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Anatomy education in the medical school curriculum has encountered considerable challenges during the last decade. The exponential growth of medical science has necessitated a review of the classical ways to teach anatomy to shorten the time students spend dissecting, allowing them to acquire critical, new knowledge in other disciplines. Augmented and mixed reality technologies have developed tremendously during the last few years, offering a wide variety of possibilities to deliver anatomy education to medical students. Here, we provide a methodology to develop, deliver, and assess an anatomy laboratory course using augmented reality applications. We suggest a novel approach, based on Microsoft® HoloLens II, to develop systematic sequences of holograms to reproduce human dissection. The laboratory sessions are prepared before classes and include a series of holograms revealing sequential layers of the human body, isolated structures, or a combination of structures forming a system or a functional unit. The in-class activities are conducted either as one group of students (n = 8-9) with a leading facilitator or small groups of students (n = 4) with facilitators (n = 4) joining the groups for discussion. The same or different sessions may be used for the assessment of students' knowledge. Although currently in its infancy, the use of holograms will soon become a substantial part of medical education. Currently, several companies are offering a range of useful learning platforms, from anatomy education to patient encounters. By describing the holographic program at our institution, we hope to provide a roadmap for other institutions looking to implement a systematic approach to teaching anatomy through holographic dissection. This approach has several benefits, including a sequential 3D presentation of the human body with varying layers of dissection, demonstrations of facilitator-selected three-dimensional (3D) anatomical regions or specific body units, and the option for classroom or remote facilitation, with the ability for students to review each session individually.
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BACKGROUND: Paclitaxel (PTX) is touted as an essential medicine due to its extensive use as a chemotherapeutic agent for various cancers and an antiproliferative agent for endovascular applications. Emerging studies in cardio-oncology implicate various vascular complications of chemotherapeutic agents. METHODS: We evaluated the inflammatory response induced by the systemic administration of PTX. The investigation included RNAseq analysis of primary human endothelial cells (ECs) treated with PTX to identify transcriptional changes in pro-inflammatory mediators. Additionally, we used dexamethasone (DEX), a well-known antiinflammatory compound, to assess its effectiveness in counteracting these PTX-induced changes. Further, we studied the effects of PTX on monocyte chemoattractant protein-1 (MCP-1) levels in the media of ECs. The study also extended to in vivo analysis, where a group of mice was injected with PTX and subsequently harvested at different times to assess the immediate and delayed effects of PTX on inflammatory mediators in blood and aortic ECs. RESULTS: Our RNAseq analysis revealed that PTX treatment led to significant transcriptional perturbations in pro-inflammatory mediators such as MCP-1 and CD137 within primary human ECs. These changes were effectively abrogated when DEX was administered. In vitro experiments showed a marked increase in MCP-1 levels in EC media following PTX treatment, which returned to baseline upon treatment with DEX. In vivo, we observed a threefold increase in MCP-1 levels in blood and aortic ECs 12 h post-PTX administration. Similar trends were noted for CD137 and other downstream mediators like tissue factor, vascular cell adhesion molecule 1, and E-selectin in aortic ECs. CONCLUSION: Our findings illustrate that PTX exposure induces an upregulation of atherothrombotic mediators, which can be alleviated with concurrent administration of DEX. Considering these observations, further long-term investigations should focus on understanding the systemic implications associated with PTX-based therapies and explore the clinical relevance of DEX in mitigating such risks.
