Phagocytosis Of Latex Beads Research Articles

Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes

The hemocytes of Octopus vulgaris were morphologically and functionally characterized. Light and electron microscopy (TEM and SEM), and flow cytometry analyses revealed the existence of two hemocyte populations. Large granulocytes showed U-shaped nucleus, a mean of 11.6μm±1.2 in diameter with basophilic granules, polysaccharide and lysosomic deposits in the cytoplasm. Small granulocytes measured a mean of 8.1μm±0.7 in diameter, and have a round nucleus occupying almost the entire cell and few or not granules in the cytoplasm. Flow cytometry analysis showed that large granulocytes are the principal cells that develop phagocytosis of latex beads (rising up to 56%) and ROS after zymosan stimulation. Zymosan induced the highest production of both ROS and NO. This study is the first tread towards understanding the O. vulgaris immune system by applying new tools to provide a most comprehensive morpho-functional study of their hemocytes.

Autologous Transplantation Of Thrombopoietin Derived-Hepatic Hematopoietic Progenitor Cells In Xenopus Laevis

Thrombopoietin (TPO) is required for megakaryopoiesis generating platelets via acting through its receptor c-Mpl, and supports hematopoietic stem cell maintenance. It is unknown whether there are evolutionary conserved signalling pathways and molecular functions governed by TPO/c-Mpl in non-vertebrates. We therefore attempt to develop a non-mammalian animal model in order to investigate thrombocyte development in adult African clawed frog (Xenopus laevis: X. laevis). We previously reported that Xenopus hepatic cells were differentiated to polyploid megakaryocyte-like cells and thrombocyte under X. laevis TPO (xlTPO) stimulation. The aim of this study is to determine the cellular characteristics of TPO-induced immature cells by establishing in vitro and in vivo Xenopus models.Hepatic cells were cultured in a 35mm diameter dish at 23°C under 5% CO2 with minimum essential medium containing 0.87% methylcellulose and 20% FCS in the presence of xlTPO (10 ng/ml). After 24 days of culture, the number of colonies reached 9.0±3.0 per dish. The size of colonies was heterogeneous, and a typical large colony was composed of more than 512 cells. Cells recovered from the large colonies at 24-day culture were then serially replated to a 96-well plate at a density of 1x103 cells/well with semi-solid medium in the presence of xlTPO (10 ng/ml) or X. laevis erythropoietin (xlEPO) (10 ng/ml). At day 8, a significant number of colonies (125±10/100μl culture well) were emerged in the xlTPO culture; but a few colonies in the xlEPO culture (3±1/100μl culture). The colony formation was inhibited by the addition of recombinant xlc-Mpl-FC fusion protein in the culture, indicating that the colony formation was directly stimulated by xlTPO. It was notable that xlTPO alone maintained and expanded the culture for over four months. The component cells in xlTPO-induced colonies resembled megakaryocytes or thrombocytic cells with spindle shapes, suggesting that xlTPO exerted the ability to support long-term maintenance and proliferation of thrombocytic progenitors. We then collected the cells after 24 days of liver cell culture in the presence of xlTPO. The RT-PCR revealed that the cells did not express hepatic markers of albumin and tpo; while they expressed not only mRNAs of thrombocytic markers such as c-mpl, fli-1, integrinαIIb, but also mRNAs of mpo, g-csfr, γ—globin , gata-1, esam and c-kit. The result proved that xlTPO expanded multilineage hematopoietic progenitors; not restricted to cells in the thrombocyte lineage. Subsequently we cultured cells collected from the day-24 colonies in conditioned medium of liver and spleen. After 8 days of culture with xlTPO, approximately 5% of the cells developed morphologies resembling mature eosinophils in the spleen-conditioned medium. As well, about 18% of the cells appeared to be macrophage-like cells, and they phagocytosed latex beads. Taken together, we concluded that xlTPO supported long-term maintenance of multipotent heamatopoietic cells in the Xenopus liver via TPO-c-Mpl signalling. Because inbred strains of X. laevis are not abundantly available, transplantation studies in X. laevis are limited. We therefore attempted to develop autologous cell transplantation of xlTPO-induced cells in X. laevis. Liver cells obtained by partial hepatic resection were cultured in the semi-solid culture system in the presence of xlTPO. After 24 days, the cells labeled with PKH26 were autologously transplanted by intracardiac injection. PKH26-positive cells were detected in the liver and the spleen of the auto-transplanted frogs after 12 days, though the cells were diminished from the body within 4 to 6 days in the allo-transplanted Xenopus. Flow cytometric analysis showed that PKH26-high cells contained much RNA and displayed lower forward scatter (FSC); while PKH 26-low cells were detected in the intermediate FSC. The findings suggested that a subpopulation of long-term maintained liver cells by xlTPO could barely proliferate and differentiate in the liver and the spleen, and the characteristics of these cells agrees with the features of hematopoietic stem cells or progenitors, as in mammals and zebrafish. This unique culture system developed in this study has clues to resolve the functional evolution of TPO-cMpl system shared by thrombocytic development and earlier hematopoietic progenitors. Disclosures:No relevant conflicts of interest to declare.

