Pancreatic adenocarcinoma is characterized by late diagnosis due to lack of early symptoms, extensive metastasis, and high resistance to chemo/radiation therapy. Recently, a subpopulation of cells within pancreatic cancers, termed cancer stem cells (CSCs), has been characterized and postulated to be the drivers for pancreatic cancer and responsible for metastatic spread. Further studies on pancreatic CSCs are therefore of particular importance to identify novel diagnosis markers and therapeutic targets for this dismal disease. Herein, the malignant phenotype of pancreatic cancer stem-like CD24+CD44+ cells was isolated from a human pancreatic carcinoma cell line (PANC-1) and demonstrated 4-fold increased invasion ability compared to CD24-CD44+ cells. Using lectin microarray and nano LC-MS/MS, we identified a differentially expressed set of glycoproteins between these two subpopulations. Lectin microarray analysis revealed that fucose- and galactose-specific lectins, UEA-1 and DBA, respectively, exhibit distinctly strong binding to CD24+CD44+ cells. The glycoproteins extracted by multilectin affinity chromatography were consequently analyzed by LC-MS/MS. Seventeen differentially expressed glycoproteins were identified, including up-regulated Cytokeratin 8/CK8, Integrin β1/CD29, ICAM1/CD54, and Ribophorin 2/RPN2 and down-regulated Aminopeptidase N/CD13. Immunohistochemical analysis of tissue microarrays showed that CD24 was significantly associated with late-stage pancreatic adenocarcinomas, and RPN2 was exclusively coexpressed with CD24 in a small population of CD24-positive cells. However, CD13 expression was dramatically decreased along with tumor progression, preferentially present on the apical membrane of ductal cells and vessels in early stage tumors. Our findings suggest that these glycoproteins may provide potential therapeutic targets and promising prognostic markers for pancreatic cancer.

Loss of E-cadherin triggers peritoneal dissemination, leading to an adverse prognosis for most patients with epithelial ovarian carcinoma (EOC). Because TWIST mainly regulates the epithelial-to-mesenchymal transition and is one of the E-cadherin repressors, we investigated the possibility that TWIST expression affects peritoneal metastasis of EOC using siRNA technique. In the present study, we showed a correlation between TWIST expression and EOC cellular morphology. Furthermore, we demonstrated that the suppression of TWIST expression in EOC cells (HEY) alters the cellular morphology from a fibroblastic and motile phenotype to an epithelial phenotype, and inhibits the adhesion of these cells to mesothelial monolayers. To investigate the mechanism by which down-regulation of TWIST leads to inhibition of adhesion to mesothelial cells (MCs), expression of adhesion molecules (CD29, CD44 and CD54) were observed. Moreover, matrix metalloproteinase 2 and membrane type 1 matrix metalloproteinase, important markers associated with invasive and metastatic potential, were remarkably reduced. This findings suggests that reduced expression of TWIST suppresses the multistep process of peritoneal dissemination (detachment from the primary lesion, adhesion to MCs and invasion of MCs) and may be a potential therapeutic target for the treatment of this carcinoma.

Primary immune responses are thought to be induced by dendritic cells. To promote such responses, dendritic cells must be activated by exogenous agonists, such as LPS, or by products of activated leukocytes, such as TNF-alpha and IL-1. How dendritic cells might be activated in the absence of exogenous stimuli, or without the immediate presence of activated leukocytes, as might occur in immunity to tumor cells or transplants, is unknown. We postulated that heparan sulfate, an acidic, biologically active polysaccharide associated with cell membranes and extracellular matrices, which is rapidly released under conditions of inflammation and tissue damage, might provide such a stimulus. Incubation of immature murine dendritic cells with heparan sulfate induced phenotypic maturation evidenced by up-regulation of I-A, CD40, CD54 (ICAM-1), CD80 (B7-1), and CD86 (B7-2). Dendritic cells exposed to heparan sulfate exhibited a markedly lowered rate of Ag uptake and increased allostimulatory capacity. Stimulation of dendritic cells with heparan sulfate induced release of TNF-alpha, IL-1beta, and IL-6, although the maturation of dendritic cells was independent of these cytokines. These results suggest that soluble heparan sulfate chains, as products of the degradation of heparan sulfate proteoglycan, might induce maturation of dendritic cells without exogenous stimuli, thus contributing to the generation and maintenance of primary immune responses.

