Effect of cell-surface components and metabolites of lactic acid bacteria and probiotic organisms on cytokine production and induction of CD25 expression in human peripheral mononuclear cells

In the current study, the relative contribution of cell-surface components (CSC) and cell-free supernatants (CFS) in the immuno-modulatory properties of 17 strains of probiotic and lactic acid bacteria (LAB) was assessed. The production of pro- and antiinflammatory cytokines including IL-2, IL-4, IL-10, IL-12 p70, IFN-γ, tumor necrosis factor-α (TNF-α), and transforming growth factor-β was measured at different time points after stimulation of buffy coat derived-peripheral blood mononuclear cells (PBMC) from healthy donors with CSC and CFS of probiotic and LAB. Results showed that CSC of probiotic and LAB strains induced production of T helper 1 and 2 type cytokines. Transforming growth factor-β was stimulated at highest concentrations, followed by IL-10 and TNF-α. The CFS of all tested bacterial strains induced PBMC for significantly high levels of IL-10 secretion compared with unstimulated cells, but the values were less than lipopolysaccharide-stimulated cells. Cytokines due to CFS stimulation showed declined concentration for IL-2, TNF-α, and IL-4, and complete disappearance of IL-12, IFN-γ, and transforming growth factor-β in the cultured medium at 96 h of incubation. Results of cytokine data demonstrate proinflammatory TNF-α immune responses are mainly directed through cell-surface structures of probiotic and LAB, but antiinflammatory immune responses are mediated both by metabolites and cell-surfaces of these bacteria. The induction of CD4(+)CD25(+) regulatory T cells after stimulation of PBMC with CSC and CFS of probiotic and LAB showed regulatory T cell activity appeared to be influenced both by the CSC and metabolites, but was principally triggered by cell surfaces of probiotic and LAB strains.

Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides brasiliensis is a cell surface protein involved in fungal adhesion to extracellular matrix proteins and interaction with cells

The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. The P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.

The N terminus of the human alpha(1D)-adrenergic receptor prevents cell surface expression

We previously reported that truncation of the N-terminal 79 amino acids of alpha(1D)-adrenoceptors (Delta(1-79)alpha(1D)-ARs) greatly increases binding site density. In this study, we determined whether this effect was associated with changes in alpha(1D)-AR subcellular localization. Confocal imaging of green fluorescent protein (GFP)-tagged receptors and sucrose density gradient fractionation suggested that full-length alpha(1D)-ARs were found primarily in intracellular compartments, whereas Delta(1-79)alpha(1D)-ARs were translocated to the plasma membrane. This resulted in a 3- to 4-fold increase in intrinsic activity for stimulation of inositol phosphate formation by norepinephrine. We determined whether this effect was transplantable by creating N-terminal chimeras of alpha(1)-ARs containing the body of one subtype and the N terminus of another (alpha(1A) NT-D, alpha(1B) NT-D, alpha(1D) NT-A, and alpha(1D)NT-B). When expressed in human embryonic kidney 293 cells, radioligand binding revealed that binding densities of alpha(1A)- or alpha(1B)-ARs containing the alpha(1D)-N terminus decreased by 86 to 93%, whereas substitution of alpha(1A)- or alpha(1B)-N termini increased alpha(1D)-AR binding site density by 2- to 3-fold. Confocal microscopy showed that GFP-tagged alpha(1D)NT-B-ARs were found only on the cell surface, whereas GFP-tagged alpha(1B)NT-D-ARs were completely intracellular. Radioligand binding and confocal imaging of GFP-tagged alpha(1D)- and Delta(1-79)alpha(1D)-ARs expressed in rat aortic smooth muscle cells produced similar results, suggesting these effects are generalizable to cell types that endogenously express alpha(1D)-ARs. These findings demonstrate that the N-terminal region of alpha(1D)-ARs contain a transplantable signal that is critical for regulating formation of functional bindings, through regulating cellular localization.

