Characterization of the zebrafish matrix metalloproteinase 9 gene and its developmental expression pattern

Members of the matrix metalloproteinase (MMP) family are important for the remodeling of the extracellular matrix in a number of biological processes including a variety of immune responses. Two members of the family, MMP2 and MMP9, are highly expressed in specific myeloid cell populations in which they play a role in the innate immune response. To further expand the repertoire of molecular reagents available to study zebrafish myeloid cell development, the matrix metalloproteinase 9 (mmp9) gene from this organism has been identified and characterized. The encoded protein is 680 amino acids with high homology to known MMP9 proteins, particularly those of other teleost fish. Maternal transcripts of mmp9 are deposited in the oocyte and dispersed throughout the early embryo. These are replaced by specific zygotic transcripts in the notochord from 12 h post fertilization (hpf) and also transiently in the anterior mesoderm from 14 to 16 h post fertilization. From 24 h post fertilization, mmp9 expression was detected in a population of circulating white blood cells that are distinct from macrophages, and which migrate to the site of trauma, and so likely represent zebrafish heterophils. In the adult, mmp9 expression was most prominent in the splenic cords, a site occupied by mature myeloid cells in other species. These results suggest that mmp9 will serve as a useful marker of mature myeloid cells in the zebrafish.

SEPS1 protects RAW264.7 cells from pharmacological ER stress agent-induced apoptosis

Selenoprotein S (SEPS1) is a novel endoplasmic reticulum (ER) resident protein and it is known to play an important role in production of inflammatory cytokines. Here, we show evidence that SEPS1 is stimulated by pharmacological ER stress agents in RAW264.7 macrophages as well as other cell types. Overexpression studies reveal a protective action of SEPS1 in macrophages against ER stress-induced cytotoxicity and apoptosis, resulting in promoting cell survival during ER stress. The protective action of SEPS1 is largely dependent on ER stress-mediated cell death signal with less effect on non-ER stress component cell death signals. Conversely, suppression of SEPS1 in macrophages results in sensitization of cells to ER stress-induced cell death. These findings suggest that SEPS1 could be a new ER stress-dependent survival factor that protects macrophage against ER stress-induced cellular dysfunction.

Antitumor activity and underlying mechamisms of ganopoly, the refined polysaccharides extracted from ganodrema lucidum, in mice

Ganopoly is an aqueous polysaccharide fraction extracted from G. lucidum by patented biochemical technique and has been marketed as an over-the-counter product for chronic diseases including cancer and hepatopathy in many Asian countries. This study was undertaken to explore the anti-tumour effect and the underlying mechanisms of Ganopoly in mice and human tumor cell lines. The maximum tolerateddose (MTD) of Ganopoly in mice was estimated to be 100 mg/kg from a pilot study. Treatment of mice with oral Ganopoly for 10 days significantly reduced the tumour weight of sarcoma-180 in a dose-dependent manner, with inhibition rates of 32.3, 48.2 and 84.9% and growth delays of 1.5, 3.5, and 13.1 days at 20, 50, and 100 mg/kg, respectively. Incubation of Ganopoly at 0.05-1.0 mg/ml for 48 hours showed little or negligible cytotoxicity against human tumor CaSki, SiHa, Hep3B, HepG2, HCT116, HT29, and MCF7 cells in vitro. In contrast, 10 mg/ml of Ganopoly caused significant cytotoxicity in all tumour cells tested except MCF7, with marked apoptotic effects observed in CaSki, HepG2, and HCT116 cells, as indicated by nuclear staining and DNA fragmentation. In addition, Ganopoly enhanced concanavalin A-stimulated proliferation of murine splenocytes by 35.3% at 10 mg/ml, and stimulated the production of nitric oxide in thioglycollate-primed murine peritoneal macrophages in a concentration-dependent manner over 0.05-10 mg/ml. Addition of Ganopoly at 1 mg/ml to murine peritoneal macrophages also potentiated lipopolysaccharide-induced nitric oxide production by 64.2%. Treatment of healthy mice or mice bearing sarsoma-180 with oral Ganopoly over 20-100 mg/kg for 7 day significantly increased the expression of both TNF-α and IFN-γ (at both mRNA and protein levels) in splenocytes in a dose-dependent manner. Moreover, treatment of Ganopoly over 20-100 mg/kg significantly increased cytotoxic T lymphocyte cytotoxicity and NK activity in mice. The overall findings indicated that Ganopoly had antitumor activity with a broad spectrum of immuno-modulating activities and may represent a novel promising immunotherapeutic agent in cancer treatment.

