The application of a flow cytometric assay for evaluation of phagocytosis of neutrophils.

Phagocytosis and the release of oxidative products generated by the respiratory burst have been studied in vitro under the influence of non-steroidal anti-inflammatory drugs: naproxen and ibuprofen, using phagocytes of peripheral blood from healthy human donors. Phagocytosis was monitored by flow cytometry in order to investigate the uptake of propidium iodide-labelled bacteria (Staphylococcus aureus) by polymorphonuclear leucocytes. In addition, the phagocytic capacity and percentage of killed bacteria was measured in isolated neutrophils using the Pantazis & Kniker method. It was found that naproxen and ibuprofen affect the phagocytic function and hydrogen peroxide production in the examined granulocytes. These methods might be useful in investigations on neutrophil functions.

[Sialic acid level in blood platelets after cryopreservation with DMSO]. / Poziom kwasów sialowych krwinek plytkowych przechowywanych po kriopreparatyce z DMSO.

Plasma platelet concentrates of man were subjected to preparatory procedures with dimethylsulphoxide (DMSO) as a cryoprotective agent. It was demonstrated that DMSO acting directly on the platelets in concentrations used in routine preparation had no effect decreasing the level of sialic acids in the platelets. Cryopreservation of platelets in presence of DMSO caused the loss of 30% of the sialic acids from the platelets. Further storage at room temperature of defrosted platelet concentrates caused no further decrease of sialic acid level in these cells. ++Cryopreservation decreased also the aggregation ability of the platelets after induction with ADP but simultaneous action of ADP and succinate increased the aggregation of ++cryopreserved platelets.

[Effect of prednisone and typhoid vaccine on the size of systemic neutrophil granulocyte pool]. / Wplyw prednisonu i szczepionki durowej na wielkosc ustrojowej puli granulocytów obojetnochlonnych

Changes of total granulocyte pool in the organism were evaluated after administration of prednisone or typhoid vaccine. For this purpose the unsaturated capacity of serum vitamin B12 binding was determined. After administration of prednisone the rise of granulocytes in peripheral blood was associated with a rise in the unsaturated capacity of vitamin B12 binding by the serum which was not observed after administration of typhoid vaccine. This observation confirmed the suggestions that corticosteroids not only mobilize the bone marrow reserve of granulocytes but prolong also their survival in the organism.

[Lysozyme activity (LZM) and unsaturated vitamin B-12-binding capacity (NZW vit B-12) in the serum following prednisone administration in patients with bone marrow aplasia with pancotypenia]. / Zachowanie sie aktywnosci lizozymu (LZM) i nienasyconej zdolnosci wiazania witaminy B12 (NZW wit. B12) w surowicy pod wplywem prednisonu u chorych z aplazja szpiku z pancytopenia.

In 18 patients with bone marrow aplasia with pancytopenia lysozyme activity and unsaturated vitamin B12-binding capacity in the serum were determined. These investigations, together with determinations of peripheral blood polymorphonuclear count, were done again after prednisone administration. In all cases a significant fall was found in the NZW vit B12 and LZM activity in the serum. A slight rise in the polymorphonuclear count in the 24th hour of the study was associated with a rise in the NZW wit. B12 in the serum, and decreased LZM activity. This confirmed the previously demonstrated complex character of corticosteroid action on the system of polymorphonuclears. These results point also to the usefulness of determination of unsaturated vitamin B12-binding capacity for evaluating the value of the total granulocyte pool in granulocytopenia.

[Usefulness of hydrocortisone test for assessment of bone marrow reserve in neutrophils]. / Przydatnosc testu hydrokortzonowego dla oceny wielkosci rezerwy szpikowej granulocytów obojetnochlonnych.

The activity of lysozyme and unsaturated binding capacity of vitamin B12 in the serum were determined in hydrocortisone test for calculating the reserve of granulocytes in bone marrow. A significant rise in the count of neutrophils in peripheral blood 3 hours after the beginning of the test was not associated with statistically significant changes in the determined parameters. This in an evidence that prolongation of the survival of cells in the circulation as one of the mechanisms of corticosteriod action on the granulocyte system is without any greater importance. This is connected, most probably, with short duration of the test. The rise of neutrophil count in peripheral blood after hydrocortisone administration may be regarded, therefore, as a measure of the value of one marrow reserve of these cells.

