Senescence markers in focal nodular hyperplasia of the liver: pathogenic considerations on the basis of immunohistochemical results.

Focal nodular hyperplasia (FNH) is a polyclonal tumour-like hepatic lesion characterised by parenchymal nodules, connective tissue septa without interlobular bile ducts, pronounced ductular reaction and inflammation. It may represent a response to local arterial hyperperfusion and hyperoxygenation resulting in oxidative stress. We aimed at obtaining closer insight into the pathogenesis of FNH with its characteristic morphologic features. Immunohistochemistry and immunofluorescence microscopy was performed on FNH specimens using antibodies against keratins (K) 7 and 19, neural cell adhesion molecule (NCAM), lamin B1, senescence markers (CDK inhibitor 1/p21Cip1, CDK inhibitor /p16Ink4a, senescence-associated (SA) ß- galactosidase activity), proliferation markers (Ki-67, proliferating-cell nuclear antigen (PCNA)), and the abnormally phosphorylated histone γ-H2AX, indicating DNA double strand breaks; moreover SA ß- galactosidase activity was determined histochemically. Ductular metaplasia of hepatocytes indicated by K7 expression in the absence of K19 plays a major role in the development of ductular reaction in FNH. Moreover, the expression of senescence markers (p21Cip1, p16Ink4a, γ-H2AX, SA ß-galactosidase activity) in hepatocytes and cholangiocytes suggests that stress-induced cellular senescence contributes to fibrosis and inflammation via production of components of the senescence-associated secretory phenotype. Expression of proliferation markers (Ki-67, PCNA) was not enhanced in hepatocytes and biliary cells. Senescence and ductular metaplasia of hepatocytes may thus be involved in inflammation, fibrosis and apoptosis resistance. Hence, fibrosis, inflammation and reduced apoptotic cell death, rather than proliferation (hyperplasia) may be responsible for increased tissue mass and tumour-like appearance of FNH.

Decidual CXCR4+ CD56bright NK cells as a novel NK subset in maternal-foetal immune tolerance to alleviate early pregnancy failure.

Natural killer (NK) cells preferentially accumulate at maternal-foetal interface and are believed to play vital immune-modulatory roles during early pregnancy and related immunological dysfunction may result in pregnant failure such as recurrent miscarriage (RM). However, the mechanisms underlying the establishment of maternal-foetal immunotolerance are complex but clarifying the roles of decidual NK (dNK) cells offers the potential to design immunotherapeutic strategies to assist RM patients. In this report, we analysed RNA sequencing on peripheral NK (pNK) and decidual NK cells during early pregnancy; we identified an immunomodulatory dNK subset CXCR4+ CD56bright dNK and investigated its origin and phenotypic and functional characteristics. CXCR4+ CD56bright dNK displayed a less activated and cytotoxic phenotype but an enhanced immunomodulatory potential relative to the CXCR4 negative subset. CXCR4+ CD56bright dNK promote Th2 shift in an IL-4-dependent manner and can be recruited from peripheral blood and reprogramed by trophoblasts, as an active participant in the establishment of immune-tolerance during early pregnancy. Diminished CXCR4+ dNK cells and their impaired ability to induce Th2 differentiation were found in RM patients and mouse models of spontaneous abortion. Moreover, adoptive transfer of CXCR4+ dNK cells to NK-deficient (Nfil3-/-) mice showed great therapeutic potential of CXCR4+ dNK via recovering the Th2/Th1 bias and reducing embryo resorption rates. The identification of this new dNK cell subset may lay the foundation for understanding NK cell mechanisms in early pregnancy and provide potential prognostic factors for the diagnosis and therapy of RM.

Polysialic acid masks neural cell adhesion molecule antigenicity.

