Lauretta Massai

The effect of LHRH and TRH on human interferon-gamma production in vivo and in vitro.

Accumulating evidence suggests that hypothalamic luteinizing hormone-releasing hormone (LHRH) and thyrotropin-releasing hormone (TRH) are two hypophysiotropic factors which modulate the immune response. The aim of the present study was to determine the in vivo effects of an intravenous bolus of LHRH and TRH on plasma interferon (IFN)-gamma production in five normoprolactinemic women with irregular menstrual cycles. We also determined prolactin (PRL), thyrotropin (TSH), follicle stimulating hormone (FSH), and luteinizing hormone (LH) levels before and after intravenous administration of LHRH and TRH. The results demonstrate that intravenous bolus of LHRH/TRH increases plasma IFN-gamma levels, with the maximum response 45 min after in vivo administration of hypothalamic peptides and after peak levels of adenohypophyseal hormones (PRL: 15 min; TSH: 30 min; FSH: 30 min; LH: 30 min). In order to investigate a possible direct action of hypothalamic hormones on immune cells, we also evaluated, in the same subjects, the influence of LHRH and TRH on IFN-gamma production by human peripheral blood mononuclear cells (PBMCs), collected before the intravenous administration of the peptides and stimulated in vitro with bacterial superantigen staphylococcal enterotoxin A (SEA) and concanavalin A (Con A). LHRH and TRH, separately and together, significantly enhanced in vitro IFN-gamma production by SEA- and ConA-activated PBMCs. The present results suggest that hypothalamic peptides (LHRH and TRH) directly, and/or indirectly pituitary hormones (PRL, TSH, FSH, and LH) or IL-2, have stimulatory effect on IFN-gamma producing cells and are further evidence of interactions between the neuroendocrine and immune systems.

Estrogen and μ-opioid receptor antagonists counteract the 17β-estradiol-induced licking increase and interferon-γ reduction occurring during the formalin test in male rats

&NA; Women have a higher incidence of chronic pain syndromes than men and are generally more sensitive to experimental pain. Numerous studies have shown that the female gonadal hormones, estrogens, can profoundly affect the nervous and immune systems, including mechanisms involved in pain and nociception. In the present study, we used antagonists of estrogen receptors (ER) or &mgr;‐opioid receptors (&mgr;OR) to evaluate the effects of estrogens on formalin‐induced behavioural and immune responses in male rats. After two days of priming with 17&bgr;‐estradiol or saline (i.c.v.), animals were subjected to the formalin test; 15 min prior to formalin (50 &mgr;l, 5%) or sham injection in the hind paw, animals were treated with an ER antagonist (ICI 182,780, ICI) or a &mgr;OR antagonist (&bgr;‐funaltrexamine, FNA) or saline. The spontaneous behaviours, pain‐related behaviours and interferon‐&ggr; (IFN‐&ggr;) production by peripheral blood mononuclear cells were studied in all groups. We found that central administration of estradiol increased the amount of licking of the formalin‐injected paw in the second phase of the formalin test. Whereas ICI and FNA had no effect on pain behaviour in saline‐pre‐treated animals, both antagonists reversed the estradiol‐induced increase in licking. The immune system was differently affected by formalin and estradiol treatment. Indeed, formalin injection per se decreased IFN‐&ggr; production; estradiol had no effect on sham‐injected animals but strongly reduce the decrease of IFN‐&ggr; production in formalin‐injected animals. The results demonstrate that centrally acting estrogens affect ER‐ and &mgr;OR‐mediated pain processing and influence immune function.

Differential expression of follistatin and FLRG in human breast proliferative disorders

BackgroundActivins are growth factors acting on cell growth and differentiation. Activins are expressed in high grade breast tumors and they display an antiproliferative effect inducing G0/G1 cell cycle arrest in breast cancer cell lines. Follistatin and follistatin- related gene (FLRG) bind and neutralize activins. In order to establish if these activin binding proteins are involved in breast tumor progression, the present study evaluated follistatin and FLRG pattern of mRNA and protein expression in normal human breast tissue and in different breast proliferative diseases.MethodsParaffin embedded specimens of normal breast (NB - n = 8); florid hyperplasia without atypia (FH - n = 17); fibroadenoma (FIB - n = 17); ductal carcinoma in situ (DCIS - n = 10) and infiltrating ductal carcinoma (IDC - n = 15) were processed for follistatin and FLRG immunohistochemistry and in situ hybridization. The area and intensity of chromogen epithelial and stromal staining were analyzed semi-quantitatively.ResultsFollistatin and FLRG were expressed both in normal tissue and in all the breast diseases investigated. Follistatin staining was detected in the epithelial cytoplasm and nucleus in normal, benign and malignant breast tissue, with a stronger staining intensity in the peri-alveolar stromal cells of FIB at both mRNA and protein levels. Conversely, FLRG area and intensity of mRNA and protein staining were higher both in the cytoplasm and in the nucleus of IDC epithelial cells when compared to NB, while no significant changes in the stromal intensity were observed in all the proliferative diseases analyzed.ConclusionThe present findings suggest a role for follistatin in breast benign disease, particularly in FIB, where its expression was increased in stromal cells. The up regulation of FLRG in IDC suggests a role for this protein in the progression of breast malignancy. As activin displays an anti-proliferative effect in human mammary cells, the present findings indicate that an increased FST and FLRG expression in breast proliferative diseases might counteract the anti-proliferative effects of activin in human breast cancer.

