Alternative dispute resolution and the physician--the use of mediation to resolve hospital-medical staff conflicts.

The use of adversarial methods to resolve disputes arising out of medical staff matters can be time-consuming, costly, and disruptive to the hospital-medical staff relationship. As this article suggests, mediation is the preferred method of alternative dispute resolution for reaching mutually acceptable solutions with minimal harm to relationships.

Use of multiple T cell-directed intact ricin immunotoxins for autologous bone marrow transplantation.

The monoclonal antibodies (MoAb) T101, G3.7, 35.1, and TA-1 were conjugated to intact ricin using a thioether linkage. These MoAb detect, respectively, the CD5[gp67], CD7[p41], CD2[p50], and [gp95, 170] determinants that are found in the vast majority of cases of T cell acute lymphocytic leukemia (T-ALL). The resulting immunotoxins (ITs) and an equimolar mixture of these ITs were evaluated as potential purgative reagents for autologous transplantation in T-ALL. Leukemic cell lines were used to compare the kinetics of protein synthesis inactivation mediated by each IT. The cells were treated with IT in the presence of lactose in order to block the native binding of ricin. The observed rates of protein synthesis inactivation correlated with target antigen expression detected by fluorescence-activated cell sorter analysis. Of the four ITs, T101-ricin (T101-R) exhibited the fastest rate of inactivation, followed in order by G3.7-ricin, TA-1-ricin, and 35.1-ricin. At concentrations greater than 300 ng/mL, a cocktail containing an equimolar amount of all four ITs (referred to as the four-IT cocktail) exhibited kinetics that were as fast or faster than those of T101-R. The long-term cytotoxic effects of individual ITs and the four-IT cocktail were evaluated using a sensitive clonogenic assay. Each IT was specifically cytotoxic and inhibited 1 to 4 logs of clonogenic leukemic cells at doses (300 to 600 ng/mL) that can be used clinically. The four-IT cocktail was highly cytotoxic; a concentration of 300 ng/mL inhibited greater than 4 logs of leukemic cells while sparing the majority of committed (CFU-GM, CFU-E) and pluripotent (CFU-GEMM) hematopoietic stem cells. The determination of both short-term kinetics of protein synthesis inactivation and longer-term inhibition of clonogenic growth allowed new insight into cell killing by IT. Our results suggest that ITs continue to act on clonogenic target cells for a period of three to five days. Interestingly, the four-IT cocktail was not as potent against clonogenic leukemic cells as T101-R alone, although it exhibited kinetics of protein synthesis inhibition that were as fast as those of T101-R alone. This finding suggests that internalized ITs may differ in the length of time they remain active within the cell. Our results also demonstrate the importance of using several different assays to evaluate IT reagents.

In vitro culture of leukemic cells in t(4;11) acute leukemia.

In the present study we utilized a semisolid culture system with feeder cells and enriched media to evaluate the growth of acute leukemia associated with the 4;11 chromosomal translocation. We compared growth of t(4;11) leukemia to typical acute nonlymphocytic leukemia (ANL) and acute lymphocytic leukemia (ALL). The two cases of t(4;11) leukemia tested exhibited the highest cloning efficiency of cells tested. The growth characteristics of t(4;11) leukemia were more similar to ANL than ALL.

Combined ex vivo treatment with immunotoxins and mafosfamid: a novel immunochemotherapeutic approach for elimination of neoplastic T cells from autologous marrow grafts.

We evaluated a novel ex vivo "purging" protocol for selective elimination of neoplastic T cells from human marrow by using a sensitive clonogenic assay. Immunotoxins (IT) were synthesized by conjugating ricin (R) to four different monoclonal antibodies (MoAb) directed against distinct markers of T cell lineage. Treatment with anti-p67-R produced effective elimination of leukemic T cells from human marrow. The cyclophosphamide congener mafosfamid (ASTA Z 7577) markedly enhanced the target cell cytotoxicity of IT and extended the final level of clonogenic kill 2 to 3 logs. Our data show that anti-p67-R in combination with mafosfamid resulted in a maximum elimination of 6.2 logs of neoplastic T cells with minimal toxicity to normal bone marrow progenitors. The efficiency of this protocol was not reduced in the presence of excess normal bone marrow cells. Similar findings were obtained by using a cocktail of four different anti-T cell IT. This approach is unique in combining both immunologic (IT) and chemical (mafosfamid) strategies for more effective ex vivo bone marrow purging in autologous bone marrow transplantation for T cell acute lymphoblastic leukemia/lymphoblastic lymphoma.

