ABSTRACT
The mammalian liver possesses a remarkable regenerative ability. Two modes of damage response have been described: (1) The "oval cell" response emanates from the biliary tree when all hepatocytes are affected by chronic liver disease. (2) A massive, proliferative response of mature hepatocytes occurs upon acute liver damage such as partial hepatectomy (PHx). While the oval cell response has been captured in vitro by growing organoids from cholangiocytes, the hepatocyte proliferative response has not been recapitulated in culture. Here, we describe the establishment of a long-term 3D organoid culture system for mouse and human primary hepatocytes. Organoids can be established from single hepatocytes and grown for multiple months, while retaining key morphological, functional and gene expression features. Transcriptional profiles of the organoids resemble those of proliferating hepatocytes after PHx. Human hepatocyte organoids proliferate extensively after engraftment into mice and thus recapitulate the proliferative damage-response of hepatocytes.
Subject(s)
Cell Proliferation , Hepatocytes/metabolism , Organoids/metabolism , Animals , Cell Culture Techniques , Cells, Cultured , Hepatocytes/cytology , Humans , Mice , Mice, Inbred BALB C , Mice, Knockout , Organoids/cytology , Stem Cells/cytology , Stem Cells/metabolism , Time FactorsABSTRACT
Chronic hepatitis B (CHB), caused by hepatitis B virus (HBV), remains a major medical problem. HBV has a high propensity for progressing to chronicity and can result in severe liver disease, including fibrosis, cirrhosis, and hepatocellular carcinoma. CHB patients frequently present with viral coinfection, including human immunodeficiency virus type (HIV) and hepatitis delta virus. About 10% of chronic HIV carriers are also persistently infected with HBV, which can result in more exacerbated liver disease. Mechanistic studies of HBV-induced immune responses and pathogenesis, which could be significantly influenced by HIV infection, have been hampered by the scarcity of immunocompetent animal models. Here, we demonstrate that humanized mice dually engrafted with components of a human immune system and a human liver supported HBV infection, which was partially controlled by human immune cells, as evidenced by lower levels of serum viremia and HBV replication intermediates in the liver. HBV infection resulted in priming and expansion of human HLA-restricted CD8+ T cells, which acquired an activated phenotype. Notably, our dually humanized mice support persistent coinfections with HBV and HIV, which opens opportunities for analyzing immune dysregulation during HBV and HIV coinfection, and preclinical testing of novel immunotherapeutics.
Subject(s)
Coinfection , HIV Infections , Hepatitis B, Chronic , Hepatitis B , Humans , Mice , Animals , Hepatitis B virus/genetics , HIV , HIV Infections/complications , Liver , Fibrosis , CD8-Positive T-LymphocytesABSTRACT
With the development of genomic technologies, the isolation of genomic DNA (gDNA) from clinical samples is increasingly required for clinical diagnostics and research studies. In this study, we explored the potential of utilizing various leftover blood samples obtained from routine clinical tests as a viable source of gDNA. Using an automated method with optimized pre-treatments, we obtained gDNA from seven types of clinical leftover blood, with average yields of gDNA ranging from 3.11 ± 0.45 to 22.45 ± 4.83 µg. Additionally, we investigated the impact of storage conditions on gDNA recovery, resulting in yields of 8.62-68.08 µg when extracting gDNA from EDTA leftover blood samples stored at 4 °C for up to 13 weeks or -80 °C for up to 78 weeks. Furthermore, we successfully obtained sequenceable gDNA from both Serum Separator Tube and EDTA Tube using a 96-well format extraction, with yields ranging from 0.61 to 71.29 µg and 3.94-215.98 µg, respectively. Our findings demonstrate the feasibility of using automated high-throughput platforms for gDNA extraction from various clinical leftover blood samples with the proper pre-treatments.
Subject(s)
DNA , Genome , Edetic Acid , DNA/genetics , Blood Specimen Collection , GenomicsABSTRACT
Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.
