Multiple medical problems following agent orange exposure.

A patient exposed to agent orange and a gunshot wound during the Vietnam War has developed multiple medical problems including nocardiosis, onychomycosis (Trichophyton rubrum), multiple thromboembolic episodes, hemochromatosis, diabetes mellitus type 2, diabetic neuropathy, activated protein C resistance (without Leyden V 1st mutation), degree A-V block, lung cancer (metastatic adenocarcinoma), carpal tunnel syndrome and arthritis.

Treatment of lead poisoning with an immobilized chelator comparison with conventional therapy.

Lead poisoning is a global public health problem. In pregnant women it may result in developmental delays of the fetus, in children it my produce learning disability. Available chelators are nephrotoxic when eliminated as lead-chelator complexes. For safe removal of lead from the body we developed a "Lead-Hemopurifier" (L-HP), a device with an immobilized chelator. In vitro, applied to lead solutions, this device reduced the lead concentration. Applied to dogs with lead intoxication, Lead-HP-s removed lead from the blood; this was continuously replaced by lead from the bones until the skeleton was cleared from lead deposit. Treatment of lead poisoning in dogs with Lead-HP-s compared favorably with Versenate treatment of children with lead toxicity. This report demonstrates the in vivo efficiency and safety of this new detoxfication method. Methods to induce lead poisoning in dogs and procedures to identify lead released from skeletal deposits are described.

Thromboembolic disease susceptibility related to red cell membrane fluidity in patients with polycythemia vera and effect of phlebotomies.

Thromboembolic disorders are frequent complications in polycythemia vera. In addition to thrombocytosis with hyperaggregability, leukocytosis, and high hematocrit, hyperviscosity syndrome, a new component, is described in the pathophysiology of this phenomenon. There is decreased red cell membrane fluidity with decreased deformability which increases the susceptibility to microvascular occlusion and also increases the chance of disseminated intravascular coagulation (DIC). Periodic phlebotomies improved the hematologic picture in these patients and results in the removal of the "stiff" red cells with an increased production of young red cells, greater membrane fluidity, deformability and less chance of microvascular occlusion.

Myocardial release of non-transferrin-bound iron during cardio-pulmonary bypass surgery.

Myocardium reperfusion following coronary artery bypass grafting (CABG) may result in "reperfusion injury" by free radical generations. Since desferrioxamine administration attenuates this syndrome, non-transferrin-bound-iron (NTBI) released into the perfusing medium during CABG was implicated as a catalyst for oxygen radical formation. From 13 patients with "redo" CABG, specimens were collected from the coronary sinus (influx) and the aortic vent (efflux) after each distal coronary anastomosis. Specimens were subjected to sieving chromatography, and fractions were analyzed for total iron and NTBI using atomic absorption spectrometry (AAS). A statistically significant increase in NTBI was measured in influx (p = 0.002) and efflux samples (p = 0.023) collected after each graft. The combined amount of NTBI measured in these specimen was proportional to the CK-MB increase measured in the patients' sera on the day of surgery and the subsequent day. NTBI which accumulated in the circulatory bypass fluid during CABG may catalyze the generation of free radicals in the myocardium when body temperature is restored. This may aggravate myocardial damage as reflected by a post-surgical increase in CK-MB concentrations. Studies are in progress to develop new methods for the removal of NTBI during cardiac surgery. Tissue injury occurs with reperfusion during ischemia. This has been attributed to oxygen-derived free radicals that are generated by substances released from hypoxic areas (Kloner, Przyklenk et al., 1989; McCord, 1998). Reperfusion injury, i.e. the "reperfusion syndrome," occurs after coronary artery bypass grafting (CABG) when the ischemic myocardium is again provided with a supply of blood. Its most serious manifestations are arrhythmia and myocardial stunning (Ar"Rajab, Dawidson et al., 1996; Ferrari, Ceconi et al, 1996). The role of iron in reperfusion injury has been implicated by indirect evidence: during the reperfusion syndrome, the binding of iron with the chelator desferrioxamine (Ambrosio, Zweier et al., 1987; Bel, Martinod et al., 1996), or the administration of exogenous apo-transferrin, improved cardiac contractility and delayed manifestations of cardiac injury (Tiede, Sareen et al., 1990). Iron, as a transition metal, is able to catalyze free radical formation when released into the circulation from endogenous stores as non-transferrin-bound-iron (NTBI). This iron may be bound to small proteins or inorganic ligands (Halliwell and Gutteridge, 1984; Pollock and Campana, 1980; Zweier, 1992). A method for the measurement of NTBI was recently developed (Ambrus, Stadler et al., 1999). The purpose of this study was to explore whether a correlation exists among (a) the amount of NTBI released during CABG surgery, (b) the length of time of myocardial ischemia, and (c) the myocardial damage that occurs during cardiopulmonary bypass.

