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1.
Ann Palliat Med ; 11(5): 1762-1773, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35672893

RESUMO

BACKGROUND: The proteinuria remission in hepatitis B virus-associated glomerulonephritis (HBV-GN) patients with massive proteinuria treated with antiviral therapy was low. Tacrolimus (TAC) is effective in primary nephropathy and can inhibit HBV infection by inhibiting HBV binding to sodium taurocholate cotransporting polypeptide on liver cells. This study evaluated the efficacy and safety of TAC combined with ETV compared with entecavir (ETV) monotherapy in HBV-GN. METHODS: Patients diagnosed with HBV-GN were recruited for this prospective, randomized, controlled, multicenter, single-blinded study in China. Patients were given TAC and ETV therapy (the TAC+ETV group) or placebo and ETV therapy (the ETV group) for 26 weeks. The efficacy endpoints included proteinuria remission, including complete and partial remission (CR and PR), the change of 24-hour proteinuria (24 h UP) and HBV DNA titer. The safety endpoints were the incidence of HBV virologic breakthrough and adverse events. RESULTS: There were 14 patients in the TAC+ETV group and 17 patients in the ETV group. In the intention-to-treat analyses, 64.3% (9/14) of patients in the TAC+ETV group and 58.8% (10/17) in the ETV group achieved PR or CR at 26 weeks (P=0.38). At week 14, 42.9% (6/14) and 41.2% (7/17) of patients in the TAC+ETV group and the ETV group, respectively, achieved PR or CR (P=0.23). At week 26, the 24 h UP had decreased by 2.63±6.33 g from baseline in the TAC+ETV group and 1.42±4.34 g in the ETV group (P=0.55). The serum albumin increased by 11.1±7.30 g/L from baseline in the TAC+ETV group and 3.81±5.09 g/L in the ETV group (P<0.001). Log10 HBV DNA decreased by 1.49±2.04 from baseline in the TAC+ETV group and 2.47±2.08 in the ETV group (P=0.37); 28.6% (4/14) patients had HBV DNA virologic breakthrough in the ETV group, while none in the TAC+ETV group (P=0.29). CONCLUSIONS: In adult HBV-GN patients, TAC and ETV combination therapy may significantly improve serum albumin levels without increasing the risk of HBV reactivation compared with entecavir monotherapy. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT03062813.


Assuntos
Glomerulonefrite , Hepatite B Crônica , Adulto , Antivirais/uso terapêutico , DNA Viral/farmacologia , DNA Viral/uso terapêutico , Glomerulonefrite/induzido quimicamente , Glomerulonefrite/tratamento farmacológico , Guanina/análogos & derivados , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Humanos , Estudos Prospectivos , Proteinúria/induzido quimicamente , Proteinúria/tratamento farmacológico , Albumina Sérica/farmacologia , Albumina Sérica/uso terapêutico , Método Simples-Cego , Tacrolimo/farmacologia , Tacrolimo/uso terapêutico , Resultado do Tratamento
2.
mBio ; 13(3): e0104922, 2022 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-35604094

RESUMO

Calcineurin is an essential virulence factor that is conserved across human fungal pathogens, including Cryptococcus neoformans, Aspergillus fumigatus, and Candida albicans. Although an excellent target for antifungal drug development, the serine-threonine phosphatase activity of calcineurin is conserved in mammals, and inhibition of this activity results in immunosuppression. FK506 (tacrolimus) is a naturally produced macrocyclic compound that inhibits calcineurin by binding to the immunophilin FKBP12. Previously, our fungal calcineurin-FK506-FKBP12 structure-based approaches identified a nonconserved region of FKBP12 that can be exploited for fungus-specific targeting. These studies led to the design of an FK506 analog, APX879, modified at the C-22 position, which was less immunosuppressive yet maintained antifungal activity. We now report high-resolution protein crystal structures of fungal FKBP12 and a human truncated calcineurin-FKBP12 bound to a natural FK506 analog, FK520 (ascomycin). Based on information from these structures and the success of APX879, we synthesized and screened a novel panel of C-22-modified compounds derived from both FK506 and FK520. One compound, JH-FK-05, demonstrates broad-spectrum antifungal activity in vitro and is nonimmunosuppressive in vivo. In murine models of pulmonary and disseminated C. neoformans infection, JH-FK-05 treatment significantly reduced fungal burden and extended animal survival alone and in combination with fluconazole. Furthermore, molecular dynamic simulations performed with JH-FK-05 binding to fungal and human FKBP12 identified additional residues outside the C-22 and C-21 positions that could be modified to generate novel FK506 analogs with improved antifungal activity. IMPORTANCE Due to rising rates of antifungal drug resistance and a limited armamentarium of antifungal treatments, there is a paramount need for novel antifungal drugs to treat systemic fungal infections. Calcineurin has been established as an essential and conserved virulence factor in several fungi, making it an attractive antifungal target. However, due to the immunosuppressive action of calcineurin inhibitors, they have not been successfully utilized clinically for antifungal treatment in humans. Recent availability of crystal structures of fungal calcineurin-bound inhibitor complexes has enabled the structure-guided design of FK506 analogs and led to a breakthrough in the development of a compound with increased fungal specificity. The development of a calcineurin inhibitor with reduced immunosuppressive activity and maintained therapeutic antifungal activity would add a significant tool to the treatment options for these invasive fungal infections with exceedingly high rates of mortality.


