Cellular clot formation in a sipunculan worm: entrapment of foreign particles, cell death and identification of a PGRP-related protein

Clotting in animals having open or closed circulatory system comprises humoral and cellular mechanisms. Sipunculans are a small phylum of non-segmented marine worms that do not have a true circulatory system. These worms can form a cellular clot without transforming cell-free coelomic fluid into an insoluble mass. The clot may also contribute to immune response by entrapping foreign particles. We evaluated the formation of a cellular clot ex vivo in the sipunculan Themiste petricola after activation through glass contact and sea water, the ability to entrap magnetic beads and non-pathogen cyanobacteria particles within the clot, and the presence of a peptidoglycan recognition protein S (PGRP-S) antigen in cells forming the clot. Our results showed that the clot was formed by homotypic aggregation of large granular leukocytes (LGLs), a subtype of cells found in the coelomic fluid. Aggregated LGLs served to entrap magnetic beads and non-pathogen cyanobacteria particles, and PGRP-S antigen was detected both in non-activated LGLs and in activated homotypic aggregates through immunofluorescence, Western blot and flow cytometry. Cellular clots were found to be positive to Annexin V-FITC labelling. Complete disintegration of cytoplasm with shedding of microparticles, nuclear isolation and degradation were also observed by light microscopy and flow cytometry. In conclusion, cellular clot formation in Themiste petricola may serve both haemostatic and immune functions entailing rapid activation changes in LGLs, entrapment of foreign particles within a homotypic aggregate, and further cell death.     

NMR fragment-based screening for development of the CD44-binding small molecules

The cell-surface protein CD44, a primary receptor for hyaluronic acid (HA), is one of the most promising targets for cancer therapies. It is prominently involved in the process of tumor growth and metastasis. The possibility of modulating the CD44-HA interaction with a pharmacological inhibitor is therefore of great importance, yet until now there are only few small molecules reported to bind to CD44. Here, we describe the results of the NMR fragment-based screening conducted against CD44 by which we found eight new hit compounds that bind to the receptor with the affinity in milimolar range. The NMR-based characterization revealed that there are two possible binding modes for these compounds, and for some of them the binding is no longer possible in the presence of hyaluronic acid. This could provide an interesting starting point for the development of new high-affinity ligands targeting the CD44-HA axis.     

The CD44 Receptor of Lymphoma Cells: Structure-Function Relationships and Mechanism of Activation

Migration of some tumor cells, and their lodgment in target organs, is dependent on the activation of cell surface CD44 receptor, usually detected by its ability to bind hyaluronic acid (HA) or other ligands. In an attempt to reveal the mechanism of tumor cell CD44 activation, we compared the physical and chemical properties of CD44 in nonactivated LB cell lymphoma with those in phorbol 12-myristate 13-acetate (PMA)-activated LB cells and of an LB cell subline (designated HA9) expressing constitutively-active CD44. In contrast to nonactivated LB cells, PMA-activated LB cells and HA9 cells displayed a CD44-dependent ability to bind HA. The ability of activated cell CD44 to bind HA was not dependent on microfilament or microtubule integrity or on changes in CD44 mobility on the membrane plane, indicating that the CD44 activation status is not associated with cytoskeleton function. Aside from the increased expression of CD44 on the surface of PMA-activated LB cells and HA9 cells, qualitative differences between the CD44 of nonactivated and activated LB cells were also detected: the CD44 of the activated lymphoma was (i) larger in molecular size, (ii) displayed a broader CD44 isoform repertoire, including a CD44 variant that binds HA, and (iii) its glycoprotein contained less sialic acid. Indeed, after removal of sialic acid from their cell surface by neuraminidase, LB cells acquired the ability to bind HA. However, a reduced dose of neuraminidase did not confer HA binding on LB cells, unless they were also activated by a low concentration of PMA, which by itself was ineffective. Similarly, under suboptimal conditions, a synergistic effect was obtained with tunicamycin and PMA: each one alone was ineffective but in combination they induced the acquisition of HA binding by the lymphoma cells, while their CD44 expression was not enhanced. Unveiling of the activation mechanism of CD44, by exposing the cells to PMA stimulation or to deglycosylation, is not only academically important, but it also has practical implications, as activated CD44 may be involved in the support of tumor progression.     

