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1.
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
2.
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
3.
Arq Bras Cir Dig ; 33(4): e1551, 2021.
Artigo em Inglês, Português | MEDLINE | ID: mdl-33503111

RESUMO

BACKGROUND: Tacrolimus and mycophenolate mofetil are immunosuppressive agents widely used on the postoperative period of the transplants. AIM: To evaluate the influence of the association of them on the abdominal wall healing in rats. METHODS: Thirty-six Wistar rats were randomly assigned in three groups of 12. On the early postoperative period, four of the control group and three of the experimental groups died. The three groups were nominated as follow: control group (GC, n=8); group I (GI, n=11, standard operation, mycophenolate mofetil and tacrolimus); group II (GII, n=10, standard operation, mycophenolate mofetil and tacrolimus). The standard operation consisted of right total nephrectomy and 20 min ischemia of the left kidney followed by reperfusion. Both NaCl 0.9% and the immunosuppressive agents were administered starting on the first postoperative day and continuing daily until the day of death on the 14th day. On the day of their deaths, two strips of the anterior abdominal wall were collected and submitted to breaking strength measurement and histological examination. RESULTS: There were no significant differences in wound infection rates (p=0,175), in the breaking strength measurement and in the histological examination among the three groups. CONCLUSION: The combination of the immunosuppressive agents used in the study associated with renal ischemia and reperfusion does not interfere in the abdominal wall healing of rats.


Assuntos
Parede Abdominal/cirurgia , Imunossupressores/farmacologia , Rim/irrigação sanguínea , Ácido Micofenólico/farmacologia , Traumatismo por Reperfusão/complicações , Tacrolimo/farmacologia , Animais , Isquemia , Ácido Micofenólico/administração & dosagem , Ratos , Ratos Wistar , Reperfusão , Tacrolimo/administração & dosagem
4.
Medicine (Baltimore) ; 99(44): e22894, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-33126341

RESUMO

BACKGROUND: Positioning infliximab (IFX), cyclosporine and tacrolimus (TAC) for treating ulcerative colitis (UC) is in great debate. METHODS: A literature search identified studies that investigated IFX vs. cyclosporine or IFX vs TAC in UC patients. Short-term remission, short-term, 1-year and 3-year colectomy rate were employed as primary end-points to assess efficacy. Odds ratios (ORs) with 95% confidence intervals (CIs) were analyzed. RESULTS: Overall, 15 studies comprised 596 patients in IFX group and 866 in calcineurin inhibitors group (644 received cyclosporine and 222 received TAC). No significant difference was seen between IFX and calcineurin inhibitors with regard to short-term remission. IFX led to a lower short-term (OR: 0.59, 95% CI: 0.43-0.82, P:.001), 1-year (OR: 0.53, 95% CI: 0.38-0.73, P < .001), 3-year colectomy (OR: 0.41, 95% CI: 0.20-0.84, P:.02) than calcineurin inhibitors. IFX led to a lower short-term (OR: 0.51, 95% CI: 0.36-0.71, P < .001), 1-year (OR: 0.53, 95% CI: 0.37-0.74, P:.003) colectomy and a trend of lower 3-year colectomy (OR: 0.49, 95% CI: 0.22-1.06, P:.07) than cyclosporine while no significant difference was seen between IFX and TAC. Results of network meta-analysis showed that the order was cyclosporine, TAC and IFX from high rate to low with regard to short-term and 1-year colectomy. CONCLUSION: IFX treatment leads to a lower short-term, 1-year colectomy rate and a trend of lower 3-year colectomy rate in UC patients than cyclosporine while no significant difference is seen between IFX and TAC. TAC may be superior than cyclosporine with regard to efficacy based on indirect comparisons. Randomized trials with fixed protocol are warranted to identify the optimal medical strategy in patients with UC.


Assuntos
Colite Ulcerativa/tratamento farmacológico , Ciclosporina/farmacologia , Infliximab/farmacologia , Tacrolimo/farmacologia , Colectomia/estatística & dados numéricos , Colite Ulcerativa/imunologia , Colite Ulcerativa/cirurgia , Humanos , Imunossupressores/farmacologia , Indução de Remissão/métodos , Resultado do Tratamento
5.
PLoS Genet ; 16(6): e1008865, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32603360

