Phenotypic and functional profile of peripheral blood mononuclear cells isolated from melanoma patients undergoing combined immunotherapy and chemotherapy

In the present study we tested the phenotypic profile as well as several immunological responses of peripheral blood mononuclear cells (PBMC) isolated from melanoma patients. These patients underwent chemotherapy with dacarbazine and carboplatin from day 1 to day 22, followed by immunotherapy of low-dose recombinant interleukin-2 and recombinant interferon administered subeutaneously from day 36 to day 75. The PBMC from 14 patients were isolated on day 0 before chemotherapy. on day 36 after chemotherapy and on day 76 after immunotherapy. After chemotherapy, a decrease in CD16⁺ cells and increase in CD3⁺ and CD4⁺ cells correlated with a significant decrease in the generation of lymphokine-activated killer (LAK) activity. After immunotherapy, an increase in CD16⁺ cells correlated with an increase in the induction of LAK activity. A comparison between responding and non-responding patients revealed statistically significant differences in LAK activity of PBMC and response to concanavalin A following chemotherapy, and in the percentage of CD8⁺ cells following immunotherapy. Our results point toward the value of continuing such a study on a larger population of cancer patients in order to select the appropriate bioassays for monitoring and predicting the clinical responsiveness to combined therapies.     

The Role of Ligand Exchange in the Uptake of Iron from Microbial Siderophores by Gramineous Plants

The siderophore rhizoferrin, produced by the fungus Rhizopus arrhizus, was previously found to be as an efficient Fe source as Fe-ethylenediamine-di(o-hydroxphenylacetic acid) to strategy I plants. The role of this microbial siderophore in Fe uptake by strategy II plants is the focus of this research. Fe-rhizoferrin was found to be an efficient Fe source for barley (Hordeum vulgare L.) and corn (Zea mays L.). The mechanisms by which these Gramineae utilize Fe from Fe-rhizoferrin and from other chelators were studied. Fe uptake from 59Fe-rhizoferrin, 59Fe-ferrioxamine B, 59Fe-ethylenediaminetetraacetic acid, and 59Fe-2[prime]-deoxymugineic acid by barley plants grown in nutrient solution at pH 6.0 was examined during periods of high (morning) and low (evening) phytosiderophore release. Uptake and translocation rates from Fe chelates paralleled the diurnal rhythm of phytosiderophore release. In corn, however, similar uptake and translocation rates were observed both in the morning and in the evening. A constant rate of the phytosiderophore's release during 14 h of light was found in the corn cv Alice. The results presented support the hypothesis that Fe from Fe-rhizoferrin is taken up by strategy II plants via an indirect mechanism that involves ligand exchange between the ferrated microbial siderophore and phytosiderophores, which are then taken up by the plant. This hypothesis was verified by in vitro ligand-exchange experiments.     

Modification of the hypothermic circadian cycles induced by DSIP and melatonin in pinealectomized and hypophysectomized rats

Both melatonin and DSIP (a nine amino acid peptide) effects have been previously shown to be (a) circadian rhythm related and (b) involved in inducing hypothermic effects in rats. In this study we report the hypothermia effects by each of these drugs alone and in combination when studied in normal (unoperated), pinealectomized, and hypophysectomized rats at various time points of the corresponding circadian cycle. A clear differential effect of drugs × time × preparation was found. While both DSIP and melatonin hypothermic effects were both circadian cycle dependent in intact rats the rhythmicity of melatonin hypothermic effect in pinealectomized rats, and DSIP hypothermic effect in hypophysectomized rats was missing. Although several hypotheses have been offered to account for the physiological mechanism(s) that govern the effects of the drugs, it is not yet possible to reliably relate the findings to existing neurochemical theory.     

Protein synthesis in resting and growth-stimulated human peripheral lymphocytes : Evidence for regulation by a non-messenger RNA

