Streptavidin-biotin immunotoxins: a new approach to purging bone marrow.

Immunotoxins have been used both experimentally and clinically to purge bone marrow of tumor cells or T cells before transplantation. We describe the synthesis of a streptavidin-biotin-toxin conjugate using whole ricin. Streptavidin-biotin-ricin (SA-BR) conjugates were synthesized by biotinylation of whole ricin, which was then complexed with streptavidin. Hybrid molecules consisting of a single biotinylated ricin moiety linked to a streptavidin molecule were separated by gel filtration. This SA-BR conjugate was used in an indirect cytotoxicity assay. The assay involved sensitizing of target cells with biotinylated monoclonal antibody (B-MCAB) followed by treatment with dilutions of SA-BR conjugate. The assay demonstrated a specific antibody-directed cytotoxicity. The strength of this SA-BR system is that a single conjugate was able to be used in conjunction with a library of B-MCABs to selectively target phenotypically different cell types. The application of the SA-BR conjugate is thus only restricted by the availability of B-MCABs specific for the desired target cells. The high affinity of avidin for biotin (Kd approximately 10(-15)) and the ability of a single conjugate to target phenotypically different cells through utilization of a library of B-MCABs gives SA-BR conjugates great potential in the selective targeting of individual cell types.

The synthesis of a peanut agglutinin-ricin A chain conjugate: potential as an in vitro purging agent for autologous bone marrow in multiple myeloma.

The lectin peanut agglutinin (PNA) and CD19 monoclonal antibody have been covalently linked to magnetic beads and utilized in an in vitro purging system for autologous bone marrow in multiple myeloma (MM). An alternative to mechanical purging involves the use of immunotoxins to provide specifically targeted cellular toxicity; however, no studies to date have examined the utility of a lectin-ricin A chain (RCA) combination as a purging agent in MM. Initially, we studied the internalization of PNA by target cells (Raji) using flow cytometry. The surface fluorescence intensity of PNA-treated Raji cells was reduced upon incubation at 37 degrees C, and subsequent studies with fixed cells detected the endocytosed PNA. Complete internalization occurred within 120 minutes, indicating the potential of PNA as a purging agent. We manufactured a novel PNA-RCA conjugate and demonstrated its strong and specific binding to PNA reactive cell targets. Subsequent experiments assessed the toxicity of the conjugate to Raji cells and normal bone marrow progenitor cells. 3H-leucine uptake assays showed that PNA-RCA was capable of reducing protein synthesis in Raji cells and that the toxic effects were specific. In addition, at concentrations of conjugate achieving greater than 99% selective cytotoxicity for Raji cells, adequate CFU-GM were preserved in normal marrow. These studies suggest that PNA-RCA may be of value as an in vitro purging agent for MM.

Coagulant and anticoagulant actions of Australian snake venoms.

A systematic study was made of the action on the plasma coagulation system of 20 Australian and Papuan Elapid and Hydrophiid snake venoms and compared with 4 Crotalid venoms and 1 Viper. The majority of Australian venoms were shown to be prothrombin activators with variable dependence on the presence of factor V phospholipid and calcium. None of these venoms had strong thrombin like activity in contrast to the Crotalid venoms which were powerfully thrombin like. The Crotalid venoms were also strongly fibrinolytic unlike the Elapid venoms which showed no or minimal evidence of fibrinolytic activity. Four Elapid venoms and 2 Crotalid venoms showed anticoagulant activity which contained neither antithrombin nor fibrinogenolytic activity and may act upon the prothrombin complex.

Plasma thrombin assay using a chromogenic substrate in disseminated intravascular coagulation due to snake bite envenomation.

Using the chromogenic substrate, Tos-Gly-Pro-Arg-pNA-HCl (Chromozym TH, Boehringer Mannheim) plasma thrombin was estimated in six cases of envenomation by Australian elapid snakes. All patients manifested findings characteristic of defibrination due to envenomation by these snakes. Fibrin-fibrinogen degradation products were grossly elevated, as was plasma thrombin in all cases. Following treatment with antivenene, all abnormal coagulation parameters returned rapidly towards normal by 24 hours and plasma thrombin disappeared.

PER-117: a new human ALL cell line with an immature thymic phenotype.

A new cell line, PER-117, was established from bone marrow cells of an eighteen months old boy with an acute lymphoblastic leukaemia (ALL). The leukaemic origin of cell line PER-117 is indicated by its cytochemical, immunological and cytogenetic similarity to the patient's fresh leukaemic cells. PER-117 carries a marker chromosome which was identified as a translocation between chromosomes 1 and 11. The surface marker analysis revealed that the phenotype of PER-117 is RFB-1+, RFT-1+ (CD5), 3A1+ (CD7), OKT 9+, OKT 10+ and HLA-DR-. Thus, this cell line appears to represent a prothymocyte or stage I thymocyte and preliminary data suggest that it can be induced in vitro to further differentiate.

