Human granulocyte generation of hydroxyl radical.

Human granulocytes were capable of oxidizing 2-keto-4 thiomethylbutyric acid to ethylene during phagocytosis or membrane perturbation. The reaction required hydrogen peroxide and superoxide and in addition was inhibited by various hydroxyl radical (OH) scavengers. These observations represent direct evidence for the generation of OH by human granulocytes. Further, inhibition of ethylene generation by azide and cyanide suggests that OH generation in granulocytes may be linked to myeloperoxidase.

Activation of human complement by immunoglobulin G antigranulocyte antibody.

The ability of antigranulocyte antibody to fix the third component of complement (C3) to the granulocyte surface was investigated by an assay that quantitates the binding of monoclonal anti-C3 antibody to paraformaldehyde-fixed cells preincubated with Felty's syndrome serum in the presence of human complement. The sera from 7 of 13 patients with Felty's syndrome bound two to three times as much C3 to granulocytes as sera from patients with uncomplicated rheumatoid arthritis. The complement-activating ability of Felty's syndrome serum seemed to reside in the monomeric IgG-containing serum fraction. For those sera capable of activating complement, the amount of C3 fixed to granulocytes was proportional to the amount of granulocyte-binding IgG present in the serum. Thus, complement fixation appeared to be a consequence of the binding of antigranulocyte antibody to the cell surface. These studies suggest a role for complement-mediated injury in the pathophysiology of immune granulocytopenia, as has been demonstrated for immune hemolytic anemia and immune thrombocytopenia.

Argatroban anticoagulant therapy in patients with heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is an immune-mediated syndrome caused by heparin. Complications range from thrombocytopenia to thrombocytopenia with thrombosis. We report a prospective, historical- controlled study evaluating the efficacy and safety of argatroban, a direct thrombin inhibitor, as anticoagulant therapy in patients with HIT or HIT with thrombosis syndrome (HITTS). METHODS AND RESULTS: Patients with HIT (isolated thrombocytopenia, n=160) or HITTS (n=144) received 2 microgram. kg(-1). min(-1) IV argatroban, adjusted to maintain the activated partial thromboplastin time 1.5 to 3.0 times baseline value. Treatment was maintained for 6 days, on average. Clinical outcomes over 37 days were compared with those of 193 historical control subjects with HIT (n=147) or HITTS (n=46). The incidence of the primary efficacy end point, a composite of all-cause death, all-cause amputation, or new thrombosis, was reduced significantly in argatroban-treated patients versus control subjects with HIT (25.6% versus 38.8%, P=0.014). In HITTS, the composite incidence in argatroban-treated patients was 43.8% versus 56.5% in control subjects (P=0.13). Significant between-group differences by time-to-event analysis of the composite end point favored argatroban treatment in HIT (P=0.010) and HITTS (P=0.014). Argatroban therapy, relative to control subjects, also significantly reduced new thrombosis and death caused by thrombosis (P<0.05). Argatroban-treated patients achieved therapeutic activated partial thromboplastin times generally within 4 to 5 hours of starting therapy and, compared with control subjects, had a significantly more rapid rise in platelet counts (P=0.0001). Bleeding events were similar between groups. CONCLUSIONS: Argatroban anticoagulation, compared with historical control subjects, improves clinical outcomes in patients who have heparin-induced thrombocytopenia, without increasing bleeding risk.

Phagocytosis of technetium-99m sulfur colloid by human polymorphonuclear leukocytes.

This study characterizes a new phagocytic assay system utilizing technetium-99m sulfur colloid as the phagocytic particle. Uptake of sulfur colloid by human polymorphonuclear leukocytes is a time and temperature dependent process that requires glucose for optimal uptake. In contrast to many other systems, sulfur colloid phagocytosis appears to be serum and divalent cation independent. An attractive feature of this system is the 10-fold increase in particle uptake with phagocytosis as compared to that at zero time.

Complement-activating antineutrophil antibody in systemic lupus erythematosus.

