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PURPOSE: Programmed death receptor ligand-1 (PD-L1) expression and tumor mutational burden (TMB) are approved screening biomarkers for immune checkpoint inhibition (ICI) in advanced triple negative breast cancer. We examined these biomarkers along with characterization of the tumor microenvironment (TME) between breast tumors (BrTs), axillary metastases (AxMs), liver metastases (LvMs), non-axillary lymph node metastases, and non-liver metastases to determine differences related to site of metastatic disease. METHODS: 3076 unpaired biopsies from breast cancer patients were analyzed using whole transcriptome sequencing and NextGen DNA depicting TMB within tumor sites. The PD-L1 positivity was determined with VENTANA PD-L1 (SP142) assay. The immune cell fraction within the TME was calculated by QuantiSeq and MCP-counter. RESULTS: Compared to BrT, more LvM samples had a high TMB (≥ 10 mutations/Mb) and fewer LvM samples had PD-L1+ expression. Evaluation of the TME revealed that LvM sites harbored lower infiltration of adaptive immune cells, such as CD4+, CD8+, and regulatory T-cells compared with the BrT foci. We saw differences in innate immune cell infiltration in LvM compared to BrT, including neutrophils and NK cells. CONCLUSIONS: LvMs are less likely to express PD-L1+ tumor cells but more likely to harbor high TMB as compared to BrTs. Unlike AxMs, LvMs represent a more immunosuppressed TME and demonstrate lower gene expression associated with adaptive immunity compared to BrTs. These findings suggest biopsy site be considered when interpreting results that influence ICI use for treatment and further investigation of immune composition and biomarkers expression by metastatic site.
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Antígeno B7-H1 , Biomarcadores de Tumor , Neoplasias de la Mama , Neoplasias Hepáticas , Microambiente Tumoral , Humanos , Microambiente Tumoral/inmunología , Femenino , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Antígeno B7-H1/metabolismo , Antígeno B7-H1/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Mutación , Metástasis Linfática , Persona de Mediana Edad , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismoRESUMEN
AIMS/HYPOTHESIS: Previous meta-analyses have suggested that diabetes confers a greater excess risk of coronary heart disease, stroke, vascular dementia, and heart failure in women compared to men. While the underlying mechanism that explains such greater excess risk is unknown, in the current meta-analysis we hypothesized that we would find a similar sex difference in the relationship between diabetes and peripheral arterial disease (PAD). METHODS: PubMed MEDLINE, the Cochrane Database of Systematic Reviews, and Embase were systematically searched for prospective population-based cohort studies, with no restriction on publication date, language, or country. We included studies that reported the relative risk (RR), and its variability, for incident PAD associated with diabetes in both sexes. We excluded studies that did not adjust at least for age, and in which participants had pre-existing PAD. In cases where sex-specific results were not reported, study authors were contacted. Random-effects meta-analyses with inverse variance weighting were used to obtain summary sex-specific RRs and the women: men ratio of RRs for PAD. The Newcastle-Ottawa scale was used to assess study quality. RESULTS: Data from seven cohorts, totalling 2071,260 participants (49.8% women), were included. The relative risk for incident PAD associated with diabetes compared with no diabetes was 1.96 (95% CI 1.29-2.63) in women and 1.84 (95% CI 1.29-2.86) in men, after adjusting for potential confounders. The multiple-adjusted RR ratio was 1.05 (95% CI 0.90-1.22), with virtually no heterogeneity between studies (I2 = 0%). All studies scored 6-8, on the Newcastle-Ottawa scale of 0-9, indicating good quality. Eleven of the 12 studies that met review inclusion criteria did not report sex-specific relative risk, and these data were collected through direct correspondence with the study authors. CONCLUSION/INTERPRETATION: Consistent with other studies, we found evidence that diabetes is an independent risk factor for PAD. However, in contrast to similar studies of other types of cardiovascular disease, we did not find evidence that diabetes confers a greater excess risk in women compared to men for PAD. More research is needed to explain this sex differential between PAD and other forms of CVD, in the sequelae of diabetes. In addition, we found that very few studies reported the sex-specific relative risk for the association between diabetes and PAD, adding to existing evidence for the need for improved reporting of sex-disaggregated results in cardiovascular disease research.
