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Long-term clinical outcome of islet transplantation is hampered by the rejection and recurrence of autoimmunity. Accurate monitoring may allow for early detection and treatment of these potentially compromising immune events. Islet transplant outcome was analyzed in 59 consecutive pancreatic islet recipients in whom baseline and de novo posttransplant autoantibodies (GAD antibody, insulinoma-associated protein 2 antigen, zinc transporter type 8 antigen) and donor-specific alloantibodies (DSA) were quantified. Thirty-nine recipients (66%) showed DSA or autoantibody increases (de novo expression or titer increase) after islet transplantation. Recipients who had a posttransplant antibody increase showed similar initial performance but significantly lower graft survival than patients without an increase (islet autoantibodies P < 0.001, DSA P < 0.001). Posttransplant DSA or autoantibody increases were associated with HLA-DR mismatches (P = 0.008), induction with antithymocyte globulin (P = 0.0001), and pretransplant panel reactive alloantibody >15% in either class I or class II (P = 0.024) as independent risk factors and with rapamycin as protective (P = 0.006) against antibody increases. DSA or autoantibody increases after islet transplantation are important prognostic markers, and their identification could potentially lead to improved islet cell transplant outcomes.

Ex vivo lung perfusion (EVLP) has been validated as a valuable technique to increase the pool of organs available for lung transplantation.After a preclinical experience, we obtained permission from the Ethics Committee of our institution to transplant lungs after EVLP reconditioning. ABO compatibility, size match, and donor arterial oxygen pressure (PaO(2))/fraction of inspired oxygen (FiO(2)) ≤ 300 mm Hg were considered to be inclusion criteria, whereas the presence of chest trauma and lung contusion, evidence of gastric content aspiration, pneumonia, sepsis, or systemic disease were exclusion criteria. We only considered subjects on an extra corporeal membrane oxygenation (ECMO) bridge to transplantation with rapid functional deterioration. Using Steen solution with packed red blood cells oxygenated with 21% O(2), 5% to 7% CO(2) was delivered, targeted with a blood flow of approximately 40% predicted cardiac output. Once normothermic, the lungs were ventilated with a tidal volume of 7 mL/kg a PEEP of 5 cmH(2)O and a respiratory rate of 7 bpm. Lungs were considered to be suitable for transplantation if well oxygenated [P(v-a) O(2)350 mm Hg on FiO(2) 100%], in the absence of deterioration of pulmonary vascular resistance and lung mechanics over the perfusion time.From March to September 2011, six lung transplantations were performed, including two with EVLP. The functional outcomes were similar between groups: at T72 posttransplantation, the median PaO(2)/FiO(2) were 306 mm Hg (range, 282 to 331 mm Hg) and 323 mm Hg (range, 270 to 396 mm Hg) (P = 1, EVLP versus conventional). Intensive care unit ICU and hospital length of stay were similar (P = .533 and P = .663, respectively) with no mortality at 60 days in both groups. EVLP donors were older (49 ± 6 y versus 21 ± 7 y, P.05), less well oxygenated (184 ± 6 mm Hg versus 570 ± 30, P.05), displaying higher Oto scores (9.5 ± 0.7 versus 1.7 ± 1.5, P.05).The first 6 months of the EVLP program allowed us to increase the number of organs available for transplantation with short-term outcomes comparable to conventional transplantations.

The development of solid-phase assays for antibody detection has aided in the frequent detection of human leukocyte antigen (HLA) antibodies in nonalloimmunized males. Some scientists have reported that these HLA antibodies are produced to pathogens or allergens and the reactivity with HLA coated beads is the result of cross-reactive epitopes. These antibodies may also be directed toward cryptic epitopes exposed on the denatured beads. In this report, we describe the case of a heart transplanted patient who exhibited anti-HLA-A*02:01 donor-specific antibodies detected with a bead-based assay (Luminex) and undetected with the complement-dependent cytotoxicity (CDC) test. Posttransplant monitoring, carried out with CDC and with Luminex on sera from this patient collected at the 2nd, 4th, 8th, and 12th posttransplant weeks and at 1 year confirmed the presence of anti-HLA-A*02:01 in all serum samples. Additional tests carried out with denatured and intact HLA molecules using single antigen beads demonstrated that the antibody was directed toward a cryptic epitope. One year after transplantation the patient is doing well. No sign of antibody-mediated rejection was observed throughout the follow-up. A comprehensive evaluation of the anamnesis and of antibodies is critical to avoid needless exclusion of organ donors.

The Italian Bone Marrow Donor Register is the institutional organization for management of unrelated hematopoietic stem cell donors. The law requires only a donor's clinical history, but not a psychosocial profile for registration. We have studied the donor's motivation for enlistment on the donor registry and the medical staff's need for this information to interact correctly with the donor. For this purpose we distributed a questionnaire to new donors at the 20 centers in the Lombardy Region over a period of 1 year. The analysis of the responses revealed a prevalence of extrinsic motivations that would not ensure continued registration for donation. Therefore, it is necessary that the donor be well informed and better educated about all aspects of donation, in order to produce a shift to an intrinsic motivation. This objective can be facilitated via professional training of health workers in communication.

BACKGROUND: The antigens of the Colton blood group system, Co(a) and Co(b), are encoded by a single gene that produces the aquaporin-1 (AQP1) protein, a water channel-forming protein, and are characterized by a single nucleotide polymorphism (SNP). A healthy Caucasoid blood donor originally typed as Co(a-b-) with commercial anti-Co(b) typed Co(a-b+) when retested with another anti-Co(b). Retyped with two different molecular biology methods, the sample came out Co(a)/Co(b). With the aim of understanding these discrepancies, serological, cytometric and molecular biology tests were carried out. METHODS: Absorption/elution studies with propositus red cells and controls were performed. The region spanning exon 1 to exon 4 of the Colton gene was sequenced, and flow cytometry analyses were carried out. RESULTS: Absorption/elution studies showed the absence of Co(a) and a weak expression of Co(b). DNA sequencing confirmed a CT heterozygosity at nucleotide position 134 (i.e. Co(a)/Co(b)), and an additional heterozygous CT was found at position 112. The presence of the Co(b) allele that encodes for the Co(b) antigen was confirmed. The new allele has the base cytosine at nucleotide 134 (Co(a)), in cis with the new nucleotide 112T. The nucleotide substitution 112C>T causes a missense mutation leading to an amino acid change from proline (CCG) to serine (TCG) at codon 38. CONCLUSION: The substitution found at codon 38 results in a modified AQP1 protein which explains the Co(a-b+) phenotype and possibly the weak expression of Co(b).

