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The assembly of organized colonies is the earliest manifestation in the derivation or induction of pluripotency in vitro. However, the necessity and origin of this assemblance is unknown. Here, we identify human pluripotent founder cells (hPFCs) that initiate, as well as preserve and establish, pluripotent stem cell (PSC) cultures. PFCs are marked by N-cadherin expression (NCAD+) and reside exclusively at the colony boundary of primate PSCs. As demonstrated by functional analysis, hPFCs harbor the clonogenic capacity of PSC cultures and emerge prior to commitment events or phenotypes associated with pluripotent reprogramming. Comparative single-cell analysis with pre- and post-implantation primate embryos revealed hPFCs share hallmark properties with primitive endoderm (PrE) and can be regulated by non-canonical Wnt signaling. Uniquely informed by primate embryo organization in vivo, our study defines a subset of founder cells critical to the establishment pluripotent state.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Linhagem da Célula , Desenvolvimento Embrionário , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Análise de Célula Única , Via de Sinalização WntRESUMO
Transcription factors (TFs) are thought to function with partners to achieve specificity and precise quantitative outputs. In the developing heart, heterotypic TF interactions, such as between the T-box TF TBX5 and the homeodomain TF NKX2-5, have been proposed as a mechanism for human congenital heart defects. We report extensive and complex interdependent genomic occupancy of TBX5, NKX2-5, and the zinc finger TF GATA4 coordinately controlling cardiac gene expression, differentiation, and morphogenesis. Interdependent binding serves not only to co-regulate gene expression but also to prevent TFs from distributing to ectopic loci and activate lineage-inappropriate genes. We define preferential motif arrangements for TBX5 and NKX2-5 cooperative binding sites, supported at the atomic level by their co-crystal structure bound to DNA, revealing a direct interaction between the two factors and induced DNA bending. Complex interdependent binding mechanisms reveal tightly regulated TF genomic distribution and define a combinatorial logic for heterotypic TF regulation of differentiation.
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Fator de Transcrição GATA4/metabolismo , Proteínas de Homeodomínio/metabolismo , Miocárdio/citologia , Organogênese , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Cristalografia por Raios X , Embrião de Mamíferos/metabolismo , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Miocárdio/metabolismo , Regiões Promotoras Genéticas , Domínios e Motivos de Interação entre Proteínas , Proteínas com Domínio T/genética , Fatores de Transcrição/genéticaRESUMO
Vascular endothelial growth factor and its receptors, FLK1/KDR and FLT1, are key regulators of angiogenesis. Unlike FLK1/KDR, the role of FLT1 has remained elusive. FLT1 is produced as soluble (sFLT1) and full-length isoforms. Here, we show that pericytes from multiple tissues produce sFLT1. To define the biologic role of sFLT1, we chose the glomerular microvasculature as a model system. Deletion of Flt1 from specialized glomerular pericytes, known as podocytes, causes reorganization of their cytoskeleton with massive proteinuria and kidney failure, characteristic features of nephrotic syndrome in humans. The kinase-deficient allele of Flt1 rescues this phenotype, demonstrating dispensability of the full-length isoform. Using cell imaging, proteomics, and lipidomics, we show that sFLT1 binds to the glycosphingolipid GM3 in lipid rafts on the surface of podocytes, promoting adhesion and rapid actin reorganization. sFLT1 also regulates pericyte function in vessels outside of the kidney. Our findings demonstrate an autocrine function for sFLT1 to control pericyte behavior.
Assuntos
Glomérulos Renais/citologia , Glomérulos Renais/metabolismo , Podócitos/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Gangliosídeos/metabolismo , Humanos , Técnicas In Vitro , Metabolismo dos Lipídeos , Lipídeos/química , Camundongos , Camundongos Transgênicos , Pericitos/metabolismo , Proteinúria/metabolismo , Transdução de Sinais , Sindecanas/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Alternative splicing (AS) is a key process underlying the expansion of proteomic diversity and the regulation of gene expression. Here, we identify an evolutionarily conserved embryonic stem cell (ESC)-specific AS event that changes the DNA-binding preference of the forkhead family transcription factor FOXP1. We show that the ESC-specific isoform of FOXP1 stimulates the expression of transcription factor genes required for pluripotency, including OCT4, NANOG, NR5A2, and GDF3, while concomitantly repressing genes required for ESC differentiation. This isoform also promotes the maintenance of ESC pluripotency and contributes to efficient reprogramming of somatic cells into induced pluripotent stem cells. These results reveal a pivotal role for an AS event in the regulation of pluripotency through the control of critical ESC-specific transcriptional programs.
