Organ Transplant Rejection Research Articles

Recent insights how combined inhibition of immuno/proteasome subunits enables therapeutic efficacy.

The proteasome is a multicatalytic protease in the cytosol and nucleus of all eukaryotic cells that controls numerous cellular processes through regulated protein degradation. Proteasome inhibitors have significantly improved the survival of multiple myeloma patients. However, clinically approved proteasome inhibitors have failed to show efficacy against solid tumors, neither alone nor in combination with other therapies. Targeting the immunoproteasome with selective inhibitors has been therapeutically effective in preclinical models for several autoimmune diseases and colon cancer. Moreover, immunoproteasome inhibitors prevented the chronic rejection of allogeneic organ transplants. In recent years, it has become apparent that inhibition of one single active center of the proteasome is insufficient to achieve therapeutic benefits. In this review we summarize the latest insights how targeting multiple catalytically active proteasome subunits can interfere with disease progression in autoimmunity, growth of solid tumors, and allograft rejection.

The rapamycin analog Everolimus reversibly impairs male germ cell differentiation and fertility in the mouse†.

Sirolimus, also known as rapamycin, and its closely related rapamycin analog (rapalog) Everolimus inhibit "mammalian target of rapamycin complex 1" (mTORC1), whose activity is required for spermatogenesis. Everolimus is Food and Drug Administration approved for treating human patients to slow growth of aggressive cancers and preventing organ transplant rejection. Here, we test the hypothesis that rapalog inhibition of mTORC1 activity has a negative, but reversible, impact upon spermatogenesis. Juvenile (P20) or adult (P>60) mice received daily injections of sirolimus or Everolimus for 30days, and tissues were examined at completion of treatment or following a recovery period. Rapalog treatments reduced body and testis weights, testis weight/body weight ratios, cauda epididymal sperm counts, and seminal vesicle weights in animals of both ages. Following rapalog treatment, numbers of differentiating spermatogonia were reduced, with concomitant increases in the ratio of undifferentiated spermatogonia to total number of remaining germ cells. To determine if even low doses of Everolimus can inhibit spermatogenesis, an additional group of adult mice received a dose of Everolimus ∼6-fold lower than a human clinical dose used to treat cancer. In these animals, only testis weights, testis weight/body weight ratios, and tubule diameters were reduced. Return to control values following a recovery period was variable for each of the measured parameters and was duration and dose dependent. Together, these data indicate rapalogs exerted a dose-dependent restriction on overall growth of juvenile and adult mice and negative impact upon spermatogenesis that were largely reversed; following treatment cessation, males from all treatment groups were able to sire offspring.

CD19-targeted CAR regulatory T cells suppress B cell pathology without GvHD.

Regulatory T cells (Tregs) play essential roles in maintaining immunological self-tolerance and preventing autoimmunity. The adoptive transfer of antigen-specific Tregs has been expected to be a potent therapeutic method for autoimmune diseases, severe allergy, and rejection in organ transplantation. However, effective Treg therapy has not yet been established because of the difficulty in preparing a limited number of antigen-specific Tregs. Chimeric antigen receptor (CAR) T cells have been shown to be a powerful therapeutic method for treating B cell lymphomas, but application of CAR to Treg-mediated therapy has not yet been established. Here, we generated CD19-targeted CAR (CD19-CAR) Tregs from human PBMCs (hPBMCs) and optimized the fraction of the Treg source as CD4+CD25+CD127loCD45RA+CD45RO-. CD19-CAR Tregs could be expanded in vitro while maintaining Treg properties, including high expression of the latent form of TGF-β. CD19-CAR Tregs suppressed IgG antibody production and differentiation of B cells via a TGF-β-dependent mechanism. Unlike conventional CD19-CAR CD8+ T cells, CD19-CAR Tregs suppressed antibody production in immunodeficient mice that were reconstituted with hPBMCs, reducing the risk of graft-versus-host disease. Therefore, the adoptive transfer of CD19-CAR Tregs may provide a novel therapeutic method for treating autoantibody-mediated autoimmune diseases.

The Monitoring of Donor-derived Cell-free DNA in Kidney Transplantation.