Urate metabolism during bone marrow transplantation.

We studied urate metabolism in 36 patients undergoing both allogeneic and autologous bone marrow transplantation (BMT) without allopurinol. Most patients had low tumour burdens. Three different preparative regimens were used; busulphan/cyclophosphamide (BUCY); BCNU, etoposide, ara-C and melphalan (BEAM) and cyclophosphamide/total body irradiation (CY/TBI). Urate excretion rose during each of the regimens but the pattern of excretion varied with each regimen. Urate excretion remained elevated 72 h after completion of BEAM and BUCY, possibly reflecting the prolonged action of some of the agents used, e.g. melphalan, busulphan and etoposide. Urinary urate concentrations were unchanged compared with pre-chemotherapy levels reflecting the adequacy of the hydration protocol. No significant rise in creatinine was seen and no cases of tumour lysis syndrome occurred. Serum uric acid levels were a poor reflection of urate production, falling in most patients, and are an unreliable end-point for decisions regarding prophylaxis. BMT can be safely undertaken in patients with low tumour loads without allopurinol if an adequate urine volume is maintained. In this series, high levels of urate excretion often persisted for 72 h after the completion of conditioning and adequate hydration should be ensured during this period.

Prompt haemopoietic reconstitution following hyperthermia purged autologous marrow and peripheral blood stem cell transplantation in acute myeloid leukaemia.

We report a case of acute myeloid leukaemia in a 35-year-old woman where marrow and chemotherapy-mobilized peripheral blood stem cells (PBSC) were obtained in first complete remission and heat treated at 42 degrees C for 1 h to minimize the likelihood of leukaemic relapse. Transplantation of marrow and PBSC was undertaken following busulphan/cyclophosphamide conditioning 9 months after diagnosis in CR1. Hyperthermic purging did not impair the rate of engraftment and the patient was independent of blood product support by day 21. Studies on this patient's committed normal granulocyte-macrophage progenitor cells (CFU-GM) on samples from marrow and peripheral blood following treatment at 42 degrees C for 1 h, showed minimal and acceptable loss of activity comparable with the loss seen in other marrow progenitor activity treated in a similar fashion in our laboratory. We conclude that hyperthermia-purged PBSC can be used to hasten recovery in autologous haemopoietic progenitor transplantation without compromising rate of engraftment. This is part of an ongoing pilot study to evaluate the role of hyperthermic purging to reduce the risk of leukaemic relapse.

Infections in patients managed at home during autologous stem cell transplantation for lymphoma and multiple myeloma.

A group of 51 patients with multiple myeloma, non-Hodgkin's lymphoma or Hodgkin's disease receiving high-dose chemotherapy and autologous peripheral blood stem cell rescue received chemotherapy and clinical care in the peritransplant period at home. This group was compared with 88 cases with the same diagnoses, receiving the peripheral stem cell transplant over the same time period as an inpatient in a high efficiency particulate air filtered bone marrow transplant unit. Patients were treated at home based on choice, geographic accessibility, availability of an educated care giver and a clean home environment, and comprehension of the concepts of infection and aseptic techniques. Febrile neutropenia and sepsis were not increased in the home group and no episodes of septic shock were seen in this group. Patients at home received prophylactic oral ciprofloxacin and roxithromycin during the phase when the absolute neutrophil count was < 1 x 10(9)/l. Fewer gram-negative infections, but no diminution in gram-positive infections or in the rate of fever were seen in patients at home. Empiric therapy with a third generation cephalosporin, teicoplanin and tobramycin was instituted in 31 patients who developed a fever greater than 38.5 degrees C. Of this group of 31, 18 required admission to hospital, 12 because of febrile neutropenia which persisted or was considered unsuitable for management at home due to sepsis. The remaining 13 with febrile neutropenia remained at home throughout, as did the 20 cases not developing neutropenic fever. This study demonstrates the feasibility of managing carefully selected patients in their home environment when at risk from febrile neutropenia or other septic complications following autologous peripheral stem cell support.

Mycobacterial central venous catheter tunnel infection: a difficult problem.

