A peripheral blood mononuclear cell-based in vitro model: A tool to explore indoleamine 2, 3-dioxygenase-1 (IDO1).

BACKGROUND: Proinflammatory cytokines powerfully induce the rate-limiting enzyme indoleamine 2, 3-dioxygenase-1 (IDO-1) in dendritic cells (DCs) and monocytes, it converts tryptophan (Trp) into L-kynurenine (KYN), along the kynurenine pathway (KP). This mechanism represents a crucial innate immunity regulator that can modulate T cells. This work explores the role of IDO1 in lymphocyte proliferation within a specific pro-inflammatory milieu. METHODS: Peripheral blood mononuclera cells (PBMCs) were isolated from buffy coats taken from healthy blood donors and exposed to a pro-inflammatory milieu triggered by a double-hit stimulus: lipopolysaccharide (LPS) plus anti-CD3/CD28. The IDO1 mRNA levels in the PBMCs were measured by RT-PCR; the IDO1 activity was analyzed using the KYN/Trp ratio, measured by HPLC-EC; and lymphocyte proliferation was measured by flow cytometry. Trp and epacadostat (EP) were used as an IDO1 substrate and inhibitor, respectively. KYN, which is known to modulate Teffs, was tested as a positive control in lymphocyte proliferation. RESULTS: IDO1 expression and activity in PBMCs increased in an in vitro pro-inflammatory milieu. The lymphoid stimulus increased IDO1 expression and activity, which supports the interaction between the activated lymphocytes and the circulating myeloid IDO1-expressing cells. The addition of Trp decreased lymphocyte proliferation but EP, which abrogated the IDO1 function, had no impact on proliferation. Additionally, incubation with KYN seemed to decrease the lymphocyte proliferation. CONCLUSION: IDO1 inhibition did not change T lymphocyte proliferation. We present herein an in vitro experimental model suitable to measure IDO1 expression and activity in circulating myeloid cells.

Reduction of IL-6, IL-8 and ß2-ADRENOCEPTOR mRNA levels in circulating polymorphonuclear leukocytes after adenotonsillectomy in children with Obstructive Sleep Apnea Syndrome.

BACKGROUND: Obstructive Sleep Apnea Syndrome (OSAS) affects approximately 1-5% of children and is linked to cardiovascular, metabolic, and neurobehavioral complications. Dysregulation of inflammatory process and sympathetic nervous system overstimulation leading to increased catecholamine production may contribute to OSAS pathogenesis. Polymorphonuclear Neutrophils (PMN), key cells in the inflammatory process, express adrenergic receptors, including ß2-adrenergic receptor (ADRB2), which modulate their functions through an autocrine/paracrine loop. In this pilot study, we aimed to investigate the relationship between OSAS severity, ADRB2 expression in PMN and patient's inflammatory profile before and after adenotonsillectomy. PATIENTS/METHODS: In this pilot study we enrolled OSAS pediatric patients in which ADRB2, IL-6 and IL-8 mRNA expression levels were evaluated in circulating PMN by RT-PCR. RESULTS: 9 OSAS pediatric patients, ranged from 3 to 8 years of age, were enrolled in the study. We found that adenotonsillectomy significantly reduced ADRB2 as well as IL-6, IL-8 mRNA expression levels in PMN. CONCLUSIONS: These findings offer valuable insights into the underlying immune and inflammatory mechanisms of OSAS and open the way for the development of novel therapeutic approaches.

Effect of Cannabidiol on Human Peripheral Blood Mononuclear Cells and CD4+ T Cells.

