The Therapeutic Potential of the Essential Oil of Thymbra capitata (L.) Cav., Origanum dictamnus L. and Salvia fruticosa Mill. And a Case of Plant-Based Pharmaceutical Development.

This review performs a comprehensive assessment of the therapeutic potential of three native herbs of Crete (Thymbra capitata (L.) Cav., Salvia fruticosa Mill. and Origanum dictamnus L.), their phytochemical constituents, health benefits and issues relevant to their safety, within a translational context. Issues discussed comprise: 1) Ethnopharmacological uses of the three herbs, reviewed through an extensive search of the literature; 2) Systematic analysis of the major phytochemical constituents of each plant, and their medicinal properties; 3) To what extent could the existing medicinal properties be combined and produce an additive or synergistic effect; 4) Possible safety issues. We conclude with a specific example of the use of a combination of the essential oils of these plants as an effective anti-viral product and the experience gained in a case of a plant-based pharmaceutical development, by presenting the major steps and the continuum of the translational chain.

Reporting effectiveness of an extract of three traditional Cretan herbs on upper respiratory tract infection: results from a double-blind randomized controlled trial.

ETHNOPHARMACOLOGICAL RELEVANCE: Observations from the island of Crete, Greece suggest that infusions of traditional Cretan aromatic plants, well known for their ethnopharmacological use in Eastern Mediterranean region and Near East, could be effective in the prevention and treatment of upper respiratory tract infections, including viral-induced infections. The aim of this study was to report the effectiveness of an essential-oil extract of three Cretan aromatic plants in the treatment of cases with an upper respiratory tract infection. MATERIALS AND METHODS: A double blind randomized controlled trial was implemented between October 2013 and February 2014. An essential-oil extract of Cretan aromatic plants in olive oil (total volume of 15ml of essential oil per litre of olive oil) was administered as 0.5ml soft gel capsules, twice a day, for 7 days. Placebo treatment was 0.5ml olive oil in soft gel capsules. Eligible patients were those presenting for clinical examination in the selected setting with signs and symptoms of upper respiratory tract infection that had begun within the previous 24 hours. Real-Time Polymerase Chain Reaction (PCR) was used for the detection of respiratory viruses. The primary outcome was the severity and duration of symptoms of upper respiratory tract infection, assessed using the Wisconsin Upper Respiratory System Survey (WURSS-21) questionnaire. A secondary outcome of interest was the change in C-reactive protein (CRP) status. RESULTS: One hundred and five patients completed the study: 51 in the placebo group, and 54 in the intervention (treated) group. Baseline characteristics were similar in the two groups. No statistically significant differences were found in symptom duration or severity between the two groups, although small and clinically favorable effects were observed. When the analysis was restricted to subjects with a laboratory-documented viral infection, the percentage of patients with cessation of symptoms after 6 days of treatment was 91% in the intervention group and 70% in the control group (p=0.089). At baseline, one third of the patients in each group had elevated CRP levels. At follow-up, the respective proportions were 0% in the intervention group and 15% in the placebo group (p=0.121). The data were also in a favorable direction when 50% and 80% symptom reduction points were considered for specific virus types. CONCLUSIONS: Compared with placebo the essential-oil extract of three Cretan aromatic plants provided no detectable statistically significant benefit or harm in the patients with upper respiratory illness, although descriptive differences were identified in favorable direction mainly in the virus-positive population.

Identification of polyproline II regions derived from the proline-rich nuclear receptor coactivators PNRC and PNRC2: new insights for ERα coactivator interactions.

