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Desmoplasia in breast cancer leads to heterogeneity in physical properties of the tissue, resulting in disparities in drug delivery and treatment efficacy among patients, thus contributing to high disease mortality. Personalized in vitro breast cancer models hold great promise for high-throughput testing of therapeutic strategies to normalize the aberrant microenvironment in a patient-specific manner. Here, tumoroids assembled from breast cancer cell lines (MCF7, SKBR3, and MDA-MB-468) and patient-derived breast tumor cells (TCs) cultured in microphysiological systems including perfusable microvasculature reproduce key aspects of stromal and vascular dysfunction causing impaired drug delivery. Models containing SKBR3 and MDA-MB-468 tumoroids show higher stromal hyaluronic acid (HA) deposition, vascular permeability, interstitial fluid pressure (IFP), and degradation of vascular HA relative to models containing MCF7 tumoroids or models without tumoroids. Interleukin 8 (IL8) secretion is found responsible for vascular dysfunction and loss of vascular HA. Interventions targeting IL8 or stromal HA normalize vascular permeability, perfusion, and IFP, and ultimately enhance drug delivery and TC death in response to perfusion with trastuzumab and cetuximab. Similar responses are observed in patient-derived models. These microphysiological systems can thus be personalized by using patient-derived cells and can be applied to discover new molecular therapies for the normalization of the tumor microenvironment.
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The knowledge of the blood microvasculature and its functional role in health and disease has grown significantly attributable to decades of research and numerous advances in cell biology and tissue engineering; however, the lymphatics (the secondary vascular system) has not garnered similar attention, in part due to a lack of relevant in vitro models that mimic its pathophysiological functions. Here, a microfluidic-based approach is adopted to achieve precise control over the biological transport of growth factors and interstitial flow that drive the in vivo growth of lymphatic capillaries (lymphangiogenesis). The engineered on-chip lymphatics with in vivo-like morphology exhibit tissue-scale functionality with drainage rates of interstitial proteins and molecules comparable to in vivo standards. Computational and scaling analyses of the underlying transport phenomena elucidate the critical role of the three-dimensional geometry and lymphatic endothelium in recapitulating physiological drainage. Finally, the engineered on-chip lymphatics enabled studies of lymphatic-immune interactions that revealed inflammation-driven responses by the lymphatics to recruit immune cells via chemotactic signals similar to in vivo, pathological events. This on-chip lymphatics platform permits the interrogation of various lymphatic biological functions, as well as screening of lymphatic-based therapies such as interstitial absorption of protein therapeutics and lymphatic immunomodulation for cancer therapy.
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Vasos Linfáticos , Microfluídica , Humanos , Microfluídica/métodos , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patologia , Linfangiogênese , Microvasos , Inflamação/metabolismoRESUMO
During bloodstream infections, neutrophils home to the liver as part of an intravascular immune response to eradicate blood-borne pathogens, but the mechanisms regulating this crucial response are unknown. Using in vivo imaging of neutrophil trafficking in germ-free and gnotobiotic mice, we demonstrate that the intestinal microbiota guides neutrophil homing to the liver in response to infection mediated by the microbial metabolite D-lactate. Commensal-derived D-lactate augments neutrophil adhesion in the liver independent of granulopoiesis in bone marrow or neutrophil maturation and activation in blood. Instead, gut-to-liver D-lactate signaling primes liver endothelial cells to upregulate adhesion molecule expression in response to infection and promote neutrophil adherence. Targeted correction of microbiota D-lactate production in a model of antibiotic-induced dysbiosis restores neutrophil homing to the liver and reduces bacteremia in a model of Staphylococcus aureus infection. These findings reveal long-distance traffic control of neutrophil recruitment to the liver by microbiota-endothelium crosstalk.
