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OBJECTIVES: There is limited data on the incidence, prevalence, and treatments for myelofibrosis (MF) in Germany. This retrospective study examined claims data from 3.3 million insured individuals, spanning from 2010 to 2021. METHODS: Four sensitivity scenarios were explored to identify cases of MF. Point prevalence and cumulative incidence of MF were determined as of December 31, 2021, and within 2021, respectively. A cross-sectional analysis used the main scenario definition of MF to identify cases and evaluate the period prevalence of patients receiving treatment for symptoms and/or splenomegaly, including first-line (1L) Janus kinase inhibitor (JAKi), second-line, or further (2L+) MF-related treatment therapies during 2021. The prevalence of anemia treatment was also reported. RESULTS: The estimated standardized point prevalence of MF on December 31, 2021, was 9.9-12.4 cases per 100 000 persons, and cumulative incidence in 2021 was 1.2-1.8 cases per 100 000 persons. Standardized period prevalence in 2021 for MF patients receiving 1L JAKi and/or 2L+ MF-related treatment was 4.0 cases per 100 000. Among these patients, 47.1%-53.7% required treatment for anemia, resulting in a period prevalence of 1.9-2.2 cases per 100 000 individuals. CONCLUSION: The data reveal gaps in MF treatments and the need to improve patient quality of life.
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The approved dose of bosutinib in chronic phase CML is 400 mg QD in first-line and 500 mg QD in later-line treatment. However, given that gastrointestinal (GI) toxicity typically occurs early after treatment initiation, physicians often tend to start therapy with lower doses although this has never been tested systematically in prospective trials in the Western world. The Bosutinib Dose Optimization (BODO) Study, a multicenter phase II study, investigated the tolerability and efficacy of a step-in dosing concept of bosutinib (starting at 300 mg QD) in chronic phase CML patients in 2nd or 3rd line who were intolerant and/or refractory to previous TKI treatment. Of 57 patients included until premature closure of the study due to slow recruitment, 34 (60%) reached the targeted dose level of 500 mg QD following the 2-weekly step-in dosing regimen. While the dosing-in concept failed to reduce GI toxicity (grade II-IV, primary study endpoint) to < 40% (overall rate of 60%; 95% CI: 45-74%), bosutinib treatment (mean dosage: 403 mg/day) showed remarkable efficacy with a cumulative major molecular remission (MMR) rate of 79% (95% CI: 66 to 88%) at month 24. Of thirty patients refractory to previous therapy and not in MMR at baseline, 19 (64%) achieved an MMR during treatment. GI toxicity did not significantly impact on patient-reported outcomes (PRO) and led to treatment discontinuation in only one patient. Overall, the results of our trial support the efficacy and safety of bosutinib after failure of second-generation TKI pre-treatment. Trial registration: NCT02577926.
Assuntos
Leucemia Mieloide de Fase Crônica , Humanos , Estudos Prospectivos , Compostos de Anilina/efeitos adversos , Leucemia Mieloide de Fase Crônica/tratamento farmacológicoRESUMO
The pandemic of the severe acute respiratory syndrome coronavirus (SARS-CoV)-2 at the end of 2019 marked the third outbreak of a highly pathogenic coronavirus affecting the human population in the past twenty years. Cross-species zoonotic transmission of SARS-CoV-2 has caused severe pathogenicity and led to more than 655,000 fatalities worldwide until July 28, 2020. Outbursts of this virus underlined the importance of controlling infectious pathogens across international frontiers. Unfortunately, there is currently no clinically approved antiviral drug or vaccine against SARS-CoV-2, although several broad-spectrum antiviral drugs targeting multiple RNA viruses have shown a positive response and improved recovery in patients. In this review, we compile our current knowledge of the emergence, transmission, and pathogenesis of SARS-CoV-2 and explore several features of SARS-CoV-2. We emphasize the current therapeutic approaches used to treat infected patients. We also highlight the results of in vitro and in vivo data from several studies, which have broadened our knowledge of potential drug candidates for the successful treatment of patients infected with and discuss possible virus and host-based treatment options against SARS-CoV-2.