IMMUNOCYTOCHEMICAL DIFFERENTIATION OF REACTIVE HYPERPLASIA FROM FOLLICULAR LYMPHOMA USING MONOCLONAL-ANTIBODIES TO CELL-SURFACE AND PROLIFERATION-RELATED MARKERS

We tested the hypothesis that a panel of antibodies to cell surface, cytoplasmic, and nuclear antigens could reliably distinguish the cells composing reactive germinal centers from those composing follicular lymphoma. Immunocytochemistry was performed on deparaffinized sections of methacarn-fixed lymph node and tonsil (15 cases of reactive hyperplasia and 14 cases of follicular lymphoma) using antibodies to the nerve growth factor receptor (NGFR5), bcl-2 protein (124), proliferating cell nuclear antigen (PCNA; 19A2), and CD45RA (MT2). In 100% of cases of reactive hyperplasia, both MT2 and 124 showed positive immunostaining of mantle zone and scattered interfollicular lymphocytes, but in all cases there was a sharply demarcated absence of immunostaining of germinal center cells. However, diffuse immunostaining of follicular centers with MT2 (64%) and 124 (93%) and scattered intervening cells were seen in follicular lymphoma. The combination of antibodies to CD45RA and bcl-2 yielded positive immunostaining of follicular center cells in 93% of follicular lymphomas. The germinal center cells of reactive hyperplasia showed >75% nuclear positivity with antibodies to PCNA, in contrast to the follicular lymphoma cells, which showed variable PCNA indices ranging from 25 to >75%. A minority of follicular lymphoma cases (29%) showed PCNA indices comparable with those seen in cases of reactive hyperplasia. Antibodies to NGFR were positive in all cases of reactive hyperplasia and in 79% of cases of follicular hyperplasia, although the immunostaining intensity was generally decreased in follicular hyperplasia. In summary, antibodies to bcl-2 appear to be superior to those to CD45RA in distinguishing reactive hyperplasia from follicular lymphoma. Reactive hyperplasia cannot be discriminated from follicular hyperplasia using antibodies to PCNA or to nerve growth factor receptor.

Design, synthesis and the effect of 1,2,3-triazole sialylmimetic neoglycoconjugates on Trypanosoma cruzi and its cell surface trans-sialidase

This work describes the synthesis of a series of sialylmimetic neoglycoconjugates represented by 1,4-disubstituted 1,2,3-triazole-sialic acid derivatives containing galactose modified at either C-1 or C-6 positions, glucose or gulose at C-3 position, and by the amino acid derivative 1,2,3-triazole fused threonine-3-O-galactose as potential TcTS inhibitors and anti-trypanosomal agents. This series was obtained by Cu(I)-catalysed azide-alkyne cycloaddition reaction ('click chemistry') between the azido-functionalized sugars 1-N(3)-Gal (commercial), 6-N(3)-Gal, 3-N(3)-Glc and 3-N(3)-Gul with the corresponding alkyne-based 2-propynyl-sialic acid, as well as by click chemistry reaction between the amino acid N(3)-ThrOBn with 3-O-propynyl-GalOMe. The 1,2,3-triazole linked sialic acid-6-O-galactose and the sialic acid-galactopyranoside showed high Trypanosoma cruzi trans-sialidase (TcTS) inhibitory activity at 1.0 mM (approx. 90%), whilst only the former displayed relevant trypanocidal activity (IC(50) 260 mu M). These results highlight the 1,2,3-triazole linked sialic acid-6-O-galactose as a prototype for further design of new neoglycoconjugates against Chagas' disease. (C) 2011 Elsevier Ltd. All rights reserved.

Bladder expression of CD cell surface antigens and cell-type-specific transcriptomes

Many cell types have no known functional attributes. In the bladder and prostate, basal epithelial and stromal cells appear similar in cytomorphology and share several cell surface markers. Their total gene expression (transcriptome) should provide a clear measure of the extent to which they are alike functionally. Since urologic stromal cells are known to mediate organ-specific tissue formation, these cells in cancers might exhibit aberrant gene expression affecting their function. For transcriptomes, cluster designation (CD) antigens have been identified for cell sorting. The sorted cell populations can be analyzed by DNA microarrays. Various bladder cell types have unique complements of CD molecules. CD9(+) urothelial, CD104(+) basal and CD13(+) stromal cells of the lamina propria were therefore analyzed, as were CD9(+) cancer and CD13(+) cancer-associated stromal cells. The transcriptome datasets were compared by principal components analysis for relatedness between cell types; those with similarity in gene expression indicated similar function. Although bladder and prostate basal cells shared CD markers such as CD104, CD44 and CD49f, they differed in overall gene expression. Basal cells also lacked stem cell gene expression. The bladder luminal and stromal transcriptomes were distinct from their prostate counterparts. In bladder cancer, not only the urothelial but also the stromal cells showed gene expression alteration. The cancer process in both might thus involve defective stromal signaling. These cell-type transcriptomes provide a means to monitor in vitro models in which various CD-isolated cell types can be combined to study bladder differentiation and bladder tumor development based on cell-cell interaction.