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Diabetes Mellitus/epidemiología , Enfermedad Arterial Periférica/epidemiología , Femenino , Humanos , Incidencia , Masculino , Factores de Riesgo , Factores SexualesRESUMEN
Schistosomiasis is a major cause of portal hypertension worldwide. It associates with portal fibrosis that develops during chronic infection. The mechanisms by which the pathogen evokes these host responses remain unclear. We evaluated the hypothesis that schistosome eggs release factors that directly stimulate liver cells to produce osteopontin (OPN), a pro-fibrogenic protein that stimulates hepatic stellate cells to become myofibroblasts. We also investigated the utility of OPN as a biomarker of fibrosis and/or severity of portal hypertension. Cultured cholangiocytes, Kupffer cells and hepatic stellate cells were treated with soluble egg antigen (SEA); OPN production was quantified by quantitative reverse transcriptase polymerase chain reaction (qRTPCR) and ELISA; cell proliferation was assessed by BrdU (5-bromo-2'-deoxyuridine). Mice were infected with Schistosoma mansoni for 6 or 16 weeks to cause early or advanced fibrosis. Liver OPN was evaluated by qRTPCR and immunohistochemistry (IHC) and correlated with liver fibrosis and serum OPN. Livers from patients with schistosomiasis mansoni (early fibrosis n=15; advanced fibrosis n=72) or healthy adults (n=22) were immunostained for OPN and fibrosis markers. Results were correlated with plasma OPN levels and splenic vein pressures. SEA-induced cholangiocyte proliferation and OPN secretion (P<0.001 compared with controls). Cholangiocytes were OPN (+) in Schistosoma-infected mice and humans. Liver and serum OPN levels correlated with fibrosis stage (mice: r=0.861; human r=0.672, P=0.0001) and myofibroblast accumulation (mice: r=0.800; human: r=0.761, P=0.0001). Numbers of OPN (+) bile ductules strongly correlated with splenic vein pressure (r=0.778; P=0.001). S. mansoni egg antigens stimulate cholangiocyte proliferation and OPN secretion. OPN levels in liver and blood correlate with fibrosis stage and portal hypertension severity.
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Proliferación Celular , Hipertensión Portal/metabolismo , Cirrosis Hepática/metabolismo , Osteopontina/metabolismo , Esquistosomiasis mansoni/metabolismo , Adolescente , Adulto , Animales , Antígenos Helmínticos/farmacología , Conductos Biliares/citología , Conductos Biliares/efectos de los fármacos , Conductos Biliares/metabolismo , Línea Celular , Células Cultivadas , Femenino , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Interacciones Huésped-Parásitos , Humanos , Hipertensión Portal/genética , Hipertensión Portal/parasitología , Inmunohistoquímica , Macrófagos del Hígado/efectos de los fármacos , Macrófagos del Hígado/metabolismo , Cirrosis Hepática/genética , Cirrosis Hepática/parasitología , Masculino , Ratones , Persona de Mediana Edad , Osteopontina/sangre , Osteopontina/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Schistosoma/fisiología , Esquistosomiasis mansoni/genética , Esquistosomiasis mansoni/parasitología , Adulto JovenRESUMEN
Personalized medicine is a broad and rapidly advancing field of health care that is informed by each person's unique clinical, genetic, genomic, and environmental information. Personalized medicine depends on multidisciplinary health care teams and integrated technologies (e.g., clinical decision support) to utilize our molecular understanding of disease in order to optimize preventive health care strategies. Human genome information now allows providers to create optimized care plans at every stage of a disease, shifting the focus from reactive to preventive health care. The further integration of personalized medicine into the clinical workflow requires overcoming several barriers in education, accessibility, regulation, and reimbursement. This review focuses on providing a comprehensive understanding of personalized medicine, from scientific discovery at the laboratory bench to integration of these novel ways of understanding human biology at the bedside.
