Carcinoembryonic Antigen Cell Adhesion Molecule Research Articles

Maintenance and repair of the pancreas of the adult rat by primitive stem cells

Stage‐specific antigen‐4 (SSEA‐4) positive stem cells and carcinoembryonic antigen‐cell adhesion molecule‐1 (CEA‐CAM‐1) positive stem cells have been isolated from the skeletal muscle of adult mammals, including humans. The current study was undertaken to determine the location of these cells within the pancreas of the adult rat. Adult rats were euthanized following the guidelines of Mercer University's IACUC. The pancreases were fixed, cryosectioned and stained with two antibodies diagnostic for adult pluripotential stem cells, i.e. SSEA‐4 for pluripotent stem cells and CEA‐CAM‐1 for totipotent stem cells. Populations of both stem cells were located in tissues of the pancreas based on apparent repair processes. Heavy infiltrations of SSEA‐4 positive stem cells and CEA‐CAM‐1 positive stem cells were located among both pancreatic acinar cells of the exocrine pancreas and islet cells of the endocrine pancreas apparently undergoing repair, whereas other areas of the same pancreases were void of either stem cell population. Studies are ongoing to address the functional significance of these populations of primitive stem cells in pancreatic injury and repair.

CEA‐CAM‐1 positive stem cells from the brain of the adult pig

Carcinoembryonic antigen‐cell adhesion molecule‐1 (CEA‐CAM‐1) positive stem cells have been visualized in the white matter and gray matter in the cerebral cortex of adult rats. Similar cells have also been isolated and characterized from the skeletal muscle of and peripheral vasculature of adult mammals, including humans. The current study was undertaken to determine the location of these cells with respect to the brains of the adult pig. Adult pigs were euthanized following the guidelines of Fort Valley State University's IACUC. The heads were fixed by immersion fixation, the brains isolated, cryosectioned and stained with an antibody diagnostic for adult pluripotential stem cells, i.e. CEA‐CAM‐1. The current study will discuss the location with respect to the white and gray natter in the adult pig. Studies are ongoing to address their functional significance in neuronal injury and repair.

CEACAM1 : modulateur central du métabolisme, de la progression tumorale,de l’angiogenèse et de l’immunité

CEACAM1 (carcinoembryonic antigen cell adhesion molecule 1), a member of the immunoglobulin (Ig) superfamily, is a heavily glycosylated protein. This glycoprotein exhibits an intracytoplasmic region that can be either long (71-73 amino acids) with two inhibitory tyrosine-phosphorylated motifs and several phosphorylated serine residues, or short (10 amino acid). CEACAM1 is a multifunctional protein that plays a role in intercellular adhesion, as an inhibitor of tumor development, as a bacterial adhesin, and as a receptor for the mouse hepatitis virus. Moreover, CEACAM1 is an active regulator of cell signaling, modulating the insulin or EGF receptor pathways in epithelial cells or the Zap-70 pathway in hematopoietic cells. The recent development of genetically modified mouse models altering the Ceacam1 gene corroborates most of these data, but also highlights CEACAM1's functional complexity. Thus, in addition to the functions identified previously, CEACAM1 is an important regulator of lipid metabolism, of tumor progression as a regulator of the Wnt signaling pathway, of normal and tumor neo-angiogenesis and of immunity.

Direct Interaction of Tumor Suppressor CEACAM1 with Beta Catenin: Identification of Key Residues in the Long Cytoplasmic Domain

CEACAM1-4L (carcinoembryonic antigen cell adhesion molecule 1, with 4 extracellular Ig-like domains and a long, 71 amino acid cytoplasmic domain) is expressed in epithelial cells and activated T-cells, but is down-regulated in most epithelial cell cancers and T-cell leukemias. A highly conserved sequence within the cytoplasmic domain has ca 50% sequence homology with Tcf-3 and -4, transcription factors that bind beta-catenin, and to a lesser extent (32% homology), with E-cadherin that also binds beta-catenin. We show by quantitative yeast two-hybrid, BIAcore, GST-pull down, and confocal analyses that this domain directly interacts with beta-catenin, and that H-469 and K-470 are key residues that interact with the armadillo repeats of beta-catenin. Jurkat cells transfected with CEACAM1-4L have 2-fold less activity in the TOPFLASH reporter assay, and in MCF7 breast cancer cells that fail to express CEACAM1, transfection with CEACAM1 and growth in Ca2+ media causes redistribution of beta-catenin from the cytoplasm to the cell membrane, demonstrating a functional role for the long cytoplasmic domain of CEACAM1 in regulation of beta-catenin activity.