Assuntos
Cryptococcus neoformans , Tacrolimo , Animais , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Calcineurina/metabolismo , Inibidores de Calcineurina/farmacologia , Cryptococcus neoformans/metabolismo , Imidazóis , Imunossupressores/metabolismo , Imunossupressores/farmacologia , Mamíferos/metabolismo , Camundongos , Sulfonamidas , Tacrolimo/farmacologia , Proteína 1A de Ligação a Tacrolimo/metabolismo , Tiofenos , Fatores de Virulência/metabolismo
3.
Int J Mol Sci ; 23(9)2022 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-35563498

RESUMO

Increasing extracellular osmolarity 100 mOsm/kg above plasma level to the physiological levels for cartilage induces chondrogenic marker expression and the differentiation of chondroprogenitor cells. The calcineurin inhibitor FK506 has been reported to modulate the hypertrophic differentiation of primary chondrocytes under such conditions, but the molecular mechanism has remained unclear. We aimed at clarifying its role. Chondrocyte cell lines and primary cells were cultured under plasma osmolarity and chondrocyte-specific in situ osmolarity (+100 mOsm, physosmolarity) was increased to compare the activation of nuclear factor of activated T-cells 5 (NFAT5). The effects of osmolarity and FK506 on calcineurin activity, cell proliferation, extracellular matrix quality, and BMP- and TGF-ß signaling were analyzed using biochemical, gene, and protein expression, as well as reporter and bio-assays. NFAT5 translocation was similar in chondrocyte cell lines and primary cells. High supraphysiological osmolarity compromised cell proliferation, while physosmolarity or FK506 did not, but in combination increased proteoglycan and collagen expression in chondrocytes in vitro and in situ. The expression of the TGF-ß-inducible protein TGFBI, as well as chondrogenic (SOX9, Col2) and terminal differentiation markers (e.g., Col10) were affected by osmolarity. Particularly, the expression of minor collagens (e.g., Col9, Col11) was affected. The inhibition of the FK506-binding protein suggests modulation at the TGF-ß receptor level, rather than calcineurin-mediated signaling, as a cause. Physiological osmolarity promotes terminal chondrogenic differentiation of progenitor cells through the sensitization of the TGF-ß superfamily signaling at the type I receptor. While hyperosmolarity alone facilitates TGF-ß superfamily signaling, FK506 further enhances signaling by releasing the FKBP12 break from the type I receptor to improve collagenous marker expression. Our results help explain earlier findings and potentially benefit future cell-based cartilage repair strategies.


Assuntos
Inibidores de Calcineurina , Tacrolimo , Calcineurina/metabolismo , Inibidores de Calcineurina/farmacologia , Diferenciação Celular , Células Cultivadas , Condrócitos/metabolismo , Condrogênese , Tacrolimo/farmacologia , Fator de Crescimento Transformador beta/metabolismo
4.
Ann Plast Surg ; 88(3 Suppl 3): S235-S238, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35513326

RESUMO

PURPOSE: Our previous rodent studies demonstrated significantly decreased full-thickness necrosis in pedicled dorsal skin flaps with topical tacrolimus as compared with petroleum jelly. Histologically, we found that topical tacrolimus was correlated with increased vascular growth in areas more susceptible to ischemic damage. The purpose of this study was to investigate the potential benefits of pretreatment with tacrolimus. By applying tacrolimus in advance of raising the dorsal skin flaps, we hoped to increase vascularity and thus increase the overall viability of the flaps. METHODS: Twenty Sprague-Dawley rats were initially randomized to 4 groups based on timing of tacrolimus treatment (presurgical/postsurgical treatment): control/control (C/C), control/tacrolimus (C/T), tacrolimus/control (T/C), and tacrolimus/tacrolimus (T/T). Treatments consisted of 0.2 g of the control (topical petroleum jelly) and 0.1% topical tacrolimus to the rat dorsum twice per day. After 7 days of presurgical treatment, a cranially based dorsal skin flap measuring 3 × 10 cm was created. Two rats perished during surgery and were excluded for further analysis. Each rat was treated for a further 7 days and sacrificed. Two blinded reviewers marked the total skin flap area as well as areas of viable tissue, reversible ischemia, and full-thickness necrosis. Percentage areas were calculated using Fiji/ImageJ, and statistical analysis was performed in R. RESULTS: The average viable areas for C/C, C/T, T/C, and T/T were 31.4%, 31.9%, 35.6%, and 22.6%, respectively. The average reversible ischemic area for C/C, C/T, T/C, and T/T was 53.1%, 54.0%, 54.1%, and 71.5%, respectively. The average necrotic area for C/C, C/T, T/C, and T/T was 15.4%, 14.0%, 10.2%, and 5.9%, respectively. For areas of reversible ischemia, T/T arm had higher areas compared with C/T (P = 0.004) and T/C (P = 0.044). There was no significance between treatment arms for areas of viable and necrotic tissue. CONCLUSIONS: We observed higher areas of reversible ischemia for continuous tacrolimus treatment compared with only pre-tacrolimus application or post-tacrolimus application. This suggests that tacrolimus application before and after surgical insult may be associated with improved ischemic survival of the skin. Although we did not observe decreased areas of necrosis for tacrolimus treatment compared with control, this was likely due to the limited number of rats available in each arm to reach significance. Further study is needed to fully elucidate the encouraging trends that were observed.


