Vitamin C and E supplementation effects on secretory and molecular aspects of vascular endothelial growth factor derived from peritoneal fluids of patients with endometriosis.

Endometriosis is an extremely heterogeneous disease and affects about ten percent of the female population during their reproductive years. Recent studies showed that endometriosis is an angiogenesis-dependent disease. Peritoneal macrophages are a well-characterised source of vascular endothelial growth factor (VEGF). The aim of this study was to determine the VEGF gene expression and production in peritoneal macrophages of patients with endometriosis under the effects of vitamins C and E in comparison with control. The lab trial study carried out on 50 patients undergoing laparoscopy and peritoneal fluid samples were collected from them. We compared the VEGF gene expression and production in peritoneal macrophages among groups by using real-time polymerase chain reaction and enzyme-linked immunosorbent assay methods, respectively. Our results showed that gene expressions influenced by vitamin C increased in different concentrations and incubation times, except for the incubation time after 48 h. In the case of vitamin E, this was evident with the exception of vitamin E 50 µM after 24 h and vitamin E 100 µM after 48 h. Our findings indicated that vitamin C and E in different concentrations and incubation times altered VEGF gene expression in the peritoneal macrophages but they had not affected on VEGF productions. Impact statement What is already known on this subject? Previous studies showed that antioxidants play a key role in the inhibition of oxidative stress-induced damages and the reduction of pelvic pain in patients with endometriosis. Vitamin E and vitamin C are the main components in neutralising free radicals. Also, antioxidant consumption such as vitamin C and vitamin E in women with endometriosis showed an inverse correlation between antioxidant intake and endometriosis pathology. What do the results of this study add? Vitamin C and E in different concentrations and times of incubation altered vascular endothelial growth factor gene expression and production in peritoneal macrophages. What are the implications of these findings for clinical practice and/or further research? Further studies are needed to determine the effects of C and E vitamins in different concentrations on vascular endothelial growth factor gene expression and production in peritoneal macrophages and the possible roles of these vitamins in treating endometriosis.

Antileishmanial and immunomodulatory effects of the essential oil from Tetradenia riparia (Hochstetter) Codd.

Cutaneous leishmaniasis usually presents therapeutic resistance to antimonials, and the existing therapies for leishmaniasis have many adverse effects and toxicity. Natural products may be regarded as possible candidates for alternative leishmaniasis treatment. The plant Tetradenia riparia has shown promise for the treatment of infectious diseases in folk medicine. We evaluated the antileishmanial activity of an essential oil from T. riparia (TrEO) and the modulatory effects of TrEO on cytokine modulation by peritoneal fluid cells that were infected with L. (L.) amazonensis. Peritoneal fluid cells were infected with Leishmania and incubated with TrEO (30 ng/mL) for 3, 6, and 24 h. Cytokines were screened using semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and flow cytometry. Antileishmanial activity was evaluated at 24 h by microscopic counting and quantitative PCR (qPCR). TrEO treatment induced the death of 50% of Leishmania amastigotes (indicated by microscopic counting) and 91% of the parasite load (indicated by qPCR). TrEO inhibited some of the most critical cytokines for parasite growth and the establishment of infection, including granulocyte-macrophage colony-stimulating factor, interleukin-4 (IL-4), IL-10, and tumour necrosis factor. The parasite inhibited interferon-γ and IL-12, and TrEO blocked this inhibition, indicating that these cytokines are critical for activating mechanisms associated with the death and elimination of the parasite. These results suggest that TrEO may be an alternative leishmaniasis therapy when considering its antileishmanial and immunomodulatory activity.

Evaluation of nanoparticles loaded with benzopsoralen in rat peritoneal exudate cells.

