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The immune system of cancer patients is often suppressed. Accumulating evidence suggests that exosomes released from tumor cells may play an essential role in this process but the mechanisms are not fully understood. Here we review recent papers showing that exosomes trigger the release of cytokines/chemokines from immune cells. We suggest that this process will either result in the stimulation of anti-tumor immune reactions or in a systemic immunosuppression. The direction appears to be largely dependent on the duration of interactions between immune cells and exosomes leading to the accumulation of inflammatory factors, i.e. on the length of the exposure to these factors. We propose that a long-term interaction of the immune system with elevated levels of tumor exosomes contributes to the development of immunosuppression in cancer patients.

The first prototype of the protease activated receptor (PAR) family, the thrombin receptor PAR1, plays a central role both in the malignant invasion process of breast carcinoma metastasis and in the physiological process of placental implantation. The molecular mechanism underlying PAR1 involvement in tumor invasion and metastasis, however, is poorly defined. Here we show that PAR1 increases the invasive properties of tumor cells primarily by increased adhesion to extracellular matrix components. This preferential adhesion is accompanied by the cytoskeletal reorganization of F-actin toward migration-favoring morphology as detected by phalloidin staining. Activation of PAR1 increased the phosphorylation of focal adhesion kinase and paxillin, and the induced formation of focal contact complexes. PAR1 activation affected integrin cell-surface distribution without altering their level of expression. The specific recruitment of alpha(v)beta(5) to focal contact sites, but not of alpha(v)beta(3) or alpha(5)beta(1), was observed by immunofluorescent microscopy. PAR1 overexpressing cells showed selective reciprocal co-precipitation with alpha(v)beta(5) and paxillin but not with alpha(v)beta(3) that remained evenly distributed under these conditions. This co-immunoprecipitation failed to occur in cells containing the truncated form of PAR1 that lacked the entire cytoplasmic portion of the receptor. Thus, the PAR1 cytoplasmic tail is essential for conveying the cross-talk and recruiting the alpha(v)beta(5) integrin. While PAR1 overexpressing cells were invasive in vitro, as reflected by their migration through a Matrigel barrier, invasion was further enhanced by ligand activation of PAR1. Moreover, the application of anti-alpha(v)beta(5) antibodies specifically attenuated this PAR1 induced invasion. We propose that the activation of PAR1 may lead to a novel cooperation with the alpha(v)beta(5) integrin that supports tumor cell invasion.

Background: The pathomechanisms underlying disseminated intravascular coagulation (DIC) following amniotic fluid (AF) embolism remain to be fully elucidated. Highly procoagulant phosphatidylserine (PS)- and tissue factor (TF) expressing extracellular vesicles (EVs) might play a central role. Objective: To perform extensive analyses of the procoagulant properties of AF with a panel of functional coagulation assays and flow cytometry to investigate the pathogenesis of AF induced DIC. Methods: A prothrombinase assay, an EV-TF dependent factor Xa (FXa) generation assay, a modified thrombin- and fibrin-generation assay, a whole blood clotting model and flow cytometry were applied in AF- and control plasma. Results: Phosphatidylserine expression was 21-fold increased in AF compared to plasma. Factor Xa generation was extremely high when TF-expressing EVs from AF were co-incubated with recombinant FVIIa. In the thrombin- and fibrin generation assay AF-derived EVs strongly activated the blood coagulation cascade via PS and TF. In a whole blood clotting model AF-derived TF-expressing EVs significantly shortened the clotting time from 734 ± 139 seconds in the presence- to 232 ± 139 seconds in the absence of an anti-TF antibody. The contact activation pathway via factor FXII was not affected. Applying flow cytometry, a sub-population of PS- and TF co-expressing EVs was clearly identified in AF. Conclusions: We thoroughly investigated the effect of AF on blood coagulation and found that PS+ and TF+ EVs determine its procoagulant potential. Taken together our data further delineate the pathomechanisms underlying AF-induced coagulopathy, which could improve diagnostic- and treatment modalities. Disclosures No relevant conflicts of interest to declare.

A characteristic feature of integrin-ligand interactions is the requirement for divalent cations. Putative cation binding sites have been identified in the alpha and beta subunit of the alpha4 integrins, alpha4beta1 and alpha4beta7, and within their ligands which display the tripeptide LDV in fibronectin and homologous motifs in VCAM-1 and MAdCAM-1. The extracellular domain of the murine and human alpha4-subunit contains three conserved LDV motifs, designated LDV-1 to -3. Using site directed mutagenesis and transfection studies, we now examined the functional relevance of the LDV motifs for alpha4beta7 integrins. We present evidence that LDV-1 mutants (D489N) behave like alpha4 wt cells, but LDV-3 mutants (D811N) are impaired in alpha4beta7 integrin-triggered homotypic cell aggregation and in adhesion and spreading on alpha4 specific ligands. Further characterization of LDV-3 mutants revealed a defect in mAb-induced alpha4beta7-cell surface cluster formation. Mutation of the LDV-2 motif (D698N) caused loss of alpha4beta7 integrin cell surface expression. Our results indicate: (i) that LDV-3, located proximal to the cell membrane, is important for alpha4beta7 integrin-triggered functions and for lateral clustering and (ii) that LDV-2 affects alpha4beta7 heterodimer stability.

The use of anti-L1-cell adhesion molecule monoclonal antibodies (anti-L1CAM-mAb) in an endometriosis epithelial cell line Z12 led to a statistically significant decrease in cell proliferation and cell invasion and to an inhibition of the adhesion compared with unspecific IgG-Ab treated and untreated cells. Because it increases the cell invasion and adhesion which consequently aggravates the disease, L1 could possibly promote endometriosis development; thus, further studies should evaluate the possible use of anti-L1-mAb in an animal endometriosis model.

The L1 cell adhesion molecule (L1CAM) is overexpressed in many human cancers and can serve as a biomarker for prognosis in most of these cancers (including type I endometrial carcinomas). Here we provide an optimized immunohistochemical staining procedure for a widely used automated platform (VENTANA™), which has recourse to commercially available primary antibody and detection reagents. In parallel, we optimized the staining on a semi-automated BioGenix (i6000) immunostainer. These protocols yield good stainings and should represent the basis for a reliable and standardized immunohistochemical detection of L1CAM in a variety of malignancies in different laboratories.

