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Biomedical subjects

A M Mastro

Publications and source records attributed to A M Mastro.

At least 37 records · Page 2Linked to original sources
PubMed1996

The S-oxalin, N-acetyl-S-oxalylcysteamine, inhibits lymphocyte proliferation, IL-2 production and utilization.

S-Oxalins are a recently described class of cell metabolites that appear to function as negative regulators of proliferation. Previously we have shown that exogenous S-oxalylglutathione (GS-Ox) inhibits the proliferation of lymphocytes by inhibiting the production and utilization of IL-2. In the present study the synthetic S-oxalin, N-acetyl-S-oxalylcysteamine (ACS-Ox), was utilized in similar experiments to determine whether GS-Ox itself, or possibly some metabolite formed following initial conversion of GS-Ox by gamma-glutamyltransferase (GGT), is responsible for the effects (ACS-Ox is not metabolized by GGT). ACS-Ox inhibited DNA synthesis in lymphocytes stimulated by concanavalin A similarly to GS-Ox. IL-2 production and utilization and IL-2R expression were inhibited as well. ACS-Ox also inhibited the proliferation of IL-2 dependent cells at the same concentration as GS-Ox. Because the effects of GS-Ox and ACS-Ox are so similar, presumably the S-oxalin itself, rather than some metabolite, is responsible for the observed effects. Transfer of oxalyl groups from S-oxalins to various proteins thiols is the most likely mechanism involved.

Animals↗
PubMed1996

Prolactin receptor gene expression in rat splenocytes and thymocytes from birth to adulthood.

In vivo and in vitro studies have indicated that the anterior pituitary hormone prolactin (PRL) is an immunoregulator and functions in the development of the neonatal immune system. In this study, prolactin receptor (PRL-R) expression from birth to adulthood as well as the effect of milk ingestion on the PRL-R expression were examined in splenocytes and thymocytes of neonatal rats. Three approaches were taken to measure PRL-R expression: (i) polymerase chain reaction (RT-PCR); (ii) antibody to PRL-R and Western blotting; (iii) antibody to PRL-R and flow cytometry. RT-PCR analysis revealed the short and long form of PRL-R mRNA in both spleen and thymus at every age tested. However, the long form of PRL-R mRNA was always more abundant than that of the short form. In addition, antipeptide antibody against the long form of PRL-R recognized 84 and 42 kD proteins in the spleen, but only the 84 kD protein in the thymus. A monoclonal antibody U6 recognized 38 and 40 kD proteins in both the spleen and thymus. Although the mRNA level of PRL-R was relatively low at birth and increased with age in both the spleen and thymus, the levels of protein bands detected with both antibodies correlated with development in the spleen; whereas the levels remained steady in the thymus. Therefore, we concluded that the expression of PRL-R at the protein level is developmentally regulated in the spleen but not in the thymus. Finally, milk ingestion in the first seven hours decreased the percentage of cells expressing cell surface PRL-R, suggesting that milk-borne PRL may have a direct effect on lymphocytes.

Amino Acid Sequence↗
PubMed1996

Modulation of levels of a negative transcription factor for IL-2 by 12-O-tetradecanoyl phorbol-13-acetate and okadaic acid.

A negatively acting transcription factor, negative regulatory element-A (NREA) that suppresses the transcription of interleukin 2 (IL-2) mRNA, has been described previously. We found that treatment of primary bovine lymphocytes with 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of protein kinase C, for at least 18 h both increased the levels of the factor and blocked concanavalin A (Con A)-induced proliferation. In contrast, treatments of less than 18 h with TPA decreased NREA levels and increased Con A-induced proliferation. NREA binding activity also increased over basal levels during the first 4 h of stimulation of lymphocytes with Con A in the absence of TPA; after 4 h, NREA levels fell. Phosphorylation of the NREA protein was required for binding to its DNA consensus sequence. Furthermore, incubation of lymphocytes with okadaic acid (OKA), a phosphatase inhibitor, led to increased levels of NREA binding activity and to decreased cell proliferation. Because exposure of lymphocytes to either OKA or TPA should lead to an increase in the phosphorylation and binding of the NREA protein, and a decrease in IL-2 production, proliferation should be decreased. Incubation of lymphocytes with either TPA or OKA inhibited proliferation. However, the mechanisms of action of OKA and TPA appeared to be different because exogenous IL-2 reversed the inhibition of proliferation caused by TPA but not by OKA.