Utilization of a colony assay to assess the variables influencing elimination of leukemic cells from human bone marrow with monoclonal antibodies and complement.

We have previously used a chromium-release assay to demonstrate that the cocktail of monoclonal antibodies BA-1, BA-2, BA-3, and complement can effectively lyse human leukemic cells in the presence of excess bone marrow. Using a leukemic cell colony assay, we have reinvestigated the variables influencing lysis of human leukemic cells (KM-3, HPB-NULL, NALM-6) in bone marrow using BA-1, BA-2, BA-3, and complement. Specific variables addressed included the concentration of excess bone marrow cells, the number of treatments, the presence or absence of DNase during the treatment, the combination of antibodies, and the sensitivity of different leukemic cell lines to lysis. Using the colony assay, the BA-1,2,3 cocktail was shown to be more effective than any single antibody or combination of two antibodies. We also determined that the concentration of excess bone marrow cells and number of treatments had a direct bearing on leukemic cell lysis. Although two cycles of treatment were significantly superior to one cycle, three cycles were not significantly superior to two cycles. Inclusion of DNase (10 micrograms/mL) was a critical adjunct that eliminated clumping and facilitated plating cells in the colony assay. Finally, we could show that striking differences existed in the sensitivity of the leukemic cell lines to lysis with the BA-1,2,3 cocktail and complement. NALM-6 cells were the most sensitive (approximately four logs of kill), and KM-3 cells were the most resistant (less than two logs of kill). Our results strongly support the utility of sensitive leukemic cell colony assays in the analysis of marrow treatment variables in autologous bone marrow transplantation.

Human acute leukemia cell line with the t(4;11) chromosomal rearrangement exhibits B lineage and monocytic characteristics.

A cell line, designated RS4;11, was established from the bone marrow of a patient in relapse with an acute leukemia that was characterized by the t(4;11) chromosomal abnormality. The cell line and the patient's fresh leukemic cells both had the t(4;11)(q21;q23) and an isochromosome for the long arm of No. 7. Morphologically, all cells were lymphoid in appearance. Ultrastructurally and cytochemically, approximately 30% of the cells possessed myeloid features. The cells were strongly positive for terminal deoxynucleotidyl transferase. They were HLA-DR positive and expressed surface antigens characteristic for B lineage cells, including those detected by anti-B4, BA-1, BA-2, and PI153/3. Immunoglobulin gene analysis revealed rearrangements of the heavy chain and kappa chain genes. The cells lacked the common acute lymphoblastic leukemia antigen and antigenic markers characteristic of T lineage cells. The cells reacted with the myeloid antibody 1G10 but not with other myeloid monoclonal antibodies. Treatment with 12-O-tetradecanoyl-phorbol-13-acetate induced a monocyte-like phenotype demonstrated by cytochemical, functional, immunologic, and electron microscopic studies. The expression of markers of both early lymphoid and early myeloid cells represents an unusual phenotype and suggests that RS4;11 represents a cell with dual lineage capabilities. To our knowledge, RS4;11 is the first cell line established from t(4;11)-associated acute leukemia.

Transplacental alloimmunization to fetal erythrocytes in rhesus monkeys (Macaca mulatta).

Evidence of transplacental immunization in rhesus monkeys was obtained by testing postpartum sera from 252 females for antierythrocyte agglutinins. One-third of the sera contained antibodies reactive with the mates' or the newborns' cells. Fetal erythrocytes were detected in the maternal circulation as early as 8 weeks after conception and as late as 12 days postpartum. Maternal antibodies were detected as early as 2 months after conception and persisted for more than 3 weeks postpartum. It was concluded that the fetal cells stimulated antibody production. Several features of transplacental immunization differ between rhesus monkeys and humans. A parity effect was not observed in rhesus. In fact, 33% of primiparous rhesus females produced antibodies. Also, several of the different allogeneic blood group factors appeared to be immunogenic but differed in immunopotency. Finally, direct antiglobulin tests indicated that erythrocytes of 11% of newborns were coated with maternal antibodies. Nevertheless, in contrast to humans, hemolytic disease was not observed.

Mononuclear phagocyte receptors in rhesus monkeys (Macaca mulatta) and their role in hemolytic disease of the newborn.

