Determination of growth kinetics in a renal sarcoma of the rat by double labeling with 14C- and 3H-thymidine

An in vitro double-labeling technique for determination of kinetic growth parameters was tested; 13 renal sarcomas of rats were incubated after excision under standardized conditions with 14C- and 3H-thymidine successively. Another ten tumors were evaluated after in vivo double injection of 14C and 3H-thymidine into sarcoma-bearing animals. In both groups histoautoradiography was performed using a simple stripping-film method. Results including labeling indices, mitotic indices, length of DNA-synthesis phase and cell-production rate obtained by both methods were in excellent agreement. All values fitted those determined in earlier experiments using a continuous labeling and a fraction-labeled-mitoses method in vivo. Theoretical considerations on the calculation of kinetic parameters by double labeling have been added. The experimental results found in this study represent a base for evaluation of biopsies from human tumors.     

On the biochemical mechanism of tumorigenesis in mouse skin. VII. The effects of tumor promoters on 3H-choline and 3H-glycerol incorporation into mouse epidermal phosphatidylcholine in relation to their effects on 3H-thymidine incorporation into DNA.

The kinetics of the stimulation of phospholipid and DNA biosynthesis in mouse epidermis after treatment with various tumor promoting agents has been investigated. 3H-choline and 3H-glycerol were used as precursors for phosphatidylcholine. An early stimulation of 3H-choline incorporation into phosphatidylcholine is observed which always precedes the stimulation of 3H-thymidine incorporation into epidermal DNA. This is interpreted as being a manifestation of the proliferation of cellular membranes in preparation for cell division. Appreciable differences are observed in the incorporation of 3H-choline and 3H-glycerol into phosphatidylcholine, the possible reasons for which are discussed. It is concluded that 3H-choline incorporation is a more reliable parameter for the measurement of phosphatidylcholine biosynthesis than 3H-glycerol incorporation. The relationship between those effects on phospholipid synthesis and the tumor promoting activity of the substances tested is discussed.     

Stimulation of sugar uptake and thymidine incorporation in mouse 3T3 cells by calcium phosphate and other extracellular particles.

Evidence is presented that the marked stimulation of sugar uptake and thymidine incorporation by addition of extra Ca2+ to stationary phase mouse 3T3 cells in culture is phosphate dependent and due to the action of the calcium phosphate precipitate formed in the medium. The cells are similarly stimulated by a variety of particulate materials, including calcium pyrophosphate, barium sulfate, kaolin, and polystrene beads. The precipitate effects on sugar uptake are of the same magnitude as those seen with certain hormones (insulin, epidermal growth factor) or with fresh 10% calf serum. The effect of barium sulfate on thymidine incorporation is also of the same magnitude as seen with these hormones, but much less than half that found with fresh calf serum. The stimulation by barium sulfate or hormones of thymidine incorporation is not phosphate dependent.     

The nitroblue tetrazolium (NBT) reduction of neutrophil granulocytes from newborn infants and their mothers in relation to the occurrence of bacteria in amnion fluid and cord blood.

The nitroblue tetrazolium (NBT) reduction of granulocytes from cord blood, when measured without in vitro stimulation with bacteria, was significantly higher than that of maternal granulocytes. This elevated activity may be an adequate response to the presence in cord blood of degradation products from the placenta but also of potentially pathogenic bacteria, found in cord blood of infants with high NBT values. On in vitro stimulation cord blood granulocytes displayed the same high NBT activity as those of the mothers and of healthy non-pregnant women. All but one blood culture from the infants 25--48 h after delivery were sterile. The only child with a low non-stimulated NBT activity of cord blood granulocytes was also the only one presenting a clinical picture of neonatal septicemia, though not bacteriologically verified.     

