Primary hyperoxaluria: Long-term outcomes of isolated kidney versus simultaneous liver/kidney transplant.

OBJECTIVES: To compare long-term transplant outcomes (organ rejection and retransplant) of simultaneous liver/kidney transplant (SLK) versus isolated kidney transplant (IK) for patients with primary hyperoxaluria (PH). METHODS: The Rare Kidney Stone Consortium PH registry was queried to identify patients with PH who underwent SLK or IK from 1999 to 2021. Patient characteristics and long-term transplant outcomes were abstracted and analyzed. Statistical comparisons were performed with Kaplan-Meier plots and Cox proportional hazards models. RESULTS: We identified 250 patients with PH, of whom 35 received care at Mayo Clinic and underwent SLK or IK. Patients who underwent SLK as their index transplant had lower odds of kidney rejection than did those who underwent IK (hazard ratio [HR], 0.29; 95% confidence interval [CI], 0.08-0.99; p = .048). The immunoprotective effect of concomitant liver and kidney transplant appeared to enhance outcomes for patients with PH. Additionally, the odds of retransplant were significantly lower for patients who underwent SLK as their index transplant than for those who underwent IK (HR, 0.08; 95% CI, 0.02-0.42; p = .003). Of five patients who underwent IK and had maintained graft function for at least 5 years after transplant, three (60%) had documented vitamin B6 responsiveness. CONCLUSIONS: Patients with PH who underwent SLK had a lower risk of kidney rejection and retransplant than those who underwent IK. Accurate genetic assessment for vitamin B6 responsiveness may optimize IK allocation. Novel therapeutics, such as lumasiran, have been introduced as promising agents for the management of PH.

The Importance of the FUT2 rs602662 Polymorphism in the Risk of Cardiovascular Complications in Patients after Kidney Transplantation.

The FUT2 gene encodes an enzyme called α-1,2-fucosyltransferase, which is involved in the formation of blood group antigens AB0(H) and is also involved in the processes of vitamin B12 absorption and its transport between cells. FUT2 gene polymorphisms are associated with vitamin B12 levels in the body. Vitamin B12 deficiency associated with hyperhomocysteinemia is a major risk factor for cardiovascular diseases (CVDs), which are one of the main causes of death in patients after kidney transplantation. The aim of our study was to determine the impact of the rs602662 (G>A) polymorphism of the FUT2 gene on the functionality of transplanted kidneys and the risk of CVD in patients after kidney transplantation. The study included 402 patients treated with immunosuppression (183 patients taking cyclosporine (CsA) and 219 patients taking tacrolimus (TAC)). The analysis of the FUT2 rs602662 (G>A) polymorphism was performed using real-time PCR. Patients with CsA were more likely to be underweight (1.64% vs. 0.91%) and obese (27.87% vs. 15.98%), while those taking TAC were more likely to be of normal weight (39.27%) or overweight (43.84%). No statistically significant differences were observed comparing the mean blood pressure, both systolic and diastolic. The renal profile showed a higher median urea nitrogen concentration in patients with CsA (26.45 mg/dL (20.60-35.40) vs. 22.95 mg/dL (17.60-33.30), p = 0.004). The observed frequency of rs602662 alleles of the FUT2 gene was similar in the analyzed groups. The A allele was present in 43.7% of patients with CsA and 41.1% of those taking TAC (OR = 0.898; 95% CI: 0.678-1.189; p = 0.453). In the group with CsA, the GG genotype was present in 32.2% of patients, the GA in 48.1% and the AA in 19.7%. A similar distribution was obtained in the TAC group: GG-33.8%, GA-50.2%, and AA-16.0%. An association of genotypes containing the G allele with a higher incidence of hypertension was observed. The G allele was present in 65% of people with hypertension and in 56% of patients with normal blood pressure (p = 0.036). Moreover, the evaluation of the renal parameters showed no effect of the FUT2 polymorphism on the risk of organ rejection because the levels of creatinine, eGFR, potassium, and urea nitrogen were prognostic of successful transplantation. Our results suggest that the rs6022662 FUT2 polymorphism may influence the risk of cardiovascular diseases.