Phosphatidylinositol 4,5-Bisphosphate Clusters the Cell Adhesion Molecule CD44 and Assembles a Specific CD44-Ezrin Heterocomplex,as Revealed by Small Angle Neutron Scattering

The cell adhesion molecule CD44 regulates diverse cellular functions, including cell-cell and cell-matrix interaction, cell motility, migration, differentiation, and growth. In cells, CD44 co-localizes with the membrane-cytoskeleton adapter protein Ezrin that links the CD44 assembled receptor signaling complexes to the cytoskeletal actin network, which organizes the spatial and temporal localization of signaling events. Here we report that the cytoplasmic tail of CD44 (CD44ct) is largely disordered. Upon binding to the signaling lipid phosphatidylinositol 4,5-bisphosphate (PIP2), CD44ct clusters into aggregates. Further, contrary to the generally accepted model, CD44ct does not bind directly to the FERM domain of Ezrin or to the full-length Ezrin but only forms a complex with FERM or with the full-length Ezrin in the presence of PIP2. Using contrast variation small angle neutron scattering, we show that PIP2 mediates the assembly of a specific heterotetramer complex of CD44ct with Ezrin. This study reveals the role of PIP2 in clustering CD44 and in assembling multimeric CD44-Ezrin complexes. We hypothesize that polyvalent electrostatic interactions are responsible for the assembly of CD44 clusters and the multimeric PIP2-CD44-Ezrin complexes.     

The hyaluronic acid receptor (CD44) is expressed in bovine oocytes and early stage embryos

Hyaluronic acid (HA) is a high molecular weight polysaccharide found in the extracellular matrix of most animal tissues, that exerts a profound influence on cell behavior. HA is one of the most abundant glycosaminoglycans (GAGs) in the uterine, oviductal and follicular fluids in mouse, pig, human and cattle. CD44, the principal cell membrane receptor for HA, is expressed from the 1- to 8-cell stage in human embryos, during post-implantation mouse embryogenesis and on the surface of differentiated embryonic stem cells. In the present study, we have analyzed by immunofluorescence, whether CD44 is present in bovine oocytes, fertilized oocytes and early stage embryos. Bovine cumulus–oocyte complexes (COCs) were aspirated from follicles (2–5 mm) and were selected for IVM and incubated for 24 h. Oocytes showing an expanded cumulus (generally 90–95%) were used for IVF. Fertilized oocytes were separated for immunofluorescence assay after 16 h of sperm incubation in order to fix the eggs at the pronuclear stage. The embryos were cultured for 8 days and the different stages of development for immunofluorescence assay were separated every 24 h of culture. The CD44 receptor was detected at every observation time examined. Fluorescence-tagged HA for the internalization assay was prepared by mixing fluorescein amine, Isomer I and 1 mg of HA from umbilical cord. Fluorescence-tagged HA was internalized in 2-, 4-, 8- and 16-cell-stage embryos, morulae and blastocysts. CD44 is expressed on the surface and in the cytoplasm of bovine oocytes and embryos in different stages of development.     

Multisite and bidirectional exonic splicing enhancer in CD44 alternative exon v3

The human CD44 gene encodes multiple isoforms of a transmembrane protein that differ in their extracellular domains as a result of alternative splicing of its variable exons. Expression of CD44 is tightly regulated according to the type and physiological status of a cell, with expression of high molecular weight isoforms by inclusion of variable exons and low molecular weight isoforms containing few or no variable exons. Human CD44 variable exon 3 (v3) can follow a specific alternative splicing route different from that affecting other variable exons. Here we map and functionally describe the splicing enhancer element within CD44 exon v3 which regulates its inclusion in the final mRNA. The v3 splicing enhancer is a multisite bipartite element consisting of a tandem nonamer, the XX motif, and an heptamer, the Y motif, located centrally in the exon. Each of the three sites of this multisite enhancer partially retains its splicing enhancing capacity independently from each other in CD44 and shows full enhancing function in gene contexts different from CD44. We further demonstrate that these motifs act cooperatively as at least two motifs are needed to maintain exon inclusion. Their action is differential with respect to the splice-site target abutting v3. The first X motif acts on the 3' splice site, the second X motif acts on both splice sites (as a bidirectional exonic splicing enhancer), and the Y motif acts on the 5' splice site. We also show that the multisite v3 splicing enhancer is functional irrespective of flanking intron length and spatial organization within v3.     

