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Encountering opsonized particles by neutrophils results in phagocytosis of the particle and generation of extracellular vesicles with antibacterial property (aEV). The aim of the present study is to compare the involvement of different receptors and receptor-proximal signaling pathways in these two parallel processes. Investigating human neutrophils from peripheral blood, we show that complement receptors are decisive for both processes whereas immunoglobulin binding Fc receptors (FcR) only participate moderately in phagocytosis and pattern recognition receptors induce mild EV production but only minimal phagocytosis. Studying bone marrow derived neutrophils of genetically modified animals we verify that the involved complement receptor is CR3, also known as the β 2 integrin Mac-1. We show that genetic deletion of the adaptor molecules FcRγ chain or DAP12 does not influence either process, suggesting potential redundant function. Combined absence of the Src family kinases Hck, Fgr, and Lyn drastically impairs phagocytosis but does not influence aEV production. In contrast, deletion of PLCγ2 has no influence on phagocytosis, but reduces aEV formation. In accord with the essential role of PLCγ2, aEV biogenesis both from murine and from human neutrophils is dependent on presence of extracellular calcium. Absence of external calcium prevented the generation of antibacterial EVs, whereas the spontaneous EV formation was not influenced. We thus show that phagocytosis and biogenesis of antibacterial EVs are independent processes and proceed on different signaling pathways although the same receptor plays the critical role in both. Our data reveal the possibility in neutrophilic granulocytes to modulate aEV production without disturbing the phagocytic process., (Copyright © 2019 Lőrincz, Szeifert, Bartos, Szombath, Mócsai and Ligeti.)

Production of extracellular vesicles (EVs) involved in intercellular communication is a common capacity of most cell types. Upon encountering opsonized microorganisms, neutrophilic granulocytes release EVs that compromise bacterial growth. We carried out a systematic investigation of the involvement of potential opsonin receptors in EV-generation from human and murine neutrophils. Applying flow cytometric, proteomic and functional analysis as well as using genetically modified mice, we demonstrate that formation of antibacterial EVs depends upon stimulation of the multifunctional Mac-1 integrin complex, also called as complement receptor 3 (CR3), whereas activation of immunoglobulin binding Fc receptors or pattern recognition receptors alone or in combination is ineffective. Mac-1/CR3 stimulation and downstream tyrosine kinase signalling affect both the numbers, the cargo content and the antibacterial capacity of the produced vesicles. In contrast, Mac-1/CR3 signalling is not required for spontaneous EV formation, clearly indicating the existence of separate molecular pathways in EV biogenesis. We propose that EVs are "tailor-made" with different composition and functional properties depending on the environmental circumstances., (© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Background: The role of myeloperoxidase (MPO) is essential in the killing of phagocytosed bacteria. Certain steroid hormones increase MPO plasma concentration. Our aim was to test the effect of MPO, its inhibitor indomethacin, and certain steroid hormones on bactericidal activity., Methods: Human polymorphonuclear leukocytes (PMN) were incubated with opsonised Escherichia coli and either MPO, indomethacin, estradiol, or hydrocortisone. Intracellular killing capacity was evaluated with UV microscopy after treatment with fluorescent dye. Next, an in vivo experiment was performed with nine groups of rats: in the first phase of the study indomethacin treatment and Pasteurella multocida infection (Ii), indomethacin treatment without infection (I0), untreated control with infection (Mi) and untreated control without infection (M0); in the second phase of the study rats with infection and testosterone treatment (NT), castration, infection and testosterone treatment (CT), castration, infection and estradiol treatment (CE), non-castrated infected control (N0), and castrated infected control (C0). After treatment bacteria were reisolated from the liver and heart blood on agar plates, and laboratory parameters were analyzed. For the comparison of laboratory results ANOVA or Kruskal-Wallis test and LSD post hoc test was used., Results: Indomethacin did not have a remarkable effect on the bacterial killing of PMNs, while the other compounds increased bacterial killing to various degrees. In the animal model indomethacin and infection caused a poor clinical state, a great number of reisolated bacteria, elevated white blood cell (WBC) count, decreased C-reactive protein (CRP) and serum albumin levels. Testosterone treatment resulted in less bacterial colony numbers in group NT, but not in group CT compared to respective controls (N0, C0). Estradiol treatment (CE) decreased colony numbers compared to control (C0). Hormone administration resulted in lower WBC counts, and in group CE, a decreased CRP., Conclusions: MPO, estradiol, and hydrocortisone improve bacterial killing activity of PMNs. Indomethacin treatment and castration weaken immune responses and clinical state of infected rats, while testosterone and estradiol have a beneficial effect.

