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BACKGROUND: Fibroblast differentiation to a myofibroblast phenotype is a feature of airway remodeling in asthma. Lung fibroblasts express the integrin receptor α4ß7 and fibronectin induces myofibroblast differentiation via this receptor. OBJECTIVES: To investigate the role of the ß7 integrin receptor subunit and α4ß7 integrin complex in airway remodeling and airway hyperresponsiveness (AHR) in a murine model of chronic allergen exposure. METHODS: C57BL/6 wild type (WT) and ß7 integrin null mice (ß7 -/-) were sensitized (days 1,10) and challenged with ovalbumin (OVA) three times a week for one or 4 weeks. Similar experiments were performed with WT mice in the presence or absence of α4ß7 blocking antibodies. Bronchoalveolar (BAL) cell counts, AHR, histological evaluation, soluble collagen content, Transforming growth factor-ß (TGFß) and Interleukin-13 (IL13) were measured. Phenotype of fibroblasts cultured from WT and ß7 -/- saline (SAL) and OVA treated mice was evaluated. RESULTS: Eosinophil numbers were similar in WT vs ß7-/- mice. Prolonged OVA exposure in ß7-/- mice was associated with reduced AHR, lung collagen content, peribronchial smooth muscle, lung tissue TGFß and IL13 expression as compared to WT. Similar findings were observed in WT mice treated with α4ß7 blocking antibodies. Fibroblast migration was enhanced in response to OVA in WT but not ß7 -/- fibroblasts. α-SMA and fibronectin expression were reduced in ß7-/- fibroblasts relative to WT. CONCLUSIONS: The ß7 integrin subunit and the α4ß7 integrin complex modulate AHR and airway remodeling in a murine model of allergen exposure. This effect is, at least in part, explained by inhibition of fibroblast activation and is independent of eosinophilic inflammation.
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Remodelação das Vias Aéreas , Cadeias beta de Integrinas , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina , Animais , Remodelação das Vias Aéreas/fisiologia , Remodelação das Vias Aéreas/imunologia , Camundongos , Ovalbumina/toxicidade , Cadeias beta de Integrinas/metabolismo , Cadeias beta de Integrinas/genética , Alérgenos/imunologia , Alérgenos/toxicidade , Células Cultivadas , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/metabolismo , Hiper-Reatividade Brônquica/fisiopatologia , Hiper-Reatividade Brônquica/patologia , Pulmão/metabolismo , Pulmão/imunologia , Pulmão/patologia , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor T-cell (CAR T) therapy shows remarkable efficacy in patients with relapsed and/or refractory (R/R) multiple myeloma (MM). HBI0101, a novel second generation optimized anti- BCMA CAR T-cell therapy, was developed in an academic setting. We conducted a phase I dose-escalation study of HBI0101 (cohort 1: 150x106 CAR T cells, n=6; cohort 2: 450x106 CAR T cells, n=7; cohort 3: 800x106 CAR T cells, n=7) in 20 heavily pre-treated R/R MM patients. Grade 1-2 cytokine release syndrome (CRS) was reported in 18 patients (90%). Neither grade 3-4 CRS nor neurotoxicity of any grade were observed. No dose-limiting toxicities were observed in any cohort. The overall response rate (ORR), (stringent) complete response (CR/sCR), and very good partial response rates were 75%, 50%, and 25%, respectively. Response rates were dose-dependent with 85% ORR, 71% CR, and 57% minimal residual disease negativity in the high-dose cohort 3. Across all cohorts, the median overall survival (OS) was 308 days (range 25-466+), with an estimated OS of 55% as of June 27th (data cut-off). The median progression-free survival was 160 days, with 6 subjects remaining progression free at the time of data cut-off. Our findings demonstrate the manageable safety profile and efficacy of HBI0101. These encouraging data support the decentralization of CAR T production in an academic setting, ensuring sufficient CAR T supply to satisfy the increasing local demand. Clinicaltrials.gov NCT04720313.
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Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Humanos , Mieloma Múltiplo/tratamento farmacológico , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Linfócitos T , AnticorposRESUMO
Chimeric antigen receptor (CAR) T-cell based immunotherapy has become a promising treatment mainly for hematological malignancies. Following the major success of CD19-targeted CAR, new potential targets for other malignancies are required. As such, B-cell maturation antigen (BCMA) is an attractive tumor-associated antigen to be targeted in multiple myeloma (MM). Herein, we aimed at assessing the function and optimal configuration of different BCMA-specific CAR, based on the same targeting moiety but with a different hinge and co-stimulatory domain. We compared their function to that of a previously characterized BCMA-CAR used in clinical trials. All constructs were expressed at high levels by primary human T cells and could trigger cytokine production and cytotoxicity upon co-culture with multiple myeloma targets. Nonetheless, critical differences were observed in off-target activation, exhaustion, and activation marker expression and in vivo antitumoral activity mediated by these different constructs. Interestingly, we noted that CD8-based hinge, combined with a 4-1BB intracellular domain, proved superior compared to IgG4-connecting regions, and/or a CD28-signaling moiety respectively. Overall, this study emphasizes the influence of CAR primary structure on its function and led to the identification of a highly efficient BCMA-specific CAR, namely H8BB, which displayed superior anti-tumoral activity both in vitro and long-term in vivo efficacy.