Advancing kidney xenotransplantation with anesthesia and surgery - bridging preclinical and clinical frontiers challenges and prospects.

Xenotransplantation is emerging as a vital solution to the critical shortage of organs available for transplantation, significantly propelled by advancements in genetic engineering and the development of sophisticated immunosuppressive treatments. Specifically, the transplantation of kidneys from genetically engineered pigs into human patients has made significant progress, offering a potential clinical solution to the shortage of human kidney supply. Recent trials involving the transplantation of these modified porcine kidneys into deceased human bodies have underscored the practicality of this approach, advancing the field towards potential clinical applications. However, numerous challenges remain, especially in the domains of identifying suitable donor-recipient matches and formulating effective immunosuppressive protocols crucial for transplant success. Critical to advancing xenotransplantation into clinical settings are the nuanced considerations of anesthesia and surgical practices required for these complex procedures. The precise genetic modification of porcine kidneys marks a significant leap in addressing the biological and immunological hurdles that have traditionally challenged xenotransplantation. Yet, the success of these transplants hinges on the process of meticulously matching these organs with human recipients, which demands thorough understanding of immunological compatibility, the risk of organ rejection, and the prevention of zoonotic disease transmission. In parallel, the development and optimization of immunosuppressive protocols are imperative to mitigate rejection risks while minimizing side effects, necessitating innovative approaches in both pharmacology and clinical practices. Furthermore, the post-operative care of recipients, encompassing vigilant monitoring for signs of organ rejection, infectious disease surveillance, and psychological support, is crucial for ensuring post-transplant life quality. This comprehensive care highlights the importance of a multidisciplinary approach involving transplant surgeons, anesthesiologists, immunologists, infectiologists and psychiatrists. The integration of anesthesia and surgical expertise is particularly vital, ensuring the best possible outcomes of those patients undergoing these novel transplants, through safe procedural practices. As xenotransplantation moving closer to clinical reality, establishing consensus guidelines on various aspects, including donor-recipient selection, immunosuppression, as well as surgical and anesthetic management of these transplants, is essential. Addressing these challenges through rigorous research and collective collaboration will be the key, not only to navigate the ethical, medical, and logistical complexities of introducing kidney xenotransplantation into mainstream clinical practice, but also itself marks a new era in organ transplantation.

Prediction of Outcomes After Heart Transplantation in Pediatric Patients Using National Registry Data: Evaluation of Machine Learning Approaches.

BACKGROUND: The prediction of posttransplant health outcomes for pediatric heart transplantation is critical for risk stratification and high-quality posttransplant care. OBJECTIVE: The purpose of this study was to examine the use of machine learning (ML) models to predict rejection and mortality for pediatric heart transplant recipients. METHODS: Various ML models were used to predict rejection and mortality at 1, 3, and 5 years after transplantation in pediatric heart transplant recipients using United Network for Organ Sharing data from 1987 to 2019. The variables used for predicting posttransplant outcomes included donor and recipient as well as medical and social factors. We evaluated 7 ML models-extreme gradient boosting (XGBoost), logistic regression, support vector machine, random forest (RF), stochastic gradient descent, multilayer perceptron, and adaptive boosting (AdaBoost)-as well as a deep learning model with 2 hidden layers with 100 neurons and a rectified linear unit (ReLU) activation function followed by batch normalization for each and a classification head with a softmax activation function. We used 10-fold cross-validation to evaluate model performance. Shapley additive explanations (SHAP) values were calculated to estimate the importance of each variable for prediction. RESULTS: RF and AdaBoost models were the best-performing algorithms for different prediction windows across outcomes. RF outperformed other ML algorithms in predicting 5 of the 6 outcomes (area under the receiver operating characteristic curve [AUROC] 0.664 and 0.706 for 1-year and 3-year rejection, respectively, and AUROC 0.697, 0.758, and 0.763 for 1-year, 3-year, and 5-year mortality, respectively). AdaBoost achieved the best performance for prediction of 5-year rejection (AUROC 0.705). CONCLUSIONS: This study demonstrates the comparative utility of ML approaches for modeling posttransplant health outcomes using registry data. ML approaches can identify unique risk factors and their complex relationship with outcomes, thereby identifying patients considered to be at risk and informing the transplant community about the potential of these innovative approaches to improve pediatric care after heart transplantation. Future studies are required to translate the information derived from prediction models to optimize counseling, clinical care, and decision-making within pediatric organ transplant centers.

