Anti-apoptotic seminal vesicle protein IV inhibits cell-mediated immunity.

The in vitro effect of seminal vesicle protein IV (SV-IV) on the cytotoxic activity of human natural or acquired cellular immunity has been investigated by standard immunological procedures, a (51)Cr-release cytotoxicity assay, and labeled-ligand binding experiments. The data obtained demonstrate that: (1) fluoresceinated or [(125)I]-labeled SV-IV binds specifically to the surface of human purified non-adherent mononuclear cells (NA-MNC); (2) SV-IV suppresses the cytotoxicity of natural killer (NK) cells against K562 target cells, that of IL-2-stimulated NK (LAK) cells against DAUDI target cells, and that of VEL antigen-sensitized cytotoxic T lymphocytes (CTLs) against VEL target cells; (3) treatment of K562 target cells alone with SV-IV decreases their susceptibility to NK-induced lysis. These findings indicate that the protein SV-IV has a marked in vitro inhibitory effect on NK, LAK and CTL cytotoxicity, providing a better understanding of its immune regulatory functions.

Effect of resveratrol on proliferation and telomerase activity of human colon cancer cells in vitro.

A number of studies performed in our laboratory and elsewhere, showed that resveratrol is able to prevent carcinogenesis and to impair tumor growth and progression. In order to provide additional information on the pleiotropic effects of resveratrol on malignant cells, the present study was performed to test the in vitro influence of the compound on the growth and TLMA of HT-29 and WiDr human colon cancer cell lines. The results confirmed that resveratrol has a direct, dose dependent, inhibitory effect on cell proliferation in both lines. In addition, for the first time, relatively high concentrations of this compound were found to be able to substantially down-regulate telomerase activity. These preliminary results further support the potential role of resveratrol in chemoprevention/chemotherapy of human colon tumor cells and provide the rational basis for novel strategies in cancer control.

The weaver mutant mouse: a model to study the ontogeny of dopamine transmission systems and their role in drug addiction.

Dopaminergic neurons and their projection-systems are important in some fundamental human activities like locomotion, feeding and sex, essential for survival and procreation, and are relevant to pathologies like Parkinson's disease and drug abuse. Three main dopaminergic projection-systems, namely the nigrostriatal, mesocortical and mesolimbic pathways are the major targets of the neuropharmacological actions of psychomotor stimulants such as cocaine and amphetamine. Studies on knockout mice for dopamine or its receptors provide substantial information but fail to reveal the role of individual dopaminergic projection-systems. Mutant animals with defects specific to one or more projection-systems might be useful for studying the role of individual dopaminergic projection-systems. We propose the weaver mutant mouse, with a defective nigrostriatal dopaminergic projection-system and dopamine depletion in the dorsal striatum but with intact mesocorticolimbic projection-systems, as a suitable model to study the role of individual dopaminergic systems in diverse biological processes including Parkinson's disease and drug abuse.

Absence of lipid gel-phase domains in seven mammalian cell lines and in four primary cell types.

Fluorescence properties of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) are used to explore gel and liquid-crystalline phase domains coexistence in membranes of various cell types and in erythrocyte ghosts. Experiments and simulations were performed using liposomes composed of equimolar gel and liquid-crystalline phases in the absence and in the presence of 30 mol% cholesterol. In this model system two distinct coexisting phases can be easily recognized in the absence of cholesterol. When cholesterol is added to this phospholipid mixture, Laurdan parameters characteristic of the gel and of the liquid-crystalline phase are no longer resolvable. Coexisting domains of gel and liquid-crystalline phase were not detected in any of the examined cell membranes as judged by Laurdan excitation and emission Generalized Polarization (GP) spectra. Both in liposomes and in cell membranes, the behaviour of GP values as a function of excitation and emission wavelength corresponds to a homogeneous liquid-crystalline phase, despite the absolute GP values being relatively high, closer to the values observed in gel phase phospholipids. The presence of cholesterol appears to be the major cause for the homogeneity of phospholipids' dynamical properties in natural membranes, properties that appear close to the liquid-ordered phase state, defined to describe model systems with cholesterol concentration > or = 30 mol%.

