First report of successful isolation of a HPR0-like variant of the infectious salmon anaemia virus (ISAV) using cell culture.

ISAV is the causative agent of the infectious salmon anaemia (ISA), a disease listed by the OIE that has caused important economic losses to the Atlantic salmon (Salmo salar) industry. ISAV variants are identified as pathogenic or non-pathogenic based on the presence or absence of a deletion in the highly polymorphic region (HPR) of segment 6 (S6). HPRΔ variants (pathogenic) are the only forms of the virus known to grow in cell culture. This is the first report of a HPR0 variant isolated in cell culture. The isolate is, however, atypical as it shows a marker of virulent variants on another segment (S5), which has never been reported for any other HPR0 variants. The significance of this finding remains unclear until more in-depth work is carried out but does challenge current knowledge.

Comparison of combination Plasmapheresis/IVIg/anti-CD20 versus high-dose IVIg in the treatment of antibody-mediated rejection.

Different strategies appear to improve the success in treatment of antibody-mediated rejection (AMR), although no one best method has yet emerged. The objective of this study was to compare the efficacy of the combination of Plasmapheresis/intravenous immunoglobulin (IVIg)/anti-CD20-based regimes versus high-dose IVIg alone in the treatment of AMR. Group A (12 patients) was treated with high-dose IVIg between January 2000 and December 2003; group B (12 patients) was treated by Plasmapheresis/IVIg/anti-CD20 between January 2004 and December 2005. Graft survival at 36 months was 91.7% in group B versus 50% in group A (p = 0.02). Donor-specific human leukocyte antigens (DSA) levels detected by Luminex single antigen (Luminex SA) and ELISA, 3 months postrejection are significantly lower in group B than in group A: DSA ELISA class 2 score 6-8 (p = 0.02), DSA mean intensity of fluorescence (MFI) max (p = 0.009) and DSA mean MFI (p = 0.0004). The persistence of elevated DSA levels posttreatment is more frequent in patients with graft loss as compared to those with preserved renal function: score 6-8 on ELISA (p = 0.04); mean MFI (p = 0.00009) and MFImax (p = 0.018). We conclude that: (1) high dose IVIg alone is inferior to Plasmapheresis/IVIg/anti-CD20 as therapy for AMR and (2)DSA postrejection can be quantified using solid phase assays, showing that 3 months after AMR, DSA levels are higher in patients with graft loss.

Clinical relevance of preformed HLA donor-specific antibodies in kidney transplantation.

Since the pioneering work of Patel and Terazaki, the presence of an anti-donor anti-body of the IgG isotype, as demonstrated by a lymphocytotoxic assay on T cells, has been a contraindication to transplantation, due to the very high rate of graft loss reported (>80% in the first few weeks posttransplant). The advent of more sensible and specific techniques of detection of anti-HLA antibodies (such as ELISA or Luminex techniques) has questioned this dogma, with a number of reports showing that transplantation, despite the presence of an donor-specific antibody (DSA), could be done without excessive graft losses, despite higher rates of rejection. We thus decided to retrospectively screen a cohort of 237 patients consecutively transplanted in our unit. This study analyzes the influence of preformed DSA, identified by HLA-specific ELISA assays, on graft survival and evaluates the incidence of antibody-mediated rejection (AMR). Kidney graft survival at 8 years was significantly worse in patients with DSA. The incidence of AMR in patients with DSA was 9-fold higher than in patients without DSA and led to a significantly worse graft survival. The prevalence for AMR in patients with DSA detected on historic serum was 32.3% and was significantly more elevated in patients with strongly positive DSA (score 6-8) and in patients with his-toric positive crossmatches. Interestingly, those patients with DSA that did not experience AMR had the same graft survival as patients without DSA. Thus, the presence of preformed DSA is strongly associated with increased graft loss in kidney transplants, related to an increased risk of AMR. Our findings demonstrate the importance of detection and charac-terization of DSA before transplantation. Stratification of this immunological risk should be used both to determine kidney allocation and to devise specific strategies for these patients.

Clinical relevance of preformed HLA donor-specific antibodies in kidney transplantation.