Assuntos
Lesões dos Tecidos Moles , Tacrolimo , Animais , Sobrevivência de Enxerto , Humanos , Isquemia , Necrose , Vaselina , Projetos Piloto , Ratos , Ratos Sprague-Dawley , Retalhos Cirúrgicos/irrigação sanguínea , Tacrolimo/farmacologia
5.
Sci Rep ; 12(1): 6120, 2022 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-35449173

RESUMO

Repurposing FDA-approved drugs is an efficient and cost-effective approach in the development of therapeutics for a broad range of diseases. However, prediction of function can be challenging, especially in the brain. We screened a small-molecule library with FDA-approved drugs for effects on behavior. The studies were carried out using zebrafish larvae, imaged in a 384-well format. We found that various drugs affect activity, habituation, startle responses, excitability, and optomotor responses. The changes in behavior were organized in behavioral profiles, which were examined by hierarchical cluster analysis. One of the identified clusters includes the calcineurin inhibitors cyclosporine (CsA) and tacrolimus (FK506), which are immunosuppressants and potential therapeutics in the prevention of Alzheimer's disease. The calcineurin inhibitors form a functional cluster with seemingly unrelated drugs, including bromocriptine, tetrabenazine, rosiglitazone, nebivolol, sorafenib, cabozantinib, tamoxifen, meclizine, and salmeterol. We propose that drugs with 'CsA-type' behavioral profiles are promising candidates for the prevention and treatment of Alzheimer's disease.


Assuntos
Doença de Alzheimer , Inibidores de Calcineurina , Animais , Calcineurina , Inibidores de Calcineurina/farmacologia , Análise por Conglomerados , Ciclosporina/farmacologia , Imunossupressores/farmacologia , Tacrolimo/farmacologia , Peixe-Zebra
6.
Am J Nephrol ; 53(5): 388-396, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35413717

RESUMO

INTRODUCTION: Thy1.1 glomerulonephritis (Thy1.1 GN) in rats is widely used as an experimental model of mesangial proliferative glomerulonephritis (GN). We previously reported that T-helper (Th) cells were accumulated in glomeruli from the early phase of this model and that not Th2 cells but Th1 cells play an important role in the development of glomerular alterations. Although Th17 is reported to be involved in the pathogenesis of several autoimmune diseases, the role of Th17 cells in the pathogenesis of mesangial alterations in Thy1.1 GN remains unclear. METHODS: The kinetics of the infiltration of subsets of Th cells and the expression of IL-17 in Thy1.1 GN were analyzed. Next, the localization and the cell types of IL-17 receptor (IL-17R)-positive cells and IL-6-positive cells were analyzed. Then, the effect of tacrolimus on the expressions of Th17-related cytokines in Thy1.1 GN was analyzed. RESULTS: Not only Th1 cells but also Th17 cells were recruited into glomeruli from the early phase of the disease. mRNA expression of IL-17 in glomeruli was elevated. The increased positive expression of IL-17R was detected in the mesangial area, and some of IL-17R-positive cells were co-stained with IL-6. Tacrolimus treatment ameliorated mesangial alterations by suppressing the expressions of Th17-related cytokines such as IL-17 and IL-6. CONCLUSION: Th17 cells participate in the development of Thy1.1 GN, a mimic of mesangial proliferative GN, and Th17 cells and their related cytokines are pertinent therapeutic targets.


Assuntos
Glomerulonefrite , Tacrolimo , Animais , Citocinas/metabolismo , Glomerulonefrite/tratamento farmacológico , Humanos , Interleucina-17 , Interleucina-6 , Ratos , Tacrolimo/farmacologia , Tacrolimo/uso terapêutico , Células Th1/metabolismo , Células Th1/patologia , Células Th17/metabolismo , Células Th17/patologia , Antígenos Thy-1
7.
Biochem Biophys Res Commun ; 611: 53-59, 2022 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-35477093