Assuntos
Processamento Alternativo , Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes/metabolismo , Proteínas Repressoras/metabolismo , Animais , DNA/metabolismo , Células-Tronco Embrionárias/citologia , Genes Homeobox , Humanos , Camundongos , Células-Tronco Pluripotentes/citologia , Isoformas de Proteínas/metabolismoRESUMO
Biological processes throughout the body are orchestrated in time through the regulation of local circadian clocks. The retina is among the most metabolically active tissues, with demands depending greatly on the light/dark cycle. Most cell types within the retina are known to express the circadian clock in rodents, however, retinal clock expression in the human has not previously been localized. Moreover, the effect of local circadian clock dysfunction on retinal homeostasis is incompletely understood. We demonstrate an age-dependent decline in circadian clock gene and protein expression in the human retina. Using an animal model of targeted Bmal1 deficiency, we identify the circadian clock of the retinal Müller glia as essential for neuronal survival, vascular integrity, and retinal function. These results suggest a potential role for the local retinal circadian clock within the Müller glia in age-related retinal disease and retinal degeneration.
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Revealing the molecular events associated with reprogramming different somatic cell types to pluripotency is critical for understanding the characteristics of induced pluripotent stem cell (iPSC) therapeutic derivatives. Inducible reprogramming factor transgenic cells or animals-designated as secondary (2°) reprogramming systems-not only provide excellent experimental tools for such studies but also offer a strategy to study the variances in cellular reprogramming outcomes due to different in vitro and in vivo environments. To make such studies less cumbersome, it is desirable to have a variety of efficient reprogrammable mouse systems to induce successful mass reprogramming in somatic cell types. Here, we report the development of two transgenic mouse lines from which 2° cells reprogram with unprecedented efficiency. These systems were derived by exposing primary reprogramming cells containing doxycycline-inducible Yamanaka factor expression to a transient interruption in transgene expression, resulting in selection for a subset of clones with robust transgene response. These systems also include reporter genes enabling easy readout of endogenous Oct4 activation (GFP), indicative of pluripotency, and reprogramming transgene expression (mCherry). Notably, somatic cells derived from various fetal and adult tissues from these 2° mouse lines gave rise to highly efficient and rapid reprogramming, with transgene-independent iPSC colonies emerging as early as 1 wk after induction. These mouse lines serve as a powerful tool to explore sources of variability in reprogramming and the mechanistic underpinnings of efficient reprogramming systems.
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Reprogramação Celular , Doxiciclina , Animais , Camundongos , Camundongos Transgênicos , Reprogramação Celular/genética , Transgenes , Células Clonais , Doxiciclina/farmacologiaRESUMO
PURPOSE: Facilitated subcutaneous immunoglobulin (fSCIG; immune globulin infusion 10% [human] with recombinant human hyaluronidase [rHuPH20]) permits high-volume subcutaneous immunoglobulin (SCIG) infusion, shorter infusion times and reduced dosing frequency relative to conventional SCIG. It is initiated by gradually increasing infusion volumes over time (dose ramp-up) to achieve target dose level (TDL). Whether ramp-up strategies have tolerability or safety advantages over direct initiation at full TDL has not been evaluated clinically. METHODS: This phase 1 open-label study assessed tolerability and safety of fSCIG 10% with accelerated or no ramp-up compared with conventional ramp-up in healthy adults (NCT04578535). Participants were assigned to one of the three ramp-up arms to achieve TDLs of 0.4 or 1.0 g/kg/infusion. The primary endpoint was the proportion of infusions completed without interruption or infusion rate reduction owing to treatment-emergent adverse events (TEAEs). Safety was assessed as a secondary endpoint. RESULTS: Of 51 participants enrolled, 50 (98.0%) tolerated all fSCIG 10% infusions initiated (n = 174). Infusion rate was reduced in one participant owing to headache in the 0.4 g/kg/infusion conventional ramp-up arm. Study discontinuations were higher in the no ramp-up arm (70%) versus the conventional (0%) and accelerated (22%) arms at the 1.0 g/kg/infusion TDL. Safety outcomes did not substantially differ between treatment arms. CONCLUSION: The favorable tolerability and safety profiles of fSCIG 10% in healthy participants support initiating treatment with fSCIG 10% with accelerated ramp-up at TDLs up to 1.0 g/kg. Data support no ramp-up at TDLs close to 0.4 g/kg but additional data are needed for higher doses.
