Macropinocytosis is an alternative pathway of cysteine acquisition and mitigates sorafenib-induced ferroptosis in hepatocellular carcinoma.

BACKGROUND: Macropinocytosis, an important nutrient-scavenging pathway in certain cancer cells, allows cells to compensate for intracellular amino acid deficiency under nutrient-poor conditions. Ferroptosis caused by cysteine depletion plays a pivotal role in sorafenib responses during hepatocellular carcinoma (HCC) therapy. However, it is not known whether macropinocytosis functions as an alternative pathway to acquire cysteine in sorafenib-treated HCC, and whether it subsequently mitigates sorafenib-induced ferroptosis. This study aimed to investigate whether sorafenib drives macropinocytosis induction, and how macropinocytosis confers ferroptosis resistance on HCC cells. METHODS: Macropinocytosis, both in HCC cells and HCC tissues, was evaluated by measuring TMR-dextran uptake or lysosomal degradation of DQ-BSA, and ferroptosis was evaluated via C11-BODIPY fluorescence and 4-HNE staining. Sorafenib-induced ferroptosis and macropinocytosis were validated in tumor tissues taken from HCC patients who underwent ultrasound-guided needle biopsy. RESULTS: Sorafenib increased macropinocytosis in human HCC specimens and xenografted HCC tissues. Sorafenib-induced mitochondrial dysfunction was responsible for activation of PI3K-RAC1-PAK1 signaling, and amplified macropinocytosis in HCC. Importantly, macropinocytosis prevented sorafenib-induced ferroptosis by replenishing intracellular cysteine that was depleted by sorafenib treatment; this rendered HCC cells resistant to sorafenib. Finally, inhibition of macropinocytosis by amiloride markedly enhanced the anti-tumor effect of sorafenib, and sensitized resistant tumors to sorafenib. CONCLUSION: In summary, sorafenib induced macropinocytosis, which conferred drug resistance by mitigating sorafenib-induced ferroptosis. Thus, targeting macropinocytosis is a promising therapeutic strategy to facilitate ferroptosis-based therapy for HCC.

Purification and comparative study of bioactivities of a natural selenized polysaccharide from Ganoderma Lucidum mycelia.

The development of selenized polysaccharides is a promising strategy for the dietary selenium supplementation. The purpose of this research is to determine the influence of selenium on the structure and bioactivity of a polysaccharide fraction (MPN) isolated from Ganoderma lucidum mycelia. After biological selenium enrichment, the selenium content in the selenized polysaccharide (SeMPN) was 18.91 ± 1.8 µg/g. SeMPN had a slightly lower molecular weight than MPN, but the carbohydrate content and monosaccharide composition remained identical. Additionally, the band at 606 cm-1 in MPN changed to 615 cm-1 in SeMPN as revealed by FT-IR spectra. No significant changes were observed in the types and ratios of glycosidic linkages, as determined by NMR spectroscopy. Extracellular and intracellular antioxidant assays demonstrated that SeMPN was more effective than MPN in scavenging free radicals, inhibiting AAPH-induced erythrocyte hemolysis, and protecting catalase (CAT) and glutathione peroxidase (GSH-Px) activity in H2O2-injured PC12 cells. Additionally, SeMPN had a higher increase effect on RAW 264.7 cells's pinocytic and phagocytic capacity, as well as their production of NO, TNF-α, and IL-6. SeMPN could be as potential functional selenium supplementation.

Structural characterization and mechanisms of macrophage immunomodulatory activity of a pectic polysaccharide from Cucurbita moschata Duch.

