Blood group A(1) and A(2) revisited: an immunochemical analysis.

BACKGROUND AND OBJECTIVE: The basis of blood group A(1) and A(2) phenotypes has been debated for many decades, and still the chemical basis is unresolved. The literature generally identifies the glycolipid chemical differences between blood group A(1) and A(2) phenotypes as being poor or no expression of A type 3 and A type 4 structures on A(2) red cells, although this assertion is not unanimous. MATERIALS AND METHODS: Using purified glycolipids and specific monoclonal antibodies, we revisited the glycolipid basis of the A(1) and A(2) phenotypes. Purified glycolipids were extracted from four individual A(1) and four individual A(2) blood units. One blood unit from an A weak subgroup was also included. Monoclonal anti-A reagents including those originally used to define the basis of A(1) and A(2) phenotypes were used in a thin layer chromatography - enzyme immunoassay to identify the presence of specific glycolipids. RESULTS: A type 3 glycolipid structures were found to be present in large amounts in all phenotypes. In contrast, the A type 4 glycolipid structure was virtually undetectable in the A(2) phenotype, but was present in the A(1) and A subgroup samples. CONCLUSION: The major glycolipid difference between the A(1) and A(2) phenotypes is the dominance of A type 4 glycolipids in the A(1) phenotype.

Glycolipid studies in small intestine and pancreas of alpha1,3-galactosyltransferase knockout miniature swine: alpha1,3GALT-KO animals lack alphaGAL antigens and contain novel blood group H compounds.

BACKGROUND: To avoid hyperacute rejection of xeno-organs, alpha1,3-galactosyltransferase knock-out (GalT-KO) pigs have been produced. However, Galalpha1,3Gal (Gal) determinant elimination may expose cryptic carbohydrate antigens and/or generate new antigens that might interfere with the human immune response. METHODS: Glycolipids isolated from small intestine and pancreas of two GalT-KO and one wild-type (WT) pig were tested for immune reactivity with antibodies on thin-layer chromatograms after separation by high-performance liquid chromatography, and selected fractions were analysed by proton NMR spectroscopy. RESULTS: Immunostaining using purified human anti-Gal Abs revealed that tissues from WT animals express large amounts of Gal-antigens whereas GalT-KO tissues lacked these antigens. Proton NMR spectroscopy on small intestine fractions revealed both linear and branched nona- and decaglycosylceramides, respectively, with terminal Gal-epitopes. In corresponding GalT-KO fractions, Gal-epitopes seemed to be replaced by terminal alpha1,2fucoses. Two novel branched blood group H compounds was found in the GalT-KO intestine. CONCLUSIONS: The structural complexity of alphaGal-terminating antigens in the WT organs is very high. Knockout of alpha1,3GalT by gene-targeting results in elimination of Gal-determinants. In addition structurally novel alpha1,2fucose-terminated blood group H compounds were identified in the GalT-KO tissue. These compounds are not expected to be recognized by the human immune system.

Successful ABO-incompatible liver transplantation using A2 donors.

INTRODUCTION: The longer waiting time for a liver graft among patients with blood group O makes it necessary to expand the donor pool for these patients. We herein have reported our experience with ABO-incompatible liver transplantation using A(2) donors to blood group O recipients. PATIENTS AND METHODS: Between 1996 to 2005, 10 adult blood group O recipients received 10 A(2) cadaveric grafts. Mean recipient age was 52 +/- 7.7 years (mean +/- SD). The initial immunosuppression was induction with antithymocyte globulin (n = 2), interleukin-2-receptor antagonists (n = 3), or anti-CD20 antibody (rituximab, n = 1), followed by a tacrolimus-based protocol. No preoperative plasmapheresis, immunoadsorption, or splenectomies were performed. RESULTS: Patient and graft survival was 10/10 and 8/10, respectively, at 8.5 months median follow-up (range 10 days to 109 months). Two patients were retransplanted because of bacterial arteritis (n = 1) and portal vein thrombosis (n = 1). The six acute rejections, which occurred in four patients, were all reversed by steroids or increased tacrolimus dosages. The pretransplant anti-A titers against A(1) red blood cells were 1:128 (NaCl technique) and 1:8 to 1024 (IAT technique). The maximum postoperative titers were 1:64 to 4000 (NaCl) and 1:256 to 32000 (IAT). CONCLUSION: The favorable outcome of A(2) to O grafting, with a patient survival of 10/10 and graft survival of 8/10, makes it possible to consider this blood group combination also in nonurgent situations. There was no hyperacute rejection or increased rate of rejections. Anti-A/B titer changes seem to not play a significant role in the monitoring of A(2) to O liver transplantation.