RESUMO

Fpr1 (FK506-sensitive proline rotamase 1), a protein of the FKBP12 (FK506-binding protein 12 kDa) family in Saccharomyces cerevisiae, is a primary target for the immunosuppressive agents FK506 and rapamycin. Fpr1 inhibits calcineurin and TORC1 (target of rapamycin complex 1) when bound to FK506 and rapamycin, respectively. Although Fpr1 is recognised to play a crucial role in the efficacy of these drugs, its physiological functions remain unclear. In a hmo1Δ (high mobility group family 1-deleted) yeast strain, deletion of FPR1 induced severe growth defects, which could be alleviated by increasing the copy number of RPL25 (ribosome protein of the large subunit 25), suggesting that RPL25 expression was affected in hmo1Δfpr1Δ cells. In the current study, extensive chromatin immunoprecipitation (ChIP) and ChIP-sequencing analyses revealed that Fpr1 associates specifically with the upstream activating sequences of nearly all RPG (ribosomal protein gene) promoters, presumably in a manner dependent on Rap1 (repressor/activator site binding protein 1). Intriguingly, Fpr1 promotes the binding of Fhl1/Ifh1 (forkhead-like 1/interacts with forkhead 1), two key regulators of RPG transcription, to certain RPG promoters independently of and/or cooperatively with Hmo1. Furthermore, mutation analyses of Fpr1 indicated that for transcriptional function on RPG promoters, Fpr1 requires its N-terminal domain and the binding surface for rapamycin, but not peptidyl-prolyl isomerase activity. Notably, Fpr1 orthologues from other species also inhibit TORC1 when bound to rapamycin, but do not regulate transcription in yeast, which suggests that these two functions of Fpr1 are independent of each other.


Assuntos
Proteínas de Grupo de Alta Mobilidade/metabolismo , Peptidilprolil Isomerase/metabolismo , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Calcineurina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Fatores de Transcrição Forkhead/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Proteínas de Grupo de Alta Mobilidade/genética , Peptidilprolil Isomerase/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Sirolimo/farmacologia , Tacrolimo/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Transcrição Genética
6.
Nat Commun ; 11(1): 2713, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483127

RESUMO

Despite their rapidly-expanding therapeutic potential, human pluripotent stem cell (hPSC)-derived cell therapies continue to have serious safety risks. Transplantation of hPSC-derived cell populations into preclinical models has generated teratomas (tumors arising from undifferentiated hPSCs), unwanted tissues, and other types of adverse events. Mitigating these risks is important to increase the safety of such therapies. Here we use genome editing to engineer a general platform to improve the safety of future hPSC-derived cell transplantation therapies. Specifically, we develop hPSC lines bearing two drug-inducible safeguards, which have distinct functionalities and address separate safety concerns. In vitro administration of one small molecule depletes undifferentiated hPSCs >106-fold, thus preventing teratoma formation in vivo. Administration of a second small molecule kills all hPSC-derived cell-types, thus providing an option to eliminate the entire hPSC-derived cell product in vivo if adverse events arise. These orthogonal safety switches address major safety concerns with pluripotent cell-derived therapies.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/genética , Edição de Genes/métodos , Células-Tronco Pluripotentes/metabolismo , Transplante de Células-Tronco/métodos , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Tacrolimo/análogos & derivados , Tacrolimo/farmacologia , Teratoma/genética , Teratoma/metabolismo , Teratoma/prevenção & controle
7.
Cell Physiol Biochem ; 54(3): 457-473, 2020 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-32369692

RESUMO

BACKGROUND/AIMS: Orthotopic liver transplantation (OLT) is the recommended treatment for patients at early stages of hepatocarcinoma (HCC) with portal hypertension and/or increased bilirubinemia, but without vascular-associated diseases. Tumor recurrence, which is the main drawback for the survival of patients submitted to OLT for HCC, has been related to tumor-related variables and the immunosuppressive therapies. We have previously shown that Tacrolimus (FK506) exerts a more potent pro-apoptotic and anti-proliferative effects than the mammalian target of rapamycin (mTOR) inhibitors (Sirolimus and Everolimus) in liver cancer cells. This study identified the role of the immunosuppressant partners such as FK506-binding proteins (FKBPs) in the induction of cell death and arrest of cell proliferation by immunosuppressants in two representative liver cancer cells. METHODS: The regulation of endoplasmic reticulum (ER) stress, apoptosis/autophagy, cell proliferation, and FKBPs expression was determined in Tacrolimus-, Sirolimus- and Everolimus-treated primary human hepatocytes, and hepatoma HepG2 and Huh7 cell lines. The functional repercussion of FKBPs on cell death and proliferation was also addressed using the siRNA technology. The assessed antitumoral properties of the immunosuppressants were associated to microRNAs (miRNAs) pattern. RESULTS: The enhanced pro-apoptotic and anti-proliferative properties of Tacrolimus versus mTOR inhibitors were associated with increased protein kinase RNA-like endoplasmic reticulum kinase (PERK)-related ER stress, Ser15P-p53/p53 ratio and p21 protein expression that may counterbalance the risk of proliferative upregulation caused by enhanced Thr172P-Cdk4/Cdk4 activation in liver cancer cells. The inhibition of the mTOR pathway by Sirolimus and Everolimus was related to an induction of autophagy; and at a high dose, these drugs impaired translation likely at a very early step of the elongation phase. Tacrolimus and mTOR inhibitors increased the protein expression of FKBP12 and FKBP51 that appeared to play pro-survival role. Interestingly, the administration of immunosuppressants yields a specific pattern of miRNAs. Tacrolimus and mTOR inhibitors decreased miR-92a-1-5p, miR-197-3p, miR-483-3p and miR-720, and increased miR-22-3p, miR-376a-3p, miR-663b, miR-886-5p, miR-1300 and miR-1303 expressions in HepG2 cells. CONCLUSION: The more potent pro-apoptotic and anti-proliferative properties of Tacrolimus versus mTOR inhibitors were associated with an increased activation of PERK and p53 signaling, and p21 protein expression. FKBP12 and FKBP51 appeared to be the most relevant partners of Tacrolimus and mTOR inhibitors exerting a pro-survival effect in HepG2 cells. The observed effects of immunosuppressants were related to a specific miRNA signature in liver cancer cells.


Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Imunossupressores/farmacologia , Neoplasias Hepáticas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Tacrolimo/farmacologia , Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Everolimo/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Interferente Pequeno , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Proteína 1A de Ligação a Tacrolimo/metabolismo , Proteína Supressora de Tumor p53/metabolismo , eIF-2 Quinase/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(18): 9973-9980, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32303657

RESUMO

When transitioning from the environment, pathogenic microorganisms must adapt rapidly to survive in hostile host conditions. This is especially true for environmental fungi that cause opportunistic infections in immunocompromised patients since these microbes are not well adapted human pathogens. Cryptococcus species are yeastlike fungi that cause lethal infections, especially in HIV-infected patients. Using Cryptococcus deneoformans in a murine model of infection, we examined contributors to drug resistance and demonstrated that transposon mutagenesis drives the development of 5-fluoroorotic acid (5FOA) resistance. Inactivation of target genes URA3 or URA5 primarily reflected the insertion of two transposable elements (TEs): the T1 DNA transposon and the TCN12 retrotransposon. Consistent with in vivo results, increased rates of mutagenesis and resistance to 5FOA and the antifungal drugs rapamycin/FK506 (rap/FK506) and 5-fluorocytosine (5FC) were found when Cryptococcus was incubated at 37° compared to 30° in vitro, a condition that mimics the temperature shift that occurs during the environment-to-host transition. Inactivation of the RNA interference (RNAi) pathway, which suppresses TE movement in many organisms, was not sufficient to elevate TE movement at 30° to the level observed at 37°. We propose that temperature-dependent TE mobilization in Cryptococcus is an important mechanism that enhances microbial adaptation and promotes pathogenesis and drug resistance in the human host.


Assuntos
Antifúngicos/farmacologia , Cryptococcus neoformans/efeitos dos fármacos , Micoses/genética , Retroelementos/genética , Animais , Antifúngicos/efeitos adversos , Cryptococcus neoformans/patogenicidade , Farmacorresistência Fúngica/genética , Interações Hospedeiro-Patógeno/genética , Humanos , Camundongos , Mutagênese/genética , Micoses/microbiologia , Ácido Orótico/efeitos adversos , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Sirolimo/farmacologia , Tacrolimo/farmacologia , Virulência/genética
9.
Biochem Pharmacol ; 177: 113975, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32298692

RESUMO

BACKGROUND AND PURPOSE: Rapamycin is a potent immunosuppressant and anti-proliferative agent used clinically to prevent organ transplant rejection and for coating coronary stents to counteract restenosis. Rapamycin complexes with the immunophilin FKBP12, which subsequently binds and inhibits mTORC1. Despite several reports demonstrating that rapamycin affects platelet-mediated responses, the underlying mechanism of how it alters platelet function is poorly characterised. This study aimed to elucidate the effect of rapamycin on platelet procoagulant responses. EXPERIMENTAL APPROACH: The effect of rapamycin on platelet activation and signalling was investigated alongside the catalytic mTOR inhibitors KU0063794 and WYE-687, and the FKBP12-binding macrolide FK506. KEY RESULTS: Rapamycin affects platelet procoagulant responses by reducing externalisation of the procoagulant phospholipid phosphatidylserine, formation of balloon-like structures and local generation of thrombin. Catalytic mTOR kinase inhibitors did not alter platelet procoagulant processes, despite having a similar effect as rapamycin on Ca2+ signalling, demonstrating that the effect of rapamycin on procoagulant responses is independent of mTORC1 inhibition and not linked to a reduction in Ca2+ signalling. FK506, which also forms a complex with FKBP12 but does not target mTOR, reduced platelet procoagulant responses to a similar extent as rapamycin. Both rapamycin and FK506 prevented the loss of mitochondria integrity induced by platelet activation, one of the central regulatory events leading to PS externalisation. CONCLUSIONS AND IMPLICATIONS: Rapamycin suppresses platelet procoagulant responses by protecting mitochondrial integrity in a manner independent of mTORC1 inhibition. Rapamycin and other drugs targeting FKBP immunophilins could aid the development of novel complementary anti-platelet therapies.