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Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Antígeno de Maturação de Linfócitos B/metabolismo , Antígenos CD28 , Citocinas , Humanos , Imunoglobulina G , Imunoterapia Adotiva , Mieloma Múltiplo/patologiaRESUMO
BACKGROUND: Omalizumab is an anti-immunoglobulin E (IgE) monoclonal antibody used in the treatment of severe asthma. Its therapeutic efficacy is primarily attributed to reduction of serum-free IgE and in the expression of high-affinity IgE receptor, fc epsilon RI. However, its effect on the low-affinity IgE receptor fc epsilon RII/CD23 in vivo has not been evaluated. AIM: To determine whether CD23 plays a role in the inflammatory process in severe uncontrolled asthma and whether anti-IgE therapy modulates fc epsilon RII/CD23 expression in these patients. METHODS: We evaluated the expression of IgE receptors fc epsilon RI, fc epsilon RII/CD23, and soluble CD23 (sCD23), and the activation state of peripheral blood monocytes (tumor necrosis factor alpha, interleukin (IL) 1-beta, transforming growth factor (TGF) beta expression) in the patients with severe asthma before and after 24 weeks of omalizumab treatment and in the healthy controls. Cytokine expression of monocytes in response to different stimulation (IL-4, IL-4 plus IgE, IL-4 plus IgE plus anti-IgE, and IL-4 plus IgE plus anti-IgE plus anti-CD23 for 72 hours) was determined by enzyme-linked immunosorbent assay. RESULTS: Treatment with omalizumab (for 24 weeks) improved disease control and pulmonary function (forced expiratory volume in the first second of expiration, 64.5 versus 74%; p = 0.021). Mean ± SE expression of fc epsilon RI on monocytes was higher in the patients with asthma versus the controls (45.7 ± 12.2% versus 18.6 ± 5.8%; p = 0.04) and was reduced after omalizumab treatment (45.7 ± 12.2% versus 15.6 ± 4.4%; p = 0.027). Mean ± SE TGF-beta levels in supernatants from monocytes were reduced in the patients treated with omalizumab (211 ± 6 pg/mL versus 184 ± 9 pg/mL; p = 0.036). CONCLUSION: Modulation of the low affinity IgE receptor CD23 in severe asthma is complex, and sCD23 may inversely reflect disease activity. Treatment with omalizumab was associated with reduced monocyte activation.
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Asma/tratamento farmacológico , Omalizumab/farmacologia , Receptores de IgE/efeitos dos fármacos , Antiasmáticos/uso terapêutico , Asma/imunologia , Estudos de Casos e Controles , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Monócitos/metabolismo , Omalizumab/uso terapêutico , Fator de Crescimento Transformador beta/efeitos dos fármacos , Resultado do TratamentoRESUMO
BACKGROUND: We have recently observed that oxidative phosphorylation-mediated ATP production is essential for mast cell function. Pyruvate dehydrogenase (PDH) is the main regulator of the Krebs cycle and is located upstream of the electron transport chain. However, the role of PDH in mast cell function has not been described. Microphthalmia transcription factor (MITF) regulates the development, number, and function of mast cells. Localization of MITF to the mitochondria and its interaction with mitochondrial proteins has not been explored. OBJECTIVE: We sought to explore the role played by PDH in mast cell exocytosis and to determine whether MITF is localized in the mitochondria and involved in regulation of PDH activity. METHODS: Experiments were performed in vitro by using human and mouse mast cells, as well as rat basophil leukemia cells, and in vivo in mice. The effect of PDH inhibition on mast cell function was examined. PDH interaction with MITF was measured before and after immunologic activation. Furthermore, mitochondrial localization of MITF and its effect on PDH activity were determined. RESULTS: PDH is essential for immunologically mediated degranulation of mast cells. After activation, PDH is serine dephosphorylated. In addition, for the first time, we show that MITF is partially located in the mitochondria and interacts with PDH. This interaction is dependent on the phosphorylation state of PDH. Furthermore, mitochondrial MITF regulates PDH activity. CONCLUSION: The association of mitochondrial MITF with PDH emerges as an important regulator of mast cell function. Our findings indicate that PDH could arise as a new target for the manipulation of allergic diseases.
