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1.  Dynamic Changes in Endothelial Cell Adhesion Molecule Nepmucin/CD300LG Expression under Physiological and Pathological Conditions 
PLoS ONE  2013;8(12):e83681.
Vascular endothelial cells often change their phenotype to adapt to their local microenvironment. Here we report that the vascular endothelial adhesion molecule nepmucin/CD300LG, which is implicated in lymphocyte binding and transmigration, shows unique expression patterns in the microvascular endothelial cells of different tissues. Under physiological conditions, nepmucin/CD300LG was constitutively and selectively expressed at the luminal surface of the small arterioles, venules, and capillaries of most tissues, but it was only weakly expressed in the microvessels of the splenic red pulp and thymic medulla. Furthermore, it was barely detectable in immunologically privileged sites such as the brain, testis, and uterus. The nepmucin/CD300LG expression rapidly decreased in lymph nodes receiving acute inflammatory signals, and this loss was mediated at least in part by TNF-α. It was also down-regulated in tumors and tumor-draining lymph nodes, indicating that nepmucin/CD300LG expression is negatively regulated by locally produced signals under these circumstances. In contrast, nepmucin/CD300LG was induced in the high endothelial venule-like blood vessels of chronically inflamed pancreatic islets in an animal model of non-obese diabetes. Interestingly, the activated CD4+ T cells infiltrating the inflamed pancreas expressed high levels of the nepmucin/CD300LG ligand(s), supporting the idea that nepmucin/CD300LG and its ligand interactions are locally involved in pathological T cell trafficking. Taken together, these observations indicate that the nepmucin/CD300LG expression in microvascular endothelial cells is influenced by factor(s) that are locally produced in tissues, and that its expression is closely correlated with the level of leukocyte infiltration in certain tissues.
PMCID: PMC3871519  PMID: 24376728
2.  Relationship between Major Histocompatibility Complex Class I Expression and Prognosis in Canine Mammary Gland Tumors 
The aim of this study was to evaluate MHC class I expression and prognosis using tumor tissues surgically removed from 9 dogs with mammary gland carcinomas and from 13 dogs with complex carcinomas. We assessed MHC class I expression and its correlation with tumor size, B2M expression, infiltration of lymphocytes, histological grade and prognosis. Hematoxylin and eosin-stained sections were histologically graded using the Elston and Ellis grading method. MHC class I expression on tumor cells was evaluated using the avidin-biotin peroxidase complex method. Loss of MHC class I expression from canine mammary gland carcinomas was significantly correlated with poor prognosis (P<0.05). Loss of MHC class I expression showed no association with poor prognosis in canine mammary gland complex carcinomas, because the data were not balanced. Only 1 of 13 (7.6%) canine mammary gland complex carcinomas showed loss of MHC class I expression. All 13 of these dogs showed good prognosis. Thus, the low frequency of MHC class I expression loss from canine mammary gland complex carcinomas may be associated with good prognosis. Taken together, these results suggest that loss of MHC class I expression may be associated with poor prognosis in canine mammary gland carcinomas.
PMCID: PMC3942927  PMID: 23728200
beta2-microglobulin; canine; mammary gland carcinoma; MHC class I
3.  Immunization with a Recombinant Vaccinia Virus That Encodes Nonstructural Proteins of the Hepatitis C Virus Suppresses Viral Protein Levels in Mouse Liver 
PLoS ONE  2012;7(12):e51656.
Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29(+/−)/MxCre(+/−) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.
PMCID: PMC3524174  PMID: 23284733
4.  Structural Basis of Alternative DNA Recognition by Maf Transcription Factors▿  
Molecular and Cellular Biology  2009;29(23):6232-6244.
