Irrelevant Antibody Research Articles

Physical and Functional Coupling of Electrogenic Na+/HCO3- Cotransport and Carbonic Anhydrases in the Myocardium

The Na+/HCO3- cotransport (NBC) is an important sarcolemmal acid extruder in cardiac muscle. Functionally electroneutral (NBC3, 1 Na+:1 HCO3-) and electrogenic (NBC1 and NBC4, 1 Na+:2 HCO3-) forms of the transporter have been characterized in the heart. Mammalian cardiac muscle expresses membrane bound carbonic anhydrases (CA) IV (CAIV), IX (CAIX) and XIV (CAXIV), and cytosolic CAII. Association of CAII and CAIV, and NBC1, was previously demonstrated in the kidney and heterologous systems. NBC1 and CA physical and functional interaction was explored in the rat heart by co-immunoprecipitation and intracellular pH measurement (pHi) experiments, respectively. CAII, CAIV, CAIX, and CAXIV were immunoprecipitated with anti-NBC1 antibody, using rat ventricular lysates. Conversely, non immune serum and irrelevant anti-glial fibrilar acidic protein antibodies failed to co-immunoprecipitated CAs with NBC1. NBC1 activity was investigated in isolated rat cardiomyocytes, using intracellular fluorescent measurements of BCECF-AM, to monitor pHi. Cardiomyocyte membrane potential depolarazing pulses (MPDP) were applied by addition of 45 mM extracellular K+, to study NBC1 activity. After 10 minutes of MPDP a significant intracellular alkalinization was detected (0.17±0.03 pH units; n=6, P<0.05). The alkalinization was fully cancelled with specific anti-NBC1 functionally inhibitory antibodies (0.02±0.02 pH units; n=5), indicating activation of NBC1 isoform. Similarly, the NBC1-mediated increase of pHi (0.17±0.02 pH units; n=11, P<0.05), was inhibited with a poorly membrane-permeant CA inhibitor, benzolamide (100 μM, 0.09±0.02 pH units; n=6, P<0.05), and a potent membrane-permeant CA inhibitor, ethoxyzolamide (100 μM, 0.05±0.01 pH units; n=6, P<0.05), demonstrating a functional coupling between NBC1 cotransport and extracellular CAs, and NBC1and intracellular CAs in the cardiac muscle, respectively. We demonstrated that the NBC1 Na+/HCO3- cotransport is functionally and physically coupled to both plasma membrane-anchored CAs and cytosolic CAII, forming a HCO3- transport metabolon in the myocardium.

Conjunctival Interleukin-13 Expression in Mucous Membrane Pemphigoid and Functional Effects of Interleukin-13 on Conjunctival Fibroblasts in Vitro

Interleukin-13 (IL-13) is the dominant effector cytokine of fibrosis in pulmonary and liver disease. Excessive conjunctival fibrosis in the immunobullous disease ocular mucous membrane pemphigoid (MMP) causes blindness; the pathogenesis of scarring in this disease is incompletely understood. To determine whether IL-13 is involved in conjunctival fibrosis in MMP, we studied the expression of IL-13 in ocular MMP patients before and after systemic immunosuppression and examined the effects of IL-13 on normal human conjunctival fibroblasts. We found high stromal cell expression of IL-13 in active ocular MMP by immunohistochemistry; 80% of these cells were CD3-positive T cells. Following immunosuppression, in clinically uninflamed, treated, ocular MMP patients, the number of IL-13 positive cells was significantly reduced, but this was still fourfold greater than in normal conjunctiva. IL-13 stimulated collagen lattice contraction and migration, and decreased production of mmp-3 and mmp-10 by human conjunctival fibroblasts. The addition of T cell culture supernatant to IL-13 synergistically augmented fibroblast migration. IL-13 also up-regulated surface expression of HLA-DR, CD80, CD40, and CD154 by conjunctival fibroblasts, suggesting a potential mechanism for fibroblast-T cell cross talk, via which fibroblasts may actively engage in perpetuating chronic inflammation and continued fibrosis. Together, these findings suggest that IL-13 is involved in conjunctival fibrosis in MMP, and that IL-13 has both profibrotic and pro-inflammatory effects on human conjunctival fibroblasts.

