Beatrice Coupes

Transforming growth factor beta 1 in renal allograft recipients.

Transforming growth factor beta (TGF beta 1) is a prosclerotic cytokine implicated in several disease processes. Recent reports have demonstrated a role for TGF beta 1 in experimental models of glomerulonephritis, focusing attention on the relevance of TGF beta to renal fibrogenesis in human disease. The study reported here was designed to investigate whether circulating, active TGF beta 1 could be detected in renal allograft recipients, and whether plasma levels correlated with episodes of rejection, a process involving both inflammation and fibrosis. We have developed an ELISA assay for active TGF beta 1 using commercially available antibodies, and measured plasma levels in 43 healthy controls, 11 patients with membranous nephropathy (MN) and impaired renal function, 17 transplant recipients with stable renal function, 27 patients with acute cellular rejection, 7 patients with chronic vascular rejection, and 10 patients with acute tubular necrosis and/or cyclosporine toxicity. In the last three groups diagnoses were biopsy-proved, and all samples were collected at the time of biopsy. TGF beta 1 was also measured in urine samples from 8, 11, 0, 9, 4, and 7 individuals, respectively, from each group. TGF beta 1 was not detected in plasma from any of the healthy controls or any of the MN patients, (detection limit of assay 0.1 ng/ml). By comparison, it was significantly increased in all groups of transplant recipients (unpaired t test, P < 0.01), but there were no significant differences between the transplant groups. Plasma TGF beta 1 level did not correlate with renal function (estimated by either serum creatinine or reciprocal creatinine), kidney donor age, recipient age, time since transplantation, or cyclosporine plasma trough level. TGF beta 1 was found in every urine sample tested from healthy controls, with a range from 1 ng/ml to 35 ng/ml. Among the 20 transplant patient urines tested, 2 were negative, 18 were positive but within the range determined for the healthy controls. There were no significant differences between the groups.

Epidemiology of posttransplantation lymphoproliferative disorder in adult renal transplant recipients.

Background There is little information in the literature describing the relationship between posttransplantation lymphoproliferative disorder (PTLD) incidence and presentation with both recipient Epstein-Barr virus (EBV) serostatus and EBV status of PTLD histology, particularly in the late posttransplantation period. Methods This study reports the largest UK single-center, single-organ analysis of PTLD to date in a retrospective cohort study of 80 cases occurring in 4189 adult renal transplant recipients. Results The incidence rate was 2.6 cases per 1000 patient-years (95% confidence interval [95% CI], 2.1–3.2) for PTLD, 1.8 (95% CI, 1.4–2.4) for non-Hodgkin’s lymphoma, and 0.2 (95% CI, 0.07–4.2) for Hodgkin’s lymphoma. Non-Hodgkin’s lymphoma occurred at a rate 7.6 times that of the adult general population in England, whereas the rate for Hodgkin’s lymphoma was 5.9 times. The incidence of PTLD was highest during the 10th to 14th posttransplantation years. Early-onset disease was associated with EBV-seronegative recipient status, EBV-positive histology, and the involvement of extranodal sites. PTLD occurring in EBV-seronegative recipients was associated with EBV nuclear antigen antibody deficiency, polymorphic disease, and the involvement of extranodal sites. EBV-negative histology occurred in 32% of cases at a median time to presentation of 109 months. PTLD involving the allograft, central nervous system, and skin was uncommon and occurred late. Conclusion The incidence of PTLD is highest in the late posttransplantation period. Close clinical surveillance and education for transplant recipients is required for the duration of time while immunosuppressed. Failure to detect EBV DNA in blood should not reassure, particularly in patients with symptoms such as abdominal pain, oropharyngeal complaints, neck lumps, and B-symptoms.

Activation of transforming growth factor-beta1 and early atherosclerosis in systemic lupus erythematosus.