Cell-free DNA (cfDNA) exists in plasma and can be measured by several techniques. It is now possible to differentiate donor-derived cfDNA (ddcfDNA) from recipient cfDNA in the plasma or urine of solid organ transplant recipients in the absence of donor and recipient genotyping. The assessment of ddcfDNA is being increasingly studied as a noninvasive means of identifying acute rejection (AR) in solid organ transplants, including subclinical AR. We herein review the literature on the correlation of ddcfDNA with AR in kidney transplantation. There have been at least 15 observational studies that have assessed ddcfDNA in urine or plasma using various methodologies with various thresholds for abnormality. Overall, elevated ddcfDNA indicates allograft injury as may occur with AR, infection, or acute tubular injury but may also be found in clinically stable patients with normal histology. Sensitivity is greater for antibody-mediated AR than for cell-mediated AR, and normal levels do not preclude significant cell-mediated rejection. Measurement of ddcfDNA is not a replacement for biopsy that remains the gold standard for diagnosing AR. Serial monitoring of stable patients may allow earlier detection of subclinical AR, but the efficacy of this approach remains to be established. Normal levels should not preclude planned protocol biopsies. There may be roles for following ddcfDNA levels to assess the adequacy of treatment of AR and to guide the intensity of immunosuppression in the individual patient. Randomized controlled trials are necessary to validate the benefit and cost-effectiveness for these various uses. No firm recommendations can be made at this time.

The suppressive effect on CD4 T cell alloresponse against endothelial HLA-DR via PD-L1 induced by anti-A/B ligation.

While donor-specific human leukocyte antigen (HLA) antibodies are a frequent cause for chronic antibody-mediated rejection in organ transplantation, this is not the case for antibodies targeting blood group antigens, as ABO-incompatible (ABO-I) organ transplantation has been associated with a favorable graft outcome. Here, we explored the role of CD4 T cell-mediated alloresponses against endothelial HLA-D-related (DR) in the presence of anti-HLA class I or anti-A/B antibodies. CD4 T cells, notably CD45RA-memory CD4 T cells, undergo extensive proliferation in response to endothelial HLA-DR. The CD4 T cell proliferative response was enhanced in the presence of anti-HLA class I, but attenuated in the presence of anti-A/B antibodies. Microarray analysis and molecular profiling demonstrated that the expression of CD274 programmed cell death ligand 1 (PD-L1) increased in response to anti-A/B ligation-mediated extracellular signal-regulated kinase (ERK) inactivation in endothelial cells that were detected even in the presence of interferon-γ stimulation. Anti-PD-1 antibody enhanced CD4 T cell proliferation, and blocked the suppressive effect of the anti-A/B antibodies. Educated CD25+ CD127- regulatory T cells (edu.Tregs ) were more effective at preventing CD4 T cell alloresponses to endothelial cells compared with naive Treg ; anti-A/B antibodies were not involved in the Treg -mediated events. Finally, amplified expression of transcript encoding PD-L1 was observed in biopsy samples from ABO-I renal transplants when compared with those from ABO-identical/compatible transplants. Taken together, our findings identified a possible factor that might prevent graft rejection and thus contribute to a favorable outcome in ABO-I renal transplantation.

PELA URGÊNCIA DE VOLTAR A “ANDAR ESPANTADO DE EXISTIR”

Inspirada pela possibilidade de começar na escola a conscientização das novas gerações, foi planeada uma intervenção para um estudo piloto que teve lugar no ano letivo 2018/19, numa escola secundária (com 3º ciclo) da cidade de Coimbra envolvendo cerca de 900 estudantes (de 1017 inscritos) a propósito da eleição para a Direção da Associação de Estudantes (DAE). Os estudantes foram divididos em dois grandes grupos para reunião de apresentação do projeto de mudança e de reflexão (primeiro o 3º ciclo, estudantes mais novos; de seguida o secundário, estudantes mais velhos e mais ligados aos processos de candidatura e esquemas de campanha).
 Da estranheza e rejeição iniciais, os estudantes perceberam as implicações da tarefa de candidatura como procedimento de apresentação de um pensamento para o coletivo, e assumiram a alteração de comportamentos. No novo ano letivo, espera-se que a apresentação de Listas candidatas decorra dentro do padrão de respeito pelos princípios de cidadania e de efetiva apresentação de ideias, e de estratégias de contacto e comunicação com os colegas de escola, potenciais eleitores. A importância do diálogo, do debate de ideias, de pensamento de cidadania construído a partir dos quadros de referência dos sujeitos é a grande conclusão deste projeto.

