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BACKGROUND AND AIMS: Peritoneal metastasis (PM) in gastric cancer (GC) is associated with poor prognosis and significant morbidity. We sought to understand the genomic, transcriptomic, and tumor microenvironment (TME) features that contribute to peritoneal organotropism in GC. METHODS: We conducted a comprehensive multi-omic analysis of 548 samples from 326 patients, including primary tumors, matched normal tissues, peritoneal metastases, and adjacent-normal peritoneal tissues. We employed whole exome sequencing, whole transcriptome sequencing, and digital spatial profiling to investigate molecular alterations, gene expression patterns, and TME characteristics associated with PM. RESULTS: Our analysis identified specific genomic alterations in primary tumors, including mutations in ELF3, CDH1, and PIGR, and TME signatures, such as stromal infiltration and M2 macrophage enrichment, associated with increased risk of PM. We observed distinct transcriptional programs and immune compositions in GCPM compared to liver metastases, highlighting the importance of the TME in transcoelomic metastasis. We found differential expression of therapeutic targets between primary tumors and PM, with lower CLDN18.2 and FGFR2b expression in PM. We unravel the roles of the TME in niche reprogramming within the peritoneum, and provide evidence of pre-metastatic niche conditioning even in early GC without clinical PM. These findings were further validated using a humanized mouse model, which demonstrated niche remodeling in the peritoneum during transcoelomic metastasis. CONCLUSION: Our study provides a comprehensive molecular characterization of GCPM and unveils key biological principles underlying transcoelomic metastasis. The identified predictive markers, therapeutic targets, and TME alterations offer potential avenues for targeted interventions and improved patient outcomes.
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Encouraged by the observations of significant B7-H3 protein overexpression in many human solid tumors compared to healthy tissues, we directed our focus towards targeting B7-H3 using chimeric antigen receptor (CAR) T cells. We utilized a nanobody as the B7-H3-targeting domain in our CAR construct to circumvent the stability issues associated with single-chain variable fragment-based domains. In efforts to expand patient access to CAR T-cell therapy, we engineered our nanobody-based CAR into human Epstein-Barr virus-specific T cells (EBVST), offering a readily available off-the-shelf treatment. B7H3.CAR-armored EBVSTs demonstrated potent in vitro and in vivo activities against multiple B7-H3-positive human tumor cell lines and patient-derived xenograft models. Murine T cells expressing a murine equivalent of our B7H3.CAR exhibited no life-threatening toxicities in immunocompetent mice bearing syngeneic tumors. Further in vitro evaluation revealed that while human T, B, and natural killer cells were unaffected by B7H3.CAR EBVSTs, monocytes were targeted because of upregulation of B7-H3. Such targeting of myeloid cells, which are key mediators of cytokine release syndrome (CRS), contributed to a low incidence of CRS in humanized mice after B7H3.CAR EBVST treatment. Notably, we showed that B7H3.CAR EBVSTs can target B7-H3-expressing myeloid-derived suppressor cells (MDSC), thereby mitigating MDSC-driven immune suppression. In summary, our data demonstrate that our nanobody-based B7H3.CAR EBVSTs are effective as an off-the-shelf therapy for B7-H3-positive solid tumors. These cells also offer an avenue to modulate the immunosuppressive tumor microenvironment, highlighting their promising clinical potential in targeting solid tumors. SIGNIFICANCE: Clinical application of EBVSTs armored with B7-H3-targeting CARs offer an attractive solution to translate off-the-shelf CAR T cells as therapy for solid tumors.
