Synergistic antiproliferative activity of quercetin and cisplatin on ovarian cancer cell growth

It has been demonstrated that the flavonoid quercetin (3,3',4',5-7-pentahydroxyflavone) (Q) inhibits the growth of several cancer cell lines and that the antiproliferative activity of this substance is mediated by a so-called type II estrogen binding site (type II EBS). We investigated the effects of quercetin and cisplatin (CDDP) alone and in combination on the proliferation of the ovarian cancer cell line OVCA 433. Both drugs exhibited a dose-related growth inhibition in a range of concentrations between 0.01 and 2.5 microM and 0.01 and 2.5 micrograms/ml for Q and CDDP respectively. The combination of the two drugs resulted in a synergistic antiproliferative activity. Two other flavonoids tested, i.e., rutin (3-rhamnosylglucoside of quercetin) and hesperidin [7-b rutinoside of hesperetin (3'-5-3-hydroxy-4-methoxyflavone)] were ineffective both alone and in combination with CDDP. Since both rutin and hesperidin do not bind to type II EBS it can be hypothesized that Q synergizes with CDDP by acting through an interaction with these binding sites.     

Effect of age and asthma duration upon elastase and alpha1-antitrypsin levels in adult asthmatics.

In asthmatic subjects an imbalance between elastase and alpha1-antitrypsin (alpha1-PI) exists. This study aims to evaluate whether ageing per se affects the levels of elastase. Both young and elderly asthmatics with comparable severity and duration of disease, as well as young and elderly healthy subjects, underwent an induced sputum procedure to measure levels of elastase and alpha1-PI. The percentage of sputum neutrophils and eosinophils was higher in young and elderly asthmatics than in young and elderly controls. The levels of both total and active elastase were significantly higher in young and elderly asthmatics than in young and elderly controls, and directly correlated with the percentage of neutrophils. In addition, in both young and elderly asthmatics the levels of total and active elastase were negatively correlated with forced expiratory volume in one second values, but positively correlated with the duration of the disease. This study indicates that ageing per se does not necessarily lead to a progressive elastase/alpha1-antitrypsin imbalance in asthma, and suggests that an important variable in the development of airway remodelling in both young and elderly asthmatics is represented by the duration of the disease.     

Effect of rigid contact lens oxygen transmissibility on stromal pH in the living human eye

Corneal stromal pH was measured fluorometrically in nine human subjects after 1.5 hours of eye closure while wearing hard gas permeable contact lenses. Six lens types providing a wide range in oxygen transmissibilities (Dk/L)O2 from 0.15 to 55.0 X 10(-9) (cm ml O2/seconds of ml mmHg) were used. Stromal pH upon opening the eyes was directly related to (Dk/L)O2 and ranged from 7.01 to 7.26. These pH values were all significantly lower than the control (no lens), which had a pH of 7.38 (P less than 0.005). After eye opening (lens still on) stromal pH increased and reached a new steady-state (range, 7.11-7.45) in approximately 30 minutes; however, these pH levels were still below the control (no lens) (pH 7.54; P less than 0.005). The rate of pH change after eye opening also increased directly with (Dk/L)O2. The authors conclude that the lenses presently available provide insufficient gas exchange to prevent alteration of corneal pH and suggest that the measurement of stromal pH may provide a sensitive index for evaluating the metabolic effects of contract lens wear.     

Intracellular pH regulation in fresh and cultured bovine corneal endothelium. I. Na+/H+ exchange in the absence and presence of HCO3-.

A detailed comparison of intracellular pH (pHi) regulatory mechanisms was made between fresh (FBCE) and cultured (CBCE) bovine corneal endothelium to: (1) identify the ion transport mechanisms that could directly or indirectly affect transendothelial HCO3- transport; and (2) determine if cultured cells could serve as a model for studying transendothelial bicarbonate transport. We used the pH-sensitive fluorescent probe BCECF-AM to measure pHi. FBCE and CBCE readily incorporated the dye and showed pHi calibration curves that were not significantly different with respect to pK (7.39 for FBCE and 7.35 for CBCE). Resting pHi in bicarbonate free Ringer's (pH 7.5) was significantly lower in cultured cells (7.17 +/- .02, n = 50) than in fresh cells (7.30 +/- .02, n = 54). Steady-state pHi was reduced by addition of 0.5 mmol/l amiloride, a Na+/H+ exchange blocker (-.16 pH U for FBCE, -.18 for CBCE) or removal of Na+ (-.47 pH U for FBCE, -.51 for CBCE). Recovery from an (NH4)2SO4-induced acid load was blocked by Na+ removal, and the rate of recovery was inhibited 74% and 79% in the presence of amiloride for FBCE and CBCE, respectively. The dependence of proton efflux on Na+0 showed simple saturating kinetics (apparent Km = 30 and 31 mmol/l for FBCE and CBCE, respectively), consistent with the presence of Na+/H+ exchange in FBCE and CBCE. Na+/H+ exchange activity, as measured by amiloride-sensitive acid recovery, was inversely proportional to pHi. The activity in FBCE was about twice that in CBCE. Furthermore, the zero flux point for Na+/H+ exchange was at least 0.1 pH U higher in FBCE. Changing from bicarbonate-free Ringer's to bicarbonate Ringer's (5% CO2/28 mmol/l HCO3-, pH 7.5) induced a rapid and short acidification followed by an alkalinization .09 and .18 pH U above the starting pHi for FBCE (final pHi 7.37) and CBCE (final pHi 7.33), respectively. This transition was unaffected by amiloride. Similarly, amiloride had no effect on resting pHi in bicarbonate Ringer's for FBCE or CBCE, indicating that Na+/H+ exchange does not contribute to the maintenance of the steady-state resting pHi in bicarbonate Ringer's. Although most of the characteristics of Na+/H+ exchange for FBCE and CBCE were similar, the differences in overall activity and the low levels of activity in resting cells must be considered when using CBCE to model ion coupled fluid transport in BCE.     