Evidence for A Macromolecular Complex in Poor Prognostic CLL That Contains CD38, CD49d, CD44, MMP-9 and ZAP-70.

Abstract 2334Poster Board II-311The outcome for patients with Chronic Lymphocytic Leukemia (CLL) is highly variable and correlates with clinical parameters such as disease stage, as well as various biological markers that reflect the genomic, phenotypic and functional properties of the tumor and its microenvironment. CD38, Cd49d and ZAP-70 are three such markers that are associated with a poor clinical outcome and which have previously been shown to play a role in migration. Progressive disease is invariably associated with tissue invasion. In the present study we investigated the possibility that the markers CD38, CD49d and ZAP-70 might be physically associated as well as functionally linked. Using 4-color flow cytometry we measured the level of expression of CD38 and CD49d by CD19+/CD5+ CLL cells in patients with CLL presenting to our routine diagnostic laboratory. A correlation was observed between both the percentage positivity and mean fluorescent intensity of CD38 and CD49d expression (r2=0.42, p=<0.0001, n=234; r2=0.28, p=<0.0001, n=61 respectively). Expression levels were not always linked, with some patients globally expressing a high percentage of either CD38 or CD49d positive cells with a low percentage of the other. However, within individual cells, a strong correlation was apparent, so that cells expressing a higher CD38 Mean Fluorescence Intensity (MFI) also had a high CD49d MFI and vice-versa. Since previous studies have shown that CD49d, CD44 and matrix metalloproteinase-9 (MMP-9) are physically associated in the CLL cell membrane and are involved in tissue invasion, we investigated the possibility that CD38 and ZAP-70 might also form part of this complex. CLL cell lysates were immunoprecipitated using anti-CD38 and then western blotted for CD49d, CD44, MMP-9 and ZAP-70. In 9/9 CD38+ cases, these immunoprecipitates contained both CD49d and CD44, whilst control immunoprecipitates on the same lysates and performed with an irrelevant antibody did not. In addition, the CD38 immunoprecipitate from 5/5 patients tested also contained MMP-9 and in 4/5 cases ZAP-70. CD5 was not detected in any of the samples, further confirming the specificity of these findings. The spatial relationship between CD38 and CD49d was next investigated by immunofluorescence confocal microscopy. CLL cells from 5 different patients known to express both CD38 and CD49d were adhered to poly-L-lysine coated slides, stained by indirect immunofluorescence and analyzed using confocal microscopy. Pixel counts showed that a mean of 64% of CD38 co-localised with CD49d (SEM 5.1%) and a mean of 72% of CD49d with CD38 (SEM 4.8%). These results thus show that the membrane of some CLL cells contains a macromolocular complex comprising CD38, CD49d, MMP9, CD44 and ZAP70, which is likely to play a role in mediating migration across the endothelial barrier into the tissues. Our findings also provide a possible explanation for the correlation between the level of expression of these molecules and adverse outcome since, as noted above, infiltration of the tissues is generally observed in patients with progressive disease. As normal B-lymphocytes do not contain the CD44/CD49d/MMP-9 complex and express neither CD38 nor ZAP-70, this structure would also seem to be an ideal target for therapies aimed at inhibiting tissue infiltration and disease progression in CLL. Disclosures:No relevant conflicts of interest to declare.