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Transplantation of growth factor-mobilized peripheral blood progenitor cells (PBPC) is widely used in the treatment of several neoplastic diseases. While in PBPC harvests the presence of several accessory immune and tumor cells has been documented, that of stromal cells has not been reported. In the present study, we investigated for the presence of stromal cells in growth factor-mobilized PBPC harvests from breast cancer patients. Low-density cells from PBCP harvests in culture gave rise to an adherent layer containing fibroblast-like and large flat round cells. These cells express positive immunofluorescence staining for collagen I, collagen III, fibronectin, VCAM-1 (CD106), ICAM-1 (CD54) and mesenchymal antigens recognized by monoclonal antibodies, SH2 and SH3. PBPC-derived stromal cells do not express antigens CD34, CD45 and CD14. Stromal cells were detected in the PBPC harvests of 11/14 patients (median 0.63%; range 0.02-2.32) and their concentration correlates with the number of CD34+ cells in PBPC.

Invasion of distant tissues by tumor cells is the primary cause of therapeutic failure in the treatment of malignant lung cancer cells. Receptor activator of nuclear factor-κB ligand (RANKL) and its receptor, RANK, play a key role in osteoclastogenesis and tumor metastasis. Intercellular adhesion molecule-1 (ICAM-1, also called CD54), a member of the immunoglobulin supergene family, is an inducible surface glycoprotein that mediates adhesion-dependent cell-to-cell interactions. The effects of RANKL on cell migration and ICAM-1 expression in human lung cancer cells are largely unknown. We found that RANKL directed the migration and increased ICAM-1 expression in human lung cancer (A549) cells. Pretreatment of A549 cells with the MAPK kinase (MEK) inhibitor PD98059 or U0126 inhibited RANKL-mediated migration and ICAM-1 expression. Stimulation of cells with RANKL increased the phosphorylation of MEK and extracellular signal-regulating kinase (ERK). In addition, an NF-κB inhibitor (PDTC) and IκB protease inhibitor (TPCK) also inhibited RANKL-mediated cell migration and ICAM-1 up-regulation. Taken together, these results suggest that the RANKL and RANK interaction acts through MEK/ERK, which in turn activates NF-κB, resulting in the activation of ICAM-1 and contributing to the migration of human lung cancer cells.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) have immunosuppressive properties and have been used to treat steroid-refractory acute graft-versus-host disease (GVHD) in stem cell transplant patients. Cells with similar capacities can also be found in term placental tissue. We have isolated stromal cells from term fetal membrane (FMSCs), umbilical cords (UCSCs) and placental villi (PVSCs) as well as from bone marrow and compared their immunoregulatory capacity in allogeneic settings. We found that FMSCs and UCSCs suppressed proliferation significantly in mixed lymphocyte reactions (MLRs), whereas PVSCs showed inconsistent suppressive effects. When added to MLR cultures, FMSCs suppressed the production of interferon (IFN)-γ and interleukin (IL)-17, whereas UCSCs and PVSCs promoted the production of IL-17 instead. Secretion of IL-10 was increased after addition of FMSCs and UCSCs. In this setting, BM-MSCs had no significant effect on secretion of IFN-γ, IL-17 or IL-10 in MLR cultures. When analysing the expression of adhesion markers, we noted that FMSCs expressed the highest levels of CD29 (β1), CD49d (α4) and CD54 (ICAM-1) compared to the other types of stromal cells. Thus, our data indicate that stromal cells isolated from term fetal membrane have great immunosuppressive capacity in terms of proliferation and production of proinflammatory cytokines from alloreactive T cells, and also promote anti-inflammatory IL-10. They express high levels of integrins that may be of importance in homing to inflamed tissues. Fetal membrane may provide a valuable source of cells with immunosuppressive properties and could possibly be used for treatment of acute GVHD and other inflammatory disorders.

