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1.
Life Sci ; 284: 119906, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34478761

RESUMO

The present study was performed to investigate the effects of Cd exposure on lipid metabolism and mitochondrial dysfunction and to explore the role of mitophagy in Cd-induced dysregulation of lipid metabolism in chicken embryo liver tissues and hepatocytes. To this end, seven-day-old chicken embryos were exposed to different concentrations of Cd for 7 days, and primary chicken embryo hepatocytes were treated with Cd at four different concentrations for 6 h. Furthermore, the mitophagy inhibitor cyclosporine A (CsA) was used to investigate the role of mitophagy in Cd-induced disruption of lipid metabolism. Lipid accumulation, the expression levels of genes involved in lipid metabolism, mitochondrial dysfunction, and mitophagy were measured. The results demonstrated that Cd exposure increases hepatic triglyceride (TG) accumulation and the expression levels of lipogenic genes while decreasing those of lipolytic genes. Furthermore, Cd exposure was observed to alter mitochondrial morphology in terms of reduced size, excessive mitochondrial damage, and the formation of mitophagosomes. The co-localization of lysosome-associated membrane glycoprotein 2 and LC3 puncta was significantly increased in primary chicken embryo hepatocytes after Cd exposure. Moreover, Cd exposure increased LC3, PINK1, and Parkin protein expression levels. CsA effectively alleviated Cd-induced mitochondrial dysfunction, blocked mitochondrial membrane potential collapse, and suppressed PINK1/Parkin-mediated mitophagy. Furthermore, CsA treatment reversed the Cd-induced TG accumulation in liver tissues but further increased it in hepatocytes. Taken together, our findings demonstrate (for the first time) the importance of mitochondrial dysfunction and mitophagy via the PINK1/Parkin pathway in Cd-induced disruption of lipid metabolism.


Assuntos
Cádmio/toxicidade , Metabolismo dos Lipídeos , Fígado/metabolismo , Mitocôndrias Hepáticas/patologia , Mitofagia , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Embrião de Galinha , Ciclosporina/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/embriologia , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/ultraestrutura , Mitofagia/efeitos dos fármacos , Modelos Biológicos
2.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445139

RESUMO

Recent evidence pinpoints extracellular vesicles (EVs) as key players in intercellular communication. Given the importance of cholesterol and sphingomyelin in EV biology, and the relevance of mitochondria-associated endoplasmic reticulum membranes (MAMs) in cholesterol/sphingomyelin homeostasis, we evaluated if MAMs and sphingomyelinases (SMases) could participate in ethanol-induced EV release. EVs were isolated from the extracellular medium of BV2 microglia treated or not with ethanol (50 and 100 mM). Radioactive metabolic tracers combined with thin layer chromatography were used as quantitative methods to assay phospholipid transfer, SMase activity and cholesterol uptake/esterification. Inhibitors of SMase (desipramine and GW4869) and MAM (cyclosporin A) activities were also utilized. Our data show that ethanol increases the secretion and inflammatory molecule concentration of EVs. Ethanol also upregulates MAM activity and alters lipid metabolism by increasing cholesterol uptake, cholesterol esterification and SMase activity in microglia. Notably, the inhibition of either SMase or MAM activity prevented the ethanol-induced increase in EV secretion. Collectively, these results strongly support a lipid-driven mechanism, specifically via SMases and MAM, to explain the effect of ethanol on EV secretion in glial cells.


Assuntos
Retículo Endoplasmático/efeitos dos fármacos , Etanol/farmacologia , Vesículas Extracelulares/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Esfingomielina Fosfodiesterase/metabolismo , Compostos de Anilina/farmacologia , Animais , Compostos de Benzilideno/farmacologia , Células Cultivadas , Colesterol/metabolismo , Ciclosporina/farmacologia , Retículo Endoplasmático/metabolismo , Vesículas Extracelulares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Fosfolipídeos/metabolismo
3.
Cell Prolif ; 54(8): e13084, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34170049

