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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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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
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
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
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.
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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.
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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
Arthritogenic alphaviruses, including Ross River virus (RRV) and chikungunya virus (CHIKV), are responsible for explosive epidemics involving millions of cases. These mosquito-transmitted viruses cause inflammation and injury in skeletal muscle and joint tissues that results in debilitating pain. We previously showed that arginase 1 (Arg1) was highly expressed in myeloid cells in the infected and inflamed musculoskeletal tissues of RRV- and CHIKV-infected mice, and specific deletion of Arg1 from myeloid cells resulted in enhanced viral control. Here, we show that Arg1, along with other genes associated with suppressive myeloid cells, is induced in PBMCs isolated from CHIKV-infected patients during the acute phase as well as the chronic phase, and that high Arg1 expression levels were associated with high viral loads and disease severity. Depletion of both CD4 and CD8 T cells from RRV-infected Arg1-deficient mice restored viral loads to levels detected in T cell-depleted wild-type mice. Moreover, Arg1-expressing myeloid cells inhibited virus-specific T cells in the inflamed and infected musculoskeletal tissues, but not lymphoid tissues, following RRV infection in mice, including suppression of interferon-γ and CD69 expression. Collectively, these data enhance our understanding of the immune response following arthritogenic alphavirus infection and suggest that immunosuppressive myeloid cells may contribute to the duration or severity of these debilitating infections.
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Infecções por Alphavirus/imunologia , Arginase/imunologia , Células Mieloides/imunologia , Linfócitos T/imunologia , Carga Viral/imunologia , Transferência Adotiva , Animais , Western Blotting , Febre de Chikungunya/imunologia , Vírus Chikungunya , Citometria de Fluxo , Humanos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Ross River virus/imunologiaRESUMO
Chikungunya virus (CHIKV) infection is a re-emerging pandemic human arboviral disease. CD4+ T cells were previously shown to contribute to joint inflammation in the course of CHIKV infection in mice. The JES6-1 anti-IL-2 antibody selectively expands mouse regulatory T cells (Tregs) by forming a complex with IL-2. In this study, we show that the IL-2 JES6-1-mediated expansion of Tregs ameliorates CHIKV-induced joint pathology. It does so by inhibiting the infiltration of CD4+ T cells due to the induction of anergy in CHIKV-specific CD4+ effector T cells. These findings suggest that activation of Tregs could also become an alternative approach to control CHIKV-mediated disease. IMPORTANCE: Chikungunya virus (CHIKV) has re-emerged as a pathogen of global significance. Patients infected with CHIKV suffer from incapacitating joint pain that severely affects their daily functioning. Despite the best efforts, effective treatment is still inadequate. While T cells-mediated immunopathology in CHIKV infections has been reported, the role of regulatory T cells (Tregs) has not been explored. The JES6-1 anti-IL-2 antibody has been demonstrated to selectively expand mouse Tregs by forming a complex with IL-2. We reveal here that IL-2 JES6-1-mediated expansion of Tregs ameliorates the CHIKV-induced joint pathology in mice by neutralizing virus-specific CD4+ effector T (Teff) cells. We show that this treatment abrogates the infiltration of pathogenic CD4+ T cells through induction of anergy in CHIKV-specific CD4+ Teff cells. This is the first evidence where the role of Tregs is demonstrated in CHIKV pathogenesis and its expansion could control virus-mediated immunopathology.