Protein-protein interactions are crucial for signal transductions required for cell differentiation and proliferation. Their modulation is therefore key to the development of therapeutic alternatives, particularly in the context of cancer. According to literature data, the polyproline-rich nuclear receptor coactivators PNRC and PNRC2 interact with estrogen receptor (ERα) through their PxxP SH3-binding motifs. In a search to identify the molecular features governing this interaction, we explored using electronic circular dichroism (ECD) spectroscopy and molecular dynamics (MD) calculations, the capacity of a range of putative biologically active peptides derived from these proteins and containing this PxxP motif(s) to form polyproline II (PPII) domains. An additional more exhaustive structural study on a lead PPII peptide was also performed using 2D nuclear magnetic resonance (NMR) spectroscopy. With the exception of one of all the investigated peptides (PNRC-D), binding assays failed to detect any affinity for Grb2 SH3 domains, suggesting that PPII motifs issued from Grb2 antagonists have a binding mode distinct from those derived from Grb2 agonists. Instead, the peptides revealed a competitive binding ability against a synthetic peptide (ERα17p) with a putative PPII-cognate domain located within a coregulator recruitment region of ERα (AF-2 site). Our work, which constitutes the first structure-related interaction study concerning PNRC and PNRC2, supports not only the existence of PxxP-induced PPII sequences in these coregulators, but also confirms the presence of a PPII recognition site in the AF-2 of the steroid receptor ERα, a region important for transcription regulation.

Polyphenols and cancer cell growth.

Polyphenols constitute an important group of phytochemicals that gained increased research attention since it was found that they could affect cancer cell growth. Initial evidence came from epidemiologic studies suggesting that a diet that includes regular consumption of fruits and vegetables (rich in polyphenols) significantly reduces the risk of many cancers. In the present work we briefly review the effects of polyphenols on cancer cell fate, leading towards growth, differentiation and apoptosis. Their action can be attributed not only to their ability to act as antioxidants but also to their ability to interact with basic cellular mechanisms. Such interactions include interference with membrane and intracellular receptors, modulation of signaling cascades, interaction with the basic enzymes involved in tumor promotion and metastasis, interaction with oncogenes and oncoproteins, and, finally, direct or indirect interactions with nucleic acids and nucleoproteins. These actions involve almost the whole spectrum of basic cellular machinery--from the cell membrane to signaling cytoplasmic molecules and to the major nuclear components--and provide insights into their beneficial health effects. In addition, the actions justify the scientific interest in this class of compounds, and provide clues about their possible pharmaceutical exploitation in the field of oncology.

kappa-opioids induce a reversible inhibition of CFU-GM from CD133(+) cord blood cells.

BACKGROUND: Opioid agonists have been shown to exert an inhibitory action on a number of malignant and non-malignant cell types. However, there are no reports dealing with their effect on hemopoietic progenitors. Based upon our previous experience of opioid agonists we examined whether opioids could interfere with the growth of CFU-GM from CD133(+) cord blood cells. METHODS: Cord blood samples were subjected to CD133(+) column selection, with subsequent exposure to opioid agonists and antagonists or both, in semi-solid cultures for CFU-GM growth. Colonies of day 7 of culture were replated in fresh medium in the absence of opioids. The colonies were evaluated at 7 and 14 days of culture. RT-PCR was performed for the detection of opioid and somatostatin receptors. Apoptosis tests and immunophenotypic evaluations were employed in liquid cultures in conditions identical to those of the semi-solid ones. RESULTS AND DISCUSSION: Our results suggest that opioids can induce a significant inhibition of CFU-GM growth, which is reversible and not mediated through opioid or somatostatin receptors, while apoptosis is not implicated. Whether this finding could be used for clinical intervention remains to be examined.

Rapid effects of 17beta-estradiol and progesterone on sheep visceral and parietal pleurae via a nitric oxide pathway.

