Allogeneic Renal Transplantation Research Articles

CD154-CD40 T-cell co-stimulation pathway is a key mechanism in kidney ischemia-reperfusion injury

Ischemia-reperfusion occurs in a great many clinical settings and contributes to organ failure or dysfunction. CD154-CD40 signaling in leukocyte–endothelial cell interactions or T-cell activation facilitates tissue inflammation and injury. Here we tested a siRNA anti-CD40 in rodent warm and cold ischemia models to check the therapeutic efficacy and anti-inflammatory outcome of in vivo gene silencing. In the warm ischemia model different doses were used, resulting in clear renal function improvement and a structural renoprotective effect. Renal ischemia activated the CD40 gene and protein expression, which was inhibited by intravenous siRNA administration. CD40 gene silencing improved renal inflammatory status, as seen by the reduction of CD68 and CD3 T-cell infiltrates, attenuated pro-inflammatory, and enhanced anti-inflammatory mediators. Furthermore, siRNA administration decreased a spleen pro-inflammatory monocyte subset and reduced TNFα secretion by splenic T cells. In the cold ischemia model with syngeneic and allogeneic renal transplantation, the most effective dose induced similar functional and structural renoprotective effects. Our data show the efficacy of our siRNA in modulating both the local and the systemic inflammatory milieu after an ischemic insult. Thus, CD40 silencing could emerge as a novel therapeutic strategy in solid organ transplantation.

Prolonged ischemia elicits acute allograft rejection involved in CXCR3 activation in rat kidney transplants

Acute rejection is a major obstacle in patients with prolonged ischemia in deceased-donor renal transplantation. Chemokines and their receptors play a critical role in leukocyte trafficking, resulting in allograft rejection; therefore, the role of chemokine receptor CXCR3 in acute rejection induced by prolonged ischemia in rat kidney transplantation models was evaluated. Syngeneic and allogeneic renal transplantations were performed. For cold ischemia, grafts were stored in 4.0°C University of Wisconsin solution for 12 or 16 h. Serum and renal tissues were harvested 7.0 d after surgery and serum TNF-α, IL-6, and renal function were measured. Graft histology was stained with periodic acid-Schiff and immunohistochemical staining and further evaluated for signs of acute rejection. CXCR3 proteins were quantified by Western blot. The transplanted rats were divided into 4 groups as follows: iso-12-h = isogeneic transplant with 12-h CIT graft; iso-16-h = isogeneic kidney transplant with 16-h CIT graft; allo-12-h = allogeneic renal transplant with 12-h CIT graft; allo-16 h = allogeneic renal transplant with 16-h CIT graft; and 16 h+T = allogeneic 16-h CIT graft received tacrolimus. Prolonged cold ischemia time (CIT; 16 h) enhanced acute glomerular damage, interstitial inflammation, and tubulointerstitial cellular infiltration in allografts with and without immunosuppressant tacrolimus; but it was not apparent in the isografts. The expression of CXCR3 protein and the proportion of CXCR3-positive cells were significantly higher in the allo-16 h and 16 h +T groups than that in the allo-12 h group 7d post-surgery. CIT triggered acute rejection in allogeneic, but not in isogeneic, kidney transplants, accompanied by an elevation of leukocyte recruitment and damaged graft function. The upregulated expression of chemokine receptor CXCR3 promoted inflammatory infiltration and acute allograft rejection.

Hydrogen Sulfide Treatment Mitigates Renal Allograft Ischemia-Reperfusion Injury during Cold Storage and Improves Early Transplant Kidney Function and Survival Following Allogeneic Renal Transplantation

