ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

Simultaneous Targeting of IL-1-Signaling and IL-6-Trans-Signaling Preserves Human Pulmonary Endothelial Barrier Function During a Cytokine Storm-Brief Report.

BACKGROUND: Systemic inflammatory diseases, such as sepsis and severe COVID-19, provoke acute respiratory distress syndrome in which the pathological hyperpermeability of the microvasculature, induced by uncontrolled inflammatory stimulation, causes pulmonary edema. Identifying the inflammatory mediators that induce human lung microvascular endothelial cell barrier dysfunction is essential to find the best anti-inflammatory treatments for critically ill acute respiratory distress syndrome patients. METHODS: We have compared the responses of primary human lung microvascular endothelial cells to the main inflammatory mediators involved in cytokine storms induced by sepsis and SARS-CoV2 pulmonary infection and to sera from healthy donors and severely ill patients with sepsis. Endothelial barrier function was measured by electric cell-substrate impedance sensing, quantitative confocal microscopy, and Western blot. RESULTS: The human lung microvascular endothelial cell barrier was completely disrupted by IL (interleukin)-6 conjugated with soluble IL-6R (IL-6 receptor) and by IL-1ß (interleukin-1beta), moderately affected by TNF (tumor necrosis factor)-α and IFN (interferon)-γ and unaffected by other cytokines and chemokines, such as IL-6, IL-8, MCP (monocyte chemoattractant protein)-1 and MCP-3. The inhibition of IL-1 and IL-6R simultaneously, but not separately, significantly reduced endothelial hyperpermeability on exposing human lung microvascular endothelial cells to a cytokine storm consisting of 8 inflammatory mediators or to sera from patients with sepsis. Simultaneous inhibition of IL-1 and JAK (Janus kinase)-STAT (signal transducer and activator of transcription protein), a signaling node downstream IL-6 and IFN-γ, also prevented septic serum-induced endothelial barrier disruption. CONCLUSIONS: These findings strongly suggest a major role for both IL-6 trans-signaling and IL-1ß signaling in the pathological increase in permeability of the human lung microvasculature and reveal combinatorial strategies that enable the gradual control of pulmonary endothelial barrier function in response to a cytokine storm.

Family-based psychosocial interventions for adult Latino patients with cancer and their caregivers: A systematic review.

Objective: This review aimed to systematically examine the characteristics and outcomes of family-based psychosocial interventions offered to adult Latino patients with cancer and their caregivers. Methods: We searched six databases from their inception dates through June 2022. Studies were eligible for inclusion if they (1) targeted both adult Latino patients diagnosed with cancer and their adult caregivers or reported subgroup analyses of Latino patients and caregivers; (2) included family-based psychosocial interventions; (3) used randomized controlled trial (RCT) or quasi-experimental designs; and (4) were published in English, Spanish or Portuguese. Members of our multidisciplinary team assessed the risk of bias in the reviewed studies using the Cochrane Collaboration's Risk of Bias Tool. Results: Our database searches yielded five studies. The studies were conducted in the U.S. and Brazil. Three studies were RCTs, and two used quasi-experimental designs. The sample sizes ranged from 18 to 230 patient-caregiver dyads. These studies culturally adapted the intervention contents and implementation methods and involved bilingual interventionists. The interventions had beneficial effects on multiple aspects of psychosocial outcomes for both patients and caregivers. We also identified methodological limitations in the reviewed studies. Conclusions: Findings from this systematic review help deepen our understanding of family-based psychosocial interventions for Latinos affected by cancer. The small number of psychosocial interventions focused on adult Latino cancer patients and their caregivers is concerning, considering that Latino populations are disproportionally burdened by cancer. Future research needs to design and evaluate culturally-appropriate interventions to support Latino patients and families who cope with cancer. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=274993, identifier CRD42021274993.

Robust, maintainable, emergency invasive mechanical ventilator. / Ventilador mecânico invasivo de emergência resistente e de fácil manutenção.

