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PubMed2006

Growth hormone, VEGF and FGF: involvement in rheumatoid arthritis.

Adult rheumatoid arthritis (RA), a systemic autoimmune disorder of unknown etiology, is characterized by dysfunctional cellular and humoral immunity, enhanced migration and attachment of peripheral macrophages and pro-inflammatory leukocytes to the synovium and articular cartilage of diarthrodial joints. The progressive destruction of cartilage and bone in RA is a result of elevated pro-inflammatory cytokine gene expression, synovial neovascularization, proteinase-mediated dissolution of articular cartilage matrix and osteoclast-mediated subchondral bone resorption. Juvenile chronic arthritis (JCA) is disease with manifestations similar to adult RA that occurs in childhood. JCA usually causes precocious joint destruction and often also presents with evidence of growth plate anomalies and reduced stature. Three proteins play an integral role in both adult RA and JCA. These are somatotropin (also called pituitary growth hormone (GH)), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). GH is responsible for regulating long bone growth and skeletal maturation through its capacity to stimulate insulin-like growth factor-I (IGF-1) synthesis by hepatocytes. Mechanisms responsible for growth plate disturbances and short stature in children with JCA include deficient GH production, GH-insensitivity resulting from defects in the GH receptor, suppressed IGF-1 synthesis or neutralization of IGF-1 action by IGF-1 binding proteins (IGFBPs). In addition, GH has also been implicated in perpetuating inflammation and pain in adult RA. VEGF has been shown to be the critical angiogenesis factor responsible for vascular proliferation and blood vessel invasion of the synovial lining membrane in RA. Acidic FGF (FGF-1) and basic FGF (FGF-2) have also been implicated in aberrant synoviocyte proliferation (i.e. synovial hyperplasia) and apoptosis resistance in adult RA.

Animals↗
PubMed2001

Arterial compliance to stratify cardiovascular risk: more precision in therapeutic decision making.

The focus of attention in preventing and treating cardiovascular (CV) disease today is shifting toward the arterial wall. Evidence has been accumulating for several years that protecting the endothelium is key to reducing CV risk. Endothelial dysfunction results in reduced compliance, or increased arterial stiffness, particularly in the smaller arteries. This abnormality is characteristic of patients with hypertension but may also be seen in normotensive patients before the appearance of clinical disease. Reduced arterial compliance is also seen in patients with diabetes and in smokers, and is part of a vicious cycle that further elevates blood pressure, aggravates atherosclerosis, and leads to increased CV risk. Although other factors are involved, the damage to the endothelium results in reduced secretion of nitric oxide, which influences smooth muscle growth, migration, and contraction, as well as influencing inflammation and clotting. Arterial compliance can be measured by several techniques, most of which are invasive or otherwise not clinically appropriate. Pulse contour analysis is a newly developed noninvasive method that allows for easy, in-office measurement of arterial elasticity to identify patients at risk for CV events before disease becomes clinically apparent. Further research is needed to confirm whether this method offers a means of improving risk stratification and therapeutic decision making.

Arteries↗
PubMed2006

Small polaron hopping in Li(x)FePO4 solid solutions: coupled lithium-ion and electron mobility.

Transition metal phosphates such as LiFePO(4) have been recognized as very promising electrodes for lithium-ion batteries because of their energy storage capacity combined with electrochemical and thermal stability. A key issue in these materials is to unravel the factors governing electron and ion transport within the lattice. Lithium extraction from LiFePO(4) results in a two-phase mixture with FePO(4) that limits the power characteristics owing to the low mobility of the phase boundary. This boundary is a consequence of low solubility of the parent phases, and its mobility is impeded by slow migration of the charge carriers. In principle, these limitations could be diminished in a solid solution, Li(x)FePO(4). Here, we show that electron delocalization in the solid solution phases formed at elevated temperature is due to rapid small polaron hopping and is unrelated to consideration of the band gap. We give the first experimental evidence for a strong correlation between electron and lithium delocalization events that suggests they are coupled. Furthermore, the exquisite frequency sensitivity of Mössbauer measurements provides direct insight into the electron hopping rate.

Journal Article↗
PubMed2005

Role of pro-IGF-II processing by proprotein convertase 4 in human placental development.