Subject(s)
Hepatocytes/drug effects , Hepatocytes/metabolism , Liver Diseases/genetics , Pyrrolizidine Alkaloids/pharmacology , Animals , Cell Transplantation , Chimera , Disease Models, Animal , Female , Genetic Therapy , Hepatitis B , Hepatitis B virus , Hepatocytes/transplantation , Homeodomain Proteins/genetics , Humans , Hydrolases/genetics , Interleukin Receptor Common gamma Subunit/genetics , Liver/pathology , Liver Diseases/pathology , Malaria , Male , Mice , Mice, Inbred NOD , Mice, Knockout , Plasmodium falciparumABSTRACT
The canonical Wnt-ß-catenin pathway is important in normal development. Mutations in ß-catenin or proteins involved with regulating its phosphorylation or localization result in its nuclear accumulation where it activates its target genes and stimulates cell proliferation. This pathway is dysregulated in many different types of cancer, including gastric cancer (GC). Chibby (Cby) is a 14-kDa protein that inhibits ß-catenin localization to the nucleus and represses ß-catenin-induced transcriptional activity. In the current study, we examined the expression and function of Cby in normal and cancerous human gastric tissue. Reverse-transcription polymerase chain reaction and immunohistochemistry revealed that Cby is expressed in human stomach and localized to glandular elements. Immunohistochemical staining intensity of Cby was decreased in GC tissue when compared with normal gastric epithelium. In AGS cells, a human gastric carcinoma cell line, Cby expression was low. Stable AGS cell transfectants overexpressing Cby were prepared. Cby overexpression did not affect proliferation rates or ß-catenin levels. However, confocal microscopy and subcellular fractionation studies revealed that Cby overexpression resulted in a small decrease in nuclear ß-catenin. Moreover, Cby overexpression caused a molecular weight shift in nuclear ß-catenin and resulted in decreased ß-catenin signaling in AGS cells as measured by the TopFlash assay. However, Cby overexpression did not affect c-Myc protein levels. To conclude, Cby expression was decreased in GC samples and Cby expression altered ß-catenin localization in cultured GC cells. However, Cby did not affect cell proliferation rates or ß-catenin-induced protein expression. Cby may be involved in the early events in the pathogenesis of GC.
Subject(s)
Adenocarcinoma/metabolism , Carrier Proteins/metabolism , Gastric Mucosa/metabolism , Nuclear Proteins/metabolism , Stomach Neoplasms/metabolism , Wnt Signaling Pathway , beta Catenin/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Carrier Proteins/genetics , Cell Line, Tumor , Gastric Mucosa/pathology , Gene Expression Regulation, Neoplastic , Humans , Nuclear Proteins/genetics , Stomach Neoplasms/genetics , Stomach Neoplasms/pathology , beta Catenin/geneticsABSTRACT
Resistance to antiangiogenic therapy in glioblastoma (GBM) patients may involve hypoxia-induced expression of C-X-C motif chemokine receptor 4 (CXCR4) on invading tumor cells, macrophage/microglial cells (MGCs), and glioma stem cells (GSCs). We determined whether antagonizing CXCR4 with POL5551 disrupts anti-vascular endothelial growth factor (VEGF) therapy-induced glioma growth and dissemination. Mice bearing orthotopic CT-2A or GL261 gliomas received POL5551 and/or anti-VEGF antibody B20-4.1.1. Brain tissue was analyzed for tumor volume, invasiveness, hypoxia, vascular density, proliferation, apoptosis, GSCs, and MGCs. Glioma cells were evaluated for CXCR4 expression and polymorphism and POL5551's effects on CXCR4 ligand binding, cell viability, and migration. No CXCR4 mutations were identified. POL5551 inhibited CXCR4 binding to its ligand, stromal cell-derived factor-1α, and reduced hypoxia- and stromal cell-derived factor-1α-mediated migration dose-dependently but minimally affected cell viability. In vivo, B20-4.1.1 increased hypoxic foci and invasiveness, as seen in GBM patients receiving anti-VEGF therapy. Combination of POL5551 and B20-4.1.1 reduced both glioma invasiveness by 16% to 39% and vascular density compared to B20-4.1.1 alone in both glioma models. Reduced populations of GSCs and MGCs were also seen in CT-2A tumors. POL5551 concentrations, evaluated by mass spectrometry, were higher in tumors than in neighboring brain tissues, likely accounting for the results. Inhibition of CXCR4-regulated tumoral, stem cell, and immune mechanisms by adjunctive CXCR4 antagonists may help overcome antiangiogenic therapy resistance, benefiting GBM patients.
