Phenylalanine depletion for the management of phenylketonuria: use of enzyme reactors with immobilized enzymes.

Multitubular enzyme reactors with immobilized phenylalanine ammonia lyase were tested in vitro and in vivo for depletion of phenylalanine in circulating blood. Sustained reduction of phenylalanine was achieved in less than 30 minutes. A 50% decrease of phenylalanine was obtained with a 2-hour application of enzyme reactors and was maintained for more than 2 days. Similar enzyme reactors have therapeutic potential for temporary management of phenylketonuric patients when their circulating phenylalanine becomes exceedingly high because of infection, fever, or pregnancy.

Protection of normal tissues with 2-aminoethylisothiouronium during local pelvic radiation in monkeys.

Intestinal and bladder injury are the main limiting factors to radiation therapy in patients with pelvic neoplasms. 2-Amino-ethylisothiouronium (AET) is a radiation-protective agent when given systemically but absorbs poorly from the intestines. Accordingly, it was explored for the local protection of the bowel and bladder during radiation to the pelvis. Radiation localized to the pelvis in various high fractionated doses and various schedules was applied to pairs of stumptailed monkeys (Macaca arctoides): one was always a control; and the other was treated with AET. AET was applied to the bladder through a catheter and to the rectum with a cotton tampon during the time of radiation. After radiation, AET was removed by repeated washings. Control animals developed hemorrhage, diarrhea, and emaciation and died at various times after completion of the radiation course; biopsy of rectal mucosa showed severe radiation damage. AET-treated animals had only occult blood in the stools and suffered slight weight loss; rectal biopsies showed normal tissues 2 weeks after radiation.

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.

Clinical and experimental studies on adenine, various nucleosides and their analogs in hematology.

In red blood cells as well as in platelets there appears to be a decrease in adenine nucleotides during storage under blood bank conditions. This can be decreased by use of anticoagulant preservatives with higher phosphate content than the standard ACD solution, through the addition of adenine and inosine. Maintenance of higher ATP levels appears to be related to longer circulating life span after transfusion into patients in the case of red blood cells but not platelets. Inosine and more alkaline preservative medium also contribute to the maintenance of 2,3-DPG levels in red blood cells, and with it to the maintenance of normal hemoglobin dissociation curves and thus oxygen-carrying capacity. Certain nucleoside analogs may contribute to the preservation of platelets and of whole blood by their platelet-aggregation inhibitory activity. Platelet-aggregation inhibitors may also be useful in preventing thromboembolic episodes with potentially greater safety than anticoagulants.

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.

Studies on osteoporosis X. Effect of estrogen-progestin combination on heparin-induced osteoporosis.

Neutron activation analysis was employed to determine total body calcium in C3H/St(Ha) female mice. As 99% of body calcium is in bone, loss of calcium was used as an index of bone loss (osteoporosis). Heparin (500 U/kg b.i.d. caused bone loss in 3 months. Premarin (2 mg/kg q.d.) or norethindrone (40 mg/kg q.d.) alone prevented this osteoporosis. A Premarin-norethindrone combination (2 mg - 10 mg q.d. respectively) appeared to be somewhat more effective than either agent alone, but this difference was not significant at the 5% level. Combinations of estrogen and progestins may prevent metabolic bone disease at the same time reducing the danger of estrogen induced neoplasia.

The role of fibrinolysis in the therapy of peripheral vascular disease.

In vivo thrombolytic studies in stumptailed monkeys indicated that pentoxifylline potentiates thrombolysis induced by urokinase activated human plasmin. Pentoxifylline as well as prostaglandin E1 released plasminogen activators and activated the fibrinolysin system. From this point of view pentoxifylline and prostaglandin E1 synergized with each other. Pentoxifylline potentiated the thrombolytic effect of prostaglandin E1 in vivo.

Red cell flexibility and platelet aggregation in patients with chronic obstructive vascular disease (COAD) and study of therapeutic approaches.

The red blood cell flexibility was studied in the blood of twenty patients with severe peripheral vascular disease and twenty matched controls. Patients exhibited significantly less red cell flexibility than controls. In both groups there was an inverse relationship between age and red cell flexibility. No correlation was found between red cell flexibility and sex or smoking history. Pentoxifylline, a xanthine derivative which inhibits phosphodiesterases and platelet aggregation, was found to increase red cell flexibility. This effect of the drug was greater on red cells with imparied flexibility than on normal cells. Various prostaglandins by contrast were found to decrease red cell flexibility, this could be compensated for by pentoxifylline. Forty patients with COAD were treated intravenously with PgE1. Significant inhibition of platelet aggregation and clinical improvement was noticed. It is suggested that combinations of PgE1 and pentoxifylline should be explored in clinical studies.

Burkitt's lymphoma.

Clinical and epidemiologic features of Burkitt's lymphoma are reviewed. Epidemiologic studies suggest that simultaneous infection with Epstein-Barr (E-B) virus and malaria may be involved as etiologic agents. On the other hand we have found that in the Amazon region of Brazil and Peru both malaria and E-B virus infection is common among children, yet Burkitt's lymphoma is rare. The possibility exists that other concomitant etiologic agents and genetic factors are also involved. Several investigators suggested the possible involvement of Reo 3 virus. We have found antibodies against Yaba virus. A laboratory worker who accidentally inoculated himself with Yaba virus developed a histiocytoma which when inoculated into Asiatic monkeys produced typical Yaba tumors. This was the first case that Koch's postulates were fulfilled in a virus induced neoplasm in man. Therapeutically, the best clinical results were obtained in those patients who were treated with small doses of cyclophosphamide. On the basis of somewhat inadequate follow-up studies, it is estimated that "five year cures" were obtained in about 10% of the patients.

Myeloid regeneration after whole body irradiation, autologous bone marrow transplantation, and treatment with an anabolic steroid.

Stumptail monkeys (Macaca speciosa) received lethal whole body radiation. Autologous bone marrow injection resulted in survival of the majority of the animals. Treatment with Deca-Durabolin, an anabolic steroid, caused more rapid recovery of colony-forming cell numbers in the bone marrow than in control animals. Both the Deca-Durabolin-treated and control groups were given autologous bone marrow transplantation. Anabolic steroid effect on transplanted bone marrow colonyforming cells may explain the increased rate of leukopoietic regeneration in anabolic steroid-treated animals as compared to controls.

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.

Effect of phosphodiesterase inhibitors on platelet aggregation and tumor metastasis.

Recent studies suggest a relationship of platelet aggregation to metastatic spread of neoplasia. In the present experiment pentoxifylline appears to be a highly effective platelet aggregation inhibitor: a dose-response curve is apparent in monkey between 6 and 24 mg/kg i.v. Besides pentoxifylline significantly reduces metastasis rates in Wilms' tumor and neuroblastoma in rodents: 18 mg/kg significantly increase polyploid Ehrlich ascites tumor cell circulation time; on the contrary it has no effect on metastasis in the NIH renal adenocarcinoma. It is thus possible that platelet factor related blood coagulation processes play a different role in the fate of these tumor cells. This may be related to cell membrane characteristics. Phosphodiesterase inhibitors alter membrane fluidity and cell deformability of cancer cells which may pass the microcirculation easier and are less likely to settle and form metastases. Agents with both platelet aggregation inhibitory and red cell deformability increasing effect may find a place in our therapeutic armamentarium in oncology.