Membrane-bound Tumor Research Articles

P2X7 Receptor and Cytokines Contribute to Extra-territorial Facial Pain

The whisker pad area (WP) is innervated by the second branch of the trigeminal nerve and experiences allodynia and hyperalgesia following transection of the mental nerve (MN; the third branch of the trigeminal nerve). However, the mechanisms of this extra-territorial pain remain unclear. The ionotropic P2X7 ATP receptor (P2X7) in microglia is known to potentiate, via cytokines, the perception of noxious stimuli, raising the possibility that P2X7 and cytokines are involved in this extra-territorial pain. One day after MN transection (MNT), WP allodynia/hyperalgesia developed, which lasted for > 8 wks. Activation of microglia and up-regulation of P2X7, membrane-bound tumor necrosis factor (TNF)-α (mTNF-α), and soluble TNF-α (sTNF-α) in the trigeminal sensory nuclear complex (TNC) were evident for up to 6 wks after MNT. Allodynia/hyperalgesia after MNT was blocked by intracisternal administration of etanercept, a recombinant TNF-α receptor (p75)-Fc fusion protein. Intracisternal A438079, a P2X7 antagonist, also attenuated allodynia/hyperalgesia and blocked up-regulation of mTNF-α and sTNF-α in the TNC. We conclude that sTNF-α released by microglia following P2X7 activation may be important in both the initiation and maintenance of extra-territorial pain after MNT.

Differential Expression of Growth Factor Receptors and Membrane-Bound Tumor Markers for Imaging in Male and Female Breast Cancer

IntroductionMale breast cancer accounts for 0.5–1% of all breast cancers and is generally diagnosed at higher stage than female breast cancers and therefore might benefit from earlier detection and targeted therapy. Except for HER2 and EGFR, little is known about expression of growth factor receptors in male breast cancer. We therefore investigated expression profiles of growth factor receptors and membrane-bound tumor markers in male breast cancer and gynecomastia, in comparison with female breast cancer.MethodsTissue microarrays containing 133 male breast cancer and 32 gynecomastia cases were stained by immunohistochemistry for a panel of membrane-bound targets and compared with data on 266 female breast cancers.ResultsGrowth factor receptors were variably expressed in 4.5% (MET) up to 38.5% (IGF1-R) of male breast cancers. Compared to female breast cancer, IGF1-R and carbonic anhydrase 12 (CAXII) were more frequently and CD44v6, MET and FGFR2 less frequently expressed in male breast cancer. Expression of EGFR, HER2, CAIX, and GLUT1 was not significantly different between male and female breast cancer. Further, 48.1% of male breast cancers expressed at least one and 18.0% expressed multiple growth factor receptors. Since individual membrane receptors are expressed in only half of male breast cancers, a panel of membrane markers will be required for molecular imaging strategies to reach sensitivity. A potential panel of markers for molecular imaging, consisting of EGFR, IGF1-R, FGFR2, CD44v6, CAXII, GLUT1, and CD44v6 was positive in 77% of male breast cancers, comparable to female breast cancers.ConclusionsExpression patterns of growth factor receptors and hypoxia membrane proteins in male breast cancer are different from female breast cancer. For molecular imaging strategies, a putative panel consisting of markers for EGFR, IGF1-R, FGFR2, GLUT1, CAXII, CD44v6 was positive in 77% of cases and might be considered for development of molecular tracers for male breast cancer.

Surveillance of spontaneous breast cancer metastasis by TRAIL-expressing CD34+ cells in a xenograft model