Lack of correlation between morphological index and viability as assessed by the uptake of 3H-thymidine by macrophage resident M. leprae

Thirty strains of M. leprae derived from skin biopsies of lepromatous leprosy patients were scored for Morphological Index (MI) and concurrently maintained for 2 weeks in macrophage cultures containing 3H thymidine. Selective and significant incorporation of the radioactive label was observed in cultures containing freshly extracted M. leprae as compared to control cultures with autoclaved bacilli from the same biopsy. The percentage incorporation of 3H thymidine ranged from 103 to 1140%. Morphological Index of the bacilli from these individuals varied from zero to 8. Six cultures containing bacilli with MI of less than or equal to 1 and 3 containing bacilli with MI of zero showed significant incropration of 3H thymidine. There was no correlation between the percent of solid or beaded bacilli in the inoculum and the ability of M. leprae to incorporate 3H thymidine in the macrophage cultures.     

Detection by immunofluorescence of common antigenic determinants in unrelated gram-negative bacteria and their lipopolysaccharides.

Various gram-negative bacteria were subjected to mild acid hydrolysis. The acid-treated bacteria exhibited strong cross-reactivity with fluorescein isothiocyanate-conjugated antiserum to the Re mutant of Salmonella minnesota. Hydrolyzed bacteria showed considerably stronger fluorescence than heat-treated bacteria. It is assumed that acid hydrolysis uncovers shared glycolipid determinants that are responsible for cross-reactivity. Isolated homologous and heterologous lipopolysaccharides were allowed to react with antibody to S. minnesota Re insolublized by covalent binding to aminohexyl Sepharose 4B. The resulting antigen-antibody complexes were visualized by exposure to the fluorescent antiserum. This treatment allows the demonstration of glycolipid structures of intact lipopolysaccharides.     

The effect and possible clinical efficacy of in vivo inhibition of neutrophil extracellular traps by blockade of PI3K-gamma on the pathogenesis of microscopic polyangiitis

Objective: Neutrophil extracellular traps (NETs) are peculiar structures composed of the externalized chromatin with intracellular proteins and formed by activated neutrophils in a reactive oxygen species (ROS)-dependent manner. Aberrant NETs are considered to be autoantigens for anti-neutrophil cytoplasmic antibodies (ANCAs) underling the development of microscopic polyangiitis (MPA). However, little is known regarding the therapeutic efficacy of in vivo inhibition of NET formation (NETosis) on MPA pathogenesis. This study determines whether reducing NETosis prevents ANCA production and improves characteristic involvement.

Methods: A mouse model of MPA induced by administering a novel extract from Candida albicans was devised. By applying this method to mice lacking phosphoinositide 3-kinase gamma (PI3K-gamma), which is indispensable for ROS production in neutrophils, we investigated the levels of in vivo NETs, ANCA titers and histological damage.

Results: Our model exhibited accumulation of NETs in vivo, elevation of ANCA titers and characteristic pathologies mimicking human MPA, including small-vessel vasculitis and crescentic glomerulonephritis. Strikingly, these abnormalities were reduced by genetically and/or pharmacologically blocking PI3K-gamma. Moreover, a pharmacological PI3K-gamma blockade decreased the levels of human NETs.

Conclusion: Our results suggest that in vivo inhibition of NETosis by blocking PI3K-gamma could be a promising therapeutic strategy for the pathogenesis of MPA.     

Modulation of human neutrophil chemotactic responses by cyclic 3',5'-guanosine monophosphate and cyclic 3',5'-adenosine monophosphate.

Cyclic 3',5'-guanosine monophosphate (cGMP) and cyclic 3',5'-adenosine monophosphate (cAMP) and compounds known to effect the intracellular concentrations of these nucleotides were examined for their ability to effect human neutrophil (PMN) responsiveness to chemotactic stimulation. Incubation of neutrophils with agents recognized to promote increases in intracellular cAMP in a variety of tissues (i.e., epinephrine, norepinephrine, isoproterenol, histamine, cholera toxin, and prostaglandin E-1 and E-2) or with cAMP inhibited the leukotactic response to a bacterial chemotactic factor. In contrast, cGMP and compounds which have been shown to promote increases in intracellular cGMP concentration (i.e., acetylcholine, carbamylcholine, phorbol myristate acetate, and prostaglindin F-2-alpha) markedly enhanced the neutrophil chemotactic response. The inhibitory or stimulatory influences on chemotactic responsiveness promoted by several of the agents could be shown to be blocked by a specific pharmacologic antagonist of the particular compound tested. These data support the hypothesis that cGMP and cAMP can provide opposing regulatory influences on certain cellular functions; in this case, directed motility of leukocytes.     

