Chromosome abnormalities in Down's syndrome patients with acute leukemia

Chromosome and cytologic studies were performed on three Down's syndrome (DS) patients with acute nonlymphocytic leukemia (ANLL). All three patients had an aneuploid clone in their leukemic cells: 50, XX, +6, +19, +21, +22, +8, XX, +21, and 47,XY, +8, - 21 +dic(21;21)(p13;p11). Every patient appeared to have acute undifferentiated leukemia when the blast cells were examined with Wright-Giemsa stain; cytochemistry studies, however, showed that the leukemic blasts were in an early stage of myeloid differentiation. The two patients with +8 had a preleukemic phase; the blast cells of the patient with an extra no. 19 and no.22 could not be differentiated morphologically from those of the two patients with an extra no. 8. Our findings and a review of data on 40 other patients suggest that most DS children with ANLL have hyperdiploidy, which is usually related to gains of C, F, and /or G chromosomes, and that the abnormalities of +8 and of +19, +22 in DS children may be associated with acute leukemia (AL) in an early stage of myeloid differentiation.     

Regulation of fibrinolysis by platelet-released plasminogen activator inhibitor 1: light scattering and ultrastructural examination of lysis of a model platelet-fibrin thrombus

We have investigated the role of plasminogen activator inhibitor 1 (PAI- 1) in the regulation of fibrinolysis using a model thrombus composed of thrombin-stimulated platelets, fibrin(ogen), plasminogen, and recombinant tissue-type plasminogen activator. Laser light scattering kinetic measurements showed that clot lysis was significantly delayed both by thrombin-stimulated platelets and their cell-free releasate. This delay in lysis was almost fully reversed by the addition of a PAI- 1-specific monoclonal antibody that blocks the ability of PAI-1 to inhibit plasminogen activators. Lysis half-times exhibited a linear dependence on the concentration of PAI-1 antigen present, as determined by enzyme-linked immunosorbent assay (ELISA). Sodium dodecylsulfate- polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting confirmed the presence of PAI-1 antigen in the platelet releasates. Scanning electron micrographs of the model thrombus components sampled late in lysis showed considerable unproteolyzed fibrin still attached to platelets. Immunogold cytochemistry detected large amounts of PAI-1 antigen in the partially lysed platelet-fibrin thrombi. This PAI-1 appeared to be bound to the fibrin network rather than to the platelet surface itself. We conclude that the residual clots observed late in lysis represent platelet-associated fibrin to which platelet-released PAI-1 has bound, rendering it less susceptible to degradation.     

Type 2M:Milwaukee-1 von Willebrand disease: an in-frame deletion in the Cys509-Cys695 loop of the von Willebrand factor A1 domain causes deficient binding of von Willebrand factor to platelets

This report examines the genetic basis of a variant form of moderately severe von Willebrand disease (vWD) characterized by low plasma von Willebrand factor antigen (vWF:Ag) levels and normal multimerization, typical of type 1 vWD, but disproportionately-low agonist-mediated platelet-binding activity. We identified an in-frame deletion in vWF exon 28 in three generations of affected family members, who are heterozygous for this mutation. The deletion of nucleotides 4,173-4,205 results in the loss of amino acids Arg629-Gln639 in the Cys509-Cys695 loop of the A1 domain in mature vWF. The secreted mutant vWF showed a normal multimeric profile but did not bind to platelets in the presence of optimal concentrations of either ristocetin or botrocetin. The mutant vWF also failed to interact with heparin, and with vWF monoclonal antibody AvW3, which blocks the binding of vWF to GPlb. In addition, mutant vWF showed reduced secretion from transfected cells concomitant with increased intracellular levels. These results confirm that the deletion is the genetic defect responsible for the reduced interaction of vWF with platelets. We have designated this new variant type 2M:Milwaukee-1 vWD. Our analysis suggests that the potential frequency of this phenotype in individuals diagnosed with type 1 vWD is about 0.5%.     