Animals↗
PubMed1996

Milk-borne prolactin and neonatal development.

Milk is primarily regarded as a food furnishing essential nutrients for infant growth and development, but milk can also serve as a vehicle for mother to neonate transfer of molecules that regulate development. A wide array of biologically active compounds such as hormones, cytokines and enzymes are present in milk, especially early milk. The premise that prolactin (PRL) in milk is an important and possibly essential developmental factor for the newborn is explored. Both PRL and structurally modified isoforms are abundant in early milk and gradually diminish with the progression of lactation. Milk PRL is absorbed and biologically active in the neonate. Assays of PRL variants, experimental paradigms to test them as developmental regulators and the body of evidence supporting the hypothesis that milk PRL regulates differentiation and maturation of neonatal neuroendocrine, reproductive, and immune systems is presented.

Breast Feeding↗
PubMed1995

The mechanism of sphingosine enhancement of phorbol ester-mediated phospholipase D activation in lymphocytes.

Tumor-promoting phorbol esters stimulate the production of phosphatidylethanol (PEt) in bovine lymph node lymphocytes through the activation of Phospholipase D (PLD). Sphingosine enhanced the 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated formation of PEt in lymphocytes, while it had no effect by itself. Interestingly, the incorporation of labeled arachidonic acid into phospholipids was increased by sphingosine. Examination of the effect of sphingosine along with TPA on the incorporation of [1-14C]labeled-arachidonic acid into individual phospholipids revealed that they enhanced significantly the specific activity of only PC and lyso-PC fractions. These observations indicate that sphingosine does not activate PLD directly as TPA does. It appears that sphingosine increases the specific radioactivity of the substrate pool for PLD which results in an apparent increase in PEt when both sphingosine and TPA are present.

Animals↗
PubMed1995

Mechanisms of activation and suppression in rat Nb 2 lymphoma cells: a model for interactions between prolactin and the immune system.

Rat Nb 2 lymphoma cells have been widely used to bioassay human growth hormone and many species of prolactin. Because their morphologic characterization suggests a T-cell lineage, Nb 2 cells were examined for their response to the T-cell mitogens concanavalin A, pokeweed mitogen, and phytohemagglutinin P. As expected, a dose-response to rat prolactin was observed; however, attempts to induce proliferation using the conventional T-cell mitogens failed at concentrations normally stimulatory for rat primary lymphocytes. Moreover, when Nb 2 cells were simultaneously incubated with lectin plus a suboptimal concentration of prolactin, a dose-dependent suppression of the stimulatory effects of prolactin was observed with phytohemagglutinin P and pokeweed mitogen, although not with concanavalin A. Culture medium of prolactin-stimulated Nb 2 cells also contained a factor which inhibited normal rat lymphocyte activation by concanavalin A. The factor did not block induction of the IL-2 receptor and proliferation of IL-2-dependent CTLL-2 cells could be restored by exogenous IL-2. Because Nb 2 cells evolved from a lactogen-dependent lymph node tumor, these results may have implications for further understanding the role of pituitary hormones, particularly prolactin, in the immune response to hormone-dependent tumor progression.

Animals↗
PubMed1995

The effect of a 10-day space flight on the function, phenotype, and adhesion molecule expression of splenocytes and lymph node lymphocytes.

