Blockchain-Based Authentication and Trust Management Mechanism for Smart Cities.

Security has always been the main concern for the internet of things (IoT)-based systems. Blockchain, with its decentralized and distributed design, prevents the risks of the existing centralized methodologies. Conventional security and privacy architectures are inapplicable in the spectrum of IoT due to its resource constraints. To overcome this problem, this paper presents a Blockchain-based security mechanism that enables secure authorized access to smart city resources. The presented mechanism comprises the ACE (Authentication and Authorization for Constrained Environments) framework-based authorization Blockchain and the OSCAR (Object Security Architecture for the Internet of Things) object security model. The Blockchain lays out a flexible and trustless authorization mechanism, while OSCAR makes use of a public ledger to structure multicast groups for authorized clients. Moreover, a meteor-based application is developed to provide a user-friendly interface for heterogeneous technologies belonging to the smart city. The users would be able to interact with and control their smart city resources such as traffic lights, smart electric meters, surveillance cameras, etc., through this application. To evaluate the performance and feasibility of the proposed mechanism, the authorization Blockchain is implemented on top of the Ethereum network. The authentication mechanism is developed in the node.js server and a smart city is simulated with the help of Raspberry Pi B+. Furthermore, mocha and chai frameworks are used to assess the performance of the system. Experimental results reveal that the authentication response time is less than 100 ms even if the average hand-shaking time increases with the number of clients.

Long- and short-term outcomes in renal allografts with deceased donors: A large recipient and donor genome-wide association study.

Improvements in immunosuppression have modified short-term survival of deceased-donor allografts, but not their rate of long-term failure. Mismatches between donor and recipient HLA play an important role in the acute and chronic allogeneic immune response against the graft. Perfect matching at clinically relevant HLA loci does not obviate the need for immunosuppression, suggesting that additional genetic variation plays a critical role in both short- and long-term graft outcomes. By combining patient data and samples from supranational cohorts across the United Kingdom and European Union, we performed the first large-scale genome-wide association study analyzing both donor and recipient DNA in 2094 complete renal transplant-pairs with replication in 5866 complete pairs. We studied deceased-donor grafts allocated on the basis of preferential HLA matching, which provided some control for HLA genetic effects. No strong donor or recipient genetic effects contributing to long- or short-term allograft survival were found outside the HLA region. We discuss the implications for future research and clinical application.

Investigating the relationship between class I HLA-specific immunoglobulin-G subclasses, Pan-IgG single antigen bead assays and complement mediated interference in sera from renal transplant recipients.

INTRODUCTION: Antibody mediated rejection is the leading cause of kidney transplant failure. Not all antibodies are harmful and some may be protective. Immunoglulin Gs, of which there are four subtypes, are detected by single antigen bead testing. The aims of this study were to characterise the IgG subclass profiles for class I HLA-specific antibodies in an uncensored post-transplant population and to determine the underlying relationship between reactivity patterns and MFI cut-offs with the pan-IgG assay. METHODS: Patients were recruited to the study who were transplanted in our centre between 2009 and 2014. Prospectively stored post-transplant serum initially underwent a Labscreen Mixed assay and those positive for class I HLA-specific antibody underwent standard SAB testing, EDTA, 1 in 10 dilution and IgG subclass modifications using the Luminex platform. A total of 4947 bead reactions from 51 patients were analysed. RESULTS: A 1 in 10 dilution was used as a comparator pan-IgG assay for summed subclass and individual subclass linear regression analyses. Using a dilution to standard assay ratio we characterised all reactions for prozone potential i.e. how likely there is to be inhibition related to complement complex formation. We stratified samples into degrees of association and were able to determine suggested MFI thresholds of Log 5.35 for the dilution assay and Log 5.05 for the summed subclass assay when considering a Log MFI of 6.9 (1000) in the standard assay. Using individual subclass dominant reactions (>70%) we were able to determine linear relationships between the 1 in 10 dilution pan-IgG assay and the individual subclass assays (excluding prozone potential reactions for IgG1/3) enabling us to suggest Log MFI thresholds of 5.03, 3.58, 4.3 and 4.05 respectively for IgG1-4. DISCUSSION: We recommend a 1 in 10 dilution as the optimum pan-IgG comparator assay for a subclass analysis. We advocate the utilisation of the summed subclass assay to determine overall relationships and potential subclass failures. Following others, we recommend serum pre-treatment of the subclass assays to mitigate prozone. We suggest cut-offs for each IgG subclass which should be used with caution given the many inhibitory influences which may include competitive inhibition for bead binding, IgM and IgA interference and under-representation of specific subclasses on the bead panel.