Umbilical cord tissue represents a unique source of cells with potential for cell therapy applications for multiple diseases. Human umbilical tissue-derived cells (hUTC) are a developmentally early stage, homogenous population of cells that are HLA-ABC dim, HLA-DR negative, and lack expression of co-stimulatory molecules in the unactivated state. The lack of HLA-DR and co-stimulatory molecule expression on unactivated hUTC may account for their reduced immunogenicity, facilitating their use in allogeneic settings. However, such approaches could be confounded by host innate cells such as natural killer (NK) cells. Here, we evaluate in vitro NK cell interactions with hUTC and compare them with human mesenchymal stem cells (MSC). Our investigations show that hUTC suppress NK activation, through prostaglandin-E2 secretion in a contact-independent manner. Prestimulation of hUTC or human MSC with interferon gamma (IFN-γ) induced expression of the tryptophan degrading enzyme indoleamine 2, 3 dioxygenase, facilitating enhanced suppression. However, resting NK cells of different killer immunoglobulin-like receptor haplotypes did not kill hUTC or MSC; only activated NK cells had the ability to kill nonstimulated hUTC and, to a lesser extent, MSC. The cell killing process involved signaling through the NKG2D receptor and the perforin/granzyme pathway; this was supported by CD54 (ICAM-1) expression by hUTC. IFN-γ-stimulated hUTC or hMSC were less susceptible to NK killing; in this case, protection was associated with elevated HLA-ABC expression. These data delineate the different mechanisms in a two-way interaction between NK cells and two distinct cell therapies, hUTC or hMSC, and how these interactions may influence their clinical applications.

T cells secrete bioactive exosomes (EXO), but the potential immunoregulatory effect of T-cell EXO is largely unknown. In this study, we generated activated ovalbumin (OVA)-specific CD4(+) T cells in vitro via coculture of OVA-pulsed dendritic cells (DC(OVA)) with naive CD4(+) T cells derived from OVA-specific T-cell receptor (TCR) transgenic OTII mice. CD4(+) T-cell EXO were then purified from the CD4(+) T-cell culture supernatants by differential ultracentrifugation. CD4(+) T-cell EXO exhibited the 'saucer' shape that is characteristic of EXO with a diameter between 50 and 100 nm, as assessed by electron microscopy, and contained the EXO-associated proteins LAMP-1, TCR and lymphocyte function associated antigen-1 (LFA-1), as determined by western blot. Flow cytometric analysis showed that CD4(+) T-cell EXO expressed CD4(+) T-cell markers (CD4, TCR, LFA-1, CD25 and Fas ligand), but to a lesser extent than CD4(+) T cells. We demonstrated that DC(OVA) took up CD4(+) T-cell EXO via peptide/major histocompatibility complex (pMHC) II/TCR and CD54/LFA-1 interactions. OVA-specific CD4(+) T-cell EXO from OTII mice, but not ConA-stimulated polyclonal CD4(+) T-cell EXO from wild-type C57BL/6 mice inhibited DC(OVA)-stimulated in vitro CD4(+) T-cell proliferation and in vivo CD8(+) cytotoxic T lymphocyte (CTL) responses and antitumor immunity against OVA-expressing B16 melanoma BL6-10(OVA) cells. In addition, EXO derived from a T-cell hybridoma cell line, MF72.2D9, expressing an OVA-specific CD4(+) TCR, had a similar inhibitory effect as OTII CD4(+) T-cell EXO on CTL-mediated antitumor immunity. Taken together, our data indicate that antigen-specific T-cell EXO may serve as a new type of immunosuppressive reagent for use in transplant rejection and treatment of autoimmune diseases.

