Phagocytosis Of Yeast Cells Research Articles

Blocking Serum Amyloid-P Component from Binding to Macrophages and Augmenting Fungal Functional Amyloid Increases Macrophage Phagocytosis of Candida albicans.

Candida-macrophage interactions are important immune defense responses associated with disseminated and deep-seated candidiasis in humans. Cells of Candida spp. express functional amyloids on their surfaces during the pathogenesis of disseminated candidiasis. These amyloids become decorated with serum amyloid P-component (SAP) that binds to Candida cells and macrophages and downregulates the cellular and cytokine response to the fungi. In this report, further characterization of the interactions of SAP and fungal functional amyloid are demonstrated. Blocking the binding of SAP to macrophage FcγR1 receptors increases phagocytosis of yeast cells; seeding a pro-amyloid-forming peptide on the yeast cell surface also increases phagocytosis of yeasts by macrophages; and, lastly, miridesap, a small palindromic molecule, prevents binding of SAP to yeasts and removes SAP that is bound to C. albicans thus, potentially increasing phagocytosis of yeasts by macrophages. Some, or all, of these interventions may be useful in boosting the host immune response to disseminated candidiasis.

A CLINICOPATHOLOGICAL STUDY ON DISSEMINATED HISTOPLASMOSIS WITH SPECIAL REFERENCE TO ATYPICAL CASES IN A TERTIARY CARE CENTRE -IN EASTERN INDIA.

INTRODUCTION: Untreated Histoplasmosis is fatal for all, immunocompetent or immunocompromised. Histoplasmosis has seen a steady increase in incidence from a single recorded case in Kolkata, India in 1954 to 388 cases from 2004-17 and 161 from 2017-till date all over India. Yet, it has been consistently relegated to a niche of opportunistic infections without a proper diagnostic protocol. To study the clinical OBJECTIVES: features and natural history of disseminated histoplasmosis(DH) and analysis of cases with atypical clinical presentation of Histoplasmosis simulating malignancy in search of a consolidated and efcient diagnostic algorithm in a tertiary care centre. MATERIALS AND METHODS: The retrospective institution-based study was conducted in the Department of Pathology, CSTM from February 2011 to July 2019 in collaboration of Dept. of Tropical Medicine on specimens collected from 33 cases of Skin biopsy, Lymph node biopsy and FNAC aspirate from enlarged lymphnodes, slit skin smear followed by BM Aspirate analysis done in cases of disseminated histoplasmosis, Bone Marrow Biopsy was done in selected cases. Histoplasma capsulatum like yeast cells were detected with Leishman Giemsa stained slit skin smears in 9 cases and in FNA smears in 13cases and they were PAS positive. Diagnosis were conrmed in Histopathological examination along with special stain for fungus in all cases. Radiological investigations (CXR, USG, CT), Routine investigations (Haemogram, LFT, RFT, Electrolytes) along with CD4 count in ART clinic and microbiological tests like Gram stain of SSS, India Ink, KOH preparation, Cryptococcal Ag testing, urine C/S) were corroborated with. RESULTS: All 33 cases FNAC (13),slit skin smear(9) and HPE on rest of the specimen show small, oval yeast cells on LG stain. Biopsy showed histiocytic phagocytosis of yeast cells with foamy macrophages and PMN inltration with NO granulomas in seroposive cases. Gomori Methenamine Silver stain of BM biopsy showed black intracellular yeast consistent with . PAS stained FNAC smears Histoplasma capsulatum showed yeast cells with chromatin dot and surrounding halos with no kinetoplasts. Mycological cultures were in agreement. Here, in most cases, CD4 count and Disseminated Histoplasmosis ndings were not in accordance. DH CONCLUSION: is not uncommon in India and is being increasing day by day and it should be considered in the diagnosis of patients with prolonged fever, weight loss, oropharyngeal ulcers, hepatosplenomegaly, lymphadenopathy and adrenal enlargement. Correct diagnosis by well designed analytical studies utilising appropriate diagnostic modalities in an immunocompromised patient even with minimum suspicion of opportunistic infection, a consolidation of selective investigations like FNAC, HPE and relevant microbiological tests into a diagnostic design for maximum utilization of nances, resources and time, all of which can help to understand the epidemiology of this neglected disease for accurate treatment leading to a favourable outcome.

