RESUMO
Frontotemporal dementia (FTD) because of MAPT mutation causes pathological accumulation of tau and glutamatergic cortical neuronal death by unknown mechanisms. We used human induced pluripotent stem cell (iPSC)-derived cerebral organoids expressing tau-V337M and isogenic corrected controls to discover early alterations because of the mutation that precede neurodegeneration. At 2 months, mutant organoids show upregulated expression of MAPT, glutamatergic signaling pathways, and regulators, including the RNA-binding protein ELAVL4, and increased stress granules. Over the following 4 months, mutant organoids accumulate splicing changes, disruption of autophagy function, and build-up of tau and P-tau-S396. By 6 months, tau-V337M organoids show specific loss of glutamatergic neurons as seen in individuals with FTD. Mutant neurons are susceptible to glutamate toxicity, which can be rescued pharmacologically by the PIKFYVE kinase inhibitor apilimod. Our results demonstrate a sequence of events that precede neurodegeneration, revealing molecular pathways associated with glutamate signaling as potential targets for therapeutic intervention in FTD.
Assuntos
Cérebro/patologia , Proteína Semelhante a ELAV 4/genética , Ácido Glutâmico/metabolismo , Mutação/genética , Neurônios/patologia , Organoides/metabolismo , Splicing de RNA/genética , Proteínas tau/genética , Autofagia/efeitos dos fármacos , Autofagia/genética , Biomarcadores/metabolismo , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Morte Celular/efeitos dos fármacos , Linhagem Celular , Humanos , Hidrazonas/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Morfolinas/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Organoides/efeitos dos fármacos , Organoides/ultraestrutura , Fosforilação/efeitos dos fármacos , Pirimidinas/farmacologia , Splicing de RNA/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Grânulos de Estresse/efeitos dos fármacos , Grânulos de Estresse/metabolismo , Sinapses/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Nuclear pore complexes (NPCs) regulate nuclear-cytoplasmic transport, transcription, and genome integrity in eukaryotic cells. However, their functional roles in cancer remain poorly understood. We interrogated the evolutionary transcriptomic landscape of NPC components, nucleoporins (Nups), from primary to advanced metastatic human prostate cancer (PC). Focused loss-of-function genetic screen of top-upregulated Nups in aggressive PC models identified POM121 as a key contributor to PC aggressiveness. Mechanistically, POM121 promoted PC progression by enhancing importin-dependent nuclear transport of key oncogenic (E2F1, MYC) and PC-specific (AR-GATA2) transcription factors, uncovering a pharmacologically targetable axis that, when inhibited, decreased tumor growth, restored standard therapy efficacy, and improved survival in patient-derived pre-clinical models. Our studies molecularly establish a role of NPCs in PC progression and give a rationale for NPC-regulated nuclear import targeting as a therapeutic strategy for lethal PC. These findings may have implications for understanding how NPC deregulation contributes to the pathogenesis of other tumor types.
Assuntos
Fator de Transcrição E2F1/metabolismo , Glicoproteínas de Membrana/metabolismo , Poro Nuclear/fisiologia , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Carcinogênese , Núcleo Celular/metabolismo , Proliferação de Células , Fator de Transcrição GATA2/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Membrana Nuclear , Complexo de Proteínas Formadoras de Poros Nucleares , Transdução de SinaisRESUMO
BACKGROUND: Vacuolar protein sorting 13 homolog A (VPS13A) disease, historically known as chorea-acanthocytosis, is a rare neurodegenerative disorder caused by biallelic mutations in VPS13A, usually resulting in reduced or absent levels of its protein product, VPS13A. VPS13A localizes to contact sites between subcellular organelles, consistent with its recently identified role in lipid transfer between membranes. Mutations are associated with neuronal loss in the striatum, most prominently in the caudate nucleus, and associated marked astrogliosis. There are no other known disease-specific cellular changes (eg, protein aggregation), but autopsy reports to date have been limited, often lacking genetic or biochemical diagnostic confirmation. OBJECTIVE: The goal of this study was to characterize neuropathological findings in the brains of seven patients with VPS13A disease (chorea-acanthocytosis). METHODS: In this study, we collected brain tissues and clinical data from seven cases of VPS13A for neuropathological analysis. The clinical diagnosis was confirmed by the presence of VPS13A mutations and/or immunoblot showing the loss or reduction of VPS13A protein. Tissues underwent routine, special, and immunohistochemical staining focused on neurodegeneration. Electron microscopy was performed in one case. RESULTS: Gross examination showed severe striatal atrophy. Microscopically, there was neuronal loss and astrogliosis in affected regions. Luxol fast blue staining showed variable lipid accumulation with diverse morphology, which was further characterized by electron microscopy. In some cases, rare degenerating p62- and ubiquitin-positive cells were present in affected regions. Calcifications were present in four cases, being extensive in one. CONCLUSIONS: We present the largest autopsy series of biochemically and genetically confirmed VPS13A disease and identify novel histopathological findings implicating abnormal lipid accumulation. © 2023 International Parkinson and Movement Disorder Society.