Assuntos
Betacoronavirus , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/genética , Betacoronavirus/fisiologia , COVID-19 , Vacinas contra COVID-19 , Coronaviridae/patogenicidade , Infecções por Coronaviridae/epidemiologia , Infecções por Coronaviridae/virologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/terapia , Infecções por Coronavirus/transmissão , Síndrome da Liberação de Citocina/etiologia , Síndrome da Liberação de Citocina/prevenção & controle , Citocinas/antagonistas & inibidores , Sistemas de Liberação de Medicamentos , Endocitose/efeitos dos fármacos , Previsões , Genoma Viral , Saúde Global , Humanos , Imunidade Coletiva , Imunização Passiva , Pandemias/prevenção & controle , Peptídeo Hidrolases/farmacologia , Peptídeo Hidrolases/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/epidemiologia , Pneumonia Viral/prevenção & controle , Pneumonia Viral/transmissão , RNA Viral/genética , Receptores de Coronavírus , Receptores Virais/antagonistas & inibidores , Receptores Virais/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/metabolismo , Vacinas Virais , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Zoonoses , Tratamento Farmacológico da COVID-19 , Soroterapia para COVID-19Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Melanoma Experimental/imunologia , Neoplasias Cutâneas/imunologia , Linfócitos T Citotóxicos/imunologia , Proteína Supressora de Tumor Von Hippel-Lindau/imunologia , Animais , HumanosRESUMO
Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau (VHL) gene. Since this discovery, additional VHL mutations have been identified in patients with congenital erythrocytosis, in a homozygous or compound-heterozygous state. VHL is a major tumor suppressor gene, mutations in which were first described in patients presenting with VHL disease, which is characterized by the development of highly vascularized tumors. Here, we identify a new VHL cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. More importantly, we identify mutations in E1' in 7 families with erythrocytosis (1 homozygous case and 6 compound-heterozygous cases with a mutation in E1' in addition to a mutation in VHL coding sequences) and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, we show that the mutations induced a dysregulation of VHL splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In addition, we demonstrate a pathogenic role for synonymous mutations in VHL exon 2 that altered splicing through E2-skipping in 5 families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially affected splicing, correlating with phenotype severity. This study demonstrates that cryptic exon retention and exon skipping are new VHL alterations and reveals a novel complex splicing regulation of the VHL gene. These findings open new avenues for diagnosis and research regarding the VHL-related hypoxia-signaling pathway.
Assuntos
Éxons , Predisposição Genética para Doença , Mutação , Policitemia/genética , Splicing de RNA , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Doença de von Hippel-Lindau/genética , Adolescente , Adulto , Criança , Feminino , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Policitemia/classificação , Policitemia/patologia , Adulto Jovem , Doença de von Hippel-Lindau/patologiaRESUMO
BACKGROUND: The identification of pathologically altered neutrophil granulocyte migration patterns bears strong potential for surveillance and prognostic scoring of diseases. We recently identified a strong correlation between impaired neutrophil motility and the disease stage of myelodysplastic syndrome (MDS). Here, we apply this assay to study quantitively increased neutrophils of a patient suffering from a rare leukemia subtype, atypical chronic myeloid leukemia (aCML). METHODS: A 69-year-old male was analyzed in this study. Besides routine analyses, we purified the patient's neutrophils from peripheral whole blood and studied their migration behavior using time-lapse video microscopy in a standardized assay. These live cell migration analyses also allowed for the quantification of cell morphology. Furthermore, the cells were stained for the markers CD15, CD16, fMLPR, CXCR1 and CXCR2. RESULTS: Despite cytoreductive therapy with hydroxyurea, the patient's WBC and ANC were poorly controlled and severe dysgranulopoiesis with hypogranularity was observed. Neutrophils displayed strongly impaired migration when compared to healthy controls and migrating cells exhibited a more flattened-out morphology than control neutrophils. Because of a detected CSF3R (p.T618I) mutation and constitutional symptoms treatment with ruxolitinib was initiated. Within 1 week of ruxolitinib treatment, the cell shape normalized and remained indistinguishable from healthy control neutrophils. However, neutrophil migration did not improve over the course of ruxolitinib therapy but was strikingly altered shortly before a sinusitis with fever and bleeding from a gastric ulcer. Molecular work-up revealed that under ruxolitinib treatment, the CSF3R clone was depleted, yet the expansion of a NRAS mutated subclone was promoted. CONCLUSION: These results demonstrate the usefulness of neutrophil migration analyses to uncover corresponding alterations of neutrophil migration in rare myeloid neoplasms. Furthermore, in addition to monitoring migration the determination of morphological features of live neutrophils might represent a useful tool to monitor the effectiveness of therapeutic approaches.