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Cetona Oxirredutases/imunologia , Mastócitos/imunologia , Fator de Transcrição Associado à Microftalmia/imunologia , Trifosfato de Adenosina/metabolismo , Alérgenos/imunologia , Animais , Asma/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Degranulação Celular , Linhagem Celular Tumoral , Células Cultivadas , Exocitose , Feminino , Células HEK293 , Humanos , Masculino , Mastócitos/metabolismo , Mastócitos/fisiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Fator de Transcrição Associado à Microftalmia/genética , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Ovalbumina/imunologia , RatosRESUMO
ABSTRACT: HBI0101 is an academic chimeric antigen receptor T-cell (CART)-targeted to B-cell maturation antigen (BCMA) for the treatment of relapsed and refractory multiple myeloma (R/RMM) and light chain amyloidosis. Herein, we present the phase 1b/2 results of 50 heavily pretreated patients with R/RMM dosed with 800 × 106 CART cells. Inclusion criteria were relatively permissive (i.e., performance status and baseline organ function) and consequently, approximately half of the enrolled patients would have been ineligible for pivotal clinical trials. The median time elapsed from patient enrollment until CART delivery was 25 days (range, 14-65). HBI0101-related toxicities included grade 1 to 3 cytokine release syndrome, grade 3 to 4 hematologic toxicities, and grade 1 to 2 immune effector cell-associated neurotoxicity syndrome. Responses were achieved in 90% of the patients, 56% achieved stringent and complete response, and 70% reached a minimal residual disease negativity. Within a median follow-up of 12.3 months, the median progression-free survival (PFS) was 11.0 months (95% confidence interval [CI], 6.2-14.6), and the overall survival was not reached (95% CI, 13.3 to not reached). Multivariable analysis on patient/disease and CART-related characteristics revealed that high-risk cytogenetic, extramedullary disease, and increased number of effector-memory T cells in CART products were independently associated with inferior PFS. In conclusion, comprehensive analyses of the parameters affecting the response to CART therapy are essential for improving patients' outcome. This trial was registered at www.ClinicalTrials.gov as #NCT04720313.
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Antígeno de Maturação de Linfócitos B , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/terapia , Antígeno de Maturação de Linfócitos B/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Idoso , Feminino , Adulto , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Resultado do Tratamento , Receptores de Antígenos Quiméricos/uso terapêutico , Recidiva , Idoso de 80 Anos ou mais , Anticorpos Monoclonais HumanizadosRESUMO
PURPOSE: Although chimeric antigen receptor T therapy (CAR-T) cells are an established therapy for relapsed/refractory multiple myeloma (RRMM), there are no established models predicting outcome to identify patients who may benefit the most from CAR-T. PATIENTS AND METHODS: This is an international retrospective observational study including patients with RRMM infused with currently available commercial or academically produced anti-B-cell maturation antigen (BCMA) CAR-T. We describe characteristics and outcomes in Europe (n = 136) and the United States (n = 133). Independent predictors of relapse/progression built a simple prediction model (Myeloma CAR-T Relapse [MyCARe] model) in the training cohort (Europe), which was externally validated (US cohort) and tested within patient- and treatment-specific subgroups. RESULTS: The overall response rate was 87% and comparable between both cohorts, and complete responses were seen in 48% (Europe) and 49% (the United States). The median time to relapse was 5 months, and early relapse <5 months from infusion showed poor survival across cohorts, with the 12-month overall survival of 30% (Europe) and 14% (the United States). The presence of extramedullary disease or plasma cell leukemia, lenalidomide-refractoriness, high-risk cytogenetics, and increased ferritin at the time of lymphodepletion were independent predictors of early relapse or progression. Each factor received one point, forming the three-tiered MyCARe model: scores 0-1 (low risk), scores 2-3 (intermediate risk), and a score of 4 (high risk). The MyCARe model was significantly associated with distinct 5-month incidence of relapse/progression (P < .001): 7% for low-risk, 27% for intermediate-risk, and 53% for high-risk groups. The model was validated in the US cohort and maintained prognostic utility for response, survival, and outcomes across subgroups. CONCLUSION: Outcomes of patients with RRMM after CAR-T are comparable between Europe and the United States. The MyCARe model may facilitate optimal timing of CAR-T cells in patient-specific subgroups.