Assuntos
Neuroacantocitose , Humanos , Autopsia , Núcleo Caudado/metabolismo , Gliose , Lipídeos , Neuroacantocitose/genética , Neuroacantocitose/diagnóstico , Neuroacantocitose/patologia , Proteínas de Transporte Vesicular/genéticaRESUMO
BACKGROUND: The Girinka program in Rwanda has contributed to an increase in milk production, as well as to reduced malnutrition and increased incomes. But dairy products can be hazardous to health, potentially transmitting diseases such as bovine brucellosis, tuberculosis, and cause diarrhea. We analyzed the burden of foodborne disease due to consumption of raw milk and other dairy products in Rwanda to support the development of policy options for the improvement of the quality and safety of milk. METHODS: Disease burden data for five pathogens (Campylobacter spp., nontyphoidal Salmonella enterica, Cryptosporidium spp., Brucella spp., and Mycobacterium bovis) were extracted from the 2010 WHO Foodborne Disease Burden Epidemiology Reference Group (FERG) database and merged with data of the proportion of foodborne disease attributable to consuming dairy products from FERG and a separately published Structured Expert Elicitation study to generate estimates of the uncertainty distributions of the disease burden by Monte Carlo simulation. RESULTS: According to WHO, the foodborne disease burden (all foods) of these five pathogens in Rwanda in 2010 was like or lower than in the Africa E subregion as defined by FERG. There were 57,500 illnesses occurring in Rwanda owing to consumption of dairy products, 55 deaths and 3,870 Disability Adjusted Life Years (DALYs) causing a cost-of-illness of $3.2 million. 44% of the burden (in DALYs) was attributed to drinking raw milk and sizeable proportions were also attributed to traditionally (16-23%) or industrially (6-22%) fermented milk. More recent data are not available, but the burden (in DALYs) of tuberculosis and diarrheal disease by all causes in Rwanda has declined between 2010 and 2019 by 33% and 46%, respectively. CONCLUSION: This is the first study examining the WHO estimates of the burden of foodborne disease on a national level in Rwanda. Transitioning from consuming raw to processed milk (fermented, heat treated or otherwise) may prevent a considerable disease burden and cost-of-illness, but the full benefits will only be achieved if there is a simultaneous improvement of pathogen inactivation during processing, and prevention of recontamination of processed products.
Assuntos
Criptosporidiose , Cryptosporidium , Doenças Transmitidas por Alimentos , Animais , Bovinos , Humanos , Ruanda/epidemiologia , Doenças Transmitidas por Alimentos/epidemiologia , Doenças Transmitidas por Alimentos/microbiologia , Leite/microbiologia , Efeitos Psicossociais da DoençaRESUMO
Recent epidemiological studies suggest that some patients with diabetes progress to kidney failure without significant albuminuria and glomerular injury, suggesting a critical role of kidney tubular epithelial cell (TEC) injury in diabetic kidney disease (DKD) progression. However, the major risk factors contributing to TEC injury and progression in DKD remain unclear. We previously showed that expression of endoplasmic reticulum-resident protein Reticulon-1A (RTN1A) increased in human DKD, and the increased RTN1A expression promoted TEC injury through endoplasmic reticulum (ER) stress response. Here, we show that TEC-specific RTN1A overexpression worsened DKD in mice, evidenced by enhanced tubular injury, tubulointerstitial fibrosis, and kidney function decline. But RTN1A overexpression did not exacerbate diabetes-induced glomerular injury or albuminuria. Notably, RTN1A overexpression worsened both ER stress and mitochondrial dysfunction in TECs under diabetic conditions by regulation of ER-mitochondria contacts. Mechanistically, ER-bound RTN1A interacted with mitochondrial hexokinase-1 and the voltage-dependent anion channel-1 (VDAC1), interfering with their association. This disengagement of VDAC1 from hexokinase-1 resulted in activation of apoptotic and inflammasome pathways, leading to TEC injury and loss. Thus, our observations highlight the importance of ER-mitochondrial crosstalk in TEC injury and the salient role of RTN1A-mediated ER-mitochondrial contact regulation in DKD progression.