HLA Class I Antibodies Provoke Graft Arteriosclerosis in Human Arteries Transplanted into SCID/Beige Mice

Antibodies toward HLA class I and/or MICA are commonly observed in transplanted patients suffering from allograft arteriosclerosis, also called chronic vascular rejection (CVR). The relative importance of cellular versus humoral alloreactivity for CVR is still disputed. We demonstrate that antibodies toward HLA class I provoke lesions typical for CVR in human arteries in vivo in the absence of cellular immunity. To show this, we grafted segments of human mesenteric arteries from 8 deceased organ donors into 36 immunodeficient SCID/beige mice in the infrarenal aortic position. Three mice died postoperatively. The remaining 33 mice received weekly i.v. injections of either a monoclonal antibody toward HLA class I, toward MICA or an irrelevant monoclonal antibody. At sacrifice after 6 weeks, mice receiving the HLA antibody showed a significant neointimal thickening in the grafted artery due to smooth muscle cell (SMC) proliferation while control mice receiving anti-MICA or irrelevant antibody showed little or no thickening. Whereas antibodies toward HLA class I were mitogenic to SMC in vitro, those directed toward MICA did not have any effect. Humoral alloreactivity toward HLA may thus play a causal role for the development of CVR and this opens new possibilities for the treatment of CVR.

Stability validation of paraformaldehyde-fixed samples for the assessment of the platelet PECAM-1, P-selectin, and PAR-1 thrombin receptor by flow cytometry

Sample fixation for storage and/or transportation represents an unsolved challenge for multicenter clinical trials assessing serial changes in platelet activity, or monitoring various antiplatelet regimens. Whole blood flow cytometry represents a major advance in defining platelet function, although special training and expensive equipment is required. We sought to determine how fixation with 2% paraformaldehyde (PFA), and storage of blood samples over 1 week affects the flow cytometry readings for both intact and thrombin-activating four major surface platelet receptors. Whole blood platelet expression of PECAM-1, P-selectin, PAR-1 inactive receptor (SPAN-12), and cleaved (WEDE-15) epitope was assessed immediately after blood draw, after staining with 2% PFA, and at day 1, 3, 5, and 7. The study was performed in 6 volunteers with multiple risk factors for vascular disease, not receiving any antiplatelet agents. Staining with PFA resulted in a slight decrease of fluorescence intensity, especially for PECAM-1, while antigen expression at day 1, 3 and 5 remains consistent, and highly reproducible. At day 7 there was a small but inconsistent trend towards diminished fluorescence intensity. The platelet data were consistent while validated with the isotype-matched irrelevant antibody. These data suggest that there is a 5 day window to perform final flow cytometry readings of whole blood PFA-fixed inactivated platelet samples. In contrast, thrombin activation cause gradual loss of flow cytometry signal, and cannot be recommended for long-term storage. This is critical logistic information for conducting multicenter platelet substudies within the framework of major clinical trials.

IN VIVO NEAR-INFRARED FLUORESCENCE IMAGING OF HUMAN COLON ADENOCARCINOMA BY SPECIFIC IMMUNOTARGETING OF A TUMOR-ASSOCIATED MUCIN

Background and Aims: Accurate endoscopic detection of premalignant lesions and early cancers in the colon is essential for cure, since prognosis is closely related to lesion size and stage. Although it has great clinical potential, autofluorescence endoscopy has limited tumor-to-normal tissue image contrast for detecting small preneoplastic lesions. We have developed a molecularly specific, near-infrared fluorescent monoclonal antibody (CC49) bioconjugate which targets tumor-associated glycoprotein 72 (TAG72), as a contrast agent to improve fluorescence-based endoscopy of colon cancer. Methods: The fluorescent anti-TAG72 conjugate was evaluated in vitro and in vivo in athymic nude mice bearing human colon adenocarcinoma (LS174T) subcutaneous tumors. Autofluorescence, a fluorescent but irrelevant antibody and the free fluorescent dye served as controls. Fluorescent agents were injected intravenously, and in vivo whole body fluorescence imaging was performed at various time points to determine pharmacokinetics, followed by ex vivo tissue analysis by confocal fluorescence microscopy and histology. Results: Fluorescence microscopy and histology confirmed specific LS174T cell membrane targeting of labeled CC49 in vitro and ex vivo. In vivo fluorescence imaging demonstrated significant tumor-to-normal tissue contrast enhancement with labeled-CC49 at three hours post injection, with maximum contrast after 48 h. Accumulation of tumor fluorescence demonstrated that modification of CC49 antibodies did not alter their specific tumor-localizing properties, and was antibody-dependent since controls did not produce detectable tumor fluorescence. Conclusions: These results show proof-of-principle that our near-infrared fluorescent-antibody probe targeting a tumor-associated mucin detects colonic tumors at the molecular level in real time, and offer a basis for future improvement of image contrast during clinical fluorescence endoscopy.