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Medicina de Precisión , Enfermedad/genética , Predisposición Genética a la Enfermedad , Genoma Humano , Humanos , Farmacogenética , Medicina PreventivaRESUMEN
BACKGROUND: Alcohol consumption promotes hepatocellular carcinoma (HCC). The responsible mechanisms are not well understood. Hepatocarcinogenesis increases with age and is enhanced by factors that impose a demand for liver regeneration. Because alcohol is hepatotoxic, habitual alcohol ingestion evokes a recurrent demand for hepatic regeneration. The alcohol-preferring (P) rat model mimics the level of alcohol consumption by humans who habitually abuse alcohol. Previously, we showed that habitual heavy alcohol ingestion amplified age-related hepatocarcinogenesis in P rats, with over 80% of alcohol-consuming P rats developing HCCs after 18 months of alcohol exposure, compared with only 5% of water-drinking controls. METHODS: Herein, we used quantitative real-time PCR and quantitative immunocytochemistry to compare liver tissues from alcohol-consuming P rats and water-fed P rat controls after 6, 12, or 18 months of drinking. We aimed to identify potential mechanisms that might underlie the differences in liver cancer formation and hypothesized that chronic alcohol ingestion would activate Hedgehog (HH), a regenerative signaling pathway that is overactivated in HCC. RESULTS: Chronic alcohol ingestion amplified age-related degenerative changes in hepatocytes, but did not cause appreciable liver inflammation or fibrosis even after 18 months of heavy drinking. HH signaling was also enhanced by alcohol exposure, as evidenced by increased levels of mRNAs encoding HH ligands, HH-regulated transcription factors, and HH target genes. Immunocytochemistry confirmed increased alcohol-related accumulation of HH ligand-producing cells and HH-responsive target cells. HH-related regenerative responses were also induced in alcohol-exposed rats. Three of these processes (i.e., deregulated progenitor expansion, the reverse Warburg effect, and epithelial-to-mesenchymal transitions) are known to promote cancer growth in other tissues. CONCLUSIONS: Alcohol-related changes in Hedgehog signaling and resultant deregulation of liver cell replacement might promote hepatocarcinogenesis.
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Carcinogénesis/efectos de los fármacos , Depresores del Sistema Nervioso Central/efectos adversos , Etanol/efectos adversos , Proteínas Hedgehog/metabolismo , Neoplasias Hepáticas Experimentales/inducido químicamente , Animales , Transición Epitelial-Mesenquimal , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Distribución Aleatoria , RatasRESUMEN
OBJECTIVE: Vascular remodelling during liver damage involves loss of healthy liver sinusoidal endothelial cell (LSEC) phenotype via capillarisation. Hedgehog (Hh) signalling regulates vascular development and increases during liver injury. This study therefore examined its role in capillarisation. DESIGN: Primary LSEC were cultured for 5 days to induce capillarisation. Pharmacological, antibody-mediated and genetic approaches were used to manipulate Hh signalling. Effects on mRNA and protein expression of Hh-regulated genes and capillarisation markers were evaluated by quantitative reverse transcription PCR and immunoblot. Changes in LSEC function were assessed by migration and tube forming assay, and gain/loss of fenestrae was examined by electron microscopy. Mice with acute or chronic liver injury were treated with Hh inhibitors; effects on capillarisation were assessed by immunohistochemistry. RESULTS: Freshly isolated LSEC expressed Hh ligands, Hh receptors and Hh ligand antagonist Hhip. Capillarisation was accompanied by repression of Hhip and increased expression of Hh-regulated genes. Treatment with Hh agonist further induced expression of Hh ligands and Hh-regulated genes, and upregulated capillarisation-associated genes; whereas Hh signalling antagonist or Hh ligand neutralising antibody each repressed expression of Hh target genes and capillarisation markers. LSEC isolated from Smo(loxP/loxP) transgenic mice that had been infected with adenovirus expressing Cre-recombinase to delete Smoothened showed over 75% knockdown of Smoothened. During culture, Smoothened-deficient LSEC had inhibited Hh signalling, less induction of capillarisation-associated genes and retention of fenestrae. In mice with injured livers, inhibiting Hh signalling prevented capillarisation. CONCLUSIONS: LSEC produce and respond to Hh ligands, and use Hh signalling to regulate complex phenotypic changes that occur during capillarisation.
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Acción Capilar , Células Endoteliales/fisiología , Proteínas Hedgehog/fisiología , Hígado/citología , Animales , Western Blotting , Movimiento Celular , Células Cultivadas , Enfermedad Crónica , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Inmunohistoquímica , Hepatopatías/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Rastreo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiologíaRESUMEN
Breast cancer metastases exhibit many different genetic alterations, including copy number amplifications (CNA). CNA are genetic alterations that are increasingly becoming relevant to breast oncology clinical practice. Here we identify CNA in metastatic breast tumor samples using publicly available datasets and characterize their expression and function using a metastatic mouse model of breast cancer. Our findings demonstrate that our organoid generation can be implemented to study clinically relevant features that reflect the genetic heterogeneity of individual tumors.