Cannabidiol (CBD), the main non-psychoactive component of Cannabis sativa L., is widely used in therapy for the treatment of different diseases and as an adjuvant drug. Our aim was to assess the effects of CBD on proinflammatory cytokine production and cell proliferation in human peripheral blood mononuclear cells (PBMCs) and on CD4+ T lymphocyte differentiation, and, furthermore, to test CBD's ability to affect the functional properties of regulatory T cells (Treg). Experiments were performed on isolated PBMCs and purified CD4+ T lymphocytes obtained from the buffy coats of healthy subjects. Cytokines produced by CD4+ T cells were evaluated by flow cytometry and intracellular cytokine staining techniques. PBMC cytokine production was measured by an ELISA assay. Real-time PCR was used to assess the mRNA expression of cytokines and the key transcription factors (TFs) of CD4+ T cells. Finally, the proliferation of PBMC and CD4+ T effector cells (Teff), alone and in the presence of Treg, was assessed by flow cytometry. Results showed that CBD affects both the frequency of IL-4-producing CD4+ and of IFN-γ/IL-17-producing cells and dramatically decreases the mRNA levels of all TFs. Stimuli-induced cytokine mRNA expression was decreased while protein production was unaffected. CBD was unable to affect the ability of Treg to prevent Teff cell proliferation while it slightly increased PBMC proliferation. In conclusion, CBD may inhibit the expression of proinflammatory cytokines; however, the effect of CBD on cell proliferation suggests that this cannabinoid exerts a complex activity on human PBMCs and CD4+ T cells which deserves further investigation.

Effect of Cannabidiol on Cyclooxygenase Type 1 and 2 Expression and Function in Human Neutrophils.

Introduction: In this study, the effects of the cannabinoid CBD were assessed on cyclooxygenase (COX)-1 and COX-2 expression and activity in resting and activated human neutrophils (polymorphonuclear [PMN] leukocytes). Methods: COX expression was measured at the mRNA levels, whereas COX activity was assessed by enzyme-linked immunosorbent assay measurement of prostaglandin (PG)E2. In vitro experiments in a standard commercial acellular assay of COX-1/COX-2 activity completed the study. Results: Results show that CBD profoundly inhibits expression of COX-1 and COX-2 mRNA in activated PMN, however, without any significant consequences for PGE2 production. CBD, however, was able to induce a slight but significant direct inhibition of COX-2 in the acellular model. Conclusion: The effects of CBD occur in the µM concentration range, which is attained in humans with therapeutic doses of the drug, suggesting the clinical relevance of these findings.

Dopaminergic inhibition of human neutrophils is exerted through D1-like receptors and affected by bacterial infection.

Dopamine (DA) affects immune functions in healthy subjects (HS) and during disease by acting on D1-like (D1 and D5) and D2-like (D2, D3 and D4) dopaminergic receptors (DR); however, its effects on human polymorphonuclear leukocytes (PMN) are still poorly defined. We investigated DR expression in human PMN and the ability of DA to affect cell migration and reactive oxygen species (ROS) production. Experiments were performed on cells from HS and from patients (Pts) with bacterial infections as well, during the acute phase and after recovery. Some experiments were also performed in mice knockout (KO) for the DRD5 gene. PMN from HS express both D1-like and D2-like DR, and exposure to DA results in inhibition of activation-induced morphological changes, migration and ROS production which depend on the activation of D1-like DR. In agreement with these findings, DA inhibited migration of PMN obtained from wild-type mice, but not from DRD5KO mice. In Pts with bacterial infections, during the febrile phase D1-like DRD5 on PMN were downregulated and DA failed to affect PMN migration. Both D1-like DRD5 expression and DA-induced inhibition of PMN migration were however restored after recovery. Dopaminergic inhibition of human PMN is a novel mechanism which is likely to play a key role in the regulation of innate immunity. Evidence obtained in Pts with bacterial infections provides novel clues for the therapeutic modulation of PMN during infectious disease.

Immune profile of hyaluronic acid hydrogel polyethylene glycol crosslinked: An in vitro evaluation in human polymorphonuclear leukocytes.

