Acute leukemia associated with the t(4;11) chromosome rearrangement: ultrastructural and immunologic characteristics

The acute leukemia associated with the t(4;11) chromosome rearrangement is characterized by relatively consistent clinical features: occurrence primarily in young individuals, hyperleukocytosis, and poor response to therapy. This study describes the morphological, ultrastructural, and immunologic characteristics of the leukemic cells from ten patients with this type of leukemia. The morphological features of the leukemic blasts vary from lymphoid-appearing to monocytic. Ultrastructurally and cytochemically, some of the lymphoid-appearing blasts possess features of myeloid origin. The immunologic phenotype is characteristically E- SIg- CALLA- BA-1- BA-2+ HLA-DR+ and TdT+. These findings suggest that the t(4;11)-associated acute leukemia represents a proliferation of an early myeloid progenitor cell.     

Protein phosphorylation in neutrophils from patients with p67-phox- deficient chronic granulomatous disease

Neutrophils are known to contain a major 67-kD protein that undergoes enhanced phosphorylation and translocation to the membrane during cell stimulation. Recent studies have assumed that this 67-kD phosphoprotein is the 67-kD subunit of the phagocyte oxidase (p67-phox). We compare here the protein phosphorylation patterns in lysates of normal neutrophils and neutrophils from patients with chronic granulomatous disease (CGD) that are completely deficient in p67-phox. The phosphoproteins were labeled by incubation of the cells with radioactive inorganic phosphate (32Pi) or by the addition of [gamma- 32P]ATP to electropermeabilized neutrophils. With either method, stimulation of the normal or CGD cells always resulted in an enhanced incorporation of 32p into two proteins in the 67-kD area. The extent of phosphorylation of these two proteins was very similar in the normal and CGD cells when permeabilized neutrophils loaded with [gamma - 32P]ATP were compared. Moreover, no overall differences in the protein phosphorylation patterns were observed between the normal and CGD cells. Our data indicate that the major 67-kD phosphoproteins observed in stimulated neutrophils are clearly different from p67-phox.     

Effect of surfaces on fluid-phase prekallikrein activation

The activation of prekallikrein by factor XII fragments (XIIf), during incubation in plastic tubes was previously noted to be increased by high molecular weight (HMW) kininogen as well as other plasma proteins. In this report, we investigated the mechanism responsible for this increase. Although we confirmed that HMW kininogen, bovine serum albumin, fibrinogen, cold insoluble globulin, and mixed phospholipids apparently increased prekallikrein activation, we found that the product of prekallikrein activation (kallikrein) lost substantial activity in less than 0.5 min after exposure to a variety of fresh surfaces. This loss was partially prevented by the presence of various proteins and phospholipids. Similar protection against inactivation of XIIf, the enzyme in this reaction, was also found. In contrast, no loss of the substrate, prekallikrein, was observed during incubation. The loss of kallikrein activity was found to be proportional to the surface area of the incubation vessel as well as the concentration of kallikrein. Further loss of kallikrein activity could also be prevented by pretreating the vessel with kallikrein. We therefore conclude that various substances apparently affect prekallikrein activation in a purified system by preventing the enzyme and product in the reaction mixture from losing activity due to adsorption to a surface.     

Inhibition of factor XII in septic baboons attenuates the activation of complement and fibrinolytic systems and reduces the release of interleukin-6 and neutrophil elastase

In previous studies, we have shown that administration of monoclonal antibody (MoAb) C6B7 against human factor XII to baboons challenged with a lethal dose of Escherichia coli abrogates activation of the contact system and modulates secondary hypotension. To evaluate the contribution of activated contact proteases to the appearance of other inflammatory mediators in this experimental model of sepsis, we studied the effect of administration of MoAb C6B7 on activation of complement and fibrinolytic cascades, stimulation of neutrophil degranulation, and release of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Activation of the complement system, as reflected by circulating C3b/c and C4b/c levels, was significantly reduced in five animals that had received MoAb C6B7 before a lethal dose of E coli as compared with five control animals that had been given a lethal challenge only. Inhibition of contact activation also modulated the fibrinolytic response, since the release of tissue-type plasminogen activator (t-PA) and the appearance of plasmin-alpha2-antiplasmin (PAP) complexes into the circulation was significantly attenuated upon pretreatment with anti-factor XII MoAb. In contrast, plasma levels of plasminogen activator inhibitor (PAI) were modestly enhanced in the treatment group. Degranulation of neutrophils, as assessed by circulating elastase-alpha1-protease inhibitor complexes, and release of IL-6 but not of TNF-alpha was decreased in anti-factor XII-treated animals. Observed differences in the inflammatory response between treatment and control groups were not likely due to different challenges, since the number of E coli that had been infused, as well as circulating levels of endotoxin after the challenge, were similar for both groups. These data suggest that activation of the contact system modulates directly or indirectly various mediator systems involved in the inflammatory response during severe sepsis in nonhuman primates.     