Assuntos
Diabetes Mellitus , Nefropatias Diabéticas , Retículo Endoplasmático , Mitocôndrias , Proteínas do Tecido Nervoso , Albuminúria/metabolismo , Animais , Apoptose , Diabetes Mellitus/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Células Epiteliais/metabolismo , Hexoquinase/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/genéticaRESUMO
BACKGROUND & AIMS: Given that gastrointestinal (GI) symptoms are a prominent extrapulmonary manifestation of COVID-19, we investigated intestinal infection with SARS-CoV-2, its effect on pathogenesis, and clinical significance. METHODS: Human intestinal biopsy tissues were obtained from patients with COVID-19 (n = 19) and uninfected control individuals (n = 10) for microscopic examination, cytometry by time of flight analyses, and RNA sequencing. Additionally, disease severity and mortality were examined in patients with and without GI symptoms in 2 large, independent cohorts of hospitalized patients in the United States (N = 634) and Europe (N = 287) using multivariate logistic regressions. RESULTS: COVID-19 case patients and control individuals in the biopsy cohort were comparable for age, sex, rates of hospitalization, and relevant comorbid conditions. SARS-CoV-2 was detected in small intestinal epithelial cells by immunofluorescence staining or electron microscopy in 15 of 17 patients studied. High-dimensional analyses of GI tissues showed low levels of inflammation, including down-regulation of key inflammatory genes including IFNG, CXCL8, CXCL2, and IL1B and reduced frequencies of proinflammatory dendritic cells compared with control individuals. Consistent with these findings, we found a significant reduction in disease severity and mortality in patients presenting with GI symptoms that was independent of sex, age, and comorbid illnesses and despite similar nasopharyngeal SARS-CoV-2 viral loads. Furthermore, there was reduced levels of key inflammatory proteins in circulation in patients with GI symptoms. CONCLUSIONS: These data highlight the absence of a proinflammatory response in the GI tract despite detection of SARS-CoV-2. In parallel, reduced mortality in patients with COVID-19 presenting with GI symptoms was observed. A potential role of the GI tract in attenuating SARS-CoV-2-associated inflammation needs to be further examined.
Assuntos
COVID-19/virologia , Gastroenteropatias/virologia , Imunidade nas Mucosas , Mucosa Intestinal/virologia , SARS-CoV-2/patogenicidade , Idoso , Idoso de 80 Anos ou mais , COVID-19/diagnóstico , COVID-19/imunologia , COVID-19/mortalidade , Estudos de Casos e Controles , Células Cultivadas , Citocinas/sangue , Feminino , Gastroenteropatias/diagnóstico , Gastroenteropatias/imunologia , Gastroenteropatias/mortalidade , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/sangue , Mucosa Intestinal/imunologia , Itália , Masculino , Pessoa de Meia-Idade , Cidade de Nova Iorque , Prognóstico , Medição de Risco , Fatores de Risco , SARS-CoV-2/imunologia , Carga ViralRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated clinical syndrome COVID-19 are causing overwhelming morbidity and mortality around the globe and disproportionately affected New York City between March and May 2020. Here, we report on the first 100 COVID-19-positive autopsies performed at the Mount Sinai Hospital in New York City. Autopsies revealed large pulmonary emboli in six cases. Diffuse alveolar damage was present in over 90% of cases. We also report microthrombi in multiple organ systems including the brain, as well as hemophagocytosis. We additionally provide electron microscopic evidence of the presence of the virus in our samples. Laboratory results of our COVID-19 cohort disclose elevated inflammatory markers, abnormal coagulation values, and elevated cytokines IL-6, IL-8, and TNFα. Our autopsy series of COVID-19-positive patients reveals that this disease, often conceptualized as a primarily respiratory viral illness, has widespread effects in the body including hypercoagulability, a hyperinflammatory state, and endothelial dysfunction. Targeting of these multisystemic pathways could lead to new treatment avenues as well as combination therapies against SARS-CoV-2 infection.