Neauvia hydrogel (N-Gel) is a hyaluronic acid-based dermal filler, cross-linked with polyethylene glycol. This filler contains sodium hyaluronate at different concentrations, poly(ethylene glycol) diglycidyl ether cross-linked, glycine, and l-prolyne. Assessing any effects of N-Gel on immunity and inflammation is of crucial importance. The aim of the study was to characterize the ability of N-Gel to modulate human polymorphonuclear leukocyte (PMN) functions, including migration, oxidative metabolism, and production of proinflammatory mediators. N-Gel was tested on isolated human PMN. Spontaneous and N-formylmethionyl-leucyl-phenylalanine (fMLP)-stimulated migration were examined using the Boyden Chamber technique, whereas the oxidative metabolism was assessed through spectrofluorometric measurement of reactive oxygen species (ROS) production under resting conditions and after stimulation with fMLP. Tumor necrosis factor (TNF)-α and interleukin (IL)-8 mRNA levels were measured by real-time PCR after stimulation with fMLP or Escherichia coli lipopolysaccharide. This study showed that N-Gel reduced fMLP-induced migration and ROS production without affecting these functions in resting cells. In addition, incubation of PMN with N-Gel effectively reduced both TNF-α and IL-8 mRNA levels. N-Gel modulates critical functions of human PMN such as migration and oxidative metabolism, indicating its potential as an anti-inflammatory agent.

Beta1- and Beta2-Adrenoceptors Expression Patterns in Human Non-small Cell Lung Cancer: Relationship with Cancer Histology.

Assessment of Beta-AR protein expression on tumour tissues might be a plausible strategy to select cancer patients who can benefit from Beta-blockers therapy. The aim of this study is to evaluate the differences between resected tissue specimens from primary lung cancer (adenocarcinoma (ADC) and squamous cell carcinoma (SCC)) in terms of expression pattern of Beta1- and Beta2-AR in both tumour and adjacent surrounding non-tumour tissue. This retrospective study was based on the analysis of 80 patients with histologically confirmed diagnosis of primary Non-Small Cell Lung Cancer (NSCLC) who received surgical treatment. The cases were carefully selected in order to obtain the most homogeneous sample in terms of histologic subtype (40 ADCs and 40 SCCs) and clinical stage (10 each). Beta1- and Beta2-AR expression was determined by immunohistochemistry and the staining evaluated by semi-quantitative scoring using the H-score method. In our NSCLC series, Beta1- and Beta2-AR are differentially expressed. Beta1-AR expression is present at low levels in both SCC and ADC. Likewise, when compared with the matched surrounding non-tumour tissues, Beta1-AR expression level was significantly lower in both histologic subtypes. Conversely, Beta2-AR is highly expressed in both histologic subtypes, but clearly highly expressed in ADC when compared with SCC and with their matched surrounding non-tumour tissue. Overall, this clinicopathological study highlights the differential expression of Beta1- and Beta2-AR in ADC and SCC. Repurposing non-selective Beta-blockers in oncologic setting might be a suitable therapeutic strategy for lung ADC. Graphical abstract.

A Novel Standardized Cannabis sativa L. Extract and Its Constituent Cannabidiol Inhibit Human Polymorphonuclear Leukocyte Functions.

Cannabis and cannabinoids offer significant therapeutic benefits for a wide scope of pathological conditions. Among them, the clinical issues rooted in inflammation stand out, nonetheless, the underlying mechanisms are not yet plainly understood. Circumstantial evidence points to polymorphonuclear leukocytes (PMN) as targets for the anti-inflammatory effects of cannabis. Therefore, we conducted this study to assess the effects of CM5, a novel Cannabis sativa L. extract standardized in 5% cannabidiol (CBD), on human PMN functions, including cell migration, oxidative metabolism and production of tumour necrosis factor (TNF)-α. We then sought to investigate whether such effects could be ascribed to its content in CBD. Cell migration was assessed by the Boyden chamber assay, oxidative metabolism by means of spectrofluorimetric measurement of reactive oxygen species (ROS) production, and TNF-α was measured by real time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). Results show that both CM5 and CBD inhibit PMN migration, ROS and TNF-α production, indicating that CBD may be the main item responsible for the effects of CM5. CM5 is however more potent than CBD on cell migration and TNF-α production, and less effective on ROS production, suggesting that beyond CBD, other components of the cannabis plant may contribute to the biological effects of the extract. As a whole, such results support the use of cannabis standardized extract and CBD to stem inflammation; however, they also warrant in-depth investigation of the underlying cellular and molecular mechanisms to better exploit their therapeutic potential.