Loss of the C terminus of melanocortin receptor 2 (MC2R) results in impaired cell surface expression and ACTH insensitivity

Mutations in melanocortin receptor 2 (MC2R) and its related melanocortin receptor accessory protein (MRAP) cause familial glucocorticoid deficiency. We identified a novel MC2R mutation, K289fs. This unique mutation in the C terminus of MC2R is located in the intracellular part of the protein for which the exact function is unknown.

Stimulated platelets use serotonin to enhance their retention of procoagulant proteins on the cell surface

Activated platelets bind numerous adhesive and procoagulant proteins by receptor-mediated processes. Although there is little evidence to suggest that these processes are heterogeneous in platelets, we previously found that platelets co-stimulated with collagen and thrombin express functional alpha-granule factor V only on a subpopulation of cells. Here we show that these cells, referred to as 'COAT-platelets', bind additional alpha-granule proteins, including fibrinogen, von Willebrand factor, thrombospondin, fibronectin and alpha2-antiplasmin. These proteins are all transglutaminase substrates, and inhibitors of transglutaminase prevent the production of COAT-platelets. A synthetic transglutaminase substrate (CP15) also binds to COAT-platelets, and analysis by high performance liquid chromatography/mass spectrometry shows that a product is formed with a relative molecular mass (Mr) equal to CP15 plus 176. Serotonin, an abundant component of platelet-dense granules, has an Mr of 176, and fibrinogen isolated from COAT-platelets contains covalently linked serotonin. Synthetic bovine serum albumin-(serotonin)6 binds selectively to COAT-platelets and also inhibits the retention of procoagulant proteins on COAT-platelets. These data indicate that COAT-platelets use serotonin conjugation to augment the retention of procoagulant proteins on their cell surface through an as yet unidentified serotonin receptor.

The cell surface receptor FGFRL1 forms constitutive dimers that promote cell adhesion

FGFRL1 is a novel member of the fibroblast growth factor (FGF) receptor family. Utilizing the FRET (fluorescence resonance energy transfer) technique, we demonstrate that FGFRL1 forms constitutive homodimers at cell surfaces. The formation of homodimers was verified by co-precipitation of differentially tagged FGFRL1 polypeptides from solution. If overexpressed in cultivated cells, FGFRL1 was found to be enriched at cell-cell contact sites. The extracellular domain of recombinant FGFRL1 promoted cell adhesion, but not cell spreading, when coated on plastic surfaces. Adhesion was mediated by heparan sulfate glycosaminoglycans located at the cell surface. It could specifically be blocked by addition of soluble heparin but not by addition of other glycosaminoglycans. When the amino acid sequence of the putative heparin-binding site was modified by in vitro mutagenesis, the resulting protein exhibited decreased affinity for heparin and reduced activity in the cell-binding assay. Moreover, a synthetic peptide corresponding to the heparin-binding site was able to neutralize the effect of heparin. With its dimeric structure and its adhesion promoting properties, FGFRL1 resembles the nectins, a family of cell adhesion molecules found at cell-cell junctions.

Distinct requirements for activation-induced cell surface expression of preformed Fas/CD95 ligand and cytolytic granule markers in T cells

Fas (CD95/Apo-1) ligand is a potent inducer of apoptosis and one of the major killing effector mechanisms of cytotoxic T cells. Thus, Fas ligand activity has to be tightly regulated, involving various transcriptional and post-transcriptional processes. For example, preformed Fas ligand is stored in secretory lysosomes of activated T cells, and rapidly released by degranulation upon reactivation. In this study, we analyzed the minimal requirements for activation-induced degranulation of Fas ligand. T cell receptor activation can be mimicked by calcium ionophore and phorbol ester. Unexpectedly, we found that stimulation with phorbol ester alone is sufficient to trigger Fas ligand release, whereas calcium ionophore is neither sufficient nor necessary. The relevance of this process was confirmed in primary CD4(+) and CD8(+) T cells and NK cells. Although the activation of protein kinase(s) was absolutely required for Fas ligand degranulation, protein kinase C or A were not involved. Previous reports have shown that preformed Fas ligand co-localizes with other markers of cytolytic granules. We found, however, that the activation-induced degranulation of Fas ligand has distinct requirements and involves different mechanisms than those of the granule markers CD63 and CD107a/Lamp-1. We conclude that activation-induced degranulation of Fas ligand in cytotoxic lymphocytes is differently regulated than other classical cytotoxic granule proteins.