Fetal growth restriction (intrauterine growth restriction, IUGR) is a leading cause of perinatal mortality. However, the causes of aberrant development of the placenta and, thus, of the fetus, are not currently known. Insulin-like growth factor II (IGF-II) has been shown to be an important regulator of fetoplacental growth. This growth factor must undergo posttranslational processing, and, thus, we hypothesized that aberrant processing of pro-IGF-II to IGF-II may be a cause of IUGR. Here, we have found that the proprotein convertase PC4 is expressed in the human placenta and that it cleaves pro-IGF-II to generate the intermediate processed form, IGF-II (1-102) and, subsequently, mature IGF-II (1-67), which are accounted for by the removal of terminal basic residues by carboxypeptidases. This processing confers the ability of IGF-II to activate invasive trophoblast cells through AKT phosphorylation, whereas inhibition of PC4 by a PC4-specific inhibitor blocks pro-IGF-II processing and reduces trophoblast cell migration, which can be partly restored by addition of mature IGF-II. Consistent with the hypothesis that IGF-II processing is implicated in IUGR, sera of patients carrying IUGR fetuses displayed elevated levels of pro-IGF-II. Thus, abnormal processing of IGF-II by PC4 may represent a previously uncharacterized mechanism involved in the pathophysiology of fetoplacental growth restriction, and elevated pro-IGF-II may be a useful clinical marker for risk of IUGR.

Cell Line↗
PubMed1999

Mechanisms by which elevated intracellular calcium induces S49 cell membranes to become susceptible to the action of secretory phospholipase A2.

Exposure of S49 lymphoma cells to exogenous group IIA or V secretory phospholipase A2 (sPLA2) caused an initial release of fatty acid followed by resistance to further hydrolysis by the enzyme. This refractoriness was overcome by exposing cells to palmitoyl lysolecithin. This effect was specific in terms of lysophospholipid structure. Induction of membrane susceptibility by lysolecithin involved an increase in cytosolic calcium and was duplicated by incubating the cells with calcium ionophores such as ionomycin. Lysolecithin also activated cytosolic phospholipase A2 (cPLA2). Inhibition of this enzyme attenuated the ability of lysolecithin (but not ionomycin) to induce susceptibility to sPLA2. Lysolecithin or ionomycin caused concurrent hydrolysis of both phosphatidylethanolamine and phosphatidylcholine implying that transbilayer movement of phosphatidylethanolamine occurred upon exposure to these agents but that susceptibility is not simply due to exposure of a preferred substrate (i.e. phosphatidylethanolamine) to the enzyme. Microvesicles were apparently released from the cells upon addition of lysolecithin or ionomycin. Both these vesicles and the remnant cell membranes were susceptible to sPLA2. Together these data suggest that lysolecithin induces susceptibility through both cPLA2-dependent and -independent pathways. Whereas elevated cytosolic calcium was required for both pathways, it was sufficient only for the cPLA2-independent pathway. This cPLA2-independent pathway involved changes in cell membrane structure associated with transbilayer phospholipid migration and microvesicle release.

Calcium↗
PubMed2005

Role of ICAM1 in invasion of human breast cancer cells.

We identified previously a region on chromosome 19p13.2 spanning the genes encoding the intercellular adhesion molecules (ICAM), ICAM1, ICAM4 and ICAM5 as a breast cancer susceptibility locus. Genetic variants in this region were also associated with indicators of disease severity, including higher rates of metastases to other organs. Based on this association, we set out to explore the role of ICAM1 in proliferation and invasion of human breast cancer cells. We observed that ICAM1 downregulation at the mRNA and protein levels led to a strong suppression of human breast cell invasion through a matrigel matrix. Under the same conditions, no significant effect on cell proliferation in vitro was seen. Incubation of cells with an antibody against ICAM1 blocked invasion of the highly metastatic MDA-MB-435 cell line in a dose-dependent manner without affecting cell migration. We also demonstrated that the level of ICAM1 protein expression on the cell surface positively correlated with metastatic potential of five human breast cancer cell lines and that ICAM1 mRNA levels were elevated in breast tumor compared with adjacent normal tissue. These results corroborate our previous genetic finding that variations in the ICAM region are associated with the occurrence of metastases and establish a causal role of ICAM1 in invasion of metastatic human breast carcinoma cell lines.