A survey of extracorporeal photopheresis treatment in pediatric patients in the United Kingdom.

Extracorporeal photopheresis (ECP) is a second‐line therapy in acute and chronic GVHD and solid organ transplant rejection. We report ECP use in 98 pediatric patients in seven UK centers from 2010 to 2017, the majority treated for aGVHD (73.5%). ECP was safe and well tolerated including in low body weight patients. Most patients were on multiple immunosuppressive therapies prior to ECP; 45.9% were able to reduce or stop immunosuppression with treatment. Complete or partial response was reported in almost 60%. This study supports the need to include ECP treatment data to national transplant databases to provide accurate information regarding service provision, patient outcomes, and safety.

A comprehensive review of the impact of tacrolimus intrapatient variability on clinical outcomes in kidney transplantation.

Tacrolimus (Tac) is widely used to prevent rejection and graft loss in solid organ transplantation. A limiting characteristic of Tac is the high intra and interpatient variability associated with its use. Routine therapeutic drug monitoring (TDM) is necessary to facilitate Tac management and to avoid undesirable clinical outcomes. However, whole blood trough concentrations commonly utilized in TDM are not strong predictors of the detrimental clinical outcomes of interest. Recently, researchers have focused on Tac intrapatient variability (Tac IPV) as a novel marker to better assess patient risk. Higher Tac IPV has been associated with a number of mechanisms leading to shortened graft survival. Medication nonadherence (MNA) is considered to be the primary determinant of high Tac IPV and perhaps the most modifiable risk factor. An understanding of the methodology behind Tac IPV is imperative to its recognition as an important prognostic measure and integration into clinical practice. Therapeutic interventions targeting MNA and reducing Tac IPV are crucial to improving long-term graft survival.

P0647ACUTE KIDNEY INJURY SECONDARY TO SUCROSE CONTAING INTAVENOUS IMMUNOGLOBULIN

Abstract Background and Aims Intravenous immunoglobulins (IVIG) are pooled polyvalent IgG antibodies extracted from the human plasma. While the initial indications were mainly immune deficiency states and some autoimmune diseases, the usage has been widened to include several immune mediated diseases, viral infections, and organ transplant rejection. Stabilizers in IVIG may include sugars, such as sucrose, glucose, or maltose. Sucrose in IVIG preparations may cause acute kidney injury. We report the case of a renal transplant patient who developed acute kidney injury due to sucrose nephropathy following the administration of sucrose containing IVIG. Method Four months after transplantation he was referred to our Hospital for deterioration of kidney function with eGFR (by MDRD formula) of 27ml/min. Cytomegalovirus virus (CMV) PCR turned positive (3300 copies/ml). Cyclosporine levels were high (C2: 2937 ng/ml) and hence, cyclosporine dose was adjusted. Induction therapy with Injection Ganciclovir for 2 weeks, followed by therapeutic dose of oral Valganciclovir was administered for the treatment of CMV infection. Skin examination revealed annular purple patches, suspicious of Kaposi Sarcoma, on the upper limbs. Skin biopsy confirmed the diagnosis. It was planned to give total IVIG of 2 gm/ kg in four daily divided doses. After completion of the second dose, serum creatinine increased to 370 µmol/L. He was clinically asymptomatic, euvolumic, vital signs were stable, and his urine output remained normal and his urinalysis was inactive. Results The ultrasound of the transplant kidney was normal with normal resistivity index. IVIG was stopped. He was well hydrated and underwent ultrasound guided biopsy. The graft biopsy showed acute tubular injury with flattening and vacuolation of tubular epithelial cells. Mitosis indicating tubular regeneration was seen. There was mild focal interstitial inflammation (20%) with mild lymphocytic tubulitis not amounting to graft rejection. Immunohistochemistry for C4d and polyomavirus (BKV) were both negative. The features were most consistent with sucrose induced nephropathy (Figure 1). Subsequent visits showed a decrease in BKV-PCR serum level and eventually undetected serum level of BKV-PCR at follow up about a month later. Conclusion In this paper, we presented a case of a living unrelated kidney transplant recipient who developed BKV nephropathy and developed impaired kidney function. The patient also had new onset diabetes mellitus after kidney transplantation (NODAT) but was otherwise in good general health. Treatment included sucrose containing IVIG. The patient subsequently developed acute kidney injury. The outcome was favorable with recovery of filtration rate to the baseline within 21 days without the need for dialysis. We conclude that the administration of sucrose containing IVIG may lead to acute kidney injury. We recommend the use of sucrose-free IVIG whenever possible. In all cases, caution is required when administrating IVIG.

