Low Risk Of Rejection Research Articles

Abstract 13935: Steroids Wean Failures After Heart Transplantation - Does It Lead to Mortality?

Introduction: It is estimated that approximately 90% of heart transplant (HTx) patients who are low risk can be weaned off steroids after 6 months post-transplant. However, for those 10% who fail weaning subsequent outcome is not known. There is concern that failure to wean off steroids may lead to subsequent greater morbidity/mortality. Methods: Between 2010 and 2014, 178 HTx patients at low rejection risk were initiated to be weaned off steroids after 6 months post-transplant. Our protocol includes decreasing prednisone by 1mg per month from a baseline of 5mg daily. Monthly heart biopsy or use of Allomap are used to exclude rejection during weaning. 15 patients failed the corticosteroid wean due to either rejection (n=8), abnormal echocardiogram (n=2) or severe corticosteroid withdrawal symptoms (n=5). Study endpoints include subsequent 5-year survival, freedom from cardiac allograft vasculopathy (CAV) and non-fatal major adverse cardiac events (NF-MACE); and 1-year freedom from any treated rejection (ATR), antibody-mediated rejection (AMR), acute cellular rejection (ACR). Results: The failed wean group compared to the successful wean group had a trend for lower subsequent 5-year survival. There was no significant difference between the two groups in subsequent 5-year freedom from CAV or NF-MACE. Subsequent 1 Year freedom all rejection (ATR, ACR, AMR) were significantly lower in the failed steroid wean group compared to the successfully weaned group. Conclusions: HTx patients who are deemed low risk who failed corticosteroid weaning may be at risk to develop more rejection with compromised 5-year outcome, including a possible lower survival rate. It is unclear whether the act of corticosteroid weaning activated a deleterious response in these patients. Caution must be given to those patients who fail corticosteroid weaning.

Clinical and immunological follow-up of very long-term kidney transplant recipients treated with calcineurin inhibitors indicates dual phenotypes

Operationally tolerant kidney transplant recipients harbor an immunological signature, associated with low rejection risk, and focused on B lymphocytes. Here, we investigated whether patients with long-term transplantation and still on immunosuppressive therapy would present such a signature of low immunological rejection risk, compared to more recently transplanted patients. Of 114 kidney transplant recipients enrolled, 38 with more than 25 years of graft survival and stable graft function under calcineurin inhibitors, were matched with two different groups of transplanted patients (10-15 and 5-7 years after transplantation). Three phenotypes associated with low immunological rejection risk (Tfh, B and regulatory T cells), initially found in operationally tolerant kidney transplant recipients, and the composite score of tolerance (combination of six transcriptomic markers, age at transplantation and age at sampling) were analyzed. We found that very long-term patients were characterized by a significantly lower percentage of total B cells, a significantly higher proportion of CD24HiCD38Lo memory B cells, significantly fewer CD24LoCD38Lo naive B cells, and a significantly lower proportion of PD1HiCCR7Lo Tfh lymphocytes than more recently transplanted patients. This phenotype is associated with a positive composite score of tolerance in patients transplanted for more than 25 years. Thus, our study suggests a dual phenotype in very long-term kidney transplanted patients with an immunological profile associated with low rejection risk.

High Dose of Intravenous Allogeneic Umbilical Cord-Derived Mesenchymal Stem Cells (CLV-100) Infusion Displays Better Immunomodulatory Effect among Healthy Volunteers: A Phase 1 Clinical Study.

Background Mesenchymal stem cells (MSCs) express growth factors and other cytokines that stimulate repair and control the immune response. MSCs are also immunoprivileged with low risk of rejection. Umbilical cord-derived MSCs (UCMSCs) are particularly attractive as an off-the-shelf allogeneic treatment in emergency medical conditions. We aim to determine the safety and efficacy of intravenous allogeneic infusion of UCMSCs (CLV-100) by Cytopeutics® (Selangor, Malaysia) in healthy volunteers, and to determine the effective dose at which an immunomodulatory effect is observed. Methodology. Umbilical cord samples were collected after delivery of full-term, healthy babies with written consent from both parents. All 3 generations (newborn, parents, and grandparents) were screened for genetic mutations, infections, cancers, and other inherited diseases. Samples were transferred to a certified Good Manufacturing Practice laboratory for processing. Subjects were infused with either low dose (LD, 65 million cells) or high dose (HD, 130 million cells) of CLV-100 and followed up for 6 months. We measured cytokines using ELISA including anti-inflammatory cytokines interleukin 1 receptor antagonist (IL-1RA), interleukin 10 (IL-10), pro-/anti-inflammatory cytokine interleukin 6 (IL-6), and the proinflammatory cytokine tumor necrosis factor-alpha (TNF-α). Results 11 healthy subjects (LD, n = 5; HD, n = 6; mean age of 55 ± 13 years) were recruited. All subjects tolerated the CLV-100 infusion well with no adverse reaction throughout the study especially in vital parameters and routine blood tests. At 6 months, the HD group had significantly higher levels of anti-inflammatory markers IL1-RA (705 ± 160 vs. 306 ± 36 pg/mL; p = 0.02) and IL-10 (321 ± 27 vs. 251 ± 28 pg/mL; p = 0.02); and lower levels of proinflammatory marker TNF-α (74 ± 23 vs. 115 ± 15 pg/mL; p = 0.04) compared to LD group. Conclusion Allogeneic UCMSCs CLV-100 infusion is safe and well-tolerated in low and high doses. Anti-inflammatory effect is observed with a high-dose infusion.