Background: In our earlier studies both corticosterone and cortisol had antioxidant effect in vitro., Objectives: Our aim was to clarify whether corticosterone and cortisol oral administration results in beneficial antioxidant changes in Sprague-Dawley adult male rats in vivo., Methods: Experimental animals were fed a lipid rich diet and treated with corticosterone or cortisol in the drinking fluid. Control group was fed only lipid rich diet with untreated drinking water. The untreated group was feda normal diet with untreated water. Total scavenger capacity (TSC) was measured before and after 4 weeks of treatment in blood samples using a chemiluminometric assay., Results: Both corticosterone and cortisol treatment caused increased TSC. The control group and the untreated group showed no significant changes in TSC., Conclusion: Our results support the hypothesis that corticosterone and cortisol administration can improve the antioxidant status not only in vitro but also in vivo.

Selegiline is a selective irreversible inhibitor of the B-type of monoamine oxidase (MAO-B). The spectrum of its pharmacological activity is wide, possesses antioxidant, antiapoptotic and neuroprotective properties and, additionally, we found it is effective on the total scavenger capacity (TSC), and the regulation of fat content in rat liver kept on lipid-rich diet. Our aim was to clarify whether the oral treatment with selegiline is protective on oxidative damage of Sprague-Dawley adult rats in vivo. Four groups of rats (five animals in a group) were examined: (1) lipid-rich diet, (2) normal rat food, (3) lipid-rich diet + selegiline and (4) normal rat food + selegiline. Selegiline solution (2.5 µg/ml) was supplied with the drinking water, which was freely available for the animals. Regarding the drinking habit of the rats (20-30 ml/day), the daily dose was roughly equal with that used in the human therapy (5-10 mg/day). TSC was determined both at the beginning (0 day) and at the end of the study (28 days), when the blood samples were taken for chemiluminometric assay. Fat content of the liver was determined in the freshly frozen tissue by Sudan staining. TSC was increased in both the selegiline-treated groups. Selegiline treatment prevented the increase of liver fat in the group fed with lipid-rich diet. Our results led us to the conclusion that prolonged selegiline administration can raise the antioxidant capacity of the animals and prevents the accumulation of fat in their livers.

Background: Weak androgens have an antioxidant effect in vitro which is represented as a beneficial change in the antioxidant status., Objective: Our aim was to clarify whether dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulphate (DHEAS) oral administration results in beneficial antioxidant changes in Sprague-Dawley adult male rats in vivo., Methods: Groups of experimental animals were fed a high-fat or a normal-fat diet and treated with DHEA or DHEAS in the drinking fluid. The control group was fed a high-fat diet together with untreated drinking fluid. Total scavenger capacity (TSC) was measured before and after 4 weeks of treatment in blood samples using a chemiluminometric assay. Fat content, superoxide dismutase (SOD), catalase and glutathione S-transferase (GST) activity in the liver were determined by Sudan staining and spectrophotometric assessments, respectively, from the fresh frozen tissue., Results: DHEA and the DHEAS treatment showed significantly increased TSC in the groups fed a high-fat diet. The control group and the DHEA- or DHEAS-treated groups on normal diets showed no significant changes in TSC. The total score of liver fat content in the high-fat diet groups showed a marked positivity with Sudan staining, and the groups treated with DHEA or DHEAS had a markedly decreased amount of fat in the liver slides compared to the untreated group on the high-fat diet. Liver SOD activity was decreased in all high-fat diet groups and elevated only in the groups on a normal diet with DHEA or DHEAS treatment. Liver catalase and GST activities were decreased in the groups where TSC was significantly increased., Conclusion: Our results support the hypothesis that DHEA and DHEAS supplementation can improve the antioxidant status in lipid-rich dietary habits., (Copyright © 2010 S. Karger AG, Basel.)

Our earlier studies have shown that some steroids increase myeloperoxidase enzyme (MPO) release from human granulocytes, and that MPO plasma levels are significantly lower in postclimacteric people. Moreover, we have proven that MPO inhibits production of atherogenic free radical superoxide anion and MPO-inhibitors increase superoxide release. The aim of the present study was to investigate the effect of MPO-inhibitors on the early phase of aortic atherosclerosis, namely the extent of intimal plaques and the thickening of the medial layer. Adult male rabbits were fed with lipid rich food (cholesterol: 1.3%, peanut oil: 8%) for 8 weeks. During this period MPO-inhibitors were also given (4-aminobenzoicacid-hydrazide/ABAH/-13.3 mg/kg/day or indometacin-5 mg/kg/day). All animals developed intimal lipid plaques (raised fatty streaks). The relative plaque-covered areas of the aortas were compared and the media thickness of the aorta was measured on plaque-free as well as plaque-containing areas. The medial smooth muscle density and peroxidase activity of the aortic media were also determined. The media thickness increased (p<0.05) in the cholesterol+ABAH as well as in the cholesterol+indometacin groups up to 375.7 (+/-60.5) and 442.5 (+/-123.4) microm, respectively, compared to the control group (cholesterol feeding alone) where it measured only 308.4 (+/-51.67) microm. The medial peroxidase activity decreased significantly in the indometacin treated group and showed a decreasing tendency using ABAH. In parallel to this there was a tendency of increase in the relative plaque covered areas. The smooth muscle density showed no significant modifications, while inhibitors of the MPO seemed to enhance aortic medial thickness, i.e. the grade of a pre-atherosclerotic lesion, in our animal model. Collectively, the anti-atherogenic effect of certain steroid hormones might be realized through the impact on MPO activity.