Removal of non-transferrin-bound iron from blood with iron overload using a device with immobilized desferrioxamine.

An extracorporeal hollow-fiber device with immobilized desferrioxamine (DFO) was developed for the removal of nontransferrin-bound iron (NTBI) from blood, without the toxicity of parenteral chelation. When blood circulates through the fibers having pores with 30 kD cut-off, non-transferrin-bound-iron (NTBI) crosses the fiber pores and is chelated by the immobilized desferrioxamine. Removal of circulating iron stimulates iron release from larger proteins and tissue stores, establishing continuous iron flow to the immobilized chelator. During in vitro circulation through a device, iron removed from blood of hemodialysis or sickle cell patients was proportional to, but always in less than 50% of the initial iron level. We attribute the inability to remove more serum iron to irreversible iron binding by transferrin. To investigate where removable and fixed iron was bound, iron binding proteins were analyzed in sera from six patients with genetic anemias and iron overload. Sera separated by sieving chromatography contained 1-14% of the iron in the < 30 kD protein pool, 26-48% was in the combined non-transferrin pools. Sera from hemochromatosis patients without iron overload did not contain NTBI. Circulation of hemochromatosis blood through the device removed one third of the iron, this came from all molecular weight fractions. Iron removal by the device from the < 30 kD pool appears to establish a disequilibrium, that stimulates continuous iron release from ligands with low iron affinity, renewing the pool in the < 30 kD range, which includes potentially toxic NTBI. Therapy with the chelator device having immobilized desferrioxamine should be beneficial for treatment of patients with iron overload.

Treatment of aluminum overload using a cartridge with immobilized desferrioxamine.

Intravenous desferrioxamine (DFO) is the method commonly used to treat aluminum toxicity. This laboratory has developed a hollow fiber device with immobilized DFO, an "Aluminum DFO-HP" (DFO-HP), for the purpose of removing aluminum without the chelator (DFO) entering the blood. With Food and Drug Administration approval, a polysulfone DFO-HP, placed in the extracorporeal circuit in series with the patient's customary dialyzer, was tested for its safety and ability to remove aluminum in patients with ESRD who had aluminum overload. During treatment with this device, no toxic reactions, side effects, or hematologic or clinical laboratory changes were seen other than those associated with dialysis. Average aluminum clearance with the DFO-HP device was 25.3 mL/min with a range of 7.2 to 52.4 mL/min, whereas aluminum clearance with the F-60 polysulfone high-flux dialyzer was 8.4 mL/min. Aluminum clearance of the cuprophane dialyzers in series with the DFO-HP was negligible. The amount of aluminum removed over a 2-h treatment with DFO-HP ranged from 94 to 628 micrograms, which corresponded to 32 to 199% of the initial aluminum in the circulation before that particular treatment. The excess 99% was provided from aluminum released from tissue sites into the circulation throughout the duration of the treatment. It is expected that, because of the efficiency and safety of the DFO-HP device, the time presently needed for aluminum depletion using intravenous DFO will be greatly shortened and the potential toxicity of intravenous DFO will be eliminated.

