Phase I trial of intravenous infusion of ex-vivo-activated autologous blood-derived macrophages in patients with non-small-cell lung cancer: Toxicity and immunomodulatory effects

Summary The purpose of this phase I study was to evaluate the toxicity and biological activity of autologous blood-derived macrophages activated ex-vivo with recombinant human interferon (rhuIFN) [monokine-activated killer (MAK) cells] and administered intravenously to 11 lung cancer patients once a week for 6 consecutive weeks. Peripheral blood monocytes were collected by leukapheresis and then purified by counterflow elutriation. The MAK cells were generated by culturing the purified monocytes in Teflon bags for 7 days and adding rhuIFN to the cultured cells for the last 18 h. These MAK cells expressed differentiation-associated surface antigen MAX1, and were cytotoxic in vitro against tumour cell line U937. The MAK cells were infused at dose levels from 1 × 10⁷ to 5 × 10⁸ on an intrapatient dose-escalating schedule. No severe adverse side-effects occurred. Toxicity was mild to moderate [primarly fever (75%) and chills (32%)], non-dose-dependent, and non-cumulative. No consistent change in haemostatic function, or liver or renal function was observed. Dose-limiting toxicity was not reached at 5 × 10⁸ cells (optimal dose reproduced for each patient). The maximum tolerated dose was not determined. The immunomodulatory activity of i.v. infused MAK cells was demonstrated both in vivo by significant increases in granulocyte count and neopterin level in the patients' peripheral blood postinfusion and in vitro by secretory products (IL-1. TNF, neopterin, and thromboplastin-like substance) in the culture supernatants. The in vivo traffic patterns of autologous MAK cells labelled ex-vivo with¹¹¹In oxine were studied in 7 patients. Gamma imaging showed an immediate but transient lung uptake (<24 h), and a progressive uptake of radioactivity in the liver and spleen was seen from 6 h to 72 h post-infusion. Our results indicate that the preparation of high numbers of autologous, blood-derived MAK cells is a feasible procedure, and their transfusion is safe for patients. This immunotherapeutic approach seems to be encouraging from the point of view of establishing an adjuvant therapeutic modality in cancer patients with minimal residual disease.This work was supported in part by a grant 6911 from the Association pour la Recherche contre le Cancer (ARC), grants from the Ligue Nationale contre le cancer and the Ligues Regionales (Bas-Rhin, Haut-Rhin) contre le cancer, and contract 891013 from the Institut National pour la Santé et la Recherche Médicale (INSERM), France     

The involvement of ethanol in the free radical reaction of 6-hydroxydopamine

ESR spin trapping technique was used to detect and analyze free radical formation. When 6-hydroxydomine (6-OHDA) was incubated alone or in the presence of a free radical generating system (H₂O₂ and FeSO₄), hydroxyl free radicals were observed in a concentration-dependent manner. Glutathione was found to be the most effective scavenger of the ESR signal when compared with vitamin E or Mannitol. The addition of ethanol resulted in the formation of the pure hydroxyethyl free radicals. The amount of hydroxyethyl free radicals in the system was dependent upon the concentration of ethanol and the formation of hydroxyethyl free radicals correlated well with the extent of lipid peroxidation and the loss of enzymic activity of the membrane-bound (Na⁺, K⁺)-ATPase. We suggest that in the biological system ethanol may potentiate the neurotoxicity of 6-OHDA with the formation of hydroxyethyl free radicals, which are longer-lived and far more damaging to membranes that the hydroxyl radicals. These data lead us to further hypothesize that the neuronal degeneration caused by 6-OHDA and other compounds that generate free radicals could be potentiated in the presence of ethanol.     