Melanin as a Virulence Factor in Different Species of Genus Paracoccidioides.

Paracoccidioidomycosis (PCM) is a granulomatous systemic mycosis caused by the thermo-dimorphic fungi of the genus Paracoccidioides. Melanin production by fungi can affect their pathogenesis and virulence. This study evaluates the production of melanin by different isolates of genus Paracoccidioides and examines how the presence of this polymer affects yeast cell phagocytosis, as well as laccase enzyme production. The results obtained showed that the isolates of genus Paracoccidioides: P. lutzii (Pb01, Pb66, ED01, Pb1578, and Pb8334), P. restrepiensis (PS3-Pb60855), P. brasiliensis (S1-Pb18), and P. americana (PS2-Pbcão) produce melanin in the presence of L-3,4-dihydroxyphenylalanine (L-DOPA). Phagocytosis assays were carried out with peritoneal macrophages from C57Bl/6 mice that were challenged with Pb18, Pb60855, and Pb01. We observed that melanin interferes with phagocytosis in the presence or absence of complement or heat-inactivated serum. This article confirms that different species of the genus Paracoccidioides produce melanin in different magnitudes and that the polymer functions as a virulence factor.

Immunostimulatory properties of fructans derived from raw garlic (Allium sativum L.)

Garlic (Allium sativum L.), commonly used as a spice and traditional medicine has innumerable biological activities including immunomodulation. Fructans are present in abundance (14–17%, fresh wt.) in garlic. Although fructans from aged garlic extract have been shown to exert immunomodulatory properties, such information is lacking for fructans from raw garlic. The main objective of this study is to isolate raw garlic fructans (RGF) and investigate its effects on the cells of the immune system in vitro. RGF (detected by cold anthrone assay) was isolated, in a yield of 13.8g/100g (fresh weight) garlic, from the flow-through pool of Q-Sepharose chromatography of raw garlic extract. Proliferation of murine splenocytes by RGF was assessed by MTT assay. Activation of macrophages in peritoneal exudates cells was followed by the release of nitric oxide (NO) and phagocytosis of yeast cells. RGF stimulated murine splenocytes to a similar extent as seen in the case of established polysaccharide immunostimulators (galactan, arabinogalactan, mannan and zymosan); 4–5 fold stimulation was seen compared to untreated cells (p<0.01). Further, RGF activated macrophages present in rat peritoneal exudates to release NO which was significant at both 24h and 48h (p<0.01). Yeast cell phagocytosis by activated macrophages was significantly enhanced (p<0.05) by RGF in comparison to untreated cells. In conclusion, RGF causes significant immunostimulation of murine lymphocytes and macrophages in vitro. Since fructans are present in abundance in raw garlic, it appears to be promising for the development of functional foods for immunomodulation by its inclusion in processed foods.

The Membrane Phospholipid Binding Protein Annexin A2 Promotes Phagocytosis and Nonlytic Exocytosis of Cryptococcus neoformans and Impacts Survival in Fungal Infection.

Cryptococcus neoformans is a fungal pathogen with a unique intracellular pathogenic strategy that includes nonlytic exocytosis, a phenomenon whereby fungal cells are expunged from macrophages without lysing the host cell. The exact mechanism and specific proteins involved in this process have yet to be completely defined. Using murine macrophages deficient in the membrane phospholipid binding protein, annexin A2 (ANXA2), we observed a significant decrease in both phagocytosis of yeast cells and the frequency of nonlytic exocytosis. Cryptococcal cells isolated from Anxa2-deficient (Anxa2(-/-)) bone marrow-derived macrophages and lung parenchyma displayed significantly larger capsules than those isolated from wild-type macrophages and tissues. Concomitantly, we observed significant differences in the amount of reactive oxygen species produced between Anxa2(-/-) and Anxa2(+/+) macrophages. Despite comparable fungal burden, Anxa2(-/-) mice died more rapidly than wild-type mice when infected with C. neoformans, and Anxa2(-/-) mice exhibited enhanced inflammatory responses, suggesting that the reduced survival reflected greater immune-mediated damage. Together, these findings suggest a role for ANXA2 in the control of cryptococcal infection, macrophage function, and fungal morphology.