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Voluntários Saudáveis , Hialuronoglucosaminidase , Infusões Subcutâneas , Humanos , Hialuronoglucosaminidase/administração & dosagem , Hialuronoglucosaminidase/efeitos adversos , Masculino , Feminino , Adulto , Adulto Jovem , Pessoa de Meia-Idade , Imunoglobulinas/administração & dosagem , Imunoglobulinas/efeitos adversos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/efeitos adversos , AdolescenteRESUMO
While oncotherapy has made rapid progress in recent years, side effects of anti-cancer drugs and treatments have also come to the fore. These side effects include cardiotoxicity, which can cause irreversible cardiac damages with long-term morbidity and mortality. Despite the continuous in-depth research on anti-cancer drugs, an improved knowledge of the underlying mechanisms of cardiotoxicity are necessary for early detection and management of cardiac risk. Although most reviews focus on the cardiotoxic effect of a specific individual chemotherapeutic agent, the aim of our review is to provide comprehensive insight into various agents that induced cardiotoxicity and their underlying mechanisms. Characterization of these mechanisms are underpinned by research on animal models and clinical studies. In order to gain insight into these complex mechanisms, we emphasize the role of inflammatory processes and oxidative stress on chemotherapy-induced cardiac changes. A better understanding and identification of the interplay between chemotherapy and inflammatory/oxidative processes hold some promise to prevent or at least mitigate cardiotoxicity-associated morbidity and mortality among cancer survivors.
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Human pluripotent cell lines hold enormous promise for the development of cell-based therapies. Safety, however, is a crucial prerequisite condition for clinical applications. Numerous groups have attempted to eliminate potentially harmful cells through the use of suicide genes1, but none has quantitatively defined the safety level of transplant therapies. Here, using genome-engineering strategies, we demonstrate the protection of a suicide system from inactivation in dividing cells. We created a transcriptional link between the suicide gene herpes simplex virus thymidine kinase (HSV-TK) and a cell-division gene (CDK1); this combination is designated the safe-cell system. Furthermore, we used a mathematical model to quantify the safety level of the cell therapy as a function of the number of cells that is needed for the therapy and the type of genome editing that is performed. Even with the highly conservative estimates described here, we anticipate that our solution will rapidly accelerate the entry of cell-based medicine into the clinic.
Assuntos
Proteína Quinase CDC2/genética , Divisão Celular/genética , Terapia Baseada em Transplante de Células e Tecidos/métodos , Genes Transgênicos Suicidas/genética , Segurança do Paciente , Animais , Proliferação de Células , Terapia Baseada em Transplante de Células e Tecidos/normas , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Ganciclovir/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simplexvirus/enzimologia , Simplexvirus/genética , Timidina Quinase/genética , Timidina Quinase/metabolismoRESUMO
Glucagon-like peptide-1 receptor agonists (GLP-1 RA) are novel antihyperglycemic agents. By acting through the central nervous system, they increase satiety and reduce food intake, thus lowering body weight. Furthermore, they increase the secretion of insulin while decreasing the production of glucagon. However, recent studies suggest a more complex metabolic impact through the interaction with various other tissues. In our present review, we aim to provide a summary of the effects of GLP-1 RA on serum lipids, adipose tissue, and muscle metabolism. It has been found that GLP-1 RA therapy is associated with decreased serum cholesterol levels. Epicardial adipose tissue thickness, hepatic lipid droplets, and visceral fat volume were reduced in obese patients with cardiovascular disease. GLP-1 RA therapy decreased the level of proinflammatory adipokines and reduced the expression of inflammatory genes. They have been found to reduce endoplasmic reticulum stress in adipocytes, leading to better adipocyte function and metabolism. Furthermore, GLP-1 RA therapy increased microvascular blood flow in muscle tissue, resulting in increased myocyte metabolism. They inhibited muscle atrophy and increased muscle mass and function. It was also observed that the levels of muscle-derived inflammatory cytokines decreased, and insulin sensitivity increased, resulting in improved metabolism. However, some clinical trials have been conducted on a very small number of patients, which limits the strength of these observations.