Granulocyte-macrophage colony-stimulating factor expression by human fibroblasts is both upregulated and subsequently downregulated by interleukin-1

Interleukin-1 (IL-1) treatment of human WI-38 lung fibroblasts results in granulocyte-macrophage colony-stimulating factor (GM-CSF) expression as well as a delayed increase in prostaglandin E2 (PGE2) production that closely correlates with the decline of GM-CSF mRNA levels. Pretreatment with PGE2 reduces the IL-1 induced GM-CSF mRNA and protein expression to 10% to 15% of control values at concentrations of PGE2 that are endogenously produced after IL-1 stimulation. Inhibition of PGE2 synthesis by indomethacin prolongs the IL-1 induced GM-CSF mRNA expression and increases the cumulative GM-CSF protein secretion. Exposure of WI-38 fibroblasts to PGE2 results in an increase in intracellular cyclic adenosine monophosphate (cAMP) levels. The inhibition of GM-CSF expression by PGE2 can be mimicked by stable cAMP analogs as well as cAMP elevating agents such as cholera toxin, forskolin, and isobutylmethylxanthine. Thus the inhibition exerted by PGE2 is mediated via cAMP. Taken together, these results suggest that IL-1 stimulation of human fibroblasts provides not only the upregulatory signal for GM-CSF expression but also a delayed and indirect downregulatory signal that serves to limit GM-CSF expression in the continued presence of IL-1.     

The effect of recombinant GM-CSF on the recovery of monkeys transplanted with autologous bone marrow

The regulatory function of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) on granulocyte production in vivo was evaluated in an autologous bone marrow transplantation model using rhesus monkeys. Monkeys were exposed to 9.0 Gy total body irradiation and then transplanted with 5.0 x 10(7) low-density bone marrow cells/kg. Alzet miniosmotic pumps were subcutaneously implanted to deliver rhGM-CSF at a rate of 50,400 U/kg/d. Minipumps, containing either rhGM-CSF or saline, were implanted between zero and five days after transplantation for seven days. Kinetic recoveries of peripheral blood cells after either saline or rhGM-CSF treatment were compared. Treatment with rhGM-CSF accelerated the recovery of neutrophils. Neutrophils in rhGM-CSF-treated animals recovered to 80% (3.4 x 10(3)/mm3) pre-irradiation control levels by day 20, in comparison with only 33% (0.9 x 10(3)/mm3) recovery for saline control monkeys. In addition, the recovery of neutrophils was enhanced over that of the controls, reaching 140% v 70% on day 30. Another prominent feature of rhGM-CSF-treated monkeys was the accelerated recovery of platelets, reaching near 50% normal levels by day 24 in comparison with 20% of normal levels for controls. The infusion of rhGM-CSF was shown to be an effective regulator of early hematopoietic regeneration, leading to the accelerated recovery of both neutrophils and platelets and then providing a consistent sustained increase of neutrophils even in the absence of rhGM-CSF.     

A comparative study of the iron-clearing properties of desferrithiocin analogues with desferrioxamine B in a Cebus monkey model

A comparative study of the iron-clearing properties of subcutaneously administered desferrioxamine B (DFO) with those of orally administered desferrithiocin sodium salt (1), desmethyl desferrithiocin (2), desazadesmethyl desferrithiocin sodium salt (3), desazadesmethyl desferrithiocin pivaloyloxymethyl ester (4), and desazadesmethyl-5,5- dimethyl desferrithiocin (5) in an iron-loaded Cebus monkey model and a non-iron overloaded bile duct-cannulated rat model is presented. All six drugs, which performed well in rodent studies, demonstrated increased efficiency in the Cebus monkey model. When administered to rodents at a daily dosage of 384 mumol/kg over a period of 10 days, drug 1 demonstrated severe renal toxicity. whereas drugs 3, 4, and 5 exhibited severe gastrointestinal (GI) toxicity. Under the same experimental protocol, drug 2 did not show significant toxic side effects. In addition, to further evaluate the iron-clearing properties of analogue 2, a dose-response study was performed in the primates that showed that iron excretion increased in a dose-dependent fashion.     

Orientation and specificity of fibrin protofibril binding to ADP- stimulated platelets

We have investigated the molecular basis of platelet:fibrin binding by studying interactions between platelets and protofibrils, soluble two- stranded polymers of fibrin, which are intermediates on the fibrin assembly pathway. The specificity of these interactions was examined with transmission electron microscopy (TEM), which clearly showed thin fibers with lengths to 150 nm attached to the cell surface of normal, stimulated platelets. Immunogold electron microscopy using rabbit anti- human fibrinogen as the first stage antibody verified the identity of the surface-bound molecules, and the immunogold distribution paralleled that observed with the fibrin/fibrinogen molecules alone. Contacts between the ends of the fibers and the platelets were frequently observed, but lateral contacts were also evident. Given the diameter at the point of fibrin contact (18.2 +/- 1.3 nm), it is possible that several glycoprotein receptors were involved in binding each protofibril. Morphometric analyses demonstrated that normal platelets stimulated by ADP in the absence of exogenous fibrin(ogen) or in the presence of fibrin protofibrils and antibodies directed against the GPIIb/IIIa complex lacked this molecular layer on the surface. Neither protofibrils nor fibrin fibers adhered to the surface of Glanzmann's thrombasthenic platelets, as demonstrated by TEM and microfluorimetry. Synthetic peptides of sequence RGDS and HHLGGAKQAGDV effectively blocked the binding of protofibrils to the surface of normal, stimulated platelets while synthetic GHRP had no effect. These results provide direct evidence for multiple points of attachment between fibrin protofibrils and the glycoprotein IIb/IIIa complexes present in a functional conformation on the surface of normal, stimulated platelets.     