Elevated plasma endothelial microparticles (EMP) have been documented in MS during exacerbation. However, the role of EMP in pathogenesis of MS remains unclear. We investigated the formation of EMP-monocyte conjugates (EMP-MoC) and their potential role in transendothelial migration of inflammatory cells in MS. EMP-MoC were assayed in 30 MS patients in exacerbation, 20 in remission and in 35 controls. EMP-leukocyte conjugation was investigated flowcytometrically by employing alpha-CD54 or alpha-CD62E for EMP, and alpha-CD45 for leukocytes. EMP-MoC were characterized by identifying adhesion molecules involved and their effect on monocyte function. In vivo (clinical): EMP-MoC were markedly elevated in exacerbation vs. remission and controls, correlating with presence of GD+ MRI lesions. Free CD54+ EMP were not elevated but free CD62E+ EMP were. In vitro: EMP bound preferentially to monocytes, less to neutrophils, but little to lymphocytes. Bound EMP activated monocytes: CD11b expression increased 50% and migration through cerebral endothelial cell layer increased 2.6-fold. Blockade of CD54 reduced binding by 80%. Most CD54+ EMP bound to monocytes, leaving little free EMP, while CD62+ EMP were found both free and bound. These results demonstrated that phenotypic subsets of EMP interacted differently with monocytes. Based on our observations, EMP may enhance inflammation and increase transendothelial migration of monocytes in MS by binding to and activating monocytes through CD54. EMP-MoC were markedly increased in MS patients in exacerbation compared to remission and may serve as a sensitive marker of MS disease activity.

The B7 molecule is expressed by APC that can costimulate T cells by binding the T cell surface receptors CD28 and CTLA-4. The human epidermal Langerhans cell (LC) is one of the most potent APC, yet B7 expression by this cell type has not previously been assessed. We used a CTLA4-Ig fusion protein that binds B7 with high avidity to probe cell surface expression of B7 by cultured and noncultured LC. LC cultured for 1 or more days were specifically stained with biotinylated CTLA4-Ig and fluorescent streptavidin. In contrast, binding of CTLA4-Ig to freshly isolated LC was not detected. The cell surface distributions of B7 and of HLA-DR on cultured LC differed, as CTLA4-Ig binding was localized to discrete foci, whereas anti-DR mAb uniformly stained the LC plasma membrane. Analyses of epidermal cell (EC) mRNA indicated that the B7 gene is expressed by these cells. Thus, B7 gene probes specifically hybridized to polymerase chain reaction-amplified B7 mRNA isolated from cultured and noncultured EC. As LC are the only normal epidermal cell type that induces proliferation of allogeneic T cells, the role of B7 in this LC function was studied by coculturing highly purified resting CD4+ T cells and allogeneic EC in the presence of CTLA4-Ig, anti-CD54 (RR/1, anti-intercellular adhesion molecule-1) mAb, or both. CTLA4-Ig and RR/1 each inhibited CD4+ T cell responses to freshly isolated allogeneic EC, and cooperative inhibition of more than 90% was observed in cultures treated with both CTLA4-Ig and RR/1 at 5 micrograms/ml. CTLA4-Ig inhibited stimulation by either fresh EC or cultured EC, suggesting that the increased potency of cultured LC vs noncultured LC may reflect the time needed for noncultured LC to express cell surface B7 in vitro. These studies indicate that B7 is expressed on the cell surface of cultured LC, and that LC B7 costimulates the proliferation of resting allogeneic CD4+ T cells.

CCR6 is a chemokine receptor that is expressed at the cell surface of Th17 cells, an IL-17- and IL-22-secreting population of CD4(+) T cells with antipathogenic, as well as inflammatory, properties. In the current study, we have determined the involvement of CCR6 in human Th17 lymphocyte migration toward inflamed tissue by analyzing the capacity of its ligands to induce arrest of these cells onto inflamed endothelium in vitro under flow conditions. We show that polarized, in situ-differentiated, skin-derived Th17 clones activated via the TCR-CD3 complex produce CCL20 in addition to IL-17 and IL-22. The latter cytokines induce, in a synergic fashion, the production of human β-defensin (hBD)-2, but neither hBD-1 nor hBD-3, by epidermal keratinocytes. Both CCL20 and hBD-2 are capable of inducing the arrest of Th17 cells, but not Th1 or Th2 cells, on HUVEC in an CD54-dependent manner that is CCR6 specific and independent from the expression of CXCR4, reported to be an alternative receptor for hBD-2. In addition, Ag-specific activation induces a transient loss of CCR6 expression, both at the transcriptional and protein level, which occurs with slow kinetics and is not due to endogenous CCL20-mediated internalization of CCR6. Together, these results indicate that Ag-specific activation will initially contribute to CCR6-mediated Th17 cell trafficking toward and sequestration in inflamed tissue, but that it eventually results in a transitory state of nonresponsiveness to further stimulation of these cells with CCR6 ligands, thus permitting their subsequent migration out of the inflamed site.