Cytokeratin-positive Cells in the Bone Marrow of Breast Cancer Patients and Noncancer Donors

Detection of disseminated tumor cells in the bone marrow may provide important prognostic information in breast cancer patients. With few exceptions the number of stained cells scored as cancer is very low; there may be only 1 cell per slide. This makes definitive interpretation of cancer in marrow challenging. False-positive staining of marrow cells with cytokeratin (CK) antibody is relatively common and makes interpretation more difficult. In this report we focus on false-positive staining of marrow specimens from breast cancer patients and noncancer controls and demonstrate that the frequency of false-positive events is common. Bone marrow was collected from 23 cancer-free donors and 60 breast cancer patients. Samples were processed by Ficoll density gradient centrifugation and slides were prepared for immunocytochemical staining with CK and irrelevant (IR) antibody. Slides were evaluated manually and positive cells were categorized as tumor cells, hematopoetic cells, or questionable cells. False-positive staining events were commonly observed in noncancer cases stained with CK or IR antibodies and in breast cancer cases stained with IR antibody. There was little difference in the number of breast cancer marrow specimens scored as tumor cells regardless of whether the antibody used was CK or IR. It is important to devise improved criteria and methods for accurate detection and interpretation of disseminated tumor cells in the marrow of breast cancer patients.

Systemic and Local Interleukin-17 and Linked Cytokines Associated with Sjögren’s Syndrome Immunopathogenesis

Recently recognized as a distinct CD4(+) T helper (Th) lineage, Th17 cells have been implicated in host responses to infections and in pathogenesis associated with autoimmune diseases. This cytokine is implicated in primary Sjögren's syndrome (pSS) immunopathology because of the increased levels of circulating interleukin (IL)-17 in pSS. Plasma and minor salivary glands (MSGs) from patients with pSS were therefore evaluated for CD4(+) T cells, T regulatory cells, IL-17, and supporting cytokines by immunohistochemistry, RT-PCR, and microbead assays. MSGs from pSS patients contain IL-17-expressing cells as a dominant population within inflammatory lesions. IL-17 protein expression progressively increased with higher biopsy focus scores (P < 0.0001), in parallel with detection by RT-PCR. Transforming growth factor-beta, IL-6 and IL-23, which are requisite promoters of Th17 differentiation, were found in abundance compared with the amounts in control tissues. Although transforming growth factor-beta is also a pivotal differentiation factor for immunosuppressive Foxp3(+) T regulatory cells (Tregs), an increase in Foxp3(+) Tregs was evident in biopsy specimens with mild and moderate inflammation but this increase was disproportionate to escalating pro-inflammatory Th17 populations in advanced disease. Furthermore, the Th17-centric cytokines IL-17, IL-6, IL-23, and IL-12 were significantly elevated in pSS plasma. These data identify a profusion of IL-17-generating cells and supporting cytokines within diseased pSS MSGs without a compensatory increase in immunomodulatory Tregs; this imbalance seems to foster a pathogenic milieu that may be causative and predictive of infiltrative injury and amenable to therapeutic intervention.

Active Uptake of Dendritic Cell-Derived Exovesicles by Epithelial Cells Induces the Release of Inflammatory Mediators through a TNF-α-Mediated Pathway

Dendritic cells (DCs) can release hundreds of membrane vesicles, called exovesicles, which are able to activate resting DCs and distribute antigen. Here, we examined the role of mature DC-derived exovesicles in innate and adaptive immunity, in particular their capacity to activate epithelial cells. Our analysis of exovesicle contents showed that exovesicles contain major histocompatibility complex-II, CD40, and CD83 molecules in addition to tumor necrosis factor (TNF) receptors, TNFRI and TNFRII, and are important carriers of TNF-alpha. These exovesicles are rapidly internalized by epithelial cells, inducing the release of cytokines and chemokines, but do not transfer an alloantigen-presenting capacity to epithelial cells. Part of this activation appears to involve the TNF-alpha-mediated pathway, highlighting the key role of DC-derived exovesicles, not only in adaptive immunity, but also in innate immunity by triggering innate immune responses and activating neighboring epithelial cells to release cytokines and chemokines, thereby amplifying the magnitude of the innate immune response.