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Antiinflamatorios , Quimiocina CCL2 , Dexametasona , Células Endoteliales , Mediadores de Inflamación , Ratones Endogámicos C57BL , Paclitaxel , Paclitaxel/efectos adversos , Paclitaxel/administración & dosificación , Humanos , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/efectos adversos , Quimiocina CCL2/metabolismo , Quimiocina CCL2/genética , Dexametasona/efectos adversos , Mediadores de Inflamación/metabolismo , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Masculino , Enfermedad Iatrogénica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , RNA-Seq , Factores de Tiempo , Regulación de la Expresión Génica/efectos de los fármacos , Transducción de Señal , RatonesRESUMEN
Chronic kidney disease (CKD) is characterized by the accumulation of uremic toxins and renal tubular damage. Tryptophan-derived uremic toxins [indoxyl sulfate (IS) and kynurenine (Kyn)] are well-characterized tubulotoxins. Emerging evidence suggests that transmembrane and immunoglobulin domain-containing 1 (TMIGD1) protects tubular cells and promotes survival. However, the direct molecular mechanism(s) underlying how these two opposing pathways crosstalk remains unknown. We posited that IS and Kyn mediate tubular toxicity through TMIGD1 and the loss of TMIGD1 augments tubular injury. Results from the current study showed that IS and Kyn suppressed TMIGD1 transcription in tubular cells in a dose-dependent manner. The wild-type CCAAT enhancer-binding protein ß (C/EBPß) enhanced, whereas a dominant-negative C/EBPß suppressed, TMIGD1 promoter activity. IS down-regulated C/EBPß in primary human renal tubular cells. The adenine-induced CKD, unilateral ureteric obstruction, and deoxycorticosterone acetate salt unilateral nephrectomy models showed reduced TMIGD1 expression in the renal tubules, which correlated with C/EBPß expression. C/EBPß levels negatively correlated with the IS and Kyn levels. Inactivation of TMIGD1 in mice significantly lowered acetylated tubulin, decreased tubular cell proliferation, caused severe tubular damage, and worsened renal function. Thus, the current results demonstrate that TMIGD1 protects renal tubular cells from renal injury in different models of CKD and uncovers a novel mechanism of tubulotoxicity of tryptophan-based uremic toxins.
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Insuficiencia Renal Crónica , Triptófano , Humanos , Animales , Ratones , Tóxinas Urémicas , Riñón/fisiología , Dominios de Inmunoglobulinas , Glicoproteínas de MembranaRESUMEN
Background: Paclitaxel is touted as an essential medicine due to its extensive use as a chemotherapeutic for various cancers and an antiproliferative agent for restenosis. Due to recent concerns related to long-term mortality, paclitaxel (PTX)-based endovascular therapy is now surrounded by controversies. Objective: Examine the inflammatory mediators driven by the systemic administration of PTX and explore the means to suppress these effects. Methods: RNAseq analysis, cell and mouse models. Results: RNAseq analysis of primary human endothelial cells (ECs) treated with PTX demonstrated transcriptional perturbations of a set of pro-inflammatory mediators, including monocyte chemoattractant protein-1 (MCP-1) and CD137, which were validated in EC lysates. These perturbations were abrogated with dexamethasone, a prototypic anti-inflammatory compound. The media of ECs pre-treated with PTX showed a significant increase in MCP-1 levels, which were reverted to baseline levels with DEX treatment. A group of mice harvested at different time points after PTX injection were analyzed for immediate and delayed effects of PTX. A 3-fold increase in MCP-1 was noted in blood and aortic ECs after 12 hours of PTX treatment. Similar changes in CD137 and downstream mediators such as tissue factor, VCAM-1 and E-selectin were noted in aortic ECs. Conclusions: Our study shows that systemic PTX exposure upregulates atherothrombotic markers, and co-delivery of DEX can subdue the untoward toxic effects. Long-term studies are needed to probe the mechanisms driving systemic complications of PTX-based therapies and evaluate the clinical potential of DEX to mitigate risk.
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Murine models are employed to probe various aspects of peritoneal dialysis (PD), such as peritoneal inflammation and fibrosis. These events drive peritoneal membrane failure in humans, which remains an area of intense investigation due to its profound clinical implications in managing patients with end-stage kidney disease (ESKD). Despite the clinical importance of PD and its related complications, current experimental murine models suffer from key technical challenges that compromise the models' performance. These include PD catheter migration and kinking and usually warrant earlier catheter removal. These limitations also drive the need for a greater number of animals to complete a study. Addressing these drawbacks, this study introduces technical improvements and surgical nuances to prevent commonly observed PD catheter complications in a murine model. Moreover, this modified model is validated by inducing peritoneal inflammation and fibrosis using lipopolysaccharide injections. In essence, this paper describes an improved method to create an experimental model of PD.
