. Moreover, transduction restored cell adhesion to levels comparable to a non-EB donor culture. These data provide proof of concept for a combined cell and gene therapy approach to treat airway disease in LAMA3-affected EB.

Stem Cell-derived Respiratory Epithelial Cell Cultures as Human Disease Models.

Advances in stem cell biology and the understanding of factors that determine lung stem cell self-renewal have enabled long-term in vitro culture of human lung cells derived from airway basal and alveolar type II cells. Improved capability to expand and study primary cells long term, including in clonal cultures that are recently derived from a single cell, will allow experiments that address fundamental questions about lung homeostasis and repair, as well as translational questions in asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, and lung cancer research. Here, we provide a brief history of postnatal lung epithelial cell culture and describe recent methodological advances. We further discuss the applications of primary cultures in defining "normal" epithelium, in modeling lung disease, and in future cell therapies.

Sociodemographic and clinical factors associated with transdermal alcohol concentration from the SCRAM biosensor among persons living with and without HIV.

BACKGROUND: Transdermal alcohol biosensors can objectively monitor alcohol use by measuring transdermal alcohol concentration (TAC). However, it is unclear how sociodemographic and clinical factors that influence alcohol metabolism are associated with TAC. The main aim of this study was to examine how sociodemographic factors (sex, age, race/ethnicity) and clinical factors (body mass index, liver enzymes: alanine aminotransferase [ALT] and aspartate transaminase [AST]), alcohol use disorder, and HIV status were associated with TAC while controlling for level of alcohol use. METHODS: We analyzed data from a prospective study involving contingency management for alcohol cessation among persons living with and without human immunodeficiency virus (HIV) that used the Secure Continuous Remote Alcohol Monitoring (SCRAM) biosensor. Forty-three participants (Mage  = 56.6 years; 63% male; 58% people living with HIV) yielded 183 SCRAM-detected drinking days. Two indices derived from SCRAM: peak TAC (reflecting level of intoxication) and TAC area under the curve (TAC-AUC; reflecting alcohol volume)-were the main outcomes. Self-reported alcohol use (drinks/drinking day) measured by Timeline Followback was the main predictor. To examine whether factors of interest were associated with TAC, we used individual generalized estimating equations (GEE), followed by a multivariate GEE model to include all significant predictors to examine their associations with TAC beyond the effect of self-reported alcohol use. RESULTS: Number of drinks per drinking day (B = 0.29, p < 0.01) and elevated AST (B = 0.50, p = 0.01) were significant predictors of peak TAC. Positive HIV status, female sex, elevated AST, and number of drinks per drinking day were positively associated with TAC-AUC at the bivariate level, whereas only self-reported alcohol use (B = 0.85, p < 0.0001) and female sex (B = 0.67, p < 0.05) were significant predictors of TAC-AUC at the multivariate level. CONCLUSIONS: HIV status was not independently associated with TAC. Future studies should consider the sex and liver function of the participant when using alcohol biosensors to measure alcohol use.

Bioengineered airway epithelial grafts with mucociliary function based on collagen IV- and laminin-containing extracellular matrix scaffolds.

Current methods to replace damaged upper airway epithelium with exogenous cells are limited. Existing strategies use grafts that lack mucociliary function, leading to infection and the retention of secretions and keratin debris. Strategies that regenerate airway epithelium with mucociliary function are clearly desirable and would enable new treatments for complex airway disease.Here, we investigated the influence of the extracellular matrix (ECM) on airway epithelial cell adherence, proliferation and mucociliary function in the context of bioengineered mucosal grafts. In vitro, primary human bronchial epithelial cells (HBECs) adhered most readily to collagen IV. Biological, biomimetic and synthetic scaffolds were compared in terms of their ECM protein content and airway epithelial cell adherence.Collagen IV and laminin were preserved on the surface of decellularised dermis and epithelial cell attachment to decellularised dermis was greater than to the biomimetic or synthetic alternatives tested. Blocking epithelial integrin α2 led to decreased adherence to collagen IV and to decellularised dermis scaffolds. At air-liquid interface (ALI), bronchial epithelial cells cultured on decellularised dermis scaffolds formed a differentiated respiratory epithelium with mucociliary function. Using in vivo chick chorioallantoic membrane (CAM), rabbit airway and immunocompromised mouse models, we showed short-term preservation of the cell layer following transplantation.Our results demonstrate the feasibility of generating HBEC grafts on clinically applicable decellularised dermis scaffolds and identify matrix proteins and integrins important for this process. The long-term survivability of pre-differentiated epithelia and the relative merits of this approach against transplanting basal cells should be assessed further in pre-clinical airway transplantation models.