Psoralens are widely used for the treatment of hyperproliferative skin disease. In this work, we prepared nanoparticles (NP) containing a benzopsoralen (3-ethoxy carbonyl-2H-benzofuro[3,2-f]-1-benzopiran-2-one) by the solvent evaporation technique. We evaluated important NP parameters such as particle size, drug encapsulation efficiency, effect of the encapsulation process over the drug's photochemistry, zeta potential, external morphology, and in vitro release behavior. We also investigated the nanoparticle as a drug delivery system (DDS), as well as its target delivery to the action site, which is a very important parameter to increase the therapeutic use of psoralens and to reduce their side effects. The uptake of benzopsoralen-loaded PLGA nanoparticles by different kinds of cells found in rat peritoneal exudates was also studied. The photodamage promoted by irradiation with UV light revealed morphological characteristics of cell damage such as cytoplasmic vesiculation, mitochondrial damage, and swelling of both the granular endoplasmatic reticulum and nuclear membrane. This encapsulation method maintained the drug's properties and improved drug delivery to the target cell.

Dietary supplementation with antioxidants improves functions and decreases oxidative stress of leukocytes from prematurely aging mice.

OBJECTIVES: Aging is accompanied by chronic inflammation and oxidative stress, which lead to a marked impairment of immune function and therefore increased mortality. This study assessed the effect of dietary supplementation, for 15 wk, with 5% and 20% (w/w) of biscuits enriched with nutritional doses of vitamins C and E, zinc, selenium, and beta-carotenes on function and oxidative stress parameters of peritoneal leukocytes from middle-aged, prematurely aging mice (PAM) and non-prematurely aging mice (NPAM). METHODS: After supplementation we measured leukocyte functions (adherence, chemotaxis, phagocytosis, intracellular reactive oxygen species levels, lymphoproliferation, natural killer activity, and interleukin-2 release), antioxidant defenses (superoxide dismutase, glutathione peroxidase, and reduced glutathione), oxidant compounds (extracellular O(2)(-), glutathione disulfide, glutathione disulfide/reduced glutathione ratio, tumor necrosis factor-alpha, nitric oxide, and prostaglandin E(2)), and lipid and DNA oxidative damage, measured by malondialdehyde and 8-oxo,7,8-dihydro-2'-deoxyguanosine levels, respectively. RESULTS: In general, leukocyte functions were improved and redox homeostasis was restored after intake of antioxidants. In consequence, malondialdehyde and 8-oxo,7,8-dihydro-2'-deoxyguanosine in PAM and NPAM were strikingly decreased after 5% and 20% supplementation (malondialdehyde, P < 0.001 in PAM; P < 0.01 in NPAM after both treatments; 8-oxo,7,8-dihydro-2'-deoxyguanosine, P < 0.01 after 5% supplementation and P < 0.001 after 20% supplementation in PAM and NPAM). Moreover, the effect of the antioxidants was stronger in PAM than in NPAM, and 20% supplementation was more effective than 5%. CONCLUSION: Our data suggest that improvement of leukocyte function and restoration of redox balance after consumption of adequate levels of antioxidants from adulthood may be useful to attain healthy aging, especially in animals with premature aging.

Peritoneal exudate cells treated with calcitonin gene-related peptide suppress murine experimental autoimmune uveoretinitis via IL-10.

Immunization with retinal Ag induces experimental autoimmune uveoretinitis (EAU) in mice. We investigated the suppression of murine EAU by peritoneal exudate cells (PEC) cultured with calcitonin gene-related peptide (CGRP). PEC derived from mice were treated with CGRP and residues 1-20 of human interphotoreceptor retinoid-binding protein (hIRBP 1-20). The hIRBP 1-20-immunized mice were injected i.v. with PEC treated with CGRP and hIRBP 1-20. After immunization, Ag-specific delayed hypersensitivity (DH) was measured and EAU was assessed histopathologically. Both EAU- and Ag-specific DH were suppressed by injection of PEC treated with CGRP (100 ng/ml) and hIRBP 1-20. However, hIRBP 1-20-mediated EAU was not suppressed by injection of PEC treated with CGRP and BSA. Both EAU- and Ag-specific DH were not suppressed by injection of PEC treated with CGRP and hIRBP 1-20 into splenectomized mice. In mice adoptively transferred spleen cells from hIRBP 1-20-immunized mice, EAU was also suppressed by injection of CGRP-treated PEC. EAU was markedly inhibited in hIRBP 1-20-immunized mice adoptively transferred T cells obtained from mice injected with hIRBP 1-20-pulsed, CGRP-treated PEC. Furthermore, EAU- and Ag-specific DH were not suppressed by injection of PEC treated with CGRP and hIRBP 1-20 when the recipient mice were given anti-IL-10 Ab i.p., or when the PEC were derived from IL-10 knockout mice. The present results indicate that PEC treated with CGRP suppress murine EAU in an Ag-specific manner, even in the efferent phase, and IL-10 secreted from PEC might play an important role in the CGRP-mediated suppression of murine EAU.