Expression of natriuretic peptide receptor mRNA and functional response to atrial natriuretic peptide and C-type natriuretic peptide in rainbow trout (Oncorhynchus mykiss) head kidney leucocytes

The stimulatory effect of vasomodulatory natriuretic peptide hormones on macrophages and peripheral blood leucocytes in mammals is well-established. However, the relationship in lower vertebrates has not been characterised. Expression of atrial natriuretic peptide, ventricular natriuretic peptide and C-type natriuretic peptide-1, and the guanylyl cyclase-linked (GC) natriuretic peptide receptor-A and -B-type receptors (NPR-A and NPR-B, respectively) was determined by PCR from the mRNA of rainbow trout head kidney leucocytes yielding gene fragments with 100% homology to the same respective natriuretic peptide and NPR-A and -B sequences obtained from other rainbow trout tissues. A mixed population of isolated rainbow trout head kidney leucocytes was stimulated in vitro with trout atrial natriuretic peptide (specific NPR-A agonist) and trout C-type natriuretic peptide (NPR-A and -B agonist) as well as the cGMP agonist 8-bromo-cGMP or the GC inhibitor 8-bromo-phenyl-eutheno-cGMP. Respiratory burst was stimulated by trout atrial natriuretic peptide, trout C-type natriuretic peptide-1 and 8-bromo-cGMP in a dose dependant manner with the highest activity as a result of stimulation with trout C-type natriuretic peptide-1 in excess of that achieved by phorbol myristate acetate (PMA). Equimolar concentrations of the inhibitor, inhibited the respiratory burst caused by the natriuretic peptides and 8-bromo-cGMP. The natriuretic peptide receptors on rainbow trout head kidney leucocytes appear to have a stimulatory function with regard to respiratory burst that is activated through a cGMP second messenger pathway and the natriuretic peptides expressed in the head kidney leucocytes may well act in a paracrine/autocrine manner.

Immunostimulatory principles from Chlorella pyrenoidosa – part 1 : isolation and biological assessment in vitro

Our proprietary preparation obtained by extraction of Chlorella pyrenoidosa cells, ONC-107 (Respondin™), was recently found to selectively boost antibody response to the influenza vaccine in a human clinical trial. Respondin™ is a potent stimulator of mouse B cell proliferation and an activator of macrophages. Bioactivity-guided resolution concluded that Respondin™ is composed of a mixture of immunostimulatory principles of different chemical nature. A combination of size exclusion, anion exchange and hydrophobic interaction chromatography revealed that the bulk of the immunostimulatory activity resides in polysaccharide/protein complexes with molecular masses larger than 100 kDa that are composed primarily of galactose, rhamnose and arabinose.

Cellular adaption to repeated eccentric exercise-induced muscle damage

Eccentrically biased exercise results in skeletal muscle damage and stimulates adaptations in muscle, whereby indexes of damage are attenuated when the exercise is repeated. We hypothesized that changes in ultrastructural damage, inflammatory cell infiltration, and markers of proteolysis in skeletal muscle would come about as a result of repeated eccentric exercise and that gender may affect this adaptive response. Untrained male (n = 8) and female (n = 8) subjects performed two bouts (bout 1 and bout 2), separated by 5.5 wk, of 36 repetitions of unilateral, eccentric leg press and 100 repetitions of unilateral, eccentric knee extension exercises (at 120% of their concentric single repetition maximum), the subjects' contralateral nonexercised leg served as a control (rest). Biopsies were taken from the vastus lateralis from each leg 24 h postexercise. After bout 2, the postexercise force deficit and the rise in serum creatine kinase (CK) activity were attenuated. Women had lower serum CK activity compared with men at all times (P < 0.05), but there were no gender differences in the relative magnitude of the force deficit. Muscle Z-disk streaming, quantified by using light microscopy, was elevated vs. rest only after bout 1 (P < 0.05), with no gender difference. Muscle neutrophil counts were significantly greater in women 24 h after bout 2 vs. rest and bout 1 (P < 0.05) but were unchanged in men. Muscle macrophages were elevated in men and women after bout 1 andbout 2 (P < 0.05). Muscle protein content of the regulatory calpain subunit remained unchanged whereas ubiquitin-conjugated protein content was increased after both bouts (P < 0.05), with a greater increase after bout 2. We conclude that adaptations to eccentric exercise are associated with attenuated serum CK activity and, potentially, an increase in the activity of the ubiquitin proteosome proteolytic pathway.