The L1 adhesion molecule is a 200-220-kDa membrane glycoprotein of the Ig superfamily implicated in important neural processes including neuronal cell migration, axon outgrowth, learning, and memory formation. L1 supports homophilic L1-L1 binding that involves several Ig domains but can also bind with high affinity to the proteoglycan neurocan. It has been reported that neurocan can block homophilic binding; however, the mechanism of inhibition and the precise binding sites in both molecules have not been determined. By using fusion proteins, site-directed mutagenesis, and peptide blocking experiments, we have characterized the neurocan-binding site in the first Ig-like domain of human L1. Results from molecular modeling suggest that the sequences involved in neurocan binding are localized on the surface of the first Ig domain and largely overlap with the G-F-C beta-strands proposed to interact with the fourth Ig domain during homophilic binding. This suggests that neurocan may sterically hinder a proper alignment of L1 domains. We find that the C-terminal portion of neurocan is sufficient to mediate binding to the first Ig domain of L1, and we suggest that the sushi domain cooperates with a glycosaminoglycan side chain in forming the binding site for L1.

The ectodomain of certain transmembrane molecules can be released by proteolysis, and the solubilized antigens often exert important biological functions. We demonstrated before that the L1 adhesion molecule is shed from the cell surface. Here we show that L1 release in AR breast carcinoma cells is mediated by a member of the disintegrin metalloproteinase (ADAM) family of proteinases. Up-regulation of L1 shedding by phorbol ester or pervanadate involved distinct mechanisms. Pervanadate induced shedding and rounding-up of cells from the substrate, which was blocked by the Src kinase inhibitor PP2. Tyr phosphorylation of the L1 cytoplasmic tail and the Src kinase Fyn was observed following pervanadate treatment. Up-regulation of L1 release and activation of Fyn occurred also when cells were detached by EDTA suggesting that the regulation of L1 shedding by this pathway was linked to cell morphology and adhesion. The phorbol 12-myristate 13-acetate-induced shedding was inhibited by the protein kinase C inhibitor bisindolylmaleimide I and by PD98059, a specific inhibitor of the mitogen-activated protein kinase pathway. Soluble L1 binds to the proteoglycan neurocan and in bound form could support integrin-mediated cell adhesion and migration. We propose that the release of cell-associated adhesion molecules such as L1 may be relevant to promote cell migration.

The L1 adhesion molecule is an approx. 200–220 kDa type I membrane glycoprotein belonging to the immunoglobulin (Ig) superfamily. L1 can bind in a homotypic fashion and was shown to support integrin-mediated binding via RGDs in the 6th Ig-like domain. In addition to its cell-surface expression, L1 can occur in the extracellular matrix (ECM). Here we demonstrate that L1 is constitutively released from the cell surface by membrane-proximal cleavage. L1 shed from B16F10 melanoma cells remains intact and can serve as substrate for integrin-mediated cell adhesion and migration. The release of L1 occurs in mouse and human cells and is blocked by the metalloproteinase inhibitor TAPI (Immunex compound 3). This compound has been shown previously to block release of L-selectin and TNF-alpha which is mediated by the membrane-bound metalloproteinase TNF-alpha converting enzyme (TACE). Using CHO cells that are low in TACE expression and do not release L-selectin we demonstrate that L1 release is distinct from L-selectin shedding. We propose that cell-surface release may be necessary for the conversion of L1 from a membrane into an ECM protein.

The cell-adhesion molecule L1 was originally described in the nervous system. It has recently been detected in CD4+ T lymphocytes, peripheral B lymphocytes, and granulocytes in the human immune system and in similar leucocyte types in the murine immune system. L1 mediates neural recognition by Ca+2, Mg+2-independent homophilic binding. In the human and murine immune systems, L1 binds to the "classical" vitronectin receptor, alphaVbeta3, and fibronectin receptor, alpha5beta1, respectively, and abstains from homophilic binding. Homophilic L1 binding probably involves antiparallel alignment of several interactive domains. Integrin binding is mediated by a short segment of immunoglobulinlike domain 6, which includes two RGD repeats in rodent L1 and one RGD motif in human L1. L1 is modulated in activated leucocytes in vitro in parallel to L-selectin, and diverse cell types release intact L1 in vivo and in vitro. Released L1 can bind to laminin and adheres to the extracellular matrix of sciatic nerve, M21 melanoma, and possibly spleen and other tissues. It can support integrin-dependent cell migration and preliminary data implicate it in tumor development and transnodal lymphocyte migration.

It is generally recognized that activation through membrane effector molecules such as CD40 or the B cell receptor (BCR) is mandatory to allow B cells to proliferate and differentiate into antibody (Ab)-secreting cells in response to cytokines. We show here that purified tonsillar B cells can be stimulated directly by a cytokine combination to proliferate and secrete immunoglobulins when cultures are performed at high cell density. The contact-mediated activation of B cells in this experimental system is strongly inhibited both by anti-very late antigen (VLA)-4 monoclonal Ab and by a peptide containing the LDV sequence specifically recognized by the alpha 4 integrin binding site. These reagents also significantly suppressed the B cell responses elicited by engagement of the BCR or CD40. Our data reveal that memory B cells but not virgin or germinal center B cells are sensitive to the direct stimulatory effect of cytokines in high-density cultures. Finally, we found that the dual expression of the alpha and beta chains of VLA-4 is a distinctive feature of the memory B cell population. Collectively, our findings support the notion that VLA-4-dependent homotypic B cell interactions can mediate a co-stimulatory signal to human memory B cells and might participate in the B cell activation triggered through the BCR and CD40.

CD24 is a differentiation antigen expressed by murine hematopoietic and neural cells which is linked to the membrane via a glycosylphosphatidylinositol (GPI) anchor. In monocytic ESb-MP cells the molecule serves as a ligand for P-selectin and triggering with CD24 specific antibodies can activate VLA-5/L1-mediated cell adhesion in these cells. We report here that the aggregation is specific for CD24 and not seen with antibodies to the GPI-anchored molecule Thy-1. The Tyr-kinase inhibitor herbimycin can block the aggregation. We studied CD24 associated molecules that might be involved in signal transduction. Antibodymediated crosslinking of CD24 induced a rapid Tyr-phosphorylation of several cellular proteins in ESb-MP cells which correlated with an elevated activity of p56lck but not p60fyn or MAP-1 kinase. Several phosphorylated proteins were co-immunoprecipitated with CD24. Re-immunoprecipitation allowed the detection of p56lck, p56hck, and p54lyn but not p60fyn, PI-3k, or PLCgamma as a compenent of the CD24 detergent resistant complex. It is suggested that the CD24-associated kinases are involved in the activation of cell aggregation.

The L1 adhesion molecule is a member of the immunoglobulin superfamily which is expressed by neural and hematopoietic cells. L1 is primarily a cell surface molecule but in its released form it becomes embedded in the extracellular matrix. In addition to the established L1-L1 homotypic interaction, L1 can bind to alpha v beta 3 in the human. The 6th Ig-like domain is critical for this function. We now demonstrate that a fusion protein containing the 6th Ig-like domain of L1 (6.L1-Fc) can support the migration of human MED-B1 (alpha v beta 3+) but not of Nalm-6 cells (alpha 5 beta 1+). The migration was blocked in the presence of a mab to alpha v beta 3 and was not seen on a 6.L1-Fc in which the RGD site was mutated. Activation of human T lymphocytes in the presence of PHA and PMA led to the induction of alpha v beta 3 and alpha v beta 5 expression and concomitantly induced migration of the cells on 6.L1-Fc. The migration was blocked by mabs to alpha v beta 3 but not to alpha v beta 5. Our results suggest that L1 exposed at the cell surface or as a matrix constituent can serve as a potent substrate for alpha v beta 3 mediated cell migration.

