G Rizzo

Effects of pancreas-kidney transplantation on diabetic retinopathy

The effects of pancreas transplantation (PTx) on diabetic retinopathy (DR) are still debated. We studied the course of DR in 48 patients (age: 40 ± 7 years; males/females 26/22, body mass index (BMI): 23.0 ± 2.4 kg/m2, duration of diabetes: 24 ± 8 years) bearing a successful PTx (combined with a kidney). Follow‐up ranged 6–60 months (median: 17 months). Before transplantation, according to the Eurodiab Study classification, 12 patients (25%) had nonproliferative retinopathy (NPDR; mild, moderate or severe), and 36 patients (75%) had laser‐treated and/or proliferative retinopathy (LT/PDR). During the follow‐up, in the NPDR group improvement/deterioration was defined as regression/progression to a lower/higher retinopathy grade; in the LT/PTD group, stabilization was defined as no new neo‐vessel formation or development of new lesions requiring laser‐treatment. In the NPDR group, five (41.7%) patients improved of one or more lesion grading, three (25%) patients showed no change, and four (33.3%) patients progressed of one grade. In the LT/PDR group, the post‐transplant data were: stabilization in 35 (97%) patients, and worsening in one (3%) patient. The number of improved/stabilized patients was significantly higher in the transplanted than in a control group of nontransplanted type 1 diabetic patients. In conclusion, despite a relatively short follow‐up period, successful PTx in our cohort of patients was associated with improvement and/or stabilization of DR in the majority of recipients.

Living Kidney Transplantation: Evaluation of Renal Function and Morphology of Potential Donors

The evaluation of potential living kidney donors requires an accurate study of renal function and morphology. The gold standard to assess renal function is the measurement of glomerular filtration rate (GFR). However, GFR is often estimated from serum creatinine (SCr), cystatin C (SCys), or creatinine clearance (CCr). Otherwise, GFR is predicted using formulas based on SCr or SCys. Ultrasound scanning evaluates morphology and dimensions, while scintigraphy provides information on morphofunctional symmetry of kidneys. The aim of this study in 79 potential donors was to assess the accuracy of the tests employed to estimate GFR and the utility of renal ultrasound and scintigraphy for morphofunctional evaluation of potential donors. GFR (clearance of (99m)Tc-DTPA) was compared with estimates obtained with Cockcroft and Gault (CG-CCr) and Modification of Diet in Renal Disease (MDRD-GFR) formulas, and from SCys (Cys-GFR). The correlation with GFR was statistically significant for SCys and for all estimates, but not for SCr. CCr showed a poor agreement with GFR, with a large range of agreement and a marked and significant overestimation of GFR (33.8 mL/min). The accuracy of CG-CCr and MDRD-GFR as indicators of a GFR < 80 mL/min was better than that of Cys-GFR and CCr. However, their mean prediction errors versus GFR were relevant. Renal dimensions, particularly renal volume, showed a good correlation with GFR. The correlation was higher than that of all prediction equations. The direct measurement of GFR remains the reference method to assess renal function in potential kidney donors. The measurement of renal dimensions can provide useful information also on renal function.

Usefulness of direct sequencing in the detection of microchimerism in liver transplant recipients.

Hematopoietic microchimerism has been widely demonstrated after organ transplant operations (liver, kidney, heart, and lung) using methods based on polymerase chain reaction (PCR) amplification of the Y chromosome and the HLA-DR region of the major histocompatibility complex. However, the exact role of microchimerism in graft acceptance remains a matter of debate (1, 2). To study the actual prevalence of microchimerism in transplant patients, we randomly selected 25 patients who had undergone orthotopic liver transplantation (OLTx) from 1996 to 1998 at the General and Vascular Surgery Unit of the University of Pisa. The presence of microchimerism was assessed using a twostep nested PCR sequence-specific primer typing as previously described by other authors (3). A first round of generic amplification with primers amplifying a 274-bp region of the second exon of DRb1-9 (4) was carried out for 30 cycles on a PTC 100 thermal cycler (MJ Research, Watertown, MA). PCR products were diluted 1/10 in H2O and submitted to a second round of amplification with donor DRb1 allele-specific primers selected on the basis of published sequences (5). The specificity of the nested PCR sequence-specific primer procedure was assessed by the direct sequencing of nested PCR products resulting from the amplification of all the primer mixes used in the analysis. One-microliter aliquots of nested PCR product were sequenced using the Big Dye Terminator sequencing kit (Perkin Elmer, Foster City, CA). Cycle sequencing was performed using the same forward or reverse primer that had been utilized in the second round of nested PCR SSP typing. The samples were ethanol precipitated, loaded onto an automated ABI 310 sequencer (Perkin Elmer), and analyzed using Perkin Elmer match maker software. This sequencing procedure was adopted for two main reasons: (i) pretransplantation blood samples were not available for the study patients (6); and (ii) there is a well-known risk of false positive cases due to mispriming events (7). Indeed, from our analysis of the data on this series of 25 patients, 3 cases (12%) of false-positive results emerged. In all three cases the presence of a single band could be observed on the agarose gel, which was apparently attributable to a correct amplification of the donor’s specific alleles (DRb1*15) judging on the size of the relative amplicons (197 bp) (5). However direct sequencing showed that the PCR products did not correspond to the expected alleles but rather to DRb1*16, whose amplicon is of similar size (213 bp) (5). Based on these findings, we believe that the direct sequencing of PCR products could represent a useful addition to the procedures currently used in the evaluation of microchimerism in organ transplant patients, because it would reliably help to eliminate false-positive cases. MICHELE CURCIO MARTA MOSCA SIMONE LAPI FRANCO FILIPPONI FRANCO MOSCA SALVATORE ITALIA GAETANO RIZZO Laboratory of Immunogenetic and Transplant Biology S. Chiara Hospital 56126 Pisa, Italy General Surgery and Transplantation Unit Department of Oncology Cisanello Hospital 56124 Pisa, Italy