Potentiation of thrombolytic therapy by enzyme combinations and with aspirin or pentoxifylline.

Radioactively labeled human fibrin clots were placed into veins of Macaca arctoides monkeys. Thrombolysis was recorded by the disappearance of radioactivity and by angiography. Streptokinase (SK) and urokinase (UK) induced thrombolysis was potentiated by low dose aspirin (ASA) and pentoxifylline (PE). Studies on the mechanisms of action revealed that PE inhibits platelet aggregation, releases tissue plasminogen activator (t-PA) from the endothelium, increases red cell deformability and inhibits white cell adhesion. Thrombolysis by pro-urokinase (pro-UK) was potentiated by low dose SK probably because of streptokinase-plasmin activation of pro-UK to UK. Platelet aggregation inhibitory effects, disaggregation of platelet aggregate inducing effects, and the t-PA releasing activity of PE was demonstrated in patients with obstructive cardiovascular disease. Pharmacodynamic studies suggested that PE metabolites one and five are most effective from this point of view. These metabolites are currently studied in combination with thrombolytic enzymes.

Studies on tumor induced angiogenesis.

Methods were developed to test angiogenic response to human tumor implants and various biologic agents in the cornea of rabbits and non-human primates (Macaca arctoides). Crude PDGF preparations were found to have significant angiogenic effect. Purified, recombinant PDGF preparations were also effective inhibitors (e.g. pentoxifylline (Px) (which also were found to release PgI2 and t-PA) inhibited human tumor implant induced angiogenesis and reduced spontaneous metastases in 3 transplantable murine tumors (Furth-Columbia Wilms' tumor in Furth-Wistar rats, C-1300 neuroblastoma in A/J mice and HM-Kim mammary carcinoma in Wistar rats) but not in the NIH adenocarcinoma in Balb/c mice. Sodium diethyldithiocarbamate (DDTC), a metal complexing agent with special affinity to copper and anti-thyroid as well as, immune stimulating activity was shown to be anti-angiogenic and to potentiate the effect of Px. The anti-fibrinolytic agents epsilon amino caproic acid (EACA) and tranaxamic acid (t-AMCHA) were anti-angiogenic. DDTC and Px were synergistic from this point of view.

Prevention and treatment of osteoporosis. One of the most frequent disorders in American women: a review.

This review summarizes basic pathophysiology, epidemiology, classification and theories on the etiology of osteoporoses. Diagnostic, preventative and therapeutic aspects are discussed including the use of hormones and hormone analogues (estrogen, progestins, calcitonins, anabolic steroids), calcium, fluoride, bisphosphonates, vitamins D and their metabolites and the recently proposed "coherence therapy". Reference is made to new compounds under study including imidazoquinazolinones, methylxanthines and benzothiophenes.

Studies on tumor induced angiogenesis.

Methods were developed to test the angiogenic response to human tumor implants and various biologic agents in the cornea of rabbits and non-human primates (Macaca arctoides). Human malignant melanoma tissue and crude platelet derived growth factor (PDGF) preparations had significant angiogenic effects. Purified, recombinant PDGF preparations were also effective initiators. Hemorheologic agents which also inhibit platelet aggregation [e.g. pentoxifylline (Px) (Trental) (also found to release PgI2 and tissue plasminogen activator (t-PA)] inhibited human tumor implant-induced angiogenesis. Sodium diethyldithiocarbamate (DDTC), a metal complexing agent with special affinity to copper and anti-thyroid as well as immune stimulating activity, was shown to be anti-angiogenic and to increase the effect of Px. The anti-fibrinolytic agents epsilon amino caproic acid (EACA) and tranexamic acid (t-AMCHA) were anti-angiogenic.

Changes in the fibrinolysin system in infantile and adult respiratory distress syndrome (ARDS), caused by trauma and/or septic shock in patients and in experimental animals.