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Fallo Renal Crónico , Diálisis Peritoneal , Animales , Cateterismo/métodos , Catéteres de Permanencia , Fibrosis , Humanos , Inflamación , Ratones , Diálisis Peritoneal/efectos adversos , Diálisis Peritoneal/métodosRESUMEN
Chronic kidney disease (CKD) imposes a strong and independent risk for peripheral artery disease (PAD). While solutes retained in CKD patients (uremic solutes) inflict vascular damage, their role in PAD remains elusive. Here, we show that the dietary tryptophan-derived uremic solutes including indoxyl sulfate (IS) and kynurenine (Kyn) at concentrations corresponding to those in CKD patients suppress ß-catenin in several cell types, including microvascular endothelial cells (ECs), inhibiting Wnt activity and proangiogenic Wnt targets in ECs. Mechanistic probing revealed that these uremic solutes downregulated ß-catenin in a manner dependent on serine 33 in its degron motif and through the aryl hydrocarbon receptor (AHR). Hindlimb ischemia in adenine-induced CKD and IS solute-specific mouse models showed diminished ß-catenin and VEGF-A in the capillaries and reduced capillary density, which correlated inversely with blood levels of IS and Kyn and AHR activity in ECs. An AHR inhibitor treatment normalized postischemic angiogenic response in CKD mice to a non-CKD level. In a prospective cohort of PAD patients, plasma levels of tryptophan metabolites and plasma's AHR-inducing activity in ECs significantly increased the risk of future adverse limb events. This work uncovers the tryptophan metabolite/AHR/ß-catenin axis as a mediator of microvascular rarefaction in CKD patients and demonstrates its targetability for PAD in CKD models.
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Miembro Posterior/irrigación sanguínea , Indicán/metabolismo , Isquemia/metabolismo , Quinurenina/metabolismo , Insuficiencia Renal Crónica/metabolismo , Triptófano/metabolismo , Vía de Señalización Wnt , Anciano , Anciano de 80 o más Años , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Humanos , Isquemia/etiología , Isquemia/patología , Ratones , Persona de Mediana Edad , Receptores de Hidrocarburo de Aril/metabolismo , Insuficiencia Renal Crónica/complicaciones , Insuficiencia Renal Crónica/patologíaRESUMEN
BACKGROUND: CKD, characterized by retained uremic solutes, is a strong and independent risk factor for thrombosis after vascular procedures . Urem ic solutes such as indoxyl sulfate (IS) and kynurenine (Kyn) mediate prothrombotic effect through tissue factor (TF). IS and Kyn biogenesis depends on multiple enzymes, with therapeutic implications unexplored. We examined the role of indoleamine 2,3-dioxygenase-1 (IDO-1), a rate-limiting enzyme of kynurenine biogenesis, in CKD-associated thrombosis after vascular injury. METHODS: IDO-1 expression in mice and human vessels was examined. IDO-1-/- mice, IDO-1 inhibitors, an adenine-induced CKD, and carotid artery injury models were used. RESULTS: Both global IDO-1-/- CKD mice and IDO-1 inhibitor in wild-type CKD mice showed reduced blood Kyn levels, TF expression in their arteries, and thrombogenicity compared with respective controls. Several advanced IDO-1 inhibitors downregulated TF expression in primary human aortic vascular smooth muscle cells specifically in response to uremic serum. Further mechanistic probing of arteries from an IS-specific mouse model, and CKD mice, showed upregulation of IDO-1 protein, which was due to inhibition of its polyubiquitination and degradation by IS in vascular smooth muscle cells. In two cohorts of patients with advanced CKD, blood IDO-1 activity was significantly higher in sera of study participants who subsequently developed thrombosis after endovascular interventions or vascular surgery. CONCLUSION: Leveraging genetic and pharmacologic manipulation in experimental models and data from human studies implicate IS as an inducer of IDO-1 and a perpetuator of the thrombotic milieu and supports IDO-1 as an antithrombotic target in CKD.