The neural cell adhesion molecule (NCAM) is a transmembrane protein involved in major cellular processes. The addition of polysialic acid (PSA), a post-translational modification (PTM) almost exclusively carried by NCAM, alters NCAM properties and functions and is therefore tightly regulated. Changes in NCAM and PSA-NCAM take place during development and ageing and occur in various diseases. The presence of PTMs can reduce the accessibility of antibodies to their epitopes and lead to false negative results. Thus, it is vital to identify antibodies that can specifically detect their target regardless of the presence of PTMs. In the present study, four commercially available NCAM antibodies were characterized by western blot and immunocytochemistry. Antibody specificity was determined by decreasing NCAM expression with small interfering RNA and subsequently determining whether the antibodies still produced a signal. In addition, PSA was digested with endoneuraminidase N to assess whether removing PSA improves NCAM detection with these antibodies. Our study revealed that the presence of PSA on NCAM reduced antibody accessibility to the epitope and consequently masked NCAM antigenicity for both techniques investigated. Moreover, three of the four antibodies tested were specific for the detection of NCAM by western blot and by immunocytochemistry. Altogether, this study demonstrates the importance of choosing the correct antibody to study NCAM depending on the technique of interest and underlines the importance of taking PTMs into account when using antibody-based techniques for the study of NCAM.

Generation and intracellular trafficking of a polysialic acid-carrying fragment of the neural cell adhesion molecule NCAM to the cell nucleus.

Polysialic acid (PSA) and its major protein carrier, the neural cell adhesion molecule NCAM, play important roles in many nervous system functions during development and in adulthood. Here, we show that a PSA-carrying NCAM fragment is generated at the plasma membrane by matrix metalloproteases and transferred to the cell nucleus via endosomes and the cytoplasm. Generation and nuclear import of this fragment in cultured cerebellar neurons is induced by a function-triggering NCAM antibody and a peptide comprising the effector domain (ED) of myristoylated alanine-rich C kinase substrate (MARCKS) which interacts with PSA within the plane of the plasma membrane. These treatments lead to activation of the fibroblast growth factor (FGF) receptor, phospholipase C (PLC), protein kinase C (PKC) and phosphoinositide-3-kinase (PI3K), and subsequently to phosphorylation of MARCKS. Moreover, the NCAM antibody triggers calmodulin-dependent activation of nitric oxide synthase, nitric oxide (NO) production, NO-dependent S-nitrosylation of matrix metalloprotease 9 (MMP9) as well as activation of matrix metalloprotease 2 (MMP2) and MMP9, whereas the ED peptide activates phospholipase D (PLD) and MMP2, but not MMP9. These results indicate that the nuclear PSA-carrying NCAM fragment is generated by distinct and functionally defined signal transducing mechanisms.

Intrabodies against the Polysialyltransferases ST8SiaII and ST8SiaIV inhibit Polysialylation of NCAM in rhabdomyosarcoma tumor cells.

BACKGROUND: Polysialic acid (polySia) is a carbohydrate modification of the neural cell adhesion molecule (NCAM), which is implicated in neural differentiation and plays an important role in tumor development and metastasis. Polysialylation of NCAM is mediated by two Golgi-resident polysialyltransferases (polyST) ST8SiaII and ST8SiaIV. Intracellular antibodies (intrabodies; IB) expressed inside the ER and retaining proteins passing the ER such as cell surface receptors or secretory proteins provide an efficient means of protein knockdown. To inhibit the function of ST8SiaII and ST8SiaIV specific ER IBs were generated starting from two corresponding hybridoma clones. Both IBs αST8SiaII-IB and αST8SiaIV-IB were constructed in the scFv format and their functions characterized in vitro and in vivo. RESULTS: IBs directed against the polySTs prevented the translocation of the enzymes from the ER to the Golgi-apparatus. Co-immunoprecipitation of ST8SiaII and ST8SiaIV with the corresponding IBs confirmed the intracellular interaction with their cognate antigens. In CHO cells overexpressing ST8SiaII and ST8SiaIV, respectively, the transfection with αST8SiaII-IB or αST8SiaIV-IB inhibited significantly the cell surface expression of polysialylated NCAM. Furthermore stable expression of ST8SiaII-IB, ST8SiaIV-IB and luciferase in the rhabdomyosarcoma cell line TE671 reduced cell surface expression of polySia and delayed tumor growth if cells were xenografted into C57BL/6 J RAG-2 mice. CONCLUSION: Data obtained strongly indicate that αST8SiaII-IB and αST8SiaIV-IB are promising experimental tools to analyze the individual role of the two enzymes during brain development and during migration and proliferation of tumor cells.