Assuntos
Antígeno de Maturação de Linfócitos B , Imunoterapia Adotiva , Mieloma Múltiplo , Humanos , Mieloma Múltiplo/terapia , Mieloma Múltiplo/mortalidade , Mieloma Múltiplo/imunologia , Pessoa de Meia-Idade , Masculino , Estudos Retrospectivos , Feminino , Idoso , Imunoterapia Adotiva/métodos , Antígeno de Maturação de Linfócitos B/imunologia , Estados Unidos , Adulto , Receptores de Antígenos Quiméricos/imunologia , Europa (Continente) , Resultado do Tratamento , Recidiva Local de Neoplasia/terapiaRESUMO
BACKGROUND: Extra domain A-containing fibronectin (EDA-FN) is necessary for the development of allergen-induced lower airway fibrosis. The pathogenesis of fibrosis in allergic rhinitis has not been well studied. OBJECTIVES: To determine whether EDA-fibronectin is necessary for the development of nasal remodeling in a murine model of chronic allergic rhinitis and in human allergic rhinitis. METHODS: EDA(-/-) and wild-type (WT) C57Bl/6 mice were sensitized intraperitoneally and then challenged with inhaled ovalbumin (OVA) or saline for 2 and 5 weeks. Clinical signs of rhinitis and histological analysis of nasal tissue were evaluated. Immunohistological staining for EDA-FN was performed in human tissue of inferior nasal conchae from patients with allergic rhinitis and controls. RESULTS: After 2 weeks of allergen exposure, only goblet cell hyperplasia and perivascular eosinophilia were observed. After 5 weeks, goblet cell number, thickening of the subepithelial layer, and extent and area of collagen deposition were increased in the nasal tissue of WT OVA (ovalbumin)-challenged mice as compared with saline controls (P < .0001, P < .0001, P = .018, and P = .03, respectively). Clinical signs of rhinitis were observed only in WT OVA-challenged mice. In the EDA(-/-) mice exposed to OVA, collagen deposition, collagen area, and subepithelial thickness showed no increase and were similar to saline control mice, whereas goblet cell hyperplasia was similar to WT OVA-challenged mice. EDA-FN expression was prominent in inferior conchae from patients with allergic rhinitis but was absent in control patients. CONCLUSION: EDA-containing fibronectin is necessary for the development of nasal tissue fibrotic remodeling process in both murine and human allergic rhinitis.
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Fibronectinas/imunologia , Mucosa Nasal/imunologia , Rinite/imunologia , Adulto , Alérgenos/imunologia , Animais , Colágeno/imunologia , Eosinofilia/imunologia , Feminino , Fibronectinas/genética , Células Caliciformes/patologia , Humanos , Hiperplasia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Mucosa Nasal/patologia , Ovalbumina/imunologia , Rinite/patologiaRESUMO
Multiple myeloma (MM) is characterized by recurrent relapses. Consequently, patients receive multiple therapy lines, including alkylating agents and immune modulators, which have been associated with secondary malignancies such as myelodysplastic syndrome (MDS). Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor T cell (CART) therapy is efficacious in patients with relapsed/refractory (R/R) MM. However, the long-term complications, particularly MDS, are not well understood. Whether CART therapy causes or promotes MDS has not been thoroughly investigated. In this study, we explored the causal relationship between MDS and CART therapy. We retrospectively examined the prevalence of MDS-related morphological and mutational changes before and after administration of CART therapy in five patients. Among them, four developed MDS after CART therapy, while one had pre-existing MDS prior to CART. None of the four patients who developed post-CART MDS showed morphological MDS changes prior to CART therapy. However, all four patients exhibited molecular alterations associated with MDS in their pre-CART as well as post-CART therapy bone marrow. No new mutations were observed. Our findings provide initial evidence suggesting that anti-BCMA CART therapy in MM may promote expansion of pre-existing MDS clones rather than causing development of new clones.