Assuntos
COVID-19/fisiopatologia , Pulmão/fisiopatologia , Embolia Pulmonar/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Autopsia , Coagulação Sanguínea , COVID-19/sangue , COVID-19/patologia , COVID-19/virologia , Causas de Morte , Citocinas/sangue , Feminino , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/sangue , Pulmão/patologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Cidade de Nova Iorque , Embolia Pulmonar/sangue , Embolia Pulmonar/patologia , Embolia Pulmonar/virologia , SARS-CoV-2/patogenicidadeRESUMO
BACKGROUND: Maintenance of the intricate interdigitating morphology of podocytes is crucial for glomerular filtration. One of the key aspects of specialized podocyte morphology is the segregation and organization of distinct cytoskeletal filaments into different subcellular components, for which the exact mechanisms remain poorly understood. METHODS: Cells from rats, mice, and humans were used to describe the cytoskeletal configuration underlying podocyte structure. Screening the time-dependent proteomic changes in the rat puromycin aminonucleoside-induced nephropathy model correlated the actin-binding protein LIM-nebulette strongly with glomerular function. Single-cell RNA sequencing and immunogold labeling were used to determine Nebl expression specificity in podocytes. Automated high-content imaging, super-resolution microscopy, atomic force microscopy (AFM), live-cell imaging of calcium, and measurement of motility and adhesion dynamics characterized the physiologic role of LIM-nebulette in podocytes. RESULTS: Nebl knockout mice have increased susceptibility to adriamycin-induced nephropathy and display morphologic, cytoskeletal, and focal adhesion abnormalities with altered calcium dynamics, motility, and Rho GTPase activity. LIM-nebulette expression is decreased in diabetic nephropathy and FSGS patients at both the transcript and protein level. In mice, rats, and humans, LIM-nebulette expression is localized to primary, secondary, and tertiary processes of podocytes, where it colocalizes with focal adhesions as well as with vimentin fibers. LIM-nebulette shRNA knockdown in immortalized human podocytes leads to dysregulation of vimentin filament organization and reduced cellular elasticity as measured by AFM indentation. CONCLUSIONS: LIM-nebulette is a multifunctional cytoskeletal protein that is critical in the maintenance of podocyte structural integrity through active reorganization of focal adhesions, the actin cytoskeleton, and intermediate filaments.
Assuntos
Actinas/fisiologia , Filamentos Intermediários/fisiologia , Nefropatias/patologia , Glomérulos Renais/patologia , Podócitos/patologia , Vimentina/fisiologia , Animais , Técnicas de Cultura de Células , Proteínas do Citoesqueleto/fisiologia , Humanos , Nefropatias/etiologia , Proteínas com Domínio LIM/fisiologia , Camundongos , RatosRESUMO
Extracellular vesicles (EVs) have recently emerged as an important carrier for various genetic materials including microRNAs (miRs). Growing evidences suggested that several miRs transported by EVs were particularly involved in modulating cardiac function. However, it has remained unclear what miRs are enriched in EVs and play an important role in the pathological condition. Therefore, we established the miR expression profiles in EVs from murine normal and failing hearts and consecutively identified substantially altered miRs. In addition, we have performed bioinformatics approach to predict potential cardiac outcomes through the identification of miR targets. Conclusively, we observed approximately 63% of predicted targets were validated with previous reports. Notably, the predicted targets by this approach were often involved in both beneficial and malicious signalling pathways, which may reflect heterogeneous cellular origins of EVs in tissues. Lastly, there has been an active debate on U6 whether it is a proper control. Through further analysis of EV miR profiles, miR-676 was identified as a superior reference control due to its consistent and abundant expressions. In summary, our results contribute to identifying specific EV miRs for the potential therapeutic targets in heart failure and suggest that miR-676 as a new reference control for the EV miR studies.
Assuntos
Vesículas Extracelulares/genética , Perfilação da Expressão Gênica , Insuficiência Cardíaca/genética , MicroRNAs/genética , Animais , Regulação para Baixo/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestrutura , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Proteômica , Reprodutibilidade dos Testes , Regulação para Cima/genéticaRESUMO
Neurologic sequelae can be devastating complications of respiratory viral infections. We report the presence of virus in neural and capillary endothelial cells in frontal lobe tissue obtained at postmortem examination from a patient infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Our observations of virus in neural tissue, in conjunction with clinical correlates of worsening neurologic symptoms, pave the way to a closer understanding of the pathogenic mechanisms underlying central nervous system involvement by SARS-CoV-2.
