In vitro developmental competence of bovine demi-embryos generated by blastomere separation and blastocyst bisection.

The efficiency of bovine in vitro embryo production can be significantly improved by splitting embryos at different stages. However, the blastocyst quality of in vitro-produced demi-embryos remains unexplored. The objective of this research was to compare embryo developmental rates and quality of bovine demi-embryos produced by two different strategies: (a) embryo bisection (BSEC) and (b) 2-cell blastomere separation (BSEP). To determine demi-embryos quality, we evaluated total blastocyst cell number and proportion of SOX2+ cells. Additionally, the expression of SOX2, NANOG, OCT4, CDX2, IFNT, BAX and BCL genes and let-7a and miRNA-30c Micro RNAs was analysed. BSEP resulted in improved blastocyst development, higher ICM cells and a significantly higher expression of IFNΤ than demi-embryos produced by BSEC. Let-7a, which is associated with low pregnancy establishment was detected in BSEC, while miRNA-30c expression was observed in all treatments. In conclusion, BSEP of 2-cell embryos is more efficient to improve in vitro bovine embryo development and to produce good quality demi-embryos based on ICM cell number and the expression pattern of the genes explored compared to BSEC.

Single-Cell Transcriptomic Analysis of Kaposi Sarcoma.

Kaposi Sarcoma (KS) is a complex tumor caused by KS-associated herpesvirus 8 (KSHV). Histological analysis reveals a mixture of "spindle cells", vascular-like spaces, extravasated erythrocytes, and immune cells. In order to elucidate the infected and uninfected cell types in KS tumors, we examined skin and blood samples from twelve subjects by single cell RNA sequence analyses. Two populations of KSHV-infected cells were identified, one of which represented a proliferative fraction of lymphatic endothelial cells, and the second represented an angiogenic population of vascular endothelial tip cells. Both infected clusters contained cells expressing lytic and latent KSHV genes. Novel cellular biomarkers were identified in the KSHV infected cells, including the sodium channel SCN9A. The number of KSHV positive tumor cells was found to be in the 6% range in HIV-associated KS, correlated inversely with tumor-infiltrating immune cells, and was reduced in biopsies from HIV-negative individuals. T-cell receptor clones were expanded in KS tumors and blood, although in differing magnitudes. Changes in cellular composition in KS tumors were identified in subjects treated with antiretroviral therapy alone, or immunotherapy. These studies demonstrate the feasibility of single cell analyses to identify prognostic and predictive biomarkers. Author Summary: Kaposi sarcoma (KS) is a malignancy caused by the KS-associated herpesvirus (KSHV) that causes skin lesions, and may also be found in lymph nodes, lungs, gastrointestinal tract, and other organs in immunosuppressed individuals more commonly than immunocompetent subjects. The current study examined gene expression in single cells from the tumor and blood of these subjects, and identified the characteristics of the complex mixtures of cells in the tumor. This method also identified differences in KSHV gene expression in different cell types and associated cellular genes expressed in KSHV infected cells. In addition, changes in the cellular composition could be elucidated with therapeutic interventions.

The Incremental Cost of Incompatible Living Donor Kidney Transplantation: A National Cohort Analysis.

Incompatible living donor kidney transplantation (ILDKT) has been established as an effective option for end-stage renal disease patients with willing but HLA-incompatible living donors, reducing mortality and improving quality of life. Depending on antibody titer, ILDKT can require highly resource-intensive procedures, including intravenous immunoglobulin, plasma exchange, and/or cell-depleting antibody treatment, as well as protocol biopsies and donor-specific antibody testing. This study sought to compare the cost and Medicare reimbursement, exclusive of organ acquisition payment, for ILDKT (n = 926) with varying antibody titers to matched compatible transplants (n = 2762) performed between 2002 and 2011. Data were assembled from a national cohort study of ILDKT and a unique data set linking hospital cost accounting data and Medicare claims. ILDKT was more expensive than matched compatible transplantation, ranging from 20% higher adjusted costs for positive on Luminex assay but negative flow cytometric crossmatch, 26% higher for positive flow cytometric crossmatch but negative cytotoxic crossmatch, and 39% higher for positive cytotoxic crossmatch (p < 0.0001 for all). ILDKT was associated with longer median length of stay (12.9 vs. 7.8 days), higher Medicare payments ($91 330 vs. $63 782 p < 0.0001), and greater outlier payments. In conclusion, ILDKT increases the cost of and payments for kidney transplantation.