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Indicán/fisiología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Indolamina-Pirrol 2,3,-Dioxigenasa/sangre , Quinurenina/fisiología , Terapia Molecular Dirigida , Complicaciones Posoperatorias/enzimología , Insuficiencia Renal Crónica/enzimología , Trombosis/enzimología , Procedimientos Quirúrgicos Vasculares/efectos adversos , Animales , Aorta , Traumatismos de las Arterias Carótidas/complicaciones , Trombosis de las Arterias Carótidas/etiología , Trombosis de las Arterias Carótidas/prevención & control , Medios de Cultivo/farmacología , Inducción Enzimática/efectos de los fármacos , Retroalimentación Fisiológica , Femenino , Células HEK293 , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/deficiencia , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Quinurenina/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos del Músculo Liso/efectos de los fármacos , Complicaciones Posoperatorias/sangre , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/prevención & control , Insuficiencia Renal Crónica/tratamiento farmacológico , Tromboplastina/metabolismo , Trombosis/sangre , Trombosis/etiología , Trombosis/prevención & control , Triptófano/metabolismo , Uremia/sangreRESUMEN
OBJECTIVE: The risk of thrombosis in myeloproliferative neoplasms, such as primary myelofibrosis varies depending on the type of key driving mutation (JAK2 [janus kinase 2], CALR [calreticulin], and MPL [myeloproliferative leukemia protein or thrombopoietin receptor]) and the accompanying mutations in other genes. In the current study, we sought to examine the propensity for thrombosis, as well as platelet activation properties in a mouse model of primary myelofibrosis induced by JAK2V617F (janus kinase 2 with valine to phenylalanine substitution on codon 617) mutation. Approach and Results: Vav1-hJAK2V617F transgenic mice show hallmarks of primary myelofibrosis, including significant megakaryocytosis and bone marrow fibrosis, with a moderate increase in red blood cells and platelet number. This mouse model was used to study responses to 2 models of vascular injury and to investigate platelet properties. Platelets derived from the mutated mice have reduced aggregation in response to collagen, reduced thrombus formation and thrombus size, as demonstrated using laser-induced or FeCl3-induced vascular injury models, and increased bleeding time. Strikingly, the mutated platelets had a significantly reduced number of dense granules, which could explain impaired ADP secretion upon platelet activation, and a diminished second wave of activation. CONCLUSIONS: Together, our study highlights for the first time the influence of a hyperactive JAK2 on platelet activation-induced ADP secretion and dense granule homeostasis, with consequent effects on platelet activation properties.
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Coagulación Sanguínea , Plaquetas/enzimología , Traumatismos de las Arterias Carótidas/enzimología , Janus Quinasa 2/sangre , Megacariocitos/enzimología , Activación Plaquetaria , Mielofibrosis Primaria/enzimología , Trombosis/enzimología , Animales , Traumatismos de las Arterias Carótidas/sangre , Traumatismos de las Arterias Carótidas/genética , Modelos Animales de Enfermedad , Janus Quinasa 2/genética , Ratones Transgénicos , Mutación , Agregación Plaquetaria , Mielofibrosis Primaria/sangre , Mielofibrosis Primaria/genética , Trombopoyesis , Trombosis/sangre , Trombosis/genéticaRESUMEN
The function of site-specific phosphorylation of nucleophosmin (NPM), an essential Bax chaperone, in stress-induced cell death is unknown. We hypothesized that NPM threonine 95 (T95) phosphorylation both signals and promotes cell death. In resting cells, NPM exclusively resides in the nucleus and T95 is nonphosphorylated. In contrast, phosphorylated T95 NPM (pNPM T95) accumulates in the cytosol after metabolic stress, in multiple human cancer cell lines following γ-radiation, and in postischemic human kidney tissue. Based on the T95 phosphorylation consensus sequence, we hypothesized that glycogen synthase kinase-3ß (GSK-3ß) regulates cytosolic NPM translocation by phosphorylating T95 NPM. In a cell-free system, GSK-3ß phosphorylated a synthetic NPM peptide containing T95. In vitro, bidirectional manipulation of GSK-3ß activity substantially altered T95 phosphorylation, cytosolic NPM translocation, and cell survival during stress, mechanistically linking these lethal events. Furthermore, GSK-3ß inhibition in vivo decreased cytosolic pNPM T95 accumulation in kidney tissue after experimental ischemia. In patients with acute kidney injury, both cytosolic NPM accumulation in proximal tubule cells and NPM-rich intratubular casts were detected in frozen renal biopsy tissue. These observations show, for the first time, that GSK-3ß promotes cell death partly by phosphorylating NPM at T95, to promote cytosolic NPM accumulation. T95 NPM is also a rational therapeutic target to ameliorate ischemic renal cell injury and may be a universal injury marker in mammalian cells.
