Laurence A. Boxer

Seeding arterial prostheses with vascular endothelium. The nature of the lining.

Arterial prostheses seeded with autogenous vascular endothelium demonstrate a well-organized, cellular, inner lining. To determine the nature of the lining cells, six animals underwent replacement of the infrarenal aorta with Dacron® prostheses. During the preparation of three such grafts, endothelium was scraped from the saphenous vein with a steel wool pledget, suspended in chilled Sacks solution, and mixed with blood used to preclot the graft. This suspension was omitted from the three control grafts. After six weeks, the grafts were removed, rinsed and examined. Fluorescent Factor VIII related antigen (F VIIIRA) strongly stained the lining cells. Silver nitrate Haütchen and electron microscopy preparations revealed a lining pattern characteristic of vascular endothelium. Endothelial cell-specific Weibel-Palade bodies were identified in the lining cell cytoplasm. Massons trichrome staining revealed a relatively collagen-poor connective tissue within the seeded fabric. Transmission electron microscopy disclosed vascular smooth muscle cells between the seeded graft fabric and the lining cells. Vasa vasorum, arising from the outer capsule, penetrated the fabric to supply the inner capsules of the seeded grafts. It is concluded that the cells lining seeded canine arterial prostheses are true vascular endothelium supported by vascular smooth muscle cells, that the lining contains minimal connective tissue, and that vasa vasorum develop. Unseeded control grafts lacked these features.

Effect of nutrition staging on treatment delays and outcome in stage IV neuroblastoma

The effect of the state of nutrition of 18 children with Stage IV neuroblastoma at diagnosis and during initial therapy, was evaluated with respect to treatment delays, drug dosage alterations, tumor response, days to first event (relapse or death), and survival. All patients received similar therapy (CCSG protocol CCG 371). Based on nutrition staging at diagnosis, nine were classified as malnourished; four were randomized to receive total parenteral nutrition (TPN) and four peripheral parenteral nutrition plus enteral nutrition for 28 days (through 2 chemotherapy courses), and one died before randomization. Nine were nourished at diagnosis; seven received a comprehensive enteral nutrition program and two received TPN. By life‐table analysis, the duration of remission was significantly greater in the nourished than the malnourished (P < 0.01) and a trend towards improved survival was evident at one year (P = 0.08). The median length of survival for children nourished at diagnosis was approximately 12 months, whereas those malnourished had a median survival of only 5 months. Nine children remained nourished or were becoming renourished during the first 21 days of therapy, and one of these had treatment delays and decreased drug dosages. Seven were becoming malnourished or remained malnourished during this period and six had treatment delays (P < 0.01). These data support the idea that nutrition staging at diagnosis and during initial treatment should be an integral part of protocol design and initial evaluation of children with Stage IV neuroblastoma.

Abnormal platelet function in Chediak-Higashi syndrome.

Platelets in an infant with Chediak‐Higashi (C‐H) syndrome without bleeding manifestations and not in the accelerated phase showed abnormal function consistent with storage pool disorder as shown by abnormal aggregation, decreased storage capacity and release of [14C]5‐HT, low endogenous 5‐HT, reduced ATP and ADP with an increased ATP/ADP ratio, increased specific radioactivity of ADP after [14C]adenine labelling, decreased release of adenine nucleotides after stimulation, impaired secretion of acid hydrolases despite normal stores, and decreased calcium content. Incorporation of [14C]adenine into metabolic pool adenine nucleotides was normal. Nucleotide conversion to hypoxanthine in stimulated platelets was mildly impaired. Platelet cyclic‐AMP (c‐AMP) was initially elevated, but even when c‐AMP returned to normal levels after ascorbate treatment, platelet function was not improved. Elevated intracellular c‐AMP was not solely responsible for the abnormal platelet function.

Impaired Microtubule Assembly and Polymorphonuclear Leucocyte Function in the Chediak‐Higashi Syndrome Correctable by Ascorbic Acid

Summary. It was previously shown that the abnormal surface characteristics and defective bactericidal function of polymorphonuclear leucocytes (PMN) in the Chediak‐Higashi syndrome (CHS) are correlated with impaired microtubule assembly, and in one patient direct electron microscopic evidence for an anomaly in microtubule assembly following surface membrane activation by concanavalin A (Con A). We show here that very few microtubules are visible in CHS leucocytes from two additional patients under conditions where normal PMNs contain abundant microtubules, and that both in vivo and in vitro exposure of the CHS leucocytes to ascorbic acid promotes the assembly of microtubules. This agent, which normalizes chemotaxis and degranulation in CHS leucocytes, is shown also to correct granulocyte adherence in these leucocytes. It is suggested that the improved clinical course of patients with CHS following treatment with ascorbic acid is related at least in part to improvement of microtubule assembly and PMN function by the ascorbic acid.

Polymorphonuclear Leukocyte Species Differences in the Disposal of Hydrogen Peroxide (H2O2)

Summary Catalase and the glutathione system containing GPX and GR offer the PMN two mechanisms for disposal of peroxide during phagocytosis. Catalase requires peroxide as substrate whereas GPX requires peroxide and reduced glutathione which is regenerated by GR and NADPH from the HMP shunt. Impairment of either system may lead to accumulation of toxic amounts of peroxide potentially affecting phagocytic function. The activities of catalase compared to GPX and GR were reciprocally related in four mammalian species. Rat and mouse PMNs had negligible catalase activities and enhanced HMP shunt activity reflecting their relative dependence on the more active GPX and GR enzyme systems compared to human and guinea pig PMN which depend more heavily on the cytoplasmic catalase for disposal of peroxide.