Intestinal tumor progression is promoted by decreased apoptosis and dysregulated Wnt signaling in Ceacam1−/− mice

The carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is downregulated in colonic and intestinal hyperplastic lesions as well as in other cancers, where it functions as a tumor suppressor. To investigate the functions of CEACAM1 in the normal intestine and in intestinal tumors, we generated a compound knockout mouse model and examined both Ceacam1(-/-) and Apc(1638N/+):Ceacam1(-/-) mice. Ceacam1(-/-) intestinal cells exhibited a significant decrease in apoptosis, with no change in proliferation or migration, however. Compound Apc(1638N/+):Ceacam1(-/-) mice demonstrated an increase in intestinal tumor multiplicity and tumor progression. Increases in intussusceptions and desmoid lesions were also observed. We have shown that CEACAM1-L associates with beta-catenin by co-immunoprecipitation and colocalization in CEACAM1-L-transfected CT26 and CT51 mouse colon carcinoma cells. Ceacam1(-/-) enterocytes displayed decreased glycogen synthase kinase 3-beta activity with corresponding nuclear localization of beta-catenin. Increased T-cell factor/Lef transcriptional activity was observed in CEACAM1-null CT51 colonic cells and in Caco2 colon cancer cells in which CEACAM1 was downregulated. A significant increased expression in c-Myc and cyclin D1 targets of the Wnt signaling pathway was also revealed in the Ceacam1(-/-) intestine. CEACAM1 therefore actively participates in Wnt signaling in intestinal cells and its downregulation in intestinal tissue contributes to malignancy by augmenting tumor multiplicity and progression.

Carcinoembryonic Antigen Cell Adhesion Molecule 6 Predicts Breast Cancer Recurrence following Adjuvant Tamoxifen

Tamoxifen remains therapy of choice for premenopausal estrogen receptor alpha-positive breast cancer. However, resistance and recurrence are serious problems. Our previous work indicated that carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) was significantly up-regulated in tamoxifen-resistant (TAMr) MCF-7 derivatives. The aim of this study was to determine the functional role of CEACAM6 in endocrine-resistant breast cancer and to retrospectively test whether it was predictive of resistance in a large cohort of breast cancers with long-term follow-up. siRNA silencing of CEACAM6 was done in TAMr cells and effects on clonogenicity and endocrine sensitivity were determined. CEACAM6 immunohistochemistry was done on a tissue microarray comprising 108 relapsed primary human breast cancers and 243 tamoxifen-sensitive controls. siRNA-mediated silencing of CEACAM6 reduced both clonogenicity and anchorage-dependent and anchorage-independent growth of TAMr cells. Importantly, CEACAM6 silencing restored sensitivity of TAMr cells to 4-hydroxytamoxifen and proliferative response to 17beta-estradiol. Immunohistochemistry showed significantly more CEACAM expression in the relapsed group compared with nonrelapsed controls [35 of 108 (33.3%) and 32 of 243 (13.2%), respectively; odds ratio, 3.16 (95% confidence interval, 1.83-5.47); P < 0.0001]. Additionally, we derived an outcome predictor model based on CEACAM expression that restratified patients in the Nottingham prognostic index intermediate-risk group into either higher-risk or lower-risk group. Our data support an important role for CEACAM6 in endocrine resistance, which can serve as a powerful predictor of future recurrence.