RESUMO

OBJECTIVES: Autoimmune diseases are a heterogeneous group of diseases which lose the immunological tolerance to self-antigens. It is well recognized that irregularly provoked T cells participate in the pathological immune responses. As a novel nanomaterial with promising applications, tetrahedral framework nucleic acid (TFNA) nanostructure was found to have immune regulatory effects on T cells in this study. MATERIALS AND METHODS: To verify the successful fabrication of TFNA, the morphology of TFNA was observed by atomic force microscopy (AFM) and dynamic light scattering. The regulatory effect of TFNA was evaluated by flow cytometry after cocultured with CD3+ T cells isolated from healthy donors. Moreover, the associated signaling pathways were investigated. Finally, we verified our results on the T cells from patients with neuromyelitis optica spectrum disorder (NMOSD), which is a typical autoimmune disease induced by T cells. RESULTS: We revealed the alternative regulatory functions of TFNA in human primary T cells with steady status via the JNK signaling pathway. Moreover, by inhibiting both JNK and ERK phosphorylation, TFNA exhibited significant suppressive effects on IFNγ secretion from provoking T cells without affecting TNF secretion. Similar immune regulatory effects of TFNA were also observed in autoreactive T cells from patients with NMOSD. CONCLUSIONS: Overall, our results revealed a potential application of TFNA in regulating the adaptive immune system, as well as shed a light on the treatment of T cell-mediated autoimmune diseases.


Assuntos
Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ácidos Nucleicos/farmacologia , Adulto , Células Cultivadas , Ciclosporina/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Interferon gama/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Pessoa de Meia-Idade , Nanoestruturas/química , Neuromielite Óptica/metabolismo , Neuromielite Óptica/patologia , Ácidos Nucleicos/síntese química , Ácidos Nucleicos/química , Fosforilação/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto Jovem
4.
Anticancer Res ; 41(6): 2781-2793, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34083268

RESUMO

BACKGROUND/AIM: This study explored the mechanisms of the allogeneic graft versus leukemia effect in acute lymphoblastic leukemia (ALL) cells by examining whether they change gene expression in the post-transplant environment containing cytokines and the immunosuppressant cyclosporine, and if such changes affect ALL cell survival. MATERIALS AND METHODS: RNASeq was used to assess leukemia global gene expression and flow cytometry to measure ALL survival in the presence of T cells, NK cells, cytokines, and cyclosporine. RESULTS: A total of 4,805 genes were differentially expressed. Gene set enrichment analysis demonstrated up-regulation of biological processes related to cytokine responses, control of viral infection, and regulation of leukocyte function including proliferation. Down-regulated genes were related to mesenchymal tissue morphogenesis. ALL cells exposed to cytokines and cyclosporine retained susceptibility to T and NK cell killing, and also exhibited increased cell death without exposure to killer cells. CONCLUSION: A significant portion of the graft versus leukemia effect may be mediated by cytokines and cyclosporine.


Assuntos
Sobrevivência Celular/genética , Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adulto , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Criança , Ciclosporina/farmacologia , Citocinas/sangue , Citocinas/fisiologia , Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Transplante Homólogo
5.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064311

RESUMO

Dephosphorylation of target proteins at serine/threonine residues is one of the most crucial mechanisms regulating their activity and, consequently, the cellular functions. The role of phosphatases in synaptic plasticity, especially in long-term depression or depotentiation, has been reported. We studied serine/threonine phosphatase activity during the protein synthesis blocker (PSB)-induced impairment of long-term potentiation (LTP). Established protein phosphatase 2B (PP2B, calcineurin) inhibitor cyclosporin A prevented the LTP early phase (E-LTP) decline produced by pretreatment of hippocampal slices with cycloheximide or anisomycin. For the first time, we directly measured serine/threonine phosphatase activity during E-LTP, and its significant increase in PSB-treated slices was demonstrated. Nitric oxide (NO) donor SNAP also heightened phosphatase activity in the same manner as PSB, and simultaneous application of anisomycin + SNAP had no synergistic effect. Direct measurement of the NO production in hippocampal slices by the NO-specific fluorescent probe DAF-FM revealed that PSBs strongly stimulate the NO concentration in all studied brain areas: CA1, CA3, and dentate gyrus (DG). Cyclosporin A fully abolished the PSB-induced NO production in the hippocampus, suggesting a close relationship between nNOS and PP2B activity. Surprisingly, cyclosporin A alone impaired short-term plasticity in CA1 by decreasing paired-pulse facilitation, which suggests bi-directionality of the influences of PP2B in the hippocampus. In conclusion, we proposed a minimal model of signaling events that occur during LTP induction in normal conditions and the PSB-treated slices.