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Células Endoteliais , Microbiota , Animais , Camundongos , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Fígado/metabolismo , Endotélio , Lactatos/metabolismoRESUMO
Despite surviving a SARS-CoV-2 infection, some individuals experience an intense post-infectious Multisystem Inflammatory Syndrome (MIS) of uncertain etiology. Children with this syndrome (MIS-C) can experience a Kawasaki-like disease, but mechanisms in adults (MIS-A) are not clearly defined. Here we utilize a deep phenotyping approach to examine immunologic responses in an individual with MIS-A. Results are contextualized to healthy, convalescent, and acute COVID-19 patients. The findings reveal systemic inflammatory changes involving novel neutrophil and B-cell subsets, autoantibodies, complement, and hypercoagulability that are linked to systemic vascular dysfunction. This deep patient profiling generates new mechanistic insight into this rare clinical entity and provides potential insight into other post-infectious syndromes.
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COVID-19 , Doenças do Tecido Conjuntivo , Criança , Humanos , Adulto , Neutrófilos , SARS-CoV-2RESUMO
Epithelial ovarian cancer has the highest mortality rate of any gynecologic malignancy and most frequently metastasizes to the peritoneal cavity. Intraperitoneal metastases are highly associated with ascites, the pathologic accumulation of peritoneal fluid due to impaired drainage, increased peritoneal permeability, and tumor and stromal cytokine secretion. However, the relationship between ascites, vascular and mesothelial permeability, and ovarian cancer intraperitoneal metastases remains poorly understood. In this study, a vascularized in vitro model of the human peritoneal omentum and ovarian tumor microenvironment (TME) was employed to study stromal cell effects on tumor cell (TC) attachment and growth, as well as TC effects on vascular and mesothelial permeability in models of both early- and late-stage metastases. Control over the number of TCs seeded in the vascularized peritoneum revealed a critical cell density requirement for tumor growth, which was further enhanced by stromal adipocytes and endothelial cells found in the peritoneal omentum. This tumor growth resulted in both a physically-mediated decrease and cytokine-mediated increase in microvascular permeability, emphasizing the important and potentially opposing roles of tumor cells in ascites formation. This system provides a robust platform to elucidate TC-stromal cell interactions during intraperitoneal metastasis of ovarian cancer and presents the first in vitro vascularized model of the human peritoneum and ovarian cancer TME.
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Neoplasias Ovarianas , Peritônio , Ascite/patologia , Carcinoma Epitelial do Ovário/patologia , Linhagem Celular Tumoral , Citocinas , Células Endoteliais/patologia , Feminino , Humanos , Dispositivos Lab-On-A-Chip , Microfluídica , Omento/patologia , Neoplasias Ovarianas/patologia , Peritônio/patologia , Microambiente TumoralRESUMO
BACKGROUND: Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is completed through reverse transcriptase-PCR (RT-PCR) from either oropharyngeal or nasopharyngeal swabs, critically important for diagnostics but also from an infection control lens. Recent studies have suggested that COVID-19 patients can demonstrate prolonged viral shedding with immunosuppression as a key risk factor. CASE PRESENTATION: We present a case of an immunocompromised patient with SARS-CoV-2 infection demonstrating prolonged infectious viral shedding for 189 days with virus cultivability and clinical relapse with an identical strain based on whole genome sequencing, requiring a multi-modal therapeutic approach. We correlated clinical parameters, PCR cycle thresholds and viral culture until eventual resolution. CONCLUSIONS: We successfully demonstrate resolution of viral shedding, administration of COVID-19 vaccination and maintenance of viral clearance. This case highlights implications in the immunosuppressed patient towards infection prevention and control that should consider those with prolonged viral shedding and may require ancillary testing to fully elucidate viral activity. Furthermore, this case raises several stimulating questions around complex COVID-19 patients around the role of steroids, effect of antiviral therapies in absence of B-cells, role for vaccination and the requirement of a multi-modal approach to eventually have successful clearance of the virus.