Investigating complement mediated interference in class I HLA-specific antibodies following renal transplantation.

INTRODUCTION: Single antigen bead testing (SAB) for HLA-specific antibody enables efficient organ allocation and aids in the diagnosis of antibody mediated rejection. In this retrospective cohort study, a population of kidney transplant recipients possessing HLA Class I antibodies was used to evaluate the best method for resolving complement interference, the so called "prozone" effect. The aim was to compare the use of EDTA versus a Biotin-Streptavidin Complex as methodological approaches for abating the prozone effect using a fixed 1 in 10 dilution as validation. METHODS: One hundred and seventeen patients transplanted in our centre between 2009 and 2014 were identified as having class I HLA-specific antibody(-ies) using a Labscreen® Mixed assay. Positive sera underwent class I HLA-specific SAB testing; for comparison a standard SAB with and without EDTA, BSC and dilution (1 in 10) modifications were utilised. Samples were processed on the Luminex platform generating 11,349 bead reactions for analysis. RESULTS: We identified sera from 23 patients giving rise to 170 bead reactions showing complement interference. Using linear modelling, we observed slightly higher MFIs on average in both EDTA and BSC modifications when compared to the standard assay, allowing the nominal threshold MFI of 2000 in the standard assay to be adjusted to 2097 and 2033 in the EDTA and BSC assays respectively. We calculated 99% prediction intervals to establish outlier bead reactions for each assay. The 1 in 10 dilution was used as a crosscheck for determining which prozone reactions were overcome by EDTA and BSC. Using ROC curve analysis, EDTA was found to be ~90% sensitive and 100% specific compared to BSC which was ~60% sensitive and 100% specific in ameliorating prozone positive reactions at the thresholds defined by linear models. DISCUSSION: Our data indicates that both EDTA and BSC are suitable assays in overcoming CMI. We recommend that all clinical laboratories adopt a validated assay designed specifically to abrogate CMI for all potential renal transplant recipients, as the standard assay is inhibited in nearly 20% of a post-transplant cohort.

Stent extrusion on the external surface of the transplanted kidney: unusual occurrence.

Here we present the case of a 40-year-old man, who underwent deceased donor renal transplantation. Towards the end of this operation, open-ended double J stent was inserted in the transplanted kidney. Modified Lich-Gregoir ureterovesical anastomosis was performed. Prior to the abdominal closure, it was discovered that proximal end of the stent had pierced the renal parenchyma and extruded on the external surface of the transplanted kidney. We contemplated removing the stent and reinserting it but decided against that due to various reasons. The stent was left as such. The patient was managed conservatively with satisfactory outcome in the postoperative period. To the best of our knowledge, this is first such report of conservative management of stent extrusion in transplanted kidney in the literature.

Induction of IL-8(CXCL8) and MCP-1(CCL2) with oxidative stress and its inhibition with N-acetyl cysteine (NAC) in cell culture model using HK-2 cell.

Renal transplantation can often be complicated due to delayed graft function, which is a direct sequel of ischaemia reperfusion injury. The adverse outcome of delayed graft function is not only short term but the long-term function of the graft is also affected. Therefore, it is important to understand the mechanisms of ischaemia reperfusion injury. Reactive oxygen species are the key mediators in ischaemia reperfusion injury causing direct cell damage which also initiate inflammation by inducing chemokines. The presence of inflammation is a marker of severe delayed graft function. However, the effect of oxidative stress on the expression of key chemokines has not been fully established yet. Therefore, the aim of this study was to measure the oxidative stress response and the secretion of chemokines in a cell culture model that mimics the effects of ischaemia reperfusion injury in immortalised human renal proximal tubular epithelial cells, HK-2. Cells were treated with varying concentrations of hydrogen peroxide and markers of oxidative stress response and chemokine release were measured. Exposure to hydrogen peroxide induced a significant increase in the activity of the antioxidant enzyme glutathione peroxidase and the levels of the chemokines Interleukin-8 (IL-8; CXCL8) and MCP-1 (CCL2). A dose related increase of chemokine secretion was also observed. The cytokine Interleukin-1ß (IL-1ß) at 1 ng/ml significantly potentiated the expression of both IL-8 (CXCL8) and MCP-1 (CCL2) which showed synergistic response in the presence of hydrogen peroxide. Pre-incubation of the cells with the anti-oxidant N-acetyl cysteine (NAC) strongly suppressed the induction of both IL-8 and MCP-1 when stimulated with hydrogen peroxide and IL-1ß. This study demonstrates the potential of anti-oxidants like N-acetyl cysteine in ameliorating the effects of ischaemia reperfusion injury thus suggesting a new therapeutic approach in renal transplantation. These findings can have potential implications for clinical use to prevent ischaemia reperfusion injury in renal transplantation.