CD44与HA特异性结合在肿瘤靶向给药中的应用

设计肿瘤靶向性抗癌药物一直是研究热点。因为CD44在许多肿瘤细胞表面过表达,所以基于CD44与其配体透明质酸（hyaluronic acid,HA）的相互作用设计肿瘤靶向药物成为一个新的研究方向。本文介绍了CD44的基本结构7LCD44与HA之间的相互作用,并对以CD44为靶点的靶向抗癌药物研究进展进行了简介。     

Interaction of CD44 with different forms of hyaluronic acid. Its role in adhesion and migration of tumor cells

Interaction between hyaluronic acid (HA) and CD44 has been considered a key event in tumor invasion and metastasis. HA is a linear, high molecular weight glycosaminoglycan in its native state, but fragmented low molecular forms are found at sites of neoplastic or inflammatory infiltrates. Both high and low molecular weights HA are involved in diverse biological functions. In this study, we used two clonal variants of a T cell murine lymphoma designated LBLa and LBLc. These cell lines were found to differ in their in vivo and in vitro growth rates. LBLa grew faster and exhibited an enhanced invasive capacity as compared to LBLc. In contrast, cell lines did not differ in the expression of surface markers (CD8, CD24, CD25, CD44, and CD18), or in their capacity to bind HA. However, LBLa cells exhibited higher capacity to migrate to low molecular weight HA than did LBLc. Migration was mediated by CD44 since it was abrogated by anti-CD44 monoclonal antibody as well as by hyaluronidase. We suggest that interaction between CD44 and low molecular weight HA may trigger migration mechanisms in LBLa cells, thus contributing to enhanced invasive cell capacity.     

Effects of the novel anthelmintic emodepside on the locomotion, egg-laying behaviour and development of Caenorhabditis elegans

Emodepside, a cyclooctadepsipeptide, is a broad-spectrum anthelmintic previously shown to paralyse body wall muscle and pharyngeal muscle in the model nematode Caenorhabditis elegans. We demonstrate that wild-type C. elegans L4 are less sensitive than adults to emodepside in two independent assays of locomotor behaviour: body bend generation on agar (adult IC(50) 3.7 nM, L4 IC(50) 13.4 nM) and thrashing behaviour in liquid (thrashing behaviour as a % of controls after 1h in 10 microM emodepside: adults 16%, L4 worms 48%). We also show that continuous exposure of wild-type C. elegans to emodepside throughout the life-cycle from egg onwards, slows worm development, an effect that is emodepside concentration-dependent. The rate of worm-hatching from eggs on agar plates containing emodepside was not significantly different from controls, suggesting that it is development post-hatching rather than hatching itself that is affected by the drug. Emodepside also inhibits wild-type C. elegans egg-laying, with acute exposure to the drug at 500 nM resulting in an almost total inhibition within the first hour. However, the rate of egg production was not inhibited and therefore emodepside-treated worms became bloated with eggs, eventually rupturing. This suggests that the effect of emodepside on reproduction is not due to an inhibition of egg production but rather a paralytic effect on the egg-laying muscles. These results, when coupled with previous research, suggest that emodepside interferes with signalling at the neuromuscular junction on the body-wall muscles (Willson et al., 2003), pharynx (Willson et al., 2004) and egg-laying muscles and thus inhibits three important physiological functions: locomotion, feeding and reproduction.     

Leishmania major: species specific delayed hypersensitivity reaction induced by exogenous secreted antigen in the guinea pig

The cellular response to Leishmania major (L. major) is usually evaluated in vivo by the delayed-type-hypersensitivity (DTH) test using leishmanin. Leishmanin can give false-positive reactions in areas where there is a background of leishmaniasis. In a previous study, it was shown that a 56 kDa antigen purified from promastigote and culture supernatant of L. major induce strong DTH reactions in sensitized guinea pigs. In this study, the species-specificity of this antigen was further investigated. Three groups of guinea pigs were sensitized with L. major, L. tropica, and L. infantum and both flanks of sensitized animal were injected intradermally with purified 56 kDa antigen or soluble leishmania antigen (SLA). The extent of indurations were measured after 24, 48, and 72 h. In animals which were sensitized with three species of leishmania, only those immunized with L. major showed skin reactions to purified antigen by an increase in skin thickness. Since complex antigen mixtures such as SLA and leishmanin show cross-reactivity and can be non-specific, the result obtained here suggest that 56 kDa antigen may be a useful diagnostic tool for species specific diagnosis in field studies of leishmaniasis.     