High molecular weight kininogen: localization in the unstimulated and activated platelet and activation by a platelet calpain(s)

High mol wt kininogen (HMWK), the major cofactor-substrate of the contact phase of coagulation, is contained within and secreted by platelets. Studies have been performed to localize platelet HMWK in both the unstimulated and activated platelet and to ascertain the effect of platelet enzymes on HMWK itself. On platelet subcellular fractionation, platelet HMWK was localized to alpha-granules, and platelets from a patient with a deficiency of these granules (gray platelet syndrome) had 28% normal platelet HMWK. Platelet HMWK, in addition to being secreted from the platelet, was also localized to the surface of the platelet when activated. Using a competitive enzyme- linked immunosorbent assay for HMWK as an indirect antibody consumption assay, the external membrane of thrombin-activated platelets as well as the releasate from these stimulated platelets had 17 ng HMWK antigen/10(8) platelets available, whereas unstimulated platelets and their supernatant had only 4.9 and 4.2 ng HMWK/10(8) platelets present, respectively. The anti-HMWK antibody consumption by activated normal platelets was specific for membrane-expressed platelet HMWK, since activated platelets from a patient with total kininogen deficiency did not adsorb the anti-HMWK antibody. Enzymes in the cytosolic fraction of platelets cleaved 125I-HMWK (mol wt 120,000) into a mol wt 100,000 polypeptide as well as smaller products at mol wt 74,000, mol wt 62,000, mol wt 47,000, and a few components below mol wt 45,000. No cleavage products were observed when DFP and leupeptin were present. The cleavage of HMWK was specifically prevented by inhibitors of calcium-activated cysteine proteases (leupeptin, N-ethylmaleimide, iodoacetamide, and EDTA) but not by inhibitors of serine proteases (DFP, benzamidine, soybean trypsin inhibitor, or aprotinin). Platelet cytosol increased the coagulant activity of exogenous purified HMWK with maximum HMWK coagulant activity (35-fold) occurring within ten minutes of exposure to platelet cytosol. Treatment of platelet cytosol with leupeptin prevented the increase in the coagulant activity of exogenous HMWK. These studies indicate that activated platelets express platelet HMWK on their external membrane and platelet enzymes can cleave and increase the coagulant activity of exogenous HMWK.     

Minactivin expression in human monocyte and macrophage populations

Adherent monolayer cultures of human blood monocytes, peritoneal macrophages, bone marrow macrophages, and colonic mucosa macrophages were examined for their ability to produce and secrete minactivin, a specific inactivator of urokinase-type plasminogen activator. All except colonic mucosa macrophages produced and secreted appreciable amounts of minactivin, but only blood monocytes were stimulated by muramyl dipeptide (adjuvant peptide) to increase production. The minactivin from each of these populations could be shown to preferentially inhibit urokinase-type plasminogen activator and not trypsin, plasmin, or "tissue"-type plasminogen activator (HPA66). A plasminogen-activating enzyme present in monocyte cultures appeared unaffected by the presence of minactivin and could be shown to be regulated independently by dexamethasone.     

Development and characterization of monoclonal antiplatelet autoantibodies from autoimmune thrombocytopenic purpura-prone (NZW x BXSB)F1 mice

Male (NZW x BXSB)F1 (W/BF1) mice develop systemic autoimmunity involving autoantibodies, progressive thrombocytopenia, lupus nephritis, and degenerative coronary vascular disease with myocardial infarction. Platelet-associated IgG (PAIgG) on the platelet surface mediates platelet destruction by the reticuloendothelial system in the autoimmune thrombocytopenic purpura (ATP) of W/BF1 mice. Because the epitopes targeted in ATP by PAIgG have not been identifiable using serum from thrombocytopenic W/BF1 mice, we developed seven hybridomas secreting antiplatelet monoclonal antibodies (MoAbs) using splenocytes of thrombocytopenic W/BF1 mice. Epitopes recognized by three MoAbs were similar to those recognized by PAIgG, because eluted IgG from platelets of thrombocytopenic W/BF1 mice inhibited platelet binding by MoAbs in competitive micro-enzyme-linked immunosorbent assay. Hybridoma cells or purified Ig from the ascites of two clones (2A12 and 6A6), when injected into nude mice produced acute thrombocytopenia, elevated the levels of PAIgG, purpura, and megakaryocytosis. MoAbs of two clones also reacted with single-stranded DNA or double-stranded DNA, and one of these clones (4-13) bound to cardiolipin (CL) but was nonpathogenic in nude mice, suggesting that anti-CL and antiplatelet autoantibodies can be distinct. On immunoblotting analysis, antiplatelet MoAbs frequently bound a 100-Kd platelet protein. These MoAbs contribute to an understanding of the etiopathogenesis of ATP and the several antigens and autoantibodies involved.     