Assuntos
Biomarcadores Tumorais/genética , Movimento Celular , Granulócitos/patologia , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/tratamento farmacológico , Neutrófilos/patologia , Pirazóis/efeitos adversos , Idoso , Estudos de Casos e Controles , Feminino , Granulócitos/efeitos dos fármacos , Granulócitos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia Mieloide Crônica Atípica BCR-ABL Negativa/patologia , Estudos Longitudinais , Masculino , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Nitrilas , Prognóstico , PirimidinasRESUMO
Farber disease (FD) is a rare lysosomal storage disorder resulting from acid ceramidase deficiency and subsequent ceramide accumulation. No treatments are clinically available and affected patients have a severely shortened lifespan. Due to the low incidence, the pathogenesis of FD is still poorly understood. Here, we report a novel acid ceramidase mutant mouse model that enables the study of pathogenic mechanisms of FD and ceramide accumulation. Asah1tmEx1 mice were generated by deletion of the acid ceramidase signal peptide sequence. The effects on lysosomal targeting and activity of the enzyme were assessed. Ceramide and sphingomyelin levels were quantified by liquid chromatography tandem-mass spectrometry (LC-MS/MS) and disease manifestations in several organ systems were analyzed by histology and biochemistry. We show that deletion of the signal peptide sequence disrupts lysosomal targeting and enzyme activity, resulting in ceramide and sphingomyelin accumulation. The affected mice fail to thrive and die early. Histiocytic infiltrations were observed in many tissues, as well as lung inflammation, liver fibrosis, muscular disease manifestations and mild kidney injury. Our new mouse model mirrors human FD and thus offers further insights into the pathogenesis of this disease. In the future, it may also facilitate the development of urgently needed therapies.
Assuntos
Modelos Animais de Doenças , Lipogranulomatose de Farber/patologia , Animais , Ceramidas/análise , Ceramidas/metabolismo , Cromatografia Líquida , Lipogranulomatose de Farber/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Esfingomielinas/análise , Esfingomielinas/metabolismo , Espectrometria de Massas em TandemRESUMO
Neutrophil granulocyte biology is a central issue of immunological research, but the lack of animal models that allow for neutrophil-selective genetic manipulation has delayed progress. By modulating the neutrophil-specific locus Ly6G with a knock-in allele expressing Cre recombinase and the fluorescent protein tdTomato, we generated a mouse model termed Catchup that exhibits strong neutrophil specificity. Transgene activity was found only in very few eosinophils and basophils and was undetectable in bone marrow precursors, including granulomonocytic progenitors (GMPs). Cre-mediated reporter-gene activation allowed for intravital two-photon microscopy of neutrophils without adoptive transfer. Homozygous animals were Ly6G deficient but showed normal leukocyte cellularity in all measured organs. Ly6G-deficient neutrophils were functionally normal in vitro and in multiple models of sterile or infectious inflammation in vivo. However, Cre-mediated deletion of FcγRIV in neutrophils reduced the cells' recruitment to immune-complex-mediated peritonitis, suggesting a cell-intrinsic role for activating Fc receptors in neutrophil trafficking.
