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Chronic pain is related to gastrointestinal (GI) functions because food components affect inflammation and pain through their action on the GI immune and/or neural system and because many analgesics interact with the gut to alter its structure and function. Immunoglobulin G4 (IgG4) are food-specific antibodies resulting from exposure of the gut immune system to nutrients. High IgG4 levels have been found to be associated with inflammation.IgG4 were determined (both with the rapid test and enzyme-linked immunosorbent assay, ELISA) in men and women outpatients with chronic pain. All subjects were asked to exclude for 4 weeks all foods to which they had high blood levels of IgG4 antibodies. Pain and quality of life questionnaires were administered before (visit 1) and after (visit 2) the personalized exclusion diet period. Visual analogue scale (VAS), Italian Pain Questionnaire (QUID) and Margolis (MA) questionnaires were administered to determine pain intensity, pain features and pain extent, while Short Form Health Survey (SF-36) and Profile of Mood States (POMS) were used to test the quality of life and mood state. The nutritional status was evaluated in all subjects. Subject groups were women of reproductive age (pre-MW), women in menopause for at least 1 year (MW) and men.Fifty-four subjects with chronic pain (n = 12 neuropathic, n = 14 diffuse pain, n = 11 headache, n = 17 low back pain) completed the two visits and the 1-month exclusion diet. At visit 1, 47 (87%) subjects showed medium/high levels of IgG4 to at least one food. The foods showing the highest IgG4 values were eggs, dairy products, cereals and dried fruit. At visit 2, IgG4 levels were decreased, increased or unchanged. In all groups, the 4-week exclusion diet resulted in a significant reduction in all pain measures and an improvement of quality of life parameters. In particular, at visit 2, the VAS score determined in the morning decreased by more than 50%.A food elimination diet based on IgG4 antibody levels may be effective in reducing pain and improving quality of life in patients with chronic pain.

Menopause is the last step in the reproductive history of a woman. The ovaries stop producing hormones and the body reacts by lowering its functions, including the neuronal one. Phytoestrogens are plant products with estrogen-like activity able to affect many body functions. The aim of the present experiment was to study the effects of 30 days of regular consumption of a soy-enriched bread containing a known amount of phytoestrogens (genistein and daidzein). Women at climacteric, within 5 years or more than 5 years of menopause, were asked to include in their diet 200 g/day of a bread containing 40 mg of phytoestrogens. The effect on common menopausal symptoms and neurophysiological, hormonal and antioxidant parameters were determined before and after 30 days through questionnaires and experimental tests. Phytoestrogens were measured in the urine. In all groups, there was a significant increase of phytoestrogens in the urine and a decrease of the classical symptoms of menopause as well as a significant improvement in attentional performance tests, the quality of life index and pain intensity. Phytoestrogens present in the soy-enriched bread, are an important supplement in aging women due to their ability to induce estrogen-like effects without the potential side effects of estrogens.

Vitamin C (ascorbic acid) is an essential nutrient with a number of beneficial effects on the human body. Although the majority of mammals can synthesize their own Vitamin C, humans and a few other species, do not produce it and depend on dietary sources for their Vitamin C supply. Among its many effects on cell function and metabolism, Vitamin C has shown, in vitro, a powerful anticancer effect against a number of human tumour cell lines, including myeloid leukaemia.

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

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.

Arsenic Trioxide (ATO) is widely acknowledged as the treatment of choice for Acute Promyelocytic Leukemia (APL). It is a “two-sided” drug since it can induce differentiation or kill APL and other tumor cells according to the dosage. Part of the cytotoxic effects of ATO on APL cells is due to its pro-oxidant activity, a characteristic which ATO shares with a number of other compounds, including high doses of ascorbate (ASC). In a comparative investigation on the cytotoxic effects of both ATO and ASC on HL60 (APL) cell lines, in Vitro, we have been able to confirm the known cytotoxic effects of ATO, but, more importantly, we have demonstrated that ASC is significantly more effective than ATO, in killing these cancer cells in Vitro, when the concentrations are maintained within the millimolar (mM) range, i.e. the range of plasma concentrations at which ASC induces oxidative damage to tumor cells. Since these plasma levels can be reached only by the intravenous administration of high doses of ASC, we propose that intravenous high doses of ASC may represent a potentially revolutionary new approach in the management of APL.