It has been demonstrated, that the H2-receptor antagonists: cimetidine and ranitidine-administered in doses of the same order of magnitude as reported for the inhibition of rat gastric secretion and development of the experimentally induced gastric and duodenal ulcer-are capable of increasing significantly the parotid secretion evoked by the cholinergic stimulant bethanechol. The salivary carbonic anhydrase activity was also elevated after combined treatment of bethanechol(+)-cimetidine. The similar action of the two H2-antagonists appears to suggest that their effect on salivation is connected probably with the cholinergic activity and it is concluded that the combination of bethanechol with these blockers may improve the esophageal clearance, an action that may be beneficial in the treatment of the gastroesophageal reflux disease.

Effects of intravenous cimetidine, ranitidine and intragastric prostaglandin E1 (alprostadil) treatments on the transmucosal potential difference (PD) of the stomach were compared. It was also investigated whether the above-mentioned drugs influenced the decrease of PD which followed both intragastric administration of 30% alcohol or Ca++ solution in 5 Mm final concentration and intravenous administration of pentagastrin. Both cimetidine and ranitidine treatments led to significant (p < 0.05) increase of PD, the effect of ranitidine was dose dependent. Prostaglandin E1 in a dose of 40 micrograms/kg led to significant decrease of PD (< 0.05). Both intragastric administration of prostaglandin E1 in a dose of 40 micrograms/kg and intravenous administration of ranitidine in a dose of 10 mg/kg significantly diminish the effect of Ca++ and alcohol to decrease PD. Neither prostaglandin E1, nor ranitidine pretreatment had any effect on the rapid and highly significant (p < 0.01) decrease of PD following i.v. pentagastrin administration. It is hypothesized that transmucosal PD of the stomach provides information not only on the actual condition of the mucosal barrier but on the electrophysiology of gastric secretion as well.

Studies from our laboratory have previously shown that two opioid agonists (morphine and [D-Met2,Pro5]enkephalinamide) aggravate, whereas naloxone inhibits cold-restraint stress-induced ulceration in rats. In the present study the effects of these substances were examined using the Shay-model. Contrary to expectations, both naloxone and the opioid agonists decreased gastric acid output. Naloxone in combination with either opioid agonist failed to reverse their inhibitory action. Thus the secretory activity of the stomach may be modulated by opioids in both naloxone-reversible and irreversible ways.

During Shay-ulcer formation damages to the barrier of the gastric mucosa develop even before the appearance of macroscopic ulceration. Proximal selective vagotomy prevents these damages. Following pyloric ligation the prostaglandin content of the mucosa changes in parallel with the injuries of the mucosal barrier: TXB2 content of the forestomach increases, while PGF2 alpha content of both the forestomach and the antrum decreases. Following PSV operation the 6-keto-PGF1 alpha content of the mucosa decreases, whereas PGF2 alpha and TXB2 contents exhibit no alteration. As a combined effect of proximal selective vagotomy pretreatment and pyloric ligation the 6-keto-PGF1 alpha and PGF2 alpha contents of the mucosa remain low and the TXB2 increase, otherwise detectable after pyloric ligation, does not take place.

Rats were exposed to whole body vibration horizontally for four hours at 5 Hz frequency and 2 cm amplitude. Of the components of the plasma kallikrein-kinin system the free (spontaneous) and kaolin-activated kallikrein (prekallikrein) activities, the concentration of bradykinin, the bradykinin splitting total kininase activity, total kallikrein-inhibitor activity and the concentration of alpha 2-macroglobulin, a major plasma kallikrein inhibitor, were estimated. Results showed that in response to acute vibration plasma free kallikrein activity was increased significantly in association with a significant reduction of prekallikrein concentration. The concentrations of bradykinin and total kininase activity were significantly elevated, too. Neither total kallikrein-inhibitor activity nor the concentration of alpha 2-macroglobulin were changed indicating that the plasma kallikrein inhibitors did not play a role in the alterations of plasma free kallikrein and prekallikrein activities. During acute vibration the plasma kallikrein-kinin system was activated probably by the enhanced catecholamine secretion. We suggest that the biological importance of this phenomenon is in the defense against the impaired microcirculation caused by catecholamines.