OBJECTIVE: To develop a simple, robust, safe and efficient invasive mechanical ventilator that can be used in remote areas of the world or war zones where the practical utility of more sophisticated equipment is limited by considerations of maintainability, availability of parts, transportation and/or cost. METHODS: The device implements the pressure-controlled continuous mandatory ventilation mode, complemented by a simple assist-control mode. Continuous positive airway pressure is also possible. The consumption of compressed gases is minimized by avoiding a continuous flow of oxygen or air. Respiratory rates and inspiration/expiration time ratios are electronically determined, and an apnea/power loss alarm is provided. RESULTS: The pressure profiles were measured for a range of conditions and found to be adjustable within a ± 2.5cmH2O error margin and stable well within this range over a 41-hour period. Respiratory cycle timing parameters were precise within a few percentage points over the same period. The device was tested for durability for an equivalent period of four months. Chemical and biological tests failed to identify any contamination of the gas by volatile organic compounds or microorganisms. A ventilation test on a large animal, in comparison with a well established ventilator, showed that the animal could be adequately ventilated over a period of 60 minutes, without any noticeable negative aftereffects during the subsequent 24-hour period. CONCLUSION: This ventilator design may be viable, after further animal tests and formal approval by the competent authorities, for clinical application in the abovementioned atypical circumstances.

OBJETIVO: Desenvolver um ventilador mecânico invasivo simples, resistente, seguro e eficiente que possa ser utilizado em áreas remotas do mundo ou zonas de guerra, em que a utilidade prática de equipamentos mais sofisticados é limitada por questões de manutenção, disponibilidade de peças, transporte e/ou custo. MÉTODOS: O dispositivo implementa o modo de ventilação mandatória contínua com pressão controlada, complementado por um simples modo assisto-controlado. Pode-se também utilizar a pressão positiva contínua nas vias aéreas. Ao se evitar o fluxo contínuo de oxigênio ou ar, minimiza-se o consumo de gases comprimidos. As taxas respiratórias e as relações de tempo de inspiração e expiração são determinadas eletronicamente. Além disso, conta com um alarme de apneia/falta de energia. RESULTADOS: Os perfis de pressão foram medidos para uma série de condições, sendo considerados ajustáveis dentro de uma margem de erro de ± 2,5cmH2O, e foram considerados bem estáveis dentro dessa variação durante um período de 41 horas. Os parâmetros de tempo do ciclo respiratório foram precisos dentro de alguns pontos percentuais durante o mesmo período. O dispositivo foi testado quanto à durabilidade por um período equivalente a 4 meses. Os testes químicos e biológicos não conseguiram identificar qualquer contaminação do gás por compostos orgânicos voláteis ou micro-organismos. Em comparação com um ventilador bem estabelecido, o teste de ventilação em um animal de grande porte mostrou que este poderia ser ventilado adequadamente durante um período de 60 minutos, sem quaisquer efeitos negativos perceptíveis durante o período subsequente de 24 horas. CONCLUSÃO: Este projeto de ventilador pode ser viável após novos testes em animais e aprovação formal pelas autoridades competentes, para aplicação clínica nas circunstâncias atípicas anteriormente mencionadas.

Ventilador mecânico invasivo de emergência resistente e de fácil manutenção / Robust, maintainable, emergency invasive mechanical ventilator