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Antígenos B7 , Herpesvirus Humano 4 , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Linfócitos T , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Humanos , Antígenos B7/imunologia , Antígenos B7/metabolismo , Camundongos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Herpesvirus Humano 4/imunologia , Imunoterapia Adotiva/métodos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linhagem Celular Tumoral , Neoplasias/terapia , Neoplasias/imunologia , Feminino , Anticorpos de Domínio Único/imunologiaRESUMO
BACKGROUND: Mesenchymal stromal cells (MSCs) have broad potential as a cell therapy including for the treatment of drug-resistant inflammatory conditions with abnormal T cell proliferation such as graft-versus-host disease (GVHD). Clinical success, however, has been complicated by the heterogeneity of culture-expanded MSCs as well as donor variability. Here, we devise culture conditions that promote expansion of MSCs with enhanced immunomodulatory functions both in vitro and in animal models of GVHD. METHODS: Human bone marrow-derived MSCs were expanded at high-confluency (MSCHC) and low-confluency state (MSCLC). Their immunomodulatory properties were evaluated with in vitro co-culture assays based on suppression of activated T cell proliferation and secretion of pro-inflammatory cytokines from activated T cells. Metabolic state of these cells was determined, while RNA sequencing was performed to explore transcriptome of these MSCs. Ex vivo expanded MSCHC or MSCLC was injected into human peripheral blood mononuclear cells (PBMC)-induced GVHD mouse model to determine their in vivo therapeutic efficacy based on clinical grade scoring, human CD45+ blood count and histopathological examination. RESULTS: As compared to MSCLC, MSCHC significantly reduced both the proliferation of anti-CD3/CD28-activated T cells and secretion of pro-inflammatory cytokines upon MSCHC co-culture across several donors even in the absence of cytokine priming. Mechanistically, metabolic analysis of MSCHC prior to co-culture with activated T cells showed increased glycolytic metabolism and lactate secretion compared to MSCLC, consistent with their ability to inhibit T cell proliferation. Transcriptome analysis further revealed differential expression of immunomodulatory genes including TRIM29, BPIFB4, MMP3 and SPP1 in MSCHC as well as enriched pathways including cytokine-cytokine receptor interactions, cell adhesion and PI3K-AKT signalling. Lastly, we demonstrate in a human PBMC-induced GVHD mouse model that delivery of MSCHC showed greater suppression of inflammation and improved outcomes compared to MSCLC and saline controls. CONCLUSION: Our study provides evidence that ex vivo expansion of MSCs at high confluency alters the metabolic and transcriptomic states of these cells. Importantly, this approach maximizes the production of MSCs with enhanced immunomodulatory functions without priming, thus providing a non-invasive and generalizable strategy for improving the use of MSCs for the treatment of inflammatory diseases.
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Leucócitos Mononucleares , Células-Tronco Mesenquimais , Animais , Camundongos , Humanos , Medula Óssea , Fosfatidilinositol 3-Quinases , Citocinas , Modelos Animais de Doenças , Proteínas de Ligação a DNA , Fatores de Transcrição , Peptídeos e Proteínas de Sinalização IntercelularRESUMO
Cancer therapeutics have undergone immense research over the past decade. While chemotherapies remain the mainstay treatments for many cancers, the advent of new molecular techniques has opened doors for more targeted modalities towards cancer cells. Although immune checkpoint inhibitors (ICIs) have demonstrated therapeutic efficacy in treating cancer, adverse side effects related to excessive inflammation are often reported. There is a lack of clinically relevant animal models to probe the human immune response towards ICI-based interventions. Humanized mouse models have emerged as valuable tools for pre-clinical research to evaluate the efficacy and safety of immunotherapy. This review focuses on the establishment of humanized mouse models, highlighting the challenges and recent advances in these models for targeted drug discovery and the validation of therapeutic strategies in cancer treatment. Furthermore, the potential of these models in the process of uncovering novel disease mechanisms is discussed.
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BACKGROUND: Currently available anti-leukemia drugs have shown limited success in the treatment of acute myeloid leukemia (AML) due to their poor access to bone marrow niche supporting leukemic cell proliferation. RESULTS: Herein, we report a bone marrow-targetable green tea catechin-based micellar nanocomplex for synergistic AML therapy. The nanocomplex was found to synergistically amplify the anti-leukemic potency of sorafenib via selective disruption of pro-survival mTOR signaling. In vivo biodistribution study demonstrated about 11-fold greater bone marrow accumulation of the nanocomplex compared to free sorafenib. In AML patient-derived xenograft (AML-PDX) mouse model, administration of the nanocomplex effectively eradicated bone marrow-residing leukemic blasts and improved survival rates without noticeable off-target toxicity. CONCLUSION: This study may provide insights into the rational design of nanomedicine platforms enabling bone marrow-targeted delivery of therapeutic agents for the treatment of AML and other bone marrow diseases.