We investigated the effects of 17beta-estradiol and progesterone on transepithelial electrical resistance (R(TE)) in sheep visceral and parietal pleurae. Specimens of intact pleurae from adult female sheep were used. The samples were transferred to the laboratory within 30 min after death of the animal in a Krebs-Ringer solution at 4 degrees C. The pleura was then mounted as a planar sheet in Ussing-type chambers, and electrical measurements were made. There was an increase in R(TE) in all of the samples examined after addition of 17beta-estradiol and progesterone in visceral and parietal pleurae. This increase was rapid within 1 min, lasted for ~15 min, returned to the basal level within 30-45 min, and was dose dependent. Tamoxifen, an estrogen receptor antagonist, did not significantly eliminate the effect of 17beta-estradiol. Furthermore, no steroid receptors were identified in cytosolic preparations of visceral and parietal pleura with ligand binding assays. The estrogen- and progesterone-induced increase in R(TE) in both visceral and parietal pleurae was affected by addition of an inhibitor of nitric oxide synthase. Indeed, previous administration of N(omega)-nitro-L-arginine methyl ester prevented the increase in R(TE) by 17beta-estradiol and progesterone. These results suggest that 17beta-estradiol and progesterone induce an increase in R(TE) in both visceral and parietal pleura and thus alter the transepithelial permeability. The effect of steroids may be accounted for by rapid release of nitric oxide in pleura.

Matrix metalloproteinases and their inhibitors in acute viral hepatitis.

Matrix metalloproteases (MMPs) and their inhibitors are effector molecules involved in extracellular matrix remodelling. The serum profile for these proteolytic enzymes and their inhibitors during acute self-limiting viral hepatitis has not been studied. We therefore determined serum concentrations of MMP-1, MMP-3, MMP-2, MMP-9 and their inhibitors (tissue inhibitors of metalloproteinase) TIMP-1, TIMP-2 and alpha2 macroglobulin (AMG) in the serum of patients during the icteric stage of self-limiting acute viral hepatitis. Transforming growth factor-beta (TGF-beta) and interleukin (IL)-10, two cytokines involved in the regulation of MMPs and TIMPs were also assessed. Nineteen patients (12 men, seven women) with a mean age of 29.9 years (range 16-65 years) participated in the study. Fifteen had hepatitis B virus (HBV, two HCV and two HAV infection. The values of patients were compared with those obtained from 15 blood donor controls (eight men, seven women), mean age 36.2 years (range 18-55 years). Serum levels of TGF-beta, IL-10, MMP-1, MMP-3, MMP-2, MMP-9, TIMP-1 and TIMP-2 were assessed by ELISA. MMP-2 and MMP-9 were also measured by a zymogram protease assay. alpha2 macroglobulin (AMG) was measured by nephelometry. Compared with the healthy controls the mean serum concentrations of all MMPs were significantly decreased in the acute hepatitis patients. There was no difference in the serum concentration of TIMP-1 between patients and the controls. Serum levels of TIMP-2 (P < 0001), TGF-beta (P < 0.05), IL-10 (P < 0.001) and AMG (P < 0001) were increased in patients compared to healthy controls. A statistically significant negative correlation by linear regression analysis was found between AMG and MMP-1 (P=0003). The decreased levels of MMPs observed, together with normal and increased levels of TIMP-1 and TIMP-2, may indicate an attempt to limit matrix degradation at this stage of disease resolution. The increased levels of the anti-inflammatory cytokines IL-10 and TGF-beta might be the underlying mechanism responsible for the above effect. AMG inhibition especially for MMP-1 may play an additional important role.

Receptorphin: a conserved peptide derived from the sequence of the opioid receptor, with opioid displacement activity and potent antiproliferative actions in tumor cells.

BACKGROUND: In addition to endogenous opioids, a number of peptide sequences, derived from endogenous (hemorphins, alphaS1-casomorphin), and exogenous proteins (casomorphins, exorphins) have been reported, possessing opioid activity. In the present work, we report the identification of a new peptide, receptorphin (Tyr-Ile-Phe-Asn-Leu), derived from the sequence of the second transmembrane loop of the opioid receptor. This sequence is unique for the opioid receptor, and conserved in all species and receptor-types. RESULTS AND DISCUSSION: Receptorphin competes for opioid binding, presenting a kappa-receptor interaction, while it binds equally to delta- and mu- opioid and somatostatin-binding sites, and inhibits the cell proliferation of a number of human cancer cell lines, in a dose-dependent and reversible manner, at the picomolar or the nanomolar range. Receptorphin shows a preferential action on prostate cancer cells. CONCLUSION: Our work identifies, for the first time a peptide, in a receptor sequence, possessing ligand-agonistic activities. A hypothesis, based on receptorphin liberation after cell death, is presented, which could tentatively explain the time-lag observed during opioid antiproliferative action.