Stabilization of transcobalamins against heating on sephadex G-50.

The human serum stabilized on Sephadex G-50 did not show any changes in the distribution of radioactive vitamin B12 bound to transcobalamins during incubation at 56 degrees C (30 and 120 min.) and 80 degrees C (20 min.), whereas, in the non-stabilized serum, heating increased the vitamin 57CoB12 in transcobalamin I and III fraction and decreased or completely abolished the amount of radioactive vitamin B12 bound to transcobalamin II.

Effect of ethylenediamine-tetra-acetate, sodium, iodoacetate and potassium cyanide on the release of cobalophilins from polymorphonuclear granulocytes during phagocytosis.

The influence of ethylenediamine-tetra-acetate (EDTA, 10(-3) M, 10(-5) M and 10(-7) M), sodium iodoacetate (CH2 . I. COONa, 10(-4) M and 10(-6) M) and potassium cyanide (KCN, 10(-2) M, 10(-3) M and 10(-5) M) on the release of cobalophilins (vitamin B12 binding proteins) from polymorphonuclear granulocoytes (PMN) was studied. The agents mentioned above reduced the release of cobalophilins from resting and functionally stimulated granulocytes. This effect increased with the growth of concentration of these agents in the sample. The inhibitory effect of EDTA, CH2 . I. COONa and KCN on phagocytosis-activated cobalophilins release occurred irrespective of the time of granulocytes stimulation. This could be observed in these experiments, where granulocytes were first affected by these chemical agents and then stimulated functionally, as well as in those samples where EDTA, CH2 . I . COONa, and KCN influenced the cells after incubation with latex particles. The inhibitory effect of EDTA was diminished in the presence of a higher concentration of calcium ions in an incubation medium. On the contrary, CH2 . I . COONa reduced the release of cobalophilins from PMN during phagocytosis irrespective of the concentration of calcium ions in the medium.

The influence of phagocytosis on transcobalamins releasing from polymorphonuclear granulocytes of patients with Hodgkin's disease.

The release of transcobalamins I and III (TC-I/III) during phagocytosis of latex particles from polymorphonuclear granulocytes of healthy persons and patients with Hodgkin's disease were studied. Our investigations indicate that phagocytosis stimulates the release of these proteins in both examined groups. The values of the patients with I and II stage of disease did not differ distinctly from the results of healthy persons. This preliminary studies will be extended to patients with advanced Hodgkin's disease. In addition, our results may suggest the influence of phagocytosis on the activation of TC-I/III synthesis.

Inhibitory influence of methotrexate and vincristine on the release of cobalophilins from polymorphonuclear granulocytes.

The studies have evaluated the effect of methotrexate and vincristine on the release of cobalophilins (vitamin B12 binding proteins) from resting and functionally stimulated polymorphonuclear granulocytes (PMN). Methotrexate (2.5 micrograms/ml; 5.0 micrograms/ml; 20.0 micrograms/ml; and 50.0 micrograms/ml) and vincristine (0.3 microgram/ml; 0.6 microgram/ml; 2.4 micrograms/ml; and 6.0 micrograms/ml) inhibited the cobalophilins release from resting granulocytes. This effect increased with growing concentrations of these drugs. Stimulated PMN could be shown to release cobalophilins more actively than resting granulocytes. Methotrexate (2.5 micrograms/ml; 5.0 micrograms/ml and 20.0 micrograms/ml) and vincristine (0.3 microgram/ml; 0.6 microgram/ml and 2.4 micrograms/ml) inhibited the phagocytosis-activated release of cobalophilins irrespective of the time of PMN stimulation, i.e. before or after being incubated with latex particles.

Inhibition of the vitamin B12 binding capacity of proteins by the hydrolysis product of cyclophosphamide.