Why Cell-Free DNA Can Be a "Game Changer" for Lung Allograft Monitoring for Rejection and Infection.

Purpose of Review: Although there has been improvement in short-term clinical outcomes for patients following lung transplant (LT), advances have not translated into longer-term allograft survival. Furthermore, invasive biopsies are still standard of practice for monitoring LT recipients for allograft injury. We review the relevant literature supporting the role of using plasma donor-derived cell-free DNA (dd-cfDNA) as a non-invasive biomarker for LT allograft injury surveillance and discuss future research directions. Recent Findings: Accumulating data has demonstrated that dd-cfDNA is associated with molecular and cellular injury due to acute (cellular and antibody-mediated) rejection, chronic lung allograft dysfunction, and relevant infectious pathogens. Strong performance in distinguishing rejection and allograft injury from stable patients has set the stage for clinical trials to assess dd-cfDNA utility for surveillance of LT patients. Research investigating the potential role of dd-cfDNA methylation signatures to map injured tissue and cell-free DNA in detecting allograft injury-related pathogens is ongoing. Summary: There is an amassed breadth of clinical data to support a role for dd-cfDNA in monitoring rejection and other forms of allograft injury. Rigorously designed, robust clinical trials that encompass the diversity in patient demographics are paramount to furthering our understanding and adoption of plasma dd-cfDNA for surveillance of lung allograft health.

Histopathological examination of the ectocervical biopsy in non-transplanted uteri: A study contributing to the provisional scoring system of subclinical graft rejection after uterus transplantation.

INTRODUCTION: Uterus transplantation is a causal treatment for absolute uterine factor infertility. Assessing rejection signs using a histopathological examination of the ectocervical biopsy from the transplanted uterus is common practice in all human uterus transplants worldwide to date. A provisional scoring system was used for the histopathological assessment of subclinical rejection signs in uterus recipients. Here we hypothesized that histopathological and immunohistochemical findings in the normal uteri would differ from the borderline category of subclinical rejection in uterine transplants. MATERIAL AND METHODS: This prospective observational study included ectocervical biopsies of 54 women who underwent hysterectomy for benign reasons. All biopsy samples were assessed histopathologically and immunohistochemically. RESULTS: Most of the ectocervical biopsies showed clustering lymphocytic infiltrates affecting the stromal-epithelial interface with the epithelial influx of lymphocytes, primarily CD45RO-positive activated T-cells with CD8 T-lymphocyte predominance. CD4-positive T-lymphocytes and B-cells were rarely detected in the ectocervix. These morphological findings and immunoprofiles of lymphocytic populations overlapped with the so-called borderline changes defined in the provisional scoring system for rejection in the transplanted uteri. The immunoprofiles of ectocervical and endocervical lymphocytic populations differed, with strikingly prominent B-cell participation in the endocervix vs the rare detection of B-cells in the ectocervix. CONCLUSIONS: The histopathological and immunohistochemical findings in the uteri of premenopausal women were similar to the borderline category of the currently used provisional scoring system of subclinical uterine rejection utilized in all uterine transplant studies. However, future similar studies are required to validate our findings.

Educational Case: Kidney Transplant Rejection.

The following fictional case is intended as a learning tool within the Pathology Competencies for Medical Education (PCME), a set of national standards for teaching pathology. These are divided into three basic competencies: Disease Mechanisms and Processes, Organ System Pathology, and Diagnostic Medicine and Therapeutic Pathology. For additional information, and a full list of learning objectives for all three competencies, see http://journals.sagepub.com/doi/10.1177/2374289517715040.1.