Gut health immunomodulatory and anti-inflammatory functions of gut enzyme digested high protein micro-nutrient dietary supplement-Enprocal

Background: Enprocal is a high-protein micro-nutrient rich formulated supplementary food designed to meet the nutritional needs of the frail elderly and be delivered to them in every day foods. We studied the potential of Enprocal to improve gut and immune health using simple and robust bioassays for gut cell proliferation, intestinal integrity/permeability, immunomodulatory, anti-inflammatory and anti-oxidative activities. Effects of Enprocal were compared with whey protein concentrate 80 (WPC), heat treated skim milk powder, and other commercially available milk derived products.

Results: Enprocal (undigested) and digested (Enprocal D) selectively enhanced cell proliferation in normal human intestinal epithelial cells (FHs74-Int) and showed no cytotoxicity. In a dose dependent manner Enprocal induced cell death in Caco-2 cells (human colon adencarcinoma epithelial cells). Digested Enprocal (Enprocal D: gut enzyme cocktail treated) maintained the intestinal integrity in transepithelial resistance (TEER) assay, increased the permeability of horseradish peroxidase (HRP) and did not induce oxidative stress to the gut epithelial cells. Enprocal D upregulated the surface expression of co-stimulatory (CD40, CD86, CD80), MHC I and MHC II molecules on PMA differentiated THP-1 macrophages in coculture transwell model, and inhibited the monocyte/lymphocyte (THP-1/Jurkat E6-1 cells)-epithelial cell adhesion. In cytokine secretion analyses, Enprocal D down-regulated the secretion of proinflammatory cytokines (IL-1β and TNF-α) and up-regulated IFN-γ, IL-2 and IL-10.

Conclusion: Our results indicate that Enprocal creates neither oxidative injury nor cytotoxicity, stimulates normal gut cell proliferation, up regulates immune cell activation markers and may aid in the production of antibodies. Furthermore, through downregulation of proinflammatory cytokines, Enprocal appears to be beneficial in reducing the effects of chronic gut inflammatory diseases such as inflammatory bowel disease (IBD). Stimulation of normal human fetal intestinal cell proliferation without cell cytotoxicity indicates it may also be given as infant food particularly for premature babies.

Ascophyllum compositions and methods

The invention provides extracts of brown seaweed of the genus Ascophyllum that have valuable pharmacological properties. In particular, compositions of the invention are useful for e.g.: inhibiting alpha-glucosidase activity; preventing or treating conditions mediated by alpha-glucosidase activity; reducing blood glucose levels; preventing or treating diabetes; modulating glucose uptake in adipocytes; preventing or treating obesity; scavenging free radicals; stimulating the immune system; activating macrophages; preventing or treating condition mediated by macrophage activation; and modulating nitric oxide production by macrophages. Methods for using the Ascophyllum extracts of the invention are provided, as are kits comprising Ascophyllum extracts of the invention and instructions for using the extracts.

Temporal expression of heat shock proteins 60 and 70 at lesion-prone sites during atherogenesis in ApoE-deficient mice