Assuntos
Ageusia/diagnóstico , Ataxia/diagnóstico , Betacoronavirus/patogenicidade , Infecções por Coronavirus/diagnóstico , Transtornos do Olfato/diagnóstico , Pneumonia Viral/diagnóstico , Convulsões/diagnóstico , Idoso , Ageusia/complicações , Ageusia/fisiopatologia , Ageusia/virologia , Ataxia/complicações , Ataxia/fisiopatologia , Ataxia/virologia , Betacoronavirus/genética , COVID-19 , Teste para COVID-19 , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/complicações , Infecções por Coronavirus/fisiopatologia , Infecções por Coronavirus/virologia , Células Endoteliais/patologia , Células Endoteliais/virologia , Evolução Fatal , Lobo Frontal/irrigação sanguínea , Lobo Frontal/patologia , Lobo Frontal/virologia , Hospitalização , Humanos , Pulmão/irrigação sanguínea , Pulmão/patologia , Pulmão/virologia , Masculino , Neurônios/patologia , Neurônios/virologia , Transtornos do Olfato/complicações , Transtornos do Olfato/fisiopatologia , Transtornos do Olfato/virologia , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/fisiopatologia , Pneumonia Viral/virologia , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2 , Convulsões/complicações , Convulsões/fisiopatologia , Convulsões/virologiaRESUMO
AIMS: Myocardial fibrosis is associated with profound changes in ventricular architecture and geometry, resulting in diminished cardiac function. There is currently no information on the role of the delta-like homologue 1 (Dlk1) in the regulation of the fibrotic response. Here, we investigated whether Dlk1 is involved in cardiac fibroblast-to-myofibroblast differentiation and regulates myocardial fibrosis and explored the molecular mechanism underpinning its effects in this process. METHODS AND RESULTS: Using Dlk1-knockout mice and adenoviral gene delivery, we demonstrate that overexpression of Dlk1 in cardio-fibroblasts resulted in inhibition of fibroblast proliferation and differentiation into myofibroblasts. This process is mediated by TGF-ß1 signalling, since isolated fibroblasts lacking Dlk1 exhibited a higher activation of the TGF-ß1/Smad-3 pathway at baseline, leading to an earlier acquisition of a myofibroblast phenotype. Likewise, Dlk1-null mice displayed increased TGF-ß1/Smad3 cardiac activity, resulting in infiltration/accumulation of myofibroblasts, induction and deposition of extra-domain A-fibronectin isoform and collagen, and activation of pro-fibrotic markers. Furthermore, these profibrotic events were associated with disrupted myofibril integrity, myocyte hypertrophy, and cardiac dysfunction. Interestingly, Dlk1 expression was down-regulated in ischaemic human and porcine heart tissues. Mechanistically, miR-370 mediated Dlk1's regulation of cardiac fibroblast-myofibroblast differentiation by directly targeting TGFß-R2/Smad-3 signalling, while the Dlk1 canonical target, Notch pathway, does not seem to play a role in this process. CONCLUSION: These findings are the first to demonstrate an inhibitory role of Dlk1 of cardiac fibroblast-to-myofibroblast differentiation by interfering with TGFß/Smad-3 signalling in the myocardium. Given the deleterious effects of continuous activation of this pathway, we propose Dlk1 as a new potential candidate for therapy in cases where aberrant TGFß signalling leads to chronic fibrosis.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Fibroblastos/metabolismo , Fibrose/genética , Miocárdio/patologia , Miofibroblastos/metabolismo , Animais , Diferenciação Celular , Regulação para Baixo , Humanos , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/metabolismo , Proteína Smad3/genética , Suínos , Fator de Crescimento Transformador beta1/genéticaRESUMO
Primary cilia have been linked to signaling pathways involved in cell proliferation, cell motility and cell polarity. Defects in ciliary function result in developmental abnormalities and multiple ciliopathies. Patients affected by severe ciliopathies, such as Meckel syndrome, present several ocular surface disease conditions of unclear pathogenesis. Here, we show that primary cilia are predominantly present on basal cells of the mouse corneal epithelium (CE) throughout development and in the adult. Conditional ablation of cilia in the CE leads to an increase in proliferation and vertical migration of basal corneal epithelial cells (CECs). A consequent increase in cell density of suprabasal layers results in a thicker than normal CE. Surprisingly, in cilia-deficient CE, cilia-mediated signaling pathways, including Hh and Wnt pathways, were not affected but the intensity of Notch signaling was severely diminished. Although Notch1 and Notch2 receptors were expressed normally, nuclear Notch1 intracellular domain (N1ICD) expression was severely reduced. Postnatal development analysis revealed that in cilia-deficient CECs downregulation of the Notch pathway precedes cell proliferation defects. Thus, we have uncovered a function of the primary cilium in maintaining homeostasis of the CE by balancing proliferation and vertical migration of basal CECs through modulation of Notch signaling.