Opioids are non-competitive inhibitors of nitric oxide synthase in T47D human breast cancer cells.

Opioids and nitric oxide (NO) interact functionally in different systems. NO-generating agents decrease the activity of opioid agonists, prevent opioid tolerance, and are used in opioid withdrawal syndromes. There exist, however, few reports indicating a direct interaction of the two systems. T47D human breast cancer cells in culture express opioid receptors, and opioid agonists inhibit their growth, while they release high amounts of the NO-related molecules NO(2-)/NO(3-)to the culture medium. We have used this system to assay a possible direct interaction of opiergic and nitric oxide systems. Our results show that delta- or mu-acting opioid agonists do not modify the release of NO(2-)/NO(3-). In contrast, kappa-acting opioid agonists (ethylketocyclazocine, and alpha(S1)-casomorphine) decrease the release of NO(2-)/NO(3-), in a time- and dose-dependent manner. The general opioid antagonist diprenorphine (10(-6) M) produce a similar NO(2-)/NO(3-)release inhibition, indicating a possible non-opioid-receptor mediated phenomenon. In addition, ethylketocyclazocine, alpha(S1)-casomorphin and diprenorphine directly inhibit NOS activity: agonists, interact with both calcium-dependent and independent NOS-isoforms, while the antagonist diprenorphine modifies only the activity of the calcium-dependent fraction of the enzyme. Analysis of this interaction revealed that opioids modify the dimeric active form of NOS, through binding to the reductase part of the molecule, acting as non-competitive inhibitors of the enzyme. This interaction opens interesting new possibilities for tumor biology and breast cancer therapy.

Anti-Saccharomyces cerevisiae mannan antibodies and antineutrophil cytoplasmic autoantibodies in Greek patients with inflammatory bowel disease.

OBJECTIVES: The combined measurement of perinuclear antineutrophil cytoplasmic autoantibodies (pANCA) and anti-Saccharomyces cerevisiae mannan antibodies (ASCA) has recently been suggested as a valuable diagnostic approach in inflammatory bowel disease (IBD). The aim of this study was to assess the value of detecting pANCA and ASCA in the differentiation between ulcerative colitis (UC) and Crohn's disease (CD) in a Greek population with IBD. METHODS: Sera were collected from 157 patients with IBD (97 with UC, 56 with CD, and four with indeterminate colitis) and 150 healthy controls. Determination of pANCA was performed by a standard indirect immunofluorescence technique on ethanol-fixed granulocytes and ASCA by an ELISA assay. RESULTS: In patients with UC, sensitivity, specificity, positive predictive value, and negative predictive value of the pANCA test was 67%, 84%, 93%, and 46% respectively. These values did not change significantly when the combination of positive pANCA and negative ASCA was used. ASCA test in diagnosing CD yielded a sensitivity, specificity, positive predictive value, and negative predictive value of 39%, 89%, 54%, and 81%. The combination of pANCA negative and ASCA positive increased the positive predictive value to 77% and it was associated with small bowel disease. CONCLUSIONS: A positive pANCA test in Greek patients has a diagnostic value in confirming a diagnosis of UC. Measurement of pANCA and ASCA together has a rather limited value in the differential diagnosis between UC and CD but may be of help in studying disease heterogeneity.

Somatostatin and opioid receptors in mammary tissue. Role in cancer cell growth.