Subject(s)
Angiogenesis Inhibitors/therapeutic use , Antibodies/therapeutic use , Glioma/drug therapy , Receptors, CXCR4/antagonists & inhibitors , Vascular Endothelial Growth Factor A/immunology , Animals , Apoptosis/drug effects , Calcium/metabolism , Cell Movement/drug effects , Dose-Response Relationship, Drug , Glioma/metabolism , Glioma/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , Proteins/pharmacology , Signal Transduction/drug effectsABSTRACT
BACKGROUND & AIMS: Human liver chimeric mice are useful models of human hepatitis virus infection, including hepatitis B and C virus infections. Independently, immunodeficient mice reconstituted with CD34(+) hematopoietic stem cells (HSC) derived from fetal liver reliably develop human T and B lymphocytes. Combining these systems has long been hampered by inefficient liver reconstitution of human fetal hepatoblasts. Our study aimed to enhance hepatoblast engraftment in order to create a mouse model with syngeneic human liver and immune cells. METHODS: The effects of human oncostatin-M administration on fetal hepatoblast engraftment into immunodeficient fah(-/-) mice was tested. Mice were then transplanted with syngeneic human hepatoblasts and HSC after which human leukocyte chimerism and functionality were analyzed by flow cytometry, and mice were challenged with HBV. RESULTS: Addition of human oncostatin-M enhanced human hepatoblast engraftment in immunodeficient fah(-/-) mice by 5-100 fold. In contrast to mice singly engrafted with HSC, which predominantly developed human T and B lymphocytes, mice co-transplanted with syngeneic hepatoblasts also contained physiological levels of human monocytes and natural killer cells. Upon infection with HBV, these mice displayed rapid and sustained viremia. CONCLUSIONS: Our study provides a new mouse model with improved human fetal hepatoblast engraftment and an expanded human immune cell repertoire. With further improvements, this model may become useful for studying human immunity against viral hepatitis. LAY SUMMARY: Important human pathogens such as hepatitis B virus, hepatitis C virus and human immunodeficiency virus only infect human cells which complicates the development of mouse models for the study of these pathogens. One way to make mice permissive for human pathogens is the transplantation of human cells into immune-compromised mice. For instance, the transplantation of human liver cells will allow the infection of these so-called "liver chimeric mice" with hepatitis B virus and hepatitis C virus. The co-transplantation of human immune cells into liver chimeric mice will further allow the study of human immune responses to hepatitis B virus or hepatitis C virus. However, for immunological studies it will be crucial that the transplanted human liver and immune cells are derived from the same human donor. In our study we describe the efficient engraftment of human fetal liver cells and immune cells derived from the same donor into mice. We show that liver co-engraftment resulted in an expanded human immune cell repertoire, including monocytes and natural killer cells in the liver. We further demonstrate that these mice could be infected with hepatitis B virus, which lead to an expansion of natural killer cells. In conclusion we have developed a new mouse model that could be useful to study human immune responses to human liver pathogens.