BACKGROUND

The benefit of the potent chemotherapeutic agent cisplatin in treating neoplasms is limited by nephrotoxicity. We tested the hypothesis that CD54 [intercellular adhesion molecule-1 (ICAM-1)] is an important mediator in cisplatin-mediated renal failure.

METHODS

The effect of a monoclonal anti-CD54 antibody was evaluated in a rat model of cisplatin toxicity. Renal function, histopathology, renal myeloperoxidase activity, and mortality were determined in the anti-CD54 and placebo groups.

RESULTS

Renal CD54 mRNA expression was markedly increased by 24 hours after exposure to cisplatin in mice. An improvement in renal function, mortality, and histological abnormalities was evident in animals exposed to cisplatin and treated with anti-CD54 antibody (mAb). Seven days after the administration of cisplatin, the mean creatinine was 0.65+/-0.05 mg/dl in the rats that received anti-CD54 mAb and 4.76+/-1.42 in control animals (P<0.02). Mortality was lower in experimental animals (0 vs. 29% in control rats seven days following cisplatin, P<0.04). Histological evidence of cell injury was markedly attenuated (P<0.04) in the treated compared with the control rats.

CONCLUSIONS

CD54 may be critical in the pathophysiology of renal injury following cisplatin, perhaps by its effects on leukocyte-endothelial interactions.

Fucoidan is a polysaccharide purified from the brown seaweed Fucus vesiculosus. Although some effects of fucoidan on immune functions have been elucidated, there have been no studies concerning the immunomodulatory effects of fucoidan on dendritic cells (DCs), which are powerful antigen-presenting cells. In this study, fucoidan increased the viability of DCs, the production of interleukin-12 and tumor necrosis factor-alpha, and the expression of major histocompatibility complex class I, class II, CD54, and CD86 molecules. Furthermore, fucoidan-treated DCs showed decreased antigen uptake and increased proliferation of allogeneic splenocytes. In a study of the transcriptional regulation effects of fucoidan, translocation of p65 molecules of nuclear factor-kappaB (NF-kappaB) from the cytosol to the nucleus was clearly observed in fucoidan-treated DCs. Taken together, the results suggest that fucoidan has immunostimulating and maturing effects on DCs, via a pathway involving at least NF-kappaB.

OBJECTIVE

Identification, quantification and isolation of subpopulations with characteristics of mesenchymal progenitor cells (MPC) from the synovial membrane (SM) from patients with osteoarthritis (OA).

METHODS

Cells from the SM of patients with end stage OA who underwent total knee joint replacement were enzymatically isolated. One aliquot was directly analyzed by fluorescence automated cell sorting (FACS) using various combinations of surface markers of bone marrow MPC (CD9, CD44, CD54, CD90, and CD166). Remaining cells were cultivated on plastic, expanded over several passages, analyzed by FACS again and tested for their osteo- and chondrogenic potential. The differentiation was analyzed by immuno-/histochemistry and by RT-PCR for the expression of lineage related marker genes.

RESULTS

Using FACS analysis we could show that the relative proportion of subpopulations expressing triplicate combinations of CD9, CD44, CD54, CD90 and CD166 in the SM from OA patients varies between 3 and 10%. Upon cultivation their relative amount markedly increased to values between 24 and 48%. Within the heterogeneous cell populations it was possible to induce osteogenic and chondrogenic differentiation. Initial sorting for CD9/CD90/CD166 triplicate positive cells proved that this subpopulation contains cells with multipotency for mesenchymal differentiation and thus characteristics of MPC.

CONCLUSIONS

Our results show that SM from OA patients contains cells that express typical combinations of MPC surface markers and have the potency of osteogenic and chondrogenic differentiation. Their relative enrichment during in vitro cultivation and the possibility of cell sorting to get more homogenous populations offer interesting perspectives for possible future therapeutic applications.