New-onset diabetes after kidney transplantation

Introduction. The occurrence of the new-onset diabetes after kidney transplantation is common and represents a risk factor for decreased survival of the graft and the patient. Case presentations. We report 6 cases of diabetes appeared in post renal transplantation. Treatment after kidney transplantation was based on corticosteroids associated with immunosuppressive therapy in all cases. Diabetes appeared after renal transplantation in 30% of cases, it was discovered during a systematic examination. The treatment consisted of insulin therapy for 3 patients, oral anti-diabetic for a patient and 2 patients had normalized their blood glucose levels after discontinuation of corticosteroids and immunosuppressive therapy adjustment. Conclusion. Immunosuppressive therapy associated with multiple risk factors presented by patients favor the occurrence of this diabetes.

Association of donor-specific microchimerism with graft dysfunction in kidney transplant patients

The biological significance of donor-specific microchimerism (DSM) in solid organ transplantation is unresolved. It has been reported both as a favourable feature, which may facilitate induction and maintenance of tolerance, and as a sign of graft-vs-host disease. Here, we applied a quantitative real-time PCR assay (qRT-PCR) to a selected series of kidney transplant recipients to measure the level of microchimerism in relation to allograft function and survival. DSM level was assessed by scoring the HLA-DRB1 locus in 54 patients (42 males, 12 females) with more than 2 years of follow-up after transplantation; 38 patients were considered to have stable renal function (SRF) and 16 had allograft dysfunction (AD). Among patients with AD, 12 (75%) showed detectable level of microchimerism, compared to 11 (29%) SRF patients (Odds Ratio 7.36, 95% CI 1.7-35.2; p<0.01). In addition, AD patients showed a higher mean donor genome equivalents (6.5×10(-5) vs. 2.4×10(-5); p<0.001). SRF patients were re-evaluated two years later; 2 out of 27 DSM negative vs. 2 out of 11 DSM positive had lost their transplanted organ. In conclusion, qRT-PCR applied to peripheral blood shows significant association between DSM and allograft dysfunction in kidney transplant patients.

Use of new ADA and WHO criteria for the diagnosis of impaired fasting glycemia and diabetes in kidney graft recipients

Sir: Post-transplant alterations of glucose metabolism, in particular post-transplant diabetes mellitus (PTDM), are serious complications of transplantation therapy and are mainly due to the effect of immunosuppression [1]. The reported prevalences of glucose intolerance and PTDM vary widely, and are approximately 3%–30% in renal and liver transplantation [2]. Most of the published studies suffer from methodogical inconsistencies, in particular regarding the criteria used for the diagnosis of diabetes and other forms of glucose intolerance. Recently, the American Diabetes Association (ADA) and the World Health Organization (WHO) have proposed new diagnostic criteria [3]. To our knowledge, no study has used such criteria to assess the prevalence of glucose metabolism alterations in transplanted patients. With permission of our local ethics committee, we studied 130 kidney recipients (97 men) with pretransplant fasting plasma glucose concentrations less than 110 mg/dl. Based on their post-transplant fasting plasma glucose, we found that the prevalences of impaired fasting glycemia (IFG) and PTDM were 14% and 7%, respectively. When compared with kidney recipients with normal fasting plasma glucose, patients with IFG and PTDM had similar age (48±10 vs. 43±10 years), but higher body mass index (26.2±4.1 vs. 24.5±3.7 kg/m 2 , p<0.02). The dosages of immunosuppressants and, in particular, of prednisone (8.1±2.8 vs. 8.4±2.9 mg/day) were similar in the two groups. Insulin levels did not differ significantly in patients with altered glucose levels (12.2±6.7 μU/ml) when compared with recipients with normal fasting glucose levels (10.5±7.0 μU/ml). However, when homeostasis model assessment (HOMA) index was calculated, it was significantly higher in the former (3.6±2.0 vs. 2.4±1.6, p<0.05). Of the 130 kidney recipients, all were being treated with steroids and either azathioprine or mycophenolate mofetil. Furthermore, the patients were receiving either cyclosporine (n=101) or tacrolimus (n=29). In the subgroup of patients on tacrolimus, the prevalence of IFG was 14%, similar to that of patients on cyclosporine (16%). However, patients on tacrolimus showed a higher prevalence of PTDM (16% vs. 2%, p<0.05). Steroid daily dose in patients on cyclosporine was similar to that of tacrolimus groups (7.3±0.3 mg vs. 7.2±0.2 mg).