Biomedical subjects

A M Mastro

Publications and source records attributed to A M Mastro.

At least 55 records · Page 3Linked to original sources

PubMed1992

Selenium deficiency alters the lipoxygenase pathway and mitogenic response in bovine lymphocytes.

We investigated the effect of altered selenium (Se) nutrition on arachidonic acid oxidation in immune cells. Experiments were conducted with peripheral blood lymphocytes obtained from dairy cattle fed diets either supplemented with or deficient in Se. The results indicate that the concanavalin A-stimulated lymphocyte proliferation was significantly lower in Se-deficient cows. When stimulated by calcium ionophore A23187, the lymphocytes derived from Se-deficient cows produced significantly less 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4) than those obtained from Se-supplemented cows. When included in cell cultures from animals fed +Se diets, 5-HETE and LTB4 elicited a partial reversal of the inhibition of lymphocyte proliferation by either hydrocortisone or nordihydroguaiaretic acid. Based on this information, we postulate that dietary Se status, which in turn determines tissue Se concentration, plays an important role in the regulation of arachidonate metabolism by way of the 5-lipoxygenase pathway. This may be one of the biochemical mechanisms underlying the inhibition of lymphocyte proliferation and the decrease in resistance to infectious diseases observed in Se-deficient animals.

Animals↗

PubMed1992

Effect of spaceflight on lymphocyte proliferation and interleukin-2 production.

Studies of peripheral blood lymphocytes from astronauts indicate that microgravity depresses T-cell responsiveness. However, this effect has not been examined in cells of peripheral lymphatic tissue, where most lymphocytes are located. In this study, inguinal lymph node lymphocytes from rats flown on the COSMOS 2044 mission were tested for proliferation and interleukin-2 (IL-2) production. Cells cultured with mitogenic lectins, phorbol ester, and calcium ionophore, or T-cell mitogen and lymphokine, were assayed for DNA synthesis by [3H]thymidine incorporation. Lymphocytes incubated with a T-cell mitogen alone also were tested for IL-2 production. Proliferation of lymphocytes from flight rats was not significantly different from controls for any of the mitogens tested. Furthermore, lymph node lymphocytes from flight rats was not significantly different from controls for any of the mitogens tested. Furthermore, lymph node lymphocytes from control and flown rats produced similar amounts of IL-2. Thus microgravity may act on lymphocytes in a tissue-specific manner, a new finding that could impact on the evaluation of spaceflight effects on immunocompetence.

Animals↗

PubMed1992

Characterization of the inhibition of interleukin 2 mRNA accumulation by 12-O-tetradecanoylphorbol-13-acetate in primary lymphocytes.

We found previously that bovine lymph node cells (LNC) incubated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 18 h proliferate only to a limited degree on subsequent stimulation with concanavalin A (Con A), or with the comitogenic combination of Con A plus TPA. The lack of proliferation was traced to a lack of secretion of interleukin 2 (IL-2). Lack of secretion was paralleled by a decrease in IL-2 mRNA levels. In this study we further characterized how TPA pretreatment affected IL-2 mRNA production. We found that TPA depressed IL-2 mRNA accumulation in a dose-dependent manner after at least 10 h of pretreatment. In contrast, pretreatment from 4 to 6 h augmented IL-2 mRNA accumulation. Furthermore, LNC stimulated with ionomycin plus TPA were less susceptible to inhibition by pretreatment with TPA, most likely because this mitogenic combination caused a much greater amount of IL-2 mRNA than did Con A or Con A plus TPA. Finally, a protein synthesis inhibitor, cycloheximide, partially counteracted the negative effects of TPA on IL-2 mRNA accumulation and on proliferation. These results suggest that TPA, probably acting through protein kinase C, initially augments the production of IL-2 mRNA but subsequently induces mechanisms to decrease the level of mRNA.

Animals↗

PubMed1991

Increased 12-HETE production in bovine lymphocytes during selenium deficiency.

