Distinct gene expression pathways in islets from individuals with short- and long-duration type 1 diabetes.

AIMS: Our current understanding of the pathogenesis of type 1 diabetes (T1D) arose, in large part, from studies using the non-obese diabetic (NOD) mouse model. In the present study, we chose a human-focused method to investigate T1D disease mechanisms and potential targets for therapeutic intervention by directly analysing human donor pancreatic islets from individuals with T1D. MATERIALS AND METHODS: We obtained islets from a young individual with T1D for 3 years and from an older individual with T1D for 27 years and performed unbiased functional genomic analysis by high-depth RNA sequencing; the T1D islets were compared with islets isolated from 3 non-diabetic donors. RESULTS: The islets procured from these T1D donors represent a unique opportunity to identify gene expression changes in islets after significantly different disease duration. Data analysis identified several inflammatory pathways up-regulated in short-duration disease, which notably included many components of innate immunity. As proof of concept for translation, one of the pathways, governed by IL-23(p19), was selected for further study in NOD mice because of ongoing human trials of biologics against this target for different indications. A mouse monoclonal antibody directed against IL-23(p19) when administered to NOD mice resulted in a significant reduction in incidence of diabetes. CONCLUSION: While the sample size for this study is small, our data demonstrate that the direct analysis of human islets provides a greater understanding of human disease. These data, together with the analysis of an expanded cohort to be obtained by future collaborative efforts, might result in the identification of promising novel targets for translation into effective therapeutic interventions for human T1D, with the added benefit of repurposing known biologicals for use in different indications.

Re-exposure to beta cell autoantigens in pancreatic allograft recipients with preexisting beta cell autoantibodies.

Re-exposure to beta cell autoantigens and its relevance in the presence of donor-specific antibodies (DSA) in pancreatic allograft recipients is not well known. Thirty-three patients requiring a pancreas transplant were enrolled in an IRB approved study. They underwent prospective monitoring for DSA and beta cell autoantibody (BCAA) levels to GAD65, insulinoma-associated antigen 2 (IA-2), insulin (micro-IAA [mIAA]), and islet-specific zinc transporter isoform-8 (ZnT8). Twenty-five (75.7%) had pre-transplant BCAA. Twenty had a single antibody (mIAA n = 15, GAD65 n = 5); five had two or more BCAA (GAD65 + mIAA n = 2, GAD65 + mIAA+IA-2 n = 2, GA65 + mIAA+IA-2 + ZnT8 = 1). No changes in GAD65 (p > 0.29), IA-2 (>0.16), and ZnT8 (p > 0.07) were observed between pre-transplant and post-transplant at 6 or 12 months. A decrease in mIAA from pre- to post-6 months (p < 0.0001), 12 months (p < 0.0001), and from post-6 to post-12 months (p = 0.0002) was seen. No new BCAA was observed at one yr. Seven (21.0%) developed de novo DSA. The incidence of DSA was 24% in patients with BCAA vs. 25% in patients without BCAA (p = 0.69). Pancreatic allograft function of patients with vs. without BCAA, and with and without BCAA + DSA was comparable until last follow-up (three yr). Re-exposure to beta cell autoantigens by pancreas transplant may not lead to increased levels or development of new BCAA or pancreatic allograft dysfunction.

Pre-transplant angiotensin receptor II type 1 antibodies and risk of post-transplant focal segmental glomerulosclerosis recurrence.