A quantitative method for measuring innate phagocytosis by human monocytes using real‐time flow cytometry

Phagocytosis is central to immunity however a rapid and standardized method is much needed for quantitative assessment of the phagocytic process. We describe a real-time flow cytometric method to quantitate the phagocytosis of fluorescent latex beads by human monocytes in serum-free conditions. Effects of buffer composition, temperature, pH, and bead surface on phagocytic rate are described. The innate phagocytic ability of human monocytes from single subjects measured by this method was relatively stable over many months although phagocytosis rate varied as much as two-fold between individuals. Comparable results were obtained with a simplified method using several mL of whole blood which is suitable for routine clinical application. This method also allows two-color flow cytometric measurement of cytosolic calcium levels during the phagocytic uptake of fluorescent beads.

Isolation and Identification of Active Compound of n-hexane Fraction from Kasturi (Mangifera casturi Konsterm.) against Antioxidant and Immunomodulatory Activity

: A research had been conducted to isolated, to identify and to test biological activity of active isolates from n-hexane extracts of casturi (Mangifera casturi Konsterm.) fruit. The research aims at isolating active isolates from n-hexane fractions and testing for antioxidant and immunomodulatory activities. n-hexane fractions were isolated using column chromatographic method by using n-hexane-ethylacetate as eluents with eluent gradient (20:1; 15:1; 10:1; 8:2; 7:3; 6:4 and 5:5) v/v. The isolated compounds (purified isolates) were white crystals identified as terpenoid compound. Identification of chemical structures found a compound, namely, 1-isopropenyl-3a, 5a, 5b, 8, 8, 11a-hexamethyl-eicosahydro-cyclo penta [α]chrysen-9-ol (lupeol). The isolated compound was tested for antioxidant activity using DPPH (1,1-diphenyl -2-picrylhydrazyl) method with an IC50 value of 14384.71-lower than that of quercetin control (IC50 = 2.962). The finding shows that the isolate is not a potent compound against DPPH free radical inhibition. In vitro immunomodulatory activity test was conducted for the isolated compound. The isolates were given at dosages of 6.25, 12.5, 25, 50 and 100 μg. The test revealed increasing macrophage activity against latex bead phagocytosis. The highest phagocytosis activity and capacity were found at a dosage of 100 μg, namely, latex-bead consuming macrophage by 26% and macrophage-eaten latex bead by 32% (significant, compared to the negative control, namely, latex-bead eating macrophage by 13% and macrophage-eaten latex bead by 15%). Keywords : Mangifera casturi Kosterm, n-hexane fraction, lupeol, antioxidant, immuonomodulatory

Interplay between human microglia and neural stem/progenitor cells in an allogeneic co‐culture model

Experimental neural cell therapies, including donor neural stem/progenitor cells (NPCs) have been reported to offer beneficial effects on the recovery after an injury and to counteract inflammatory and degenerative processes in the central nervous system (CNS). The interplay between donor neural cells and the host CNS still to a large degree remains unclear, in particular in human allogeneic conditions. Here, we focused our studies on the interaction of human NPCs and microglia utilizing a co-culture model. In co-cultures, both NPCs and microglia showed increased survival and proliferation compared with mono-cultures. In the presence of microglia, a larger subpopulation of NPCs expressed the progenitor cell marker nestin, whereas a smaller group of NPCs expressed the neural markers polysialylated neural cell adhesion molecule, A2B5 and glial fibrillary acidic protein compared with NPC mono-cultures. Microglia thus hindered differentiation of NPCs. The presence of human NPCs increased microglial phagocytosis of latex beads. Furthermore, we observed that the expression of CD200 molecules on NPCs and the CD200 receptor protein on microglia was enhanced in co-cultures, whereas the release of transforming growth factor-β was increased suggesting anti-inflammatory features of the co-cultures. To conclude, the interplay between human allogeneic NPCs and microglia, significantly affected their respective proliferation and phenotype. Neural cell therapy including human donor NPCs may in addition to offering cell replacement, modulate host microglial phenotypes and functions to benefit neuroprotection and repair.