When peripheral blood lymphocytes were incubated with arachidonic acid in the presence of Ca++ ionophore (A23187), the cells from the selenium-deficient dairy cows produced significantly greater quantities of 12-hydroxyeicosatetraenoic acid (12-HETE) than the cells from the selenium-supplemented animals. The major product of reaction was verified as 12-HETE by cochromatography with a 12-HETE standard on an HPLC and structural analysis by GC-MS. Additionally, concanavalin A-stimulated lymphocyte proliferation was significantly decreased in cells from the Se-deficient cows. Furthermore, 12-HETE generated by the A23187-stimulated lymphocytes inhibited lymphocyte proliferation when added to Se-supplemented cell cultures. These observations suggest that self-regulation of lymphocyte proliferation might be mediated by 12-HETE production, especially during an altered nutritional state such as Se deficiency.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗

PubMed1991

Differential activation and inhibition of lymphocyte proliferation by phorbol esters, mezerein, teleocidin, and okadaic acid.

Lymphocytes can be stimulated to proliferate in vitro by mitogens such as concanavalin A. The tumor-promoting phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) can enhance this proliferation, partly because of an increase in interleukin 2 (IL-2) production. However, if lymphocytes are treated with TPA for 24 h before concanavalin A exposure, IL-2 production and proliferation are depressed. The target of the action of TPA is protein kinase C, which is activated after a short exposure but down-regulated after a longer one. This study was designed to determine if the modulation of IL-2 was separable from the modulation of protein kinase C. When phorbol esters phorbol 12-retinoate-13-acetate, phorbol 12,13-dibutyrate, 12-deoxyphorbol 13-phenylacetate, and 12-deoxyphorbol 13-phenylacetate-20-acetate, as well as nonphorbol tumor promoters mezerein, telocidin, and okadaic acid, were tested, all but okadaic acid reproduced the effects of TPA. However, 12-deoxyphorbol 13-phenylacetate and 12-deoxyphorbol 13-phenylacetate-20-acetate were required at nearly 100-fold higher concentrations than TPA to suppress IL-2 production, suppress mitogenesis, and cause down-regulation of protein kinase C. A comparison of structures indicated that an R group at the 12-position was less important for IL-2 production and mitogenesis than for down-regulation of protein kinase C and the suppression of mitogenesis. In no case was the modulation of protein kinase C separated from the effects on IL-2 production and proliferation.

Animals↗

PubMed1991

Effect of hindlimb suspension simulation of microgravity on in vitro immunological responses.

The effects of microgravity on the immune system are largely unknown, but understanding such effects becomes increasingly important as space exploration continues and mission duration increases. Reductions in postflight human T cell reactivity to mitogens is well documented. Similar results have been obtained using a clinostat as an in vitro model of microgravity. In this study, a rat tail suspension model of weightlessness was used to examine in vitro lymphocyte proliferation in response to mitogens. Experiments were designed to uncover potential deficits in events related to proliferation including cell surface protein and IL-2 receptor (IL-2R) expression, interleukin-2 (IL-2) production, and accessory cells. Suspension of rats for 1 week led to a significant depression in [3H]thymidine incorporation by mitogen-stimulated peripheral blood lymphocytes (PBL) but only a small decrease in the proliferation of lymph node lymphocytes and splenocytes. There were no changes in the percentages of cells expressing CD4, CD5, CD8 or immunoglobulin. Moreover, no changes in IL-2 production or IL-2R expression were observed. More esterase-positive macrophages were detected in all lymphatic tissues of suspended rats, but there was no corresponding increase in the percentage of cells bearing the macrophage markers OX41 or OX42. This increase in the number of macrophages may be related to the observed suppression of lymphocyte proliferation. The tissue specificity of the decrease in mitogen activation indicates that there may be a compartmentalized response in the rats tested in the hindlimb suspension model.