The biochemical properties of the molecular interactions mediating viral-cell recognition are poorly characterized. In this study, we use surface plasmon resonance to study the affinity and kinetics of the interaction of echovirus 11 with its cellular receptor decay-accelerating factor (CD55). As reported for interactions between cell-cell recognition molecules, the interaction has a low affinity (KD approximately 3.0 microM) as a result of a very fast dissociation rate constant (kon approximately 10(5) M-1.s-1, koff approximately 0.3 s-1). This contrasts with the interaction of soluble ICAM-1 (sICAM-1, CD54) with human rhinovirus 3 which has been reported to have a similar affinity but 10(2)-10(3)-fold slower kinetics (Casasnovas, J. M., and Springer, T. A. (1995) J. Biol. Chem. 270, 13216-13224). The extracellular portion of decay-accelerating factor comprises four short consensus repeat domains (domains 1-4) and a mucin-like stalk. By comparison of the binding affinity for echovirus 11 of various fragments of decay-accelerating factor, we are able to conclude that short consensus repeat domain 3 contributes approximately 80% of the binding energy.

OBJECTIVE

To assess the ability of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) to function as antigen-presenting cells (APCs) for arthritogenic autoantigens found within inflamed joint tissues.

METHODS

Human class II major histocompatibility complex (MHC)-typed FLS were used as APCs for murine class II MHC-restricted CD4 T cell hybridomas. Interferon-gamma (IFNgamma)-treated, antigen-loaded FLS were cocultured with T cell hybridomas specific for immunodominant portions of human cartilage gp-39 (HC gp-39) or human type II collagen (CII). T cell hybridoma activation was measured by enzyme-linked immunosorbent assay of culture supernatants for interleukin-2. Both synthetic peptide and synovial fluid (SF) were used as sources of antigen. APC function in cocultures was inhibited by using blocking antibodies to human class II MHC, CD54, or CD58, or to murine CD4, CD11a, or CD2.

RESULTS

Human FLS could present peptides from the autoantigens HC gp-39 and human CII to antigen-specific MHC-restricted T cell hybridomas. This response required pretreatment of FLS with IFNgamma, showed MHC restriction, and was dependent on human class II MHC and murine CD4 for effective antigen presentation. Furthermore, FLS were able to extract and present antigens found within human SF to both the HC gp-39 and human CII T cell hybridomas in an IFNgamma-dependent and MHC-restricted manner.

CONCLUSIONS

RA FLS can function as APCs and are able to present peptides derived from autoantigens found within joint tissues to activated T cells in vitro. In the context of inflamed synovial tissues, FLS may be an important and hitherto overlooked subset of APCs that could contribute to autoreactive immune responses.

Dendritic cells (DCs), the most effective antigen-presenting cells, are being studied as adjuvants or antigen delivery vehicles for eliciting T-cell-mediated antitumor immunity. Gene delivery to DCs provides an intracellular source of antigen for efficient and persistent loading to MHC class I molecules capable of activating CD8(+) CTLs, which play a central role in antitumor immunity. We previously reported that the fiber-mutant adenovirus vector (Ad) harboring the Arg-Gly-Asp (RGD) sequence in the HI loop of its fiber knob could more efficiently transduce the LacZ gene into both murine DC lines and normal human DCs than conventional Ad. In the present study, we compared immunological properties and vaccine efficacy of DC2.4 cells, an immature murine DC line, transduced with an ovalbumin (OVA) gene by fiber-mutant Ad (Ad-RGD-OVA) or conventional Ad (Ad-OVA). Ad-RGD-OVA-infected DC2.4 cells could more efficiently present OVA peptides via MHC class I molecules in a vector particle-dependent manner and induce OVA-specific CTL response by vaccination than Ad-OVA-infected DC2.4 cells. This result was correlated with the efficiency of gene transduction into DC2.4 cells by both types of Ad. Moreover, vaccination with Ad-RGD-OVA-infected DC2.4 cells could achieve an equal or greater antitumor effect against challenge with E.G7-OVA tumor cells with lower doses of Ad on infection or fewer cells for immunization than the vaccination procedure using Ad-OVA-infected DC2.4 cells. In addition, the maturation of DC2.4 cells was promoted by efficient expression of the antigen gene by the Arg-Gly-Asp fiber-mutant Ad. Flow cytometric analysis indicated enhanced expression of MHC class I and II molecules as well as CD80, CD86, CD40, and CD54, and reverse transcription-PCR analysis revealed increased levels of interleukin 12 p40 mRNA. However, infection by Ad-OVA or Ad that did not contain the cDNA of interest (Ad-Null and Ad-RGD-Null) contributed little to phenotypical changes in DC2.4 cells. On the basis of these results, we propose that DC manipulation using the Arg-Gly-Asp fiber-mutant Ad system could advance the development of more effective vaccines and allow for more convenient administration of DC-based gene immunotherapy.