A pectic polysaccharide (named CMDP-4b) with a molecular weight of 31.97 kDa was extracted from Cucurbita moschata Duch and purified by column chromatography. On the basis of methylation, Fourier-transform infrared, monosaccharide composition, and one- and two-dimensional nuclear magnetic resonance spectroscopy analyses, the structure of CMDP-4b was determined to be composed of an α-1,4-linked homogalacturonan backbone, which was slightly acetylated and highly methyl-esterified, and branched at the O-3 position of the →4)-α-D-GalpA-6-OMe-(1→. Immunomodulatory assays showed that CMDP-4b not only induced the secretion of nitrous oxide and cytokines (i.e. IL-1ß, TNF-α, and IL-6) but also promoted pinocytic and phagocytic activities of macrophages, suggesting that CMDP-4b possessed immunomodulatory activity. Moreover, toll-like receptor 4 and complement receptor 3 may play a critical role in CMDP-4b-induced macrophage activation through the NF-κB and the MAPKs signaling pathways. Our study provides the molecular basis for the potential use of CMDP-4b as a natural immunostimulant.

Immunomodulatory effects of fucosylated chondroitin sulfate from Stichopus chloronotus on RAW 264.7 cells.

Sea cucumbers were nutritional food and traditional Chinese medicine. In this study, fucosylated chondroitin sulfate from sea cucumber Stichopus chloronotus (fCS-Sc), a potential anticoagulant agent and immunological adjuvant, was investigated for its immune activation effects on RAW 264.7 macrophage for the first time. The results indicated that fCS-Sc could significantly promote the proliferation, the pinocytic activity of RAW 264.7 cells, and the production of NO, TNF-α, IL-1ß, and IL-6. The fluorescence labeling assay indicated that fCS-Sc could bind to the macrophage. Moreover, the specific pattern recognition receptor inhibition assays showed that toll-like receptor 4 (TLR4) and TLR2 were involved in the recognition of fCS-Sc. Western blot assays indicated that fCS-Sc could induce degradation of cytoplasm IκB-α, and promotion of NF-κB p65 subunit translocation to nucleus, leading to a functional improvement of macrophage through NF-κB pathway. The results suggested that fCS-Sc might served as a promising candidate of immunomodulator.

Dietary supplementation with Lactobacillus plantarum and ß-glucan affects immune parameters in the tench (Tinca tinca) fry.

The aim of the experiment was to examine the effect of a diet enriched with Lactobacillus plantarum and/or ß-glucan on the immune parameters in the juvenile tench (Tinca tinca). Fish were fed for 14 days different diets (phase 1 of the experiment), a dry commercial starter feed in the control group or the same feed supplemented with: 1% ß-1,3/1,6-glucan in group G, 108 cfu L. plantarum g-1 in group L, 1% ß-1,3/1,6-glucan + 108 cfu L. plantarum g-1 in group G+L. During consecutive 14 days all fish were fed the commercial feed alone (phase 2). The stimulating effects of the tested preparations was evaluated twice, at the end of each experimental phase. Dietary supplementation of ß-1,3/1,6-glucan considerably improved the humoral innate immune response (activity of lysozyme and total Ig) and the pinocytotic activity of phagocytes. Supplement of L. plantarum improved the ability of the head kidney phagocytes (RBA) to carry out oxygen burst in L and G+L groups. A similar effect was observed for the killing activity of phagocytes (PKA) from the head kidney after the stimulation of A. hydrophila, and the effect persisted for two weeks after the commercial feed regime was resumed. A significant increase in the proliferative activity of B lymphocytes originating from the head kidney was observed in groups L and G+L. The study has revealed that the addition of the tested G+L synbiotic to dry diet stimulates the innate immune response mechanisms in the juvenile tench.

Identification of novel macropinocytosis inhibitors using a rational screen of Food and Drug Administration-approved drugs.