Use of Mycophenolate Mofetil Suspension as Part of Induction Therapy After Living-Donor Liver Transplant

The aim of this study was to evaluate recipient safety, tolerability, and pharmacokinetics of mycophenolate mofetil suspension compared with mycophenolate mofetil capsules as part of induction therapy after living-donor liver transplant. Between July 2017 and April 2019, we retrospectively enrolled 20 adult primary living-donor liver transplant recipients. Recipients were divided into 3 groups: group 1 received mycophenolate mofetil suspension of 3000 mg (n = 6), group 2 received 3000 mg mycophenolate mofetil via opened capsules (n = 8), and group 3 received mycophenolate mofetil suspension of 2000 mg (n = 6). Administration was started on postoperative day 1, with tacrolimus administered on postoperative day 2 or day 3. The values of area under the plasma concentration time curve for 0 to 12 hours were significantly higher in the 3000 mg/day mycophenolate mofetil suspension group than in the 2000 mg/day mycophenolate mofetil suspension group (P = .024) and in the 3000mg/day mycophenolate mofetil capsule group (P = .013). Significant positive correlations were shown between blood concentration at 8 hours after administration and the plasma concentration time curve for 0 to 12 hours (r2 = 0.96; P < .001) in patients in the suspension group. No patients required mycophenolate mofetil reduction because of leukopenia and diarrhea. Only 1 biopsy-proven acute cellular rejection was recognized in the mycophenolate mofetil suspension group (at 2000 mg/day). There were no significant differences in frequency of opportunistic infections among the 3 groups. Mycophenolate mofetil suspension is useful as part of immunosuppressive induction therapy after living-donor liver transplant because its concentration increases greater than that of mycophenolate mofetil capsules and because of the low risk of rejection and adverse events.

Lymphocytes cross-matching as retrospective tool for hyper-acute rejection diagnosis in heart transplantation.

BackgroundLymphocyte cross-matching (CDC) is considered as important screening test that is unfrequently performed preoperatively in heart transplantation procedures. This test is desired to identify the risk for hyper-acute rejection in immediate postoperative period. Patient and methodThere were 57 patients (51 men and 6 women) in mean age of 44 +/- 14 between 2010 and 2019 screened for CDC tests. All patients were low risk for rejection including negative panel reactive antibody preoperative test. Preoperative donor's blood samples and lymph nodes were obtained during retrieval. Recipients blood samples were collected before surgery. CDC test were obtained retrospectively. Endomyocardial biopsies including immunopathological staining were performed on 6th and 14th postoperative day. ResultsThe results obtained from cross-match test revealed 4 patients with lymphocytic T and B incompatibility (positive for cellular and humoral hyper-acute rejection) and 3 more for Lymphocytes B hyper-acute rejection (humoral rejection). All patients were hemodynamically stable. Successful therapy including high doses of polyvalent immunoglobulins and steroids parenteral infusions were proven by endomyocardial biopsies samples. ConclusionThe lymphocytic cross-match testing is useful as postoperative examination. It help to find high risk patients for hyper-acute rejection. It allows for introduction immediate immunosuppressive therapeutic adjustment.

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Systematic Review and Meta-Analysis of Clinical Outcomes of Penetrating Keratoplasty Versus Deep Anterior Lamellar Keratoplasty for Keratoconus.