[Fibrotic remodeling of the lung following lung and stem-cell transplantation]. / Fibrotischer Lungenparenchymumbau nach Lungen- und Stammzelltransplantation.

Transplantation of solid organs and hematopoietic stem cells represents an important therapeutic option for a variety of end-stage pulmonary diseases, aggressive hematopoietic neoplasms, or severe immunodeficiencies. Although the overall survival following transplantation has generally improved over recent decades, long-time survival of lung and stem-cell transplant recipients is still alarmingly low with an average 5­year survival rate of only 50-60%. Chronic allo-immunoreactions in general and pulmonary allo-immunoreactions with subsequent fibrosis in particular are major reasons for this poor outcome. Comparable patterns of fibrotic lung remodeling are observed following both lung and hematopoietic stem-cell transplantation. Besides the meanwhile well-established obliterative and functionally obstructive remodeling of the small airways - obliterative bronchiolitis - a specific restrictive subform of fibrosis, namely alveolar fibroelastosis, has been identified. Despite their crucial impact on patient outcome, both entities can be very challenging to detect by conventional histopathological analysis. Their underlying mechanisms are considered overreaching aberrant repair attempts to acute lung injuries with overactivation of (myo-) fibroblasts and excessive and irreversible deposition of extracellular matrix. Of note, the underlying molecular mechanisms are widely divergent between these two morphological entities and are independent of the underlying clinical setting.Further comprehensive investigations of these fibrotic alterations are key to the development of much-needed predictive diagnostics and curative concepts, considering the high mortality of pulmonary fibrosis following transplantation.

Predisposing factors for late mortality in heart transplant patients.

BACKGROUND: Because of the growing prevalence of terminal heart failure on the one hand and organ shortage on the other hand, an optimal care of heart transplant recipients based on the knowledge of potential risk factors not only early, but also in a long-term course after heart transplantation is of great importance. Therefore, the aim of the present study was to identify predisposing factors for late mortality in this patient collective. METHODS: Data from long-term heart transplant patients collected during follow-up visits in the current center were retrospectively analyzed. Clinical, laboratory, including immune monitoring and apparative examination results were studied with regard to all-cause mortality. RESULTS: One hundred and seventy-two patients after heart transplantation (mean: 13.2 ± 6.4 years) were divided into two groups: survivors (n = 133) and non-survivors (n = 39). In comparison with survivors, non-survivors were characterized by significantly more pronounced renal insufficiency with more frequent dialysis, anemia and worse functional status. Additionally, non-survivors obtained hearts from relevantly more obese donors. In a multivariate Cox regression analysis the following parameters were shown to be independent risk factors for increased mortality: CD4 percentage < 42%, C-reactive protein ≥ 0.5 mg/dL, presence of rejections requiring therapies in the past, onset of cardiac allograft vasculopathy < 5 years following heart transplantation and no use of beta-blockers. CONCLUSIONS: Low CD4+ cell percentages, sustained inflammation, relevant organ rejections, early onset of transplant vasculopathy and no use of beta-blockers are risk factors for higher mortality in a long-term follow-up after heart transplantation.

Gastroenterological complications in kidney transplant patients.

Kidney transplantation is the surgical operation by which one of the two original kidneys is replaced with another healthy one donated by a compatible individual. In most cases, donors are recently deceased. There is the possibility of withdrawing a kidney from a consenting living subject. Usually, living donors are direct family members, but they could be volunteers completely unrelated to the recipient. A much-feared complication in case of kidney transplantation is the appearance of infections. These tend to arise due to immune-suppressor drugs administered as anti-rejection therapy. In this review, we describe the gastrointestinal complications that can occur in subjects undergoing renal transplantation associated with secondary pathogenic microorganisms or due to mechanical injury during surgery or to metabolic or organic toxicity correlated to anti-rejection therapy. Some of these complications may compromise the quality of life or pose a significant risk of mortality; fortunately, many of them can be prevented and treated without the stopping the immunosuppression, thus avoiding the patient being exposed to the risk of rejection episodes.