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COVID-19/patologia , Rituximab/farmacologia , SARS-CoV-2/efeitos dos fármacos , Eliminação de Partículas Virais/efeitos dos fármacos , COVID-19/prevenção & controle , Vacinas contra COVID-19/administração & dosagem , Humanos , Hospedeiro Imunocomprometido , Masculino , Pessoa de Meia-Idade , Nasofaringe , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Carga Viral , Tratamento Farmacológico da COVID-19RESUMO
Although critical for host defense, innate immune cells are also pathologic drivers of acute respiratory distress syndrome (ARDS). Innate immune dynamics during Coronavirus Disease 2019 (COVID-19) ARDS, compared to ARDS from other respiratory pathogens, is unclear. Moreover, mechanisms underlying the beneficial effects of dexamethasone during severe COVID-19 remain elusive. Using single-cell RNA sequencing and plasma proteomics, we discovered that, compared to bacterial ARDS, COVID-19 was associated with expansion of distinct neutrophil states characterized by interferon (IFN) and prostaglandin signaling. Dexamethasone during severe COVID-19 affected circulating neutrophils, altered IFNactive neutrophils, downregulated interferon-stimulated genes and activated IL-1R2+ neutrophils. Dexamethasone also expanded immunosuppressive immature neutrophils and remodeled cellular interactions by changing neutrophils from information receivers into information providers. Male patients had higher proportions of IFNactive neutrophils and preferential steroid-induced immature neutrophil expansion, potentially affecting outcomes. Our single-cell atlas (see 'Data availability' section) defines COVID-19-enriched neutrophil states and molecular mechanisms of dexamethasone action to develop targeted immunotherapies for severe COVID-19.
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COVID-19/imunologia , Citocinas/imunologia , Dexametasona/uso terapêutico , Glucocorticoides/uso terapêutico , Neutrófilos/imunologia , Pneumonia Bacteriana/imunologia , Síndrome do Desconforto Respiratório/imunologia , Adulto , Idoso , COVID-19/complicações , COVID-19/genética , Comunicação Celular , Cromatografia Líquida , Regulação para Baixo , Feminino , Redes Reguladoras de Genes , Humanos , Imunidade Inata/imunologia , Interferons/imunologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/metabolismo , Pneumonia Bacteriana/complicações , Pneumonia Bacteriana/tratamento farmacológico , Pneumonia Bacteriana/genética , Prostaglandinas/imunologia , Proteômica , RNA-Seq , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/genética , SARS-CoV-2 , Índice de Gravidade de Doença , Fatores Sexuais , Análise de Célula Única , Espectrometria de Massas em Tandem , Tratamento Farmacológico da COVID-19RESUMO
Monocytes (Mos) are immune cells that critically regulate cancer, enabling tumor growth and modulating metastasis. Mos can give rise to tumor-associated macrophages (TAMs) and Mo-derived dendritic cells (moDCs), all of which shape the tumor microenvironment (TME). Thus, understanding their roles in the TME is key for improved immunotherapy. Concurrently, various biological and mechanical factors including changes in local cytokines, extracellular matrix production, and metabolic changes in the TME affect the roles of monocytic cells. As such, relevant TME models are critical to achieve meaningful insight on the precise functions, mechanisms, and effects of monocytic cells. Notably, murine models have yielded significant insight into human Mo biology. However, many of these results have yet to be confirmed in humans, reinforcing the need for improved in vitro human TME models for the development of cancer interventions. Thus, this chapter (1) summarizes current insight on the tumor biology of Mos, TAMs, and moDCs, (2) highlights key therapeutic applications relevant to these cells, and (3) discusses various TME models to study their TME-related activity. We conclude with a perspective on the future research trajectory of this topic.
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Monócitos/patologia , Neoplasias/patologia , Microambiente Tumoral , Animais , Humanos , Imunoterapia , Macrófagos/patologia , Neoplasias/imunologia , Neoplasias/terapia , Microambiente Tumoral/imunologiaRESUMO
Drug discovery and efficacy in cancer treatments are limited by the inability of pre-clinical models to predict successful outcomes in humans. Limitations remain partly due to their lack of a physiologic tumor microenvironment (TME), which plays a considerable role in drug delivery and tumor response to therapy. Chemotherapeutics and immunotherapies rely on transport through the vasculature, via the smallest capillaries and stroma to the tumor, where passive and active transport processes are at play. Here, a 3D vascularized tumor on-chip is used to examine drug delivery in a relevant TME within a large bed of perfusable vasculature. This system demonstrates highly localized pathophysiological effects of two tumor spheroids (Skov3 and A549) which cause significant changes in vessel density and barrier function. Paclitaxel (Taxol) uptake is examined through diffusivity measurements, functional efflux assays and accumulation of the fluorescent-conjugated drug within the TME. Due to vascular and stromal contributions, differences in the response of vascularized tumors to Taxol (shrinkage and CD44 expression) are apparent compared with simpler models. This model specifically allows for examination of spatially resolved tumor-associated endothelial dysfunction, likely improving the representation of in vivo drug distribution, and has potential for development into a more predictable model of drug delivery.