β1- and β3- voltage-gated sodium channel subunits modulate cell surface expression and glycosylation of Nav1.7 in HEK293 cells

Voltage-gated sodium channels (Navs) are glycoproteins composed of a pore-forming α-subunit and associated β-subunits that regulate Nav α-subunit plasma membrane density and biophysical properties. Glycosylation of the Nav α-subunit also directly affects Navs gating. β-subunits and glycosylation thus comodulate Nav α-subunit gating. We hypothesized that β-subunits could directly influence α-subunit glycosylation. Whole-cell patch clamp of HEK293 cells revealed that both β1- and β3-subunits coexpression shifted V ½ of steady-state activation and inactivation and increased Nav1.7-mediated I Na density. Biotinylation of cell surface proteins, combined with the use of deglycosydases, confirmed that Nav1.7 α-subunits exist in multiple glycosylated states. The α-subunit intracellular fraction was found in a core-glycosylated state, migrating at ~250 kDa. At the plasma membrane, in addition to the core-glycosylated form, a fully glycosylated form of Nav1.7 (~280 kDa) was observed. This higher band shifted to an intermediate band (~260 kDa) when β1-subunits were coexpressed, suggesting that the β1-subunit promotes an alternative glycosylated form of Nav1.7. Furthermore, the β1-subunit increased the expression of this alternative glycosylated form and the β3-subunit increased the expression of the core-glycosylated form of Nav1.7. This study describes a novel role for β1- and β3-subunits in the modulation of Nav1.7 α-subunit glycosylation and cell surface expression.

Cell surface galactosyltransferase structure and function during mesenchymal cell migration

In order to more fully understand the function of surface GalTase on mesenchymal cells, anti-GalTase IgG was used to (a) examine the role of surface GalTase during mouse mesenchymal cell migration on laminin and fibronectin; (b) define the plasma membrane distribution of GalTase by indirect immunofluorescence on migrating cells; (c) quantitate the level of surface GalTase on migrating cells; and (d) determine whether GalTase is associated with the cytoskeleton.^ Results show that anti-GalTase IgG was able to inhibit migration (48-80% as compared to basal rate) when cells were migrating on laminin-containing matrices. Monovalent Fab fragments inhibited migration on laminin by 90% after 4 hours. On the other hand, anti-GalTase IgG had no effect on cells migrating on fibronectin. This illustrates the substrate specificity of GalTase mediated-migration. When anti-GalTase IgG was used to localize surface GalTase on cells migratory on laminin, the enzyme was restricted to the leading and trailing edges of the cell. Assays indicate that GalTase is elevated approximately 3-fold when cells are migrating on laminin-containing matrices as compared to migratory cells on plastic or fibronectin, or as compared to stationary cells on any substrate. Laminin appears to recruit GalTase from preexisting intracellular pools to the growing lamellipodia.^ Double-label indirect immunofluorescence studies indicate that there is an apparent co-localization between some of the surface GalTase and some actin filaments. This relationship was explored by extracting cells prelabeled with anti-GalTase IgG and quantitated by radiolabeled second antibodies. Results show that 79% of the surface GalTase is associated with the cytoskeleton (as judged by detergent insolubility) when monovalent antibodies (Fab) are used. However virtually all (80-100%) of the surface GalTase can be induced to associate with the cytoskeleton when cross-linked with bivalent antibodies. Furthermore, when cells in suspension are incubated with divalent antibodies, an additional 66% of the surface GalTase can be induced to associate with the cytoskeleton. The elevated levels of surface GalTase detectable on cells migrating on laminin also appear to be associated with the cytoskeleton.^ Several lines of evidence suggest that GalTase is associated with F-actin. Data suggest that laminin induces the expression of surface GalTase to the growing lamellipodia where it becomes associated with the cytoskeleton leading to cell spreading and migration. (Abstract shortened with permission of author.) ^