Assuntos
Neutrófilos/citologia , Neutrófilos/fisiologia , Animais , Antígenos Ly/genética , Antígenos Ly/metabolismo , Morte Celular , Movimento Celular , Feminino , Regulação da Expressão Gênica/fisiologia , Técnicas de Transferência de Genes , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Peritonite/patologia , Espécies Reativas de Oxigênio , Transgenes/genéticaRESUMO
Long-term repopulating (LT) hematopoietic stem cells (HSCs) are the most undifferentiated cells at the top of the hematopoietic hierarchy. The regulation of HSC pool size and its contribution to hematopoiesis are incompletely understood. We depleted hematopoietic stem and progenitor cells (HSPCs) in adult mice in situ and found that LT-HSCs recovered from initially very low levels (<1%) to below 10% of normal numbers but not more, whereas progenitor cells substantially recovered shortly after depletion. In spite of the persistent and massive reduction of LT-HSCs, steady-state hematopoiesis was unaffected and residual HSCs remained quiescent. Hematopoietic stress, although reported to recruit quiescent HSCs into cycle, was well tolerated by HSPC-depleted mice and did not induce expansion of the small LT-HSC compartment. Only upon 5-fluorouracil treatment was HSPC-depleted bone marrow compromised in reconstituting hematopoiesis, demonstrating that HSCs and early progenitors are crucial to compensate myeloablation. Hence, a contracted HSC compartment cannot recover in situ to its original size, and normal steady-state blood cell generation is sustained with <10% of normal LT-HSC numbers without increased contribution of the few residual cells.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas/citologia , Estresse Fisiológico , Animais , Contagem de Células , Proliferação de Células , Camundongos Endogâmicos C57BL , Nicho de Células-TroncoRESUMO
The hepatocyte NF (HNF) family of transcription factors regulates the complex gene networks involved in lipid, carbohydrate, and protein metabolism. In humans, HNF1A mutations cause maturity onset of diabetes in the young type 3, whereas murine HNF6 participates in fetal liver B lymphopoiesis. In this study, we have identified a crucial role for the prototypical member of the family HNF1A in adult bone marrow B lymphopoiesis. HNF1A(-/-) mice exhibited a clear reduction in total blood and splenic B cells and a further pronounced one in transitional B cells. In HNF1A(-/-) bone marrow, all B cell progenitors-from pre-pro-/early pro-B cells to immature B cells-were dramatically reduced and their proliferation rate suppressed. IL-7 administration in vivo failed to boost B cell development in HNF1A(-/-) mice, whereas IL-7 stimulation of HNF1A(-/-) B cell progenitors in vitro revealed a marked impairment in STAT5 phosphorylation. The B cell differentiation potential of HNF1A(-/-) common lymphoid progenitors was severely impaired in vitro, and the expression of the B lymphopoiesis-promoting transcription factors E2A, EBF1, Pax5, and Bach2 was reduced in B cell progenitors in vivo. HNF1A(-/-) bone marrow chimera featured a dramatic defect in B lymphopoiesis recapitulating that of global HNF1A deficiency. The HNF1A(-/-) lymphopoiesis defect was confined to B cells as T lymphopoiesis was unaffected, and bone marrow common lymphoid progenitors and hematopoietic stem cells were even increased. Our data demonstrate that HNF1A is an important cell-intrinsic transcription factor in adult B lymphopoiesis and suggest the IL-7R/STAT5 module to be causally involved in mediating its function.
Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Fator 1-alfa Nuclear de Hepatócito/imunologia , Linfopoese/imunologia , Animais , Linfócitos B/citologia , Separação Celular , Citometria de Fluxo , Células Progenitoras Linfoides/citologia , Células Progenitoras Linfoides/imunologia , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Fatores de TranscriçãoRESUMO
BACKGROUND: The blood-brain barrier (BBB) formed by brain endothelial cells interconnected by tight junctions is essential for the homeostasis of the central nervous system. Although studies have shown the importance of various signaling molecules in BBB formation during development, little is known about the molecular basis regulating the integrity of the adult BBB. METHODS AND RESULTS: Using a mouse model with tamoxifen-inducible endothelial cell-restricted disruption of ctnnb1 (iCKO), we show here that endothelial ß-catenin signaling is essential for maintaining BBB integrity and central nervous system homeostasis in adult mice. The iCKO mice developed severe seizures accompanied by neuronal injury, multiple brain petechial hemorrhages, and central nervous system inflammation, and all had postictal death. Disruption of endothelial ß-catenin induced BBB breakdown and downregulation of the specific tight junction proteins claudin-1 and -3 in adult brain endothelial cells. The clinical relevance of the data is indicated by the observation of decreased expression of claudin-1 and nuclear ß-catenin in brain endothelial cells of hemorrhagic lesions of hemorrhagic stroke patients. CONCLUSIONS: These results demonstrate the prerequisite role of endothelial ß-catenin in maintaining the integrity of adult BBB. The results suggest that BBB dysfunction secondary to defective ß-catenin transcription activity is a key pathogenic factor in hemorrhagic stroke, seizure activity, and central nervous system inflammation.