Effect of Influenza Vaccination Inducing Antibody Mediated Rejection in Solid Organ Transplant Recipients.

Introduction: Our goal was to study whether influenza vaccination induced antibody mediated rejection in a large cohort of solid organ transplant recipients (SOTR). Methods: Serum anti-Human Leukocyte Antigen (HLA) antibodies were determined using class I and class II antibody-coated latex beads (FlowPRATM Screening Test) by flow cytometry. Anti-HLA antibody specificity was determined using the single-antigen bead flow cytometry (SAFC) assay and assignation of donor specific antibodies (DSA) was performed by virtual-crossmatch. Results: We studied a cohort of 490 SOTR that received an influenza vaccination from 2009 to 2013: 110 (22.4%) received the pandemic adjuvanted vaccine, 59 (12%) within the first 6 months post-transplantation, 185 (37.7%) more than 6 months after transplantation and 136 (27.7%) received two vaccination doses. Overall, no differences of anti-HLA antibodies were found after immunization in patients that received the adjuvanted vaccine, within the first 6 months post-transplantation, or based on the type of organ transplanted. However, the second immunization dose increased the percentage of patients positive for anti-HLA class I significantly compared with patients with one dose (14.6% vs. 3.8%; P = 0.003). Patients with pre-existing antibodies before vaccination (15.7% for anti-HLA class I and 15.9% for class II) did not increase reactivity after immunization. A group of 75 (14.4%) patients developed de novo anti-HLA antibodies, however, only 5 (1.02%) of them were DSA, and none experienced allograft rejection. Only two (0.4%) patients were diagnosed with graft rejection with favorable outcomes and neither of them developed DSA. Conclusion: Our results suggest that influenza vaccination is not associated with graft rejection in this cohort of SOTR.

Prognosis of heart transplant patients in Mashhad University of Medical Sciences.

INTRODUCTION: Heart transplant is the ultimate treatment for patients with end-stage heart failure. AIM: To assess 50 heart transplant patients for underlying diseases, transplantation outcome and mortality rate during a 5-year follow-up program. MATERIAL AND METHODS: Fifty heart transplant patients who underwent heart transplantation from 2012 to 2017 were assessed for underlying diseases, organ rejection, duration of hospitalization, extubation time, cardiac output and survival. Biopsy samples were obtained after surgery for evaluation of rejection. RESULTS: Dilated cardiomyopathy (DCM) and ischemic cardiomyopathy (ICM) were the most common underlying diseases with prevalence of 56% and 12%, respectively. Significant improvement in ejection fraction was observed following heart transplant. Minimum and maximum extubation and hospitalization times were 3-408 hours and 1-51 days, respectively. Organ rejection evaluation 10 days after heart transplantation revealed that 50% of patients did not show any rejection while 10% had severe rejection. At 30 days post-operatively the number of patients with grade III rejection decreased to 2% while 56% of patients had no sign of rejection. The 5-year survival rate was 66% while infection and arrhythmia were the most common causes of death. CONCLUSIONS: DCM and ICM are considered the most prevalent underlying diseases in heart transplant candidates. Ejection fraction reached normal ranges following transplant, which provides good quality of life. Low incidence of severe acute rejection demonstrates the effectiveness of our immunosuppressive therapy. In the cases of increased rejection, the patient's immunosuppressive regimen was re-assessed accordingly.

Development and validation of a combined enzymatic-digestion/mass spectrometry assay for Tacrolimus quantitation in cardiac biopsies.