Hemolytic disease of the newborn does not develop in rhesus monkeys because placentally-transferred maternal antibodies do not induce immune clearance of the newborn's erythrocytes. In an in vitro RBC adherence assay, rhesus peripheral blood monocytes did not bind newborn's RBC which had been coated in utero or in vitro with maternal antibodies. Nevertheless, rhesus phagocytes possess receptors that are specific for the Fc portion of IgC and for the C3b. Using purified human IgG subclasses as inhibitors of RBC adherence, rhesus Fc receptors preferentially bind IgG1 and IgG3. Thus, it may be that maternal antibodies are non-opsonic because they belong to IgG subclasses that do not bind effectively to rhesus Fc receptors. Also, RBC adherence appears to be controlled by the level of antibody coating which in turn is determined by avidity of the antibodies and by the number of RBC membrane determinants. The failure of maternal antibodies to opsonize the newborn's RBC and thus cause hemolytic disease is very likely due to the low avidity of antibodies and to the weak expression of blood group determinants on the membranes of these RBC.

Elimination of clonogenic T-leukemic cells from human bone marrow using anti-Mr 65,000 protein immunotoxins.

Two anti-Mr 65,000 protein (p65) murine monoclonal antibodies, T101 and VIII-1, were conjugated to intact ricin. Toxicity of the resulting immunotoxins (IT) was measured against leukemic cell lines treated alone and in the presence of excess bone marrow using a highly sensitive colony inhibition assay. Cells were pretreated with IT in the presence of lactose to block the native binding of ricin. The IT proved to be potent cytotoxins for the p65-positive cell lines, CEM and MOLT-4. Treatment with T101-ricin (1000 ng/ml) inhibited clonogenic activity of these lines by more than 5.1 logs. Less than 1 log of the inhibition at this dose was due to nonspecific killing by IT. Notably, the presence of excess bone marrow did not reduce IT toxicity against the leukemic populations. Comparison of IT concentrations which inhibited 50% of clonogenic activity showed that T101-ricin was 140- to 540-fold and VIII-1-ricin was 12- to 192-fold more toxic to p65-positive than to p65-negative cell lines. Neither unconjugated anti-p65 nor IT prepared with an irrelevant antibody inhibited clonogenic activity. Blocking of IT toxicity by unconjugated antibody further demonstrated that the antibody moiety of the IT directed the selective toxicity. We found that T101-ricin was more toxic for CEM cells than was VIII-1-ricin, even though blocking studies indicated that the two antibodies bind to proximal or identical epitopes. This report is unique in that an IT was shown to specifically eliminate greater than 99.99% of leukemic cells from human bone marrow. These findings indicate the utility of T101-ricin as an in vitro reagent for autologous bone marrow transplantation in treatment of T-cell leukemia.

The absence of hemolytic disease in the newborn rhesus monkey (Macaca mulatta).

Hemolytic disease of the newborn has not been observed in rhesus monkeys even though the newborn's erythrocytes may be coated with maternal antibodies. Using a 51chromium-erythrocyte survival assay, we found that maternal antibodies do not mediate immune elimination of newborn's red blood cells. However, certain allogeneic or xenogeneic antibodies mediate clearance via sequestration by the reticuloendothelial system, or by intravascular hemolysis, or by a combination of these. The class of antibody plays a major role in elimination since red cells coated with IgG but not with IgM were rapidly cleared. In addition, the quantity of antibody controls the rate and extent of clearance. A comparison of rhesus alloantisera suggests that coating of multiple antigenic sites is necessary for clearance. Passive immunization with selected high-titered anti-erythrocyte alloantisera can induce some hematologic signs of erythrocyte destruction in newborn monkeys.

The human Duffy blood group in rhesus monkeys.

There is good evidence that susceptibility to Plasmodium vivax infection and to P. knowlesi erythrocyte invasion is influenced by certain human Duffy (Fy) blood group antigens. Since P. knowlesi readily infects rhesus monkeys (Macaca mulatta), it was not surprising to find an Fy-like antigen on rhesus erythrocytes. Using human Fy antisera in elution and absorption experiments, we found that all 40 rhesus monkeys tested displayed the Fy(a-b+) phenotype. Furthermore, the rhesus Fyb antigen was inactivated by chymotrypsin but not by trypsin, suggesting that it is homologous to the human Fyb antigen. Preliminary serological analyses and enzyme hydrolysis experiments suggest that none of the 13 blood group systems that we have described in rhesus are analogous to the human Fy system. Thus, it appears that there is no Duffy-like polymorphism in rhesus monkeys.