Assuntos
Gânglios da Base/metabolismo , Barreira Hematoencefálica/fisiologia , Hemorragia Cerebral/metabolismo , beta Catenina/deficiência , beta Catenina/fisiologia , Adulto , Idoso , Animais , Ataxia/etiologia , Encéfalo/patologia , Hemorragia Cerebral/etiologia , Claudina-1/biossíntese , Claudina-1/deficiência , Claudina-1/genética , Claudina-3/biossíntese , Claudina-3/genética , Cruzamentos Genéticos , Citocinas/biossíntese , Citocinas/genética , Regulação para Baixo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Genes Reporter , Homeostase , Humanos , Hiperestesia/etiologia , Inflamação , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Especificidade de Órgãos , Interferência de RNA , Convulsões/etiologia , Junções Íntimas , Transgenes , beta Catenina/biossíntese , beta Catenina/genéticaRESUMO
BACKGROUND/AIMS: Rheumatoid arthritis is a chronic autoimmune disease hallmarked by inflammation in synovial joints. Treatment is hampered by the lack of a cure and current disease-modifying drugs are associated with potentially severe toxicities. METHODS: We investigated arthritis severity by measuring joint swelling and pro-inflammatory cytokine production in a murine experimental model of inflammatory arthritis (antigen-induced arthritis). We analyzed acid sphingomyelinase knock-out mice and wild-type littermates, as well as mice treated with the pharmacological acid sphingomyelinase inhibitor amitriptyline. RESULTS: Genetic ablation or pharmacological inhibition of acid sphingomyelinase reduced joint swelling and levels of pro-inflammatory cytokines in the arthritic joint. CONCLUSION: We identified acid sphingomyelinase as a novel druggable target in rheumatoid arthritis. Functional inhibitors of acid sphingomyelinase have been clinically used for decades, are well tolerated and suitable for long-term treatment. They would be immediately available for clinical development as a novel rheumatoid arthritis therapy.
Assuntos
Artrite Experimental/genética , Artrite Experimental/patologia , Articulações/patologia , Esfingomielina Fosfodiesterase/genética , Amitriptilina/uso terapêutico , Animais , Artrite Experimental/tratamento farmacológico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Modelos Animais de Doenças , Deleção de Genes , Articulações/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esfingomielina Fosfodiesterase/antagonistas & inibidoresRESUMO
RATIONALE: Lymphotoxin ß receptor (LTbR) regulates immune cell trafficking and communication in inflammatory diseases. However, the role of LTbR in atherosclerosis is still unclear. OBJECTIVE: The aim of this study was to elucidate the role of LTbR in atherosclerosis. METHODS AND RESULTS: After 15 weeks of feeding a Western-type diet, mice double-deficient in apolipoprotein E and LTbR (apoE(-/-)/LTbR(-/-)) exhibited lower aortic plaque burden than did apoE(-/-) littermates. Macrophage content at the aortic root and in the aorta was reduced, as determined by immunohistochemistry and flow cytometry. In line with a decrease in plaque inflammation, chemokine (C-C motif) ligand 5 (Ccl5) and other chemokines were transcriptionally downregulated in aortic tissue from apoE(-/-)/LTbR(-/-) mice. Moreover, bone marrow chimeras demonstrated that LTbR deficiency in hematopoietic cells mediated the atheroprotection. Furthermore, during atheroprogression, apoE(-/-) mice exhibited increased concentrations of cytokines, for example, Ccl5, whereas apoE(-/-)/LTbR(-/-) mice did not. Despite this decreased plaque macrophage content, flow cytometric analysis showed that the numbers of circulating lymphocyte antigen 6C (Ly6C)(low) monocytes were markedly elevated in apoE(-/-)/LTbR(-/-) mice. The influx of these cells into atherosclerotic lesions was significantly reduced, whereas apoptosis and macrophage proliferation in atherosclerotic lesions were unaffected. Gene array analysis pointed to chemokine (C-C motif) receptor 5 as the most regulated pathway in isolated CD115(+) cells in apoE(-/-)/LTbR(-/-) mice. Furthermore, stimulating monocytes from apoE(-/-) mice with agonistic anti-LTbR antibody or the natural ligand lymphotoxin-α1ß2, increased Ccl5 mRNA expression. CONCLUSIONS: These findings suggest that LTbR plays a role in macrophage-driven inflammation in atherosclerotic lesions, probably by augmenting the Ccl5-mediated recruitment of monocytes.
