Modulation of human concanavalin A-induced lymphocyte proliferative response by physiological concentrations of beta-endorphin

The effects of physiological concentrations of beta-endorphin on the proliferative response to concanavalin A of human peripheral blood mononuclear cells from a large series of healthy donors are reported. These effects are also compared with those obtained employing beta-endorphin and phytohemagglutinin under the same experimental conditions. The donors (32), aged between 20 and 48 years, chosen among military personnel of the Italian Air Force, underwent clinical and laboratory investigations to exclude any detectable disturbance in their psychophysical fitness. Our results show that beta-endorphin is not mitogenic per se and is unable to modify the response of mononuclear cells to phytohemagglutinin irrespective of the concentration of opioid or mitogen used. beta-Endorphin is also unable to alter the PBMC response to low concentrations of concanavalin A, but significantly increases such a response when higher concentrations of concanavalin A and concentrations of beta-endorphin similar to those found in human plasma under physiological conditions are used. The effect is not reverted by naloxone, the specific opiate antagonist. When the activity of beta-endorphin on the mononuclear cell response to concanavalin A is examined at the single donor level, it is noted that some of the donors fail to show the opioid-dependent increase. The baseline levels of the response to concanavalin A of such subjects, compared to those of the donors whose response is augmented by the opioid, are significantly higher, thus demonstrating that beta-endorphin can selectively modulate concanavalin A-induced mitogenesis with a behavior depending on the individual characteristics of the donor's response. The process involves non-opioid cell receptors.     

Suppression of polyclonal immunoglobulin production by a soluble factor produced by a human thymus hybridoma

A human thymus cell hybridoma was established using thymus cells obtained from a patient with common variable hypogammaglobulinemia and associated thymoma. This hybridoma secreted a suppressor factor for polyclonal antibody synthesis. Supernatants of this hybrid showed 40-80% suppression of both IgM and IgG synthesis by pokeweed mitogen-stimulated human peripheral blood lymphocytes. Hybridoma supernatants were suppressive for immunoglobulin production only if added within the initial 48 h of the seven-day culture period. Suppression of antibody production by the hybridoma supernatant was prevented by preabsorption with T lymphocytes. Further, the suppressor factor was shown to inhibit antibody production in reconstructed cultures containing T4+ cells and B cells, yet the suppression could be abrogated by increasing the number of T4+ cells. The hybrid supernatant had no affect on the proliferation of human mononuclear cells in response to pokeweed mitogen, lipopolysaccharide, concanavalin A or alloantigen but inhibited phytohemagglutinin-induced proliferation. The target cell population for the inhibition of phytohemagglutinin responsiveness was shown to be a T4+ lymphocyte (helper inducer T cell). These results suggest that thymus hybridoma cells can produce immunoregulatory products that act through the modulation of T4+ lymphocyte function. To our knowledge this is the first human thymus cell hybridoma to be reported. Studies on such cell lines may provide important information on immunoregulatory thymic factors.     

Angiotensin II suppression of human mononuclear cell reactivity is associated with enhanced OKT8+ lymphocyte thymidine incorporation

Recent evidence suggests that angiotensin II may participate in the regulation of inflammation. We previously reported that angiotensin II inhibits human peripheral blood mononuclear cell reactivity and acts directly on lymphocytes. These observations are again confirmed. In addition, purified OKT8+ but not OKT4+ T cell suspensions stimulated with phytohemagglutinin revealed increased thymidine incorporation when simultaneously cultured for 48 hours with angiotensin II. These findings suggest that angiotensin II may inhibit mononuclear cell thymidine uptake through stimulation of suppressor lymphocytes contained within the OKT8+ subpopulation.     