RESUMO

Delivery of cerebroprotective agents using liposomes has been demonstrated to be useful for treating cerebral ischemia/reperfusion (I/R) injury. We previously reported that intravenous administration of liposomes with diameters of 100 nm showed higher accumulation in the I/R region compared with larger liposomes (>200 nm) by passage through the disintegrated blood-brain barrier, suggesting a size-dependence for liposome-mediated drug delivery. Based on these findings, we hypothesized that regulation of liposomal particle size (<100 nm) may enhance the therapeutic efficacy of encapsulated drugs on cerebral I/R injury. Herein, we prepared lipid nanoparticles (LNP) with particle sizes <100 nm by the microfluidics method and compared their therapeutic potential with LNP exhibiting sizes >100 nm in cerebral I/R model rats. Intravenously administered smaller LNP (ca. 60 nm) exhibited wider accumulation and diffusivity in the brain parenchyma of the I/R region compared with larger LNP (>100 nm). Importantly, treatment with LNP encapsulating the cerebroprotective agent FK506 (FK-LNP) with particle sizes <100 nm showed greater cerebroprotective effects than FK-LNP with sizes >100 nm, and also significantly ameliorated brain injury. These results suggest that particle size regulation of LNP to sizes <100 nm can enhance the therapeutic effect of encapsulated drugs for treatment of cerebral I/R injury, and that FK-LNP could be a promising cerebroprotective agent.


Assuntos
Isquemia Encefálica , Nanopartículas , Fármacos Neuroprotetores , Traumatismo por Reperfusão , Animais , Isquemia Encefálica/tratamento farmacológico , Lipossomos/uso terapêutico , Fármacos Neuroprotetores/farmacologia , Tamanho da Partícula , Ratos , Ratos Wistar , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/prevenção & controle , Tacrolimo/farmacologia , Tacrolimo/uso terapêutico
8.
Biocontrol Sci ; 27(1): 31-39, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35314558

RESUMO

Calcineurin (CN) is a conserved Ca2+-calmodulin activated protein phosphatase, which plays important roles in immune regulation, cardiac hypertrophy, and apoptosis in humans. In pathogenic fungi, CN is essential for stress survival, sexual development, and virulence. The immunosuppressant tacrolimus (FK506) is a specific inhibitor of CN in humans and fungi including nonpathogenic fission yeast. Although calcineurin inhibition by FK506 or CN deletion in fission yeast does not induce growth defects, treatment with some anti-fungal drugs such as micafungin and valproic acid, induced synthetic lethality with calcineurin inhibition. Here, we searched for the compounds that induce synthetic growth defects with CN inhibition in fission yeast. We found that ellagic acid (EA) preferentially induced growth inhibition in CN deletion cells. Consistently, co-treatment with EA and FK506 induced severe growth inhibition in the wild-type cells, whereas neither of the single treatment with each compound did so. Moreover, deletion of the calcineurin-regulated transcription factor Prz1 also induced a marked EA sensitivity. Intriguingly, EA also enhanced the growth inhibitory effect of other anti-fungal drugs, including micafungin and miconazole. Thus, our data suggesting the synergistic growth inhibitory effect of the calcineurin inhibitor FK506 and EA may be useful to understand the mechanism to overcome the antifungal resistance.


Assuntos
Schizosaccharomyces , Tacrolimo , Calcineurina/metabolismo , Calcineurina/farmacologia , Inibidores de Calcineurina/metabolismo , Inibidores de Calcineurina/farmacologia , Ácido Elágico/metabolismo , Ácido Elágico/farmacologia , Humanos , Schizosaccharomyces/metabolismo , Tacrolimo/metabolismo , Tacrolimo/farmacologia
9.
BMC Ophthalmol ; 22(1): 101, 2022 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-35246084

RESUMO

BACKGROUND: To evaluate the efficacy of the topical administration of immunosuppressants and corticosteroids in tapering doses in the management of patients with high-risk keratoplasty. METHODS: One hundred and six patients treated with topical immunosuppressants (50 eyes in the FK506 group and 56 eyes in the CsA group) and corticosteroid eye drops in tapering doses were enrolled in the study. The rates of rejection episodes, irreversible rejection, graft survival, and related influential factors were evaluated. RESULTS: The mean follow-up period was 48.1 ± 7.9 months (range, 36-60 months). The rates of rejection episodes and irreversible rejection were 14.0% and 6.00% in the FK506 group and 37.5% and 7.1% in the CsA group, respectively. Kaplan-Meier survival analysis demonstrated a significantly higher graft survival rate in the FK506 group (81.6%±5.3%, 71.1%±6.3%) compared with that in the CsA group (71.1%±6.3%, 57.5%±7.5%) at 3 and 5 years after surgery (P = 0.006). Multivariate logistic regression revealed that preoperative risk score ≥ 3 (P = 0.016) and endothelial immune rejection (P = 0.033) were risk factors associated with graft survival. CONCLUSIONS: Topical administration of tacrolimus and corticosteroids in tapering doses is effective in decreasing the incidence of immune rejection in high-risk keratoplasty. Careful instruction of patients on the reasonable use of topical tacrolimus is critical to avoid immune rejection induced by sudden discontinuation of medication.