The cell adhesion molecule L1 is known to mediate neuronal adhesion, neurite fasciculation, and stimulation of fibroblastin growth factor (FGF)-receptor-dependent neurite outgrowth by homophilic interaction. Recent findings have also revealed heterophilic interactions between L1 and two "classical" integrin matrix receptors, murine alpha 5 beta 1 and human alpha V beta 3. The homophilic binding mechanism of L1 involves multiple domains and has been conserved in chicken neuron-glia cell adhesion molecule (NgCAM) and mammalian L1. The integrin-binding site of L1 contains the tripeptide Arg-Gly-Asp but varies among different species. L1-integrin binding predominates in leucocyte subsets and in several tumours. It can mediate homotypic and heterotypic cell-cell adhesion and haptotactic cell movement on substrate-embedded L1. L1 is released in response to cytokines by at least some neuronal and leucocyte types. It can be detected in the extracellular matrix and conceivably contributes to cell and axonal navigation. Antibody perturbation studies indicate that the integrin-binding site of L1 is important for granule cell migration and neurite outgrowth in postnatal murine cerebellum, possibly via modulation of the src signal transduction pathway. The model of L1 binding seems to be governed by the cell, but it does not correlate with known alternative splicing or glycosylation patterns of L1.

Extracellular membrane vesicles derived from the endosomal compartments and released by the fusion of the multivesicular bodies with the cell membrane are referred as exosomes (Exo) [Van Niel et al., J Biochem 140:13-21, 2006]. They function as mediators of intercellular communication and are employed by the organism in the regulation of systemic and local processes. Meantime, Exo are recognized as an indispensable entity of physiological fluids [Caby et al., Int Immunol 17:879-887, 2005; Lasser et al., J Transl Med 9:9, 2011; Lasser et al., Am J Rhinol Allergy 25:89-93, 2011]. Exo and other types of extracellular vesicles, e.g., exosome-like vesicles [van Niel et al., Gastroenterology 121:337-349, 2001] and microvesicles (MV) [Daveloose et al., Thromb Res 22:195-201, 1981], contain multiple functional molecules including lipids [Vidal et al., J Cell Physiol 140:455-462, 1989]; proteins [Simpson et al., Expert Rev Proteomics 6:267-283, 2009]; mRNA [Valadi et al., Nat Cell Biol 9:654-659, 2007]; DNA [Waldenstrom et al., PLoS One 7:e34653, 2012]; noncoding RNA, e.g., miRNA [Simpson et al., Expert Rev Proteomics 6:267-283, 2009]; and retrotransposon elements [Balaj et al., Nat Commun 2:180, 2011]. Assessment of the biological functions of Exo showed that they deliver specifically their cargo from the donor to recipient cells. Albeit the molecular mechanisms of this process are not fully understood, approaches for the application of Exo and MV as a tool for a cell-specific delivery of signalling molecules were successfully tested in in vitro and in vivo models [Maguire et al., Mol Ther 20:960-971, 2012]. Ovarian cancer cells release Exo, which bind stroma cells as well as donor cancer cells [Escrevente et al., BMC Cancer 11:108, 2011]. Here we describe an experimental approach for the assessment of Exo interaction and uptake by target cells. Methods for the isolation and purification of Exo from cell culture supernatants are included. To allow visualization of vesicle uptake, labelling of Exo with different fluorescent dyes, such as CFSE, PKH, DHPE, and DiOC18, is presented. Finally, we explain qualitative and quantitative analysis of Exo uptake by immunofluorescence and flow cytometry, respectively.

Background/Aims The neural cell adhesion molecule L1CAM is a transmembrane glycoprotein abnormally expressed in tumors and previously associated with cell proliferation, adhesion and invasion, as well as neurite outgrowth in endometriosis. Being an attractive target molecule for antibody-based therapy, the present study assessed the ability of the monoclonal anti-L1 antibody (anti-L1 mAb) to impair the development of endometriotic lesions in vivo and endometriosis-associated nerve fiber growth. Methods and Results Endometriosis was experimentally induced in sexually mature B6C3F1 (n=34) and CD-1 nude (n=21) mice by autologous and heterologous transplantation, respectively, of endometrial fragments into the peritoneal cavity. Transplantation was confirmed four weeks post-surgery by in vivo magnetic resonance imaging and laparotomy, respectively. Mice were then intraperitoneally injected with anti-L1 mAb or an IgG isotype control antibody twice weekly, over a period of four weeks. Upon treatment completion, mice were sacrificed and endometrial implants were excised, measured and fixed. Endometriosis was histologically confirmed and L1CAM was detected by immunohistochemistry. Endometriotic lesion size was significantly reduced in anti-L1-treated B6C3F1 and CD-1 nude mice compared to mice treated with control antibody (P

Extracellular membrane vesicles derived from the endosomal compartments and released by the fusion of the multivesicular bodies with the cell membrane are referred as exosomes (Exo) [Van Niel et al., J Biochem 140:13-21, 2006]. They function as mediators of intercellular communication and are employed by the organism in the regulation of systemic and local processes. Meantime, Exo are recognized as an indispensable entity of physiological fluids [Caby et al., Int Immunol 17:879-887, 2005; Lasser et al., J Transl Med 9:9, 2011; Lasser et al., Am J Rhinol Allergy 25:89-93, 2011]. Exo and other types of extracellular vesicles, e.g., exosome-like vesicles [van Niel et al., Gastroenterology 121:337-349, 2001] and microvesicles (MV) [Daveloose et al., Thromb Res 22:195-201, 1981], contain multiple functional molecules including lipids [Vidal et al., J Cell Physiol 140:455-462, 1989]; proteins [Simpson et al., Expert Rev Proteomics 6:267-283, 2009]; mRNA [Valadi et al., Nat Cell Biol 9:654-659, 2007]; DNA [Waldenstrom et al., PLoS One 7:e34653, 2012]; noncoding RNA, e.g., miRNA [Simpson et al., Expert Rev Proteomics 6:267-283, 2009]; and retrotransposon elements [Balaj et al., Nat Commun 2:180, 2011]. Assessment of the biological functions of Exo showed that they deliver specifically their cargo from the donor to recipient cells. Albeit the molecular mechanisms of this process are not fully understood, approaches for the application of Exo and MV as a tool for a cell-specific delivery of signalling molecules were successfully tested in in vitro and in vivo models [Maguire et al., Mol Ther 20:960-971, 2012]. Ovarian cancer cells release Exo, which bind stroma cells as well as donor cancer cells [Escrevente et al., BMC Cancer 11:108, 2011]. Here we describe an experimental approach for the assessment of Exo interaction and uptake by target cells. Methods for the isolation and purification of Exo from cell culture supernatants are included. To allow visualization of vesicle uptake, labelling of Exo with different fluorescent dyes, such as CFSE, PKH, DHPE, and DiOC18, is presented. Finally, we explain qualitative and quantitative analysis of Exo uptake by immunofluorescence and flow cytometry, respectively.