Maf transcription factors constitute a family of the basic region-leucine zipper (bZip) factors and recognize unusually long DNA motifs (13 or 14 bp), termed the Maf recognition element (MARE). The MARE harbors extended GC sequences on each side of its core motif, which is similar to TRE or CRE (7 or 8 bp) recognized by the AP1 and CREB/ATF families, respectively. To ascertain the structural basis governing the acquirement of such unique DNA recognition, we determined the crystal structure of the MafG-DNA complex. Each MafG monomer consists of three helices in which the carboxyl-terminal long helix organizes one DNA-contacting element and one coiled-coil dimer formation element. To our surprise, two well-conserved residues, Arg57 and Asn61 in the basic region, play critical roles in Maf-specific DNA recognition. These two residues show unique side-chain orientations and interact directly with the extended GC bases. Maf-specific residues in the amino-terminal and basic regions appear to indirectly stabilize MARE recognition through DNA backbone phosphate interactions. This study revealed an alternative DNA recognition mechanism of the bZip factors that bestows specific target gene profiles upon Maf homodimers or Maf-containing heterodimers.
PMCID: PMC2786689  PMID: 19797082
5.  Regulation of leukocyte migration across endothelial barriers by ecto-5’-nucleotidase-generated adenosine 
CD73-deficient mice are valuable for evaluating the ability of CD73-generated adenosine to modulate adenosine receptor-mediated responses. Here we report the role of CD73 in regulating lymphocyte migration across two distinct barriers. In the first case, CD73-generated adenosine restricts the migration of lymphocytes across high endothelial venules (HEV) into draining lymph nodes after an inflammatory stimulus, apparently by triggering A2B receptors on HEV. Secondly, CD73 promotes the migration of pathogenic T cells into the central nervous system during experimental autoimmune encephalomyelitis. Experiments are in progress to determine whether this effect is also adenosine receptor-mediated and to identify the relevant adenosine receptor.
PMCID: PMC2776682  PMID: 18600537
CD73; ecto-5’-nucleotidase; leukocyte migration; experimental autoimmune encephalomyelitis
6.  CD73-generated Adenosine Restricts Lymphocyte Migration into Draining Lymph Nodes1 
After an inflammatory stimulus, lymphocyte migration into draining lymph nodes increases dramatically to facilitate the encounter of naïve T cells with antigen-loaded dendritic cells. Here we show that CD73 (ecto-5′-nucleotidase) plays an important role in regulating this process. CD73 produces adenosine from AMP and is expressed on high endothelial venules (HEV) and subsets of lymphocytes. Cd73-/- mice have normal sized lymphoid organs in the steady state, but approximately 1.5-fold larger draining lymph nodes and 2.5-fold increased rates of L-selectin-dependent lymphocyte migration from the blood through HEV compared to wild type mice 24 hours after LPS administration. Migration rates of cd73+/+ and cd73-/- lymphocytes into lymph nodes of wild type mice are equal, suggesting that it is CD73 on HEV that regulates lymphocyte migration into draining lymph nodes. The A2B receptor is a likely target of CD73-generated adenosine, as it is the only adenosine receptor expressed on the HEV-like cell line KOP2.16 and it is up regulated by TNFα. Furthermore, increased lymphocyte migration into draining lymph nodes of cd73-/- mice is largely normalized by pretreatment with the selective A2B receptor agonist BAY 60-6583. Adenosine receptor signaling to restrict lymphocyte migration across HEV may be an important mechanism to control the magnitude of an inflammatory response.
PMCID: PMC2709497  PMID: 18424752
Rodent; Cell Trafficking; Spleen and lymph nodes; Inflammation
7.  Nepmucin, a novel HEV sialomucin, mediates L-selectin–dependent lymphocyte rolling and promotes lymphocyte adhesion under flow 
The Journal of Experimental Medicine  2006;203(6):1603-1614.
Lymphocyte trafficking to lymph nodes (LNs) is initiated by the interaction between lymphocyte L-selectin and certain sialomucins, collectively termed peripheral node addressin (PNAd), carrying specific carbohydrates expressed by LN high endothelial venules (HEVs). Here, we identified a novel HEV-associated sialomucin, nepmucin (mucin not expressed in Peyer's patches [PPs]), that is expressed in LN HEVs but not detectable in PP HEVs at the protein level. Unlike conventional sialomucins, nepmucin contains a single V-type immunoglobulin (Ig) domain and a mucin-like domain. Using materials affinity-purified from LN lysates with soluble L-selectin, we found that two higher molecular weight species of nepmucin (75 and 95 kD) were decorated with oligosaccharides that bind L-selectin as well as an HEV-specific MECA-79 monoclonal antibody. Electron microscopic analysis showed that nepmucin accumulates in the extended luminal microvillus processes of LN HEVs. Upon appropriate glycosylation, nepmucin supported lymphocyte rolling via its mucin-like domain under physiological flow conditions. Furthermore, unlike most other sialomucins, nepmucin bound lymphocytes via its Ig domain, apparently independently of lymphocyte function–associated antigen 1 and very late antigen 4, and promoted shear-resistant lymphocyte binding in combination with intercellular adhesion molecule 1. Collectively, these results suggest that nepmucin may serve as a dual-functioning PNAd in LN HEVs, mediating both lymphocyte rolling and binding via different functional domains.