Experimental modeling of desensitization: What have we learned about preventing AMR?

During the past 5 decades, short-term outcomes in kidney transplant have significantly improved, in large part due to reduced rates and severity of acute rejection. Development of better immunosuppressive maintenance agents, as well as new induction therapies, helped make these advances. Nonhuman primate models provided a rigorous testing platform to evaluate candidate biologics during this process. However, antibody-mediated rejection remains a major cause of late failure of kidney allografts despite advances made in pharmacologic immunosuppression and strategies developed to facilitate improved donor-recipient matching. Our laboratory has been actively working to develop strategies to prevent and treat antibody-mediated rejection and immunologic sensitization in organ transplant, relying largely on a nonhuman primate model of kidney transplant. In this review, we will cover outcomes achieved by managing antibody-mediated rejection or sensitization in nonhuman primate models and discuss promises, limitations, and future directions for this model.

1688-P: Peak Insulin Drip Rate Associated with Decreased Infections Post–Heart Transplant

Hyperglycemia is associated with infection and rejection in post-solid organ transplant (Tx) patients (pts), but little is known about peak insulin requirements and morbidity. We previously reported an unexpected finding in a retrospective cohort study of 207 liver Tx pts that evaluated the relationship to peak IV insulin requirements and subsequent morbidity. Pts were separated into quartiles (Q) by peak insulin drip rate [U/hr]. In that study, pts in Q4 of insulin requirements had significantly reduced infections out to 6 months post-Tx compared to Q1-3. In the present study, we sought to replicate these findings in 188 heart Tx pts. Peak drip rate Qs are shown in the Figure below. Compared to Q1-3, Q4 pts were similar in age (53 ± 10.7 vs. 50.3 ± 9.4) and BMI (30.4 ± 6.4 vs. 29.2 ± 6.8), but more had DM (42.6% vs. 38.3%, p=0.003). Q4 pts had significantly fewer infections (19.1% vs. 53.9%, p<0.000001) up to 6 mos but similar rejections (4.3% vs. 11.3%, p=0.15). Also, we showed that avg blood glucose levels were not significantly different between Q1-3 and Q4 (p = 1.03). These data in heart Tx pts confirm our findings in liver Tx pts that pts requiring higher insulin doses for glycemic control have decreased infections out to 6 months post Tx. We hypothesize that pts able to mount a greater counterregulatory hormone response to surgery may be healthier and able to better respond to infection. Alternatively, higher insulin doses could be protective. Disclosure G. Upadrasta: None. S.J. Kumar: None. A. Wallia: Research Support; Self; Eli Lilly and Company, Novo Nordisk Inc., UnitedHealth Group. M. Molitch: Advisory Panel; Self; Janssen Pharmaceuticals, Inc., Merck & Co., Inc., Pfizer Inc. Consultant; Self; AstraZeneca. Research Support; Self; Novartis Pharmaceuticals Corporation, Novo Nordisk Inc.

Analysis of Immunosuppressant Drugs in Whole Blood by Liquid Chromatography–Tandem Mass Spectrometry (LC‐MS/MS)

Immunosuppressant medications help suppress the immune system response through inhibition of various checkpoints in the regulatory biochemical pathway. This is useful in prevention of organ rejection in transplantation or in the treatment of autoimmune diseases such as lupus or rheumatoid arthritis. Quantification of immunosuppressive drugs in blood is needed clinically for optimization of treatment and to avoid toxicity or unwanted side effects. Here, we describe a quantitative method to determine the concentration of cyclosprine A, tacrolimus, sirolimus, and everolimus in whole blood. This method has been used for many years clinically to support patient care. © 2020 by John Wiley & Sons, Inc.