We present Monte Carlo simulation results for the dynamical critical exponent $z$ of the two-dimensional kinetic Ising model using a lattice of size $10^6 \times 10^6$ spins. We used Glauber as well as Metropolis dynamics. The $z$-value of $2.16\pm 0.005$ was calculated from the magnetization and energy relaxation from an ordered state towards the equilibrium state at $T_c$., Comment: 6 pages + 2 figures as separate uuencoded compressed tar file, Postscipt also available at http://wwwcp.tphys.uni-heidelberg.de/papers/

Infection of metastatic lymphoma cells (ESbL) by a low dose of a non-lytic strain of Newcastle disease virus (NDV) leads to viral replication followed by strong cell surface expression of viral antigens, especially hemagglutinin neuraminidase (HN). The expressed HN was functional and facilitated cell-cell interactions and cell attachment. This was shown for NDV infected tumor cells, lymphocytes, fibroblasts and endothelial cells. The interactions could be strongly inhibited by antibodies against the viral HN protein. Increased binding was also seen with HN c-DNA transfectants expressing the HN as the only viral protein. Viral infection did not influence proliferation and lysability of the infected tumor cells. Following intravenous injection of tumor cells, the number of hepatic metastases was significantly reduced when the cells had been pre-infected with NDV. This reduction of metastases correlated with an increased survival time of the animals. As potential mechanisms of these NDV effects we propose augmentation of cell-eel interactions and immune functions and reduction of invasive capacity of NDV infected, as compared to non-infected tumor cells.

The aggregation rate of resuspended neuroblastoma N2A cells depends on the density of the cells in culture prior to their resuspension: isolated, fast growing cells have a weak tendency to aggregate whereas confluent, slowly growing cells reaggregate very strongly. L1 antibody 557 strongly inhibited the slow aggregation of isolated, fast growing cells but not the reaggregation of confluent cells. CD24 (nectadrin) antibodies did not affect the aggregation of isolated or confluent cells but stimulated the aggregation of subconfluent cells. In all stages aggregation was not inhibited when antibody 557 was used together with CD24 antibodies at 37 degrees C in the presence of divalent cations. EA-1 antibody to alpha 6 integrin chain stimulated the aggregation of subconfluent cells but inhibited the reaggregation of confluent cells. Therefore, L1 appears to be an early recognition molecule mediating weak primary adhesion. CD24 appears to participate in activating secondary adhesion mechanisms during primary adhesion, possibly in cooperation with L1, and alpha 6 integrin seems to serve as a secondary, strong adhesion molecule that in early adhesion phases also mediates the activation of itself or of other adhesion mechanisms. These results indicate that neural cells might employ a strategy of adhesion cascade in establishing stable contacts.

We present a new approach for investigating very large lattices of the Ising type with local update algorithms for short time scales. Instead of the conventional storing of configurations and iterating through time, we calculate spins for all time steps simultaneously and propagate through the lattice. We apply the algorithm to the two-dimensional Ising model with Metropolis dynamics and simulate systems of up to (4 × 10 5 ) 2 lattice sites for 10 MC steps.

Overexpression of CD24 has a negative impact on breast cancer prognosis. We have recently reported that the CD24 codon 57 Val/Val genotype (rs52812045) is associated with pathologic complete response after neoadjuvant chemotherapy for primary breast cancer and correlates with intratumoral lymphocyte infiltrates. This study was performed to investigate the influence of CD24 polymorphisms on breast cancer prognosis and risk. A total of 2,514 patients and 4,858 controls recruited as part of the MARIE study, a population-based case-control study, were genotyped for two CD24 polymorphisms (rs52812045, rs3838646) using TaqMan custom genotyping assays. Associations with overall and breast cancer-specific survival were assessed using uni- and multivariable Cox regression models stratified by age at diagnosis and adjusted for prognostic factors. Conditional logistic regression analysis adjusted for major risk factors was used to estimate multivariable odds ratios for risk of putative allele carriers compared to wildtype carriers. CD24 Ala/Val was significantly associated with breast cancer prognosis [Val/Val hazard ratio (HR)(adjusted) = 1.52; 95 % confidence interval (CI): 1.00-2.30, p = 0.05 and HR(adjusted) = 1.83; 95 % CI: 1.10-3.05, p = 0.018 for all-cause and breast cancer-specific mortality, respectively). The association was significant only in patients with a BMI 25 and in those who received adjuvant chemotherapy. None of the CD24 alleles was associated with breast cancer risk. These results provide further evidence of the CD24 Val/Val genotype influencing outcome in primary breast cancer. Together with previous data of CD24 overexpression as a poor prognostic marker, the findings underline the biological importance of CD24 for breast cancer.

We describe an algorithm for dynamic load balancing of geometrically parallelized synchronous Monte Carlo simulations of physical models. This algorithm is designed for a (heterogeneous) multiprocessor system of the MIMD type with distributed memory. The algorithm is based on a dynamic partitioning of the domain of the algorithm, taking into account the actual processor resources of the various processors of the multiprocessor system., 12 pages, uuencoded figures included, 75.93.08

Nectadrin, the heat-stable antigen (HSA), is a highly glycosylated GPI-linked glycoprotein that can undergo homophilic and heterophilic binding. In the present work we have examined short-term effects of nectadrin antibodies on splenic B lymphoblast aggregation and signal transduction. Monoclonal antibody 79 inhibited cell aggregation and induced an intracellular Ca++ signal in the absence of cross-linking. Both these effects were perturbed in the presence of LFA-1 antibodies. Nectadrin antibody M1/69 and polyclonal nectadrin antibodies stimulated cell aggregation, did not induce a Ca++ signal, and their effects were functionally independent of LFA-1. These results suggest that nectadrin may concomitantly mediate primary and activate secondary adhesion mechanisms whereby each of these processes may be related to a different signal transduction pathway.

