Exogenous VIP limits zymosan-induced generalized inflammation (ZIGI) in mice.

Vasoactive intestinal peptide (VIP) was administered in a model of zymosan-induced generalized inflammation (ZIGI). Its beneficial action was associated with reduced TNF-alpha and increased IL-10 production, lowered levels of creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and bilirubin in circulation. VIP diminished the level of RANTES and MIP-1alpha in peritoneal exudate and circulation. The neuropeptide inhibited NO release from stimulated peritoneal macrophages. Decreased spleen, liver and kidney enlargement and less pathological changes in liver were observed. The effect of VIP was attenuated by pretreatment with VIP antagonist (anti-VIP) before the induction of shock.

Downstream components of RhoA required for signal pathway of superoxide formation during phagocytosis of serum opsonized zymosans in macrophages.

Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47(PHOX). Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47(PHOX) may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.

Role of peroxynitrite and activation of poly (ADP-ribose) synthase in the vascular failure induced by zymosan-activated plasma.

1. Zymosan is a wall component of the yeast Saccharomyces Cerevisiae. Injection of zymosan into experimental animals is known to produce an intense inflammatory response. Recent studies demonstrated that the zymosan-induced inflammatory response in vivo can be ameliorated by inhibitors of nitric oxide (NO) biosynthesis. The cytotoxic effects of NO are, in part, mediated by the oxidant preoxynitrite and subsequent activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS). In the present in vitro study, we have investigated the cellular mechanisms of vascular failure elicited by zymosan-activated plasma and the contribution of peroxynitrite production and activation of PARS to the changes. 2. Incubation of rat aortic smooth muscle cells with zymosan-activated plasma (ZAP) induced the production of nitrite, the breakdown product of NO, due to the expression of the inducible isoform of NO synthase (iNOS) over 6 24 h. In addition, ZAP triggered the production of peroxynitrite in these cells, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123 and by nitrotyrosine Western blotting. 3. Incubation of the smooth muscle cells with ZAP induced DNA single strand breakage and PARS activation. These effects were reduced by inhibition of NOS with NG-methyl-L-arginine (L-NMA, 3 mM), and by glutathione (3 mM), a scavenger of peroxynitrite. The PARS inhibitor 3-aminobenzamide (1 mM) inhibited the ZAP-induced activation of PARS. 4. Incubation of thoracic aortae with ZAP in vitro caused a reduction of the contractions of the blood vessels to noradrenaline (vascular hyporeactivity) and elicited a reduced responsiveness to the endothelium-dependent vasodilator acetylcholine (endothelial dysfunction). 5. Preincubation of the thoracic aortae with L-NMA (1 mM), glutathione (3 mM) or by the PARS inhibitor 3-aminobenzamide (1 mM) prevented the development of vascular hyporeactivity in response to ZAP. Moreover, glutathione and 3-aminobenzamide treatment protected against the ZAP-induced development of endothelial dysfunction. The PARS-related loss of the vascular contractility was evident at 30 min after incubation in endothelium-intact, but not in endothelium-denuded vessels and also manifested at 6 h after incubation with ZAP in endothelium-denuded rings. The acute response is probably related, therefore, to peroxynitrite formation (involving the endothelial NO synthase), whereas the delayed response may be related to the expression of iNOS in the smooth muscle. 6. The data obtained suggest that zymosan-activated plasma causes vascular dysfunction by inducing the simultaneous formation of superoxide and NO. These radicals combine to form peroxynitrite, which, in turn causes DNA injury and PARS activation. The protective effect of 3-aminobenzamide demonstrates that PARS activation contributes both to the development of vascular hyporeactivity and endothelial dysfunction during the vascular failure induced by ZAP.

Peroxidase activity in relation to iodide, 17 beta-oestradiol and thioureylene drug uptake in human polymorphoneutrophils.