TNF-alpha is a pleiotropic cytokine activating several signaling pathways initiated at distinct intracellular domains of the TNF receptors. Although the C-terminal region is believed to be responsible for apoptosis induction, the functions of more membrane-proximal domains, including the domain that couples to neutral sphingomyelinase activation, are not yet fully elucidated. The roles of this region and of the associated adapter protein FAN (factor associated with neutral SMase activation) in the cytotoxic response to TNF have been investigated. We have now shown that stable expression in human fibroblasts of a dominant negative form of FAN abrogates TNF-induced ceramide generation from sphingomyelin hydrolysis and reduces caspase processing, thus markedly inhibiting TNF-triggered apoptosis. However, the cytotoxic responses to daunorubicin and exogenous ceramide remain unaltered, as do the TNF-induced p42/p44 MAPK activation and CD54 expression. Fibroblasts from FAN-knockout mice also proved to be resistant to TNF toxicity. These findings highlight the previously unrecognized role of the adapter protein FAN in signaling cell death induction by TNF.

Dendritic cells (DC) are important APCs that play a key role in the induction of an immune response. The signaling molecules that govern early events in DC activation are not well understood. We therefore investigated whether DC express carcinoembryonic Ag-related cell adhesion molecule 1 (CEACAM1, also known as BGP or CD66a), a well-characterized signal-regulating cell-cell adhesion molecule that is expressed on granulocytes, monocytes, and activated T cells and B cells. We found that murine DC express in vitro as well as in vivo both major isoforms of CEACAM1, CEACAM1-L (having a long cytoplasmic domain with immunoreceptor tyrosine-based inhibitory motifs) and CEACAM1-S (having a short cytoplasmic domain lacking phosphorylatable tyrosine residues). Ligation of surface-expressed CEACAM1 on DC with the specific mAb AgB10 triggered release of the chemokines macrophage inflammatory protein 1alpha, macrophage inflammatory protein 2, and monocyte chemoattractant protein 1 and induced migration of granulocytes, monocytes, T cells, and immature DC. Furthermore, the surface expression of the costimulatory molecules CD40, CD54, CD80, and CD86 was increased, indicating that CEACAM1-induced signaling regulates early maturation and activation of dendritic cells. In addition, signaling via CEACAM1 induced release of the cytokines IL-6, IL-12 p40, and IL-12 p70 and facilitated priming of naive MHC II-restricted CD4(+) T cells with a Th1-like effector phenotype. Hence, our results show that CEACAM1 is a signal-transducing receptor that can regulate early maturation and activation of DC, thereby facilitating priming and polarization of T cell responses.

Human cytosolic thioredoxin (Trx), which is the 12-kDa protein disulfide reductase with the Cys-Gly-Pro-Cys active site and a key component of cellular redox biochemistry and regulation, acts as cocytokine upon leaderless secretion. A 10-kDa C-terminally truncated thioredoxin (Trx80) comprising the 80 or 84 N-terminal amino acids is also secreted and present in plasma, where it originally was purified and identified as eosinophilic cytotoxicity enhancing factor. Recombinant Trx80 was discovered to be a potent mitogenic cytokine that stimulates growth of resting human peripheral blood mononuclear cells (PBMC) in a synthetic medium, an effect that Trx lacks. Trx80 is very different from Trx because it is a dimer lacking reductase activity and the cytokine activity is not dependent on the Cys residues of the Trx active-site motif. The primary targets of Trx80 in PBMC are monocytes that are activated to proliferate and increase expression of CD14, CD40, CD54, and CD86. Trx80 induces secretion of interleukin (IL)-12 in CD40+ monocytes from PBMC. Trx80 and IL-2 together were strongly synergistic to induce secretion of interferon-gamma in PBMC. Trx80 is a potent cytokine for monocytes directing the immune system to a Th1 response via IL-12 production.