The inhibitory effect of cyclophosphamide hydrolysis product (CPHP) on vitamin B12 binding ability to proteins has been established. The ester N-(2-chloroethyl)-N'-(3-phosphopropyl)-ethylenediamine hydrochloride is probably responsible, in vitro, for blocking the protein binding sites. Preincubation of proteins with vitamin B12 prevents the inhibitory effect of CPHP.

Urinary glycosaminoglycans in patients with hypothyroidism and in healthy subjects.

Although the changes in urinary glycosaminoglycans have been investigated in several endocrinopathies, no information was hitherto available on the content and composition of urinary glycosaminoglycans in hypothyroidism. Urinary glycosaminoglycans were therefore investigated in patients with hypothyroidism and in healthy subjects. The total daily excretion of urinary glycosaminoglycans was found to be significantly increased (by 41%) in hypothyroidism. Two electrophoretic bands were always detected in both examined groups: a major band of chondroitin sulphate and a minor band of heparan sulphate. Heparan sulphate and chondroitin sulphate levels were respectively 114% and 42% higher in patients with hypothyroidism than in controls. The respective increases in chondroitin-4-sulphate and chondroitin-6-sulphate were 31% and 41%. The relative quantities of chondroitin-4-sulphate, dermatan sulphate, chondroitin-6-sulphate and non-sulphated chondroitin sulphate were unchanged in the two examined groups. The changes observed in the levels of the excreted glycosaminoglycans may reflect the altered metabolism of connective tissue in hypothyroidism.

Effect of tannic acid on benzo[a]pyrene-DNA adduct formation in mouse epidermis: comparison with synthetic gallic acid esters.

Tannic acid, a naturally occurring plant phenol, was shown to inhibit the mutagenicity and/or tumorigenicity of several polycyclic aromatic hydrocarbons in mouse skin. In this study the effect of topical application of tannic acid on epidermal aryl hydrocarbon hydroxylase, glutathione S-transferase, and binding of benzo[a]pyrene (B[a]P) to epidermal DNA was compared with the activity of synthetic gallic acid esters. Single topical application of 8 mumol octyl and dodecyl gallate had no effect on the induction of aryl hydrocarbon hydroxylase, whereas propyl gallate and tannic acid increased the enzyme activity by nearly 200%. Application of the phenolics one hour before 0.2 mumol of B[a]P enhanced the enzyme activity, but the observed differences were not significant in comparison with a B[a]P-treated group of mice. Application of dodecyl and octyl gallates to mouse skin resulted in three- and twofold increases, respectively, in the activity of glutathione S-transferase. Combined treatment with dodecyl gallate and B[a]P also resulted in significant enhancement of this enzyme activity. Application of the same dose of tannic acid to mouse skin one hour before the application of 0.2 or 1 mumol of B[a]P afforded 60% inhibition of covalent benzo[a]pyrene-diol-epoxide binding to epidermal DNA. Gallic acid esters with the exception of dodecyl gallate were less effective inhibitors of benzo[a]pyrene-diol-epoxide binding, especially when the higher dose of B[a]P was used. These results indicate that the antitumorigenic activity of tannic acid involves the interaction of the ultimate carcinogen with DNA rather than an altered metabolism. The linkage between gallic acid and glucose in natural plant phenols is also more effective at inhibiting B[a]P binding to epidermal DNA than the linkage with the alkyl group in synthetic gallates.

Unsaturated vitamin B12 binding capacity and serum lysozyme activity during the marrow granulocytes reserve tests.

Unsaturated vitamin B12 binding capacity (UBBC) and serum lysozyme activity (LZM) were estimated durinng the endotoxin, prednisone and hydrocortisone marrow granulocyte reserve (MGR) pool tests. Our results showed, that no additional mechanism except the shift of MGR from marrow caused granulocytosis after typhoid vaccine administration. While the prednisone, when given orally diminished additionally the number of the physiologically destroyed neutrophils. The hydrocortisone, however, showed the results very similar to those obtained after typhoid vaccine adminstration. Thus the hydrocortisone test seems to be most useful. It gives as good information as typhoid vaccine test but does not show its side-effects.