Effect of zinc on the proliferative response of human lymphocytes: mechanism of its mitogenic action

To study the effect of Zn on the proliferative response of normal human lymphocytes, ZnCl2 at a final concentration of 10(-4) M was added to cultures of peripheral blood mononuclear cells (PBMC) stimulated with concanavalin A (Con A) and to autologous mixed lymphocyte cultures of responder T lymphocytes and irradiated autologous non-T cells. Addition of Zn increased by about 50% the synthesis of DNA in cultures stimulated with either 10 or 20 micrograms/ml of Con A and markedly enhanced the autologous mixed lymphocyte reaction, which increased about 5-fold in the presence of Zn. In a narrow dose range, Zn induced per se the incorporation of [3H]thymidine by PBMC, with maximal effects in cultures stimulated with 10(-4) M ZnCl2. The percentage of cells expressing receptors for IL-2 and transferrin as assessed by immunofluorescence with the monoclonal antibodies (mAb) anti-Tac and OKT9, respectively, significantly increased when PBMC were stimulated with 10(-4) M ZnCl2 alone. Maximal [3H]thymidine incorporation and maximal percentage of cells bearing those activation markers were observed on day 6 of culture. Thus, the increase in the uptake of [3H]thymidine induced by Zn is not artifactual but due to progression in the cell cycle. Incubation with the mAb anti-Tac significantly inhibited the proliferative response to Zn, indicating that this requires binding of IL-2 to its receptor. However, addition of human recombinant IL-2 did not increase [3H]thymidine incorporation by PBMC cultured in the presence of ZnCl2.     

Apoptotic effect of cyanobacterial extract on rat hepatocytes and human lymphocytes

Toxic cyanobacterial blooms are an increasing problem in Poland. The production of cyanobacterial toxins and their presence in drinking and recreational waters represent a growing danger to human and animal health. This is connected with the increase of cyanobacterial biomass caused by excessive eutrophication of the water ecosystem. There is evidence that cyanobacterial hepatotoxins can act as a potent promoter of primary liver cancer. The apoptotic effect of microcystins in Polish cyanobacterial bloom samples on rat hepatocytes and human lymphocytes was observed using light and fluorescence microscopy, flow cytometry, and electrophoretic analysis. The incubation time needed to observe the first morphological apoptotic changes in hepatocytes was approximately 30 min; however, the characteristic biochemical changes in DNA were not observed even after 120 min. In lymphocyte cultures the morphological changes characteristic for apoptosis were observed after 24 h of incubation and a 48‐h incubation was found to be optimal for analysis of internucleosomal DNA fragmentation, which is one of the main biochemical hallmarks of programmed cell death. These cells are an easily isolated and inexpensive material for medical diagnostics. Therefore the apoptotic changes, together with the clastogenic effect seen in lymphocyte cultures, are proposed as a future analytical method for these toxins. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 225–233, 2001     

Pharmacodynamics of FTY720, the first member of a new class of immune-modulating therapeutics in transplantation medicine

FTY is a novel immunomodulator currently undergoing clinical investigation and has the potential of improving immunosuppressive therapy after organ transplantation. Previous experimental studies in animals have shown that FTY has a unique mechanism of action. We have studied the pharmacodynamic effects of FTY in stable renal allograft recipients taking part in a phase I clinical trial. As in various animal models including non-human primates, a single oral dose of FTY (0.25 - 3.5 mg) significantly reduced peripheral lymphocyte count by 30 - 70%. The peripheral lymphocyte count returned to baseline within 24 hours. Only in those patients treated with the highest dose of FTY (3.5 mg), did peripheral lymphopenia persist for more than 96 hours. FTY reduced all lymphocyte subsets, T cells more than B cells and CD4+ cells more than CD8+ cells. The reduction in CD3+CD62L+ cell counts was more pronounced, whereas CD3+CCR5+ cell counts were less affected in comparison to the total number of CD3+ lymphocytes. We found only slightly increased apoptosis rates (< 5%) in peripheral lymphocytes, and this change does not explain the marked reduction in lymphocyte count. In cultured human lymphocytes only suprapharmacological doses of 10 microM FTY induced apoptosis (20.6 +/- 2.8%) after a 4-h incubation. More important, clinically relevant doses of 0.1 microM FTY increased lymphocyte mobility 2-fold. No effect of FTY on anti-CD3mAb-stimulated lymphocyte proliferation was detected and there was no change in phagocytosis rates in whole-blood cultures incubated with FTY. Further studies are necessary to investigate the mechanism of action of FTY in detail.     