Somatostatin and opioid systems, are the two main inhibitory systems in mammals. Both classes of substances have been identified in normal and malignant mammary gland, as well as their cognitive receptors. They have been implied in the inhibition of cell growth of cancer cells and cell lines, in a dose-dependent and reversible manner. Somatostatin acts through homologous receptors (SSTRs), belonging to five distinct classes (SSTR1-5). We, and others have identified SSTR2 and 3 as been the only SSTRs present in the breast. Furthermore, opioids act through the three classes of opioid receptors (mu, delta,kappa). In the breast, kappa opioid receptor subtypes (kappa 1-kappa 3) are the most widely expressed. We further have shown that opioids, in addition to their binding to opioid receptors, compete for binding to SSTRs. This functional interaction, together with other identified modes of opioid action in the breast (modulation of steroid receptors, proteases' secretion, interaction with cytoskeletal elements), will be discussed, taking into consideration also the possible local production of casomorphins (casein-derived opioids), which are very potent antiproliferative agents.

Potent inhibitory action of red wine polyphenols on human breast cancer cells.

Breast cancer (one of the most common malignancy in Western societies), as well as esophagus, stomach, lung, bladder, and prostate cancer, depend on environmental factors and diet for growth and evolution. Dietary micronutriments have been proposed as effective inhibitory agents for cancer initiation, progression, and incidence. Among them, polyphenols, present in different foods and beverages, have retained attention in recent years. Red wine is a rich source of polyphenols, and their antioxidant and tumor arresting effects have been demonstrated in different in vitro and in vivo systems. In the present study, we have measured the antiproliferative effect of red wine concentrate, its total polyphenolic pool, and purified catechin, epicatechin, quercetin, and resveratrol, which account for more than 70% of the total polyphenols in red wine, on the proliferation of hormone sensitive (MCF7, T47D) and resistant (MDA-MB-231) breast cancer cell lines. Our results indicate that polyphenols, at the picomolar or the nanomolar range, decrease cell proliferation in a dose- and a time-dependant manner. In hormone sensitive cell lines, a specific interaction of each polyphenol with steroid receptors was observed, with IC(50)s lower than previously described. Interaction of polyphenols with steroid receptors cannot fully explain their inhibitory effect on cell proliferation. In addition, discrete antioxidant action on each cell line was detected under the same concentrations, both by modifying the toxic effect of H(2)O(2), and the production of reactive oxygen species (ROS), after phorbol ester stimulation. Our results suggest that low concentrations of polyphenols, and consecutively, consumption of wine, or other polyphenol-rich foods and beverages, could have a beneficial antiproliferative effect on breast cancer cell growth.

Kappa opioids and TGFbeta1 interact in human endometrial cells.

The transforming growth factor beta1 (TGFbeta1) is a major regulator of human endometrial function. Human endometrium possesses specific opioid binding sites, the majority of which belong to the kappa type, for which the prodynorphin-derived opioids are the endogenous ligands. Since these two systems interact in several other tissues we postulated that opioids may affect the production of TGFbeta1 in human endometrium. We have found that kappa opioids exerted a time- and dose-dependent inhibitory effect on TGFbeta1 production from endometrial stromal and epithelial cells and from the Ishikawa human endometrial adenocarcinoma cell line. This effect was reversible by the specific opioid antagonist diprenorphine. To examine if this effect represents a paracrine endometrial response to locally produced kappa opioids we searched for the presence of the endogenous kappa opioid receptor ligands. Indeed, the prodynorphin transcript was detectable on Northern blots from normal and tumoral human endometrial cells; its size was that of the pituitary transcript, i.e. approximately 2.4 kb long. Most immunoreactive dynorphin from human endometrium had a molecular weight of 8 kDa. Finally, immunofluorescence staining of normal and tumoral human endometrial cells revealed the presence of dynorphin-positive cytoplasmic secretory granules. Taken together, our data suggest that in human endometrium, kappa opioids and the TGFbeta1 form a paracrine network which appears to be retained by the Ishikawa human endometrial adenocarcinoma cell line.

Wine antioxidant polyphenols inhibit the proliferation of human prostate cancer cell lines.