Assuntos
Transplante de Córnea , Tacrolimo , Administração Tópica , Corticosteroides , Ciclosporina/uso terapêutico , Seguimentos , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Humanos , Imunossupressores/uso terapêutico , Ceratoplastia Penetrante , Tacrolimo/farmacologia , Tacrolimo/uso terapêutico
10.
Carcinogenesis ; 43(4): 338-348, 2022 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-35136987

RESUMO

One key reason for T cell exhaustion is continuous antigen exposure. Early exhausted T cells can reverse exhaustion and differentiate into fully functional memory T cells if removed from persisting antigen stimulation. Therefore, this study viewed T cell exhaustion as an over-activation status induced by chronic antigen stimuli. This study hypothesized that blocking TCR signal intermittently to terminate over-activation signal can defer the developmental process of T cell exhaustion. In this study, melanoma-bearing mice were treated with tacrolimus (FK506) every 5 days. The tumor size and tumor-infiltrating lymphocytes (TILs) were analyzed. We found that intermittent administration of tacrolimus significantly inhibited tumor growth, and this effect was mediated by CD8+T cells. Intermittent tacrolimus treatment facilitated the infiltration of CD8+TILs. RNA-seq and quantitative RT-PCR of sorted CD8+TILs showed the expression of Nr4a1 (an exhaustion-related transcription factor) and Ctla4 (a T cell inhibitory receptor) was remarkably downregulated. These results indicated that intermittently blocking TCR signal by tacrolimus can promote anti-tumor immunity and inhibit the tumor growth in melanoma-bearing mice, inhibiting the transcription of several exhaustion-related genes, such as Nr4a1 and Ctla4.


Assuntos
Melanoma , Tacrolimo , Animais , Linfócitos T CD8-Positivos , Antígeno CTLA-4/metabolismo , Linfócitos do Interstício Tumoral , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Camundongos , Tacrolimo/metabolismo , Tacrolimo/farmacologia
11.
J Transl Med ; 20(1): 104, 2022 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-35216600

RESUMO

BACKGROUND: Graft-versus-host disease (GvHD) is a critical complication after allogeneic hematopoietic stem cell transplantation (HSCT). The immunosuppressants given to patients undergoing allogeneic HSCT disturb the microbiome and the host immune system, potentially leading to dysbiosis and inflammation, and may affect immune function and bone marrow transplantation. The intestinal microbiome is a target for the development of novel therapies for GvHD. Lactobacillus species are widely used supplements to induce production of antimicrobial and anti-inflammatory factors. METHODS: We determined the effect of the combination of Lactobacillus acidophilus and FK506 on GvHD following major histocompatibility complex-mismatched bone marrow transplantation. RESULTS: The combination treatment suppressed IFN-γ and IL-17-producing T cell differentiation, but increased Foxp3+Treg differentiation and IL-10 production. Also, the combination treatment and combination treated-induced Treg cells modulated the proliferation of murine alloreactive T cells in vitro. Additionally, the combination treatment upregulated Treg-related genes-Nt5e, Foxp3, Ikzf2, Nrp1 and Itgb8-in murine CD4+-T cells. The combination treatment also alleviated GvHD clinically and histopathologically by controlling the effector T cell and Treg balance in vivo. Moreover, the combination treatment decreased Th17 differentiation significantly and significantly upregulated Foxp3 and IL-10 expression in peripheral blood mononuclear cells from healthy controls and liver transplantation (LT) patients. CONCLUSIONS: Therefore, the combination of L. acidophilus and FK506 is effective and safe for patients undergoing allogeneic hematopoietic stem cell transplantation.


Assuntos
Doença Enxerto-Hospedeiro , Linfócitos T Reguladores , Doença Aguda , Animais , Doença Enxerto-Hospedeiro/tratamento farmacológico , Humanos , Lactobacillus acidophilus , Leucócitos Mononucleares , Camundongos , Camundongos Endogâmicos C57BL , Tacrolimo/farmacologia , Tacrolimo/uso terapêutico
12.
Int J Biol Macromol ; 206: 670-680, 2022 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-35218805

RESUMO

Chemically induced dimerization (CID) is used to induce proximity and result in artificial complex formation between a pair of proteins involved in biological processes in cells to investigate and regulate these processes. The induced heterodimerization of FKBP fusion proteins by rapamycin and FK506 has been extensively exploited as a chemically induced dimerization system to regulate and understand highly dynamic cellular processes. Here, we report the crystal structure of the AtFKBP53 FKBD in complex with rapamycin. The crystal packing reveals an unusual feature whereby two rapamycin molecules appear to mediate homodimerization of the FKBD. The triene arm of rapamycin appears to play a significant role in forming this dimer. This forms the first structural report of rapamycin-mediated homodimerization of an FKBP. The structural information on the rapamycin-mediated FKBD dimerization may be employed to design and synthesize covalently linked dimeric rapamycin, which may subsequently serve as a chemically induced dimerization system for the regulation and characterization of cellular processes.


