Sergio Barocci

Role of major histocompatibility complex class I expression and natural killer-like T cells in the genetic control of endometriosis

OBJECTIVE To evaluate whether the expression of human leukocyte antigen (HLA) class I on eutopic and ectopic endometrial cells modify the susceptibility to lysis mediated by lymphocytes. DESIGN Evaluation of T lymphocyte cytotoxic activity and HLA class I expression on endometrial cells. SETTING Subjects were recruited at laparoscopy. PATIENTS Patients with endometriosis (n = 7). Healthy women as controls (n = 10). MAIN OUTCOME MEASURES Human leukocyte antigen class I molecule analysis of endometrial cells was carried out by immunofluorescence and flow cytometry. Phenotyping of T lymphocytes was performed to analyze T-cell subsets. Cytotoxicity was performed to determine cytolytic activity against endometrial cells. RESULTS In vitro culture of endometrial cells down-regulates the expression of HLA class I molecules and enhances the susceptibility to lysis mediated by natural killer (NK)-like T lymphocytes. Cytolytic T-cell clones, expressing the CD94 antigen, are inhibited by the HLA-B7 allele on endometrial cells. Ectopic endometrial cells modulate the expression of HLA class I molecules. CONCLUSIONS The resistance to lysis of endometrial cells is related to expression of surface HLA class I molecules, which send a negative signal for lysis mediated by NK-like T lymphocytes. The HLA-B7 allele inhibits the cytotoxic activity, suggesting that the growth of ectopic endometrial cells might be under a genetic control.

HLA class-I-soluble antigen serum levels in liver transplantation : a predictor marker of acute rejection

The serum levels of sHLA-I have been determined in 16 patients following liver transplantation. sHLA-I levels did not show remarkable variations in six patients without evidence of transplant-related complications. sHLA-I levels strongly increased in 10 patients undergoing acute rejection episodes. In these patients, an average 20% daily increase of sHLA-I levels was detected on the 6 days preceding and on the 2 days following the rejection episode. A fast decrease of sHLA-I levels was observed in seven patients following treatment of acute rejection with anti-CD3 mAb. The serum level of sHLA-I antigens positively correlated with ALT serum level and inversely correlated with PT. The determination of sHLA-I in serum may therefore be proposed as a useful marker in the monitoring of patients following liver transplantation. The increase of sHLA-I antigens may predict the onset of acute rejection whereas their decrease may be related to a good response of acute rejection to immunosuppressive treatment.

Long-Term Outcome on Kidney Retransplantation: A Review of 100 Cases From a Single Center

Renal transplantation has become an effective form of treatment for end-stage renal failure. Unfortunately, as a consequence of immunological and nonimmunological pathogenic mechanisms, chronic allograft nephropathy is responsible for the loss of a large proportion of kidney grafts after several years and return to dialysis. We have reported herein our 24 years of experience with second kidney transplantations. Of 1,302 kidney transplantations between January 1983 and June 2007 performed in our transplantation center, 100 were second transplantations. Kidney retransplantation was performed in 74 men and 26 women of overall mean age of 35.4 +/- 12.6 years. Cadaveric donor grafts were transplanted in 92 patients, whereas the remaining 8 were living-related donor kidneys. At 1, 5, and 10 years after kidney transplantation, patient survival rates were 100%, 96%, and 92%, respectively, whereas graft survival rates were 85%, 72%, and 53%, respectively. Immunosuppressive therapy included induction therapy with polyclonal anti-lymphocyte antibodies (ALG/ATG) or (starting from 1999) monoclonal anti CD 25 antibody. Our results demonstrated good outcomes for kidney retransplantations with allocation based on anti- HLA antibody identification together with induction immunosuppression.

Recognition of donor fibroblast antigens by lymphocytes homing in the human grafted kidney

A human transplanted kidney, surgically removed because of untreatable chronic rejection, was used as the source of lymphocytes (K-L) of recipient origin that were expanded with interleukin-2 (IL-2), and of kidney fibroblasts (K-F) of donor origin that were maintained as an established line. Cytotoxicity assays were performed using K-L and peripheral blood lymphocytes (PBL) as effectors, and K-F and donor PBL as targets. From the results the following conclusions can be drawn: (1) cytotoxic lymphocytes, presumably involved in the process of chronic graft rejection, home in the kidney (from which they can be recovered) but are not detected in the circulation; (2) cytotoxic lymphocytes can be generated from peripheral lymphocytes by mixed lymphocyte culture (MLC) and further expansion in vitro with IL-2 (MLC-L); and (3) although both K-L and MLC-L are cytotoxic toward K-F, the former are not cytotoxic toward donor PBL. This suggests that although MLC-L recognize antigens shared by K-F and PBL, K-L recognize antigens specific for K-F only. These results, if confirmed, indicate that antigens not present on PBL, and possibly tissue-restricted are important in graft rejection. Thus, while monitoring transplanted patients, a lack of cytotoxicity in the recipient PBL may be misleading because the relevant cytotoxic effector cells may have disappeared from the periphery and the appropriate antigenic target may be absent on donor PBL.