Subject(s)
Killer Cells, Natural , Monocytes , Animals , Hepatitis B , Hepatocytes , Humans , Mice , Mice, SCIDABSTRACT
UNLABELLED: At least 170 million people are chronically infected with hepatitis C virus (HCV). Owing to the narrow host range of HCV and restricted use of chimpanzees, there is currently no suitable animal model for HCV pathogenesis studies or the development of a HCV vaccine. To identify cellular determinants of interspecies transmission and establish a novel immunocompetent model system, we examined the ability of HCV to infect hepatocytes from a small nonhuman primate, the rhesus macaque (Macaca mulatta). We show that the rhesus orthologs of critical HCV entry factors support viral glycoprotein-dependent virion uptake. Primary hepatocytes from rhesus macaques are also permissive for HCV-RNA replication and particle production, which is enhanced when antiviral signaling is suppressed. We demonstrate that this may be owing to the diminished capacity of HCV to antagonize mitochondrial antiviral-signaling protein-dependent innate cellular defenses. To test the ability of HCV to establish persistent replication in vivo, we engrafted primary rhesus macaque hepatocytes into immunocompromised xenorecipients. Inoculation of resulting simian liver chimeric mice with either HCV genotype 1a or 2a resulted in HCV serum viremia for up to 10 weeks. CONCLUSION: Together, these data indicate that rhesus macaques may be a viable model for HCV and implicate host immunity as a potential species-specific barrier to HCV infection. We conclude that suppression of host immunity or further viral adaptation may allow robust HCV infection in rhesus macaques and creation of a new animal model for studies of HCV pathogenesis, lentivirus coinfection, and vaccine development.
Subject(s)
Disease Models, Animal , Hepacivirus/physiology , Hepatitis C , Hepatocytes/virology , Host-Pathogen Interactions , Animals , Humans , Immunity, Innate , Macaca mulatta , Mice , Virus Internalization , Virus ReplicationABSTRACT
BACKGROUND: New biomarkers are needed to detect pleural mesothelioma at an earlier stage and to individualize treatment strategies. We investigated whether fibulin-3 in plasma and pleural effusions could meet sensitivity and specificity criteria for a robust biomarker. METHODS: We measured fibulin-3 levels in plasma (from 92 patients with mesothelioma, 136 asbestos-exposed persons without cancer, 93 patients with effusions not due to mesothelioma, and 43 healthy controls), effusions (from 74 patients with mesothelioma, 39 with benign effusions, and 54 with malignant effusions not due to mesothelioma), or both. A blinded validation was subsequently performed. Tumor tissue was examined for fibulin-3 by immunohistochemical analysis, and levels of fibulin-3 in plasma and effusions were measured with an enzyme-linked immunosorbent assay. RESULTS: Plasma fibulin-3 levels did not vary according to age, sex, duration of asbestos exposure, or degree of radiographic changes and were significantly higher in patients with pleural mesothelioma (105±7 ng per milliliter in the Detroit cohort and 113±8 ng per milliliter in the New York cohort) than in asbestos-exposed persons without mesothelioma (14±1 ng per milliliter and 24±1 ng per milliliter, respectively; P<0.001). Effusion fibulin-3 levels were significantly higher in patients with pleural mesothelioma (694±37 ng per milliliter in the Detroit cohort and 636±92 ng per milliliter in the New York cohort) than in patients with effusions not due to mesothelioma (212±25 and 151±23 ng per milliliter, respectively; P<0.001). Fibulin-3 preferentially stained tumor cells in 26 of 26 samples. In an overall comparison of patients with and those without mesothelioma, the receiver-operating-characteristic curve for plasma fibulin-3 levels had a sensitivity of 96.7% and a specificity of 95.5% at a cutoff value of 52.8 ng of fibulin-3 per milliliter. In a comparison of patients with early-stage mesothelioma with asbestos-exposed persons, the sensitivity was 100% and the specificity was 94.1% at a cutoff value of 46.0 ng of fibulin-3 per milliliter. Blinded validation revealed an area under the curve of 0.87 for plasma specimens from 96 asbestos-exposed persons as compared with 48 patients with mesothelioma. CONCLUSIONS: Plasma fibulin-3 levels can distinguish healthy persons with exposure to asbestos from patients with mesothelioma. In conjunction with effusion fibulin-3 levels, plasma fibulin-3 levels can further differentiate mesothelioma effusions from other malignant and benign effusions. (Funded by the Early Detection Research Network, National Institutes of Health, and others.).