The effect of different wine antioxidant polyphenols (catechin, epicatechin, quercetin, and resveratrol) on the growth of three prostate cancer cell lines (LNCaP, PC3, and DU145) was investigated. A dose- and time-dependent inhibition of cell growth by polyphenols was found at nanomolar concentrations. The proliferation of LNCaP and PC3 cells was preferentially inhibited by flavonoids (catechin, epicatechin, and quercetin), whereas resveratrol was the most potent inhibitor of DU145 cell growth. Possible mechanisms of action were investigated: 1) The competition of polyphenols for androgen binding in LNCaP cells revealed significant interaction only in the case of high concentrations of quercetin, at least at five orders of magnitude higher than the concentrations needed for cell growth inhibition. All other phenols showed low interactions. 2) Oxygen species production after mitogen stimulation and H2O2 sensitivity of these cell lines did not correlate with the observed antiproliferative effects, ruling out such a mode of action. 3) NO production revealed two different patterns: LNCaP and DU145 cells produced high concentrations of NO, whereas PC3 cells produced low concentrations. Phorbol ester stimulation of cells did not reveal any additional effect in LNCaP and DU145 cells, whereas it enhanced the secretion of NO in PC3 cells. Polyphenols decreased NO secretion. This effect correlates with their antiproliferative action and the inhibition of inducible NO synthase. It is therefore proposed that the antiproliferative effect of polyphenols is mediated through the modulation of NO production. In conclusion, our data show a direct inhibitory effect of low concentrations of antioxidant wine phenols on the proliferation of human prostate cancer cell lines mediated by the production of NO, further suggesting potential beneficial effects of wine and other phenol-containing foods or drinks for the control of prostate cancer cell growth.

Safety of the ArF193 excimer laser for the removal of dental plaque and calculi: an in vitro histological study.

OBJECTIVE: To assay the safety of the ArF excimer laser in the integrity of human pulp elements. BACKGROUND DATA: The use of lasers in dentistry remains controversial, in spite of their increasing application in medical practice. The main reason for this discrepancy is the frequent report of damage to surrounding tissues and the dental pulp, due to the energy transfer, from the site of laser impact. The progress made on laser technology during the last 10 years, could overcome this obstacle and allow the use of lasers in dentistry. METHODS AND RESULTS: The present study reports the use of the ArF 193 excimer laser, under conditions of strict control of frequency and fluency, for the ablation of dental carries, plaque, and calculi, by the use of a new, articulated arm. We have tested 10 teeth, extracted for prosthetic reasons, immediately after extraction. Our in vitro results show that the ArF193 excimer laser does not produce any harm to the dental pulp (at least at the photo- or electronic microscopy level), whereas in a matter of seconds, it can be effective in removing all dental deposits. In addition, the use of the flexible articulated arm, makes this treatment comfortable and easier for both the dentist and patient. CONCLUSION: Under a strict control of laser technology, and the use of the new articulated arm presented, the use of the ArF excimer laser in dentistry is safe and comfortable.

Opioid agonists modify breast cancer cell proliferation by blocking cells to the G2/M phase of the cycle: involvement of cytoskeletal elements.

Opioids decrease cell proliferation in different systems including breast, prostate, lung, kidney, and intestine, through an interaction with opioid as well as other membrane-receptor systems (somatostatin, cholinergic), through an unidentified mechanism. Recently, we have reported an interaction of taxol with opioid membrane sites (BBRC 235, 201-204, 1997), and an involvement of opioids to the modification of actin cytoskeleton in renal OK cells (J Cell Biochem. [19981 70:60-69), indicating a possible action of the opioid effect. In the present work, we have examined the effect of two general opioid agonists (ethylketocyclazocine and etorphine) on the cell cycle, in human breast cancer T47D cells, as well as a possible modification of the cellular cytoskeleton under their action, in order to explain the antiproliferative effect of these agents. These two opioids produce a dose-dependent and reversible decrease of the proliferation of T47D cells, with a maximum attained at 10(-8) M. The addition of 10(-8) M of either opioid produced a significant increase of the number of cells arrested in the G2/M phase. Confocal laser microscopy revealed a modification of the actin and tubulin microfilaments, with a clear redistribution at the periphery of the cell, reversed by the addition of the general opioid antagonist diprenorphine. Furthermore, differences between the two opioids were obvious, attributed to the different receptor affinity of each agent. The observed redistribution of actin and tubulin cytoskeletal elements gives therefore a possible answer of the antiproliferative action of opioids. The modification of the cytoskeleton, directly involved to cell division, might provoke a "mechanical" obstacle, which could be the reason of the antiproliferative effect of these agonists. Furthermore, the observed tubulin-opioid interaction by opioids provides a possible explanation of the arrest at the G2/M phase of T47D cells under opioid treatment. Nevertheless, although the observed interaction of opioids with cytoskeletal elements gives a plausible answer of the antiproliferative effects of the agents, this might not be the only action of these agents in cell proliferation. Other, direct or indirect, genomic actions, which which remains to be elucidated, might be taken into consideration.