Expression of Carcinoembryonic Antigen Cell Adhesion Molecule 6 Oncoprotein in Atypical Ductal Hyperplastic Tissues is Associated with the Development of Invasive Breast Cancer

Expression of Carcinoembryonic Antigen Cell Adhesion Molecule 6 Oncoprotein in Atypical Ductal Hyperplastic Tissues is Associated with the Development of Invasive Breast Cancer

Haplotypic diversity in human CEACAM genes: effects on susceptibility to meningococcal disease

Adhesion between the opacity-associated adhesin (Opa) proteins of Neisseria meningitidis and human carcino-embryonic antigen cell adhesion molecule (CEACAM) proteins is an important stage in the pathogenesis of meningococcal disease, a globally important bacterial infection. Most disease is caused by a small number of meningococcal genotypes known as hyperinvasive lineages. As these are also carried asymptomatically, acquisition of them alone cannot explain why only some hosts develop meningococcal disease. Our aim was to determine whether genetic diversity in CEACAM is associated with susceptibility to meningococcal disease. Frequency distributions of alleles, genotypes and haplotypes were compared in four CEACAM genes in 384 case samples and 190 controls. Linkage disequilibrium among polymorphic sites, haplotype structures and relationships were also analysed. A number of polymorphisms were observed in CEACAM genes but the diversity of CEACAM1, to which most Opa proteins bind, was lower, and a small number of high-frequency haplotypes were detected. Dose-dependent associations of three CEACAM haplotypes with meningococcal disease were observed, with the effect of carrying these haplotypes amplified in homozygous individuals. Two haplotypes were protective while one haplotype in CEACAM6 was associated with a twofold increase in disease susceptibility. These data imply that human CEACAM may be one determinant of human susceptibility to meningococcal disease.

A transplantable human medullary thyroid carcinoma as a model for RET tyrosine kinase-driven tumorigenesis

Hereditary medullary thyroid carcinoma (MTC) is caused by germline mutations in the RET proto-oncogene, resulting in constitutive activation of the RET tyrosine kinase. A substantial proportion of sporadic MTCs also have RET mutations, making the RET tyrosine kinase a potential therapeutic target in MTC. We have established a transplantable MTC in nude mice from a sporadic human MTC carrying a RET C634R mutation. Transplanted tumors had an exponential growth rate with an approximate doubling time of about 3 weeks, and expressed a neuroendocrine phenotype characteristic of MTC, e.g., expression of calcitonin, chromogranin A (CgA), synaptophysin, synaptic vesicle protein 2 (SV2), vesicular monoamine transporter-1 and -2, carcinoembryonic antigen, cytokeratin 8/18, epithelial cadherin, and neural cell adhesion molecule. Plasma calcitonin and CgA levels were elevated in tumor-bearing mice and correlated with tumor size. Cytogenetic analysis, including spectral karyotyping, confirmed the human origin of the xenografted tumors and demonstrated an abnormal, near triploid karyotype. Treatment of tumor-bearing nude mice with the tyrosine kinase inhibitor ZD6474, which specifically inhibits RET, epidermal growth factor receptor (EGFR), and vascular endothelium growth factor receptor (VEGFR) tyrosine kinases, resulted in a dose-dependent inhibition of tumor growth. Oral ZD6474 given once daily (250 mg/kg, 5 days/week) reduced tumor volume to 11% when compared with controls after 4 weeks. Our results show that this transplantable MTC, designated GOT2, represents a novel and useful model for studies of MTC and RET tyrosine kinase-dependent tumor growth.

Mutation Analysis of the Short Cytoplasmic Domain of the Cell-Cell Adhesion Molecule CEACAM1 Identifies Residues That Orchestrate Actin Binding and Lumen Formation

CEACAM1-4S (carcinoembryonic antigen cell adhesion molecule 1, with 4 ectodomains and a short, 12-14 amino acid cytoplasmic domain) mediates lumen formation via an apoptotic and cytoskeletal reorganization mechanism when mammary epithelial cells are grown in a three-dimensional model of mammary morphogenesis. We show by quantitative yeast two-hybrid, BIAcore, NMR HSQC and STD, and confocal analyses that amino acids phenylalanine (Phe(454)) and lysine (Lys(456)) are key residues that interact with actin orchestrating the cytoskeletal reorganization. A CEACAM1 membrane model based on vitamin D-binding protein that predicts an interaction of Phe(454) at subdomain 3 of actin was supported by inhibition of binding of actin to vitamin D-binding protein by the cytoplasmic domain peptide. We also show that residues Thr(457) and/or Ser(459) are phosphorylated in CEACAM1-transfected cells grown in three-dimensional culture and that mutation analysis of these residues (T457A/S459A) or F454A blocks lumen formation. These studies demonstrate that a short cytoplasmic domain membrane receptor can directly mediate substantial intracellular signaling.