Serum samples from 18 patients with systemic lupus erythematosus (SLE) were tested for neutrophil C3-fixing ability and neutrophil-binding lgG by the binding of radioiodinated monoclonal anti-C3 antibody and staphylococcal protein A to paraformaldehyde-fixed allogeneic neutrophils sensitized with serum. Serum from patients with SLE resulted in the binding of significantly greater amounts of lgG to neutrophils than normal serum, but this lgG binding did not correlate with the degree of neutropenia. In contrast, serum samples from 10 neutropenic patients with SLE resulted in the binding of significantly greater amounts of C3 to neutrophils when compared with serum samples from eight non-neutropenic patients with SLE. Fixation of C3 to neutrophils by serum from patients with SLE appeared to be due to the binding of complement-activating monomeric antineutrophil lgG autoantibody. A significant negative correlation (r = -0.78) between the neutrophil count and the C3-fixing ability of serum from patients with SLE suggested that antineutrophil antibody-mediated activation of complement may be important in the pathophysiology of neutropenia in SLE.

Hemorrhagic disorder due to an isoniazid-associated acquired factor XIII inhibitor in a patient with Waldenström's macroglobulinemia.

A case is described of a 75-year-old woman with a history of pulmonary tuberculosis and Waldenström's macroglobulinemia who developed an inhibitor of coagulation factor XIII while taking isoniazid. The patient presented with a subcutaneous hematoma of the abdominal wall that extended from the xiphoid process to the symphysis pubis and measured 20 cm in diameter. Results of routine coagulation studies were normal with the exception of an increased solubility of the patient's plasma clot in 5M urea consistent with a deficiency of factor XIII activity. Persistence of the deficiency following a 1:2 dilution of the patient's plasma in normal plasma indicated the presence of an inhibitor. A sample of the patient's plasma was depleted of IgG by streptococcal protein G adsorption. The IgG-depleted plasma did not inhibit factor XIII activity, indicating that the inhibitory activity was not attributable to the underlying IgM paraprotein. The patient's purified IgG, on the other hand, inhibited factor XIII activity and the inhibitory activity could be neutralized by anti-IgG antibody. The patient's IgG also inhibited factor XIII-mediated incorporation of fluorescent monodansylcadaverine into casein. Binding of the patient's IgG to factor XIII concentrate was demonstrated by enzyme-linked immunosorbent assay and the IgG that bound to the factor XIII was demonstrated to be polyclonal. Isoniazid was discontinued after the patient was admitted to the hospital. Cryoprecipitate infusion controlled bleeding and reduced the inhibitor titer by 50%. Treatment with cyclophosphamide and prednisone, followed by extracorporeal immunoadsorption over a staphylococcal protein A column, did not reduce the inhibitor titer further. Plasma exchange therapy reduced the inhibitor titer to undetectable levels but failed to restore factor XIII activity. Infusions of factor XIII concentrate reproducibly restored factor XIII activity and were not associated with an anamnestic rise in the inhibitor titer. This represents the seventh reported case of an acquired inhibitor to factor XIII associated with the ingestion of isoniazid.

Effects of synthetic oligonucleotides on human complement and coagulation.

Oligodeoxynucleotide phosphorothioates (PS-oligos) are being studied as novel therapeutic agents based on their ability to inhibit gene expression. Preclinical studies produced unanticipated complement and coagulation effects in monkeys receiving high-dose PS-oligo. In the present in vitro studies, PS-oligo inhibited normal human blood clotting as well as subsequent assays for prothrombin fragment PF(1+2) and hemolytic complement. PS-oligo treatment of normal donor plasma produced concentration-dependent prolongations of clotting times, with the activated partial thromboplastin time more sensitive than prothrombin time or thrombin clotting time. PS-oligo treatment of normal donor serum similarly reduced hemolytic complement activity in a concentration-dependent manner. Reduced hemolysis correlated with increased levels of complement fragment C4d. The anti-heparin drug protamine sulfate inhibited in vitro effects of PS-oligo in both complement and coagulation assays, suggesting that charged residues in internucleotide linkages of PS-oligo mediated the observed activities. Therefore, oligonucleotides with varying internucleotide linkages, nucleotide sequence, or secondary structure were compared. Both complement and coagulation effects appeared to be independent of nucleotide sequence but were strongly related to the nature of internucleotide linkages. Several of these modified oligonucleotides have been shown previously to retain potent antisense activity and thus may represent viable alternatives for antisense therapeutics.