Assuntos
Sirolimo , Tacrolimo , Dimerização , Sirolimo/farmacologia , Tacrolimo/farmacologia , Proteínas de Ligação a Tacrolimo/química
13.
Cell Death Dis ; 13(1): 34, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-35013114

RESUMO

iCasp9 suicide gene has been widely used as a promising killing strategy in various cell therapies. However, different cells show significant heterogeneity in response to apoptosis inducer, posing challenges in clinical applications of killing strategy. The cause of the heterogeneity remains elusive so far. Here, by simultaneously monitoring the dynamics of iCasp9 dimerization, Caspase3 activation, and cell fate in single cells, we found that the heterogeneity was mainly due to cell-to-cell variability in initial iCasp9 expression and XIAP/Caspase3 ratio. Moreover, multiple-round drugging cannot increase the killing efficiency. Instead, it will place selective pressure on protein levels, especially on the level of initial iCasp9, leading to drug resistance. We further show this resistance can be largely eliminated by combinatorial drugging with XIAP inhibitor at the end, but not at the beginning, of the multiple-round treatments. Our results unveil the source of cell fate heterogeneity and drug resistance in iCasp9-mediated cell death, which may enlighten better therapeutic strategies for optimized killing.


Assuntos
Caspase 9/farmacologia , Morte Celular/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 9/química , Caspase 9/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistência a Medicamentos/efeitos dos fármacos , Sinergismo Farmacológico , Células HEK293 , Células HeLa , Humanos , Multimerização Proteica , Tacrolimo/análogos & derivados , Tacrolimo/farmacologia , Fatores de Tempo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo
14.
Int Immunopharmacol ; 105: 108547, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35066448

RESUMO

BACKGROUND: Tissue kallikrein offers a wide spectrum of biological activity in the protection against various types of injury. However, information on its role in tacrolimus (TAC)-induced renal injury is limited. OBJECTIVES: This study aimed to assess the beneficial effects of pancreatic kininogenase (PK) in a rat model of chronic TAC nephrotoxicity and in vitro. METHODS: Sprague Dawley rats were treated daily with either TAC or PK or a combination of the two for four weeks. The influence of PK on renal injury was examined in terms of renal function, histopathology, cytokine expression, oxidative stress, intracellular organelles, programmed cell death, and PI3K/AKT signaling. Human kidney proximal tubular (HK-2) cells and mouse mesangial (SV40 MES13) cells treated with TAC and PK were also studied. RESULTS: PK treatment improved renal function and histopathology. This effect was paralleled by downregulation of proinflammatory and profibrotic cytokine expression. TAC-induced oxidative stress was closely associated with endoplasmic reticulum stress and mitochondrial dysfunction, resulting in excessive programmed cell death (apoptosis and autophagy) that was significantly abrogated by concurrent PK interference with PI3K/AKT signaling. PK also stimulated bradykinin receptor 1 (B1R) and B2R mRNA synthesis and increased bioactive nitric oxide (NO) and cAMP concentrations in TAC-treated kidneys. Blockade of either B1R or B2R eliminated the renoprotective effects of PK. In HK-2 and SV40 MES13 cells, PK decreased TAC-induced overproduction of intracellular reactive oxygen species and inhibited apoptotic cells, whereas cell viability was improved. Moreover, activated PI3K/AKT signaling in HK-2 cells was inhibited by PK and the PI3K inhibitor, LY294002. CONCLUSIONS: These findings indicate that PK treatment protects against chronic TAC nephrotoxicity via inhibition of PI3K/AKT signaling.


Assuntos
Fosfatidilinositol 3-Quinases , Tacrolimo , Animais , Apoptose , Rim , Camundongos , Estresse Oxidativo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores da Bradicinina/metabolismo , Tacrolimo/farmacologia , Calicreínas Teciduais/metabolismo , Calicreínas Teciduais/farmacologia
15.
J Biol Chem ; 298(3): 101589, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35033536

RESUMO

Current immunosuppressive strategies in organ transplantation rely on calcineurin inhibitors cyclosporine A (CsA) or tacrolimus (Tac). Both drugs are nephrotoxic, but CsA has been associated with greater renal damage than Tac. CsA inhibits calcineurin by forming complexes with cyclophilins, whose chaperone function is essential for proteostasis. We hypothesized that stronger toxicity of CsA may be related to suppression of cyclophilins with ensuing endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in kidney epithelia. Effects of CsA and Tac (10 µM for 6 h each) were compared in cultured human embryonic kidney 293 (HEK 293) cells, primary human renal proximal tubule (PT) cells, freshly isolated rat PTs, and knockout HEK 293 cell lines lacking the critical ER stress sensors, protein kinase RNA-like ER kinase or activating transcription factor 6 (ATF6). UPR was evaluated by detection of its key components. Compared with Tac treatment, CsA induced significantly stronger UPR in native cultured cells and isolated PTs. Evaluation of proapoptotic and antiapoptotic markers suggested an enhanced apoptotic rate in CsA-treated cells compared with Tac-treated cells as well. Similar to CsA treatment, knockdown of cyclophilin A or B by siRNA caused proapoptotic UPR, whereas application of the chemical chaperones tauroursodeoxycholic acid or 4-phenylbutyric acid alleviated CsA-induced UPR. Deletion of protein kinase RNA-like ER kinase or ATF6 blunted CsA-induced UPR as well. In summary, inhibition of cyclophilin chaperone function with ensuing ER stress and proapoptotic UPR aggravates CsA toxicity, whereas pharmacological modulation of UPR bears potential to alleviate renal side effects of CsA.