Assuntos
Plaquetas/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Plaquetas/citologia , Plaquetas/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Proteínas de Transporte/farmacologia , Regulação da Expressão Gênica , Humanos , Ionomicina/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Mitocôndrias/metabolismo , Morfolinas/farmacologia , Peptídeos/farmacologia , Fosfatidilserinas/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Cultura Primária de Células , Pirazóis/farmacologia , Pirimidinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Tacrolimo/farmacologia , Trombina/metabolismo , Trombina/farmacologia
10.
PLoS Pathog ; 16(4): e1008477, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251475

RESUMO

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication after organ transplantation frequently associated with the Epstein-Barr virus (EBV). Immunosuppressive treatment is thought to allow the expansion of EBV-infected B cells, which often express all eight oncogenic EBV latent proteins. Here, we assessed whether HLA-A2 transgenic humanized NSG mice treated with the immunosuppressant FK506 could be used to model EBV-PTLD. We found that FK506 treatment of EBV-infected mice led to an elevated viral burden, more frequent tumor formation and diminished EBV-induced T cell responses, indicative of reduced EBV-specific immune control. EBV latency III and lymphoproliferation-associated cellular transcripts were up-regulated in B cells from immunosuppressed animals, akin to the viral and host gene expression pattern found in EBV-PTLD. Utilizing an unbiased gene expression profiling approach, we identified genes differentially expressed in B cells of EBV-infected animals with and without FK506 treatment. Upon investigating the most promising candidates, we validated sCD30 as a marker of uncontrolled EBV proliferation in both humanized mice and in pediatric patients with EBV-PTLD. High levels of sCD30 have been previously associated with EBV-PTLD in patients. As such, we believe that humanized mice can indeed model aspects of EBV-PTLD development and may prove useful for the safety assessment of immunomodulatory therapies.


Assuntos
Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/virologia , Tacrolimo/farmacologia , Animais , Linfócitos B/metabolismo , DNA Viral , Modelos Animais de Doenças , Infecções por Vírus Epstein-Barr/virologia , Feminino , Perfilação da Expressão Gênica/métodos , Antígeno HLA-A2 , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Herpesvirus Humano 4/patogenicidade , Humanos , Hospedeiro Imunocomprometido , Imunossupressores/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Transplante de Órgãos/efeitos adversos , Transcriptoma/genética , Carga Viral
11.
Biochem Biophys Res Commun ; 526(1): 48-54, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32192767

RESUMO

The 12-kDa FK506-binding protein (FKBP12) is the target of the commonly used immunosuppressive drug FK506. The FKBP12-FK506 complex binds to calcineurin and inhibits its activity, leading to immunosuppression and preventing organ transplant rejection. Our recent characterization of crystal structures of FKBP12 proteins in pathogenic fungi revealed the involvement of the 80's loop residue (Pro90) in the active site pocket in self-substrate interaction providing novel evidence on FKBP12 dimerization in vivo. The 40's loop residues have also been shown to be involved in reversible dimerization of FKBP12 in the mammalian and yeast systems. To understand how FKBP12 dimerization affects FK506 binding and influences calcineurin function, we generated Aspergillus fumigatus FKBP12 mutations in the 40's and 50's loop (F37 M/L; W60V). Interestingly, the mutants exhibited variable FK506 susceptibility in vivo indicating differing dimer strengths. In comparison to the 80's loop P90G and V91C mutants, the F37 M/L and W60V mutants exhibited greater FK506 resistance, with the F37M mutation showing complete loss in calcineurin binding in vivo. Molecular dynamics and pulling simulations for each dimeric FKBP12 protein revealed a two-fold increase in dimer strength and significantly higher number of contacts for the F37M, F37L, and W60V mutations, further confirming their varying degree of impact on FK506 binding and calcineurin inhibition in vivo.