Subject(s)
Asbestos , Extracellular Matrix Proteins/blood , Mesothelioma/diagnosis , Occupational Exposure , Pleural Neoplasms/diagnosis , Aged , Asbestos/adverse effects , Biomarkers/blood , Case-Control Studies , Diagnosis, Differential , Female , Humans , Kaplan-Meier Estimate , Male , Mesothelioma/blood , Middle Aged , Pleural Effusion/blood , Pleural Effusion/diagnosis , Pleural Effusion, Malignant/blood , Pleural Effusion, Malignant/diagnosis , Pleural Neoplasms/blood , ROC Curve , Sensitivity and SpecificityABSTRACT
Adenosine has an important role in inflammation and tissue remodeling and promotes dermal fibrosis by adenosine receptor (A2AR) activation. Adenosine may be formed intracellularly from adenine nucleotides or extracellularly through sequential phosphohydrolysis of released ATP by nucleoside triphosphate diphosphohydrolase (CD39) and ecto-5'-nucleotidase (CD73). Because the role of these ecto-enzymes in fibrosis appears to be tissue specific, we determined whether these ectonucleotidases were directly involved in diffuse dermal fibrosis. Wild-type and mice globally deficient in CD39 knockout (CD39KO), CD73 (CD73KO), or both (CD39/CD73DKO) were challenged with bleomycin. Extracellular adenosine levels and dermal fibrosis were quantitated. Adenosine release from skin cultured ex vivo was increased in wild-type mice after bleomycin treatment but remained low in skin from CD39KO, CD73KO, or CD39/CD73DKO bleomycin-treated mice. Deletion of CD39 and/or CD73 decreased the collagen content, and prevented skin thickening and tensile strength increase after bleomycin challenge. Decreased dermal fibrotic features were associated with reduced expression of the profibrotic mediators, transforming growth factor-ß1 and connective tissue growth factor, and diminished myofibroblast population in CD39- and/or CD73-deficient mice. Our work supports the hypothesis that extracellular adenosine, generated in tandem by ecto-enzymes CD39 and CD73, promotes dermal fibrogenesis. We suggest that biochemical or biological inhibitors of CD39 and/or CD73 may hold promise in the treatment of dermal fibrosis in diseases such as scleroderma.
Subject(s)
5'-Nucleotidase , Adenosine , Antigens, CD , Apyrase , Dermis , Scleroderma, Systemic , 5'-Nucleotidase/genetics , 5'-Nucleotidase/metabolism , Adenosine/genetics , Adenosine/metabolism , Animals , Antibiotics, Antineoplastic/adverse effects , Antibiotics, Antineoplastic/pharmacology , Antigens, CD/genetics , Antigens, CD/metabolism , Apyrase/genetics , Apyrase/metabolism , Bleomycin/adverse effects , Bleomycin/pharmacology , Dermis/metabolism , Dermis/pathology , Fibrosis/chemically induced , Fibrosis/genetics , Fibrosis/metabolism , Fibrosis/pathology , Mice , Mice, Knockout , Scleroderma, Systemic/chemically induced , Scleroderma, Systemic/genetics , Scleroderma, Systemic/metabolism , Scleroderma, Systemic/pathology , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolismABSTRACT
Cancer diagnosis and management depend upon the extraction of complex information from microscopy images by pathologists, which requires time-consuming expert interpretation prone to human bias. Supervised deep learning approaches have proven powerful, but are inherently limited by the cost and quality of annotations used for training. Therefore, we present Histomorphological Phenotype Learning, a self-supervised methodology requiring no labels and operating via the automatic discovery of discriminatory features in image tiles. Tiles are grouped into morphologically similar clusters which constitute an atlas of histomorphological phenotypes (HP-Atlas), revealing trajectories from benign to malignant tissue via inflammatory and reactive phenotypes. These clusters have distinct features which can be identified using orthogonal methods, linking histologic, molecular and clinical phenotypes. Applied to lung cancer, we show that they align closely with patient survival, with histopathologically recognised tumor types and growth patterns, and with transcriptomic measures of immunophenotype. These properties are maintained in a multi-cancer study.