BACKGROUND AND PURPOSE: Macropinocytosis is involved in many pathologies, including cardiovascular disorders, cancer, allergic diseases, viral and bacterial infections. Unfortunately, the currently available pharmacological inhibitors of macropinocytosis interrupt other endocytic processes and have non-specific endocytosis-independent effects. Here we have sought to identify new, clinically relevant inhibitors of macropinocytosis, using an FDA-approved drug library. EXPERIMENTAL APPROACH: In the present study, 640 FDA-approved compounds were tested for their ability to inhibit macropinocytosis. A series of secondary assays were performed to confirm inhibitory activity, determine IC50 values and investigate cell toxicity. The ability of identified hits to inhibit phagocytosis and clathrin-mediated and caveolin-mediated endocytosis was also investigated. Scanning electron microscopy and molecular biology techniques were utilized to examine the mechanisms by which selected compounds inhibit macropinocytosis. KEY RESULTS: The primary screen identified 14 compounds that at ~10 µM concentration inhibit >95% of macropinocytotic solute internalization. Three compounds - imipramine, phenoxybenzamine and vinblastine - potently inhibited (IC50  ≤ 131 nM) macropinocytosis without exerting cytotoxic effects or inhibiting other endocytic pathways. Scanning electron microscopy imaging indicated that imipramine inhibits membrane ruffle formation, a critical early step leading to initiation of macropinocytosis. Finally, imipramine has been shown to inhibit macropinocytosis in several cell types, including cancer cells, dendritic cells and macrophages. CONCLUSIONS AND IMPLICATIONS: Our results identify imipramine as a new pharmacological tool to study macropinocytosis in cellular and biological systems. This study also suggests that imipramine could be a good candidate for repurposing as a therapeutic agent in pathological processes involving macropinocytosis.

Effect of Tinospora cordifolia (Guduchi) on the phagocytic and pinocytic activity of murine macrophages in vitro.

Tinospora cordifolia (Guduchi) is a widely used herb in Ayurvedic system of medicine known to possess immunomodulatory properties. The present study was aimed to study the activation of macrophages after in vitro guduchi treatment. The aqueous extract of T. cordifolia was found to enhance phagocytosis and pinocytosis in vitro. The rate of pinocytosis by macrophages when measured by uptake of horseradish peroxidase was significantly increased after guduchi treatment as compared to medium alone. The macrophages demonstrated an increased phagocytosis to non-infective microorganisms (heat killed yeast) and live infective microorganisms (E. coli) after guduchi treatment. The results demonstrate that Guduchi enhances macrophage activation as analyzed by cytochemical parameters.

Vitamin D-binding protein controls T cell responses to vitamin D.

BACKGROUND: In vitro studies have shown that the active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), can regulate differentiation of CD4+ T cells by inhibiting Th1 and Th17 cell differentiation and promoting Th2 and Treg cell differentiation. However, the serum concentration of 1,25(OH)2D3 is far below the effective concentration of 1,25(OH)2D3 found in in vitro studies, and it has been suggested that 1,25(OH)2D3 must be produced locally from the inactive precursor 25-hydroxyvitamin D3 (25(OH)D3) to affect ongoing immune responses in vivo. Although it has been reported that activated T cells express the 25(OH)D-1α-hydroxylase CYP27B1 that converts 25(OH)D3 to 1,25(OH)2D3, it is still controversial whether activated T cells have the capacity to produce sufficient amounts of 1,25(OH)2D3 to affect vitamin D-responsive genes. Furthermore, it is not known how the vitamin D-binding protein (DBP) found in high concentrations in serum affects T cell responses to 25(OH)D3. RESULTS: We found that activated T cells express CYP27B1 and have the capacity to produce sufficient 1,25(OH)2D3 to affect vitamin D-responsive genes when cultured with physiological concentrations of 25(OH)D3 in serum-free medium. However, if the medium was supplemented with serum or purified DBP, DBP strictly inhibited the production of 1,25(OH)2D3 and 25(OH)D3-induced T cell responses. In contrast, DBP did not inhibit the effect of exogenous 1,25(OH)2D3. Actin, arachidonic acid and albumin did not affect the sequestration of 25(OH)D3 by DBP, whereas carbonylation of DBP did. CONCLUSIONS: Activated T cells express CYP27B1 and can convert 25(OH)D3 to 1,25(OH)2D3 in sufficiently high concentrations to affect vitamin D-responsive genes when cultured in serum-free medium. However, DBP sequesters 25(OH)D3 and inhibits the production of 1,25(OH)2D3 in T cells. To fully exploit the immune-regulatory potential of vitamin D, future studies of the mechanisms that enable the immune system to exploit 25(OH)D3 and convert it to 1,25(OH)2D3 in vivo are required.

