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Treatment of Innate Immune Arthritis With a Toll-Like Receptor 7 Agonist Requires Type I Interferon

Abstract
T. Hayashi, B. Crain, S. Yao M. Chan, H.B. Cottam, J. Yang, D.A. Carson, M. Corr
Arthritis Rheum 2013;65 Suppl 10 :1731 DOI: 10.1002/art.2013.65.issue-s10

We previously demonstrated that repeated administration of the low molecular weight Toll-like receptor (TLR) 7 agonist (1V136) substantially reduces arthritic inflammation in mice. Here we investigated the mechanisms contributing to this potent anti-inflammatory effect using the K/BxN passive transfer model of murine arthritis. The joint inflammation and vascular permeability observed in the passive serum transferred arthritis were significantly attenuated by 1V136 treatment. The drug requires TLR7 on bone marrow derived cells, and an intact type I interferon pathway to be fully effective.

Alternative activation mechanisms of Protein Kinase B trigger distinct downstream signaling responses

Paper
D. Balzano, M-A. Fawal, JV Velazquez, CM Santiveri, , J. Yang, J. Pastor, R. Campos-Olivas, N. Djouder, D. Lietha
J Biol Chem 2015:jbc.M115.651570.

Protein kinase B (PKB/Akt) is an important mediator of signals that control various cellular processes including cell survival, growth, proliferation and metabolism. PKB promotes these processes by phosphorylating many cellular targets, which trigger distinct downstream signaling events. However, how PKB is able to selectively target its substrates to induce specific cellular functions remains elusive. Here we perform a systematic study to dissect mechanisms that regulate intrinsic kinase activity versus mechanisms that specifically regulate activity towards specific substrates. We demonstrate that activation loop phosphorylation and the C-terminal hydrophobic motif (HM) are essential for high PKB activity in general. On the other hand we identify membrane targeting, which for decades has been regarded as an essential step in PKB activation, as a mechanism mainly affecting substrate selectivity. Further, we show that PKB activity in cells can be triggered independently of PI3K by initial HM phosphorylation, presumably through a mechanism analogous to other AGC kinases. Importantly, different modes of PKB activation result in phosphorylation of distinct downstream targets. Our data indicate that specific mechanisms have evolved for signaling nodes, like PKB, to select between various downstream events. Targeting such mechanisms selectively could facilitate the development of therapeutics that might limit toxic side effects.

Continuous Wave Ultrasonic Doppler Tonometry

Paper
A. Akinin, J. Yang, A. Williams, A. Lee, P. Pourhoseini, A. Fronek, G. Cauwenberghs
Biomedical Circuits and Systems (BioCAS), 2014 IEEE , vol., no., pp.328,331, 22-24 Oct. 2014 doi: 10.1109/BioCAS.2014.6981729

A variety of cardiovascular conditions such as hypertension are related to the decay in the mechanical properties of blood vessels. We combine continuous wave Doppler ultrasound measurement of blood velocity with high-resolution compressive force sensing to design a sensor to analyze the hemodynamic characteristics of blood vessels. We test the specifications of our device through an ultrasonic phantom we developed and a set of calibration routines for the tonometer, the Doppler velocity analysis and the design of a blood mimicking fluid. Finally we demonstrate the application of our Doppler tonometer in the analysis of veins and arteries. We show the change in hemodynamic parameters typical to a veins and artery during Doppler Tonometry.

Self-antigens and Rejection: A Proteomic Anaysis

Review
Joshua Y.C. Yang, Tara K. Sigdel, Minnie M. Sarwal
Curr Opin Organ Transplant (2016) 21:362–367. doi:10.1097/MOT.0000000000000328

Allo- and auto-antibodies have been found to play important roles in both acute and chronic allograft rejection in organ transplantation, although only recently have non-HLA, non-donor specific antibodies been given a more in-depth treatment. This review summarizes recent reports about investigations and proteomic approaches to identify self-antigens and corresponding auto-antibodies that are associated with acute and chronic allograft rejection. Finally, we discuss the insights gained from these, challenges, and future prospects.

Transplant Genetics and Genomics

Review
Joshua Y.C. Yang, Minnie M. Sarwal
Nature Reviews Genetics (2017). doi: 10.1038/nrg.2017.12

Ever since the discovery of the major histocompatibility complex, scientific and clinical understanding in the field of transplantation has been advanced through genetic and genomic studies. Candidate-gene approaches and recent genome-wide association studies (GWAS) have enabled a deeper understanding of the complex interplay of the donor–recipient interactions that lead to transplant tolerance or rejection. Genetic analysis in transplantation, when linked to demographic and clinical outcomes, has the potential to drive personalized medicine by enabling individualized risk stratification and immunosuppression through the identification of variants associated with immune-mediated complications, post-transplant disease or alterations in drug-metabolizing genes.