Insights into the prey of Vespa mandarinia (Hymenoptera: Vespidae) in Washington state, obtained from metabarcoding of larval feces.

The northern giant hornet, Vespa mandarinia (Hymenoptera: Vespidae), was detected for the first time in North America in 2019. Four nests have since been located and removed in northwestern Washington State as part of an extensive survey and eradication program. This recent introduction into North America has prompted new research on the biology and ecology of V. mandarinia to help inform management strategies. In its native range, V. mandarinia is known to prey on a variety of insects including the economically important honey bee species Apis cerana and Apis mellifera. Although A. cerana has developed defense mechanisms against attack by V. mandarinia, A. mellifera have no such defenses and an entire hive can be quickly destroyed by only a few hornets. In North America the hornet has been observed foraging on paper wasps (Polistes dominula) and honey bees, but little else is known about prey use in its novel range. To address this knowledge gap, we employed a DNA metabarcoding approach to characterize species detected in larval feces collected from 3 of the 4 Washington V. mandarinia nests found to date. Sequences were recovered for 56 species across fourteen orders, of which 36 species were likely prey items and 20 were suspected inquilines. The most frequently detected species were other social Hymenoptera, with Dolichovespula maculata, P. dominula, and A. mellifera present in most samples. All of the species detected, except for A. mellifera, represent new prey records for V. mandarinia, with eight families of insects newly associated with giant hornets. These results suggest that V. mandarinia in Washington preys on an assortment of insects similar to those documented in its native range, and that this new invader has readily incorporated novel species into its foraging and diet.

Acceptability, Suitability, and Feasibility of an Evidence-Based Intervention to Reduce HIV Risk Behaviors: Engaging Comadronas in HIV Prevention in Rural Guatemala.

This study addresses rural Guatemala's poor maternal health and HIV status by culturally adapting an evidence-based HIV intervention, SEPA (Self-Care, Education, Prevention, Self-Care), to extend the capacity of comadronas (Mayan birth attendants) as HIV prevention providers. This mixed-method study examined the acceptability, suitability, and feasibility of SEPA presented to traditional elder and a younger cohort of comadronas over three sessions. Outcome variables were reported as mean scores. Open-ended qualitative responses were categorized under central themes. Session 1, 2, and 3 acceptability (4.6/5, 4.6/5, 4.8/5), suitability (4.7/5, 4.6/5, 4.9/5), and feasibility (4.4/5, 4.7/5, 4.8/5) remained high across sessions. While comadronas reported that information was difficult, they reported high levels of understanding and comfort with SEPA content and they also found it to be culturally appropriate, increasing their confidence to discuss HIV with their community. The broader utilization of comadronas could create a pathway to enhance reproductive health among indigenous women.

Lung viral infection modelling in a bioengineered whole-organ.

Lung infections are one of the leading causes of death worldwide, and this situation has been exacerbated by the emergence of COVID-19. Pre-clinical modelling of viral infections has relied on cell cultures that lack 3D structure and the context of lung extracellular matrices. Here, we propose a bioreactor-based, whole-organ lung model of viral infection. The bioreactor takes advantage of an automated system to achieve efficient decellularization of a whole rat lung, and recellularization of the scaffold using primary human bronchial cells. Automatization allowed for the dynamic culture of airway epithelial cells in a breathing-mimicking setup that led to an even distribution of lung epithelial cells throughout the distal regions. In the sealed bioreactor system, we demonstrate proof-of-concept for viral infection within the epithelialized lung by infecting primary human airway epithelial cells and subsequently injecting neutrophils. Moreover, to assess the possibility of drug screening in this model, we demonstrate the efficacy of the broad-spectrum antiviral remdesivir. This whole-organ scale lung infection model represents a step towards modelling viral infection of human cells in a 3D context, providing a powerful tool to investigate the mechanisms of the early stages of pathogenic infections and the development of effective treatment strategies for respiratory diseases.