Characterization of the expression of variant and standard CD44 in prostate cancer cells: Identification of the possible molecular mechanism of CD44/MMP9 complex formation on the cell surface

CD44 is a glycosylated adhesion molecule and osteopontin is one of its ligand. CD44 undergoes alternative splicing to produce variant isoforms. Our recent studies have shown an increase in the surface expression of CD44 isoforms (sCD44 and v4–v10 variant CD44) in prostate cancer cells over‐expressing osteopontin (PC3/OPN). Formation of CD44/MMP9 complex on the cell surface is indispensable for MMP9 activity. In this study, we have characterized the expression of variant CD44 using RT‐PCR, surface labeling with NHS–biotin, and immunoblotting. Expression of variant CD44 encompassing v4–v10 and sCD44 at mRNA and protein levels are of the same levels in PC3 and PC3/OPN cells. However, an increase in the surface expression of v6, v10, and sCD44 in PC3/OPN cells suggest that OPN may be a ligand for these isoforms. We then proceeded to determine the role of sCD44 in MMP9 activation. Based on our previous studies in osteoclasts, we hypothesized that phosphorylation of CD44 has a role on its surface expression and subsequent activation of MMP9. We have prepared TAT‐fused CD44 peptides comprising unphosphorylated and constitutively phosphorylated serine residues at positions Ser323 and Ser325. Transduction of phosphopeptides at Ser323 and Ser323/325 into PC3 cells reduced the surface levels of CD44, MMP9 activity, and cell migration; but had no effect on the membrane localization of MMP9. However, MMP9 knock‐down PC3 cells showed reduced CD44 at cellular and surface levels. Thus we conclude that surface expression of CD44 and activation of MMP9 on the cell surface are interdependent. J. Cell. Biochem. 108: 272–284, 2009. © 2009 Wiley‐Liss, Inc.     

CD147 mediates the CD44s-dependent differentiation of myofibroblasts driven by transforming growth factor-β1

Progressive fibrosis leads to loss of organ function and affects many organs as a result of excessive extracellular matrix production. The ubiquitous matrix polysaccharide hyaluronan (HA) is central to this through association with its primary receptor, CD44, which exists as standard CD44 (CD44s) or multiple splice variants. Mediators such as profibrotic transforming growth factor (TGF)-β1 and proinflammatory interleukin (IL)-1β are widely associated with fibrotic progression. TGF-β1 induces myofibroblast differentiation, while IL-1β induces a proinflammatory fibroblast phenotype that promotes fibroblast binding to monocyte/macrophages. CD44 expression is essential for both responses. Potential CD44 splice variants involved, however, are unidentified. The TGF-β1-activated CD44/epidermal growth factor receptor complex induces differentiation of metastatic cells through interactions with the matrix metalloproteinase inducer, CD147. This study aimed to determine the CD44 variants involved in TGF-β1- and IL-1β-mediated responses and to investigate the potential profibrotic role of CD147. Using immunocytochemistry and quantitative PCR, standard CD44s were shown to be essential for both TGF-β1-induced fibroblast/myofibroblast differentiation and IL-1β-induced monocyte binding. Co-immunoprecipitation identified that CD147 associated with CD44s. Using CD147-siRNA and confocal microscopy, we also determined that incorporation of the myofibroblast marker, αSMA, into F-actin stress fibers was prevented in the absence of CD147 and myofibroblast-dependent collagen gel contraction was inhibited. CD147 did not associate with HA, but removal of HA prevented the association of CD44s with CD147 at points of cell–cell contact. Taken together, our data suggest that CD44s/CD147 colocalization is essential in regulating the mechanical tension required for the αSMA incorporation into F-actin stress fibers that regulates myofibroblast phenotype.     