The efficiency of activating latent transforming growth factor (TGF)-β1 in systemic lupus erythematosus (SLE) may control the balance between inflammation and fibrosis, modulating the disease phenotype. To test this hypothesis we studied the ability to activate TGF-β1 in SLE patients and control individuals within the context of inflammatory disease activity, cumulative organ damage and early atherosclerosis. An Activation Index (AI) for TGF-β1 was determined for 32 patients with SLE and 33 age-matched and sex-matched control individuals by quantifying the increase in active TGF-β1 under controlled standard conditions. Apoptosis in peripheral blood mononuclear cells was determined by fluorescence-activated cell sorting. Carotid artery intima-media thickness was measured using standard Doppler ultrasound. These measures were compared between patients and control individuals. In an analysis conducted in patients, we assessed the associations of these measures with SLE phenotype, including early atherosclerosis. Both intima-media thickness and TGF-β1 AI for SLE patients were within the normal range. There was a significant inverse association between TGF-β1 AI and levels of apoptosis in peripheral blood mononuclear cells after 24 hours in culture for both SLE patients and control individuals. Only in SLE patients was there a significant negative correlation between TGF-β1 AI and low-density lipoprotein cholesterol (r = -0.404; P = 0.022) and between TGF-β1 AI and carotid artery intima-media thickness (r = -0.587; P = 0.0004). A low AI was associated with irreversible damage (SLICC [Systemic Lupus International Collaborating Clinics] Damage Index ≥1) and was inversely correlated with disease duration. Intima-media thickness was significantly linked to total cholesterol (r = 0.371; P = 0.037). To conclude, in SLE low normal TGF-β1 activation was linked with increased lymphocyte apoptosis, irreversible organ damage, disease duration, calculated low-density lipoprotein levels and increased carotid IMT, and may contribute to the development of early atherosclerosis.

Subcutaneous interstitial pressure and volume characteristics in renal impairment associated with edema.

The kidneys and the interstitial compartment play a vital role in body fluid regulation. The latter may be significantly altered in renal dysfunction, but experimental studies are lacking. To help define this we measured the subcutaneous interstitial pressure, bioimpedance volumes, and edema characteristics in 10 healthy subjects and 21 patients with obvious edema and chronic kidney disease (CKD). Interstitial edema was quantified by the time taken for a medial malleolar thumb pit to refill and termed the edema refill time. Interstitial pressure was significantly raised in CKD compared to healthy subjects. Total body water (TBW), extracellular fluid volume (ECFV), interstitial fluid volume, the ratio of the ECFV to the TBW, and segmental extracellular fluid volume were raised in CKD. The ratio of the ECFV to the TBW and the interstitial fluid volume were the best predictors of interstitial pressure. Significantly higher interstitial pressures were noted in edema of 2 weeks or less duration. A significant nonlinear relationship defined interstitial pressure and interstitial fluid volume. Edema refill time was significantly inversely related to interstitial pressure, interstitial compartment volumes, and edema vintage. Elevated interstitial pressure in CKD with obvious edema is a combined function of accumulated interstitial compartment fluid volumes, edema vintage, and tissue mechanical properties. The edema refill time may represent an important parameter in the clinical assessment of edema, providing additional information about interstitial pathophysiology in patients with CKD and fluid retention.

Epstein-Barr virus infection in adult renal transplant recipients

Epstein–Barr virus (EBV) DNAemia in the first year posttransplantation has been studied extensively. There is a paucity of information on prevalence and sequelae of EBV infection in adult renal transplantation beyond the first year. This single‐center study examines the relationship between EBV DNAemia and demographic, immunosuppressive, hematologic and infection‐related parameters in 499 renal transplant recipients between 1 month and 33 years posttransplant. Participants were tested repeatedly for EBV DNAemia detection over 12 months and clinical progress followed for 3 years. Prevalence of DNAemia at recruitment increased significantly with time from transplant. In multivariate adjusted analyses, variables associated with DNAemia included EBV seronegative status at transplant (p = 0.045), non‐White ethnicity (p = 0.014) and previous posttransplant lymphoproliferative disease (PTLD) diagnosis (p = 0.006), while low DNAemia rates were associated with mycophenolate mofetil use (p < 0.0001) and EBV viral capsid antigen positive Epstein–Barr nuclear antigen‐1 positive serostatus at transplant (p = 0.044). Patient and graft survival, rate of kidney function decline and patient reported symptoms were not significantly different between EBV DNAemia positive and negative groups. EBV DNAemia is common posttransplant and increases with time from transplantation, but EBV DNAemia detection in low‐risk (seropositive) patients has poor specificity as a biomarker for future PTLD risk.

Post-Transplant Lymphoproliferative Disorder in Adult Renal Transplant Recipients: Survival and Prognosis.