Efficacy and safety of chemical thromboprophylaxis in renal transplantation – A systematic review

IntroductionThe benefit of administering chemical thromboprophylaxis to chronic kidney disease patients undergoing renal transplantation is unclear and no previous systematic review has addressed this as reflected by variations in national guidelines. MethodsA literature search was performed using MEDLINE, Embase, Cochrane, CINAHL, World Health Organisation (WHO) International Clinical Trials Registry Platform and ClinicalTrials.gov databases to December 2019. Studies included participants undergoing renal transplantation only with no contra-indication to thromboprophylaxis, no history/clinical suspicion of acute organ rejection and those describing a form of chemical thromboprophylaxis intervention compared with another form, no intervention or placebo. ResultsThirteen studies with 1600 patients were included. There was wide variation concerning type of thromboprophylaxis, time of onset, dosing and duration. Reports of symptomatic/asymptomatic venous thromboembolism and mortality were limited. Seven studies reported on renal allograft thrombosis. When comparing thromboprophylaxis to no intervention, there was no evidence of difference for thrombosis risk (risk ratio 0.2; [95% CI 0.01–4.63]), however all studies were underpowered to answer this question. Six studies reported on major bleeding but type of intervention, timing of onset and duration of thromboprophylaxis varied significantly, making it difficult to pool data for further analysis. ConclusionThere is insufficient evidence to advise on efficacy and safety of chemical thromboprophylaxis in patients undergoing renal transplantation or to determine whether one chemical thromboprophylaxis is better than another thromboprophylaxis.

The Role of Innate Lymphoid Cells in Immune Regulation and Fetal Protection During Allogeneic Pregnancies

Abstract Maternal immune tolerance against paternal alloreactive antigens expressed by the fetus is essential in the development of successful pregnancies. Disturbances of immunoregulatory mechanisms by genetic modifications or viral infections results in aberrant placental development, intrauterine growth restriction (IUGR), and fetal loss. Mechanisms of immune regulation during pregnancy has not been fully elucidated. Our lab and others were the first to uncover a novel subset of regulatory innate lymphoid cells (ILCregs) that suppressed immune responses in chronic viral infections, organ transplant rejection, and cancer. In this study, we identified a subset of decidual NK1.1+ ILC1s in healthy murine pregnancies that expressed surface markers and intracellular cytokines reminiscent of an ILCreg phenotype. Interestingly, these characteristics were more abundant early in gestation compared to mid-late gestation. Furthermore, depletion of NK1.1+ ILC1s using neutralizing antibodies induced fetal destruction in allogeneic (C57BL/6J female X BALB/c male), but not in syngeneic pregnancies (C57BL/6J female X C57BL/6J male). This highlights the importance of allogenicity in fetal destruction in the absence of NK1.1+ ILC1s. Decidual ILC1s have been shown to promote trophoblast invasion, placental angiogenesis, and in utero fetal growth during pregnancy. However, we are one of the first groups to thoroughly investigate potential immunosuppressive role of decidual ILC1s to prevent immune-mediated fetal destruction. Further elucidation of immune tolerance mechanisms during pregnancy may uncover novel therapeutic targets for pregnancy-related complications such as preeclampsia and spontaneous miscarriages.

MAPC® cell therapy enhances the ex-vivo expansion of polyclonal, regulatory T cells

Background & Aim Regulatory T (Treg) cells are a naturally occurring population of lymphocytes that maintain immune homeostasis and resolve excessive immune activation. They act through a spectrum of cell-mediated and soluble mechanisms. Preclinical data in humanized mouse models has firmly established that adoptive transfer or in vivo generation of Treg cells can remedy autoimmunity, GVHD and solid organ transplant rejection; a few clinical trials have already demonstrated safety and suggest some limited efficacy. Therefore, clinical adoptive Treg cell therapy (the isolation, ex vivo expansion and re-infusion of Treg cells) has been heralded as a promising strategy for the treatment of auto-immune and inflammatory disorders. As such, a living drug cell therapy may have greater specificity and more complex therapeutic benefits than conventional immunosuppressive drugs and could potentially cure autoimmune diseases by restoring the immune tolerance. However, several major roadblocks in the widespread clinical implementation of Treg therapy remain, mainly the low number of Treg cells that can be harvested from peripheral blood (PB) and the limited expansion potential, while maintaining purity and function. Methods, Results & Conclusion ReGenesys has developed a platform technology for the expansion of Treg cells derived from PB while fully maintaining their functional characteristics. This platform is based on the use of our proprietary MAPC stromal stem cell therapy which consists of non-hematopoietic stem cells derived from bone marrow that secrete various growth factors and have potent immunomodulatory effects. When CD4+CD25+127low-sorted Treg cells from PB were expanded on MAPC cells, a robust and high Treg cell yield was obtained, reaching the critical threshold of clinical batch production (1 billion cells) in all donors. Furthermore, a higher purity was observed (less potential CD8+ T cell contamination) and a specific signature was seen in terms of phenotype (low skin-homing/ high gut-homing marker expression). The immunosuppressive function was confirmed in both in vitro and in vivo models. Finally, successful Treg expansions have also been performed from PB of Crohn's Disease patients.

