Isabel Aguilera

Antibodies against glutathione S-transferase T1 (GSTT1) in patients with de novo immune hepatitis following liver transplantation

Four patients of 283 liver‐transplant recipients (1·4%) developed de novo immune‐mediated hepatitis approximately 2 years after transplantation. Antibodies showing an unusual liver/kidney cytoplasmic staining pattern were detected in the sera of all four patients and one of them was used to screen a human liver cDNA expression library with the aim of identifying the antigenic target of these newly developed antibodies. After cloning and sequencing the gene, it was identified as the gene encoding the glutathion‐S‐transferase T1 (GSTT1), a 29‐kD molecular weight protein, expressed abundantly in liver and kidney. Sera from the other three patients also contained anti‐GSTT1 antibodies, two of them demonstrated by immunoblot analysis against the recombinant antigen and the other, which was negative by immunoblot, gave a positive reaction when used directly to screen the same library, suggesting it to be directed to a conformational epitope. The GSTT1 enzyme is the product of a single polymorphic gene that is absent from 20% of the Caucasian population. When we analysed the GSTT1 genotype of the four patients described above, we found that this gene is absent from all of them. Three donor paraffin embedded DNA samples were available and were shown to be positive for GSSTT1 genotype. In accordance with these results, we suggest that this form of post‐transplant de novo immune hepatitis, that has been reported as autoimmune hepatitis by others, could be the result of an antigraft reaction in individuals lacking the GSTT1 phenotype, in which the immune system recognizes the GSTT1 protein as a non‐self antigen, being the graft dysfunction not the result of an autoimmune reaction, but the consequence of an alo‐reactive immune response.

Glutathione S‐transferase T1 mismatch constitutes a risk factor for de novo immune hepatitis after liver transplantation

A new form of autoimmune hepatitis referred to as de novo, has been reported after liver transplantation during the past 5 years. The features are identical to those of classical autoimmune hepatitis (AIH), but the facts involved in the onset and outcome of this type of graft dysfunction are still unclear. The identification of antibodies directed to glutathione S‐transferase T1 (GSTT1) in the sera of patients with de novo immune hepatitis led us to the description of an alloimmune reaction due to a GSTT1 genetic incompatibility between donor and recipient. We analyzed a cohort of 110 liver transplant patients treated in the liver transplant unit of our hospital during a period of 1 year, from September 2002 to October 2003. We found the following distribution of the GSTT1 genotypes (recipient/donor): +/+ = 66, +/− = 23, −/+ = 15, −/− = 6. Six of these patients were diagnosed with de novo immune hepatitis; all of them belong to the group of negative recipients with positive donors, and all produced anti‐GSTT1 antibodies. This genetic combination is associated with a statistically significant increased risk of de novo immune hepatitis (IH) in liver transplant patients (P < .0001 by the Fisher exact test). In conclusion, our results clearly establish the importance of the GSTT1 genotype from donor and recipient of a liver transplant as a predictive marker for de novo IH. At the same time, we confirmed our initial results that only this particular donor/recipient combination triggers the anti‐GSTT1 antibody production. (Liver Transpl 2004;10:1166–1172.)

Molecular structure of eight human autoreactive monoclonal antibodies

The heavy (H) and light (L) chain V‐region sequences of eight human autoreactive immunoglobulin M (IgM) monoclonal antibodies (mAbs: BY‐4, BY‐7, BY‐12, IRM‐3, IRM‐7, IRM‐8, IRM‐10 and CDC‐1) were determined at the cDNA level. All VH and VL families were identified. Four different VH families were represented, VH3 being the most common as five of the mAbs (BY‐7, BY‐12, IRM‐3, IRM‐8 and CDC‐1) used genetic elements of this family, whereas VH1, VH2 and VH4 were only present in IRM‐7, BY‐4 and IRM‐10, respectively. BY‐4, BY‐7, BY‐12, IRM‐7 and IRM‐10 reacted with a variety of self as well as non‐self antigens, thus exhibiting polyreactive behaviour. Comparison of the gene segments utilized by these mAbs with their germline counterparts revealed that the gene segments were close to germline configuration. The length of H‐CDR3 was found to be relatively long (27–60 nucleotides) among the polyreactive mAbs and the presence of Tyr and Trp residues in this region seems to be of vital importance for polyreactivity. We have analysed the utilization of gene elements and the presence of amino acid residues in regions particularly important for antigen binding, such as CDR. Common molecular features relating to the function of the mAbs are discussed.