CD14-Dependent Monocyte Isolation Enhances Phagocytosis of Listeria monocytogenes by Proinflammatory, GM-CSF-Derived Macrophages

Macrophages are an important line of defence against invading pathogens. Human macrophages derived by different methods were tested for their suitability as models to investigate Listeria monocytogenes (Lm) infection and compared to macrophage-like THP-1 cells. Human primary monocytes were isolated by either positive or negative immunomagnetic selection and differentiated in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF) into pro- or anti-inflammatory macrophages, respectively. Regardless of the isolation method, GM-CSF-derived macrophages (GM-Mφ) stained positive for CD206 and M-CSF-derived macrophages (M-Mφ) for CD163. THP-1 cells did not express CD206 or CD163 following incubation with PMA, M- or GM-CSF alone or in combination. Upon infection with Lm, all primary macrophages showed good survival at high multiplicities of infection whereas viability of THP-1 was severely reduced even at lower bacterial numbers. M-Mφ generally showed high phagocytosis of Lm. Strikingly, phagocytosis of Lm by GM-Mφ was markedly influenced by the method used for isolation of monocytes. GM-Mφ derived from negatively isolated monocytes showed low phagocytosis of Lm whereas GM-Mφ generated from positively selected monocytes displayed high phagocytosis of Lm. Moreover, incubation with CD14 antibody was sufficient to enhance phagocytosis of Lm by GM-Mφ generated from negatively isolated monocytes. By contrast, non-specific phagocytosis of latex beads by GM-Mφ was not influenced by treatment with CD14 antibody. Furthermore, phagocytosis of Lactococcus lactis, Escherichia coli, human cytomegalovirus and the protozoan parasite Leishmania major by GM-Mφ was not enhanced upon treatment with CD14 antibody indicating that this effect is specific for Lm. Based on these observations, we propose macrophages derived by ex vivo differentiation of negatively selected human primary monocytes as the most suitable model to study Lm infection of macrophages.

OP0056 12/15-lipoxygenase mediates GC-induced anti-inflammatory mechanisms in monocytes

BackgroundGlucocorticoids (GC) are used for the treatment of many chronic inflammatory diseases. We have previously shown that GC treatment does not simply suppress monocyte functions but rather induces a distinct...

Effect of okadaic acid on carpet shell clam (Ruditapes decussatus) haemocytes by in vitro exposure and harmful algal bloom simulation assays

Okadaic acid (OA), produced by dinoflagellates during harmful algal blooms (HAB), belongs to the Diarrheic Shellfish Poisoning toxins that cause gastrointestinal symptoms in humans after consumption. In the present work, Ruditapes decussatus haemocytes were selected to evaluate the effect of OA on cell viability, enzymatic status and immune capacity through the measure by flow cytometry of apoptosis-cell death, non-specific esterase activity and phagocytosis. In order to compare different exposure conditions, two experiments were developed: in vitro exposure to OA and HAB simulation by feeding clams with the OA producer, Prorocentrum lima. Apoptosis was not OA dose-dependent and cell death increased in both assays. Phagocytosis of latex beads and esterase activity decreased in haemocytes incubated with OA. In contrast, esterases increased during the feeding with P. lima. Our results showed that OA and the simulated HAB caused damages on haemocyte functions and viability.

Alterations in the secretory pattern of dermal dendritic cells following melanin uptake

Under a variety of circumstances, melanin occurs in the dermal compartment of the skin, being mostly observed in cells that have been termed melanophages, some of which have been identified as dermal dendritic cells. We analysed changes in the expression and secretion pattern of cytokines by dendritic cells after the uptake of melanin from various sources. Dendritic cells were derived from human primary blood monocytes or from the human monocytic cell line THP-1. Melanin uptake increased the secretion of the chemokines MIP-1β (CCL4) and MCP-1 (CCL2). The higher MIP-1β secretion was accompanied by higher MIP-1β gene expression. Elevation of MIP-1β secretion was dependent on the uptake of melanin but could not be induced by the phagocytosis of latex beads, indicating that the phagocytic process itself was not sufficient to increase the secretion of this cytokine. The data thus show that the uptake of melanin changes the cytokine expression and secretion pattern of dendritic-like cells.

