Immunotoxic effects on freshwater mussels of a primary-treated wastewater before and after ozonation: a pilot plant study

The immunotoxic potential of a primary-treated municipal effluent following enhanced disinfection by ozonation was studied in the freshwater mussel Elliptio complanata. Mussels were exposed to increasing concentrations (0%, 1%, 3%, 10%, and 20% v/v) of the effluent before and after ozone treatment (approximately 10 mg/L of purged O(3)) in a continuous flow-through laboratory for 7 weeks. Immunocompetence was appraised by measuring phagocytosis, cell viability (fluorescein retention), cell adherence to polystyrene micro-wells, cyclooxygenase (COX) activity and total nitrite levels in peripheral hemocytes from the hemolymphs. The results showed that phagocytosis was significantly inhibited by the primary-treated effluent at a threshold concentration of 1.7% v/v. Cell viability was also significantly reduced three-fold compared to controls at the same effluent threshold concentration, but hemocyte adherence was unchanged. COX activity was increased 1.3-fold at a threshold concentration of 14% v/v. Total nitrite levels were significantly increased 2.2-fold at a threshold concentration of 5.5% v/v. Ozone treatment of the effluent was not successful in removing phagocytic inhibition, but did completely remove cytotoxicity from hemocytes. Ozonation also reduced cell adherence at a threshold concentration of 1.7% v/v. The inflammatory properties of the effluent appeared to be accentuated by the ozone, as evidenced by an increase in COX activity, which reached 2.6-fold activity compared to controls, as compared to the 1.3-fold increase witnessed in the primary-treated effluent. Furthermore, total nitrite levels reached a two-fold induction at a threshold concentration of 1.7% v/v in the ozone-enhanced effluent compared to 5.5% v/v in the primary-treated effluent. In conclusion, ozonation of a primary-treated effluent successfully reduced microbial loading and completely removed cytotoxicity, but increased the inflammatory properties of the effluent. Investigations aimed at examining the impacts of sustained inflammation on the host's capacity to remove potentially pathogenic bacteria are recommended.     

A technique for isolating heterophils from blood of orange-winged Amazon parrots (Amazona amazonica amazonica)

Blood samples from adult orange-winged Amazon parrots (Amazona amazonica amazonica) were collected to develop a rapid and efficient technique for isolating pure populations of morphologically intact and functional heterophils. In addition, normal haematological parameters for the orange-winged Amazon parrots (n=20) were established and found to be within the reported range of other Amazon species. Heterophil isolation (n=16) was maximally achieved by concentrating the white blood cells with 3% hetastarch prior to running the sample through a polysucrose and disodium diatrizoate centrifugation. The isolation procedure yielded an average heterophil recovery of 61.9%, the purity exceeded 92.6% and viability was 98.9%. Cell integrity was evaluated utilising flow cytometry, cytochemistries, and light microscopy. In this study, isolated heterophils exhibited morphological and cytochemical characteristics similar to whole blood heterophils and were phagocytically active. The results of this study demonstrate that an intact, viable and functionally active heterophil population can successfully be isolated from relatively small volumes of blood and that these cell populations can be further studied employing in vitro bioassaying techniques.     

Human phagocytic cell response to histamine derived from potential probiotic strains of Lactobacillus reuteri