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Placental vasculopathies are associated with a number of pregnancy-related diseases, including pre-eclampsia (PE)-a leading cause of maternal-fetal morbidity and mortality worldwide. Placental presentations of PE are associated with endothelial dysfunction, reduced vessel perfusion, white blood cell infiltration, and altered production of angiogenic factors within the placenta (a candidate mechanism). Despite maintaining vascular quiescence in other tissues, how pericytes contribute to vascular growth and signaling in the placenta remains unknown. Here, pericytes are hypothesized to play a detrimental role in the pathogenesis of placental vascular growth. A perfusable triculture model is developed, consisting of human endothelial cells, fibroblasts, and pericytes, capable of recapitulating growth and remodeling in a system that mimics inflamed placental microvessels. Placental pericytes are shown to contribute to growth restriction of microvessels over time, an effect that is strongly regulated by vascular endothelial growth factor and Angiopoietin/Tie2 signaling. Furthermore, this model is capable of recapitulating essential processes including tumor necrosis factor alpha (TNFα)-mediated vascular leakage and leukocyte infiltration, both important aspects associated with placental PE. This placental vascular model highlights that an imbalance in endothelial-pericyte crosstalk can play a critical role in the development of vascular pathology and associated diseases.
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Recent therapeutic success of large-molecule biologics has led to intense interest in assays to measure with precision their transport across the vascular endothelium and into the target tissue. Most current in vitro endothelial models show unrealistically large permeability coefficients due to a non-physiological paracellular transport. Thus, more advanced systems are required to better recapitulate and discern the important contribution of transcellular transport (transcytosis), particularly of pharmaceutically-relevant proteins. Here, a robust platform technology for the measurement of transport through a human endothelium is presented, which utilizes in vitro microvascular networks (MVNs). The self-assembled MVNs recapitulate the morphology and junctional complexity of in vivo capillaries, and express key endothelial vesicular transport proteins. This results in measured permeabilities to large molecules comparable to those observed in vivo, which are orders of magnitude lower than those measured in transwells. The permeability of albumin and immunoglobulin G (IgG), biopharmaceutically-relevant proteins, is shown to occur primarily via transcytosis, with passage of IgG regulated by the receptor FcRn. The physiological relevance of the MVNs make it a valuable tool to assess the distribution of biopharmaceuticals into tissues, and may be used to prioritize candidate molecules from this increasingly important class of therapeutics.
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Permeabilidade Capilar/fisiologia , Dispositivos Lab-On-A-Chip , Microcirculação/fisiologia , Proteínas/metabolismo , Albuminas/metabolismo , Endotélio Vascular/metabolismo , Glicocálix/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Imunoglobulina G/metabolismo , Microvasos/fisiologia , Perfusão , Receptores Fc/metabolismo , TranscitoseRESUMO
Background: Eggerthella lenta is a anaerobic gram-positive bacilli associated with polymicrobial intraabdominal infections. Recently, E. lenta was recognized as an important cause of anaerobic bloodstream infections (BSIs) associated with high mortality. Eggerthella lenta has been reported to have high minimal inhibitory concentrations (MICs) to piperacillin-tazobactam (TZP), a broad-spectrum antibiotic with anaerobic coverage commonly used in multiple centers for empiric treatment of abdominal sepsis. Methods: We describe a retrospective population-based analysis of invasive E. lenta infections from 2009 through 2015. A logistic regression analysis for 30-day mortality risk factors was conducted. Results: We identified 107 E. lenta infections, 95 (89%) were BSIs, 11 (10%) skin and soft tissue infections, and 1 intraabdominal abscess. Polymicrobial infections were found in 40%; 72% of isolates were from a gastrointestinal source, most commonly appendicitis (33%) of which two-thirds were perforated. TZP MIC50 and MIC90 for E. lenta isolates were 32 µg/mL and 64 µg/mL, respectively. The overall 30-day mortality for BSI was 23% and was independently associated with empiric TZP monotherapy (odds ratio [OR], 4.4; 95% confidence interval [CI], 1.2-16; P = .02) and intensive care unit stay (OR, 6.2; 95% CI, 1.4-27.3; P = .01). Thirty-day mortality rates were significantly influenced by the use of different TZP MIC breakpoints. Conclusions: Our results demonstrate the increased recognition of E. lenta as an anaerobic opportunistic pathogen and highlight the need for improved empiric antimicrobial guidelines and TZP MIC breakpoints with better correlation to clinical outcomes to guide appropriate management of invasive E. lenta infections.