We report our experience of non-tuberculous mycobacterial infection associated with the tunnel of Hickman-Broviac central venous catheters in immunosuppressed patients with haematological malignancies undergoing high-dose chemotherapy supported by BMT. The problem is rare and difficult to treat. Our cases are unique in developing tunnel site mycobacterial infection well after the tunnelled catheters were removed. We diagnosed one case of Mycobacterium chelonae, which is a well-documented cause of such infections, and two cases of Mycobacterium haemophilum, which are the first reported cases in this setting. Early wide surgical excision of the infected tunnel site and prolonged antibiotic therapy is necessary. Despite these measures recurrence occurred in two cases. Close liaison with the microbiology laboratory is needed to ensure the appropriate culture media and conditions are used for these fastidious organisms. Empiric antibiotic regimens should be based on the likely organism. Drugs active against M. chelonae and M. haemophilum should be included.

The MHC influences acute graft versus host disease in MHC matched adults undergoing allogeneic bone marrow transplantation.

To determine whether the MHC plays an antigen non-specific role in the development of acute GVHD, the prevalence of acute GVHD in relation to MHC genotype was examined in 51 adult patients undergoing allogeneic BM grafting. The majority of patients received grafts from HLA-identical siblings. HLA-B7 haplotypes were associated with a decreased risk of acute GVHD (2 of 15, p = 0.005) whereas HLA-B44 haplotypes were associated with a higher risk of acute GVHD (11 of 14, p = 0.02). As these alleles have been reported previously as having opposite effects in relation to inflammatory mediators, these findings may have important implications with respect to donor selection and patient management.

Immunohistochemical changes in sigmoid colon after allogeneic and autologous bone marrow transplantation.

AIM: To determine whether there are characteristic immunohistological changes in the colonic mucosa in acute graft versus host disease (GvHD). METHODS: Consecutive allogeneic (n = 11) and autologous (n = 11) bone marrow transplant recipients underwent endoscopic biopsy of sigmoid mucosa before transplant and on day 30 post-transplant. Immunohistochemical staining and quantitation of intraepithelial and lamina propria mononuclear cells were undertaken using a panel of monoclonal antibodies and a Streptavidin-biotin alkaline phosphatase staining technique. RESULTS: In the allogeneic group (nine of whom had clinical acute GvHD) there was a fivefold increase in lamina propria CD16+ mononuclear cells (3.1 +/- 4.3 to 17.0 +/- 12.2 per 100 lamina propria nucleated cells), compared with autologous transplant recipients in whom this rise was twofold (5.5 +/- 4.6 to 10.6 +/- 7.1 per 100 lamina propria nucleated cells). The CD16+ mononuclear cells had morphological appearances of tissue macrophages, but in neither the allogeneic nor autologous groups was there an increase in total macrophage numbers (CD14+). In patients with acute GvHD the lamina propria CD4+:CD8+ lymphocyte ratio fell (1.97 +/- 1.12 to 1.07 +/- 1.01), primarily because of a fall in the number of lamina propria CD4+ lymphocytes. In both allogeneic and autologous groups there was a fall in intraepithelial lymphocyte numbers, but there was no change in CD19+ (B cell), CD25+ (interleukin-2 receptor positive) or CD56+ (natural killer) cell numbers. CONCLUSION: Following bone marrow transplantation, there appears to be upregulation of lamina propria tissue macrophage CD16 (an Fc receptor for IgG), a change which is more noticeable after allogeneic transplantation and which may be related to the development of acute GvHD. In patients with acute GvHD there was a fall in the lamina propria CD4+:CD8+ lymphocyte ratio. If these changes are functionally important, they may have significant implications for understanding the pathogenesis of GvHD.

Hyperthermia potentiates the activity of immunotoxin conjugates against common acute lymphoblastic leukaemia cells in vitro.

We studied the in-vitro cytotoxic effect of hyperthermia at 42 degrees C, both alone and in combination with ricin-linked immunotoxins, reactive with the common acute lymphoblastic leukaemia cell lines Reh and KM-3. Assessment of cytotoxicity was by incorporation of 3H-leucine and limiting dilutions analysis. The effect of immunotoxins alone and in combination with hyperthermia on normal human marrow progenitor cells was assessed by conventional colony forming units-granulocyte macrophage (CFU-GM) assay. We found that incubation of either of the cell lines with a mixture of the two immunotoxins, RPH-7-ricin and PHM-6-ricin, at 42 degrees C for one hour (h) potentiated the cytotoxic activity of the immunotoxins at 37 degrees C. At a concentration of 10(-8) mol/L, a 2.2-log kill was seen with KM-3 leukaemic cells at 37 degrees C and a 3.3-log kill at 42 degrees C, an increase of approximately 10 fold in cytotoxic activity. Survival of CFU-GM following treatment at 42 degrees C for one h with a similar concentration of immunotoxins was 26.2% (+/- 13.7%) (equivalent to 0.6 log kill) and 76.0% (+/- 1.83%) (0.1 log kill) when normal marrow was incubated with immunotoxins at 37 degrees C. This suggests relative sparing of normal marrow cells compared with the leukaemic cells tested as indicated by the 2.1-log kill difference (approximately 100 fold) between normal and leukaemic cells at 37 degrees C and the 2.7-log kill (approximately 500-fold) difference at 42 degrees C. We conclude that hyperthermia may have a role in addition to immunotoxins in the purging of marrow ex vivo to remove leukaemic cells.