Immunostimulatory properties of the major protein from the stem of the Ayurvedic medicinal herb, guduchi (Tinospora cordifolia)

Ethnopharmacological relevanceGuduchi (Tinospora cordifolia), a widely used plant in folk and Ayurvedic systems of medicine is well known for its immunomodulatory activity; however, the presence of an immunomodulatory protein (ImP) in guduchi has not been investigated. Materials and methodsGuduchi ImP was purified from dry stem powder extract by anion-exchange chromatography on Q-Sepharose. Characterization of guduchi ImP was performed by SDS-PAGE, periodic acid-Schiff staining, HPLC, and immunochemical analyses. Immunostimulatory activity was assessed by lymphocyte proliferation and macrophage activation assays. Fresh guduchi stem/leaf, guduchi satwa and guduchi capsules were also analyzed for the presence of guduchi ImP. ResultsGuduchi ImP was purified to homogeneity from dry stem powder extract (∼150mg protein per 100g guduchi stem powder) as a single chain acidic protein (25kDa) without glycans; it was noticeably absent in guduchi leaf. Guduchi satwa and guduchi capsule preparations also lacked this protein. Guduchi ImP showed ∼3-fold mitogenic activity compared to untreated murine splenocytes in the 1–10μg/mL concentration range; 5–7-fold increase in mitogenic activity was seen in the case of murine thymocytes vs. control. The purified protein also induced nitric oxide production from macrophages present in isolated murine peritoneal exudates cells. Guduchi ImP displays enhanced phagocytosis of yeast cells by macrophages. Guduchi ImP does not possess hemagglutination activity (towards rabbit and human erythrocytes of all blood groups) indicating that the immunomodulatory protein is not a lectin. ConclusionsThe confirmation of an immunomodulatory protein in guduchi stem showing lymphoproliferative and macrophage-activating properties reinforces the rationale of the use of guduchi preparations in several Ayurvedic medicines for immunomodulation. To our knowledge, this is the first report of an immunomodulatory protein isolated from guduchi.

Interplay between amphioxus complement with sea bass macrophages: Opsonic activity of amphioxus humoral fluids

Previous studies have shown the existence of a complement system in the amphioxus Branchiostoma japonicum. However, whether it has an opsonic activity similar to that of vertebrates remains unknown. We demonstrated that the humoral fluid (HF) of amphioxus promoted the phagocytosis of yeast cells with sea bass (Lateolabrax japonicus) macrophages, whereas the C3-depleted and heated HF significantly lost the phagocytosis-promoting capacity. In addition, the precipitation of factor B (Bf) led to a marked loss of opsonic activity. Moreover, C3 fragments in the HF were found to bind to yeast cell surfaces. The results indicate that the amphioxus complement system is an important element involved in the opsonic activity, which promotes the sea bass macrophage phagocytosis by tagging yeast cells with C3 fragments via the activation of alternative complement pathway.

Blockades of phospholipase A2 and platelet-activating factor receptors reduce the hemocyte phagocytosis in Rhodnius prolixus: In vitro experiments

The hemocytes phagocytosis in response to microorganisms may play an important role in the cellular immune responses of insects. Here, we have evaluated the effects of the platelet-activating factor (PAF) and eicosanoids in the phagocytosis of hemocyte monolayers of Rhodnius prolixus to the yeast Saccharomyces cerevisiae. Experiments showed that the phagocytosis of yeast cells by Rhodnius hemocytes is very efficient in both controls and cells treated with PAF and arachidonic acid. Phagocytosis of yeast particles is significantly blocked when the specific phopholipase A2 inhibitor, dexamethasone, is applied on the hemocytes. By contrast, dexamethasone-pretreated hemocyte monolayers exhibit a drastic increase in the quantity of yeast cell-hemocyte internalization when the cells are treated by arachidonic acid. In addition, phagocytosis presents significant reduction in hemocyte monolayers treated with a specific PAF receptor antagonist, WEB 2086. Nevertheless, inhibition of phagocytosis with WEB 2086 is counteracted by the treatment of the hemocyte monolayers with PAF. In conclusion, phagocytosis of yeast cells by hemocytes is related to the activation of PAF receptors and eicosanoid pathways in the bloodsucking bug, R. prolixus.