Assuntos
Inibidores de Calcineurina , Ciclosporina , Estresse do Retículo Endoplasmático , Túbulos Renais , Animais , Calcineurina/metabolismo , Inibidores de Calcineurina/farmacologia , Ciclofilinas/metabolismo , Ciclosporina/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Células HEK293 , Humanos , Imunossupressores/farmacologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/imunologia , Proteínas Quinases , RNA , Ratos , Tacrolimo/farmacologia , Resposta a Proteínas não Dobradas
16.
J Cell Mol Med ; 26(2): 507-514, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34889045

RESUMO

Lipopolysaccharide (LPS)-induced sepsis-associated acute kidney injury (SA-AKI) is a model of clinical serious care syndrome, with high morbidity and mortality. Tacrolimus (TAC), a novel immunosuppressant that inhibits inflammatory response, plays a pivotal role in kidney diseases. In this study, LPS treated mice and cultured podocytes were used as the models of SA-AKI in vivo and in vitro, respectively. Medium- and high-dose TAC administration significantly attenuated renal function and renal pathological manifestations at 12, 24 and 48 h after LPS treatment in mice. Moreover, the Toll-like receptor 4 (TLR4)/myeloid differential protein-88 (MyD88)/nuclear factor-kappa (NF-κB) signalling pathway was also dramatically inhibited by medium- and high-dose TAC administration at 12, 24 and 48 h of LPS treatment mice. In addition, TAC reversed LPS-induced podocyte cytoskeletal injury and podocyte migratory capability. Our findings indicate that TAC has protective effects against LPS-induced AKI by inhibiting TLR4/MyD88/NF-κB signalling pathway and podocyte dysfunction, providing another potential therapeutic effects for the LPS-induced SA-AKI.


Assuntos
Injúria Renal Aguda , Receptor 4 Toll-Like , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/metabolismo , Animais , Lipopolissacarídeos/farmacologia , Camundongos , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Tacrolimo/farmacologia , Receptor 4 Toll-Like/metabolismo
17.
Am J Transplant ; 22(4): 1014-1030, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34510717

RESUMO

Our understanding of the involvement of the gut microbiota (GM) in human health has expanded exponentially over the last few decades, particularly in the fields of metabolism, inflammation, and immunology. Immunosuppressive treatment (IST) prescribed to solid organ transplant (SOT) recipients produces GM changes that affect these different processes. This review aims at describing the current knowledge of how IST changes the GM. Overall, SOT followed by IST results in persistent changes in the GM, with a consistent increase in proteobacteria including opportunistic pathobionts. In mice, Tacrolimus induces dysbiosis and metabolic disorders, and alters the intestinal barrier. The transfer of the GM from Tacrolimus-treated hosts confers immunosuppressive properties, suggesting a contributory role for the GM in this drug's efficacy. Steroids induce dysbiosis and intestinal barrier alterations, and also seem to depend partly on the GM for their immunosuppressive and metabolic effects. Mycophenolate Mofetil, frequently responsible for digestive side effects such as diarrhea and colitis, is associated with pro-inflammatory dysbiosis and increased endotoxemia. Alemtuzumab, m-TOR inhibitors, and belatacept have shown more marginal impact on the GM. Most of these observations are descriptive. Future studies should explore the underlying mechanism of IST-induced dysbiosis in order to better understand their efficacy and safety characteristics.


Assuntos
Microbioma Gastrointestinal , Transplante de Órgãos , Animais , Disbiose , Camundongos , Transplante de Órgãos/efeitos adversos , Tacrolimo/farmacologia
18.
Int J Oral Maxillofac Surg ; 51(2): 279-287, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34090756

RESUMO

This study was performed to investigate the effect of platelet-rich fibrin membrane (PRFM), alone and with topical tacrolimus application, on regeneration of the crushed facial nerve (FN). Thirty healthy 7-week-old albino rats were used. The left FN was damaged by crushing in all rats. Three random groups of rats were formed: group 1, untreated; group 2, treated with PRFM; group 3, treated with PRFM plus topical tacrolimus. Functional recovery and histological and immunohistochemical evaluations were performed 4 and 8 weeks later. Anti-S100 was used to detect myelin sheath. At 4 weeks, blinking reflex recovery was more rapid in group 3 than in groups 2 and 1 (4.30 ± 0.48, 3.40 ± 0.52, and 2.20 ± 0.42, respectively); the difference was statistically significant (P = 0.001). Histologically, group 3 showed more apparent normal FN structures than the other groups. Immunohistochemical caspase-3 evaluation of the axon area revealed a significant difference between group 2 (PRFM alone; 8.67 ± 0.029) and group 3 (PRFM plus topical tacrolimus; 4.42 ± 0.028) (P = 0.001). Group 3 showed the greatest positive staining in the myelin sheath. Based on the results of this animal study, clinical studies should be performed to determine whether the combination of PRF and tacrolimus also improves the outcome of nerve regeneration in humans.