Amyloid-β-Induced Astrocytic Phagocytosis is Mediated by CD36, CD47 and RAGE

Astrocytes, the most numerous glial cell in the brain, have multiple functions and are key to maintenance of homeostasis in the central nervous system. Microglia are the resident immunocompetent cells in the brain and share several functions with macrophages, including their phagocytic ability. Indeed microglia are the resident phagocytes in the brain and express numerous cell surface proteins which act to enable receptor-mediated phagocytosis. However recent evidence suggests that astrocytes express some genes which permit phagocytosis of phosphatidylserine-decorated cells and this probably explains sporadic reports in the literature which suggest that astrocytes become phagocytic following brain trauma. Here we examined the potential of astrocytes to phagocytose fluorescently-labelled latex beads and amyloid-β (Aβ) and report that they competently engulf both in a manner that relies on actin polymerization since it was inhibited by cytochalasin D. The data indicate that incubation of cultured astrocytes or microglia with Aβ increased phagocytosis and markers of activation of both cell types. Aβ was found to markedly increase expression of the putative Aβ-binding receptors CD36 and CD47 in astrocytes, while it decreased expression of the receptor for advanced glycation endproducts (RAGE). It is demonstrated that blocking these receptors using a neutralizing antibody attenuated Aβ-induced phagocytosis of latex beads by astrocytes. Interestingly blocking these receptors also decreased uptake of beads even in the absence of Aβ. Here we demonstrate that astrocytes are competent phagocytes and are capable of engulfing Aβ.

Indirubin-3′-(2,3 dihydroxypropyl)-oximether (E804) is a potent modulator of LPS-stimulated macrophage functions

Indirubin is a deep-red bis-indole isomer of indigo blue, both of which are biologically active ingredients in Danggui Longhui Wan, an ancient Chinese herbal tea mixture used to treat neoplasia and chronic inflammation and to enhance detoxification of xenobiotics. Multiple indirubin derivatives have been synthesized and shown to inhibit cyclin-dependent kinases (CDKs) and glycogen-synthase kinase (GSK-3β) with varying degrees of potency. Several indirubins are also aryl hydrocarbon receptor (AhR) agonists, with AhR-associated activities covering a wide range of potencies, depending on molecular structure. This study examined the effects of indirubin-3′-(2,3 dihydroxypropyl)-oximether (E804), a novel indirubin with potent STAT3 inhibitory properties, on basal and LPS-inducible activities in murine RAW264.7 macrophages. Using a focused commercial qRT-PCR array platform (SuperArray®), the effects of E804 on expression of a suite of genes associated with stress and toxicity were determined. Most genes up-regulated by LPS treatment were suppressed by E804; including LPS-induced expression of pro-inflammatory cytokines and receptors, apoptosis control genes, and oxidative stress response genes. Using qRT-PCR as a follow up to the commercial arrays, E804 treatment suppressed LPS-induced COX-2, iNOS, IL-6 and IL-10 gene expression, though the effects on iNOS and COX-2 protein expression were less dramatic. E804 also inhibited LPS-induced secretion of IL-6 and IL-10. Functional endpoints, including iNOS and lysozyme enzymatic activity, phagocytosis of fluorescent latex beads, and intracellular killing of bacteria, were also examined, and in each experimental condition E804 suppressed activities. Collectively, these results indicate that E804 is a potent modulator of pro-inflammatory profiles in LPS-treated macrophages.