The anticancer properties of Vitamin C (ascorbic acid o sodium ascorbate) are known since at least four decades, However, being a cheap and "natural" product, Vitamin C is not patentable and therefore has never been developed as an anticancer molecule. Recent in vitro investigations have confirmed the extraordinary antitumor properties of high doses of Vitamin C (sodium ascorbate), particularly when administered by the intravenous route, and phase I/II randomized, controlled clinical trials have been started to verify its anticancer properties in vivo. Unfortunately, the controlled clinical trials performed so far, do not confirm the extraordinary results obtained with Vitamin C (sodium ascorbate) in vitro. However, this may depend on a number of different factors, such as the pharmaceutical preparation (Sodium ascorbate may be more suitable than buffered ascorbic acid), the schedule of administration (slow infusion better than rapid infusion), tumor tissue oxygenation (Cancer tissue oxygenation is lower that oxygenation of tumor cell lines, in vitro), etc., which deserve further in depth investigation. Even with these limitations, Vitamin C (sodium ascorbate) in high doses, administered by intravenous route, beyond being extremely effective in vitro, against a number of human tumor cell lines, is safe, has minimal contraindications, improves the quality of life of patients, and is highly selective for cancer cells. The Authors discuss these important aspects and suggest possible solutions to improve the in vivo anticancer effects of Vitamin C (sodium ascorbate).

Background and purpose: 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. Experimental approach: In this study we used human astrocytoma U-373MG cells to investigate the role of nitric oxide (NO), intracellular Ca2+ concentration ([Ca2+]i), and extracellular signal-regulated protein kinase (ERK) in the signalling pathway mediating IL-1β-induced astrocyte proliferation. Key results: Low IL-1β concentrations induced dose-dependent ERK activation which paralleled upregulation of cell division, whereas higher concentrations gradually reversed both these responses by promoting apoptosis. Pretreatment with the nonspecific NOS inhibitor, N-ω-nitro-l-arginine methyl ester (L-NAME) or the selective iNOS inhibitor, N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide dihydrochloride (1400W), antagonized ERK activation and cell proliferation induced by IL-1β. Inhibition of cGMP formation by the guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), partially inhibited ERK activation and cell division. Functionally blocking Ca2+ release from endoplasmic reticulum with ryanodine or 2-aminoethoxydiphenylborane (2-APB), inhibiting calmodulin (CaM) activity with N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide hydrochloride (W7) or MAPK kinase activity with 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthiol]butadiene (U0126) downregulated IL-1β-induced ERK activation as well as cell proliferation. The cytokine induced a transient and time-dependent increase in intracellular NO levels which preceded elevation in [Ca2+]i. Conclusions and implications: These data identified the NO/Ca2+/CaM/ERK signalling pathway as a novel mechanism mediating the mitogenic effect of IL-1β in human astrocytes. As astrocyte proliferation is a hallmark of reactive astrogliosis, our results reveal a new potential target for therapeutic intervention in neuroinflammatory disorders. British Journal of Pharmacology (2008) 153, 1706–1717; doi:10.1038/bjp.2008.40; published online 25 February 2008

The effect of high doses of intravenous (sodium) ascorbate (ASC) in the treatment of cancer has been controversial although there is growing evidence that ASC in high (pharmacologic) concentrations induces dose-dependent pro-apoptotic death of tumor cells, in vitro. Very few data are available on the role of ASC in the treatment of acute myeloid leukemia (AML). Ascorbate behaves as an antioxidant at low (physiologic), and as pro-oxidant at pharmacologic, concentrations, and this may account for the differences reported in different experimental settings, when human myeloid cell lines, such as HL60, were treated with ASC. Considering the myeloid origin of HL60 cells, and previous literature reports showing that some cell lines belonging to the myeloid lineage could be sensitive to the pro-apoptotic effects of high concentrations of ASC, we investigated in more details the effects of high doses (0.5 to 7 mM) of ASC in vitro, on a variety of human myeloid cell lines including the following: HL60, U937, NB4, NB4-R4 (retinoic acid [RA]-resistant), NB4/AsR (ATO-resistant) acute promyelocytic leukemia (APL)-derived cell lines, and K562 as well as on normal CD34+ progenitors derived from human cord blood. Our results indicate that all analyzed cell lines including all-trans retinoic acid (ATRA)- and arsenic trioxide (ATO)-resistant ones are highly sensitive to the cytotoxic, pro-oxidant effects of high doses of ASC, with an average 50 % lethal concentration (LC50) of 3 mM, depending on cell type, ASC concentration, and time of exposure. Conversely, high doses of ASC neither did exert significant cytotoxic effects nor impaired the differentiation potential in cord blood (CB) CD34+ normal cells. Since plasma ASC concentrations within the millimolar (mM) range can be easily and safely reached by intravenous administration, we conclude that phase I/II clinical trials using high doses of ASC should be designed for patients with advanced/refractory AML and APL.