Kappa1-opioid binding sites are the dominant opioid binding sites in surgical specimens of human pheochromocytomas and in a human pheochromocytoma (KAT45) cell line.

The adrenal medulla produces opioids which exert paracrine effects on adrenal cortical and chromaffin cells and on adrenal splanchnic nerves, via specific binding sites. The opioid binding sites in the adrenals are detectable mainly in the medullary part of it and differ in type between species. Thus, the bovine adrenal medulla contains mostly kappa-opioid binding sites and fewer delta- and mu-opioid binding sites while primate adrenals contain mainly delta sites and few kappa-opioid binding sites. Most chromaffin cell tumors, the pheochromocytomas, produce opioids which suppress catecholamine production by the tumor. The aim of the present work was to identify the types of opioid binding sites in human pheochromocytomas. For this purpose, we characterized the opioid binding sites on crude membrane fractions prepared from 14 surgically excised pheohromocytomas and on whole KAT45 cells, a recently characterized human pheochromocytoma cell line. Our data showed that human pheohromocytomas are heterogeneous, as expected, with regard to the production of catecholamines and the distribution and profile of their opioid binding sites. Indeed, only one out of the 14 pheochromocytomas expressed exclusively delta and mu opioid sites, while in the remaining 13 tumors kappa-type binding sites were dominant. The KAT45 cell line possessed a significant number of kappa1 binding sites, fewer kappa2-opioid binding sites and kappa3-opioid binding sites, and minimal binding capacity for delta- and mu-opioid receptor agonists sites. More specifically, the kappa1 sites/cell were approximately 18,000, the kappa2 4500/cell and the kappa3 sites 2000/cell. Our findings for the surgical specimens and the cell line combined with previously published pharmacological data obtained from KAT45 cells suggest that kappa sites appear to be the most prevalent opioid binding sites in pheochromocytomas. Finally, in normal bovine adrenals the profile of opioid binding sites differs in adrenaline and noradrenaline producing chromaffin cells. To test the hypothesis that the type of catecholamine produced by a pheochromocytoma depends on its cell of origin, we compared our binding data with the catecholamine content of each pheochromocytoma examined. We found no correlation between the type of the predominant catecholamine produced and the opioid binding profile of each tumor suggesting that this hypothesis may not be valid.

Modulation of the estrogen-regulated proteins cathepsin D and pS2 by opioid agonists in hormone-sensitive breast cancer cell lines (MCF7 and T47D): evidence for an interaction between the two systems.