In polymorphoneutrophils (PMNs) phagocytosis is accompanied by an increase in peroxidase activity. Accumulation of iodide, thioureylene antithyroid drugs and 17 beta-oestradiol also occurs during the process. There is no evidence of an active iodide transport system in the PMNs as pertechnetate is not concentrated and neither ouabain nor perchlorate abolishes iodide accumulation. The uptakes of 125I, [35S]PTU and [3H]-17 beta-oestradiol were compared in phagocytosing PMNs and the effects of various compounds examined. In addition, chemiluminescence generation from luminol by PMNs and by horseradish peroxidase was studied. This indicated that uptake of all three compounds could be associated with activation of the peroxidase system, and inhibition of this enzyme system caused a reduction in their accumulation.

Zymosan-activated plasma induces paw oedema by nitric oxide and prostaglandin production.

Injection of zymosan-activated plasma into the rat paw induced oedema formation. Subplantar injection of the non isoform- selective inhibitors of nitric oxide (NO) synthase, N(G)-nitro-L-Arginine methyl ester and N(G)-methyl-L-Arginine, and of a scavenger of NO, haemoglobin, inhibited the early phase of oedema development. Inhibition of cyclooxygenase activity by indomethacin reduced the late increase in paw volume after the injection of zymosan-activated plasma. Methylene blue, an inhibitor of the soluble guanylate cyclase, had no effect. Our results suggest that in paw oedema induced by zymosan-activated plasma, the inflammatory response is dependent on NO (for the early phase) and prostaglandins (for the late phase). The effect of NO is likely to be mediated by a pathway which does not involve cyclic GMP.

Dependence of whole blood luminol chemiluminescence on PMNL and RBC count.

Luminol chemiluminescence (LCL) of whole blood as response to zymosan stimulation was used to assess the activity of polymorpho-nuclear leukocytes (PMNLs). Since PMNL and red blood cells (RBCs) are the primary components determining LCL response, the influence of PMNL and RBC count on the response was investigated over wide ranges of cell count and time interval. A linear relationship was found between maximum LCL intensity (Imax) (reflecting PMNL activity) and PMNL count. This allows a proper correction of Imax to be made as related to the sample PMNL count. This correction was valid for PMNL concentrations from 3.10(3) PMNL/ml to 6.10(5) PMNL/ml. When concentrations range from 6.10(3) PMNL/ml to 3.10(4) PMNL/ml the relationship was valid for a time interval round the peak. During the same interval the inhibitory effect of RBC count was studied. A correction of LCL was found possible for different RBC concentration ranging from 2.5.10(6) RBC/ml to 10(8) RBC/ml. These independent corrections of LCL response to PMNL and RBC counts were applied to diluted whole blood samples. The common correction of Imax was applicable in dilutions not less than 1:50. To correct light intensity for the time interval around Imax, the dilutions used had to be not less than 1:200. The results obtained permit an objective assessment of PMNL activity to be made by testing whole blood samples from different individuals and/or samples diluted to different extents.

In vitro aggregation of bovine neonatal neutrophils. A comparative study with adult cattle.

Deficient in vitro functions of neonatal neutrophils have been reported in various species. They may be functionally related to the well-known susceptibility of newborn individuals to microbial infections. To evaluate an early step in the sequence of neutrophil activation, neutrophils from adult cows (A-PMN) and newborn calves (N-PMN) were stimulated with zymosan-activated plasma (ZAP) or with the lipid mediator platelet-activating factor (PAF): Aggregation was recorded kinetically in a standard aggregometer and measured quantitatively as the area under the aggregation curve (AUAC). The mean +/- SEM of the AUAC of the first 2.5 min of the reaction induced with ZAP was similar in N-PMN and A-PMN. However, N-PMN deaggregated only partially, whereas A-PMN deaggregated almost completely (P less than 0.05). This may indicate a mechanism of microvascular sequestration in vivo with the potential to inhibit chemotaxis. PAF (10(-5)-10(-10) M) aggregated N- and A-PMNs similarly and dose-dependently with a maximal reaction at 10(-6) M. Inhibition of aggregation induced by 10(-6) M PAF was evaluated by preincubation with four antiinflammatory drugs: dexamethasone (Dex: 5.1, 51.0, 510.0 microM), flumethasone (Flu: 12.2 and 122.0 microM), phenylbutazone (PB: 0.33 and 3.3 mM), and flunixin meglumine (Flxin: 51 and 510 microM). Dex and Flu each inhibited (P less than 0.05) PAF-induced N-PMN aggregation at the highest dose, and A-PMN aggregation at the two higher doses. PB and Flxin each inhibited aggregation of N- and A-PMNs at all doses used. We compared the inhibition rate in both age groups and could demonstrate that Dex, Flu, and Flxin each at the highest dose, and PB at all doses used, inhibited PAF-induced aggregation less (P less than 0.05) in N-PMNs than in A-PMNs. These functional differences indicate hyperirritability of N-PMNs, and they need further elucidation to help understand mechanisms of increased neonatal susceptibility.