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Antibacterianos/uso terapêutico , Bacteriemia/mortalidade , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Infecções por Bactérias Gram-Positivas/mortalidade , Combinação Piperacilina e Tazobactam/uso terapêutico , Actinobacteria/efeitos dos fármacos , Actinobacteria/isolamento & purificação , Idoso , Bacteriemia/tratamento farmacológico , Bactérias Anaeróbias/efeitos dos fármacos , Bactérias Anaeróbias/isolamento & purificação , Gerenciamento Clínico , Feminino , Humanos , Modelos Logísticos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Infecções Oportunistas/tratamento farmacológico , Infecções Oportunistas/microbiologia , Vigilância em Saúde Pública , Estudos Retrospectivos , Fatores de Risco , Resultado do TratamentoRESUMO
Candida albicans bloodstream infection causes fungal septicaemia and death in over half of afflicted patients. Polymorphonuclear leukocytes (PMN) mediate defense against invasive candidiasis, but their role in protection versus tissue injury and sepsis is unclear. We observe PMN intravascular swarming and subsequent clustering in response to C. albicans yeast in a lethal septic mouse and human pulmonary circulation model. Live C. albicans sequester to the endothelium and are immediately captured by complement-dependent PMN chemotaxis, which is required for host survival. However, complement activation also leads to Leukotriene B4 (LTB4)-mediated intravascular PMN clustering and occlusion, resulting in capillaritis with pulmonary hemorrhage and hypoxemia. This clustering is unique to fungi and triggered by fungal cell wall components. PMN clustering is absent in mice lacking LTB4-receptor, and capillaritis is attenuated upon pharmacological LTB4 blockade without affecting phagocytosis. Therefore, therapeutically disrupting infection-induced capillaritis may limit organ injury without impairing host defense during fungal sepsis.
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Arteriopatias Oclusivas/microbiologia , Candida albicans/imunologia , Candidíase/imunologia , Leucotrieno B4/imunologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Sepse/imunologia , Animais , Arteriopatias Oclusivas/imunologia , Candidíase/microbiologia , Candidíase/patologia , Células Cultivadas , Feminino , Humanos , Pulmão/irrigação sanguínea , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sepse/microbiologia , Sepse/patologiaRESUMO
Plasmodial species are protozoan parasites that infect erythrocytes. As such, they are in close contact with microvascular endothelium for most of the life cycle in the mammalian host. The host-parasite interactions of this stage of the infection are responsible for the clinical manifestations of the disease that range from a mild febrile illness to severe and frequently fatal syndromes such as cerebral malaria and multi-organ failure. Plasmodium falciparum, the causative agent of the most severe form of malaria, is particularly predisposed to modulating endothelial function through either direct adhesion to endothelial receptor molecules, or by releasing potent host and parasite products that can stimulate endothelial activation and/or disrupt barrier function. In this review, we provide a critical analysis of the current clinical and laboratory evidence for endothelial dysfunction during severe P. falciparum malaria. Future investigations using state-of-the-art technologies such as mass cytometry and organs-on-chips to further delineate parasite-endothelial cell interactions are also discussed.