Regulation of Phagocytosis in Dictyostelium by the Inositol 5‐Phosphatase OCRL Homolog Dd5P4

Phosphoinositides are involved in endocytosis in both mammalian cells and the amoeba Dictyostelium discoideum. Dd5P4 is the Dictyostelium homolog of human OCRL (oculocerebrorenal syndrome of Lowe); both have a RhoGAP domain and a 5-phosphatase domain that acts on phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). Inactivation of Dd5P4 inhibits growth on liquid medium and on bacteria. Dd5p4-null cells are impaired in phagocytosis of yeast cells. In wild-type cells, PI(3,4,5)P3 is formed and converted to PI(3,4)P2 just before closure of the phagocytic cup. In dd5p4-null cells, a phagocytic cup is formed upon contact with the yeast cell, and PI(3,4,5)P3 is still produced, but the phagocytic cup does not close. We suggest that Dd5P4 regulates the conversion of PI(3,4,5)P3 to PI(3,4)P2 and that this conversion is essential for closure of the phagocytic cup. Phylogenetic analysis of OCRL-like 5-phosphatases with RhoGAP domains reveal that D. discoideum Dd5P4 is a surprisingly close homolog of human OCRL, the protein responsible for Lowe syndrome. We expressed human OCRL in dd5p4-null cells. Growth on bacteria and axenic medium is largely restored, whereas the rate of phagocytosis of yeast cells is partly restored, indicating that human OCRL can functionally replace Dictyostelium Dd5P4.

Scuticociliate cysteine proteinases modulate turbot leucocyte functions

The effects exerted by cysteine proteinases isolated from the histiophagous ciliate Philasterides dicentrarchi on the phagocytic functions of turbot pronephric leucocytes (PL) were investigated. The enzymes were tested at concentrations of 125, 250 and 500 μg ml −1, and it was found that the viability of the leucocytes was not affected after treatment for 24 h. Leucocyte migration was inhibited by the cysteine proteinases in a dose-dependent manner, whereas the ascitic fluid obtained from turbot experimentally infected with P. dicentrarchi induced high chemotactic activity in the turbot PL. The proteinases did not affect yeast cell phagocytosis but increased intracellular production of the superoxide anion (O 2 −). Stimulation with the proteinases did not alter the PGE 2 levels in supernatants from 24-h cultures of PL, however, β-glucans (100 μg ml −1) provoked a large increase in PGE 2 levels, which were inhibited after addition of 10 μg ml −1 of indomethacin, a non-selective inhibitor of COX2 enzymatic activity. The mean PGE 2 level in ascitic fluid from turbot, experimentally infected with P. dicentrarchi, was 500 pg ml −1, and the addition of low levels of PGE 2 (62.5 pg ml −1) to PL cultures stimulated O 2 − production, although addition of PGE 2 at concentrations higher than 250 pg ml −1 blocked the increase in stimulation. Addition of cysteine proteinases to 24-h cultures of PL also increased mRNA levels in the pro-inflammatory cytokine interleukin-1β. The results revealed the capacity of cysteine proteinases isolated from P. dicentrarchi to modulate the innate immune response of turbot, which together with the inflammation mediators produced during infection, may play an important role in pathogenesis of the disease and in the survival of the parasite.

Phagocytosis by Trichomonas vaginalis: new insights

The parasitic protozoan Trichomonas vaginalis is the causative agent of trichomoniasis, a sexually transmitted disease. The phagocytic activity of this parasite has not been completely elucidated. In order to better understand the mechanisms of trichomonal phagocytosis, we have studied the in vitro capacity of T. vaginalis to phagocytose and degrade Saccharomyces cerevisiae cells. To analyse the phagocytic ability and capacity, two isolates of T. vaginalis presenting different virulence grades were used. Complementary techniques, such as fluorescence microscopy, computer-based fluorescence analysis, scanning and transmission electron microscopy and the use of drugs that interfere with the actin microfilaments, were used in order to follow the behaviour of the actin cytoskeleton during phagocytosis of yeast cells by T. vaginalis. It was concluded that: (1) T. vaginalis changes its shape rapidly and engulfs the yeast cells, which are almost as large as the parasite; (2) long-term and fresh cultures are able to phagocytose, although the low-virulence strain JT demonstrated a lower activity when compared with the highly virulent T016 isolate; (3) the T016 strain exhibited an amoeboid morphology during the internalization of yeast cells in contrast with the JT strain; (4) attachment of yeast cells to the parasite occurs via the whole cell surface, including both anterior and recurrent flagella; (5) two forms of phagocytosis were observed: a 'sinking' process without any apparent participation of plasma membrane extensions and the classical phagocytosis where pseudopodia are extended toward the target cell; (6) the internalized S. cerevisiae are digested in lysosomes; (7) competitor sugars D-mannose or L-fucose inhibit the phagocytosis, and inhibition was 1.67 times higher in long-term cultured JT than that of the parasites from fresh isolate T016; (8) a thick layer of actin microfilaments was present underlying the plasma membrane, and especially in the pseudopodia and around the phagocytosed particles; (9) a dramatic change in the distribution pattern of fibrillar actin occurred during phagocytosis; (10) cytochalasin D depressed the phagocytosis; (11) a non-specific recognition and phagocytosis of yeast cells by T. vaginalis is mediated by a mannose receptor present on the parasite surface; (12) the phagocytic process may occur simultaneously during mitosis of the parasite.