Although human leukocyte antigen (HLA) crossmatching is often thought to be unnecessary for liver transplants (LTs), we provide evidence that for retransplants, it is essential. Sera from 139 retransplant patients who had received livers from deceased donors were retrospectively analyzed with single antigen beads on a Luminex platform for HLA antibodies. Each patient received at least 2 transplants and was followed up for at least 6 months from the second LT, which was deemed to have failed if the patient had a third LT or died. Second LT survival was calculated from the date of the second LT to the date of the third LT or death. Our study cohort consisted of 118 adult patients (≥18 years old) as well as 21 pediatric patients (

In 20% to 30% of infected individuals, hepatitis C virus (HCV) can cause cirrhosis and hepatocellular carcinoma, for which liver transplantation is the best treatment available. HCV re-infection is universal, and hepatitis disease recurrence occurs in most cases with a 30% probability of progression to graft cirrhosis at 5 years post-transplant. The immunological response to HCV involves natural killer (NK) cells and killer cell immunoglobulin-like receptors (KIRs), which specifically recognize human leukocyte antigen (HLA) class I antigens present on target cells. The effector functions of NK cells are influenced by inhibitory KIR interaction with self-HLA class I ligands, with HLA-C being the most predominant. This study examines the roles of KIR genotypes and their HLA ligands in both HCV disease recurrence and its progression. A total of 151 patients were included in the cohort, and their clinical details were recorded. Liver biopsies were used to define the absence/presence of recurrent hepatitis, the degree of fibrosis, and the progression to cirrhosis over a 10-year period. Mismatching of KIR-HLA-C ligands between donor-recipient pairs was associated with the recurrence of hepatitis (P = 0.008). The presence of KIR2DL3 in the recipient correlated with progression to liver fibrosis (P = 0.04). The mismatching of HLA-KIR ligands favored the progression of the recurrent hepatitis to fibrosis only in the presence of KIR2DL3 (P = 0.04). These preliminary results indicate that the KIR genotype and KIR-HLA-C ligand compatibility play roles in the recurrence and progression of hepatitis C disease in liver transplant recipients.

BACKGROUND: Traditionally, blood group typing has been performed with serologic techniques, the classical method being the hemagglutination test. Serotyping, however, may present important limitations such as scarce availability of rare antisera, typing of recently transfused patients, and those with a positive direct antiglobulin test. Consequently, serologic tests are being complemented with molecular methods. The aim of this study was to develop a low-cost, high-throughput method for large-scale genotyping of red blood cells (RBCs). STUDY DESIGN AND METHODS: Single-nucleotide polymorphisms associated with some clinically important blood group antigens, as well as with certain rare blood antigens, were evaluated: Jk a /Jk b , Fy a /Fy b , S/s, K/k, Kp a /Kp b , Js a /Js b , Co a /Co b , and Lu a /Lu b . Polymerase chain reaction (PCR)-amplified targets were detected by direct hybridization to microspheres coupled to allele-specific oligonucleotides. Cutoff values for each genotype were established with phenotyped and/or genotyped samples. RESULTS: The method was validated with a blind panel of 92 blood donor samples. The results were fully concordant with those provided by hemagglutination assays and/or sequence-specific primer (SSP)-PCR. The method was subsequently evaluated with approximately 800 blood donor and patient samples. CONCLUSION: This study presents a flexible, quick, and economical method for complete genotyping of large donor cohorts for RBC alleles.

We describe here the isolation and the full-length sequence of the coding region of the HLA new variant at the HLA-A locus officially named A*68020102. This variant shows an 11 base pairs deletion within the 5' UTR region. The exon sequence is identical to that of A*6802 and the commercially available anti-A68 typing sera react with the antigen coded by the allele A*68020102. This variant was originally identified in two unrelated Caucasoid families because of discrepant HLA typing results between serology, Sequence Specific Oligonucleotide Probe (SSOP), and SBT. In fact, the A68 assigned by serology was undetectable with the molecular techniques. This has occurred because the deletion present in A*68020102 prevents specific amplification of HLA-A locus by some commercially available typing kits.

The recent introduction of new technologies such as Luminex has provided alternative methods to the Complement Dependent Cytotoxicity (CDC) test for HLA specific antibody detection. In this study we compared the results obtained with CDC to those obtained using a Luminex method with the aim of evaluating the impact of this new technology on antibody screening policies in our transplant setting.A total of 1,421 sera, acquired from patients on the waiting list for a kidney transplant or following transplantation, were tested by both methodologies. CDC was performed using a whole lymphocyte population comprising a panel of 52 cells. The percentage panel reactive antibodies (PRA) and antibody specificity were evaluated using Lambda Scan Analysis software. For the Luminex method sera screening and identification of antibody specificity were carried out using the LABScreen Mixed and LABScreen PRA respectively. The overall concordance between the results obtained using the CDC and the Luminex methods was 85%. HLA antibody specificity was confirmed in 96% of the sera which tested positive using the Luminex system and serum positivity corresponded with a previous sensitisation event in these individuals. Using the Luminex method 18% of patients on the waiting list were considered and managed as sensitised as compared to 7% when testing with CDC alone. The Luminex method was able to detect a number of antibody specificities significantly more frequently than the CDC method and in addition the CDC method failed to detect some of the antibody specificities detected by the Luminex system. Based on this comparison study we have incorporated the Luminex methodology into our screening strategy.