Mixed lymphocyte cultures can predict TCR Vbeta repertoires of T cells infiltrating kidney transplants during acute rejection episodes.

Alloreactive T cell populations can show skewing of T-cell antigen receptor (TCR) Vbeta gene usage. The aims of the experiments were to compare in vivo and in vitro T cell alloresponses against donor alloantigens for TCR Vbeta gene usage. T-cell cultures from renal biopsies taken during acute rejection and pretransplant mixed lymphocyte cultures (MLC) were established from five renal transplant patients. TCR Vbeta gene usage, assessed with Vbeta family specific antibodies, showed that up to five different Vbeta families were significantly expanded. In four of five cases, there was close concordance between Vbeta families expanded from the biopsy and in MLC. T-cell clones from one renal biopsy were specific for the mismatched donor alloantigen and showed similar TCR Vbeta gene usage to the original T-cell line. The results show very similar patterns of TCR Vbeta gene usage in alloreactive T cells generated ex vivo or in vitro.

Calcineurin inhibitors and proximal renal tubular injury in renal transplant patients with proteinuria and chronic allograft nephropathy.

BACKGROUND: Chronic allograft nephropathy (CAN) is commonly associated with proteinuria. In native nephropathies, proteinuria is linked with proximal renal tubular damage. This study uses regression analysis to link proteinuria with urinary N-acetyl-beta-d-glucosaminidase (NAG) as a marker of tubular injury or hyperfunction in renal transplant patients. METHODS: Proteinuria and urinary NAG were measured and regression analysis applied in 105 transplant patients (42 with CAN). Most were receiving calcineurin inhibitor-based immunosuppression (cyclosporine, n=60; tacrolimus, n=26; and neither drug, n=19). Patients with native nephropathies (n=96) and volunteers (n=21) were also studied. RESULTS: Urinary NAG increased with increasing proteinuria. However, patients taking calcineurin inhibitors had higher urinary NAG at any level of urinary protein than those on alternative therapy, or in native nephropathies. CONCLUSIONS: In groups of transplant patients taking different immunosuppressive regimens, regression analysis of urinary NAG against urinary protein can identify the separate effects of drug-related tubular injury or hyperfunction from that of proteinuria.

Oxidative stress in kidney transplant biopsies.

OBJECTIVES: Kidney allograft biopsies are performed after kidney transplant to determine graft dysfunction. We aimed to define and measure the oxidative stress occurring in these biopsies and compared these biopsies with donor pretransplant biopsies. MATERIALS AND METHODS: The biopsy procedure was done according to the unit protocol. A core of tissue was taken for research purposes only when it was safe enough to proceed for an extra core. Common indications for biopsy were acute or chronic graft dysfunction, delayed graft function, acute cellular rejection, and calcineurin toxicity. There were 17 pretransplant biopsies taken from deceased-donor kidneys. Biopsy specimens were snap frozen immediately in liquid nitrogen and stored at -70 °C. Samples were processed for Western blot and tested for markers of oxidative stress. RESULTS: There were 61 biopsies analyzed. Oxidative stress enzymes were evaluated by Western blot including catalase, manganese superoxide dismutase, copper zinc superoxide dismutase, thioredoxin reductase, and thioredoxin. Upregulation of most antioxidant enzymes was observed in pretransplant biopsies. Increased expression of manganese superoxide dismutase was observed in donor kidneys and kidneys with acute cellular rejection and calcineurin toxicity. Copper zinc superoxide dismutase and catalase were elevated in donor and acute cellular rejection biopsies. Thioredoxin was elevated in donor biopsies and thioredoxin reductases were elevated in donor biopsies and biopsies with acute cellular rejection and calcineurin toxicity. CONCLUSIONS: The kidney allograft biopsies showed that oxidative stress levels were elevated during allograft dysfunction in all biopsies regardless of diagnosis, but not significantly. The levels also were elevated in pretransplant biopsies. The study showed that oxidative stress is involved in various acute injuries occurring within the allograft.

Forgotten ureteric stents in renal transplant recipients: three case reports.

Ureteric stents are widely used in renal transplantation to minimize the early urological complications. Ureteric stents are removed between two and 12 weeks following trans-plantation, once the vesico-ureteric anastomosis is healed. Ureteric stents are associated with considerable morbidity due to complications such as infection, hematuria, encrustations and migration. Despite the patient having a regular follow-up in the renal transplant clinic, ureteric stents may be overlooked and forgotten. The retained or forgotten ureteric stents may adversely affect renal allograft function and could be potentially life-threatening in immunocompromised transplant recipients with a single transplant kidney. Retrieving these retained ureteric stents could be challenging and may necessitate multimodal urological treatments. We report three cases of forgotten stents in renal transplant recipients for more than four years. These cases emphasize the importance of patient education about the indwelling ureteric stent and possibly providing with a stent card to the patient. Maintaining a stent register, with a possible computer tracking system, is highly recommended to prevent such complications.