Immunopotentiation of Pleurotus eryngii (DC. ex Fr.) Quel.

ETHNOPHARMACOLOGICAL RELEVANCE: Pleurotus eryngii (DC. ex Fr.) Quel has been collected from the wild, cultivated and used in traditional medicines to treat various disorders and diseases since antiquity. In traditional Chinese medicine, the powdered fruiting bodies of Pleurotus eryngii were used for immunostimulation, skin-care, wound-healing, cancer and lumbago treatment. In the current study, we investigated the antiproliferative activity of Pleurotus eryngii powder on A549, BGC-823, HepG2 and HGC-27 cancer cells and its immunomodulating activity on macrophage, RAW 264.7 cells based on its active compound. MATERIALS AND METHODS: A novel bioactive protein (PEP) was extracted from Pleurotus eryngii fruiting bodies powder and purified on DEAE-52, CM-52 and Superdex 75 column chromatographies using an ÄKTA purifier. Its cytotoxicity on A549, BGC-823, HepG2, HGC-27 and RAW 267.4 cell lines was then evaluated using MTT, alamar blue (AB), trypan blue (TB), neutral red (NR), lactate dehydrogenase (LDH), Annexin V FITC/PI and morphological change assays. Moreover, lysosomal enzyme activity, pinocytosis, nitric oxide (NO) and hydrogen peroxide (H2O2) production assays were used to examine immunomostimulatory activity of PEP on RAW 267.4 cells. RESULTS: Based on high performance gel permeation chromatography (HPGPC), Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) analyses, the isolated protein (PEP) had a molecular weight of 63 kDa, a secondary (α-helical) structure and was mainly composed of arginine, serine and glycine. PEP significantly (P<0.05) inhibited A549, BGC-823, HepG2 and HGC-27 tumor cells proliferation dose-dependently with an IC50 range of 36.5 ± 0.84 to 229.0 ± 1.24 µg/ml. Contrarily, PEP stimulated the proliferation of macrophages. CONCLUSION: Pleurotus eryngii fruiting bodies powder has a potential application as a natural antitumor agent with immunomodulatory activity, proposedly, by targeting the lysosomes of cancerous cells and stimulating macrophage-mediated immune responses.

Biological effects of aqueous extract from Turkey vulture Cathartes aura (Cathartidae) meat.

ETHNOPHARMACOLOGICAL RELEVANCE: Cathartes aura is a bird used in the Mexican traditional medicine for the empirical treatment of cancer, injuries, infections and burns. MATERIALS AND METHODS: The in vitro immunomodulatory effects of Cathartes aura extract (CAE) were evaluated estimating its effects on proliferation of human peripheral blood mononuclear cells and murine splenocytes. The effects of CAE (1-200 µg/ml) on NO production, pinocytosis and lysosomal enzyme activity were assayed in murine macrophages RAW 264.7. The cytotoxic effects of CAE (1-500 µg/ml) on tumorigenic and non tumorigenic cells were evaluated using the MTT assay. RESULTS: In the absence of LPS, CAE induced the proliferation of murine splenocytes (119%), enhanced the pinocytosis (113%) and lysosomal enzyme activity (141%) in murine macrophages with a similar potency than lypopolisaccharides 1 µg/ml. In addition, CAE exerted cytotoxic effects mainly on human cervical cancer cells (IC(50)=117 µg/ml) but lacked toxic effects on non tumorigenic cells (IC(50)>500 µg/ml). CONCLUSION: Cathartes aura exerts immunostimulatory and cytotoxic activities.

Phenotypic and functional maturation of murine dendritic cells (DCs) induced by purified Glycyrrhizin (GL).