Assuntos
Região CA1 Hipocampal/metabolismo , Região CA3 Hipocampal/metabolismo , Calcineurina/genética , Potenciação de Longa Duração/genética , Potenciais Sinápticos/genética , Animais , Anisomicina/farmacologia , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/efeitos dos fármacos , Região CA3 Hipocampal/citologia , Região CA3 Hipocampal/efeitos dos fármacos , Calcineurina/metabolismo , Inibidores de Calcineurina/farmacologia , Cicloeximida/farmacologia , Ciclosporina/farmacologia , Giro Denteado/citologia , Giro Denteado/efeitos dos fármacos , Giro Denteado/metabolismo , Regulação da Expressão Gênica , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Microtomia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/genética , Óxido Nítrico/química , Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Inibidores da Síntese de Proteínas/farmacologia , Ratos , Ratos Wistar , S-Nitroso-N-Acetilpenicilamina/química , S-Nitroso-N-Acetilpenicilamina/farmacologia , Potenciais Sinápticos/efeitos dos fármacos , Técnicas de Cultura de Tecidos
6.
Acta Cir Bras ; 36(4): e360402, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33978094

RESUMO

PURPOSE: To assess the influence of prolonged cyclosporine use on the macro- and microscopic morphology of the spleen. METHODS: 16 adult rabbits were divided into two groups (n = 8): group 1 - a placebo group, which was followed-up over a period of nine months; group 2 - which had taken an oral dose of cyclosporine (10 mg·kg-1·day-1) over nine months. At the end of this period, the splenic histoarchitecture of all animals was evaluated and the splenic corpuscles were measured. RESULTS: The spleens of the first group presented normal characteristics and dimensions. The second group, however, had a reduction in all dimensions and its tissue texture had become soft. The white pulp and the perivascular sheath had become reduced in size and the number of lymphoid follicles had also fallen (p = 0.002), manifesting less splenic corpuscles (p = 0.0012) and lymphocyte nuclear pigments (p = 0.03). CONCLUSIONS: Prolonged use of cyclosporine reduces the spleen size, transforming it into a soft organ associated with a decrease in white pulp, perivascular sheath, lymphoid follicles and nuclear pigments in rabbits.


Assuntos
Ciclosporina , Baço , Animais , Ciclosporina/farmacologia , Coelhos
7.
J Clin Invest ; 131(11)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33822776

RESUMO

Inhibitors of calcineurin phosphatase activity (CNIs) such as cyclosporin A (CsA) are widely used to treat tissue transplant rejection and acute graft-versus-host disease (aGVHD), for which inhibition of gene expression dependent on nuclear factor of activated T cells (NFAT) is the mechanistic paradigm. We recently reported that CNIs inhibit TCR-proximal signaling by preventing calcineurin-mediated dephosphorylation of LckS59, an inhibitory modification, raising the possibility of another mechanism by which CNIs suppress immune responses. Here we used T cells from mice that express LckS59A, which cannot accept a phosphate at residue 59, to initiate aGVHD. Although CsA inhibited NFAT-dependent gene upregulation in allo-aggressive T cells expressing either LckWT or LckS59A, it was ineffective in treating disease when the T cells expressed LckS59A. Two important NFAT-independent T cell functions were found to be CsA-resistant in LckS59A T cells: upregulation of the cytolytic protein perforin in tissue-infiltrating CD8+ T cells and antigen-specific T/DC adhesion and clustering in lymph nodes. These results demonstrate that effective treatment of aGVHD by CsA requires NFAT-independent inhibition of TCR signaling. Given that NFATs are widely expressed and off-target effects are a major limitation in CNI use, it is possible that targeting TCR-associated calcineurin directly may provide effective therapies with less toxicity.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Inibidores de Calcineurina/farmacologia , Ciclosporina/farmacologia , Doença Enxerto-Hospedeiro/tratamento farmacológico , Fatores de Transcrição NFATC/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Doença Aguda , Animais , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genética
8.
Cell Rep ; 35(1): 108959, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33811811

RESUMO

There is an urgent need for antivirals to treat the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To identify new candidates, we screen a repurposing library of ∼3,000 drugs. Screening in Vero cells finds few antivirals, while screening in human Huh7.5 cells validates 23 diverse antiviral drugs. Extending our studies to lung epithelial cells, we find that there are major differences in drug sensitivity and entry pathways used by SARS-CoV-2 in these cells. Entry in lung epithelial Calu-3 cells is pH independent and requires TMPRSS2, while entry in Vero and Huh7.5 cells requires low pH and triggering by acid-dependent endosomal proteases. Moreover, we find nine drugs are antiviral in respiratory cells, seven of which have been used in humans, and three are US Food and Drug Administration (FDA) approved, including cyclosporine. We find that the antiviral activity of cyclosporine is targeting Cyclophilin rather than calcineurin, revealing essential host targets that have the potential for rapid clinical implementation.