Improved solubility and increased biological activity of NeoSol™RCL40, a novel Red Clover Isoflavone Aglycones extract preparation.

Red clover (Trifolium pratense L., Fabaceae; RCL), a perennial plant rich in isoflavones, is a natural alternative for menopausal symptoms, as well as antiaging and antioxidant. Isoflavone preparations usually contain aglycones and ß-glycosides. Aglycones, the active moieties, are absorbed slowly and unevenly due to reduced water solubility and biotransformation from ß-glycosides. NeoSol™RCL40 is a novel RCL isoflavone aglycones preparation based on active solubilization technologies. In the present study, NeoSol™RCL40 was shown to induce solubilization of isoflavones and to increase estrogenic and antioxidative effects in comparison to a standard RCL extract (RCLE). NeoSol™RCL40 was prepared from RCLE using as host molecules either 2-pyrrolidone, 1-ethenyl homopolymer (PVP), γ-cyclodextrin, or maltodextrin. Solubilisation assays, performed by means of HPLC-UV, showed that solubilization of isoflavone aglycones was highest with RCLE processed with PVP, which was therefore selected for functional assays. In comparison to RCLE, NeoSol™RCL40 containing the same amount of isoflavone aglycones displayed 3.4 times higher estrogenicity in MCF-7 cell, 1.9-2.0 higher antioxidant activity in the DPPH and in the FRAP assay, and was cytoprotective in PC12 cells. As a whole, results support the ability of NeoSol™RCL40 to promote isoflavones solubilization leading to increased biological activity. NeoSol™RCL40 is therefore an interesting novel preparation providing improved availability of active isoflavones aglycones.

ß2 -Adrenoceptors inhibit neutrophil extracellular traps in human polymorphonuclear leukocytes.

This study tests the hypothesis that in isolated human polymorphonuclear leukocytes (PMN) adrenergic ligands can affect neutrophil extracellular trap (NET) formation. We have previously shown that, in PMN, adrenaline (A), through the activation of adrenergic receptors (AR), reduces stimulus-dependent cell activation; we have, therefore, planned to investigate if AR are involved in NET production. PMN were obtained from venous blood of healthy subject. The ability of adrenergic ligands to affect reactive oxygen species (ROS) production, NET production, and cell migration was investigated in cells cultured under resting conditions or after activation with N-formyl-methionyl-leucyl-phenylalanine (fMLP), LPS, or IL-8. Stimuli-induced NET production measured as ROS, microscopic evaluation, and elastase production was reverted by A and this effect was blocked by the selective ß2 -AR antagonist ICI-118,551. The stimulus-induced ROS generation and migration was prevented by A and by isoprenaline (ISO), and these effects were counteracted only by ICI-118,551 and not by the other two selective ligands for the ß1 and ß3 -AR. Finally, the presence of the ß-ARs on PMN was confirmed, by means of microscopy and flow cytometry. The data of the present study suggest that adrenergic compounds, through the interaction of mainly ß2 -AR, are able to affect neutrophil functions. These data are suggestive of a possible therapeutic role of ß2 -AR ligands (in addition to their classical use), promoting the possible therapeutic relevance of adrenergic system in the modulation of innate immunity proposing their possible use as anti-inflammatory drugs.

Characterization of human leukocyte-HUVEC adhesion: Effect of cell preparation methods.