Heat-stable antigen (HSA)/CD24 is a cell surface molecule expressed by many cell types in the mouse. The molecule has an unusual structure because of its small protein core and extensive glycosylation. In order to study the functional role of the HSA-associated glycoconjugates we have isolated different forms of HSA. Using lectin analysis we provide evidence for extensive heterogeneity in carbohydrate composition and sialic acid linkage. Several HSA forms were recognized by mouse P-selectin-IgG but not E-selectin-IgG in ELISA. As expected, P-selectin-IgG also bound to L2/HNK-1-positive neural glycoproteins (L2-glycoproteins) and sulfatides but not to gangliosides and other control glycoproteins. The binding of P-selectin-IgG to L2-glycoproteins and HSA required bivalent cations. The reactivity to HSA was sensitive to sialidase treatment whereas the binding to L2-glycoproteins was not. Studies with alpha 2-6 sialytransferase indicated that alpha 2-6 linked sialic acid was not involved in the P-selectin binding to HSA. Surprisingly, an L2/HNK-1 specific antibody was found to cross-react with some HSA glycoforms and its binding correlated with P-selectin-IgG reactivity. L2/HNK-1-positive or L2/HNK-1-negative HSA glycoforms were also analyzed after coating to polystyrene beads. Only the L2/HNK-1-positive HSA coated beads were reactive with P-selectin-IgG and could bind to activated bend3 endothelioma cells expressing P-selectin whereas the L2/HNK-1-negative HSA beads did not. It is suggested that in its L2/HNK-1 modified form the HSA molecule on leukocytes could represent a ligand for P-selectin on endothelial cells or platelets.

CD24 is a glycosyl-phosphatidylinositol-anchored membrane protein that is frequently over-expressed in a variety of human carcinomas and is correlated with poor prognosis. In cancer cell lines, changes of CD24 expression can alter several cellular properties in vitro and tumor growth in vivo. However, little is known about how CD24 mediates these effects. Here we have analyzed the functional consequences of CD24 knock-down or over-expression in human cancer cell lines. Depletion of CD24 reduced cell proliferation and adhesion, enhanced apoptosis, and regulated the expression of various genes some of which were identified as STAT3 target genes. Loss of CD24 reduced STAT3 and FAK phosphorylation. Diminished STAT3 activity was confirmed by specific reporter assays. We found that reduced STAT3 activity after CD24 knock-down was accompanied by altered Src phosphorylation. Silencing of Src, similar to CD24, targeted the expression of prototype STAT3-regulated genes. Likewise, the over-expression of CD24 augmented Src-Y416 phosphorylation, the recruitment of Src into lipid rafts and the expression of STAT3-dependent target genes. An antibody to CD24 was effective in reducing tumor growth of A549 lung cancer and BxPC3 pancreatic cancer xenografts in mice. Antibody treatment affected the level of Src-phosphorylation in the tumor and altered the expression of STAT3 target genes. Our results provide evidence that CD24 regulates STAT3 and FAK activity and suggest an important role of Src in this process. Finally, the targeting of CD24 by antibodies could represent a novel route for tumor therapy.

The up-regulation of the cell adhesion molecule L1 has been associated with impaired prognosis in several cancers. This study aimed to identify potential prognostic markers, including L1, in adenoid cystic carcinoma of the salivary glands (ACCs), which might give additional insight into the molecular mechanisms underlying malignant progression.The expression of L1 was analysed in 34 primary ACCs (nine tubular, 15 cribriform, nine solid, one mixed) and correlated with recurrence, metastasis, overall survival and clinicopathological parameters. Independent of the histological subtype, intense L1 expression in the primary tumours was associated significantly with metastasis (P = 0.02) and death (P = 0.044). In the subgroup of cribriform ACCs, 10 of 15 tumours contained pseudocysts, which were associated with significantly lower recurrence rates (P = 0.003), lower metastasis rates (P = 0.009) and a prolonged overall survival (P =0.004).Determination of L1 expression in primary ACCs improves risk estimations. As up-regulation of L1 expression predicts fatal prognosis, L1 might be involved functionally in growth and spread of ACC and might thus present a molecular target for future therapeutic strategies.

The malignancy of a tumor depends on the capability of cancer cells to metastasize. The process of metastasis involves cell invasion through connective tissue and transmigration through endothelial monolayers. The expression of the glycosylphosphatidylinositol-anchored receptor CD24 is increased in several tumor types and is consistently associated with increased metastasis formation in patients. Furthermore, the localization of β1-integrins in lipid rafts depends on CD24. Cell invasion is a fundamental biomechanical process and usually requires cell adhesion to the extracellular matrix (ECM) mainly through β1 heterodimeric integrin receptors. Here, we studied the invasion of A125 human lung cancer cells with different CD24 expression levels in three-dimensional ECMs. We hypothesized that CD24 expression increases cancer cell invasion through increased contractile forces. To analyze this, A125 cells (CD24 negative) were stably transfected with CD24 and sorted for high and low CD24 expression. The invasiveness of the CD24(high) and CD24(low) transfectants was determined in three-dimensional ECMs. The percentage of invasive cells and their invasion depth was increased in CD24(high) cells compared with CD24(low) cells. Knockdown of CD24 and of the β1-integrin subunit in CD24(high) cells decreased their invasiveness, indicating that the increased invasiveness is CD24- and β1-integrin subunit-dependent. Fourier transform traction microscopy revealed that the CD24(high) cells generated 5-fold higher contractile forces compared with CD24(low) cells. To analyze whether contractile forces are essential for CD24-facilitated cell invasion, we performed invasion assays in the presence of myosin light chain kinase inhibitor ML-7 as well as Rho kinase inhibitor Y27632. Cell invasiveness was reduced after addition of ML-7 and Y27632 in CD24(high) cells but not in CD24(neg) cells. Moreover, after addition of lysophosphatidic acid or calyculin A, an increase in pre-stress in CD24(neg) cells was observed, which enhanced cellular invasiveness. In addition, inhibition of the Src kinase or STAT3 strongly reduced the invasiveness of CD24(high) cells, slightly reduced that of CD24(low) cells, and did not alter the invasiveness of CD24(neg) cells. Taken together, these results suggest that CD24 enhances cell invasion through increased generation or transmission of contractile forces.

Background Exosomes are small membrane vesicles with a size of 40-100 nm that are released by different cell types from a late endosomal cellular compartment. They can be found in various body fluids including plasma, malignant ascites, urine, amniotic fluid and saliva. Exosomes contain proteins, miRNAs and mRNAs (exosome shuttle RNA, esRNA) that could serve as novel platform for diagnosis. Method We isolated exosomes from amniotic fluid, saliva and urine by differential centrifugation on sucrose gradients. Marker proteins were identified by Western blot and FACS analysis after adsorption of exosomes to latex beads. We extracted esRNA from exosomes, carried out RT-PCR, and analyzed amplified products by restriction length polymorphism. Results Exosomes were positive for the marker proteins CD24, CD9, Annexin-1 and Hsp70 and displayed the correct buoyant density and orientation of antigens. In sucrose gradients the exosomal fractions contained esRNA that could be isolated with sufficient quantity for further analysis. EsRNAs were protected in exosomes from enzymatic degradation. Amniotic fluid esRNA served as template for the typing of the CD24 single nucleotide polymorphism (rs52812045). It also allowed sex determination of the fetus based on the detection of the male specific ZFY gene product. Conclusions Our data demonstrate that exosomes from body fluids carry esRNAs which can be analyzed and offers access to the transcriptome of the host organism. The exosomal lipid bilayer protects the genetic information from degradation. As the isolation of exosomes is a minimally invasive procedure, this technique opens new possibilities for diagnostics.