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Catequina , Leucemia Mieloide Aguda , Camundongos , Animais , Humanos , Medula Óssea , Catequina/farmacologia , Micelas , Sorafenibe , Distribuição Tecidual , Leucemia Mieloide Aguda/tratamento farmacológico , Modelos Animais de Doenças , CháRESUMO
OBJECTIVES: Epigenomic alterations in cancer interact with the immune microenvironment to dictate tumour evolution and therapeutic response. We aimed to study the regulation of the tumour immune microenvironment through epigenetic alternate promoter use in gastric cancer and to expand our findings to other gastrointestinal tumours. DESIGN: Alternate promoter burden (APB) was quantified using a novel bioinformatic algorithm (proActiv) to infer promoter activity from short-read RNA sequencing and samples categorised into APBhigh, APBint and APBlow. Single-cell RNA sequencing was performed to analyse the intratumour immune microenvironment. A humanised mouse cancer in vivo model was used to explore dynamic temporal interactions between tumour kinetics, alternate promoter usage and the human immune system. Multiple cohorts of gastrointestinal tumours treated with immunotherapy were assessed for correlation between APB and treatment outcomes. RESULTS: APBhigh gastric cancer tumours expressed decreased levels of T-cell cytolytic activity and exhibited signatures of immune depletion. Single-cell RNAsequencing analysis confirmed distinct immunological populations and lower T-cell proportions in APBhigh tumours. Functional in vivo studies using 'humanised mice' harbouring an active human immune system revealed distinct temporal relationships between APB and tumour growth, with APBhigh tumours having almost no human T-cell infiltration. Analysis of immunotherapy-treated patients with GI cancer confirmed resistance of APBhigh tumours to immune checkpoint inhibition. APBhigh gastric cancer exhibited significantly poorer progression-free survival compared with APBlow (median 55 days vs 121 days, HR 0.40, 95% CI 0.18 to 0.93, p=0.032). CONCLUSION: These findings demonstrate an association between alternate promoter use and the tumour microenvironment, leading to immune evasion and immunotherapy resistance.
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Neoplasias Gastrointestinais , Neoplasias Gástricas , Animais , Epigênese Genética , Epigenômica , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/terapia , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia , Camundongos , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/terapia , Microambiente TumoralRESUMO
Advancements in science enable researchers to constantly innovate and create novel biologics. However, the use of non-human animal models during the development of biologics impedes identification of precise in vivo interactions between the human immune system and treatments. Due to lack of this understanding, adverse effects are frequently observed in healthy volunteers and patients exposed to potential biologics during clinical trials. In this study, we evaluated and compared the effects of known immunotoxic biologics, Proleukin®/IL-2 and OKT3 in humanized mice (reconstituted with human fetal cells) to published clinical outcomes. We demonstrated that humanized mice were able to recapitulate in vivo pathological changes and human-specific immune responses, such as elevated cytokine levels and modulated lymphocytes and myeloid subsets. Given the high similarities of immunological side effects observed between humanized mice and clinical studies, this model could be used to assess immunotoxicity of biologics at a pre-clinical stage, without placing research participants and/or patients at risk.
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Interleucina-2/análogos & derivados , Modelos Imunológicos , Animais , Avaliação de Medicamentos , Feto , Humanos , Interleucina-2/efeitos adversos , Interleucina-2/imunologia , Interleucina-2/farmacologia , Camundongos , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologiaRESUMO
Non-alcoholic fatty liver disease (NAFLD) has been on a global rise. While animal models have rendered valuable insights to the pathogenesis of NAFLD, discrepancy with patient data still exists. Since non-alcoholic steatohepatitis (NASH) involves chronic inflammation, and CD4+ T cell infiltration of the liver is characteristic of NASH patients, we established and characterized a humanized mouse model to identify human-specific immune response(s) associated with NAFLD progression. Immunodeficient mice engrafted with human immune cells (HIL mice) were fed with high fat and high calorie (HFHC) or chow diet for 20 weeks. Liver histology and immune profile of HIL mice were analyzed and compared with patient data. HIL mice on HFHC diet developed steatosis, inflammation and fibrosis of the liver. Human CD4+ central and effector memory T cells increased within the liver and in the peripheral blood of our HIL mice, accompanied by marked up-regulation of pro-inflammatory cytokines (IL-17A and IFNγ). In vivo depletion of human CD4+ T cells in HIL mice reduced liver inflammation and fibrosis, but not steatosis. Our results highlight CD4+ memory T cell subsets as important drivers of NAFLD progression from steatosis to fibrosis and provides a humanized mouse model for pre-clinical evaluation of potential therapeutics.