Because the precise immunopathological events occurring in appendicitis are not completely understood, possible local production of endothelin-1 (ET-1) in human appendix was investigated. We used immunohistochemistry and in situ hybridization to detect the presence, distribution, and phenotype of ET-1-positive cells and prepro-ET-1 (pp-ET-1) mRNA-expressing cells. ET-1-positive stromal cells and pp-ET-1 mRNA-expressing cells were detected with different distributions and relative frequencies in normal control appendix, histologically normal appendix, and inflamed appendix. Six of 20 histologically normal appendixes from patients with a clinical diagnosis of acute appendicitis had many ET-1-positive stromal cells and high pp-ET-1 mRNA expression, similar to inflamed appendix. Forty percent of the pp-ET-1 mRNA-expressing cells were neutrophils, and the other positive cells were mast cells and macrophages. We suggest that local production of ET-1 by neutrophils and other inflammatory cells could be a molecular sign of focal inflammation in histologically normal appendixes and that ET-1 could be implicated, with other cytokines, in the pathogenesis of appendicitis by inducing appendiceal ischemia through vasoconstriction.

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.

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.

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.

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.

Background Activins 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. Methods Paraffin 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. Results Follistatin 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. Conclusion The 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.

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 mu-opioid receptors (mu OR) to evaluate the effects of estrogens on formalin-induced behavioural and immune responses in male rats. After two days of priming with 17 beta-estradiol or saline (i.c.v.), animals were subjected to the formalin test; 15 min prior to formalin (50 microl, 5%) or sham injection in the hind paw, animals were treated with an ER antagonist (ICI 182,780, ICI) or a mu OR antagonist (beta-funaltrexamine, FNA) or saline. The spontaneous behaviours, pain-related behaviours and interferon-gamma (IFN-gamma) 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-gamma production; estradiol had no effect on sham-injected animals but strongly reduce the decrease of IFN-gamma production in formalin-injected animals. The results demonstrate that centrally acting estrogens affect ER- and mu OR-mediated pain processing and influence immune function.

Freely interacting male rabbits were studied to establish the effect of exogenous testosterone on interferon-gamma (IFN-gamma) 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-gamma 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-gamma 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-gamma 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-gamma production and GcR binding capacity.

PROBLEM Plasma interferon (IFN)-beta levels, lymphocyte responsiveness, and evaluation of the relationship between circulating antiviral activity (AA) and IFN-gamma production were studied in pregnant women and nonpregnant age-matched controls with the objective of elucidating the downregulation of IFN-gamma production in successful pregnancy. METHOD OF STUDY In plasma and supernatants of peripheral blood mononuclear cell (PBMC) cultures, stimulated with staphylococcal enterotoxin A (SEA) superantigen, from 43 pregnant women with a history of normal pregnancy and 30 healthy nonpregnant age-matched controls, levels of AA were measured in a micromethod by inhibition of the cytopathic effect (CPE) caused by vesicular stomatitis virus (VSV) in the human amnionic cell line (WISH). RESULTS Significantly higher plasma AA (60% was IFN-gamma and residual activity was acid-labile IFN-like) was present in pregnant women than controls. On the other hand, SEA-activated PBMCs from pregnant women produced significantly lower IFN-gamma levels than those of nonpregnant women. Furthermore, maternal plasma AA levels correlated negatively with IFN-gamma production by SEA-stimulated PBMCs. CONCLUSION The hypothesis that successful pregnancy requires downregulation of IFN-gamma is only partially sustained, suggesting that the immunology of pregnancy is more complex and that murine and human pregnancy have different cytokine profiles.