LTB4 is present in exudative pleural effusions and contributes actively to neutrophil recruitment in the inflamed pleural space

The pleural space is a virtual compartment between the lung and chest wall that becomes filled with fluid and inflammatory cells during a variety of respiratory diseases. Here, we study the potential role of the eicosanoid metabolite leukotriene B4 (LTB4) in disparate diseases leading to acute (pneumonia) or chronic (tuberculosis, cancer) inflammation of the pleural space. LTB4 concentrations were significantly higher in pleural fluid due to pneumonia, tuberculosis and cancer with respect to congestive heart failure and correlated with neutrophil elastase, which is used as an indication of state of activation of neutrophils in the pleural space. Moreover, pleural LTB4 was biologically active, as an anti-LTB4 antibody partially neutralized the chemotactic activity of parapneumonic, tuberculous and cancer effusions. Macrophages, neutrophils, lymphocytes, mesothelial cells and cancer cells all expressed mRNA for 5-lipoxygenase, the enzyme that initiates leukotriene synthesis leading to the production of LTB4, in exudative pleural effusions. Upon stimulation in transudative pleural effusions, pleural macrophages produced, in a time-dependent fashion, a significantly higher concentration of LTB4 than mesothelial cells. These studies demonstrate that different cell types are capable of producing LTB4 in the inflamed pleural space and that this mediator may play a crucial role in the recruitment of neutrophils into the pleural space.     

Continuing bonds and adjustment at 5 years after the death of a spouse

Thirty-nine bereaved individuals completed the Continuing Bonds Scale (CBS), assessing various aspects of the ongoing attachment to the deceased, at 60 months postloss in a longitudinal conjugal bereavement study. They also completed symptom measures at 6, 14, 25, and 60 months postloss. Higher CBS scores were associated with a more elevated grief-specific symptom pattern over the 5-year postloss period. Moreover, those who expressed greater helplessness and less blame toward the deceased during a monologue role-play involving their deceased spouse at 6 months postloss had higher CBS scores. Finally, greater satisfaction in the past relationship with the spouse was predictive of higher CBS scores. The results were discussed in relation to existing literature on the adaptiveness of continuing bonds.     

Reciprocal stimulation of gammadelta T cells and dendritic cells during the anti-mycobacterial immune response

Gammadelta T cells and dendritic cells (DC) are two distinct cell types of innate immunity that participate in early phases of immune response against Mycobacterium tuberculosis infection. Here we show that a close functional relationship exists between these cell populations. Using an in vitro coculture system, Vgamma1 T cells from Tcrb(-/- )mice were found to be activated by DC infected in vitro with BCG, as indicated by the elevated CD69 expression, IFN-gamma secretion and cytotoxic activity. This activation process was due to a non-cognate mechanism since it required neither cell to cell contact nor interaction between the TCR and a specific antigen, but was mediated by DC-derived IL-12. Reciprocally, Vgamma1 T cells provided a key cytokine, IFN-gamma, which increased IL-12 production by BCG-infected DC. Moreover, exposure of BCG-infected DC to Vgamma1 T cells conditioned the former to prime a significantly stronger anti-mycobacterial CD8 T cell response. Consequently, stimulation of gammadelta T cells and their non-cognate interaction with DC could be applied as an immune adjuvant strategy to optimize vaccine-induced CD8 T cell immunity.     