Breast Neoplasms↗
PubMed2003

Functional analysis of EBV in nasopharyngeal carcinoma cells.

A membrane invasion culture system was used to study the ability of EBV to enhance invasion and migration of nasopharyngeal carcinoma (NPC) cells. Semi-reverse transcriptase-PCR analysis of matrix proteinases and angiogenic factors from EBV-infected, or EBV-positive (EBV+), cells demonstrated different degrees of elevated gene expression. In our animal model, EBV+ tumors grew faster and larger than EBV-free, or EBV-negative (EBV-), tumors and also had clonal EBV terminal repeat sequences. Double-localization of EBV and certain host proteins in EBV+ tumors and biopsy specimens demonstrated that EBV up-regulates host genes only in cells that express those genes but not in cells that do not express them. Double-localization of EBV and host genes in NPC biopsy specimens all showed EBV- tumor cells expressing those host genes. Our data strongly suggest that EBV infection enhances progression of NPC tumor growth. They do not rule out a role for EBV infection in the induction and early promotion of NPC development. Unidentified factors may also enhance NPC tumor growth independent of the effects of EBV.

Animals↗
PubMed1979

Immunological and genetic markers in a family with Hashimoto's disease.

A family with Hashimoto's disease in three generations is described. Seven persons (all female) had Hashimoto's disease was the only thyroid disorder occurring in this family; no other autoimmune disease was observed. Circulating thyroid antibodies were detected in all seven subjects with overt thyroiditis in this family. Thyroid antibodies were also detected in low titres in about half of the healthy relatives. Evidence of thyroid antigen-directed cell-mediated immunity was demonstrated using the leucocyte migration inhibition test in four out of seven subejcts with thyroiditis and also in about half of the relatives without clinical thyroid disease. The relative number of thyroglobulin-binding circulating lymphocytes was elevated in six subjects with Hashimoto's disease. Again, the percentage of such cells was also increased in about half of the 'healthy' relatives. Thyroid-stimulating immunoglobulins were detected by the radioreceptor assay in three of the seven subjects with Hashimoto's disease and in four out of thirteen relatives without overt signs of thyroiditis. In conclusion, all subjects with Hashimoto's disease carried immunological markers of autoimmune thyroid disease in the circulation. In addition, most of the 'healthy' relatives were also positive for some or all of the markers sought in this study. The expression of these markers thus seems to be variable. No clear-cut conclusion could be drawn regarding the inheritance of these markers. HLA genotypes were assayed for thirty-five specificities of A, B and C loci and five of the D loci. There was no correlation between any individual antigen or HLA haplotype and overt Hashimoto's disease in this family.

Adolescent↗
PubMed2004

Haptoglobin expression and activity during coronary collateralization.

Coronary collateral development relies on the coordinated secretion of growth factors. However, alone they are insufficient for permanent collateral growth. We utilized proteomics to identify other important proteins in the extracellular environment that could facilitate collateralization. Chronically instrumented dogs developed coronary collaterals by the repetitive occlusion method. Subendocardial (0.19 +/- 0.04, 0.27 +/- 0.06, 0.48 +/- 0.10, and 0.81 +/- 0.11 ml x min(-1) x g(-1) on days 1, 7, 14, and 21, respectively) and subepicardial (0.14 +/- 0.01, 0.36 +/- 0.06, 0.51 +/- 0.07, and 0.71 +/- 0.08 ml x min(-1) x g(-1) on days 1, 7, 14, and 21, respectively) blood flow increased in animals subjected to repetitive occlusion. Sham animals exhibited no changes in blood flow. Myocardial interstitial fluid (MIF) from both groups was analyzed by two-dimensional electrophoresis with matrix-assisted laser desorption/ionization time-of-flight identification. The acute-phase protein haptoglobin was identified in the group subjected to repetitive occlusion. ELISA of MIF showed haptoglobin to be elevated at all time points of collateral development compared with sham, with maximal production on day 7. Purified haptoglobin dose dependently stimulated endothelial cells to form tubes and vascular smooth muscle cells to migrate. Purified haptoglobin did not stimulate proliferation of either cell type. The relative contribution of haptoglobin to the chemotactic properties of MIF was tested using a neutralizing antibody. Neutralized MIF could not stimulate smooth muscle cells to migrate at any time during collateral development. Endothelial cell tube formation was inhibited after the midpoint of collateralization. Therefore, the acute-phase protein haptoglobin plays a critical role during coronary collateralization.