Superoxide Radical-Mediated Alteration of Synaptosome Membrane Structure and High-Affinity γ-[14C]Aminobutyric Acid Uptake

Mouse cortical synaptosomal structure and function are altered when exposed to hypoxanthine/xanthine oxidase (HPX/XOD)-generated active oxygen/free radical species. The structure of both the synaptic vesicle and plasma membrane systems are altered by HPX/XOD treatment. The alteration of synaptic vesicle structure is exhibited by a significant increase in the cumulative length of nonsynaptic vesicle membrane per nerve terminal. With respect to the nerve terminal plasma membrane, the length of the perimeter of the synaptosome is increased as the membrane pulls away from portions of the terminal in blebs. The functional lesion generated by HPX/XOD treatment results in a reduction in selective high-affinity gamma-[14C]aminobutyric acid (GABA) uptake. Kinetic analysis of the reduction in high-affinity uptake reveals that the Vmax is significantly altered whereas the Km is not. Preincubation with specific active oxygen/free radical scavengers indicates that the super-oxide radical is directly involved. This radical, most probably in the protonated perhydroxyl form, initiates lipid peroxidative damage of the synaptosomal membrane systems. Low-affinity [14C]GABA transport is unaltered by the HPX/XOD treatment. The apparent ineffectiveness of free radical exposure on low-affinity [14C]GABA transport coupled with its effectiveness in reducing high-affinity transport supports the idea that two separate and different amino acid uptake systems exist in CNS tissue, with the high-affinity being more sensitive (lipid-dependent) and/or more energy-dependent (Na+,K+-ATPase) than the low-affinity system.     

The effects of antibiotics on hemolytic behavior of red cells

Human blood was sheared between rotating polyethylene disks and plasma hemoglobin measured at intervals to produce kinetic hemolysis curves (KHC), plotted as free hemoglobin concentration vs time. The KHC produced by blood samples incubated in the presence of penicillin, streptomycin, gentamicin, and amikacin lie always below those for control samples, indicating a reduction in hemolysis; this reduction was greater as the drug concentration was increased. Explanations in terms of alterations in red cell structure were sought by several characterization tests of amikacin-loaded blood samples. Drug-localization studies demonstrated that significant fractions of the total dosage were associated with the red-cell membrane. Resistive pulse spectroscopy was used to show how amikacin affected cell size, deformability, and osmotic fragility; results were sensitive to storage age of the blood. In all cases, the effect of shearing was to reduce cell size, deformability, and osmotic fragility. Mechanisms for hemolytic protection by drugs are proposed.     

大豆下胚轴线粒体产生超氧物自由基的效率

大豆下胚轴线粒体在呼吸基质存在下,显著地增加了肾上腺素氧化速率,这种氧化速率能为外源SOD抑制,表明线粒体呼吸时产生分子氧的单电子还原成O_2(?)。亚线粒体颗粒产生O_2(?)的效率略高于线粒体。大豆下胚轴线粒体吸链内O_2(?)的产生为NADH所支持并与交替途径无关。表明分子氧单电子还原的部位可能是NADH-黄素蛋白和UbQ-Cyt.B。     

The influence of level of infection of rats with Nippostrongylus brasiliensis on the haematology and phospholipase activity and mast cell numbers in the small intestine and colon

The haematology and phospholipase activity and mast cell numbers of the small intestine and colon of rats was studied 10 days after infection with various numbers of larvae of N. brasiliensis. A significant reduction in the RBC occurred after infections with 200 and 5000 larvae but not with 1000 larvae. Hb was significantly reduced after infection with 200 larvae and increases in the MCV and MCH indicated the development of a macrocytic anaemia. Reticulocyte count was increased at all levels of infection except after 200 larvae. WBC was increased at all levels of infection except in the 5000 larvae group. Lymphocytes were significantly increased in all groups except those infected with 5000 larvae. Neutrophils increased only at the lower levels of infection. The most marked changes occurred in eosinophil numbers, significant increases occurring with increasing levels of infection. However, after infection with 5000 larvae the numbers were significantly lower than after infection with 200 or 1000 larvae. Phospholipase activity, which is believed to be related to tissue eosinophil levels, was significantly increased at all infection levels in the proximal small intestine. Significant increases in the distal ileum and colon occurred mainly after infection with 1000 and 5000 larvae. Mast cell numbers did not change significantly at any infection level. It is suggested that the pathology observed, here in the form of anaemia, is multifactorial in origin and is largely a function of the immune response, the development and expression of which is dependent on the level of infection, with suppression of immune damage occurring at the high levels of infection when pathogenesis may involve a direct effect of the worms.     