Subject(s)
Lung Neoplasms , Phenotype , Supervised Machine Learning , Humans , Lung Neoplasms/pathology , Lung Neoplasms/genetics , Neoplasms/pathology , Neoplasms/genetics , Deep Learning , TranscriptomeABSTRACT
BACKGROUND: Dickkopf-related protein 1 (DKK1) is a Wingless-related integrate site (Wnt) signaling modulator that is upregulated in prostate cancers (PCa) with low androgen receptor expression. DKN-01, an IgG4 that neutralizes DKK1, delays PCa growth in pre-clinical DKK1-expressing models. These data provided the rationale for a clinical trial testing DKN-01 in patients with metastatic castration-resistant PCa (mCRPC). METHODS: This was an investigator-initiated parallel-arm phase 1/2 clinical trial testing DKN-01 alone (monotherapy) or in combination with docetaxel 75 mg/m2 (combination) for men with mCRPC who progressed on ≥1 AR signaling inhibitors. DKK1 status was determined by RNA in-situ expression. The primary endpoint of the phase 1 dose escalation cohorts was the determination of the recommended phase 2 dose (RP2D). The primary endpoint of the phase 2 expansion cohorts was objective response rate by iRECIST criteria in patients treated with the combination. RESULTS: 18 pts were enrolled into the study-10 patients in the monotherapy cohorts and 8 patients in the combination cohorts. No DLTs were observed and DKN-01 600 mg was determined as the RP2D. A best overall response of stable disease occurred in two out of seven (29%) evaluable patients in the monotherapy cohort. In the combination cohort, five out of seven (71%) evaluable patients had a partial response (PR). A median rPFS of 5.7 months was observed in the combination cohort. In the combination cohort, the median tumoral DKK1 expression H-score was 0.75 and the rPFS observed was similar between patients with DKK1 H-score ≥1 versus H-score = 0. CONCLUSION: DKN-01 600 mg was well tolerated. DKK1 blockade has modest anti-tumor activity as a monotherapy for mCRPC. Anti-tumor activity was observed in the combination cohorts, but the response duration was limited. DKK1 expression in the majority of mCRPC is low and did not clearly correlate with anti-tumor activity of DKN-01 plus docetaxel.
ABSTRACT
BACKGROUND: Young breast cancer (BC) patients less than 45 years old are at higher risk of dying from the disease when compared to their older counterparts. However, specific risk factors leading to this poorer outcome have not been identified. METHODS: One candidate is iron deficiency, as this is common in young women and a clinical feature of young age. In the present study, we used immuno-competent and immuno-deficient mouse xenograft models as well as hemoglobin as a marker of iron status in young BC patients to demonstrate whether host iron deficiency plays a pro-metastatic role. RESULTS: We showed that mice fed an iron-deficient diet had significantly higher tumor volumes and lung metastasis compared to those fed normal iron diets. Iron deficiency mainly altered Notch but not TGF-ß and Wnt signaling in the primary tumor, leading to the activation of epithelial mesenchymal transition (EMT). This was revealed by increased expression of Snai1 and decreased expression of E-cadherin. Importantly, correcting iron deficiency by iron therapy reduced primary tumor volume, lung metastasis, and reversed EMT markers in mice. Furthermore, we found that mild iron deficiency was significantly associated with lymph node invasion in young BC patients (p<0.002). CONCLUSIONS: Together, our finding indicates that host iron deficiency could be a contributor of poor prognosis in young BC patients.
Subject(s)
Breast Neoplasms/complications , Breast Neoplasms/pathology , Iron Deficiencies , Premenopause/metabolism , Animals , Blotting, Western , Disease Models, Animal , Epithelial-Mesenchymal Transition/physiology , Female , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Mice , Mice, Inbred BALB C , Polymerase Chain Reaction , Transplantation, HeterologousABSTRACT
Adenosine A2A receptor (A2AR) stimulation promotes wound healing and is required for the development of fibrosis in murine models of scleroderma and cirrhosis. Nonetheless, the role of A2AR in the formation of scars following skin trauma has not been explored. Here, we examined the effect of pharmacological blockade of A2AR, with the selective adenosine A2AR-antagonist ZM241385 (2.5 mg/ml), in a murine model of scarring that mimics human scarring. We found that application of the selective adenosine A2AR antagonist ZM241385 decreased scar size and enhanced the tensile strength of the scar. Within the scar itself, collagen alignment and composition (marked reduction in collagen 3), but not periostin, biglycan, or fibronectin accumulation, was improved by application of ZM241385. Moreover, A2AR blockade reduced the number of myofibroblasts and angiogenesis but not macrophage infiltration in the scar. Taken together, our work strongly suggests that pharmacological A2AR blockade can be used to diminish scarring while improving the collagen composition and tensile strength of the healed wound.