The aim of this study is to investigate phenotypic and functional modulation of murine dendritic cells (DCs) with use of purified Glycyrrhizin (GL). These impacts of GL on DCs both from bone marrow derived DCs and established DC cell 2.4 were assessed with conventional scanning electron microscopy (SEM), flow cytometry (FCM), transmission electron microscopy (TEM), cytochemistry assay, FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We found that the purified GL induced phenotypic maturation as evidenced by increased expression of CD86, CD40, CD80, CD83 and major histocompatibility complex II (MHC II). The functional tests showed the activity of acidic phosphatase (ACP) inside the DCs2.4 cells were down- regulated after treatment with GL (which occurs when phagocytosis of DCs2.4 cells were decreased). Finally, we proved that GL increased the production of IL-12, IL-10 and decreased the production of tumor necrosis factor alpha (TNF-α). These data indicated that GL could promote maturation of DCs and this adjuvant-like activity may have potential therapeutic value. It is therefore concluded that GL could exert positive modulation on murine DCs.

Hot water extract of the sclerotium of Polyporus rhinocerus Cooke enhances the immune functions of murine macrophages.

Treatment of hot water extract of the sclerotium of Polyporus rhinocerus (PRW) with murine macrophages including RAW 264.7 cell line and primary macrophages (PMs) could enhance their functional activities. These include a significant up-regulation of pinocytosis; an increase in the production of reactive oxygen species (ROS) and nitric oxide (NO); an increase in tumor necrosis factor alpha (TNF-alpha) production and inducible nitric oxide synthase (iNOS) expression in both RAW 264.7 cells and PMs. Cell surface receptors for yeast-derived beta-glucan, including Dectin-1, CR3, and TLR2, were determined by flow cytometry, and the expression of Dectin-1+ cells on the cell surface decreased in the responses of PMs to PRW. PRW increased phosphorylation of IkappaBalpha, which could trigger the nuclear factor kappa B (NF-kappaB) signal pathway for macrophage activation in RAW 264.7 cells. Therefore, the immunomodulatory effect of PRW could be mediated by macrophage activation via the NF-kappaB signal pathway.

Aqueous extract of Phyllanthus niruri (Euphorbiaceae) enhances the phenotypic and functional maturation of bone marrow-derived dendritic cells and their antigen-presentation function.

Decoctions of Phyllanthus niruri (PN) (Fam. Euphorbiaceae) is promoted in traditional medicine of Africa, Asia, and South America as beneficial supplement for different infectious diseases, especially for viral hepatitis, tumor, and for immune compromised patients. This stimulated the interest in understanding the mechanisms by which the whole extract of the plant could stimulate the immune system. Dendritic cells (DCs) are professional antigen-presenting cells and provide a link between the innate and the adaptive immune responses. In the present study, the effects of lyophilized aqueous extract of PN on structural and functional maturation of murine bone marrow-derived DCs (BM-DCs) were investigated. Bone marrow cells were cultured in the presence of granulocyte macrophage-colony stimulating factor and interleukin-4 (IL-4) and the generated immature DCs were stimulated with PN (25, 50, and 100 microg/mL) or lipopolysaccharide (10 microg/mL) for 48 h. Results showed that treatment with PN increased the expression of major histocompatibility complex-II and the various makers for DCs maturation (CD40), activation (CD83), and costimulation (CD86) in a concentration-dependent manner. Consistent with the increase in phenotypic makers, functional maturation assay showed that treatment of BM-DCs with PN caused a decrease in fluorescein isothiocyanate-dextran pinocytosis and an increase in IL-12 in the supernatant. In a transgenic T-cell activation model, PN-treated BM-DCs presented Ova antigen to Ova-specific CD8(+) T cells from OT-1 mice more efficiently as demonstrated by increased T-cells proliferation and IL-2 production. Therefore, PN enhances the structural and functional maturation of BM-DCs and their antigen-presenting function. These effects are relevant in immunodeficient conditions, tumor control, and in infectious diseases.

Selenium addition to colostrum increases immunoglobulin G absorption by newborn calves.