Five hundred premature infants were treated on a randomized double-blind basis with human plasminogen or placebo. We found that in premature infants plasminogen levels are low; thus, defense against intra-alveolar fibrin deposition during birth trauma is reduced. A significant decrease in the incidence of respiratory distress syndrome-hyaline membrane disease and death was seen in the treated infants. Infants with established respiratory distress syndrome were treated with human plasmin or placebo. A significant decrease in death rate was found in the treated infants. Decreased plasminogen and anti-thrombin III (AT-III) levels were found in patients with adult respiratory distress syndrome and/or septic shock. These levels returned to normal within 14 days in survivors, but remained depressed in those who died. It was thought that these parameters may have diagnostic and predictive values. In experimental animals, injection of E. coli endotoxin or oleic acid produced an adult respiratory distress syndrome type phenomenon. This was also accompanied by decreases in plasminogen levels, with recovery in the survivors. It is suggested that plasminogen and anti-thrombin III should be explored as auxiliary therapeutic agents in adult respiratory distress syndrome.

Erythrocyte filterability in normal and high-risk pregnancy.

Erythrocyte filterability was studied longitudinally in normal pregnancy and in certain categories of high-risk pregnancy. Study subjects included ten normal controls, 12 insulin-dependent diabetics, eight gestational diabetics, and five essential hypertensives. Our results indicate that erythrocyte filterability remains relatively stable over the course of normal gestation. We noted no differences between controls and essential hypertensives or gestational diabetics, although a favorable effect of insulin therapy was suggested in gestational diabetics. Erythrocyte filterability and mean arterial blood pressure were not related. Insulin-dependent diabetics demonstrated a significantly elevated and widely varying erythrocyte filterability, and individual patient trends correlated well with outcome. Fibrinogen levels in diabetics rose precipitously and were significantly higher than normal throughout gestation. Fibrinogen levels paralleled changes in erythrocyte filterability, with the two parameters positively correlated. Mean glucose control had no influence on filterability. We conclude that in the diabetic pregnancy, varying erythrocyte filterability is related to altered fibrinogen metabolism and may contribute to perinatal morbidity.

Extracorporeal enzyme reactors for depletion of phenylalanine in phenylketonuria.

Multitubular enzyme reactors with immobilized enzymes were developed to achieve depletion of circulating substrate by extracorporeal means. To act as prototypes, reactors were prepared with immobilized L-phenylalanine ammonia-lyase, an enzyme that metabolizes phenylalanine to trans-cinnamic acid and ammonia without the need for a coenzyme. We report the first application of phenylalanine ammonia-lyase reactors in an extracorporeal circulation system in a patient with phenylketonuria. A phenylalanine level of 1.82 mmol/L (for the last 6 years) decreased to 1.24 mmol/L after 5.5. hours of treatment, without the enzyme entering the circulation. Total phenylalanine depletion from blood and tissue stores was estimated at 1800 mg. The hemodialysis-like procedure proved to be without side effects, specific for phenylalanine, and suitable in the management of pregnant women with phenylketonuria and late-onset hyperphenylalaninemia. The extracorporeal use of enzyme reactors for temporary enzyme replacement represents a new, safe, and effective therapeutic modality.

Mechanism of the potentiation of thrombolysis by pentoxifylline (Trental).

Streptokinase induced thrombolysis of radioactive labeled human fibrin clots was potentiated by simultaneous treatment with pentoxifylline (Trental). This appears to be due in part to the prevention of platelet aggregation on the clot. In addition, release of t-PA and PgI2 from the endothelium and increases in red cell deformability may also play a role.

Effect of imidazoquinazolinones on tumor cells in the circulation.

Of several imidazoquinazolinones, Anagrelide was found to be the most effective compound to inhibit platelet aggregation and to increase circulation time of intravenously injected tumor cells resulting in metastasis formation.