Multilevel Determinants of Rapid Antiretroviral Treatment Implementation and Demand in Miami-Dade County.

BACKGROUND: Rapidly linking newly diagnosed HIV patients to antiretroviral treatment (ART) is the best practice for achieving optimal treatment outcomes, including viral suppression. However, rapid ART implementation varies throughout the United States, highlighting the importance of identifying rapid ART implementation determinants in US HIV epicenters, such as Miami-Dade County (MDC). METHODS: Clinic focus groups (N = 4 clinics) and patient interviews (N = 31 recently diagnosed patients) systematically and qualitatively assessed rapid ART implementation determinants in MDC. Independent coders analyzed focus groups and interviews using a directed content analysis approach guided by the Consolidated Framework for Implementation Research. RESULTS: For clinic stakeholders, key rapid ART implementation determinants included the following: complexity and adaptability (innovation characteristics); networks between clinics and patient needs rooted in structural inequities (outer setting); leadership and available resources (inner setting); staff/provider flexibility (characteristics of individuals); and appointing patient navigators and champions (process). For patients, key determinants included complexity and relative advantage of rapid treatment (innovation characteristics); patient needs and clinic networks (outer setting); provider knowledge and skills (inner setting); provider warmth and affirmation (characteristics of individuals); and need for improved outreach (process). CONCLUSIONS: Multilevel factors impact clinic implementation and patient demand for rapid ART in MDC. Informed by these factors, we identified potential implementation strategies to enhance rapid ART implementation throughout MDC. These implementation strategies can be tested in an implementation trial, enhancing the toolkit of strategies to ensure that evidence-based tools, particularly rapid ART, are readily available to the most impacted communities.

Cell-intrinsic differences between human airway epithelial cells from children and adults.

The airway epithelium is a protective barrier that is maintained by the self-renewal and differentiation of basal stem cells. Increasing age is a principle risk factor for chronic lung diseases, but few studies have explored age-related molecular or functional changes in the airway epithelium. We retrieved epithelial biopsies from histologically normal tracheobronchial sites from pediatric and adult donors and compared their cellular composition and gene expression profile (in laser capture-microdissected whole epithelium, fluorescence-activated cell-sorted basal cells, and basal cells in cell culture). Histologically, pediatric and adult tracheobronchial epithelium was similar in composition. We observed age-associated changes in RNA sequencing studies, including higher interferon-associated gene expression in pediatric epithelium. In cell culture, pediatric cells had higher colony formation ability, sustained in vitro growth, and outcompeted adult cells in a direct competitive proliferation assay. Our results demonstrate cell-intrinsic differences between airway epithelial cells from children and adults in both homeostatic and proliferative states.

The tissue diagnostic instrument.

Tissue mechanical properties reflect extracellular matrix composition and organization, and as such, their changes can be a signature of disease. Examples of such diseases include intervertebral disk degeneration, cancer, atherosclerosis, osteoarthritis, osteoporosis, and tooth decay. Here we introduce the tissue diagnostic instrument (TDI), a device designed to probe the mechanical properties of normal and diseased soft and hard tissues not only in the laboratory but also in patients. The TDI can distinguish between the nucleus and the annulus of spinal disks, between young and degenerated cartilage, and between normal and cancerous mammary glands. It can quantify the elastic modulus and hardness of the wet dentin left in a cavity after excavation. It can perform an indentation test of bone tissue, quantifying the indentation depth increase and other mechanical parameters. With local anesthesia and disposable, sterile, probe assemblies, there has been neither pain nor complications in tests on patients. We anticipate that this unique device will facilitate research on many tissue systems in living organisms, including plants, leading to new insights into disease mechanisms and methods for their early detection.