Decreased lymphocyte blastogenesis,IL2 production and NK activity following nifedipine administration to healthy humans

Summary The effects of a single oral dose of nifedipine on part of the immune response in healthy humans has been investigated in terms of two different immune functions: T lymphocyte proliferation and NK activity. Both functions are known to require calcium ions.Ten healthy subjects were bled before and 30 min, and 4 and 24 h after receiving 10 mg nifedipine. Lymphocyte proliferation, both in mitogen-activated lymphocyte cultures, and in autologous and allogeneic mixed lymphocyte reactions, was significantly reduced (up to 48%) 30 min after drug administration and reverted to normal 4 h later. The inhibition could be attributed to reduction in IL2 production by the T cells isolated 30 min following the administration of nifedipine, since they normally express IL2-receptors. The addition of recombinant IL2 of 200 U·ml^–1 to the cell cultures restored their responsiveness.NK activity was significantly reduced 30 min and 4 h after drug administration and returned to normal at the 24th h. This function was also restored by the addition of IL2.The data suggest that calcium channel blockers may inhibit, at least transiently, lymphocyte functions in vivo.List of abbreviations AMLC Autologous mixed lymphocyte cultures - CD Cluster determinants - ConA Concanavallin A - CTL Cytotoxic T lymphocytes - GAM-FITC Fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulins - HLA-class II DP-DQ-DR antigens - IL2 Interleukin 2 - mAb Monoclonal antibodies - MHC Major histocompatibility gene complex - MLC Mixed lymphocyte cultures - NK Natural killer activity - non- T/T type AMLC Autologous mixed lymphocyte culture in which non-T cells are used as stimulators - PBL Peripheral blood lymphocytes - T/T type AMLC Autologous mixed lymphocyte culture in which PHA-activated T lymphocytes are used as stimulators     

Importance of opiate receptor ligands in regulating lymphocyte proliferative activity

Effects of morphine (10(-12)-10(-6) M), metenkephalin (10(-12)-10(-6) M), beta-endorphin (10(-12)-10(-6) M), dalargin--synthetic analogue of leu-enkephalin (10(-12)-10(-7) M) and naloxone (10(-8)-10(-6) M) on lymphocytes of the human peripheral blood stimulated by polyclonal mitogens were studied. It was shown that morphine (10(-8)-10(-6) M) and dalargin (10(-12)-10(-7) M) inhibited the lymphocyte proliferative activity induced by optimal doses of phytohemagglutinin; effects of morphine and dalargin were blocked by naloxone. It was also found that beta-endorphin (10(-11)-10(9) M) inhibited the proliferative activity of lymphocytes stimulated by pokeweed mitogen; naloxone failed to block beta-endorphin effect.     

Study of the toxicity of povidone-lodine for fibroblast-like cells (BALB-3T3) and primary human chondrocytes

Povidone-iodine (polyvinyl-pyrrolidone-iodine complex, PVP-iodine, CAS 25655-41-8) is a commonly used antiseptic because of its broad spectrum of antimicrobial effect and its comparatively low allergic risk. It is also used for open joint lavage. Animal and organ culture studies provide controversial results about the risk of cartilage damage due to povidone-iodine. There is a paucity of in vitro study data concerning the effect of povidone-iodine on chondrocyte cultures are still missing. The aim of this study was therefore to investigate the effect of different concentrations and exposition times of povidone-iodine on cell growth and differentiation of human chondrocytes. Using of a vitality test (MTT) and a proliferation assay (BrdU) in the fibroblast-like cell line BALB3T3, suitable concentrations and incubation times were identified to investigate the influence of povidone-iodine on proteoglycan synthesis and DNA synthesis of primary human chondrocytes. Concentrations of up to 1% povidone-iodine had no significant effect on proteoglycan and DNA synthesis of chondrocytes after incubation for 30 min. An incubation time of 24 h did not inhibit DNA- and proteoglycan synthesis, until a concentration of 0.2% povidone-iodine was used. DNA synthesis rate was impaired after 10 min incubation with 0.2% and fully inhibited with 1% povidone iodine. BALB3T3 reacted more sensitively than chondrocytes. Vitality and proliferation rate were fully inhibited at a concentration of 0.5% after the same exposition time. However, cells recovered 24 h after 30 min incubation with 0.5% povidone-iodine. After incubation with 5% povidone iodine cells did not recover. From the results it can be concluded that low concentrations of povidone-iodine (< 1%) and short incubation times (< 30 min) have no damaging influence on chondrocytes. Previous studies have reported the antimicrobial effectiveness of low concentrations of povidone-iodine on the reduction of tissue damage by microorganisms. Data from previus studies and the current findings from this investigation support the clinical use of povidone-iodine at low concentrations and short incubation times for antiseptic treatment of cartilage tissues.     