In recent years, there have been rapid advances in the field of keratoconus. Penetrating keratoplasty and deep anterior lamellar keratoplasty are the standard surgical procedures. Nevertheless, controversy remains regarding the outcomes of both procedures in the treatment of keratoconus. Therefore, we conducted a meta-analysis comparing postoperative outcome measures of penetrating keratoplasty versus deep anterior lamellar keratoplasty for keratoconus. We searched PubMed, Embase, Web of Science, and the Cochrane Database of Systematic Reviews for eligible studies comparing best-corrected visual acuity, spherical equivalent, refractive cylinder, topography cylinder, and graft rejection episodes and complications of penetrating keratoplasty and deep anterior lamellar keratoplasty. Seven risk domains from software Review Manager 5.3 (The Cochrane Collaboration, Oxford, UK) were applied as quality assessments for the eligible studies. A random-effects model was used for data synthesis. Thirteen eligible studies were included in our meta-analysis, which encompassed 530 eyes that underwent penetrating keratoplasty and 568 eyes that underwent deep anterior lamellar keratoplasty. With regard to best-corrected visual acuity, refractive cylinder, and topography cylinder, we found no significant differences in results between the 2 procedures (P = .49 and .47, respectively). However, spherical equivalent results were significantly greater in the deep anterior lamellar keratoplasty group than in the penetrating keratoplasty group (P < .001). The risk of graft rejection episodes was more prominent in the penetrating keratoplasty than in the deep anterior lamellar keratoplasty group (odds ratio = 2.69; P = .001). The odds ratio for complications was 1.79 (P = .03). Three studies showed moderate risk of bias, and the other 10 showed high risk of bias. Deep anterior lamellar keratoplasty is preferred over penetrating keratoplasty for the treatment of keratoconus because of its low risk of rejection and complications.

Dose-dependent immunomodulatory effect of intravenous allogeneic umbilical cord-derived mesenchymal stromal cells (Cytopeutics CLV-100) infusion in healthy volunteers

Background & Aim Background Mesenchymal stromal cells (MSCs) express growth factors and other cytokines that stimulate repair and control the immune response. MSCs are also immunoprivileged with low risk of rejection. Umbilical cord derived MSC (UCMSCs) are particularly attractive as off-the-shelf allogeneic treatment in emergency medical conditions. Objective We aim to determine the safety and feasibility of intravenous allogeneic infusion of UCMSCs (CLV-100) by Cytopeutics® (Selangor, Malaysia) in healthy volunteers, and to determine the effective dose at which an immunomodulatory effect is observed. Methods, Results & Conclusion Methodology Umbilical cord samples were collected after delivery of full-term, healthy babies with written consent from both parents. All 3 generations (newborn, parents and grandparents) were screened for genetic mutations, infections, cancers and other inherited diseases. Samples were transferred to a certified Good Manufacturing Practice laboratory for processing. Subjects were infused with either low dose (LD, 65 million cells) or high dose (HD, 130 million cells) of CLV-100 followed up for 6 months. We measured cytokines level using ELISA including the acute phase reactant high-sensitivity C-reactive protein (hs-CRP), anti-inflammatory cytokines interleukin 1 receptor antigen (IL-1RA) and interleukin 10 (IL-10), and the pro-inflammatory cytokine Tumor Necrosis Factor-Alpha (TNF-α). The study was approved by the National Medical Research and Ethics Committee (MREC). Results 11 healthy subjects (LD, n=5; HD, n=6; mean age of 55±13 years) were recruited. All subjects tolerated the CLV-100 infusion well with no adverse reaction throughout the study. While there was no change in vital parameters and routine blood tests, there was a transient rise in hs-CRP at Day 3 post infusion (1.2±1.5 vs. 4.5±3.9 mg/L; p=0.03) which resolved at Day 7. The anti-inflammatory IL-1RA was significantly elevated in HD patients at 1 month post infusion (436±128 vs. 615±148 pg/mL; p=0.028). At 6 months, HD group had significantly higher levels of anti-inflammatory markers IL1-RA (705±160 vs. 306±36 pg/mL; p=0.02) and IL-10 (321±27 vs. 251±28 pg/mL; p=0.02); and lower levels of pro-inflammatory marker TNF-α (74±23 vs. 115±15 pg/mL; p=0.038) compared to LD group. Conclusion Allogeneic UCMSCs CLV-100 infusion is safe and well-tolerated in low and high doses. Anti-inflammatory effect is observed with high dose infusion.

Effective use of mesenchymal stem cells in human skin substitutes generated by tissue engineering.

Mesenchymal stem cells (MSCs) can differentiate toward epithelial cells and may be used as an alternative source for generation of heterotypical artificial human skin substitutes, thus, enhancing their development and translation potential to the clinic. The present study aimed at comparing four types of heterotypical human bioengineered skin generated using MSCs as an alternative epithelial cell source. Adipose-tissue-derived stem cells (ADSCs), dental pulp stem cells (DPSCs), Wharton's jelly stem cells (WJSCs) and bone marrow stem cells (BMSCs) were used for epidermal regeneration on top of dermal skin substitutes. Heterotypic human skin substitutes were evaluated before and after implantation in immune-deficient athymic mice for 30 d. Histological and genetic studies were performed to evaluate extracellular matrix synthesis, epidermal differentiation and human leukocyte antigen (HLA) molecule expression. The four cell types differentiated into keratinocytes, as shown by the expression of cytokeratin 10 and filaggrin 30 d post-grafting; also, they induced dermal fibroblasts responsible for the synthesis of extracellular fibrillar and non-fibrillar components, in a similar way among each other. WJSCs and BMSCs showed higher expression of cytokeratin 10 and filaggrin, suggesting these cells were more prone to epidermal regeneration. The absence of HLA molecules, even when the epithelial layer was differentiated, supports the future clinical use of these substitutes - especially ADSCs, DPSCs and WJSCs - with low rejection risk. MSCs allowed the generation of bioengineered human skin substitutes with potential clinical usefulness. According to their epidermal differentiation potential and lack of HLA antigens, WJSCs should preferentially be used.