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Linfócitos T CD4-Positivos/imunologia , Cirrose Hepática Experimental/etiologia , Cirrose Hepática Experimental/imunologia , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/imunologia , Animais , Linfócitos T CD4-Positivos/patologia , Citocinas/sangue , Dieta Hiperlipídica/efeitos adversos , Feminino , Células-Tronco Fetais/transplante , Hepatócitos/transplante , Xenoenxertos , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Cirrose Hepática Experimental/patologia , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Hepatopatia Gordurosa não Alcoólica/patologiaRESUMO
Hepatitis C virus (HCV) infection is commonly attributed as a major cause of chronic hepatotropic diseases, such as, steatosis, cirrhosis and hepatocellular carcinoma. As HCV infects only humans and primates, its narrow host tropism hampers in vivo studies of HCV-mammalian host interactions and the development of effective therapeutics and vaccines. In this context, we will focus our discussion on humanized mice in HCV research. Here, these humanized mice are defined as animal models that encompass either only human hepatocytes or both human liver and immune cells. Aspects related to immunopathogenesis, anti-viral interventions, drug testing and perspectives of these models for future HCV research will be discussed.
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Hepatite C/patologia , Hepatite C/virologia , Interações Hospedeiro-Patógeno , Animais , Modelos Animais de Doenças , Progressão da Doença , Hepacivirus/fisiologia , Hepatite C/imunologia , Humanos , Camundongos , Vacinas contra Hepatite Viral/imunologiaRESUMO
Since the discovery of enterovirus A71 (EV-A71) half a century ago, it has been recognized as the cause of large-scale outbreaks of hand-foot-and-mouth disease worldwide, particularly in the Asia-Pacific region, causing great concern for public health and economic burdens. Detailed mechanisms on the modulation of immune responses after EV-A71 infection have not been fully known, and the lack of appropriate models hinders the development of promising vaccines and drugs. In the present study, NOD-scid IL2Rγ-/- (NSG) mice with a human immune system (humanized mice) at the age of 4 weeks were found to be susceptible to a human isolate of EV-A71 infection. After infection, humanized mice displayed limb weakness, which is similar to the clinical features found in some of the EV-A71-infected patients. Histopathological examination indicated the presence of vacuolation, gliosis, or meningomyelitis in brain stem and spinal cord, which were accompanied by high viral loads detected in these organs. The numbers of activated human CD4+ and CD8+ T cells were upregulated after EV-A71 infection, and EV-A71-specific human T cell responses were found. Furthermore, the secretion of several proinflammatory cytokines, such as human gamma interferon (IFN-γ), interleukin-8 (IL-8), and IL-17A, was elevated in the EV-A71-infected humanized mice. Taken together, our results suggested that the humanized mouse model permits insights into the human immune responses and the pathogenesis of EV-A71 infection, which may provide a platform for the evaluation of anti-EV-A71 drug candidates in the future.IMPORTANCE Despite causing self-limited hand-food-and-mouth disease in younger children, EV-A71 is consistently associated with severe forms of neurological complications and pulmonary edema. Nevertheless, only limited vaccines and drugs have been developed over the years, which is possibly due to a lack of models that can more accurately recapitulate human specificity, since human is the only natural host for wild-type EV-A71 infection. Our humanized mouse model not only mimics histological symptoms in patients but also allows us to investigate the function of the human immune system during infection. It was found that human T cell responses were activated, accompanied by an increase in the production of proinflammatory cytokines in EV-A71-infected humanized mice, which might contribute to the exacerbation of disease pathogenesis. Collectively, this model allows us to delineate the modulation of human immune responses during EV-A71 infection and may provide a platform to evaluate anti-EV-A71 drug candidates in the future.