Size and stability of dipalmitoylphosphatidylcholine/cholesterol unilamellar vesicles are affected by interaction with proteins.

The effect of entrapping the enzyme ascorbate oxidase into dipalmitoylphosphatidylcholine/cholesterol vesicles, was studied by conventional transmission electron microscopy and freeze-fracture. The freeze-fracture technique has definitely demonstrated the unilamellar nature of empty and enzyme-loaded vesicles. Images of freeze-fractured and label-fractured liposomes also indicate that the observed reduction of vesicles volume could be related to the localization of ascorbate oxidase across the membrane. The membrane localization of ascorbate oxidase may explain the oxidation of externally added ascorbate by intact enzyme-loaded liposomes. Finally, the ageing of liposomes appears to be accelerated in the presence of proteins.

Inhibition of interleukin-1 release and activity by the rat seminal vesicle protein SV-IV.

SV-IV, a 9.8-kd protein isolated from rat seminal vesicle secretion, has been shown to have strong non-species-specific immunosuppressive, anti-inflammatory, antithrombotic, and antiphospholipase A2 properties. The present investigation was undertaken to determine the mechanism of action of its immunosuppressive effects. It was found that SV-IV is a potent inhibitor of interleukin-1 (IL-1) release from lipopolysaccharide (LPS)-activated human adherent monocytes and an effective inhibitor of IL-1-induced thymocyte proliferation. The ability of SV-IV to form a noncovalent dimeric association with IL-1 alpha but not with IL-1 beta, its ability to induce a marked decrease of IL-1 binding to its own receptors on the thymocyte surface, and its capacity to bind specifically to the macrophage plasma membrane might play an important role in the molecular mechanism of these inhibitory effects.

Inhibitory effect of SV-IV, a major protein secreted from the rat seminal vesicle epithelium, on phagocytosis and chemotaxis of human polymorphonuclear leukocytes.

The effect of SV-IV, one of the major proteins secreted from the rat seminal vesicle epithelium, on phagocytosis and chemotaxis of human polymorphonuclear leukocytes (PMNs) has been studied. Various cytological, biochemical, metabolic, and physical correlates of both biological activities have been found to be markedly reduced by the presence in the medium of micromolar concentrations of protein SV-IV. Moreover, the Scatchard analysis of the labeled SV-IV binding to PMN cell surface has demonstrated that such binding is specific. The binding sites contain only saturable components, completely displaceable by unlabeled SV-IV. The number of the specific sites has been calculated to be 87,000/cell, with a Kd of 1.72 X 10(-7) M. The molecular mechanism of the inhibitory effect is discussed along with the possible biological and clinical implications of the experimental findings.

Cholesterol protects the phospholipid bilayer from oxidative damage.

The measurement of fluorescence lifetime distribution of 1,6-diphenyl-1,3,5-hexatriene is used for the detection of oxidative damage produced in phospholipid membranes by ionizing radiation. The recently developed method is based on the linear relationship between the width of the probe lifetime distribution and the logarithm of the dose. The molecular origin of the damage resides in the production of hydroperoxide residues at the level of acyl chains double bonds. A chemiluminescence assay was used to quantitate the amount of produced hydroperoxides. Consequences of the produced damages include an increased disorder in the upper portion of the bilayer, accompanied by the penetration of water molecules. In the presence of the physiological concentration of cholesterol in phopholipid bilayers, the amount of hydroperoxides produced by ionizing radiation is dramatically reduced. The packing effect of cholesterol in phopholipid bilayers is well recognized, as well as its influence on the reduction of water concentration in the bilayer. The dramatic reduction of hydroperoxides concentration observed when irradiation is performed in the presence of cholesterol probably originates from a steric hindrance to the radical chain reaction through the unsaturated lipids due to the presence of cholesterol.