Polyclonal neural cell adhesion molecule antibody prolongs the effective duration time of botulinum toxin in decreasing muscle strength.

This study aimed to investigate if the effective duration time of botulinum toxin A (Btx-A) could be prolonged by polyclonal neural cell adhesion molecule antibody (P-NCAM-Ab). 175 male SD rats were randomly divided into three major groups: control group (n = 25), Btx-A group (n = 25), and P-NCAM-Ab groups. P-NCAM-Ab groups were composed of five sub-groups, with 25 rats each in the dose-response study. Muscle strength of rat lower limbs was determined using a survey system. The expressions of muscle-specific receptor tyrosine kinase (MuSK) and neural cell adhesion molecule (NCAM) were determined by real-time polymerase chain reactions (RT-PCR) and western blotting (WB). The muscle strength was significantly decreased by Btx-A in Btx-A/P-NCAM-Ab groups compared with normal control group. Besides, the muscle strength of P-NCAM-Ab group was significantly decreased compared with the Btx-A group. The recovery time of muscle strength in P-NCAM-Ab group was significantly longer compared with Btx-A group. RT-PCR and WB assay showed that PNCAM-Ab delayed the increase of MuSK and NCAM after Btx-A injection. P-NCAM-Ab prolongs the effective duration time of Btx-A in decreasing muscle strength, which could provide a novel enhancement in clinical application.

Dscam and pancrustacean immune memory - a review of the evidence.

Evidence is accumulating for a memory-like phenomenon in the immune defence of invertebrates. Down syndrome cell adhesion molecule (Dscam) has been proposed as a key candidate for a somatically diversified receptor system in the crustaceans and insects (Pancrustacea) that could enable challenge-specific protection. However, what is the evidence for an involvement of Dscam in pancrustacean immune memory, and in particular specificity? Here we review the current state of the art, and discuss hypotheses of how Dscam could be involved in immunity. We conclude that while there is increasing evidence for the involvement of Dscam in pancrustacean immunity, crucial experiments to address whether it plays a role in specificity upon secondary encounter with a pathogen still remain to be done.

Insight on the fate of CNS-targeted nanoparticles. Part II: Intercellular neuronal cell-to-cell transport.

The application of polymeric nanoparticles (NPs) has a promising future for targeting and delivering drugs into the central nervous system (CNS). However, the fate of NPs once entered in the brain after crossing the blood-brain barrier (BBB) and taken up into neuronal cells is a neglected area of study. Thus, here, we investigate the possible mechanisms of a cell-to-cell transport of poly-lactide-co-glycolide (PLGA) NPs modified with a glycopeptide (g7-NPs), already demonstrated to be able to cross the BBB after in vivo administration in rodents. We also tested antibody (Ab) -modified g7-NPs both in vitro and in vivo to investigate the possibility of specific targeting. Our results show that g7-NPs can be transported intra- and inter-cellularly within vesicles after vesicular internalization. Moreover, cell-to-cell transport is mediated by tunneling-nanotube (TNT)-like structures in cell lines and most interestingly in glial as well as neuronal cells in vitro. The transport is dependent on F-actin and can be increased by induction of TNT-like structures overexpressing M-Sec, a central factor and inducer of TNT formation. Moreover, cell-to-cell transport occurs independently from NP surface modification with antibodies. These in vitro findings were in part confirmed by in vivo evidence after i.p. administration of NPs in mice.

Soluble polysialylated NCAM: a novel player of the innate immune system in the lung.