Novel enzymatic and immunological responses to oligonucleotides.

Oligonucleotide phosphorothioates (PS-oligos) are being studied as antisense agents for viral infection and cancer. In preclinical studies, PS-oligos produced dose-dependent changes in heart rate and blood pressure and significantly reduced serum hemolytic complement, which could be avoided by slowing infusion rates. Here, in vitro PS-oligo treatment of either human, rhesus monkey or guinea pig serum reduced hemolytic complement and further inhibited in vitro coagulation when added to whole blood or citrated plasma. These effects were dependent upon both oligonucleotide dose and structure. Oligonucleotides having identical sequences but containing methylphosphonates (Chimeric), 2'-O-methyl ribonucleosides (Hybrid) or 3' hairpin loop (Self-stabilized) had altered effects on complement and coagulation in vitro.

Cardiac amyloidoma with IgM-kappa gammopathy.

A 73-year-old man presented with dyspnea and atrial flutter associated with an amyloid tumor in the heart. IgM-kappa gammopathy, hypercalcemia, and extensive cardiac and mediastinal invasion suggested a malignant lymphoid or plasma cell process. Although amyloidoma is generally considered to be a benign tumor, the aggressive features of this case mandated chemotherapy because the critical location rendered the tumor inoperable. This case provides noteworthy evidence in support of a possible pathogenic relationship between amyloidoma and plasmacytoma by virtue of dual representative features: localized amyloid infiltrated with plasma cells and the associated gammopathy. Local and systemic malignant features lend additional support to this hypothesis.

Transient protein S deficiency associated with cerebral venous thrombosis in active ulcerative colitis.

Cerebral venous thrombosis is an uncommon complication of ulcerative colitis. We report the case of a 29-yr-old female with a recent diagnosis of ulcerative colitis who suffered stroke secondary to thrombosis of the veins of Galen and straight sinus. A search for a hypercoagulable state revealed a nonfamilial transient protein S deficiency. Possible involvement of C4b-binding protein in this hypercoagulable state is discussed.

Relative risk of adolescent drug abuse: Part I. Socio-demographic and interpersonal variables.

This study was carried out on a sample of 254 male senior high school students in New Delhi. The authors found that variables contributing significantly to drug use were age, heterosexual dating, drug abuse among family members and drug abuse among friends. However, drug use was not found to be significantly associated with family income, father's occupation, family structure and place of residence. Since the sample studied was relatively small, additional confirmation is needed.

Relative risk of adolescent drug abuse: Part II. Intrapsychic variables.

The total sample size for the study of intrapsychic variables was 252 (152 non-drug users and 100 life-time users including 69 current users). Using a multiphasic personality questionnaire, the Eysenck personality inventory and an orientation questionnaire, the authors found that high orientation, extraversion, anxiety and psychopathic deviate scores were associated with a relative risk of drug use. No significant difference is reported between the mean scores of users and non-users on mania, paranoia, depression, schizophrenia, hysteria and neuroticism.

Neutropenia associated with beta-lactam antibiotics.