Glucocorticoids: Effects on prostaglandin release,cyclic AMP levels and glycosaminoglycan synthesis in fibroblast tissue cultures

Summary Glucocorticoids (GCs) reduced cyclic AMP levels and inhibited glycosaminoglycan (GAG) synthesis in secondary embryonic mouse fibroblast cultures, when cells were incubated for short periods (30 min). The order of potency was dexamethasone > prednisolone > hydrocortisone. The effect was more marked, when cyclic AMP levels and GAG synthesis were increased by addition of PGE₁.Glucocorticoids exerted no longer an inhibitory effect on cyclic AMP and GAG synthesis in cultures pretreated for 48 h with the steroids. Addition of PGE₁ caused a stronger rise in cyclic AMP and GAG synthesis than in controls without GC-preincubation. This enhancement was even more pronounced, when PGE₁ was added together with the GCs.The reversal of the inhibitory effect of the GCs into a potentiating effect following preincubation correlated to a reduction of endogenous PGE formation in the cultures. Short-term treatment with GCs did not reduce endogenous PGE levels, but prolonged incubation markedly decreased PGE levels. PGE formation recovered following addition of fresh medium after the 48 h incubation with the steroids, but the amount of PGE formed remained significantly lower than in untreated cultures. Non-glucocorticoid steroid hormones did not decrease PGE levels.The results indicate that the apparent loss of inhibitory activity of GCs on cyclic AMP and GAG synthesis observed after prolonged incubation may result from a reduction of endogenous PGE formation which renders the cells more sensitive to the stimulatory effect of exogenous PGE₁.with technical assistance of I. Klappstein     

The effects of opiates on calcium accumulation on rat peritoneal mast cells

Opiate agonists, morphine, levorphanol and beta-endorphin increased calcium accumulation in rat peritoneal mast cells. This effect was dose dependent and beta-endorphin was 10 times more potent than morphine. The stimulation was stereospecific and inhibited by naloxone. The site of the opiate action appears to be on the outer surface of the plasma membrane since lysis of the mast cell did not alter the response to morphine. Tolerance to the opiate effect was not seen after chronic morphine administration. Morphine did not stimulate histamine release even at relatively high doses in vivo or high concentrations in vitro. It is reasoned that the enhancing effects on external calcium accumulation may reduce the critical cytosol calcium level for effecting histamine release.     

Intraseptally injected opiate agents: effects on morphine-induced behaviour of cats

Behavioural effects of intraseptally administered opiate agents were analyzed in cats pretreated with an intraperitoneal injection of morphine. In this way, it became possible to investigate (1) the involvement of septal opiate receptors in the behavioural response of cats to systemic administration of morphine, and (2) the pharmacological character of septal opiate receptors. The following results were obtained with intraseptal injections 15-16 min after intraperitoneal morphine: (1) naloxone decreased frequencies of head and limb movements, and (2) morphine was ineffective. The following results were obtained with intraseptal injections 40-41 min after intraperitoneal morphine: (1) beta-endorphin and, to a lesser extent, fentanyl increased frequencies of locomotor patterns, (2) morphine and Met-enkephalin were ineffective, (3) naloxone and naltrexone decreased frequencies of locomotor patterns in a dose-dependent way, (4) naloxone and naltrexone antagonized the effects of beta-endorphin and fentanyl, and (5) morphine did not attenuate the effect of naloxone. The intraseptal injections affected only the frequencies of the systemically evoked behaviour patterns; the nature of the behaviour patterns remained unchanged. It is concluded that (1) systemically administered morphine does not affect behaviour via a direct action on septal opiate receptors, and (2) the receptors mediating the septally evoked effects are most probably epsilon-type opiate receptors. The hypothesis is put forward that systemic administration of morphine results in an increased release of beta-endorphin from hypothalamo-septal neurons and, as a consequence, changes the beta-endorphin activity at the epsilon-type opiate receptors in the septum.     