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UNLABELLED: Chikungunya virus (CHIKV) is a mosquito-borne arthralgic alphavirus that has garnered international attention as an important emerging pathogen since 2005. More recently, it invaded the Caribbean islands and the Western Hemisphere. Intriguingly, the current CHIKV outbreak in the Caribbean is caused by the Asian CHIKV genotype, which differs from the La Réunion LR2006 OPY1 isolate belonging to the Indian Ocean lineage. Here, we adopted a systematic and comparative approach against LR2006 OPY1 to characterize the pathogenicity of the Caribbean CNR20235 isolate and consequential host immune responses in mice. Ex vivo infection using primary mouse tail fibroblasts revealed a weaker replication efficiency by CNR20235 isolate. In the CHIKV mouse model, CNR20235 infection induced an enervated joint pathology characterized by moderate edema and swelling, independent of mononuclear cell infiltration. Based on systemic cytokine analysis, localized immunophenotyping, and gene expression profiles in the popliteal lymph node and inflamed joints, two pathogenic phases were defined for CHIKV infection: early acute (2 to 3 days postinfection [dpi]) and late acute (6 to 8 dpi). Reduced joint pathology during early acute phase of CNR20235 infection was associated with a weaker proinflammatory Th1 response and natural killer (NK) cell activity. The pathological role of NK cells was further demonstrated as depletion of NK cells reduced joint pathology in LR2006 OPY1. Taken together, this study provides evidence that the Caribbean CNR20235 isolate has an enfeebled replication and induces a less pathogenic response in the mammalian host. IMPORTANCE: The introduction of CHIKV in the Americas has heightened the risk of large-scale outbreaks due to the close proximity between the United States and the Caribbean. The immunopathogenicity of the circulating Caribbean CHIKV isolate was explored, where it was demonstrated to exhibit reduced infectivity resulting in a weakened joint pathology. Analysis of serum cytokine levels, localized immunophenotyping, and gene expression profiles in the organs revealed that a limited Th1 response and reduced NK cells activity could underlie the reduced pathology in the host. Interestingly, higher asymptomatic infections were observed in the Caribbean compared to the La Réunion outbreaks in 2005 and 2006. This is the first study that showed an association between key proinflammatory factors and pathology-mediating leukocytes with a less severe pathological outcome in Caribbean CHIKV infection. Given the limited information regarding the sequela of Caribbean CHIKV infection, our study is timely and will aid the understanding of this increasingly important disease.
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Febre de Chikungunya/imunologia , Febre de Chikungunya/virologia , Vírus Chikungunya/imunologia , Vírus Chikungunya/isolamento & purificação , Articulações/imunologia , Células Matadoras Naturais/imunologia , Células Th1/imunologia , Animais , Região do Caribe/epidemiologia , Febre de Chikungunya/epidemiologia , Febre de Chikungunya/patologia , Vírus Chikungunya/genética , Vírus Chikungunya/fisiologia , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Articulações/patologia , Articulações/virologia , Células Matadoras Naturais/virologia , Camundongos , Camundongos Endogâmicos C57BL , Reunião/epidemiologia , Células Th1/virologiaRESUMO
OBJECTIVE: Arthrogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus (CHIKV) circulate worldwide. This virus class causes debilitating illnesses that are characterized by arthritis, arthralgia, and myalgia. In previous studies, we identified macrophage migration inhibitory factor (MIF) as a critical inflammatory factor in the pathogenesis of alphaviral diseases. The present study was undertaken to characterize the role of CD74, a cell surface receptor of MIF, in both RRV- and CHIKV-induced alphavirus arthritides. METHODS: Mouse models of RRV and CHIKV infection were used to investigate the immunopathogenesis of arthritic alphavirus infection. The role of CD74 was assessed using histologic analysis, real-time polymerase chain reaction, flow cytometry, and plaque assay. RESULTS: In comparison to wild-type mice, CD74-/- mice developed only mild clinical features and had low levels of tissue damage. Leukocyte infiltration, characterized predominantly by inflammatory monocytes and natural killer cells, was substantially reduced in the infected tissue of CD74-/- mice, but production of proinflammatory cytokines and chemokines was not decreased. CD74 deficiency was associated with increased monocyte apoptosis, but had no effect on monocyte migratory capacity. Consistent with these findings, alphaviral infection resulted in a dose-dependent up-regulation of CD74 expression in human peripheral blood mononuclear cells, and serum MIF levels were significantly elevated in patients with RRV or CHIKV infection. CONCLUSION: CD74 appears to regulate immune responses to alphaviral infection through its effects on cellular recruitment and survival. These findings suggest that both MIF and CD74 play a critical role in mediating alphaviral disease, and blocking these factors with novel therapeutic agents could substantially ameliorate the pathologic manifestations.
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Infecções por Alphavirus/complicações , Antígenos de Diferenciação de Linfócitos B/fisiologia , Artrite Infecciosa/etiologia , Artrite Infecciosa/fisiopatologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Miosite/fisiopatologia , Miosite/virologia , Receptores Imunológicos/fisiologia , Infecções por Alphavirus/patologia , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Apoptose/fisiologia , Artrite Infecciosa/patologia , Células Cultivadas , Quimiocinas/metabolismo , Vírus Chikungunya/fisiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/patologia , Miosite/patologia , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Ross River virus/fisiologia , Índice de Gravidade de DoençaRESUMO
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: Chikungunya virus (CHIKV) and related arboviruses have been responsible for large epidemic outbreaks with serious economic and social impact. Although infected individuals clear the virus from the blood, some develop debilitating and prolonged arthralgia. METHODS: We investigated specificity and strength of antibody responses in a longitudinal study on CHIKV-infected patients and analyzed their association with viral load, cytokine profile, and severity. RESULTS: We found that CHIKV-specific response is dominated by immunoglobulin G3 (IgG3) antibodies. The antibodies were neutralizing, and patients with high viremia rapidly developed high levels of anti-CHIKV antibodies of this specific isotype. Although these patients endured a more severe disease progression during the acute viremic phase, they cleared the virus faster and did not experience persistent arthralgia. However, significant persistent arthralgia was observed in patients with low viremia who developed IgG3 at a later stage. CONCLUSIONS: Absence of early CHIKV-specific IgG3 may therefore serve as a specific marker of patients with increased risk of disease.