Post-transplant lymphoproliferative disorder (PTLD) is a rare, serious complication following solid organ transplantation, with an incidence of 2.6 cases per 1000 patient years. Optimal treatment strategies and risk stratifications specific to kidney transplantation are lacking and PTLD mortality remains high. This study investigated survival and prognosis in 89 cases of PTLD presenting over 44 years at Manchester Royal Infirmary. Patient survival following diagnosis was 72% at 6 months, 67% at 1 year and 54% at 3 years. In multivariate analysis, a poorer 3 year survival was associated with acute kidney injury at diagnosis (p = 0.0001), impaired renal function (p = 0.04), early onset (p = 0.02), T cell disease (p = 0.02) and previous treatment with anti-thymocyte globulin (p = 0.04). The inclusion of graft function adds prognostic value to risk stratification and should be explored further. Strategies to improve survival should include timing and choice of immuno-chemotherapy, preparation for dialysis and aggressive surveillance for sepsis and treatment toxicity.

Tacrolimus monotherapy in renal transplantation: four-year data

Forty-five primary allograft recipients were approached. Fifteen patients were excluded from the study for one or more of the following reasons: pregnancy or nursing mother, allergy or intolerance to steroids or tacrolimus, panel reactive antibodies greater than 50%, other organ transplant or investigational drug, delayed graft function, or acute tubular necrosis. Thirty patients were subsequently recruited into the study, 25 males and five females, with a mean age of 43 years. The mean number of HLA mismatches was 1.82, with no more than three in total, and no more than one at DR. 85% of patients had no mismatch at DR. The target trough level of tacrolimus during the first month was 10 to 20 ng/mL with a target maintenance level of 10 ng/mL. The fourth anniversary of the study has been reached, and 21 patients (70%) remain on (and have only ever received) tacrolimus monotherapy as immunosuppression, except for the transient use of intravenous methylprednisolone for the treatment of acute rejection. We have reevaluated the progress of the group. The clinical and biochemical data were abstracted from the clinical records.

A Modified in vivo Flow Variation Technique of Microdialysis for Sampling Uremic Toxins in the Subcutaneous Interstitial Compartment

Background: Uremic toxins are typically measured in plasma and little is known of their interstitial concentrations. We undertook experiments to validate a microdialysis technique for simultaneous recovery of small and large uremic toxins in the subcutaneous interstitial fluid (ISF). Methods: Microdialysis catheters were inserted into the subcutaneous interstitium of 8 subjects (controls and uremic patients) and perfused using two different solutions at incremental flow rates to determine analyte recovery and ISF concentrations of urea and protein. Results: 10% dextran-40 perfusate allowed the determination of interstitial concentrations of urea and protein reliably, by virtue of the exponential decay of their concentrations in the microdialysate with incremental flow rates (R2 = 0.63–0.99). Interstitial and plasma urea correlated well (r = 0.95), as did interstitial urea from distant anatomical sites (r = 0.96). Conclusion: Cutaneous microdialysis with dextran-40 allows measurement of small and large molecule concentrations in ISF, creating an opportunity to characterize ISF in uremia.

Immune Dysregulation in Renal Transplant Recipients With Chronic High Level EBV DNAemia.: Abstract# D2468