Assuntos
COVID-19/tratamento farmacológico , Ciclosporina/farmacologia , Reposicionamento de Medicamentos , Células Epiteliais/metabolismo , Pulmão/metabolismo , SARS-CoV-2/metabolismo , Animais , COVID-19/metabolismo , COVID-19/patologia , Chlorocebus aethiops , Células Epiteliais/patologia , Células Epiteliais/virologia , Humanos , Pulmão/patologia , Pulmão/virologia , Serina Endopeptidases/metabolismo , Estados Unidos , United States Food and Drug Administration , Células Vero
9.
Molecules ; 26(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810514

RESUMO

The aim of this work is the design, preparation and characterization of membranes based on cyclosporine A (CsA) and chitosan carboxylate (CC) to be used as an implantable subcutaneous medical device for a prolonged therapeutic effect in the treatment of breast cancer. The choice to use CsA is due to literature data that have demonstrated its possible antitumor activity on different types of neoplastic cells. To this end, CsA was bound to CC through an amidation reaction to obtain a prodrug to be dispersed in a chitosan-based polymeric membrane. The reaction intermediates and the final product were characterized by Fourier transform infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance (1H-NMR). Membranes were analyzed by differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). The data obtained showed the effective formation of the amide bond between CsA and CC and the complete dispersion of CsA inside the polymeric membrane. Furthermore, preliminary tests, conducted on MDA-MB-231, a type of breast cancer cell line, have shown a high reduction in the proliferation of cancer cells. These results indicate the possibility of using the obtained membranes as an interesting strategy for the release of cyclosporin-A in breast cancer patients.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Quitosana/uso terapêutico , Ciclosporina , Portadores de Fármacos/uso terapêutico , Administração Cutânea , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ciclosporina/administração & dosagem , Ciclosporina/farmacologia , Humanos , Membranas/química , Suínos
10.
Int J Mol Sci ; 22(9)2021 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-33926009

RESUMO

Prognosis of patients with myocardial infarction is detrimentally affected by comorbidities like diabetes mellitus. In the experimental setting, not only diabetes mellitus but also acute hyperglycemia is shown to hamper cardioprotective properties by multiple pharmacological agents. For Levosimendan-induced postconditioning, a strong infarct size reducing effect is demonstrated in healthy myocardium. However, acute hyperglycemia is suggested to block this protective effect. In the present study, we investigated whether (1) Levosimendan-induced postconditioning exerts a concentration-dependent effect under hyperglycemic conditions and (2) whether a combination with the mitochondrial permeability transition pore (mPTP) blocker cyclosporine A (CsA) restores the cardioprotective properties of Levosimendan under hyperglycemia. For this experimental investigation, hearts of male Wistar rats were randomized and mounted onto a Langendorff system, perfused with Krebs-Henseleit buffer with a constant pressure of 80 mmHg. All isolated hearts were subjected to 33 min of global ischemia and 60 min of reperfusion under hyperglycemic conditions. (1) Hearts were perfused with various concentrations of Levosimendan (Lev) (0.3-10 µM) for 10 min at the onset of reperfusion, in order to investigate a concentration-response relationship. In the second set of experiments (2), 0.3 µM Levosimendan was administered in combination with the mPTP blocker CsA, to elucidate the underlying mechanism of blocked cardioprotection under hyperglycemia. Infarct size was determined by tetrazolium chloride (TTC) staining. (1) Control (Con) hearts showed an infarct size of 52 ± 12%. None of the administered Levosimendan concentrations reduced the infarct size (Lev0.3: 49 ± 9%; Lev1: 57 ± 9%; Lev3: 47 ± 11%; Lev10: 50 ± 7%; all ns vs. Con). (2) Infarct size of Con and Lev0.3 hearts were 53 ± 4% and 56 ± 2%, respectively. CsA alone had no effect on infarct size (CsA: 50 ± 10%; ns vs. Con). The combination of Lev0.3 and CsA (Lev0.3 ± CsA) induced a significant infarct size reduction compared to Lev0.3 (Lev0.3+CsA: 35 ± 4%; p < 0.05 vs. Lev0.3). We demonstrated that (1) hyperglycemia blocks the infarct size reducing effects of Levosimendan-induced postconditioning and cannot be overcome by an increased concentration. (2) Furthermore, cardioprotection under hyperglycemia can be restored by combining Levosimendan and the mPTP blocker CsA.