Background.Solidorgantransplantedpatientshaveathree-tofourfoldhigherlifetimeriskofdevelopingacancerthan the general population. However, the incidence of a second primary cancer in transplanted patients has never been studied, despite the fact that the presence of regular follow-ups and the increased survival of these patients make them a very attractive model. Methods. We investigated the incidence of a second primary cancer (SPC) in 7,636 patients who underwent a kidney, liver, lung or heart transplant between 1970 and 2004, and were followed-up for 51,819 person-years. Results. During the follow-up, 499 subjects developed a first cancer (annual incidence: 98.610,000 PY), and 22 of them developed a SPC (annual incidence: 3.910,000 PY). The annual incidence of a SPC in the transplanted patients who developed a first cancer was 107.810,000 PY, giving a standardized incidence ratio of 1.1 (95% CI: 0.83–1.41). Conclusions. This result shows that the incidence of the SPC was the same as the incidence of a first cancer. Our study does not indicate an increased risk of SPC in transplanted subjects who already suffered a first malignancy.

Background. The risk of transmitting a hepatitis B virus (HBV) infection from donor kidneys with a past HBV serological profile may be negligible. Data on HBV transmission to kidney transplant recipients from donor organs that were anti-HBc/HBsAg - in Italy has not been previously reported. Anti-HBc testing in cadaver organ donors has been mandatory in Italy since 2002, when anti-HBc determinations were included in the National Guidelines for donor evaluation. Therefore, prior to that date kidney recipients from anti-HBc/HBsAg - donors can be identified retrospectively where stored serum is available for testing. Methods. The prevalence of anti-HBc Italian organ donors, the incidence of HBV transmission according to the recipients’ HBV status (vaccinated, recovered, or naive), and the clinical impact (5-year graft and patient survival rates) in the North Italy Transplant program was evaluated by retrospectively screening for anti-HBc antibodies in the sera of cadaver kidney donors used in transplants from 1997 to 1999. Results. Two hundred and ten donors were found to have been anti-HBc. At the time of the study, no active infection was observed in any of the 344 HBsAg - recipients, but 4/140 (2.86%) of the vaccinated recipients were found to have been anti-HBc/HBsAg - . None of these patients, however, had any biochemical or clinical history of HBV infection. Patient and graft survival rates of anti-HBc or anti-HBc - kidney recipients did not differ statistically. Conclusion. Kidney grafts from anti-HBc donors should be considered in all recipients because the benefit obtained from the transplantation out weighs the negligible risk of HBV transmission.

A specific split liver transplantation (SLT) program has been pursued in the North Italian Transplant program (NITp) since November 1997. After 5 yr, 1,449 liver transplants were performed in 7 transplant centers, using 1,304 cadaveric donors. Whole liver transplantation (WLT) and SLT were performed in 1, 126 and 323 cases, respectively. SLTs were performed in situ as 147 left lateral segments (LLS), 154 right trisegment liver (RTL) grafts, and 22 modified split livers (MSL), used for couples of adult recipients. After a median posttransplant follow-up of 22 months, SLTs achieved a 3-yr patient and graft survival not significantly different from the entire series of transplants (79.4 and 72.2% vs. 80.6 and 74.9%, respectively). Recipients receiving a WLT or a LLS showed significantly better outcomes than patients receiving RTL and MSL (P < 0.03 for patients and P < 0.04 for graft survival). At the multivariate analysis, donor age of > 60 yr, RTL transplant, < 50 annual transplants volume, urgent transplantation (United Network for Organ Sharing (UNOS) status I and IIA), ischemia time of > 7 hours, and retransplantation were factors independently related to graft failure and to significantly worst patient survival. Right grafts procured from RTL and either split procured as MSL had a similar outcome of marginal whole livers. In conclusion, in 5 yr, the increased number of pediatric transplants due to split liver donation reduced to 3% the in-list children mortality, and a decrease in the adult patient dropout rate from 27.2 to 16.2% was observed. Such results justify a more widespread adoption of SLT protocols, organizational difficulties not being a limit for the application of such technique.

Objectives Transfusion-related acute lung injury (TRALI) is a rare but serious complication which can occur after transfusion of blood components. In this report we describe our flow-cytometry approach to the laboratory diagnosis of a case of TRALI in a recipient of fresh frozen plasma containing human leukocyte antigen (HLA) class II antibodies. Methods The post-transfusion reaction work-up included the direct and indirect Granulocyte Immunofluorescence Test (GIFT) on the recipient's neutrophils collected before and after the reaction and on the serum from the recipient and from all implicated donors; flow-cytometry bead-based screening and identification assay for HLA class I and II antibodies in donor sera and flow cytometry cross-matching on T and B patient's lymphocytes. Finally, we investigated the reactivity of one donor serum, containing HLA class II antibodies, with the patient's neutrophils activated in vitro to induce expression of HLA class II. Results We found an increased level of IgG bound on patient's granulocytes collected after TRALI, in the absence of detectable granulocyte and HLA class I antibodies in the five implicated donors. One of them showed HLA-DR 1 and -DR 51 antibodies, which determined a positive cross-match with patient's B lymphocytes and in vitro activated granulocytes. Both HLA class II antigens were present in the recipient and absent in the donor. Conclusions In some pathological conditions, HLA class II antibodies can react with activated granulocytes expressing HLA-DR antigens, and activate TRALI reaction. HLA class II antibodies screening and flow cytometry cross-matching techniques should be added to the current diagnostic algorithm of TRALI.

Evaluation of outcomes is a major step in quality assessment of any health process. In the transplant field, the evaluation of outcome is extremely important for both patients' growing demand for health and for the joint commitment the transplant process requires. In this study, the outcome of 12,647 transplants, carried out between 1995 and 2000 were analysed. Graft survival at 5 years was 79% for kidney, 67% for liver, 72% for heart and 38% for lung. Patient survival was 92% for kidney, 76% for liver, 72% for heart and 38% for lung. In comparison to other international case records [Collaborative Transplant Study (CTS) and The United Network for Organ Sharing (UNOS)], results are similar or even better for all transplant programmes. As a whole, survival after solid organ transplant in Italy ranks among the best for both donations and transplantation. The quality of transplants carried out is above European standards. Nevertheless, the growing health needs of patients require improvement in both the procurement process and in the use of available organs.

