The presence and release of alpha 2-antiplasmin from human platelets

An antigen immunochemically indistinguishable from plasma alpha 2- antiplasmin, the primary plasmin inhibitor, was detected in human platelets. By radioimmunoassay, 33-114 ng alpha 2-antiplasmin antigen was quantitated in the detergent-soluble extract of 10(9) washed human platelets from 10 normal donors with a mean level of 62 +/- 24 ng/10(9) platelets. Plasma alpha 2-antiplasmin, either in the platelet suspending medium or on the surface of the platelets, could account for less than 8% of the antigen present in the platelet extracts. When stimulated with thrombin, the platelets released alpha 2-antiplasmin antigen without cell lysis, and greater than 85% of the alpha 2- antiplasmin antigen was released at a high thrombin dose. At a lower dose of thrombin, alpha 2-antiplasmin and platelet factor 4 were partially released without concomitant secretion of serotonin. No alpha 2-antiplasmin antigen was detected in extracts or red blood cells, polymorphonuclear leukocytes, and adherent and nonadherent mononuclear cells. Thus, the platelet is the only peripheral blood cell containing significant amounts of alpha 2-antiplasmin.     

Lumbar myelography with iohexol and metrizamide: a comparative multicenter prospective study

Diagnostic quality of radiographs and adverse reactions associated with the use of metrizamide and iohexol as contrast agents in lumbar myelography were compared in a prospective randomized double blind study in 350 patients at seven centers. The contrast media were administered in comparable volumes at a concentration of 180 mg I per ml. Overall quality of radiographic visualization was graded good or excellent in 95% of 175 metrizamide studies and in 98% of 175 iohexol studies. Ninety-three patients examined using metrizamide (53%) and 130 patients examined using iohexol (74%) experienced no discomfort during or after myelography. Postmyelographic headache was associated with 38% of metrizamide examinations and 21% of iohexol examinations. Nausea and vomiting were also more common with metrizamide. Five patients examined using metrizamide (3%) experienced transient confusion and disorientation following lumbar myelography. No such reactions were observed following iohexol myelography.     

Max(a), a new low-frequency platelet-specific antigen localized on glycoprotein IIb, is associated with neonatal alloimmune thrombocytopenia

We have identified a new platelet-specific alloantigen, Max(a), responsible for a typical case of neonatal alloimmune thrombocytopenic purpura. The maternal serum reacted strongly with paternal platelets in the platelet immunofluorescence test, whereas platelet alloantigen typing showed that no known human platelet antigen (HPA)-system was involved. In the monoclonal antibody (MoAb)-specific immobilization of platelet antigens (MAIPA) assay, the new antigen was located on the platelet membrane glycoprotein (GP) IIb-IIIa complex, but immunoprecipitation and immunoblot experiments to further localize the antigen failed. However, in the MAIPA assay, the binding of the anti- Max(a) antibodies from the maternal serum was blocked by two anti-GPIIb MoAbs. Thus, the antigen appeared to be located on GPIIb. Analysis of the family lead to the identification of six additional Max(a+) individuals. Three of these six individuals and the father were tested in the platelet aggregation test and were found to be normal. In the analysis of normal donors, three of 500 were typed positive for the new platelet-specific antigen, indicating a phenotype frequency of 0.6% in the normal population. Platelet RNA was isolated from the newborn's Max(a)+ father and from a healthy donor phenotyped as Max(a-), reverse- transcribed, and the entire GPIIb coding region was amplified by polymerase chain reaction. Subsequent nucleotide sequence analysis showed a single G-->A substitution at position 2,603, predicting a valine-->methionine amino acid substitution at position 837 of the mature glycoprotein. This mutation abolished a BsiYI restriction site at the cDNA level and a BstNI restriction site at genomic DNA level, respectively. The genetic association between the new antigen and this point mutation was confirmed by allele-specific restriction analysis on cDNA and on genomic DNA, as well as by allele-specific primer amplification on genomic DNA. The new mutation is 19 bp upstream of the mutation underlying the HPA-3 system. Therefore, we also evaluated the association between Mas and the HPA-3 polymorphism. So far, all Max(a+) individuals were also found to be HPA-3b, whereas 50 HPA-3a individuals were all Max(a-). This may indicate that Max(a) is a variant of the HPA- 3 allele.     

Metabolic effects of antisickling amounts of nitrogen and nor-nitrogen mustard on rabbit and human erythrocytes

Nitrogen mustard (NH2) and Nor-nitrogen mustard (Nor-HN2) both inhibit the polymerization of deoxyhemoglobin S in solution and in intact erythrocytes. Metabolic studies were undertaken to determine the feasability of an extracorporeal treatment with these or related agents. Glucose utilization, hexose monophosphate shunt activity, methemoglobin reduction, and incubation with acetylphenylhydrazine for Heinz body formation were performed, as well as specific assays for hexokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase, glutathione reductase, ATP, reduced glutathione (GSH), and survival of autologous mustard-treated cells in rabbits. HN2 was found to enter red cells rapidly and bind to intracellular contents. Metabolic studies revealed no significant inhibition or alteration of function by Nor-HN2 at 10 mg/ml of whole blood. Rabbit red cell survival was also normal. HN2, however, inhibited glutathione reductase and blocked the free sulfhydryl group of GSH by forming serveral addition products of alkylated GSH. Heinz body test with acetylphenylhydrazine became positive in HN2-treated cells, and rabbit red cell survival was shortened considerably in the concentration range used to inhibit sickling. Ascorbic acid stimulation of the hexose shunt pathway was inhibited by HN2, but methylene blue stimulation remained unaffected. 14-C-HN2 remains bound to red cells in vivo, and the disappearance of radioactivity is similar to that found with 14-C-DFP (disopropylfluorophosphate). Oxygen affinity of both HN2 and Nor-HN2 treated human red cells remains virtually the same as that found in control samples. It is concluded that Nor-HN2 may be a suitable agent for an extracorporeal therapy, and that each mustard needs to be evaluated individually for its antisickling effects and its suitability for extracorporeal use.     