A HUMANIZED PEGYLATED SINGLE CHAIN FRAGMENT VARIABLE (SCFV) TARGETING CARCINOEMBRYONIC ANTIGEN CELL ADHESION MOLECULE 6 REDUCES TUMOR BURDEN IN A SCID MOUSE MODEL BOTH ALONE AND IN COMBINATION WITH GEMCITABINE BY INCREASING APOPTOSIS AND DECREASING PROLIFERATION.

Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer and death in North America. In the year 2002, adenocarcinoma of the exocrine pancreas accounted for ≈30,300 new ases and 29,700 deaths. The current treatment strategy for localized pancreatic cancer is multimodality therapy combining pancreaticoduodenectomy with postoperative adjuvant chemotherapy and external beam radiation therapy, which results in a median overall survival of 12 to 24 months. For locally advanced and metastatic pancreatic cancer, the median survival is 6 to 12 months with currently available palliative chemotherapy. CEACAM6 (carcinoembryonic antigen cell adhesion molecule 6 [CD66c]) is an oncogene expressed on the cell surface as a GPI-linked glycoprotein comprised of 3 Ig-like domains capable of homophilic and heterophilic (CEACAM 5 and 8) interactions. It is highly expressed in > 90% of patients with pancreatic ductal adenocarcinoma. Deregulated expression of CEACAM6 inhibits differentiation and apoptosis of cells deprived of their anchorage to the extracellular matrix. We have previously demonstrated that CEACAM6-expressing pancreatic cancer cell lines treated with a murine monoclonal antibody (13-1) display reduced cellular viability by MTS assay, an indication of cytotoxicity. A murine scFv based on mAb (13-1) as well as a humanized form of the murine scFv also showed in vitro activity in CEACAM6-expressing cancer cell lines. In vivo studies using an SCID mouse model xenograft with BXPC-3 cells indicated a significant decrease in tumor burden as a result of treatment with a PEGylated form of the previously mentioned humanized scFv. The humanized scFv drug reduced the tumor burden as a stand-alone agent and in combination with gemcitabine compared with the control animal groups. Immunohistochemical analysis of tumors excised from animals treated with the humanized scFv and untreated animals indicated a marked increase in apoptosis (H&E) as well as a marked decrease in proliferation (Ki67) in the treated group relative to the untreated group. This observed in vivo effect on proliferation and apoptosis is currently being confirmed in vitro using CEACAM6-expressing cancer cell lines.

CEACAM6: A HORMONALLY REGULATED PROTEIN IN DIFFERENTIATING HUMAN FETAL LUNG TYPE II CELLS.

Ceacam6 (carcinoembryonic antigen cell adhesion molecule) is a GPI-anchored membrane glycoprotein recently identified in human fetal lung undergoing in vitro hormone-induced differentiation of type II cells. The protein is a tumor biomarker in adult lung and selected other tissues, where it has antimicrobial activity; however, expression and function in lung have not been characterized. Undifferentiated epithelial cells were isolated by collagenase/trypsin digestion from human fetal lung (16-21 wk gestation) and cultured 1-5 d in the absence (control) or presence of DCI (dexamethasone, 10 nM; 8-Br-cAMP, 0.1 mM; isobutylmethylxanthine, 0.1 mM). Some cells in control media were treated with adenovirus Ad12A2 (0.5-6 pfu/cell) to overexpress thyroid transcription factor-1 (TTF-1) or were transfected by electroporation (AMAXA nucleofection technology) with siRNAs (200 nM) for TTF-1 or Ceacam6 to knockdown expression in the presence of DCI. CEACAM6 mRNA and protein were up-regulated ≈10-fold by exposure of fetal lung epithelial cells to either DCI or to a dose of Ad12A2 that produced a level of recombinant TTF-1 comparable with endogenous TTF-1 in DCI-treated cells. By confocal immunofluorescence, CEACAM6 localized to both intracellular vesicles, costaining with SP-B (ie, lamellar bodies), and to the plasma membrane of type II cells. Knockdown of TTF-1 (46 ± 4% of control) in DCI-treated cells reduced CEACAM6 mRNA and protein by 80% (n = 4). Knockdown of CEACAM6 expression by siRNA (88 ± 3%, n = 3) did not alter expression or staining patterns of TTF-1, SP-B or DC-LAMP (a lamellar body membrane protein). Secretion rates for CEACAM6 were similar for both DCI- and Ad12A2-treated cells, with no response to treatment with secretagogues that stimulate surfactant secretion. Treatment of cells with mannosamine, an inhibitor of GPI anchor synthesis, blocked ≈50% of CEACAM6 secretion, consistent with GPI-anchor involvement in the secretory mechanism. We conclude that CEACAM6 expression in primary fetal lung epithelial cells is regulated by glucocorticoid and cAMP and is dependent on TTF-1. CEACAM6 is associated with intracellular surfactant but is secreted primarily by a constitutive pathway independent of lamellar bodies. CEACAM6 may contribute to the innate defense mechanisms of the mature type II cell.