PRELIMINARY STUDY ON THE ANTINOCICEPTIVE EFFECT OF ELEPHANT β-ENDORPHIN

1. Intraventricular administration of human beta-endorphin and elephant beta-endorphin significantly prolonged the tail flick response tested 30 min later. However, elephant beta-endorphin was about 7-8 times more potent than human beta-endorphin in the tail flick test. 2. beta-Endorphin antagonized the antinociceptive effect of both human beta-endorphin and elephant beta-endorphin by the same extent. Naloxone also antagonized the antinociceptive effects of the beta-endorphins but it was less effective than beta-endorphin. 3. Human beta-endorphin and elephant beta-endorphin were of equal potency in inhibiting the abdominal constriction response induced by intraperitoneal (i.p.) acetic acid. Both beta-endorphin and naloxone antagonized these effects of the beta-endorphins with naloxone being more effective. 4. The present study showed that different opioid receptor subtypes may be involved in the tail flick test and the abdominal constriction test. Furthermore, elephant beta-endorphin was a better antinociceptive agent than human beta-endorphin in the tail flick test.     

Cellular immunotoxicity of amyl nitrite

Functional deficits in lymphocyte interaction following occasional or chronic exposure to inhaled nitrites may be a potential contributing but not the etiologic factor in the acquired immunodeficiency syndrome (AIDS). We evaluated the effect of amyl nitrite vapors on mononuclear cell function and demonstrated functional deficits and structural alterations in these cells. In this closed, in vitro system, exposure of cells to amyl nitrite for up to 30 minutes did not effect cell viability. The functional deficits demonstrated were: inhibition of lymphocyte erythrocyte (E) rosette formation, a suppression of lymphocyte mitogen (phytohemagglutinin) and antigen (cytomegalovirus) transformation, a block in the S, G2 and M phases of cell cycling and diminished cell cytotoxicity to CMV infected cells. These effects on cellular function were demonstrated following 5, 10, and 15 minutes of amyl nitrite vapor exposure; some effect on all cellular functions was demonstrated at 5 minutes. The structural alterations seen on scanning and transmission electron micrographs were: reduction of filapodia, smoothing of the cell profile, cytoplasmic protrusions with pseudopod-like extensions, an increase in rough endoplasmic reticulum with swollen cisternae, alterations in size and distribution of golgi components and exocytotic vesicles in the outer membrane of the nuclear envelope. These vesicles and increased membrane proliferation suggests an effect on the membrane synthesis mechanism in these cells. These effects may be a potential factor in the alterations of phenotypic markers on T lymphocyte populations, as well as, a potential contributing factor in the functional deficit of mononuclear cells in patients with AIDS.     

Choline potentiates the pressor response evoked by glycyl-glutamine or naloxone in haemorrhaged rats

1. Severe blood loss initially lowers arterial pressure through a central mechanism that is thought to involve opioid and cholinergic neurons. The present study tested the hypothesis that simultaneous administration of a cholinergic agonist and an opioid receptor antagonist would produce a synergistic effect in the treatment of haemorrhage. Specifically, we tested whether choline, a precursor of acetylcholine, potentiates the pressor effect of the beta-endorphin derived peptide glycyl-glutamine (Gly-Gln) or the opioid receptor antagonist naloxone following acute haemorrhage. 2. Conscious rats were treated intracerebroventricularly (i.c.v.) with choline chloride (180 nmol) alone or combined with Gly-Gln (10 nmol) or naloxone (10 nmol) 2 min after blood withdrawal (2.5 mL/100 g bodyweight over 20 min) was completed; mean arterial pressure and heart rate were monitored for 30 min. 3. Combined treatment with choline and Gly-Gln elevated mean arterial pressure but did not affect heart rate significantly. Choline and Gly-Gln had no effect on cardiovascular function when administered alone to haemorrhaged rats or when given together to normotensive animals. Choline also potentiated the pressor and tachycardic effect of naloxone in haemorrhaged rats. 4. These data show that choline potentiates the pressor effect of Gly-Gln and naloxone in haemorrhaged rats.     