Metabolic breakdown of [3H]thymidine and the inability to measure human lymphocyte proliferation by incorporation of radioactivity.

Quantitative measurement of the incorporation of tritiated thymidine into cultures of phytohemagglutinin-stimulated lymphocytes is routinely used as an indication of the immunocompetence of the cells and of their proliferation. The present experiments show that several components of human blood catabolize nucleosides, including thymidine, extensively. Most of the radioactivity from tritiated thymidine, for example, is quickly rendered unincorporable as the compound is metabolized to thymine and further breakdown products. Thus, cells continue to proliferate without incorporating radioactivity from the medium. Furthermore, variability in the degree of catabolism has been found from person to person, so that neither measurement of the depletion of radioactivity from the medium nor measurement of the amount of label incorporated into the cultures can be used as a quantitative indicator of cell proliferation or immunocompetence.     

Formation of steroids by the pregnant mare. V. Metabolism of 14C-isopentenylpyrophosphate and 3H-dehydroisoandrosterone injected into the fetus.

A mixture of 1-14C-isopentenylpyrophosphate and 3H-dehydroisoandrosterone was injected into a horse fetus intramuscularly during laparotomy, after which maternal urine was collected for 4 days. Steroid conjugates in the urine were extracted with Amberlite XAD-2 resin, hydrolysed and separated into phenolic and neutral fractions. From the phenolic fraction estrone, 17alpha-estradiol, equilin and equilenin were isolated. Only estrone and 17alpha-estradiol contained both 3H and 14C, while the ring B unsaturated estrogens contained only 14C. From the neutral fraction 14C-labeled 3beta-hydroxy-5alpha-pregnan-20-one, 5alpha-pregnane-3beta,20beta-diol and 5alpha-pregnan-3beta, 20alpha-diol were isolated. These results demonstrate that the route of biosynthesis of both the ring B saturated and unsaturated estrogens is the same up to the stage of isopentenylpyrophosphate. Thus, the bifurcation in the classical pathway of steroid biosynthesis reported previously by us is occurring at a point after the formation of isopentenylpyrophosphate and prior to the formation of squalene.     

Myeloid-related protein 8/14 complex is released by monocytes and granulocytes at the site of coronary occlusion: a novel, early, and sensitive marker of acute coronary syndromes.

AIMS: We investigated whether myeloid-related protein 8/14 complex (MRP8/14) expressed by infiltrating monocytes and granulocytes may represent a mediator and early biomarker of acute coronary syndromes (ACS). METHODS AND RESULTS: Immunohistochemistry of coronary thrombi was done in 41 ACS patients. Subsequently, levels of MRP8/14 were assessed systemically in 75 patients with ACS and culprit lesions, with stable coronary artery disease (CAD), or with normal coronary arteries. In a subset of patients, MRP8/14 was measured systemically and at the site of coronary occlusion. Macrophages and granulocytes, but not platelets stained positive for MRP8/14 in 76% of 41 thrombi patients. In ACS, local MRP8/14 levels [22.0 (16.2-41.5) mg/L] were increased when compared with systemic levels [13.4 (8.1-14.7) mg/L, P = 0.03]. Systemic levels of MRP8/14 were markedly elevated [15.1 (12.1-21.8) mg/L, P = 0.001] in ACS when compared with stable CAD [4.6 (3.5-7.1) mg/L] or normals [4.8 (4.0-6.3) mg/L]. Using a cut-off level of 8 mg/L, MRP8/14 but not myoglobin or troponin, identified ACS presenting within 3 h from symptom onset. CONCLUSION: In ACS, MRP8/14 is markedly expressed at the site of coronary occlusion by invading phagocytes. The occurrence of elevated MRP8/14 in the systemic circulation prior to markers of myocardial necrosis makes it a prime candidate for the detection of unstable plaques and management of ACS.     