Stimulation of lymphocyte growth is accompanied by an early increase in the rate of protein synthesis. This increase is dependent upon the flow of inactive free ribosomes into polysomes, which is limited by a rate-controlling step at initiation [2]. Addition of actinomycin D (actD) to lymphocytes caused a gradual reduction in protein synthesis in resting cells, but rapidly inhibited both the elevation of protein synthesis and the activation of free ribosomes which normally follow exposure to mitogens. Since actD does not affect protein synthesis in enucleated lymphocytes [4], the effect in intact cells must be mediated by a nuclear event, which available data indicate is RNA synthesis. ActD prevented the accumulation of 80S initiation complexes which normally occurs in resting lymphocytes treated with pactamycin and cycloheximide, showing that its locus of action was at some point in initiation. The decline in rate of protein synthesis began without detectable lag when resting lymphocytes were treated with actD. However, after growth stimulation, a delay of ca 50 min occurred before the protein synthetic rate declined in response to actD. These observations agree with the hypothesis that the concentration of some moderately short-lived RNA is rate-limiting for protein synthesis in resting lymphocytes, and that an early event in growth stimulation is a rise in the amount of this component to levels which are no longer rate-limiting. This permits an increased flow of ribosomes into polysomes and a consequent rise in protein synthesis. Available evidence indicates that the regulatory RNA is neither mRNA nor rRNA, but may either be one of the small cytoplasmic RNAs whose function is unknown, or tRNA_i^met.     

Actin-activated ATPase from human erythrocytes

A fibrillar protein complex, possessing ouabain-insensitive Ca²⁺-ATPase activity was isolated from human erythrocyte membranes by using a low ionic strength extraction procedure. Mg²⁺-ATPase activity was revealed upon addition of rabbit skeletal muscle actin, thus demonstrating the presence of a myosin-like protein in the crude extract of the erythrocyte membrane. Upon sodium dodecylsulfate gel electrophoresis, the extract showed mainly the doublet of subunit molecular weight bands of 230 000 and 210 000, and more than 10 faster moving hands. Gel filtration of the erythrocyte membrane extract on Sepharose 4B furnished 4 fractions. Fraction I, containing the doublet and 80 000, 60 000 and 46 000 subunit molecular weight bands was 5-fold purified with respect to Ca²⁺-ATPase activity, but was devoid of actin-activated Mg²⁺-ATPase activity. Fraction II, containing only the doublet, was devoid of Ca²⁺ and actin-activated Mg²⁺-ATPase activity. The 210 000 subunit molecular weight protein could be phosphorylated in the presence of Mg²⁺ in the crude extract and Fraction I but not in Fraction II.     

Membrane-bound ATPase in chloroplasts of Euglena gracilis

Membrane-bound ATPase activities in chloroplasts of Euglena were examined. Ca²⁺- and Mg²⁺-dependent activities were relatively high in membrane preparations and could not be further activated by a number of procedures. The enzyme was found to be highly specific for purine nucleotides and was inhibited by the usual inhibitors of photophosphorylation. K_m values of Ca²⁺ and Mg²⁺ ATPase for ATP were 2.5 and 2.1 mM, respectively. Both activities were competitively inhibited by ADP and inorganic phosphate. A relationship was found between Ca²⁺- or Mg²⁺-dependent ATPase activities and chloroplast completeness. The possibilities that these activities result from one enzyme depending on Ca²⁺ or Mg²⁺ or from two different enzymes are discussed.     

The need for immune evaluation prior to thymosin-containing chemoimmunotherapy for melanoma

Summary In an attempt improve the response to BCG or BCG + DTIC therapy in Stage IIIB melanoma patients, we added thymosin treatment with repeated doses of 4 mg/m² or 40 mg/m². Twenty-eight patients were clinically and immunologically evaluable. Pretreatment immunological evaluation consisted of determination of delayed-type hypersensitivity to recall antigens, enumeration of E-rosettes in blood, and measurement of blood lymphocyte response to phytohemmagglutinin (PHA) and concanavalin A (Con-A). The disease-free interval was correlated with thymosin dose and parameters of immunocompetence. Immunocompetent melanoma patients treated with a high thymosin dose and BCG relapsed earlier than those treated with a low thymosin dose and BCG. Thus, 72% of the melanoma patients who had a PHA SI50 and were treated with 4 mg thymosin/m², were disease-free at 9 months, compared with only 31% of those treated with 40 mg/m² (P=0.02). When the dermatophytin delayed hypersensitivity response (>10 mm induration) was used as a parameter of immunocompetence, 86% of the patients treated with 4 mg/m², were disease-free at 9 months, as against 28% of patients treated with 40 mg thymosin/m² (P=0.07). None of the immunoincompetent patients on high thymosin dose relapsed (0/3). The results suggest that while a high thymosin dose (40 mg/m²) may be detrimental to immunocompetent patients, it may have a beneficial effect in immunoincompetent patients. A low thymosin dose is probably not detrimental to immunocompetent melanoma patients in this study. Monitoring of the immune status prior to and during the use of thymosin in cancer immunotherapy is mandatory.This paper was presented at the Thirteenth Annual Meeting of the American Society for Clinical Oncology, Denver, 1977