Bjarte G. Solheim

Vaccination with mutant ras peptides and induction of T-cell responsiveness in pancreatic carcinoma patients carrying the corresponding RAS mutation

Mutations in codon 12 of K-RAS are frequently found in pancreatic adenocarcinomas. T-cell responses specific for individual RAS mutations can be elicited in vitro by stimulation of peripheral blood mononuclear cells with synthetic peptides. Mutant ras peptides are therefore a candidate vaccine for specific immunotherapy in pancreatic carcinoma patients. When vaccinated with a synthetic ras peptide representing the K-RAS mutation in their tumours, a transient ras-specific T-cell response was induced in two of five patients treated. The vaccination protocol involved multiple infusions of large amounts of peptide-pulsed antigen-presenting-cells obtained by leucapheresis. These results indicate that specific T-cell responses against mutations uniquely harboured in tumour cells can be induced in cancer patients by vaccination.

Ex vivo ras peptide vaccination in patients with advanced pancreatic cancer: Results of a phase I/II study

In a pilot phase I/II study we have tested synthetic ras peptides used as a cancer vaccine in 5 patients with advanced pancreatic carcinoma. The treatment principle used was based on loading professional antigen‐presenting cells (APCs) from peripheral blood with a synthetic ras peptide corresponding to the ras mutation found in tumour tissue from the patient. Peptide loading was performed ex vivo and the next day APCs were re‐injected into the patients after washing to remove unbound peptide. Patients were vaccinated in the first and second week and thereafter every 4–6 weeks. In 2 of the 5 patients treated, an immune response against the immunising ras peptide could be induced. None of the patients showed evidence of a T‐cell response against any of the ras peptides before vaccination. The treatment was well tolerated and could be repeated multiple times in the same patient. Side effects were not observed even if an immunological response against the ras peptide was evident. We conclude that ras peptide vaccination according to the present protocol is safe and may result in a potentially beneficial immune response even in patients with advanced malignant disease.

Pathogen reduction of blood components.

Thanks to many blood safety interventions introduced in developed countries the risk of transfusion transmitted infections has become exceedingly small in these countries. However, emerging pathogens still represent a serious challenge, as demonstrated by West Nile virus in the US and more recently by Chikungunya virus in the Indian Ocean. In addition bacterial contamination, particularly in platelets, and protozoa transmitted by blood components still represent sizeable risks in developed countries. In developing countries the risk of all transfusion transmitted infections is still high due to insufficient funding and organisation of the health service. Pathogen reduction of pooled plasma products has virtually eliminated the risk of transfusion transmitted infections, without compromising the quality of the products significantly. Pathogen reduction of blood components has been much more challenging. Solvent detergent treatment which has been so successfully applied for plasma products dissolves cell membranes, and can, therefore, only be applied for plasma and not for cellular blood components. Targeting of nucleic acids has been another method for pathogen inactivation of plasma and the only approach possible for cellular blood products. As documented in more than 15 years track record, solvent detergent treatment of pooled plasma can yield high quality plasma. The increased risk for contamination by unknown viruses due to pooling is out weighed by elimination of TRALI, significant reduction in allergic reactions and standardisation of the product. Recently, a promising method for solvent detergent treatment of single donor plasma units has been published. Methylene blue light treatment of single donor plasma units has a similar long track record as pooled solvent detergent treated plasma; but the method is less well documented and affects coagulation factor activity more. Psoralen light treated plasma has only recently been introduced (CE marked in Europe, but not licensed by the FDA), while the method of Riboflavin light treatment of plasma still is under development. In addition to pathogen reduction the methods, however, result in some reduction of coagulation factor activity. For platelets only Psoralen and Riboflavin light treatment have been implemented. Both are CE marked products in Europe but only approved for clinical trials in the USA. The methods affect platelet activity, but result in clinically acceptable platelets with only slightly reduced CCI and increased demand for platelet transfusions. Pathogen reduction of red blood cells with FRALE (S-303) or INACTINE (PEN110) has so far resulted in the formation of antibodies against neo-epitopes on red blood cells. A promising method for Riboflavin treatment of red blood cells is under development. This manuscript reviews the current experience and discusses future trends.