RESUMO Objetivo: Desenvolver um ventilador mecânico invasivo simples, resistente, seguro e eficiente que possa ser utilizado em áreas remotas do mundo ou zonas de guerra, em que a utilidade prática de equipamentos mais sofisticados é limitada por questões de manutenção, disponibilidade de peças, transporte e/ou custo. Métodos: O dispositivo implementa o modo de ventilação mandatória contínua com pressão controlada, complementado por um simples modo assisto-controlado. Pode-se também utilizar a pressão positiva contínua nas vias aéreas. Ao se evitar o fluxo contínuo de oxigênio ou ar, minimiza-se o consumo de gases comprimidos. As taxas respiratórias e as relações de tempo de inspiração e expiração são determinadas eletronicamente. Além disso, conta com um alarme de apneia/falta de energia. Resultados: Os perfis de pressão foram medidos para uma série de condições, sendo considerados ajustáveis dentro de uma margem de erro de ± 2,5cmH2O, e foram considerados bem estáveis dentro dessa variação durante um período de 41 horas. Os parâmetros de tempo do ciclo respiratório foram precisos dentro de alguns pontos percentuais durante o mesmo período. O dispositivo foi testado quanto à durabilidade por um período equivalente a 4 meses. Os testes químicos e biológicos não conseguiram identificar qualquer contaminação do gás por compostos orgânicos voláteis ou micro-organismos. Em comparação com um ventilador bem estabelecido, o teste de ventilação em um animal de grande porte mostrou que este poderia ser ventilado adequadamente durante um período de 60 minutos, sem quaisquer efeitos negativos perceptíveis durante o período subsequente de 24 horas. Conclusão: Este projeto de ventilador pode ser viável após novos testes em animais e aprovação formal pelas autoridades competentes, para aplicação clínica nas circunstâncias atípicas anteriormente mencionadas.

ABSTRACT Objective: To develop a simple, robust, safe and efficient invasive mechanical ventilator that can be used in remote areas of the world or war zones where the practical utility of more sophisticated equipment is limited by considerations of maintainability, availability of parts, transportation and/or cost. Methods: The device implements the pressure-controlled continuous mandatory ventilation mode, complemented by a simple assist-control mode. Continuous positive airway pressure is also possible. The consumption of compressed gases is minimized by avoiding a continuous flow of oxygen or air. Respiratory rates and inspiration/expiration time ratios are electronically determined, and an apnea/power loss alarm is provided. Results: The pressure profiles were measured for a range of conditions and found to be adjustable within a ± 2.5cmH2O error margin and stable well within this range over a 41-hour period. Respiratory cycle timing parameters were precise within a few percentage points over the same period. The device was tested for durability for an equivalent period of four months. Chemical and biological tests failed to identify any contamination of the gas by volatile organic compounds or microorganisms. A ventilation test on a large animal, in comparison with a well established ventilator, showed that the animal could be adequately ventilated over a period of 60 minutes, without any noticeable negative aftereffects during the subsequent 24-hour period. Conclusion: This ventilator design may be viable, after further animal tests and formal approval by the competent authorities, for clinical application in the abovementioned atypical circumstances.

Plasmolipin regulates basolateral-to-apical transcytosis of ICAM-1 and leukocyte adhesion in polarized hepatic epithelial cells.

Apical localization of Intercellular Adhesion Receptor (ICAM)-1 regulates the adhesion and guidance of leukocytes across polarized epithelial barriers. Here, we investigate the molecular mechanisms that determine ICAM-1 localization into apical membrane domains of polarized hepatic epithelial cells, and their effect on lymphocyte-hepatic epithelial cell interaction. We had previously shown that segregation of ICAM-1 into apical membrane domains, which form bile canaliculi and bile ducts in hepatic epithelial cells, requires basolateral-to-apical transcytosis. Searching for protein machinery potentially involved in ICAM-1 polarization we found that the SNARE-associated protein plasmolipin (PLLP) is expressed in the subapical compartment of hepatic epithelial cells in vitro and in vivo. BioID analysis of ICAM-1 revealed proximal interaction between this adhesion receptor and PLLP. ICAM-1 colocalized and interacted with PLLP during the transcytosis of the receptor. PLLP gene editing and silencing increased the basolateral localization and reduced the apical confinement of ICAM-1 without affecting apicobasal polarity of hepatic epithelial cells, indicating that ICAM-1 transcytosis is specifically impaired in the absence of PLLP. Importantly, PLLP depletion was sufficient to increase T-cell adhesion to hepatic epithelial cells. Such an increase depended on the epithelial cell polarity and ICAM-1 expression, showing that the epithelial transcytotic machinery regulates the adhesion of lymphocytes to polarized epithelial cells. Our findings strongly suggest that the polarized intracellular transport of adhesion receptors constitutes a new regulatory layer of the epithelial inflammatory response.