Retrospective Evaluation of the Efficacy and Safety of Belatacept with Thymoglobulin Induction and Maintenance Everolimus: A Single Center Clinical Experience

Paper
Wojciechowski D, Chandran S, Yang J, Sarwal M, Vincenti F
Clin Transplant. 2017.

Belatacept use has been constrained by higher rates of acute rejection. We hypothesized that belatacept with low dose rATG and initial mycophenolate maintenance with conversion to everolimus at 1 month post-transplant ± corticosteroids would improve efficacy and maintain safety. Retrospective single center analysis of the first 44 low immunologic risk kidney transplant recipients treated with this regimen. The cohort was 59% male, mean age at transplant of 57 years. Diabetes was the most common cause of ESRD (39%). The mean 1 year eGFR was 61.4 (SD 18.4) mL/min/1.73 m2. There were 5 acute cellular rejections (11.4%) and occurred in patients who had changed from everolimus to mycophenolate mofetil due to side effects. 32% developed BK viremia and 12% developed CMV viremia. There were no cases of PTLD. A novel belatacept regimen with rATG induction and maintenance everolimus demonstrated a low acute rejection rate and maintained an excellent 1-year eGFR.

Polyclonal Regulatory T cell Therapy for Control of Inflammation in Kidney Transplants

Paper
Chandran S, Tang Q, Sarwal M, Laszik ZG, Putnam AL, Leung J, Nguyen V, Lee K, Sigdel T, Tavares EC, Yang J, Hellerstein M, Fitch M, Bluestone J, Vincenti F
Am J Transplant. 2017.

Early subclinical inflammation in kidney transplants is associated with later graft fibrosis and dysfunction. Regulatory T cells (Tregs) can reverse established inflammation in animal models. We conducted a pilot safety and feasibility trial of autologous Treg cell therapy in three kidney transplant recipients with subclinical inflammation noted on 6-month surveillance biopsies. Tregs were purified from peripheral blood and polyclonally expanded ex vivo using medium containing deuterated glucose to label the cells. All patients received a single infusion of ~320 X106 (319, 321 and 363.8 x 106) expanded Tregs. Persistence of the infused Tregs was tracked. Graft inflammation was monitored with follow-up biopsies and urinary biomarkers. Nearly 1×109 (0.932, 0.956, 1.565 x 109) Tregs were successfully manufactured for each patient. There were no infusion reactions or serious therapy-related adverse events. The infused cells demonstrated patterns of persistence and stability similar to those observed in non-immunosuppressed subjects receiving the same dose of Tregs. Isolation and expansion of Tregs is feasible in kidney transplant patients on immunosuppression. Infusion of these cells was safe and well tolerated. Future trials will test the efficacy of polyclonal and donor alloantigen-reactive Tregs for the treatment of inflammation in kidney transplants.

A Non-invasive Urinary Common Rejection Module (uCRM) Gene Expression Score Quantifies and Differentiates Kidney Transplant Injury

Abstract
Sigdel TK, Yang JY, Bestard O, Hsieh S-C, Roedder S, Damm I, Liberto J, Nandoe S, Sarwal MM
Am J Transplant. 2017; 17 (suppl 3).

We sought to investigate the use of the common rejection module (CRM) genes in the discrimination of AR, BKVN, and stable transplant phenotypes in urine of kidney transplant patients. Kidney transplantation (tx)is preferred for treatment of ESRD by any cause. Despite short-term improvemnts, long-term kidney allograft outcomes have not improved as expected. The most plausible explanation for this unmet achievement relies on rather poor immune-risk assessment of transplant patients in current clinical practice.

Methods

A CRM consisting of 11 genes expressed on allograft biopsies was recently shown to be highly accurate to discriminate patients undergoing acute rejection (AR), correlate with the extent of graft injury, and predict future allograft damage. Cell pellet from 117 urine samples were evaluated for expression values of all 11 using qPCR. ROC analysis was performed to assess clinical utility of these CRM genes. Symbolic regression was used to create a urinary CRM (uCRM) score from a subset of these genes.

Results

The 5-gene uCRM score calculated from gene expression in the urine cell pellet was significantly higher in AR than in stable (STA) and in BK viral nephropathy (BKVN) (9.705 ± 1.018 vs. 0.4567 ± 0.1213 and 1.45 ± 0.3917 respectively, p<0.0001) and significantly discriminated AR from STA and BKVN patients with high accuracy (AUC=0.9723; 95% CI = 0.9426 – 1.0000, p<0.0001). In a validation set, a threshold of 1.896 showed 100% sensitivity and 85.7% specificity to discriminate high-risk from low-risk patients for AR. Conclusion

By using a previously published gene list, we investigated the role of this panel as a surrogate biomarker panel. The 5-gene uCRM score in urine samples is able to non-invasively detect kidney transplant patients at high risk of AR and could provide a tool for non-invasive, prospective AR and BKVN risk assessment, with implications on tx precision medicine.