Ischemia-reperfusion injury is unavoidable during organ transplantation. Prolonged ischemia-reperfusion injury is detrimental to short-term and long-term graft function and survival. H2S is a recently characterized, endogenously produced gaseous molecule with important physiological roles that has been shown to be cytoprotective during tissue ischemia-reperfusion injury. The current study aimed to determine whether H2S could mitigate cold renal ischemia-reperfusion injury in the clinically relevant context of allogeneic renal transplantation. Following bilateral native nephrectomy Lewis rats underwent renal transplantation with kidneys from Brown Norway donor rats that were flushed with cold (4C) standard University of Wisconsin preservation solution (University of Wisconsin preservation solution group) or cold University of Wisconsin preservation solution plus 150 μM NaHS (H2S group) solution. Kidneys were stored for 6 hours at 4C in the same solution. Recipient animals were monitored for 14 days or until sacrifice using metabolic cages to assess various parameters of renal graft function. H2S treatment improved early allograft survival and function, and decreased early levels of necrosis, apoptosis and Kim-1 compared to University of Wisconsin preservation solution alone. H2S treatment did not affect allograft rejection. Rather, it modulated the early allograft transcriptome to decrease the expression of renal injury, coagulation and cellular stress response genes, and increase the expression of cellular proliferation and Ifn-γ induced genes compared to University of Wisconsin preservation solution alone. To our knowledge our findings are the first to show that H2S protects donor kidneys against cold ischemia-reperfusion injury in the context of allogeneic renal transplantation. This potentially represents a novel cost-effective therapeutic solution to mitigate ischemia-reperfusion injury and improve the clinical outcomes of renal transplantation.

Combined Central Retinal Vein and Cilioretinal Artery Occlusion in a Patient Who Underwent Allogeneic Renal Transplantation

A high incidence of venous thromboembolism has been reported in patients who have had renal transplantation especially within the early postoperative period. Herein, the management of a 31year-old renal graft recipient with the diagnosis of combined left central retinal vein occlusion (CRVO) and cilioretinal artery occlusion (CRAO) is presented. Although detailed work-up for inherited as well as acquired thrombophilic disorders was performed in our case, none of the definite prothrombotic and predisposing risk factors for venous thromboembolism was identified. The case under review or the index case had a remarkable visual recovery within 3 weeks after the referral, since anterior chamber paracentesis was immediately scheduled after performing ocular massage in order to achieve acute resolution of venous stasis.

P31 Hydrogen sulfide treatment improves graft function and survival following prolonged cold ischemia and allogeneic renal transplantation

Organ procurement is inherently associated with ischemia–reperfusion injury (IRI), resulting from loss and subsequent restoration of blood flow, and is detrimental to graft function and survival. Treatment of donor organs with small molecules such as hydrogen sulfide (H 2 S) may be a novel method of mitigating IRI during transplantation. We postulated that H 2 S treatment could mitigate IRI during cold organ storage and subsequent genetically mismatched (allogeneic) renal transplantation (RTx), improving allograft function and survival. Following bilateral native nephrectomy, recipient Lewis rats underwent RTx with kidneys obtained from Brown Norway donor rats that were flushed with either University of Wisconsin preservation solution (UW group) or UW + H 2 S (150 μM NaHS; H 2 S group) and stored for 24 h at 4°C in the same solution. Serum was obtained on subsequent post-operative (PO) days and creatinine was used as a surrogate marker of graft function. H 2 S treated animals exhibited significantly improved survival compared to UW animals ( p < 0.01), with UW animals exhibiting 0% survival by PO day 4 and H 2 S treated animals retaining 20% survival by PO day 14 (end of time course). As well, UW treated animals exhibited markedly increased serum creatinine levels at both PO days 2 and 4 compared to H 2 S animals. These findings indicate that H 2 S treatment can mitigate graft injury following prolonged periods of cold IRI, improving initial graft function and possibly delaying graft rejection. H 2 S treatment could represent a novel method of mitigating IRI during transplantation, thereby improving clinical outcomes.

(762) - Efficacy and Safety of Basiliximab as Curative Treatment of Persistent or Complicated Acute Rejection in Cardiac Transplantation

Basiliximab ( Simulect ) is a monoclonal antibody specifically directed against the alpha chain ( CD25 ) of the interleukin 2 receptor. It inhibits the activation of T lymphocytes that play a major role in the mechanism of acute rejection. It is indicated for the prevention of acute rejection in de novo allogeneic renal transplantation in adults and children and is commonly used as induction therapy in solid organ transplantation. We report a retrospective analysis of the efficacy and safety of basiliximab administered as curative treatment of acute rejection in heart transplantation.