Laparostomy management using the ABThera™ open abdomen negative pressure therapy system in a grade IV open abdomen secondary to acute pancreatitis

Wound control in laparostomy for the treatment of intra‐abdominal hypertension remains challenging and numerous techniques have been described. We report the first UK experience with a new commercially available device specifically designed to facilitate management of the open abdomen. A 44‐year‐old gentleman presented with a 3‐day history of constant severe epigastric pain and associated vomiting. Amylase was markedly elevated and he was admitted for supportive management of pancreatitis, with subsequent transfer to intensive care due to severe systemic inflammatory syndrome. The patient decompensated, developing intra‐abdominal hypertension with renal and respiratory failure. This was successfully managed by performing a laparostomy and using an ABThera? Open Abdomen Negative Pressure Therapy System (KCI, San Antonio, TX). We describe its use to facilitate wound control, including enteroatmospheric fistula, allowing granulation and eventual restoration of gastrointestinal continuity 383‐days after admission. We found the ABThera? System proved to be a useful treatment adjunct, protecting intra‐abdominal contents while removing large volumes of exudate and infected material from within the abdominal cavity. Complex cases such as this remain infrequent and this article provides a summary of our experience, including a review of indications for laparostomy and the underlying basic science in this difficult area.     

Persistence of human multilineage, self-renewing lymphohematopoietic stem cells in chimeric sheep

We have previously reported the ability of uncharacterized human bone marrow (BM) cells to engraft into preimmune fetal sheep, thereby creating sheep-human chimera suitable for in vivo examination of the properties of human hematopoietic stem cells (HSC). Adult human bone marrow CD34+ HLA-DR- cells have been extensively characterized in vitro and have been demonstrated to contain a number of primitive hematopoietic progenitor cells (PHPC). However, the capacity of such highly purified populations of human marrow CD34+ HLA-DR- cells to undergo in vivo self-renewal and multipotential lymphohematopoietic differentiation has not been previously demonstrated. To achieve that, human CD34+ HLA-DR- cells were transplanted in utero into immunoincompetent fetal sheep to investigate the BM-populating potential of these cells. Long-term chimerism, sustained human hematopoiesis, and expression of human cells belonging to all human blood cell lineages were demonstrated in two animals for more than 7 months' posttransplantation. Chimeric BM contained erythroid, granulocytic/monocytic, and megakaryocytic hematopoietic progenitor cells, as well as the primitive high proliferative potential colony- forming cell (HPP-CFC). Under a variety of in vitro experimental conditions, chimeric BM cells gave rise to human T cells expressing T- lymphocyte-specific markers, human natural killer (NK) cells, and human IgG-producing B cells. In vivo expansion and possibly self-renewal of transplanted PHPC was confirmed by the detection in chimeric BM 130 days' posttransplantation of CD34+ HLA-DR- cells, the phenotype of human cells constituting the stem-cell graft. These studies demonstrate not only the BM-populating capacity, multipotential differentiation, and most likely self-renewal capabilities of human CD34+ HLA-DR- cells, but also that this BM population contains human HSC. Furthermore, it appears that this animal model of xenogeneic stem-cell transplantation is extremely useful for in vivo examination of human hematopoiesis and the behavioral and functional characteristics of human HSC.     

Both tumor necrosis factor receptors, TNFR60 and TNFR80, are involved in signaling endothelial tissue factor expression by juxtacrine tumor necrosis factor alpha

We have investigated the role of the two distinct tumor necrosis factor (TNF) receptors (TNFR60 and TNFR80) in endothelial cell activation employing an in vitro model of tumor necrosis factor alpha (TNF-alpha)- dependent tissue factor production of human umbilical vein endothelial cells (HUVECs). In this model, tissue factor is produced either on addition of exogeneous TNF-alpha, or by induction of endogenous TNF- alpha via adhesion molecule-linked signal pathways. Under both conditions, tissue factor expression could be partially blocked by antagonistic antibodies against either TNFR60 or TNFR80 and was fully inhibited by simultaneous application of both antibodies. Selective inhibitors of either TNFR60 or TNFR80-induced signal pathways inhibited tissue factor expression, and selective triggering of either of the two TNF receptors by agonistic antibodies induced this response in HUVECs. Furthermore, a coculture system of HUVECs and Chinese hamster ovary transfectants expressing a noncleavable, exclusively membrane-bound form of TNF-alpha resulted in a potent activation of HUVECs with synergistic action of both TNF receptors. Together, these data underline the importance of juxtacrine pathways in endothelial cell activation of procoagulant functions and show that membrane TNF-alpha and both TNFR types play a critical role.