Animals↗

PubMed1991

Differential activation and inhibition of lymphocyte proliferation by modulators of protein kinase C: diacylglycerols, "rationally designed" activators and inhibitors of protein kinase C.

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) can enhance or inhibit lymphocyte proliferation. Enhancement correlated with increased interleukin 2 (IL-2) production and activation of protein kinase C while inhibition correlated with decreased IL-2 and downregulation of protein kinase C activity (D.S. Grove and A.M. Mastro, Cancer Res. 51, 82-88). In this study, various activators and inhibitors of protein kinase C were used in order to try to separate the effects of TPA on this enzyme from its effects on IL-2 production and determine if protein kinase C activity was directly or indirectly related to IL-2 production. 1,2-Dioctanoylglycerol, 1-oleoyl-2-acetyl-glycerol, phospholipase C, and two "rationally designed" activators, 6-(N-decylamino)-4-hydroxy-methylindole and 3-(N-acetylamino)-5-(N-decyl-N-methylamino)-benzyl alcohol, were tested. Some activators enhanced proliferation in the presence of a Ca2+ ionophore, ionomycin, but not concanavalin A. Some activators suppressed proliferation and downregulated protein kinase C. Others neither downregulated protein kinase C nor inhibited IL-2 production and proliferation. However, inhibition or downregulation of protein kinase C activity always correlated with decreased IL-2 and depressed proliferation. Thus, the evidence in this and the previous study suggests that activation of protein kinase C is directly related to IL-2 production in activated T cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1991

Prolactin-induced mitogenesis of lymphocytes from ovariectomized rats.

PRL can induce interleukin-2 (IL-2) receptor expression in splenocytes from ovariectomized (OVX) female rats. In this further study of the effects of PRL on lymphocytes in vitro we found that PRL induced IL-2 receptor expression, IL-2 production, and proliferation of splenocytes and thymocytes from OVX rats. Cells from male rats were not affected. The proliferative response, as measured by [3H]thymidine incorporation, depended on the concentration of PRL and the presence of adherent cells in the culture. After a 48-h incubation with PRL (1 microgram/ml), splenocytes from OVX rats incorporated essentially the same amount of [3H]thymidine as cells incubated with the polyclonal T-cell mitogen Concanavalin-A (ConA). As determined by autoradiography, approximately 40% of the splenocytes responded to PRL or to ConA. After incubation of splenocytes and thymocytes with PRL, bioactive IL-2 was detected in culture medium only from cells of OVX female rats, while incubation with ConA caused IL-2 production by lymphocytes from both male and OVX rats. However, ConA induced IL-2 activity sooner than PRL. Immunofluorescent-flow cytometric analysis revealed time-dependent increases in percentages of IL-2 receptor-positive splenocytes as well as increases in percentages of total T-cells and cells of the CD8 and, to a lesser extent, the CD4 subclass after PRL stimulation.

Animals↗

PubMed1991

Negative regulation of interleukin-2 production in primary lymphocytes by 12-O-tetradecanoylphorbol-13-acetate.

Stimulation of quiescent T lymphocytes to proliferate involves a complex series of events both between and within cells. At least 70 genes are known to be induced or activated from the time of the initial stimulation until DNA synthesis. While some of these gene products, e.g., interleukin-2 (IL-2) and IL-2 receptors, are required for proliferation, others, e.g., gamma-interferon and colony-stimulating factor, are ancillary to activated T cell function. Several biochemical signal transductions are among the early events. One of the earliest is phospholipase C-mediated hydrolysis of phosphatidylinositol leading to release of diacylglycerols and inositol phosphates, which in turn activate protein kinase C and elevate intracellular free calcium levels. The discovery that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) both enhances proliferation and activates protein kinase C strengthens the evidence for a general role of protein kinase C in proliferation. Yet, the exact consequences of stimulation of protein kinase C in regard to specific proliferation proteins is still not clear. In this study, we present evidence that protein kinase C activation is directed to production of IL-2 but not to IL-2 receptors. Under conditions of TPA treatment in which protein kinase C was chronically reduced in T lymphocytes, IL-2 production was greatly depressed as were the level of IL-2 mRNA and [3H]thymidine incorporation. In contrast, these cells still expressed high affinity IL-2 receptors and proliferated when endogenous IL-2 was added. Because neither phosphatidylinositol metabolism nor Ca2+ flux was affected, the block appeared to be mediated directly or indirectly through protein kinase C.