In many cancer cell lines, including breast, prostate, lung, brain, head and neck, retina, and the gastrointestinal tract, opioids decrease cell proliferation in a dose-dependent and reversible manner. Opioid and/or other neuropeptide receptors mediate this decrease. We report that only the steroid-hormone-sensitive cell lines MCF7 and T47D respond to opioid growth inhibition in a dose-dependent manner. Therefore, an interaction of the opioid and steroid receptor system might exist, as is the case with insulin. To investigate this interaction, we have assayed two estrogen-inducible proteins (pS2 and the lysosomal enzyme cathepsin D) in MCF7 and T47D cells. When cells were grown in the presence of FBS (in which case a minimal quantity of estrogens and/or opioids is provided by the serum), we observed either no effect of etorphine or ethylketocyclazocine (EKC) or an increase of secretion and/or production of pS2 and cathepsin D. However, when cells were cultured in charcoal-stripped serum and in the absence of phenol red, the effect of the two opioids is different: EKC decreased the production and/or secretion of pS2 and cathepsin D, whereas etorphine increased their synthesis and/or secretion. The differential effect of the two general opioids was attributed to their different receptor selectivity. Furthermore, the variations of the ratio of secreted/produced protein and the use of cycloheximide indicate that opioids selectively modify the regulatory pathway of each protein discretely. In conclusion, through the interaction with opioid and perhaps other membrane-receptor sites, opioid agonists modify in a dose-dependent manner the production and the secretion of two estrogen-regulated proteins. Opioids may therefore disturb hormonal signals mediated by the estrogen receptors. Hence, these chemicals may have potential endocrine disrupting activities.

Low stimulation of peripheral lymphocytes, following in vitro application of Emdogain.

Fast tissue regeneration after therapeutic manipulations is a central problem of periodontology, oral surgery and trauma of the periodontal tissues, including bone. Several products, which augment tissue regeneration, have been manufactured and assayed in clinical practice with positive results. Emdogain is a recent addition in this field, as a tissue-regenerating product. The substance is a derivative of amelogenin, obtained from porcine embryonic tissues. At the present time, it is not known whether the substance can induce a local (due to the uptake of the substance) or systemic immune response. The aim of the present study was to evaluate, in vitro, the ability of Emdogain to influence, in vitro, the immune system. Peripheral blood lymphocytes, isolated for 10 healthy donors, were cultured in the presence of various concentrations of the substance, in order to determine the rate of cell proliferation, the expression of surface antigens and the production of cytokines and immunoglobulins. Under our experimental conditions, Emdogain produced a slight increase of the proliferation of lymphocytes, restricted to the CD25 (IL-2 receptor) fraction of the CD4 positive T-lymphocytes, and a concomitant decrease of CD19 positive B-lymphocytes. Other cell fractions (CD8 positive T-cells, B-cells and NK-cells) were not affected. Under our conditions too, immunoglobulin and cytokin (IL-2 and IL-6) production was not modified, even after a 3-day application of concentrations much higher than those used in clinical practice. Our data suggest that Emdogain slightly induce an immune response, restricted to the activated fraction of CD4 T-lymphocytes in vitro.

Early alterations of actin cytoskeleton in OK cells by opioids.

Recently we identified and characterized opioid binding sites in OK (opossum kidney) cells and observed decreased proliferation of these cells in response to opioids. In the present study we investigated the effects of opioids on the actin cytoskeleton and explored whether their antiproliferative action may relate to alterations in the distribution or the dynamics of actin microfilaments. Exposure of OK cells to the opioids alphaS1 casomorphin and ethylketocyclazocine resulted in a rapid and substantial actin microfilament reorganization. This was documented by a significant dose-dependent decrease in the amounts of F-actin, determined by measurements of quantitative fluorescence, by immunoblot analysis and by a concomitant increase of the G/total-actin ratio measured by the DNase I inhibition assay. These changes were verified by confocal laser scanning microscopy, which showed marked redistribution of the microfilamentous structures in the presence of the opioids without affecting the organization of microtubules or vimentin intermediate filaments. The effect of opioids on actin polymerization dynamics occurred within 15 min and persisted for at least 2 h, while their restoration to control levels was accomplished 6 h later, indicating a reversible phenomenon. Northern blot analysis showed that the concentration of the actin transcript was unaffected. The addition of diprenorphine, a general opioid antagonist, prevented the effects of opioids on the actin cytoskeleton. The inhibition of OK cell proliferation, induced by ethylketocyclazocine and alphaS1 casomorphin was partially prevented in the presence of phallacidin, which stabilizes microfilaments. Our findings demonstrate that opioids, acting via kappa 1 binding sites, induce rapidly modifications in the dynamics of actin polymerization, and in the organization of microfilaments in OK cells, which may relate to their antiproliferative effect on these cells.