Assuntos
Cílios/metabolismo , Epitélio Corneano/metabolismo , Homeostase , Receptor Notch1/metabolismo , Receptor Notch2/metabolismo , Transdução de Sinais , Animais , Animais Recém-Nascidos , Movimento Celular , Proliferação de Células , Cílios/ultraestrutura , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Epitélio Corneano/ultraestrutura , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Proteínas Supressoras de Tumor/metabolismo , CicatrizaçãoRESUMO
BACKGROUND: Platelet (PLT) transfusions are the most effective treatments for patients with thrombocytopenia. The growing demand for PLT transfusion products is compounded by a limited supply due to dependency on volunteer donors, a short shelf-life, risk of contaminating pathogens, and alloimmunization. This study provides preclinical evidence that a third-party, cryopreservable source of PLT-generating cells has the potential to complement presently available PLT transfusion products. STUDY DESIGN AND METHODS: CD34+ hematopoietic stem/progenitor cells derived from umbilical cord blood (UCB) units were used in a simple and efficient culture system to generate a cell product consisting of megakaryocytes (MKs) at different stages of development. The cultures thus generated were evaluated ex vivo and in vivo before and after cryopreservation. RESULTS: We generated a megakaryocytic cell product that can be cryopreserved without altering its phenotypical and functional capabilities. The infusion of such a product, either fresh or cryopreserved, into immune-deficient mice led to production of functional human PLTs which were observed within a week after infusion and persisted for 8 weeks, orders of magnitude longer than that observed after the infusion of traditional PLT transfusion products. The sustained human PLT engraftment was accompanied by a robust presence of human cells in the bone marrow (BM), spleen, and lungs of recipient mice. CONCLUSION: This is a proof-of-principle study demonstrating the creation of a cryopreservable megakaryocytic cell product which releases functional PLTs in vivo. Clinical development of such a product is currently being pursued for the treatment of thrombocytopenia in patients with hematological malignancies.
Assuntos
Plaquetas/metabolismo , Criopreservação , Megacariócitos/citologia , Transfusão de Plaquetas/métodos , Animais , Antígenos CD34/metabolismo , Células Cultivadas , Feminino , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Camundongos , Trombocitopenia/terapiaRESUMO
Kidney podocytes represent a key constituent of the glomerular filtration barrier. Identifying the molecular mechanisms of podocyte injury and survival is important for better understanding and management of kidney diseases. KIBRA (kidney brain protein), an upstream regulator of the Hippo signaling pathway encoded by the Wwc1 gene, shares the pro-injury properties of its putative binding partner dendrin and antagonizes the pro-survival signaling of the downstream Hippo pathway effector YAP (Yes-associated protein) in Drosophila and MCF10A cells. We recently identified YAP as an essential component of the glomerular filtration barrier that promotes podocyte survival by inhibiting dendrin pro-apoptotic function. Despite these recent advances, the signaling pathways that mediate podocyte injury remain poorly understood. Here we tested the hypothesis that, similar to its role in other model systems, KIBRA promotes podocyte injury. We found increased expression of KIBRA and phosphorylated YAP protein in glomeruli of patients with biopsy-proven focal segmental glomerulosclerosis (FSGS). KIBRA/WWc1 overexpression in murine podocytes promoted LATS kinase phosphorylation, leading to subsequent YAP Ser-127 phosphorylation, YAP cytoplasmic sequestration, and reduction in YAP target gene expression. Functionally, KIBRA overexpression induced significant morphological changes in podocytes, including disruption of the actin cytoskeletal architecture and reduction of focal adhesion size and number, all of which were rescued by subsequent YAP overexpression. Conversely, constitutive KIBRA knockout mice displayed reduced phosphorylated YAP and increased YAP expression at baseline. These mice were protected from acute podocyte foot process effacement following protamine sulfate perfusion. KIBRA knockdown podocytes were also protected against protamine-induced injury. These findings suggest an important role for KIBRA in the pathogenesis of podocyte injury and the progression of proteinuric kidney disease.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Glomerulosclerose Segmentar e Focal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoproteínas/metabolismo , Podócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Animais , Biópsia , Feminino , Regulação da Expressão Gênica , Glomerulosclerose Segmentar e Focal/enzimologia , Glomerulosclerose Segmentar e Focal/patologia , Células HEK293 , Via de Sinalização Hippo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosforilação , Podócitos/patologia , Podócitos/ultraestrutura , Processamento de Proteína Pós-Traducional , Interferência de RNA , Serina/metabolismo , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
Virological synapses (VS) are adhesive structures that form between infected and uninfected cells to enhance the spread of HIV-1. During T cell VS formation, viral proteins are actively recruited to the site of cell-cell contact where the viral material is efficiently translocated to target cells into heterogeneous, protease-resistant, antibody-inaccessible compartments. Using correlative light and electron microscopy (CLEM), we define the membrane topography of the virus-containing compartments (VCC) where HIV is found following VS-mediated transfer. Focused ion beam scanning electron microscopy (FIB-SEM) and serial sectioning transmission electron microscopy (SS-TEM) were used to better resolve the fluorescent Gag-containing structures within the VCC. We found that small punctate fluorescent signals correlated with single viral particles in enclosed vesicular compartments or surface-localized virus particles and that large fluorescent signals correlated with membranous Gag-containing structures with unknown pathological function. CLEM imaging revealed distinct pools of newly deposited viral proteins within endocytic and nonendocytic compartments in VS target T cells. IMPORTANCE: This study directly correlates individual virus-associated objects observed in light microscopy with ultrastructural features seen by electron microscopy in the HIV-1 virological synapse. This approach elucidates which infection-associated ultrastructural features represent bona fide HIV protein complexes. We define the morphology of some HIV cell-to-cell transfer intermediates as true endocytic compartments and resolve unique synapse-associated viral structures created by transfer across virological synapses.