Animals↗

PubMed1991

Effect of neonatal milk-prolactin deprivation on the ontogeny of the immune system of the rat.

A growing body of evidence suggests that prolactin (PRL) is involved in regulation of the immune system in the adult. PRL provided to the neonate in mother's milk also has been shown to be important in development of the neonatal neuroendocrine regulation of PRL secretion. Therefore, in this study we asked if deprivation of the neonate of milk-PRL on days 2-5 postpartum affected the ontogeny of the immune system. Two aspects, DNA synthesis (3H-thymidine incorporation) of neonatal lymphocytes in response to polyclonal mitogens in vitro and expression of lymphoid cell surface antigens, were examined. Splenocytes and thymocytes from neonates ranging in age from 5 to 28 days were taken from mothers treated with bromocriptine or saline on days 2-5 of lactation. Splenocytes from pups of vehicle-treated mothers showed a gradual increase in surface antigen expression by day 5 to 28. Thymocyte patterns and percentages of these surface proteins were at adult levels at the earliest times tested. Thymocytes from day 5 and 10 neonates were more responsive to Con A than were splenocytes, but both thymocytes and splenocytes showed an increase in mitogenic responsiveness until day 18, a sharp decline at day 21, and an increase again at day 28. The fact that day 21 is the time of intestinal closure (cessation of absorption of macromolecules from the gut) suggested that milk-borne material plays a role in immune cell maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Evidence for protein kinase C independent activation of phospholipase D by phorbol esters in lymphocytes.

Recently it was reported that tumor-promoting phorbol esters stimulate the production of phosphatidylethanol (PEt) in lymphocytes through the activation of phospholipase D (PLD). However, it remains unclear whether this activation is mediated through protein kinase (PKC). The study reported here shows that tumor promoters 12-0-tetradecanoylphorbol-13-acetate (TPA), phorbol dibutyrate (PDBU), 12-deoxyphorbol-13-phenylacetate (DOPP), 12-deoxyphorbol-13-phenylacetate-20-acetate (DOPPA) and mezerin activated PLD, as measured by the formation of PEt, whereas Concanavalin A (ConA) had no effect. Inhibitors of PKC, sphingosine (2 x 10(-6) M - 5 x 10(-6) M), H-7, HA1004 (5 x 10(-7) - 5 x 10(-6) M) and K252a (1 x 10(-7) - 1 x 10(-6) M) failed to block the PEt synthesis induced by TPA. In fact, sphingosine increased it. Other PKC activators, 1-oleoyl-2-acetylglycerol (OAG) and dioctanoylglycerol (DiC8) had no effect on lymphocyte PLD activity. Analysis of the phospholipid contents after stimulation by TPA showed that only phosphatidylcholine (PC) was significantly decreased. Interestingly, TPA activated PLD in intact cells but not in lysates or subcellular fractions. These observations suggest that stimulation of PLD-catalyzed PEt synthesis by TPA is not solely mediated through PKC activation.

Animals↗

PubMed1990

An evaluation of the mononuclear cells derived from bovine mammary gland dry secretions using leukocyte antigen specific monoclonal antibodies, light scattering properties and non-specific esterase staining.