318. Ideal interval between renal transplantation and pregnancy regarding obstetric outcomes

The optimal timing for pregnancy after kidney transplant remains uncertain, due to the risk of allograft failure. In the last consensus of American Transplant Society, this interval decreased from 2 years to 1 year, due to advanced maternal age with fewer childbearing years and lower risk of rejection with the more recent and potent immunosuppressive strategies. Analyze whether the interval between transplantation and pregnancy (TTPI) influences obstetric outcomes. Medical records from a retrospective cohort of pregnancies following kidney transplanted in our department, since 1989 (n = 41), were analyzed. Obstetric and neonatal outcomes were compared according to transplantation-to-pregnancy interval (TTPI). Statistical analysis was performed using SPSS® version 22.0 (p = 0,05). The study includes 41 pregnant patients after kidney transplant, 4 (10%) in the first year, 7 (17%) in second year and 30 (73%) after 24 months. Within the first year after transplantation, we observed a higher incidence of fetal growth restriction (66,7% vs 18,2%, p = 0,06), preterm labor (100% vs 54,5%, p = 0,06) and low and very low birth weight (100% vs 51,5%, p = 0,05 and 66,7% vs 6,1%, p = 0,013). Mean gestational age (32,3 ± 0,6 [32–33] vs 35,8 ± 2,5 [29–39] weeks, p = 0,04) and weight at delivery (1500 ± 282[1300–1700] vs 2523 ± 642[885–3740], p = 0,04) were significantly lower 1 year after transplant. In the second year, the incidence of gestational hypertension (57%) is similar to first year (33%), but significantly higher when compared with TTPI > 24 months (15%, p = 0,03). With a transplantation-to-pregnancy interval higher than 2 years, the incidence of urinary infections is higher (27% vs 0%, p = 0,02). Regarding the obstetric outcomes and according to our results, the ideal time for pregnancy after transplantation is between 12 and 24 months, with a lower risk of urinary infections, restriction of fetal growth, preterm birth and low birth weight.

Identifying risk profiles in liver transplant candidates and implications for induction immunosuppression.

Changes in recipient and donor characteristics are redefining the role of induction in liver transplant recipients. Older recipients are more common, with greater concomitant comorbidity. Moderate or severe renal dysfunction is now estimated to affect 40% of liver transplant recipients. Donors are also becoming older, and other factors such as more frequent non-alcoholic fatty liver disease (NAFLD) compromise the quality of some grafts. Rejection rates are now relatively low (~10%) but some patients have a markedly increased risk such as younger recipients and those undergoing re-transplantation. Induction immunosuppression is associated with a significant reduction in rejection risk but due to various factors universal induction is not justified. Steroid-free therapy without induction increases the risk of biopsy-proven acute rejection (BPAR) but randomized trials have shown that induction with an interleukin-2 antagonist receptor (IL-2RA) agent or with rabbit antithymocyte globulin (rATG) maintains immunosuppressive efficacy in steroid-free regimens. Delayed calcineurin inhibitor (CNI) initiation (e.g. to days 4-5 post-transplant) can prevent deterioration of renal function during the first year post-transplant, but requires induction with an IL-2RA agent or rATG to maintain early immunosuppressive efficacy. IL-2RA induction may be inadequate to ensure a low risk of rejection in a steroid-free regimen combined with delayed tacrolimus. Randomized trials of CNI withdrawal at month 1 post-transplant have only achieved an acceptable rate of BPAR when induction is administered. In terms of safety, an increased rate of infection does not seem to be a concern. The most recent large-scale analyses have not indicated any evidence for an increased risk of malignancy, or specifically post-transplant lymphoproliferative disease. In summary, the place of induction in the management of liver transplant patients is becoming established. Selective use in high-risk individuals to avoid graft rejection is still relevant, but the key rationale for induction is to facilitate steroid-sparing and CNI-sparing regimens to reduce long-term complications.

Glucocorticosteroid-free versus glucocorticosteroid-containing immunosuppression for liver transplanted patients.