Allogene Nierentransplantation

Allogeneic renal transplantation is the best treatment for many patients with chronic renal failure and end-stage kidney disease. Especially the health-related quality of life markedly improves after renal transplantation and the side effects of dialysis treatment as well as the progression of organ and tissue deterioration related to renal failure which are not treated effectively by dialysis are greatly reduced. To achieve good results of renal transplantation, however, the best possible preoperative as well as perioperative and postoperative conditions have to be established and patients on waiting lists need to be well prepared. Interdisciplinary patient care is needed before and after renal transplantation in order to achieve durable and long-term success of renal transplantation.

PharmGKB summary

Lamba, Vishala; Sangkuhl, Katrinb; Sanghavi, Kinjala; Fish, Alyssaa; Altman, Russ B.b,c; Klein, Teri E.b Author Information

Early treatment with xenon protects against the cold ischemia associated with chronic allograft nephropathy in rats

Chronic allograft nephropathy (CAN) is a common finding in kidney grafts with functional impairment. Prolonged hypothermic storage-induced ischemia-reperfusion injury is associated with the early onset of CAN. As the noble gas xenon is clinically used as an anesthetic and has renoprotective properties in a rodent model of ischemia-reperfusion injury, we studied whether early treatment with xenon could attenuate CAN associated with prolonged hypothermic storage. Exposure to xenon enhanced the expression of insulin growth factor-1 (IGF-1) and its receptor in human proximal tubular (HK-2) cells, which, in turn, increased cell proliferation. Xenon treatment before or after hypothermia-hypoxia decreased cell apoptosis and cell inflammation after reoxygenation. The xenon-induced HK-2 cell proliferation was abolished by blocking the IGF-1 receptor, mTOR, and HIF-1α individually. In the Fischer-to-Lewis rat allogeneic renal transplantation model, xenon exposure of donors before graft retrieval or recipients after engraftment enhanced tubular cell proliferation and decreased tubular cell death and cell inflammation associated with ischemia-reperfusion injury. Compared with control allografts, xenon treatment significantly suppressed T-cell infiltration and fibrosis, prevented the development of CAN, and improved renal function. Thus, xenon treatment promoted recovery from ischemia-reperfusion injury and reduced susceptibility to the subsequent development of CAN in allografts.

P57 Hydrogen sulfide treatment reduces activation of ischemia–reperfusion injury-induced tissue inflammatory processes and resultant recipient adaptive immune response to donor tissue during allogeneic renal transplantation

Background Organ procurement is inherently associated with ischemia–reperfusion injury (IRI), resulting from loss and subsequent restoration of blood flow and is detrimental to short- and long-term graft function and survival. Treatment of donor organs with endogenously produced gasotransmitters such as hydrogen sulfide (H 2 S) is a novel method of mitigating IRI during transplantation, as previously shown in a model of murine syngeneic renal transplantation (RTx) [1] . The protective effects of H 2 S against IRI during allogeneic RTx, in which genetically dissimilar grafts are targeted by the recipient immune system causing organ rejection, have not yet been studied. Since H 2 S is known to exhibit anti-inflammatory actions during tissue IRI, we postulated that H 2 S treatment during allogeneic RTx could reduce graft inflammation and modulate the resultant recipient immune response against donor tissue. Methods Following bilateral native nephrectomy, recipient Lewis rats underwent RTx with left kidneys obtained from Brown Norway donor rats that were flushed, at the time of procurement, with either cold (4°C) University of Wisconsin standard preservation solution (UW group) or cold UW + H 2 S donor molecule (150 μ M NaHS) (H 2 S group) and stored for 6 h at 4°C in the same solution. Sham animals were also followed. Renal grafts were obtained at post-operative day 2 and RNA was isolated from renal homogenates for gene microarray analysis (Affymetrix Rat Gene 1.0 ST Microarray). Additionally, serum was collected post-operatively at day 1 to 2 and at time of sacrifice to determine the relative abundance of allo-reactive serum antibodies. TIB-120 cells (ATCC), a rat B-cell line heterozygous for Brown Norway and Lewis alleles, were incubated with serum from transplant recipients in each treatment group, stained for bound rat IgG/M antibodies and analyzed via flow cytometry. Results Upon microarray analysis both treatment groups exhibited significant enrichment in genes involved in inflammatory and innate immune responses as well as cytokine signaling and antigen processing at day 2 compared to Sham (p 2 S treated grafts had markedly reduced enrichment scores in each of these pro-inflammatory processes compared to UW. Upon allo-antibody binding analysis both UW and H 2 S treatment groups exhibited similar trends, with more cells unbound by allo-antibodies at day 1 to 2 and few unbound cells at time of sacrifice. However, while cells incubated with UW serum at day 1 to 2 exhibited markedly more allo-antibody binding (27% unbound cells) compared to cells incubated with naive Sham serum (37% unbound cells), cells incubated with H 2 S serum at day 1 to 2 exhibited similar levels of allo-antibody binding (40% cells unbound) as those incubated with Sham serum. These findings indicate that H 2 S treated animals experienced less acute graft inflammation, leading to a delayed induction of adaptive immune response to donor tissue compared to UW alone. Conclusions These findings are the first to report that H 2 S treatment may modulate the recipient immune response to donor tissue during allogeneic RTx. This phenomenon could represent an important underlying mechanism of H 2 S-mediated protection against renal injury early in the transplant curriculum, ultimately prolonging the survival and function renal allografts.