Post-kidney transplant recurrence of focal segmental glomerulosclerosis (FSGS) is a major problem. AT1R is expressed on podocyte; its expression is elevated in the proteinuric state. Using an ELISA, we tested pre-transplant sera of 28 patients with history of idiopathic FSGS for anti-AT1R levels and serum soluble urokinase-type plasminogen activator receptor (suPAR) as a biomarker for risk of recurrence of FSGS. Sera from 11 patients with polycystic kidney disease (PKD) were used as controls. Twelve patients had biopsy proven post-transplant FSGS recurrence at 1.5 months. No difference was found in the pre-transplant suPAR levels of FSGS patients (5993 ± 2292 pg/mL) vs. PKD (7334 ± 4538 pg/mL) (p = 0.23). Serum suPAR levels in patients with FSGS recurrence (5786 ± 1899 pg/mL) vs. no FSGS recurrence (6149 ± 2598 pg/mL) (p = 0.69) were not different. Anti-AT1R levels in patients with FSGS were 12.66 ± 11.85 U/mL vs. 8.69 ± 6.52 U/mL in PKD (p = 0.32); however, a difference was found in patients with and without FSGS recurrence 20.41 ± 14.36 U/mL 6.84 ± 4.181 U/mL, respectively (p < 0.01). Area under curve for suPAR and anti-AT1R to predict post-transplant FSGS recurrence was 0.51 and 0.84, respectively. Pre-transplant anti-AT1R levels appear to be a helpful biomarker in identifying patients at high risk of post-transplant FSGS recurrence.

The effectiveness of the combination of rituximab and high-dose immunoglobulin in the immunomodulation of sensitized kidney transplant candidates.

Kidney transplantation faces many challenges not the least of which is the presence of pre-formed HLA antibodies. At our institution, we have used a combination of methods to immunomodulate sensitized patients. Most recently, this has been attempted with a combination of immunoglobulin (IVIG) and rituximab (Rituxan; Genetech, CA, USA). A total of 31 patients were followed for up to one yr following treatment with IVIG (2 gm/kg on day 1 and day 30) and rituximab (1 g - day 15). Antibody levels were followed serially at designated time points via solid-phase single-antigen beads (SAB) method (One Lambda, Inc., Canoga Park, CA, USA). Concentration of antibodies was based on median fluorescence intensity (MFI). The majority of patients had both class I and class II antibodies (79%). Our results showed that this protocol appeared to be patient and antibody specific. The most pronounced MFI reduction in antibodies occurred within the 30- to 100-d period post-treatment. Calculated panel-reactive antibodies decreased but rebound tended to occur by 104 d after antibody MFI nadir. Because of this rebound, it can be inferred that the patients did not show a durable increase in their potential for transplantation. The search for a more effective method to immunomodulate patients continues.

Early findings of prospective anti-HLA donor specific antibodies monitoring study in pancreas transplantation: Indiana University Health Experience.

The significance of donor-specific antibodies (DSA) is not well known in the setting of pancreas transplantation. Since December 2009, we prospectively followed pancreas transplant patients with single-antigen-luminex-bead testing at one, two, three, six, and then every six months for the first two yr. Thirty-five of the 92 patients that underwent pancreas transplantation (13 pancreas-alone [PTA], 20 with a kidney [SPK], and two after a kidney [PAK]) agreed to participate in study. Median age at transplant was 45 yr and follow-up was 23 months. Majority were Caucasian (n = 33) and male (n = 18). Rabbit anti-thymocyte globulin induction was used. Median HLA-mismatch was 4.2 ± 1.1. Eight patients (7SPK, 1PAK) developed post-transplant DSA at median follow-up of 76 d (26-119), 1 SPK had pre-formed DSA. Seven patients had both class I and class II DSA, one with class I and one with class II only. Mean peak class I DSA-MFI was 3529 (±1456); class II DSA-MFI was 5734 (±3204) whereas cumulative DSA MFI (CI + CII) was 9264 (±4233). No difference was observed in the patient and donor demographics among patients with and without DSA. One patient in non-DSA group developed acute cellular rejection of pancreas. From our data it appears that post-transplant DSA in pancreas allograft recipients may not impact the early-pancreatic allograft outcomes. The utility of prospective DSA monitoring in pancreatic transplant patients needs further evaluation and long-term follow-up.

Effect of calcineurin inhibitors on posaconazole blood levels as measured by the MVista microbiological assay.

Calcineurin inhibitors may augment the effects of antifungal drugs in microbiological assays. We examined this interaction in a microbiological assay for posaconazole. No effect was observed. However, concurrent or recently discontinued treatment with other antifungal drugs caused false-positive results, emphasizing a limitation of microbiological assays for antifungal drug level measurement.

Utility of HbA1c in the detection of subclinical post renal transplant diabetes.