Assuntos
Aorta/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Apolipoproteínas E/deficiência , Aterosclerose/prevenção & controle , Receptor beta de Linfotoxina/deficiência , Animais , Antígenos Ly/metabolismo , Aorta/imunologia , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/diagnóstico , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Transplante de Medula Óssea , Células Cultivadas , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Quimiotaxia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Heterotrímero de Linfotoxina alfa1 e beta2/metabolismo , Receptor beta de Linfotoxina/genética , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Placa Aterosclerótica , Fatores de Tempo , Transcrição Gênica , Quimeras de TransplanteRESUMO
The close relationship between endothelial and hematopoietic precursors during early development of the vascular system suggested the possibility of a common yet elusive precursor for both cell types. Whether similar or related progenitors for endothelial and hematopoietic cells are present during organogenesis is unclear. Using inducible transgenic mice that specifically label endothelial and hematopoietic precursors, we performed fate-tracing studies combined with colony-forming assays and crosstransplantation studies. We identified a progenitor, marked by the expression of helix-loop-helix transcription factor stem cell leukemia (SCL/Tal1). During organogenesis of the kidney, SCL/Tal1(+) progenitors gave rise to endothelium and blood precursors with multipotential colony-forming capacity. Furthermore, appropriate morphogenesis of the kidney vasculature, including glomerular capillary development, arterial mural cell coating, and lymphatic vessel development, required sphingosine 1-phosphate (S1P) signaling via the G protein-coupled S1P receptor 1 in these progenitors. Overall, these results show that SCL/Tal1(+) progenitors with hemogenic capacity originate and differentiate within the early embryonic kidney by hemovasculogenesis (the concomitant formation of blood and vessels) and underscore the importance of the S1P pathway in vascular development.
Assuntos
Vasos Sanguíneos/embriologia , Rim/irrigação sanguínea , Organogênese , Receptores de Lisoesfingolipídeo/fisiologia , Animais , Endotélio Vascular/embriologia , Camundongos , Receptores de Esfingosina-1-FosfatoRESUMO
Rapid activation of immune responses is necessary for antibacterial defense, but excessive immune activation can result in life-threatening septic shock. Understanding how these processes are balanced may provide novel therapeutic potential in treating inflammatory disease. Fc receptors are crucial for innate immune activation. However, the role of the putative Fc receptor for IgM, known as Toso/Faim3, has to this point been unclear. In this study, we generated Toso-deficient mice and used them to uncover a critical regulatory function of Toso in innate immune activation. Development of innate immune cells was intact in the absence of Toso, but Toso-deficient neutrophils exhibited more reactive oxygen species production and reduced phagocytosis of pathogens compared with controls. Cytokine production was also decreased in Toso(-/-) mice compared with WT animals, rendering them resistant to septic shock induced by lipopolysaccharide. However, Toso(-/-) mice also displayed limited cytokine production after infection with the bacterium Listeria monocytogenes that was correlated with elevated presence of Listeria throughout the body. Accordingly, Toso(-/-) mice succumbed to infections of L. monocytogenes, whereas WT mice successfully eliminated the infection. Taken together, our data reveal Toso to be a unique regulator of innate immune responses during bacterial infection and septic shock.
Assuntos
Proteínas de Transporte/imunologia , Granulócitos/imunologia , Imunidade Inata/imunologia , Listeriose/imunologia , Ativação de Macrófagos/imunologia , Proteínas de Membrana/imunologia , Monócitos/imunologia , Análise de Variância , Animais , Proteínas de Transporte/genética , Cruzamentos Genéticos , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Immunoblotting , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Peroxidase/metabolismo , Fagocitose/imunologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Variants of the multidrug resistance gene (MDR1/ABCB1) have been associated with increased susceptibility to severe ulcerative colitis (UC). In this study, we investigated the role of TLR/IL-1R signaling pathways including the common adaptor MyD88 in the pathogenesis of chronic colonic inflammation in MDR1A deficiency. Double- or triple-null mice lacking TLR2, MD-2, MyD88, and MDR1A were generated in the FVB/N background. Deletion of TLR2 in MDR1A deficiency resulted in fulminant pancolitis with early expansion of CD11b(+) myeloid cells and rapid shift toward TH1-dominant immune responses in the lamina propria. Colitis exacerbation in TLR2/MDR1A double-knockout mice required the unaltered commensal microbiota and the LPS coreceptor MD-2. Blockade of IL-1ß activity by treatment with IL-1R antagonist (IL-1Ra; Anakinra) inhibited colitis acceleration in TLR2/MDR1A double deficiency; intestinal CD11b(+)Ly6C(+)-derived IL-1ß production and inflammation entirely depended on MyD88. TLR2/MDR1A double-knockout CD11b(+) myeloid cells expressed MD-2/TLR4 and hyperresponded to nonpathogenic Escherichia coli or LPS with reactive oxygen species production and caspase-1 activation, leading to excessive cell death and release of proinflammatory IL-1ß, consistent with pyroptosis. Inhibition of reactive oxygen species-mediated lysosome degradation suppressed LPS hyperresponsiveness. Finally, active UC in patients carrying the TLR2-R753Q and MDR1-C3435T polymorphisms was associated with increased nuclear expression of caspase-1 protein and cell death in areas of acute inflammation, compared with active UC patients without these variants. In conclusion, we show that the combined defect of two UC susceptibility genes, MDR1A and TLR2, sets the stage for spontaneous and uncontrolled colitis progression through MD-2 and IL-1R signaling via MyD88, and we identify commensally induced pyroptosis as a potential innate immune effector in severe UC pathogenesis.
Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Colite Ulcerativa/genética , Colite Ulcerativa/imunologia , Receptor 2 Toll-Like/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Antígeno CD11b , Caspase 1/metabolismo , Morte Celular/genética , Morte Celular/imunologia , Colo/imunologia , Colo/metabolismo , Colo/patologia , Progressão da Doença , Deleção de Genes , Humanos , Interleucina-1beta/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Antígeno 96 de Linfócito/metabolismo , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mutação , Células Mieloides/imunologia , Células Mieloides/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Receptor 2 Toll-Like/deficiênciaRESUMO
Inflammation contributes to ischemic brain injury. However, translation of experimental findings from animal models into clinical trials is still ineffective, since the majority of human stroke studies mainly focus on acute neuroprotection, thereby neglecting inflammatory mechanisms and inflammation-associated co-morbidity factors such as hypercholesterolemia. Therefore, both wildtype and ApoE(-/-) mice that exhibit increased serum plasma cholesterol levels fed with normal or high cholesterol diet were exposed to transient middle cerebral artery occlusion. Analysis of peripheral immune responses revealed an ischemia-induced acute leukocytosis in the blood, which was accompanied by enhanced myeloid cell and specifically granulocyte cell counts in the spleen and blood of ApoE(-/-) mice fed with Western diet. These cellular immune changes were further associated with increased levels of pro-inflammatory cytokines like IL-6 and TNF-α. Moreover, endogenous stroke-induced endothelial activation as well as CXCL-1 and CXCL-2 expression were increased, thus resulting in accelerated leukocyte, particularly granulocyte accumulation, and enhanced ischemic tissue damage. The latter was revealed by larger infarct volumes and increased local DNA fragmentation in ischemic brains of ApoE(-/-) mice on Western diet. These effects were not observed in wildtype mice on normal or Western diet and in ApoE(-/-) mice on normal diet. Our data demonstrate that the combination of both ApoE knockout and a high cholesterol diet leads to increased ischemia-induced peripheral and cerebral immune responses, which go along with enhanced cerebral tissue injury. Thus, clinically predisposing conditions related to peripheral inflammation such as hypercholesterolemia should be included in up-coming preclinical stroke research.
Assuntos
Isquemia Encefálica/imunologia , Córtex Cerebral/imunologia , Hipercolesterolemia/imunologia , Animais , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Quimiocinas/metabolismo , Colesterol/administração & dosagem , Células Endoteliais/metabolismo , Granulócitos/metabolismo , Hipercolesterolemia/metabolismo , Hipercolesterolemia/patologia , Infarto da Artéria Cerebral Média , Leucocitose/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismoRESUMO
OBJECTIVE: In resistance arteries, diameter adjustment in response to pressure changes depends on the vascular cytoskeleton integrity. Serum response factor (SRF) is a dispensable transcription factor for cellular growth, but its role remains unknown in resistance arteries. We hypothesized that SRF is required for appropriate microvascular contraction. METHODS AND RESULTS: We used mice in which SRF was specifically deleted in smooth muscle or endothelial cells, and their control. Myogenic tone and pharmacological contraction was determined in resistance arteries. mRNA and protein expression were assessed by quantitative real-time PCR (qRT-PCR) and Western blot. Actin polymerization was determined by confocal microscopy. Stress-activated channel activity was measured by patch clamp. Myogenic tone developing in response to pressure was dramatically decreased by SRF deletion (5.9±2.3%) compared with control (16.3±3.2%). This defect was accompanied by decreases in actin polymerization, filamin A, myosin light chain kinase and myosin light chain expression level, and stress-activated channel activity and sensitivity in response to pressure. Contractions induced by phenylephrine or U46619 were not modified, despite a higher sensitivity to p38 blockade; this highlights a compensatory pathway, allowing normal receptor-dependent contraction. CONCLUSIONS: This study shows for the first time that SRF has a major part to play in the control of local blood flow via its central role in pressure-induced myogenic tone in resistance arteries.