OBJECTIVE: Sample manipulation to obtain isolated granulocytes represents a key, and often necessary, step in the in vitro studies. We investigated by the means of flow cytometry and microscopic techniques (both optical microscopy [OM] and scanning electron microscopy [SEM]), the granulocyte-endothelium adhesion and the role of sample manipulation. METHODS: By means of a co-culture method, we have analysed the adhesion of human leukocytes, originated from two different blood samples (fresh venous blood [FB] and buffy coat [BC]), to the human umbilical venous endothelial cell (HUVEC) monolayer. Cultured HUVEC were analysed for adhesion molecule expression by means of flow cytometry, while the morphological changes were evaluated by means of SEM. Cell adhesion was evaluated by means of flow cytometry and both OM and SEM. RESULTS: HUVEC expressed under resting conditions the adhesion molecules ICAM-1, VCAM-1 and E-selectin and their expression was upregulated by stimulation with TNF-α (0.1-10ng/ml) as well as with LPS (1µg/ml). SEM analysis showed that stimulation with both stimuli profoundly affect cell morphology. Flow cytometric evaluation of cell adhesion showed that the ability of cells to adhere to HUVEC monolayer was quite different in the two preparations, with the lowest adhesion for FB in all the cell subsets analysed. Finally, isolated granulocytes were able to adhere to HUVEC monolayer more than cells identified in FB or BC and the adhesion was increased during activation of HUVEC with 10ng/ml of TNF-α. CONCLUSION: Our data showed that cell manipulation necessary for the isolation of specific immune cells from whole blood profoundly affect the ability of these cells to adhere to the HUVEC monolayer although their functional properties remain unchanged.

Adrenergic modulation of migration, CD11b and CD18 expression, ROS and interleukin-8 production by human polymorphonuclear leukocytes.

OBJECTIVES: Adrenergic modulation of immunity has been extensively characterized, however, few information exist regarding polymorphonuclear leukocytes (PMN), despite their key role in immunity and inflammation. We investigated the effect of adrenergic agents on human PMN migration, CD11b and CD18 expression, reactive oxygen species (ROS) and interleukin (IL)-8 production, and on adrenoceptor (AR) expression. METHODS: Migration was measured by the Boyden chamber assay, CD11b/CD18 expression was assessed by flow cytometry, intracellular ROS were detected by spectrofluorimetry, and IL-8 was quantitated by standard ELISA assay. AR mRNA levels were measured by real-time PCR and PMN morphology was studied by scanning electron microscopy. RESULTS: Adrenaline(A), noradrenaline and the ß-AR agonist isoprenaline reduced N-formyl-Met-Leu-Phe (fMLP)-induced migration, CD11b/CD18 expression, and ROS production, without affecting IL-8. The effect of A on CD11b was antagonized by yohimbine and propranolol, and increased by prazosin. The effect on ROS production was completely abolished by propranolol. PMN expressed α(1A)-, α(1B)-, α(1D)-, α(2A)-, α(2C)-, ß(1)-, ß(2)-, and ß(3)-AR mRNA. A prevented fMLP-induced morphological changes of PMN. CONCLUSIONS: Adrenergic agents reduced PMN responses mainly through ß-AR, although α-AR may contribute at least to CD11b expression. AR-operated pathways in PMN should be investigated in disease conditions and in the response to therapeutic agents.

Increased expression of dopamine receptors in synovial fibroblasts from patients with rheumatoid arthritis: inhibitory effects of dopamine on interleukin-8 and interleukin-6.

OBJECTIVE: Observations in both animal models of arthritis and patients with rheumatoid arthritis (RA) suggest a role for dopamine and its receptors in RA. Because synovial fibroblasts (SFs) contribute to inflammation and joint destruction in RA, the aim of this study was to investigate dopaminergic pathways in SFs obtained from patients with RA and, for comparison, in SFs from patients with osteoarthritis (OA) undergoing knee joint replacement surgery. METHODS: The expression of all dopamine receptors (D1 -D5 ) and dopamine transporter was assessed by immunofluorescence and immunohistochemical staining. The levels of dopamine receptor and tyrosine hydroxylase messenger RNA were measured by real-time polymerase chain reaction. The intracellular content of dopamine, its precursor, and its main metabolites was assayed by high-performance liquid chromatography. The influence of dopamine on proinflammatory interleukin-6 (IL-6) and IL-8, matrix metalloproteinase 3, and tissue inhibitor of metalloproteinases 1 (TIMP-1) and TIMP-2 was studied in SFs. RESULTS: SFs possess an intrinsic dopaminergic system, including dopamine receptors, dopamine transporter, and tyrosine hydroxylase, and contain dopamine, its precursor, and its main metabolites. SFs from patients with RA, in comparison with those from patients with OA, showed increased expression of dopamine receptors D1 and D5 , and exogenous dopamine strongly inhibited the production of IL-8 in patients with RA. CONCLUSION: SFs from patients with RA and patients with OA show a dopaminergic phenotype. The expression of D1-like dopamine receptors was higher in RASFs, and this increased expression may lead to antiinflammatory effects, as demonstrated by the expression of IL-8. Studies in animal models and patients with RA are needed to assess the therapeutic potential of endogenous, local production of dopamine in synoviocytes.