Papillon-Lefèvre syndrome. Analysis of neutrophil chemotaxis.

Papillon-lefèvre syndrome (PLS) is described as the association of palmar-plantar hyperkeratosis with precocious periodontal disease which results in exfoliation of primary and permanent dentitions. This study was planned to assess the chemotaxis of peripheral blood neutrophils in 7 patients (3 females and 4 males) with Papillon-Lefevre syndrome. The neutrophil chemotaxis was analyzed using the zymosan activated serum (ZAS) assay. Chemotaxis and spontaneous migration measurements were compared to those of the healthy control subjects. The peripheral blood neutrophil chemotaxis and spontaneous migration were depressed in all patients with Papillon-Lefèvre syndrome. The decreased chemotaxis of peripheral blood neutrophils strongly suggests that the neutrophils may act as one of the important key determinants in the pathogenesis of severe periodontal destruction in patients with PLS.

Optimization of the under-agarose assay of porcine neutrophil migration.

Important procedural factors in the under-agarose assay for porcine neutrophil migration were identified, and optimal conditions were established. Three factors were tested: the concentration of zymosan-activated serum inoculated into the outer well; the number of neutrophils inoculated into the center well; and the time of incubation of the agarose plates. All factors had a significant (P less than 0.0001, 0.0001, and 0.01, respectively) effect on the chemotactic index of porcine neutrophils. The optimal combination of these 3 factors was undiluted zymosan-activated serum as the chemoattractant, 8 X 10(5) neutrophils inoculated into the center well, and 5 hours of incubation. The assay was validated, using standard conditions, and the data were used to predict the number of pigs and/or repetitive assays needed to identify differences among experimental groups.

Evidences of 4-hydroxynonenal involvement in modulation of phagocyte activities.

The peroxidative breakdown of membrane polyunsaturated fatty acids leads to the production of various carbonylic compounds: among these, 4-hydroxynonenal (HNE) displays many biological properties related to neutrophil functions. It stimulates rat and human polymorphonuclear (PMN) cell migration and has been detected during inflammation. The aim of this study was to elucidate and well characterize the mechanism of action of HNE. We observed that micromolar HNE concentrations that influence migration do not stimulate differently from many other chemoattractants the human PMN chemiluminescence (CL) induced by opsonized zymosan or phorbol 12-myristate 13-acetate (PMA). Higher HNE concentrations inhibit the light emission of stimulated PMN. Addition of 0.5 mM L-arginine (L-arg), the substrate of nitric oxide synthase, into the incubation medium had the effect of modifying human CL. In fact, HNE at 10-6 M, a concentration which is ineffective in absence of L-Arg, at 10-5 M reduces CL emission of PMA-stimulated human PMN. These observations have been confirmed by electron-spin resonance (ESR) analysis. HNE, according to other stimuli, induced PMN phosphoinositide-specific phospholipase C (PL-C). All these results considered together suggest the conclusion that HNE represents an interesting endogenous molecule that plays a role as an inflammatory mediator involved a) in the recruitment of phagocytic cells at the inflamed area, and b) in the modulation of respiratory burst and of nitric oxide (NO) production.