Animals↗
PubMedDate not supplied

Secretory immune response in patients with oropharyngeal carcinoma.

The secretory immune system is a unique, local immunological mechanism which appears to be independent of systemic immunity. Secretory immunoglobulin A (SIgA), a major component in this mucosal immune system, is structurally different from the IgA immunoglobulin in serum and has a function as a first line of defense against the uptake of macromolecules and against infectious agents in the intestine, respiratory tract and genitourinary system. However, its relationship to the development and control of neoplasia is not well understood despite the fact that the major portion of human malignancies are of the epithelial type and located where the secretory immune system is vigorously functioning. In this study, the amounts of SIgA and secretory component (SC) in oral secretions and sera were measured in 20 patients with oropharyngeal carcinoma and 22 appropriate control subjects in order to elucidate the involvement of the secretory immune system as it related to oropharyngeal carcinoma. Cancer patients had increased IgA levels in oral secretions (561 +/- 161 micrograms/ml) compared to normal subjects (166 +/- 18 micrograms/ml, p less than 0.02) and to a smoker/drinker subgroup of controls (229 +/- 8.7 micrograms/ml, p less than 0.05). The elevated IgA levels in cancer patients appear to be unrelated to local inflammation or the patient's history of smoking or drinking. Density gradient analysis demonstrated that the IgA measured in oral secretions migrates at the 11S position, confirming that it is of the secretory type and indicating that the increased levels of IgA in oral secretions of patients with oropharyngeal carcinoma are not due to nonspecific leakage of 7 SIgA from sera. Secretory component levels in sera also revealed highly significant differences between cancer patients (1.65 +/- 0.73 micrograms/ml) and all control subjects (0.85 +/- 0.12 micrograms/ml, p less than 0.002). The data suggest that the increased SIgA concentration in secretions and SC level in sera accompany the existence of oropharyngeal carcinoma. The biological significance of these findings and their possible clinical application are discussed.

Adult↗
PubMed2003

Distribution of plasma membrane Ca2+ pump activity in normal human red blood cells.

The plasma membrane calcium pump (PMCA) is the only active Ca2+ transporter in human red blood cells (RBCs). Previous measurements of maximal Ca2+ extrusion rates (Vmax) reported only mean values in the RBC population. Despite early evidence for differences in Ca2+ extrusion capacity among RBCs, the precise Vmax distribution remained unknown. It was important to characterize this distribution to assess the range and modality (uni- or multimodal) of PMCA Vmax variation and the likelihood of RBCs with elevated [Ca2+]i in the circulation participating in physiologic and pathologic processes. We report here the application of a new method to investigate the detailed distribution of PMCA Vmax activity in RBCs. The migrating profile of osmotic lysis curves was used to identify and quantify the fraction of cells that extrude a uniform Ca2+ load at different rates. The results revealed that RBCs from single donors have large variations in PMCA activity that follow a unimodal, broad distribution pattern consistently skewed toward higher Vmax values, suggesting an excess of cells with Vmax higher than the mean value. The method applied may provide a way of evaluating whether the observed variation in PMCA Vmax is related to cell age.

Calcium-Transporting ATPases↗
PubMed2006

Metalloproteinases control brain inflammation induced by pertussis toxin in mice overexpressing the chemokine CCL2 in the central nervous system.