Analysis of the immune response induced by a single xenoantigen in vivo

Transgenic mice expressing human major histocompatibility complex (MHC) class II molecules would provide a valuable model system for studying murine anti-human MHC immune response. We have previously shown that skin from HLA-DR1 transgenic mice was rejected by control littermates and spleen cells from rejecting mice were able to proliferate to donor cells. The aim of this paper is to analyze the mechanism of recognition of this xenoantigen and the possible involvement of antibody response in anti-HLA-DR1 immune response. Control littermates were immunized with spleen cells from HLA-DR1 transgenic (TG) mice; at indicated times, xenoantigen-specific proliferation and IFNgamma production was assessed using APC obtained from HLA-DR1 TG mice. Mixed direct-indirect pathway of xenoantigen recognition was suggested by the following findings: i)T cell response to HLA-DR1 was inhibited adding in culture monoclonal antibodies directed either to donor (HLA-DR) or to recipient MHC (I-A); ii) APC from control mice pulsed with purified DR1 molecules were able to induce proliferation by FVB/N mice immunized with transgenic spleen cells. HLA-DR1 recognition permits DR peptide-specific T cell response by lymphocytes of control littermates immunized with the xenoantigen. In addition, we detected xenoreactive IgM and IgG2 antibodies. Our data suggest that HLA-DR1 xenoantigen may be recognized through direct or indirect pathway and provide additional information on mouse anti-human HLA immune response.     

A human leucocyte antigen-DR1 transgene confers susceptibility to experimental allergic encephalomyelitis elicited by an epitope of myelin basic protein

Much evidence now indicates that human leucocyte antigen (HLA) class I and class II transgenic (Tg) mice can be of value in analysing HLA-restricted presentation of T-cell epitopes relevant to experimental models of autoimmune diseases. One area where this has been applied is the characterization of myelin epitopes presented by HLA class II molecules in experimental model of multiple sclerosis (experimental allergic encephalomyelitis (EAE)). As a first step towards humanized disease models in HLA Tg mice, we have analysed immune response of lymph node cells of HLA-DR1 Tg mice immunized with the human myelin basic protein (MBP) peptides 13-33, 87-106 and 139-154 bound by HLA-DR1. We report here that HLA-DR1 Tg mice display a hierarchy of response in vivo and in vitro to MBP epitopes depending on the binding affinity to DRB*0101 molecule. In fact, the 13-33 epitope induced a strong T helper 1 (Th1) response accompanied by high T-cell precursor frequency and caused mild EAE, while the two other epitopes gave poor (139-154) or no disease (87-106), and these data correlate with in vitro Th1 response. These data could prove a useful tool in understanding the role played by different MBP epitopes in EAE.     

Interaction between 5-HT uptake inhibition and activation of 5-HT autoreceptors by exogenous agonists in rat cerebral cortex slices and synaptosomes

Summary Rat cerebral cortex slices or synaptosomes were labelled with ³H-5-hydroxytryptamine (³H-5-HT) and subsequently superfused. They were depolarized by electrical stimulation (slices) or with high K⁺ (slices and synaptosomes). Continuous electrical stimulation (2 Hz, 24 mA, 2 ms) and continuous or discontinuous K⁺ depolarization (15–25 mM) were used. 1. Continuous electrical stimulation or continuous K⁺-depolarization of slices evoked a steady overflow of tritium that slowly decayed with time. 2. Exposure to increasing concentrations of 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole succinate (RU 24969) (0.001–0.1 M) during continuous electrical stimulation produced a concentration-dependent decrease in tritium overflow. Citalopram (1 M) counteracted the effect of RU 24969. 3. RU 24969 inhibited the evoked ³H-overflow and citalopram reduced the effect of RU 24969 also during continuous depolarization of slices with 20 mM K⁺. Similar results were obtained by using 5-methoxytryptamine or LSD. 4. In slices 1 M citalopram increased significantly the tritium overflow evoked by electrical stimulation or by 20 mM K⁺-depolarization. 5. Increasing the K⁺ concentration from 20 mM to 25 mM mimicked the effects of 1 M citalopram both on the RU 24969 activity and on the evoked tritium overflow. 6. RU 24969 (0.001–0.1 M) decreased in a concentration-dependent way the release of tritium from cortical synaptosomes depolarized with K⁺ (15–20 mM). The presence of 1 M citalopram did not modify significantly the effect of the agonist. Citalopram was ineffective also when the serotonin uptake carrier in superfused synaptosomes was activated by tryptamine. In conclusion, in slices of rat cerebral cortex, the action of exogenous 5-HT autoreceptor agonists is inhibited by 5-HT uptake blockers independently of the depolarizing agent (electrical stimulation or high-K⁺) used to elicit ³H-5-HT release. Increasing K⁺-concentration, which probably increases serotonin in the biophase, mimics the presence of the reuptake inhibitor. These data together with the finding that, in superfused synaptosomes, 5-HT uptake inhibition did not affect the potency of autoreceptor agonists, favours the idea that, in cerebral cortex slices, inhibitors of 5-HT reuptake prevent activation of autoreceptors by exogenous agonists by increasing the concentration of 5-HT in the autoreceptor biophase. Send offprint requests to M. Raiteri at the above address