Cytokine mRNA expression in chronically rejected human renal allografts

Abstract:  Although both immunologic and non‐immunologic components may cause kidney allograft chronic rejection (KGCR), also referred to as chronic allograft nephropathy (CAN), its pathogenesis is largely not yet understood. To explore relevant immunologic mechanisms occurring in KGCR, we have analyzed in surgically removed KG the transcription of the following cytokine and apoptotic molecule genes: interleukin (IL)‐2, IL‐3, IL‐4, IL‐5, IL‐6, IL‐10, tumor necrosis factor (TNF)‐α, IFN‐γ, FAS, and FAS‐L. Semiquantitative RT‐PCR was used and KG explants were obtained from two groups of transplanted patients. Group 1 was represented by CR/CAN KG, removed for: (a) superimposed symptoms of acute lesions (SAL) due to tapering or suspension of immunosuppression (subgroup 1a, eight cases); (b) causes other than SAL (two cases, subgroup 1b). Group 2 comprised explanted kidneys with no CR/CAN (three cases – vascular thrombosis, intrarenal hemorrhage and vascular thrombosis). The results showed that in group 1 IL‐ 6 was detectable in seven of 10, IL‐10 in six of 10, IFN‐γ in five of 10, and IL‐3 in four of 10 cases with a variable pattern of reciprocal association. IL‐2 and TNF‐α were represented in one of 10 cases only. Particularly, in the subgroup 1b IL‐10 was never detected. Among the most represented cytokines of group 1, IL‐10 as well as IL‐3 were never found in group 2. The peculiar expression of IL‐10 and IL‐3 and partially IL‐6 seems to support the hypothesis that a Th2 pattern predominantly characterizes KGCR, thus indicating that Th2 cytokines, likely produced by different intragraft cell types including T cells, macrophages and natural killer (NK) cells, may represent an important component in the pathogenesis of this process. Moreover, IL‐10 seems to exquisitely characterize a group of CR/CAN kidney grafts more prone to immunologic assaults.

In vitro removal of anti-HLA IgG antibodies from highly sensitized transplant recipients by immunoadsorption with protein A and protein G sepharose columns: a comparison

Abstract. In the present study we compared the capabilities of sepharose‐bound protein A versus protein G columns to remove in vitro lymphocytotoxic anti‐HLA antibodies from sera of four highly sensitized renal transplant recipients (PRA≥70%). In none of the patients were protein A sepharose columns capable of completely removing anti‐HLA antibodies, as demonstrated by the presence of residual alloreactive lymphocytotoxic activity in IgG 3 antibodies containing fractions eluted at pH 7. In contrast, no residual anti‐HLA lymphocytotoxic antibody activity was found in fractions eluted at pH 7 from protein G columns. These data demonstrate that: (1) IgG 3 antibodies can be partly responsible for lymphocytotoxic anti‐HLA reactivity in some sensitized renal transplant recipients and (2) at least in this patient category, in vitro immunoadsorption with protein G is more efficient than protein A sepharose columns in completely removing anti‐HLA IgG antibodies from sera.

HLA matching in pediatric recipients of a first kidney graft. A single center analysis.

We retrospectively examined the effect of HLA-A, -B, and -DR serological matching on graft survival in 88 pediatric end-stage renal disease patients who underwent primary renal transplantation. Actuarial graft survivals (GS) at 2 and 6 years in patients with zero DR mismatches (MM) (12 patients) or 1 DR MM (58 patients) were significantly higher than those in patients with 2 DR MM (18 patients) (2-year GS: 100% vs. 90% vs. 59%; 6-year GS: 100% vs. 79% vs. 59%, respectively). Because of the low number of patients in the zero DR MM group, only the GS difference between 1 DR MM and 2 DR MM had a significant result at 1 year (92% vs. 68%). No clear HLA matching effect was obtained in the HLA-A and -B loci. When DR were combined with A or B antigens (0-2 MM vs. 3-4 MM), significantly higher GS at 1, 2, and 6 years persisted for patients with 0-2 MM only in the A, DR group (96%, 94%, and 85% vs. 68%, 63%, and 56%, respectively). It is suggested that avoidance of mismatching for DR alleles at the serological level, in the selection of pediatric recipients of first cadaveric renal transplantation, leads to an improvement of both short- and longterm graft outcome.

Detection and analysis of HLA class I and class II specific alloantibodies in the sera of dialysis recipients waiting for a renal retransplantation.