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), delivered as a membrane-bound molecule expressed on the surface of adenovirus-transduced CD34+ cells (CD34-TRAIL+), was analyzed for its apoptotic activity in vitro on 12 breast cancer cell lines representing estrogen receptor-positive, HER2+ and triple-negative (TN) subtypes and for its effect on tumor growth, vascularization, necrosis, and lung metastasis incidence in NOD/SCID mice xenografted with the TN breast cancer line MDA-MB-231. Mesenchymal TN cell lines, which are the richest in putative tumor stem cells among the different breast cancer cell subtypes, were the most susceptible to apoptosis induced by CD34-TRAIL+ cells. Indeed, tumor cell “stemness”, assessed based on the proportion of CD44+/CD24−/low cells, was significantly correlated with susceptibility to TRAIL. Moreover, in vitro cytotoxicity experiments showed that CD34-TRAIL+ cells selectively targeted CD44+/CD24−/low cells. Although in vivo treatment with CD34-TRAIL+ cells did not lead to tumor growth inhibition, treated mice revealed significantly larger areas of necrosis associated with damage of tumor vasculature than did control mice. Moreover, lungs from MDA-MD-231 tumor-bearing mice were completely free of metastases at 12 days after the last injection of CD34-TRAIL+ cells, whereas metastases were present in all control mouse lungs. An anti-metastatic effect of CD34-TRAIL+ cells was also observed in a model of experimental lung metastases. The correlation between in vitro susceptibility to membrane-bound TRAIL and tumor stem cell content, together with CD34-TRAIL+ cell-induced inhibition of the metastatic process, points to the selective targeting of cancer stem cells by CD34-armed cells and the potential value of such cells in eradicating tumor stem cells before the onset of overt metastases.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-012-2281-4) contains supplementary material, which is available to authorized users.

Abstract 640: The Akt inhibitor Perifosine strongly enhances the antitumor and antivascular activity of CD34+ cells engineered to express membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

Abstract Introduction. We have previously demonstrated that adenovirus-transduced CD34+ cells expressing membrane-bound (m)TRAIL (CD34-TRAIL+ cells) exert potent antitumor activity against a variety of hematopoietic tumors by targeting both tumor cells and tumor vasculature (Blood, 115:2231-40, 2010). Recently, we have identified non-Hodgkin lymphoma cell lines which are resistant to the in vivo antitumor activity of mTRAIL. Perifosine has been shown to increase the toxicity of soluble TRAIL against cancer cell lines by inhibiting the PI3K/Akt pathway as well as enhancing pro-apoptotic TRAIL receptors. We therefore investigated the efficacy of Perifosine in modulating the antitumor activity of CD34-TRAIL+ cells using as model systems the TRAIL-resistant SU-DHL-4V and the TRAIL-sensitive KMS-11 cell lines. Methods and Results. In vitro, Perifosine significantly enhanced the cytotoxic activity of CD34-TRAIL+ cells against both SU-DHL-4V and KMS-11 cell lines by increasing the expression of TRAIL receptors and down-modulating phospho-Akt, cFLIP and Mcl-1 expression. In vivo, in NOD/SCID mice bearing subcutaneous nodules, Perifosine significantly increased the antitumor activity of CD34-TRAIL+ cells against both tumor types. In fact, CD34-TRAIL+ cells used as single agent exerted a limited if any activity against TRAIL-resistant SU-DHL-4V nodules, whereas, when combined with Perifosine, transduced cells reduced SU-DHL-4V growth by 43% (p< .001) over controls. TRAIL-sensitive KMS-11 nodules were reduced by 39% (p< .001) following treatment with CD34-TRAIL+ cells, and addition of Perifosine further reduced tumor growth by 65% (p<.001) over controls. Upon in vivo treatment with Perifosine, confocal microscopy analysis revealed a strong down-modulation of phospho-Akt expression by tumor cells and tumor endothelial cells (TECs), and flow cytometry analysis of TECs revealed a strong induction of TRAIL-R2 expression. In particular, Perifosine induced de novo TRAIL-R2 expression by TECs enriched from SU-DHL-4V tumors (41.6 ± 5.7% vs 4.0 ± 4.0%, p<.05), and enhanced TRAIL-R2 expression by TECs from KMS-11 tumors (81.3 ± 7.6% vs 41.7 ± 8.2%, p<.05). Increased levels of endothelial TRAIL-R2 was associated with a significant increase of (i) tumor specific CD34-TRAIL+ cells-induced vascular damage, (ii) tumor hemorragic necrosis and (iii) tumor apoptosis. Conclusions. Our results demonstrate that: (i) TRAIL-R2 expression by TECs correlates with the in vivo antivascular activity of CD34-TRAIL+ cells; (ii) Perifosine potentiates the antitumor activity of TRAIL-armed CD34+ cells and is able to overcome in vivo resistance to mTRAIL by inducing TRAIL-R2 expression on tumor endothelial cells. These results may open new perspectives in view of clinical applications of CD34-TRAIL+ cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 640. doi:10.1158/1538-7445.AM2011-640