Combined Percutaneous Transrenal and Transfemoral Endovascular Recanalization and Angioplastic Reconstruction of a Disrupted Transplant Renal Artery Stent: A Novel Salvage Technique.

Renal artery stenosis is the most common vascular complication following renal transplantation. Percutaneous endovascular transluminal angioplasty with stenting is the treatment of choice for clinically significant renal artery stenosis. The authors present a case describing a novel combined transrenal parenchyma and transfemoral approach to repairing a disrupted transplant renal artery stent. The patient's allograft renal artery stenosis was initially managed via the standard percutaneous approach, but during follow-up the stent became disrupted and crushed, causing partial occlusion of the renal artery. This was manifested by persistently elevated serum creatinine values, lower extremity edema, and four-medication hypertension. After a failed traditional percutaneous transfemoral attempt, the authors were able to successfully access the renal arterial system via a combined transrenal and transfemoral approach, using an upper-pole artery through the renal parenchyma. This transrenal approach used a 3 Fr system, allowing the authors to get a wire across the stent, which they were previously unable to do. With wire access, they performed a balloon angioplastic reconstruction to restore the stent's patency, resulting in a reduction in serum creatinine, lower extremity edema, and blood pressure. This technique avoided a potentially difficult reoperative repair without immediate complication and provides a method for vascular access to the renal arterial system in select patients.

Vascular management during live donor nephrectomy: an online survey among transplant surgeons.

In 2006, a survey from the American Society of Transplant Surgeons disclosed significant and sometimes fatal hemorrhagic events in live donor nephrectomies (LDN) related to failure of clips, leading to the contraindication of the Weck® Hem-o-lok® clip for control of the renal artery during LDN. A survey regarding vascular control techniques, their perceived safety ratings and their failures was sent to 645 European Society for Organ Transplantation members who profiled their profession as "surgeon" and selected "kidney" as organ type. Two hundred forty-three (41%) members responded, of whom 171 (63.3%) independently perform LDN. Their responses were analyzed. For arterial and venous vascular control, the GIA™ and TA™stapler are used most frequently, and were rated the safest. Of the 121 reported hemorrhagic events, slippage and dislodgement of clips occurred at least 58 times, while stapler malfunction occurred at least 40 times. One donor death from hemorrhage related to clip dysfunction was reported. Hemorrhagic complications of LDN with fatal and non-fatal outcomes still occur. Strikingly, many surgeons do not use the vascular closing technique that they consider most safe. Failure of non-transfixion techniques is associated with greater risks for the donor. Control of major vessels in LDN must employ transfixion techniques for optimal donor safety.

Equipoise: ethical, scientific, and clinical trial design considerations for compatible pair participation in kidney exchange programs.

Compatible living donor/recipient pair participation (CPP) in kidney exchange (KE) transplantation may substantially increase transplant volumes and significantly mitigate the O blood group donor shortage in KE. Initial ethical analysis did not support CPP for two primary reasons: (1) KE would be "unbalanced," and (2) the possibility of undue influence experienced by the compatible pair living donor. Recent developments with CPP (modeling studies and small clinical experiences), have demonstrated substantial potential for increasing KE volumes. This encouraged us to reconsider initial ethical concerns, with a focus on the potential for a design of a prospective CPP clinical trial. This ethical reconsideration led us to conclude that the concept of unbalanced kidney exchanges (manifested primarily by differential benefit between compatible and incompatible pairs) is no longer as clear cut as originally conceived. In addition, application of two concepts substantially diminishes ethical concerns including: (1) "quasi-compatible" pairs, and (2) a priori definition of mitigating factors. We conclude that genuine uncertainty exists regarding whether kidney exchange is best performed with or without compatible pair participation and that a clinical trial is therefore warranted.

Changes in the influence of lymphoma- and HIV-specific factors on outcomes in AIDS-related non-Hodgkin lymphoma.