A phase I/II study of intensive dose escalation of cytarabine in combination with idarubicin and etoposide in induction and consolidation treatment of adult acute myeloid leukemia. Australian Leukaemia Study Group (ALSG).

To determine the safety and efficacy of the combination of idarubicin, cytarabine and etoposide ("ICE") for induction and consolidation treatment of acute myeloid leukemia (AML), and of dose-intensification of cytarabine in this setting, 54 previously untreated patients in three cohorts were studied by sequential dose escalation of cytarabine, in combination with standard doses of idarubicin and etoposide. Cytarabine was given to Cohort 1 at the conventional dosage of 100 mg/m2 per day by continuous infusion for 7 days in induction and 5 days in consolidation; to Cohort 2 at high-dose (HiDAC) (3 g/m2 intravenously twice daily on days 1, 3, 5 and 7) during induction with conventional dosage during consolidation; to Cohort 3 HiDAC was given for both induction and consolidation. In addition, Cohort 3 patients received lenograstim (Granocyte; rHuG-CSF) after both induction and consolidation courses. We found that there was no significant difference between the three cohorts in hematological toxicity in induction, but that HiDAC was associated with a greater incidence of gastro-intestinal toxicities. There was no difference in induction mortality between the three cohorts, which was 11% overall. Consolidation with HiDAC led to a significant increase in hematological toxicity. Overall, the complete remission (CR) rate was 80% with no significant difference between the three regimens. The estimated disease free survival at 3 years was 28%, 67% and 54% respectively for Cohorts 1, 2 and 3 with an estimated overall survival of 38%, 63% and 47%. We conclude that cytarabine dosage can be escalated safely in combination with idarubicin and etoposide in both induction and consolidation. The combination is effective for induction treatment of AML and its side-effects appear similar to those of standard regimens. Whether its use offers long-term benefits compared with standard regimens is the subject of ongoing controlled randomized studies.

Plasma cofactors necessary for Enhydrina schistosa (beaked sea snake) venom to induce platelet aggregation.

A study of the effect of the venom of the beaked sea snake (Enhydrina schistosa) was undertaken on platelet aggregation. It was found that platelet aggregation and the release reaction occurred in the presence of both venom and plasma but, not with the venom alone. No effect was observed with fixed platelets distinguishing the effect of the venom from that of platelet aggregation with ristocetin or botrocetin and indicating metabolic dependence. Studies to elucidate the plasma factor required for platelet aggregation under the release reaction indicated dependence on the presence of both Factor II and calcium ions. This venom may prove to be a useful laboratory reagent in coagulation studies because of the Factor II and calcium dependence.

Australian snake venoms and their in vitro effect on human platelets.

Thrombocytopenia is generally not associated with cases of envenomation by Australian snakes, however the clinical evidence is conflicting. The in vitro effect of these venoms upon platelets had hitherto not been studied. This study systematically examines the effect on human fresh and fixed platelets of twenty Australian snake venoms, nineteen elapid and an hydrophiid; for comparision four crotalid venoms from the Americas and S.E. Asia were also included. Electron micrographs were taken of platelets after exposure to some of the venoms. Results demonstrated that all venoms except the hydrophiid venom caused fresh platelets to irreversibly aggregate directly, and this was associated with degranulation as evidenced by electron microscopy (EM). Response to all venoms by fixed platelets was less marked and also suggests, that metabolically, active platelets are necessary for the venoms to exert their maximal effect. The hydrophiid venom's action on fresh platelets was unique, as a plasma co-factor was required before aggregation could be induced. Crotalid and hydrophiid venoms were more active against platelets than the elapid venoms. Nevertheless, platelet aggregation and degranulation in the presence of elapid venoms suggests that a platelet response in vitro may be a significant factor in the "defibrination syndrome" induced in humans by Australian snakes.

Passovoy factor deficiency in five Western Australian kindreds.