The Convenience of Flow Cytometry for HLA-B*5701 Screening to Prevent Abacavir Hypersensitivity Reactions

The Convenience of Flow Cytometry for HLA-B*5701 Screening to Prevent Abacavir Hypersensitivity Reactions

EKLF/KLF1 Controls Cell Cycle Entry via Direct Regulation of E2f2

Differentiation of erythroid cells requires precise control over the cell cycle to regulate the balance between cell proliferation and differentiation. The zinc finger transcription factor, erythroid Krüppel-like factor (EKLF/KLF1), is essential for proper erythroid cell differentiation and regulates many erythroid genes. Here we show that loss of EKLF leads to aberrant entry into S-phase of the cell cycle during both primitive and definitive erythropoiesis. This cell cycle defect was associated with a significant reduction in the expression levels of E2f2 and E2f4, key factors necessary for the induction of S-phase gene expression and erythropoiesis. We found and validated novel intronic enhancers in both the E2f2 and E2f4 genes, which contain conserved CACC, GATA, and E-BOX elements. The E2f2 enhancer was occupied by EKLF in vivo. Furthermore, we were able to partially restore cell cycle dynamics in EKLF(-/-) fetal liver upon additional genetic depletion of Rb, establishing a genetic causal link between reduced E2f2 and the EKLF cell cycle defect. Finally, we propose direct regulation of the E2f2 enhancer is a generic mechanism by which many KLFs regulate proliferation and differentiation.

Up-Regulation of Soluble Axl and Mer Receptor Tyrosine Kinases Negatively Correlates with Gas6 in Established Multiple Sclerosis Lesions

Multiple sclerosis is a disease that is characterized by inflammation, demyelination, and axonal damage; it ultimately forms gliotic scars and lesions that severely compromise the function of the central nervous system. Evidence has shown previously that altered growth factor receptor signaling contributes to lesion formation, impedes recovery, and plays a role in disease progression. Growth arrest-specific protein 6 (Gas6), the ligand for the TAM receptor tyrosine kinase family, consisting of Tyro3, Axl, and Mer, is important for cell growth, survival, and clearance of debris. In this study, we show that levels of membrane-bound Mer (205 kd), soluble Mer ( approximately 150 kd), and soluble Axl (80 kd) were all significantly elevated in homogenates from established multiple sclerosis lesions comprised of both chronic active and chronic silent lesions. Whereas in normal tissue Gas6 positively correlated with soluble Axl and Mer, there was a negative correlation between Gas6 and soluble Axl and Mer in established multiple sclerosis lesions. In addition, increased levels of soluble Axl and Mer were associated with increased levels of mature ADAM17, mature ADAM10, and Furin, proteins that are associated with Axl and Mer solubilization. Soluble Axl and Mer are both known to act as decoy receptors and block Gas6 binding to membrane-bound receptors. These data suggest that in multiple sclerosis lesions, dysregulation of protective Gas6 receptor signaling may prolong lesion activity.

Cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) against tumor cell lines characterized for EGFR expression and K-ras mutation