Using the PVM programming environment for parallel applications, we have parallelized a simulation of the two-dimensional Ising Model on a cluster of IBM RISC System/6000 workstations connected by a Token Ring (16Mb/sec) and by Serial Optical Channels (220 Mb/sec) via a NSC DX Router. The parallelization is done by dividing the lattice into sublattices, each sublattice being associated with one workstation. On each sublattice, a Metropolis algorithm using Multispin Coding techniques is used to generate new configurations. We provide numerical results concerning the number of spin updates per second, speedups, and efficiencies for various numbers of processors and lattice sizes.

The murine equivalent to CD2, previously known as a T cell marker, is expressed on mouse B cells. The monoclonal anti-CD2 antibody 12-15 was found to induce B cell homotypic adhesion. When treated with F(ab')2 fragments of 12-15, purified, resting B cells aggregate within 2 h of incubation and the response is optimal after 20 h. Anti-CD2-induced aggregation is a dose-related active process, dependent on temperature, metabolic energy and divalent cations. Aggregation is inhibited by two different Fab monomers of anti-CD2, implying that it is the CD2 molecule itself that functions as an adhesion molecule. We also report that interleukin 4-induced B cell homotypic adhesion involves CD2-mediated cell binding, since the antibodies specific for mouse, CD2, inhibited interleukin-4-induced cell aggregation.

Objective Cancer cells in the body release soluble and membranous factors that manipulate the tumor environment to facilitate growth and survival. Recent years have provided evidence that small microvesicles that are termed exosomes may play a pivotal role in this process. Exosomes are membrane vesicles with a size of 40–100nm that are released by both tumor and normal cells and can be found in various body fluids. Tumor-derived exosomes carry functional proteins, mRNAs, and miRNAs and could serve as novel platform for tumor diagnosis and prognosis. However, marker proteins that allow enrichment of tumor-derived exosomes over normal exosomes are less well defined. Methods We used Western blot analysis and antibody coupled magnetic beads to characterize CD24 and EpCAM as markers for exosomes. We investigated ovarian carcinoma ascites, pleural effusions and serum of breast carcinoma patients. As non-tumor derived control we used exosomes from ascites of liver cirrhosis patients. Results Exosomes could be isolated from all body fluids and contained marker proteins as well as miRNAs. We observed that CD24 and EpCAM were selectively present on ascites exosomes of tumor patients and copurified together on anti-EpCAM or anti-CD24 magnetic beads. In breast cancer patients CD24 was present but EpCAM was absent from serum exosomes. Instead, the intact EpCAM ectodomain was recovered in a soluble form. We provide evidence that EpCAM can be cleaved from exosomes via serum metalloproteinase(s). Conclusion Loss of EpCAM on serum exosomes may hamper enrichment by immune-affinity isolation. We suggest that CD24 could be an additional marker for the enrichment of tumor-derived exosomes from blood.

Members of the Slug/Snail family of transcription factors are thought to drive epithelial-mesenchymal-transition (EMT) in preneoplastic epithelial cells, thereby contributing to malignant transformation. One mediator in the EMT of pancreatic ductal adenocarcinoma (PDAC) cells and a potential target gene of Slug is the cellular adhesion molecule L1CAM. Using the human pancreatic ductal epithelial cell line H6c7 and the PDAC cell line Panc1, we could show that along with TGF-β1-induced EMT, L1CAM expression is increased in a Slug- but not Snail-dependent fashion. Two E-box recognition motifs in the L1CAM promoter upstream of the most distal transcriptional start site could be verified by gel shift and supershift assay to interact with Slug. ChIP assays detected an increased interaction of Slug with both recognition motifs of the human L1CAM promoter in TGF-β1-treated H6c7 cells, whereas binding of Snail was downregulated. Moreover, ChIP assays with Panc1 cells confirmed this interaction of Slug with the human L1CAM promoter and further detected an interaction of both recognition sites with RNA-polymerase II in a Slug-dependent fashion. Luciferase reporter gene assays using wild-type or single- and double-mutated variants of the L1CAM promoter confirmed transcriptional activation by Slug involving both recognition motifs. By demonstrating the direct transcriptional control of L1CAM expression through Slug during TGF-β1-induced EMT of PDAC cells, our findings point to a novel mechanism by which Slug contributes quite early to tumorigenesis. Moreover, our study is the first one describing the control of the human L1CAM promoter in tumor cells.

Ovarian carcinoma is the leading cause of death from gynecological cancers in many Western countries. Aberrant glycosylation is an important aspect in malignant transformation and consequently in ovarian cancer. In this study, a detailed structure analysis of the N-linked glycans from total glycoproteins from the SKOV3 ovarian carcinoma cell line and from a recombinantly expressed secretory glycoprotein, erythropoietin (EPO), produced from the same cells has been performed using high-performance anion exchange chromatography with pulsed amperometric detection and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Total cellular N-glycans contained high-mannose type and proximally fucosylated complex type partially agalactosylated structures. On the other hand, the recombinant human EPO secreted from SKOV3 cells contained predominantly core-fucosylated tetraantennary structures, which were partially lacking one or two galactose residues, and partially contained the LacdiNAc motif. Only minor amounts of di- and triantennary complex-type glycans were found, and high-mannose-type glycans were not present in the secreted EPO protein. A large amount of N-acetylneuraminic acid in α2,3-linkage was detected as well. Endogenous glycoproteins were also found to contain the LacdiNAc motif in N-linked glycans. This work contributes to the knowledge of the glycosylation of a human ovarian cancer cell line. It also establishes the basis to further explore high-mannose-type glycans, and the LacdiNAc motif as possible markers of ovarian carcinoma.

The promigratory molecule L1CAM is overexpressed in various tumors, often representing an unfavorable prognostic marker. Recently, we identified L1CAM expression in pancreatic ductal adenocarcinoma (PDAC) cells accounting for chemoresistance and increased cell migration. Thus, the present study aims at further elucidating the role of L1CAM in a larger cohort of PDAC specimens including precursor lesions and metastasis. L1CAM expression was determined by immunohistochemistry in tissues of 123 patients including tissues of 110 primary PDACs, 15 lymph node metastases and 14 liver metastases. The immunohistochemical analyses revealed L1CAM expression in 92.7% of primary PDACs, 80% of lymph node metastases and 100% of liver metastases. Furthermore, we have investigated PDAC precursors, pancreatic intraepithelial neoplasia (PanIN) lesions, revealing a significant increase of L1CAM expression with the PanIN grade (6.4 and 6.8% in PanIN 1A and B, 35% in PanIN 2 and 20% in PanIN 3). The elevated expression of L1CAM already found in PanINs points to a role of L1CAM quite early in tumorigenesis of PDAC. Furthermore, its broad expression in primary tumors as well as in metastases of PDAC patients provide a rationale to further explore the value of L1CAM as a therapeutic target in the treatment of this highly malignant tumor.