Passovoy factor deficiency, a coagulation abnormality affecting the intrinsic coagulation system, was discovered in 5 Western Australian kindreds. The defect is inherited as an autosomal dominant and is associated with a clinical bleeding tendency characterized by easy bruising and undue blood loss following trauma such as dental extraction and tonsillectomy. Fresh frozen plasma appears to provide effective prophylaxis during surgery. The activated partial thromboplastin time (APTT) shows a prolongation which, in most patients, is of relatively slight degree, and this may be the reason for the paucity of reports in the literature. The discovery of 5 kindreds carrying the defect suggests that it may be relatively common in the Australian community and that care should be taken to identify and follow up minor grades of abnormality of the APTT where individuals suspected of having an inherited bleeding tendency are screened. A sample from one case was distributed, with a clinical history, to participants in the Royal College of Pathologists of Australasia 1980 Quality Assurance Programme in Haematology. Approximately one-third of 175 participants failed to detect the definite abnormality present.

Peanut agglutinin (lectin from Arachis hypogaea) binding to hemopoietic cells: an immunophenotypic study using a biotin streptavidin technique.

The lectin peanut agglutinin (PNA) was used to study the surface carbohydrate expression of galactose beta 1, 3, N-acetylgalactosamine by normal and malignant hemopoietic cells. Immunostaining was performed using biotinylated PNA and a streptavidin-alkaline phosphatase staining technique on 78 patients. The study was undertaken to enlarge on previous reports of lectin binding to cells of hemopoietic origin and to establish the potential role of biotinylated PNA as a component of an immunotoxin for in vitro purging of bone marrow in patients with multiple myeloma. In normals only monocytes, macrophages, centroblasts and plasma cells showed reactivity. Of the hematological malignancies, all cases of multiple myeloma were positive and non-Hodgkin's lymphoma cases with a large cell component had positive centroblasts. Two of 5 cases of acute myelomonocytic leukemia, one case of chronic myelomonocytic leukemia and one case of pleomorphic T cell non-Hodgkin's lymphoma showed PNA positive neoplastic cells. The reactivity of biotinylated PNA with centroblasts and plasma cells suggests that it may be of potential value when linked to a streptavidin-ricin conjugate in the in vitro purging of bone marrow of patients with multiple myeloma prior to autologous bone marrow transplantation.

A unique T-cell monoclonal antibody with potential uses in autologous bone marrow transplantation.

A monoclonal antibody reactive with an antigen expressed by a T-cell subset has been produced. The antibody designated RPH-1 reacts with a subset of normal T-cells and thymocytes. The antibody was strongly reactive with blast cells from 3 patients with T-acute lymphoblastic leukemia (T-ALL) and also reacted with cells of 2 patients with non-T-ALL. The antibody did not react with leukemic cells from patients with acute myeloid leukemia (AML) or B-chronic lymphocytic leukemia (B-CLL)). RPH-1 reacted with 2 T-cell lines, no B-cell lines and no myeloid cell lines. Absence of toxicity to granulocyte macrophage colony forming units (CFU-GM) by RPH-1 in the presence of complement was demonstrated, and in view of strong reactivity with certain leukemic cells, potential application of RPH-1 as an immunological means of selectively removing tumour cells is indicated.

Cross-matched platelets in bone marrow transplantation.

Antibodies to HLA-antigens remain a problem in multiply-transfused patients. Over a 2 yr period 44 bone marrow transplant recipients were screened at weekly intervals for the presence of HLA-antibodies using a solid phase red cell adherence technique. An adaptation of this method was used to provide cross-matched random donor platelets (XMRDP) when screening proved positive. Twelve of the 44 patients were antibody positive, 6 prior to transplantation and 6 following the transplant. Those 4 patients who developed an antibody following the transplant had a significant increase in platelet increments following the change from random donor platelets (RDP) to XMRDP even though only one patient was refractory to platelets at the time the antibody was first detected. No significant bleeding occurred during the study period. The use of routine platelet antibody screening followed by platelet cross-matching allows excellent support of thrombocytopenic patients without requiring HLA-typed blood donors and avoiding clinical platelet refractoriness.

Monoclonal antibody-ricin conjugate cytotoxic to cells expressing the common acute lymphoblastic leukemia antigen (CALLA).

The monoclonal antibody PHM-6, which is specific for the common acute lymphoblastic leukemia antigen (CALLA), was conjugated to the plant toxin ricin. Binding of the PHM-6-ricin conjugate to cells via the ricin molecule was blocked by the presence of 100 mM lactose. The IC50 (concentration resulting in 50% inhibition) of the PHM-6-ricin conjugate for the CALLA-positive KM-3 cell line was 280-fold greater than for bone marrow stem cells, indicating the potential of this conjugate for immunological purging of autologous remission marrow.