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     

Potentiation of growth suppression and modulation of the antigenic phenotype in human melanoma cells by the combination of recombinant human fibroblast and immune interferons

Summary Administration of interferon as a single therapeutic regimen in cancer patients with various neoplasias has had only limited efficacy in ameliorating the negative clinical course of their disease. In the present study, we have evaluated the effect of recombinant human fibroblast (IFN) and immune (IFN) interferon, alone and in combination, on growth, differentiation and the expression of class I and II histocompatibility locus antigens (HLA) and melanoma-associated antigens on the human melanoma cell line H0-1. The effect of combinations of interferons on the antigenic profile of human melanoma cells displaying different organ colonization and spontaneous metastatic potential in athymic nude mice was also determined. H0-1 cells were more sensitive to the antiproliferative activity of IFN than to IFN and the combination of interferons resulted in a potentiation of growth suppression. The antiproliferative effect of both interferons was greater in later-passage than in earlier-passage H0-1 cells, possibly reflecting alterations in the evolving tumor cell population as a result of long-term in vitro propagation and/or the selective outgrowth of cells with an increased growth rate. The enhanced growth suppression observed in H0-1 cells treated with the combination of IFN plus IFN was not associated with a significant increase in the level of melanin, a marker of melanoma differentiation, above that observed with either interferon used alone. IFN and IFN differentially modulated the expression of class I and II HLA and melanoma-associated antigens in H0-1 cells and a series of melanoma cells with different organ colonization and metastatic potential, including MeWo, MeM 50-10, MeM 50-17, 3S5 and 70W. No consistent potentiation or antagonism in the expression of any specific antigen was observed in any of the melanoma cell lines exposed to the combination of interferons. The present study demonstrates that the combination of IFN plus IFN can potentiate growth suppression in H0-1 human melanoma cells and that this effect is not associated with an increase in differentiation or a potentiation in antigenic modulation. In addition, no direct correlation between the expression of any specific antigen or its modulation by IFN or IFN, alone or in combination, and organ colonization and metastatic potential in nude mice was observed in the different melanoma cell lines.     

Paradise lost: Mitochondrial eve refuted

Being based solely on neontological data, all «unique parent» evolutionary hypotheses, of which «Mitochondrial Eve» is one, fall into the category ofscala naturae. Mathematical treatment of neontological data bases, using cladistic approaches does not confer the status of scientific hypotheses onto such scenarios. Apart from these fundamental problems, such hypotheses are flawed on a number of other bases, including the fact that there is a proportion of parental contribution to mitochondrial lineages, despite widely publicised statements that mithocondrial DNA in mammals is «strictly» maternally inherited. Other weaknesses of «unique mother» hypotheses on that their proponents endeavour to describe the evolution of diploid organisms on the basis of variability in extant haploid organelles, the evolution of which is delinked from that of the diploid organism. A further difficulty is that it is not possible to reconstruct interspecific relationships on the basis of intraspecific variability. There is a general ignorance among proponents of «unique mother» hypotheses regarding the distribution of biological variability on the surface of the globe, a fact which renders the molecular clock inaccurate, and which upsets the simplistic proposal that molecular diversity equates with time. «Unique mother» scenarios are also invalidated by the presence of shared chromosome and other polymorphisms in african great apes and humans at similar percentages in the different lineages, a fact which indicates that these evolving populations did not experience «bottlenecks». These and other difficulties effectively refute the «Mitochondrial Eve» hypothesis, which in any case much resembles creationism of a special kind, in which the offspring of a breeding pair are visualised as belonging to a species different from its parents. Such extreme examples of the punctuational mode of evolution are highly likely to be incorrect.     

Issues related to mineralized tissue biology in human evolutionary research

This comunication has two primary aims concerned with mineralized tissue biology (e.g. hard tissue biology of bone and tooth) research in human evolutionary studies: First, to introduce the literature and the methods (at the time of this symposium) so that one has an idea of the nature of this research and where one can go for details of the methodologies, etc; Second — and of primary concern here — to discuss issues that have come to light as a result of these studies mainly because of its recent beginnings as a subfield within paleoanthropology. Issues related to skeletal studies include; 1) whether different cortical surface pattens and bone tissue types influence the appearance and interpretation of bone growth activity states; 2) if SEM analyses of cortical surfaces in fossil hominids allow one to construct meaningful representations of remodeling patterns; 3) whether these representations can be used in phylogenetic arguments; and 4) how intraspecific variability would affect these issues. Issues related to dental studies include: 1) the relationship between the rate and pattern of eraly hominid dental development; 2) experimental support for the calibration of eraly hominid dental developmental rates; and 3) whether replica techniques are suitable for microanatomical studies of these sorts.     