Posttranslational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) is well studied in the nervous system and described as a dynamic modulator of plastic processes like precursor cell migration, axon fasciculation, and synaptic plasticity. Here, we describe a novel function of polysialylated NCAM (polySia-NCAM) in innate immunity of the lung. In mature lung tissue of healthy donors, polySia was exclusively attached to the transmembrane isoform NCAM-140 and located to intracellular compartments of epithelial cells. In patients with chronic obstructive pulmonary disease, however, increased polySia levels and processing of the NCAM carrier were observed. Processing of polysialylated NCAM was reproduced in a mouse model by bleomycin administration leading to an activation of the inflammasome and secretion of interleukin (IL)-1ß. As shown in a cell culture model, polySia-NCAM-140 was kept in the late trans-Golgi apparatus of lung epithelial cells and stimulation by IL-1ß or lipopolysaccharide induced metalloprotease-mediated ectodomain shedding, resulting in the secretion of soluble polySia-NCAM. Interestingly, polySia chains of secreted NCAM neutralized the cytotoxic activity of extracellular histones as well as DNA/histone-network-containing "neutrophil extracellular traps", which are formed during invasion of microorganisms. Thus, shedding of polySia-NCAM by lung epithelial cells may provide a host-protective mechanism to reduce tissue damage during inflammatory processes.

Nodal proteins are target antigens in Guillain-Barré syndrome.

Neurofascin-186 (NF186), neuronal cell adhesion molecule (NrCAM), and gliomedin are adhesion molecules playing a central role in the formation of nodes of Ranvier. In Guillain-Barré syndrome (GBS), immune attack toward the nodes may participate in the disabilities. Autoantibodies to NF186 and gliomedin have been detected in a rat model of GBS. Here, we investigated the prevalence of antibodies against nodal adhesion molecules in patients with GBS or chronic inflammatory demyelinating polyneuropathy (CIDP). Sera from 100 GBS patients, 50 CIDP patients, 80 disease controls, and 50 healthy controls were tested for their ability to bind the nodes of Ranvier. To characterize the antigens, we performed cell binding assays against NF186, gliomedin, contactin, and NrCAM. We found that 43% of patients with GBS and 30% of patients with CIDP showed IgG fixation at nodes or paranodes. In eight patients with GBS or CIDP, we identified that IgG antibodies recognized the native extracellular domain of NF186, gliomedin, or contactin. Also, 29 patients showed IgM against nodal adhesion molecules. However, we did not detect IgM fixation at nodes or paranodes. Antibodies to gliomedin or NF186 were mostly detected in demyelinating and axonal GBS, respectively. The adsorption of the antibodies to their soluble antigens abolished IgG deposition at nodes and paranodes in nerves, indicating these were specific to NF186, gliomedin, and contactin. In conclusion, gliomedin, NF186, and contactin are novel target antigens in GBS. At nodes, additional epitopes are also the targets of IgG. These results suggest that antibody attack against nodal antigens participates in the etiology of GBS.

Physical and functional cooperation of neural cell adhesion molecule and ß1-integrin in neurite outgrowth induction.

Neural cell adhesion molecule (NCAM) and ß1-integrin are both involved in cell differentiation, with changes in the expression of these two molecules correlating with changes in the malignancy of tumor cells. There is a known functional correlation between NCAM and ß1-integrin in adhesion and also neurite outgrowth in tumor cells. In the present study, we used immunostaining and immunoprecipitation studies to demonstrate that isoform 120 of NCAM associates physically as well as functionally with ß1-integrin in the induction of neurite outgrowth in SH-SY5Y-human neuroblastoma cells. The interaction between these two molecules is mandatory for neurite outgrowth. NCAM blockage completely inhibits the effects of ß1-integrin on neurite outgrowth. These findings further our understanding of the interactions between NCAMs and integrins in malignancy.

Neural cell-cell and cell-substrate adhesion through N-cadherin, N-CAM and L1.