Expression of Carcinoembryonic Antigen Cell Adhesion Molecule 6 Oncoprotein in Atypical Ductal Hyperplastic Tissues Is Associated with the Development of Invasive Breast Cancer

Epidemiologic studies have established that women with prior atypical ductal hyperplastic (ADH) lesions have a 5-fold increased risk of developing invasive breast cancer (IBC). However, there is currently no means of identifying a subclass of ADH from women who will most likely develop cancer. The purpose of this study is to investigate whether elevated expression of carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) in ADH tissues is associated with the development of IBC. A retrospective study was conducted with archival ADH tissues and clinical information on the development/nondevelopment of IBC. The control group was ADH from patients who had no prior history of IBC and did not develop cancer within 5 years after the diagnosis of ADH (n = 44). The test group was ADH from patients who either developed cancer concurrently or subsequently after diagnosis (ADHC; n = 44). The expression of CEACAM6 was studied by immunohistochemistry and the results were statistically analyzed for significant association to develop cancer (P value), specificity, sensitivity, positive predictive value, and negative predictive value. Of the 44 control ADH tissues from patients with no history of cancer, 9 were positive for CEACAM6. Among the ADHC tissues, 40 of 44 samples were positive. Statistical analysis of CEACAM6 expression data showed a significant association between its expression and cancer development, high sensitivity, specificity, positive predictive value, and negative predictive value. The expression of CEACAM6 in ADH lesions is strongly associated with the development of IBC, therefore, it can be applied as a diagnostic marker either singly or in combination with other marker(s) to predict IBC development in women with ADH lesions. It could also be a potential molecular therapeutic target for preventing IBC.

Immunomodulatory Roles of the Carcinoembryonic Antigen Family of Glycoproteins

One of the most remarkable aspects of the immune system is its ability to fashion an immune response most appropriate to the activating stimulus. Although the immune system possesses a number of adaptations to accomplish this, an important theme is local immune regulation by site-specific expression of receptors and ligands. One family of molecules that is gaining attention as modulators of the immune system is the carcinoembryonic antigen cell-adhesion molecule family (CEACAM). Functionally, the carcinoembryonic antigen family can mediate cell-cell contact, host-pathogen interactions, and immune regulation. For example, biliary glycoprotein (CEACAM1) can have direct activity on T cells, leading to the inhibition of helper or cytotoxic T cell function. The expression of carcinoembryonic antigen (CEACAM5) on intestinal epithelial cells is involved in the activation of populations of regulatory CD8(+) T cells, while a distinct subset of regulatory CD8+ T cells is activated by nonspecific cross-reacting antigen (CEACAM6) on placental trophoblasts. Interestingly, the function and phenotype of these cells depend upon the specific member of the carcinoembryonic antigen family expressed, as well as the antigen-presenting molecule with which it associates. Thus, these glycoproteins comprise a family of molecules whose functions can depend on their nature and context.