Two patients who developed neutropenia while receiving beta-lactam antibiotics are presented, and the literature on beta-lactam-induced neutropenia is reviewed. A 55-year-old white woman was admitted to the hospital with a white blood cell (WBC) count of 8700/cu mm (68% neutrophils, 12% neutrophil bands, 0% eosinophils, 14% lymphocytes, 5% monocytes). Moxalactam 2 g i.v. (as the disodium salt) every eight hours was started on hospital day 15 after a postoperative fever failed to respond to a regimen of intravenous tobramycin and clindamycin. The patient again had surgery on hospital day 27, and the moxalactam regimen was continued postoperatively. Approximately one week later the patient's WBC count had dropped to 1900/cu mm (8% neutrophils, 14% neutrophil bands, 6% eosinophils, 54% lymphocytes, 16% monocytes); moxalactam was discontinued, and the WBC count gradually increased after substitution of tobramycin and clindamycin for moxalactam. The second patient was a 75-year-old white man who was being treated with intravenous tobramycin and cefoxitin for a hospital-acquired pneumonia. Ticarcillin 3 g i.v. (as the disodium salt) every four hours was added to this regimen on hospital day 23 after sputum cultures revealed Pseudomonas aeruginosa; four days previously, the WBC count had been 25,100/cu mm (64% neutrophils, 31% neutrophil bands, 1% eosinophils, 3% lymphocytes, 0% monocytes). The WBC count on hospital day 36 was 11,900/cu mm (39% neutrophils, 33% neutrophil bands, 11% eosinophils, 10% lymphocytes, 6% monocytes). Two days later it had dropped to 3700/cu mm (2% neutrophils, 0% neutrophil bands, 53% eosinophils, 24% lymphocytes, 16% monocytes), and ticarcillin was discontinued. The WBC count gradually increased and returned to normal within three days after discontinuing ticarcillin. Neutropenia associated with the administration of beta-lactam antibiotics appears to result from an immunologic reaction characterized by rapid destruction of peripheral neutrophils. Among penicillin analogs, penicillinase-resistant penicillins are involved most frequently, especially in pediatric patients receiving dosages of 150 mg/kg/day or greater. Two case reports have implicated ticarcillin as a cause of neutropenia; moxalactam has not been associated with this adverse effect in previous literature reports. Discontinuation of the suspected agent and initiation of an alternative antibiotic regimen is recommended as initial treatment of this condition since recovery usually occurs within days after discontinuing the offending drug.

Antibodies to granulocyte precursors in selective myeloid hypoplasia and other suspected autoimmune neutropenias: use of HL-60 cells as targets.

Patients with syndromes of autoantibody-mediated hematocytopenias may manifest signs of increased cell destruction and/or decreased cell production, depending on the maturity of the target cell and the effects of antibody binding. The purpose of this study was to use a cultured human cell line of hematopoietic origin for in vitro assays of antibody binding to overcome the relative inaccessibility of natural human marrow progenitor cells. This report describes the detection, using radioiodinated staphylococcal protein A (SPA), of antibodies binding to a human promyelocytic cell line (HL-60) in sera from three patients with chronic idiopathic granulocytic hypoplasia ("pure white cell aplasia," PWCA) and 22 patients with other syndromes of suspected immune neutropenia. Bone marrow from patients with increased IgG binding to HL-60 cells showed less than 15% granulocytic lineage cellularity in 11 of 17 cases. In vitro differentiation of HL-60 cells by retinoic acid resulted in increased IgG binding for sera that had shown increased IgG binding to mature granulocytes but not undifferentiated HL-60 cells; in contrast, for sera with antibodies to untreated HL-60 cells and for normal serum, in vitro differentiation had little effect on IgG binding. Antibodies eluted from mature granulocytes were similar to the parent serum regarding the ratio of IgG binding to mature cells v HL-60 cells. No sera from 19 patients with febrile transfusion reactions showed increased IgG binding to HL-60 cells in the absence of increased IgG binding to mature granulocytes, although two sera had antibodies to both cell types. The use of HL-60 cells as targets may permit measurement of serum antibodies associated with granulocytic hypoplasia. In combination with assays to detect antibody binding to mature granulocytes, these techniques may discriminate among autoantibody specificities for antigens that are gained, conserved, or lost during myeloid maturation.