BACKGROUND Viaspan (University of Wisconsin [UW]) solution is the gold standard for abdominal organ preservation. Celsior (CEL) is an extracellular-type, low-potassium, low-viscosity solution, initially used for heart and lung preservation. We have performed a prospective multicenter study to compare the role of these cold-storage solutions on kidney and liver recovery after transplantation. PATIENTS AND METHODS From March 15, 2000 to December 31, 2001, 441 (172 CEL and 269 UW) renal transplants (RT) and 175 (79 CEL and 96 UW) liver transplants (LT) were included in the study. RESULTS Perfusate volume used was significantly lower in the UW group, being 4,732 +/- 796 mL versus 5,826 + 834 mL in the CEL group (P < 0.001). In LT, median total bilirubin serum levels were significantly higher at 5 and 7 posttransplant days in the UW group (90.6 and 92.3 micromol/L, respectively) as compared with CEL (51.3 and 63.4 micromol/L, respectively). After LT, primary nonfunction (PNF) rates in the CEL and UW groups were 3.8% and 4.2% (P = NS) respectively, with 1-year graft and patient survival being 83.3% versus 85.4% (P = NS) and 89.9% versus 90.6% (P = NS). After RT, delayed graft function (DGF) rates were 23.2% and 22.7% (P = NS), respectively; PNF rates were 1.9% and 1.7% (P = NS) respectively, with 1-year graft and patient survival being 92.3% versus 94.2% (P = NS) and 99.4% versus 97.7% (P = NS). CONCLUSIONS CEL solution was shown to be as effective as UW in both liver and kidney preservation. In LT patients, biliary function recovery is significantly better in the CEL group. CEL solution represents an efficacious option in multiorgan harvesting.

Recent evidence indicates that neural stem cell properties can be found among a mammalian skin-derived multipotent population. A major barrier in the further characterization of the human skin-derived neural progenitors is the inability to isolate this population based on expression of cell surface markers. Our work has been devoted to purified human skin-derived stem cells that are capable of neural differentiation, based on the presence or absence of the AC133 cell surface marker. The enriched skin-derived AC133+ cells express the CD34 and Thy-1 antigens. These cells cultured in a growth medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) proliferate, forming spheres, and differentiate in vitro into neurons, astrocytes, and rarely into oligodendrocytes. Single cells from sphere cultures initiated from human purified AC133+ cells were replated as single cells and were able to generate new spheres, demonstrating the self-renewing ability of these stem cell populations. Brain engraftment of cells obtained from human purified AC133+-derived spheres generated different neural phenotypes: immature neurons and a most abundant population of well differentiated astrocytes. The AC133-derived astrocytes assumed perivascular locations in the frontal cortex. No donor-derived oligodendrocytes were found in the transplanted mouse brains. Several donor small, rounded cells that expressed endothelial markers were found close to the host vessel and near the subventricular zone. Thus, mammalian skin AC133-derived cells behave as a multipotent population with the capacity to differentiate into neural lineages in vitro and, prevalently, endothelium and astrocytes in vivo, demonstrating the great plasticity of these cells and suggesting potential clinical application. © 2004 Wiley-Liss, Inc.

BACKGROUND Patients undergoing kidney transplantation demonstrate a higher risk of developing cancer as the result of immunosuppressive treatment and concurrent infections. METHODS The incidence of cancer in a cohort of patients who underwent kidney transplantation between 1990 and 2000, and who survived the acute phase (10 days), was analyzed as part of the North Italy Transplant program. RESULTS A total of 3,521 patients underwent transplantation during a 10-year period in 10 of 13 participating centers; the length of follow-up after kidney transplant was 67.7+/-36.0 months. During the follow-up, 172 patients developed cancer (39 with Kaposi sarcoma, 38 with lymphoproliferative diseases, and 95 with carcinomas [17 kidney, 11 non-basal cell carcinoma of the skin, 10 colorectal, 8 breast, 7 gastric, 7 lung, 6 bladder, and 3 mesothelioma]). The average time to cancer development after transplant was 40.1+/-33.4 months (range 0-134 months). Twenty-four patients developed cancer within 6 months from the transplant (10 with carcinomas, 7 with Kaposi sarcoma, and 7 with lymphoproliferative diseases). Three patients demonstrated a second primary cancer. The average cancer incidence was 4.9%. The incidence of cancer was 0.01 per year. Independent determinants of cancer development were age, gender, and immunosuppressive protocol including induction. Ten-year mortality was significantly higher in patients with cancer (33.1%) than among patients without cancer (5.3%). The relative risk of death in subjects with cancer was 5.5 (confidence interval 4.1-7.4). CONCLUSIONS These preliminary data underline the importance of long-term surveillance of transplant recipients, choice of immunosuppressive treatment, and careful donor selection.

In this brief communication we report the characterization of two new HLA-B variants officially named HLA-B*0817 and HLA-B*1311. The HLA-B*0817 allele was identified in a Caucasoid male candidate for renal transplantation in the North Italy Transplant program. The nucleotidic sequence of exons 2, 3 and 4 of this novel allele is identical to that of HLA-B*0804 except for three point mutations in exon 2: from A to G at position 259, from C to G at position 261 and from G to A at position 302. These mutations are responsible for two aminoacidic substitutions [Asn (r) Glu, codon 63, and Ser (r) Asn, codon 77]. HLA-B*1311 was found in a volunteer donor belonging to National Marrow Donor Program®. This new variant is identical to that of HLA-B*1301 except for three nucleotide substitutions at positions 353, 355 and 369 leading to two aminoacidic variations from Ile to Thr at codon 94 and from Ile to Leu at codon 95 and a silent mutation at codon 99.