Donor-specific antibodies against HLA, MICA, and GSTT1 in patients with allograft rejection and C4d deposition in renal biopsies.

Background. Production of antibodies against donor-specific antigens is one of the central mechanisms of allograft rejection. This antibody-mediated rejection (AMR) is evidenced by the presence of circulating donor-specific antibodies and deposition of complement component C4d on renal endothelium. Although anti-human leukocyte antigen (HLA) antibodies account for a high proportion of AMR, in many cases anti-HLA antibodies cannot be demonstrated. In liver transplant, antibodies against glutathione-S-transferase T1 (GSTT1) expressed on the graft may induce an antibody response leading to a severe graft dysfunction. In addition, presence of antibodies against major-histocompatibility-complex class I chain-related gene A (MICA) has been associated with a poor graft survival in kidney transplantation. Methods. Pre- and posttransplantation sera from 19 patients fulfilling the criteria for AMR including C4d deposition in renal biopsies were included. Donor-specific antibodies against HLA-I and -II and MICA were studied using Luminex. Anti-GSTT1 antibodies were analyzed by indirect immunofluorescence and by an ELISA method. A control group of 39 patients with graft dysfunction negative for C4d was also included. Results. At the time of the biopsy, 4 (21%) patients had only anti-HLA class I antibodies; 3 (15.8%) had anti-GSTT1, 2 (10.5%) had anti-HLA-class II, and 2 (10.5%) had anti-MICA; four patients had combination of antibodies: HLA-I+MICA (n=1), HLA-I+GSTT1 (n=2), and GSTT1+MICA (n=1). No antibodies were found in 4 (21%) patients. In total, 6 (31.6%) C4d+ patients had anti-GSTT1 antibodies, whereas, among the 39 C4d-negative patients, only 3 (7.7%) had anti-GSTT1 antibodies (P=0.027). Conclusion. Besides anti-HLA antibodies, donor-specific antibodies against MICA and GSTT1 antigens could be responsible for the occurrence of antibody-mediated kidney graft rejection.

LIPOMATOSIS, PROXIMAL MYOPATHY, AND THE MITOCHONDRIAL 8344 MUTATION. A LIPID STORAGE MYOPATHY?

Multiple symmetric lipomatosis (MSL) has been related in some cases to the 8344 point mutation of the tRNA‐lysine gene of the mitochondrial DNA, mainly in the context of families with classic myoclonic epilepsy with ragged‐red fibers (MERRF) and exceptionally in patients with proximal myopathy as the only manifestation of mitochondrial disease. We report on two families harboring the 8344 mutation. The patients presented with MSL and myopathy, expressed as limb girdle weakness in index cases and as exercise intolerance in the others. All muscle biopsies performed showed lipid storage apart from RRF and respiratory chain complexes deficiency. A possible explanation for both adipose proliferation and lipid storage myopathy in these cases is a disturbance in intermediary lipid metabolism secondary to mitochondrial respiratory chain deficiency that could be related via carnitine deficiency.

Complement component 4d immunostaining in liver allografts of patients with de novo immune hepatitis

De novo immune hepatitis (DNIH) is a form of late graft dysfunction after liver transplantation. The fine mechanisms leading to the development of DNIH are not known, and whether this hepatitis is a form of rejection or a result of an auto/alloimmune injury has not been established. In our patients, DNIH was always preceded by the production of donor‐specific antibodies against the glutathione S‐transferase T1 (GSTT1) enzyme because of a genetic mismatch in which the donors carried the wild‐type gene and the recipients displayed the null genotype. Complement component 4d (C4d) immunopositivity in 12 paraffin‐embedded liver biopsy samples from 8 patients diagnosed with DNIH associated with anti‐GSTT1 antibodies was retrospectively evaluated. Six patients with a diagnosis of chronic rejection (CR) and 7 patients with hepatitis C virus recurrence were included as control groups. Among the patients with DNIH, 7 showed C4d‐positive immunostaining localized in the portal tracts, whereas in the tested biopsy samples of the 2 control groups, this staining pattern was absent. Four biopsy samples of the CR group showed C4d‐positive sinusoidal staining. This study confirms the activation of the complement pathway in the presence of donor‐specific antibodies, which was shown by the deposition of C4d elements in liver biopsy samples of patients with DNIH. The use of C4d as a marker of antibody‐mediated rejection in liver allografts in the presence of antidonor antibodies is discussed, and it may contribute to improved differential diagnoses based on biopsy findings. Liver Transpl 17:779‐788, 2011.