D2468 Immune Dysregulation in Renal Transplant Recipients With Chronic High Level EBV DNAemia. M. Morton,1 J. Adams,2 B. Coupes,1 S. Roberts,3 P. Klapper,4 P. Vallely,4 J. Burthem,2 M. Picton.1 1Dept of Renal Medicine, Manchester Royal Infi rmary, Manchester, United Kingdom; 2Dept of Haematology, Manchester Royal Infi rmary, Manchester, United Kingdom; 3Centre for Biostatistics, Institute of Population Health, University of Manchester, Manchester, United Kingdom; 4Microbiology and Virology, University of Manchester, Manchester, United Kingdom. We previously investigated EBV DNAemia in 500 renal transplant recipients and found 46% of patients positive, with chronic high viral loads (CHVL) in 7%. Risk factors for DNAemia included time from transplant and recipient seronegative status. Mycophenolate was associated with low rates of DNAemia. We now report a nested case control study recruiting 60 patients from the original 500, matched for age and time from transplant and stratifi ed by viral load into; group 1, undetectable (n=19), group 2 low (<75% samples >1000copies/ml) (n=20) and group 3 CHVL ( ≥75% samples >1000copies/ml or ≥3 samples over >6 months with ≥10,000 copies/ml) (n=21). Methods We analysed serology, DNAemia in whole blood and plasma, lymphocyte subsets, ultrasound examination of cervical lymph nodes and immunosuppression burden. Results All participants were EBV seropositive. Anti-VCA levels were signifi cantly higher in those with a CHVL. 7 patients had DNA detectable in plasma as well as whole blood (6/7 were in Group 3). This small sub-group had lower CD4:8 ratios than those with DNA in whole blood only (p=0.041), with higher CD8 counts (p=0.09). Group 3 had more individuals with ≥2 lymph nodes measuring >0.5mm than Group 1 (62% v 26%; OR 4.6; CI 1.2-17.5; p=0.03). A “heavy” immunosuppression burden was identifi ed in 0% of Group 1 and 29% of Group 3 (p=0.012). No association between mycophenolate and lymphocyte counts was identifi ed. PTLD has not been identifi ed in any of the 3 patient groups to date. Conclusion Chronic immunosuppression and immune dysregulation may predispose to EBV DNAemia, with similarities to the HIV setting. There may be scope to optimise immunosuppression in the setting of high level EBV DNAemia. A planned 5 year follow-up of the study patients will provide outcome data in relation to immune function and lymph node fi ndings. Abstract# D2469 Long-Term Effect of Splenectomy On Post-Transplant Infection Rates in ABO-Incompatible Kidney Transplant Patients. N. Ichimaru, J. D2469 Long-Term Effect of Splenectomy On Post-Transplant Infection Rates in ABO-Incompatible Kidney Transplant Patients. N. Ichimaru, J. Kaimori, Y. Kakuta, M. Okumi, T. Abe, R. Imamura, N. Nonomura, S. Takahara. Osaka University, Suita, Japan. Background: ABO-incompatible (ABOi) kidney transplantation commonly included splenectomy to reduce B-cell abundance and antibody titers. However, recent advances have made splenectomy unnecessary; pharmacological treatments (e.g., rituximab) are now used in place of splenectomy to achieve complete depletion of B-cells in peripheral blood. Patients and Methods:Our hospital began performing ABOi kidney transplantation with splenectomy in July 1992.In this retrospective study, we evaluated the rate of adverse events associated with splenectomy in ABOi kidney transplant patients to determine whether ABOi kidney transplantation with splenectomyis associated with an increased rate of infection. Between July 1992 and December 2008, 40 cases of ABOi transplantation with splenectomy and 198 cases of ABO-compatible (ABOc) transplantation were performed in our hospital. We compared the fi ve-year infection free rate in these patients from 2009 and 2013 using Kaplan-Meier method and log-rank test. Results:Between 1992 and December 2008, 6 patients in the ABOi group and 44 patients in the ABOc group were readmitted for dialysis and/or had died. The remaining 34 patients in the ABOi group and 154 patients in the ABOc group were included in the study. A total of 18 patientsin the ABOi group and 62 patientsin the ABOc group were treated for infection. Five-year infection free rates were 47.0% in the ABOi group and 59.7% in the ABOc group (p=0.151). The splenectomy group was vaccinated against pneumococcal infection, and no increase in the rate of infection was observed post-splenectomy. Conclusions: No signifi cant difference was seen in the infection rate among patients receiving ABOi kidney transplants with splenectomycompared with that among patients receiving ABOc kidney transplants. DISCLOSURES: Ichimaru, N.: Grant/Research Support, Astellas, Novarits, Chugai. Abstract# D2470 Hyposplenism Increases The Risk of Recurrent Urinary Infection in Renal Transplant Recipients. S. Rioja,1 E. Kasai,2 M. Orlando,2 S. Lucena,3 N. Araujo.1 1Nephrology, University of the State of Rio de Janeiro, Rio de Janeiro, Brazil; 2Nuclear Medicine, University of the State of Rio de Janeiro, Rio