Previous studies have indicated that space flight affects the activation of lymphocytes from humans, monkeys, and rodents. In rats, where lymphocytes from blood, spleen, and lymph nodes have been tested, the accumulated data suggest that the effects of flight on various cells are lymphoid organ-specific. Thus, cells may be affected by variations in trafficking brought about by fluid shifts in microgravity (< 10(-3) g). In this study we examined lymphocyte activation (IL-2 production) as well as the expression of surface differentiation antigens and of adhesion molecules by splenocytes and lymph node lymphocytes (LNL) after a 10-day flight (Space Shuttle Mission STS-57). For splenocytes and LNL from flight (FLT) animals, IL-2 production decreased in response to the T cell receptor-independent mitogen 12-O-tetradecanoylphorbol-13-acetate plus ionomycin, but was not affected by stimulation with the T cell receptor-dependent mitogens Concanavalin A or phytohemagglutinin. In addition, the percentage, as well as fluorescent intensity, of splenocytes which expressed CD8, CD4, or kappa increased after flight. The percentage of LNL expressing CD2 also increased but those expressing CD5 decreased. The percentage of cells expressing the integrins LFA-1 alpha and beta increased with splenocytes from FLT animals but decreased for LNL. In contrast, FLT animals showed a decrease in the percentage of selectin-positive splenocytes. ICAM-1 expression did not change. In summary, these data are consistent with a model in which microgravity affects lymphocyte redistribution among organs, which in turn influences the activation potential of the cells.

Analysis of Variance↗
PubMed1994

Characterization of resting and phorbol ester or concanavalin A activated bovine lymph node cells with leukocyte specific monoclonal antibodies.

Bovine lymph node cells (LNC) have been used as a model to study cell activation and proliferation. Because monoclonal antibodies to bovine lymphoid-specific surface antigens have only recently become available, these cells have not been previously characterized in regard to subpopulations. Furthermore, it was not known how expression of lymphoid differentiation antigens and subset proportionalities might change following different modes of activation of LNC. Therefore, the distribution of cell-surface differentiation antigens in unstimulated LNC as well as in LNC incubated with the mitogen concanavalin A (Con A) or the phorbol ester, phorbol dibutyrate (PDBU), was measured using a series of leukocyte-specific monoclonal antibodies and flow cytometry. Unstimulated LNC were found to have similar proportions of T cells, B cells (sIgM positive), and MHC Class II positive cells similar to bovine peripheral blood mononuclear leukocytes (reviewed by Baldwin et al., 1988a). Treatment of the LNC with PDBU or mitogenic doses of Con A induced changes in the expression of surface antigens consistent with the changes observed with human and mouse cells after similar activation. However, these two compounds did not cause identical effects. After treatment with PDBU, the percentage of cells expressing CD4 as well as the density of surface expression decreased. An increase in the percentage of cells expressing and/or density of surface expression of the pan T cell antigens CD2, CD5, CD6, MHC Class II and J5, a T cell activation antigen, also occurred. PDBU treatment also increased the percentage of CD8 positive cells. The change in CD6 following PDBU treatment has not been reported previously. Con A treatment led to a significant increase in the percentage of cells bearing CD8, CD6, MHC Class II and J5, but it had no effect on the percentages of cells positive for the other T cell markers CD5, CD4, or CD2. Because Con A is a complete mitogen and PDBU is not, the changes observed following Con A stimulation probably reflected an expansion of a particular subpopulation. In contrast, PDBU most likely modifies surface antigen expression directly. Neither treatment affected the B cell subpopulation.

Animals↗
PubMed1994

IL-2 mRNA levels and degradation rates change with mode of stimulation and phorbol ester treatment of lymphocytes.

Transient expression of interleukin 2 (IL-2) in activated T lymphocytes may be due to transcriptional and post-transcriptional regulation. As incubation of lymphocytes with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) prior to mitogenic stimulation results in decreased levels of IL-2 mRNA, we asked if IL-2 mRNA stability was affected. We found that in TPA-treated cells, IL-2 mRNA was degraded more rapidly than in untreated ones whether the mitogenic stimulus was Concanavalin A (Con A), Con A plus TPA, or TPA plus ionomycin. The degradation was blocked if the TPA pre-incubation included cycloheximide. In contrast, when TPA was included as a co-mitogen, i.e. added at the same time as the mitogen, the IL-2 mRNA levels and stability significantly increased. Compared to the levels found in Con A stimulated cells, TPA plus Con A increased IL-2 mRNA levels by as much as 20-fold and the half-life by 5-fold. TPA plus ionomycin increased the message levels at least 100-fold and half-life by nearly 10-fold. These effects on IL-2 mRNA were not general because IL-2 receptor mRNA stability was not changed even though it also is transiently expressed during the course of lymphocyte activation.