Background. The aim of this study was to evaluate the effects of islet transplantation on patient survival and diabetic vascular complications. Methods. Thirty-seven type 1 uremic diabetic kidney transplant patients underwent islet transplantation (KI group). Uremic type 1 diabetic kidney-pancreas (KP group, n=162), kidney-alone (KD group, n=42) transplant patients, and uremic type 1 diabetic patients still on hemodialysis (HD+DM group, n=196) constituted the control groups for survival and endothelial morphology. Results. Patient survival was similar in the KI and KP groups and higher than in the HD+DM group (P

Shortage of kidney donor is still a major limitation for renal transplantation programs. This review focuses on the emerging practices, adopted to increase transplant activities, of expanding the criteria for donor and recipient selection without exposing the recipient to the drawbacks of a graft with inadequate nephron mass. Expanding the donor pool inevitably led to consideration for kidney transplantation of organs from older donors or from donors with hypertension, diabetes or other renal diseases. To fit the reduced performance of these suboptimal organs with the renal requirement of the recipient, selection of recipients with reduced metabolic requirements or increase of nephron mass by simultaneous transplantation of two suboptimal kidneys in the same recipient have been pursued. However, a critical aspect of both approaches is to quantify functioning nephron mass provided to the recipient by pre-transplant kidney biopsies. Morphological parameters assessed on kidney biopsies at the time of donor evaluation may serve to quantify the preserved tissue and to discriminate chronic irreversible lesions from acute changes that may account for a transiently impaired renal function in the donor, but that may recover after transplant.

We describe here the sequences of 3 new HLA-DRB1 variants officially named DRB1*03:05:03, DRB1*11:10:02, and DRB1*14:86. These novel alleles have been detected in 3 Caucasoid individuals by sequence-based typing. The first and second alleles are the result of a silent mutation, which does not imply any amino acid change. The sequence of DRB1*14:86 exhibits a single nucleotide difference with the allele DRB1*14:01:01 at position 239.

Polymorphisms in the regulatory and intronic regions of several cytokines have been associated with differential cytokine production. In this paper we genotyped, using the polymerase chain reaction-sequence-specific primers (PCR-SSP) method, a series of 363 healthy Italian Caucasians with the aim of obtaining a reference population for further studies on the role of cytokines in the inflammatory and immune responses. We also compared the results to those for other populations. The polymorphisms analysed were those of tumour necrosis factor alpha (TNFA), interleukin 6 (IL-6), interleukin 10 (IL-10) and interferon gamma (IFNG). We found that the frequency of allele TNFA*1 at position -380 was 87.7% and that of TNFA*2 was 12.4%, significantly different from those of the UK and Japanese populations but not different from that of a population in Gambia. For IL-10 the frequencies of alleles -1082A and -1082G were 63.0% and 37.0% and those of alleles -819C, - 819T, -592C and -592A were 70.8, 29.2, 70.8 and 29.2%, respectively, significantly different from those observed in south-east England, in Manchester and in an Oriental population from southern China. The frequencies of IL-6 alleles - 174C and -174G were 29.0 and 71.0%, respectively; for IFNG polymorphisms at position -874, in the population under evaluation, the alleles -874T and -874A were present in 44.7 and 55.3% of the subjects, respectively. Genotype frequencies of IL-6 were significantly different from those observed in populations from Germany and from the UK. The analysis carried out by our group indicates that there is heterogeneity in the frequencies of the cytokine polymorphisms among the different Caucasian populations, and this underlines the importance of a 'local' reference population when evaluating the clinical relevance of cytokine gene polymorphisms.

Background. A possible link between coeliac disease and dilated cardiomyopathy has recently been suggested. Aims. To assess the frequency of anti-endomysial antibodies, the marker for coeliac disease, in patients with different forms of heart failure, and to establish the clinical features of those endomysial antibody positive. Subjects and Methods. A total of 642 consecutive patients entering the waiting list for heart transplantation from 1995 through 1997 were studied. The prevalence of endomysial IgA antibodies, determined by indirect immunofluorescence, was compared to that observed in three surveys conducted in the Italian general population. Results. Of the 642 patients, 12 (1.9%; 95% confidence interval 0.97–3.2) resulted endomysial antibody positive, versus 349720 healthy controls (0.35%; 95% confidence interval, 0.23–0.47), accounting for a relative risk of 5.3 (95% confidence interval, 2.8–10.3). Anti-endomysial antibodies were found in 6275 patients with dilated cardiomyopathy and 6367 with other forms of heart failure (2.2% versus 1.6%; 95% confidence interval 0.8–4.7 and 0.6–3.5), with no statistical difference. The 12 endomysial antibody positive patients were leaner (body mass index, 22.0±1.9 vs 24.2±3.1, p

Long-term outcomes of renal transplantation using kidneys from donors >60 years old are generally considered to be poor. This retrospective study included 265 living donor (LD) transplants in adult recipients with a mean follow-up of 13.1 ± 6.1 years (range, 1.3–25.8), all of them under CNI. They were grouped according to the donor age at least (n = 49) or less (n = 216) than 60 years. Graft and patient survivals were compared using the Kaplan-Meier method and Cox multiple regression. At 1, 3, and 10 years, postoperatively patient survivals in the group of older LD recipients were 97%, 96%, and 93%, versus 98%, 97% and 92% among the younger LD recipients. At 1, 3 and 10 years, postoperatively graft survivals uncensored for death were 94%, 92%, and 81% among the older LD recipients versus 93%, 89%, 75% among the control group, respectively, despite a slightly increased creatininemia observed at 10 years among the older LD recipients. Deaths censored graft survivals were 96%, 96%, and 87% among the older versus 94%, 91% and 78% among the younger LD recipients, respectively. Therefore, significantly better noncensored death-censored graft survivals, were observed among the recipients of older LD compared with recipients of the younger donor group.

