Gram-negative bacteria and phagocytic cell interaction mediated by complement receptor 3

Complement receptor 3 (CR3) is an integrin that recognizes several different ligands. Binding to CR3 in phagocytic cells activates signaling pathways involved in cytoskeleton rearrangement, regulation of cell motility, alteration of gene expression and phagocytosis of complement-opsonized as well as of some non-opsonized particles and pathogenic bacteria. However, CR3-mediated phagocytosis of some Gram-negative bacteria does not induce bacterial clearance. Pseudomonas aeruginosa, Salmonella and Escherichia coli are eliminated after phagocytic cell-bacteria interaction mediated by CR3. However, Bordetella takes advantage of the CR3 function and uses it to enter into macrophages leading to bacterial survival. The final fate of the pathogen is determined by combinations of host and bacterial factors, in which molecular interactions between CR3 and bacterial ligands are involved.     

The influence of Conidiobolus coronatus on phagocytic activity of insect hemocytes

An essential component of the insect cellular response is phagocytosis. Analyses of the in vitro phagocytosis could be useful for the studies of the relationship between insects and their pathogens. Fungal metabolites are known to inhibit phagocytosis whereas components of the fungal cell wall stimulate phagocytosis. To achieve a better understanding of fungal pathogenesis in insects, haemocyte populations of two insect species susceptible to Conidiobolus coronatus infection (Galleria mellonella, Dendrolimus pini ) were compared with haemocytes of the resistant species (Calliphora erythrocephala ). Fungal infection increased phagocytic activity of G. mellonella plasmatocytes 3.3 times and this of D. pini plasmatocytes 2.1 times. Analysis of infected C. erythrocephala larvae did not reveal any influence of C. coronatus upon phagocytic activity.     

Morphofunctional parameters of hemocytes in the assessment of the physiological status of bivalves

This review presents data on the morphofunctional properties of bivalve hemocytes and considers the effects of the most significant biotic and abiotic factors on the hemolymph cell population dynamics and the immunological parameters of hemocytes. The prospects for the use of hematological criteria in assessing the health status of bivalves are discussed.     

Autoradiographic and cytochemical studies of phagocytic cells in selected fibre tracts of the mouse periodontium

Proliferative and protein synthetic activities of phagocytic cells of specific fibre tracts of the periodontium of C57Bl mice were employing autoradiographic techniques; these were combined with a histochemical technique for horseradish peroxidase (HRP) as a marker for phagocytic activity. Animals were injected either with [3H]thymidine as a marker for proliferative activity, or with [3H]proline as a marker for protein synthetic activity prior to HRP injection. Blocks from the maxillae of experimental and control animals were fixed, decalcified, and sectioned at 50 micrometers. These were incubated with HRP localization media, dehydrated and flat embedded in Epon 812 wafers. The entire length of the periodontium, including adjacent tooth and bone, were selectively cut from the wafers, mounted on epoxy blocks and serially sectioned at 2 micrometers. Slides containing these sections were then dipped in NTB-3 nuclear track emulsion, and after appropriate exposure times, were developed and post-stained. Sections were examined microscopically, employing an ocular grid, and phagocytic cells within each area examined were delineated as either 'fibroblast-like' (FL cells) or 'endothelial/macrophage-like' (EML cells) according to criteria such as morphology, location, orientation and proximity to a vascular channel. They were then subclassified as labelled or unlabelled with respect to the autoradiographic markers. The thymidine labelling index obtained for non-phagocytic FL cells was 3.09%; this was more than twice that for phagocytic FL cells (1.35%). Similarly phagocytic FL cells in all regions studied incorporated less than half as much [3H]proline as did their non-phagocytic counterparts. This was determined by silver grain counts over HRP-stained and unstained cells using a matched pair system. In addition, the variation of the relative number of phagocytic FL cells in specific fibre tracts suggested a relationship to functional demand. The distribution of these cells was closely related to experimentally determined rates of protein turnover. Phagocytic FL cells have a markedly reduced proliferative rate and synthesize proline-containing proteins at a reduced rate. This may reflect protein production primarily for the purpose of cell maintenance. These findings are consistent with the presence of subpopulations of fibroblasts (or fibrocytes) developmentally or functionally modified for phagocytosis; alternatively, this could signify modulation of fibroblasts from primarily biosynthetic activities to degradative functions in response to varying microenvironmental conditions.     