Recent studies report strategies for analysing immunosuppressive drugs in brain, liver and renal tissue, mostly in animals: we developed and validated a two steps combined enzymatic digestion/mass spectrometry assay to quantify Tacrolimus (TAC) in heart biopsies. Our aims were to avoid sample loss and sample contamination during the laboratory preparation, and to limit matrix effects in the electrospray ionization source (ESI) of the mass spectrometer. Enzymatic tissue digestion followed by a liquid-liquid drug extraction in the same vial of reaction allowed us to reach both our aims. The assay was assessed for selectivity, matrix effect, linearity, Lower Limit of Quantification (LLOQ) and Detection (LOD), accuracy and precision, according to the "Guideline on Bioanalytical Method Validation (EMA). A stable isotopically labelled (SIL) analogue (13CD2-TAC) was used as internal standard. The chromatographic separation of the analyte took 6 min. The observed linear range of quantification was 0.0162-0.520 ng in terms of TAC added to the biopsies (by 50 µL of the corresponding working solutions). The limit of detection and the lower limit of quantification (LLOQ) were 0.008 and 0.0162 ng, respectively. Both the mobile phases contained ammonium acetate and formic acid that promote the formation of ammoniated precursor ions that can be easily fragmented ([M + NH4]+, TAC m/z 821.3; 13CD2-TAC m/z 824.3). The calibration curves were generated by plotting analyte-to-internal standard peak area ratios versus TAC amount (ng) added to the biopsies, and using a weighted (1/x) linear regression. Curves were not forced to pass through the origin. Swine hearts were employed as blank matrix for all the analytical method validation procedures but, after approval by the ethics committee (by "Fondazione IRCCS Policlinico San Matteo": Protocol 20190032933), TAC was also quantified in endomyocardial biopsies from informed and consenting heart transplant patients. The study was funded by Fondazione IRCCS Policlinico San Matteo (RC08017617), as a part of the clinical studies on the maintenance of immunosuppressive therapy in cardiac transplant patients. Tacrolimus concentrations in patients biopsies were expressed as ratio between the detected amount of TAC (ng) in the tissue and the weight of the tissue itself (mg).

Skin Transplantation and Lymphoid Organ Analysis in Mice.

Skin transplantation in mice is an important procedure to evaluate immune responses generated against heterologous grafts, especially given its highly immunogenic nature. In fact, skin is one of the most challenging organs in terms of allograft retention. In this protocol, we provide a detailed procedure for skin grafting using the tail skin as donor organ that is grafted on the dorsal site of thoracic cage in a recipient mouse. We also provide protocols for the systematic analysis of lymphoid organ analysis in transplanted mice. Together these protocols may be valuable for evaluation of parameters that affect skin grafting, including genetic factors, immune cell activation as well as the analysis of compounds that may be useful in allowing graft tolerance.

Mast cell-mediated mechanistic pathways in organ transplantation.

Adaptive immunity has gained importance in transplant immunology for years, based on models in which T-cells orchestrate the immune responses during rejection. Most recently, researches revealed that innate immune cells, including mast cells (MCs) also play a pivotal role in allograft rejection. MC mediated immunoregulatory responses influence the innate and adaptive immune responses. Their capability to produce an array of both pro-inflammatory and anti-inflammatory mediators, expressing a wide range of costimulatory molecules in addition to acting as antigen-presenting cells (APCs), make them effective immune cells far beyond their classical role as primary orchestrator cells of allergy. Activated regulatory Tcells (Treg) cells contribute to MC recruitment into grafts by releasing interleukin (IL)-9. Tregs are capable of stabilizing MCs and suppressing IgE mediated degranulation through interaction of Treg expressing OX40 with MCs expressing OX40L. MCs in turn release transforming growth factor (TGF)-ß and IL-10 which possess suppressive properties. Thus, these cells can suppress the proliferation of T-cells and support the generation of Tregs. MCs in addition to orchestrating immune responses in grafts by cell-to-cell interactions with variety of immune cells, cause histologic changes, mainly fibrosis by releasing mediators such as histamine, fibroblast growth factor-2 (FGF-2), TGF-ß, chymase, and cathepsin G. The role of MCs in transplant rejection remains controversial. The accumulation of MCs in rejected grafts suggests that they play a role in preventing graft tolerance, and contribute to the progression of chronic rejection of allografts. However, high expression of MC-related gene products in tolerant grafts and their known interaction with Tregs on the other hand, support the notion that they are an integral component in achieving peripheral tolerance.