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Apoptosis/fisiología , Proteínas Nucleares/metabolismo , Lesión Renal Aguda , Animales , Femenino , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Túbulos Renales Proximales/citología , Masculino , Ratones , Proteínas Nucleares/química , Nucleofosmina , Fosforilación , Conformación Proteica , Estrés FisiológicoRESUMEN
Casitas B-lineage lymphoma (c-Cbl) is a recently identified ubiquitin ligase of nuclear ß-catenin and a suppressor of colorectal cancer (CRC) growth in cell culture and mouse tumor xenografts. We hypothesized that reduction in c-Cbl in colonic epithelium is likely to increase the levels of nuclear ß-catenin in the intestinal crypt, augmenting CRC tumorigenesis in an adenomatous polyposis coli (APCΔ14/+) mouse model. Haploinsufficient c-Cbl mice (APCΔ14/+ c-Cbl+/-) displayed a significant (threefold) increase in atypical hyperplasia and adenocarcinomas in the small and large intestines; however, no differences were noted in the adenoma frequency. In contrast to the APCΔ14/+ c-Cbl+/+ mice, APCΔ14/+ c-Cbl+/- crypts showed nuclear ß-catenin throughout the length of the crypts and up-regulation of Axin2, a canonical Wnt target gene, and SRY-box transcription factor 9, a marker of intestinal stem cells. In contrast, haploinsufficiency of c-Cbl+/- alone was insufficient to induce tumorigenesis regardless of an increase in the number of intestinal epithelial cells with nuclear ß-catenin and SRY-box transcription factor 9 in APC+/+ c-Cbl+/- mice. This study demonstrates that haploinsufficiency of c-Cbl results in Wnt hyperactivation in intestinal crypts and accelerates CRC progression to adenocarcinoma in the milieu of APCΔ14/+, a phenomenon not found with wild-type APC. While emphasizing the role of APC as a gatekeeper in CRC, this study also demonstrates that combined partial loss of c-Cbl and inactivation of APC significantly contribute to CRC tumorigenesis.
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Adenocarcinoma/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Neoplasias del Colon/genética , Neoplasias Colorrectales/genética , Haploinsuficiencia , Linfoma/genética , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Adenocarcinoma/patología , Proteína de la Poliposis Adenomatosa del Colon/genética , Animales , Carcinogénesis , Neoplasias del Colon/patología , Neoplasias Colorrectales/patología , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Linfoma/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-cbl/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Emerging evidence in animal models of chronic kidney disease (CKD) implicates Aryl Hydrocarbon Receptor (AHR) signaling as a mediator of uremic toxicity. However, details about its tissue-specific and time-dependent activation in response to various renal pathologies remain poorly defined. Here, a comprehensive analysis of AHR induction was conducted in response to discrete models of kidney diseases using a transgenic mouse line expressing the AHR responsive-promoter tethered to a ß-galactosidase reporter gene. Following validation using a canonical AHR ligand (a dioxin derivative), the transgenic mice were subjected to adenine-induced and ischemia/reperfusion-induced injury models representing CKD and acute kidney injury (AKI), respectively, in humans. Indoxyl sulfate was artificially increased in mice through the drinking water and by inhibiting its excretion into the urine. Adenine-fed mice showed a distinct and significant increase in ß-galactosidase in the proximal and distal renal tubules, cardiac myocytes, hepatocytes, and microvasculature in the cerebral cortex. The pattern of ß-galactosidase increase coincided with the changes in serum indoxyl sulfate levels. Machine-learning-based image quantification revealed positive correlations between indoxyl sulfate levels and ß-galactosidase expression in various tissues. This pattern of ß-galactosidase expression was recapitulated in the indoxyl sulfate-specific model. The ischemia/reperfusion injury model showed increase in ß-galactosidase in renal tubules that persisted despite reduction in serum indoxyl sulfate and blood urea nitrogen levels. Thus, our results demonstrate a relationship between AHR activation in various tissues of mice with CKD or AKI and the levels of indoxyl sulfate. This study demonstrates the use of a reporter gene mouse to probe tissue-specific manifestations of uremia in translationally relevant animal models and provide hypothesis-generating insights into the mechanism of uremic toxicity that warrant further investigation.