Histamine derived from lactobacilli isolates is considered to be a potential immunomodulator able to interact with the host immune system. We tested the effect of pure histamine (0.413?mM) together with the effect of cell-culture supernatants (CCS) containing different concentration of histamine produced by two of Lactobacillus reuteri isolates on the activities of antioxidant enzyme, as well as on the phagocytic activity of human leucocytes (HL). Phagocytic activity represents the non-specific immune response of HL homogenate, in vitro. Analysed histamine-producers were represented by a goatling isolate named L. reuteri KO5 and a lamb isolate named L. reuteri E and histamine production was determined using HPLC method connected with UV detection. Concretely, the samples contained the mixture of isolated HL and the addition of lactobacilli CCS at three different final concentrations of histamine ～ 0.1, 1.8 and 5.4?mM. It was found that pure histamine (0.413?mM) did not significantly influence the oxidant-antioxidant balance in HL demonstrated by unchanged degree of HL lipid peroxidation. However, at the same time, the final activity of catalase and superoxide dismutase were significantly changed (p?≤?0.001).Moreover, the phagocytic index (p?≤?0.01), lysozyme (p?≤?0.05) and peroxidase activity (p?≤?0.001), and production of IL-1β significantly decreased. CCS containing different concentration of produced histamine were also able to modulate the host non-specific immune response together with the enzymatic activity of SOD and catalase too. However, our findings indicated that the impact of lactobacilli histamine is strictly strain-dependent and concentration dependent. Moreover, it seems that histamine is not the only one lactobacilli metabolite, which may play an important role in overall immunomodulatory and antioxidant potential of tested lactobacilli.     

Phagocytosis and cellular metabolism

Summary The uptake of foreign particles by mouse and human macrophages influenced by various metabolic inhibitors was examined in order to obtain further informations about the energy-dependent mechanisms which are involved in the phagocytic process. The inhibitors employed were iodoacetate, fluoroacetate, fluoride, malonate, sodium azide, 2-4-dinitrophenol, cycloheximide and ouabain. These substances were rested on monolayer cultures and the phagocytosis assay was performed by using zymosan suspension in the nutrient media. The quantitation of phagocytosis was obtained by the direct count of intracellular zymosan particles (immersion microscopy, 100x) and the results were evaluated and compared by biometrical analysis. The effects of these inhibitors on phagocytosis and their relation with the metabolic intracellular pathways are discussed.     

The Evolving Biology of Microglia in Alzheimer’s Disease

Alzheimer’s disease (AD) is typified by a robust microglial-mediated inflammatory response within the brain. Indeed, microglial accumulation around plaques in AD is one of the classical hallmarks of the disease pathology. Although microglia have the capacity to remove β-amyloid deposits and alleviate disease pathology, they fail to do so. Instead, they become chronically activated and promote inflammation-mediated impairment of cognition and cytotoxicity. However, if microglial function could be altered to engage their phagocytic response, promote their tissue maintenance functions, and prevent release of factors that promote tissue damage, this could provide therapeutic benefit. This review is focused on the current knowledge of microglial homeostatic mechanisms in AD, and mechanisms involved in the regulation of microglial phenotype in this context.

Electronic supplementary material

The online version of this article (doi:10.1007/s13311-014-0316-8) contains supplementary material, which is available to authorized users.     

Modeling microglial activation in Alzheimer’s disease with human postmortem microglial cultures

Alzheimer’s disease (AD) is a uniquely human disorder. Despite intense research, the lack of availability of model systems has hindered AD studies though in recent years transgenic mouse models have been produced, which develop AD-like amyloid beta peptide (Aβ) plaques. For the study of inflammatory changes in AD brains, these transgenic mice may have limitations due to differences in the innate immune system of humans and rodents. Many studies of inflammatory processes in AD have focused on the role of activated microglia. Over the last 8 years, our research has focused on the properties of human microglia cultured from brain tissues of AD and non-demented (ND) individuals. As these are the cells observed to be activated in AD tissues, they represent a useful system for modeling the inflammatory components of AD.

In this review, we summarize data by our group and others on the use of microglia for AD-related inflammatory research, with emphasis on results using human postmortem brain microglia. A range of products have been shown to be produced by human postmortem microglia, both constitutively and in response to treatment with Aβ, including proinflammatory cytokines such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF) , and macrophage colony stimulating factor (M-CSF), along with complement proteins, especially C1q, superoxide radicals and neurotoxic factors. In our studies, we have demonstrated that there was a significant difference between AD and ND microglia in terms of their secretion of M-CSF and C1q. We also discuss the role of putative Aβ microglial receptors, particular recent data showing a role for the receptor for advanced glycation endproducts (RAGE) in mediating the responses of human microglia to Aβ. Finally, our studies on the use of an Aβ spot paradigm to model microglia interactions with plaques demonstrated that many of the features of AD inflammation can be modeled with postmortem brain derived microglia.     