Abstract:  The objective of this study was to evaluate the specificities of HLA class I (‐A,‐B) and class II (‐DR,‐DQ) antibodies (Ab) detected in the sera of alloimmunized patients waiting for a subsequent renal transplantation. The study group consisted of 62 dialysis patients (42 men and 20 women, mean age: 43 ± 18 yr) on waiting list for a subsequent kidney transplant (52 for a second and 10 for a third transplant) at S. Martino Hospital Transplant Centre in Genoa/Italy, who were enrolled from 2002 to 2004 for HLA antibody screening. Complement dependent cytotoxicity (CDC) technique was used firstly to select anti‐HLA class I sensitized patients; indeed sera from 50 individuals out of 62 (80.6%) were found to display persistent HLA class I PRA (panel reactive antibody) values >4% (range: 20–100). ELISA technique was subsequently adopted to analyze HLA class I Ab positive sera for the presence also of HLA class II Ab and to characterize class I and class II Ab specificities. Anti‐class I immunized patients were divided in three groups according to the type of class I Ab specificities, that were classified as private, public, and multispecific. The first group included 35 patients (70% of the total number of positive patients) showing only antibodies directed against private HLA class I specificities, represented in 33 cases by those expressed by graft donors (first or second transplant). In this group anti‐class I PRA% values ranged from 20% to 60%. HLA class II Ab, with an heterogeneous specificity pattern (private, public or multispecific), were present in 25 (78.1%) out of the 32 patients, whose sera were also available for this analysis. The second group comprised 12 patients (24%) who displayed antibodies directed against class I public epitopes belonging to CREGs (Cross reactive Groups) or an association of anti‐private and anti‐public antibodies. In this group PRA values ranged from 25% to 90%. Five patients (46.7%) were positive for HLA class II Ab, whose specificity pattern appeared also heterogeneous (private or multispecific). The third group was represented by three patients (6%) displaying multispecific antibodies with PRA values ≥90%. No multispecific class II Ab were found in this group, where only two patients had class II Ab showing anti‐private or anti‐private plus public specificities. Globally, 74% of anti‐class I Ab positive patients, having at least one HLA class II antigen mismatch, appeared also positive for class II Ab. These results indicate that: (i) a large proportion of patients, waiting for a kidney retransplantation, display in their sera alloantibodies specific for graft mismatched HLA class I (80.6%) and class II antigens (54.2); (ii) the immunogenic determinants, mainly involved in HLA class I and II specific Ab production, were, in a significant rate, private specificities of mismatched HLA antigens (70% for class I and 59.4% for class II), and in a lesser percentage by public (CREG) epitopes (24% for class I and 34.3% for class II). In a few patients only no HLA class I and class II Ab specificities could be determined, as they displayed multispecific antibodies (6% for class I and 6.2% for class II). These findings may have important implications to improve donor–recipient matching in dialysis recipients waiting for a subsequent renal transplantation.

Difference in sensitivity to cyclosporine in vitro of human alloreactive lines and clones.

The effective of cyclosporine (CsA) as immuno-suppressive agent in human kidney graft rejection is well established. However, in spite of efforts to maintain optimal plasma levels, a fraction of transplanted patients undergo rejection episodes and/or irreversible chronic rejection. This suggests that immunosuppression by CsA cannot control the alloreactive response if there is a high degree of histoincompatibility for HLA or non-HLA antigens, or it has little effect on the “high responder” patient. Both possibilities are difficult to test in the human system. A third hypothesis, the existence of individual CsA resistance, was tested by evaluating the in vitro inhibitory activity of CsA on alloreactive T cell lines from several individuals. A different degree of in vitro sensitivity to the drug was observed among alloreactive lines generated from different individuals and among clones obtained from the same bulk line. The variability at the individual level and at the clonal level may account for the onset of CsA-resistant rejection assuming that in vivo a positive selection in the presence of the drug occurs and allows for the resistant clones, if present, to dominate the sensitive ones.

Expression of common acute lymphoblastic leukemia antigen (CD 10) by myelinated fibers of the peripheral nervous system

The common acute lymphoblastic leukemia antigen (CALLA), CD10, is a 100-kDa surface glycoprotein endowed with neutral endopeptidase activity, shared by a number of hemopoietic and non-hemopoietic cells. In this report, immunohistochemical and Western blot techniques, using different anti-CD10 monoclonal antibodies, were utilized to demonstrate that CD10 is also expressed by myelin sheaths of the human peripheral nervous system (PNS), but not of the central nervous system. CD10-positive immunoreactivity appeared to be localized in the outer and inner borders of myelinated fibers, in nodes of Ranvier and in the Schmidt-Lantermann clefts, thus showing a distribution pattern very similar to that of myelin-associated glycoprotein (MAG). The above findings suggest that CD10 antigen, through its enzymatic activity, may have a functional role in the assembly and maintenance of PNS myelin. In addition, it is not known whether CD10, similarly to MAG, may be a target antigen in some PNS immune-mediated disorders.