Liver transplantation is an established treatment option for end-stage liver failure. Now that newer, more potent immunosuppressants have been developed, glucocorticosteroids may no longer be needed and their removal may prevent adverse effects. To assess the benefits and harms of glucocorticosteroid avoidance (excluding intra-operative use or treatment of acute rejection) or withdrawal versus glucocorticosteroid-containing immunosuppression following liver transplantation. We searched the Cochrane Hepato-Biliary Group Controlled Trials Register, Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, Embase, Science Citation Index Expanded and Conference Proceedings Citation Index - Science, Literatura Americano e do Caribe em Ciencias da Saude (LILACS), World Health Organization International Clinical Trials Registry Platform, ClinicalTrials.gov, and The Transplant Library until May 2017. Randomised clinical trials assessing glucocorticosteroid avoidance or withdrawal versus glucocorticosteroid-containing immunosuppression for liver transplanted people. Our inclusion criteria stated that participants should have received the same co-interventions. We included trials that assessed complete glucocorticosteroid avoidance (excluding intra-operative use or treatment of acute rejection) versus short-term glucocorticosteroids, as well as trials that assessed short-term glucocorticosteroids versus long-term glucocorticosteroids. We used RevMan to conduct meta-analyses, calculating risk ratio (RR) for dichotomous variables and mean difference (MD) for continuous variables, both with 95% confidence intervals (CIs). We used a random-effects model and a fixed-effect model and reported both results where a discrepancy existed; otherwise we reported only the results from the fixed-effect model. We assessed the risk of systematic errors using 'Risk of bias' domains. We controlled for random errors by performing Trial Sequential Analysis. We presented our results in a 'Summary of findings' table. We included 17 completed randomised clinical trials, but only 16 studies with 1347 participants provided data for the meta-analyses. Ten of the 16 trials assessed complete postoperative glucocorticosteroid avoidance (excluding intra-operative use or treatment of acute rejection) versus short-term glucocorticosteroids (782 participants) and six trials assessed short-term glucocorticosteroids versus long-term glucocorticosteroids (565 participants). One additional study assessed complete post-operative glucocorticosteroid avoidance but could only be incorporated into qualitative analysis of the results due to limited data published in an abstract. All trials were at high risk of bias. Only eight trials reported on the type of donor used. Overall, we found no statistically significant difference for mortality (RR 1.15, 95% CI 0.93 to 1.44; low-quality evidence), graft loss including death (RR 1.15, 95% CI 0.90 to 1.46; low-quality evidence), or infection (RR 0.88, 95% CI 0.73 to 1.05; very low-quality evidence) when glucocorticosteroid avoidance or withdrawal was compared with glucocorticosteroid-containing immunosuppression. Acute rejection and glucocorticosteroid-resistant rejection were statistically significantly more frequent when glucocorticosteroid avoidance or withdrawal was compared with glucocorticosteroid-containing immunosuppression (RR 1.33, 95% CI 1.08 to 1.64; low-quality evidence; and RR 2.14, 95% CI 1.13 to 4.02; very low-quality evidence). Diabetes mellitus and hypertension were statistically significantly less frequent when glucocorticosteroid avoidance or withdrawal was compared with glucocorticosteroid-containing immunosuppression (RR 0.81, 95% CI 0.66 to 0.99; low-quality evidence; and RR 0.76, 95% CI 0.65 to 0.90; low-quality evidence). We performed Trial Sequential Analysis for all outcomes. None of the outcomes crossed the monitoring boundaries or reached the required information size. Hence, we cannot exclude random errors from the results of the conventional meta-analyses. Many of the benefits and harms of glucocorticosteroid avoidance or withdrawal remain uncertain because of the limited number of published randomised clinical trials, limited numbers of participants and outcomes, and high risk of bias in the trials. Glucocorticosteroid avoidance or withdrawal appears to reduce diabetes mellitus and hypertension whilst increasing acute rejection, glucocorticosteroid-resistant rejection, and renal impairment. We could identify no other benefits or harms of glucocorticosteroid avoidance or withdrawal. Glucocorticosteroid avoidance or withdrawal may be of benefit in selected patients, especially those at low risk of rejection and high risk of hypertension or diabetes mellitus. The optimal duration of glucocorticosteroid administration remains unclear. More randomised clinical trials assessing glucocorticosteroid avoidance or withdrawal are needed. These should be large, high-quality trials that minimise the risk of random and systematic error.

Immunologic Stromal Rejection After Deep Anterior Lamellar Keratoplasty With Grafts of a Larger Size (9 mm) for Various Stromal Diseases.