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Insuficiencia Renal Crónica , Uremia , Animales , Indicán , Ratones , Ratones Transgénicos , Receptores de Hidrocarburo de Aril/genética , Insuficiencia Renal Crónica/genéticaRESUMEN
PURPOSE: Cancer patients are at a higher risk of venous thromboembolism (VTE) than the general population. In the general population, blacks are at a higher risk of VTE compared with whites. The influence of race on cancer-associated VTE remains unexplored. We examined whether black cancer patients are at a higher risk of VTE and whether these differences are present in specific cancer types. DESIGN: A retrospective study was performed in the largest safety net hospital of New England using a cohort of cancer patients characterized by a substantial number of nonwhites. RESULTS: We identified 16,498 subjects with solid organ and hematologic malignancies from 2004 to 2018. Among them, we found 186 unique incident VTE events, of which the majority of the events accrued within the first 2 years of cancer diagnosis. Overall, blacks showed a 3-fold higher incidence of VTE (1.8%) compared with whites (0.6%; P<0.001). This difference was observed in certain cancer types such as lung, gastric and colorectal. In lung cancer, the odds of developing VTE in blacks was 2.77-times greater than those in white patients (confidence interval, 1.33-5.91; P=0.007). Despite the greater incidence of cancer-associated VTE in blacks, their Khorana risk score of VTE was not higher. CONCLUSIONS: In a diverse cancer cohort, we observed a higher incidence of cancer-associated VTE in blacks compared with patients from other races. This study indicates the consideration of race in the risk assessment of cancer-associated VTE. It could also lead to future mechanistic studies aiming at identifying reasons for differential VTE risk depending on cancer type.
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Negro o Afroamericano/estadística & datos numéricos , Neoplasias/etnología , Tromboembolia Venosa/etnología , Población Blanca/estadística & datos numéricos , Anticoagulantes/uso terapéutico , Neoplasias de la Mama/complicaciones , Neoplasias de la Mama/etnología , Neoplasias Colorrectales/complicaciones , Neoplasias Colorrectales/etnología , Femenino , Humanos , Incidencia , Neoplasias Pulmonares/complicaciones , Neoplasias Pulmonares/etnología , Masculino , Neoplasias/complicaciones , Neoplasias de la Próstata/complicaciones , Neoplasias de la Próstata/etnología , Estudios Retrospectivos , Estados Unidos/epidemiología , Tromboembolia Venosa/etiologíaRESUMEN
Patients with malignancy are at 4- to 7-fold higher risk of venous thromboembolism (VTE), a potentially fatal, yet preventable complication. Although general mechanisms of thrombosis are enhanced in these patients, malignancy-specific triggers and their therapeutic implication remain poorly understood. Here we examined a colon cancer-specific VTE model and probed a set of metabolites with prothrombotic propensity in the inferior vena cava (IVC) ligation model. Athymic mice injected with human colon adenocarcinoma cells exhibited significantly higher IVC clot weights, a biological readout of venous thrombogenicity, compared with the control mice. Targeted metabolomics analysis of plasma of mice revealed an increase in the blood levels of kynurenine and indoxyl sulfate (tryptophan metabolites) in xenograft-bearing mice, which correlated positively with the increase in the IVC clot size. These metabolites are ligands of aryl hydrocarbon receptor (AHR) signaling. Accordingly, plasma from the xenograft-bearing mice activated the AHR pathway and augmented tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) levels in venous endothelial cells in an AHR-dependent manner. Consistent with these findings, the endothelium from the IVC of xenograft-bearing animals revealed nuclear AHR and upregulated TF and PAI-1 expression, telltale signs of an activated AHR-TF/PAI-1 axis. Importantly, pharmacological inhibition of AHR activity suppressed TF and PAI-1 expression in endothelial cells of the IVC and reduced clot weights in both kynurenine-injected and xenograft-bearing mice. Together, these data show dysregulated tryptophan metabolites in a mouse cancer model, and they reveal a novel link between these metabolites and the control of the AHR-TF/PAI-1 axis and VTE in cancer.
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Neoplasias del Colon/complicaciones , Modelos Animales de Enfermedad , Metaboloma , Inhibidor 1 de Activador Plasminogénico/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Tromboplastina/metabolismo , Tromboembolia Venosa/etiología , Animales , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Femenino , Humanos , Masculino , Ratones , Ratones Desnudos , Transducción de Señal , Triptófano/metabolismo , Tromboembolia Venosa/metabolismo , Tromboembolia Venosa/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Casitas B lymphoma (c-Cbl) is an E3 ubiquitin ligase and a negative regulator of colorectal cancer (CRC). Despite its high expression in immune cells, the effect of c-Cbl on the tumor microenvironment remains poorly understood. Here we demonstrate that c-Cbl alters the tumor microenvironment and suppresses Programmed cell death-1 (PD-1) protein, an immune checkpoint receptor. Using syngeneic CRC xenografts, we observed significantly higher growth of xenografts and infiltrating immune cells in c-Cbl+/- compared to c-Cbl+/+ mice. Tumor-associated CD8+ T-lymphocytes and macrophages of c-Cbl+/- mice showed 2-3-fold higher levels of PD-1. Functionally, macrophages from c-Cbl+/- mice showed a 4-5-fold reduction in tumor phagocytosis, which was restored with an anti-PD-1 neutralizing antibody suggesting regulation of PD-1 by c-Cbl. Further mechanistic probing revealed that C-terminus of c-Cbl interacted with the cytoplasmic tail of PD-1. c-Cbl destabilized PD-1 through ubiquitination- proteasomal degradation depending on c-Cbl's RING finger function. This data demonstrates c-Cbl as an E3 ligase of PD-1 and a regulator of tumor microenvironment, both of which were unrecognized components of its tumor suppressive activity. Advancing immune checkpoint and c-Cbl biology, our study prompts for probing of PD-1 regulation by c-Cbl in conditions driven by immune checkpoint abnormalities such as cancers and autoimmune diseases.