A Novel Belatacept Regimen to Optimize Efficacy and Safety

Abstract
Wojciechowski D, Chandran S, Sarwal M, Yang J, Vincenti F
Am J Transplant. 2017;17 (suppl 3).

Despite superior long term graft survival when compared to cyclosporine, the use of the FDA approved belatacept regimen in clinical practice has been constrained by higher rates of acute rejection. We hypothesized that the use of rATG and an mTORi with belatacept would improve efficacy and provide comparable safety.

Methods

Retrospective single center analysis of the first 44 EBV seropositive, low immunologic risk kidney transplant recipients treated with belatacept combined with rATG induction (3 mg/Kg) and initial MMF maintenance with conversion to everolimus at 1 month post-transplant ± corticosteroids. Biopsies were performed for cause. GFR was estimated by the MDRD equation. A subset of patients who agreed to immune monitoring were classified by 2 different gene expression assays: (1) the kidney solid organ response test/kSORT (peripheral blood) and (2) urine common rejection module/uCRM as being positive (kSORT ≥9, uCRM ≥4; high risk for rejection) or negative (immune quiescence).

Results

The cohort was 59% male, 43% Caucasian, with a mean age of 57 years and low immunologic risk (mean cPRA 6%). Diabetes was the most common cause of ESRD (39%) followed by PKD (23%). 54% received a deceased donor kidney transplant; 28% developed DGF. The mean 1 year eGFR was 61.4 (SD 18.4) mL/min/1.73 m2. 12 cause biopsies were performed with 5 acute cellular rejections (11.4%).

Cause Biopsy, n 12
No rejection, n 2
Borderline, n 4
ACR, n 5
1a 2
1b 1
2b 2
BKVAN, n 1

All rejections occurred in patients still on MMF or changed from everolimus; 2 rejections occurred in patients off steroids. There was 1 patient death. 32% developed BK viremia and 12% developed CMV viremia. No fungal infections were seen. There were no cases of PTLD. 27 patients underwent kSORT and uCRM testing, with samples obtained at a mean of 731 (range 169-1335) days post-transplant.Conclusion

A novel belatacept regimen with low dose rATG induction and maintenance everolimus demonstrated a low acute rejection rate and maintained an excellent 1-year eGFR with evidence of immune quiescence in a majority of patients.

Novel biomarkers for segregating lung transplant rejection phenotypes

Abstract
Yang JY, Vanaudenaerde B, Sigdel TK, Liberto J, Verleden S, Sarwal MM
Am J Transplant. 2017;17 (suppl 3).

We sought to investigate biomarkers in BAL fluid and tissue for segregating BOS and RAS chronic rejection phenotypes in lung allografts. We had previously identified a common response module (CRM) of 11 genes that define acute rejection (AR) across different engrafted tissues. The CRM score quantifies injury and stratifies patients at increased risk of future progressive fibrosis. We investigated if this CRM score could be applied for progressive rejection post lung transplantation with special consideration for the different phenotypes of chronic lung rejection.

Methods: We collected and analyzed BAL fluid cell pellet and tissues from patients with different phenotypes of end-stage chronic lung rejection (Bronchiolitis Obliterans Syndrome, BOS, n=13 vs. Restrictive allograft syndrome, RAS, n=18) and compared this to unused donor lungs for tissue or stable non-rejecting patients for BAL (n=15). RNA was extracted and the CRM gene expressions were measured.

Results: We found several genes that were differentially regulated in tissue. CXCL10 was increased in tissue of BOS vs. control patients (p < 0.0001) and in BOS vs. RAS (p = 0.0077). CXCL9 was increased in tissue of RAS vs. control patients (p = 0.0021) and in RAS vs. BOS (p = 0.0232). The geometric mean CRM score was higher in RAS versus control (p=0.0272) and trending in BOS (p = 0.0652). Neither the CRM score or individual genes in BAL fluid cell pellet were significantly different. Conclusion: A proportion of the CRM genes were upregulated in RAS compared to BOS and control. Since RAS shows typical interstitial fibrosis, it is of interest that the gene expression signature is similar to rejection post kidney transplantation which could demonstrate that similar mechanisms are in place causing the progressive tissue fibrosis. A study including more tissue or a prospective study is further needed to validate these findings.