Specific amino acids moderate the effects on Ni2+ on the testosterone production of mouse leydig cells in vitro

The purpose of this investigation was to study the effectiveness of two nickel-binding amino acids, histidine (His) and cysteine (Cys), to prevent the inhibitory action of Ni2+ on testosterone (T) production by mouse primary Leydig cell culture. The maximal human chorionic gonadotropin (hCG)-stimulated T response was measured by radioimmunoassay (RIA) in the culture media. Three types of experiments were performed. In a concentration-response study, Ni2+ (62.5 to 1,000 microM) was added to the cells simultaneously with equimolar or twice the equimolar concentrations of His or Cys and the cultures were maintained for 48 h. Nickel-induced reduction in T production was completely prevented by equimolar concentrations of His at Ni2+ concentrations of 125, 250, and 500 microM; equimolar or twice the equimolar concentrations of His were only partially effective at 1,000 microM Ni2+. Protective action of Cys was complete only at the lowest concentration of Ni2+ (125 microM). In a second series, the cells were incubated for various times (0.5 to 48 h) with 1,000 microM Ni2+ in the presence of 2,000 microM His or Cys. Increasing the time of incubation, the protective effect of both amino acids against Ni2+ was reduced. In a third series, attempts were made to reverse the action of 1,000 microM Ni2+ after incubation with cells for various times (0.5 to 24 h), followed by exposure to 2,000 microM His or Cys. Cell cultures were maintained for 48 h. A partial recovery of hCG-stimulated T production could be observed only if the amino acid was added not later than 4 h after the metal. This time was also required to elicit the T depression produced by Ni2+. Administration of either His or Cys at later times had no effect. Our results show that both His and Cys are able to moderate the effects of Ni2+ on Leydig cell T production, depending on the concentration of this metal ion, as well as on amino acid. However, at higher Ni2+ concentrations the complete protection by His or Cys is only temporary. Administration of these amino acids after the Ni2+-produced decrease in T production was not able to reverse the process.     

Suppressive activity of epinastine hydrochloride on TARC production from human peripheral blood CD4+ T cells in-vitro

Thymus- and activation-regulated chemokine (TARC) is an important molecule in the development and maintenance of allergic diseases. However, there is little information about the influence of anti-allergic agents on TARC production. The aim of this study is to examine the influence of epinastine hydrochloride, an H1-receptor antagonist, on TARC production from human peripheral blood CD4+ T cells using an in-vitro cell culture technique. CD4+ T cells prepared from healthy subjects were cultured in wells coated with a combination of OKT3 and anti-CD28 monoclonal antibody in the presence or absence of epinastine HCl for 24 h. The cells were also stimulated with interleukin (IL)-4 in a similar manner. Levels of TARC and IL-4 in culture supernatants were examined by ELISA. The addition of epinastine HCl exerted a dose-dependent suppressive effect on the production of both TARC and IL-4 from CD4+ T cells under co-stimulatory molecule stimulation. The minimum concentration of the agent showing a significant suppressive effect on TARC and IL-4 production was 5.0 microM and 2.5 microM, respectively. Epinastine HCl also suppressed the ability of cells to produce TARC in response to IL-4 stimulation, when the agent was added to cell cultures at more than 2.5 microM. It was concluded that this inhibitory action of epinastine HCl may be partially responsible for epinastine's attenuating effect on allergic diseases.     