The distribution of mononuclear cells isolated from the bovine mammary gland during the nonlactating (dry) period was examined using monoclonal antibodies against leukocyte cell surface antigens, cellular light scattering properties, and the presence of nonspecific esterase. Most of the mononuclear cells isolated during the dry period were lymphocytes. T cells predominated until about 1 week prior to parturition. During the week prior to calving, the percentage of B cells increased until it approximated T cells. The ratio of CD4:CD8 cells was 2-3:1 for mammary gland T cells. This was similar to the ratio found in peripheral blood. At dry-off, about 12% of mammary mononuclear cells were macrophages. The macrophage percentage increased (to about 30%) at mid-dry and remained at this levels until parturition. PMN's were isolated with the mononuclear cells during the first 2 weeks dry and the week prior to calving. Three methods were used to identify mammary macrophages. Esterase staining (as an enzymatic method), forward angle/90 degrees light scatter (based on size and internal complexity), and MHC class II/forward angle light scatter (based on size and surface markers) were compared. Each method yielded similar specificity for macrophage identification. Non-adherent cell fractions, obtained by passage of the cells over Sephadex G-10 columns, were enriched in CD4 positive T cells, somewhat depleted of B cells, and depleted of macrophages and PMN's. Cells eluted from G-10 columns, with lidocaine, were mostly lymphocytes, but reflected the cells loaded onto the column.

Animals↗

PubMed1990

Prolactin induction of interleukin-2 receptors on rat splenic lymphocytes.

A case for the involvement of PRL in the regulation of the immune system is strong. However, no mechanism by which PRL exerts this regulation has yet been identified. We studied the in vitro effects of PRL on splenocytes from ovariectomized (OVX) rats and discovered that PRL induced the formation of interleukin-2 (IL-2) cell surface receptors. However, PRL did not induce IL-2 secretion. This response, which was dependent on the concentration of PRL, also depended upon the estrogen status of the splenocyte donor; thus, splenocytes from OVX rats or rats in diestrus responded to PRL, whereas those from estrogen-treated OVX rats or rats in estrus did not. We propose that in vivo exposure of PRL, under certain physiological conditions, may prime a pool of splenocytes to express IL-2 cell surface receptors, allowing these cells to be responsive to variations in local concentrations of IL-2.

Animals↗

PubMed1989

Detection and quantitation of interleukin-2 from individual cells.

In this report we present the use of cell blotting for the detection of interleukin-2 (IL-2)-producing lymphocytes. This is a rapid and sensitive immunochemical method analogous to Western blotting of proteins. When combined with image analysis one can determine the percentages of IL-2 positive cells as well as quantitate the amount of IL-2 surrounding each cell. When bovine lymph node cells (bLNC) were stimulated with the combination of concanavalin A (ConA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 h, 46.4 +/- 0.6% stained positive for IL-2 and, on average, each cell produced 0.92 +/- 0.6 pg of IL-2 in 24 h. Phytohemagglutinin (PHA) and TPA-stimulated human peripheral blood mononuclear cells (hPBMC) produced approximately the same amount. 0.86 +/- 0.4 pg of IL-2 per cell in 24 h; 45.6 +/- 3.6% stained positive for IL-2.

Animals↗

PubMed1989

Effect of macrophages on the translocation of protein kinase C in concanavalin A-stimulated lymphocytes.

The concanavalin A (Con A)-induced proliferation of lymph node lymphocytes is dependent on the presence of macrophages. When lymphocytes are depleted of macrophages, Con A is no longer mitogenic. Either 12-0-tetradecanoylphorbol-13-acetate (TPA), interleukin 1 (IL1), or macrophages in combination with Con A can restore proliferation. To establish where the proliferation process is blocked in the absence of macrophages, an early step in the signalling pathway, the activation of protein kinase C, was examined. It was found that although Con A caused translocation of protein kinase C from the cytosol to the membrane of lymph node cells, when the lymph node cells were depleted of macrophages and exposed to Con A, this translocation of protein kinase C did not occur. Instead, protein kinase C activity decreased in the membrane fraction and increased in the cytosol. On the other hand, TPA caused translocation of protein kinase C (PKC) from the cytosol to the membrane regardless of the presence of macrophages. However, the macrophage product, IL1, alone or in combination with Con A did not cause translocation of protein kinase C. In a reconstitution experiment, in which lymph node cells were depleted of macrophages and then macrophages were added back, the addition of Con A again lead to translocation of protein kinase C from the cytosol to the membrane. This combination also restored cell proliferation. Therefore, the Con A induced PKC translocation in T lymphocytes is macrophage mediated. TPA overcomes the macrophage requirement by directly activating PKC, while IL1 appears to act at a different step in proliferation.