A nitric oxide/Ca2+/calmodulin/ERK1/2 mitogen-activated protein kinase pathway is involved in the mitogenic effect of IL-1β in human astrocytoma cells

Evidence is accumulating to support a role for interleukin‐1β (IL‐1β) in astrocyte proliferation. However, the mechanism by which this cytokine modulates this process is not fully elucidated.

Glucocorticoid receptors in human peripheral blood mononuclear cells in relation to explosive performance in elite handball players.

Ten handball players, members of the Italian National Team (aged 20-25 years), were studied in two sessions corresponding to different performance levels. The first session occurred one week after the end of the regular season of the Italian Handball Federation: it corresponded to the beginning of the training cycle for the European Handball Championship. The second session occurred ten weeks after the first session. During this period, training consisted of 3 weeks of active recovery and 7 weeks of increasing workload. For each session, jumping performances (maximal height in a single jump, average mechanical power for a 15-s set of consecutive jumps) were evaluated. Venous blood samples were collected in resting conditions immediately before jumping performances to assess cortisol and testosterone plasma concentrations and glucocorticoid receptors (GcR) binding capacity and affinity in peripheral blood mononuclear cells (PBMCs). All the parameters, except GcR binding affinity, increased in the second session. The trends of variation in jumping performances, steroid hormone levels and GcR binding capacity were similar. For testosterone, this agrees with the hypothesis that an adequate level of this hormone is a prerequisite for improvement in explosive performances. For cortisol, higher GcR binding capacity after 10 weeks of training (with respect to initial values) indicated an up-regulation of GcR concomitant with the increase in hormone levels and performances. These findings suggest that the adaptation to training, confirmed by the improvement in performance, is characterized by a high value of GcR binding capacity and that it is mediated, among other factors, by the hormone levels and up-regulation of the receptors.

High-dose ascorbate and arsenic trioxide selectively kill acute myeloid leukemia and acute promyelocytic leukemia blasts in vitro

The use of high-dose ascorbate (ASC) for the treatment of human cancer has been attempted several decades ago and has been recently revived by several in vitro and in vivo studies in solid tumors. We tested the cytotoxic effects of ASC, alone or in combination with arsenic trioxide (ATO) in acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL). Leukemic cell lines and primary blasts from AML and APL patients were treated with graded concentrations of ASC, alone or in association with standard concentration (1 μM) of ATO. The ASC/ATO combination killed myeloid blasts, including leukemic CD34+ cells, while sparing CD34+ progenitors obtained from normal cord blood and bone marrow. Actually, approximately one-third (11/36) of primary AML cases were highly sensitive to the ASC/ATO combination. The mechanism of cell killing appeared to be related to increased oxidative stress and overproduction of ROS in a non-quantitative fashion, which resulted in induction of apoptosis. These effects were reverted by the addition of the antioxidant N-Acetyl-Cysteine (NAC). In the APL NB4 model, ASC induced direct degradation of the PML and PML/RARA proteins via caspase activation, while the transcriptional repressor DAXX was recruited in re-constituted PML nuclear bodies. Our findings encourage the design of pilot studies to explore the potential clinical benefit of ASC alone or in combination with ATO in advanced AML and APL.

High Doses of Ascorbate Kill Y79 Retinoblastoma Cells In vitro

Objectives: To tests the sensitivity of Y79 retinoblastoma cell lines to high doses of ascorbate, in vitro, and compare its effects with those of some chemotherapeutic agents routinely employed in the treatment of retinoblastoma.Methods: Y79 retinoblastoma cells have been exposed to increasing doses of either sodium ascorbate (SA) or Melphalan (MEL), to define a dose-response curve around the peak plasma concentrations reached by both chemicals when administered according to the existing therapeutic procedures and protocols. The assessment of cell number and viability was performed, before and after exposure, with both the manual (Trypan Blue Exclusion Test) and automated (flow cytometry) methods. Fluorescence microscopy and direct observation of cells in culture, with inverted microscope, were also performed.Results: Y79 cells are highly sensitive to the cytotoxic effect of SA, with cell viability reduced of over 90% in some experiments. As reported in the literature, this effect is directly cytotoxic and most probably mediated by acute oxidative stress on different cellular components. The same does not apply to Melphalan which, at the doses commonly used for therapeutic purposes, did not show any significant effect on cell viability, in vitro.Conclusion: To our knowledge, this is the first report showing that high doses of SA can actively kill retinoblastoma cells in vitro. While it is not surprising for SA, to show direct cytotoxic effect on tumor cells, the data reported herein represent the first evidence in favor of the possible clinical use of high doses of intravenous SA, to treat children affected by retinoblastoma. Given the many advantages of SA over the chemotherapeutic agents commonly employed to treat cancer (including its almost total absence of toxic or side effects, and its exclusive specificity for cancer cells), it is reasonable to assume, from the data reported herein, that the high doses of intravenous ascorbate, have the potential to represent a real revolution in the treatment of retinoblastoma.