A study of zearalenone cytotoxicity on human peripheral blood mononuclear cells

The mycotoxin zearalenone (ZEA) is a common contaminant of all major cereal grains worldwide with estrogenic and anabolic activity. We investigated the in vitro cytopathic effects of ZEA on freshly isolated human peripheral blood mononuclear cells (PBMC) in relation to proliferation and cell death patterns of untreated and mitogen-activated cells. The higher concentration of 30microg/ml ZEA was found to totally inhibit T and B lymphocyte proliferation from the stimulation with phytohemagglutinin and pokeweed mitogen. The inhibitory effects of ZEA were further related to cell necrosis/apoptosis. Flow cytometry analysis showed a distinct necrotic effect on PBMC, irrespective of mitogen stimulation, whereas apoptotic activity was less evident. Necrosis was observed in both the lymphocyte and monocyte/granulocyte gates. Measurements of ZEA-induced intracellular calcium ion (Ca(2+)) mobilization showed an increase of both Ca(2+) levels and the number of cells with high Ca(2+) only in the monocyte/granulocyte gated cells. Using phenylmethyl sulfonyl fluoride (PMSF), a serine protease inhibitor, and ammonium chloride (NH(4)Cl), a lysosomal inhibitor, both associated with cell necrosis inhibition, we showed that PMSF at 0.05mM and NH(4)Cl at 1 and 10mM reduced the cytopathic effects induced by 30microg/ml ZEA, whereas apoptosis was less affected. Expose of PBMC to 1microg/ml ZEA did not alter the viability of the cells. Our results suggest that high ZEA concentrations in the blood may well exert cytotoxic effects that merit further investigation.     

Antiviral effects of Melia azedarach L. leaves extracts on Sindbis virus-infected cells

Partially purified extracts from leaves of Melia azedarach L. (MA) exert a broad range of antiviral effects on DNA and RNA viruses. The effect of MA on different stages of Sindbis virus replicative cycle in BHK cells was investigated. Under one-step growth conditions MA afforded a greater than 90% inhibition in virus yield if added to the cell cultures 2 h before or after infection, and when added 4 h after infection MA still caused a greater than 80% inhibition. Analysis of early events following Sindbis virus infection showed that MA did not affect viral adsorption to or penetration in BHK cell. In contrast, viral RNA and protein synthesis was almost totally inhibited in cells pretreated with MA 2 h before infection, while cellular macromolecular synthesis was similar in MA-treated and untreated cell cultures.     

Morphine-induced kinetic alterations of choline acetyltransferase of the rat caudate nucleus

1. In order to explain the decrease of choline acetyltransferase (2.3.1.6.) activity observed in the caudate nucleus of morphine-treated rats, partially purified preparations of the enzyme were used in kinetic studies, with choline as substrate.2. The apparent Michaelis constant for the enzyme obtained from normal rats was found to be 0.9 mM choline; this value doubled when the animals were killed one hour after a single injection of morphine (30 mg/kg). When the rats were injected daily for 4 or 15 days, and killed one hour after the last injection, the apparent Km value was 2.1 mM in each case. Prolonged daily treatment with morphine, followed by 48 h withdrawal, or by administration of 4 mg/kg of naloxone (given half an hour after the last injection of morphine) resulted in apparent Km values of 1.3-1.5 mM of choline, suggesting a gradual return to the lower, normal substrate requirement. Vmax changes were insignificant.3. The effect of morphine added in vitro to different enzyme preparations was also studied. The Km values of 0.9 mM, in the enzyme isolated from normal rats, increased to 2.0 after incubation in vitro with 12.5 mM morphine. Similar increases were found in enzymes obtained from rats 48 h after the withdrawal of morphine or from rats injected with naloxone after prolonged morphine treatment. The high apparent Km values, found in enzyme obtained from animals killed one hour after the last dose of morphine, did not change upon incubation with 12.5 mM morphine. A similar pattern of Km changes was noticed after incubation with 25 mM acetylcholine.4. An increase of 32% in acetylcholine (ACh) level was found in the caudate nucleus one hour after subcutaneous injection of 30 mg/kg of morphine. Return to normal values was observed when morphine was administered daily. After two to three weeks of daily treatment and subsequent withdrawal from morphine for 48 h, the levels of ACh were normal. If the daily treated rats were given naloxone within half an hour of the last injection of morphine, and killed 30 min later, the levels of ACh remained normal.5. Fifty per cent inhibition of enzyme activity was observed upon in vitro incubation with 75 mM acetylcholine, or with 25 mM morphine. The same degree of inhibition was noticed when the enzyme was obtained from normal or from morphine-treated rats.