To evaluate the rate and outcomes of immunologic stromal rejection occurring after large deep anterior lamellar keratoplasty (DALK) and the effect of the underlying disease on the cumulative probability of rejection. This was a retrospective chart review of all eyes that underwent a 9-mm DALK at Ospedali Privati Forlì (Forlì, Italy). On the basis of preoperative diagnosis, eyes were assigned to group 1: keratoconus, group 2: stromal disease with a low risk for rejection, or group 3: stromal disease with a high risk for rejection. The cumulative probability of experiencing a rejection episode over time was assessed by Kaplan-Meier analysis and was compared among the 3 groups using the log-rank test. Values of corrected distance visual acuity, central corneal thickness, and endothelial cell density before and 6 months after rejection were compared. Twenty of 377 eyes (5.3%) experienced an episode of stromal rejection [17/265 (6.4%) in group 1, 2/71 (2.8%) in group 2, and 1/41 (2.4%) in group 3]. The mean time interval between DALK and the rejection onset was 11.8 months (range 1-24). The cumulative probability of rejection episodes did not differ significantly among the 3 groups. All episodes resolved within 6 months after the onset, with no significant differences between prerejection and 6-month postrejection values of corrected distance visual acuity, central corneal thickness, and endothelial cell density. The rate of immunologic stromal rejection after large 9-mm DALK is within the range reported in the literature for conventional DALK, regardless of the indication for surgery.

Very Long-term Survival of the Transplanted Kidney—Characteristics of Recipients

BackgroundAfter kidney transplantation (KTx), donor- and recipient-dependent factors, as well as the immunosuppression protocol, may have an impact long-term graft function. The aim of this retrospective study was to identify and describe recipients from a single center who had their transplanted kidney survive for more than 20 years. MethodsThe database of KTx recipients was searched to find identify patients with a functioning kidney graft for >20 years. Clinical, demographic, and immunologic data were recorded and analyzed. Moreover, the Charlson Comorbidity Index was calculated. ResultsWe identified 25 patients, with graft survival of 23.9 ± 3.2 years (maximum, 31.5 years), with following characteristics: age at time of transplantation 36.2 ± 11.9 years; median of 4 human leukocyte antigen (HLA) mismatches; low risk of rejection (panel-reactive antibodies [PRA] 0%); and 14 recipients had delayed graft function (DGF) and 9 had a single episode of acute rejection successfully treated with steroid pulses. In 24 cases there was a deceased donor. There was a predominance of males aged <54 years. At 1 year after KTx, serum creatinine was 1.36 ± 0.26 mg/dL. All recipients were given cyclosporine + azathioprine + prednisone as primary immunosuppression. The majority of recipients have continued to visit the clinic on an oupatient basis, with a most recent creatinine average of 1.5 ± 0.82 mg/dL. ConclusionVery long-term kidney graft survival is most likely associated with a low risk of rejection (0% PRA pre-KTx), a relatively weak immunosuppression protocol, and optimal function at 12 months post-KTx.

Low Immunogenic Endothelial Cells Maintain Morphological and Functional Properties Required for Vascular Tissue Engineering.

The limited availability of native vessels suitable for the application as hemodialysis shunts or bypass material demands new strategies in cardiovascular surgery. Tissue-engineered vascular grafts containing autologous cells are considered ideal vessel replacements due to the low risk of rejection. However, endothelial cells (EC), which are central components of natural blood vessels, are difficult to obtain from elderly patients of poor health. Umbilical cord blood represents a promising alternative source for EC, but their allogeneic origin corresponds with the risk of rejection after allotransplantation. To reduce this risk, the human leukocyte antigen class I (HLA I) complex was stably silenced by lentiviral vector-mediated RNA interference (RNAi) in EC from peripheral blood and umbilical cord blood and vein. EC from all three sources were transduced by 93.1% ± 4.8% and effectively, HLA I-silenced by up to 67% compared to nontransduced (NT) cells or transduced with a nonspecific short hairpin RNA, respectively. Silenced EC remained capable to express characteristic endothelial surface markers such as CD31 and vascular endothelial cadherin important for constructing a tight barrier, as well as von Willebrand factor and endothelial nitric oxide synthase important for blood coagulation and vessel tone regulation. Moreover, HLA I-silenced EC were still able to align under unidirectional flow, to take up acetylated low-density lipoprotein, and to form capillary-like tube structures in three-dimensional fibrin gels similar to NT cells. In particular, addition of adipose tissue-derived mesenchymal stem cells significantly improved tube formation capability of HLA I-silenced EC toward long and widely branched vascular networks necessary for prevascularizing vascular grafts. Thus, silencing HLA I by RNAi represents a promising technique to reduce the immunogenic potential of EC from three different sources without interfering with EC-specific morphological and functional properties required for vascular tissue engineering. This extends the spectrum of available cell sources from autologous to allogeneic sources, thereby accelerating the generation of tissue-engineered vascular grafts in acute clinical cases.

The epigenetic promise to improve prognosis of heart failure and heart transplantation.