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Linfócitos T CD8-Positivos/patologia , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/patologia , Feto/patologia , Carga Viral/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Enterovirus Humano A/isolamento & purificação , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Feto/imunologia , Feto/virologia , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Bats are an important animal model with long lifespans, low incidences of tumorigenesis and an ability to asymptomatically harbour pathogens. Currently, in vivo studies of bats are hampered due to their low reproduction rates. To overcome this, we transplanted bat cells from bone marrow (BM) and spleen into an immunodeficient mouse strain NOD-scid IL-2R-/- (NSG), and have successfully established stable, long-term reconstitution of bat immune cells in mice (bat-mice). Immune functionality of our bat-mouse model was demonstrated through generation of antigen-specific antibody response by bat cells following immunization. Post-engraftment of total bat BM cells and splenocytes, bat immune cells survived, expanded and repopulated the mouse without any observable clinical abnormalities. Utilizing bat's remarkable immunological functions, this novel model has a potential to be transformed into a powerful platform for basic and translational research.
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Transplante de Medula Óssea/métodos , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Linfócitos/imunologia , Imunodeficiência Combinada Severa/terapia , Quimeras de Transplante/imunologia , Animais , Quirópteros , Rejeição de Enxerto/prevenção & controle , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Imunodeficiência Combinada Severa/imunologiaRESUMO
With an increasing human population, medical research is pushed to progress into an era of precision therapy. Humanized mice are at the very heart of this new forefront where it is acutely required to decipher human-specific disease pathogenesis and test an array of novel therapeutics. In this review, "humanized" mice are defined as immunodeficient mouse engrafted with functional human biological systems. Over the past decade, researchers have been conscientiously making improvements on the development of humanized mice as a model to closely recapitulate disease pathogenesis and drug mechanisms in humans. Currently, literature is rife with descriptions of novel and innovative humanized mouse models that hold a significant promise to become a panacea for drug innovations to treat and control conditions such as infectious disease and cancer. This review will focus on the background of humanized mice, diseases, and human-specific therapeutics tested on this platform as well as solutions to improve humanized mice for future clinical use.
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Doenças Transmissíveis/imunologia , Imunoterapia/métodos , Camundongos SCID , Neoplasias/imunologia , Animais , Doenças Transmissíveis/terapia , Modelos Animais de Doenças , Patrimônio Genético , Humanos , Camundongos , Neoplasias/terapia , Pesquisa Translacional BiomédicaRESUMO
The arthropod-transmitted chikungunya virus (CHIKV) causes a flu-like disease that is characterized by incapacitating arthralgia. The re-emergence of CHIKV and the continual risk of new epidemics have reignited research in CHIKV pathogenesis. Virus-specific antibodies have been shown to control virus clearance, but antibodies present at sub-neutralizing concentrations can also augment virus infection that exacerbates disease severity. To explore this occurrence, CHIKV infection was investigated in the presence of CHIKV-specific antibodies in both primary human cells and a murine macrophage cell line, RAW264.7. Enhanced attachment of CHIKV to the primary human monocytes and B cells was observed while increased viral replication was detected in RAW264.7 cells. Blocking of specific Fc receptors (FcγRs) led to the abrogation of these observations. Furthermore, experimental infection in adult mice showed that animals had higher viral RNA loads and endured more severe joint inflammation in the presence of sub-neutralizing concentrations of CHIKV-specific antibodies. In addition, CHIKV infection in 11 days old mice under enhancing condition resulted in higher muscles viral RNA load detected and death. These observations provide the first evidence of antibody-mediated enhancement in CHIKV infection and pathogenesis and could also be relevant for other important arboviruses such as Zika virus.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Artralgia/virologia , Febre de Chikungunya/virologia , Vírus Chikungunya/imunologia , Índice de Gravidade de Doença , Animais , Artralgia/imunologia , Artralgia/patologia , Células Cultivadas , Febre de Chikungunya/imunologia , Febre de Chikungunya/patologia , Humanos , Interferon gama/fisiologia , Macrófagos/imunologia , Macrófagos/patologia , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de IgG/metabolismo , Carga Viral , Replicação ViralRESUMO
Mouse models have contributed to the bulk of knowledge on Systemic Lupus Erythematosus (SLE). Nevertheless, substantial differences exist between human and mouse immune system. We aimed to establish and characterise a SLE model mediated by human immune system. Injection of pristane into immunodeficient mice reconstituted with human immune system (humanised mice) recapitulated key SLE features, including: production of human anti-nuclear autoantibodies, lupus nephritis, and pulmonary serositis. There was a reduction in the number of human lymphocytes in peripheral blood, resembling lymphopenia in SLE patients. Concurrently, B cells and T cells were systemically hyperactivated, with a relative expansion of CD27+ and CD27-IgD- memory B cells, increased number of plasmablasts/plasma cells, and accumulation of effector memory T cells. There was also an increased production of human pro-inflammatory cytokines, including: IFN-γ, IL-8, IL-18, MCP-1, and IL-6, suggesting their role in SLE pathogenesis. Increased expression of type I IFN signature genes was also found in human hepatocytes. Altogether, we showed an SLE model that was mediated by human immune system, and which recapitulated key clinical and immunological SLE features. The advancements of humanised mice SLE model would provide an in vivo platform to facilitate translational studies and pre-clinical evaluations of human-specific mechanisms and immunotherapies.