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We present an integrated single-cell RNA sequencing atlas of the primary breast tumor microenvironment (TME) containing 236,363 cells from 119 biopsy samples across eight datasets. In this study, we leverage this resource for multiple analyses of immune and cancer epithelial cell heterogeneity. We define natural killer (NK) cell heterogeneity through six subsets in the breast TME. Because NK cell heterogeneity correlates with epithelial cell heterogeneity, we characterize epithelial cells at the level of single-gene expression, molecular subtype, and 10 categories reflecting intratumoral transcriptional heterogeneity. We develop InteractPrint, which considers how cancer epithelial cell heterogeneity influences cancer-immune interactions. We use T cell InteractPrint to predict response to immune checkpoint inhibition (ICI) in two breast cancer clinical trials testing neoadjuvant anti-PD-1 therapy. T cell InteractPrint was predictive of response in both trials versus PD-L1 (AUC = 0.82, 0.83 vs. 0.50, 0.72). This resource enables additional high-resolution investigations of the breast TME.
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Neoplasias de la Mama , Inhibidores de Puntos de Control Inmunológico , Células Asesinas Naturales , Análisis de la Célula Individual , Microambiente Tumoral , Humanos , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Neoplasias de la Mama/genética , Femenino , Microambiente Tumoral/inmunología , Análisis de la Célula Individual/métodos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/farmacología , Células Asesinas Naturales/inmunología , Células Epiteliales/inmunología , Células Epiteliales/patología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , Receptor de Muerte Celular Programada 1/inmunología , Regulación Neoplásica de la Expresión Génica , Linfocitos T/inmunología , Heterogeneidad GenéticaRESUMEN
Metastatic disease results from the dissemination of tumor cells beyond their organ of origin to grow in distant organs and is the primary cause of death in patients with advanced breast cancer. Preclinical murine models in which primary tumors spontaneously metastasize are valuable tools for studying metastatic progression and novel cancer treatment combinations. Here, we characterize a novel syngeneic murine breast tumor cell line that provides a model of spontaneously metastatic neu-expressing breast cancer with quicker onset of widespread metastases after orthotopic mammary implantation in immune-competent NeuN mice. The NT2.5-lung metastasis (-LM) cell line was derived from serial passaging of tumor cells that were macro-dissected from spontaneous lung metastases after orthotopic mammary implantation of parental NT2.5 cells. Within one week of NT2.5-LM implantation, metastases are observed in the lungs. Within four weeks, metastases are also observed in the bones, spleen, colon, and liver. We demonstrate that NT2.5-LM metastases are positive for NeuN-the murine equivalent of human epidermal growth factor 2 (HER2). We further demonstrate altered expression of markers of epithelial-to-mesenchymal transition (EMT), suggestive of their enhanced metastatic potential. Genomic analyses support these findings and reveal enrichment in EMT-regulating pathways. In addition, the metastases are rapidly growing, proliferative, and responsive to HER2-directed therapy. The new NT2.5-LM model provides certain advantages over the parental NT2/NT2.5 model, given its more rapid and spontaneous development of metastases. Besides investigating mechanisms of metastatic progression, this new model may be used for the rationalized development of novel therapeutic interventions and assessment of therapeutic responses.
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Preclinical murine models in which primary tumors spontaneously metastasize to distant organs are valuable tools to study metastatic progression and novel cancer treatment combinations. Here, we characterize a novel syngeneic murine breast tumor cell line, NT2.5-lung metastasis (-LM), that provides a model of spontaneously metastatic neu-expressing breast cancer with quicker onset of widespread metastases after orthotopic mammary implantation in immune-competent NeuN mice. Within one week of orthotopic implantation of NT2.5-LM in NeuN mice, distant metastases can be observed in the lungs. Within four weeks, metastases are also observed in the bones, spleen, colon, and liver. Metastases are rapidly growing, proliferative, and responsive to HER2-directed therapy. We demonstrate altered expression of markers of epithelial-to-mesenchymal transition (EMT) and enrichment in EMT-regulating pathways, suggestive of their enhanced metastatic potential. The new NT2.5-LM model provides more rapid and spontaneous development of widespread metastases. Besides investigating mechanisms of metastatic progression, this new model may be used for the rationalized development of novel therapeutic interventions and assessment of therapeutic responses targeting distant visceral metastases.