Therapeutic prospects of mycophenolate mofetil for the treatment of neurodermatitis

Mycophenolate mofetil (MMF), an inhibitor of inosine 5′-monophosphate dehydrogenase, serves to suppress the immune response against alloantigens by hindering purine nucleotide synthesis in immune cells. At present, MMF is US FDA approved for the prevention of allogeneic renal transplant rejection. In this report, the authors demonstrate, to their knowledge, the first case of successful treatment of neurodermatitis complicated by lichen simplex chronicus and prurigo nodularis with MMF.

PET with 18F-FDG–Labeled T Lymphocytes for Diagnosis of Acute Rat Renal Allograft Rejection

We proposed small-animal PET with (18)F-FDG-labeled T lymphocytes as a new method for image-based diagnosis of acute allogeneic renal transplant rejection (AR) established in a rat model. One and 2 h after tail vein injection of 30 × 10(6) ex vivo (18)F-FDG-labeled human T cells into male 10-wk-old uninephrectomized, allogeneically transplanted rats (aTX; Lewis-brown Norway [LBN] to Lewis), whole-body radioactivity distribution was assessed in vivo by small-animal PET (postoperative day 4), and percentage injected dose (%ID) as a parameter of T-cell infiltration was assessed and compared between graft and native kidney. In vivo results were confirmed by autoradiography and staining of human CD3 after postmortem dissection. Syngeneically transplanted rats (sTX) (LBN to LBN), rats with ischemia-reperfusion injury (IRI) (45-min warm ischemia), and rats subjected to acute cyclosporine A (CSA) toxicity (50 mg/kg for 2 d intraperitoneally) served as controls. The accumulation of labeled cells was significantly elevated in allografts with AR (1.07 ± 0.28 %ID), compared with native control kidneys (0.49 ± 0.18 %ID) (P < 0.0001). No differences were found among native controls, sTX, CSA toxicity, and kidneys with IRI. In vivo uptake of (18)F-FDG cells measured in the PET scanner correlated with results obtained by autoradiography, histologic evaluation, and polymerase chain reaction. We proposed graft PET imaging using (18)F-FDG-labeled T cells as a new option to detect rat renal AR with a low dose of (18)F-FDG in a noninvasive, fast, and specific manner in rats.

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using &lt;sup&gt;18&lt;/sup&gt;F-FDG PET

The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function (1). At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models. Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR (2). We herein present in addition small animal positron emission tomography (PET) using (18)F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation (3). Further, this method can be applied for follow-up to improve monitoring of transplant rejection (4).

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using &lt;sup&gt;18&lt;/sup&gt;F-FDG PET

The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function 1. At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models. Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR 2. We herein present in addition small animal positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation 3. Further, this method can be applied for follow-up to improve monitoring of transplant rejection 4.

Prolonged hypocalcaemia following a single dose of 60 mg denosumab in two patients with CKD 4/5 on cinacalcet treatment for tertiary hyperparathyroidism.