Isolation of viable antigen-specific CD8 + T cells based on membrane-bound tumor necrosis factor (TNF)-α expression

Current technology to isolate viable cytokine-producing antigen-specific primary human T cells is limited to bi-specific antibody capture systems, which suffer from limited sensitivity and high background. Here, we describe a novel procedure for isolating antigen-specific human T cells based on their ability to produce tumor necrosis factor (TNF)-α. Unlike many cytokines, TNF-α is initially produced in a biologically active membrane-bound form that is subsequently cleaved by TNF-α converting enzyme (TACE) to release the soluble form of TNF-α. By preventing this cleavage event, we show that TNF-α can be ‘trapped’ on the surface of the T cells from which it originates and directly labeled for viable isolation of these antigen-specific T cells. Together with other existing sorting procedures to isolate activated T cells, this new technique should permit the direct isolation of multi-functional T lymphocytes for further protein and gene expression analyses, as well as a detailed functional assessment of the potential role that TNF-α producing T cells play in the adaptive immune system.

Membrane‐bound HSP70‐engineered myeloma cell‐derived exosomes stimulate more efficient CD8+ CTL‐ and NK‐mediated antitumour immunity than exosomes released from heat‐shocked tumour cells expressing cytoplasmic HSP70

Exosomes (EXO) derived from tumour cells have been used to stimulate antitumour immune responses, but only resulting in prophylatic immunity. Tumour-derived heat shock protein 70 (HSP70) molecules are molecular chaperones with a broad repertoire of tumour antigen peptides capable of stimulating dendritic cell (DC) maturation and T-cell immune responses. To enhance EXO-based antitumour immunity, we generated an engineered myeloma cell line J558HSP expressing endogenous P1A tumour antigen and transgenic form of membrane-bound HSP70 and heat-shocked J558HS expressing cytoplasmic HSP70, and purified EXOHSP and EXOHS from J558HSP and J558HS tumour cell culture supernatants by ultracentrifugation. We found that EXOHSP were able to more efficiently stimulate maturation of DCs with up-regulation of Iab, CD40, CD80 and inflammatory cytokines than EXOHS after overnight incubation of immature bone-marrow-derived DCs (5 × 106 cells) with EXO (100 μg), respectively. We also i.v. immunized BALB/c mice with EXO (30 μg/mouse) and assessed P1A-specific T-cell responses after immunization. We demonstrate that EXOHSP are able to stimulate type 1 CD4+ helper T (Th1) cell responses, and more efficient P1A-specific CD8+ cytotoxic T lymphocyte (CTL) responses and antitumour immunity than EXOHS. In addition, we further elucidate that EXOHSP-stimulated antitumour immunity is mediated by both P1A-specific CD8+ CTL and non-P1A-specific natural killer (NK) responses. Therefore, membrane-bound HSP70-expressing tumour cell-released EXO may represent a more effective EXO-based vaccine in induction of antitumour immunity.

E0154 Expression of tumour necrosis factor-α converting enzyme and tumour necrosis factor-α in rats with alcoholic cardiomyopathy

PurposeChronic excessive consumption of alcohol causes ventricular remodelling and eventually leads to alcoholic cardiomyopahty (ACM). Tumour necrosis factor-α converting enzyme (TACE) has been identified to cleave membrane-bound tumour necrosis factor-α...