Interaction between intestinal dendritic cells and bacteria translocated from the gut in rats with cirrhosis

Cirrhosis with ascites is associated with a high rate of gut bacterial translocation (GBT) and spontaneous bacterial infections of enteric origin. We addressed the activation state and role of intestinal dendritic cells (DCs) in experimental ascitic cirrhosis and their relationship with GBT. Cirrhosis with ascites was CCl(4) induced in rats. To examine their activation state and functions, DCs (CD103(+) RT1B(+) CD3(-) CD45RA(-) ) were isolated from the intestinal lamina propria and mesenteric lymph nodes (MLNs), and the following parameters were determined by flow cytometry: surface antigen expression; spontaneous or lipopolysaccharide-stimulated tumor necrosis factor alpha (TNF-α) production; and in vitro capacity to phagocytose latex beads and to migrate toward the chemokine (C-C motif) ligand 21. GBT was defined as the growth of bacteria in MLNs culture. Bacterial DNA (Bact-DNA) in MLNs was identified by polymerase chain reaction. In rats with Bact-DNA in MLNs without GBT, intestinal and MLNs CD103(+) -DCs showed features of activation, expansion of the proinflammatory CD4(+) -DC subpopulation, augmented TNF-α production, and increased phagocytic and migratory capacities. In contrast, in rats with GBT, CD103(+) -DCs showed the absence of an activated phenotype, lowered TNF-α production, and relatively deficient phagocytosis and migration capacities. The CD103(+) -DC of rats without Bact-DNA in MLNs or GBT were similar to controls. In cirrhotic rats, bowel decontamination with antibiotics eliminated Bact-DNA in MLNs and GBT, normalized the activation state and functions of CD103(+) -DCs, and increased their TNF-α production. In experimental cirrhosis with ascites, continuous pressure of gut bacteria shapes the phenotypic and functional profile of intestinal DCs to produce effects that range from their activation and enhanced functions to their exhaustion and tolerance.

Validation of Aedes aegypti Aag-2 cells as a model for insect immune studies

BackgroundThe understanding of mosquito immune responses can provide valuable tools for development of novel mosquito control strategies. Aiming the study at insect innate immunity, continuous insect cell lines have been established and used as research tools due to the fact that they constitute more homogeneous, sensitive, and reproducible systems than the insects from which they originated. More recently, Aag-2, an Aedes aegypti cell lineage, began to be frequently used as a model for studies of mosquito immunity. Nevertheless, to our knowledge, no study has systematically characterized the responses of Aag-2 cell line against different kinds of pathogens and compared its response to those exhibited by whole mosquitoes. For this reason, in this study we characterized gene expression profiles of the Aag-2 cell line in response to different kinds of immune challenges, such as Gram negative and positive bacteria, fungi and viruses, comparing the obtained results with the ones already described in the literature for whole mosquitoes.MethodsAedes aegypti Aag-2 cells were exposed to different immune stimuli (gram-positive and gram negative heat inactivated bacteria, zymosan or Sindbis virus) for 24 hours and the expression of selected marker genes from toll, IMD and Jak/STAT pathways was analyzed by qPCR. Also, cells were incubated with fluorescent latex beads for evaluation of its phagocytosis capacity.ResultsAag-2 cells were stimulated with two concentrations of heat-killed Gram negative (Enterobacter cloacae) or Gram positive (Micrococcus luteus) bacteria, Zymosan or infected with Sindbis virus and the expression of key genes from the main immune related pathways, Toll, IMD and Jak/STAT, were investigated. Our results suggest that Toll and IMD pathways are activated in response to both Gram positive and negative bacteria and Zymosan in Aag-2 cells, displaying an immune profile similar to those described in the literature for whole mosquitoes. The same stimuli were also capable of activating Jak/STAT pathway in Aag-2 cells. Infection with Sindbis virus led to an up-regulation of the transcription factor STAT but was not able to induce the expression of any other gene from any of the pathways assayed. We also showed that this cell line is able to phagocytose latex beads in culture.ConclusionsOur results characterize the expression profile of Aag-2 cells in response to different immune stimuli and demonstrate that this cell lineage is immune-competent and closely resembles the response described for whole Ae. aegypti mosquitoes. Hence, our findings support the use of Aag-2 as a tool to comprehend Ae. aegypti immune response both at cellular and humoral levels.

NADPH oxidase is involved in H2O2‐induced differentiation of human promyelocytic leukaemia HL‐60 cells

The expression and activity of NADPH oxidase increase when HL-60 cells are induced into terminally differentiated cells. However, the function of NADPH oxidase in differentiation is not well elucidated. With 150-500 μM H2O2 inducing differentiation of HL-60 cells, we measured phagocytosis of latex beads and investigated cell electrophoresis. Two inhibitors of NADPH oxidase, DPI (diphenyleneiodonium) and APO (apocynin), blocked the differentiation potential of cells induced by 200 μM H2O2. However, H2O2 stimulated the generation of intracellular superoxide (O2•-), which decreased in the presence of the two inhibitors. DPI also inhibited H2O2-induced ERK (extracellular-signal-regulated kinase) activation, as detected by Western blotting. Furthermore, PD98059, the inhibitor of the ERK pathway, inhibited the differentiation of HL-60 cells induced by H2O2. This shows that H2O2 can activate NADPH oxidase, leading to O2•- production, followed by ERK activation and ultimately resulting in the differentiation of HL-60 cells. The data indicate that NADPH oxidase is an important cell signal regulating cell differentiation.