Heart transplantation is still the only possible life-saving treatment for end-stage heart failure, the critical epilogue of several cardiac diseases. Epigenetic mechanisms are being intensively investigated because they could contribute to establishing innovative diagnostic and predictive biomarkers, as well as ground-breaking therapies both for heart failure and heart transplantation rejection. DNA methylation and histone modifications can modulate the innate and adaptive immune response by acting on the expression of immune-related genes that, in turn, are crucial determinants of transplantation outcome. Epigenetic drugs acting on methylation and histone-modification pathways may modulate Treg activity by acting as immunosuppressive agents. Moreover, the identification of non-invasive and reliable epigenetic biomarkers for the prediction of allograft rejection and for monitoring immunosuppressive therapies represents an attractive perspective in the management of transplanted patients. MiRNAs seem to fit particularly well to this purpose because they are differently expressed in patients at high and low risk of rejection and are detectable in biological fluids besides biopsies. Although increasing evidence supports the involvement of epigenetic tags in heart failure and transplantation, further short and long-term clinical studies are needed to translate the possible available findings into clinical setting.

Properties Analysis of Biological Grafts Used in Pelvic Surgery

Pelvic surgery addresses congenital malformations like Mullerian anomalies, pelvic organ prolapse or after radical intervention, while constructive surgery involves treatment of congenital/iatrogenic absence of organs: uterus or vagina. Intuitively, inert and nondegradable biomaterials appear ideal for this purpose, but surgical reality is that the persistence of a permanent foreign body in the wound has a lot of clinical postoperative disadvantages. This paper aims to discuss the properties of the biologic grafts, detailing the structure of biologic extracellular matrix, the biomechanical properties of biological grafts and the use of extracellular matrix in reconstructive pelvic surgery. A biologic graft used in pelvic surgery is a tridimensional extracellular matrix, acellular and of animal origin. It has as a low risk of transmission of viral and prionic infections and determines reduced inflammatory reaction and a low risk of rejection. The absence of crosslinking facilitates the colonization of the mesh by the host´s cells, thus avoids the foreign body reaction represented by the encapsulation of the mesh by the host. Our experience with clinical use of biological acellular grafts in reconstructive pelvic surgery includes oncoplastic surgery like in cases of vulvar cancer or neovagina reconstruction for Rokintansky syndrome, complications after prolapse surgery treated with polypropylene meshes or relapsing recto-vaginal fistulas. The biomechanical properties evaluated by tensile stress and elastic modulus revealed that biologic grafts with moderate collagen infiltration are the strongest. The degree of cross-linkage influences the rate of degradation and the degree of the inflammatory response triggered by the host organ. Cross-linked collagenous matrices induce little cell infiltration hence there is limited collagen remodeling and graft degradation. On the other hand, in non-cross- linked xenografts cell infiltration is greater with faster degradation rate and collagen production. The decrease in the mechanical strength of the graft materials is related to the lack of collagen infiltration into the material. In conclusion, taking into account the biomechanical properties of biologic grafts, these may be used in reconstructive pelvic surgery and oncoplastic surgery with little complications and good clinical results. They can be attached to large mucosal defects and on potentially septic tissue, they are mechanically resistant and they can be sutured on surrounding healthy tissue.

Failure of Calcineurin Inhibitor (Tacrolimus) Weaning Randomized Trial in Long-Term Stable Kidney Transplant Recipients.

Long-term renal transplant outcome is limited by side effects of immunosuppressive drugs, particularly calcineurin inhibitor (CNI). We assumed that some patients selected for a "low immunological risk of rejection" could be eligible and benefit from a CNI weaning strategy. We designed a prospective, randomized, multicenter, double-blind placebo-controlled clinical study (Eudract: 2010-019574-33) to analyze the benefit-risk ratio of tacrolimus weaning on highly selected patients (≥4 years of transplantation, normal histology, stable graft function, no anti-HLA immunization). The primary endpoint was improvement of renal function. Fifty-two patients were scheduled in each treatment arm, placebo compared to the CNI maintenance arm. Only 10 patients were eligible and randomized. Five patients were assigned to the placebo arm and five were assigned to the tacrolimus maintenance arm. In the tacrolimus maintenance arm, all patients maintained stable graft function and no immunological events occurred. Contrastingly, in the placebo arm, all five patients had to reintroduce a full dose of tacrolimus since three of them presented an acute rejection episode (one humoral, one mixed, and one borderline) and two displayed anti-HLA antibodies without histological lesion (one donor-specific antibodies [DSA] and one non-DSA). Clearly, tacrolimus withdrawal must be avoided even in long-term highly selective stable kidney recipients.

Glucocorticosteroid-free versus glucocorticosteroid-containing immunosuppression for liver transplanted patients.