Inflammatory leukocytes infiltrate the CNS parenchyma in neuroinflammation. This involves cellular migration across various structures associated with the blood-brain barrier: the vascular endothelium, the glia limitans, and the perivascular space between them. Leukocytes accumulate spontaneously in the perivascular space in brains of transgenic (Tg) mice that overexpress CCL2 under control of a CNS-specific promoter. The Tg mice show no clinical symptoms, even though leukocytes have crossed the endothelial basement membrane. Pertussis toxin (PTx) given i.p. induced encephalopathy and weight loss in Tg mice. We used flow cytometry, ultra-small superparamagnetic iron oxide-enhanced magnetic resonance imaging, and immunofluorescent staining to show that encephalopathy involved leukocyte migration across the glia limitans into the brain parenchyma, identifying this as the critical step in inducing clinical symptoms. Metalloproteinase (MPs) enzymes are implicated in leukocyte infiltration in neuroinflammation. Unmanipulated Tg mice had elevated expression of tissue inhibitor of metalloproteinase-1, matrix metalloproteinase (MMP)-10, and -12 mRNA in the brain. PTx further induced expression of tissue inhibitor of metalloproteinase-1, metalloproteinase disintegrins-12, MMP-8, and -10 in brains of Tg mice. Levels of the microglial-associated MP MMP-15 were not affected in control or PTx-treated Tg mice. PTx also up-regulated expression of proinflammatory cytokines IL-1beta and TNF-alpha mRNA in Tg CNS. Weight loss and parenchymal infiltration, but not perivascular accumulation, were significantly inhibited by the broad-spectrum MP inhibitor BB-94/Batimastat. Our finding that MPs mediate PTx-induced parenchymal infiltration to the chemokine-overexpressing CNS has relevance for the pathogenesis of human diseases involving CNS inflammation, such as multiple sclerosis.

Animals↗
PubMed1999

Elevated constitutive IkappaB kinase activity and IkappaB-alpha phosphorylation in Hs294T melanoma cells lead to increased basal MGSA/GRO-alpha transcription.

The basal transcription of the CXC chemokine, melanocyte growth stimulatory activity (MGSA)/growth-regulated protein (GRO)-alpha, is up-regulated in Hs294T melanoma cells compared with the normal retinal pigment epithelial (RPE) cells. Previous studies characterized a cytokine-inducible, functional nuclear factor (NF)-kappaB consensus element in the immediate 5' regulatory region of the MGSA/GRO-alpha gene at -78 bp. Although the cytokine-inducible mechanisms for transcription of this gene are fairly well delineated, the mechanisms involved in its basal up-regulation of transcription in Hs294T melanoma cells are poorly understood. Recently, we demonstrated an increased rate of IkappaB-alpha degradation in Hs294T cells, which leads to an increased nuclear localization of NF-kappaB (R. L. Shattuck-Brandt and A. Richmond. Cancer Res., 57: 3032-3039, 1997). Here we demonstrate that Hs294T melanoma cells have elevated basal IkappaB kinase (IKK) activity relative to RPE cells, causing an increased constitutive IkappaB-alpha phosphorylation and degradation. We also show here that the resultant elevated nuclear NF-kappaB (p50/p65) in these cells is responsible for the increased basal transcription of MGSA/GRO-alpha. Pretreatment of Hs294T or RPE cells with proteasome inhibitors MG115 or MG132 captures the slower migrating, constitutively phosphorylated form of IkappaB-alpha in Hs294T melanoma cells, but not in RPE cells. In addition, a phospho-specific antibody that specifically recognizes the inhibitory form of IkappaB that is phosphorylated at Ser-32 reacted with IkappaB-alpha in Hs294T cell, but not in unstimulated RPE cells. Although the basal level of protein expression of IKK-alpha or IKK-beta are the same in both Hs294T and RPE cells, immunoprecipitation with IKK-alpha antibody combined with activity assay reveal a constitutively active IKK complex in Hs294T melanoma cells. Cotransfection of a 350-bp MGSA/GRO-alpha promoter-luciferase reporter construct with either the dominant negative IKK-alpha or the repressors of NF-kappaB, the IkappaB-alpha wild type or mutants lacking the inducible phosphorylation sites, demonstrates that the increased basal MGSA/GRO-alpha transcription in the Hs294T cells is due to the enhanced nuclear activation of NF-kappaB.

Chemokine CXCL1↗
PubMed1999

Reduced cadherin/catenin complex expression in celiac disease can be reproduced in vitro by cytokine stimulation.