Pivotal Advance: Peritoneal cavity B-1 B cells have phagocytic and microbicidal capacities and present phagocytosed antigen to CD4+ T cells

Breaking the long-held paradigm that primary B cells are not phagocytic, several studies have demonstrated recently that B cells from fish, amphibians, and reptilians have a significant phagocytic capacity. Whether such capacity has remained conserved in certain mammalian B cell subsets is presently an enigma. Here, we report a previously unrecognized ability of PerC B-1a and B-1b lymphocytes to phagocytose latex beads and bacteria. In contrast, B-2 lymphocytes had an almost negligible ability to internalize these particles. Upon phagocytosis, B-1a and B-1b cells were able to mature their phagosomes into phagolysosomes and displayed the ability to kill internalized bacteria. Importantly, B-1a and B-1b cells effectively present antigen recovered from phagocytosed particles to CD4(+) T cells. However, these cells showed a much lower competence to present soluble antigen or antigen from large, noninternalized particles. B-1 B cells presented particulate and soluble antigen to CD4(+) T cells more efficiently than macrophages, whereas DCs were the most potent APCs. The novel phagocytic and microbicidal abilities identified in B-1 B lymphocytes strengthen the innate nature that has long been attributed to these cells. In the context of adaptive immunity, we show that these innate immune processes are relevant, as they enable B-1 B cells to present phagocytosable particulate antigen. These capacities position these cells at the crossroads that link innate with adaptive immune processes. In a broader context, these newly identified capacities of B-1 B cells further support the previously recognized functional, developmental, and evolutionary relationships between these cells and macrophages.

A new organotypic model containing dermal-type macrophages

Human skin equivalents (SEs) are popular three-dimensional (D) cell culture systems in fundamental and applied dermatology. They have been made to contain dendritic cells, but so far no study on the incorporation of potentially anti-inflammatory dermal macrophages has been performed. Here, we show that monocyte-derived dermal-type macrophages can be introduced into a rigid scaffold with dermal fibroblasts. They maintain their cell surface markers CD163, DC-SIGN/CD209 and HLA-DR, which discriminate them from monocytes and dendritic cells. They retain the ability to produce the anti-inflammatory cytokine IL-10 in response to lipopolysaccharide (LPS) and to phagocytose latex beads. We thus demonstrate the feasibility of creating macrophage-fibroblast 3D cultures as a first step towards generating SEs with dermal macrophages.

Green tea extract ameliorates reperfusion injury to rat livers after warm ischemia in a dose‐dependent manner

Polyphenolic constituents of green tea (Camellia sinensis) have been shown to be potent scavengers of reactive oxygen species (ROS). Thus, this study was designed to assess its effects after liver ischemia-reperfusion. Fasted Sprague-Dawley rats were gavaged with different concentrations of green tea extract (GTE) 2 h before 90 min of warm ischemia of the left lateral liver lobe (30% of liver). Controls were given the same volume of Ringer's solution. A preparation of pentobarbital sodium (intraperitoneal) and ketamine (intramuscular) was used for anesthesia. After reperfusion, transaminases, liver histology, hepatic microcirculation, and both phagocytosis of latex bead particles as well as the expression of tumor necrosis alpha (TNF-α) to index cellular activation were investigated. Furthermore, the expression of superoxide dismutase (Mn-SOD) was assessed. After 90 min of warm ischemia aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) increased dramatically to 1946 ± 272/3244 ± 757 U/L, 1680 ± 134/2080 ± 379 U/L, and 7857 ± 1851/2036 ± 1193 U/L at 2/6 h, respectively. GTE (200 mg/kgbody weight) significantly prevented this increase in a dose-dependent manner by 21-51% at 2 h and 29-34% at 6 h, respectively. Histology confirmed the protective effects while both TNF-α expression and phagocytosis of latex beads by Kupffer cells (KCs) were significantly reduced. GTE intake significantly increased the expression of manganese superoxide dismutase. In vivo microscopy revealed improved acinar and sinusoidal perfusion after GTE. Preconditioning with a single oral dose of GTE ameliorates ischemia-reperfusion injury in liver. Decreased cellular activation and improved microcirculation are the proposed mechanisms.