Compensatory increase of VE-cadherin expression through ETS1 regulates endothelial barrier function in response to TNFα.

VE-cadherin plays a central role in controlling endothelial barrier function, which is transiently disrupted by proinflammatory cytokines such as tumor necrosis factor (TNFα). Here we show that human endothelial cells compensate VE-cadherin degradation in response to TNFα by inducing VE-cadherin de novo synthesis. This compensation increases adherens junction turnover but maintains surface VE-cadherin levels constant. NF-κB inhibition strongly reduced VE-cadherin expression and provoked endothelial barrier collapse. Bacterial lipopolysaccharide and TNFα upregulated the transcription factor ETS1, in vivo and in vitro, in an NF-κB dependent manner. ETS1 gene silencing specifically reduced VE-cadherin protein expression in response to TNFα and exacerbated TNFα-induced barrier disruption. We propose that TNFα induces not only the expression of genes involved in increasing permeability to small molecules and immune cells, but also a homeostatic transcriptional program in which NF-κB- and ETS1-regulated VE-cadherin expression prevents the irreversible damage of endothelial barriers.

Activation of Rac1 and RhoA Preserve Corneal Endothelial Barrier Function.

PURPOSE: The corneal endothelium is responsible for the correct hydration of the corneal stroma. Corneal endothelial cells have a low proliferative capacity, so preserving their barrier function under suboptimal conditions that cause osmotic imbalance, such as those arising from corneal pathologies, age, cryopreservation, and transplantation, is essential for maintaining corneal transparency. We have investigated the signaling induced by hyperosmotic shock that reversibly disrupts corneal endothelial barriers in human endothelial cells and in murine corneas. METHODS: Endothelial barrier properties were analyzed in vitro by electric cell substrate impedance sensing (ECIS) and confocal microscopy of the human endothelial cell line B4G12-HCEC, and, ex vivo, by confocal microscopy and stimulated emission-depletion (STED) super-resolution microscopy of murine corneas. Cell signaling in response to hyperosmotic stress, induced with an excess of sodium chloride, was investigated in B4G12-HCECs. Rho GTPase activity was detected by pulldown assays with recombinant GST proteins fused to the Rho binding domains of Rho effectors. RESULTS: Hyperosmotic stress increased actin polymerization and activated the Rho GTPases Rac1 and RhoA, but not Cdc42. Rac1- and RhoA-mediated pathway inhibition had a minor effect on barrier disruption but partially delayed barrier reformation after stress withdrawal. In contrast, Rac1 and RhoA activation enhanced constitutive endothelial barrier function and accelerated barrier repair. CONCLUSIONS: Our results indicate that Rac1 and RhoA activation do not mediate stress-induced cell contraction but are endothelial responses that act to restore and maintain barrier homeostasis. Therefore, pharmacological activation of these two GTPases could be a therapeutic strategy for preserving corneal endothelial barrier function.

A human cellular system for analyzing signaling during corneal endothelial barrier dysfunction.