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Infecções por Alphavirus/imunologia , Infecções por Alphavirus/virologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vírus Chikungunya/imunologia , Imunoglobulina G/sangue , Adulto , Idoso , Infecções por Alphavirus/patologia , Biomarcadores , Citocinas/metabolismo , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Prognóstico , Medição de Risco , Índice de Gravidade de Doença , Carga ViralRESUMO
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: 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
Chikungunya virus (CHIKV) is an alphavirus that causes chronic and incapacitating arthralgia in humans. To date, interactions between the immune system and the different stages of the virus life cycle remain poorly defined. We demonstrated for the first time that CHIKV Ags could be detected in vivo in the monocytes of acutely infected patients. Using in vitro experimental systems, whole blood and purified monocytes, we confirmed that monocytes could be infected and virus growth could be sustained. CHIKV interactions with monocytes, and with other blood leukocytes, induced a robust and rapid innate immune response with the production of specific chemokines and cytokines. In particular, high levels of IFN-alpha were produced rapidly after CHIKV incubation with monocytes. The identification of monocytes during the early phase of CHIKV infection in vivo is significant as infected monocyte/macrophage cells have been detected in the synovial tissues of chronically CHIKV-infected patients, and these cells may behave as the vehicles for virus dissemination. This may explain the persistence of joint symptoms despite the short duration of viremia. Our results provide a better understanding on the basic mechanisms of infection and early antiviral immune responses and will help in the development of future effective control strategies.
Assuntos
Infecções por Alphavirus/imunologia , Vírus Chikungunya/imunologia , Imunidade Inata , Monócitos/imunologia , Monócitos/virologia , Doença Aguda , Infecções por Alphavirus/sangue , Animais , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/virologia , Células Cultivadas , Chlorocebus aethiops , Doença Crônica , Células Dendríticas/imunologia , Células Dendríticas/virologia , Modelos Animais de Doenças , Humanos , Insetos Vetores/imunologia , Insetos Vetores/virologia , Macaca , Macrófagos/imunologia , Macrófagos/virologia , Monócitos/metabolismo , Células Mieloides/imunologia , Células Mieloides/virologia , Células Vero , Viremia/imunologiaRESUMO
BACKGROUND: Chikungunya virus (CHIKV) infection induces arthralgia. The involvement of inflammatory cytokines and chemokines has been suggested, but very little is known about their secretion profile in CHIKV-infected patients. METHODS: A case-control longitudinal study was performed that involved 30 adult patients with laboratory-confirmed Chikungunya fever. Their profiles of clinical disease, viral load, and immune mediators were investigated. RESULTS: When patients were segregated into high viral load and low viral load groups during the acute phase, those with high viremia had lymphopenia, lower levels of monocytes, neutrophilia, and signs of inflammation. The high viral load group was also characterized by a higher production of pro-inflammatory cytokines, such as interferon-α and interleukin (IL)-6, during the acute phase. As the disease progressed to the chronic phase, IL-17 became detectable. However, persistent arthralgia was associated with higher levels of IL-6 and granulocyte macrophage colony-stimulating factor, whereas patients who recovered fully had high levels of Eotaxin and hepatocyte growth factor. CONCLUSIONS: The level of CHIKV viremia during the acute phase determined specific patterns of pro-inflammatory cytokines, which were associated with disease severity. At the chronic phase, levels of IL-6, and granulocyte macrophage colony-stimulating factor found to be associated with persistent arthralgia provide a possible explanation for the etiology of arthralgia that plagues numerous CHIKV-infected patients.
Assuntos
Artralgia/etiologia , Artrite/virologia , Vírus Chikungunya/imunologia , Vírus Chikungunya/patogenicidade , Adulto , Idoso , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/patologia , Artrite/imunologia , Artrite/patologia , Sangue/imunologia , Sangue/virologia , Estudos de Casos e Controles , Febre de Chikungunya , Citocinas/sangue , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Carga ViralRESUMO
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.
Assuntos
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.
Assuntos
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.
Assuntos
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
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.