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Modelos Animais de Doenças , Lúpus Eritematoso Sistêmico/etiologia , Animais , Autoanticorpos/sangue , Autoanticorpos/imunologia , Biomarcadores , Biópsia , Citocinas/metabolismo , Perfilação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Hepatócitos/metabolismo , Humanos , Isotipos de Imunoglobulinas/sangue , Isotipos de Imunoglobulinas/imunologia , Imuno-Histoquímica , Imunofenotipagem , Mediadores da Inflamação , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Nefrite Lúpica/etiologia , Nefrite Lúpica/patologia , Ativação Linfocitária/imunologia , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND: Xenotransplantation of patient-derived AML (acute myeloid leukemia) cells in NOD-scid Il2rγ null (NSG) mice is the method of choice for evaluating this human hematologic malignancy. However, existing models constructed using intravenous injection in adult or newborn NSG mice have inferior engraftment efficiency, poor peripheral blood engraftment, or are difficult to construct. METHODS: Here, we describe an improved AML xenograft model where primary human AML cells were injected into NSG newborn pups intrahepatically. RESULTS: Introduction of primary cells from AML patients resulted in high levels of engraftment in peripheral blood, spleen, and bone marrow (BM) of recipient mice. The phenotype of engrafted AML cells remained unaltered during serial transplantation. The mice developed features that are consistent with human AML including spleen enlargement and infiltration of AML cells into multiple organs. Importantly, we demonstrated that although leukemic stem cell activity is enriched and mediated by CD34+CD117+ subpopulation, CD34+CD117- subpopulation can acquire CD34+CD117+ phenotype through de-differentiation. Lastly, we evaluated the therapeutic potential of Sorafenib and Regorafenib in this AML model and found that periphery and spleen AML cells are sensitive to these treatments, whereas BM provides a protective environment to AML. CONCLUSIONS: Collectively, our improved model is robust, easy-to-construct, and reliable for pre-clinical AML studies.
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Leucemia Mieloide Aguda/genética , Transplante Heterólogo/métodos , Animais , Modelos Animais de Doenças , Humanos , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
Hepatitis C is a liver disease caused by infection of the Hepatitis C virus (HCV). Many individuals infected by the virus are unable to resolve the viral infection and develop chronic hepatitis, which can lead to formation of liver cirrhosis and cancer. To understand better how initial HCV infections progress to chronic liver diseases, we characterised the long term pathogenic effects of HCV infections with the use of a humanised mouse model (HIL mice) we have previously established. Although HCV RNA could be detected in infected mice up to 9 weeks post infection, HCV infected mice developed increased incidences of liver fibrosis, granulomatous inflammation and tumour formation in the form of hepatocellular adenomas or hepatocellular carcinomas by 28 weeks post infection compared to uninfected mice. We also demonstrated that chronic liver inflammation in HCV infected mice was mediated by the human immune system, particularly by monocytes/macrophages and T cells which exhibited exhaustion phenotypes. In conclusion, HIL mice can recapitulate some of the clinical symptoms such as chronic inflammation, immune cell exhaustion and tumorigenesis seen in HCV patients. Our findings also suggest that persistence of HCV-associated liver disease appear to require initial infections of HCV and immune responses but not long term HCV viraemia.