Subject(s)
Adenosine A2 Receptor Antagonists/therapeutic use , Cicatrix/drug therapy , Cicatrix/metabolism , Receptor, Adenosine A2A/metabolism , Triazines/therapeutic use , Triazoles/therapeutic use , Animals , Blotting, Western , Collagen/metabolism , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Receptor, Adenosine A2A/genetics , Reverse Transcriptase Polymerase Chain Reaction , Wound Healing/drug effects , Wound Healing/geneticsABSTRACT
Multiplexed Imaging technologies are powerful techniques that enable ultrahigh-plex spatial phenotyping of whole tissue sections at single cell spatial resolution. Co-Detection by Indexing (CODEX) multiplexing can detect up to 100 proteins using cyclic detection of DNA conjugated antibodies applied to tissue sections. However, it is necessary to correlate multiplexed fluorescent (mIF) spatial images with Hematoxylin and Eosin (H&E) stained sections post analysis. To effectively correlate mIF spatial images with H&E morphology, an (H&E) staining protocol was developed that is directly applied to the CODEX Fusion flow-cell slide after analysis allowing for direct H&E correlation and annotation with mIF images.
Subject(s)
Coloring Agents , Proteins , Eosine Yellowish-(YS)/pharmacology , Hematoxylin/pharmacology , Staining and Labeling , Coloring Agents/pharmacologyABSTRACT
BACKGROUND: Mitral valve (MV) elongation is a primary hypertrophic cardiomyopathy (HCM) phenotype and contributes to obstruction. The residual MV leaflet that protrudes past the coaptation point is especially susceptible to flow-drag and systolic anterior motion. Histopathological features of MVs in obstructive hypertrophic cardiomyopathy (OHCM), and of residual leaflets specifically, are unknown. OBJECTIVES: The purpose of this study was to characterize gross, structural, and cellular histopathologic features of MV residual leaflets in OHCM. On a cellular-level, we assessed for developmental dysregulation of epicardium-derived cell (EPDC) differentiation, adaptive endocardial-to-mesenchymal transition and valvular interstitial cell proliferation, and genetically-driven persistence of cardiomyocytes in the valve. METHODS: Structural and immunohistochemical staining were performed on 22 residual leaflets excised as ancillary procedures during myectomy, and compared with 11 control leaflets from deceased patients with normal hearts. Structural components were assessed with hematoxylin and eosin, trichrome, and elastic stains. We stained for EPDCs, EPDC paracrine signaling, valvular interstitial cells, endocardial-to-mesenchymal transition, and cardiomyocytes. RESULTS: The residual leaflet was always at A2 segment and attached by slack, elongated and curlicued, myxoid chords. MV residual leaflets in OHCM were structurally disorganized, with expanded spongiosa and increased, fragmented elastic fibers compared with control leading edges. The internal collagenous fibrosa was attenuated and there was collagenous tissue overlying valve surfaces in HCM, with an overall trend toward decreased leaflet thickness (1.09 vs 1.47 mm, P = 0.08). No markers of primary cellular processes were identified. CONCLUSIONS: MV residual leaflets in HCM were characterized by histologic findings that were likely secondary to chronic hemodynamic stress and may further increase susceptibility to systolic anterior motion.
ABSTRACT
Pathogen encounter results in long-lasting epigenetic imprinting that shapes diseases caused by heterologous pathogens. The breadth of this innate immune memory is of particular interest in the context of respiratory pathogens with increased pandemic potential and wide-ranging impact on global health. Here, we investigated epigenetic imprinting across cell lineages in a disease relevant murine model of SARS-CoV-2 recovery. Past SARS-CoV-2 infection resulted in increased chromatin accessibility of type I interferon (IFN-I) related transcription factors in airway-resident macrophages. Mechanistically, establishment of this innate immune memory required viral pattern recognition and canonical IFN-I signaling and augmented secondary antiviral responses. Past SARS-CoV-2 infection ameliorated disease caused by the heterologous respiratory pathogen influenza A virus. Insights into innate immune memory and how it affects subsequent infections with heterologous pathogens to influence disease pathology could facilitate the development of broadly effective therapeutic strategies.