The objective of this study was to show a new function of Se in IgG absorption from colostrum by newborn calves. The same amount and quality of colostrum with or without Se addition was fed to paired calves (n = 60) 4 times at <2, 12, 24, and 36 h after birth, respectively. Four-time feeding of colostrum containing 1.0 ppm Se significantly increased IgG amount in the blood plasma of calves 24 h after birth; however, its effect was small (about 20% increase). Although the addition of 3.0 ppm Se once at the first colostrum feeding was more effective on IgG absorption, its significant effect was a 42% increase on average. The increased IgG concentration of blood plasma continued for about 2 wk. It is known that the absorption of colostrum IgG is mediated by intestinal pinocytosis, which continues for only 24 h after birth. The addition of Se to colostrum might directly activate this physiological pinocytosis of intestinal epithelial cells because of the rapidity of the reaction. This effect is not nutritional but rather pharmacological. Supplemented Se also resulted in its increased concentration in blood plasma. Selenium is an essential mineral for animals; however, newborn calves are always deficient in Se at birth. Application of this method in calves would also provide an immediate supply of Se and might contribute to the development of the immune system of calves. This study showed that Se supplementation to colostrum increased IgG amount and Se concentration in blood plasma in newborn calves.

Association of alphaB-crystallin, a small heat shock protein, with actin: role in modulating actin filament dynamics in vivo.

Disruption of cytoskeletal assembly is one of the early effects of any stress that can ultimately lead to cell death. Stabilization of cytoskeletal assembly, therefore, is a critical event that regulates cell survival under stress. alphaB-crystallin, a small heat shock protein, has been shown to associate with cytoskeletal proteins under normal and stress conditions. Earlier reports suggest that alphaB-crystallin could prevent stress-induced aggregation of actin in vitro. However, the molecular mechanisms by which alphaB-crystallin stabilizes actin filaments in vivo are not known. Using the H9C2 rat cardiomyoblast cell line as a model system, we show that upon heat stress, alphaB-crystallin preferentially partitions from the soluble cytosolic fraction to the insoluble cytoskeletal protein-rich fraction. Confocal microscopic analysis shows that alphaB-crystallin associates with actin filaments during heat stress and the extent of association increases with time. Further, immunoprecipitation experiments show that alphaB-crystallin interacts directly with actin. Treatment of heat-stressed H9C2 cells with the actin depolymerzing agent, cytochalasin B, failed to disorganize actin. We show that this association of alphaB-crystallin with actin is dependent on its phosphorylation status, as treatment of cells with MAPK inhibitors SB202190 or PD98059 results in abrogation of this association. Our results indicate that alphaB-crystallin regulates actin filament dynamics in vivo and protects cells from stress-induced death. Further, our studies suggest that the association of alphaB-crystallin with actin helps maintenance of pinocytosis, a physiological function essential for survival of cells.

The antioxidative and immunostimulating properties of D-glucosamine.

The objective of the present study was to investigate the antioxidant activity and immunostimulating property of glucosamine (GlcN) using various in vitro and in vivo tests. Results showed that GlcN possessed excellent antioxidant activities as manifested by strong chelating effect on ferrous ions and protection of macromolecules such as protein, lipid, and deoxyribose from oxidative damage induced by hydroxyl radicals. The immunostimulating effects of GlcN were further evaluated through various immunological tests. GlcN showed excellent activity of enhancing splenocyte proliferation. Neutral red pinocytosis and NO production in mouse peritoneal macrophages were significantly augmented. Oral administration of GlcN to mice for 20 days significantly enhanced the serum antibody level in mice in response to sheep red blood cells (SRBC), increased the relative organ weight of spleen and thymus tissue, and promoted the delayed-type hypersensitivity (DTH) against SRBC as compared with control group. In conclusion, the present investigation reveals GlcN is biologically functional in antioxidative activities and immunostimulating properties.

Endocytosis of cell surface material mediates cell plate formation during plant cytokinesis.

Dividing plant cells perform a remarkable task of building a new cell wall within the cytoplasm in a few minutes. A long-standing paradigm claims that this primordial cell wall, known as the cell plate, is generated by delivery of newly synthesized material from Golgi apparatus-originated secretory vesicles. Here, we show that, in diverse plant species, cell surface material, including plasma membrane proteins, cell wall components, and exogenously applied endocytic tracers, is rapidly delivered to the forming cell plate. Importantly, this occurs even when de novo protein synthesis is blocked. In addition, cytokinesis-specific syntaxin KNOLLE as well as plasma membrane (PM) resident proteins localize to endosomes that fuse to initiate the cell plate. The rate of endocytosis is strongly enhanced during cell plate formation, and its genetic or pharmacological inhibition leads to cytokinesis defects. Our results reveal that endocytic delivery of cell surface material significantly contributes to cell plate formation during plant cytokinesis.