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Carcinoma Hepatocelular/etiologia , Transformação Celular Neoplásica , Hepacivirus , Hepatite C Crônica/complicações , Hepatite C Crônica/imunologia , Neoplasias Hepáticas/etiologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/imunologia , Citocinas/sangue , Modelos Animais de Doenças , Hepacivirus/imunologia , Hepatite C Crônica/metabolismo , Hepatite C Crônica/virologia , Testes de Função Hepática , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Monócitos/imunologia , Monócitos/metabolismo , Albumina Sérica/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Viremia/imunologia , Viremia/virologiaRESUMO
Chikungunya virus (CHIKV) is one of the many rheumatic arthropod-borne alphaviruses responsible for debilitating joint inflammation in humans. Despite the severity in many endemic regions, clinically approved intervention targeting the virus remains unavailable. CD4+ T cells have been shown to mediate CHIKV-induced joint inflammation in mice. We demonstrate here that transfer of splenic CD4+ T cells from virus-infected C57BL/6 mice into virus-infected T cell receptor-deficient (TCR-/-) mice recapitulated severe joint pathology including inflammation, vascular leakages, subcutaneous edema, and skeletal muscle necrosis. Proteome-wide screening identified dominant CD4+ T cell epitopes in nsP1 and E2 viral antigens. Transfer of nsP1- or E2-specific primary CD4+ T cell lines into CHIKV-infected TCR-/- recipients led to severe joint inflammation and vascular leakage. This pathogenic role of virus-specific CD4+ T cells in CHIKV infections led to the assessment of clinically approved T cell-suppressive drugs for disease intervention. Although drugs targeting interleukin-2 pathway were ineffective, treatment with fingolimod, an agonist of sphingosine 1-phosphate receptor, successfully abrogated joint pathology in CHIKV-infected animals by blocking the migration of CD4+ T cells into the joints without any effect on viral replication. These results set the stage for further clinical evaluation of fingolimod in the treatment of CHIKV-induced joint pathologies.
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Artralgia/tratamento farmacológico , Linfócitos T CD4-Positivos/citologia , Febre de Chikungunya/tratamento farmacológico , Cloridrato de Fingolimode/uso terapêutico , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Artralgia/virologia , Linfócitos T CD4-Positivos/virologia , Vírus Chikungunya , Epitopos/química , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , NecroseRESUMO
BACKGROUND: Dengue virus infection typically causes mild dengue fever, but, in severe cases, life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) occur. The pathophysiological hallmark of DHF and DSS is plasma leakage that leads to enhanced vascular permeability, likely due to a cytokine storm. METHODS: Ninety patients with dengue during 2010-2012 in Singapore were prospectively recruited and stratified according to their disease phase, primary and secondary infection status, and disease severity, measured by plasma leakage. Clinical parameters were recorded throughout the disease progression. The levels of various immune mediators were quantified using comprehensive multiplex microbead-based immunoassays for 46 immune mediators. RESULTS: Associations between clinical parameters and immune mediators were analyzed using various statistical methods. Potential immune markers, including interleukin 1 receptor antagonist, interferon γ-inducible protein 10, hepatocyte growth factor, soluble p75 tumor necrosis factor α receptor, vascular cell adhesion molecule 1, and matrix metalloproteinase 2, were significantly associated with significant plasma leakage. Secondary dengue virus infections were also shown to influence disease outcome in terms of disease severity. CONCLUSIONS: This study identified several key markers for exacerbated dengue pathogenesis, notably plasma leakage. This will allow a better understanding of the molecular mechanisms of DHF and DSS in patients with dengue.