[Age-related immunological characteristics in typhoid fever and their significance in the clinical manifestations of the disease]. / Vozrastnye immunologicheskie osobennosti pri briushnom tife i ikh znachenie v klinicheskikh proiavleniiakh bolezni.

The results of the comparison of the pattern of specific typhoid exanthema, as well as the time of its appearance, in elderly and senile patients and in young patients are presented; besides, some characteristics of humoral immunity in these groups of patients are compared. This comparison has revealed that in typhoid fever in elderly and senile patients roseola typhosa is more rare, while the phenomenon of eruption is more frequent than in young patients. The differences in the manifestation of exanthema, observed in patients of different age groups, are due to less intensive and regular antibody formation in elderly and senile patients, as well as to the decreased activity of their altered blood vessels of the skin.

New inhibitors of ROS generation and T-cell proliferation from Myrtus communis.

Phytochemical investigation on Myrtus communis Linn. afforded myrtucommuacetalone (1) with an unprecedented carbon skeleton and a new phloroglucinol-type compound, myrtucommulone M (2), along with four known constituents 3-6. Their structures were established by extensive analyses of NMR and mass spectral data as well as by single-crystal X-ray diffraction studies. These constituents were evaluated for their ability to modulate the immune response, based on their effects on various components of immune system. Compounds 1 and 5 exhibited significant inhibitory effect against nitric oxide (NO(•)) production. Compound 1 also exhibited significant antiproliferative activity (IC50 < 0.5 µg/mL) against T-cell proliferation. Myricetin (3) exerted a significant inhibition (IC50 = 1.6 µg/mL) on zymosan-stimulated whole blood phagocytes ROS production. Compounds 1 and 3 were active against PMA-stimulated ROS generation.

Oxidative burst inhibitory and cytotoxic amides and lignans from the stem bark of Fagara heitzii (Rutaceae).

Two amides, heitziamide A and heitziamide B and two phenylethanoids, heitziethanoid A and heitziethanoid B together with thirteen known compounds were isolated from F. heitzii (Letouzey). The structures of all compounds were established by spectroscopic analysis. Nine compounds were evaluated for oxidative burst inhibitory activity in a chemoluminescence assay and for cytotoxicity against PC-3 prostate cancer cells. All compounds exhibited a clear suppressive effect on phagocytosis response upon activation with serum opsonized zymosan at the range of IC(50)=2.0-6.5 microM, but no cytotoxic effect was observed (IC(50)>100 microM).

Human leukocyte phagocytosis of zymosan particles measured by flow cytometry.

Human leukocyte phagocytosis of fluorescein-isothiocyanate (FITC)-labelled zymosan particles was studied by a flow cytometric (FCM) assay allowing discrimination of adhered and ingested zymosan particles. Free zymosan particles, non-phagocytes and phagocytes could be discriminated and quantified by simultaneous registration of fluorescence and light scatter. All leukocytes capable of phagocytosis were phagocytosing, and within 15 min 80% of the zymosan particles were adhered or ingested. Compared to the FITC-fluorescence of free zymosan particles, the mean fluorescence of phagocyte-associated zymosan particles was reduced by about 35%, indicating ingestion and processing of zymosan particles. Abolishing the FITC-fluorescence of extracellular zymosan particles by crystal violet, the number of zymosan particles adhered and ingested could be calculated from FCM measurements of phagocyte fluorescence. This showed that in 15 min 83% of the phagocyte-associated zymosan particles were actually ingested.

A properdin system intermediate formed by zymosan and serum at 0 degrees C.

The interaction of guinea pig or rat serum with Z at 0 degrees C leads to the formation of properdin pathway intermediate, ZPI, whose activity is assessed by its capacity to deplete the titer of cobra venom inducible lysis in diluted rat serum. The formation of ZPI does not require C1 or C2 and is not diminished by prior absorption of the serum with Z. In contrast, removal of P suppresses ZPI formation. Both Ca++ and Mg++ are essential cofactors for the formation of this intermediate whose function is abrogated in the presence of anti-C3, anti-P, but not by anti-Factor B. Since C4-deficient guinea pig serum is also effective in ZPI formation, the data suggest that ZPI is an alternative pathway intermediate containing both P and C3.