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BACKGROUND & AIMS: The pathogenesis of cirrhosis, a disabling outcome of defective liver repair, involves deregulated accumulation of myofibroblasts derived from quiescent hepatic stellate cells (HSCs), but the mechanisms that control transdifferentiation of HSCs are poorly understood. We investigated whether the Hedgehog (Hh) pathway controls the fate of HSCs by regulating metabolism. METHODS: Microarray, quantitative polymerase chain reaction, and immunoblot analyses were used to identify metabolic genes that were differentially expressed in quiescent vs myofibroblast HSCs. Glycolysis and lactate production were disrupted in HSCs to determine if metabolism influenced transdifferentiation. Hh signaling and hypoxia-inducible factor 1α (HIF1α) activity were altered to identify factors that alter glycolytic activity. Changes in expression of genes that regulate glycolysis were quantified and localized in biopsy samples from patients with cirrhosis and liver samples from mice following administration of CCl(4) or bile duct ligation. Mice were given systemic inhibitors of Hh to determine if they affect glycolytic activity of the hepatic stroma; Hh signaling was also conditionally disrupted in myofibroblasts to determine the effects of glycolytic activity. RESULTS: Transdifferentiation of cultured, quiescent HSCs into myofibroblasts induced glycolysis and caused lactate accumulation. Increased expression of genes that regulate glycolysis required Hh signaling and involved induction of HIF1α. Inhibitors of Hh signaling, HIF1α, glycolysis, or lactate accumulation converted myofibroblasts to quiescent HSCs. In diseased livers of animals and patients, numbers of glycolytic stromal cells were associated with the severity of fibrosis. Conditional disruption of Hh signaling in myofibroblasts reduced numbers of glycolytic myofibroblasts and liver fibrosis in mice; similar effects were observed following administration of pharmacologic inhibitors of Hh. CONCLUSIONS: Hedgehog signaling controls the fate of HSCs by regulating metabolism. These findings might be applied to diagnosis and treatment of patients with cirrhosis.
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Transdiferenciación Celular/genética , Regulación de la Expresión Génica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Células Estrelladas Hepáticas/metabolismo , Miofibroblastos/metabolismo , Transducción de Señal/genética , Actinas/genética , Actinas/metabolismo , Animales , Conductos Biliares , Tetracloruro de Carbono , Células Cultivadas , Perfilación de la Expresión Génica , Glucólisis/genética , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/enzimología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Ácido Láctico/metabolismo , Ligadura , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias , Miofibroblastos/enzimología , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , ARN Mensajero/metabolismo , Ratas , Factores de TiempoRESUMEN
BACKGROUND: Schistosomiasis mansoni is a major cause of portal fibrosis and portal hypertension. The Hedgehog pathway regulates fibrogenic repair in some types of liver injury. AIMS: Determine if Hedgehog pathway activation occurs during fibrosis progression in schistosomiasis and to determine if macrophage-related mechanisms are involved. METHODS: Immunohistochemistry was used to characterize the cells that generate and respond to Hedgehog ligands in 28 liver biopsies from patients with different grades of schistosomiasis fibrosis staged by ultrasound. Cultured macrophages (RAW264.7 and primary rat Kupffer cells) and primary rat liver sinusoidal endothelial cells (LSEC) were treated with schistosome egg antigen (SEA) and evaluated using qRT-PCR. Inhibition of the Hedgehog pathway was used to investigate its role in alternative activation of macrophages (M2) and vascular tube formation. RESULTS: Patients with schistosomiasis expressed more ligands (Shh and Ihh) and target genes (Patched and Gli2) than healthy individuals. Activated LSEC and myofibroblasts were Hedgehog responsive [Gli2(+)] and accumulated in parallel with fibrosis stage (P < 0.05). Double IHC for Ihh/CD68 showed that Ihh(+) cells were macrophages. In vitro studies demonstrated that SEA-stimulated macrophages to express Ihh and Shh mRNA (P < 0.05). Conditioned media from such macrophages induced luciferase production by Shh-LightII cells (P < 0.001) and Hedgehog inhibitors blocked this effect (P < 0.001). SEA-treated macrophages also up-regulated their own expression of M2 markers, and Hh pathway inhibitors abrogated this response (P < 0.01). Inhibition of the Hedgehog pathway in LSEC blocked SEA-induced migration and tube formation. CONCLUSION: SEA stimulates liver macrophages to produce Hh ligands, which promote alternative activation of macrophages, fibrogenesis and vascular remodelling in schistosomiasis.