Regulatory mechanisms in cell-mediated immune responses. VI. Interaction of H-2 and non-H-2 genes in elaboration of mixed leukocyte reaction suppressor factor

Previous studies have shown that alloantigen-activated spleen T cells produce a soluble factor which suppresses mixed lymphocyte reaction proliferative responses, and that the interaction between suppressor and responder cells is controlled by genes of the H-2 complex. However a defect in the expression of suppressor activity was identified in the mouse strain C57BL/6J. Factor prepared from alloactivated B6 spleen cells failed to suppress MLR responses of syngeneic or H-2 compatible responder cells. Unimpaired suppressor factor production by other H-2 (b) strains and failure of suppressor factor production by a B6 congenic strain, B6.C-H-2(d) isolated the defective gene to the non-H-2 portion of the genome. In addition, the defect appeared to be related specifically to inability to produce an active factor, while the capacity to respond to suppressor molecules was unimpaired. The genetic character of the non-H-2 gene action was identified in F1 hybrid studies. Initially F(1) hybrids of the nondefective histoincompatible strains were studied. Suppressor factor from F1 cells suppressed the responses of both parental strains, and parental factors each suppressed the response of F(1) cells. Adsorption of F(1) factor with Con A-activated thymocytes of either parental strain removed suppressor activity specific for that strain, leaving activity against the other parental strain intact. The data support cedominant expression and production of distinct, parental H-2 haplotype-specific suppressor molecules by F(1) suppressor cells. An F(1) hybrid of the defective B6 strain with nondefective BALB/c produced suppressor factor which was also capable of suppressing both parental strains. Production of a suppressive B6-reactive factor by F(1) cells was verified by adsorption studies. Thus it appears that non-H-2 genes of the BALB/c parent acted in a genetically dominant fashion to provide the function required for expression of B6 suppressor molecules. We conclude that multiple genes control the expression of alloactivated suppressor cell activity, with at least one gene mapped to the I-C subregion of the murine major histocompatibility complex and one or more genes mapped to the non-H-2 gene complement.     

Fatal outcome in a patient with autoimmune hemolytic anemia associated with an IgM bithermic anti-ITP

BACKGROUND: Several cold autoantibodies (usually IgG) with IT specificity have been reported previously, as have autoantibodies with joint I and P blood group specificities (IP1, ITP1, iP1, IP). A fatal outcome associated with an IgM cold autoantibody of ITP specificity is reported. CASE REPORT: A 54-year-old man suffered from progressively severe cold autoimmune hemolytic anemia for 9 months. Hemoglobin concentration ranged from 6 to 7 g per dL (60-70 g/L) and reticulocytes from 3 to 5 percent (0.030-0.050). The direct antiglobulin test was weakly positive for IgM and strongly positive for C3d. The serum contained a cold agglutinin that reacted strongest with cord i red cells (RBCs) > adult I RBCs > adult i RBCs, which is consistent with IT specificity. The Donath-Landsteiner test was positive; the reaction was neutralized by globoside. The serum reacted weakly or was negative with RBCs from five group p blood donors, which suggests anti-ITP specificity. Dithiothreitol treatment of the serum abolished the cold agglutinin reactivity, which suggests that the anti-IT was IgM. The patient received > 40 RBC transfusions and failed to respond to oral steroids, oral cytoxan, high-dose pulse intravenous steroids, and plasma exchange at room temperature and at 35 degrees C. He died of sepsis following an unsuccessful trial of chlorambucil. Autopsy revealed unsuspected disseminated non-Hodgkin's lymphoma. CONCLUSION: Serologic studies are consistent with our patient's having a single IgM cold autoantibody with IT and P specificities (anti-ITP) and requiring both specificities on the same RBC to permit maximal antibody expression.