In the study, we investigate whether the expressions of heat shock protein (hsp)60 (a potential autoantigen) and the stress-inducible form of cytoprotector hsp70 are correlated with the development of atherosclerotic lesions in the aortic tree of apolipoprotein E–deficient (apoE^-/-) mice. The apoE^-/- mouse model is advantageous because the stress-inducible form of hsp70 is not constitutively expressed in mice, unlike primates; hence, tissues under stress can be clearly defined. Both mammalian hsps were detected newly expressed (before mononuclear cell infiltration) on aortic valves and endothelia at lesion-prone sites of 3-week-old apoE^-/- mice. In 8- and 20-week-old mice, they were strongly and heterogeneously expressed in early to advanced fibrofatty plaques, with levels correlating with lesion severity. Expression was markedly downregulated in advanced collagenous, acellular, calcified plaques of 40- and 69-week-old mice and was absent in control aortas of normocholesterolemic wild-type (apoE^+/+) mice. Western blot analysis of tissue homogenates confirmed the temporal expression of the hsps. Double immunostaining revealed that both hsps were expressed by lesional endothelial cells, macrophages, smooth muscle cells, and CD3⁺ T lymphocytes. This study provides evidence that hsp60 and hsp70 are temporally expressed on all major cell types in lesion-prone sites during atherogenesis, suggesting that few cells escape the toxic environment of the atherosclerotic plaque.

Anti-inflammatory immunotherapy for multiple sclerosis/experimental autoimmune encephalomyelitis (EAE) disease

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are inflammatory diseases of the central nervous system (CNS) characterized by localized areas with demyelination. Disease is believed to be an autoimmune disorder mediated by activated immune cells such as T- and B-lymphocytes and macrophages/microglia. Lymphocytes are primed in the peripheral tissues by antigens, and clonally expanded cells infiltrate the CNS. They produce large amounts of inflammatory cytokines, nitric oxide (NO) that lead to demyelination and axonal degeneration. Although several studies have shown that oligodendrocytes (OLGs), the myelin-forming glial cells in the CNS, are sensitive to cell death stimuli, such as cytotoxic cytokines, anti-myelin antibodies, NO, and oxidative stress, in vitro, the mechanisms underlying injury to the OLGs in MS/EAE remain unclear. The central role of glutamate receptors in mediating excitotoxic neuronal death in stroke, epilepsy, trauma and MS has been well established. Glutamate is the major excitatory amino acid transmitter within the CNS and it's signaling is mediated by a number of postsynaptic ionotropic and metabotropic receptors. Inflammation can be blocked with anti-cell adhesion molecules MAb, simultaneously protected oligodendrocytes and neurons against glutamate-mediated damage with the AMPA/kainate antagonist NBQX, and the NMDA receptor antagonist GPE, could thus be effective therapies for multiple sclerosis.

Legionella pneumophila multiplication is enhanced by chronic AMPK signalling in mitochondrially diseased dictyostelium cells

Human patients with mitochondrial diseases are more susceptible to bacterial infections, particularly of the respiratory tract. To investigate the susceptibility of mitochondrially diseased cells to an intracellular bacterial respiratory pathogen, we exploited the advantages of Dictyostelium discoideum as an established model for mitochondrial disease and for Legionella pneumophila pathogenesis. Legionella infection of macrophages involves recruitment of mitochondria to the Legionella-containing phagosome. We confirm here that this also occurs in Dictyostelium and investigate the effect of mitochondrial dysfunction on host cell susceptibility to Legionella. In mitochondrially diseased Dictyostelium strains, the pathogen was taken up at normal rates, but it grew faster and reached counts that were twofold higher than in the wild-type host. We reported previously that other mitochondrial disease phenotypes for Dictyostelium are the result of the activity of an energy-sensing cellular alarm protein, AMP-activated protein kinase (AMPK). Here, we show that the increased ability of mitochondrially diseased cells to support Legionella proliferation is suppressed by antisense-inhibiting expression of the catalytic AMPKα subunit. Conversely, mitochondrial dysfunction is phenocopied, and intracellular Legionella growth is enhanced, by overexpressing an active form of AMPKα in otherwise normal cells. These results indicate that AMPK signalling in response to mitochondrial dysfunction enhances Legionella proliferation in host cells.

A question of self-preservation : immunopathology in influenza virus infection

Influenza A viruses that circulate normally in the human population cause a debilitating, though generally transient, illness that is sometimes fatal, particularly in the elderly. Severe complications arising from pandemic influenza or the highly pathogenic avian H5N1 viruses are often associated with rapid, massive inflammatory cell infiltration, acute respiratory distress, reactive hemophagocytosis and multiple organ involvement. Histological and pathological indicators strongly suggest a key role for an excessive host response in mediating at least some of this pathology. Here, we review the current literature on how various effector arms of the immune system can act deleteriously to initiate or exacerbate pathological damage in this viral pneumonia. Generally, the same immunological factors mediating tissue damage during the anti-influenza immune response are also critical for efficient elimination of virus, thereby posing a significant challenge in the design of harmless yet effective therapeutic strategies for tackling influenza virus.