Isoflavone content and estrogenic activity of different batches of red clover (Trifolium pratense L.) extracts: an in vitro study in MCF-7 cells.

The estrogenicity of different batches of red clover (Trifolium pratense L., Fabaceae; RCL) extracts and its relationship with the isoflavone content were assessed by measuring MCF-7 cell proliferation by flow cytometry and propidium iodide staining. RCL extracts were compared to estradiol (E2) and to the main RCL isoflavones biochanin A, daidzein, genistein and formononetin. Isoflavone content in the extracts was assayed by HPLC. E2 and isoflavones increased MCF-7 proliferation in a concentration-dependent fashion, with the following potency order: E2>>>genistein>biochanin A=daidzein>formononetin. Extracts increased MCF-7 proliferation with different potencies, which in four out of five extracts correlated with the ratios 5,7-dihydroxyisoflavones/7-hydroxyisoflavones. The efficacy of all extracts increased with decreasing genistein contents. A solution containing the main isoflavones at the average concentration of RCL extracts increased MCF-7 proliferation with higher potency and steeper concentration-response curve. The effects of E2, of RCL extracts and of the isoflavone solution were inhibited by the estrogen receptor antagonist 4-hydroxytamoxifen. Flow cytometric analysis of MCF-7 proliferation is a suitable bioassay for the estrogenicity of RCL extracts, thus expanding the characterization of individual batches beyond assessment of chemical composition and contributing to improved standardization of quality and activity.

The essential oil of bergamot stimulates reactive oxygen species production in human polymorphonuclear leukocytes.

Bergamot (Citrus aurantium L. subsp. bergamia) essential oil (BEO) is used in folk medicine as an antiseptic and anthelminthic and to facilitate wound healing. Evidence indicates that BEO has substantial antimicrobial activity; however its effects on immunity have never been examined. We studied the effects of BEO on reactive oxygen species (ROS) production in human polymorphonuclear leukocytes (PMN) and the role of Ca(2+) in the functional responses evoked by BEO in these cells. Results show that BEO increased intracellular ROS production in human PMN, an effect that required the contribution of extracellular (and, to a lesser extent, of intracellular) Ca(2+) . Bergamot essential oil also significantly increased ROS production induced by the chemotactic peptide N-formyl-Met-Leu-Phe and reduced the response to the protein kinase C activator phorbol myristate acetate. In conclusion, this is the first report showing the ability of BEO to increase ROS production in human PMN. This effect could both contribute to the activity of BEO in infections and in tissue healing as well as underlie an intrinsic proinflammatory potential. The relevance of these findings for the clinical uses of BEO needs careful consideration.

Antioxidant and cytoprotective properties of infusions from leaves and inflorescences of Achillea collina Becker ex Rchb.

Plants are the main source of molecules with antioxidant and radical scavenging properties that aid the natural defence systems of cells and may be involved in the preservation of human health, particularly preventing all the physiopathological conditions where oxidative damage is a hallmark. Achillea collina Becker ex Rchb. is a medicinal plant of the Achillea millefolium aggregate (yarrow) traditionally used, particularly in mountain areas, as an infusion or alcohol extract for its digestive, antiinflammatory, analgesic, antipyretic and wound healing properties. The aim of this study was to investigate the antioxidant capacity and cytoprotective activity against oxidative stress of infusions obtained from the leaves and inflorescences of Achillea collina Becker ex Rchb., assessed by chemical (free radical scavenging activity by DPPH and Folin Ciocalteu assay) and biological assays (in vitro model of cytotoxicity and lipid peroxidation in PC12 cells line). Infusions of leaves had the highest antioxidant properties and cytoprotective activity. The antioxidant capacity was significantly correlated with the total phenolic content but not with the cytoprotective profile. Achillea collina Becker ex Rchb. has good antioxidant and cytoprotective properties, suggesting further investigations on its chemical composition and potential health value, particularly for traditionally prepared infusions of leaves.