Celiac disease is characterized by a chronic immune response to dietary gluten, in which T cell responses result in elevated mucosal levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, interferon (IFN)-gamma, and transforming growth factor (TGF)-beta, which induce profound mucosal remodeling associated with increased enterocyte proliferation, apoptosis, and migration. Reduced intestinal expression of the morphoregulatory cell adhesion molecule E-cadherin, which forms complexes with beta-catenin, can increase enterocyte proliferation and migration. However, its mechanism of action in gastrointestinal inflammatory conditions and any involvement in celiac disease is unknown. In this study, we describe changes in E-cadherin and beta-catenin expression in celiac disease tissue and determine the effect of cytokines on their expression in an in vitro model. We assessed E-cadherin and beta-catenin expression in intestinal biopsies from 24 patients with celiac disease, 12 patients with treated celiac disease, and 10 healthy patients by immunohistochemistry, Western blotting, and confocal microscopy. Using Caco-2 cells, we examined the effect of TNF-alpha, IL-1, IFN-gamma, and TGF-beta on E-cadherin expression. E-cadherin transcription was assessed in both intestinal biopsies and Caco-2 cells by in situ hybridization and RT-PCR, respectively. A marked reduction in protein expression of E-cadherin and beta-catenin that returns to normal levels after treatment was observed in celiac disease; this reduction was associated with reduced levels of E-cadherin mRNA. E-cadherin expression in Caco-2 cells was significantly reduced after TNF-alpha, IL-1, and IFN-gamma stimulation. The effect of TNF-alpha on E-cadherin expression was maximal after stimulation for 48 hours and also induced modest reductions in beta-catenin expression. The action of TNF-alpha on E-cadherin was reversible and was shown to act at the transcriptional level. These results demonstrate the novel findings that E-cadherin and beta-catenin expression are reversibly down-regulated in celiac disease and that such changes in epithelial cadherin/catenin complexes may be mediated by cytokines acting on cadherin transcription.

Base Sequence↗
PubMed1989

Detection of factor X activation in humans.

A sensitive radioimmunoassay (RIA) for the fragment that is liberated from factor X when this zymogen is activated by factor VII/VIIa-tissue factor or factor IXa was developed. Antisera were raised in rabbits to a synthetic 15 amino acid peptide containing the COOH-terminal sequence of the activation fragment coupled to bovine serum albumin with glutaraldehyde. The reactivity of the antibody population obtained toward the factor X zymogen was negligible (less than 1/36,000 that of the activation peptide on a molar basis). However, because other plasma constituents contributed to a nonspecific basal signal in the RIA, a procedure by which the peptide could be reproducibly extracted from plasma was developed. The mean level of this species in normal individuals younger than the age of 40 was 66.4 pmol/L, and elevations up to 550 pmol/L were observed in patients with evidence of disseminated intravascular coagulation. The validity of these measurements of factor X activation is supported by the fact that the RIA signal migrates on reverse-phase high pressure liquid chromatography in a manner identical to that of the native peptide and can be quantitatively recovered. The mean concentration of the activation fragment was markedly decreased to 25.7 pmol/L in patients with hereditary factor VII deficiency (P = .0001 v normal controls), whereas the mean level in subjects with factor VIII deficiency was 61.1 pmol/L (P greater than .1 v normal controls). These data indicate that the basal (ie, in the absence of thrombosis or provocative stimuli) levels of FXP under in vivo conditions result mainly from the activity of the extrinsic pathway.

Animals↗
PubMed2003

Plasminogen activator inhibitor type-1 deficiency does not influence the outcome of murine pneumococcal pneumonia.