Animals↗
PubMed1994

Prolactin-immune interactions in carcinogen-induced rat mammary tumors.

Because many mammary tumors are prolactin (PRL) dependent, tumor-bearing animals are immunocompromised, and PRL directly affects the immune system, we examined the endocrine and immune systems of rats initiated with nitrosomethylurea (NMU) to cause mammary tumors. We tested: a) PRL cells in the pituitary; b) pituitary PRL as detected by radioimmunoassay (RIA), Nb2 bioassay, and induction of interleukin-2 receptors on splenocytes; c) induction of IL-2R on lymphocytes in response to a standard PRL; d) CD phenotype of the splenocytes and tumor infiltrating lymphocytes. We found that 80% of all NMU-treated animals developed mammary tumors 10 to 13 weeks post-injection. PRL cell number, size, and granule content were unaffected. When tested by RIA or by the Nb2 bioassay, there appeared to be approximately 50% less PRL secreted (2 weeks post-injection) by cells of the NMU-treated than the vehicle-treated animals. However, when tested by IL-2R assay, PRL cells of NMU-treated animals secreted 50% more activity. Splenocytes from the treated animals, 2-6 weeks post-injection, expressed fewer IL-2R in response to standard PRL. NMU treatment (12 wks post-injection) increased the numbers of T-cytotoxic cells by 49%, had no effect on T-helpers, and increased the number of IL-2R positive splenocytes by 81%. Our interpretation is that NMU treatment interferes with the feedback of lymphokines on the pituitary with a decrease in the form of PRL detected by the RIA and Nb2 assays and an increase in the form which activates splenocytes, and thus changes the composition and function of the immune system.

Animals↗
PubMed1993

Activation of primary lymphocytes requires prolonged lectin stimulation.

T lymphocytes are activated by a complex series of events, but the mechanisms remain unclear. One uncertainty is the time of receptor-ligand interaction necessary for commitment to DNA synthesis and proliferation. Although this issue has broad implications for the interpretation of T cell activation data, it remains unresolved. Therefore, we examined the temporal activation requirements of rat splenocytes stimulated with concanavalin A (Con A) by measuring proliferation, as well as interleukin-2 (IL-2) production and IL-2 receptor IL-2R) expression. Splenocytes stimulated with various Con A concentrations for 3 h did not incorporate significantly more [3H]thymidine than unstimulated splenocytes. Some increase occurred after 6 h of lectin exposure but maximum proliferation occurred only after the 52-h stimulation. Furthermore, Con A incubations of 6 h or more were required for significant increases in IL-2 or IL-2R. Maximum lymphokine production and receptor expression were observed after the 52-h stimulation. Thus, activation of some primary lymphocytes required only 6 h of stimulation, but much longer mitogen contact was necessary for maximum recruitment.

Animals↗
PubMed1993

Induction of interleukin-2 receptor alpha-chain gene expression by cytochalasin B and 12-O-tetradecanoylphorbol-13-acetate in T lymphocytes.