BACKGROUND: We hypothesized that the use of HbA1c testing would help identify postrenal transplant diabetes (PTDM). METHODS: In all, 199 adult kidney transplant recipients at least 3 months posttransplant without previous history of diabetes or elevated fasting blood sugar were studied. Medical history, a fasting blood glucose, calcineurin inhibitor blood level, and HbA1c were obtained. Primary outcome was the incidence of subjects with HbA1c > or =6.1%. The covariates were use of cyclosporine or tacrolimus, time posttransplant, body mass index (BMI) at transplant and change since transplant, current steroid dose, history of graft rejection, current fasting glucose, age, and race. Proportions were compared between HbA1c <6 and > or =6.1% using Fisher's exact test. Means were compared using Student's t test. Logistic regression was used to identify risk factors associated with elevated HbA1c. RESULTS: Twenty subjects (10.1%) had an elevated HbA1c. High normal fasting glucose (P=0.003) and African American race (P=0.08, marginally significant) were found to be associated with an elevated HbA1c. Subjects with normal and abnormal HbA1c levels were otherwise similar. There was no difference in HbA1c in tacrolimus versus cyclosporine treated subjects or in the percent of subjects with elevated HbA1c between these groups. CONCLUSIONS: HbA1c levels were found to be more a more sensitive test than fasting blood glucose levels in PTDM, with 10.1% of all patients and 19.4% of blacks found to have an elevated HbA1c. HbA1c testing should be considered as a screening test for PTDM, especially in African Americans.

Removal of therapeutic anti-lymphocyte antibodies from human sera prior to anti-human leukocyte antibody testing.

Both monoclonal (e.g. Orthoclone (OKT3), rituximab) and polyclonal (e.g. ATGAM, Thymoglobulin (Thymo)) anti-lymphocyte Abs (ALAs) are used extensively in organ transplantation for immunosuppression induction, desensitization, and treatment of acute rejection. ALAs often interfere with post transplant immunologic monitoring. We describe a method that uses magnetic beads to selectively remove ALAs from patient serum. Rabbit anti-mouse Fc-specific (180 mug), or rabbit anti-mouse Fab-specific (180 microg), or rabbit anti-horse heavy and light chain-specific and rabbit anti-horse F(ab')2 (200 microg) (Jackson Immunoresearch) was adsorbed to 6.7 x 10(8) Dynabeads M-280 conjugated with sheep anti-rabbit IgG (Dynal Biotech). Fifty microliters of normal human serum (NHS) with 2 microg/ml of OKT3 or 100 microg/ml ATGAM, Thymo, or rituximab were incubated with conjugated beads for several incubations. NHS containing ALAs before and after treatment by the protocol were incubated with human lymphocytes and labeled with FITC-antibody to immunoglobulin of the species used to produce the particular ALA. Residual ALA was determined using flow cytometry. Average median channel for serum with or without ALA was 11.1 and 0.120, respectively for OKT3; 64.4 and 0.344 for ATGAM; 108.5 and 0.200 for Thymo; and 1022.5 and 11.4 for rituximab. Treatment lowered the median channel for serum with OKT3 to 0.103, 0.309 for ATGAM, 0.199 for Thymo, and 12.1 for rituximab. ALAs can be effectively removed from serum by the use of magnetic beads conjugated with Ab specific for ALA thereby permitting immunologic monitoring without interference.

Rituximab inhibits the in vivo primary and secondary antibody response to a neoantigen, bacteriophage phiX174.

The response to primary immunization in patients treated with Rituximab (RIT) is not clear. We studied the in vivo antibody response of chronic renal failure (CRF) patients to the neoantigen bacteriophage phiX174 given alone or after ablation with RIT. Eighteen CRF subjects received two immunizations with phiX174 separated by 6 weeks. Nine subjects received a single dose of RIT. The intensity and immunoglobulin isotype of the antibody response (K(v)) were measured post-infusion. In addition, three subjects previously immunized and treated with RIT underwent a third and fourth immunization with phiX174 and a tetanus control 2 years later. RIT significantly decreased peak K(v) responses when compared to both historic non-CRF controls and to CRF subjects. CRF itself decreased peak K(v) responses compared to non-CRF controls. Percent-ratio of anti-phage IgM to IgG was significantly decreased in RIT treated subjects. One of three subjects treated with RIT was found to have developed a partial B cell tolerance to phiX174 administration 2 years later. RIT decreases antibody production and isotype switching to neoantigens and might be useful to prevent antibody response to therapeutic drugs and to newly transplanted organs.