Use of magnetic beads with immobilized monoclonal antibodies for isolation of highly pure plasma membranes

In plasma membrane proteome research, contamination of the isolated plasma membrane fraction with proteins from other organelles is still a problem. Even if highly specific isolation methods are used, such as density gradient centrifugation combined with selective extraction, contaminating proteins cannot be completely removed. To solve this problem, a protocol for the isolation of highly pure plasma membrane fractions from rat liver and two different hepatocellular carcinoma cell lines was developed. Magnetic beads with immobilized mAb's against highly expressed membrane proteins were used for specific binding of membrane vesicles and their separation from other organelles. Isolated plasma membranes were further selectively solubilized with different reagents and analyzed by use of different methods, such as Western blotting, 1- and 2-DE, and MS. Purification and further selective solubilization was validated by use of mAb's against the marker integral plasma membrane protein carcinoembryonic antigen cell adhesion molecule 1, and identification of isolated proteins by MS. The method presented here minimizes contamination with other organelles and enables further identification of membrane proteins.

In Vivo Selection for Neisseria gonorrhoeae Opacity Protein Expression in the Absence of Human Carcinoembryonic Antigen Cell Adhesion Molecules

The neisserial opacity (Opa) proteins are phase-variable, antigenically distinct outer membrane proteins that mediate adherence to and invasion of human cells. We previously reported that Neisseria gonorrhoeae Opa protein expression appeared to be selected for or induced during experimental murine genital tract infection. Here we further defined the kinetics of recovery of Opa variants from the lower genital tracts of female mice and investigated the basis for this initial observation. We found that the recovery of different Opa phenotypes from mice appears cyclical. Three phases of infection were defined. Following intravaginal inoculation with primarily Opa- gonococci, the majority of isolates recovered were Opa+ (early phase). A subsequent decline in the percentage of Opa+ isolates occurred in a majority of mice (middle phase) and was followed by a reemergence of Opa+ variants in mice that were infected for longer than 8 days (late phase). We showed the early phase was due to selection for preexisting Opa+ variants in the inoculum by constructing a chloramphenicol-resistant (Cm(r)) strain and following Cm(r) Opa+ populations mixed with a higher percentage of Opa- variants of the wild-type (Cm(s)) strain. Reciprocal experiments (Opa- Cm(r) gonococci spiked with Opa+ Cm(s) bacteria) were consistent with selection of Opa+ variants. Based on the absence in mice of human carcinoembryonic antigen cell adhesion molecules, the major class of Opa protein adherence receptors, we conclude the observed selection for Opa+ variants early in infection is not likely due to a specific adherence advantage and may be due to Opa-mediated evasion of innate defenses.

Carcinoembryonic Antigen and Related Molecules in Normal and Transformed Trophoblast

Carcinoembryonic antigen (CEA, CD66e) and CEA-related cell adhesion molecules (CEACAMs) are important mediators in remodeling of diverse human tissues, and modulators of cell proliferation and differentiation. Expression by normal and transformed trophoblast of gestational trophoblastic diseases (GTDs), isolated cytotrophoblast and choriocarcinoma cell lines is presented here. Immunocyto/histochemistry of normal placenta ( n = 9), invasive mole ( n = 8), choriocarcinoma ( n = 7), a placental site trophoblastic tumor, cytotrophoblast in primary culture and JAr and JEG-3 cells was performed using polyclonal anti-CEA and specific monoclonal anti-CEA antibodies. Data were analyzed and scored using Mann–Whitney Test. CEA and CEA-related molecules were identified by Western blot and immunoaffinity chromatography in JAr and JEG-3 cells and extracts of 1st and 3rd trimester of pregnancy tissue and cytotrophoblast cell lysates. CEA is expressed throughout pregnancy, in first trimester predominantly in syncytiotrophoblast, but also in villous cytotrophoblast and extravillous trophoblast. Data presented here demonstrate that CEA is significantly increased in transformed trophoblast of GTDs ( p < 0.05). Both cytotrophoblast in primary culture and choriocarcinoma cell lines express CEA, with staining of granular deposits in JAr and cell membrane in JEG-3. The results suggest that CEA (CD66e) and other CEA-related protein(s) could be involved in trophoblast differentiation.