Immunostimulating activity of the hot water-soluble polysaccharide extracts of Anacyclus pyrethrum, Alpinia galanga and Citrullus colocynthis.

Hot water polysaccharide extracts of Anacyclus pyrethrum (L.) Link. (family Compositae) Citrullus colocynthis (L.) Schrad. (family Cucurbitaceae) and Alpinia galanga (L.) Willd. (family Zingiberaceae) were tested for their immunostimulating activity in mice. The fractions from Anacyclus pyrethrum and Alpinia galanga showed a marked stimulating effect on the reticulo-endothelial system (RES) and increased the number of peritoneal exudate cells (PEC), and spleen cells of mice. In this case, the optimum doses were 50 and 25 mg/kg for the two fractions, respectively. On the other hand, the polysaccharide extracts of both Anacyclus pyrethrum and Alpinia galanga markedly enhanced the proliferation of the murine spleen cells in vitro using two tests (in vitro and in vivo effect). The results of the in vivo effect at a doses of 50 and 25 mg/kg, showed a stimulation index better than obtained with the in vitro effect at 50 and 25 microg/ml for Anacyclus pyrethrum and Alpinia galanga, respectively. While the extract of Citrullus colocynthis showed much weaker and variable immunostimulating activity.

Effects of wogonin, wogonoside, and 3,5,7,2',6'-pentahydroxyflavone on chemical mediator production in peritoneal exduate cells and immunoglobulin E of rat mesenteric lymph node lymphocytes.

Wogonin (WG), wogonoside (WGS), and 3,5,7,2',6'-pentahydroxyl flavanone (PHF) were isolated from Scutellaria baicalensis, and their effects on histamine, leukotriene B(4) (LTB(4)), and immunoglobulin E (IgE) were examined in rats, observing for a manifestation of a type I allergic reaction. WG and WGS in the amounts of 10 and 100 microM were shown to markedly inhibit histamine release in cells stimulated with calcium ionophore A23187 or compound 48/80. PHF exerted inhibitory activity only at 100 microM. In the case of LTB(4), WG, WGS and PHF markedly inhibited LTB(4) production at the concentration of 100 microM. We also find that the increase in the IgE content induced by concanavalin A (ConA) was alleviated in the presence WG and WGS, while the inhibitory effect of PHF was much weaker. However, the magnitude of inhibitory effect observed on the content of lipid peroxidation induced by ConA was in order of PHF > WG > WGS, with PHF being the strongest. Interestingly, WG and WGS with the methoxyl group strongly inhibited histamine and IgE production, whereas PHF with the hydroxyl group in the B ring was much stronger than WG and WGS against lipid peroxidation. Based on data, it was concluded that the flavonoid components, WG, WGS, and PHF, may block a common pathway for the release of histamine and LTB(4), and that the IgE level is responsible for the lipid peroxidation induced by ConA.

Two phenanthraquinones from Dendrobium moniliforme.

Two phenanthraquinones were isolated from the stems of Dendrobium moniliforme. They were identified by interpretation of spectral data as 2,6-dimethoxy-1,4,5,8-phenanthradiquinone and 5-hydroxy-3,7-dimethoxy-1,4-phenanthraquinone, named moniliformin (1) and denbinobin (2), respectively. This is the first report of compound 1, which possesses a novel 1,4,5,8-diquinone moiety in the phenanthraquinone skeleton. Compound 2 showed potent antiinflammatory effects in vitro.