Assuntos
Aspergillus fumigatus/metabolismo , Inibidores de Calcineurina/farmacologia , Calcineurina/metabolismo , Proteínas Fúngicas/genética , Mutação/genética , Multimerização Proteica , Proteína 1A de Ligação a Tacrolimo/genética , Tacrolimo/farmacologia , Sequência de Aminoácidos , Simulação por Computador , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Proteína 1A de Ligação a Tacrolimo/química , Proteína 1A de Ligação a Tacrolimo/metabolismo
12.
Sci Rep ; 10(1): 3353, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32098997

RESUMO

The epigenetic regulation of microRNA (miRNA) expression related to the FK506-binding protein 5 (FKBP5) gene may contribute to the risk of stress-related disorders such as posttraumatic stress disorder (PTSD). Here, we identified candidate miRNAs derived from FKBP5 knockout mice as a potential diagnostic biomarker of PTSD. Using a translational approach, candidate miRNAs found to alter in expression within the medial prefrontal cortex of FKBP5 knockout mice were selected. Each candidate miRNA was examined in the serum of 48 recently traumatized individuals with PTSD and 47 healthy individuals. Multimodal imaging was also conducted to identify the neural correlates for the expression of candidate exosomal miRNAs in response to trauma exposure. Differential miRNA expression was found according to PTSD diagnosis in two composite marker groups. The differential miRNA expression between the composite marker groups contributed to PTSD symptom severity, which may be explained by differential recruitment of prefrontolimbic activity in brain imaging. The present study reveals that a set of circulating exosomal miRNAs showing altered expression in FKBP5 knockout mice play a potential role as epigenetic markers of PTSD. The corroborative evidence from multiple levels including molecular, brain, and behavioral indicates that these epigenetic biomarkers may serve as complementary measures for the diagnosis and prognosis prediction of PTSD in recently traumatized individuals.


Assuntos
Predisposição Genética para Doença , MicroRNAs/genética , Transtornos de Estresse Pós-Traumáticos/genética , Proteínas de Ligação a Tacrolimo/genética , Adulto , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Epigênese Genética/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Humanos , Masculino , Camundongos , Camundongos Knockout , Polimorfismo de Nucleotídeo Único/genética , Córtex Pré-Frontal/diagnóstico por imagem , Córtex Pré-Frontal/fisiopatologia , Transtornos de Estresse Pós-Traumáticos/fisiopatologia , Tacrolimo/farmacologia
13.
J Med Microbiol ; 69(3): 436-442, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32068525

RESUMO

Introduction. Malassezia folliculitis (MF) and pityriasis versicolor (PV) are common dermatoses caused by Malassezia species. Their molecular epidemiology, drug susceptibility and exoenzymes are rarely reported in China.Aim. To investigate the molecular epidemiology, drug susceptibility and enzymatic profile of Malassezia clinical isolates.Methodology. Malassezia strains were recovered from MF and PV patients and healthy subjects (HS) and identified by sequencing analysis. The minimum inhibitory concentrations (MICs) of nine antifungals (posaconazole, voriconazole, itraconazole, fluconazole, ketoconazole, miconazole, bifonazole, terbinafine and caspofungin) and tacrolimus, the interactions between three antifungals (itraconazole, ketoconazole and terbinafine) and tacrolimus, and the extracellular enzyme profile were evaluated using broth and checkerboard microdilution and the Api-Zym system, respectively.Results. Among 392 Malassezia isolates from 729 subjects (289 MF, 218 PV and 222 HS), Malassezia furfur and Malassezia globosa accounted for 67.86 and 18.88 %, respectively. M. furfur was the major species in MF and PV patients and HS. Among 60M. furfur and 50M. globosa strains, the MICs for itraconazole, posaconazole, voriconazole and ketoconazole were <1 µg ml-1. M. furfur was more susceptible to itraconazole, terbinafine and bifonazole but tolerant to miconazole compared with M. globosa (P<0.05). Synergistic effects between terbinafine and itraconazole or between tacrolimus and itraconazole, ketoconazole or terbinafine occurred in 6, 7, 6 and 9 out of 37 strains, respectively. Phosphatases, lipases and proteases were mainly secreted in 51 isolates.Conclusions. Itraconazole, posaconazole, voriconazole and ketoconazole are theagents against which there is greatest susceptibility. Synergistic effects between terbinafine and itraconazole or tacrolimas and antifungals may be irrelevant to clinical application. Overproduction of lipases could enhance the skin inhabitation of M. furfur.