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Proteínas Hedgehog/metabolismo , Cirrosis Hepática/metabolismo , Hígado/metabolismo , Macrófagos/metabolismo , Neovascularización Patológica , Esquistosomiasis mansoni/complicaciones , Transducción de Señal , Adulto , Animales , Biopsia , Línea Celular , Células Endoteliales/metabolismo , Células Endoteliales/parasitología , Femenino , Genes Reporteros , Humanos , Inmunohistoquímica , Macrófagos del Hígado/metabolismo , Ligandos , Hígado/diagnóstico por imagen , Hígado/parasitología , Hígado/patología , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/parasitología , Cirrosis Hepática/fisiopatología , Activación de Macrófagos , Macrófagos/parasitología , Macrófagos/patología , Masculino , Ratones , Persona de Mediana Edad , Miofibroblastos/metabolismo , Miofibroblastos/parasitología , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Schistosoma mansoni/metabolismo , Esquistosomiasis mansoni/diagnóstico , Esquistosomiasis mansoni/metabolismo , Esquistosomiasis mansoni/fisiopatología , Índice de Severidad de la Enfermedad , Transfección , Ultrasonografía , Adulto JovenRESUMEN
OBJECTIVE: Immune responses are important in dictating non-alcoholic steatohepatitis (NASH) outcome. We previously reported that upregulation of hedgehog (Hh) and osteopontin (OPN) occurs in NASH, that Hh-regulated accumulation of natural killer T (NKT) cells promotes hepatic stellate cell (HSC) activation, and that cirrhotic livers harbour large numbers of NKT cells. DESIGN: The hypothesis that activated NKT cells drive fibrogenesis during NASH was evaluated by assessing if NKT depletion protects against NASH fibrosis; identifying the NKT-associated fibrogenic factors; and correlating plasma levels of the NKT cell-associated factor OPN with fibrosis severity in mice and humans. RESULTS: When fed methionine-choline-deficient (MCD) diets for 8 weeks, wild type (WT) mice exhibited Hh pathway activation, enhanced OPN expression, and NASH-fibrosis. Ja18-/- and CD1d-/- mice which lack NKT cells had significantly attenuated Hh and OPN expression and dramatically less fibrosis. Liver mononuclear cells (LMNCs) from MCD diet fed WT mice contained activated NKT cells, generated Hh and OPN, and stimulated HSCs to become myofibroblasts; neutralising these factors abrogated the fibrogenic actions of WT LMNCs. LMNCs from NKT-cell-deficient mice were deficient in fibrogenic factors, failing to activate collagen gene expression in HSCs. Human NASH livers with advanced fibrosis contained more OPN and Hh protein than those with early fibrosis. Plasma levels of OPN mirrored hepatic OPN expression and correlated with fibrosis severity. CONCLUSION: Hepatic NKT cells drive production of OPN and Hh ligands that promote fibrogenesis during NASH. Associated increases in plasma levels of OPN may provide a biomarker of NASH fibrosis.
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Hígado Graso/metabolismo , Proteínas Hedgehog/fisiología , Células T Asesinas Naturales/inmunología , Osteopontina/metabolismo , Animales , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Fibrosis/inmunología , Fibrosis/metabolismo , Fibrosis/fisiopatología , Células Estrelladas Hepáticas/fisiología , Humanos , Inmunohistoquímica , Hígado/metabolismo , Activación de Linfocitos , Ratones , Enfermedad del Hígado Graso no Alcohólico , Osteopontina/sangre , Transducción de SeñalRESUMEN
The role of antibiotic overuse in intensifying selection pressures and contributing to the emergence of antimicrobial resistance is well established. The Kumbh Mela, a religious festival that occurs in 4 Indian cities of spiritual significance, is the world's largest mass gathering, attracting over 80 million pilgrims in 2013. Digital syndromic surveillance from the 2013 and 2015 Melas demonstrated a consistent pattern of antibiotic overuse, with an antibiotic prescribing rate of up to 31% for all patient encounters. As preparations for the 2025 Kumbh Mela begin, task shifting, point-of-care diagnostic and digital tools, robust clinician training, and community awareness can promote the restrained and evidence-based use of antibiotics, minimizing the potential for the emergence of antimicrobial resistance at the world's largest mass gathering.