In vivo human B-cell subset recovery after in vivo depletion with rituximab, anti-human CD20 monoclonal antibody.

Rituximab, chimeric anti-human CD20 monoclonal antibody approved for B-cell lymphoma, depletes circulating B cells. Little data exist on its use for nonmalignant diseases or B-cell subset recovery after treatment. We hypothesized that rituximab may reduce panel reactive alloantibodies (PRA) in dialysis patients awaiting renal transplantation and performed a single-dose, dose-escalation phase 1 trial. Here we report changes in lymphocyte phenotypes in subjects treated with rituximab alone. Nine subjects, 44 +/- 10 years(Yr); (5 F, 4 M) received one dose (n=3) at 50, 150, or 375 mg/m(2) without other immunosuppression. Blood was collected before dosing and intervals thereafter. No significant changes in leukocytes, total or CD3(+) lymphocytes were noted. In all, there was CD19(+) depletion by day 2(D2) (12.0 +/- 5.6 cells/mm(3) vs. 181 +/- 137, D0; p<0.01) and CD20(+) (11.0 +/- 12.0 vs. 205 +/- 116, D0; p<0.01). At 6 months (mo), CD19(+) and CD20(+) remained low (51.1 +/- 42.2, p<0.05; 75.4 +/- 38.7, p<0.05, respectively) compared to D0. CD19(+)CD5(+) cells recovered more rapidly, returning to baseline by 6mo while B memory cells (CD19(+)CD27(+)) remained low (32.3 +/- 29.0) at 1Yr (7.5 +/- 4.5; p<0.001) and 2Yr (12.1 +/- 7.9; p<0.001) after treatment. We conclude that single dose rituximab ablates B cells in high PRA dialysis patients awaiting transplantation. B-cell ablation, particularly memory B cells, was long-lasting, lagging repopulation by CD5(+) B cells.

Pronase treatment facilitates alloantibody flow cytometric and cytotoxic crossmatching in the presence of rituximab.

Rituximab (RIT), a murine/human chimeric monoclonal antibody directed against human CD20 is under investigation for its role in transplantation. RIT causes B-cell crossmatches to appear positive. Pronase, a proteolytic enzyme that targets F(c) receptors removes CD20 from B cells. After CD20 is removed, RIT should not bind, making it possible to detect class I or class II antibodies on treated B cells. In this study, we incubated RIT with normal human serum (NHS, negative control) or pooled sera from highly sensitized (>50% panel reactive antibody, HLA+) subjects awaiting renal transplantation (positive control) and then performed B-cell flow cytometric crossmatches using untreated or pronase treated B cells as targets. We observed that untreated B cells incubated with RIT-spiked NHS displayed a significant increase in surface fluorescence compared with NHS without RIT, similar to the fluorescence that occurs with a positive crossmatch. In contrast, when CD20 was cleaved from the B cells with pronase, B cells displayed a negative crossmatch with the RIT-spiked NHS. In addition, there was no change in the crossmatches of pooled high panel reactive antibody (PRA) sera after pronase treatment. RIT could be used without worry about losing the ability to perform transplant immunologic monitoring.

Rituximab, anti-CD20, induces in vivo cytokine release but does not impair ex vivo T-cell responses.