This study analyzes the influence of preformed DSA, identified by HLA-specific ELISA assays, on graft survival and evaluates the incidence of antibody-mediated rejection (AMR) in patients with and without pregraft desensitization. Kidney graft survival at 8 years was significantly worse in patients with DSA (n = 43) than in those without DSA (n = 194)(p = 0.03). The incidence of AMR in patients with DSA is 9-fold higher than in patients without DSA (p < 0.001) and their graft survival is significantly worse than in DSA patients without AMR and in non-DSA patients (p = 0.005). The prevalence for AMR in patients with DSA detected on historic serum is 32.3% in nondesensitized patients and 41.7% in desensitized patients. The risk for AMR is significantly more elevated in patients with strongly positive DSA (score 6-8) compared to those with DSA score 4 (p < 0.001), and in patients with historic DSA+/CXM+ compared to those with DSA+/CXM- (p = 0.01). The presence of preformed DSA is strongly associated with graft loss in kidney transplants, related to an increased risk of AMR. Our findings demonstrate the importance of detection and characterization of DSA before transplantation. Stratification of this risk could be used to determine kidney allocation and to devise specific strategies for these patients.

14th International HLA and Immunogenetics Workshop: report on the Prospective Chronic Rejection Project.

An international collaborative study of 45 transplant centers was undertaken at the 14th International HLA (human leukocyte antigen) and Immunogenetics Workshop to see if HLA antibodies detected posttransplant are predictive of chronic graft failure. With the newly developed assay, MICA (major histocompatibility complex class I-related chain A) antibodies were also measured and their effect analyzed. Total of 5219 sera from patients who were more than 6 months posttransplant with functioning graft were tested for HLA antibodies by enzyme-linked immunosorbent assay, flow cytometry, or Luminex. HLA antibodies were found in 27.2% of kidney patients, 23.6% in the liver, 52.7% in the heart, and 21.7% in the lung. The method of antibody testing did not have a marked influence on the frequency of antibodies detected. MICA antibodies were detected in 15% of kidney patients, 30% of heart patients, and 31% of liver patients. Among 948 kidney patients who had HLA antibodies, 7.3% had rejected their graft within 1 year of testing, compared with 1.7% in 2615 patients without HLA antibodies (P= 0.8 x 10(-17)). Death occurred in 1.4% of total kidney patients and did not correlate to the presence of antibodies. We conclude that patients with posttransplant HLA antibodies indeed have a higher rate of chronic graft failure and that posttransplant antibodies are predictive of chronic rejection.

14th International HLA and Immunogenetics Workshop Prospective Chronic Rejection Project: a three-year follow-up analysis.

The three-year follow-up of 4,144 patients of the 14th International Workshop Prospective Chronic Rejection study has reinforced the evidence that post-transplant HLA antibodies are predictive of long-term graft loss. Three years after a single testing for HLA antibodies, 10% of kidney recipients who were antibody-positive had lost their grafts, in contrast to only 5% of antibody-negative patients (p<0.0001). The adverse effect of post-transplant antibodies on graft survival was also observed in lung, heart, and liver transplants. Donor-specific antibodies and 'strong' non-DSA had stronger association with graft loss than 'moderate' non-DSA. Periodic antibody monitoring, combined with specificity and strength analysis, would help in the early identification of allograft recipients who are at high risk of graft failure.

The role of HLA genes in familial spondyloarthropathy: a comprehensive study of 70 multiplex families.

OBJECTIVES: To investigate whether HLA alleles, other than HLA-B27, influence predisposition to spondyloarthropathy (SpA) in multiplex families. METHODS: Seventy French families with at least two affected SpA members were recruited. Patients, and their first degree relatives were typed for HLA-A, B, C, and DR, and extended HLA haplotypes were determined. The distribution of HLA-A, C, and DR alleles carried on HLA-B27+ haplotypes in SpA families was compared with the distribution of these alleles among HLA-B27+ haplotypes in the French general population. Contribution to SpA susceptibility of HLA-A, B, C, and DR alleles, other than HLA-B27, was tested by transmission disequilibrium test. The contribution of HLA alleles to specific presentation features of SpA was examined. RESULTS: Frequencies of HLA-A, C, and DR alleles carried on HLA-B27+ haplotypes from SpA families were comparable with those seen in the French population, except for DR13 which was overrepresented among patients (pcorr<0.001). Most interestingly, the HLA-DR4 allele was transmitted in excess to patients with SpA, independently of linkage to HLA-B27 (pcorr=0.05), and in a direction opposite to that for HLA-B27+ unaffected siblings (pcorr=0.01). Finally, the distribution of HLA alleles was not related to the presentation feature of SpA. CONCLUSION: HLA predisposition to familial SpA appears not to be limited to HLA-B27, but some HLA-DR alleles also have a significant influence. In particular, HLA-DR4 contributes significantly to a genetic predisposition to SpA, which may have implications in our understanding of SpA pathogenesis.