N6-Cyclohexyl-adenosine but not adenosine is a modulator of teleost fish innate immune activities

The possible immuneregulatory effects of two purine nucleotides, adenosine and cyclohexyladenosine, on gilthead seabream ( Sparus aurata L.) leucocytes were investigated. Leucocytes isolated from the head-kidney were incubated with five different concentrations (ranging from 0 to 100 μM) of adenosine or cyclohexyladenosine for 30, 180 and 360 min and the effect on leucocyte viability and some of the main innate cellular immune responses was evaluated. Adenosine did not significantly affect the viability or the innate immune parameters of seabream leucocytes at any of the assayed concentrations or incubation times. High concentrations of cyclohexyladenosine, on the other hand, caused a significant drop in the respiratory burst activity of head-kidney leucocytes at all the times assayed. In addition, phagocytosis of yeast cells was significantly rapidly depressed (30 min) after incubation with 100 μM of this analogue. The present results demonstrate that cyclohexyladenosine at high concentrations is capable of down-regulating certain innate immune responses in seabream leucocytes, suggesting that telesot fish immune cells, like their mammal counterparts, possess receptors for purine nucleotides.

Cholinergic modulation of baker’s yeast cell phagocytosis by rat astrocytes

Cholinergic regulation of baker’s yeast cell phagocytosis in rat cultured astrocytes was studied. Phagocytic activity was reduced by 1×10 −5 M of atropine or pirenzepine, but not by AF-DX116 or 4-DAMP. In addition, carbachol stimulated phagocytosis in a dose-dependent manner. Furthermore, only 1×10 −5 M of atropine, pirenzepine and 4-DAMP significantly reduced enhanced activity induced by 1×10 −7 M carbachol. It was also observed that l-NMMA, staurosporine, or U-73122, reduced phagocytosis activity while TFP failed to do so. Nitrite levels in astrocyte supernatants increased after baker’s yeast cells were incorporated to astrocyte cultures, correlating with enhanced phagocytosis induced by carbachol stimulation, and were reduced by 1×10 −5 M of atropine, pirenzepine or aminopiridine, but not by AF-DX116 or 4-DAMP. Enhanced NO production triggered by astrocyte phagocytosis may have pathological consequences.

Interaction of Blastomyces dermatitidis, Sporothrix schenckii, and Histoplasma capsulatum with Acanthamoeba castellanii.

Several dimorphic fungi are important human pathogens, but the origin and maintenance of virulence in these organisms is enigmatic, since an interaction with a mammalian host is not a requisite for fungal survival. Recently, Cryptococcus neoformans was shown to interact with macrophages, slime molds, and amoebae in a similar manner, suggesting that fungal pathogenic strategies may arise from environmental interactions with phagocytic microorganisms. In this study, we examined the interactions of three dimorphic fungi with the soil amoeba Acanthameobae castellanii. Yeast forms of Blastomyces dermatitidis, Sporothrix schenckii, and Histoplasma capsulatum were each ingested by amoebae and macrophages, and phagocytosis of yeast cells resulted in amoeba death and fungal growth. H. capsulatum conidia were also cytotoxic to amoebae. For each fungal species, exposure of yeast cells to amoebae resulted in an increase in hyphal cells. Exposure of an avirulent laboratory strain of H. capsulatum to A. castellanii selected for, or induced, a phenotype of H. capsulatum that caused a persistent murine lung infection. These results are consistent with the view that soil amoebae may contribute to the selection and maintenance of certain traits in pathogenic dimorphic fungi that confer on these microbes the capacity for virulence in mammals.