The Use of Solvent/Detergent Treatment in Pathogen Reduction of Plasma

The solvent/detergent (SD) process used for plasma can safely inactivate all lipid-enveloped viruses. The introduction of a specific prion-binding ligand gel in combination with SD treatment, time-reduced from 4 to 1–1.5 h, still ensures efficient virus kill, reduces abnormal prion protein by >5 log steps, and preserves levels of plasmin inhibitor at close to the reference range. Infections with known non-enveloped viruses such as HAV or parvovirus B19 are prevented by ensuring low virus loads in the starting plasma units, dilution through pooling of single plasma units, and neutralization of immune antibodies already present in the initial plasma pools. The major advantages of SD plasma over fresh frozen plasma and the other pathogen-inactivated plasmas are its extreme safety with respect to transfusion-related acute lung injury and the significantly lower likelihood of provoking allergic reactions. Both advantages are best interpreted as results of the dilution effect of pooling. No fewer than 18 clinical studies covering all indications for plasma, and extensive clinical experience have shown that reduced levels of coagulation factors and inhibitors as a result of SD treatment do not impair significantly the clinical efficacy or tolerance of plasma. Properly standardized clotting factor and inhibitor potencies and low batch-to-batch variations when compared with single-donor plasma units makes SD plasma more suitable for standardized treatment.

Practical guidelines for the clinical use of plasma

Despite differences in the composition of fresh frozen plasma (FFP) and solvent/detergent-treated plasma, prospective controlled clinical trials have not revealed any significant difference in clinical efficacy and tolerance between the two types of plasma. Evidence of the clinical efficacy of plasma is mainly based on expert opinion, case reports, and on controlled and uncontrolled observational studies. The application of plasma without laboratory analysis to verify the coagulopathy is normally not justified. With the exception of emergency situations when timely clotting assay results are not available, the administration of plasma in coagulopathy must be verified both clinically and by laboratory analysis before plasma is administered. The rapid infusion of at least 10 ml plasma/kg of body weight is required to increase the respective plasma protein levels significantly. Based on the present state of knowledge, plasma is indicated for complex coagulopathy associated with manifest or imminent bleeding in massive transfusion, disseminated intravascular coagulation, and liver disease. Therapeutic plasma exchange with 40 ml plasma/kg of body weight is the treatment of first choice in acute thrombotic-thrombocytopenic purpura-adult hemolytic uremic syndrome (TTP-HUS). A rare indication is the treatment or prevention of bleeding in congenital factor V or factor XI deficiency, plasma exchange in neonates with severe hemolysis or hyperbilirubinemia, and filling of the oxygenator in extracorporeal membrane oxygenation (ECMO) in neonates. Prothrombin complex concentrates should be preferred to plasma for the reversal of oral anticoagulation in emergency situations, since controlled studies have shown a minor efficacy of plasma. Side effects resulting from the administration of plasma are rare but have to be considered.

Serological typing of HLA-D: Predictive value in mixed lymphocyte cultures (MLC)

Locally produced antisera and antisera received through the Seventh International Histocompatibility Workshop exchange were investigated for specific B-cell cytotoxic activity in a panel of 95 unrelated HLA-D-typed donors. A number of sera formed clusters defining eight B-cell specificities which were strongly associated (p<0.001) to the HLA-D determinants Dw1–8. In panel investigations, only four triplets occurred. In five HLA recombinant families, these B-cell specificities followed the HLA-B-D chromosomal region, and in one —B/D recombination, DRw1 traveled with —Dw1. In MLCs between panel donors sharing zero, one, or two HLA-D-related B-cell specificities, significantly weaker MLC stimulation was observed with increasing compatibility, the median relative responses being 100, 52, and 17 percent, respectively. It is concluded that B cell-specificities HLA-DRw1–7 and WIA8 are probably coded for by HLA-D; they are excellent markers for the HLA-D determinants, which can thus be typed for by serological means; and serological typing for HLA-D has great value in predicting the outcome of MLCs.