Torregrosa reports a rare case of dramatic increase in intact parathyroid hormone (iPTH) and hypocalcaemia after administration of denosumab in a kidney-transplanted patient [1]. However, the author does not comment on another important finding of this case which is the unusually prolonged hypocalcaemia. In the only study to have investigated this issue so far, Block et al. report that in the chronic kidney disease (CKD) 4 subgroup, adjusted serum calcium (ACa) levels return to normal within 15 days [2], but in Torregrosa's report the patient still remains hypocalcaemic 6 months after denosumab [1]. We report a similar case of a 64-year-old female with an allogeneic renal transplant 3 years prior to presenting to the clinic with stable CKD4 and tertiary hyperparathyroidism, and presented with sustained multiple fragility fractures secondary to osteoporosis. Her medication included alfacalcidol 500 ng daily, cinacalcet 60 mg daily, tacrolimus, and prednisolone. Biochemistry was as follows: ACa 2.37 mmol/L (9.48 mg/dL) (always within the normal range in the last year), estimated glomerular filtration rate (eGFR) 16 mL/min/1.73 m2, iPTH >190 pmol/L (1.79 ng/dL) and 25(OH)vitamin D 34.2 nmol/L (13.7 ng/mL). Following administration of denosumab 60 mg, ACa concentrations decreased to 1.96 mmol/L (7.84 mg/dL) at Day 16, returning to normal at Day 93. iPTH remained persistently elevated at >100 pmol/L (943 pg/mL). Throughout this period, the patient had adequate dietary calcium intake and did not change medication. Block also reports that in the CKD 5 subgroup, ACa returns to normal by Day 60 after denosumab [2]. We report an unusually prolonged (almost 5 months) and poor response to treatment for hypocalcaemia following administration of 60 mg denosumab in a patient with CKD5. A 64-year-old female presented with osteoporosis and a femoral fragility fracture. Past medical history included CKD5 secondary to IgA nephropathy, 11 years of haemodialysis treatment, tertiary hyperparathyroidism and treated non-metastatic breast cancer. Medication included cinacalcet 60 mg daily, alfacalcidol 1 µg daily and letrozole (all taken long term in an unchanged dose). Biochemistry was as follows: ACa 2.45 mmol/L (9.8 mg/dL) (always within the normal range in the last 12 months), eGFR 9 mL/min/1.73 m2 and PTH 54.8 pmol/L (517 pg/dL). Following denosumab, ACa decreased to 1.97 mmol/L (7.88 mg/dL) at Day 30 and remained low for the following 3 months [2.04 mmol/L (8.16 mg/dL) at Day 91]. Similarly to Torregrosa's case, iPTH rose from 54.8 to 108 pmol/L (1.02 ng/dL) at the nadir of hypocalcaemia. Throughout this period, medication and dialysis settings were not changed and the patient had adequate dietary calcium intake. On Day 91, alfacalcidol was increased to 4 µg daily and cinacalcet was reduced to 30 mg daily, but this resulted in only a modest improvement in ACa to 2.26 mmol/L (9.04 mg/dL) at Day 139. From our two cases and from Torregrosa's case, it appears that hypocalcaemia following denosumab can be more prolonged than previously thought even with adequate calcium and vitamin D supplementation. We hypothesized that in our two cases, the prolonged hypocalcaemia was due to the combined effect of denosumab and cinacalcet, both of which are known to lower serum calcium levels. However, Torregrosa's case points out that there might be other possible causes and there is scope for further studies.

Ganciclovir Prophylaxis Modifies Long-Term Murine Cytomegalovirus Induced Renal Allograft Injury