ABSTRACT
Whereas the cellular and molecular features of human inflammatory skin diseases are well characterized, their tissue context and systemic impact remain poorly understood. We thus profiled human psoriasis (PsO) as a prototypic immune-mediated condition with a high predilection for extracutaneous involvement. Spatial transcriptomics (ST) analyses of 25 healthy, active lesion, and clinically uninvolved skin biopsies and integration with public single-cell transcriptomics data revealed marked differences in immune microniches between healthy and inflamed skin. Tissue-scale cartography further identified core disease features across all active lesions, including the emergence of an inflamed suprabasal epidermal state and the presence of B lymphocytes in lesional skin. Both lesional and distal nonlesional samples were stratified by skin disease severity and not by the presence of systemic disease. This segregation was driven by macrophage-, fibroblast-, and lymphatic-enriched spatial regions with gene signatures associated with metabolic dysfunction. Together, these findings suggest that mild and severe forms of PsO have distinct molecular features and that severe PsO may profoundly alter the cellular and metabolic composition of distal unaffected skin sites. In addition, our study provides a valuable resource for the research community to study spatial gene organization of healthy and inflamed human skin.
Subject(s)
Ecosystem , Psoriasis , Humans , Transcriptome , Skin/pathology , Psoriasis/genetics , Patient AcuityABSTRACT
Approximately 30% of early-stage lung adenocarcinoma patients present with disease progression after successful surgical resection. Despite efforts of mapping the genetic landscape, there has been limited success in discovering predictive biomarkers of disease outcomes. Here we performed a systematic multi-omic assessment of 143 tumors and matched tumor-adjacent, histologically-normal lung tissue with long-term patient follow-up. Through histologic, mutational, and transcriptomic profiling of tumor and adjacent-normal tissue, we identified an inflammatory gene signature in tumor-adjacent tissue as the strongest clinical predictor of disease progression. Single-cell transcriptomic analysis demonstrated the progression-associated inflammatory signature was expressed in both immune and non-immune cells, and cell type-specific profiling in monocytes further improved outcome predictions. Additional analyses of tumor-adjacent transcriptomic data from The Cancer Genome Atlas validated the association of the inflammatory signature with worse outcomes across cancers. Collectively, our study suggests that molecular profiling of tumor-adjacent tissue can identify patients at high risk for disease progression.
Subject(s)
Adenocarcinoma of Lung , Lung Neoplasms , Humans , Adenocarcinoma of Lung/genetics , Inflammation/genetics , Lung Neoplasms/genetics , Lung , Disease ProgressionABSTRACT
The central tenet of scientific research is the rigorous application of the scientific method to experimental design, analysis, interpretation, and reporting of results. In order to confer validity to a hypothesis, experimental details must be transparent and results must be reproducible. Failure to achieve this minimum indicates a deficiency in rationale, design, and/or execution, necessitating further experimental refinement or hypothesis reformulation. More importantly, rigorous application of the scientific method advances scientific knowledge by enabling others to identify weaknesses or gaps that can be exploited by new ideas or technology that inevitably extend, improve, or refine a hypothesis. Experimental details, described in manuscript materials and methods, are the principal vehicle used to communicate procedures, techniques, and resources necessary for experimental reproducibility. Recent examination of the biomedical literature has shown that many published articles lack sufficiently detailed methodological information to reproduce experiments. There are few broadly established practice guidelines and quality assurance standards in basic biomedical research. The current paper provides a framework of best practices to address the lack of reporting of detailed materials and methods that is pervasive in histological slide-based assays. Our goal is to establish a structured framework that highlights the key factors necessary for thorough collection of metadata and reporting of slide-based assays.