The involvement of NK cells in ankylosing spondylitis

A role for NK cells in the regulation of autoimmunity has been demonstrated. Since there is a strong association between Ankylosing Spondylitis (AS) and HLA-B27, which is specifically recognized by the NK-inhibitory receptor KIR3DL1, this study evaluated the potential involvement of NK cells in AS. We studied 19 AS patients and 22 healthy volunteer donors and assessed the percentage, activity and receptor expression of peripheral blood NK cells. We also evaluated candidate-inflammatory mediators in sera. We found that AS patients have significantly higher percentages of NK cells. However, we found no differences between the ability of NK cells derived from AS and healthy controls to recognize target cells expressing HLA-B27. Remarkably, we observed that the NK-inhibitory receptor CEACAM1 (carcino-embryonic antigen-cell adhesion molecule) is highly expressed among AS-derived NK cells. Furthermore, engagement of CEACAM1 inhibited NK activity in these patients. Finally, we demonstrated that CEACAM1 expression is induced by IL-8 and SDF-1 (stromal cell derived factor), both of which are present in high levels in the sera of AS patients. These results may indicate that NK cells and CEACAM1 play a role in AS pathogenesis and implicate chemokines in the mechanism of CEACAM1 expression.

Receptor-Independent Spread of a Highly Neurotropic Murine Coronavirus JHMV Strain from Initially Infected Microglial Cells in Mixed Neural Cultures

Although neurovirulent mouse hepatitis virus (MHV) strain JHMV multiplies in a variety of brain cells, expression of its receptor carcinoembryonic antigen cell adhesion molecule 1 (CEACAM 1) (MHVR) is restricted only in microglia. The present study was undertaken to clarify the mechanism of an extensive JHMV infection in the brain by using neural cells isolated from mouse brain. In contrast to wild-type (wt) JHMV, a soluble-receptor-resistant mutant (srr7) infects and spreads solely in an MHVR-dependent fashion (F. Taguchi and S. Matsuyama, J. Virol. 76:950-958, 2002). In mixed neural cell cultures, srr7 infected a limited number of cells and infection did not spread, although wt JHMV induced syncytia in most of the cells. srr7-infected cells were positive for GS-lectin, a microglia marker. Fluorescence-activated cell sorter analysis showed that about 80% of the brain cells stained with anti-MHVR antibody (CC1) were also positive for GS-lectin. Pretreatment of those cells with CC1 prevented virus attachment to the cell surface and also blocked virus infection. These results show that microglia express functional MHVR that mediates JHMV infection. As expected, in microglial cell-enriched cultures, both srr7and wt JHMV produced syncytia in a majority of cells. Treatment with CC1 of mixed neural cell cultures and microglia cultures previously infected with wt virus failed to block the spread of infection, indicating that wt infection spreads in an MHVR-independent fashion. Thus, the present study indicates that microglial cells are the major population of the initial target for MHV infection and that the wt spreads from initially infected microglia to a variety of cells in an MHVR-independent fashion.

Angiogenic activation of valvular endothelial cells in aortic valve stenosis

Here, we demonstrate the angiogenic response of valvular endothelial cells to aortic valve (AV) stenosis using a new ex vivo model of aortic leaflets. Histological analysis revealed neovascularization within the cusps of stenotic but not of non-stenotic aortic valves. Correspondingly, the number of capillary-like outgrowth in 3D collagen gel was significantly higher in stenotic than in non-stenotic valves. Capillary-like sprouting was developed significantly faster in stenotic than in non-stenotic valves. New capillary sprouts from stenotic aortic valves exhibited the endothelial cell markers CD31, CD34 and von-Willebrand factor (vWF) as well as carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1), Tie-2 and angiogenesis inhibitor endostatin. Western blot analyses revealed a significant increase of CEACAM1 and endostatin in stenotic aortic valve tissue. Electron microscopic examinations demonstrate that these capillary-like tubes are formed by endothelial cells containing Weibel–Palade bodies. Remarkably, inter-endothelial junctions are established and basement membrane material is partially deposited on the basal side of the endothelial tubes. Our data demonstrate the capillary-like sprout formation from aortic valves and suggest a role of angiogenesis in the pathogenesis of aortic valve stenosis. These data provide new insights into the mechanisms of valvular disorders and open new perspectives for prevention and early treatment of calcified aortic stenosis.