Alabama at Birmingham, Genetics, Birmingham, United States Background: Human cytomegalovirus (CMV) infection is associated with adverse outcomes in renal transplantation, but the pathogenesis remains cryptic. Ganciclovir prophylaxis is associated with improved survival in some but not all clinical studies. In a murine CMV (MCMV) donor positive/recipient negative allogeneic renal transplant model, MCMV exacerbated innate infiltrates and early histologic injury at 2 weeks post-transplant, and these parameters improved with ganciclovir prophylaxis. The effect of ganciclovir prophylaxis on late allograft histology has not been examined. Methods: Murine CMV (MCMV) infected Balb/c donor kidneys were transplanted orthotopically into C57Bl/6 recipients with cyclosporine immunosuppression (CMV group). Control transplants were performed using uninfected Balb/c donors (control group). Ganciclovir (GCV) prophylaxis was administered for 14 days post-transplant to one cohort of MCMV infected transplants (GCV group). Animals were sacrificed at day 42 post-transplant. Histology was scored according to an established grading scale (grade 0-3 for 9 criteria, maximum score 27) by a veterinary pathologist blinded to sample identity. Immune infiltrates were examined by flow cytometry in allograft kidneys, livers, and spleens. Results: At day 42, CMV transplants had higher aggregate damage scores (average 14.5) compared to control uninfected transplants (average 10.25). Allografts with GCV prophylaxis had lower damage scores (average 9.5) and appeared histologically more similar to uninfected grafts than to CMV infected grafts. Analysis of leukocyte infiltrates by flow cytometry revealed greater frequencies of CD45+ cells in CMV infected compared to uninfected grafts. Of the CD45+ population, CMV infected and uninfected grafts had similar frequencies of CD4+, CD8+, and CD19+ lymphocytes, but CMV infected grafts contained higher frequencies of CD49b+ and Gr-1+ cells compared to uninfected grafts. GCV prophylaxis reduced the frequency of Gr-1+ infiltrates but not CD49b+ infiltrates, and also contained a lower frequency of CD11b+/CD204+ cells. Grafts with GCV prophylaxis contained greater frequencies of CD4+ cells compared to both CMV and control grafts. Analysis of infiltrates in livers and spleens from allograft recipients showed no differences in frequencies of any of these leukocyte subsets between control, CMV, and GCV treated recipients. Conclusion: CMV infection exacerbates late renal allograft damage by histologic criteria and is associated with ongoing infiltration of innate leukocyte subsets including NK cells and myeloid cells. Allografts with GCV prophylaxis had less severe allograft injury and appeared histologically similar to uninfected grafts, even though prophylaxis had been discontinued for 28 days prior to terminal sacrifice. GCV prophylaxis was associated with reduction of myeloid and macrophage infiltrates but not NK infiltrates, and additionally was associated with increased CD4+ infiltrates. These results suggest that ganciclovir prophylaxis may modulate long-term CMV effects upon allograft injury, which may possibly be related to differences in recruitment of innate and CD4+ leukocytes even after cessation of prophylaxis. Intragraft responses differed from leukocyte populations in the liver and spleen, suggesting that systemic responses may not necessarily serve as surrogates for the intragraft inflammatory response.

Killer Immunoglobulin-like Receptor (KIR) and HLA Genotypes Affect the Outcome of Allogeneic Kidney Transplantation

BackgroundRecipient NK cells may detect the lack of recipient's (i.e., self) HLA antigens on donor renal tissue by means of their killer cell immunoglobulin-like receptors (KIRs). KIR genes are differently distributed in individuals, possibly contributing to differences in response to allogeneic graft.Methodology/Principal FindingsWe compared frequencies of 10 KIR genes by PCR-SSP in 93 kidney graft recipients rejecting allogeneic renal transplants with those in 190 recipients accepting grafts and 690 healthy control individuals. HLA matching results were drawn from medical records. We observed associations of both a full-length KIR2DS4 gene and its variant with 22-bp deletion with kidney graft rejection. This effect was modulated by the HLA-B,-DR matching, particularly in recipients who did not have glomerulonephritis but had both forms of KIR2DS4 gene. In contrast, in recipients with glomerulonephritis, HLA compatibility seemed to be much less important for graft rejection than the presence of KIR2DS4 gene. Simultaneous presence of both KIR2DS4 variants strongly increased the probability of rejection. Interestingly, KIR2DS5 seemed to protect the graft in the presence of KIR2DS4fl but in the absence of KIR2DS4del.Conclusions/SignificanceOur results suggest a protective role of KIR2DS5 in graft rejection and an association of KIR2DS4 with kidney rejection, particularly in recipients with glomerulonephritis.

