Bjarne Nielsen

Treatment with total lymphoid irradiation, cyclosporin A and a monoclonal anti-T-cell antibody in a hamster-to-rat heart transplantation model: Graft survival and morphological analysis

Abstract. Treatment with preoperative total lymphoid irradiation and post‐transplant cyclosporin A has been shown to have a synergistic effect on graft survival in alio‐and xenotransplantation. Specific monoclonal antibodies against T cells and T cell subpopulations could offer new ways of preventing graft rejection in xenotransplantation. Graft survival and histology were examined after total lymphoid irradiation plus cyclosporin A treatment versus cyclosporin A plus a monoclonal antibody in a concordant, heterotopic, hamster‐to‐rat heart transplantation model. Preoperative total lymphoid irradiation was given at a dose of 1. 25 Gy, 12 times over a period of 3 weeks. Cyclosporin A at a dose of 12. 5 mg/kg per day was administered perorally and OX‐19, a pan T cell monoclonal antibody, was given as intraperitoneal injections at doses of 100 μg or 500 μg/kg per day from day 0 until graft rejection. While total lymphoid irradiation alone prolonged graft survival to 9. 4 days, total lymphoid irradiation plus cyclosporin A extended graft survival to a mean of 22 days. Cyclosporin alone or combined with the monoclonal antibody could not increase graft survival significantly when compared to untreated animals, which rejected their grafts within 3. 7 days. Vascular rejection was the characteristic morphological finding, even after some weeks of excellent graft function. In conclusion, total lymphoid irradiation and cyclosporin A had a synergistic effect on graft survival in this concordant xenotransplantation model, although recent impressive results from other groups could not be reproduced. Total lymphoid irradiation combined with cyclosporin A appears to delay a primary humoral graft rejection, while the mechanism of rejection, judged by histology, stays the same.

The pharmacokinetic profile of plasma-derived mannan-binding lectin in healthy adult volunteers and patients with Staphylococcus aureus septicaemia

Mannan-binding lectin (MBL) is a member of the innate immune system, and MBL-deficiency affects 10–15% of Caucasians. With development of a plasma-derived MBL, substitution has become a therapeutic option in diseases associated with MBL insufficiency. The pharmacokinetics of injected MBL is weakly described, particularly in patients with infectious diseases. The pharmacokinetic profile of MBL following administration of 0.08 mg/kg to 20 healthy MBL-deficient volunteers and 0.2 mg/kg to 2 patients with Staphylococcus aureus septicaemia was established. In the volunteers, the maximal concentration was 2849 µg/l; the mean half-life (T1/2) was 69.6 h (14.6–114.9 h). The normalized clearance was 9×10−6 l/min×kg, and the mean residence time was 82 h. In the patients the serum-MBL versus time curves were similar to those in the volunteers, and T1/2 values were 36 and 40 h. In conclusion, MBL is distributed into a median volume of 3.4 l similar to the plasma volume, and the elimination in septicaemic patients was within the range of the controls. Due to the large individual variation in T1/2, we recommend that MBL therapy, with respect to dose and infusion intervals, is based on the chosen therapeutic target (≥1000 µg/l) and MBL serum determinations following the first infusion.

Human‐human hybridoma producing monoclonal antibodies against colorectal cancer‐associated antigens

Lymphocytes from lymph nodes draining the tumor region in patients with colorectal cancer were fused with two different human B‐lymphoblastoid cell lines, LICR‐LON‐HMy‐2 (HMy‐2) and WI‐L2–729‐HF2 (729‐HF2), to generate hybridomas synthesizing antibodies reacting with tumor‐associated antigens. In this way 220 hybridomas were obtained which produce antibody reacting with colon cancer cells. All established clones produced IgM. Four human monoclonal antibodies have been furhter analyzed. The cell lines producing these antibodies are all hybrids based on DNA analysis. Three of the antibodies (G4146, B9165 and D4213) showed binding to differentiation antigens by immunocytochemical analysis on different cancer cell lines and normal human leucocytes and by immunohistochemical analysis on sections of frozen malignant and normal tissues, while the fourth (F11348) showed a reaction with all cells and tissues tested. Western blots of tumor extracts showed binding of G4146 to two components from colon cancer cells with Mr of 59 K. and 61 K, while B9165 bound to a 43 K component and F11348 to several components with Mr from 30 to 200K. D4213 showd no binding in this analysis. The results obtained demonstrate the successful application of hybridoma technology to produce human monoclonals with reactivity to differentiation antigens.