The high affinity form of interleukin-2 receptor (IL-2R) is composed of two subunits; the alpha (p55) and beta (p75). The alpha chain, unlike the beta, is expressed only on activated T lymphocytes. Therefore, high affinity binding of interleukin-2 (IL-2) is controlled by the expression of the IL-2R alpha-chain. In this study, we examined the effect of cytochalasin B (CB) plus 12-O-tetradecanoylphorbol-13-acetate (TPA) on expression of IL-2 and IL-2R. Northern blot and flow cytometric analysis showed that the IL-2R alpha-chain was expressed both at mRNA and protein levels. However, IL-2 gene expression was not induced by this treatment. Unlike the cells treated individually with CB or TPA, cells treated with CB plus TPA accumulated IL-2R mRNA at all the times examined. In order to determine the percentage of cells that incorporated tritiated thymidine ([3H]dT) in the presence of IL-2 after treatment with CB plus TPA, autoradiography was carried out. We found that about 11% of the cells were labelled. Because the percentage of labelled cells and cells expressing IL-2R alpha-chain was relatively low (11% and 9% respectively), perhaps CB plus TPA caused IL-2R expression in only a subset of T cells.

Animals↗
PubMed1993

Prolactin receptors are found on heterogeneous subpopulations of rat splenocytes.

An increasingly large body of evidence implicates PRL as an immunoregulatory molecule. While most of the data relate to PRL levels and immunocompetence in vivo, we have shown that PRL is mitogenic for splenocytes from ovariectomized rats and rats in certain other hormonal states. This finding suggests that these lymphocytes express PRL receptors. Here, we wished to determine whether all or only a subset of splenocytes were PRL receptor positive. By using polyclonal as well as monoclonal antibodies to PRL receptor, we determined that as many as 20% of the primary splenocytes expressed PRL receptors. In a culture of Nb2 cells, a PRL receptor-positive lymphoid line, as many as 70% were PRL receptor positive. Dual labeling for lymphoid-specific antigen surface markers and PRL receptor indicated that about one third of the PRL receptor-positive splenocytes were kappa-light chain-positive B-cells, while the others stained with antibodies to T-cell markers, CD4 or CD8. These data confirm that lymphocytes express PRL receptors and show for the first time that PRL receptor-positive lymphocytes are a heterogenous subset of total primary splenocytes. These cells may be the target for PRL-mediated immunoregulation.

Animals↗
PubMed1993

The expression of forms of prolactin receptors in splenocytes and thymocytes of neonatal rats: the effect of milk ingestion.

Because prolactin (PRL) plays a role in neonatal immune development, we examined the expression of prolactin receptors (PRL-R) in neonatal lymphoid tissues. We had shown previously that deprivation of milk-borne PRL, days 2-5 in the neonatal rat, leads to enhanced in vitro mitogenesis of thymocytes and splenocytes as well as a change in lymphoid-specific, cell surface antigens (GROVE et al. 1991). In this present study, we asked if neonatal lymphocytes express PRL-R; which forms of PRL-R are expressed (long vs. short form); when these forms are expressed during development; and if milk ingestion plays a role in receptor expression. Two approaches were taken using neonatal rat thymocytes and splenocytes: RNA was analyzed by polymerase chain reaction (PCR) and cells were stained with antibody to PRL-R and analyzed by flow cytometry. In regard to cell surface expression, the percentage of PRL-R positive splenocytes was greater than thymocytes at all ages tested. In the spleen, the percentage of PRL-R positive cells gradually increased to adult levels by day 10; in the thymus the percentage fell to adult levels by the first day after birth. Finally, milk ingestion in the first 7 h decreased the percentage of cells expressing cell surface PRL-R. Tissues from animals deprived of milk during this time expressed PRL-R at the same level as the newborn.

Administration, Oral↗
PubMed1992

Production of interleukin-2 mRNA by bovine lymph node lymphocytes in response to concanavalin A, 12-O-tetradecanoylphorbol-13-acetate, and ionomycin.