Effects of high doses of cortisol on innate cellular immune response of seabream ( Sparus aurata L.)

The effects of high doses of cortisol, biologically the most active corticosteroid in the circulating blood of teleost fish, on gilthead seabream ( Sparus aurata L.) leucocytes were determined. Leucocytes isolated from the head-kidney (the principal haemopoietic organ of this fish species) were incubated with five different concentrations (ranging from 0 to 10 −1 mM) of cortisol for 30, 120, 240, 360, and 480 min and their effects on leucocyte viability and some of the main innate cellular immune responses were evaluated. The viability and the cytotoxic activity of leucocytes against tumor cells were not significantly affected by in vitro incubation with cortisol, at any of the assayed concentration or incubation times. With cortisol concentrations of 10 −9, 10 −7 or 10 −5 mM, the respiratory burst activity of head-kidney leucocytes were significantly depressed after 30 min of incubation. On the other hand, with cortisol concentrations of 10 −1 mM, the phagocytosis of yeast cells and the total peroxidase content of leucocytes were significantly depressed at incubation times longer than 240 min. To corroborate that the observed effects were due to the cortisol treatment, assays were developed using both cortisol and cycloheximide, which blocked the inhibitory effects of cortisol. The present results demonstrate that cortisol plays an important role in the down-regulation of phagocytic but not of cytotoxic activity in seabream leucocytes.

Quantitative evaluation of macrophage phagocytosing capacity by a fluorometric assay.

This paper reviews sensitive and simple quantitative evaluation of macrophage phagocytosing capacity by applying fluoresecin-labeled Sacharomyces cerevisiae cells. Yeast cells were conjugated with fluoresceinisothiocyanate (FITC) and used as fluorescent particles. A time course analysis within this method showed that phagocytosis of yeast cells was temperature dependent and that the number of that ones ingested by macrophages increased rapidly during the initial 60 min of incubation at 37 degrees C. Free fluorescent cells can be effectively removed by aspiration from the well. Furthermore, yeast cells required preopsonization with serum to achieve optimal uptake of the cells. The uptake of nonopsonized yeast cells by macrophages was significantly lower than that of opsonized cells (P < 0.05). We propose that about 50% of mouse macrophages can carry functionally active FcR responsible for phagocytosis.

A mannose-receptor is possibly involved in the phagocytosis of Saccharomyces cerevisiae by seabream (Sparus aurata L.) leucocytes

In this paper the possible involvement of the mannose-receptor on the non-specific recognition and phagocytosis of heat killed yeast cells (Saccharomyces cerevisiae) by gilthead seabream (Sparus aurata L.) head-kidney leucocytes was established by studying the ability of different sugars to inhibit the uptake of the yeast cells by leucocytes. Leucocytes were preincubated for 30min with different concentrations of sugar (α-mannan, d-mannose, d-fucose, l-fucose, d-glucose, d-glucosamine and n-acetyl-glucosamine, all of them described as specific ligands of the vertebrate mannose-receptor) and afterwards incubated with FITC-labelled yeast cells for phagocytosis assays. The phagocytic ability (percentage of cells with one or more ingested yeast cells within the total cell population) and capacity (number of ingested yeast cells per cell) of leucocytes was analysed by flow cytometry. The results demonstrate the potential existence of a specific receptor-sugar or receptor-yeast cell binding process, which was saturable, specific and dose-dependent. More specifically, when leucocytes were preincubated with appropriate doses of d-mannose, d- or l-fucose, d-glucose or n-acetyl-glucosamine the phagocytosis of yeast cells by head-kidney leucocytes was partially blocked. Seabream leucocytes were also preincubated with chloroquine, a lysosomotropic drug which downregulates (in a nonspecific manner) the expression of mannose-receptors in mammals, before phagocytosis assays were performed. The results demonstrated that the phagocytosis of yeast was completely blocked by this substance. The overall results seem to corroborate the presence of the mannose-receptor in seabream phagocytes, which is involved in the non-specific binding and phagocytosis of yeast cells by head-kidney leucocytes.

