Benita K. Book

Reduced human IgG anti-ATGAM antibody formation in renal transplant recipients receiving mycophenolate mofetil.

Exposure to the equine-derived polyclonal antithymocyte preparation, ATGAM, frequently elicits human anti-ATGAM antibody formation. The influence of concomitant immunosuppressants on this antiantibody response has not been established. We therefore evaluated IgG antibody formation to ATGAM in 47 patients receiving ATGAM as part of a prospective, randomized, double-blinded study of mycophenolate mofetil versus azathioprine for maintenance immunosuppression after primary cadaveric renal transplantation. All patients received ATGAM for induction of immunosuppression plus methylprednisolone, prednisone, and cyclosporine. In addition, patients were randomized to receive maintenance immunosuppression consisting of either azathioprine (AZA) 1-2 mg/kg/day, mycophenolate mofetil 2 gm/day (MMF2), or mycophenolate mofetil 3 gm/day (MMF3). Patient sequential sera were independently tested for IgG anti-ATGAM antibody by 2 laboratories, which were blinded to treatment arm assignments, using enzyme-linked immunosorbent assays. Both laboratories found significantly greater anti-ATGAM antibody formation in group AZA compared with groups MMF2 and MMF3: laboratory 1 reported sensitization rates in the 3 groups of 94% (AZA), 50% (MMF2) (P < 0.02 vs. AZA), and 60% (MMF3) (P < 0.05 vs. AZA); and laboratory 2 reported rates of 67% (AZA), 17% (MMF2) (P < 0.02 vs. AZA), and 10% (MMF3) (P < 0.02 vs. AZA). In addition, fewer patients formed high titer antibody in the MMF arms compared to the AZA arm: 56% (AZA), 0% (MMF2) (P < 0.02 vs. AZA), and 20% (MMF3) (P < 0.02 vs. AZA) of patients for laboratory 1; and 20% (AZA), 0% (MMF2) (P < 0.05 vs. AZA), and 0% (MMF3) (P < 0.05 vs. AZA) of patients for laboratory 2. Differences in test results between the 2 laboratories were explained by differences in the sensitivity of their respective immunoassays and in the criteria used for assigning a positive result to test specimens. In this protocol, MMF at 2-3 gm/day was associated with a reduced incidence and titer of IgG anti-ATGAM antibody formation compared with standard azathioprine dosing. Although MMF previously has been reported to inhibit T cell responses that mediate acute cellular rejection, this is the first demonstration that MMF significantly inhibits human B cell responses to antigen in vivo.

Differential influence of azathioprine and mycophenolate mofetil on the disposition of basiliximab in renal transplant patients

Pharmacokinetic sampling was performed in two multicenter trials in which basiliximab (anti‐CD25 monoclonal antibody) was administered with triple immunosuppression consisting of cyclosporine microemulsion, corticosteroids, and either azathioprine or mycophenolate mofetil. Blood samples were collected over 12 wk post‐transplant from 31 azathioprine‐treated and 66 mycophenolate mofetil‐treated patients. Empirical Bayes estimates of each patients basiliximab disposition parameters were derived and the duration of CD25 saturation was estimated as the time over which serum concentrations exceeded 0.2 μg/mL as confirmed by flow cytometry measurements. Basiliximab clearance was 29±14 mL/h when coadministered with azathioprine and 18±8 mL/h with mycophenolate mofetil. Both were significantly lower compared with a clearance of 37±15 mL/h from a previous study of basiliximab with dual therapy (p<0.001). As a consequence of the lower clearance of basiliximab, the durations of CD25 saturation were prolonged in the presence of azathioprine (50±20 d; range, 13–84) and mycophenolate mofetil (59±17 d; range, 28–94) compared with dual therapy (36±14 d; range, 12–91). A total of 27 acute rejection episodes occurred during the first 6 months in the two studies. Durations of CD25 saturation were not different in these patients compared with those who remained rejection‐free in each study. A single patient among 57 who were screened developed anti‐idiotype antibodies to basiliximab. The average duration of CD25 saturation was prolonged by 39 and 64% in the presence of azathioprine and mycophenolate mofetil, respectively. This graded effect was also observed for basiliximab clearance and may be due in part to a differentially reduced humoral response to basiliximab. Nonetheless, the range of CD25 saturation durations and basiliximab clearances did not extend outside the range when basiliximab was used with dual therapy in the absence of these agents. Hence, no dosing adjustment is deemed necessary when basiliximab is used in triple immunosuppressive therapy including either azathioprine or mycophenolate mofetil.

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 ≥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 ≥6.1% using Fishers exact test. Means were compared using Students 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.

Evaluation of seven PCR-based assays for the analysis of microchimerism.