NMR studies on the conformation of oligomannosides and their interaction with banana lectin

The conformational and dynamic behaviour of three mannose containing oligosaccharides, a tetrasaccharide with α1→2, and α1→3, and a penta and a heptasaccharide with α1→2, α1→3, and α1→6 linkages has been evaluated by molecular mechanics and dynamics simulations and NMR spectroscopical methods. It is found that they display a fair amount of conformational freedom, with one major and one minor conformation per glycosidic linkage. The evaluation of their recognition by banana lectin has also been performed by STD NMR methods and a preliminary view of their putative interaction mode has been carried out by means of docking procedures.     

On the evolutionary ecology of symbioses between chemosynthetic bacteria and bivalves

Mutualistic associations between bacteria and eukaryotes occur ubiquitously in nature, forming the basis for key ecological and evolutionary innovations. Some of the most prominent examples of these symbioses are chemosynthetic bacteria and marine invertebrates living in the absence of sunlight at deep-sea hydrothermal vents and in sediments rich in reduced sulfur compounds. Here, chemosynthetic bacteria living in close association with their hosts convert CO₂ or CH₄ into organic compounds and provide the host with necessary nutrients. The dominant macrofauna of hydrothermal vent and cold seep ecosystems all depend on the metabolic activity of chemosynthetic bacteria, which accounts for almost all primary production in these complex ecosystems. Many of these enigmatic mutualistic associations are found within the molluscan class Bivalvia. Currently, chemosynthetic symbioses have been reported from five distinct bivalve families (Lucinidae, Mytilidae, Solemyidae, Thyasiridae, and Vesicomyidae). This brief review aims to provide an overview of the diverse physiological and genetic adaptations of symbiotic chemosynthetic bacteria and their bivalve hosts.     

Microscopical and spectroscopic studies on the fluorescence of a daunomycin-aluminum complex.

In this study, the spectroscopic features and microscopical applications of the fluorescent daunomycin-Al3+ complex have been analyzed. In the presence of Al3+, the absorption spectrum of daunomycin showed a deep bathochromic shift and new peaks at 529 and 566 nm, whereas the fluorescence emission was considerably modified. The emission of daunomycin alone (peak at 560 nm under optimal excitation at 470 nm) decreased continuously from 0.5 to 24h after addition of Al3+ ions, and a new emission peak appeared at 580 nm (optimal excitation at 530 nm). Under the fluorescence microscope using green exciting light, nuclei from chicken blood smears and paraffin sections of rat embryos stained with daunomycin showed a weak emission, which greatly increased after treatment with Al3+ ions. The bright and stable fluorescence of chromatin DNA induced by daunomycin-Al3+ could be a valuable labelling method in fluorescence microscopy and DNA cytochemistry.     

Immunogen-dependent quantitative and qualitative differences in phagocytic responses of the circulating hemocytes of the lobster Homarus americanus

Phagocytic responses in circulating hemocytes of the lobster Homarus americanus were measured before and after treatment of lobsters with 2 different immunogens: (1) lipolysaccharide (LPS) or endotoxin from a non-pathogenic Pseudomonas perolens, and (2) a vancomycin/live Gram-positive pathogen (Aerococcus viridans [var.] homari) combination, essentially attenuated cells, shown previously to induce a high degree of resistance to this pathogen. The responses elicited by each of the immunogens were markedly different. Hemocytes drawn from LPS-treated lobsters showed significant, largely non-specific, increases in phagocytic responses over baseline values against sheep red blood cells and an array of test bacteria, with the notable exception of the pathogen. In marked contrast, induction with the vancomycin/live pathogen combination resulted in highly significant and specific increases in phagocytic responses to the pathogen and to the related, (but avirulent) strains of the pathogen, as well as inducing in the lobsters the usual high degree of resistance to the pathogen. These results suggest that quantitative and qualitative variations in phagocytic and resistance levels induced in at least 1 crustacean genus are determined largely by the particular characteristics of the immunogen.     