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Programas de Optimización del Uso de los Antimicrobianos , Reuniones Masivas , Humanos , Aglomeración , India/epidemiología , Antibacterianos/uso terapéutico , TecnologíaRESUMEN
Antibody-drug conjugates (ADCs) have emerged as a revolutionary therapeutic class, combining the precise targeting ability of monoclonal antibodies with the potent cytotoxic effects of chemotherapeutics. Notably, ADCs have rapidly advanced in the field of breast cancer treatment. This innovative approach holds promise for strengthening the immune system through antibody-mediated cellular toxicity, tumor-specific immunity, and adaptive immune responses. However, the development of upfront and acquired resistance poses substantial challenges in maximizing the effectiveness of these therapeutics, necessitating a deeper understanding of the underlying mechanisms. These mechanisms of resistance include antigen loss, derangements in ADC internalization and recycling, drug clearance, and alterations in signaling pathways and the payload target. To overcome resistance, ongoing research and development efforts are focused on urgently identifying biomarkers, integrating immune therapy approaches, and designing novel cytotoxic payloads. This Review provides an overview of the mechanisms and clinical effectiveness of ADCs, and explores their unique immune-boosting function, while also highlighting the complex resistance mechanisms and safety challenges that must be addressed. A continued focus on how ADCs impact the tumor microenvironment will help to identify new payloads that can improve patient outcomes.
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Neoplasias de la Mama , Inmunoconjugados , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Anticuerpos Monoclonales , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Inmunidad , Microambiente TumoralRESUMEN
Breast cancer metastases exhibit many different genetic alterations, including copy number amplifications. Using publicly available datasets, we identify copy number amplifications in metastatic breast tumor samples and using our organoid-based metastasis assays, and we validate FGFR1 is amplified in collectively migrating organoids. Because the heterogeneity of breast tumors is increasingly becoming relevant to clinical practice, we demonstrate our organoid method captures genetic heterogeneity of individual tumors.
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Natural killer (NK) cells are innate immune cells that are critical to the body's antitumor and antimetastatic defense. As such, novel therapies are being developed to utilize NK cells as part of a next generation of immunotherapies to treat patients with metastatic disease. Therefore, it is essential for us to examine how metastatic cancer cells and NK cells interact with each other throughout the metastatic cascade. In this Review, we highlight the recent body of work that has begun to answer these questions. We explore how the unique biology of cancer cells at each stage of metastasis alters fundamental NK cell biology, including how cancer cells can evade immunosurveillance and co-opt NK cells into cells that promote metastasis. We also discuss the translational potential of this knowledge.
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Células Asesinas Naturales , Neoplasias , Humanos , Inmunoterapia , Neoplasias/patologíaRESUMEN
Metastasis is a complex process that has been historically difficult to model in culture. Host immune responses play critical roles in restraining and promoting metastatic tumor cells. Here we describe a method of 3D organotypic co-culture of natural killer cells and tumor organoids to capture interactions between the two cellular populations. These assays can be used to model key aspects of metastatic biology and to screen for the effectiveness of agents that stimulate natural killer cell cytotoxicity.
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Neoplasias , Organoides , Técnicas de Cocultivo , Humanos , Células Asesinas NaturalesRESUMEN
Including patient advocates in basic cancer research ensures that breast cancer research is intentional, supports effective communication with broader audiences, and directly connects researchers with those who they are striving to help. Despite this utility, many cancer research scientists do not work with patient advocates. To understand barriers to engagement and build a framework for enhanced interactions in the future, we hosted a workshop with patient advocates and researchers who do engage, then discussed findings at an international metastatic breast cancer conference to solicit additional feedback and suggestions. Findings demonstrate that researchers are uncertain about how to initiate and maintain relationships with advocates. We offer actionable steps to support researchers working with patient advocates to improve cancer research and accomplish our collective goal of improving lives of those who have been diagnosed with breast cancer. We hope that this initiative will facilitate such collaborative efforts.
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Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.