In this study neural (N)-cadherin, neural cell adhesion molecule (N-CAM) and L1 proteins and their antibody equivalents were covalently immobilized on a polyethylene-imine (PEI)-coated glass surface to form neuron-adhesive coatings. Impedance sensing and (supplementary) image analysis were used to monitor the effects of these CAMs. Immobilization of high concentrations of both N-cadherin protein and antibody led to good adhesion of neurons to the modified surface, better than surfaces treated with 30.0 and 100.0 µg ml(-1) N-CAM protein and antibody. L1 antibody and protein coating revealed no significant effect on neuronal cell-substrate adhesion. In a second series of combinatorial experiments, we used the same antibodies and proteins as medium-additives to inhibit cell-cell adhesion between neurons. Adhesion of neurons cultured on N-cadherin protein or antibody-modified surfaces was lowered by the addition of a soluble N-cadherin protein and antibody to the culturing medium, accelerating neuronal aggregation. The presence of a soluble N-CAM antibody or protein had no effect on the adhesion of neuronal cells on a N-cadherin protein-modified surface. On a N-cadherin antibody-coated surface, the addition of a soluble N-CAM protein led to cell death of neurons after 48 h, while a N-CAM antibody had no effect. In the presence of a soluble N-cadherin protein and antibody the aggregation of neurons was inhibited, both on N-CAM protein and N-CAM antibody-modified surfaces. Neurons cultured on immobilized antibodies were less affected by the addition of soluble CAM blockers than neurons cultured on immobilized proteins, indicating that antibody-protein bonds are more stable compared to protein-protein bonds.

Rostral growth of commissural axons requires the cell adhesion molecule MDGA2.

BACKGROUND: Long-distance axonal growth relies on the precise interplay of guidance cues and cell adhesion molecules. While guidance cues provide positional and directional information for the advancing growth cone, cell adhesion molecules are essential in enabling axonal advancement. Such a dependence on adhesion as well as guidance molecules can be well observed in dorsal commissural interneurons, which follow a highly stereotypical growth and guidance pattern. The mechanisms and molecules involved in the attraction and outgrowth towards the ventral midline, the axon crossing towards the contralateral side, the rostral turning after midline crossing as well as the guidance along the longitudinal axis have been intensely studied. However, little is known about molecules that provide the basis for commissural axon growth along the anterior-posterior axis. RESULTS: MDGA2, a recently discovered cell adhesion molecule of the IgCAM superfamily, is highly expressed in dorsolaterally located (dI1) spinal interneurons. Functional studies inactivating MDGA2 by RNA interference (RNAi) or function-blocking antibodies demonstrate that either treatment results in a lack of commissural axon growth along the longitudinal axis. Moreover, results from RNAi experiments targeting the contralateral side together with binding studies suggest that homophilic MDGA2 interactions between ipsilaterally projecting axons and post-crossing commissural axons may be the basis of axonal growth along the longitudinal axis. CONCLUSIONS: Directed axonal growth of dorsal commissural interneurons requires an elaborate mixture of instructive (guidance) and permissive (outgrowth supporting) molecules. While Wnt and Sonic hedgehog (Shh) signalling pathways have been shown to specify the growth direction of post-crossing commissural axons, our study now provides evidence that homophilic MDGA2 interactions are essential for axonal extension along the longitudinal axis. Interestingly, so far each part of the complex axonal trajectory of commissural axons uses its own set of guidance and growth-promoting molecules, possibly explaining why such a high number of molecules influencing the growth pattern of commissural interneurons has been identified.

NCAM-induced neurite outgrowth depends on binding of calmodulin to NCAM and on nuclear import of NCAM and fak fragments.