Assuntos
Tecido Adiposo , Agonistas do Receptor do Peptídeo 1 Semelhante ao Glucagon , Animais , Humanos , Tecido Adiposo/metabolismo , Tecido Adiposo/efeitos dos fármacos , Agonistas do Receptor do Peptídeo 1 Semelhante ao Glucagon/farmacologia , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/sangue , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculos/metabolismo , Músculos/efeitos dos fármacosRESUMO
Rosmarinic acid is a well-known natural antioxidant and anti-inflammatory compound, and it is one of the polyphenolic compounds found in comfrey plants. Comfrey root also contains allantoin, which helps with new skin regeneration. This study aimed to investigate the healing and skin regeneration process of skin wounds in Wistar rats using creams based on comfrey extract and to correlate the results with active compounds in the extract. The obtained results showed that comfrey root is rich in bioactive compounds, including allantoin, salvianolic acid, and rosmarinic acid, which are known for their great free radical scavenging activity, and the high antioxidant activity of the extract may be mainly due to these compounds. The obtained extract has an antimicrobial effect on Staphylococcus aureus (1530.76/382.69), Escherichia coli (6123.01/6123.01), and Pseudomonas aeruginosa (6123.01/6123.01). The macroscopic evaluation and the histological analysis of the skin defects 14 days after the intervention showed faster healing and complete healing in the skin excisions treated with oil-in-water cream with 20% extract of comfrey as the active ingredient.
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Boraginaceae , Confrei , Ratos , Animais , Alantoína/farmacologia , Extratos Vegetais/farmacologia , Ratos Wistar , Cicatrização , Antioxidantes/farmacologiaRESUMO
PURPOSE: Hyaluronidase-facilitated subcutaneous immunoglobulin (fSCIG) 20% will allow reduced infusion volumes and frequency versus existing subcutaneous therapies such as fSCIG 10% and conventional subcutaneous immunoglobulin 20%, respectively. We assessed the tolerability, safety, and immunogenicity of warmed and unwarmed fSCIG 20%. METHODS: This phase 1, single-dose, open-label, three-arm study enrolled healthy adults aged 19-50 years (inclusive) at a single US center (NCT05059977). Post-screening, participants received a single fSCIG 20% dose comprising recombinant human hyaluronidase and varying doses of in-line warmed or unwarmed immunoglobulin G (IgG) during a 4-day treatment period in a sentinel and sequential dosing design (treatment arm 1, warmed IgG 20% 0.4 g/kg; treatment arm 2, warmed IgG 20% 1.0 g/kg; treatment arm 3, unwarmed IgG 20% 1.0 g/kg). Participants were followed for 12 (± 1) weeks post-infusion. The primary endpoint was tolerability ("tolerable" infusions were not interrupted, stopped, or reduced in rate owing to fSCIG 20%-related treatment-emergent adverse events (TEAEs)). Secondary endpoints included occurrence of TEAEs. RESULTS: Overall, 24 participants were included, 8 per treatment arm (mean age 39.0 years, 54.2% men). All participants tolerated the infusions. All TEAEs were mild (107 events, in all participants), and all participants experienced fSCIG 20%-related (105 events) and local (102 events) TEAEs. Infusion site erythema and infusion site swelling were most frequently reported. No serious TEAEs occurred, and no participants discontinued the study owing to TEAEs. CONCLUSION: fSCIG 20% was well-tolerated with a favorable safety profile in healthy adults. Future studies will evaluate fSCIG 20% in primary immunodeficiency diseases. Trial registration number (ClinicalTrials.gov): NCT05059977 (registered 28 September 2021).
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Hialuronoglucosaminidase , Imunoglobulina G , Masculino , Adulto , Humanos , Feminino , Hialuronoglucosaminidase/uso terapêutico , Imunoglobulina G/uso terapêutico , Injeções Subcutâneas , Infusões Subcutâneas , Protocolos ClínicosRESUMO
This research explores the determinants of vaccine hesitancy during the third wave of the COVID-19 pandemic in Hungary. This article utilizes data from in-person public opinion research conducted in Hungary (March 2021, N = 1000). Government supporters, older people (60 +) and COVID-19 survivors were more likely to accept vaccination, but these variables lose significance, once controlling for personal fears and pandemic-related attitudes. COVID-19 related fears and precautious behavior reduce, while general level of fears increase the probability of vaccine hesitancy. Fear from partner's aggression and higher levels of financial security negatively correlate with vaccine hesitancy. Our study separately analyzes the effect of various pandemic-related conspiratorial beliefs on vaccine hesitancy. All analyzed false beliefs have a significant positive effect on vaccine hesitancy, but the strongest predictors are vaccine-related conspiracy theories ("microchip" and "population control" theories) and virus denial.