The effect of pregnancy on polymorphonuclear leukocyte function.

Pregnancy exerts suppressive effects on a number of chronic inflammatory conditions, particularly rheumatoid arthritis. We isolated peripheral blood polymorphonuclear leukocytes (PMN) from pregnant women at 30 to 34 wk (n = 34) and showed significant reductions in respiratory burst activity compared with nonpregnant controls (n = 34), as determined by lucigenin-enhanced chemiluminescence (LUCL). Responses to FMLP were reduced by 54% (p = 0.0046) and to zymosan-activated serum (ZAS) by 69% (p = 0.0043). Following LUCL responses to these agonists in women throughout the course of their pregnancy (n = 7) revealed significantly reduced responses by the second and third trimesters (p < 0.005). Intracellular H2O2 production in PMN at 30 to 34 wk gestation was significantly reduced (p = 0.0454) in response to FMLP, compared with the nonpregnant controls. Investigation of adhesion molecule expression revealed no differences in CD11b or CD18. However, loss of CD62L from the PMN surface in response to FMLP and ZAS was significantly reduced at 30 to 34 wk, as compared with controls (FMLP, p = 0.049; ZAS, p = 0.01; n = 34). There were no significant differences in cell surface formyl peptide receptor expression, although there were statistical differences in LUCL responses to all concentrations of FMLP used (p < 0.05). Incubating PMN with TNF, IL-8, and granulocyte-macrophage CSF increased formyl peptide receptor expression but revealed no differences between the two groups. Priming of pregnancy PMN with the same cytokines gave significantly reduced LUCL when cells were subsequently stimulated with FMLP (p < 0.05; n = 6). Our results show a reduction in PMN NADPH-oxidase activity during pregnancy and may offer a partial explanation for the remission of symptoms observed in rheumatoid arthritis.

Platelet-activating factor and leukotriene biosynthesis in whole blood. A model for the study of transcellular arachidonate metabolism.

As a model to perhaps better indicate potential in vivo tissue inflammatory events, the generation of leukotriene (LT)B4, 20-OH-LTB4, sulfidopeptide LT, and platelet-activating factor (PAF) from human whole blood stimulated with zymosan was compared with that produced by isolated human neutrophils suspended either in buffer or plasma. Several reports have shown that substantial LTB4 biosynthesis could be induced after addition of zymosan to whole blood, but little was known concerning the generation of other important lipid mediators, or the cellular source of these. We have shown that, in spite of some subject variation, the zymosan-induced production of 20-OH-LTB4, LTB4, and LTE4 reached maxima within 30 to 60 min with 1.1, 2.8, and 0.60 ng/10(6) neutrophils, respectively. These concentrations would be sufficient to induce significant biologic effects. Studies with isolated cell mixtures suggested that the neutrophil was the primary source of the lipid mediators or their precursors in this system, although a number of other cell types contributed as accessory cells to the final amounts and mix of mediators produced. The ratio of neutrophils to accessory cells in mixed cell experiments dramatically modified the metabolic pattern of leukotriene generation. The concentration of LTB4 was increased in the presence of RBC and that of LTE4 when platelets were present. These results suggested that cellular cooperation and transcellular biosynthesis played a key role in the overall production of eicosanoids such as LTB4 and LTC4. The concomitant synthesis of PAF in isolated cells and in whole blood was also determined as another member of the complex lipid mediator network. Maximal production of cell-associated PAF was observed within 30 min after the initiation of phagocytosis and reached levels of 3 to 5 ng PAF/10(6) neutrophils. When other cells were present in a coincubation system, the time course for production of PAF was not altered, but maximal concentration of PAF was lower, perhaps as a result of enhanced PAF metabolism. Study of eicosanoids and other lipid mediator production in mixed cell populations provides insight into those events occurring within tissues, where cross-cell signaling and transcellular biosynthesis may occur.