BACKGROUND: We undertook the present analysis to examine the shifting influence of prognostic factors in HIV-positive patients diagnosed with aggressive non-Hodgkin lymphoma (NHL) over the last two decades. PATIENTS AND METHODS: We carried out a pooled analysis from an existing database of patients with AIDS-related lymphoma. Individual patient data had been obtained prior from prospective phase II or III clinical trials carried out between 1990 until 2010 in North America and Europe that studied chemo(immuno)therapy in HIV-positive patients diagnosed with AIDS-related lymphomas. Studies had been identified by a systematic review. We analyzed patient-level data for 1546 patients with AIDS-related lymphomas using logistic regression and Cox proportional hazard models to identify the association of patient-, lymphoma-, and HIV-specific variables with the outcomes complete response (CR), progression-free survival, and overall survival (OS) in different eras: pre-cART (1989-1995), early cART (1996-2000), recent cART (2001-2004), and contemporary cART era (2005-2010). RESULTS: Outcomes for patients with AIDS-related diffuse large B-cell lymphoma and Burkitt lymphoma improved significantly over time, irrespective of baseline CD4 count or age-adjusted International Prognostic Index (IPI) risk category. Two-year OS was best in the contemporary era: 67% and 75% compared with 24% and 37% in the pre-cART era (P < 0.001). While the age-adjusted IPI was a significant predictor of outcome in all time periods, the influence of other factors waxed and waned. Individual HIV-related factors such as low CD4 counts (<50/mm(3)) and prior history of AIDS were no longer associated with poor outcomes in the contemporary era. CONCLUSIONS: Our results demonstrate a significant improvement of CR rate and survival for all patients with AIDS-related lymphomas. Effective HIV-directed therapies reduce the impact of HIV-related prognostic factors on outcomes and allow curative antilymphoma therapy for the majority of patients with aggressive NHL.

Hypothermic machine preservation facilitates successful transplantation of "orphan" extended criteria donor livers.

Hypothermic machine preservation (HMP) remains investigational in clinical liver transplantation. It is widely used to preserve kidneys for transplantation with improved results over static cold storage (SCS). At our center, we have used HMP in 31 adults receiving extended criteria donor (ECD) livers declined by the originating United Network for Organ Sharing region ("orphan livers"). These cases were compared to ECD SCS cases in a matched cohort study design. Livers were matched for donor age, recipient age, cold ischemic time, donor risk index and Model for End-Stage Liver Disease (MELD) score. HMP was performed for 3-7 h at 4-8 °C using our previously published protocol. Early allograft dysfunction rates were 19% in the HMP group versus 30% in the control group (p = 0.384). One-year patient survival was 84% in the HMP group versus 80% in the SCS group (p = NS). Post hoc analysis revealed significantly less biliary complications in the HMP group versus the SCS group (4 vs. 13, p = 0.016). Mean hospital stay was significantly shorter in the HMP group (13.64 ± 10.9 vs. 20.14 ± 11.12 days in the SCS group, p = 0.001). HMP provided safe and reliable preservation in orphan livers transplanted at our center.

Quantifying the risk of incompatible kidney transplantation: a multicenter study.

Incompatible live donor kidney transplantation (ILDKT) offers a survival advantage over dialysis to patients with anti-HLA donor-specific antibody (DSA). Program-specific reports (PSRs) fail to account for ILDKT, placing this practice at regulatory risk. We collected DSA data, categorized as positive Luminex, negative flow crossmatch (PLNF) (n = 185), positive flow, negative cytotoxic crossmatch (PFNC) (n = 536) or positive cytotoxic crossmatch (PCC) (n = 304), from 22 centers. We tested associations between DSA, graft loss and mortality after adjusting for PSR model factors, using 9669 compatible patients as a comparison. PLNF patients had similar graft loss; however, PFNC (adjusted hazard ratio [aHR] = 1.64, 95% confidence interval [CI]: 1.15-2.23, p = 0.007) and PCC (aHR = 5.01, 95% CI: 3.71-6.77, p < 0.001) were associated with increased graft loss in the first year. PLNF patients had similar mortality; however, PFNC (aHR = 2.04; 95% CI: 1.28-3.26; p = 0.003) and PCC (aHR = 4.59; 95% CI: 2.98-7.07; p < 0.001) were associated with increased mortality. We simulated Centers for Medicare & Medicaid Services flagging to examine ILDKT's effect on the risk of being flagged. Compared to equal-quality centers performing no ILDKT, centers performing 5%, 10% or 20% PFNC had a 1.19-, 1.33- and 1.73-fold higher odds of being flagged. Centers performing 5%, 10% or 20% PCC had a 2.22-, 4.09- and 10.72-fold higher odds. Failure to account for ILDKT's increased risk places centers providing this life-saving treatment in jeopardy of regulatory intervention.

Kidneys at higher risk of discard: expanding the role of dual kidney transplantation.