Interleukin-2 (IL-2) is a lymphokine which, upon binding to its receptor, leads to the proliferation and differentiation of T-cells (helper, suppressor, and cytotoxic) and B-cells. While human and murine IL-2 have been extensively studied, less is known about bovine IL-2. In order to understand the induction of bovine IL-2 at the molecular level, we have examined IL-2 mRNA induction. The dose-responses and time courses of the production of IL-2 mRNA in response to Concanavalin A (ConA), 12-O-tetradecanoylphorbol-13-acetate (TPA), and ionomycin in lymph node lymphocytes (LNC) were determined. We found that high levels of IL-2 mRNA were produced in response to 1 microgram ml-1 ConA plus 10(-8) M TPA, but that even higher levels were produced in response to 1 microM ionomycin plus 10(-8) M TPA. We also found that LNC stimulated with ConA displayed two phases of IL-2 mRNA production, one occurring approximately 2-4 h after stimulation and one occurring approximately 10 h after stimulation. However, in the presence of ConA plus TPA or ionomycin plus TPA the response was monophasic. IL-2 mRNA was detected within 2 h of addition of ConA plus TPA (the earliest time examined), reached maximum levels within 6 h, and declined to low levels after 12 h. IL-2 mRNA from LNC incubated with ionomycin plus TPA appeared within 2 h, and reached maximum levels at about 9 h. In contrast to the decrease seen after 12 h with ConA plus TPA, IL-2 mRNA from these cells remained high for 18 h and declined to low levels after 24 h.

Animals↗
PubMed1992

Cytochalasans and PMA induce IL-2 receptors on CD8+ lymphocytes.

The cytochalasans, fungal metabolites that interact with actin, can affect lymphocyte proliferation; high concentrations inhibit lectin-induced proliferation and low concentrations augment it. The phorbol ester tumor promoter, PMA, alone is not mitogenic for primary lymphocytes but enhances the activity of mitogenic lectins. Because the cytochalasans have been reported to increase intracellular Ca2+ and because PMA activates protein kinase C, lymphocytes were treated with PMA and cytochalasin B (CyB) to determine if this combination would induce DNA synthesis. While this treatment by itself did not cause proliferation, lymphocytes cultured with PMA and CyB overnight, washed, and recultured with IL-2 proliferated to the same degree as lymphocytes stimulated with Con A. Three different cytochalasans, cytochalasin B, cytochalasin D, and chaetoglobosin C, all of which bind to cellular actin with different affinities and only one of which affects glucose transport, induced IL-2 receptors in combination with PMA. Flow cytometric analysis with an antibody to the IL-2 receptor alpha subunit confirmed the induction of receptors on CD8+ cells. However, no IL-2 was produced after the exposure of lymphocytes to the combination of cytochalasans and PMA. Therefore, there was sufficient signal to induce IL-2 receptor expression but not to induce IL-2.

Animals↗
PubMed1992

Variable lymphocyte responses in rats after space flight.

Most studies of human blood lymphocyte function following space flight have indicated that microgravity suppresses T cell proliferation. However, several other postflight experiments with animals have shown no decrease in proliferation of lymphocytes from peripheral lymphatic tissues, suggesting that different tissues may be variably affected by microgravity. Therefore, we examined the proliferation of lymphocytes from both spleen and lymph nodes of rats following a 4-day flight aboard the Space Shuttle. The experiments were designed to investigate tissue variability as well as potential mechanisms involved in suppressing proliferation. We found that proliferation of lymph node lymphocytes (LNL) from flight (FLT) animals stimulated with the antigen receptor-dependent T cell mitogen concanavalin A was depressed and could not be restored by supplementing cultures with interleukin 1 or interleukin 2 (IL-2). Response to another receptor-dependent mitogen, phytohemagglutinin, was not decreased. However, proliferation of FLT LNL following stimulation with the receptor-independent, mitogenic combination of phorbol ester and ionomycin was depressed. LNL IL-2 activity, cell surface marker expression, and B cell responses to mitogen were normal. Thus, deficits in antigen receptor/ligand interactions, cell surface marker expression, or IL-2 did not account for the suppressed lymphocyte proliferation observed postflight. In contrast to LNL, FLT splenocyte proliferation was not depressed. Assayable IL-2, IL-2 receptor expression, and cell surface marker expression likewise were unaffected by space flight. The differences between lymph node and splenic responses demonstrate the tissue-specific nature of microgravity effects on individual lymphatic tissues.

Animals↗