The neural cell adhesion molecule NCAM plays important functional roles not only during nervous system development, but also in the adult after injury and in synaptic plasticity. Homophilic binding of NCAM triggers intracellular signaling events resulting in cellular responses such as neurite outgrowth that require NCAM palmitoylation-dependent raft localization and activation of the nonreceptor tyrosine kinases fyn and fak. In this study, we show that stimulation of NCAM by a function-triggering NCAM antibody results in proteolytic processing of NCAM and fak. The C-terminal fragment of NCAM, consisting of the intracellular domain, the transmembrane domain, and a stub of the extracellular domain, and the N-terminal fragment of fak are imported into the nucleus. NCAM-stimulated fak activation, generation, and nuclear import of NCAM and fak fragments as well as neurite outgrowth are abolished by mutation of the calmodulin binding motif in the intracellular domain of NCAM that is responsible for the calcium-dependent binding of calmodulin to NCAM. This mutation interferes neither with NCAM cell surface expression, palmitoylation, and raft localization nor with fyn activation. The way by which the transmembrane NCAM fragment reaches the nucleus in a calmodulin- and calcium-dependent manner is by endocytotic transport via the endoplasmic reticulum and the cytoplasm. The generation and nuclear import of NCAM and phosphorylated fak fragments resulting from NCAM stimulation may represent a signal pathway activating cellular responses in parallel or in association with classical kinase- and phosphorylation-dependent signaling cascades.

Polymyositis, dermatomyositis and malignancy: a further intriguing link.

The association between malignancy and autoimmune myositis has been largely described and confirmed by numerous epidemiological studies. The temporal relationship between the two pathologic conditions can vary: malignancy may occur before, at the same time or following the diagnosis of myositis. Beside these observations, the molecular mechanisms underlying this association are still unknown, even though it has been demonstrated a possible antigenic similarity between regenerating myoblasts and some cancer cell populations. To better identify peculiar histopathologic features common to cancer and myositis, we screened muscle biopsies from patients affected with polymyositis, dermatomyositis, myositis in association to cancer, and from patients affected with newly diagnosed cancer, but without myositis. Similarly to the histopatologic features that were observed in the muscle from myositis patients, especially in those with cancer associated myositis, in patients affected with malignancy at the clinical onset of disease we observed early sign of myopathy, characterized by internally nucleated and regenerating myofibers, most of them expressing the neural cell adhesion molecule. The hypothesis that in a particular subset of individuals genetically predisposed to autoimmunity, an initial subclinical tumor-induced myopathy may result in an autoimmune myositis, represents a further intriguing link behind the association of these two conditions.

An antibody surface for selective neuronal cell attachment.

An optimal surface for culturing human embryonic stem cell (hESC)-derived neuronal cells is of high interest. In this study, a specific antibody to a neural cell adhesion molecule (NCAM) was immobilised on a solid surface of polystyrene and used as a selective matrix for culturing of hESC-derived neuronal cells. Thereafter, hESC-derived neurospheres were seeded on the matrix. The neurospheres did not attach to the NCAM antibody containing matrix whereas individual neuronal cells did. The neuronal cell attachment was depended on the NCAM antibody concentration. The neuronal cells were viable on the NCAM antibody containing matrix during an 8 day follow-up and exhibited typical bipolar morphology of immature neurons. Specific binding of the NCAM antigen to an immunoglobulin-polymer coated surface was verified by surface plasmon resonance (SPR) measurements. This study is to our knowledge the first demonstrating the use of an antibody layer as a selective surface for hESC-derived neuronal cells.

The novel chimeric anti-NCAM (neural cell adhesion molecule) antibody ch.MK1 displays antitumor activity in SCID mice but does not activate complement-dependent cytolysis (CDC).