Granulocyte colony-stimulating factor versus placebo in addition to penicillin G in a randomized blinded study of gram-negative pneumonia sepsis: analysis of survival and multisystem organ failure

Sepsis is a common cause of morbidity and mortality. Neutrophils are the major defense against bacterial invasion, and granulocyte colony- stimulating factor (G-CSF) augments both neutrophil number and function. In our study, 160 rabbits were inoculated transtracheally with 0.5 mL of a solution containing 10(4) colony forming units per milliliter of Pasteurella multocida. Twenty-four hours later, chest x- rays and quantitative blood cultures demonstrated pneumonia and bacteremia. Therapy was then begun with penicillin G and either recombinant human G-CSF (rG-CSF; 5 to 8 micrograms/kg subcutaneously) or placebo every day for 5 days. Arterial blood gases and 23 other parameters of organ function were performed before inoculation and serially thereafter. All rabbits underwent histologic examination of organs at the time of septic death or when sacrificed on day 6. A total of 149 rabbits survived long enough to initiate therapy. A significant increase in leukocytes by day 4 was found in the rG-CSF-treated group. There was a trend towards improved survival in the rG-CSF group (77% v 67%; P = .13, n = 149). Analysis of pretreatment variables revealed sepsis-induced leukopenia (< or = 2,800/microL) as the only predictor of significantly improved survival with rG-CSF treatment (57% v 39%; P = .04, n = 73). The majority of the survival benefit occurred within the first 24 hours of treatment. This was before the time that a significant difference in mean white blood cell (WBC) count was observed between the study groups, making intravascular leukocytosis an unlikely explanation for the survival advantage in the rG-CSF group. No significant difference in laboratory variables reflecting organ function was demonstrated between the groups. Histologic grading of inflammation (0, normal, to 6, necrosis) in seven organs revealed that the surviving rabbits had mild but statistically significant increased inflammation in the liver, spleen, and noninoculated lung in the rG-CSF versus placebo groups (liver: 2.6 v 1.5, P < or = .0001; spleen: 3.2 v 2.3, P < or = .0001; and noninoculated lung: 2.9 v 2.5, P = .04). Administration of rG-CSF, in addition to penicillin G, in immune competent rabbits with gram-negative sepsis complicated by leukopenia significantly improved survival over antibiotics alone. The administration of rG-CSF in early sepsis for a short therapeutic duration was not associated with any clinically evident toxicity. Clinical trials using rG-CSF in septic patients with leukopenia are indicated.     

Factors influencing the acceleration of human factor XIa inactivation by antithrombin III

Controversy exists in the literature concerning the potentiating effect of heparin on the inactivation rate of factor XIa by antithrombin III (AT III) in both purified systems and in plasma. We have analyzed the factors that could influence this reaction and found that ionic strength of the medium, as well as the type and concentration of the heparin preparations accounted for the major discrepancies in the literature. At I = 0.43 N, a preparation of bovine lung heparin at 1 U/mL did not augment the inactivation rate of factor XIa by inhibitors in plasma or by purified AT III. However, when ionic strength was decreased, a progressive increase in the potentiating effect was observed, reaching 6.5-fold at I = 0.15 N. At saturating concentrations of heparin, which results in the formation of 100% AT III-heparin complex, (greater than ten-fold molar excess over AT III) in purified systems, all heparin preparations (porcine, bovine, low molecular weight [LMW], and high affinity) yielded an approximately 30-fold augmentation of the factor XIa inactivation rate. However, when heparin was less than saturating, we observed that various heparin preparations affected the AT III-induced inactivation of factor XIa to different degrees even though they exhibited the same inhibitory activity (1 U/mL) against thrombin. This variation resulted from differences in the number of AT III binding sites in each heparin preparation, despite a similar Kd for each. Addition of high molecular weight kininogen (HK) to AT III-heparin complexes did not enhance their ability to inhibit factor XIa, and high concentrations of HK decreased the inactivation rate. A high therapeutic dose of heparin only permits the formation of 2.5% to 16.5% of the AT III-heparin complexes that can be achieved at saturation. We observed that 1 U/mL heparin (bovine lung heparin) (high therapeutic concentration) in virtually undiluted plasma only accelerated the inactivation rate of factor XIa (in the absence of other active enzymes) less than two-fold. These new observations further support our previous conclusion that therapeutic levels of heparin have little to no influence on the inactivation rate of factor XIa in plasma.