[Effect of borneol on the intercellular tight junction and pinocytosis vesicles in vitro blood-brain barrier model].

OBJECTIVE: To explore the mechanism of borneol in opening the blood-brain barrier (BBB). METHODS: Borneol contained serum was prepared and using Matin-Darby canine kidney epithelium (MDCKE) cell line as the in vitro BBB model to observe the effects of borneol on intercellular tight junction (ICTJ) and pinocytosis vesicles of BBB model. RESULTS: Borneol reduced the ICTJ and caused increase of the number and enlarged the diameter of vesicles. The ICTJ was opened firstly 4 hrs after borneol treatment, then the pinocytosis was affected 24 hrs later. The effects disappeared 24 hrs after removal of the borneol contained serum, indicating that the above-mentioned effects were reversible. CONCLUSION: Borneol could obviously loosen the ICTJ in BBB, accelerate the transportation of substance through the intercellular passage, it also could increase the number and volume of pinocytosis vesicles in BBB cells, thus to accelerate the transportation of substance by way of cell pinocytosis.

Dietary vitamin E and rainbow trout (Oncorhynchus mykiss) phagocyte functions: effect on gut and on head kidney leucocytes.

The effects of vitamin E (deficiency or supplementation) on the non-specific immune system in rainbow trout, Oncorhynchus mykiss, were evaluated. Rainbow trout were fed daily a semi-purified diet supplemented with vitamin E at 0, 28 and 295 mg x kg(-1) of diet. After 80 days of experimental feeding, the phagocytic function (respiratory burst evaluated by the CL response, phagocytosis) from gut leucocytes and head kidney enriched macrophages was measured; head kidney cell pinocytosis and serum lysozyme activity were also analysed. The results showed that some phagocyte functions were influenced by dietary vitamin E. When fish were fed the high dietary dose of vitamin E an enhancement of phagocytosis was found, but only significantly for the leucocytes isolated from the gut of rainbow trout; moreover, an impaired response was also observed in the fish fed no vitamin E for 80 days. However, no significant differences were noticed on the oxidative burst (CL) response of both gut and head kidney cells according to the dietary dose of vitamin E. Pinocytosis evaluated on head kidney cells was not influenced by dietary vitamin E. Fish fed vitamin E at 295 mg x kg(-1) had a lower serum lysozyme activity than those fed with vitamin E at 28 mg x kg(-1) and the fish fed no vitamin E for 80 days had an impaired activity. Thus, the present results demonstrate that altered dietary levels of vitamin E modulates the phagocytic functions of gut leucocytes in rainbow trout; moreover, the vitamin E diet effect seems to be greater on the local intestinal response as compared to systemic (head kidney). Taken together, this study confirms the crucial role of gut phagocytes in mucosal non-lymphoid defences in fish.

Immunomodulatory effect of a polysaccharide-enriched preparation of Codonopsis pilosula roots.

1. A polysaccharide-enriched fraction (CPPS) was prepared from Codonopsis pilosula root extract utilizing a procedure that entailed extraction with aqueous buffer and precipitation with ethanol. 2. After administration of CPPS in drinking water to C57BL/6 mice at a dosage of 10 mg/L for 4 weeks, the splenocytes exhibited lowered mitogenic responses to Concanavalin A (ConA) and lipopolysaccharide (LPS). The in vitro production of reactive nitrogen intermediates was inhibited. 3. However, when oral administration of CPPS was prolonged to 8 weeks, there was a potentiation of ConA-stimulated and LPS-stimulated mitogenic responses. 4. When tested under in vitro conditions, CPPS augmented the mitogenic response of splenocytes to ConA. However, there was no effect on the pinocytic activity of mouse macrophages, nor was there any proliferative activity on mouse melanoma B16 cells.