Assuntos
Ciclosporina/farmacologia , Hiperglicemia/tratamento farmacológico , Simendana/farmacologia , Animais , Cardiotônicos/metabolismo , Cardiotônicos/farmacologia , Modelos Animais de Doenças , Coração/efeitos dos fármacos , Coração/fisiologia , Hiperglicemia/complicações , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Poro de Transição de Permeabilidade Mitocondrial/metabolismo , Infarto do Miocárdio/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Miocárdio/metabolismo , Ratos , Ratos Wistar
11.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810367

RESUMO

End-stage kidney disease (ESKD) is a main public health problem, the prevalence of which is continuously increasing worldwide. Due to adverse effects of renal replacement therapies, kidney transplantation seems to be the optimal form of therapy with significantly improved survival, quality of life and diminished overall costs compared with dialysis. However, post-transplant patients frequently suffer from post-transplant diabetes mellitus (PTDM) which an important risk factor for cardiovascular and cardiovascular-related deaths after transplantation. The management of post-transplant diabetes resembles that of diabetes in the general population as it is based on strict glycemic control as well as screening and treatment of common complications. Lifestyle interventions accompanied by the tailoring of immunosuppressive regimen may be of key importance to mitigate PTDM-associated complications in kidney transplant patients. More transplant-specific approach can include the exchange of tacrolimus with an alternative immunosuppressant (cyclosporine or mammalian target of rapamycin (mTOR) inhibitor), the decrease or cessation of corticosteroid therapy and caution in the prescribing of diuretics since they are independently connected with post-transplant diabetes. Early identification of high-risk patients for cardiovascular diseases enables timely introduction of appropriate therapeutic strategy and results in higher survival rates for patients with a transplanted kidney.


Assuntos
Doenças Cardiovasculares/etiologia , Diabetes Mellitus/etiologia , Falência Renal Crônica/complicações , Falência Renal Crônica/cirurgia , Transplante de Rim , Animais , Ciclosporina/farmacologia , Fatores de Risco de Doenças Cardíacas , Humanos , Imunossupressores/uso terapêutico , Resistência à Insulina , Complicações Pós-Operatórias , Prevalência , Diálise Renal/efeitos adversos , Serina-Treonina Quinases TOR/metabolismo , Tacrolimo/uso terapêutico , Transplantados
12.
Stem Cell Res Ther ; 12(1): 238, 2021 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-33853687

RESUMO

Immunosuppressive ability in human MSC donors has been shown to be variable and may be a limiting factor in MSC therapeutic efficacy in vivo. The importance of cytokine activation of mesenchymal stromal cells (MSCs) to facilitate their immunosuppressive function is well established. This study sought to further understand the interactions between MSCs and the commonly used calcineurin inhibitor cyclosporine A (CsA). The existing literature regarding approaches that use MSCs and cyclosporine are conflicting regarding the effect of CsA on MSC potency and function. Here, we clearly demonstrate that when added at the same time as MSCs, CsA negatively affects MSC suppression of T cell proliferation. However, licencing MSCs with IFNγ before addition of CsA protects MSCs from this negative effect. Notably, adding CsA to MSCs after IFNγ pre-stimulation enhances MSC production of IDO. Mechanistically, we identified that CsA reduces SOCS1 expression to facilitate enhanced IDO production in IFNγ pre-stimulated MSCs. Importantly, CsA exposure to IFNγ pre-stimulated MSC before administration, significantly enhanced the potency of MSCs in a human relevant humanised mouse model of acute Graft versus Host Disease. In summary, this study identified a novel licencing strategy to enhance MSC potency in vitro and in vivo.


Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Ciclosporina/farmacologia , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Imunossupressores , Ativação Linfocitária , Camundongos
13.
J Med Microbiol ; 70(4)2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33915075

RESUMO

Introduction. The simultaneous use of antifungals with immunosuppressive agents has become a necessity for patients taking immunosuppressive therapy. However, antifungal drugs are problematic because of their limited target.Hypothesis. Scientists have been searching for new antifungals and some compounds with at least additive effects on antifungals. Calcineurin inhibitors used as immunosuppressive agents also attract attention due to their antifungal property.Aim. To evaluate the activity of two calcineurin inhibitors alone and in combination with amphotericin B (AMB), caspofungin (CAS), itraconazole (ITR), voriconazole (VOR) and fluconazole (FLU).Methodology. MICs of AMB, CAS, ITR, VOR, FLU and cyclosporine A (CsA) and tacrolimus (TAC) as calcineurin inhibitors were evaluated by the broth microdilution method against Candida albicans (n=13), C. krusei (n=7) and C. glabrata (n=10). Checkerboard and time-kill methods were performed to investigate the activity of combining calcineurin inhibitors with antifungal drugs.Results. The lowest MIC values were detected with VOR for all Candida isolates tested. Although we did not detect any inhibition for CsA or TAC alone at concentrations tested in this study, the combinations of CAS with CsA showed the highest synergistic activity (36.7%) by the checkerboard method, and CAS with CsA and ITR with TAC combinations exhibited apparent synergistic interaction by the time-kill method. However, the combinations of both CsA and TAC with AMB resulted in antagonistic interactions, especially against C. krusei isolate in time-kill testing.Conclusion. Synergistic interactions in the combinations of TAC or CsA with antifungal drugs, except for AMB, in many concentrations was found to be promising in terms of the treatment of patients with fungal infections.


Assuntos
Antifúngicos/farmacologia , Inibidores de Calcineurina/farmacologia , Candida/efeitos dos fármacos , Imunossupressores/farmacologia , Candida/isolamento & purificação , Candidíase/microbiologia , Ciclosporina/farmacologia , Sinergismo Farmacológico , Humanos , Testes de Sensibilidade Microbiana , Tacrolimo/farmacologia
14.
Exp Brain Res ; 239(5): 1401-1415, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33666692

RESUMO

Several brain regions, including the medial prefrontal cortex (mPFC), are important in the process of fear extinction learning. Ketamine is a glutamate N-methyl-D-aspartate (NMDA) receptor antagonist, which is shown to play a role in extinction modulation. Ketamine and calcineurin (CN), an intracellular protein phosphatase, have several common targets in the cells. Therefore, in the present study, our aim is to investigate the possible role of calcineurin in the mPFC on the enhancing effects of ketamine in fear extinction. First, different doses of a CN inhibitor, cyclosporine-A (CsA), were micro-injected into the infralimbic (IL) region of the mPFC prior to extinction training in a classical conditioning model in rats. Next, sub-effective doses of CsA (Intra-mPFC) and ketamine (i.p.) were co-administered in another cohort of rats to find their possible interactions. Enzymatic activity of calcineurin was measured in the IL-mPFC following drug administration. We used the elevated plus-maze (EPM) and open field (OF) test for further behavioral assessments. The results showed that CsA can enhance the extinction of conditioned fear and inhibit the enzyme CN at a dose of 20 nM. The combination of sub-effective doses of CsA (5 nM) and ketamine (10 mg/kg) could again enhance the extinction of fear and reduce CN activity in the region. Our results propose that inhibition of CN in the IL-mPFC is involved in the extinction of fear and ketamine enhancement of extinction is probably mediated by reducing CN activity in this part of the brain.


Assuntos
Medo , Ketamina , Animais , Condicionamento Clássico , Ciclosporina/farmacologia , Extinção Psicológica , Ketamina/farmacologia , Córtex Pré-Frontal , Ratos
15.
J Biochem Mol Toxicol ; 35(6): 1-13, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33710703