Assuntos
Antifúngicos/farmacologia , Dermatomicoses/epidemiologia , Foliculite/epidemiologia , Malassezia/isolamento & purificação , Tinha Versicolor/epidemiologia , Azóis/farmacologia , China/epidemiologia , Dermatomicoses/microbiologia , Foliculite/microbiologia , Humanos , Lipase/metabolismo , Malassezia/efeitos dos fármacos , Malassezia/enzimologia , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Pele/microbiologia , Tacrolimo/farmacologia , Terbinafina , Tinha Versicolor/microbiologia
14.
Eur J Pharm Sci ; 145: 105237, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001346

RESUMO

BACKGROUND: Diverse tacrolimus population pharmacokinetic (popPK) models in adult liver transplant recipients have been established to describe the PK characteristics of tacrolimus in the last two decades. However, their extrapolated predictive performance remains unclear. Therefore, in this study, we aimed to evaluate their external predictability and identify their potential influencing factors. METHODS: The external predictability of each selected popPK model was evaluated using an independent dataset of 84 patients with 572 trough concentrations prospectively collected from Huashan Hospital. Prediction- and simulation-based diagnostics and Bayesian forecasting were conducted to evaluate model predictability. Furthermore, the effect of model structure on the predictive performance was investigated. RESULTS: Sixteen published popPK models were assessed. In prediction-based diagnostics, the prediction error within ± 30% was below 50% in all the published models. The simulation-based normalised prediction distribution error test and prediction- and variability-corrected visual predictive check indicated large discrepancies between the observations and simulations in most of the models. Bayesian forecasting showed improvement in model predictability with two to three prior observations. Additionally, the predictive performance of the nonlinear Michaelis-Menten model was superior to that of linear one- and two-compartment models with first-order elimination, indicating the underlying nonlinear kinetics of tacrolimus in liver transplant recipients, which was consistent with the findings in adult kidney transplant recipients. CONCLUSIONS: The published models performed inadequately in prediction- and simulation-based diagnostics. Bayesian forecasting may improve the predictive performance of the models. Furthermore, nonlinear kinetics of tacrolimus may be mainly caused by the properties of the drug itself, and incorporating nonlinear kinetics may be considered to improve model predictability.


Assuntos
Imunossupressores/farmacocinética , Transplante de Fígado , Modelos Biológicos , Tacrolimo/farmacologia , Transplantados , Adulto , Humanos , Transplante de Fígado/efeitos adversos , Transplante de Fígado/tendências
15.
Int J Mol Sci ; 21(3)2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-32013193

RESUMO

Tacrolimus exhibits high inter-patient pharmacokinetics (PK) variability, as well as a narrow therapeutic index, and therefore requires therapeutic drug monitoring. Germline mutations in cytochrome P450 isoforms 4 and 5 genes (CYP3A4/5) and the ATP-binding cassette B1 gene (ABCB1) may contribute to interindividual tacrolimus PK variability, which may impact clinical outcomes among allogeneic hematopoietic stem cell transplantation (HSCT) patients. In this study, 252 adult patients who received tacrolimus for acute graft versus host disease (aGVHD) prophylaxis after allogeneic HSCT were genotyped to evaluate if germline genetic variants associated with tacrolimus PK and pharmacodynamic (PD) variability. Significant associations were detected between germline variants in CYP3A4/5 and ABCB1 and PK endpoints (e.g., median steady-state tacrolimus concentrations and time to goal tacrolimus concentration). However, significant associations were not observed between CYP3A4/5 or ABCB1 germline variants and PD endpoints (e.g., aGVHD and treatment-emergent nephrotoxicity). Decreased age and CYP3A5*1/*1 genotype were independently associated with subtherapeutic tacrolimus trough concentrations while CYP3A5*1*3 or CYP3A5*3/*3 genotypes, myeloablative allogeneic HSCT conditioning regimen (MAC) and increased weight were independently associated with supratherapeutic tacrolimus trough concentrations. Future lines of prospective research inquiry are warranted to use both germline genetic and clinical data to develop precision dosing tools that will optimize both tacrolimus dosing and clinical outcomes among adult HSCT patients.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Citocromo P-450 CYP3A/genética , Transplante de Células-Tronco Hematopoéticas , Imunossupressores/farmacocinética , Tacrolimo/farmacocinética , Adulto , Idoso , Bases de Dados Genéticas , Feminino , Genótipo , Mutação em Linhagem Germinativa , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Polimorfismo de Nucleotídeo Único , Tacrolimo/farmacologia , Tacrolimo/uso terapêutico , Transplante Homólogo , Adulto Jovem
16.
FASEB J ; 34(1): 1247-1269, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914663