alpha-Gal epitopes in animal tissue glycoproteins and glycolipids.

alpha-Gal terminated saccharides are present on the cell surface both as glycolipids and glycoproteins in all mammals except Old World monkeys and humans. The structural diversity among identified saccharides terminated by this epitope in animal tissues is steadily increasing. The majority of these saccharides have the alpha-Gal linked to lactosamine but other core saccharides exist. The alpha-Gal terminated saccharides are recognized by the immune system as a specific antigen and antibodies directed to the alpha-Gal, which do not cross-react with the classic blood group B trisaccharide, are found in man and Old World monkeys. Similar to other complex carbohydrate cell surface antigens, the alpha-Gal epitope is heterogeneously distributed in different organs and in different cells within an organ. It is present on the vascular endothelium and it is the primary target for human naturally occurring antibodies following pig to primate/man xenotransplantation leading to hyperacute rejection of the graft. Important for the future will be to further structurally characterize this antigen system, its cellular/subcellular distribution, and to identify possible of additional glycosyltransferases, related to the already described alpha 1,3galactosyltransferase that may explain the structural diversity. Such information will be of importance in the studies of, for example, the pathogenesis of autoimmune diseases and for the production of genetically modified pigs to prevent xenograft rejection.

Serological and immunochemical characterization of anti-PP1Pk (anti-Tja) antibodies in blood group little p individuals. Blood group A type 4 recognition due to internal binding.

Serum samples from 13 blood group little p individuals were tested by radioimmunoassay for their IgG antibody subclass distribution against the P, P1 and Pk antigens. There was no uniform subclass distribution pattern, although all but one had IgG3 antibodies against all the P system antigens tested. Studies were performed adsorbing anti-Tja serum sequentially to columns with synthetic carbohydrate antigenic determinants within the P system coupled to silica beads (SynsorbsR). The effect on agglutinin and indirect antiglobulin titers was determined after adsorption to SynsorbsR with different P-system antigens (P1, Pk, P). Adsorption to all the three SynsorbsR was needed to eliminate or strongly reduce antibody titers. The effect on IgM, IgG, IgA as well as IgG subclass antibody binding to P, P1 and Pk antigens was also determined by radioimmunoassay and chromatogram binding assay. Anti-PP1Pk antibodies from a little p woman with repeated abortions were shown to bind to glycosphingolipid antigens prepared from one of the aborted placentae using a chromatogram binding assay. This binding was eliminated by serum adsorption to SynsorbsR with P1, Pk and P carbohydrates. Anti-PP1Pk antibodies were also shown to bind to extended structures in the globoseries, i.e. globopentaosylceramide, globohexaosylceramide (globo-H) and globoheptaosylceramide (globo-A). This binding is most probably due to antibodies recognizing internal sequences in the carbohydrate chain. Attempts were made to visualize the binding epitope of the antibodies by computer molecular modelling.

Characterisation of the anti-A antibody response following an ABO incompatible (A2 to O) kidney transplantation.

Anti-A,B antibodies produced in a blood group OLe(a-b-) recipient receiving a kidney graft from a blood group A2Le(a-b+) donor have been analysed for their ability to bind to different glycosphingolipid antigens. Solid-phase RIA using pure glycosphingolipid antigens and a chromatogram binding assay using total nonacid glycosphingolipid fractions from erythrocytes of different human blood group phenotypes together with pure glycolipid antigens were used as assay systems. Serum antibodies were shown to bind equally well to A (types 1, 2, 3 and 4) and B (types 1 and 2) antigenic structures but no binding to H antigens (types 1, 2 and 4) was detected. After adsorption of serum antibodies on A1 Le(a-b+) erythrocytes there was a residual anti-A antibody activity which could not be adsorbed by synthetic A-trisaccharides coupled to crystalline silica (Synsorb-A). These residual antibodies, which are not present in a pretransplant serum sample, had a specificity for the A antigen with type 1 core saccharide chain and the binding epitope obviously included both the N-acetylgalactosamine and the N-acetylglucosamine. The fucose residue was apparently not obligate for binding. The conformation of the sugar units involved in the binding epitope was determined.

HLA-antigens and contact hypersensitivity.

The HLA-A, -B -C typing of 100 bricklayers was performed. 50 bricklayers had developed contact allergy to chromium while 50 were healthy bricklayers. The distribution of HLA antigens were equal in the 2 groups.

ABO-incompatible kidney transplantation (A2 to O). Qualitative and semiquantitative studies of the humoral immune response against different blood group A antigens.