Prevalence of GBV C/HGV RNA and GBV C/HGV antibodies in French volunteer blood donors: results of a collaborative study.

BACKGROUND AND OBJECTIVES: Posttransfusion hepatitis still occurs at an incidence of about 1 in 118,000 for HBV and 1 in 220,000 for HCV. This collaborative study aimed to determine the prevalence of a novel flavivirus, GBV-C/HGV, even though its role in transfusion-associated hepatitis is uncertain. MATERIALS AND METHODS: GBV-C/HGV RNA was detected by PCR using either the Boehringer detection kit or by primers previously described. HGV antibodies were detected by a serological assay from Boehringer. RESULTS: The observed GBV-C/HGV RNA frequency was 3.4%. HGV antibodies occurred in 9.5% of donors. CONCLUSION: In our study, 12. 9% of the donors had been in contact with the GBV-C/HGV virus.

[Autologous transfusion and hemoglobin SC disease]. / Transfusion autologue et drépanocytose SC.

Three autologous blood units were transfused during elective orthopaedic surgery in a patient with undiagnosed haemoglobin SC disease. The packed red blood cells had been stored at 4 degrees C on SAG-M under standard conditions for 10 to 31 days. There was no evidence of adverse clinical reactions during the perioperative period. Six months later, a blood unit was collected at the initial step of an exchange transfusion in the same patient. Haemolysis was moderate after a 12-day-storage period and more significant after 32 days. This observation, as some other case reports, suggest that autologous blood transfusion may be considered for haemorrhagic surgery in selected patients with sickle cell disease.

Predominant role of the Fab fragment in delaying hyperacute rejection in guinea pig-to-rat xenotransplantation.

BACKGROUND: Human intravenous immunoglobulin G delayed xenogeneic hyperacute rejection (HAR) in the guinea pig-to-rat combination. We investigated the respective roles of the Fc and Fab fragments of the IgG molecule in this inhibitory effect. METHODS: By using a guinea pig-to-rat heart transplantation model, the efficiency of IgG, Fab, and Fc in prolonging the grafted heart's survival time (ST) was compared. RESULTS: A dose-dependent increase in the ST was observed with Fab (r=0.74, P < 0.0001), IgG (r=0.57, P < 0.001), and Fc (r=0.51, P < 0.01). The linear regression slopes with Fab and with IgG were, respectively, sevenfold and fourfold steeper than with Fc. The ST was significantly longer than controls (23+/-7 min) after infusion of 2 g/kg IgG (147+/-42 min) or 1 g/kg Fab (176+/-38 min), whereas the highest dose of Fc (1.5 g/kg) did not induce significant prolongation of ST. In terms of equivalent functional doses, 1 g/kg Fab was significantly more potent in prolonging the ST than 1.5 g/kg IgG (87+/-25 min) or 0.5 g/kg Fc (33+/-14 min). Analysis of the rejected hearts evidenced edema, necrosis, and rat C3 deposits characteristic of HAR. CONCLUSION: These results indicated that the delaying action of intravenous immunoglobulin G on HAR in the guinea pig-to-rat combination is mostly mediated through the Fab fragment.

Use of intravenous immunoglobulin to delay xenogeneic hyperacute rejection. An in vivo and in vitro evaluation.

The delaying action of intravenous immunoglobulin (IVIG) from human origin on hyperacute xenogeneic rejection was assessed in the guinea pig-to-rat combination. IVIG (1500 mg/kg) injected i.v. into Lewis rats 1 hr before grafting significantly prolonged the mean guinea pig heart survival time (167 min, P < 0.005) compared with control injections using NaCl (12 min) or the IVIG glycine vehicle (11 min). The effect of IVIG was also assessed in vitro in the pig-to-human combination. A dose-dependent inhibition of the complement-mediated direct cytotoxicity of human serum on pig RBC was shown using IVIG. The weak direct cytotoxicity of IVIG to pig RBC, which was abolished after preincubating IVIG with pig RBC, was attributed to the anti-pig xenoreactive natural antibodies (XNA) contained in the IVIG preparation. In vitro, XNA-depleted IVIG exerted a significantly stronger inhibitory effect than non-XNA-depleted IVIG, suggesting the use XNA-depleted IVIG in the pig-to-human combination. Although the mechanism of the inhibitory effect of IVIG remains to be clarified, IVIG may represent a new and simple therapeutic modality against xenogeneic hyperacute rejection.