Interactions of dietary fats and proteins on fatty acid composition of immune cells and LTB4 production by peritoneal exudate cells of rats.

The interaction of dietary fats and proteins on lipid parameters of rats was studied using safflower oil (linoleic acid-rich), borage oil (gamma-linolenic acid-rich) or perilla oil (alpha-linolenic acid-rich) in combination with casein or soybean protein. The experiment was focused on the fatty acid composition of immune cells and the leukotriene B4 production by peritoneal exudate cells. Serum total cholesterol, triglyceride, and phospholipid levels were low in perilla oil-fed or soybean protein-fed rats. Fatty acid compositions of serum and liver phospholipids reflected those of dietary fats. However, feeding borage oil resulted in a marked increase in the proportion of dihomo-gamma-linolenic acid in phospholipids of peritoneal exudate cells, spleen lymphocytes, and mesenteric lymph node lymphocytes in relation to those of liver and serum. It is suggested that activities of metabolic n-6 polyunsaturated fatty acids are different between immune and other tissues. In addition, the magnitude of the reduction of the proportion of linoleic acid of perilla oil in immune cells was considerably more moderate than serum and liver, indicating a different degree of interference of alpha-linolenic acid with linoleic acid metabolism. Leukotriene release from peritoneal exudate cells was in the order of safflower oil > borage oil > perilla oil groups as reflecting the proportion of arachidonic acid, and tended to be lower in soybean protein-fed groups. These suggest an anti-inflammatory property of gamma-linolenic acid as well as alpha-linolenic acid tended to be strengthened when they were combined with soybean protein than with casein.

Differential activation of murine macrophages by angelan and LPS.

In our previous studies, we showed that angelan, a polysaccharide purified from Angelica gigas Nakai, is a potent LPS-mimetic in murine macrophages [Jeon, Y.J., Han, S.B., Ahn, K.S., Kim, H.M., 1999. Activation of NF-kB/Rel in angelan-stimulated macrophages. Immunopharmacology 43, 1-9]. Angelan stimulates murine macrophage to produce cytokines including iNOS and activate NF-kappaB/Rel. In the present study, we investigated the role of CD14 and complement receptor type 3 (CR3) in mediating NO production and NF-kappaB/Rel activation induced by angelan and LPS. Three major differences between angelan and LPS were observed. First, angelan does not require serum proteins for NO response and NF-kappaB/Rel activation, while the activation by LPS requires serum proteins. Second, blocking of either CD14 or CR3 decreased angelan-induced NO response, while LPS-mediated NO production was inhibited by anti-CD14 mAb only. Third, angelan induced strong NF-kappaB/Rel and slight AP-1 DNA binding, whereas LPS potently activated both NF-kappaB/Rel and AP-1. Both angelan and LPS degraded IkappaB proteins and subsequently induced the mobilization of NF-kappaB/Rel proteins (p65, c-rel and p50) into nucleus. This suggests that macrophages display a common signaling machinery leading to the NF-kappaB/Rel activation in response to different stimulants. In conclusion, angelan and LPS use the membrane receptor CD14 and CR3 differentially for signaling NF-kappaB/Rel activation and NO production.

[An experimental study of the effects of pectin substances on nonspecific defenses of the body]. / Eksperimental'noe izuchenie vliianiia pektinovykh veshchestv na nespetsificheskuiu zashchitu organizma.

Effects of water-soluble alimentary fibers on nonspecific defense system attracted the attention of scientists in recent decade. Effects of some pectins on the phagocytic activity of macrophagal population infected with Yersinia pseudotuberculosis is studied on Wistar rats. The number of peritoneal exudative cells (test system) and macrophagal activity increased (as shown by increased glycolysis and acid phosphatase activity), while the number of surviving bacteria decreased 1000 times. Comparison of several apple and sunflower pectins demonstrated the highest killing effect of highly esterified apple pectin. The results necessitate further studies aimed at discovery of the new clinical applications of water-soluble fibers.

Bioactive saponins and glycosides. X. On the constituents of zizyphi spinosi semen, the seeds of Zizyphus jujuba Mill. var. spinosa Hu (1): structures and histamine release-inhibitory effect of jujubosides A1 and C and acetyljujuboside B.

