The abnormal erythrocytes in paroxysmal nocturnal hemoglobinuria, both PNH II (the moderately abnormal cells) and PNH III (the markedly abnormal cells), lack both acetylcholinesterase (AChE) activity ...and decay-accelerating factor (DAF) activity. Both of these activities are found on glycoprotein molecules with a molecular weight of about 70 Kd. To demonstrate that these two activities are in fact on different proteins, we have shown that binding to normal red cells of antibody to DAF does not inhibit the subsequent binding of monoclonal antibody to AChE nor AChE activity. Inhibition of DAF activity by polyclonal antibody increases the susceptibility of normal erythrocytes to lysis by complement but inhibition of AChE activity by antibody does not. The rate of decay of the C3 convertase complex of the classical pathway of complement activation was inhibited by DAF added in the fluid phase but not by AChE. When DAF was exhaustively immunoprecipitated from a solution of the erythrocyte membrane proteins, AChE remained and vice versa. These studies indicate that acetylcholinesterase and decay-accelerating factor are two different proteins, both of which are lacking on PNH II and PNH III erythrocytes.
Heparin-associated thrombocytopenia and thrombosis (HATT) is a potentially fatal complication of heparin therapy which is characterized by a progressive fall in the platelet count associated with ...arterial or venous thrombosis. The etiology is consistent with the development an antibody which, in the presence of heparin, induces intravascular platelet aggregation. Biochemical analysis has demonstrated that the platelet membrane glycoprotein (GP) Ib binds heparin. Recent studies have shown that polyclonal antisera or monoclonal antibodies to GP Ib can inhibit platelet aggregation induced by HATT plasma in the presence of heparin implicating GP Ib as the site of heparin-antibody interaction. The Bernard-Soulier syndrome (BSS) is an inherited bleeding disorder in which the platelets fail to adhere to exposed subendothelium due to a deficiency of GP Ib. We have used the platelets from a patient with documented BSS to further investigate the role of GP Ib in the heparin- dependent platelet aggregation induced by the plasma of three patients with HATT. We have shown that platelet aggregation by HATT plasma in the presence of heparin occurred as readily with BSS platelets as with normal donor platelets. These results indicate that glycoprotein Ib cannot be the only site of platelet-heparin-antibody interactions.
A 2-week toxicity and toxicokinetic study of a 15-mer phosphorothioate oligonucleotide, INX-3280, against the c-myc oncogene was performed in cynomolgus monkeys. As this oligonucleotide readily ...adopts an aggregate structure, a quadruplex, which may be associated with adverse physiologic effects, this study was performed using INX-3280 that had been converted to its monomeric form. Animals received intravenous (i.v.) infusions of monomeric INX-3280 three times per week for 2 weeks at doses of 3 or 15 mg/kg per administration. The monkeys were examined for clinical signs: changes in hematology, serum chemistry, coagulation, and urinalysis parameters; complement activation; macroscopic findings at necropsy; and histopathologic alterations. In addition, the toxicokinetics of INX-3280 were evaluated, using a validated HPLC assay, after the first and last (sixth) doses. No treatment-related clinical signs of any adverse effects were observed, and there were no test article-related changes in hematology, serum chemistry, or complement activation parameters. The only alteration in clinical pathology parameters was a minor (30%) prolongation of the activated partial thromboplastin time (aPTT), reflecting slight inhibition of the intrinsic coagulation pathway, which was less than that reported with other oligonucleotides given at similar doses. Treatment-related histopathologic alterations consisted of characteristic accumulation of basophilic material in the cytoplasm of tubular epithelial cells in the kidney, resident macrophages in the lymph nodes, and Kupffer cells in the liver. These changes were graded as minimal in all cases. The basophilic material is believed to reflect accumulation of the oligonucleotide or metabolites or both. The pharmacokinetic parameters of INX-3280 were identical on the first and sixth administrations and were similar to those reported for other phosphorothioate oligonucleotides. Maximum concentration (Cmax) values for INX-3280 (101-119 microg/ml) were in excess of the threshold plasma concentrations reported to trigger complement activation by phosphorothioate oligonucleotides. It is concluded that the safety profile of monomeric INX-3280 in cynomolgus monkeys is quite favorable relative to the known effects of other phosphorothioate oligonucleotides, particularly with respect to the blood level-related toxicities of this class of compounds, including complement activation and inhibition of coagulation. This study found no toxicities that were expected to be clinically significant.
A typical perinuclear antineutrophil cytoplasmic antibodies (p-ANCA) have been detected in most patients with ulcerative colitis and primary sclerosing cholangitis. Persistent atypical p-ANCA have ...been observed in ulcerative colitis patients with a prior proctocolectomy, especially with pouchitis, suggesting that this serological marker might be predictive of subsequent development of chronic or refractory pouchitis. This study prospectively evaluated this serological marker in 24 consecutive patients with inflammatory bowel disease and prior colectomies (12 with a clinical diagnosis of ulcerative colitis and 12 with a clinical diagnosis of Crohn's disease involving the colon). Of these, 14 were positive, including 11 with extensive ulcerative colitis and three with Crohn's disease. Although two of three ulcerative colitis patients with pouchitis were positive, eight of eight ulcerative colitis patients having a pelvic pouch with no pouchitis were also positive, as was a patient who elected to have an end-ileostomy (Brooke's ileostomy). Two patients had abnormal liver chemistry tests. Both had end-stage primary sclerosing cholangitis treated with liver transplantation and were positive for this serological marker. Although atypical p-ANCA may be a marker of persistent inflammation in pelvic pouch patients, a positive test result should not be used for prognosis or as a decision-making parameter for pelvic pouch procedures.
Acquired antibodies to factor VIII:C in nonhemophiliac patients are uncommon in adulthood and exceedingly rare in childhood. We report a girl, 9 years of age, with no personal or familial bleeding ...history who presented with hematuria and bruising 2 weeks after an upper respiratory infection. The activated partial thromboplastin time was 71.9 s (normal, 25-40 s) and did not correct by mixing 1:1 with normal plasma, suggesting the presence of an inhibitor. Factor VIII:C levels were detectable at 0.03 U/ml, but inhibition experiments demonstrated the presence of an inhibitor with an activity of 24 Bethesda U/ml. This inhibitor was localized to the immunoglobulin (IgG) fraction of the patient's plasma. Incubation of the patient's IgG with normal pooled plasma resulted in a 66% decrease in factor VIII:C activity. Unlike the antibodies found in most hemophilia patients, the autoantibody produced by this patient demonstrated type II kinetics and did not inhibit all factor VIII:C activity even at very high concentrations. In addition, the rate of factor VIII:C inactivation by this autoantibody was much slower than that seen with type I inhibitors. The treatment of the patient with prednisone, 2.5 mg/kg/day, resulted in the rapid disappearance of detectable inhibitor and a rise in factor VIII:C levels to 0.70 U/ml. Normal factor VIII:C levels persisted after the discontinuation of steroids. This case is most unusual in that it occurred in a child without any evidence of an underlying autoimmune disorder, and unlike classical hemophiliac factor VIII:C inhibitors, there was a rapid response to steroids.
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