Differential use of chondroitin sulfate to regulate hyaluronan binding by receptor CD44 in Inflammatory and Interleukin 4-activated Macrophages

CD44 is a cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is involved in processes ranging from leukocyte recruitment to wound healing. In the immune system, the binding of hyaluronan to CD44 is tightly regulated, and exposure of human peripheral blood monocytes to inflammatory stimuli increases CD44 expression and induces hyaluronan binding. Here we sought to understand how mouse macrophages regulate hyaluronan binding upon inflammatory and anti-inflammatory stimuli. Mouse bone marrow-derived macrophages stimulated with tumor necrosis factor α or lipopolysaccharide and interferon-γ (LPS/IFNγ) induced hyaluronan binding by up-regulating CD44 and down-regulating chondroitin sulfation on CD44. Hyaluronan binding was induced to a lesser extent in interleukin-4 (IL-4)-activated macrophages despite increased CD44 expression, and this was attributable to increased chondroitin sulfation on CD44, as treatment with β-d-xyloside to prevent chondroitin sulfate addition significantly enhanced hyaluronan binding. These changes in the chondroitin sulfation of CD44 were associated with changes in mRNA expression of two chondroitin sulfotransferases, CHST3 and CHST7, which were decreased in LPS/IFNγ-stimulated macrophages and increased in IL-4-stimulated macrophages. Thus, inflammatory and anti-inflammatory stimuli differentially regulate the chondroitin sulfation of CD44, which is a dynamic physiological regulator of hyaluronan binding by CD44 in mouse macrophages.     

Peptidases and gp63-like proteins in Herpetomonas megaseliae: possible involvement in the adhesion to the invertebrate host

The cell-associated and extracellular peptidases of Herpetomonas megaseliae grown in brain-heart infusion and in modified Roitman's complex media were analyzed by measuring peptidase activity on gelatin, casein and hemoglobin in zymograms. Casein was the best proteinaceous substrate for the peptidase detection on both growth conditions. However, no proteolytic activity was detected when hemoglobin was used. Our results showed that cellular cysteine peptidase (115-100, 40 and 35 kDa) and metallopeptidase (70 and 60 kDa) activities were detected on both media in casein and gelatin zymograms. Additionally, the use of casein in the gel revealed a distinct acidic metallopeptidase of 50 kDa when the parasite was cultured in the modified Roitman's complex medium. Irrespective of the culture medium composition, H. megaseliae released metallopeptidases exclusively in the extracellular environment. The presence of gp63-like molecules on the H. megaseliae surface was shown by flow cytometry using anti-gp63 antibody raised against recombinant gp63 from Leishmania mexicana. The pre-treatment of parasites with phospholipase C reduced the number of gp63-positive cells, suggesting that these molecules were glycosylphosphatidylinositol-anchored to the surface. Additionally, the supernatant obtained from phospholipase C-treated cells and probed with anti-cross-reacting determinant confirmed that at least a 52 kDa gp63-like molecule is glycosylphosphatidylinositol-anchored. Furthermore, we assessed a possible function for the gp63-like molecules in H. megaseliae on the interaction with explanted guts of its original host, Megaselia scalaris, and with an experimental model employing Aedes aegypti. Parasites pre-treated with either anti-gp63 antibody or phospholipase C showed a significant reduction in the adhesion to M. scalaris and A. aegypti guts. Similarly, the pre-treatment of the explanted guts with purified gp63 diminished the interaction process. Collectively, these results corroborate the ubiquitous existence of gp63 homologues in insect trypanosomatids and the potential adhesion of these molecules to invertebrate host tissues.     

The role of CD1d in the immune response against Listeria infection

To address the role of CD1d in mucosal immune regulation in bacterial infection, we infected CD1d KO mice with Listeria monocytogenes (Lm). A higher systemic bacterial burden associated with inflammatory lymphocytic infiltrations within the intestine was found in CD1d KO compared with wild type (WT) mice. Lm induced strong IFN-gamma mRNA expression in the liver of WT and the intestine of CD1d KO mice, thus demonstrating the dual, opposing immune activities of IFN-gamma in Lm infection that is dependent on CD1d and/or NKT cells. Analysis of hepatic T cell population demonstrated a reduction of NK1.1(+)TCRbeta+ cells in both mice, followed by recovery only in WT mice. Last, the proportion of alpha4beta1 integrin on lung lymphocytes from CD1d KO was dramatically increased compared with WT mice. Thus, the absence of CD1d resulted in increased susceptibility towards Listeria infection, induced changes in NKT cells, and increased trafficking of alpha4beta1 molecule to inflamed lung.     