Human CD34+ cells engineered to express membrane-bound tumor necrosis factor–related apoptosis-inducing ligand target both tumor cells and tumor vasculature

AbstractAdenovirus-transduced CD34+ cells expressing membrane-bound tumor necrosis factor–related apoptosis-inducing ligand (CD34-TRAIL+ cells) exert potent antitumor activity. To further investigate the mechanism(s) of action of CD34-TRAIL+ cells, we analyzed their homing properties as well as antitumor and antivascular effects using a subcutaneous myeloma model in immunodeficient mice. After intravenous injection, transduced cells homed in the tumor peaking at 48 hours when 188 plus or minus 25 CD45+ cells per 105 tumor cells were detected. Inhibition experiments showed that tumor homing of CD34-TRAIL+ cells was largely mediated by vascular cell adhesion molecule-1 and stromal cell–derived factor-1. Both CD34-TRAIL+ cells and soluble (s)TRAIL significantly reduced tumor volume by 40% and 29%, respectively. Computer-aided analysis of TdT-mediated dUTP nick end-labeling–stained tumor sections demonstrated significantly greater effectiveness for CD34-TRAIL+ cells in increasing tumor cell apoptosis and necrosis over sTRAIL. Proteome array analysis indicated that CD34-TRAIL+ cells and sTRAIL activate similar apoptotic machinery. In vivo staining of tumor vasculature with sulfosuccinimidyl-6-(biotinamido) hexanoate-biotin revealed that CD34-TRAIL+ cells but not sTRAIL significantly damaged tumor vasculature, as shown by TdT-mediated dUTP nick end-labeling+ endothelial cells, appearance of hemorrhagic areas, and marked reduction of endothelial area. These results demonstrate that tumor homing of CD34-TRAIL+ cells induces early vascular disruption, resulting in hemorrhagic necrosis and tumor destruction.

TRAIL gene-armed oncolytic poxvirus and oxaliplatin can work synergistically against colorectal cancer

SummaryWe have explored a unique combination therapy for metastatic colorectal cancer. This strategy combines a potent and new oncolytic poxvirus expressing a membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (TRAIL or TNFSF10) and oxaliplatin (Ox) chemotherapy. We hypothesized that TRAIL expression would increase the efficacy of the oncolytic poxvirus, and that the therapeutic efficacy would be further enhanced by combination with chemotherapy. The cytotoxicity to cancer cells by Ox, oncolytic VV and trail gene-armed VV alone or in combination was tested in vitro. The trail gene armed oncolytic VV expressed high levels of TRAIL in infected cancer cells and had greater potency as a cytotoxic agent compared to the parent VV. Ox alone exerted concentration-dependent cytotoxicity. In vitro, the combination of the two agents applied at suboptimal concentrations for individual therapy displayed synergy in inducing cancer cells into enhanced levels of apoptosis/necrosis. Western blot analyses confirmed the notion that TRAIL induced cancer cell death mainly through apoptosis, while Ox and vJS6 may induce cell death more through non-apoptotic death pathways. In two aggressive colorectal carcinomatosis models derived from human HCT116 and murine MC38 cells, the combination therapy displayed synergistic or additive antitumor activity and prolonged the survival of the tumor-bearing mice compared to either Ox chemotherapy or vvTRAIL-mediated oncolytic gene therapy alone. This combination strategy may provide a new avenue to treating peritoneal carcinomatosis and other types of metastases of colorectal cancer.