Fcγ receptor-mediated phagocytosis requires tyrosine kinase activity and is ligand independent

Receptors for the invariant chain of immunoglobulins (FcR) define the cellular response to specific antigens. FcγR recognize IgG and so elicit a variety of effector functions including phagocytosis. We are interested in the structural determinants for FcγR-mediated phagocytosis, specifically FcγRI(p135) and FcγRIIa isoforms. The low-affinity receptor, FcγRIIa, is found on macrophages and its cytoplasmic domain contains a tyrosine activation motif which has previously been shown to regulate endocytosis. In contrast, FcγRI has no known signaling motifs, though a functional interaction has recently been demonstrated with the γ chain of the high-affinity receptor for IgE, FcεRI. This accessory molecule has a cytoplasmic tyrosine activation motif implicated in signal transduction. Here we demonstrate that although FcγRI transiently expressed on COS-7 cells is able to rosette opsonized SRBC, it cannot phagocytose them. If the cytoplasmic domain of either γ chain or FcγRIIa replaces that of FcγRI in a chimeric receptor, efficient phagocytosis occurs. This particle ingestion is sensitive to the tyrosine kinase inhibitor genistein. Chimeric receptors where the extracellular domain of either FcγRI or FcγRIIa is replaced with that of CD2, a T cell antigen, indicate that FcγR-mediated phagocytosis is ligand independent. We conclude that phagocytosis is dependent upon close particle apposition, tyrosine kinase activity, and that the process is ligand independent.     

Effect of L‐Arginine on Age‐Related Changes in Macrophage Phagocytic Activity

Aging is associated with decline in the functioning of immune cells and reductions in serum L‐arginine and excretion of nitric oxide metabolites. Studies have shown that L‐arginine plays an important role in many physiological, biological and immunological processes. The present study was performed to determine if treatment with L‐arginine could prevent age‐related changes in phagocytic function of peritoneal macrophages. The effects of L‐arginine on phagocytic activity of peritoneal macrophages were compared between young and middle‐aged rats. Studies were performed in four groups of rats for 8 weeks: group 1 (3 month‐old) received physiological saline; group 2 (3 month‐old) received L‐arginine (160 mg/kg/day); group 3 (12 month‐old) received physiological saline; group 4 (12 month‐old) received L‐arginine (160 mg/kg/day). There were no significant differences in percentage of cells which were phagocytized. However, the phagocytosis of activated charcoal by peritoneal macrophages reduced with age. Thus, the phagocytic index was lower in macrophages of middle‐aged rats. L‐arginine treatment increased phagocytosis by peritoneal macrophages of both young and middle‐aged rats. L‐arginine‐induced augmentation in phagocytosis by macrophages were much higher in the middle‐aged rats compared with young rats. In summary, we found that L‐arginine prevented the age‐related reduction in phagocytic capability of peritoneal macrophages.     

Macrophage phagocytosis and membrane fluidity in mice: the effect of age and dietary protein

Male C57BL/6NNia mice were used to investigate the effects of age and dietary protein intake on Fc and C3b receptor-mediated phagocytosis and on membrane fluidity. Six-month-old (adult) and 24-month-old (aged) mice were fed a 6% or 25% protein diet for 3, 5, or 6 weeks at which time thioglycollate elicited peritoneal macrophages were isolated. Both binding of IgG-coated sheep red blood cells to the macrophages and ingestion via the Fc-receptor were identical in all 4 groups after 3 and 6 weeks of feeding but were decreased at 5 weeks in the aged animals fed 6% protein. Phagocytosis via the C3b receptor was not depressed in either age group fed the low protein diet; it was, however, augmented significantly in the aged animals fed the 25% protein diet for 5 and 6 weeks. Membrane fluidity of the plasma membrane outer hemileaflet was monitored with an impermeant fluorescent probe. No changes were observed between adult and aged mice maintained up to 6 weeks on the diets.