In vivo and in vitro inhibition of platelet aggregation by SV-IV, a major protein secreted from the rat seminal vesicle epithelium.

In summary, the present study documents that platelet aggregation triggered by thrombin, ADP, collagen and PAF both in vivo and in vitro, was prevented by SV-IV in a dose-dependent manner. Only platelet aggregation by AA was not affected by the protein, thus suggesting a possible involvement of PLA2 inhibition in the molecular mechanism at the basis of SV-IV anti-thrombotic effect.

Immunosuppressive and anti-inflammatory properties of a major protein secreted from the epithelium of the rat seminal vesicles.

The nonspecies specific immunosuppressive and anti-inflammatory properties of a major protein (SV-IV) secreted from the epithelium of rat seminal vesicles (SV) are described. To detect the immunosuppressive effect, peripheral blood lymphocytes (PBL) were pretreated for 2 hr at 37 degrees with SV-IV, and the protein was maintained in the incubation medium during the whole culture time. We obtained evidence that, during preincubation of PBL and SV-IV the protein was transformed by a transglutaminase (TGase) released from PBL into modified low and high molecular weight forms able to bind to PBL surfaces. It is suggested that T lymphocytes are the possible targets of the immunosuppressive effect. SV-IV seems to inhibit only the early phase of the proliferative response of T lymphocytes to mitogens without having any direct effect on the enzymatic system involved in DNA synthesis. Moreover, the protein SV-IV was also shown to possess an anti-inflammatory property due to a block of the arachidonic acid cascade at the level of the enzyme phospholipase A2 (PLA2). The physiological significance of the immunosuppressive and anti-inflammatory properties of SV-IV are discussed in relation to different aspects of the mammalian reproduction.

Lack of glutathione conjugation to adriamycin in human breast cancer MCF-7/DOX cells. Inhibition of glutathione S-transferase p1-1 by glutathione conjugates from anthracyclines.

One of the proposed mechanisms for multidrug resistance relies on the ability of resistant tumor cells to efficiently promote glutathione S-transferase (GST)-catalyzed GSH conjugation of the antitumor drug. This type of conjugation, observed in several families of drugs, has never been documented satisfactorily for anthracyclines. Adriamycin-resistant human breast cancer MCF-7/DOX cells, presenting a comparable GSH concentration, but a 14-fold increase of the GST P1-1 activity relative to the sensitive MCF-7 cells, have been treated with adriamycin in the presence of verapamil, an inhibitor of the 170 P-glycoprotein (P-gp) drug transport protein, and scrutinized for any production of GSH-adriamycin conjugates. HPLC analysis of cell content and culture broths have shown unequivocally that no GSH conjugates are present either inside the cell or in the culture broth. The only anthracycline present inside the cells after 24 hr of incubation was > 98% pure adriamycin. Confocal laser scanning microscopic observation showed that in MCF-7/DOX cells adriamycin was localized mostly in the Golgi apparatus rather than in the nucleus, the preferred site of accumulation for sensitive MCF-7 cells. These findings rule out GSH conjugation or any other significant biochemical transformation as the basis for resistance to adriamycin and as a ground for the anomalous localization of the drug in the cell. Adriamycin, daunomycin, and menogaril did not undergo meaningful conjugation to GSH in the presence of GST P1-1 at pH 7.2. Indeed, their synthetic C(7)-aglycon-GSH conjugates exerted a strong inhibitory effect on GST P1-1, with K(i) at 25 degrees in the 1-2 microM range, scarcely dependent on their stereochemistry at C(7).

In vivo inhibition of cell-mediated and humoral immune responses to cellular antigens by SV-IV, a major protein secreted from the rat seminal vesicle epithelium.