Certolizumab Pegol

Certolizumab pegol is a PEGylated humanized Fab' monoclonal antibody that targets and neutralizes both membrane-bound and soluble tumor necrosis factor (TNF)-alpha, preventing inflammation and consequently the destruction of cartilage and bone. Certolizumab pegol has a relatively long elimination half-life of approximately 2 weeks, allowing subcutaneous administration once every 2 or 4 weeks. In two randomized, phase III trials in patients with active rheumatoid arthritis despite previous methotrexate therapy (RAPID 1 and 2), the combination of subcutaneous certolizumab pegol 400 mg at weeks 0, 2, and 4, followed by a 200 or 400 mg dose every 2 weeks and a stable dosage of methotrexate, was more effective than placebo plus methotrexate for improving the signs and symptoms of arthritis at weeks 24 (RAPID 1 and 2) and 52 (RAPID 1), according to American College of Rheumatology (ACR) criteria. Improvements in ACR response rates were seen as early as 1 week and at all timepoints measured up to 52 weeks. In RAPID 1 and RAPID 2, radiographic progression was also significantly inhibited with certolizumab pegol plus methotrexate treatment compared with placebo and methotrexate according to van der Heijde modified Total Sharp Scores at 24 and 52 weeks after treatment initiation. In patients with active rheumatoid arthritis who had previously failed to respond to treatment with > or = 1 disease-modifying anti-rheumatic drug, certolizumab pegol 400 mg every 4 weeks as monotherapy effectively improved ACR responses at all measured timepoints up to 24 weeks, according to data from the randomized, phase III FAST4WARD trial. Certolizumab pegol was generally well tolerated in combination with methotrexate or as monotherapy in phase III trials in patients with rheumatoid arthritis, with most adverse events being of mild to moderate intensity. Infections were the most frequently reported adverse events.

Actinomycin D upregulates lipopolysaccharide induction of macrophage procoagulant expression and tumour necrosis factor-alpha production

The antitumour antibiotic actinomycin D (Act D) and the aminosugar D-galactosamine both enhance the sensitivity of animals to bacterial lipopolysaccharide (LPS). Lipopolysaccharide stimulates macrophage membrane-bound procoagulant activity (MPCA) and tumour necrosis factor-alpha (TNF-alpha) production in vitro. We investigated the effects of LPS combined with either Act D or D-galactosamine on procoagulant and TNF-alpha production in vitro. Actinomycin D directly induced procoagulant on the malignant monocytoid cell line WEHI 265, and synergized with LPS to enhance MPCA on both WEHI 265 cells and thioglycollate-induced peritoneal exudate macrophages. In the presence of Act D, exudate macrophages expressed procoagulant in response to concentrations of LPS 100,000-fold lower than normally required. Pulsing experiments demonstrated that LPS primed these cells within 4 h to respond to Act D, whereas 4 h priming with Act D inhibited subsequent procoagulant induction by LPS. Although its effects on TNF-alpha production were less intense, low levels of Act D more than doubled TNF-alpha produced by LPS-stimulated exudate macrophages. Procoagulant expression and TNF-alpha production were not always co-ordinately expressed; interferon-gamma (IFN-gamma) synergized with LPS to enhance both responses but when IFN-gamma was combined with Act D only procoagulant was upregulated. D-galactosamine failed to affect these macrophage responses. Results indicate different in vivo mechanisms of enhancement of LPS toxicity by these two agents.