Northward expansion of a marine parasite: Testing the role of temperature adaptation

The known range of the eastern oyster (Crassostrea virginica) parasite, Perkinsus marinus, expanded into the northeastern United States in the early 1990s. We used both in vitro and in vivo data to test the hypothesis that the northward expansion was associated with a low-temperature adapted strain of the parasite. In vitro proliferation of nine P. marinus isolates from three geographic sites, Massachusetts and New Jersey in the new range, and South Carolina in the historic southern range, was measured at seven temperatures (5 to 35 °C) using a tetrazolium blue dye assay. We wanted to determine if there were between- and within-geographic location differences in the P. marinus proliferation rate, and if so, whether they were associated with temperature. We found no evidence of low-temperature adaptation based on the fact that net proliferation rates for isolates from all three geographic locations were similar at temperatures from 5 to 20 °C. On the other hand, at temperatures of 25 to 35 °C, the South Carolina isolates exhibited higher proliferation rates than the northern isolates suggesting possible high-temperature adaptation of parasite strains that are routinely exposed to higher temperatures. Although there was significant within-location variation among isolates, the data tended to group together by geographic location supporting the hypothesis that there is an important regional component to the proliferation rate of P. marinus isolates. A survey of published data showed that the temperature at which in vivo proliferation was first observed in oysters at sites from the Gulf of Mexico to Massachusetts was typically between 20 and 23 °C with no evidence of a geographic cline. These results lend support to the hypothesis that the recent warming trend in the northeastern US is the most likely explanation for the P. marinus range extension.     

Differences between the pathogenic processes induced by Steinernema and Heterorhabditis (Nemata: Rhabditida) in Pseudaletia unipuncta (Insecta: Lepidoptera)

Larvae of Pseudaletia unipuncta are moderately susceptible to infections caused by entomopathogenic nematodes, being a desirable host to study pathogenic processes caused by Heterorhabditis bacteriophora, Steinernema carpocapsae, and Steinernema glaseri and their associated bacteria. The ability of the infective stage of these nematodes to invade hosts is quite different. S. carpocapsae invades the highest number of insects and presents the highest penetration rate, followed by H. bacteriophora. Regression analysis between the number of insects parasitized and the number of IJs counted per insect, over time, showed a high correlation for S. carpocapsae whereas for H. bacteriophora it was low. Dose-response was most evident at a concentration below 100 IJs per insect on H. bacteriophora, whereas on S. carpocapsae it was found for doses ranging from 100 to 2,000 IJs. Student's t test analysis of dose-response showed parallel, yet unequal, slopes for both strains of H. bacteriophora, whereas distinct regressions were obtained for S. carpocapsae and S. glaseri, thus, evidencing each species develop a distinct pathogenic process. Insects injected with Photorhabdus luminescens died within 50 h after injection, whereas those treated with X. nematophila died much later. Moreover, the mortality in insects exposed to H. bacteriophora complex and injected with P. luminescens was close, but insects injected with bacteria died faster. Insect mortality in treatments with complexes S. carpocapsae and S. glaseri was significantly higher than that which was observed in insects injected with symbiotic bacteria.     

Wave expansion of CD34 progenitor cells in the spleen in rodent malaria

Defense against malaria depends upon amplification of the spleen structure and function for the clearance of parasitized red blood cells (pRBC). We studied the distribution and amount of CD34⁺ cells in the spleens of mice infected with rodent malaria. We sought to identify these cells in the spleen and determine their relationship to infection. C57BL/6J mice were infected with self-resolving, Plasmodium chabaudi CR, or one of the lethal rodent malaria strains, P. chabaudi AJ and P. berghei ANKA. We then recorded parasitemia, mortality, and the presence of CD34⁺ cells in spleen, as determined by immunohistochemistry and flow cytometry. In the non-lethal strain, the spleen structure was maintained during amplification, but disrupted in lethal models. The abundance of CD34⁺ cells increased in the red pulp on the 4th and 6th days p.i. in all models, and subsided on the 8th day p.i. Faint CD34⁺ staining on the 8th day p.i., was probably due to differentiation of committed cell lineages. In this work, increase of spleen CD34⁺ cells did not correlate with infection control.