Liver transplantation is an established treatment option for end-stage liver failure. Now that newer, more potent immunosuppressants have been developed, glucocorticosteroids may no longer be needed and their removal may prevent adverse effects. To assess the benefits and harms of glucocorticosteroid avoidance (excluding intra-operative use) or withdrawal versus glucocorticosteroid-containing immunosuppression following liver transplantation. We searched the Cochrane Hepato-Biliary Group Controlled Trials Register, Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, EMBASE, Science Citation Index Expanded and Social Sciences Citation Index, The Transplant Library, and the World Health Organization (WHO) International Clinical Trials Registry Platform (ICTRP) until September 2014. Randomised clinical trials assessing glucocorticosteroid avoidance or withdrawal versus glucocorticosteroid-containing immunosuppression for liver-transplanted people. Our inclusion criteria stated that participants should have received the same co-interventions. We included trials that assessed complete glucocorticosteroid avoidance (excluding the perioperative period and excluding the occurrence of acute rejection) versus short-term glucocorticosteroids, as well as trials that assessed short-term glucocorticosteroids versus long-term glucocorticosteroids. We used RevMan to conduct meta-analyses, calculating risk ratio (RR) for dichotomous variables and mean difference (MD) for continuous variables, both with 95% confidence intervals (CIs). We used a random-effects model and a fixed-effect model and reported both results where a discrepancy existed. We assessed the risk of systematic errors using risk of bias domains. We controlled for random errors by performing Trial Sequential Analysis. We presented our results in a 'Summary of findings' table. We included 16 completed randomised clinical trials with a total of 1347 participants. We found 10 trials that assessed complete postoperative glucocorticosteroid avoidance (excluding intra-operative use and treatment of rejection) versus short-term glucocorticosteroids (782 participants) and six trials that assessed short-term glucocorticosteroids versus long-term glucocorticosteroids (565 participants). We found one ongoing trial assessing complete postoperative glucocorticosteroid avoidance versus short-term glucocorticosteroids, which is expected to enrol 300 participants. All trials were at high risk of bias. Overall, we found no statistically significant difference for mortality (RR 1.15, 95% CI 0.93 to 1.44; low-quality evidence), graft loss including death (RR 1.16, 95% CI 0.91 to 1.48; low-quality evidence), or infection (RR 0.88, 95% CI 0.73 to 1.05; low-quality evidence) when glucocorticosteroid avoidance or withdrawal was compared with glucocorticosteroid-containing immunosuppression. Acute rejection and glucocorticosteroid-resistant rejection were statistically significantly more frequent when glucocorticosteroid avoidance or withdrawal was compared with glucocorticosteroid-containing immunosuppression (RR 1.33, 95% CI 1.08 to 1.64; moderate-quality evidence; and RR 2.14, 95% CI 1.13 to 4.02; very low-quality evidence). Diabetes mellitus and hypertension were statistically significantly less frequent when glucocorticosteroid avoidance or withdrawal was compared with glucocorticosteroid-containing immunosuppression (RR 0.81, 95% CI 0.66 to 0.99; low-quality evidence; and RR 0.76, 95% CI 0.65 to 0.90; low-quality evidence). We performed Trial Sequential Analysis for all outcomes. None of the outcomes crossed the monitoring boundaries or reached the required information size. Hence, we cannot exclude random errors from the results of the conventional meta-analyses. Many of the benefits and harms of glucocorticosteroid avoidance or withdrawal remain uncertain because of the limited number of published randomised clinical trials, limited numbers of participants and outcomes, and high risk of bias in the trials. Glucocorticosteroid avoidance or withdrawal appears to reduce diabetes mellitus and hypertension whilst increasing acute rejection, glucocorticosteroid-resistant rejection, and renal impairment. We could identify no other benefits or harms of glucocorticosteroid avoidance or withdrawal. Glucocorticosteroid avoidance or withdrawal may be of benefit in selected patients, especially those at low risk of rejection and high risk of hypertension or diabetes mellitus. The optimal duration of glucocorticosteroid administration remains unclear. More randomised clinical trials assessing glucocorticosteroid avoidance or withdrawal are needed. These should be large, high-quality trials that minimise the risk of random and systematic error.

DALK versus PK for keratokonus

Advanced keratoconus usually leads to keratoplasty in young adult patients who have a long life expectancy. Repeat keratoplasty is known to be associated with shorter graft survival compared with primary graft and increased risk of glaucoma that may result in progressive loss of vision. For these reasons, keratoplasty in keratoconus should aim not only to provide patients with good vision but also to permit long‐term survival of the graft. Recent studies have raised controversies regarding survival of penetrating and deep anterior lamellar keratoplasties. In two large series of keratoplasties, graft survival was higher in PK than in DALK eyes. Conversely, a meta‐analysis recently showed that DALK is associated with lower risk of rejection and lower risk of failure compared with PK. These findings make sense if we consider the absence of endothelial rejection and the high survival rate of the corneal endothelium observed in DALK eyes. DALK and PK appear to give similar visual recovery at least in the short and mid‐term. It is not yet possible to determine which technique is associated with the highest chance of maintaining good vision in the long term. This issue depends not only on maintenance of graft transparency but also on corneal biomechanics. Progressive thinning of the peripheral inferior recipient cornea may result in severe irregular astigmatism after keratoplasty for keratoconus. This issue should be further addressed.