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Linfocitos T CD8-positivos/metabolismo , Neoplasias Colorrectales/genética , Receptor de Muerte Celular Programada 1/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-cbl/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Macrófagos/metabolismo , Ratones Noqueados , Fosforilación , Receptor de Muerte Celular Programada 1/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Carga Tumoral/genética , Microambiente Tumoral/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
INTRODUCTION: The number of glomeruli and glomerulosclerosis evaluated on kidney biopsy slides constitute standard components of a renal pathology report. Prevailing methods for glomerular assessment remain manual, labor intensive, and nonstandardized. We developed a deep learning framework to accurately identify and segment glomeruli from digitized images of human kidney biopsies. METHODS: Trichrome-stained images (n = 275) from renal biopsies of 171 patients with chronic kidney disease treated at the Boston Medical Center from 2009 to 2012 were analyzed. A sliding window operation was defined to crop each original image to smaller images. Each cropped image was then evaluated by at least 3 experts into 3 categories: (i) no glomerulus, (ii) normal or partially sclerosed (NPS) glomerulus, and (iii) globally sclerosed (GS) glomerulus. This led to identification of 751 unique images representing nonglomerular regions, 611 images with NPS glomeruli, and 134 images with GS glomeruli. A convolutional neural network (CNN) was trained with cropped images as inputs and corresponding labels as output. Using this model, an image processing routine was developed to scan the test images to segment the GS glomeruli. RESULTS: The CNN model was able to accurately discriminate nonglomerular images from NPS and GS images (performance on test data: Accuracy: 92.67% ± 2.02% and Kappa: 0.8681 ± 0.0392). The segmentation model that was based on the CNN multilabel classifier accurately marked the GS glomeruli on the test data (Matthews correlation coefficient = 0.628). CONCLUSION: This work demonstrates the power of deep learning for assessing complex histologic structures from digitized human kidney biopsies.
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The universal pathologic features implicated in the progression of chronic kidney disease (CKD) are interstitial fibrosis and tubular atrophy (IFTA). Current methods of estimating IFTA are slow, labor-intensive and fraught with variability and sampling error, and are not quantitative. As such, there is pressing clinical need for a less-invasive and faster method that can quantitatively assess the degree of IFTA. We propose a minimally-invasive optical method to assess the macro-architecture of kidney tissue, as an objective, quantitative assessment of IFTA, as an indicator of the degree of kidney disease. The method of elastic-scattering spectroscopy (ESS) measures backscattered light over the spectral range 320-900 nm and is highly sensitive to micromorphological changes in tissues. Using two discrete mouse models of CKD, we observed spectral trends of increased scattering intensity in the near-UV to short-visible region (350-450 nm), relative to longer wavelengths, for fibrotic kidneys compared to normal kidney, with a quasi-linear correlation between the ESS changes and the histopathology-determined degree of IFTA. These results suggest the potential of ESS as an objective, quantitative and faster assessment of IFTA for the management of CKD patients and in the allocation of organs for kidney transplantation.