RESUMO

Cyclosporine-A (CsA) is a powerful immunosuppressive agent and hepatotoxicity results from CsA treatment. This study aimed to elucidate the effectiveness of tyrosine kinase inhibitor nilotinib against CsA-induced hepatotoxicity and the underlying molecular mechanisms. Male Sprague-Dawley rats were allocated into four groups and received drugs for 28 days as follows: Control group: received vehicle, Nilotinib group: received nilotinib (20 mg/kg orally), CsA group: received CsA by subcutaneous injection (20 mg/kg daily), CsA-nilotinib: received nilotinib and CsA. Serum lactate dehydrogenase (LDH), liver function biomarkers, hepatic levels of oxidative stress biomarkers, nuclear factor erythroid-2 like-2 (Nrf2), total antioxidant capacity (TAC), interleukin-2 (IL-2), IL-1ß, IL-6, and cytochrome-C were assessed. Additionally, the protein levels and mRNA expression of Bcl2 associated X protein (Bax), caspase-3, nuclear factor-κB (NF-κB), hemoxygenase-1 (HO-1) were measured. Moreover, liver tissues were assessed histopathologically using hematoxylin-eosin and Masson trichrome stain. Nilotinib treatment decreased serum LDH, alanine aminotransferase, aspartate aminotransferase, and γ-glutamyltransferase (γ-GT), hepatic malondialdehyde, and cytochrome-C. It also increased superoxide dismutase, reduced glutathione, glutathione reductase, glutathione peroxidase, glutathione-S-transferase (GST), TAC, and Nrf2 compared to CsA-injected rats. In addition, nilotinib decreased NF-κB, IL-1ß, IL-6, Bax, and caspase-3, while elevated IL-2 and immunoexpression of HO-1. Additionally, mRNA expression of Bax and caspase-3 was elevated and that of HO-1 and inhibitory protein κB-α was reduced in the nilotinib-treated group. Moreover, nilotinib significantly attenuated CsA-induced histopathological alterations. Nilotinib may have a promising role as a hepato-protective through its antiapoptotic, antioxidant, and anti-inflammatory effects.


Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Ciclosporina/efeitos adversos , Citocromos c/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismo , Animais , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Ciclosporina/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley
16.
Fish Shellfish Immunol ; 112: 108-115, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33716110

RESUMO

Germ cell transplantation and testis graft represent promising biotechnologies that can be applied for the reproduction of commercial or endangered species. However, mechanisms of rejection from the host immune system might remove the transplanted donor cells/tissues and limit the surrogate production of gametes. In this work, we administered emulsion containing-immunosuppressants to verify whether they are capable to prevent immune rejection and promote survival of testis allografts in rainbow trout. In the first part of this study, we demonstrated in vitro that tacrolimus and cyclosporine were able to affect viability, inhibit leucocyte proliferation, and suppress il2 expression in vitro. In in vivo experiments, both doses of tacrolimus (0.5 and 1.5 mg/kg) and the lower dose of cyclosporine (20 mg/kg) significantly inhibited the expression of il2 in head kidney, three days post-injection. A higher dose of cyclosporine (40 mg/kg) was able to inhibit il2 expression for up to seven days post-injection. In the second part, testis allografts were conducted in fish treated weekly with emulsion containing-tacrolimus. Immunohistochemical, conventional histology, and qRT-PCR (vasa) analysis demonstrated the presence of spermatogonial cells by the fifth week, in animals treated with 0.5 mg/kg of tacrolimus similar as found in autografted group. In the group treated with the highest tacrolimus dose (1.5 mg/kg) and in the non-treated group (without immunosuppressant), no germ cells or their respective markers were detected. il2 expression in head kidney was also suppressed in grafted animals treated with tacrolimus compared to non-treated group. These results suggest that tacrolimus may be a promising immunosuppressant for testis allografts or germ cell transplantation in rainbow trout. Co-administration combining tacrolimus (at lower dose) with other immunosuppressive drugs for inhibiting other activation pathways of the immune system, as performed in human organ transplantation, could be an alternative approach to optimize the immunosuppressive effects in host organisms.


Assuntos
Aloenxertos/imunologia , Ciclosporina/farmacologia , Imunossupressores/farmacologia , Oncorhynchus mykiss/cirurgia , Espermatogônias/imunologia , Tacrolimo/farmacologia , Testículo/transplante , Transplante Homólogo/veterinária , Animais , Masculino
17.
Drugs ; 81(5): 605-610, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33788181

RESUMO

Voclosporin (Lupkynis™) is an oral calcineurin inhibitor immunosuppressant that is being developed by Aurinia Pharmaceuticals. In January 2021, based on positive results from the pivotal phases II and III trials, oral voclosporin received its first approval in the USA for use in combination with a background immunosuppressive therapy regimen for adults with active lupus nephritis. Voclosporin is also being explored for the novel coronavirus disease 2019 (COVID-19) in kidney transplant recipients. This article summarizes the milestones in the development of voclosporin leading to this first approval for lupus nephritis.