The humoral immune response against blood group A antigens with different core saccharide structures has been investigated in four blood group O recipients transplanted with kidneys from two blood group A2 donors. Radioimmunoassay and thin-layer chromatogram binding assay studies showed that different individuals responded differently to the same antigenic stimulus. Antibodies were produced in the recipient that bound to the terminal trisaccharide of the blood group A antigens. In some cases antibodies that bound to a larger antigen epitope, including the fourth and fifth sugar in the polysaccharide core chain, also occurred. Immunoglobulin class-specific, as well as subclass specific, responses were seen. The antibody response in the blood group O recipients receiving an A2 graft seem to be dependent on the antigenic expression in the transplanted kidney. In view of the recent findings of individuality of A antigen expression in kidneys within the A1 and A2 subgroups, an extended typing of A2 donors may be important. The humoral immune response in the recipient may also be dependent on earlier contacts with ABO incompatible pregnancies, vaccinations, or infections. A possible correlation between pre- and posttransplant findings was noted in one case and deserves further notice.

Increased anti-Gal activity in diabetic patients transplanted with fetal porcine islet cell clusters.

The natural anti-Gal antibody seems to create a major obstacle for discordant xenotransplantation in humans. Anti-Gal, which is produced in large amounts in humans (1% of circulating IgG), interacts specifically with the carbohydrate structure Gal alpha 1-3Gal beta 1-4Glc-NAc-R (termed the alpha-galactosyl epitope). This epitope is present in large amounts on porcine cells, as well as on cells of other nonprimate mammals (1 x 10(6) to 35 x 10(6) epitopes/cell). The interaction of anti-Gal with alpha-galactosyl epitopes on the xenograft was found to mediate the immune destruction of discordant xenografts. In the present study, the human immune response to alpha-galactosyl epitopes on xenografts was assessed by measuring changes in anti-Gal titers and affinity in sera of diabetic patients transplanted with fetal porcine islet cell clusters. The activity of this antibody was assessed by a hemagglutination assay with RBC, by ELISA with mouse laminin as a solid-phase antigen, and by equilibrium dialysis with the radiolabeled free haptenic form of the alpha-galactosyl epitope, i.e. [3H]Gal alpha 1-3Gal beta 1-4GlcNAc. All assays revealed a marked increase in anti-Gal activity after transplantation. The increase in anti-Gal titers ranged between 8- and 64-fold. A similar increase was observed in the binding of free alpha-galactosyl epitopes to anti-Gal, as assayed in equilibrium dialysis. Immunoglobulin concentration did not increase after transplantation, suggesting that the observed increase in anti-Gal activity is the result of a specific immune response against alpha-galactosyl epitopes on the xenograft. The elevation in anti-Gal activity was observed in all three immunoglobulin classes and the highest activity was found within the IgG class. Analysis of IgG binding to fixed porcine endothelial cells suggested that most of the observed increased activity against these cells in transplanted patients may be attributed to the elevation in anti-Gal activity.

Evidence of donor-specific cellular suppressor activity in donor-specific cell-mediated lympholysis unresponsiveness in renal transplant patients.

Twenty patients with well-functioning kidney grafts from one-haplotype-mismatched related donors, were studied 1-10 years after transplantation (A). Another group of six patients were studied at various times after transplantation (B). The presence of donor-specific transplantation tolerance, using mixed lymphocyte culture (MLC) and cell-mediated lympholysis (CML) tests was investigated, as well as the possible existence of cells with suppressive activity. All recipients were transfused prior to transplantation and treated with conventional immunosuppression. The patients in group A showed MLC reactivity against donor and third-party cells, indicating a allogeneic response capacity. The CML activity against the donor was low, however, and remained low also following removal of adherent cells. The CML activity toward third-party cells was within the normal range of unmatched individuals. In group B, two of six recipients and high postoperative CML activity against the donor. Both recipients showed clinical signs of rejection. In the remaining four recipients, the antidonor CML reactivity one week after transplantation was lower than the preoperative level. The decrease was even more pronounced at 12 months, although the reactivity against third-party cells was unaltered. The CML reactivity from unrelated fourth-party individuals toward donors was suppressed when cells from recipients with long-term functioning kidneys were added to the cell cultures. The results suggest the presence of a donor-specific cellular suppressor mechanism underlying the donor-specific CML unresponsiveness in recipients with long-term-functioning kidney allografts.

Esomeprazole provides improved acid control vs. omeprazole In patients with symptoms of gastro-oesophageal reflux disease.