Neutrophil functions and serum IgG in growing foals.

The aim of this study was to investigate the phagocytic and killing capacities as well as expression of CD18 of neutrophils obtained from healthy foals from birth to age 8 months. Blood was taken from 6 Standardbred foals at 7 time-points between ages 2-56 days and thereafter once a month. For comparison, cells from 16 mature horses were evaluated. Neutrophil phagocytosis of yeast cells was assessed by flow cytometry after opsonisation with mature pooled serum, autologous serum or anti-yeast IgG. The killing capacity of the neutrophils, as indicated by the oxidative burst, was monitored by chemiluminescence. Serum IgG concentration was measured by radial immunodiffusion. In addition to clinical examination, the amount of serum amyloid A and the total leucocyte count were used as markers for infection. The phagocytic ability was impaired until age 3 weeks, when autologous serum was used as opsonin. Killing capacity was also low initially but, from 3 months onwards, chemiluminescence values were equal to or higher than in mature horses. Serum IgG decreased from 10 g/l at 2 days to 5 g/l at 2 months and then increased gradually to 10 g/l at the end of the study. These findings may in part explain the increased susceptibility to bacterial infections in young horses.

Opsonization of yeast cells with equine iC3b, C3b, and IgG

The main opsonins in serum are antibodies and complement factor C3. The opsonization mechanisms including complement activation and deposition are important in studies of phagocytosis and of mechanisms of microbial immune evasion. The objective of the present study was to monitor the deposition of complement C3 and IgG from equine serum on yeast cells ( Saccharomyces cerevisiae) using a flow cytometric immunoassay. Correlations were made between the opsonic coating and phagocytic capacity using equine blood neutrophils. In addition, the bound C3 fragments were characterized by SDS–PAGE and Western blot analyses. Opsonic coating of yeast with equine C3 and IgG occurred rapidly with detectable levels with as little as 0.75% serum. C3 deposition was a result of complement activation and no passive adsorption was observed. When complement was inactivated, the fluorescence indicating IgG deposition increased 3–6-fold, indicating spatial competition between C3 and IgG at binding. Opsonization with 1.5% serum led to suboptimal equine neutrophil phagocytosis of yeast cells which was dependent on complement activation by the classical pathway. With ≥6.25% serum, IgG contributed to opsonization and phagocytosis. With 50% serum and more, C3 was deposited also by the alternative pathway. Phagocytosis rates became optimal with 3% serum, and did not increase further with higher serum concentrations. The main form of C3 on the yeast cells was iC3b and the rest was C3b without any detectable breakdown products (C3c or C3dg). The equine complement components are similar in size to the human equivalents. It may be concluded that opsonization of yeast particles leading to phagocytosis, occurs at very low serum concentrations (1.5%) and that it is dependent on activation of the classical complement pathway at this low opsonic level. This is an important finding for efficient host defense, e.g. extravascular phagocytosis at infection sites.

In vitro production of superoxide and nitric oxide (as nitrite and nitrate) by Mytilus galloprovincialis haemocytes upon incubation with PMA or laminarin or during yeast phagocytosis

The phagocytic process is one of the most important elements of the self-defence system in mammals as well as in molluscs. In mammalian phagocytes, superoxide participates in the innate defence system by combining with nitric oxide to generate peroxynitrite, a strong oxidant that possesses highly cytotoxic properties against bacteria. To evidence a role of nitric oxide in the self-defence system of the marine bivalve Mytilus galloprovincialis similar to the role observed in the mammalian defence system, we measured the generation of superoxide and nitrite/nitrate (the stable end products of nitric oxide) upon in vitro stimulation of M. galloprovincialis haemocytes with PMA, laminarin, LPS and by phagocytosis of Saccharomyces cerevisiae (yeast cells). We show that stimulation with PMA, laminarin and yeast cell phagocytosis promotes superoxide and nitrite/nitrate generation from M. galloprovincialis haemocytes. Inhibitors of NADPH oxidase and inhibitors of NO synthase decreased the nitrite/nitrate levels generated by M. galloprovincialis haemocytes showing that both NADPH oxidase and NO synthase pathways are involved in the self-defence system of M. galloprovincialis.