Molecular analysis of cell surface $\beta$1,4-galactosyltransferase function during lamellipodal formation, cell polarization, and cell migration

The rate and direction of fibroblast locomotion is regulated by the formation of lamellipodia. In turn, lamellipodal formation is modulated in part by adhesion of that region of the cell from which the lamellipodia will extend or orginate. Cell surface $\beta$1,4-galactosyltransferase (GalTase) is one molecule that has been demonstrated to mediate cellular interactions with extracellular matrices. In the case of fibroblasts, GalTase must be associated with the actin cytoskeleton in order to mediate cellular adhesion to laminin. The object of this study was to determine how altering the quantity of GalTase capable of associating with the cytoskeleton impacts cell motility. Stably transfected cell lines were generated that have increased or decreased levels of surface GalTase relative to its cytoskeleton-binding sites. Biochemical analyses of these cells reveals that there is a limited number of sites on the cytoskeleton with which GalTase can interact. Altering the ratio of GalTase to its cytoskeleton binding sites does not affect the cells' abilities to spread, nor does it affect the localization of cytoskeletally-bound GalTase. It does, however, appear to interfere with stress fiber bundling. Cells with altered GalTase:cytoskeleton ratios change their polarity of laminin more frequently, as compared to controls. Therefore, the ectopic expression of GalTase cytoplasmic domains impairs a cell's ability to control the placement of lamellipodia. Cells were then tested for their ability to respond to a directional stimulus, a gradient of platelet-derived growth factor (PDGF). It was found that the ability of a cell to polarize in response to a gradient of PDGF is directly proportional to the quantity of GalTase associated with its cytoskeleton. Finally, the rate of unidirectional cell migration on laminin was found to be directly dependent upon surface GalTase expression and is inversely related to the ability of surface GalTase to interact with the cytoskeleton. It is therefore proposed that cytoskeletal assembly and lamellipodal formation can be regulated by the altering the ratio of cytoplasmic domains for specific matrix receptors, such as GalTase, relative to their cytoskeleton-binding sites. ^

Molecular analysis of $\beta$1,4-galactosyl-transferase localization to the cell surface and participation in cell adhesion

$\beta$1,4-Galactosyltransferase (GalTase) is unusual among the glycosyltransferases in that it is found in two subcellular compartments where it performs different functions. In the trans-Golgi complex, GalTase participates in oligosaccharide biosynthesis as do other glycosyltransferases. GalTase is also found on the cell surface, where it associates with the cytoskeleton and functions as a receptor for extracellular oligosaccharide ligands. Although we know much regarding GalTase function on the cell surface, little is known about the mechanisms underlying its transport to the plasma membrane. Cloning of the GalTase gene revealed that there are two GalTase proteins (i.e., long and short) with different size cytoplasmic tails. This raises the possibility that differences in the cytoplasmic domain of GalTase may influence its subcellular distribution. The object of this study was to examine this hypothesis directly through the use of molecular, immunological, and biochemical approaches.^ To examine whether the two GalTase proteins are targeted to different subcellular compartments, F9 embryonal carcinoma cells were transfected with either long or short GalTase cDNAs and intracellular and cell surface enzyme levels measured. Cell surface GalTase activity was enriched in cells overexpressing the long, but not the form of short GalTase. Furthermore, a dominant negative mutation in cell surface GalTase was created by transfecting cells with GalTase cDNAs encoding a truncated version of long GalTase devoid of the extracellular catalytic domain. Overexpressing the complete cytoplasmic and transmembrane domains of long GalTase led to a loss of GalTase-dependent cellular adhesion by specifically displacing surface GalTase from its cytoskeletal associations. In contrast, overexpressing the analogous truncated protein of short GalTase had no effect on cell adhesion. Finally, chloramphenicol acetyltransferase (CAT) reporter proteins were used to determine directly whether the cytoplasmic domains of long and short GalTase were responsible for differential subcellular distribution. The cytoplasmic and transmembrane domains of long GalTase led to CAT expression on the ceil surface and its association with the detergent-insoluble cytoskeleton; the analogous fusion protein containing short GalTase was restricted to the Golgi compartment. These results suggest that the cytoplasmic domain unique to long GalTase is responsible for targeting a portion of this protein to the cell surface and associating it with the cytoskeleton, enabling it to function as a cell adhesion molecule. ^