Viral safety of solvent/detergent-treated plasma

BACKGROUND: Pooling of plasma donations increases the risk for blood‐borne infections. In solvent/detergent (SD)‐treated plasma, lipid‐enveloped viruses are efficiently inactivated. This method, however, does not affect non‐lipid‐enveloped viruses. The current study investigated the viral safety of SD‐treated plasma (Octaplas) and paid particular attention to the transmission of non‐lipid‐enveloped viruses.

Antiserum inhibition of the mixed lymphocyte culture (MLC) interaction. Inhibitory effect of antibodies reactive with HLA-D-associated determinants.

Abstract Two antisera, procured by immunization within HLA-A- and HLA-B-identical and HLA-D-incompatible unrelated combinations, were cytotoxic to B lymphocytes from the immunizing donor and from persons sharing his HLA-D-incompatible phenotype(s). The sera strongly and specifically inhibited lymphocytes from these donors when used as stimulating cells in mixed lymphocyte culture (MLC) reactions, while specific responding cell inhibition was less evident. The inhibitory effect was retained in the immunoglobulin G (IgG) and the F (ab)′ 2 fractions of these antisera. Inhibition was observed when the antisera were added within 48 hr after initiating the MLC. We conclude that these antisera contain antibodies reactive with structures closely associated with HLA-D determinants and that these may be human analogs of the mouse Ia antigens.

Importance of HLA-DR Matching in Cadaveric Renal Transplantation: A Prospective One-Center Study of 170 Transplants

To investigate the influence of matching for HLA-DR antigens in renal transplantation, we assessed the outcome of 170 prospectively HLA-typed cadaveric kidney transplantations performed since 1977 in one center. We found a beneficial effect on graft survival of HLA-DR compatibility between donor and recipient (P < 0.05). A possible effect of matching for the HLA-A and B antigens could be seen only in the HLA-DR-mismatched combinations. Pretransplantation blood transfusions were associated with increased graft survival only in patients receiving HLA-DR mismatched transplants (P < 0.02). We conclude that major emphasis should be placed on obtaining HLA-DR compatibility renal transplantation. (N Engl J Med. 1980; 303:850-4).

The Norwegian plasma fractionation project––a 12 year clinical and economic success story

The establishment of the Norwegian Fractionation Project (Project) was of major importance in preserving national self-sufficiency when plasma, cryoprecipitate and small batch factor IX-concentrates were replaced by virus inactivated products in the last part of the 1980s. Fractionation was performed abroad by contract with Octapharma after tenders on the European market. All Norwegian blood banks (>50) participated in the Project. Total yearly production was 50-60 tons of mainly recovered plasma. From 1993 solvent detergent (SD) treated plasma has replaced other plasma for transfusion. The blood banks paid for the fractionation and/or viral inactivation process, while the plasma remained the property of the blood banks and the final products were returned to the blood banks. The Project sold surplus products to other Norwegian blood banks and the majority of the coagulation factor concentrates to The Institute of Haemophilia and Rikshospitalet University Hospital. Both plasma and blood bank quality was improved by the Project. Clinical experience with the products has been satisfactory and self-sufficiency has been achieved for all major plasma proteins and SD plasma, but a surplus exceeding 3 years consumption of albumin has accumulated due to decreasing clinical use.The Project has secured high yields of the fractionated products and the net income from the produced products is NOK 1115 (140 Euros or US dollars) per litre plasma. An increasing surplus of albumin and the possibility of significant sales abroad of currently not fractionated IVIgG, could lead to a reorganisation of the Project from that of a co-ordinator to a national plasma handling unit. This unit could buy the plasma from the blood banks and have the plasma fractionated by contract after tender, before selling the products back for cost recovery. The small blood banks could produce plasma for products for the Norwegian market, while surplus products from the larger blood banks which are certified for delivery of plasma for fractionation of products to be consumed in the European Community, could be sold on the international market.