Hope for successful xenografting by immunosuppression with monoclonal antibody against CD4, total lymphoid irradiation and cyclosporine: six months' survival of hamster heart transplanted into rat

Hamster hearts were transplanted to rats, and the effects of combinations of total lymphoid irradiation (TLI), cyclophosphamide, cyclosporine A (CyA) and monoclonal antibodies (MAB) were investigated. Controls not immunosuppressed rejected their xenograft in 3 to 5 days, while combination immunosuppression including MABs against CD4 or IL-2-receptors extended graft survival significantly. In one case, the graft was still functioning 180 days after transplantation, which is the longest survival seen in this model. The use of specific MABs may open a new era for both xeno- and allo-transplantation.

An ultrastructural analysis of concordant and discordant cardiac xenografts in unmodified recipients

Ultrastructural changes in endothelial cells in a hamster‐to‐rat and guinea pig‐to‐rat heart transplantation model are described. In the hamster‐to‐rat model, changes were minimal with an increase in cytoplasmic vesicles 4–6 h after transplantation. 16–24 h after transplantation larger vesicles appeared and the basement and cell membranes were less well defined. 42–48 h after transplantation the changes had progressed with destruction of cell membranes and the appearance of extravasated erythrocytes. At the time of complete rejection, changes had further progressed with widespread endothelial cell destruction and infiltration of neutrophilic granulocytes and macrophages, and large amounts of fibrin were present. In the guinea pig‐to‐rat model, changes were characterized by the appearance of platelets in close contact with the endothelium of the capillaries 1–3 min after transplantation. 4–6 min after transplantation the basement membranes as well as the cell membranes were affected with indistinct borders and interruption. Occasionally fusion of platelets and endothelial cell membranes was demonstrated. In the grafts examined 7–9 min after transplantation, changes had further progressed. Massive aggregation of platelets now appeared in relation to remnants of endothelical cells. Signs of microvascular damage appeared in both models, but with different morphology. In hamster grafts, endothelial cell activation is indicated by gradual changes in the cell membranes resulting in vascular damage and infiltration of the grafts by macrophages and neutrophilic granulocytes. In the guinea pig grafts, activation of endothelial cells results in platelet aggregation, formation of microthrombi, and subsequent tissue damage. Even though antibody and complement are involved in both types of rejection the basic mechanisms are different.

A method for blocking antigen-independent binding of human IgM to frozen tissue sections when screening human hybridoma antibodies.

Using an indirect immunoperoxidase technique antigen independent binding of both human monoclonal and polyclonal IgM was found to a wide range of frozen sections of normal and malignant human glandular epithelia. Identical binding was found using dimeric human IgA (dIgA), whereas no binding was found with monomeric human IgA or human IgG. Secretory component (SC) was found to be the component in these tissues mediating antigen-independent binding of human IgM and dIgA antibodies. A method for the blocking of this antigen-independent binding of human IgM and dIgA was evaluated. Frozen sections of tissues containing SC were blocked with antibody to endogenous immunoglobulin and preincubated with rabbit anti-secretory component antibody (anti-SC) before applying the human monoclonal antibody. This treatment blocked the binding of control polyclonal and monoclonal human IgM to sections of SC-containing tissues such as respiratory and colonic epithelia. The influence of anti-SC on the binding of dIgA could not be established due to interactions between the anti-SC antibody and the IgA preparation. Using this method human hybridoma supernatants containing IgM could be readily screened for reactivity with frozen tissue sections from patients with colo-rectal cancer. This approach is recommended for the screening of human IgM monoclonal antibodies on frozen human tissue sections.