Microgram amounts of protein SV-IV, a major secretory protein produced by adult rat seminal vesicle epithelium, markedly decrease the mouse humoral immune response to cellular xenogeneic or allogeneic antigens (sheep red blood cells (SRBC) or mouse epididymal spermatozoa). The significant reduction in the total number of splenocytes and their main cell subsets in SRBC-immunized mice, the dramatic decrease in the number of Ia+ splenic T cells and the marked inhibition of splenocyte ability to respond in vitro to polyclonal mitogen stimuli suggest that the macrophage accessory cells are the primary target of the SV-IV immunosuppressive activity in vivo. Moreover, the infection of SV-IV-treated mice with Salmonella typhimurium produced an increased mortality of the experimental animals associated with a marked decrease of the phagocytic and intracellular killing activities of their peritoneal macrophages.

Inhibition of macrophage phagocytic activity by SV-IV, a major protein secreted from the rat seminal vesicle epithelium.

The protein SV-IV, one of the major secretory proteins produced by the rat seminal vesicle epithelium, has been found to possess a marked ability to inhibit in vitro the phagocytic properties of activated peritoneal rat macrophages, by a mechanism that apparently involves phagocytes and target cells. Although SV-IV is a substrate for transglutaminase (TGase), an enzyme secreted by activated macrophages, TGase does not seem to play any significant role either in the binding of the protein to the cells participating in the phagocytic process or in the inhibition of macrophage phagocytosis by SV-IV. The significance of the findings in relation to the reproductive process and their possible clinical implications are discussed.

Comparison of nephelometric and hemolytic techniques for determination of antistreptolysin O antibodies.

The sensitivity of a newly devised nephelometric method for determining antistreptolysin O antibodies was compared with the hemolytic inhibition assay. Three hundred-thirty single serum samples from children with and without evidence of group A streptococcal infection were analyzed by the two techniques. The nephelometric method results correlated well with those of the reference test (concordance: r = 0.88). Furthermore, 134 pairs of acute and convalescent phase sera from patients with culture-proven GAS infection and 50 pairs from children who served as control subjects were examined. The nephelometric assay was more sensitive in detecting significant ASO antibody rises than the hemolytic assay. The automated nephelometric method appears to be a much simpler and sensitive procedure for testing ASO antibodies.

Susceptibility of human liver cell cultures to hepatitis C virus infection.

To develop a cell culture system susceptible to infection by hepatitis C virus (HCV), human fetal hepatocytes, grown in serum-free medium, were inoculated with serum samples from two HCV-infected patients. Viral RNA sequences were detected by polymerase chain reaction, using primers specific for the 5' noncoding region of HCV, in extracts prepared from the hepatocyte cultures as early as 5 days after inoculation. Virus was also released from the infected cells into the medium. The HCV strains could be serially passaged three times into fresh liver cell cultures using intracellular virus as inoculum. Evidence that HCV replication really took place in primary human fetal hepatocytes was also obtained by detection of minus-strand viral RNA (replication intermediate) in cell extracts and of viral antigens in the infected cells.

Effect of acclimatisation to altitude on learning.

Long-term exposure to high altitude has been reported to impair cognitive functions, possibly resulting in an increased risk of mountain accidents. To assess the modification of cognitive functions during acclimatisation to altitude, 17 climbers were studied at 5350 m a.s.l. by means of a neuropsychological learning test. The results clearly show that by extending the period spent at elevations above 5350 m to more than 15 days, the response to a memory task was significantly enhanced. The improvements resulting from acclimatisation were more evident in the organisation of information than in information storage. We suggest that inappropriate acclimatisation has a detrimental effect on cognitive functions and the resulting impairment may particularly affect the more demanding technical tasks.

Modulation and dynamics of phase properties in phospholipid mixtures detected by Laurdan fluorescence.