Correct corneal endothelial barrier function is essential for maintaining corneal transparency. However, research on cell signaling pathways mediating corneal endothelial barrier dysfunction has progressed more slowly than that involving other cellular barriers because of the lack of human corneal endothelial cell models. Here we have optimized the culture of the human corneal endothelial cell (HCEC) line B4G12 as a model for studying paracellular permeability. We show that B4G12-HCECs form confluent monolayers with stable cell-cell junctions when cultured on plastic, but not glass, surfaces precoated with various extracellular matrix components. Cell morphometry and measuring intercellular spaces and transendothelial electric resistance indicate that B4G12-HCECs form optimal monolayers on collagen and fibronectin. Based on the use of specific inhibitors, it has been proposed that the Rho-regulated kinases, ROCK-I and ROCK-II, mediate actomyosin-induced contraction in corneal endothelial cell barriers. ROCKs are effectors of RhoA, RhoB and RhoC. We show that the GTPase RhoA and its effector ROCK-II are predominantly expressed in B4G12-HCECs and primary human corneal endothelial cells. The activation of Rho GTPases during acute barrier disruption has not been investigated in corneal endothelial cells. RhoA, but not other related GTPases that are highly expressed in B4G12-HCECs, such as Rac1 and Cdc42, is transiently activated during barrier disruption in response to the inflammatory mediator thrombin. Pharmacological inhibition of RhoA and ROCK reduces B4G12-HCEC acute contraction. We propose that exploiting B4G12-HCECs is a useful experimental strategy for gaining further insight into the signaling pathways involved in human corneal endothelial barrier function.

RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border.

Endothelial barrier dysfunction underlies chronic inflammatory diseases. In searching for new proteins essential to the human endothelial inflammatory response, we have found that the endosomal GTPase RhoB is up-regulated in response to inflammatory cytokines and expressed in the endothelium of some chronically inflamed tissues. We show that although RhoB and the related RhoA and RhoC play additive and redundant roles in various aspects of endothelial barrier function, RhoB specifically inhibits barrier restoration after acute cell contraction by preventing plasma membrane extension. During barrier restoration, RhoB trafficking is induced between vesicles containing RhoB nanoclusters and plasma membrane protrusions. The Rho GTPase Rac1 controls membrane spreading and stabilizes endothelial barriers. We show that RhoB colocalizes with Rac1 in endosomes and inhibits Rac1 activity and trafficking to the cell border during barrier recovery. Inhibition of endosomal trafficking impairs barrier reformation, whereas induction of Rac1 translocation to the plasma membrane accelerates it. Therefore, RhoB-specific regulation of Rac1 trafficking controls endothelial barrier integrity during inflammation.

CD2v Interacts with Adaptor Protein AP-1 during African Swine Fever Infection.

African swine fever virus (ASFV) CD2v protein is believed to be involved in virulence enhancement, viral hemadsorption, and pathogenesis, although the molecular mechanisms of the function of this viral protein are still not fully understood. Here we describe that CD2v localized around viral factories during ASFV infection, suggesting a role in the generation and/or dynamics of these viral structures and hence in disturbing cellular traffic. We show that CD2v targeted the regulatory trans-Golgi network (TGN) protein complex AP-1, a key element in cellular traffic. This interaction was disrupted by brefeldin A even though the location of CD2v around the viral factory remained unchanged. CD2v-AP-1 binding was independent of CD2v glycosylation and occurred on the carboxy-terminal part of CD2v, where a canonical di-Leu motif previously reported to mediate AP-1 binding in eukaryotic cells, was identified. This motif was shown to be functionally interchangeable with the di-Leu motif present in HIV-Nef protein in an AP-1 binding assay. However, we demonstrated that it was not involved either in CD2v cellular distribution or in CD2v-AP-1 binding. Taken together, these findings shed light on CD2v function during ASFV infection by identifying AP-1 as a cellular factor targeted by CD2v and hence elucidate the cellular pathways used by the virus to enhance infectivity.

Motivation, management, and mastery: a theory of resilience in the context of HIV infection.

BACKGROUND: Clients with HIV infection have been conceptualized as a resilient population. Although a few researchers have documented resilience among clients with HIV infection, a theory of resilience in the context of HIV infection has not been developed. The purpose of this study was to describe the process by which resilience occurs for clients in the context of HIV infection. METHOD: Grounded theory methodology was used to sample and analyze data from 15 qualitative interviews with adults with HIV infection. Data were collected until saturation was reached. RESULTS: A theory, motivation, management, and mastery, a description of the process by which resilience occurs in the context of HIV infection, emerged from the data. CONCLUSION: Many clients living with HIV infection are resilient, despite the physical, psychological, and social challenges of this chronic illness. Nursing interventions to promote resilience among clients with HIV infection should be directed toward identification of client motivation factors and disease management strategies that may influence health outcomes of people living with HIV infection.