OBJECTIVE The presence of small numbers of cells of donor origin in the circulation of recipients of organ transplants (microchimerism) may correlate with immunologic tolerance. As part of our ongoing studies on microchimerism, we evaluated the utility of seven PCR-based assays for the detection of the less abundant DNA in paired mixtures (100 ng total DNA). DESIGN AND METHODS DNA samples were screened to identify pairs informative for one or more PCR assays. DNA mixtures from the informative pairs were then analyzed using at least one assay. The assays were based on the X-Y homologous region; a Y chromosome microsatellite locus; three autosomal microsatellite loci; the D1S80 minisatellite locus; and sequence specific oligonucleotide probe (SSOP) analysis of the HLA DRB1 locus. RESULTS About 0.1% of male DNA against a background of female DNA was detectable using primers for the X-Y homologous region, but the sensitivity was increased to 0.0001% using nested primers for the Y chromosome microsatellite marker. Analysis of the minor DNA component was difficult with the three autosomal microsatellite assays because of the presence of shadow bands. Similar problems with the D1S80 assay were resolved using more stringent PCR conditions, and the sensitivity was 0.1%. Using the DRB1 locus, we were able to detect 1% DNA in the mixed samples. CONCLUSIONS These studies show that: (a) nested PCR for the Y chromosome is the most sensitive assay for the detection of microchimerism; (b) D1S80 is a useful marker for microchimerism; (c) additional optimization of analytical conditions is required if autosomal microsatellite markers and the SSOP assay are to be used for microchimerism analysis.

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 Treatment With Tabalumab, a B Cell-Activating Factor Inhibitor, on Highly Sensitized Patients With End-Stage Renal Disease Awaiting Transplantation.

B cell–activation factor (BAFF) is critical for B cell maturation. Inhibition of BAFF represents an appealing target for desensitization of sensitized end‐stage renal disease (ESRD) patients. We conducted a Phase 2a, single‐arm, open‐label exploratory study investigating the effect of tabalumab (BAFF inhibitor) in patients with ESRD and calculated panel reactive antibodies (cPRAs) >50%. The treatment period duration was 24 weeks. Eighteen patients received tabalumab, at doses of 240‐mg subcutaneous (SC) at Week 0 followed by 120‐mg SC monthly for 5 additional months. Patients were followed for an additional 52 weeks. Immunopharmacologic effects were characterized through analysis of blood for HLA antibodies, BAFF concentrations, immunoglobulins, T and B cell subsets, as well as pre‐ and posttreatment tonsil and bone marrow biopsies. Significant reductions in cPRAs were observed at Weeks 16 (p = 0.043) and 36 (p = 0.004); however, absolute reductions were small (<5%). Expected pharmacologic changes in B cell subsets and immunoglobulin reductions were observed. Two tabalumab‐related serious adverse events occurred (pneumonia, worsening of peripheral neuropathy), while the most common other adverse events were injection‐site pain and hypotension. Three patients received matched deceased donor transplants during follow‐up. Treatment with a BAFF inhibitor resulted in statistically significant, but not clinically meaningful reduction in the cPRA from baseline (NCT01200290, Clinicaltrials.gov).

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.

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.

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

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.

Differences in Alloimmune Response Between Elderly and Young Mice

OBJECTIVE The upper age of renal transplant recipients is rising on the transplant wait list. Age-dependent immune responsiveness to new antigens has not been thoroughly studied. This study used a mouse model of alloantibody response to neoalloantigen to study age-related differences. METHODS Transgenic huCD20-C57BL/6 mice were immunized intraperitoneally with BALB/c splenocytes (2.5 × 10(7)) at baseline and 1 month. Plasma samples were collected at baseline and 1 and 2 months after inoculation, frozen, and tested in a batch run (n = 22). Samples were tested by flow cytometric crossmatch for alloantibody with 2-fold serial dilution from neat to 1:640 using BALB/c splenocytes as targets. The sum of the median fluorescence intensity of the tested sample was calculated after subtracting that of an autologous serum control. Elderly mice (ELD; 42-103 weeks) at inoculation were compared with younger mice (YOU; 11-15 weeks). Statistical analysis was performed with 2-sample t test. RESULTS Mean age (weeks) between the groups was significantly different (ELD 69.3 ± 9.6 vs YOU 13.4 ± 1.4; P < .001). There was no difference in alloantibody between groups at baseline (ELD 0.7 ± 3.1 vs YOU 0.6 ± 0.4; P = .93). There was a higher alloantibody response at 1 month for YOU (52.9 ± 31.78) compared with ELD (5.12 ± 8.18). There was a greater difference after the 2 month (YOU 109.38 ± 66.43 vs ELD 21.97 ± 27.14; P < .0024). CONCLUSIONS There was a difference in response to new alloantigen in this animal model. Older animals had significantly decreased responses to new alloantigen stimulation 1 month after inoculation and even more profound decreases at 2 months compared with young animals. This model may be used to study differences in immune refractoriness to antigen signaling. It may be important to adapt clinical immunosuppression in the aged population to possible decreased responses to immune stimulation.