The effects of [D-Met2,Pro5]enkephalinamide, morphine and naloxone have been examined in two different models of experimentally elicited gastric mucosal lesions. One of them was the classic cold-restraint stress-induced ulceration. The other was a less frequently applied procedure, involving the measurement of decreases in the transmucosal potential difference, which is also a sensitive indicator of mucosal damage. The opioid agonists studied, [D-Met2,Pro5]enkephalinamide and morphine, aggravated, whereas naloxone, the pure opiate antagonist, mitigated the lesions in both models. The protective action of naloxone points to an eventual role of endogenous opioids in the generation of these types of mucosal lesions. Morphine is selective ligand of mu-opiate receptors. The enkephalin analogue, however, binds to both mu- and delta-receptors. Therefore, the potent pro-ulcerogenic action of the enkephalin analogue indicates that both the mu- and delta-receptors were involved in these models of experimental gastric lesions. The clarification of the eventual role of kappa-receptors requires further experimental work with a selective ligand of this subtype of opiate receptors.

The changes in the difference in the transmucosal potential of the stomach, in the ion flux of the mucosa and the prostaglandin content of the mucosa were studied after pyloric ligation combined with truncal vagotomy in rats. Results indicate that 10 hours following insertion of the pyloric ligation, and still prior to appearance of macroscopic ulceration, the mucosal barrier is damaged indicated by the decrease in PD value and by a changed ion flux. The development of these changes is prevented by truncal vagotomy performed simultaneously with pyloric ligation. Following pyloric ligation, simultaneously with the damage of the mucosal barrier, the PGF2 alpha concentration of the antrum and the rumen decreased. The 6-keto-PGF1 alpha content remained unchanged, while TXB2 content of the rumen increased. After truncal vagotomy the 6-keto-PGF1 alpha level of the antrum and the rumen and the TXB2 level of the rumen are increased. After truncal vagotomy performed simultaneously with pyloric ligation, the TXB2 content of the gastric mucosa increases with an increase also in the 6-keto-PGF1 alpha level of the rumen and the antrum and one in the PGF1 alpha content of the antrum, too. It is assumed that the beneficial effect of truncal vagotomy on the mucosal barrier is due to factors which are the known (i) decrease in H-ion secretion (ii) the unchanged bicarbonate secretion and (iii) the influencing of the prostaglandin level of the mucosa.

The changes of the transmucosal potential difference, the ionic flow, and the prostaglandin content were investigated after pylorus ligation, truncus vagotomy, and pylorus ligation with truncus vagotomy in Shay rats. A damage of the mucosa appeared already 10 hours after the pylorus ligation. This was presented in a clear decrease of the transmucosal potential difference and a change of the ionic flow. These damages could be prevented by simultaneous truncus vagotomy. A decrease of the PGF2 alpha content in the gastric antrum and rumen as well as an increase of the TXB2 in the gastric rumen were observed after additional pylorus ligation. The 6-keto-PGF1 alpha and the TXB2 content in the gastric antrum and rumen increased after truncus vagotomy. TXB2 in the rumen, 6-keto-PGF1 alpha in the rumen and antrum, as well as PGF2 alpha in the antrum ascended after pylorus ligation with simultaneous truncus vagotomy. The truncus vagotomy shows additionally a protective effect for the mucosa and influenced synergistically the content in prostaglandin after these results besides the notorious importance for the secretion of H+ion and bicarbonate.

The effect of truncal vagotomy on the gastric bicarbonate-ion, H-ion and chloride-ion secretions of the rat was studied experimentally. In the first half hour after truncal vagotomy, besides the known decrease of the H-ion, also the bicarbonate-ion and chloride-ion secretions were found reduced. The decrease in bicarbonate ion secretion was smaller as compared to the H-ion secretion. After two hours, there was no difference in bicarbonate-ion and chloride-ion secretions as compared to the controls and to the vagotomized groups. According to their results, following truncal vagotomy applied in the surgical management of ulcer, the bicarbonate-ion secretion of the stomach decreases immediately after operation. This decrease is, however, only transitory the bicarbonate-ion secretion becoming normal within a short time.

Mucosa damage, these appear in the Shay ulcer model before the macroscopic ulceration, can be prevented by the selective proximal vagotomy. Changes of the potential difference and the prostaglandin content were discovered after pylorus ligation, and Thromboxane was increased, PGF2 alpha and TXB2 were nearly constant, whereas 6-keto-PGF1 alpha increased clearly in the rumen. The 6-keto-PGF1 alpha and the PGF2 alpha content and Thromboxane remained unchanged and the potential difference was normalized in case of selective proximal vagotomy and pylorus ligation. The SPV is significant as you know for the secretion of H+ion and bicarbonate, but also for the normalization of increased TXB2 on the basis of our investigation results.