Assuntos
Traumatismos dos Nervos Periféricos , Fibrina Rica em Plaquetas , Animais , Nervo Facial , Regeneração Nervosa , Ratos , Tacrolimo/farmacologia
19.
Front Cell Infect Microbiol ; 12: 864912, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35493742

RESUMO

Scedosporium and Lomentospora infections in humans are generally chronic and stubborn. The use of azoles alone cannot usually inhibit the growth of these fungi. To further explore the combined effect of multiple drugs and potential mechanisms of action, we tested the antifungal effects of tacrolimus (FK506) and everolimus in combination with azoles in vitro and in vivo on 15 clinical strains of Scedosporium/Lomentospora species and detected the level of Rhodamine 6G, ROS activity, and apoptosis. The in vitro results showed that the combinations of tacrolimus with itraconazole, voriconazole, and posaconazole showed synergistic effects on 9 strains (60%), 10 strains (73%), and 7 strains (47%), respectively, and the combinations of everolimus with itraconazole, voriconazole, and posaconazole showed synergistic effects on 8 strains (53%), 8 strains (53%), and 7 strains (47%), respectively. The synergistic effects might correspond to the elevated ROS activity (the tacrolimus + itraconazole group compared to the itraconazole group, (P < 0.05)), early apoptosis (itraconazole (P < 0.05) and voriconazole (P < 0.05) combined with everolimus), and late apoptosis (the tacrolimus + itraconazole group compared to the itraconazole group, (P < 0.01); the tacrolimus + posaconazole group compared to the posaconazole group, (P < 0.05)), but not inhibition of efflux pump activity. Our in vitro results suggested that a combination of tacrolimus or everolimus and azoles have a synergistic effect against Scedosporium/Lomentospora. The synergistic mechanisms of action might be triggering excessive ROS activity and apoptosis. In vivo, the survival rate of G. mellonella (sixth instar larvae) was significantly improved by tacrolimus alone, everolimus alone, azoles alone, and tacrolimus and everolimus combined with azoles separately (P < 0.05 for the tacrolimus group; P < 0.01 for the everolimus group and the itraconazole group; P = 0.0001 for the tacrolimus and posaconazole group; P < 0.0001 for other groups except the everolimus and itraconazole group, everolimus and posaconazole group, and tacrolimus and itraconazole group). From the results, we infer that the combination of tacrolimus or everolimus with azoles has obvious synergistic effect on Scedosporium/Lomentospora, and might enhance the level of apoptosis and necrosis. However, the synergistic effects were not related to the efflux pump. In conclusion, from our in vitro and in vivo study, tacrolimus and everolimus combined with azoles may have a synergistic effect in the treatment against Scedosporium/Lomentospora, improving the drug activity of azoles and promoting a better prognosis for patients.


Assuntos
Ascomicetos , Scedosporium , Azóis/farmacologia , Everolimo/farmacologia , Humanos , Itraconazol/farmacologia , Testes de Sensibilidade Microbiana , Espécies Reativas de Oxigênio , Tacrolimo/farmacologia , Voriconazol/farmacologia
20.
Ophthalmic Res ; 65(2): 196-209, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34915515

RESUMO

INTRODUCTION: Corneal xenotransplantation is an effective solution for human corneal shortage. We investigated the feasibility and efficacy of different postoperative protocols on xeno-Descemet's stripping automated endothelial keratoplasty (DSAEK) grafts. METHODS: Thirty rhesus monkeys were randomly divided into three groups: control group (C) and only Descemet's membrane (DM) stripping, DSAEK 1 (D1) and DSAEK 2 (D2) groups, DM stripping followed by endothelial keratoplasty. Betamethasone 3.5 mg was subconjunctivally injected in groups control and D1 postoperatively, whereas rhesus monkeys in group D2 received topical 0.1% tacrolimus and topical steroids. All groups were evaluated by slit lamp, anterior segment optical coherence tomography, and laser scanning confocal microscopy for at least 9 months. RESULTS: Twenty-four monkeys met the inclusion criteria. Nine months after the DSAEK surgery, most corneas were transparent. Graft rejection was observed in 25% and 28.57% of the cases in group D1 and group D2 (p > 0.05), respectively. Corneal endothelium densities in DSAEK groups were 2,715.83 ± 516.20/mm2 (D1) and 2,220.00 ± 565.13/mm2 (D2) (p > 0.05). CONCLUSIONS: Xenogeneic corneal endothelial grafts can survive and function in rhesus monkey eyes for a prolonged period of time with subconjunctival steroid or topical tacrolimus and steroid treatment. Furthermore, topical drugs are more suitable for clinical use.


Assuntos
Lâmina Limitante Posterior , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior , Animais , Ceratoplastia Endotelial com Remoção da Lâmina Limitante Posterior/métodos , Endotélio Corneano , Sobrevivência de Enxerto , Xenoenxertos , Humanos , Macaca mulatta , Suínos , Tacrolimo/farmacologia , Transplante Heterólogo
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