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Long-term heat acclimation (LTHA; 30 days, 34°C) causes phenotypic adaptations that render protection against ischemic/reperfusion insult (I/R, 30 min global ischemia and 40 min reperfusion) via heat acclimation-mediated cross-tolerance (HACT) mechanisms. Short-term acclimation (STHA, 2 days, 34 °C), in contrast, is characterized by cellular perturbations, leading to increased susceptibility to insults. Here, we tested the hypothesis that enhanced mitochondrial respiratory function is part of the acclimatory repertoire and that the 30-day regimen is required for protection via HACT. We subjected isolated hearts and mitochondria from controls (C), STHA, or LTHA rats to I/R, hypoxia/reoxygenation, or Ca2+ overload insults. Mitochondrial function was assessed by measuring O2 consumption, membrane potential (ΔΨm), mitochondrial Ca2+ ([Ca2+]m), ATP production, respiratory chain complex activities, and molecular markers of mitochondrial biogenesis. Our results, combining physiological and biochemical parameters, confirmed that mitochondria from LTHA rats subjected to insults, in contrast to C, preserve respiratory functions (e.g., upon I/R, C mitochondria fueled by glutamate-malate, demonstrated decreases of 81%, 13%, 25%, and 50% in O2/P ratio, ATP production, ΔΨm, and complex I activity, respectively, whereas the corresponding LTHA parameters remained unchanged). STHA mitochondria maintained ΔΨm but did not preserve ATP production. LTHA [Ca2+]m was significantly higher than that of C and STHA and was not affected by the hypoxia/reoxygenation protocol compared with C. Enhanced mitochondrial biogenesis markers, switched-on during STHA coincidentally with enhanced membrane integrity (ΔΨm), were insufficient to confer intact respiratory function upon insult. LTHA was required for respiratory complex I adaptation and HACT. Stabilized higher basal [Ca2+]m and attenuated Ca2+ overload are likely connected to this adaptation.
Assuntos
Aclimatação , Cálcio/metabolismo , Temperatura Alta , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Aclimatação/genética , Adaptação Fisiológica , Trifosfato de Adenosina/metabolismo , Animais , DNA Mitocondrial/metabolismo , Modelos Animais de Doenças , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético , Regulação da Expressão Gênica , Masculino , Potencial da Membrana Mitocondrial , Mitocôndrias Cardíacas/patologia , Renovação Mitocondrial/genética , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Consumo de Oxigênio , Fenótipo , RNA Mensageiro/metabolismo , Ratos , Fatores de TempoRESUMO
PURPOSE: AL amyloidosis (AL) treatments are generally based on those employed for multiple myeloma. Anti-B-cell maturation antigen (BCMA) chimeric antigen receptor T (CART)-cell therapy, already approved for multiple myeloma, might be too toxic for patients with AL. EXPERIMENTAL DESIGN: Here we describe the ex vivo applicability of a novel in-house, academic anti-BCMA CAR construct on AL primary cells, as well as the safety and efficacy in 4 patients with relapsed/refractory (RR) primary AL, treated in a phase I clinical trial (NCT04720313). RESULTS: Three had MAYO stage IIIa cardiac involvement at enrollment. The treatment proved relatively safe, with a short and manageable grade 3 cytokine release syndrome evident in 2 patients and no neurotoxicity in any. Cardiac decompensations, observed in 2 patients, were also short and manageable. The overall hematologic response and complete response rates were observed in all patients with an organ response evident in all four. Within a median follow-up period of 5.2 (2.5-9.5) months, all 4 patients maintained their responses. CONCLUSIONS: BCMA-CART cells provide a first proof-of-concept that this therapy is safe enough and highly efficacious for the treatment of patients with advanced, RR AL.
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Amiloidose de Cadeia Leve de Imunoglobulina , Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Humanos , Estudos de Viabilidade , Amiloidose de Cadeia Leve de Imunoglobulina/tratamento farmacológico , Amiloidose de Cadeia Leve de Imunoglobulina/etiologia , Imunoterapia Adotiva/efeitos adversos , Mieloma Múltiplo/tratamento farmacológicoRESUMO
Over the last decades, several studies demonstrated the possibility of lung regeneration through transplantation of various lung progenitor populations. Recently, we showed in mice that fetal or adult lung progenitors could potentially provide donor cells for transplantation, provided that the lung stem cell niche in the recipient is vacated of endogenous lung progenitors by adequate conditioning. Accordingly, marked lung regeneration could be attained following i.v. infusion of a single cell suspension of lung cells into recipient mice conditioned with naphthalene (NA) and 6Gy total body irradiation (TBI). As clinical translation of this approach requires the use of allogenic donors, we more recently developed a novel transplantation modality based on co-infusion of hematopoietic and lung progenitors from the same donor. Thus, by virtue of hematopoietic chimerism, which leads to immune tolerance toward donor antigens, the lung progenitors can be successfully engrafted without any need for post-transplant immune suppression. In the present study, we demonstrate that it is possible to replace NA in the conditioning regimen with Cyclophosphamide (CY), approved for the treatment of many diseases and that a lower dose of 2 GY TBI can successfully enable engraftment of donor-derived hematopoietic and lung progenitors when CY is administered in 2 doses after the stem cell infusion. Taken together, our results suggest a feasible and relatively safe protocol that could potentially be translated to clinical transplantation of lung progenitors across major MHC barriers in patients with terminal lung diseases.