Assuntos
Linfócitos T CD4-Positivos/ultraestrutura , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , HIV-1/ultraestrutura , Interações Hospedeiro-Patógeno , Humanos , Vírion/fisiologia , Internalização do Vírus , Replicação Viral , Desenvelopamento do VírusRESUMO
Venous thromboembolism (VTE) is a significant problem in the perioperative period, increasing patient morbidity, mortality, and health care costs. It is also considered the most preventable of the major postoperative complications. Despite widespread adoption of prophylaxis guidelines, it appears that morbidity from the disease has not substantially changed within the past 2 decades. It is becoming clear that current prophylaxis efforts are not sufficient. Using more potent anticoagulants may decrease the incidence of VTE, but increase the risk for bleeding and infection. Much has been learned about the pathophysiology of venous thrombogenesis in recent years. Beyond the "traditional coagulation cascade," which anticoagulants modulate, there is a growing appreciation for the roles of tissue factor, monocytes, neutrophils, neutrophil extracellular traps, microvesicles, and platelets in thrombus initiation and propagation. These recent studies explain to some degree why aspirin appears to be remarkably effective in preventing thrombus propagation. Endothelial dysfunction, traditionally thought of as a risk factor for arterial thrombosis, plays an important role within the cusps of venous valves, a unique environment where the majority of venous thrombi originate. This suggests a role for newer treatment modalities such as statins. Not all patients have an equal likelihood of experiencing a VTE, even when undergoing high-risk procedures, and better tools are required to accurately predict VTE risk. Only then will we be able to effectively individualize prophylaxis by balancing the risks for VTE against the risks associated with treatment. Given the different cell types and pathways involved in thrombogenesis, it is likely that multimodal treatment regimens will be more effective, enabling the use of lower and safer doses of hemostatic modulating therapies such as anticoagulants, antithrombotics, and antiplatelet medications.
Assuntos
Anticoagulantes/efeitos adversos , Hemorragia/induzido quimicamente , Período Perioperatório , Embolia Pulmonar/diagnóstico , Tromboembolia Venosa/diagnóstico , Anticoagulantes/administração & dosagem , Anticoagulantes/uso terapêutico , Aspirina/administração & dosagem , Fibrinolíticos/uso terapêutico , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Hipóxia , Incidência , Lidocaína/administração & dosagem , Inibidores da Agregação Plaquetária/uso terapêutico , Complicações Pós-Operatórias/epidemiologia , Embolia Pulmonar/complicações , Medição de Risco , Fatores de Risco , Trombose/tratamento farmacológico , Tromboembolia Venosa/complicaçõesRESUMO
Therapeutic and diagnostic nanomaterials are being intensely studied for several diseases, including cancer and atherosclerosis. However, the exact mechanism by which nanomedicines accumulate at targeted sites remains a topic of investigation, especially in the context of atherosclerotic disease. Models to accurately predict transvascular permeation of nanomedicines are needed to aid in design optimization. Here we show that an endothelialized microchip with controllable permeability can be used to probe nanoparticle translocation across an endothelial cell layer. To validate our in vitro model, we studied nanoparticle translocation in an in vivo rabbit model of atherosclerosis using a variety of preclinical and clinical imaging methods. Our results reveal that the translocation of lipid-polymer hybrid nanoparticles across the atherosclerotic endothelium is dependent on microvascular permeability. These results were mimicked with our microfluidic chip, demonstrating the potential utility of the model system.