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Riñón/patología , Insuficiencia Renal Crónica/patología , Espectrofotometría/métodos , Adenina/administración & dosificación , Animales , Atrofia , Nitrógeno de la Urea Sanguínea , Dieta/veterinaria , Modelos Animales de Enfermedad , Femenino , Fibrosis , Riñón/química , Masculino , Ratones , Ratones Endogámicos C57BL , Insuficiencia Renal Crónica/metabolismoRESUMEN
Casitas B lineage lymphoma (c-Cbl) is a multifunctional protein with a ubiquitin E3 ligase activity capable of degrading diverse sets of proteins. Although previous work had focused mainly on c-Cbl mutations in humans with hematological malignancies, recent emerging evidence suggests a critical role of c-Cbl in angiogenesis and human solid organ tumors. The combination of its unique structure, modular function, and ability to channelize cues from a rich network of signaling cascades, empowers c-Cbl to assume a central role in these disease models. This review consolidates the structural and functional insights based on recent studies that highlight c-Cbl as a target with tantalizing therapeutic potential in various models of angiogenesis and tumorigenesis.
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Carcinogénesis/metabolismo , Neovascularización Patológica/enzimología , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Animales , Humanos , Ratones , Terapia Molecular Dirigida , Fosfolipasa C gamma/metabolismo , Proteínas Proto-Oncogénicas c-cbl/química , Ubiquitinación , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
The proto-oncogene ß-catenin drives colorectal cancer (CRC) tumorigenesis. Casitas B-lineage lymphoma (c-Cbl) inhibits CRC tumor growth through targeting nuclear ß-catenin by a poorly understood mechanism. In addition, the role of c-Cbl in human CRC remains largely underexplored. Using a novel quantitative histopathologic technique, we demonstrate that patients with high c-Cbl-expressing tumors had significantly better median survival (3.7 years) compared with low c-Cbl-expressing tumors (1.8 years; P = 0.0026) and were more than twice as likely to be alive at 3 years compared with low c-Cbl tumors (P = 0.0171). Our data further demonstrate that c-Cbl regulation of nuclear ß-catenin requires phosphorylation of c-Cbl Tyr371 because its mutation compromises its ability to target ß-catenin. The tyrosine 371 (Y371H) mutant interacted with but failed to ubiquitinate nuclear ß-catenin. The nuclear localization of the c-Cbl-Y371H mutant contributed to its dominant negative effect on nuclear ß-catenin. The biological importance of c-Cbl-Y371H was demonstrated in various systems, including a transgenic Wnt-8 zebrafish model. c-Cbl-Y371H mutant showed augmented Wnt/ß-catenin signaling, increased Wnt target genes, angiogenesis, and CRC tumor growth. This study demonstrates a strong link between c-Cbl and overall survival of patients with CRC and provides new insights into a possible role of Tyr371 phosphorylation in Wnt/ß-catenin regulation, which has important implications in tumor growth and angiogenesis in CRC.
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Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/mortalidad , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Tirosina/metabolismo , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Estudios de Casos y Controles , Proliferación Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Mutación , Neovascularización Patológica , Fosforilación , Pronóstico , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-cbl/genética , Tasa de Supervivencia , Células Tumorales Cultivadas , Proteína Wnt1/genética , Pez Cebra , beta Catenina/genéticaRESUMEN
INTRODUCTION: Chronic kidney damage is routinely assessed semiquantitatively by scoring the amount of fibrosis and tubular atrophy in a renal biopsy sample. Although image digitization and morphometric techniques can better quantify the extent of histologic damage, we need more widely applicable ways to stratify kidney disease severity. METHODS: We leveraged a deep learning architecture to better associate patient-specific histologic images with clinical phenotypes (training classes) including chronic kidney disease (CKD) stage, serum creatinine, and nephrotic-range proteinuria at the time of biopsy, and 1-, 3-, and 5-year renal survival. Trichrome-stained images processed from renal biopsy samples were collected on 171 patients treated at the Boston Medical Center from 2009 to 2012. Six convolutional neural network (CNN) models were trained using these images as inputs and the training classes as outputs, respectively. For comparison, we also trained separate classifiers using the pathologist-estimated fibrosis score (PEFS) as input and the training classes as outputs, respectively. RESULTS: CNN models outperformed PEFS across the classification tasks. Specifically, the CNN model predicted the CKD stage more accurately than the PEFS model (κ = 0.519 vs. 0.051). For creatinine models, the area under curve (AUC) was 0.912 (CNN) versus 0.840 (PEFS). For proteinuria models, AUC was 0.867 (CNN) versus 0.702 (PEFS). AUC values for the CNN models for 1-, 3-, and 5-year renal survival were 0.878, 0.875, and 0.904, respectively, whereas the AUC values for PEFS model were 0.811, 0.800, and 0.786, respectively. CONCLUSION: The study demonstrates a proof of principle that deep learning can be applied to routine renal biopsy images.