Glycine and Taurine Equally Prevent Fatty Livers from Kupffer Cell‐Dependent Injury: An In Vivo Microscopy Study

IRI still is a major problem in liver surgery due to warm ischemia and organ manipulation. Steatosis is not only induced by diabetes, hyperalimentation, alcohol and toxins, but also chemotherapy given before resection. Since steatotic livers are prone to Kupffer cell-dependent IRI, protection of steatotic livers is of special interest. This study was designed to compare the effect of taurine and glycine on IRI in steatotic livers. Steatosis was induced with ethanol (7 g/kg b.w.; p.o.) in female SD rats. Ten minutes after inactivation of Kupffer cells with taurine or glycine (300 mM; i.v.), left liver lobes underwent 60 minutes of warm ischemia. Controls received the same volume of valine (300 mM; i.v.) or normal saline. After reperfusion, white blood cell-endothelial interactions and latex-bead phagocytosis by Kupffer cells were investigated. Liver enzymes were measured to estimate injury. For statistical analysis, ANOVA and Student's t-test were used. Glycine and taurine significantly decreased leukocyte- and platelet-endothelium interactions and latex-bead phagocytosis (p < 0.05). Liver enzymes were significantly lower after glycine and taurine (p < 0.05). This study shows that preconditioning with taurine or glycine is equally effective in preventing injury to fatty livers most likely via Kupffer cell-dependent mechanisms.

Commercial naphthenic acids and the organic fraction of oil sands process water downregulate pro-inflammatory gene expression and macrophage antimicrobial responses

This is the first report showing that the organic fraction of oil sands process water (OSPW-OF), and commercial naphthenic acids (C-NAs), cause immunotoxicity. The exposure of mouse bone marrow-derived macrophages (BMDM) to different amounts of C-NAs or OSPW-OF, did not affect cell viability in vitro. We examined whether exposure of BMDM to C-NAs or OSPW-OF affected various antimicrobial responses of these cells. A dose-dependent decrease in nitric oxide response was observed after treatment of BMDM with OSPW-OF, but not with C-NAs. Although OSPW-OF and C-NAs both down-regulated the respiratory burst response of BMDM, the suppression of the production of reactive oxygen intermediates was more pronounced in cells treated with OSPW-OF. Treatment with OSPW-OF or C-NAs reduced BMDM phagocytosis of zymosan and latex beads. The decrease of BMDM antimicrobial response after exposure to OSPW-OF or C-NAs, was accompanied by decreased pro-inflammatory cytokine gene expression. Oral exposure of mice to OSPW-OF caused down-regulation in the expression of genes encoding pro-inflammatory cytokines IFNγ, IL-1β and CSF-1. Our findings indicated that OSPW causes immunotoxic effects that may impair the ability of an exposed host to defend against infectious disease. Furthermore, given the differences between the effects of OSPW-OF and C-NAs, C-NAs should not be assumed to be a direct surrogate for the immunotoxic chemical species in OSPW.

PMS‐1077, a PAF antagonist, induced differentiation of HL‐60 cells with its novel activity

The cell differentiation-inducing effect of 2-N,N-diethylaminocarbonyloxymethyl-1-diphenylmethyl-4-(3,4,5-trimethoxybenzoyl) piperazine, hydrochloride (PMS-1077) was determined in human leukaemic HL-60 cells with profiling of cell proliferation, analysis of cell cycling, characterization of expression of various CD molecules and determination of phagocytotic activity of differentiated HL-60 cells. After treatment with PMS-1077, HL-60 cells exhibited a decreased cell viability during which cell cycle was arrested in G₀-/G₁-phase. Flow cytometric analysis showed CD11b and CD14 were up-regulated, whereas CD15 was unaffected. Together with the finding that PMS-1077-treated HL-60 cells exhibited activities of differentiation by examining their ability of phagocytosing latex beads, an antiproliferative effect and a differentiation-inducing role were determined for PMS-1077 in HL-60 cells.