A monoclonal chimeric antibody ch.MK1 was generated by immunizing F004 mice expressing human instead of murine IgG1/kappa immunoglobulin constant regions. The novel antibody specifically binds cell surface-expressed human neural cell adhesion molecule (NCAM) as shown by immunoprecipitation, flow cytometry and cytospins. Functional analysis revealed nearly complete absence of complement-dependent cytolysis in ch.MK1 and in all other anti-NCAM antibodies tested for reference (UJ13a, ERIC1, 123C3, ch.5A2, B159), indicating an unexpected and group-specific property of anti-NCAM antibodies. As a most plausible mechanism, posttranslational modification of NCAM by complement-inhibiting polysialic acid is discussed. The antibody ch.MK1 demonstrated significant in vivo activity against NCAM-positive neuroblastoma in SCID mice in presence of human peripheral blood mononuclear cell. In absence of human peripheral blood mononuclear cell no distinct antitumor activity of the antibody alone was observed. In ch.MK1 the cellular component of the immune system seems to be the dominant effector mechanism, whereas complement-dependent cytolysis seems not to be necessarily required for antitumor activity. These observations help us to understand immunotherapeutic mechanisms of native anti-NCAM antibodies and may additionally contribute to the understanding of results of currently ongoing clinical studies with conjugated anti-NCAM antibodies.

New multiple labelling method for improved satellite cell identification in human muscle: application to a cohort of power-lifters and sedentary men.

Presently applied methods to identify and quantify human satellite cells (SCs) give discrepant results. We introduce a new immunofluorescence method that simultaneously monitors two SC markers (NCAM and Pax7), the basal lamina and nuclei. Biopsies from power-lifters, power-lifters using anabolic substances and untrained subjects were re-examined. Significantly different results from those with staining for NCAM and nuclei were observed. There were three subtypes of SCs; NCAM(+)/Pax7(+) (94%), NCAM(+)/Pax7(-) (4%) and NCAM(-)/Pax7(+) (1%) but large individual variability existed. The proportion of SCs per nuclei within the basal lamina of myofibres (SC/N) was similar for all groups reflecting a balance between the number of SCs and myonuclei to maintain homeostasis. We emphasise that it is important to quantify both SC/N and the number of SCs per fibre. Our multiple marker method is more reliable for SC identification and quantification and can be used to evaluate other markers of muscle progenitor cells.

A yeast display immunoprecipitation method for efficient isolation and characterization of antigens.

Yeast antibody display has found a wide variety of applications including antibody affinity maturation, epitope mapping, and library screening. Here we report a yeast display immunoprecipitation (YDIP) technique that employs yeast cells displaying single-chain antibody fragments (scFv) on their surface as affinity capture reagents to isolate and characterize antigens. We show that displayed single-chain antibody fragments are active in a variety of detergent solutions commonly used for immunoprecipitation and that the antigen-antibody interaction can be accurately quantified by YDIP coupled with flow cytometry. The YDIP method has also been optimized so that it is compatible with commonly used protein characterization tools such as Western blotting, silver staining, and mass spectrometry. From complex protein mixtures, we have used YDIP to isolate, analyze and sequence both soluble and plasma membrane antigens using tandem mass spectrometry. In the case of the membrane antigen, YDIP coupled with tandem mass spectrometry was successful in identifying neural cell adhesion molecule (NCAM) as the antigen for an antibody previously selected as binding to the plasma membranes of brain endothelial cells. The presented method therefore has potential to facilitate antibody-antigen characterization.

Polysialic acid, a glycan with highly restricted expression, is found on human and murine leukocytes and modulates immune responses.

Polysialic acid (polySia) is a large glycan with restricted expression, typically found attached to the protein scaffold neural cell adhesion molecule (NCAM). PolySia is best known for its proposed role in modulating neuronal development. Its presence and potential functions outside the nervous systems are essentially unexplored. Herein we show the expression of polySia on hematopoietic progenitor cells, and demonstrate a role for this glycan in immune response using both acute inflammatory and tumor models. Specifically, we found that human NK cells modulate expression of NCAM and the degree of polymerization of its polySia glycans according to activation state. This contrasts with the mouse, where polySia and NCAM expression are restricted to multipotent hematopoietic progenitors and cells developing along a myeloid lineage. Sialyltransferase 8Sia IV(-/-) mice, which lacked polySia expression in the immune compartment, demonstrated an increased contact hypersensitivity response and decreased control of tumor growth as compared with wild-type animals. This is the first demonstration of polySia expression and regulation on myeloid cells, and the results in animal models suggest a role for polySia in immune regulation.