Impact of sensitivity of human leucocyte antigen antibody detection by Luminex technology on graft loss at 1 year.

BACKGROUND: The clinical relevance of the detection of human leucocyte antigen (HLA) antibodies in sera of renal transplant recipients by highly sensitive methods such as Luminex alone is uncertain and a matter of debate. The choice of output thresholds affects antibody detection and thus organ allocation, yet there are no internationally agreed threshold levels. This study aims at evaluating our current practice of using an MFI threshold of 1000 in antibody detection. METHODS: We carried out a case-control study by looking at 761 renal transplant recipients at one unit between 2000 and 2010. Of these, there were 93 cases of graft loss within 1 year and stored serum samples of 40 cases were available for testing. Controls were selected (graft function >2 years) and individually matched according to age, sex, number of transplants and date of transplant. All 40 cases and 40 controls had negative crossmatch by complement-dependent cytotoxicity (CDC) at the time of transplant, and pre-transplant sera were re-analysed for the presence of detectable HLA and donor-specific antibodies (DSAs) using Luminex screen and single-antigen beads and MFI threshold values of 1000, 2000 and 4000. RESULTS: In nearly 48% of cases with graft loss within a year, HLA antibodies were detectable by Luminex when using a 1000 MFI threshold. This was 25% greater than in controls (P = 0.017). There was also a 15% increase in detected DSAs; however, statistical significance depends on the inclusion or exclusion of one specific case. Using MFI thresholds of 2000 and 4000, no DSAs were found in any long-term surviving grafts. CONCLUSIONS: Selection of appropriate MFI cut-off values influences the detection of DSAs and, thus, organ allocation. Using a threshold of 1000 led to the detection of DSAs in 5% of long-term graft survivors in our population and should be considered too sensitive. Using a detection threshold of 2000 is sufficiently sensitive and leads to clinically relevant detection of DSA.

Campath, calcineurin inhibitor reduction and chronic allograft nephropathy (3C) study: background, rationale, and study protocol.

BACKGROUND: Kidney transplantation is the best treatment for patients with end-stage renal failure, but uncertainty remains about the best immunosuppression strategy. Long-term graft survival has not improved substantially, and one possible explanation is calcineurin inhibitor (CNI) nephrotoxicity. CNI exposure could be minimized by using more potent induction therapy or alternative maintenance therapy to remove CNIs completely. However, the safety and efficacy of such strategies are unknown. METHODS/DESIGN: The Campath, Calcineurin inhibitor reduction and Chronic allograft nephropathy (3C) Study is a multicentre, open-label, randomized controlled trial with 852 participants which is addressing two important questions in kidney transplantation. The first question is whether a Campath (alemtuzumab)-based induction therapy strategy is superior to basiliximab-based therapy, and the second is whether, from 6 months after transplantation, a sirolimus-based maintenance therapy strategy is superior to tacrolimus-based therapy. Recruitment is complete, and follow-up will continue for around 5 years post-transplant. The primary endpoint for the induction therapy comparison is biopsy-proven acute rejection by 6 months, and the primary endpoint for the maintenance therapy comparison is change in estimated glomerular filtration rate from baseline to 2 years after transplantation. The study is sponsored by the University of Oxford and endorsed by the British Transplantation Society, and 18 centers for adult kidney transplant are participating. DISCUSSION: Late graft failure is a major issue for kidney-transplant recipients. If our hypothesis that minimizing CNI exposure with Campath-based induction therapy and/or an elective conversion to sirolimus-based maintenance therapy can improve long-term graft function and survival is correct, then patients should experience better graft function for longer. A positive outcome could change clinical practice in kidney transplantation. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01120028 and ISRCTN88894088.

CMV mismatch does not affect patient and graft survival in UK renal transplant recipients.