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Vacinas contra COVID-19 , COVID-19 , Humanos , Idoso , COVID-19/prevenção & controle , Hungria , Pandemias , Agressão , VacinaçãoRESUMO
BACKGROUND: Percutaneous transhepatic gallbladder drainage (PTGBD) plays an important role in the treatment of elderly patients and/or patients in poor health with acute cholecystitis (AC). The primary aim of this study is to determine how these factors influence the clinical outcome of PTGBD. Moreover, we assessed the timing and results of subsequent cholecystectomies. PATIENTS AND METHODS: We retrospectively examined the results of 162 patients undergoing PTGBD between 2010 and 2020 (male-female ratio: 51.23% vs. 48.77%; mean age: 71.43 ± 13.22 years). Patient's performance status and intervention outcomes were assessed with clinical success rates (CSR) and in-hospital mortality. The conversion rate (CR) of possible urgent or delayed, elective laparoscopic cholecystectomies (LC) after PTGBD were analysed. RESULTS: PTGBD was the definitive treatment in 42.18% of patients, while it was a bridging therapy prior to cholecystectomy (CCY) for the other patients. CSR was 87.97%, it was only 64.29% in grade III AC. In 9.87% of the cases, urgent LC was necessary after PTGBD, and its conversion rate was approximately equal to that of elective LC (18.18 vs. 17.46%, respectively, p = 0.2217). Overall, the post-PTGBD in-hospital mortality was 11.72%, while the same figure was 0% for grade I AC, 7.41% for grade II and 40.91% for grade III. Based on logistic regression analyses, in-hospital mortality (OR 6.07; CI 1.79-20.56), clinical progression (OR 7.62; CI 2.64-22.05) and the need for emergency CCY (OR 14.75; CI 3.07-70.81) were mostly determined by AC severity grade. CONCLUSION: PTGBD is an easy-to-perform intervention with promising clinical success rates in the treatment of acute cholecystitis. After PTGBD, the level of gallbladder inflammation played a decisive role in the course of AC. In a severe, grade III inflammation, we have to consider low CSR and high mortality.
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Colecistectomia Laparoscópica , Colecistite Aguda , Idoso , Idoso de 80 Anos ou mais , Colecistectomia Laparoscópica/métodos , Colecistite Aguda/etiologia , Colecistite Aguda/cirurgia , Drenagem/métodos , Feminino , Vesícula Biliar/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Centros de Atenção Terciária , Resultado do TratamentoRESUMO
Kunitz (KTI) and Bowman-Birk (BBI) trypsin inhibitors were characterized in soybean seeds. Cultivars having KTI/BBI (Pannónia Kincse, PK) or lacking KTI (Aries; Hilario; Bahia) were assessed with well-characterized soybean varieties having Ti-a or ti types of KTI mobility. The TIA values of Pannónia Kincse (9.8 ± 0.48 mg/g) were not significantly different (p ≤ 0.05) from Ti-a samples (10.07 ± 1.86 mg/g), while of Aires, Bahia, Hilario (6.19 ± 1.89) were identical (p ≤ 0.05) with ti samples (6.63 ± 1.99). Radiofrequency heat treatment (RF) decreased TIA values (p ≤ 0.05) at ≥ 100 °C. However, in the traditional soybean variety, the RF at 110 °C was more effective in eliminating the residual KTI activity. The remaining or the disapperaing bioactive form of trypsin inhibitors were succesfully characterized by the means of a standardized in vitro digestion model. It showed that residual BBI-originated trypsin inhibitor activity was in the stomach even after RF at 110 °C, whereas its chymotrypsin inhibitor activity was not detectable at all. Although PK and KTI null types of soybean seeds still required an energy-saving, gentle heat treatment to inactive the trypsin inhibitors before using them as food or feed, the physicochemical properties and processing quality of soybean products were protected, improved.