The large imbalance between cadaver kidney supply and demand makes the implementation of equitable and effective organ allocation systems an urgent need. This has triggered a revision of the criteria used so far for cadaver kidney allocation within the North Italy Transplant program, not least in the light of the many changes that have occurred recently with respect to broader criteria for admission of patients to the waiting list, donor selection, tissue-typing methods, organ preservation and immunosuppressive protocols. We based the critical revision of our cadaver kidney allocation algorithm on univariate and multivariate analysis of a number of immunological, clinical, social and administrative factors that impacted on the transplant outcome in 2,917 patients transplanted in the 12 transplant centers operating within our organization from 1 January 1990 to 30 September 1997. This analysis indicated that younger donor age, absence of pretransplant transfusions, patient dialysis center and level of HLA match showed statistically significant positive associations with graft survival. Younger donor age and male donor gender showed a statistically significant association with excellent graft function at 4 years. The results of this analysis were used to develop a new computer-assisted version of our adult kidney allocation algorithm. It works in two steps (local pool first, then the entire waiting list) and four levels (0–1 HLA MM, PRA + ; 2 HLA MM, PRA + ; 0–1 MM, PRA–; 2–4 HLA MM, PRA–); within each level, selection takes into account waiting time and age difference from donor age. The evaluation of 731 transplants allocated in 19 months with the new algorithm, as against 698 transplants allocated in the preceding 19 months according to the previous algorithm, showed a significantly higher proportion of recipients who had been on the waiting list for more than 3 years (33.2 % versus 22.6 %). The use of the new algorithm was also associated with a significantly increased number of transplanted alloimmunized patients (18.8 % versus 9.2 % with the previous algorithm) and recipients with 0–1 HLA mismatches (22 % versus 14.3 %). Furthermore, the number of kidneys used locally has steadily increased. Differences in 6-month graft survival and percentage of patients with excellent function at 6 months were not statistically significant in recipients transplanted with the new versus the previous algorithm. Survivals were 93.7 % versus 91.8 %. Percentages of patients with excellent renal function were 69.9 % and 71.8 %, respectively. These preliminary data suggest that the new algorithm improves HLA match and reduces the number of patients on the waiting list for 3 or more years without determining significant modifications of 6-month graft survival and function. Moreover, it facilitates the achievement of a fair local balance between organs retrieved and transplanted, the compliance of operators with objective allocation rules and the documentation of the whole allocation process.

In immunocompetent patients, specific human leukocyte antigen (HLA) class II alleles have been associated with the severity of hepatitis C virus (HCV)–related disease, in particular, HLA-DRB1*11 has been found to exert a protective effect. The authors have analyzed the role of HLA class I and II alleles in determining the frequency, timing, and progression of histologically proven recurrent hepatitis C in 89 patients who underwent a liver transplant for HCV–related cirrhosis. In addition, the influence of HLA mismatch between donor and recipient, HCV genotype, and use of steroid pulses was also evaluated. Median patient follow up was 35 months (range 4-119). HLA-DRB1 typing was performed by genomic analysis in all cases. Liver biopsies were obtained routinely and at least at yearly intervals. Histologically proven recurrent hepatitis was observed in 46 patients (52%), 10 patients progressing to stage 5-6 fibrosis in most cases within 2 years after transplant. By univariate analysis, 3 variables, HLA-B14, HLA-DRB1*04, and HLA-DRB1 donor/recipient mismatch, showed a significant effect on time to recurrent hepatitis C disease. These parameters were included in a multivariate regression model along with HCV genotype, treatment with steroid pulses and DRB1*11. HLA-B14, HLA-DRB1*04, and HLA-DRB1 donor/recipient mismatch were confirmed to provide a significant and independent contribution to the risk of hepatitic disease recurrence. As for the severity of the disease, none of the 10 patients with stage 5-6 hepatitis carried the HLA-DRB1*11 allele, in line with what was observed in nontransplant subjects. Our results suggest that in posttransplant recurrent hepatitis C, immunogenetic factors are relevant in determining HCV infection outcome.

Preliminary epidemiological and histological studies from Japan suggested that hepatitis C virus (HCV) infection has a role in the development of dilated cardiomyopathy (DCM). This multicenter study was conducted to verify this hypothesis on a large cohort of Italian patients with end-stage heart failure. Antibodies to HCV were determined in the 752 consecutive patients (608 males and 144 females; age, 53 ± 13 years) who entered the waiting list for cardiac transplantation from 1995 to 1997 at the six cardiac surgery centers participating in the North Italy Transplant program. Three hundred and nine patients (41%) had dilated, 9 (1%) restrictive, and 4 (0.5%) hypertrophic cardiomyopathy; 284 patients (38%) had ischemic, 65 (9%) valvular, and 22 (3%) congenital heart disease; 5 patients (0.5%) had primary pulmonary hypertension; 54 patients (7%) had other or nonspecified heart disease. Overall, 41 of 752 patients (5.4%) resulted anti-HCV–reactive. Serological evidence of HCV infection was found in 12 of 309 patients with DCM (3.9%; 95% CI, 1.7–6.0), and in 29 of 443 without DCM (6.5%; 95% CI, 4.2–8.8), without statistical dif- ference (difference of prevalence rate: 2.6%; 95% CI, -4.9 to 5.8). In conclusion, HCV does not seem to have a primary role in the pathogenesis of DCM. However, since our findings are in disagreement with those obtained in smaller series of patients of other ethnicity, large studies from different countries should be conducted. J. Med. Virol. 58:116–120, 1999. © 1999 Wiley-Liss, Inc.

Two novel human leucocyte antigen (HLA) class I alleles were characterized by means of sequencing-based typing techniques. HLA-A*310103 was identified in a cord blood unit from a Caucasoid individual. The sequence of this allele is identical to that of HLA-A*310102 except for a silent mutation in exon 3 at position 480 (G --> A). HLA-B*9531 was found in a Caucasoid female patient registered on the heart transplantation waiting list in the North Italy Transplant programme. This new variant differs from HLA-B*1503 at position 572 (G --> C) in exon 3. This nucleotide change leads to an amino acidic substitution at codon 167 from tryptophan to serine.

HLA class I typing by standard microcytotoxicity testing has been unsatisfactory for 14.5% of 1644 cord blood samples. In this study, we evaluated the capacity of PCR-SSP in solving problems in HLA-A,B typing with serological methods. With this aim we have compared serology with PCR-SSP in 100 cord blood samples with doubtful or unreliable HLA-A,B typing. PCR-SSP was successful in amplifying HLA-A,B alleles in all 100 cord blood samples. Forty-six typings gave discrepant results with the 2 methods (serology and PCR-SSP). Typings were considered discrepant also in the case of inability to define a split. For 19 specimens, no serological conclusion was drawn due to high mortality of the cell suspension, while PCR-SSP allowed the definition of a clear typing. In 6 cases it was necessary to infer information from serology to define the current typing. Finally, in 3 other cases it was impossible to exclude or attribute the antigen/allele B67 or B4802. PCR-SSP for HLA-A,B can improve the overall reliability of HLA-A,B typing requiring a small amount of blood although, with the set of sequence specific primers adopted, a number of alleles are still poorly defined.