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Transplante de Células-Tronco Hematopoéticas , Condicionamento Pré-Transplante , Animais , Ciclofosfamida , Humanos , Indicadores e Reagentes , Pulmão , Camundongos , Quimeras de Transplante , Condicionamento Pré-Transplante/métodosRESUMO
Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease with a poor prognosis and limited treatment options. Oxidative and nitrosative stress is implicated as one of the main pathogenic pathways in IPF. The rationale for the use of antioxidants to treat lung fibrosis is appealing, however to date a consistent beneficial effect for such an approach has not been observed. We have recently demonstrated that nitroxides, particularly 3-carbamoyl-proxyl (3-CP), markedly reduce airway inflammation, airway hyper-responsiveness, and protein nitration of the lung tissue in a mouse model of ovalbumin-induced acute asthma, thus prompting its use for the treatment of IPF. The present study investigates the effect of 3-CP on the development of lung fibrosis using the murine intratracheal bleomycin model. 3-CP was administered either intranasally or orally during the entire experiment or starting 7 days after induction of the lung injury. 3-CP was found to be both a preventive and a therapeutic drug reducing the lung fibrosis (histological score), the increase in collagen content, protein nitration, TGF-ß levels, the degree of weight loss as well as inhibiting the impairment of lung function. Nitroxides are catalytic antioxidants that preferentially detoxify radicals, and therefore the effect of 3-CP on the severity of the disease supports the involvement of reactive oxygen and nitrogen species in the disease pathology.
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Bleomicina , Óxidos N-Cíclicos , Animais , Bleomicina/toxicidade , Óxidos N-Cíclicos/farmacologia , Modelos Animais de Doenças , Pulmão , Camundongos , Camundongos Endogâmicos C57BL , Óxidos de Nitrogênio , PirrolidinasRESUMO
Asthma is characterized by airway inflammation, hyper-responsiveness, symptoms of dyspnea, wheezing and coughing. In most patients, asthma is well controlled using inhaled corticosteroids and bronchodilators. A minority of patients with asthma develop severe disease, which is frequently only partially responsive or even resistant to treatment with corticosteroids. Severe refractory asthma is associated with structural changes in the airways, termed "airway remodeling", and/or with neutrophilic rather than eosinophilic airway inflammation. While oxidative stress plays an important role in the pathophysiology of asthma, cyclic nitroxide stable radicals, which are unique and efficient catalytic antioxidants, effectively protect against oxidative injury. We have demonstrated that the nitroxide 3-carbamoyl proxyl (3-CP) attenuates airway inflammation and hyperresponsiveness in allergic asthma as well as bleomycin-induced fibrosis both using murine models, most probably through modulation of oxidative stress. The present study evaluates the effect of 3-CP on airway inflammation and remodeling using two murine models of severe asthma where mice are sensitized and challenged either by ovalbumin (OVA) or by house dust mite (HDM). 3-CP was orally administered during the entire period of the experiment or during the challenge period alone where its effect was compared to that of dexamethasone. The induced increase by OVA and by HDM of BALf cell counts, airway hyperresponsiveness, fibrosis, transforming growth factor-beta (TGF-ß) levels in BALf and protein nitration levels of the lung tissue was significantly reduced by 3-CP. The effect of 3-CP, using two different murine models of severe asthma, is associated at least partially with attenuation of oxidative stress and with TGF-ß expression in the lungs. The results of this study suggest a potential use of 3-CP as a novel therapeutic agent in different forms of severe asthma.
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Antioxidantes , Asma , Animais , Asma/tratamento farmacológico , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Óxidos de Nitrogênio , Índice de Gravidade de DoençaRESUMO
Carbenoxolone is an anti-inflammatory compound and a derivate of a natural substance from the licorice plant. We previously showed that carbenoxolone reduces the metastatic burden in the lungs of mice through its antagonistic effect on high mobility group box 1 (HMGB1). To further enhance carbenoxolone's activity and localization in the lungs, thereby reducing the potential adverse side effects resulting from systemic exposure, we developed a poly(lactic-co-glycolic acid) (PLGA) slow-release system for pulmonary delivery which maintains drug activity in-vitro, as demonstrated in the anoikis assay. Both systemic and intranasal administrations of carbenoxolone effectively minimize metastatic formation in a lung colonization model in mice. Our results show a decrease in the metastatic burden in the lung tissue. Notably, the therapeutic effect of a single intranasal administration of 25 mg/kg carbenoxolone, in the form of drug-loaded particles, had a similar effect in reducing metastatic lesions in the lungs to that of a 10-fold dose of the free drug via intraperitoneal injections, three times per week over the course of four weeks. These data offer new means to potentiate the anti-cancer activity of carbenoxolone and simultaneously reduce the requirement for high dosage administration; the upshot substantially improves therapeutic effect and avoidance of side effects.