We describe for the first time a new integral molecular pathway, linking transcription factor E2F3a to epidermal growth factor receptor (EGFR) activation in ovarian cancer cells. Investigations on the role of E2F family members in EGFR-mediated mitogenic signaling revealed that E2F3a was selectively upregulated following EGFR activation, whereas all other E2F family members remained unaffected. In contrast, EGF treatment of healthy ovarian surface epithelial and mesothelial cells yielded a selective upregulation of proliferation-promoting E2F1 and E2F2 without influencing E2F3a expression. In ovarian cancer cell lines, the extent of EGF-induced proliferative stimulus was closely related to the magnitude of E2F3a increase, and proliferation inhibition by E2F3a knockdown was not overcome by EGF exposure. Furthermore, the EGFR-E2F3a axis was found to be signal transducer and activator of transcription 1/3 dependent and the ratio of IFN-regulatory factor (IRF)-1 to IRF-2 was shown to be determinative for E2F3a control. In a pilot study on 32 primary ovarian cancer specimens, a highly significant correlation between activated EGFR and E2F3a expression was disclosed. This new integral pathway in the EGFR-driven mitogenic cell response, which through its key player E2F3a was found to be essential in triggering proliferation in ovarian cancer cells, provides new insights into EGFR signaling and could represent the basis for appealing new therapeutic approaches in ovarian cancer. Cancer Res; 70(11); 4613–23. ©2010 AACR.

Endometrial carcinomas (ECs) are classified into type 1 (less aggressive) and type 2 (aggressive) tumours that differ in genetic alterations. So far, reliable immunohistochemical markers that can identify patients with high risk for recurrence are rare. We have defined the expression of L1 cell adhesion molecule (L1CAM), a biomarker previously identified for EC, and compared its expression to oestrogen receptor (ER)/progesterone receptor (PR) and E-cadherin. We found that L1CAM was absent in normal endometrium and the vast majority of endometrioid ECs (type 1) but was strongly expressed in serous and clear-cell ECs, considered as type 2. 78/272 cases were identified as L1CAM-positive endometrioid ECs that were correlated with a poor prognosis. Strikingly, we observed an inverse relationship between L1CAM and ER/PR/E-cadherin expression in all ECs. In mixed ECs, composed of endometrioid (L1CAM(-) ER/PR(+) E-cadherin(+)) and clear-cell/serous (L1CAM(+) ER/PR(-) E-cadherin(-)), both phenotypes were co-expressed. In some of these cases L1CAM was up-regulated at the leading edge of the tumour, where ER/PR and E-cadherin expression were selectively lost. In EC cell lines treated with the epithelial-mesenchymal transition (EMT) inducer TGFbeta1, L1CAM and vimentin were strongly up-regulated, while E-cadherin expression was reduced. The treatment also resulted in an increased expression of the EMT transcription factor Slug and an enhanced cell invasion. Depletion of Slug by siRNA knockdown prevented both L1CAM up-regulation and enhanced cell invasion. According to our analysis, we suggest that L1CAM is a novel marker for EMT in ECs and that L1CAM-typing could identify endometrioid ECs that have type 2-like features and are at high risk for recurrence.

The neural cell adhesion molecule L1 is a cell surface glycoprotein of the immunoglobulin superfamily which mediates adhesion between neural cells. The possibility that similar cell-cell recognition mechanisms may be shared by the nervous and immune systems prompted us to study the expression and function of L1 in cells of the hematopoietic system. Immunofluorescence analysis using monoclonal L1 antibody revealed that the molecule is expressed in the bone marrow, spleen, and thymus of the mouse. This observation was confirmed by amplifying cDNA derived from these organs by the polymerase chain reaction with L1-specific oligonucleotide primers. Two-color fluorescence analysis indicated that bone marrow lymphoid and granulocyte precursor cells express low and high levels of L1, respectively. In the thymus L1 is primarily expressed by mature cells that have a strong expression of CD3 and in the spleen both B cells and T cells express L1. The possible function of L1 in lymphoid cells was studied using subcloned ESb-MP lymphoma cells having high or low densities of L1 on the cell surface as well as activated splenic B lymphoblasts. Parental and subcloned ESb-MP cells that strongly expressed L1 could form homotypic aggregates in the presence of low Ca2+ levels, whereas subcloned ESb-MP cells with a weak expression of L1 did not aggregate, suggesting that L1 mediates the Ca2+-independent aggregation of the parental ESb-MP cells. Furthermore, the aggregation of activated B lymphoblasts under physiological concentrations of Ca2+ and Mg2+ was inhibited by 30% in the presence of Fab fragments of polyclonal L1 antibodies, implying that L1 also mediates adhesion among normal lymphoid cells. A possible role of L1 on lymphocytes in stimulating the innervation of lymphoid organs is discussed.

Research over the last 25 years on the cell adhesion molecule L1 has revealed its pivotal role in nervous system function. Mutations of the human L1CAM gene have been shown to cause neurodevelopmental disorders such as X-linked hydrocephalus, spastic paraplegia and mental retardation. Impaired L1 function has been also implicated in the aetiology of fetal alcohol spectrum disorders, defective enteric nervous system development and malformations of the renal system. Importantly, aberrant expression of L1 has emerged as a critical factor in the development of human carcinomas, where it enhances cell proliferation, motility and chemoresistance. This discovery promoted collaborative work between tumour biologists and neurobiologists, which has led to a substantial expansion of the basic knowledge about L1 function and regulation. Here we provide an overview of the pathological conditions caused by L1 malfunction. We further discuss how the available data on gene regulation, molecular interactions and posttranslational processing of L1 may contribute to a better understanding of associated neurological and cancerous diseases.