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Endotélio Vascular/parasitologia , Plasmodium falciparum/virologia , HumanosRESUMO
UNLABELLED: Plasmodium falciparum malaria remains one of the most deadly infections worldwide. The pathogenesis of the infection results from the sequestration of infected erythrocytes (IRBC) in vital organs, including the brain, with resulting impairment of blood flow, hypoxia, and lactic acidosis. Sequestration occurs through the adhesion of IRBC to host receptors on microvascular endothelium by Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a large family of variant surface antigens, each with up to seven extracellular domains that can bind to multiple host receptors. Consequently, antiadhesive therapies directed at single endothelial adhesion molecules may not be effective. In this study, we demonstrated that the serine protease thrombin, which is pivotal in the activation of the coagulation cascade, cleaved the major parasite adhesin on the surface of IRBC. As a result, adhesion under flow was dramatically reduced, and already adherent IRBC were detached. Thrombin cleavage sites were mapped to the Duffy binding-like δ1 (DBLδ1) domain and interdomains 1 and 2 in the PfEMP1 of the parasite line IT4var19. Furthermore, we observed an inverse correlation between the presence of thrombin and IRBC in cerebral malaria autopsies of children. We investigated a modified (R67A) thrombin and thrombin inhibitor, hirugen, both of which inhibit the binding of substrates to exosite I, thereby reducing its proinflammatory properties. Both approaches reduced the barrier dysfunction induced by thrombin without affecting its proteolytic activity on PfEMP1, raising the possibility that thrombin cleavage of variant PfEMP1 may be exploited as a broadly inhibitory antiadhesive therapy. IMPORTANCE: Plasmodium falciparum malaria is the third leading cause of mortality due to a pathogen, with 214 million people infected and 438,000 deaths annually. The adhesion of Plasmodium falciparum-infected erythrocytes (IRBC) to microvascular endothelium is a major pathological process in severe malaria. While the recent implementation of artemisinin-based antimalarial therapy for severe malaria improves patient survival by targeting all parasite stages, antiparasite drugs alone may not immediately reverse pathophysiological processes in occluded vessels. Here we show that thrombin, an enzyme intimately involved in the clotting process, cleaves the main parasite adhesin expressed on the surface of IRBC, thereby preventing and reversing the binding of IRBC to endothelial cells. This beneficial effect of thrombin can be achieved by modified thrombins that cause significantly less clotting and vessel leakage while preserving the ability to cleave the parasite protein. Our results provide the basis for using modified thrombins as adjunctive therapy in severe malaria.
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Adesão Celular , Interações Hospedeiro-Patógeno , Plasmodium falciparum/fisiologia , Proteólise , Proteínas de Protozoários/metabolismo , Trombina/metabolismo , Células Cultivadas , Células Endoteliais/fisiologia , Eritrócitos/parasitologia , HumanosRESUMO
Plasmodium falciparum-infected erythrocytes (IRBC) expressing the domain cassettes (DC) 8 and 13 of the cytoadherent ligand P. falciparum erythrocyte membrane protein 1 adhere to the endothelial protein C receptor (EPCR). By interfering with EPCR anti-coagulant and pro-endothelial barrier functions, IRBC adhesion could promote coagulation and vascular permeability that contribute to the pathogenesis of cerebral malaria. In this study, we examined the adhesion of DC8- and DC13-expressing parasite lines to endothelial cells from different microvasculature, and the consequences of EPCR engagement on endothelial cell function. We found that IRBC from IT4var19 (DC8) and IT4var07 (DC13) parasite lines adhered to human brain, lung and dermal endothelial cells under shear stress. However, the relative contribution of EPCR to parasite cytoadherence on different types of endothelial cell varied. We also observed divergent functional outcomes for DC8 cysteine-rich interdomain region (CIDR)α1.1 and DC13 CIDRα1.4 domains. IT4var07 CIDRα1.4 inhibited generation of activated protein C (APC) on lung and dermal endothelial cells and blocked the APC-EPCR binding interaction on brain endothelial cells. IT4var19 CIDRα1.1 inhibited thrombin-induced endothelial barrier dysfunction in lung endothelial cells, whereas IT4var07 CIDRα1.4 inhibited the protective effect of APC on thrombin-induced permeability. Overall, these findings reveal a much greater complexity of how CIDRα1-expressing parasites may modulate malaria pathogenesis through EPCR adhesion.