Assuntos
Pressão Arterial , Músculo Liso Vascular/metabolismo , Fator de Resposta Sérica/metabolismo , Cauda/irrigação sanguínea , Resistência Vascular , Vasodilatação , Actinas/metabolismo , Animais , Pressão Arterial/efeitos dos fármacos , Artérias/metabolismo , Western Blotting , Sinalização do Cálcio , Proteínas Contráteis/metabolismo , Relação Dose-Resposta a Droga , Filaminas , Regulação da Expressão Gênica , Masculino , Mecanotransdução Celular , Potenciais da Membrana , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Microscopia Confocal , Músculo Liso Vascular/efeitos dos fármacos , Miografia , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Técnicas de Patch-Clamp , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Resposta Sérica/deficiência , Fator de Resposta Sérica/genética , Fatores de Tempo , Resistência Vascular/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The balance between bone-forming osteoblasts and bone-resorbing osteoclasts is crucial to bone homeostasis, an equilibrium that is disturbed in many bone diseases. The transcription factor Tal1 is involved in the establishment of hematopoietic stem cells in the embryo and is a master regulator of hematopoietic gene expression in the adult. Here, we show that Tal1 is expressed in osteoclasts and that loss of Tal1 in osteoclast progenitors leads to altered expression of >1200 genes. We found that DC-STAMP, a key regulator of osteoclast cell fusion, is a direct target gene of Tal1 and show that Tal1 represses DC-STAMP expression by counteracting the activating function of the transcription factors PU.1 and MITF. The identification of Tal1 as a factor involved in cell fusion contributes to the understanding of osteoclast-associated diseases, including osteoporosis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Osteoclastos/citologia , Osteoclastos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Sítios de Ligação , Remodelação Óssea , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Diferenciação Celular/fisiologia , Fusão Celular , Células Cultivadas , Expressão Gênica , Técnicas de Silenciamento de Genes , Hematopoese , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Fator de Transcrição Associado à Microftalmia/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Transativadores/metabolismoRESUMO
Genomic profiling revealed the identity of at least 5 subtypes of diffuse large B-cell lymphoma (DLBCL), including the MCD/C5 cluster characterized by aberrations in MYD88, BCL2, PRDM1, and/or SPIB. We generated mouse models harboring B cell-specific Prdm1 or Spib aberrations on the background of oncogenic Myd88 and Bcl2 lesions. We deployed whole-exome sequencing, transcriptome, flow-cytometry, and mass cytometry analyses to demonstrate that Prdm1- or Spib-altered lymphomas display molecular features consistent with prememory B cells and light-zone B cells, whereas lymphomas lacking these alterations were enriched for late light-zone and plasmablast-associated gene sets. Consistent with the phenotypic evidence for increased B cell receptor signaling activity in Prdm1-altered lymphomas, we demonstrate that combined BTK/BCL2 inhibition displays therapeutic activity in mice and in five of six relapsed/refractory DLBCL patients. Moreover, Prdm1-altered lymphomas were immunogenic upon transplantation into immuno-competent hosts, displayed an actionable PD-L1 surface expression, and were sensitive to antimurine-CD19-CAR-T cell therapy, in vivo. SIGNIFICANCE: Relapsed/refractory DLBCL remains a major medical challenge, and most of these patients succumb to their disease. Here, we generated mouse models, faithfully recapitulating the biology of MYD88-driven human DLBCL. These models revealed robust preclinical activity of combined BTK/BCL2 inhibition. We confirmed activity of this regimen in pretreated non-GCB-DLBCL patients. See related commentary by Leveille et al., p. 8. This article is highlighted in the In This Issue feature, p. 1.