Engagement with Medication Among Kidney Transplant Recipients - A Descriptive Cross-Sectional Study.

The aim of this study was to determine the level of engagement with medication among kidney transplant recipients in South Africa. A descriptive cross-sectional study design with random sampling was used. Data were collected through structured face-to-face interviews. Total engagement with medication scores among kidney transplant recipients ranged from 60% to 100%, with a mean score of 80%. Although over half of the participants (55%) scored above the mean, only 19% had high levels of engagement with medication. Engagement with medication was significantly associated with race (Chi-square value = 66.225, df=42, P=0.010) and years post-kidney transplantation (Chi-square value = 415.423, df=364, P=0.032).

Human Immunodeficiency Virus Organ Transplantation.

Human immunodeficiency virus (HIV) has become a chronic disease with a near normal life span resulting in increased risk of organ failure. HIV organ transplantation is a proven and accepted intervention in appropriately selected cases. HIV-positive organ transplantation into HIV-positive recipients is in its nascent stages. Hepatitis C virus, high rates of organ rejection, and immune dysregulation are significant remaining barriers to overcome. This article provides an overview of the transplantation needs in the HIV population focusing on kidney and liver transplants.

Development and validation of a liquid chromatography-mass spectrometric assay for simultaneous determination of tacrolimus and 13-O-desmethyl tacrolimus in rat kidney tissue.

A sensitive and robust LC-MS/MS method has been developed and validated to determine the concentrations of tacrolimus and its major metabolite 13-O-desmethyl tacrolimus (13-ODMT) in kidney tissue from rats who received tacrolimus intra-peritoneally at doses of 0.5mg/kg and 2mg/kg. The samples were prepared by a liquid-liquid extraction procedure using ethyl ether as the extraction solvent and ascomycin as the internal standard. Chromatographic separation was achieved using Phenomenex Kinetex column (2.6µm C18 100Å, 100×2.1mm, Phenomenex, Torrance CA) and a gradient mobile phase of water and methanol-acetonitrile (50:50, v/v) both containing 0.1% formic acid. The limit of quantification was 0.25ng/ml and the calibration curves covered a concentration range from 0.25 to 50ng/ml. Intra-and inter-assay precision and accuracy for both tacrolimus and 13-ODMT were all within FDA guidelines for bioanalysis. Extraction efficiency for tacrolimus ranged from 67.00 to 74.90% and from 66.70 to 78.40% for 13-ODMT. Several challenges interfering with the performance of the method such as phospholipid build-up have also been addressed. Kidney tissue samples from six rats receiving either 0.5 or 2mg/kg dose were analyzed and resulted in a median concentration of 11.54 and 0.72ng/ml for tacrolimus and 13-ODMT, respectively, for the lower dose level, and a median concentration of 8.89ng/ml and 1.50ng/ml for tacrolimus and 13-ODMT, respectively, at the higher dose level.

Evaluation of the State of Transplanted Liver Health by Monitoring of Organ-Specific Genomic Marker in Circulating DNA from Receptor.

The evaluation of the transplanted liver health by non-invasive approaches may offer an improvement in early clinical intervention. As transplanted organs have genomes that are distinct from the host's genome, the quantification of the specific DNA of the donated liver in the patient serum will allow us to obtain information about its damage. We evaluated the state of transplanted liver health by monitoring the RH gene in serum circulating DNA (cirDNA) from 17 recipient and donor mismatched for this gene. cirDNA RH gene was quantified by RT- PCR before, at the moment of transplantation (day 0) and during the stay at the intensive care unit. Beta-globin cirDNA was quantified as a general cellular damage marker. Patients were grouped based on clinical outcomes: (A) patients with no complication; (B) patients that accepted the organ but suffered other complications; (C) patients that suffered organ rejection. All patients showed an increased cirDNA levels at day 0 that decreased until patient stabilization. Patients from groups A and B showed low levels of the RH gene cDNA during the follow-up, with an increase of beta-globin gene at the moment of any clinical complication. Patients from group C showed an increase in the RH gene during rejection.