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Antígenos CD/metabolismo , Adesão Celular , Células Endoteliais/fisiologia , Eritrócitos/parasitologia , Interações Hospedeiro-Patógeno , Plasmodium falciparum/fisiologia , Receptores de Superfície Celular/metabolismo , Células Cultivadas , Receptor de Proteína C Endotelial , Humanos , Ligadura , Resultado do TratamentoRESUMO
The adhesion of Plasmodium falciparum-infected erythrocytes (IRBC) to receptors on different host cells plays a divergent yet critical role in determining the progression and outcome of the infection. Based on our ex vivo studies with clinical parasite isolates from adult Thai patients, we have previously proposed a paradigm for IRBC cytoadherence under physiological shear stress that consists of a recruitment cascade mediated largely by P-selectin, ICAM-1 and CD36 on primary human dermal microvascular endothelium (HDMEC). In addition, we detected post-adhesion signaling events involving Src family kinases and the adaptor protein p130CAS in endothelial cells that lead to CD36 clustering and cytoskeletal rearrangement which enhance the magnitude of the adhesive strength, allowing adherent IRBC to withstand shear stress of up to 20 dynes/cm². In this study, we addressed whether CD36 supports IRBC adhesion as part of an assembly of membrane receptors. Using a combination of flow chamber assay, atomic force and confocal microscopy, we showed for the first time by loss- and gain-of function assays that in the resting state, the integrin α5ß1 does not support adhesive interactions between IRBC and HDMEC. Upon IRBC adhesion to CD36, the integrin is recruited either passively as part of a molecular complex with CD36, or actively to the site of IRBC attachment through phosphorylation of Src family kinases, a process that is Ca²âº-dependent. Clustering of ß1 integrin is associated with an increase in IRBC recruitment as well as in adhesive strength after attachment (â¼40% in both cases). The adhesion of IRBC to a multimolecular complex on the surface of endothelial cells could be of critical importance in enabling adherent IRBC to withstand the high shear stress in the microcirculations. Targeting integrins may provide a novel approach to decrease IRBC cytoadherence to microvascular endothelium.
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Antígenos CD36/metabolismo , Endotélio Vascular/metabolismo , Eritrócitos/metabolismo , Integrina alfa5beta1/metabolismo , Malária Falciparum/metabolismo , Plasmodium falciparum/metabolismo , Antígenos CD36/genética , Cálcio/metabolismo , Adesão Celular/genética , Células Cultivadas , Endotélio Vascular/patologia , Eritrócitos/parasitologia , Eritrócitos/patologia , Feminino , Humanos , Integrina alfa5beta1/genética , Malária Falciparum/genética , Malária Falciparum/patologia , Masculino , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosforilação/genética , Plasmodium falciparum/genética , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Plasmodium falciparum is a protozoan parasite of human erythrocytes that causes the most severe form of malaria. Severe P. falciparum infection is associated with endothelial activation and permeability, which are important determinants of the outcome of the infection. How endothelial cells become activated is not fully understood, particularly with regard to the effects of parasite subcomponents. We demonstrated that P. falciparum histones extracted from merozoites (HeH) directly stimulated the production of IL-8 and other inflammatory mediators by primary human dermal microvascular endothelial cells through a signaling pathway that involves Src family kinases and p38 MAPK. The stimulatory effect of HeH and recombinant P. falciparum H3 (PfH3) was abrogated by histone-specific antibodies. The release of nuclear contents on rupture of infected erythrocytes was captured by live cell imaging and confirmed by detecting nucleosomes in the supernatants of parasite cultures. HeH and recombinant parasite histones also induced endothelial permeability through a charge-dependent mechanism that resulted in disruption of junctional protein expression and cell death. Recombinant human activated protein C cleaved HeH and PfH3 and abrogated their proinflammatory effects. Circulating nucleosomes of both human and parasite origin were detected in the plasma of patients with falciparum malaria and correlated positively with disease severity. These results support a pathogenic role for both host- and pathogen-derived histones in P. falciparum-caused malaria.