THE INFLUENCE OF VITAMIN E ON HUMAN POLYMORPHONUCLEAR CELL METABOLISM AND FUNCTION

These studies on the effect of administration of 1,600 units of vitamin E to humans indicated the following responses to the PMNs (TABLE 6). Functional alterations occur with an increased ability to ingest particles but a mild decrease in bactericidal potency of the PMN. Although the respiratory burst is slightly enhanced as is superoxide anion release, H2O2 release from the PMN is markedly impaired. The hexose monophosphate shunt activity, which is dependent on intracellular H2O2 is decreased during phagocytosis. Membrane responses such as changes in order parameter during phagocytosis as reported by the stearic acid analogue probe 5DS are similar to those of normal PMNs. The release of arachidonic acid from membranes of vitamin E PMNs during phagocytosis of opsonized zymosan is slightly enhanced, indicating normal phospholipase A2 activation. NADH oxidase-derived H2O2 is not impaired within phagocytic generated by NADPH oxidase in phagocytic vesicles, accounting for impairment in HMPS activity and bactericidal activity in these cells.

In Vitro Inhibition of Granulopoiesis by 4-Amino-4′-Hydroxylaminodiphenyl Sulfone

Summary The antimalarial drug, 4,4′‐diaminophenylsulfone (dapsone) has been associated clinically with the production of agranulocytosis. A metabolite of dapsone, 4′‐amino‐4′‐hydroxylaminodiphenyl sulfone (DDS‐NOH) generates superoxide anion and hydrogen peroxide in vitro. To link oxidant injury of committed myeloid stem cells to agranulocytosis, the effects of DDS‐NOH on altering human bone marrow CFUc proliferation and in vitro reduced glutathione levels were examined in the presence and absence of superoxide dismutase and catalase. Bone marrow cells were preincubated for 60 min with either 0·01, 0·1 or 1·0 mm DDS‐NOH, washed and then 2×105 cells/plate were cultured for 10–14 d. Leucocyte colony forming unit cell (CFUc) proliferation was markedly suppressed by 0·1 mm and 1·0 mm DDS‐NOH. With 0·1 mm DDS‐NOH, addition of superoxide dismutase to the preincubation mixture further decreased colony numbers, while addition of catalase partially restored colony formation indicating involvement of hydrogen peroxide. After exposure to 0·1 mm DDS‐NOH, trypan blue exclusion by nucleated marrow cells was 98%. At the same DDS‐NOH concentration, addition of lactoperoxidase, sodium iodide and superoxide dismutase to the preincubation mixture resulted in a further decrease in CFUc proliferation. After incubation with 1·0 or 2·0 mm DDS‐NOH, bone marrow reduced glutathione levels were markedly decreased from 325 nmol/108 cells to 116·3 nmol/108 cells. Bone marrow reduced glutathione levels in guinea‐pig cells also fell after exposure to 1·0 and 2·0 mm DDS‐NOH. In turn catalase but no superoxide dismutase partially prevented the fall. Thus, these studies demonstrate that oxidant‐induced suppression of human marrow CFUc proliferation in vitro can be mediated by a metabolite of dapsone, DDS‐NOH.

Immunofluorescent staining for factor VIII-related antigen: A tool for study of healing in vascular prostheses

Abstract Localization of Factor VIII-related antigen by indirect immunofluorescent microscopy reliably identifies endothelium in frozen sections of canine blood vessels, including vasa vasorum. Using this technique, endothelium was found to line 6-cm-long porous Dacron arterial grafts in place in dogs for 6 months and 1 year. Grafts seeded with autogenous, venous endothelial cells at implantation showed specific fluorescence in the midportions of their flow surfaces after 1 month. Unseeded grafts were devoid of specific fluorescence beyond the region of pannus ingrowth. Vasa vasorum were very well delineated, even when no more than a row of single cells. We conclude that localization of Factor VIII-related antigen reliably identifies true endothelium in vascular grafts and will be useful in the study of vascular prosthetic healing.

Changes in Energy Metabolism, Structure and Function in Alveolar Macrophages under Anaerobic Conditions

To determine whether the change in energy metabolism of guinea‐pig alveolar macrophages (AM) would alter their function to resemble peritoneal macrophages (PM), AM were exposed to strict anaerobic environments for 96 h. Exposure of cultivated AM to hypoxic conditions resulted in a decreased activity of cytochrome oxidase, a key enzyme in oxidative phosphorylation, whereas pyruvate kinase activity, a key enzyme in glycolysis, increased to levels observed in PM. Under hypoxic conditions the total mitochondrial structure available for respiratory activity also decreased in the glycolytic energy‐dependent AM.

Improvement of Polymorphonuclear Leucocyte Oxidative and Bactericidal Functions in Chronic Granulomatous Disease with 4-Amino-4′-4 Hyndroxylaminodiphenyl Sulphone

Summary. In an effort to restore oxidant‐dependent capabilities to chronic granulomatous disease (CGD) polymorphonuclear leucocytes (PMN), we studied a dapsome derivative, 4‐amino‐4′‐hydroxylaminodiphenyl sulphone (DDS‐NOH), known to generate H2O2. After incubation of CGD PMN with 0.2 and 1.0 mm DDS‐NOH for 30 min, the rate of glucose‐I‐14C oxidation via hexose monophosphate (HMP) shunt increased 2‐4‐fold and ‐ that of iodination of ingested zymosan particles 1.5‐2.7‐fold. Both effects could be further enhanced by superoxide dismutase (SOD) but inhibited by catalase. In three patients, 0.2 mM DDS‐NOH improved in vitro killing of Staph. aureus. DDS‐NOH 0.02 mM induced capping of Concanavalin A (Con A) receptor complexs suggesting interference by the drug with microtubule‐associated function. Thus, optimal concentrations of DDS‐NOH my be employed as an oxidant to improve metabolic and bactericidal functions of PMN from patients with CGD.