Assuntos
Permeabilidade Capilar , Quimiocina CXCL10/sangue , Vírus da Dengue/imunologia , Dengue/imunologia , Dengue/fisiopatologia , Fator de Crescimento de Hepatócito/sangue , Metaloproteinases da Matriz/sangue , Adulto , Coinfecção/imunologia , Coinfecção/virologia , Citocinas/sangue , Citocinas/imunologia , Dengue/virologia , Vírus da Dengue/patogenicidade , Vírus da Dengue/fisiologia , Feminino , Humanos , Proteína Antagonista do Receptor de Interleucina 1/sangue , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Masculino , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 9 da Matriz/sangue , Pessoa de Meia-Idade , Receptores do Fator de Necrose Tumoral/sangue , Receptores do Fator de Necrose Tumoral/imunologia , Sorogrupo , Dengue Grave/imunologia , Dengue Grave/fisiopatologia , Dengue Grave/virologia , Singapura , Molécula 1 de Adesão de Célula Vascular/sangue , Molécula 1 de Adesão de Célula Vascular/imunologiaRESUMO
OBJECTIVE: The nature of the tumour-infiltrating leucocytes (TILs) is known to impact clinical outcome in carcinomas, including hepatocellular carcinoma (HCC). However, the role of tumour-infiltrating B cells (TIBs) remains controversial. Here, we investigate the impact of TIBs and their interaction with T cells on HCC patient prognosis. DESIGN: Tissue samples were obtained from 112 patients with HCC from Singapore, Hong Kong and Zurich and analysed using immunohistochemistry and immunofluorescence. RNA expression of CD19, CD8A, IFNG was analysed using quantitative PCR. The phenotype of freshly isolated TILs was analysed using flow cytometry. A mouse model depleted of mature B cells was used for functional study. RESULTS: Tumour-infiltrating T cells and B cells were observed in close contact with each other and their densities are correlated with superior survival in patients with HCC. Furthermore, the density of TIBs was correlated with an enhanced expression of granzyme B and IFN-γ, as well as with reduced tumour viability defined by low expression of Ki-67, and an enhanced expression of activated caspase-3 on tumour cells. CD27 and CD40 costimulatory molecules and TILs expressing activation marker CD38 in the tumour were also correlated with patient survival. Mice depleted of mature B cells and transplanted with murine hepatoma cells showed reduced tumour control and decreased local T cell activation, further indicating the important role of B cells. CONCLUSIONS: The close proximity of tumour-infiltrating T cells and B cells indicates a functional interaction between them that is linked to an enhanced local immune activation and contributes to better prognosis for patients with HCC.
Assuntos
Antígenos CD/análise , Linfócitos B/imunologia , Carcinoma Hepatocelular/imunologia , Neoplasias Hepáticas/imunologia , Linfócitos do Interstício Tumoral , Linfócitos T/imunologia , ADP-Ribosil Ciclase 1/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígenos CD19/genética , Antígenos CD20/análise , Linfócitos B/química , Linfócitos B/patologia , Complexo CD3/análise , Antígenos CD40/análise , Antígenos CD8/análise , Antígenos CD8/genética , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/patologia , Caspase 3/análise , Progressão da Doença , Feminino , Expressão Gênica , Granzimas/análise , Humanos , Interferon gama/genética , Antígeno Ki-67/análise , Neoplasias Hepáticas/química , Neoplasias Hepáticas/patologia , Depleção Linfocítica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fenótipo , Estudos Retrospectivos , Taxa de Sobrevida , Linfócitos T/química , Linfócitos T/patologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análise , Adulto JovemRESUMO
Chikungunya fever (CHIKF) is a global infectious disease which can affect a wide range of age groups. The pathological and immunological response upon Chikungunya virus (CHIKV) infection have been reported over the last few years. However, the clinical profile and immune response upon CHIKV infection in children remain largely unknown. In this study, we analyzed the clinical and immunological response, focusing on the cytokine/chemokine profile in a CHIKV-infected pediatric cohort from Sarawak, Malaysia. Unique immune mediators triggered upon CHIKV infection were identified through meta-analysis of the immune signatures between this pediatric group and cohorts from previous outbreaks. The data generated from this study revealed that a broad spectrum of cytokines/chemokines is up-regulated in a sub-group of virus-infected children stratified according to their viremic status during hospitalization. Furthermore, different immune mediator profiles (the levels of pro-inflammatory cytokines, chemokines and growth and other factors) were observed between children and adults. This study gives an important insight to understand the immune response of CHIKV infection in children and would aid in the development of better prognostics and clinical management for children.