African swine fever virus uses macropinocytosis to enter host cells.

African swine fever (ASF) is caused by a large and highly pathogenic DNA virus, African swine fever virus (ASFV), which provokes severe economic losses and expansion threats. Presently, no specific protection or vaccine against ASF is available, despite the high hazard that the continued occurrence of the disease in sub-Saharan Africa, the recent outbreak in the Caucasus in 2007, and the potential dissemination to neighboring countries, represents. Although virus entry is a remarkable target for the development of protection tools, knowledge of the ASFV entry mechanism is still very limited. Whereas early studies have proposed that the virus enters cells through receptor-mediated endocytosis, the specific mechanism used by ASFV remains uncertain. Here we used the ASFV virulent isolate Ba71, adapted to grow in Vero cells (Ba71V), and the virulent strain E70 to demonstrate that entry and internalization of ASFV includes most of the features of macropinocytosis. By a combination of optical and electron microscopy, we show that the virus causes cytoplasm membrane perturbation, blebbing and ruffles. We have also found that internalization of the virions depends on actin reorganization, activity of Na(+)/H(+) exchangers, and signaling events typical of the macropinocytic mechanism of endocytosis. The entry of virus into cells appears to directly stimulate dextran uptake, actin polarization and EGFR, PI3K-Akt, Pak1 and Rac1 activation. Inhibition of these key regulators of macropinocytosis, as well as treatment with the drug EIPA, results in a considerable decrease in ASFV entry and infection. In conclusion, this study identifies for the first time the whole pathway for ASFV entry, including the key cellular factors required for the uptake of the virus and the cell signaling involved.

Predictors of eating attitudes and behaviors among gay Hispanic men.

Gay men are a vulnerable population at risk for a number of health disparities, but little is known about eating disorders among gay Hispanic men. The purpose of this study was to examine the relationship of eating attitudes and behaviors with alcohol abuse, body image, depression, self-esteem, and sexual behaviors to determine predictors of eating attitudes and behaviors in a community sample of gay Hispanic men. Significant numbers of the participants were at risk for eating disorders (13%), alcohol abuse (18%), body image disturbance (29%), depression (25%), low self-esteem (12%), and high-risk sexual behaviors (34%). Alcohol abuse, body image, depression, self-esteem, and sexual behaviors were significant predictors of eating attitudes and behaviors and accounted for 38% of the variance in eating attitudes and behaviors. Nurses providing care to this population of gay men must be aware of this cluster of related mental health conditions that are experienced by these men. Addressing and treating these health conditions as a group of related mental health conditions are necessary. More research is needed to further explore this cluster of health issues among gay Hispanic men.

Living in silence: a grounded theory study of vulnerability in the context of HIV infection.

Clients with HIV infection have been conceptualized as a vulnerable population. Although some researchers have examined vulnerability with clients with HIV infection, a theory of vulnerability in the context of HIV infection is non-existent. The purpose of this study was to describe, using qualitative methodology, the process by which vulnerability occurs in the context of HIV infection. Grounded theory methodology was used to sample and analyze data from 15 qualitative interviews of adults with HIV infection. Data were collected until data saturation was reached. A theory that describes the process by which vulnerability occurs in the context of HIV infection, Living in Silence, emerged from the data. Living in Silence consists of four categories: Confronting Mortality and Illness, Struggling with Change, Encountering a Lack of Psychosocial Support, and Experiencing Vulnerability. Clients living with HIV experience vulnerability. Nursing interventions to decrease the risks of vulnerability should be directed toward the holistic needs of clients and toward increasing psychosocial support.