This study is the largest Italian survey on liver retransplantations (RET). Data report on 167 adult patients who received 2 grafts, 16 who received 3 grafts, and one who received 4 grafts over a 11 yr period. There was no statistically significant difference in graft survival after the first or the second RET (52, 40, and 29% vs 44, 36, and 18% at 1,5,and 10 yr, respectively: Log-Rank test, p = 0.30). Survivals at 1, 5, and 10 years of patients who underwent 2 (n = 151) or 3 (n = 15) RETs, were 65, 48,and 39% vs 59, 44, and 30%, respectively (p = 0.59). Multivariate analysis of survival showed that only the type of graft (whole vs reduced) was associated with a statistically significant difference (HR = 3.77, Wald test p = 0. 05); the donor age appeared to be a relevant factor as well, although the difference was not statistically significant (HR = 1.91, Wald test p = 0.08). Though late RETs have better results on long term survival relative to early RETs, no statistically significant difference can be found in early results, till three years after RET. Considering late first RETs (interval>30 days from previous transplantation) with whole grafts the difference in graft survival in RETs due to HCV recurrence (n = 17) was not significantly different from RETs due to other causes (n = 53) (65-58 and 31% vs 66-57 and 28% respectively at 1-5 and 10 years, p = 0.66). © 2012 Maggi et al

The feasibility and utility of a lung donor score that has been recently proposed was tested among a pool of lung donors referred to the Nord Italia Transplant program (NITp) organ procurement organization.Each lung donor was assigned an Oto score including, age, smoking history, chest X-ray, secretions and ratio of arterial oxygen tension to inspired oxygen fraction (PaO(2)/FiO(2)). Based on clinical compromise, each variable received a score between 0 and 3, except for PaO(2)/FiO(2), which was scored between 0 and 6 given its overall relevance.Throughout 2010, 201 multiorgan donors were initially considered to be potential lung donors. Among these, 59 (29.4%) eventually yielded 67 lung transplantations (named "Used group"). Among the 142 (70.6%) refused lungs, 28 were not used due to logistic or medical problems ("general exclusion" group, GE) and 114, because of poor lung function ("lung exclusion" group, LE). Median lung donor scores were 1 (range, 0 to 3), 4 (range, 2.5 to 6.5), and 7 (range, 5 to 9) in the Used, GE, and LE groups, respectively (one-way analysis of variance, P.001). Some donors with Oto scores ≤7 worsened over time so that the score had significantly increased by the time of organ retrieval. Overall, subjects who died after lung transplantation were characterized by higher lung donor scores, (2 [1-4] versus 0.5 [0-3], P = .003).Our analysis suggested that the use of a donor score as a dynamic tool over the donation process was of great utility to describe and analyze a pool of lung donors.

We describe here the sequence and the molecular modeling of a new variant of HLA-A*32 allele officially named A*32:22. This novel allele has been detected in an Italian cord blood sample by sequence-based typing (SBT). The mutation (CAT →CGT), which has occurred at codon 151, at nucleotide position 524, implies an amino acidic change from Histidine to Arginine. Residue 151 is located on top of the molecule inside the region contacted by T cell receptor (TCR) and it is possibly involved in docking TCR. A positively charged residue is maintained on this position determining a slight change of electrostatic potential on the molecular surface. This suggests a limited functional relevance of the amino acid substitution encoded by A*32:22.

Summary Background In a previous publication we described a method for Jk a /Jk b , Fy a /Fy b , S/s, K/k, Kp a /Kp b , Js a /Js b , Co a / Co b , and Lu a /Lu b genotyping based on a microsphere suspension array. Here, an improved version of the assay is presented. Methods: Two multiplex polymerase chain reactions (PCR) were developed: one for amplification of samples routinely tested and the other for those systems that are tested less frequently. Each biotinylated PCR product is hybridized in a single multiplex assay. A total of 2,020 samples were analyzed, and the genotypes were compared to the blood group phenotypes. Results: There have been no discrepancies with the serology results other than null and/or weak phenotypes. Conclusion: In its present form, the method presented here has the capacity to genotype hundreds of a samples in few hours with a high concordance rate with serology. Schlusselworter

Allogeneic hematopoietic SCT (HSCT) increases the risk of hepatitis B virus (HBV) reactivation in hepatitis B surface antigen (HBsAg) carriers but the incidence, risk factors and course of HBV reactivation after HSCT in HBsAg-negative/anti-hepatitis B core antigen (anti-HBc)-positive recipients are not well known. A total of 50 HBsAg-negative/anti-HBc-positive HSCT recipients with onco-hematological diseases, underwent sequential clinical and laboratory examinations, including serum HBsAg, during follow-up. Serum HBV DNA collected at HSCT was retrospectively amplified by a sensitive PCR assay. During 17 months of follow-up, six (12%) patients had seroreverted to HBsAg, 7-32 months after HSCT, with 1- and 5-year cumulative rates of 13 and 22%. HBsAg seroreversion was associated with serum HBeAg higher than 8 log₁₀ copies per ml HBV DNA and a 1.5 to 36 fold increase of serum alanine aminotransferase leading to HBeAg-positive chronic hepatitis B in all patients. Patients with chronic onco-hematological disease and long-lasting immunosuppression following HSCT had a higher risk of HBsAg seroreversion independently of serum HBV DNA levels at HSCT. HBsAg-negative/anti-HBc-positive HSCT recipients with chronic onco-hematological disease carry a significant risk of HBsAg seroreversion and HBeAg-positive chronic hepatitis B, independently of serum levels of HBV DNA at transplantation.

