Optimisation of phagocytosis assay in rainbow trout (Oncorhynchus mykiss)

A phagocytosis assay is one of the most commonly used functional immunological methods. There are many possible ways of assessing leucocytes and their ability to ingest different particles. The aim of this study was to optimise the phagocytosis assay in rainbow trout (Oncorhynchus mykiss) using labelled zymosan particles (Alexa Fluor 488 and Texas Red conjugate). Whole blood was incubated with the particles under different conditions and leucocytes were subsequently isolated by haemolysis in a hypotonic environment. The effect of the different incubation time, temperature, blood volume and dilution on the phagocytic activity was evaluated by flow cytometry. Our experiments showed that the incubation for at least 2 h at 15 °C provided optimal results, while the blood volume and dilution had no significant effect. The optimised assay will be used for the examination of fish health and in further experimental studies.

Effect of memantine, an anti-Alzheimer’s drug, on rodent microglial cells in vitro

The pathophysiology of Alzheimer’s disease (AD) is related to neuroinflammatory responses mediated by microglia. Memantine, an antagonist of N-methyl-d-aspartate (NMDA) receptors used as an anti-Alzheimer’s drug, protects from neuronal death accompanied by suppression of proliferation and activation of microglial cells in animal models of AD. However, it remains to be tested whether memantine can directly affect microglial cell function. In this study, we examined whether pretreatment with memantine affects intracellular NO and Ca2+ mobilization using DAF-2 and Fura-2 imaging, respectively, and tested the effects of memantine on phagocytic activity by human β-Amyloid (1–42) phagocytosis assay in rodent microglial cells. Pretreatment with memantine did not affect production of NO or intracellular Ca2+ elevation induced by TNF in rodent microglial cells. Pretreatment with memantine also did not affect the mRNA expression of pro-inflammatory (TNF, IL-1β, IL-6 and CD45) or anti-inflammatory (IL-10, TGF-β and arginase) phenotypes in rodent microglial cells. In addition, pretreatment with memantine did not affect the amount of human β-Amyloid (1–42) phagocytosed by rodent microglial cells. Moreover, we observed that pretreatment with memantine did not affect 11 major proteins, which mainly function in the phagocytosis and degradation of β-Amyloid (1–42), including TREM2, DAP12 and neprilysin in rodent microglial cells. To the best of our knowledge, this is the first report to suggest that memantine does not directly modulate intracellular NO and Ca2+ mobilization or phagocytic activity in rodent microglial cells. Considering the neuroinflammation hypothesis of AD, the results might be important to understand the effect of memantine in the brain.

MARBOFLOXACIN INDUCES LEISHMANICIDAL ACTIVITY AND LESS INFLAMMATORY RESPONSE IN Leishmania chagasi INFECTED MACROPHAGES

Marbofloxacin has promising leishmanicidal activity due to the direct action on the Leishmania chagasi amastigotes. It was developed only for veterinary medicine uses, and it could be used as an drug for the treatment of Canine Visceral Leishmaniasis (CVL). In the present study, we evaluated the leishmanicidal effect and macrophage modulation of marbofloxacin in macrophages infected with amastigotes of L. chagasi. Macrophages were collected from peritoneum of BALB/c mice and infected with promastigotes of L. chagasi. After internalization and transformation into amastigotes forms, cells were treated with marbofloxacin in concentration of 100, 500 and 750 μg/mL for 18 hours. The leishmanicidal effect was evaluated by morphological spects of amastigotes inside of macrophages by phagocytosis assay and detection of death amastigotes and macrophages by TUNEL assay. Macrophage modulation was evaluated by release of cytotoxic metabolites and cytokine production. The results showed that L. chagasi-infected macrophages treated with the highest concentration of the drug showed lower amount of amastigotes into the macrophages (p<0,0632) than untreated infected cells. There was pronounced presence of cellular vacuoles in the treated infected-cells, and more apoptotic amastigotes in alive macrophages. It was observed decreased levels of H2O2, IL-1β, IL-6, and TNF-α dose-dependent of marbofloxacin in infected macrophages. The results indicate the leishmanicidal effect of marbofloxacin in infected macrophages and immunomodulation actions in these cells.

Rosettes integrity protects Plasmodium vivax of being phagocytized

Plasmodium vivax is the most prevalent cause of malaria outside of Africa. P. vivax biology and pathogenesis are still poorly understood. The role of one highly occurring phenotype in particular where infected reticulocytes cytoadhere to noninfected normocytes, forming rosettes, remains unknown. Here, using a range of ex vivo approaches, we showed that P. vivax rosetting rates were enhanced by plasma of infected patients and that total immunoglobulin M levels correlated with rosetting frequency. Moreover, rosetting rates were also correlated with parasitemia, IL-6 and IL-10 levels in infected patients. Transcriptomic analysis of peripheral leukocytes from P. vivax-infected patients with low or moderated rosetting rates identified differentially expressed genes related to human host phagocytosis pathway. In addition, phagocytosis assay showed that rosetting parasites were less phagocyted. Collectively, these results showed that rosette formation plays a role in host immune response by hampering leukocyte phagocytosis. Thus, these findings suggest that rosetting could be an effective P. vivax immune evasion strategy.

microspheres made by crosslinking of polyvinyl alcohol with glutaraldehyde functionalized with yeast lysate

Microspheres comparable with yeast cells in size were obtained by the glutaraldehyde crosslinking of polyvinyl alcohol (PVA), whose 25 g/l water solution containing magnetite particles was dispersed in the isooctane/Span 85/Tween 85 medium. yeast cell walls were partially hydrolysed by sonication in formic acid near the boiling point. The microspheres were made targets for phagocytosis by the addition of yeast lysate to the crosslinkable PVA solution and a subsequent treatment of the freshly crosslinked microspheres with diluted yeast lysate. The microspheres were also made fluorescent when the emulsification medium saturated with fluorescent 2-aminopyrimidine derivatives was used. A protocol for phagocytosis assay using the thus modified microspheres was developed.

Inhibition of platelet phagocytosis as an in vitro predictor for therapeutic potential of RBC antibodies in murine ITP

Polyclonal anti-D is a first-line therapy for immune thrombocytopenia (ITP). Monoclonal antibodies are desirable alternatives, but none have yet proven successful despite their ability to opsonize erythrocytes (or red blood cells, RBCs) and cause anemia. Here, we examined 12 murine erythrocyte–specific antibodies of different specificity and subtypes and found that 8 of these antibodies could induce anemia in antigen-positive mice. Of these 8 antibodies, only 5 ameliorated ITP. All antibodies were examined for their in vitro ability to support macrophage-mediated phagocytosis of erythrocytes. Antibodies which supported erythrocyte phagocytosis in vitro successfully ameliorated ITP in vivo. To examine the ability of each antibody to inhibit phagocytosis of platelets, the antibodies were used to sensitize erythrocytes in vitro and these were added to a platelet phagocytosis assay. Antibodies that inhibited platelet phagocytosis in vitro also all ameliorated ITP in vivo. We conclude that inducing anemia is not a sufficient condition for amelioration of ITP but that the antibody’s ability to prevent platelet phagocytosis in vitro predicted its ability to ameliorate ITP. We suggest that inhibition of in vitro platelet phagocytosis may prove to be a valuable tool for determining which erythrocyte antibodies would likely be candidates for clinical use in ITP.