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Repair of injured lungs represents a longstanding therapeutic challenge. We recently demonstrated that human and mouse embryonic lung tissue from the canalicular stage of development are enriched with lung progenitors, and that a single cell suspension of canalicular lungs can be used for transplantation, provided that lung progenitor niches in the recipient mice are vacated by strategies similar to those used in bone marrow transplantation. Considering the ethical limitations associated with the use of fetal cells, we investigated here whether adult lungs could offer an alternative source of lung progenitors for transplantation. We show that intravenous infusion of a single cell suspension of adult mouse lungs from GFP+ donors, following conditioning of recipient mice with naphthalene and subsequent sublethal irradiation, led to marked colonization of the recipient lungs, at 6-8 weeks post-transplant, with donor derived structures including epithelial, endothelial, and mesenchymal cells. Epithelial cells within these donor-derived colonies expressed markers of functionally distinct lung cell types, and lung function, which is significantly compromised in mice treated with naphthalene and radiation, was found to be corrected following transplantation. Dose response analysis suggests that the frequency of patch forming cells in adult lungs was about threefold lower compared to that found in E16 fetal lungs. However, as adult lungs are much larger, the total number of patch forming cells that can be collected from this source is significantly greater. Our study provides proof of concept for lung regeneration by adult lung cells after preconditioning to vacate the pulmonary niche. Stem Cells Translational Medicine 2018;7:68-77.
Assuntos
Células Epiteliais/transplante , Regeneração Tecidual Guiada/métodos , Lesão Pulmonar/terapia , Pulmão/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Animais , Células Cultivadas , Células Epiteliais/citologia , Feminino , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Naftalenos/toxicidadeRESUMO
BACKGROUND: Sarcoidosis is a systemic inflammatory disease of unknown etiology. Osteopontin (SPP1, OPN) is an extra cellular matrix glycoprotein and cytokine with a known role in granuloma formation and in autoimmune and inflammatory diseases. OBJECTIVE: To determine whether plasma OPN levels are elevated in patients with sarcoidosis and compare the frequency of four single nucleotide polymorphism (SNPs) variants in the OPN gene in sarcoidosis patients compared to healthy controls. METHODS: Demographic and clinical information, radiological studies and pulmonary function tests were evaluated in 113 patients with sarcoidosis and in 79 healthy controls. Blood samples were analyzed for SNPs of the OPN gene and for plasma OPN and CRP levels. Association between clinical features of disease and OPN levels as well as SNP frequencies was determined. RESULTS: Plasma OPN levels were higher in sarcoidosis patients than in healthy subjects, (median: 217 vs 122ng/ml, p<0.001). Area under the curve for receiver operator curves (ROC) was 0.798 (0.686-0.909 95% CI.) No differences were observed between sarcoidosis patients and controls in the frequency of any of the SNPs evaluated. Presence of lung parenchymal involvement was associated with SNP distribution at rs1126772 (p = 0.02). We found no correlation between SNPs distribution and plasma OPN levels. CONCLUSIONS: Osteopontin protein levels are elevated in sarcoidosis. We found no evidence for an association between SNPs on the osteopontin gene and plasma OPN levels or the presence of sarcoidosis, however, an association between genotype and several phenotypic clinical parameters of disease was observed.
Assuntos
Regulação da Expressão Gênica/genética , Osteopontina/genética , Polimorfismo de Nucleotídeo Único , Sarcoidose/genética , Regiões 3' não Traduzidas/genética , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteopontina/sangue , Sarcoidose/sangue , Adulto JovemRESUMO
The effects of stable cyclic nitroxide radicals have been extensively investigated both in vivo and in vitro demonstrating anti-inflammatory, radioprotective, anti-mutagenic, age-retardant, hypotensive, anti-cancer and anti-teratogenic activities. Yet, these stable radicals have not been evaluated in asthma and other airway inflammatory disorders. The present study investigated the effect of 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl (TPL) and 3-carbamoyl-proxyl (3-CP) in a mouse model of ovalbumin (OVA)-induced allergic asthma. Both 3-CP and TPL were non-toxic when administered either orally (1% w/w nitroxide-containing chow) or via intraperitoneal (IP) injection (â¼300 mg/kg). Feeding the mice orally demonstrated that 3-CP was more effective than TPL in reducing inflammatory cell recruitment into the airway and in suppressing airway hyper-responsiveness (AHR) in OVA-challenged mice. To characterize the optimal time-window of intervention and mode of drug administration, 3-CP was given orally during allergen sensitization, during allergen challenge or during both sensitization and challenge stages, and via IP injection or intranasal instillation for 3 days during the challenge period. 3-CP given via all modes of delivery markedly inhibited OVA-induced airway inflammation, expression of cytokines, AHR and protein nitration of the lung tissue. Oral administration during the entire experiment was the most efficient delivery of 3-CP and was more effective than dexamethasone a potent corticosteroid used for asthma treatment. Under a similar administration regimen (IP injection before the OVA challenge), the effect of 3-CP was similar to that of dexamethasone and even greater on AHR and protein nitration. The protective effect of the nitroxides, which preferentially react with free radicals, in suppressing the increase of main asthmatic inflammatory markers substantiate the key role played by reactive oxygen and nitrogen species in the molecular mechanism of asthma. The present results demonstrate the therapeutic potential of nitroxides for the treatment of asthma.