Although human leukocyte antigen (HLA) crossmatching is often thought to be unnecessary for liver transplants (LTs), we provide evidence that for retransplants, it is essential. Sera from 139 retransplant patients who had received livers from deceased donors were retrospectively analyzed with single antigen beads on a Luminex platform for HLA antibodies. Each patient received at least 2 transplants and was followed up for at least 6 months from the second LT, which was deemed to have failed if the patient had a third LT or died. Second LT survival was calculated from the date of the second LT to the date of the third LT or death. Our study cohort consisted of 118 adult patients (or = 18 years old) as well as 21 pediatric patients (18 years old). Class I HLA antibodies were associated with significantly poorer regraft survival in adults [survival differences of 21.3% (P = 0.046), 22.1% (P = 0.042), and 23.7% (P = 0.033) at 1, 3, and 5 years, respectively]; however, the presence of these antibodies was not associated with significant survival differences in the pediatric population. A univariate analysis of the effect of class I antibodies on second LT survival in adults showed a hazard ratio of 2.0 (95% confidence interval = 1.0-3.8, P = 0.028). Graft survival in patients with and without HLA antibodies or class II antibodies was similar. Because class I antibodies have a deleterious effect on liver regraft survival, crossmatch testing should be performed before liver retransplantation.

Transplantation activity is dependent upon organ procurement; although great efforts are made to enlarge the cadaver donors' pool, it still remains far too small to meet the recipients' need. Waiting time is a particular problem for paediatric patients, and mortality on the waiting list for liver transplantation is very high. The number of paediatric donors is far too small to satisfy the request. To enlarge the liver pool, the split-liver procedure was introduced in several Transplant Centers. In November 1997, the North Italy Transplant program (NITp) Working Group for Liver Transplantation decided to start an official Split-liver Program. A protocol was therefore defined and criteria for donor's and recipient's eligibility were established to minimize the risk. The Working Group also standardized the technical procedure and defined collaboration between centers. Out of 410 cadaver liver donors used in the NITp, from 1 November 1997 until 31 May 1999, 49 patients (37 males and 12 females) were chosen for the split-liver procedure. Mean age was 29.9 +/- 17.5 years. Mean ICU stay of the donors was considerably short (2.5 +/- 2.1 days), and the other conditions foreseen for donor eligibility were met. In all cases (except two) an "in situ" technique was performed. Forty-nine adult recipients and 43 children were transplanted by the split-liver technique in our Transplant Centers. One right lobe and five left liver lobes were sent to Transplant Centers outside the NITp. Adult recipient age ranged from 18 to 60 years (mean 46.4 +/- 11.7 years), and the paediatric one from 2 to 144 months (mean 24.8). Mean patient follow-up was 8.3 +/- 5.5 months. In the paediatric group, the graft was successful in 34 cases (79%), five patients (10.2%) died and four (9.3%) were re-transplanted. In the adult group, graft survival was 67.3%, 11 (22%) patients died and 5 (10%) were re-transplanted. On 1 November 1997, 30 paediatric patients were on the liver waiting list. In the preceding 19 months, 52 patients were newly enrolled, and 36 transplants were performed. The mean waiting time of paediatric patients was 259 days (range 1-919 says). From 1 November 1997 to 31 May 1999, 61 paediatric patients were newly enrolled. In this period 70 patients were transplanted. The mean waiting time was 185 days (1-1010 days). At present, the liver waiting list includes eight paediatric patients. Split-liver transplantation is a successful procedure, effective in reducing waiting time for paediatric patients. It should be established if this may be a tool to enlarge the organ pool also for adult liver transplantation.

Correct definition of clinically relevant anti-HLA antibodies is important for transplant organ allocation and outcome. We describe a candidate for kidney transplantation who was treated with isoniazid because of active tuberculosis. The patient's serum gave a positive antibody result on screening with the complement-dependent cytotoxicity (CDC) test but a negative result on screening with a bead-based assay (Luminex). The clinical history indicated no immunologic stimuli. Subsequent testing on fresh serum samples confirmed the discrepancy between CDC and Luminex results. An autologous cross-match test gave negative results, and the antibodies were sensitive to dithiothreitol treatment. We postulated that nonspecific binding of drug-antibody complexes to panel lymphocytes in the CDC test may have caused the observed lympholysis. This case, although isolated, emphasizes the importance of the combined use of CDC and solid phase assays. The CDC results alone would have led to the erroneous conclusion that the patient was highly sensitized.

We report herein the identification of a new HLA-A null allele. This allele, A*0105N, was detected during histocompatibility testing of a cord blood donor and the respective mother. Serologic typing results contrasted those obtained with DNA typing that alone showed the presence of HLA-A*01. ThisA*0105N was due to a nucleotide deletion in exon 4 that altered the reading frame, causing a premature termination Note.

Traditionally, blood group typing has been performed with serologic techniques, the classical method being the hemagglutination test. Serotyping, however, may present important limitations such as scarce availability of rare antisera, typing of recently transfused patients, and those with a positive direct antiglobulin test. Consequently, serologic tests are being complemented with molecular methods. The aim of this study was to develop a low-cost, high-throughput method for large-scale genotyping of red blood cells (RBCs).Single-nucleotide polymorphisms associated with some clinically important blood group antigens, as well as with certain rare blood antigens, were evaluated: Jk(a)/Jk(b), Fy(a)/Fy(b), S/s, K/k, Kp(a)/Kp(b), Js(a)/Js(b), Co(a)/Co(b), and Lu(a)/Lu(b). Polymerase chain reaction (PCR)-amplified targets were detected by direct hybridization to microspheres coupled to allele-specific oligonucleotides. Cutoff values for each genotype were established with phenotyped and/or genotyped samples.The method was validated with a blind panel of 92 blood donor samples. The results were fully concordant with those provided by hemagglutination assays and/or sequence-specific primer (SSP)-PCR. The method was subsequently evaluated with approximately 800 blood donor and patient samples.This study presents a flexible, quick, and economical method for complete genotyping of large donor cohorts for RBC alleles.

Hand surgeons and reconstructive microsurgeons have been “transplanting” composite tissues for a long time. Every time we perform a free flap, we are actually performing a true transplant. Most of the time there is no choice other than to adapt a flap coming from a certain donor area to a recipient area with different characteristics. Normally this happens in cases of complex multitissue defects or when the tissue that needs reconstruction carries some unique features. So while we are now exploring the area of prefabrication or prelamination of flaps, we are aware that we are far from perfection, hence the need to look at using possible allogeneic tissues [1]. With the advent of microsurgery, the long-term results of autologous replantation of the upper extremity, especially after a clean-cut amputation, have become extremely good. In a recent long-term review study, it was shown that replanted hands and forearms had a mean loss of range of motion of 17.5% [2].