PMCID: PMC2118321  PMID: 16754720
8.  Toll-Like Receptor Adaptor Molecules Enhance DNA-Raised Adaptive Immune Responses against Influenza and Tumors through Activation of Innate Immunity 
Journal of Virology  2006;80(13):6218-6224.
Toll-like receptors (TLRs) recognize microbial components and trigger the signaling cascade that activates the innate and adaptive immunity. TLR adaptor molecules play a central role in this cascade; thus, we hypothesized that overexpression of TLR adaptor molecules could mimic infection without any microbial components. Dual-promoter plasmids that carry an antigen and a TLR adaptor molecule such as the Toll-interleukin-1 receptor domain-containing adaptor-inducing beta interferon (TRIF) or myeloid differentiation factor 88 (MyD88) were constructed and administered to mice to determine if these molecules can act as an adjuvant. A DNA vaccine incorporated with the MyD88 genetic adjuvant enhanced antigen-specific humoral immune responses, whereas that with the TRIF genetic adjuvant enhanced cellular immune responses. Incorporating the TRIF genetic adjuvant in a DNA vaccine targeting the influenza HA antigen or the tumor-associated antigen E7 conferred superior protection. These results indicate that TLR adaptor molecules can bridge innate and adaptive immunity and potentiate the effects of DNA vaccines against virus infection and tumors.
PMCID: PMC1488967  PMID: 16775309
10.  Keap1 Recruits Neh2 through Binding to ETGE and DLG Motifs: Characterization of the Two-Site Molecular Recognition Model 
Molecular and Cellular Biology  2006;26(8):2887-2900.
The expression of the phase 2 detoxification enzymes and antioxidant proteins is induced at the transcriptional level by Nrf2 and negatively regulated at the posttranslational level by Keap1 through protein-protein interactions with and subsequent proteolysis of Nrf2. We found that the Neh2 domain of Nrf2 is an intrinsically disordered but biologically active regulatory domain containing a 33-residue central α-helix followed by a mini antiparallel β-sheet. Isothermal calorimetry analysis indicated that one Neh2 molecule interacts with two molecules of Keap1 via two binding sites, the stronger binding ETGE motif and the weaker binding DLG motif. Nuclear magnetic resonance titration study showed that these two motifs of the Neh2 domain bind to an overlapping site on the bottom surface of the β-propeller structure of Keap1. In contrast, the central α-helix of the Neh2 domain does not have any observable affinity to Keap1, suggesting that this region may serve as a bridge connecting the two motifs for the association with the two β-propeller structures of a dimer of Keap1. Based on these observations, we propose that Keap1 recruits Nrf2 by the ETGE motif and that the DLG motif of the Neh2 domain locks its lysine-rich central α-helix in a correct position to benefit ubiquitin signaling.
PMCID: PMC1446969  PMID: 16581765
11.  Capsaicin-enhanced Ribosomal Protein P2 Expression in Human Intestinal Caco-2 Cells 
Cytotechnology  2005;47(1-3):89-96.