Carbonic acid as the host signal for the development of parasitic stages of nematodes

Petronijevic T., Rogers W. P. and Sommerville R. I. 1985. Carbonic acid as the host signal for the development of parasitic stages of nematodes. International Journal for Parasitology15: 661–667. This paper gives results on which may be based an identification of the component of the system CO₂ + H₂O ai H₂CO₃ ai H⁺ HCO₃^? which acts as the stimulus from the animal host for some nematodes. Using infective juveniles of Nematospiroides dubius and Haemonchus contortus, the effects on exsheathment of (1) low pCO₂ values, (2) the presence of carbonic anhydrase in the stimulating medium, and (3) the inhibition of carbonic anhydrase within the juveniles have been examined. The results lead to the suggestion that it is the “readily available” undissociated H₂CO₃, or H₂CO₃ + HCO₃^? which is the critical factor in the stimulus for development. The wide range of [H⁺]s over which “readily available” H₂CO₃ is present in physiological environments suggests that this host signal may be important for infection with many species.     

Spin probe clustering in human erythrocyte ghosts

Summary A model has been developed for 5-nitroxide stearate, I(12,3), distribution in human erythrocyte ghosts which accurately predicts ESR spectral alterations observed with increased probe/total lipid (P/L) at 37°C. This spin probe occupies a class of high-affinity, noninteracting sites at low loading. Saturation occurs with increasing probe concentration, and, at higher loading, the probe inserts itself at initially dilute sites to form membranebound clusters of variable size. No low probe remains at high P/L where all I(12,3) clusters in a concentrated phase. This model allows determination of the dilute/clustered probe ratio, and shows that I(12,3) segregates in erythrocytes at what might otherwise be considered low P/L (e.g., 1/359). These findings validate the earlier use of empirical parameters to estimate probe sequestration in biological membranes.     

Various proteinase inhibitors decrease prolactin and growth hormone release by anterior pituitary cells

Proteinase inhibitors were tested for their ability to inhibit prolactin (PRL) and growth hormone (GH) release by cultured anterior pituitary cells of the rat. Inhibitors of microbial origin (chymostatin, elastatinal, leupeptin) had either no or a moderate effect on hormone release while some tripeptide aldehydes, especially those with lysine at their C terminus, inhibited markedly PRL and to a lesser extent GH release. Boc-DPhe-Phe-lysinal was the most effective on lactotrophs inhibiting PRL release more than 50% at 10(-4) M. The site(s) of action of tripeptide aldehydes remain to be elucidated.     

Deficiency of immunoreactive somatostatin in the median eminence of Snell dwarf mice

Snell dwarf mice (dw/dw) are characterized by a genetically determined, congenital lack of pituitary GH, TSH and prolactin. Given that hypothalamic somatostatin is involved in the regulation of pituitary GH and TSH release, it was decided to investigate the content of immunoreactive somatostatin (IRS) in the median eminence of dw/dw and phenotypically normal mice of the same strain. The content of IRS in the pyloric antrum and pineal gland of these animals was also examined. The effects of ovariectomy and of hyperprolactinemia (induced by a pituitary graft under the kidney capsule) on the median eminence content of IRS were also studied in both normal and dwarf mice. Median eminence IRS content was significantly lower in the dw/dw (23.6 +/- 1.8 ng) than in normal mice (57.4 +/- 7.1 ng); no difference was found in the pyloric IRS content of dw/dw (16.9 +/- 1.6 ng/mg of protein) and normal animals (13.8 +/- 1.9 ng/mg of protein), nor in the pineal content of IRS (639.4 +/- 64.4 pg/gland in the dw/dw; 732 +/- 265 pg/gland in normals). Neither ovariectomy nor hyperprolactinemia were found to affect the IRS content in the tissues studied in normal or dwarf mice. Treatment of an additional group of 9 dwarf mice with L-thyroxine (L-T4 2 micrograms/48 h. s.c. for 2 weeks) significantly increased the animals weight (10.2 +/- 0.4 g versus 7.4 +/- 0.3 g) and produced maturation of facial features; however, it did not change the IRS content in any of the tissues studied. It is concluded that the content of IRS in the median eminence of mice with a congenital lack of GH, TSH and prolactin is significantly reduced and that this is unlikely to be related to the deficiency of thyroid hormones in these animals.