Journal of biomedical science 17, 3 (2010). doi:10.1186/1423-0127-17-3, Published by BioMed Central, London

Pancreatic ductal adenocarcinoma (PDAC) is thought to originate from ductal structures, exhibiting strong desmoplastic reaction with stromal pancreatic myofibroblasts (PMF), which are supposed to drive PDAC tumorigenesis. Previously, we observed high expression of the adhesion molecule L1CAM (CD171) in PDAC cells accounting for chemoresistance. Thus, this study aimed to investigate whether PMFs are involved in the induction of tumoral L1CAM and whether this contributes to malignant transformation of pancreatic ductal cells and PDAC tumorigenesis. Immunohistochemistry of tissues from chronic pancreatitis specimens revealed considerable L1CAM expression in ductal structures surrounded by dense fibrotic tissue, whereas no L1CAM staining was seen in normal pancreatic tissues. Using the human pancreatic duct cell line H6c7, we show that coculture with PMFs led to a transforming growth factor-β1 (TGF-β1)–dependent up-regulation of L1CAM expression. Similarly, L1CAM expression increased in monocultured H6c7 cells after administration of exogenous TGF-β1. Both TGF-β1– and PMF-induced L1CAM expression were independent of Smad proteins but required c-Jun NH2-terminal kinase activation leading to the induction of the transcription factor Slug. Moreover, Slug interacted with the L1CAM promoter, and its knockdown abrogated the TGF-β1– and PMF-induced L1CAM expression. As a result of L1CAM expression, H6c7 cells acquired a chemoresistant and migratory phenotype. This mechanism of TGF-β1–induced L1CAM expression and the resulting phenotype could be verified in the TGF-β1–responsive PDAC cell lines Colo357 and Panc1. Our data provide new insights into the mechanisms of tumoral L1CAM induction and how PMFs contribute to malignant transformation of pancreatic duct cells early in PDAC tumorigenesis. [Cancer Res 2009;69(10):4517–26]

Junctional adhesion molecule-A (JAM-A) is one component of tight junctions which are involved in important processes like paracellular permeability, cell polarity, adhesion, migration, and angiogenesis. Here we describe JAM-A expression in distal convoluted tubule, connecting tubule, and in cells of the collecting duct of the healthy human kidney. In addition, JAM-A was weakly expressed in cells of the proximal tubule. Using immunofluorescence, FACS and Western blot analysis we investigated JAM-A expression in tubular cells in vitro. Interestingly, treatment of HK-2 cells with IFN-gamma and TNF-alpha resulted in a metalloproteinase mediated downregulation of JAM-A. Importantly, in a tissue micro-array JAM-A protein expression was significantly downregulated in patients with clear cell renal cell carcinoma. Furthermore, knockdown of JAM-A with JAM-A specific siRNA induced the migration of RCC4 cells. In summary, downregulation of JAM-A is an early event in the development of renal cancer and increases the migration of renal cancer cells.

The antimurine CD2 mAb 12-15 was administered intravenously to investigate the role of CD2 in cell-mediated immunity in vivo. The anti-CD2 mAb was able to diminish the contact sensitivity response to the hapten trinitrophenyl and was most effective when administered during the efferent or elicitative phase of immunity. Antibody treatment was also able to inhibit in vivo priming for subsequent generation of secondary, TNP-specific CTL in vitro. This inhibitory effect was most effective in the afferent or early phase of immunity. Indeed antibody could be injected at least 3 weeks prior to in vivo antigen priming, and the subsequent CTL response was still suppressed. Additional experiments showed a well-defined dose-response relationship between the amount of anti-CD2 administered and subsequent immunosuppression. Control experiments showed that other isotype-matched antibodies were not suppressive and that the anti-CD2 was not merely shifting the kinetics of the CTL response. Further experiments revealed that in vivo mAb treatment could also inhibit the subsequent development of primary, alloantigen-specific CTL in vitro while the mixed lymphocyte reaction (MLR) remained unchanged. FACS analysis revealed a marked downmodulation of CD2 in vivo, a small and variable decrease in CD8, and essentially no change in CD3 or CD4 after treatment with anti-CD2. The F(ab')2 fragment was not able to downmodulate CD2 or to suppress CTL activity at the doses tested. These results support a major role for CD2 in diverse aspects of cell-mediated immunity affecting both CD4+ and CD8+ effector T cells. The anti-CD2 mAb functions not by deleting or depleting relevant cell populations but rather by altering the array of cell surface receptors and subsequent responses to antigenic challenge.

We recently showed that the adhesion molecule L1CAM (CD171) is overexpressed in pancreatic adenocarcinoma (PDAC) essentially contributing to chemoresistance of PDAC cells. In search of the mechanisms of this effect we now identified alpha5-integrin as the L1CAM ligand being essential for L1CAM-mediated chemoresistance of these highly malignant tumor cells. Thus, blockade or knock-down of alpha5-integrin in the L1CAM expressing PDAC cell lines PT45-P1res, Colo357 and Panc1 increased anti-cancer drug sensitivity. In line with the previously reported NO-dependent caspase inhibition resulting from L1CAM induced iNOS expression, the loss of chemoresistance upon alpha5-integrin inhibition was preceded by decreased iNOS expression and enhanced caspase-3/-7 activation. Accordingly, the loss of anti-cancer drug protection by alpha5-integrin inhibition could be overcome by administration of the NO-donor SNAP. Moreover, the gain of chemoresistance of parental PT45-P1 cells when transfected with L1CAM was abrogated by alpha5-integrin inhibition, whereas transfection of PT45-P1 cells with an integrin binding-deficient L1CAM mutant (L1mutRGE) did neither induce chemoresistance or iNOS expression nor conferred sensitivity to alpha5-integrin inhibition as seen upon transfection with wild-type L1CAM. Thus, mutational loss of the integrin binding site in the L1CAM molecule or the blockade of alpha5-integrin abolished the induction of iNOS expression and chemoresistance by L1CAM, indicating that both a functional L1CAM and alpha5-integrin are indispensable of L1CAM-induced drug resistance in PDAC cells.

Activation of CD4+ T cells by APCs occurs by multiple Ag recognition events including the exchange of costimulatory signals and cytokines. Additionally, the T cells acquire APC-derived surface molecules. Herein, we describe for the first time the transfer of human and murine T cell surface receptors to APCs after Ag-specific interaction. This transfer occurs in two qualitatively different phases. The first group of molecules (e.g., CD2) derived from the T cell surface was transferred rapidly after 2 h of interaction, was strongly bound on the DC surface (acid wash-resistant), was strictly dependent on dendritic cell-T cell contact, and transferred independently of T cell activation. The second group, including the CD3/TCR complex, CD27, and OX40, was of intracellular origin, transferred later after 10–16 h in a cell-cell contact-independent fashion, was noncovalently bound, and was strictly dependent on Ag-specific T cell activation. Functionally, murine dendritic cells that received TCR molecules from OVA-specific CD4+ T cells after Ag-specific interaction were less efficient in priming naive CD4+ T cells of the same specificity without losing their ability for CD8+ T cell stimulation, indicating that the transferred TCR molecules mask the Ag-bearing MHC II molecules, thereby reducing their accessibility to following Ag-specific CD4+ T cells. While the first group of transferred T cell surface molecules might facilitate the detachment of the CD4+ T cell from the dendritic cell during the early scanning phases, the second group could play an important immunomodulatory role in intraclonal competition of T cells for APC access, making the physical presence of CD4+ T cells unnecessary.