Human CD34+ Cells Expressing Membrane-Bound Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) Exert a Potent Anti-Lymphoma Effects by Targeting Tumor Vasculature.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expected to play a key role in cancer therapy due to its high cancer cell-specificity and potent antitumor activity. We have previously demonstrated that CD34+ cells transduced with an adenovirus encoding the full-length human TRAIL gene (CD34-TRAIL+) exert a potent anti-lymphoma effect in NOD/SCID mice. To investigate the mechanism of action of CD34-TRAIL+ cells, in vivo experiments were perfomed to analyze1.the tumor homing capacity of CD34-TRAIL+ cells and2.the effects of transduced cells on tumor vasculature.Tumor homing of CD34-TRAIL+ cells was investigated in NOD/SCID mice bearing subcutaneous lymphoid tumors. Following a single intravenous injection of CD34-TRAIL+ cells (5 x 106), nodules were excised at different time-points and immunostained with an anti-human CD45 antibody. Sections were digitally recorded and the total number of CD45+ cells per tissue section was then counted using the imaging software ImageJ (http://rsb.info.nih.gov/ij/). Tumor homing of CD34-TRAIL+ cells peaked 24 hours after cell injection when a mean of 200 CD34-TRAIL+ cells was recorded per 1 x 105 tumor cells (0.2% CD45+ cells per 1 x 105 tumor cells). Mice pretreatment with the anti-VCAM-1 antibody or the CXCR4 antagonist AMD3100 significantly reduced tumor homing of CD34-TRAIL+ cells, with 0.09% and 0.05% CD45+ cells being recorded per 1 x 105 tumor cells, respectively. To analyze the effects of CD34-TRAIL+ cells on tumor vasculature, mice were injected with sulfo-biotin (0.1 mg, IV, 15 min) and tumor endotelial cells (TEC) were then revealed by staining frozen sections with horseradish peroxidase (HRP)-conjugated streptavidin. As compared with CD34-mock- or soluble (s)TRAIL-treated mice, treatment with CD34-TRAIL+ cells induced within 24 hours a significant (P ≤.001) increase of the thickness of the vessell wall (3.7 ± 1 μm vs 3.4 ± 1 μm vs 6 ± 1 μm, respectively) as well as the endothelial surface (10 ± 4% vs 7 ± 4% vs 21 ± 6% of total tissue section, respectively), suggesting that CD34-TRAIL+ cells induce an early vascular disruption. Confocal microscopic imaging of tumor sections double-stained with anti-CD31 and anti-TRAIL-R2 showed that this receptor was expressed by 8–12% of large tumor vessels. Interestingly, upon treatment with CD34-TRAIL+ cells, but not sTRAIL, TUNEL staining revealed an extensive apoptosis of CD31+/TRAIL-R2+ TEC. Forty-eight hours following injection of CD34-TRAIL+ cells, a 21-fold increase of apoptotic index was detected, which was associated with extensive necrotic areas (20% to 25% of tissue section). These data show that:1.tumor homing of CD34-TRAIL+ cells induces extensive vascular damage, hemorrhagic necrosis and tumor destruction;2.the antitumor effect of CD34-TRAIL+ cells is mediated by both indirect vascular-disrupting mechanisms and direct tumor cell killing.

Matrix metalloproteinases in brain development and remodeling: Synaptic functions and targets

Matrix metalloproteinases (MMPs) play critical roles in egg fertilization, embryonic development, wound repair, cancer, and inflammatory and neurologic diseases. This subfamily of metzincin peptidases can cleave extracellular matrix (ECM) and pericellular proteins that have profound effects on cell behavior. Among known MMP substrates are several proteins that play important roles in synaptogenesis, synaptic plasticity, and long-term potentiation (LTP). In this Mini-Review we discuss how MMP-directed cleavage of these proteins can impact the formation and function of synapses within the brain. Pyramidal neurons in the hippocampus, and other large neurons, are surrounded by perineuronal nets that are composed of brevican, tenascin-R, and laminin, each of which is subject to proteolytic cleavage by MMPs. Tenascin-R knockout mice show deficits in learning and memory and LTP, as do at least two MMP knockouts. Impaired LTP is also seen in brain-derived neurotrophic factor (BDNF) knockout mice, which is interesting in that pro-BDNF can be processed into mature BDNF by several MMPs and thereby regulate activation of the high-affinity BDNF receptor TrkB. At the synaptic level, MMP substrates also include ephrins, Eph receptors, and cadherins, which are also involved in synapse development and plasticity. MMPs can also process membrane-bound tumor necrosis factor-alpha into a potent soluble cytokine that is increasingly implicated in neuron-glial signaling, particularly in neurologic disease. Finally, we discuss how the development of therapeutics to attenuate MMP activity in neurodegenerative disorders may become powerful tools for future studies of synaptic formation and function within the developing and mature brain.