Anti-glutathione S-transferase T1 antibody-mediated rejection in C4d-positive renal allograft recipients

BACKGROUND Chronic humoral rejection is a progressive form of graft injury, with defined diagnostic criteria, the crucial one being the evidence of circulating anti-donor antibodies. These antibodies are mainly directed against human leucocyte antigens (HLA), but other targets have also been described. We previously reported that antibodies against the Glutathione S-transferase T1 (GSTT1) enzyme appear in recipients without the GSTT1 gene who receive a graft from a GSTT1-positive donor. The primary aim of this study was to analyse the role of GSTT1 in cases of antibody-mediated rejection (AMR) in the absence of anti-HLA antibodies. A second objective was to describe the distribution of the GSTT1 enzyme in the human kidney. METHODS Four renal biopsies from four renal transplanted patients with declined renal function and circulating anti-donor GSTT1 antibodies were studied for C4d deposits in sections of paraffin-embedded tissue samples. Anti-donor-specific HLA and MICA antibody detection was done with the Luminex platform and anti-GSTT1 antibodies were tested by indirect immunofluorescence on rat tissues and ELISA assay. DNA of the patients was extracted for GSTT1 genotyping. RESULTS Four patients with the GSTT1 donor/recipient mismatch developed anti-GSTT1 antibodies 32, 42, 48 and 60 months after the transplant. One patient also had donor-specific anti-HLA antibodies. Their biopsies showed pathologic lesions compatible with chronic antibody-mediated rejection (CAMR), along with positive C4d deposition in peritubular capillaries in three of them, being no valuable in the other case. CONCLUSION This is the first study reporting an association between the appearance of chronic antibody-mediated renal allograft rejection and the occurrence of de novo production of anti-GSTT1 antibodies, in the absence of anti-HLA donor-specific antibodies. This fact suggests a potential role of the GSTT1 system in anti-graft immune response.

Positive association of anticytoskeletal endothelial cell antibodies and cardiac allograft rejection

Using an indirect immunofluorescence method on human umbilical vein endothelial cells (HUVEC), we investigated the presence of antiendothelial cell antibodies (AECA) in 136 pre- and posttransplant serum samples sequentially collected from 31 patients during the first year after cardiac transplantation. A healthy control group was also included (n = 87). Colocalization studies demonstrated a positive staining pattern of different cytoskeletal components (cytoskeletal-antiendothelial cell antibodies, CSK-AECA) including antivimentin, antiactin, antitubulin, and anticytokeratin among heart transplanted patients. Frequency of CSK-AECA in the control group and at day 0 in the transplant group was 18.3 and 22.5%, respectively (p = NS). A progressive increase in the frequency of CSK-AECA was observed after cardiac transplantation: 13.3% at day 15; 22.2% at day 30; 53.8% at day 90, and 58% at day 360. Interestingly, rejection episodes within the first year after transplantation occurred in 83.3% of CSK-AECA-positive and in 30.7% of CSK-AECA-negative patients (p = 0.0045). The presence of antibodies was detected prior to the rejection event and was associated with a poor clinical outcome: rejection episodes occurred at a mean of 36.14 +/- 17 days after transplantation in patients with preexisting AECA and 174.25 +/- 51.9 days after de novo antibody appearance in patients with no antibodies at day 0 (p = 0.029). In conclusion, a progressive increase in the frequency of CSK-AECA was observed following cardiac transplantation; the presence of these antibodies is strongly associated and precedes the rejection episodes. Thus, CSK-AECA could be a good marker for acute graft rejection.

Choice of calcineurin inhibitor may influence the development of de novo immune hepatitis associated with anti-GSTT1 antibodies after liver transplantation

Aguilera I, Sousa JM, Praena JM, Gómez‐Bravo MA, Núñez‐Roldan A. Choice of calcineurin inhibitor may influence the development of de novo immune hepatitis associated with anti‐GSTT1 antibodies after liver transplantation. 
Clin Transplant 2011: 25: 207–212.

Antibodies against glutathione S-transferase T1 in non-solid organ transplanted patients

BACKGROUND: This article describes the presence of antibodies against glutathione S‐transferase T1 (GSTT1) in a group of patients who never received a solid organ graft. These antibodies have been previously detected in liver and kidney transplant subjects with donor‐recipient mismatch for this enzyme at the genetic level. In liver‐grafted subjects, the appearance of these antibodies correlated with de novo immune hepatitis.