e14583 Background: Mutated K-ras protein is a strong predictive factor of cetuximab resistance, bypassing the classical direct inhibitory effect on epidermal growth factor receptor (EGFR) signaling. However, cetuximab is also able to mediate ADCC, which may be part of the clinical response. The aim of this ex-vivo study was to quantify cetuximab-mediated ADCC on various human cancer cell lines characterized for EGFR-expression and K-ras mutation. Methods: Two K-ras mutated cell lines over-expressing EGFR and resistant to anti-EGFR tyrosine kinase inhibitor were tested (Capan-1 and Capan-2, pancreatic), along with 2 K-ras wild-type cell lines over- expressing EGFR (CAL166, head and neck; A431, epidermoid carcinoma) and an EGFR-negative cell line (OCM1, uveal melanoma). The tested monoclonal antibodies (mAbs) were: cetuximab (Merck, anti-EGFR IgG1 mAb), panitumumab (Amgen, anti-EGFR IgG2 mAb), and as a negative control, rituximab (Roche, IgG1 anti-CD20 mAb). ADCC (51Cr release assay) was performed using freshly- isolated peripheral blood mononuclear cells from a healthy donor. Results were expressed as % of potentially maximum 51Cr release. Results: Cetuximab mediates ADCC against EGFR-over-expressing cell lines CAL166 (38.4 ± 3.1 %), A431 (13.5 ± 1.7 %), Capan-1 (31.2 ± 0.8 %) and Capan-2 (27.8 ± 8.6 %) irrespective of the K-ras mutational status, but not against EGFR-negative OCM-1 (6.2 ± 1 %). Conversely, unlike IgG1 cetuximab, the anti-EGFR IgG2 panitumumab and the irrelevant antibody rituximab were both unable to induce significant ADCC (&lt; 10 % on all tested cell lines). Conclusions: Cetuximab-mediated ADCC is independent of the K-ras mutational status of the tumor cell lines. Present data suggest that cetuximab may remain of clinical interest in K-ras-mutated patients. Immunostimulation, as well as new generation anti-EGFR mAbs with improved ability to induce ADCC, may be promising in the management of K-ras-mutated patients. No significant financial relationships to disclose.

Erbin and the NF2 Tumor Suppressor Merlin Cooperatively Regulate Cell-Type-Specific Activation of PAK2 by TGF-β

Transforming growth factor beta (TGF-beta) family ligands are pleotropic proteins with diverse cell-type-specific effects on growth and differentiation. For example, PAK2 activation is critical for the proliferative/profibrotic action of TGF-beta on mesenchymal cells, and yet it is not responsive to TGF-beta in epithelial cells. We therefore investigated the regulatory constraints that prevent inappropriate PAK2 activation in epithelial cultures. The results show that the epithelial-enriched protein Erbin controls the function of the NF2 tumor suppressor Merlin by determining the output of Merlin's physical interactions with active PAK2. Whereas mesenchymal TGF-beta signaling induces PAK2-mediated inhibition of Merlin function in the absence of Erbin, Erbin/Merlin complexes bind and inactivate GTPase-bound PAK2 in epithelia. These results not only identify Erbin as a key determinant of epithelial resistance to TGF-beta signaling, they also show that Erbin controls Merlin tumor suppressor function by switching the functional valence of PAK2 binding.

A mouse model for ricin poisoning and for evaluating protective effects of antiricin antibodies

Background. Ricin is a potential bioterrorism agent and no specific antidote or treatment exists for ricin poisoning. For this reason, we developed ricin-specific antibodies that were tested in a murine model of ricin poisoning for use as antidotes against symptoms of ricin poisoning. Methods. Mice were poisoned with a lethal dose of ricin (5 μg) and their temperature and general condition were monitored for determination of a surrogate and humane end point. Mice were then treated with injections of ricin and different combinations of polyclonal anti-ricin antibodies. Antibody effect was evaluated for various doses and using various time points from ricin to antibody injection. Also, the effect of adjuvant symptomatic treatment was examined. Brain, heart, intestines, kidney, liver, lung, pancreas, spleen, and stomach tissues were sampled for histopathological analysis. Results. The mouse model was reproducible and easy to use. A clear protective effect of both anti-ricin A-chain and anti-ricin B-chain antibodies–but not of irrelevant antibodies–was demonstrated with no added effect of symptomatic treatment. Conclusions. These data suggest that specific polyclonal antibodies against ricin A- and B-chain may reproducibly protect mice against ricin poisoning, even when the antibodies are administered up to 1.5 h after poisoning.