Upregulation of ADAM-17 expression in active lesions in multiple sclerosis

ADAM-17, a disintegrin and metalloproteinase, is the major proteinase responsible for the cleavage of membrane-bound tumour necrosis factor (TNF) as well as being an active sheddase of other cytokines, cytokine receptors, growth factors and adhesion molecules. TNF is a major proinflammatory cytokine that has been identified as having a pathogenic role in inflammatory diseases within the CNS including multiple sclerosis (MS). Here we report the cellular origin and distribution of ADAM- 17 expression within clinically and neuropathologically confirmed MS and normal control white matter, assessed by immunohistochemistry, western blotting and PCR. ADAM-17 expression was associated with the blood vessel endothelium, activated macrophages/microglia and parenchymal astrocytes in MS white matter. Increased levels of ADAM-17 immunoreactivity were displayed in active lesions with evidence of recent myelin breakdown. Further studies into the functional role of ADAM-17 in the pathogenesis of MS and other inflammatory conditions are required.

Tumour necrosis factor-alpha stimulates expression of TNF-alpha converting enzyme in endothelial cells.

Tumor necrosis factor-alpha converting enzyme (ADAM17) is a major metalloproteinase involved in the shedding of several membrane-bound cytokines and cytokine receptors. Interplay of cytokines and their soluble receptors might be an important regulatory element in the network of interactions responsible for maintaining homeostasis in the immune system. ADAM17 thus has the potential to participate in a broad range of immune reactions. We studied the mechanisms of ADAM17 activation in endothelial cells and found that pro-inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, interferon-gamma) and growth factors (epidermal growth factor, vascular endothelial growth factor) are able to upregulate transcription of ADAM17 and expression of ADAM17 protein. This process might constitute an important mechanism of regulation of ADAM17 activity. Stimulation of transcription, rather than increased ADAM17 mRNA stability, was responsible for increased levels of ADAM17 mRNA. Importantly, the increase in ADAM17 was accompanied by increased shedding of TNF-Receptor I (p55) in tumor necrosis factor-alpha-stimulated endothelial cells. Therefore, ADAM17-dependent depletion of membrane-bound tumor necrosis factor receptors from endothelial cells might constitute a mechanism of self-protection in states of prolonged immunostimulation.

Periodontal disease, matrix metalloproteinases and chemically modified tetracyclines

Matrix metalloproteinases (MMPs) are crucial in the degradation of the main components in the extracellular matrix and thereby play important roles in cell migration, wound healing and tissue remodelling. MMPs have pathogenic roles in arthritis, periodontitis, hepatitis, glomerulonephritis, atherosclerosis and cancer cell invasion. MMPs are activators of pro-inflammatory mediators that occur in latent forms, such as interleukin (IL)-1β, membrane-bound tumour necrosis factor (TNF) and different MMPs. To regulate their action, MMPs are secreted in latent, inactive pro-enzyme forms, with plasmin and other MMPs as likely activators. MMP activity is further modulated by the tissue inhibitors of metalloproteinases (TIMP-1, -2, -3 and -4), which partly control and stabilize MMPs. In normal steady-state tissues, only low levels of MMPs are detected intra- and extra-cellularly because MMPs are tightly regulated at the level of transcription and secretion. In inflamed periodontal tissue, numbers of cells expressing MMPs are increased as compared with healthy gingival tissue. Recently, novel non-antimicrobial MMP-inhibitory effects of tetracyclines were discovered, and subsequently a panel of chemically modified non-antimicrobial tetracyclines (CMTs) was developed. This paper discusses these new drugs and their possible biological effects in periodontal disease.

Periodontal Disease, Matrix Metalloproteinases and Chemically Modified Tetracyclines

Matrix metalloproteinases (MMPs) are crucial in the degradation of the main components in the extracellular matrix and thereby play important roles in cell migration, wound healing and tissue remodelling. MMPs have pathogenic roles in arthritis, periodontitis, hepatitis, glomerulonephritis, atherosclerosis and cancer cell invasion. MMPs are activators of pro-inflammatory mediators that occur in latent forms, such as interleukin (IL)-1β, membrane-bound tumour necrosis factor (TNF) and different MMPs. To regulate their action, MMPs are secreted in latent, inactive pro-enzyme forms, with plasmin and other MMPs as likely activators. MMP activity is further modulated by the tissue inhibitors of metalloproteinases (TIMP-1, -2, -3 and -4), which partly control and stabilize MMPs. In normal steady-state tissues, only low levels of MMPs are detected intra- and extra-cellularly because MMPs are tightly regulated at the level of transcription and secretion. In inflamed periodontal tissue, numbers of cells expressing MMPs are increased as compared with healthy gingival tissue. Recently, novel nonantimicrobial MMP-inhibitory effects of tetracyclines were discovered, and subsequently a panel of chemically modified non-antimicrobial tetracyclines (CMTs) was developed. This paper discusses these new drugs and their possible biological effects in periodontal disease.