IFN-γ production in rabbits: Behavioral and endocrine correlates

Freely interacting male rabbits were studied to establish the effect of exogenous testosterone on interferon-γ (IFN-γ) production in peripheral blood mononuclear cells (PBMCs) and to evaluate if this effect is related to season, social rank, plasma corticosterone and glucocorticoid receptors (GcR) in PBMCs. Dominance behavior increases after testosterone propionate (TP) administration only in rank 1 animals, while submission behavior increases after TP only in rank 4 animals, indicating a reinforcing effect of TP on the behavior. Corticosterone and IFN-γ production are higher and GcR binding capacity is lower in spring than in autumn, suggesting that seasonal fluctuations in the immune system may be related to the pattern of secretion of immunomodulatory hormones. In autumn, corticosterone decreases after TP treatment and increases after social interaction, while GcR binding capacity decreases after TP treatment and social interaction. IFN-γ production decreases in spring and increases in autumn after TP treatment plus social interaction, indicating that the modulating action of testosterone is related to the current immune status. The relationship between dominance, testosterone and the immune system in spring is suggested by the finding that GcR binding capacity after TP treatment is directly related to social rank, as confirmed by the positive correlation with dominance behavior frequency. The dominance index is positively correlated with GcR binding capacity and negatively with IFN-γ production before TP treatment, indicating that high receptor activity in immunocompetent cells and low immunoreactivity could be prerequisites for dominance behavior. The immunosuppressive effect of corticosterone and the mechanism of down-regulation on GcR are confirmed by the negative correlations with IFN-γ production and GcR binding capacity.

Megadoses of ascorbate as a new chemotherapeutic approach in uveal melanoma: a preliminary in vitro investigation

Background: Despite the more recent advances, there is still no effective systemic therapy for Uveal Melanoma (UM). However, a better understanding of the role of oxidative stress in cancer, has more recently led to a completely new approach to the systemic therapy of cancer, and modulators of the oxidative balance, such as sodium ascorbate (ASC) or arsenic trioxide (ATO), have already entered advanced phases of preclinical and clinical development. Since high doses of ASC have already demonstrated a strong cytotoxic effect on different human cancer cell lines, we have undertaken the present investigation in order to test the sensitivity of OCM1 and C918 uveal melanoma (UM) cell lines to high doses of ASC, in vitro, as compared to ATO, a pro-oxidant drug which has already undergone extensive in vitro and pre-clinical investigation in UM. Methods: Both OCM1 and C918 UM cell lines have been exposed to increasing doses of either ASC or ATO, to build a dose-response curve around the peak plasma concentrations reached by both chemicals. The assessment of cell count and viability was performed with flow cytometry. Results: Both OCM1 and C918 UM cell lines are highly sensitive to ASC in the range of millimolar (mM) concentrations which can be usually reached by the intravenous injection of high doses of the compound. ATO at the dosages used in this study, never reached the LC50. When the exposure to ASC was reduced to two hours, it still had significant effects on survival of both OCM1 and C918 UM cells. Conclusions: This report shows that ASC is highly cytotoxic for both OCM1 and C918 UM cells, when used in high, pro-oxidant doses. To our knowledge, this is the first report showing that UM cells can be efficiently killed, in vitro, with high doses of ASC.

Expression, localization and control of activin A release from human umbilical vein endothelial cells.

Abstract Activin-A is a member of the TGFβ superfamily found in maternal and umbilical cord blood throughout gestation. We investigated whether human umbilical vein endothelial cells (HUVEC) express activin-A in vivo and tested the effects of vasoactive (endothelin-1), pro-inflammatory (interferon-γ, interleukin-8) and anti-inflammatory (dexamethasone, urocortin) factors on activin-A release by isolated HUVEC in vitro. Activin βA subunit protein and mRNA were strongly localized in the endothelial cells of umbilical veins and were also detectable in scattered cells of the cord connective tissue. Dimeric activin-A was detected in the HUVEC culture medium at picomolar concentrations. Activin-A release by HUVEC decreased after cell incubation with urocortin (p < 0.01), whereas no effect was observed with interleukin-8, interferon-γ, endothelin-1 or dexamethasone. In summary, activin-A is present in the human umbilical vein endothelium in vivo and is produced and released by isolated HUVEC. Activin-A secretion is inhibited in vitro by urocortin, a neuropeptide with predominantly anti-inflammatory action.