Assuntos
COVID-19/tratamento farmacológico , Inibidores de Calcineurina/uso terapêutico , Ciclosporina/uso terapêutico , Imunossupressores/uso terapêutico , Transplante de Rim , Animais , COVID-19/complicações , Inibidores de Calcineurina/farmacocinética , Inibidores de Calcineurina/farmacologia , Ciclosporina/farmacocinética , Ciclosporina/farmacologia , Aprovação de Drogas , Humanos , Imunossupressores/farmacocinética , Imunossupressores/farmacologia , Nefrite Lúpica/tratamento farmacológico
18.
J Exp Med ; 218(4)2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33533918

RESUMO

The low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic and cell signaling transmembrane protein. Endothelial LRP1 clears proteinaceous toxins at the blood-brain barrier (BBB), regulates angiogenesis, and is increasingly reduced in Alzheimer's disease associated with BBB breakdown and neurodegeneration. Whether loss of endothelial LRP1 plays a direct causative role in BBB breakdown and neurodegenerative changes remains elusive. Here, we show that LRP1 inactivation from the mouse endothelium results in progressive BBB breakdown, followed by neuron loss and cognitive deficits, which is reversible by endothelial-specific LRP1 gene therapy. LRP1 endothelial knockout led to a self-autonomous activation of the cyclophilin A-matrix metalloproteinase-9 pathway in the endothelium, causing loss of tight junctions underlying structural BBB impairment. Cyclophilin A inhibition in mice with endothelial-specific LRP1 knockout restored BBB integrity and reversed and prevented neuronal loss and behavioral deficits. Thus, endothelial LRP1 protects against neurodegeneration by inhibiting cyclophilin A, which has implications for the pathophysiology and treatment of neurodegeneration linked to vascular dysfunction.


Assuntos
Doença de Alzheimer/metabolismo , Barreira Hematoencefálica/metabolismo , Ciclofilina A/metabolismo , Células Endoteliais/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Transdução de Sinais/genética , Doença de Alzheimer/terapia , Animais , Células Cultivadas , Disfunção Cognitiva/genética , Disfunção Cognitiva/metabolismo , Ciclofilina A/antagonistas & inibidores , Ciclosporina/farmacologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Técnicas de Inativação de Genes , Terapia Genética/métodos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo , Transdução de Sinais/efeitos dos fármacos
20.
J Pharm Biomed Anal ; 196: 113921, 2021 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-33548873

RESUMO

The number of approved peptide therapeutics has increased significantly in recent years. Peptide therapeutics have many advances over small molecule drugs, such as higher affinity to target and lower toxicity profiles. Although peptide-like drugs are mainly metabolized/catabolized in the body for smaller peptides and amino acids, metabolite identification still has an essential part of in their development, especially if their structure contains modified amino acids, and also to identify the metabolic soft spots enabling modification to more stable sequence. The use of human derived in vitro systems is an important tool when investigating metabolism of peptide drugs, and comparison of results by various hepatic systems was investigated here. Peptides were incubated in several different in vitro human liver-derived subcellular and cellular incubation systems, i.e. liver S9 fraction, suspended cryo-preserved human primary hepatocytes and plated Upcyte hepatocytes. Samples were collected at different time points and analysed by UPLC/HR-MS-method developed for the purpose. Both substrate disappearance and metabolite formation were monitored, and the systems were compared. S9 fraction formed the highest number of metabolites for leuprorelin and cetrorelix, while for desmopressin and cyclosporin, primary hepatocytes and liver S9 produced similar metabolite profiles. Interestingly, not only cyclosporin, but also leuprorelin and cetrorelix showed metabolites whose formation was CYP (NADPH) dependent in liver S9. For leuprorelin and cetrorelix, the metabolites that showed NADPH dependency with liver S9, were not detected with hepatocytes, even though for leuprorelin these reactions played a major role in liver S9. The hydrolytic metabolic reactions were very similar between liver S9 and hepatocytes, i.e. the metabolite profiles in hepatocytes matched better with liver S9 profiles without NADPH, which may be caused by cell uptake rate limitation with hepatocytes, or then hydrolytic processes are more stressed in peptide metabolism with hepatocytes, in comparison to CYP-mediated processes.


Assuntos
Ciclosporina , Leuprolida , Ciclosporina/farmacologia , Desamino Arginina Vasopressina , Hormônio Liberador de Gonadotropina/análogos & derivados , Hepatócitos , Humanos , Fígado , Microssomos Hepáticos
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