Discovery and Validation of Novel Peptide Agonists for G-protein-coupled Receptors

G-protein-coupled receptors (GPCRs) represent an important group of targets for pharmaceutical therapeutics. The completion of the human genome revealed a large number of putative GPCRs. However, the identification of their natural ligands, and especially peptides, suffers from low discovery rates, thus impeding development of therapeutics based on these potential drug targets. We describe the discovery of novel GPCR ligands encrypted in the human proteome. Hundreds of potential peptide ligands were predicted by machine learning algorithms. In vitro screening of selected 33 peptides on a set of 152 GPCRs, including a group of designated orphan receptors, was conducted by intracellular calcium measurements and cAMP assays. The screening revealed eight novel peptides as potential agonists that specifically activated six different receptors in a dose-dependent manner. Most of the peptides showed distinct stimulatory patterns targeted at designated and orphan GPCRs. Further analysis demonstrated a significant in vivo effect for one of the peptides in a mouse inflammation model.

Involvement of Highly Sulfated Chondroitin Sulfate in the Metastasis of the Lewis Lung Carcinoma Cells

The altered expression of cell surface chondroitin sulfate (CS) and dermatan sulfate (DS) in cancer cells has been demonstrated to play a key role in malignant transformation and tumor metastasis. However, the functional highly sulfated structures in CS/DS chains and their involvement in the process have not been well documented. In the present study, a structural analysis of CS/DS from two mouse Lewis lung carcinoma (3LL)-derived cell lines with different metastatic potentials revealed a higher proportion of Delta(4,5)HexUA-GalNAc(4,6-O-disulfate) generated from E-units (GlcUA-GalNAc(4, 6-O-disulfate)) in highly metastatic LM66-H11 cells than in low metastatic P29 cells, although much less CS/DS is expressed by LM66-H11 than P29 cells. This key finding prompted us to study the role of CS-E-like structures in experimental lung metastasis. The metastasis of LM66-H11 cells to lungs was effectively inhibited by enzymatic removal of tumor cell surface CS or by preadministration of CS-E rich in E-units in a dose-dependent manner. In addition, immunocytochemical analysis showed that LM66-H11 rather than P29 cells expressed more strongly the CS-E epitope, which was specifically recognized by the phage display antibody GD3G7. More importantly, this antibody and a CS-E decasaccharide fraction, the minimal structure recognized by GD3G7, strongly inhibited the metastasis of LM66-H11 cells probably by modifying the proliferative and invading behavior of the metastatic tumor cells. These results suggest that the E-unit-containing epitopes are involved in the metastatic process and a potential target for the diagnosis and treatment of malignant tumors.

CD74 Is a Regulator of Fas-Mediated Apoptotic Signaling

Resistance to Fas-mediated apoptosis in hematopoietic cancers interferes with the efficacy of currently available chemotherapy. Our prior studies of human herpesvirus 8 oncoprotein K1 showed that K1 binds to Fas and interferes with activation of Fas-mediated apoptotic signaling (Wang, W, et al, Blood 2007; 109:5455–62). Herpesvirus proteins often mimic host proteins or their functions, we thus searched for cellular proteins associated with inactive Fas in order to identify potential regulators of Fas signaling. We identified CD74, the invariant light chain of the major histocompatibility class II complex, associated exclusively with inactive/activation resistant Fas in B-cell lymphoma-derived BJAB cell line. Interestingly, overexpression of CD74 has been previously reported in ~ 90% of hematopoietic cancers and derived cell lines (Stein, R, et al., Ciln Cancer Res. 2007; 13: 5556s–63s), as well as in ~ 80% of non-small-cell lung carcinoma (Ioachim, H.L., Am J Surg Pathol. 1996; 20: 64–71). The role of CD74 in carcinogenesis was thus suspected. Through siRNA suppression of CD74 expression in BJAB cells we have determined that CD74 positive cells are more resistant to agonistic antibody CH-11-induced Fas-mediated apoptosis then the CD74 siRNA transfected cells (38 ± 7.8 % vs. 54 ± 9.8 %; P = 0.045). In addition, a challenge with agonistic anti-Fas antibody showed a significant survival advantage of the mice expressing CD74 in their livers over the vector-transfected mice (83% vs. 0 %; P < 0.016). We have mapped domain of CD74 required for its association with Fas and for significant protection of mice to a membrane-proximal extracellular region of CD74. Treatment of BJAB cells for 24 hours with humanized anti-CD74 antibody (hLL1 + crosslinking antibody), FasL-Fc, or anti-Fas antibody CH-11 induced apoptosis in 19%, 25% and 13% of cells, respectively. Combination of hLL1+crosslinking antibody with FasL-Fc or CH-11 increased apoptosis to ~ 45% of cells in both cases (P < 0.01), while combination of FasL-Fc or CH-11 with hLL1 + irrelevant antibody had no significant effect on the extent of apoptosis. Our results support the idea of an endogenous regulatory system of Fas-mediated apoptosis that utilizes transmembrane proteins interacting with Fas. We anticipate that specific blocking of the CD74-Fas interaction will sensitize CD74 overexpressing cancer cells to Fas-mediated apoptosis and thus will lead to a more effective chemotherapy for hematopoietic cancers.