Steady-state and dynamic fluorescence properties of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) have been used to ascertain the coexistence of separate phase domains and their dynamic properties in phospholipid vesicles composed of different mole ratios of dilauroyl- and dipalmitoyl-phosphatidylcholine (DLPC and DPPC, respectively). The recently introduced generalized polarization together with time-resolved emission spectra have been utilized for detecting changes. The results indicate the coexistence of phospholipid phase domains in vesicle compositions in the range between 30 mol% and 70 mol% DPPC in DLPC. Below and above these concentrations a homogeneous phase is observed, with averaged properties. In the case of coexisting phase domains, the properties of each individual phase are largely influenced by the presence of the other phase. Implications on fluctuations between the coexisting phases and on the size and shape of domains are discussed.

Use of octadecylrhodamine fluorescence dequenching to study vesicular stomatitis virus fusion with human aged red blood cells.

Human erythrocytes were separated into five fractions representing different age groups. In each group phospholipid inside-outside translocation was determined by quantitation of the amino phospholipids phosphatidylserine and phosphatidylethanolamine and their lyso-derivatives by thin layer chromatography. To assess the role of transbilayer phospholipid distribution in the recognition and fusion of vesicular stomatitis virus (VSV) and human aged erythrocytes, we monitored the fusion kinetics using the octadecylrhodamine dequenching assay. Fusion of VSV with each single group of red blood cells (RBC) was not detectable with the youngest cells (F1 group) but increased with RBC aging (F2-F5 groups). The same increase in fusion was observed with microvesicles generated from RBC in which aging was mimicked by incubating the cells with Ca2+ in the presence of the Ca2+ ionophore A23187. Conversion of the aminophospholipids to the trinitrophenyl derivative by reaction with trinitrobenzensulfonate completely inhibits fusion on ghosts in which aging was artificially induced by translocation of aminophospholipids in the outer leaflet (symmetric ghosts). These results indicate that RBC become susceptible to VSV fusion during aging and in all pathology related to the aging process.

Effects of resveratrol on human immune cell function.

Resveratrol (3,5,4'-trihydroxystilbene), a polyphenol found in grapes and grape products such as red wine, has been reported to exhibit a wide range of biological and pharmacological activities both in vitro and in vivo. Because many of the biological activities of resveratrol, like the inhibition of cyclooxygenase, induction of CD95 signaling-dependent apoptosis, effects on cell division cycle and modulation of NF-kB activation, suggest a possible effect on the immune system, we evaluated the in vitro effects of resveratrol in three immune response models: i) development of cytokine-producing CD4+ and CD8+ T cells induced by stimulation of peripheral blood mononuclear cells (PBMC) with anti-CD3/anti-CD28; ii) specific antigen-induced generation of cytotoxic T lymphocytes; iii) natural killer (NK) activity of PBMC. The results showed that in vitro exposure to resveratrol produces a biphasic effect on the anti-CD3/anti-CD28-induced development of both IFN-gamma- IL2- and IL4-producing CD8+ and CD4+ T cells, with stimulation at low resveratrol concentrations and suppression at high concentrations. Similarly, the compound was found to induce a significant enhancement at low concentrations and suppression at high concentrations of both CTL and NK cell cytotoxic activity. On the whole, the results of the study indicate that resveratrol modulates several human immune cell functions and suggest that this activity may be related to its effects on cytokine production by both CD4+ and CD8+ T cells.

Differential effects of short-term or prolonged cocaine exposure on peripheral blood cells in mice.

The effects of cocaine on the number of murine peripheral blood cells and on phenotypic expression of lymphocyte subsets were examined using different regimens of drug exposure. Cocaine administered to mice for 7 consecutive days at 1 and 10 mg/kg/day reduced the absolute number of circulating white blood cells and the relative number of cells stained as lymphocytes, polymorphonuclear cells and monocytes. Cocaine did not modify the relative proportion between lymphocytes, polymorphonuclear cells and monocytes. Parallely, there were no changes in the percentage of circulating CD4+ and CD8+ lymphocytes, while the number of natural killer cells increased. In sharp contrast, we didn't observe any significant change in peripheral blood cells after 30 days of consecutive drug administrations.