Pre-formed HLA antibodies (Ab), reported as panel-reactive antibody (PRA), prolong transplant waiting time. We hypothesized that rituximab (RIT) could reduce PRA via B-cell depletion. As part of a Phase I study of single RIT dose, we studied in vivo and ex vivo effects on T-cell immune responses. Nine subjects (n = 3) were treated at 50, 150, and 375 mg/m(2). Serum interleukin-1alpha (IL-1alpha), IL-6, IL-12, tumor necrosis factor beta (TNF-beta), and interferon-gamma (IFN-gamma) were measured by enzyme-linked immunosorbent assay (ELISA). T-cell function was monitored with T-cell proliferation assays. IL-6 levels rose in eight patients (7.15 +/- 4.38 pg/mL to 86.22 +/- 77.08, p = 0.021). The high-dose group had detectable TNF-betapost rituximab infusion (874.7 +/- 1466.5 pg/mL). There was no decline in T-cell proliferation in response to phytohemagglutinin or allogeneic lymphocyte stimuli. Stimulation indices in the presence of both concentrations of tetanus toxoid rose significantly at 4 weeks.

Rituximab for reduction of anti-HLA antibodies in patients awaiting renal transplantation: 1. Safety, pharmacodynamics, and pharmacokinetics.

BACKGROUND: Preformed HLA antibodies (Ab), reported as panel-reactive antibody (PRA), prolong patient waiting time for kidney transplantation. We hypothesized that rituximab (RTX) could reduce PRA via B-cell depletion. This initial study reports the safety, pharmacokinetics, and pharmacodynamics of RTX in patients with end-stage renal failure. METHODS: The study was an investigator-initiated single-dose, dose-escalation phase I trial of RTX in chronic dialysis patients (PRA >50%). It was approved by the Institutional Review Board and the Food and Drug Administration. Nine subjects were treated with a single dose of RTX (n=3 per group) at 50, 150, or 375 mg/m. Peripheral lymphocyte cell surface markers and HLA Ab levels (%PRA and titers) were tested using flow cytometry. RESULTS: There were four significant adverse events: a suspected histoplasmosis infection; two Tenchkoff dialysis catheter infections; and fever (38.7 degrees C) during infusion. At 2 days after RTX therapy, there was depletion of CD19 cells (pre-RTX 181+/-137 vs. post-RTX 12+/-5.6, P =0.006). In 2 (22%) of 9 subjects, there was no appreciable change in PRA. Among the other seven patients, one had a decrease in PRA from 87% to 51% with a concurrent decrease in fluorescence intensity; five patients had changes in histogram architecture suggesting loss of antibody specificity; and one patient had a fourfold decrease in PRA titer from 1:64 to 1:16 at 6 months after treatment. In addition, one of the seven patients converted a donor-specific crossmatch to negative and underwent a successful living donor kidney transplantation. CONCLUSIONS: RTX can be safely administered and may be an effective agent to reduce high-titer anti-HLA Abs in subjects awaiting kidney transplantation.

The addition of sirolimus to cyclosporine and steroids inhibits the anti-equine antibody response in renal transplant recipients treated with antithymocyte globulin.

Polyclonal antibodies, such as equine antithymocyte globulin (ATGAM), are known to induce antibody formation. This study evaluated the in vivo effect of sirolimus on antibody formation associated with the use of equine antithymocyte globulin in renal transplant recipients. Recipients of either a living-related donor or cadaveric renal allograft received azathioprine (AZA) (n = 15), mycophenolate mofetil (MMF) (n = 12), or sirolimus (n = 15) in addition to baseline immunosuppression with corticosteroids, cyclosporine, and equine antithymocyte globulin. Immediately before transplantation and weekly for at least 1 month, sequential serum specimens were tested for the presence of human anti-equine antibody using an enzyme-linked immunosorbent assay (ELISA). Anti-equine antibody formation was significantly different among the three treatment groups. Fewer patients receiving MMF (17%, p = 0.007 vs. AZA) and sirolimus (13%, p = 0.003 vs. AZA) developed anti-equine antibody compared with AZA (66%). There was no significant difference (p = 0.81) in the sensitization to equine antithymocyte globulin when comparing the patients receiving MMF or sirolimus. In the sensitized patients, high anti-equine antibody titers (>1 : 500) were more common in those receiving AZA (n = 3) than MMF (n = 0) or sirolimus (n = 1). Compared to AZA, sirolimus, when given in combination with cyclosporine A, significantly reduced anti-equine antibody formation to a degree similar to MMF.