Half of the recovered expanded criteria donor (ECD) kidneys are discarded in the United States. A new kidney allocation system offers kidneys at higher risk of discard, Kidney Donor Profile Index (KDPI)>85%, to a wider geographic area to promote broader sharing and expedite utilization. Dual kidney transplantation (DKT) based on the KDPI is a potential option to streamline allocation of kidneys which otherwise would have been discarded. To assess the clinical utility of the KDPI in kidneys at higher risk of discard, we analyzed the OPTN/UNOS Registry that included the deceased donor kidneys recovered between 2002 and 2012. The primary outcomes were allograft survival, patient survival and discard rate based on different KDPI categories (<80%, 80-90% and >90%). Kidneys with KDPI>90% were associated with increased odds of discard (OR=1.99, 95% CI 1.74-2.29) compared to ones with KDPI<80%. DKTs of KDPI>90% were associated with lower overall allograft failure (HR=0.74, 95% CI 0.62-0.89) and better patient survival (HR=0.79, 95% CI 0.64-0.98) compared to single ECD kidneys with KDPI>90%. Kidneys at higher risk of discard may be offered in the up-front allocation system as a DKT. Further modeling and simulation studies are required to determine a reasonable KDPI cutoff percentile.

Risk factors associated with Clostridium difficile infection after kidney and pancreas transplantation.

BACKGROUND: Clostridium difficile infection (CDI) is a common cause of nosocomial antibiotic-associated diarrhea with an increased incidence reported in solid organ transplant recipients. We sought to determine if kidney and/or pancreas transplant recipients possess unique risk factors for CDI. METHODS: Between January 2009 and February 2011, 942 kidney and 56 pancreas transplants were performed at the 2 centers. Of these, 28 recipients (kidney, n = 24; pancreas, n = 4) developed CDI. Cases were matched to controls (n = 56) in a 1:2 ratio. RESULTS: Those with CDI were mostly male patients (82% vs. 48%, P = 0.003), deceased-donor organ recipients (86% vs. 64%, P = 0.045), more likely to have leukopenia (18% vs. 4%, P = 0.038), and had undergone a gastrointestinal procedure within 3 months preceding CDI diagnosis (18% vs. 4%, P = 0.038). Cases had higher cumulative and restricted antimicrobial exposure in days (37 ± 79 vs. 8 ± 12, P = 0.009 and 27 ± 69 vs. 7 ± 10, P = 0.032). Cephalosporin use was more common among cases (43% vs. 16%, P = 0.008). CONCLUSION: Careful antimicrobial selection and assurance of optimal treatment duration in the kidney and pancreas transplant population is prudent. Clinicians should have a heightened awareness of CDI risk particularly during periods of leukopenia and in the setting of gastrointestinal procedures.

Availability, utilization and outcomes of deceased diabetic donor kidneys; analysis based on the UNOS registry.

The number of kidneys obtained from deceased diabetic donors available for transplantation has increased >eightfold increase in the past 15 years. We assessed allograft outcomes associated with deceased diabetic donors and compared them with that of standard and extended criteria donors (ECD) in the UNOS data registry. We identified 1982 recipients of diabetic standard criteria donors over a 10-year period from 1995 through 2004. Both overall and death-censored survival of organs from diabetic standard criteria donors was significantly better than that of organs obtained from nondiabetic ECD while inferior to that from nondiabetic standard criteria donors. Compared with ECD donors, diabetic donors had lower serum creatinine, less cold ischemia and these kidneys were less likely to be pump-perfused. Recipients of diabetic kidneys were younger and less likely to experience delayed graft function compared with recipient of ECD kidneys. More recently, many diabetic donor kidneys have been given to diabetic recipients with early graft survival being similar to that among nondiabetic recipients. These findings demonstrate the potential to expand and to improve utilization of this resource without compromising outcomes for recipients. Improved, evidence-based evaluation and allocation of deceased diabetic donor kidneys is needed to optimize their use.

Regulatory failure contributing to deaths of live kidney donors.

Hemorrhagic deaths of living kidney donors from failure of vascular clips used on the renal artery, first documented in 2006, have continued due to postoperative Hem-o-lok clip failure with sudden, massive bleeding. While the FDA issued a Class II recall of the Hem-o-lok clip for laparoscopic donor nephrectomies in 2006, two live kidney donors in the United States and one in India have since died. Compliance in timely reporting of deaths by the manufacturer and donor hospitals has not been enforced. Oversight agencies did not inform practitioners that donors died due to clip failures. A February 2011 survey disclosed that Hem-o-lok or other clips are still used by some surgeons as a sole means of arterial control in laparoscopic donor nephrectomy; thus, a practice with documented fatal outcomes persists. We conclude that systems failures by oversight-regulatory agencies in communication to active clinicians led, at least in part, to preventable deaths. Information which was disseminated was neither complete nor timely. A corrective plan, funded by oversight agencies and the Hem-o-lok manufacturer, is proposed. All surgeons operating on a living organ donor must select vascular control techniques that entail tissue transfixion and assure a safe operative recovery. The Hem-o-lok and other surgical clips must not be used to control the donor renal artery.