RESUMO

Glycogen synthase kinase 3 (GSK3) was identified as an enzyme regulating sperm protein phosphatase. The GSK3α paralog, but not GSK3ß, is essential for sperm function. Sperm lacking GSK3α display altered motility and are unable to undergo hyperactivation, which is essential for fertilization. Male mice lacking sperm-specific calcineurin (PP2B), a calcium regulated phosphatase, in testis and sperm, are also infertile. Loss of PP2B results in impaired epididymal sperm maturation and motility. The phenotypes of GSK3α and PP2B knockout mice are similar, prompting us to examine the interrelationship between these two enzymes in sperm. High calcium levels must exist to permit catalytically active calcineurin to function during epididymal sperm maturation. Total and free calcium levels are high in immotile compared to motile epididymal sperm. Inhibition of calcineurin by FK506 results in an increase in the net phosphorylation and a consequent decrease in catalytic activity of sperm GSK3. The inhibitor FK506 and an isoform-selective inhibitor of GSK3α, BRD0705, also inhibited fertilization of eggs in vitro. Interrelated functions of GSK3α and sperm PP2B are essential during epididymal sperm maturation and during fertilization. Our results should enable the development of male contraceptives targeting one or both enzymes.


Assuntos
Calcineurina/metabolismo , Fertilização , Quinase 3 da Glicogênio Sintase/metabolismo , Motilidade Espermática , Espermatozoides/enzimologia , Animais , Calcineurina/genética , Inibidores de Calcineurina/farmacologia , Epididimo/metabolismo , Epididimo/patologia , Feminino , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Masculino , Camundongos , Camundongos Knockout , Tacrolimo/farmacologia
17.
Diabetes ; 69(4): 699-712, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31974141

RESUMO

Current therapeutic strategies for diabetic foot ulcer (DFU) have focused on developing topical healing agents, but few agents have controlled prospective data to support their effectiveness in promoting wound healing. We tested a stem cell mobilizing therapy for DFU using a combination of AMD3100 and low-dose FK506 (tacrolimus) (AF) in streptozocin-induced type 1 diabetic (T1DM) rats and type 2 diabetic Goto-Kakizaki (GK) rats that had developed peripheral artery disease and neuropathy. Here, we show that the time for healing back wounds in T1DM rats was reduced from 27 to 19 days, and the foot wound healing time was reduced from 25 to 20 days by treatment with AF (subcutaneously, every other day). Similarly, in GK rats treated with AF, the healing time on back wounds was reduced from 26 to 21 days. Further, this shortened healing time was accompanied by reduced scar and by regeneration of hair follicles. We found that AF therapy mobilized and recruited bone marrow-derived CD133+ and CD34+ endothelial progenitor cells and Ym1/2+ M2 macrophages into the wound sites, associated with enhanced capillary and hair follicle neogenesis. Moreover, AF therapy improved microcirculation in diabetic and neuropathic feet in GK rats. This study provides a novel systemic therapy for healing DFU.


Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Pé Diabético/fisiopatologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Mobilização de Células-Tronco Hematopoéticas/métodos , Compostos Heterocíclicos/farmacologia , Tacrolimo/farmacologia , Cicatrização/efeitos dos fármacos , Animais , Feminino , Masculino , Ratos , Ratos Sprague-Dawley
18.
Sci Transl Med ; 12(528)2020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996467

RESUMO

Preclinical studies have shown that persistent mixed chimerism is linked to acceptance of organ allografts without immunosuppressive (IS) drugs. Mixed chimerism refers to continued mixing of donor and recipient hematopoietic cells in recipient tissues after transplantation of donor cells. To determine whether persistent mixed chimerism and tolerance can be established in patients undergoing living donor kidney transplantation, we infused allograft recipients with donor T cells and hematopoietic progenitors after posttransplant lymphoid irradiation. In 24 of 29 fully human leukocyte antigen (HLA)-matched patients who had persistent mixed chimerism for at least 6 months, complete IS drug withdrawal was achieved without subsequent evidence of rejection for at least 2 years. In 10 of 22 HLA haplotype-matched patients with persistent mixed chimerism for at least 12 months, reduction of IS drugs to tacrolimus monotherapy was achieved. Withdrawal of tacrolimus during the second year resulted in loss of detectable chimerism and subsequent rejection episodes, unless tacrolimus therapy was reinstituted. Posttransplant immune reconstitution of naïve B cells and B cell precursors was more rapid than the reconstitution of naïve T cells and thymic T cell precursors. Robust chimerism was observed only among naïve T and B cells but not among memory T cells. No evidence of rejection was observed in all surveillance graft biopsies obtained from mixed chimeric patients withdrawn from IS drugs, and none developed graft-versus-host disease. In conclusion, persistent mixed chimerism established in fully HLA- or haplotype-matched patients allowed for complete or partial IS drug withdrawal without rejection.