de Janeiro, Brazil; 3Haematology, University of the State of Rio de Janeiro, Rio de Janeiro, Brazil. BACKGROUND: In our unit, we noticed that patients with long-standing functioning renal graft compared to those soon after post-transplant showed smaller spleen on ultrasound along with haematological pictures of splenic dysfunction. To address this issue, we submited them to Sn-colloide hepatosplenic scintigraph to confi rm the presumed reduction of splenic function. Hypsplenism is known to increase susceptibility to infection. Accordingly, the authors decided to investigate if transplant recipients would have higher risk to recurrent urinary tract infection (UTI) caused by encapsulated bacteria due to poor splenic function. METHODS: The study was a cross-sectional survey based on single samples. Hepatosplenic scintigraphy was performed at 20 min following i.v. injection of 5mCi of 99mstannous colloid on an out patients basis. The splenic uptake of the colloid on the posterior view was classifi ed in relation to the liver uptake into decreased or equal. In accordance with the results, the patients were assigned to normal or reduced spleen function group. Further, the episodes of UTI were drawn from medical records. Immunosuppressive regimen use corticosteroid, azathioprine or mycophenolate and calcineurin inhibitors or m-TOR inhibitors. RESULTS: We enrolled 98 patients (62 men; 36 women; 57 from a deceased donor and 41 from a living donor); the mean age was 48 years (range 23.0-74.0 years). Mean transplant duration was 2446 days (range 181-8978 days). According to the scintigraphy, the prevalence of hyposplenism was 30.6% for the whole group. 42 out 98 patients developed recurrent UTI (defi ned as two episodes in six months or three episodes in a year). The risk for the disease was higher in the group with reduced spleen function (OR=2.47; IC 95% 1.02-5.98; p=0.043). In 27% of these patients, Escherichia coli was the single infecting organism, in all UTI episodes. In the majority, E. coli took turns with different Enterobacteriaceae or gram positive bacteria. DISCUSSION: Our fi nding shows that renal transplant recipients may develop signifi cant poor splenic function. Accordingly, we speculate that the increased UTI episodes would be due a defi ciency of IgM memory B cells and IL-10, known to be related to the impairment of splenic function. D2470 Hyposplenism Increases The Risk of Recurrent Urinary Infection in Renal Transplant Recipients. S. Rioja,1 E. Kasai,2 M. Orlando,2 S. Lucena,3 N. Araujo.1 1Nephrology, University of the State of Rio de Janeiro, Rio de Janeiro, Brazil; 2Nuclear Medicine, University of the State of Rio de Janeiro, Rio de Janeiro, Brazil; 3Haematology, University of the State of Rio de Janeiro, Rio de Janeiro, Brazil. BACKGROUND: In our unit, we noticed that patients with long-standing functioning renal graft compared to those soon after post-transplant showed smaller spleen on ultrasound along with haematological pictures of splenic dysfunction. To address this issue, we submited them to Sn-colloide hepatosplenic scintigraph to confi rm the presumed reduction of splenic function. Hypsplenism is known to increase susceptibility to infection. Accordingly, the authors decided to investigate if transplant recipients would have higher risk to recurrent urinary tract infection (UTI) caused by encapsulated bacteria due to poor splenic function. METHODS: The study was a cross-sectional survey based on single samples. Hepatosplenic scintigraphy was performed at 20 min following i.v. injection of 5mCi of 99mstannous colloid on an out patients basis. The splenic uptake of the colloid on the posterior view was classifi ed in relation to the liver uptake into decreased or equal. In accordance with the results, the patients were assigned to normal or reduced spleen function group. Further, the episodes of UTI were drawn from medical records. Immunosuppressive regimen use corticosteroid, azathioprine or mycophenolate and calcineurin inhibitors or m-TOR inhibitors. RESULTS: We enrolled 98 patients (62 men; 36 women; 57 from a deceased donor and 41 from a living donor); the mean age was 48 years (range 23.0-74.0 years). Mean transplant duration was 2446 days (range 181-8978 days). According to the scintigraphy, the prevalence of hyposplenism was 30.6% for the whole group. 42 out 98 patients developed recurrent UTI (defi ned as two episodes in six months or three episodes in a year). The risk for the disease was higher in the group with reduced spleen function (OR=2.47; IC 95% 1.02-5.98; p=0.043). In 27% of these patients, Escherichia coli was the single infecting organism, in all UTI episodes. In the majority, E. coli took turns with different Enterobacteriaceae or gram positive bacteria. DISCUSSION: Our fi nding shows that renal transplant recipients may develop signifi cant poor splenic function. Accordingly, we speculate that the increased UTI episodes would be due a defi ciency of IgM memory B ce