Human temporal bone from the lower Paleolithic site of Castel di Guido, near Rome, Italy

The Castel di Guido site, with an estimated age of approximately 300,000 years, has yielded abundant animal remains, Acheulian stone and bone bifaces, and small tools. On the surface of the original deposit, turned over by agricultural activities, fragments of human remains were discovered between 1980 and 1986, including a temporal bone described here.     

Human heart cytochrome c oxidase subunit VIII. Purification and determination of the complete amino acid sequence

Subunit VIII was purified from a preparation of the human heart cytochrome c oxidase and its complete amino acid sequence was determined. The sequence proved to be much more related to that of the bovine liver oxidase subunit VIII than to that found in bovine heart. Our finding of a ‘liver-type’ subunit VIII in the human heart enzyme suggests that either there are no isoforms of human subunit VIII or the human oxidase does not show the type of tissue specificity that has been reported for the oxidase in other mammals.     

Phosphate-Activated Glutaminase in the Crude Mitochondrial Fraction (P2 Fraction) from Human Brain Cortex

The kinetics and other properties of phosphate-activated glutaminase have for the first time been studied in the crude mitochondrial fraction (P2 fraction) from human brain. The enzyme is for unexplained reasons inactivated postmortem. The enzyme activity decreases by storing the tissue or homogenate at 37 degrees C. The inactivation is not caused by formation of a dialysable inhibiting compound. No large proteolytic degradation has occurred, since the phosphate-activated glutaminase-like immunoreactive band did not disappear during the storage. The molecular weight of the subunit of the enzyme as determined by immunoblots of sodium dodecyl sulfate-treated homogenates from human brain is estimated to be approximately 64 K. The enzyme has been shown to have a pH optimum of 8.6; it is activated by phosphate, inhibited by glutamate, and partially inhibited by ammonia. Double-inverse plots of enzyme activity against phosphate are concave-upward, and more so in the presence of an inhibitor. The inhibition by glutamate appears to be noncompetitive with the substrate glutamine, and competitive with the activator phosphate. These kinetic properties are not significantly different from our earlier observations concerning phosphate-activated glutaminase from pig brain and pig kidney.     

Studies on Muscarinic Binding Sites in Human Brain Identified with [3H]Pirenzepine

Pirenzepine, a potent antimuscarinic agent with apparent selectivity for a subtype (M1) of muscarinic receptors, was used in tritiated form to characterize its binding to human brain tissue. Specific [3H]pirenzepine binding showed rapid association and dissociation. From kinetic and competitive binding experiments, its KD was 5.5 nM and 9 nM, respectively. Regional distribution of [3H]pirenzepine binding determined in parallel with [3H]quinuclidinyl benzilate binding, a nonselective muscarinic antagonist, indicated a significant correlation for the maximum number of binding sites for the two radioligands in 13 brain regions, with the highest amount of binding for each in the putamen and the least in the cerebellum. Binding for [3H]pirenzepine averaged 57% of that for [3H]quinuclidinyl benzilate, with a range of 20% (cerebellum) to 77% (frontal cortex). Most antidepressants and neuroleptics tested had affinities for [3H]pirenzepine binding sites that were not significantly different from their previously reported values obtained with the use of [3H]quinuclidinyl benzilate.     

Quantitation of the Myelin-Associated Glycoprotein in Human Nervous Tissue from Controls and Multiple Sclerosis Patients

Myelin-associated glycoprotein (MAG) was measured by radioimmunoassay in the human CNS and peripheral nervous system (PNS). The level of MAG, expressed as ng/microgram of total protein, was approximately 20-fold higher in whole homogenates of cerebral white matter (4.7 +/- 0.60) than of peripheral nerve (0.12-0.28). MAG concentrations were only slightly higher in the isolated myelin fractions from these tissues: CNS myelin, 5.6 ng/microgram; PNS myelin, 0.37 ng/microgram. The levels of MAG were measured in nine plaques, periplaque regions, and areas of macroscopically normal-appearing white matter (NAWM) from six separate multiple sclerosis brains and compared with the levels of other myelin proteins in the same samples. MAG and other myelin proteins were reduced to very low levels in plaques. The levels of MAG and basic protein (BP) and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in periplaque areas were significantly lower than those in control white matter, and MAG and BP levels were also significantly reduced in NAWM. In a periplaque region and NAWM from the most rapidly progressing case of multiple sclerosis examined, the MAG content was between 30 and 35% of the control level, whereas BP and PLP levels and CNP activity were between 50 and 85% of control values. The reduction of MAG content in periplaque regions from all nine multiple sclerosis plaques examined was significantly greater than the reductions of BP level and CNP activity. In NAWM samples, the mean reduction of MAG content was also greater than the reductions of BP level and CNP activity, but the difference was only statistically significant in comparison to CNP.(ABSTRACT TRUNCATED AT 250 WORDS)