New dammarane-type triterpene oligoglycosides, jujubosides A1 and C and acetyljujuboside B1 were isolated from Zizyphi Spinosi Semen, the seeds of Zizyphus jujuba MILL. var. spinosa Hu, together-with three known saponins. The structures of jujubosides A1 and C and acetyljujuboside B were determined on the basis of chemical and physicochemical evidence. Jujubosides A1 and C and acetyljujuboside B were found to inhibit the histamine release from rat peritoneal exudate cells induced by antigen-antibody-reaction.

Inhibitory effect of tea polyphenols on histamine and leukotriene B4 release from rat peritoneal exudate cells.

The effect of tea polyphenols on the release of chemical mediators, histamine and leukotriene B4 (LTB4), from rat peritoneal exudate cells (PEC) was studied. Among polyphenols, (-)-epigallocatechin gallate (EGCG) most strongly inhibited the histamine release from the cells stimulated with a calcium ionophore, A23187 or compound 48/80. Though (+)-catechin (C) and (-)-epicatechin (EC) had no effect, (-)-epigallocatechin (EGC) and (-)-epicatechin gallate (ECG) moderately inhibited the histamine release. Similarly, EGCG, ECG, and EGC inhibited LTB4 release from PEC, whereas C and EC were not effective. The magnitude of the inhibitory effect on the release of these mediators of tea polyphenols was in the order of EGCG > ECG > EGC. These results indicated an important role of the triphenol structure in the inhibitory activity. Therefore, the possible antiallergic effect of tea polyphenols can be expected.

Induction of type IV hypersensitivity to contact allergens in guinea pigs by in vitro haptenized allogenic peritoneal exudate cells.

The induction of type IV hypersensitivity to contact allergens in guinea pigs has been studied by using allogenic peritoneal exudate cells (> 90% macrophages), which had been incubated primarily in vitro with dinitrochlorobenzene, formaldehyde, potassium dichromate, nickel II sulphate or para-aminobenzoic acid. In these guinea pig sensitization experiments Freund's complete adjuvant was used. In all haptens investigated the sensitization rates of the presented method were parallel to the known contact allergenicity in humans and, apart from the potassium dichromate results, comparable with those of the guinea pig maximization test. Because of its alternative immunization procedure, in which only few or no allergen molecules escape the effective presentation pathway, the authors conclude that this method could be developed into a predictive test assay for the evaluation of the contact allergenicity of water-soluble substances.

Mouse peritoneal macrophage prostaglandin E1 synthesis is altered by dietary gamma-linolenic acid.

The ability of dietary gamma-linolenic acid [18:3(n-6)] to modulate prostaglandin biosynthesis in mouse resident peritoneal macrophages was determined. Mice were fed diets containing corn oil, borage oil or evening primrose oil or a mixture of borage and fish oils. After 2 wk, resident peritoneal macrophages were isolated and stimulated with unopsonized zymosan to induce prostaglandin synthesis. Borage oil, primrose oil and fish-borage oil mixture dietary groups (containing 25.6, 11.9 and 19.5 g gamma-linolenic acid/100 g fatty acids, respectively) had significantly (P less than 0.05) enhanced prostaglandin E1 synthesis (39.7, 29.4 and 73.0 nmol prostaglandin E1/mg protein, respectively) compared with corn oil-fed (containing less than 0.1 g gamma-linolenic acid/100 g fatty acids) animals, which synthesized less than 0.1 nmol prostaglandin E1/mg protein. Borage oil- and fish-borage oil mixture-fed mice had the highest biosynthetic ratio of prostaglandin E1/prostaglandin E2 (E1/E2 approximately 0.2). Macrophages from borage oil-fed mice synthesized the lowest amount of prostacyclin (198.7 nmol 6-keto-prostaglandin F1 alpha/mg protein) compared with corn oil-, primrose oil- and fish-borage oil mixture-fed mice (379.7, 764.8 and 384.2 nmol 6-keto-prostaglandin F1 alpha/mg protein, respectively). In addition, borage oil-, primrose oil- and fish-borage oil mixture-fed mice had significantly (P less than 0.05) higher levels of dihomo-gamma-linolenic acid [20:3(n-6)] in membrane phospholipids (5.5, 3.5 and 5.7 mol/100 mol, respectively) relative to corn oil-fed mice (2.0 mol/100 mol).(ABSTRACT TRUNCATED AT 250 WORDS)