On the basis of transepithelial electrical resistance (TER) measurements, we found that capsaicin (100 μM)-treated human intestinal Caco-2 cells show a momentary increase in tight-junction (TJ) permeability (decrease in TER) followed by a complete recovery. We used proteome analysis to search for proteins that are associated with the recovery of TJ permeability in capsaicin-treated Caco-2 cells. A protein with a relative molecular mass of 14 kDa was found to be expressed more highly in capsaicin-treated cells than in nontreated cells. Mass spectrometry and sequence analyses revealed that the protein that is expressed significantly upon capsaicin treatment is the ribosomal protein P2; its cDNA sequence was identical to that found in the human genome database. An increase in the amount of cellular filamentous actin (F-actin) was shown after 8 h of incubation with capsaicin. It has been reported that P2 activates elongation factor 2, which stabilizes F-actin filaments, and that the depolymerization of F-actin is associated with the increase in TJ permeability (decrease in TER). Consequently, these results suggest that P2 plays an important role in the recovery of the TJ permeability in capsaicin-treated human intestinal cells.
PMCID: PMC3449817  PMID: 19003048
Caco-2 Cells; Capsaicin; Elongation factor 2; F-actin; Proteome analysis; Ribosomal protein P2; Tight-junction permeability
12.  VLA-4-dependent and -independent pathways in cell contact-induced proinflammatory cytokine production by synovial nurse-like cells from rheumatoid arthritis patients 
Arthritis Research  2002;4(6):R10.
Nurse-like stromal cell lines from the synovial tissue of patients with rheumatoid arthritis (RA-SNC) produce, on coculture with lymphocytes, large amounts of proinflammatory cytokines. In the present paper, we analyze the molecular events necessary for the induction of cytokine release from RA-SNC cells, and particularly the roles played by cell adhesion and the transmigration (also known as pseudoemperipolesis) of lymphocytes. For this purpose, the effects of various mAbs on the binding and transmigration of a human B-cell line, MC/car, were examined using a cloned RA-SNC line, RA-SNC77. To analyze the role of lymphocyte binding and transmigration on upregulated cytokine production by the RA-SNC77 cells, we used C3 exoenzyme-treated MC/car cells, which could bind to RA-SNC77 cells but could not transmigrate. Treatment with anti-CD29 or anti-CD49d mAb significantly reduced binding and transmigration of the MC/car cells. In contrast, the neutralizing anti-CD106/vascular cell adhesion molecule 1 mAb did not show any inhibitory effect. Likewise, none of the neutralizing mAbs against CD11a, CD18, CD44, CD49e, or CD54 showed significant effects. Binding of C3-treated or untreated MC/car cells to RA-SNC77 cells induced comparable levels of IL-6 and IL-8 production. In addition, the enhanced cytokine production by RA-SNC77 cells required direct lymphocyte contact via a very late antigen-4 (VLA-4)-independent adhesion pathway. These results indicate that, although both the VLA-4-dependent/vascular cell adhesion molecule 1-independent and the VLA4-independent adhesion pathways are involved in MC/car binding and subsequent transmigration, only the VLA4-independent adhesion pathway is necessary and sufficient for the enhanced proinflammatory cytokine production by RA-SNC77 cells. The transmigration process, which is dependent on Rho-GTPase, is not a prerequisite for this phenomenon.
PMCID: PMC153839  PMID: 12453313
cell adhesion; cytokine production; nurse cells; rheumatoid arthritis; transmigration
13.  Experimental Study of Thallium 201 Redistribution in Transient Myocardial Ischemia 
The influence of differential washout on the redistribution phenomenon of thallium 201 chloride deposited in ischemic myocardium was investigated. Two groups of dogs had serial scintigraphic images and tissue counting performed. The first group had ischemia produced prior to the injection of the thallium and, following the control image after production of ischemia, the occlusion was released and serial images obtained over a three-hour period. The second group received thallium initially and after a control scintigraph was taken, ischemia was created and maintained for three hours via transthoracic ligature. Activity distribution was followed by serial images. Thallium content of ischemic areas was compared to the normal area by computer assisted data analysis. Tissues from the ischemic and normal areas from both groups were obtained after the serial images and counted. In the first group, prompt redistribution of activity into the ischemic areas was seen within 30 minutes of releasing the occlusion and was verified by tissue counting. Neither scintigraphic image changes nor tissue uptake differences were observed in the second group. The restoration of blood flow and consequently increased avidity for thallium probably accounted for the redistribution seen in the first group as there appeared to be no differential washout of activity in the second group from either the ischemic or normal areas to contribute to the redistribution phenomenon.
PMCID: PMC2552817  PMID: 7120444

Results 1-13 (13)