Mesenchymal Stem Cells for the Treatment of Diabetes

The field of regenerative medicine is rapidly evolving, paving the way for novel therapeutic interventions through cellular therapies and tissue engineering approaches that are reshaping the biomedical field. The remarkable plasticity of different cell subsets obtained from human embryonic and adult tissues from disparate sources (including bone marrow, umbilical cord, amniotic fluid, placenta, and adipose tissue) has sparked research endeavors evaluating use of these cells for numerous conditions, including diabetes and its complications (1). A readily accessible source for multipotent stem cells is the bone marrow, which comprises progenitors of hematopoietic, endothelial, and mesenchymal stem cells (MSCs). Unfractioned and fractioned bone marrow–derived stem cells have been used in experimental and clinical settings to improve diabetes and diabetes complications. Bone marrow–derived MSCs are stromal, nonhematopoietic cells generally obtained from iliac crest aspirates following enrichment based on their preferential adhesion on culture vessels in defined media. MSC characterization relies on expression of specific surface markers and on their ability to differentiate into fat, bone, and cartilage when exposed to appropriate culture conditions (2). Recent clinical trials have demonstrated powerful immunomodulatory effects of the inoculum of MSCs to treat graft-versus-host disease (3,4), to improve allogeneic renal transplant outcomes using lower immunosuppressive regimens (5), and to reduce immune cell activation in patients with multiple sclerosis and amyotrophic lateral sclerosis (6). Autologous MSCs were shown to improve Crohn disease lesions refractory to …

Atorvastatin donor pre-treatment in a model of brain death and allogeneic kidney transplantation in rat

The aim of the present study was to evaluate the effect of donor pre-treatment with atorvastatin in a model of brain death followed by prolonged cold preservation and allogeneic kidney transplantation in rats. Donor rats were pre-treated with atorvastatin or vehicle 2 days prior to induction of brain death. After a brain death period of 6h kidneys were explanted and stored for 24 h at 4°C in UW solution and transplanted into allogeneic recipients. Non brain dead rats treated with vehicle were ventilated for 6h prior to explantation and served as controls. Grafts were harvested after 10 days. Donor treatment of brain dead organ donors with atorvastatin had no influence on renal histology (Banff score) or renal inflammation compared to vehicle treated brain dead rats 10 days after cold preservation and allogeneic transplantation. Grafts from brain dead organ donors showed severe signs of vasculopathy compared to grafts from non brain dead organ donors 10 days after prolonged cold preservation and allogeneic kidney transplantation. Kidneys harvested after brain death before cold preservation and allogeneic transplantation showed an increased infiltration of ED1 and MHCII positive cells compared to kidneys of non-brain dead animals (NBD). Atorvastatin donor pre-treatment of brain dead organ donors combined with 24h of cold preservation has no influence on graft rejection and infiltration with ED1 or MHCII positive cells in an allogeneic rat renal transplantation model. The detrimental combination of brain death and cold preservation might have overcome the possible protective effect of atorvastatin donor pre-treatment.

Mycophenolate Mofetil: Use of a Simple Dissolution Technique to Assess Generic Formulation Differences

Mycophenolate mofetil (MMF) is an immunosuppressive agent indicated for the prophylaxis of acute rejection in patients r eceiving allogeneic renal, cardiac, or hepatic transplants. It is a Biopharmaceutics Classification System Class II substance that has a strongly pH-dependent solubility profile. Consequently, differences in solid-state properties, formulation, and manufacturing processes of MMF can lead to disparities in bioavailability between brands of the same drug. This study was conducted to compare the in vitro dissolution profile of the original MMF innovator brand (CellCept, Roche) with available generic products. Two representative batches of CellCept 500-mg tablets and 14 different generic formulations were tested using different dissolution testing scenarios simulating conditions in the proximal gastrointestinal tract. These scenarios took into account stomach and small intestine media composition, surface tension, pH, increased buffer capacity, and osmolarity after food intake. Eight of the generic formulations tested passed the quality control dissolution test (pH 1.1) according to the specification Q = 75% after 5 min (i.e., all single units >80% dissolved), and 12 passed the specification Q = 85% after 15 min (i.e., all single units >90% dissolved). This suggests an almost homogenous dissolution rate between formulations in an acidic environment. However, at pH 4.5, large variations in the in vitro dissolution performance between generic formulations were observed (extremes resulting in greater than 60% dissolved difference after 30 min). Marked variability was seen among the different generic formulations and the innovator brand, CellCept. In conclusion, important differences exist among the different generic formulations with regard to in vitro performance. As MMF is required for life-long use, changes in drug performance as a result of switching between formulations may have serious clinical consequences (e.g., organ rejection). Therefore, clinical testing is necessary to evaluate the pharmacokinetics and the impact on clinical safety of a change of brands.