Establishment of phagocytic cell lines from larval hemocytes of the beet armyworm, Spodoptera exigua

Hemolymph was taken from beet armyworm (Spodoptera exigua) larvae and a new hemocyte cell line (SeHe920-1a) was established by supplementing the culture medium with a reduced form of glutathione to avoid the activation of prophenoloxidase cascade. To evaluate the phagocytic ability of the SeHe920-1a cells, polystyrene microspheres of two sizes (6.14 +/- 0.45 microm and 2.84 +/- 0.14 microm in diameter) and inactivated spores of an entomopathogenic microsporidium, Vairimorpha sp. NIS M12 (5.10 +/- 0.21 microm x 2.00 +/- 0.11 microm), were introduced into the cell culture. The SeHe920-1a cells had higher phagocytic ability than other lepidopteran cell lines that were not derived from the hemocytes. When microsporidian spores were inoculated, 27% of SeHe920-1a cells were observed to take up spores (average 1.7 spores per cell). By cloning SeHe920-1a cells, 12 cell lines were established and designated SeHe920Y1 to SeHe920Y12. In comparison with the parental cell line, phagocytic activity was enhanced in SeHe920Y6, SeHe920Y10, and SeHe920Y11 cell lines and especially in the SeHe920Y7 cell line, where approximately 50% of cells were phagocytic and the average number of microsporidian spores engulfed per cell was twice that of the SeHe920-1a cell line.     

Root border-like cells of Arabidopsis. Microscopical characterization and role in the interaction with rhizobacteria

Plant roots of many species produce thousands of cells that are released daily into the rhizosphere. These cells are commonly termed border cells because of their major role in constituting a biotic boundary layer between the root surface and the soil. In this study, we investigated the occurrence and ultrastructure of such cells in Arabidopsis (Arabidopsis thaliana) using light and electron microscopy coupled to high-pressure freezing. The secretion of cell wall molecules including pectic polysaccharides and arabinogalactan-proteins (AGPs) was examined also using immunofluorescence microscopy and a set of anticarbohydrate antibodies. We show that root tips of Arabidopsis seedlings released cell layers in an organized pattern that differs from the rather randomly dispersed release observed in other plant species studied to date. Therefore, we termed such cells border-like cells (BLC). Electron microscopical results revealed that BLC are rich in mitochondria, Golgi stacks, and Golgi-derived vesicles, suggesting that these cells are actively engaged in secretion of materials to their cell walls. Immunocytochemical data demonstrated that pectins as well as AGPs are among secreted material as revealed by the high level of expression of AGP-epitopes. In particular, the JIM13-AGP epitope was found exclusively associated with BLC and peripheral cells in the root cap region. In addition, we investigated the function of BLC and root cap cell AGPs in the interaction with rhizobacteria using AGP-disrupting agents and a strain of Rhizobium sp. expressing a green fluorescent protein. Our findings demonstrate that alteration of AGPs significantly inhibits the attachment of the bacteria to the surface of BLC and root tip.     

Light and electron microscopic studies of crayfish hemocytes

Using light and electron microscopy, three hemocyte types are described in the hemolymph of the crayfish. The coagulocyte comprises 65% of the total hemocyte number and contains medium-sized cytoplasmic granules, abundant dilated rough endoplasmic reticulum, and a highly developed Golgi complex. It rapidly undergoes cytolysis in vitro and participates in coagulation by releasing the contents of its granules to the hemolymph. The granulocyte comprises 31% of the total hemocyte number and is capable of phagocytosis. It contains large, irregularly shaped cytoplasmic granules, a moderately developed Golgi complex, and moderate amounts of non-dilated rough endoplasmic reticulum. During coagulation in vitro, the cell attaches and spreads onto the substratum; this is followed by a slow intracellular granule breakdown and cytolysis. The amebocyte comprises 4% of the total hemocyte number and it is also capable of phagocytosis. It possesses small cytoplasmic granules, many vacuoles, a moderately developed Golgi complex, and large amounts of smooth endoplasmic reticulum. It is distinguished from the other two cell types by being stable and motile in vitro.     