Augmentation of portal blood flow improves function of human cirrhotic liver.

In cirrhotic livers, the intrahepatic resistance is increased and drug elimination and portal transhepatic flow are decreased. The aim of our work was to study the effect of a twofold increase in portal blood flow during 2 hr on the hemodynamic parameters, drug elimination and hepatic viability in eight isolated perfused human cirrhotic livers. Using an oxygenated recirculating system with independent arterial and portal flows, we perfused livers with Kreb's buffer bicarbonate solution, bovine serum albumin (20 gm.L-1) and human red blood cells (hematocrit 20%). The flow was maintained at a basal level of 0.713 +/- 0.19 L/min for 1 hr and then increased and maintained for 2 hr at twice the basal flow. Portal pressure-portal flow curve slopes were linear (27.04 +/- 21.06 mm Hg.L-1 x min; range = 6.43 to 60.8) and correlated with intrahepatic resistance during the basal-flow period (r = 0.87, p < 0.01). Parameters registered during the basal- and high-flow periods were compared by use of Student's t test: portal pressure increased from 23.5 +/- 7 to 37.3 +/- 16.7 mm Hg (p < 0.05); arterial pressure increased from 80.3 +/- 19 to 103.5 +/- 26 mm Hg (p < 0.005); hepatic artery flow resistance increased 31.9% (from 690.1 +/- 218 to 899.4 +/- 269 mm Hg.L-1 x min; p < 0.005); indocyanine green clearance increased by 28.2% (from 86.0 +/- 58.3 to 109.2 +/- 74.8 ml.min-1 x kg liver-1; p < 0.04). No significant differences were observed in enzyme release, biliary flow (n = 5) and oxygen consumption. Histological examinations demonstrated sinusoidal dilatations in six of eight cases.(ABSTRACT TRUNCATED AT 250 WORDS)

[Immuno-hematologic aspects of xenotransplantation]. / Aspects immuno-hématologiques de la xénotransplantation.

The main barrier to pig organ xenograft in humans, is the hyperacute rejection, which is provoked by the fixation of human xenoantibodies on the graft endothelial cells, and by complement activation. Because pig red blood cells and endothelial cells carry common xenogeneic epitopes, human anti-pig xenoantibodies can be detected and titered by pig red blood cells direct agglutination. Individuals of AB blood group have lower anti-pig hemagglutinins titers than those of other blood groups. Anti-A and anti-B alloantibodies do not agglutinate pig red blood cells. The anti-B (and not the anti-A) alloantibodies can be adsorbed on pig red blood cells. A linear B type 2 epitope recognized by human natural antibodies has been previously described on pig cells. If the role of xenoantibodies is prominent in the rejection of pig organ xenografts in humans, the role of alloantibodies remains to be precise.

[Natural human anti-pig agglutinins. Role in the hyperacute rejection of xenogeneic graft in the pig-human donor-recipient combination]. / Les anticorps agglutinants naturels humains anti-porc. Rôle dans le rejet hyperaigu de greffe xenogénique dans la combinaison donneur-receveur porc-homme.

Binding of human natural xenoantibodies to the graft endothelial cells is considered as one of the initial steps in the hyperacute rejection of a pig liver by human. In addition to binding to endothelial cells, human xenoantibodies also agglutinate pig red blood cells. We have therefore compared the anti-pig hemagglutinin titers with the cytotoxic activity to pig endothelial cells of a series of human sera. In order to determine the localisation of the human xenoantibodies fixation in the pig liver, we have studied by immunoperoxidase staining, deposits of IgG, IgM and IgA, using an ex vivo model of isolated pig liver perfused with human blood. Moreover, we have measured hemagglutinin titers in the perfusate along the perfusion. We have found a strong correlation between the anti-pig hemagglutinin titers and the anti-pig endothelial cell cytotoxic activity of human serum. Anti-pig hemagglutinin become undetectable in the perfusate within 4 minutes of perfusion of the pig liver with human blood. The sinusoids in the pig liver were the only structures with positive peroxidase staining. It is therefore suggested that human anti-pig hemagglutinins are in great part the same than human anti-pig endothelial cell cytotoxic antibodies. Pig red blood cells could be used to easily detect anti-pig xenoantibodies in human, and to adsorb xenoantibodies from the recipient before a xenograft.