In vivo conversion of tryptophan to nicotinic acid in rats studied by simultaneous incorporation of [3H]-tryptophan and [14C]-nicotinic acid into liver NAD and NADP

This study was carried out with three groups of weanling rats. One group was fed a high-protein (20%) diet, another group a low-protein (2.5%) diet, the third group a high-protein diet in restricted amounts. After 4 weeks of feeding, rats were injected simultaneously with L-[G-3H]-tryptophan and [carboxyl-14C]-nicotinic acid. The ratio of incorporation of [3H]-tryptophan to that of [14C]-nicotinic acid into liver NAD and NADP was found to be higher in protein-restricted rats. On the other hand, the ratio was found to be reduced in diet-restricted group of rats compared with ad libitum fed or low-protein diet fed groups. These results suggest that the efficiency of conversion of tryptophan to NAD is increased in protein deficiency, but reduced in the diet restriction. These observations are in line with our earlier findings on the changes in liver quinolinate phosphoribosyltransferase (EC 2.4.2.19) activity following feeding of low-protein or restricted diets. It is suggested that this technique of measuring the incorporation of two isotopes from the substrates labelled with two different isotopes can be conveniently used as a tool to measure the relative contribution of tryptophan and nicotinic acid to the synthesis of nicotinamide nucleotides.     

Enhancement of [3H-methyl]thymidine incorporation and replication of rat chondrocytes grown in tissue culture by plasma, tissue extracts and vasopressin.

A pituitary mitogenic peptide, which stimulates cellular replication of a variety of cells maintained in tissue culture, has been identified by other investigators. To study this mitogenic substance, we developed an assay to measure mitogenic substances utilizing fetal rat chondrocytes grown in monolayer culture. Mitogenic activity of added test substances was determined by [3H-methyl]thymidine incorporation into trichloroacetic acid insoluble cell products and increase in total cell number after 24 h exposure. Extracts of whole pituitary glands were more potent in stimulating these cellular indices than either those of liver or muscle, confirming that the chondrocytes are sensitive to the described mitogen. Identically prepared extracts of either anterior or posterior pituitary lobes were mitogenic indicating the presence of two or more mitogenic substances in crude pituitary extracts. Synthetic lysine vasopressin and a beef pitressin concentrate stimulated thymidine incorporation into chondrocytes in the absence of calf serum and this effect was additive to that of calf serum, suggesting that the mitogenic substance of posterior pituitary extracts was vasopressin. The maximum effective dose of vasopressin leading to an increase in either thymidine incorporation or total cell number was between 100 to 500 pg/ml, and as little as 50 pg/ml of hormone elicited an increase in total cell number. The mitogenic effect of both vasopressin and calf serum on chondrocytes was partially inhibited by 1 X 10(-4)M N, O'dibutryl cyclic adenosine 3',5' monophosphate suggesting that cell division of chrondrocytes may be under tonic control by the andenylyl cyclase system. We conclude that vasopressin is a potent mitogen for chondrocytes maintained in tissue culture and its presence must be rigorously excluded in evaluating mitogenic activity of pituitary or serum concentrates.     

Activation of human eosinophil and neutrophil functions by haematopoietic growth factors: comparisons of IL-1, IL-3, IL-5 and GM-CSF.

We compared the effect of haematopoietic growth factors granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-1, IL-3, and IL-5 on the functional activation of human eosinophils and neutrophils from the same donor. All four colony-stimulating factors (CSF) enhanced the phagocytosis of Candida albicans by eosinophils and increased staphylococcal, but not Candida, killing. GM-CSF and IL-5 had a profound stimulating effect on eosinophil staphylocidal activity. GM-CSF and IL-3 enhanced the generation of leukotriene C4 (LTC4) induced by calcium ionophore A23187 and the release of arylsulphatase and beta-glucuronidase from specific and small granules of eosinophils. In contrast, IL-1 and IL-5 had no effect on degranulation. GM-CSF and IL-1 enhanced phagocytosis of C. albicans by neutrophils, and GM-CSF stimulated degranulation and the release of the enzymes beta-glucuronidase and arylsulphatase from neutrophils while IL-1 stimulated the release of arylsulphatase only. This study indicates that the eosinophil-active colony-stimulating factors can markedly enhance the host defence function of the eosinophil and even make it the equal of the neutrophil in staphylocidal activity. The CSF-activated eosinophil, however, may cause inappropriate inflammation and normal tissue damage.