Suppressor of cytokine signaling 1 regulates embryonic myelopoiesis independently of its effects on T cell development

Suppressor of cytokine signaling 1 (SOCS1) has been shown to play important roles in the immune system. It acts as a key negative regulator of signaling via receptors for IFNs and other cytokines controlling T cell development, as well as Toll receptor signaling in macrophages and other immune cells. To gain further insight into SOCS1, we have identified and characterized the zebrafish socs1 gene, which exhibited sequence and functional conservation with its mammalian counterparts. Initially maternally derived, the socs1 gene showed early zygotic expression in mesodermal structures, including the posterior intermediate cell mass, a site of primitive hematopoiesis. At later time points, expression was seen in a broad anterior domain, liver, notochord, and intersegmental vesicles. Morpholino-mediated knockdown of socs1 resulted in perturbation of specific hematopoietic populations prior to the commencement of lymphopoiesis, ruling out T cell involvement. However, socs1 knockdown also lead to a reduction in the size of the developing thymus later in embryogenesis. Zebrafish SOCS1 was shown to be able to interact with both zebrafish Jak2a and Stat5.1 in vitro and in vivo. These studies demonstrate a conserved role for SOCS1 in T cell development and suggest a novel T cell-independent function in embryonic myelopoiesis mediated, at least in part, via its effects on receptors using the Jak2-Stat5 pathway.

Aging and its effects on inflammation in skeletal muscle at rest and following exercise-induced muscle injury

The world's elderly population is expanding rapidly, and we are now faced with the significant challenge of maintaining or improving physical activity, independence, and quality of life in the elderly. Counteracting the progressive loss of muscle mass that occurs in the elderly, known as sarcopenia, represents a major hurdle in achieving these goals. Indirect evidence for a role of inflammation in sarcopenia is that markers of systemic inflammation correlate with the loss of muscle mass and strength in the elderly. More direct evidence is that compared with skeletal muscle of young people, the number of macrophages is lower, the gene expression of several cytokines is higher, and stress signaling proteins are activated in skeletal muscle of elderly people at rest. Sarcopenia may also result from inadequate repair and chronic maladaptation following muscle injury in the elderly. Macrophage infiltration and the gene expression of certain cytokines are reduced in skeletal muscle of elderly people compared with young people following exercise-induced muscle injury. Further research is required to identify the cause(s) of inflammation in skeletal muscle of elderly people. Additional work is also needed to expand our understanding of the cells, proteins, and transcription factors that regulate inflammation in the skeletal muscle of elderly people at rest and after exercise. This knowledge is critical for devising strategies to restrict sarcopenia, and improve the health of today's elderly population.

Membrane nanotubes in myeloid cells in the adult mouse cornea represent a novel mode of immune cell interaction

Membrane nanotubes (MNTs) are newly discovered cellular extensions that are either blind-ended or can connect widely separated cells. They have predominantly been investigated in cultured isolated cells, however, previously we were the first group to demonstrate the existence of these structures in vivo in intact mammalian tissues. We previously demonstrated the frequency of both cell–cell or bridging MNTs and blind-ended MNTs was greatest between major histocompatibility complex (MHC) class II+ cells during corneal injury or TLR ligand-mediated inflammation. The present study aimed to further explore the dynamics of MNT formation and their size, presence in another tissue, the dura mater, and response to stress factors and an active local viral infection of the murine cornea. Confocal live cell imaging of myeloid-derived cells in inflamed corneal explants from Cx3cr1GFP and CD11ceYFP transgenic mice revealed that MNTs form de novo at a rate of 15.5 μm/min. This observation contrasts with previous studies that demonstrated that in vitro these structures originate from cell–cell contacts. Conditions that promote formation of MNTs include inflammation in vivo and cell stress due to serum starvation ex vivo. Herpes simplex virus-1 infection did not cause a significant increase in MNT numbers in myeloid cells in the cornea above that observed in injury controls, confirming that corneal epithelium injury alone elicits MNT formation in vivo. These novel observations extend the currently limited understanding of MNTs in live mammalian tissues.