C-reactive protein induces M-CSF release and macrophage proliferation

Inflammation is pivotal in atherosclerosis. M-CSF regulates macrophage growth and differentiation and plays a role in atherogenesis. C-reactive protein (CRP), a cardiovascular risk marker, may promote atherogenesis. However, the effects of CRP on M-CSF release and subsequent macrophage proliferation have not been examined previously. Human aortic endothelial cells (HAEC) were incubated with boiled CRP or native CRP 12.5, 25, and 50 microg/mL for 12-15 h, and M-CSF release was examined by flow cytometry and ELISA. CRP resulted in a significant and dose-dependent increase in M-CSF mRNA and secretion from HAEC as well as human monocyte-derived macrophages (HMDM; P<0.01). Furthermore, conditioned medium (5%) from HAEC pretreated with CRP, when incubated with HMDM, increased macrophage proliferation significantly. This was blocked with M-CSF antibody but not irrelevant antibody. Inhibition of NF-kappaB resulted in significant abrogation of CRP-induced M-CSF release and subsequent macrophage proliferation. Antibodies to CD32 and CD64 but not CD16 abrogated CRP-induced M-CSF release. Thus, CRP up-regulates M-CSF release from HMDM and HAEC and increased macrophage proliferation. These effects appear to be mediated via activation of NF-kappaB via CD32 and CD64. These studies provide further evidence for a proatherogenic role for CRP.

Identification of Aurora-A as a Direct Target of E2F3 during G2/M Cell Cycle Progression

Aurora-A is a centrosome kinase and plays a pivotal role in G(2)/M cell cycle progression. Expression of Aurora-A is cell cycle-dependent. Levels of Aurora-A mRNA and protein are low in G(1)/S, accumulate during G(2)/M, and decrease rapidly after mitosis. Previous studies have shown regulation of the Aurora-A protein level during the cell cycle through the ubiquitin-proteasome pathway. However, the mechanism of transcriptional regulation of Aurora-A remains largely unknown. Here, we demonstrated that E2F3 modulates Aurora-A mRNA expression during the cell cycle. Ectopic expression of E2F3 induces Aurora-A expression. Stable knockdown of E2F3 decreases mRNA and protein levels of Aurora-A and delays G(2)/M entry. Further, E2F3 directly binds to Aurora-A promoter and stimulates the promoter activity. Deletion and mutation analyses of the Aurora-A promoter revealed that a region located 96-bp upstream of the transcription initiation site is critical for the activation of the promoter by E2F3. In addition, expression of E2F3 positively correlates with the protein level of Aurora-A in human ovarian cancer examined. These results indicate for the first time that Aurora-A is transcriptionally regulated by E2F3 during the cell cycle and that E2F3 is a causal factor for up-regulation of Aurora-A in a subset of human ovarian cancer. Thus, the E2F3-Aurora-A axis could be an important target for cancer intervention.