Assuntos
Asma/tratamento farmacológico , Óxidos N-Cíclicos/administração & dosagem , Radicais Livres/administração & dosagem , Inflamação/tratamento farmacológico , Hipersensibilidade Respiratória/tratamento farmacológico , Alérgenos/imunologia , Alérgenos/toxicidade , Animais , Asma/induzido quimicamente , Asma/patologia , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , Humanos , Inflamação/patologia , Masculino , Camundongos , Óxidos de Nitrogênio/administração & dosagem , Ovalbumina/imunologia , Ovalbumina/toxicidade , Hipersensibilidade Respiratória/induzido quimicamente , Hipersensibilidade Respiratória/patologiaRESUMO
Repair of injured lungs represents a longstanding therapeutic challenge. We show that human and mouse embryonic lung tissue from the canalicular stage of development (20-22 weeks of gestation for humans, and embryonic day 15-16 (E15-E16) for mouse) are enriched with progenitors residing in distinct niches. On the basis of the marked analogy to progenitor niches in bone marrow (BM), we attempted strategies similar to BM transplantation, employing sublethal radiation to vacate lung progenitor niches and to reduce stem cell competition. Intravenous infusion of a single cell suspension of canalicular lung tissue from GFP-marked mice or human fetal donors into naphthalene-injured and irradiated syngeneic or SCID mice, respectively, induced marked long-term lung chimerism. Donor type structures or 'patches' contained epithelial, mesenchymal and endothelial cells. Transplantation of differentially labeled E16 mouse lung cells indicated that these patches were probably of clonal origin from the donor. Recipients of the single cell suspension transplant exhibited marked improvement in lung compliance and tissue damping reflecting the energy dissipation in the lung tissues. Our study provides proof of concept for lung reconstitution by canalicular-stage human lung cells after preconditioning of the pulmonary niche.
Assuntos
Células-Tronco Embrionárias/transplante , Pulmão/embriologia , Condicionamento Pré-Transplante , Animais , Bromodesoxiuridina/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos SCID , Regeneração , Quimeras de Transplante , Transplante HeterólogoRESUMO
Faster reinduction of heat acclimation (AC) after its decline indicates "AC memory." Our previous results revealed involvement of epigenetic mechanisms of transcriptional regulation. We hypothesized that the decline of AC (DeAC) is a period of "dormant memory" during which many processes are alerted to enable rapid reacclimation (ReAC). Using a genomewide approach we studied the AC, DeAC, and ReAC transcriptomes, to uncover hallmark pathways linked to "molecular memory" in the cardioacclimatome. Fifty rats subjected to heat acclimation [34°C for 2d (AC2d) or 30d (AC30)], DeAC (24°C, 30 days), ReAC (34°C, 2 days), and untreated controls were used. The GeneChip Rat Gene 1.0 ST Array was employed for left ventricular (cardiac) mRNA hybridization. Three independent bioinformatic analyses showed that 1) during AC2d enrichment of DNA impair/repair-linked genes is seen, and this is the molecular on-switch of acclimation; 2) genes activated in AC30 underlie the qualitative physiological adaptations of cardiac performance; 3) particular molecular programs encompassing constitutive upregulation of p38 MAPK, Jak/Stat, and Akt pathways and targets are specifically activated during DeAC and ReAC; and 4) epigenetic markers such as linker histones (histones H1 cluster), associated with nucleosome spacing, transcriptional chromatin modifiers, poly-(ADP-ribose) polymerase-1 (PARP1) linked to chromatin compaction, and microRNAs are only altered during DeAC/ReAC. The latter are newcomers to the AC/DeAC puzzle. We suggest that these transcriptional responses maintain euchromatin and proteostasis and enable faster physiological recovery upon ReAC by rapidly reestablishing the protected acclimated cardiophenotype. We propose that the cardiac AC model can be applied to acclimation processes in general.