Assuntos
Quimerismo , Imunossupressores/farmacologia , Transplante de Rim , Suspensão de Tratamento , Adulto , Linfócitos B/imunologia , Feminino , Sobrevivência de Enxerto/imunologia , Haplótipos/genética , Teste de Histocompatibilidade , Humanos , Isoantígenos/imunologia , Teste de Cultura Mista de Linfócitos , Masculino , Pessoa de Meia-Idade , Análise de Sobrevida , Linfócitos T/imunologia , Tacrolimo/farmacologia , Doadores de Tecidos , Resultado do Tratamento , Adulto Jovem
19.
Nat Commun ; 11(1): 264, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31937758

RESUMO

Paralleling the activation of dorsal horn microglia after peripheral nerve injury is a significant expansion and proliferation of macrophages around injured sensory neurons in dorsal root ganglia (DRG). Here we demonstrate a critical contribution of DRG macrophages, but not those at the nerve injury site, to both the initiation and maintenance of the mechanical hypersensitivity that characterizes the neuropathic pain phenotype. In contrast to the reported sexual dimorphism in the microglial contribution to neuropathic pain, depletion of DRG macrophages reduces nerve injury-induced mechanical hypersensitivity and expansion of DRG macrophages in both male and female mice. However, fewer macrophages are induced in the female mice and deletion of colony-stimulating factor 1 from sensory neurons, which prevents nerve injury-induced microglial activation and proliferation, only reduces macrophage expansion in male mice. Finally, we demonstrate molecular cross-talk between axotomized sensory neurons and macrophages, revealing potential peripheral DRG targets for neuropathic pain management.


Assuntos
Gânglios Espinais/imunologia , Macrófagos/fisiologia , Neuralgia/imunologia , Animais , Comunicação Celular , Proliferação de Células/efeitos dos fármacos , Feminino , Hiperalgesia/imunologia , Imunossupressores/farmacologia , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microglia/metabolismo , Microglia/fisiologia , Traumatismos dos Nervos Periféricos/imunologia , Gravidez , Células Receptoras Sensoriais/metabolismo , Fatores Sexuais , Tacrolimo/análogos & derivados , Tacrolimo/farmacologia
20.
Eur J Pharm Sci ; 144: 105229, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31958581

RESUMO

Tacrolimus, also known as FK506, is a first-line drug for the topical treatment of immune-mediated inflammatory anterior ocular diseases (IIAODs). However, due to its limited water solubility, hydrophobic nature and relatively high molecular weight, topical application of FK506 features poor bioavailability. Numbers of formulations have been attempted to enhance the erratic bioavailability of FK506 through various techniques. But until now, none of them could satisfy the clinical needs completely. Here, a novel formulation of FK506, FK506-loaded cationic nanoemulsions (FK506 CNE), was developed to prolong the precorneal residence time of FK506, thereby enhancing the bioavailability of FK506 for IIAODs therapy. FK506 CNE was prepared by high-pressure homogenization, and its composition was screened and optimized by single-factor experiments. The FK506 CNE showed spherical morphology with a mean diameter of 178.8 ± 2.7 nm and a zeta potential of +25.6 ± 0.6 mV. Results from in vivo gamma scintigraphy studies proved that the precorneal residence time of FK506 CNE was significantly increased, compared with FK506-loaded neutral nanoemulsions (FK506 NE) and saline. The data of aqueous humor pharmacokinetic study in rabbits showed that the relative bioavailability of FK506 CNE was 1.68-fold and 1.77-fold of FK506 NE and the marketed FK506 eye drops (Talymus®), respectively. Finally, hematoxylin and eosin staining images and in vitro cytotoxicity data confirmed the safety of the FK506 CNE. Taking all these into consideration, we propose that FK506 CNE is a promising topical ophthalmic nanoformulation for the management of IIAODs.


Assuntos
Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Imunossupressores/administração & dosagem , Imunossupressores/farmacologia , Tacrolimo/administração & dosagem , Tacrolimo/farmacologia , Administração Oftálmica , Administração Tópica , Animais , Humor Aquoso/metabolismo , Disponibilidade Biológica , Cátions , Linhagem Celular , Sobrevivência Celular , Córnea , Portadores de Fármacos/farmacologia , Emulsões/química , Olho/metabolismo , Humanos , Imunossupressores/farmacocinética , Nanopartículas/química , Coelhos , Solubilidade , Tacrolimo/farmacocinética , Distribuição Tecidual
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