In previous studies we have demonstrated that syngeneic and xenogeneic pancreatic islet grafts are revascularized within a 10 to 14-day period after transplantation. With the combined use of intravital and electron microscopy, as well as immunohistochemistry using a set of species-specific or -crossreacting antibodies to endothelial cell antigens, we investigated 1) the origin of the endothelium of the newly formed capillaries in free pancreatic islet isografts (hamster->>hamster) and xenografts (rat->>hamster), and 2) the ultrastructural characteristics of these microvessels. Intravital microscopy demonstrated that newly formed microvessels grow from the vascular bed of the host muscle tissue into the islet grafts. Immunohistochemical analysis of host tissue and transplanted islets with antibodies against factor VIII (recognizing both hamster and rat factor VIII), bovine PECAM-1 (CD31; endoCAM, crossreacting with hamster but not rat PECAM-1), and rat ICAM-1 (CD54, non-crossreacting with hamster ICAM-1) showed that the transplanted rat islets were revascularized by endothelium of hamster (host) origin. At an ultrastructural level, the endothelial lining of the newly formed microvessels showed diaphragmatic fenestration, a characteristic feature of endothelial cells of pancreatic islets in situ. On the basis of these findings we suggest that pancreatic islet transplantation may take a unique position in the field of organ transplantation, since the generally proposed mechanisms of endothelial cell-dependent antigen recognition as a trigger of graft rejection may not be transferred to islet grafts, containing microvessels lined by endothelial cells of host origin.

Intercellular adhesion molecule-1 (ICAM-1) is a crucial receptor in the cell-cell interaction, a process central to the reaction to all forms of injury. Its expression is upregulated in response to a variety of inflammatory/immune mediators, including cellular stresses. The NF-kappaB signalling pathway is known to be important for activation of ICAM-1 transcription. Here we demonstrate that ICAM-1 induction represents a new cellular response to p53 activation and that NF-kappaB inhibition does not prevent the effect of p53 on ICAM-1 expression after DNA damage. Induction of ICAM-1 is abolished after treatment with the specific p53 inhibitor pifithrin-alpha and is abrogated in p53-deficient cell lines. Furthermore, we map two functional p53-responsive elements to the introns of the ICAM-1 gene, and show that they confer inducibility to p53 in a fashion similar to other p53 target genes. These results support an NF-kappaB-independent role for p53 in ICAM-1 regulation that may link p53 to ICAM-1 function in various physiological and pathological settings.

The ability of stem cells to self-renew as well as their multilineage differentiation potential makes them ideal candidates for skin regeneration strategies. Mesenchymal stem cells residing in human adult dermis, in contrast to adipose tissue, have not yet been described. The objective of this study was to determine the stemness and chemokine-mediated homing potential of dermal stromal cells (DSC) and to compare this with adipose stem cells (ASC). DSC have a less stellate form than ASC, confirming that DSC and ASC are two different types of mesenchymal cell populations. However, DSC display a mesenchymal stem cell phenotype (CD31(-), CD34(+), CD45(-), CD54(+), CD90(+), CD105(+), and CD166(+) similar to ASC and are also multipotent in their ability to differentiate into adipocytes, chondrocytes, and osteoblasts. Both ASC and DSC display a similar set of chemokine receptors (CCR3, CCR4, CCR6, CCR10, CXCR1, and CXCR2). Several ligands for these receptors, with CCL5/RANTES being the most potent, can induce migration of ASC and DSC in an in vitro wound-healing assay. Taken together, these results show that a population of mesenchymal stem cells resides in the dermis of human adult skin and these dermal-derived stem cells have a phenotypic and chemokine-mediated homing potential similar to adipose stem cells, which to our knowledge is previously unreported.