Role of bivalves in the depuration of seawaters contaminated by bacteria

Bacterial contamination the inshore areas of seas, is currently one of the most common manifestations of increasing anthropogenic pressure on aquatic ecosystems. Permanent sources of allochthonic microflora in the inshore areas of seas, are polluted with untreated or insufficiently treated domestic and industrial sewage [5, 8, 12, 45, 66] and river run-off from areas of active water use [22, 28], and suffer the effects of over-exploitation of beaches during the bathing season (in the summer).     

Enhancing phagocytic activity of hemocytes and disease resistance in the prawn Penaeus merguiensis by feeding Spirulina platensis

Exposing the prawn Penaeus merguiensis to the bacteria Vibrio harveyi and Escherichia coli for an hour or feeding the prawns with Spirulina (Arthrospira) platensis (0.3% w/w feed) enhanced the phagocytic activity of their hemocytes. Improvement of the phagocytic activity was primarily through the activation of the hemocytes. The activated phagocytic hemocytes had a higher capacity to engulf foreign agents, such as bacteria, and a higher rate of phagocytosis. The phagocytic enhancement effect peaked on the fourth day of feeding with Spirulina. In the in vitro study, the granular cells from prawns took 45–60 min to complete the process of degranulation. Pre-exposure to Salmonella typhimurium and Bacillus subtilis did not result in enhancement of phagocytic activity of hemocytes. Only 10% prawns fed with Spirulina died in the first 14 days when challenged by V. harveyi at a concentration of 1 × 10⁴CFUs mL^–1, while all control prawns (basal feed without Spirulina) died within 14 days.     

Effect of preinjection of Crassostrea virginica with bacteria on subsequent chemotactic response by its hemocytes

It has been demonstrated that hemocytes collected from the American oyster, Crassostrea virginica, 2 hr postchallenge with live Bacillus megaterium are significantly less chemotactic to this bacterium. Since chemotaxis between hemocytes and non-self materials is an integral part of the cellular internal defense mechanism in mollusks, this finding may be important in our understanding of reduced cellular reactions in prechallenged animals.     

Photothermal studies of modulating effect of photoactivated chlorin on interaction of blood cells with bacteria.

BACKGROUND: Bactericidal application of photosensitizers (PS) is a new and promising area. Up to now the action of PS against bacteria was studied without regard to immunocompetent cells, doses of drugs and radiation being used usually are not safe for such cells and therefore there is still no efficient model for PS application. METHODS: Action of chlorin, a plant derived photosensitizer, on a system of interacting cells and microbes was studied. Cell monitoring was done with a photothermal microscopy method. The two bacteria used were gram-positive (S. aureus) and gram-negative (E. coli) in a mixture of basic blood cells and neutrophils. The latter were used to model phagocytosis as fast cell response reaction. RESULTS: The strongest bactericidal effect was found when the cells and photoactivated chlorin act together. Selective bactericidal regime of PS application which does not affect cell viability was demonstrated. CONCLUSIONS: The results obtained suggest that this photosensitizer may enhance cell natural defense capabilities.     

Specific interaction of murine colony-stimulating factor with mononuclear phagocytic cells

L-cell colony-stimulating factor (CSF) is identical to macrophage growth factor and stimulates macrophage proliferation (Stanley et al., 1976, J. Exp. Med. 143: 631-647). The nature of the interaction of iodinated L-cell CSF (125I-CSF) with murine peritoneal exudate macrophages was studied. On incubation with 10 pM 125I-CSF at 0 degrees C, cellular binding of 125I-CSF reaches a stable maximum within 15 h. This is in contrast to the association behavior at higher temperatures. At 37 degrees C, cell-associated 125I-CSF levels reach, within 45 min, an unstable maximum which is up to 10-fold less than that occurring under the same conditions at 0 degrees C. At 0 degrees C, binding is saturated (approximately 5 X 10(4) sites/cell) at CSF concentrations of 1 nM. A comparison of binding and competition experiments indicates that iodinated L-cell CSF binds as effectively as L-cell CSF and that human urinary CSF and L-cell CSF equipotently compete for 125I-CSF binding. Specificity of the CSF-binding site is demonstrated by the failure of other known growth factors and hormones to compete for 125I- CSF binding. These studies and other findings suggest that 125I-CSF binding is restricted to macrophages and their precursors and to macrophage cell lines and that the binding site(s) is the receptor mediating the biological action of this CSF.