Assuntos
Aterosclerose/fisiopatologia , Sistemas de Liberação de Medicamentos , Endotélio/metabolismo , Nanopartículas Metálicas/química , Animais , Aterosclerose/tratamento farmacológico , Modelos Animais de Doenças , Ouro/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Imageamento por Ressonância Magnética , Masculino , Microcirculação , Microfluídica , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Modelos Teóricos , Permeabilidade , Placa Aterosclerótica , Coelhos , Resistência ao CisalhamentoRESUMO
Acute intermittent porphyria (AIP) is an autosomal-dominant hepatic disorder caused by the half-normal activity of hydroxymethylbilane (HMB) synthase. Symptomatic individuals experience life-threatening acute neurovisceral attacks that are precipitated by factors that induce the hepatic expression of 5-aminolevulinic acid synthase 1 (ALAS1), resulting in the marked accumulation of the putative neurotoxic porphyrin precursors 5-aminolevulinic acid (ALA) and porphobilinogen (PBG). Here, we provide the first detailed description of the biochemical and pathologic alterations in the explanted liver of an AIP patient who underwent orthotopic liver transplantation (OLT) due to untreatable and debilitating chronic attacks. After OLT, the recipient's plasma and urinary ALA and PBG rapidly normalized, and her attacks immediately stopped. In the explanted liver, (a) ALAS1 mRNA and activity were elevated approximately ~3- and 5-fold, and ALA and PBG concentrations were increased ~3- and 1,760-fold, respectively; (b) uroporphyrin III concentration was elevated; (c) microsomal heme content was sufficient, and representative cytochrome P450 activities were essentially normal; (d) HMB synthase activity was approximately half-normal (~42%); (e) iron concentration was slightly elevated; and (f) heme oxygenase I mRNA was increased approximately three-fold. Notable pathologic findings included nodular regenerative hyperplasia, previously not reported in AIP livers, and minimal iron deposition, despite the large number of hemin infusions received before OLT. These findings suggest that the neurovisceral symptoms of AIP are not associated with generalized hepatic heme deficiency and support the neurotoxicity of ALA and/or PBG. Additionally, they indicate that substrate inhibition of hepatic HMB synthase activity by PBG is not a pathogenic mechanism in acute attacks.
Assuntos
5-Aminolevulinato Sintetase/genética , Hidroximetilbilano Sintase/biossíntese , Fígado/metabolismo , Porfiria Aguda Intermitente/genética , 5-Aminolevulinato Sintetase/biossíntese , Adulto , Ácido Aminolevulínico/sangue , Ácido Aminolevulínico/urina , Feminino , Heme/metabolismo , Humanos , Hidroximetilbilano Sintase/antagonistas & inibidores , Fígado/patologia , Transplante de Fígado , Porfobilinogênio/sangue , Porfobilinogênio/urina , Porfiria Aguda Intermitente/enzimologia , Porfiria Aguda Intermitente/patologia , RNA Mensageiro/biossíntese , Uroporfirinas/metabolismoRESUMO
Cardiac experimental biology and translational research would benefit from an in vitro surrogate for human heart muscle. This study investigated structural and functional properties and interventional responses of human engineered cardiac tissues (hECTs) compared to human myocardium. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs, >90% troponin-positive) were mixed with collagen and cultured on force-sensing elastomer devices. hECTs resembled trabecular muscle and beat spontaneously (1.18 ± 0.48 Hz). Microstructural features and mRNA expression of cardiac-specific genes (α-MHC, SERCA2a, and ACTC1) were comparable to human myocardium. Optical mapping revealed cardiac refractoriness with loss of 1:1 capture above 3 Hz, and cycle length dependence of the action potential duration, recapitulating key features of cardiac electrophysiology. hECTs reconstituted the Frank-Starling mechanism, generating an average maximum twitch stress of 660 µN/mm(2) at Lmax, approaching values in newborn human myocardium. Dose-response curves followed exponential pharmacodynamics models for calcium chloride (EC50 1.8 mM) and verapamil (IC50 0.61 µM); isoproterenol elicited a positive chronotropic but negligible inotropic response, suggesting sarcoplasmic reticulum immaturity. hECTs were amenable to gene transfer, demonstrated by successful transduction with Ad.GFP. Such 3-D hECTs recapitulate an early developmental stage of human myocardium and promise to offer an alternative preclinical model for cardiology research.
Assuntos
Miocárdio/citologia , Engenharia Tecidual/métodos , Linhagem Celular , Eletrofisiologia , HumanosRESUMO
The role of the anesthesiologist in reducing the incidence of surgical-site infections by the administration of prophylactic parenteral beta-lactam antibiotics is reviewed. Suggestions are made with regard to timing, dosing, and method of administration of these drugs to potentially reduce the risk of surgical-site infection.