Reduced fracture risk with early corticosteroid withdrawal after kidney transplant.

Corticosteroid use after kidney transplantation results in severe bone loss and high fracture risk. Although corticosteroid withdrawal in the early posttransplant period has been associated with bone mass preservation, there are no published data regarding corticosteroid withdrawal and risk of fracture. We hypothesized lower fracture incidence in patients discharged from the hospital without than with corticosteroids after transplantation. From the United States Renal Data System (USRDS), 77, 430 patients were identified who received their first kidney transplant from 2000 to 2006. Fracture incidence leading to hospitalization was determined from 2000 to 2007; discharge immunosuppression was determined from United Networks for Organ Sharing forms. Time-to-event analyses were used to evaluate fracture risk. Median (interquartile range) follow-up was 1448 (808-2061) days. There were 2395 fractures during follow-up; fracture incidence rates were 0.008 and 0.0058 per patient-year for recipients discharged with and without corticosteroid, respectively. Corticosteroid withdrawal was associated with a 31% fracture risk reduction (HR 0.69; 95% CI 0.59-0.81). Fractures associated with hospitalization are significantly lower with regimens that withdraw corticosteroid. As this study likely underestimates overall fracture incidence, prospective studies are needed to determine differences in overall fracture risk in patients managed with and without corticosteroids after kidney transplantation.

When disaster strikes: death of a living organ donor.

Donor safety is of paramount importance in living donor transplantation. Yet, living donor deaths occur. We believe that problems exist in our system of live donor transplantation that can be summarized in a series of simple statements: (1) Donor mortality can never be completely eliminated; (2) Live donor risk has not been mitigated so that it is as low as possible; (3) After a donor death, systematic reviews are not routinely performed to identify correctable causes; (4) The lessons learned from any donor death are not adequately communicated to other programs and (5) The administrative mechanisms and resources are not universally available at all transplant centers to implement lessons learned. To rectify these problems, we propose the following: (1) A national living donor death task force be established with the purpose of performing systematic reviews of any donor death. (2) Findings of these reviews be disseminated to all institutions performing live donor transplants on a secure, password-protected website. (3) A no-fault donor death indemnity fund be established to provide a financial imperative for institutions to cooperate with this external peer-review. These measures will serve the best interests of the involved institutions, the transplant community, and most importantly, the patients and their families.

Recurrent focal segmental glomerulosclerosis in the renal allograft: single center experience in the era of modern immunosuppression.

FSGS is an important cause of ESRD and tends to recur in allografts (rFSGS). Older series suggest recurrence rates of 30-60%. In the modern era of transplant immunosuppression, recurrence rates are unknown. There are also few data regarding prevalence of known genetic mutations in adult FSGS patients who undergo transplantation. Recently, FSGS has been subdivided into histological variants, which may predict renal outcomes; there is little information on patterns of recurrence and outcomes in these variants. Finally, treatment for rFSGS relies upon up-titrating calcineurin inhibitors and plasmapheresis. Insufficient information exists on the use of these regimens for rFSGS in the era of modern immunosuppression. We conducted a retrospective chart review involving all renal transplant recipients at Columbia University Medical Center from December 1999 to March 2007. Those with biopsy confirmed primary FSGS were included and information regarding baseline characteristics, histologic variants, genetics, treatment, and clinical outcomes were collected. FSGS recurred in 23% of patients. Those with collapsing histology on native kidney biopsy, tended to recur with the same histology. No known genetic mutations were identified among those with recurrence. Plasmapheresis resulted in complete or partial remission in 75% of those with recurrence. Recurrent FSGS resulted in a trend toward the combined outcome of ESRD or death compared to those without recurrence (27% vs. 12%). Modern immunosuppression does not reduce the rate of rFSGS, known genetic mutations are uncommon in such adult patients, collapsing FSGS tends to recur with the same histology, and plasmapheresis may be helpful in the treatment of recurrence.