Immunomodulatory properties of Achyrocline satureioides (Lam.) D.C. infusion: a study on human leukocytes.

AIM OF THE STUDY: Achyrocline satureioides (Lam.) D.C. is a South American native medicinal herb known by the popular name of "Marcela". Its infusion is widely utilized for the treatment of several digestive ailments, as an anti-inflammatory preparation, as a sedative and anti-atherosclerotic. Circumstantial evidence suggests that extracts of Achyrocline satureioides may have immunomodulatory properties. The present study was therefore devised to investigate the in vitro effects Achyrocline satureioides infusion on human peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs). MATERIALS AND METHODS: Experiments were performed on cells isolated from venous blood obtained from healthy donors. PBMC proliferation and cytokine production were assessed by standard ELISA methods. Reactive oxygen species (ROS) production by PMNs was evaluated by spectrofluorimetry. RESULTS: In PBMCs, Achyrocline satureioides infusion in the 0.06-0.24microg/ml quercetin equivalent (QE) concentration range concentration-dependently reduced PHA-induced proliferation and production of interferon (IFN)-gamma and interleukin (IL)-4. Lower concentrations of the infusion (0.006-0.03microg/ml QE), which were ineffective on cell proliferation, significantly increased the production of both IFN-gamma and IL-4 and decreased the ratio IFN-gamma/IL-4. In PMNs, Achyrocline satureioides infusion slightly increased the spontaneous generation of ROS only at concentrations > or =0.06microg/ml QE. On the contrary, in the 0.0012-0.03microg/ml QE concentration range the infusion profoundly inhibited fMLP-induced ROS generation as well as spontaneous and fMLP-induced IL-8 production. CONCLUSIONS: The present results provide evidence that Achyrocline satureioides infusion may exert several immunomodulatory effects, in line with its traditional use as an anti-inflammatory agent in many disease conditions. Further studies are warranted to better characterize such effects and to assess their therapeutic relevance.

Estrogenic activity of 7-hydroxymatairesinol potassium acetate (HMR/lignan) from Norway spruce (Picea abies) knots and of its active metabolite enterolactone in MCF-7 cells.

Lignans are plant polyphenols which may possess anticancer, antioxidant, antimicrobial, anti-inflammatory and immunomodulatory activities. In particular, the lignan 7-hydroxymatairesinol (HMR/lignan, HMR) is a novel precursor of the mammalian lignan enterolactone (EL). In the present study, we investigated the estrogenicity of HMR and of EL in comparison to estradiol (E2), by measuring their effects on growth and apoptotic markers in the human estrogen-sensitive cell line MCF-7. HMR, EL and E2 concentration-dependently increased the percentage of MCF-7 cells in the S phase of the cell cycle, with the following relative potencies: E2 congruent with EL>>HMR, and efficacies: E2>HMR>>EL. Treatment of MCF-7 cells with either HMR, EL or E2 also increased the Bcl-2/Bax mRNA ratio. The effects of HMR and EL were reduced in the presence of the estrogen receptor (ER) antagonist tamoxifene. We conclude that both HMR and its metabolite EL are endowed with estrogenic activity, which is likely to be exerted through the contribution of ER-dependent pathways and to target the same intracellular mechanisms acted upon by E2. The estrogenicity of HMR and EL is however milder than that of E2, as indicated by the lower potencies and efficacies of both lignans. The present results support the notion that dietary supplementation with HMR may result in a mild estrogenic activity, both directly and by providing a suitable source for endogenous EL.