Animals↗

PubMed1989

Oxalyl thiolester concentrations decreased in lectin and phorbol ester-stimulated lymphocytes.

Oxalyl thiolesters (OTEs) are a newly discovered class of mammalian metabolites that are believed to function in controlling animal metabolism and possibly serve as intracellular mediators for some hormones. Previous correlations had suggested that the concentrations of OTEs might be decreased when cells are stimulated to proliferate, and in our research that was found to be the case. Thus, when bovine lymph node lymphocytes are stimulated either with concanavalin A (Con A) or with a combination of phytohemagglutinin (PHA) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), the concentration of OTEs in the lymphocytes decreases within 3 h by a factor of approximately two. With either PHA or TPA alone, the decrease in OTE concentration is considerably smaller. With Con A as stimulant, the OTE levels decrease within 1 h and remain low for at least 24 h. It was also noted that the concentration of OTEs in unstimulated isolated lymphocytes is significantly lower in lymphocytes obtained from 2-year-old animals than in lymphocytes obtained from older animals. The results of the current investigation, when considered in conjunction with other recent results, suggest that OTEs may be natural cell proliferation inhibitors.

Animals↗

PubMed1988

Lectin-induced phosphatidylinositol metabolism in lymphocytes is potentiated by macrophages.

The generation and hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) have been shown to occur early after the stimulation of a variety of cells and to trigger an array of responses, including proliferation in lymphocytes. In this study we examined the requirement for macrophages in the production and metabolism of PIP2 in lectin-stimulated lymph node lymphocytes. When lymph node cells (LNC) were stimulated by the mitogenic lectin, Con A, a two- to threefold increase in [3H]inositol-labeled PIP2 occurred within 15 min, followed by a decrease to the level of unstimulated cells by 30 min. In addition, there was also a two- to threefold increase in the accumulation of [3H]inositol-labeled inositol monophosphate (IP) 30 min after the addition of lectin. However, when the LNC were depleted of macrophages by adherence to plastic and passage over cotton columns before the addition of Con A, the PIP2 response was not seen and the IP accumulation was reduced by 40%. The macrophage-depleted, Con A-stimulated cultures failed to synthesize DNA. [3H]TdR incorporation was only 15% of that of Con A-stimulated unseparated LNC cultures. Soluble IL-2 production was also depressed to 20% of that observed in macrophage-containing cultures. Furthermore, when macrophages (2%) were added to the macrophage-depleted cultures, PIP2 production, IP accumulation, and [3H]TdR incorporation were restored to levels observed in the stimulated unseparated LNC cultures. The addition of IL-1 or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in combination with Con A also restored both [3H]TdR incorporation and IL-2 synthesis in macrophage-depleted cultures. However, they did so without a detectable increase in phosphatidylinositol metabolism. Collectively, these data suggested that the production and degradation of PIP2 in Con A-stimulated lymphocytes was potentiated by the presence of macrophages.

Animals↗

PubMed1988

Prevention of the TPA-mediated down-regulation of protein kinase C.

Protein kinase C activity in lymphocytes was down-regulated upon exposure of the cells to 12-0-tetradecanoylphorbol-13 acetate. This down-regulation was prevented by preincubating the cells with sphingosine, a reported protein kinase C inhibitor. Two other protein kinase C inhibitors, palmitoylcarnitine and phloretin, were ineffective in preventing down-regulation by the phorbol ester.

Animals↗