[Composition and immunological activity of membrane-bound surface glycoprotein from Crithidia oncopelti]. / Sostav i immunologicheskaia aktivnos't membranosviazannogo poverkhnostnogo glikoproteina Crithidia oncopelti.

A membrane-bound glycoprotein with a molecular weight of 10000-12000 was isolated from Crithidia oncopelti and purified. The glycoprotein contained peptide, carbohydrate and lipid fragments and phosphorus. The peptide fragment was represented by 10 amino acids. The carbohydrate fragment was represented by 7 monosaccharides. The lipid part was mainly represented by stearic acid. The glycoprotein showed immunostimulating properties. It had a comitogenic effect on murine spleen cells in vitro and induced tumoricidal activity in murine peritoneal macrophages in vitro and in vivo.

Activation of macrophages to inhibit proliferation of Mycobacterium tuberculosis: comparison of the effects of recombinant gamma-interferon on human monocytes and murine peritoneal macrophages.

When cultured in 20% heat-inactivated human serum, human monocytes from seven donors were not on average significantly different from non-activated murine peritoneal cells (cultured simultaneously and in an identical manner) in their ability to inhibit BCG and, when calculated relative to growth of bacilli in the same medium without macrophages, to enhance the growth of Mycobacterium tuberculosis. Recombinant gamma-interferon caused marked inhibition of virulent M. tuberculosis by murine (BALB/c) peritoneal macrophages. This effect was seen, whether the cells were cultured in 10% fetal calf serum or in 20% heat-inactivated normal human serum, with or without the addition of iron supplements. However, unlike murine cells, the addition of crude lymphokine or recombinant gamma-interferon to human monocytes caused only weak inhibition of M. tuberculosis, and in some instances, gamma-interferon caused enhancement of growth of the bacilli. Monocytes were only slightly more effective if precultured for 4-8 days before the addition of the activating stimulus. This relative failure to develop anti-mycobacterial mechanisms occurred in spite of the activation of the cells as shown by a massive increase in reduction of nitro-blue tetrazolium inducible by phorbol myristate acetate.

Inhibitory effect of the CA2+ antagonist nifedipine on histamine release from rat peritoneal mast cells.

45Ca uptake and histamine release was examined in mast cells from rats sensitized with ovalbumin and Bordetella Bertussis as an adjuvant. The uptake of 45Ca by the mast cells was significantly increased by stimulation with ovalbumin as was the release of histamine from the mast cells. Nifedipine, a calcium antagonist, inhibited the increase in both 45Ca uptake and histamine release stimulated by ovalbumin, though the effect on 45Ca uptake was stronger than that on histamine release.

Effects of cobalt-chrome alloy wear particles on the morphology, viability and phagocytic activity of murine macrophages in vitro.

Metallic wear particles were prepared from orthopaedic cobalt-chrome alloy by milling in medium supplemented with 20% foetal calf serum to maintain particle dispersion. The size distribution of particles was determined by sedimentation and centrifugation and particle concentration was assessed using light extinction. Monolayers of mouse peritoneal macrophages were exposed to metal particles at different concentrations for varying periods. After 4 h of exposure to particle concentrations exceeding 30 micrograms/ml there was a progressive decline in cell viability, and light and electron microscopy showed that surviving cells had assumed remarkably smooth profiles and contained abundant endocytosed metal. Phagocytic uptake of polystyrene spherules was inhibited markedly by exposure to metal particles even at concentrations at which macrophages remained 100% viable, and preceded the reduction of viability at higher concentrations. The findings are consistent with a pathological role for the metallic wear particles observed frequently within macrophages in the synovial tissues around loose artificial joints in humans.