Effect of RBCs on the activation of human complement by heparin-protamine complexes.

Complement activation on red cells by heparin-protamine complexes was studied by using whole human serum. C3 bound to red cells was measured by radiolabeled monoclonal antibody to C3, and fluid-phase C5a was determined by radioimmunoassay. Heparin and protamine in clinically relevant concentrations caused the binding of C3 to red cell membranes, and the measurement of C3 binding provided a sensitive indicator of complement activation produced by these complexes. Complement activation by these reagents occurred at concentration ratios of protamine and heparin at which protamine neutralized the anticoagulant effect of heparin. Heparin-protamine complexes appeared to bind to red cells and produce complement activation by the classic pathway. C5a generation with heparin-protamine complexes in serum was greatly enhanced in the presence of red cells and increased with increasing red cell concentration. This enhancement of complement activation in the presence of red cells was also seen as measured by depletion of available C3 hemolytic complement units in the fluid phase. Thus red cells seem to play an important role in activation of complement by heparin-protamine complexes.

T-lymphocytes escape membrane defect in paroxysmal nocturnal haemoglobinuria.

Erythrocytes, granulocytes and platelets from patients with paroxysmal nocturnal haemoglobinuria (PNH) are abnormally sensitive to lysis by complement. We studied T-lymphocytes from PNH patients for abnormal complement lysis sensitivity. T-lymphocytes free of other contaminating blood cells were prepared by sedimentation, nylon wool filtration, and density gradient centrifugation. The lymphocytes were then labelled with 51Cr and lysis induced by antithymocyte globulin and rabbit complement. PNH lymphocytes were no more susceptible to complement-mediated lysis than lymphocytes from normal individuals. The unusual sensitivity of PNH erythrocytes could still be demonstrated when rabbit serum was a source of complement so the lack of any difference in the sensitivity of normal and PNH lymphocytes was probably not attributable to the inability of rabbit serum to elicit the membrane defect. PNH erythrocytes and granulocytes also acquire more membrane-bound C3 when human complement is activated. Therefore we also searched for increased membrane C3 binding on PNH lymphocytes using anti-I antibody and human serum as a complement source. C3 binding was measured using 125I labelled monoclonal mouse anti-human C3. While we verified increased membrane C3 binding on PNH granulocytes during complement activation we were unable to show similar differences between PNH and normal T-lymphocytes. Thus PNH T-lymphocytes do not share the membrane abnormalities of PNH-erythrocytes and granulocytes.

Type IIB mutation His-505-->Asp implicates a new segment in the control of von Willebrand factor binding to platelet glycoprotein Ib.

Type IIB von Willebrand disease is characterized by increased affinity of mutant von Willebrand factor (vWF) for platelet glycoprotein Ib. Eight different missense mutations that cause this phenotype have been reported within the disulfide loop defined by Cys-509 and Cys-695 of the mature vWF subunit; this disulfide loop is required for normal binding of vWF to platelet glycoprotein Ib. A new mutation was identified in a patient with type IIB von Willebrand disease (vWD) characterized by a lifelong bleeding disorder, mild thrombocytopenia, normal levels of factor VIII, vWF antigen, and ristocetin cofactor activity but increased ristocetin-induced platelet agglutination at low concentrations of ristocetin. Exon 28 of the patient's vWF gene was amplified, cloned, and sequenced. At nucleotide 3802 (numbering the cDNA from translation initiation), a C to G transversion was identified, which changes the encoded amino acid sequence from His-505 to Asp. The corresponding mutant recombinant vWF was expressed in transiently transfected COS cells. Relative to wild type vWF, the mutant vWF exhibited markedly increased binding to platelets at low concentrations of ristocetin, confirming the association between the His-505-->Asp substitution and the type IIB vWD phenotype. The His-505-->Asp mutation lies outside the disulfide loop affected by other type IIB vWD mutations and implicates a new segment of vWF in the regulation of platelet glycoprotein Ib binding.