BACKGROUND: Cytomegalovirus (CMV) is one of the major infections encountered posttransplantation. UK Guidelines (2003) recommend CMV prophylaxis or screening with preemptive treatment for all high risk recipients. Studies predating the widespread use of CMV prophylaxis have shown that CMV seronegative recipients (R-) receiving a renal allograft from a CMV seropositive donor (D+) have worse outcomes than those avoiding primary CMV infection. Therefore, it has been suggested that CMV matching should be a part of the UK national deceased donor kidney allocation scheme. METHODS: We examined patient and allograft survival according to donor and recipient CMV serostatus in 10,190 UK adult and pediatric deceased donor renal transplant recipients transplanted between 2000 and 2007. We also ascertained CMV prophylaxis strategies in all UK renal transplant units. RESULTS: Twenty-one of the 22 UK renal transplant centers used prophylactic oral valganciclovir for 3 months posttransplant in the D+R- transplants, having done so for a median of 4 years. Unadjusted data showed that D+R+ rather than D+R- transplants had the lowest patient and allograft survivals at 3 years posttransplant. However, after adjustment for donor age, there was no significant effect of donor and recipient CMV serostatus on allograft or patient survival. CONCLUSIONS: These findings suggest that in an era where CMV prophylaxis is used routinely in D+R- transplants, the previously noted adverse effects of primary CMV infection on allograft and patient survival can be avoided (perhaps through a reduction in the incidence and/or severity of primary CMV infection), without using a CMV-matching allocation scheme.

Cytomegalovirus-specific CD8+ T cells do not develop in all renal transplant patients at risk of virus infection.

Cytomegalovirus (CMV) is an important pathogen in immunosuppressed renal transplant patients. At greatest risk are CMV IgG seronegative recipients (R-) of kidneys from CMV IgG seropositive donors (D+), although not all develop CMV disease. The aims of the study were to determine whether D+/R- patients who do or do not go on to develop CMV disease differ in their CD8+ T cell responses to CMV. Responses to the immunodominant NLVPMVATV peptide from the CMV structural protein pp65 in HLA-A2+ renal transplant patients were quantified using HLA tetramers/pentamers. Most D+/R+ patients had detectable tetramer+ cells while most D-/R- patients did not. Around 50% of D+/R- patients had some CD8+ tetramer+ cells and there was a strong correlation between % tetramer+ cells and the occurrence of a CMV infection post-transplantation (P<0.005). 18/41 (44%) of CMV negative patients receiving a kidney from a CMV+ donor failed to develop a detectable CMV infection, or significant numbers of tetramer+ cells. There was no relationship between CMV infection and acute cellular rejection. There was a tendency for patients who were given pre-emptive antiviral therapy to have lower levels of tetramer+ cells but this was not statistically significant. Hence the results show that CMV- patients receiving a kidney from a CMV+ donor do not inevitably acquire CMV infection. Those without CMV disease did not show any T cell response while most patients with detectable CMV developed specific CD8+ T cells.

A randomized controlled trial of immunosuppression conversion for the treatment of chronic allograft nephropathy.

BACKGROUND: This study was conducted to assess the effect of immunosuppression conversion on progression of chronic allograft nephropathy (CAN). METHODS: Forty-two cyclosporin-treated renal transplant recipients were studied. Patients were included if they had a negatively sloping reciprocal of creatinine vs time (ROCT) plot for >6 months and biopsy-proven CAN. Patients were excluded if they had previously been treated with tacrolimus/mycophenolate mofetil (MMF) or their serum creatinine was >400 micromol/l. Subjects were randomly treated with either: (A) MMF/reduced dose cyclosporin [MMF for azathioprine 0.5-1.0 g bd; cyclosporin trough level (C(0)): 75-100 ng/ml]; (B) tacrolimus for cyclosporin (C(0): 5-10 ng/ml); or (C) continuation of standard therapy. Glomerular filtration rate (GFR) was measured at baseline and after 6 months. RESULTS: Two patients started dialysis within 6 months (one each from groups A and B). One patient in group A was intolerant of MMF, six others reported gastrointestinal symptoms and three developed anaemia. Cyclosporin dose was reduced by 24% [interquartile range (IQR): 14-27%] in group A [end-of-study C(0): 99 ng/ml (IQR: 90-113 ng/ml)]. In group B, the end-of-study tacrolimus C(0) was 7 ng/ml (5-9 ng/ml). The end-of-study cyclosporin C(0) in group C was 163 ng/ml (145-215 ng/ml). Comparison of ROCT slopes before and after intervention revealed a treatment advantage for group A (P<0.05). The GFR analysis was supportive (P = 0.05). When patients with GFR <20 ml/min/1.73 m(2) at enrollment were excluded from the analysis, the treatment advantage for group A reached statistical significance (n = 27, P<0.05). CONCLUSIONS: MMF/reduced dose cyclosporin is superior to tacrolimus-for-cyclosporin and standard dose cyclosporin in patients with CAN, at least in the short term. The cyclosporin dose reduction component is likely to be of particular importance. Other findings suggest that early intervention is beneficial.