BACKGROUND: Esomeprazole (Nexium) is a new proton pump inhibitor for the treatment of acid-related diseases. METHODS: In this double-blind crossover study, 38 patients with gastro-oesophageal reflux disease (GERD) symptoms were randomized to esomeprazole 40 and 20 mg and omeprazole 20 mg once daily for 5 days. On day 5 of each dosing period, 24-h intragastric pH and pharmacokinetic variables were measured. RESULTS: Thirty-six patients aged 29-58 (mean 45) years completed the study. Esomeprazole 40 and 20 mg maintained intragastric pH > 4 for (mean) 16.8 and 12.7 h, respectively, vs. 10.5 h for omeprazole 20 mg (P < 0.001 and P < 0. 01). Twenty-four-hour median intragastric pH was significantly higher with esomeprazole 40 mg (4.9) and 20 mg (4.1) than with omeprazole 20 mg (3.6) (P < 0.001 and P < 0.01). Area under the plasma concentration-time curve (AUC) was 80% higher for esomeprazole 20 mg vs. omeprazole, while that for esomeprazole 40 mg was more than five times higher (each P < 0.0001). Interpatient variability in intragastric pH and AUC was less with esomeprazole than with omeprazole. Esomeprazole was well tolerated and there were no safety concerns. CONCLUSIONS: Esomeprazole provides more effective acid control than omeprazole, with reduced interpatient variability, thereby offering the potential for improved efficacy in acid-related diseases.

Blood group lewis phenotype on erythrocytes and in saliva in alcoholic pancreatitis and chronic liver disease.

The distributions of ABO, rhesus, and Lewis blood group antigens were studied in patients with alcoholic cirrhosis, alcoholic pancreatitis, chronic active hepatitis, and primary biliary cirrhosis. There were no differences in frequencies of ABO and rhesus blood group antigens between the groups or in comparison with a control group of blood donors. Lewis phenotype Le (a- b-), however, was more common on erythrocytes than in saliva in patients with alcoholic cirrhosis, alcoholic pancreatitis, and severe renal disease but equally common in saliva and on red blood cells in patients with non-alcoholic liver disease. It is suggested that Lewis typing should be performed on saliva because blood typing may give misleading results in some patients.

Characteristics of secondary oesophageal peristalsis in operated and non-operated patients with chronic gastro-oesophageal reflux disease.

BACKGROUND AND AIMS: Secondary oesophageal peristalsis contributes to oesophageal volume clearance and may be impaired in a significant proportion of patients with chronic gastro-oesophageal reflux disease (GORD). This study aimed to investigate the triggering of secondary peristalsis in chronic GORD patients compared to those previously operated on with anti-reflux surgery. PATIENTS AND METHODS: Healthy volunteers, chronic GORD patients with proven oesophagitis and patients successfully operated on with anti-reflux surgery (> 3 years ago) were investigated. Secondary peristalsis was elicited by oesophageal distension by a bolus of air (10 ml) injected rapidly into the mid-portion of the oesophagus. The peristaltic characteristics in the distal oesophagus were assessed by use of stationary manometry. RESULTS: The primary peristaltic amplitude in the distal third of the oesophagus was significantly higher (P < 0.002) in the non-operated GORD cases than in those recruited for surgery. Furthermore, a difference in the frequency of failed primary peristalsis was revealed (2.1 versus 8.4%) between the non-operated and operated patients. Secondary peristalsis occurred in 65 +/- 13.2% (mean +/- SE) of the healthy subjects on stimulation, which was a higher figure than in the GORD patients. In patients investigated after successful anti-reflux surgery, a secondary peristaltic wave was elicited in only 26 +/- 7.2% of the attempts, which was significantly lower than the 46 +/- 7.7% seen in non-operated GORD patients (P < 0.05). A direct comparison between motor characteristics of primary and secondary peristalsis revealed that the latter amplitudes were significantly lower both in the non-operated and in the operated cases (P < 0.005). CONCLUSIONS: The triggering of secondary peristalsis seems to be impaired in chronic GORD patients. Investigating similar patients > 3 years after successful anti-reflux surgery revealed an even lower prevalence of secondary peristaltic waves, implying persistence of the abnormality after surgery and consistent with other evidence that GORD is associated with a primary defect in oesophageal motor function.

Kidney transplantation--a 46-year experience from the Transplant Institute, Sahlgrenska University Hospital, Gothenburg, Sweden.

The limiting factor in organ transplantation is the availability of organs. Ongoing work to improve donation rates both at the public and the organizational level in donating hospitals is essential. We also think that encouragement of live donation is important, and the possibility of ABO incompatible transplantation has increased the number of LD transplantations. The one-year graft survival rate is excellent and focus has shifted towards achieving long-term results to reduce the attrition rate. There is also an increasing interest in studying and working to reduce comorbidities on a long-term basis and thus, improve survival rates and recipient quality of life.