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Endotélio Vascular/metabolismo , Histonas/farmacologia , Interleucina-8/biossíntese , Merozoítos , Plasmodium falciparum , Animais , Permeabilidade Capilar/fisiologia , Células Cultivadas , Endotélio Vascular/parasitologia , Humanos , Estágios do Ciclo de Vida , Pulmão/irrigação sanguínea , Pulmão/parasitologia , Sistema de Sinalização das MAP Quinases/fisiologia , Malária Falciparum/parasitologia , Microvasos , Proteína C/farmacologia , Proteínas Recombinantes , Pele/irrigação sanguínea , Pele/parasitologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Quinases da Família src/fisiologiaRESUMO
The adhesion of infected red blood cells (IRBCs) to microvascular endothelium is critical in the pathogenesis of severe malaria. Here we used atomic force and confocal microscopy to examine the adhesive forces between IRBCs and human dermal microvascular endothelial cells. Initial contact of the cells generated a mean ± sd adhesion force of 167 ± 208 pN from the formation of single or multiple bonds with CD36. The strength of adhesion increased by 5- to 6-fold within minutes of contact through a signaling pathway initiated by CD36 ligation by live IRBCs, or polystyrene beads coated with anti-CD36 or PpMC-179, a recombinant peptide representing the minimal binding domain of the parasite ligand PfEMP1 to CD36. Engagement of CD36 led to localized phosphorylation of Src family kinases and the adaptor protein p130CAS, resulting in actin recruitment and CD36 clustering by 50-60% of adherent beads. Uninfected red blood cells or IgG-coated beads had no effect. Inhibition of the increase in adhesive strength by the Src family kinase inhibitor PP1 or gene silencing of p130CAS decreased adhesion by 39 ± 12 and 48 ± 20%, respectively, at 10 dyn/cm(2) in a flow chamber assay. Modulation of adhesive strength at PfEMP1-CD36-actin cytoskeleton synapses could be a novel target for antiadhesive therapy.
Assuntos
Antígenos CD36/metabolismo , Proteína Substrato Associada a Crk/metabolismo , Citoesqueleto/metabolismo , Eritrócitos/metabolismo , Plasmodium falciparum/metabolismo , Actinas/metabolismo , Antígenos CD36/genética , Adesão Celular/efeitos dos fármacos , Comunicação Celular , Células Cultivadas , Proteína Substrato Associada a Crk/genética , Células Endoteliais/metabolismo , Eritrócitos/parasitologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interações Hospedeiro-Parasita , Humanos , Recém-Nascido , Masculino , Microscopia de Força Atômica , Microscopia Confocal , Fosforilação , Plasmodium falciparum/fisiologia , Ligação Proteica , Proteínas de Protozoários/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Interferência de RNA , Análise de Célula Única/métodos , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/metabolismoRESUMO
The response of leukocytes to lipoteichoic acid (LTA), a TLR2-dependent major cell wall component of Staphylococcus aureus, is linked to the outcome of an infection. In this study we investigated the role of nonhematopoietic TLR2 in response to LTA and S. aureus by creating bone marrow chimeras. Significant leukocyte recruitment in response to LTA required IFN-gamma priming in WT C57BL/6 and TLR2(-/-)-->WT mice, but was not observed in TLR2(-/-) or WT-->TLR2(-/-) animals. LTA also induced a proinflammatory response in IFN-gamma primed primary human microvascular endothelial cells leading to leukocyte recruitment in vitro. When mice were infected with S. aureus, the most profound elevation of TNF-alpha and IL-6 was seen in TLR2(-/-) and TLR2(-/-)-->WT mice. TLR2(-/-), but not chimeric mice, demonstrated increased IL-17, blood leukocytosis and pulmonary neutrophilia compared to WT mice. Collectively, the results suggest an essential role for IFN-gamma and nonhematopoietic TLR2 for leukocyte recruitment in response to LTA. In contrast, TLR2 on both hematopoietic and nonhematopoietic cells appears to orchestrate an inhibitory response to S. aureus such that in complete TLR2 deficiency, there is an exaggerated proinflammatory response and/or skewing of the immune response towards a Th17 phenotype that may contribute to the decreased survival of TLR2(-/-) mice.