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Several lines of evidence indicate that the propagation of misfolded α-synuclein (α-syn) plays a central role in the progression and manifestation of Parkinson's disease. Pathogenic α-syn species can be present in the extracellular space. Thus, the identification and modulation of the key enzymes implicated in extracellular α-syn turnover becomes vital. Kallikrein peptidase 6 has been identified as one of the major α-syn degrading enzymes and has been implicated in the clearance of extracellular α-syn. However, the physiological role of this enzyme in regulating α-syn, in vivo, still remains elusive. Here, by utilizing Klk6 knock-out (Klk6-/- ) mice as our experimental model, we provide insight into the physiologic relevance of endogenous KLK6 expression on α-syn processing. Behavioral phenotyping showed that Klk6-/- mice display no gross behavioral abnormalities. Further in vivo characterization of this mouse model, in the context of α-syn accumulation, showed that KLK6 deletion had no impact on the protein levels of intracellular or extracellular α-syn. Upon in vivo administration of α-syn pre-formed fibrils (PFF), α-syn pathologic accumulations were evident both in the brains of Klk6-/- mice and wt mice without significant differences. Intrastriatal delivery of active KLK6, did not affect secreted α-syn levels observed in the A53T α-syn over-expressing mice. These findings suggest that in the in vivo setting of PFF pathology induction, KLK6 alone is not able to modulate pathology transmission. Our study raises implications for the use of recombinant α-syn fibrils in α-syn turnover studies.
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Encéfalo/metabolismo , Encéfalo/patologia , Calicreínas/deficiência , Sinucleinopatias/metabolismo , Sinucleinopatias/patologia , alfa-Sinucleína/metabolismo , Animais , Células Cultivadas , Feminino , Calicreínas/genética , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sinucleinopatias/genética , alfa-Sinucleína/genéticaRESUMO
Both intravenous thrombolysis (IVT) and mechanical thrombectomy (MT) are evidence-based treatments for acute ischemic stroke (AIS) in selected cases. Recanalization may occur following IVT without the necessity of further interventions or requiring a subsequent MT procedure. IVT prior to MT (bridging-therapy) may be associated with benefits or hazards. We studied the retrieved clot area and degree of recanalization in patients undergoing MT or bridging-therapy for whom it was possible to collect thrombus material. We collected mechanically extracted thrombi from 550 AIS patients from four International stroke centers. Patients were grouped according to the administration (or not) of IVT before thrombectomy and the mechanical thrombectomy approach used. We assessed the number of passes for clot removal and the mTICI (modified Treatment In Cerebral Ischemia) score to define revascularization outcome. Gross photos of each clot were taken and the clot area was measured with ImageJ software. The non-parametric Kruskal-Wallis test was used for statistical analysis. 255 patients (46.4%) were treated with bridging-therapy while 295 (53.6%) underwent MT alone. By analysing retrieved clot area, we found that clots from patients treated with bridging-therapy were significantly smaller compared to those from patients that underwent MT alone (H1 = 10.155 p = 0.001*). There was no difference between bridging-therapy and MT alone in terms of number of passes or final mTICI score. Bridging-therapy was associated with significantly smaller retrieved clot area compared to MT alone but it did not influence revascularization outcome.