Currently Used Drugs for Prevention and Treatment of Acute Organ Rejection

Currently Used Drugs for Prevention and Treatment of Acute Organ Rejection

Linking indirect effects of cytomegalovirus in transplantation to modulation of monocyte innate immune function.

Cytomegalovirus (CMV) is an important cause of morbidity and mortality in the immunocompromised host. In transplant recipients, a variety of clinically important "indirect effects" are attributed to immune modulation by CMV, including increased mortality from fungal disease, allograft dysfunction and rejection in solid organ transplantation, and graft-versus-host-disease in stem cell transplantation. Monocytes, key cellular targets of CMV, are permissive to primary, latent and reactivated CMV infection. Here, pairing unbiased bulk and single cell transcriptomics with functional analyses we demonstrate that human monocytes infected with CMV do not effectively phagocytose fungal pathogens, a functional deficit which occurs with decreased expression of fungal recognition receptors. Simultaneously, CMV-infected monocytes upregulate antiviral, pro-inflammatory chemokine, and inflammasome responses associated with allograft rejection and graft-versus-host disease. Our study demonstrates that CMV modulates both immunosuppressive and immunostimulatory monocyte phenotypes, explaining in part, its paradoxical "indirect effects" in transplantation. These data could provide innate immune targets for the stratification and treatment of CMV disease.

Rapamycin restrains platelet procoagulant responses via FKBP-mediated protection of mitochondrial integrity

Background and PurposeRapamycin 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 ApproachThe 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 ResultsRapamycin 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 ImplicationsRapamycin 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.

Effects of Different Tacrolimus Doses on Adipose-Derived Stem Cells.

Tacrolimus is currently one of the most commonly used immunosuppressant agents to prevent rejection in organ transplant. Although modern immunosuppressive agents have been successful, rejection cannot be completely prevented. Therefore, in organ transplant research, additional treatment methods are being investigated, with the most important one being stem cell therapy. In addition to tacrolimus therapy, stem cell therapy is now clinically applicable, with frequency of concomitant use seeming to expand in the future. In this study, the effects of tacrolimus on stem cells were investigated. Adipose-derived stem cells were treated with tacrolimus at different doses and time points. We analyzed the effects of changes in stem cell proliferation using MTT analysis. Sox2, Oct3/4, and Nanog protein levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to measure stem cell pluripotency capabilities. Our observations showed that tacrolimus causes changes in stem cell proliferation and pluripotency, with changes dependent on time and dose. The dose of tacrolimus and the time of application of stem cells are important in the planning of stem cell therapy in organ transplant.

Sensitive UHPLC-MS/MS method for the determination of tacrolimus in minipig whole blood.

Background: Tacrolimus, a potent immunosuppressant drug widely used systemically to reduce the risk of organ rejection in transplants, has been repositioned for topical treatment of atopic dermatitis. Results & methodology: This work describes the optimization of a new method for the determination of tacrolimus in whole blood after topical administration. Sample treatment consisted of an automated procedure based on protein precipitation followed by solid-phase extraction. The present method showed good performance with quantitation limits of 10 pg ml-1 and intra- and interday precision and accuracy lower than 15 and 10%, respectively. Conclusion: A new highly sensitive UHPLC-MS/MS method has been developed enabling a better characterization of the minipig blood plasma pharmacokinetic behavior of tacrolimus after topical administration.