Adalimumab efficacy and safety in patients with moderate to severe chronic plaque psoriasis: preliminary findings from a 12-week dose-ranging trial

Adalimumab is a human monoclonal IgG1 antibody containing only human peptide sequences which is approved for use in the US in adults with rheumatoid arthritis. It binds specifically to soluble and membrane-bound tumor necrosis factor-alpha (TNF-alpha), thereby neutralizing the biological activities of this cytokine. In psoriasis, TNF-alpha expression is notably increased in psoriatic plaques. The efficacy and safety of two dose levels of adalimumab in the treatment of psoriasis was determined in a randomized, double blind, placebo-controlled trial (M02–528).

Endoproteolysis of औ-Secretase (औ-Site Amyloid Precursor Protein-cleaving Enzyme) within Its Catalytic Domain: A POTENTIAL MECHANISM FOR REGULATION

Sequential proteolysis of the amyloid precursor protein (APP) by beta- and gamma-secretase activities yields the amyloid beta peptide that is widely deposited in the brains of individuals with Alzheimer's disease. The membrane-anchored aspartyl protease beta-site APP-cleaving enzyme (BACE) exhibits all of the characteristics of a beta-secretase and has been shown to cleave APP at its beta-site in vitro and in vivo. We found that BACE undergoes cleavage on a surface-exposed alpha-helix between amino acid residues Leu-228 and Ala-229, generating stable N- and C-terminal fragments that remain covalently associated via a disulfide bond. The efficiency of BACE endoproteolysis was observed to depend heavily on cell and tissue type. In contrast to brain where holoprotein was predominant, BACE was found primarily as endoproteolyzed fragments in pancreas, liver, and muscle. In addition, we observed a marked up-regulation of BACE endoproteolysis in C2 myoblasts upon differentiation into multinucleated myotubes, a well established model system of muscle tissue specification. As in liver, BACE exists as endoproteolyzed fragments in the hepatic cell line, HepG2. We found that HepG2 cells are capable of generating amyloid beta peptide, suggesting that endoproteolyzed BACE retains measurable beta-secretase activity. We also found that BACE endoproteolysis occurs only after export from the endoplasmic reticulum, is enhanced in the trans-Golgi network, and is sensitive to inhibitors of vesicular acidification. The membrane-bound proteases tumor necrosis factor alpha-converting enzyme and furin were not found to be responsible for this cleavage nor was BACE observed to mediate its own endoproteolysis by an autocatalytic mechanism. Thus, we characterize a specific processing event that may serve to regulate the enzymatic activity of BACE on a post-translational level.

Tumor cells expressing membrane-bound tumor necrosis factor activate macrophages and have a compromised growth in immunosuppressed and immunodeficient mice

Tumor necrosis factor (TNF)-α producing tumors as vaccines were demonstrated to induce a therapeutic anti-tumor immune response, but their clinical use is limited by the toxicity of soluble TNF. We investigated the growth characteristics and immunomodulatory properties of HeLa cells producing an uncleavable transmembrane form of TNF (preTNF). The growth of the transformed tumors was compromised in both immunosuppressed and severe combined immunodeficient mice; no signs of TNF toxicity were detected. Macrophages co-cultured with the transformed cells showed increased phagocytosis and cytokine production, indicating that activated macrophages may be the mediators of the anti-tumor effect. preTNF producing tumor cells are promising safe anti-tumor vaccine candidates.