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Fibrinolíticos/uso terapêutico , AVC Isquêmico/terapia , Trombólise Mecânica/métodos , Trombose/terapia , Ativador de Plasminogênio Tecidual/uso terapêutico , Feminino , Fibrinolíticos/administração & dosagem , Humanos , AVC Isquêmico/patologia , Masculino , Estudos Prospectivos , Terapia Trombolítica/métodos , Trombose/patologia , Ativador de Plasminogênio Tecidual/administração & dosagem , Resultado do TratamentoRESUMO
The basic helix-loop-helix (bHLH) transcription factor ASCL2 plays essential roles in diploid multipotent trophoblast progenitors, intestinal stem cells, follicular T-helper cells, as well as during epidermal development and myogenesis. During early development, Ascl2 expression is regulated by genomic imprinting and only the maternally inherited allele is transcriptionally active in trophoblast. The paternal allele-specific silencing of Ascl2 requires expression of the long non-coding RNA Kcnq1ot1 in cis and the deposition of repressive histone marks. Here we show that Del7AI, a 280-kb deletion allele neighboring Ascl2, interferes with this process in cis and leads to a partial loss of silencing at Ascl2. Genetic rescue experiments show that the low level of Ascl2 expression from the paternal Del7AI allele can rescue the embryonic lethality associated with maternally inherited Ascl2 mutations, in a level-dependent manner. Despite their ability to support development to term, the rescued placentae have a pronounced phenotype characterized by severe hypoplasia of the junctional zone, expansion of the parietal trophoblast giant cell layer, and complete absence of invasive glycogen trophoblast cells. Transcriptome analysis of ectoplacental cones at E7.5 and differentiation assays of Ascl2 mutant trophoblast stem cells show that ASCL2 is required for the emergence or early maintenance of glycogen trophoblast cells during development. Our work identifies a new cis-acting mutation interfering with Kcnq1ot1 silencing function and establishes a novel critical developmental role for the transcription factor ASCL2.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Linhagem da Célula , Glicogênio/metabolismo , Trofoblastos/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Impressão Genômica , Técnicas de Genotipagem , Heterozigoto , Masculino , Camundongos , Placenta/citologia , Gravidez , Análise de Sequência de RNA , Células-Tronco/citologia , Trofoblastos/citologiaRESUMO
T cell immunotherapy is now a mainstay therapy for several blood-borne cancers as well as metastatic melanoma. Unfortunately, many epithelial tumors respond poorly to immunotherapy, and the reasons for this are not well understood. Cancer-associated fibroblasts (CAFs) are the most frequent non-neoplastic cell type in most solid tumors, and they are emerging as a key player in immunotherapy resistance. A range of immortalized CAF lines will be essential tools that will allow us to understand immune responses against cancer and develop novel strategies for cancer immunotherapy. To study the effect of CAFs on T cell proliferation, we created and characterized a number of novel immortalized human CAFs lines (Im-CAFs) from human breast, colon, and pancreatic carcinomas. Im-CAFs shared similar phenotypes, matrix remodeling and contraction capabilities, and growth and migration rates compared to the primary CAFs. Using primary isolates from breast carcinoma, colorectal carcinoma, and pancreatic ductal adenocarcinoma, we report that CAFs across major tumor types are able to potently suppress T cell proliferation in vitro. Im-CAFs retained this property. Im-CAFs are a key tool that will provide important insights into the mechanisms of CAF-mediated T cell suppression through techniques such as CRISPR-Cas9 modification, molecular screens, and pipeline drug testing.
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Fibroblastos Associados a Câncer/imunologia , Proliferação de Células , Neoplasias/imunologia , Linfócitos T/imunologia , Fibroblastos Associados a Câncer/patologia , Linhagem Celular Transformada , Humanos , Neoplasias/patologia , Linfócitos T/patologiaRESUMO
OBJECTIVES: Most clots retrieved from patients with acute ischemic stroke are 'red' in color. 'White' clots represent a less common entity and their histological composition is less known. Our aim was to investigate the composition, imaging and procedural characteristics of 'white' clots retrieved by mechanical thrombectomy. MATERIALS AND METHODS: Seventy five 'white' thrombi were selected by visual inspection from a cohort of 760 clots collected as part of the RESTORE registry. Clots were evaluated histopathologically. RESULTS: Quantification of Martius Scarlett Blue stain identified platelets/other as the major component in 'white' clots' (mean of 55% of clot overall composition) followed by fibrin (31%), red blood cells (6%) and white blood cells (3%). 'White' clots contained significantly more platelets/other (p<0.001*) and collagen/calcification (p<0.001*) and less red blood cells (p<0.001*) and white blood cells (p=0.018*) than 'red' clots. The mean platelet and von Willebrand Factor expression was 43% and 24%, respectively. Adipocytes were found in four cases. 'White' clots were significantly smaller (p=0.016*), less hyperdense (p=0.005*) on computed tomography angiography/non-contrast CT and were associated with a smaller extracted clot area (p<0.001*) than 'red' clots. They primarily caused the occlusion of middle cerebral artery, were less likely to be removed by aspiration and more likely to require rescue-therapy for retrieval. CONCLUSIONS: 'White' clots represented 14% of our cohort and were platelet, von Willebrand Factor and collagen/calcification-rich. 'White' clots were smaller, less hyperdense, were associated with significantly more distal occlusions and were less successfully removed by aspiration alone than 'red' clots.