Urokinase-type plasminogen activator (uPA) and its receptor uPAR are components of the fibrinolytic system and are important for an adequate immune response to respiratory tract infection, in part through their role in the migration of inflammatory cells. PA inhibitor-1 (PAI-1) is the predominant inhibitor of soluble and receptor-bound uPA. To determine the role of PAI-1 in host defense against pneumococcal pneumonia, the following studies were performed: (1) Patients with unilateral community-acquired pneumonia demonstrated elevated PAI-1 concentrations together with decreased PA activity in bronchoalveolar lavage fluid (BALF) obtained from the infected, but not from the contralateral, site. (2) Mice with Streptococcus pneumoniae pneumonia displayed elevated PAI-1 protein and mRNA levels in their lungs. (3) PAI-1 gene-deficient mice, however, had an unaltered immune response to pneumococcal pneumonia, as measured by cell recruitment into lungs, bacterial outgrowth, and survival. Furthermore, plasminogen-gene-deficient mice also had an unremarkable defense against pneumococcal pneumonia. These data indicate that pneumonia is associated with inhibition of the fibrinolytic system at the site of the infection secondary to increased production of PAI-1; an intact fibrinolytic response is not required for an adequate host response to respiratory tract infection, however, suggesting that the previously described role of uPA and uPAR are restricted to their function in cell migration.

Adult↗
PubMed1999

Comparison of HTLV-I basal transcription and expression of CREB/ATF-1/CREM family members in peripheral blood mononuclear cells and Jurkat T cells.

HTLV-I is the etiologic agent of adult T-cell leukemia/lymphoma and is associated with tropical spastic paraparesis/HTLV-I-associated myelopathy. Following integration into the host cell genome, HTLV-I replication is regulated by both host and viral mechanisms that control transcription. Low levels of viral transcription (basal transcription) occur before expression of the virally encoded Tax protein (Tax-mediated transcription). Members of the cyclic adenosine monophosphate (cAMP) response element binding (CREB)/activating transcription factor 1 (ATF-1) family of transcription factors bind three 21-bp repeats (Tax-responsive element-1, or TRE-1) within the viral promoter and are important for basal and Tax-mediated transcription. Using mitogen stimulated and quiescent peripheral blood mononuclear cells (PBMC) and Jurkat cells, we compared differences in basal transcription and amounts and binding of transcription factors with TRE-1. We demonstrate that amounts of transcriptionally active phosphorylated CREB protein (P-CREB) differ between activated PBMC and Jurkat cells. Following stimulation, P-CREB levels remain elevated in PBMC for up to 24 hours whereas CREB is dephosphorylated in Jurkat cells within 4 hours following stimulation. The differences in P-CREB levels between PBMC and Jurkat cells were directly correlated with basal transcription of HTLV-I in the two cell types. Using electrophoretic mobility shift assays, we determined that the pattern of band migration differed between the two cell types. These data demonstrate that PBMC differentially regulate basal HTLV-I transcription compared with Jurkat T cells, and this differential regulation is due, in part to differential phosphorylation and binding of CREB/ATF-1 to TRE-1 in the HTLV-I promoter. We demonstrate the utility of using primary lymphocyte models to study HTLV-I transcription in the context of cell signaling and suggest that activated PBMC maintain elevated levels of P-CREB, which promote basal HTLV-I transcription and enhance viral persistence in vivo.

Activating Transcription Factor 1↗
PubMed2003

Gene therapy delivery of endostatin enhances the treatment efficacy of radiation.

BACKGROUND AND PURPOSE: To evaluate whether sustained expression of mouse endostatin by adeno-associated virus (AAV)-mediated gene transfer can enhance the treatment efficacy of ionizing radiation. MATERIALS AND METHODS: Mouse endostatin was cloned into recombinant AAV (rAAV) under the control of CMV beta-actin promoter. Recombinant mouse endostatin expressed via AAV gene transfer was tested for biological activity in endothelial cells. The impact of elevated serum levels of endostatin on tumor-induced angiogenesis was evaluated using an in vivo angiogenesis assay. The anti-tumor efficacy of combining rAAV-mediated endostatin delivery with radiation was evaluated in a human colorectal tumor model (HT29). RESULTS: Recombinant mouse endostatin expressed through an AAV vector (rAAV-mEndo) inhibited endothelial cell proliferation (by 40-45%) and migration (by 22-33%). Intramuscular injection of rAAV-mEndo (1x10(9) i.u.) led to a sustained serum endostatin level of approximately 500 ng/ml. Compared to control animals this endostatin level was sufficient to inhibit tumor cell-induced vessel formation (37 vs. 28.5, P<0.05) and delay the growth of HT29 xenografts (time from 200 to 1,000 mm(3), 21 vs. 34.5 days, P<0.05). When combined with ionizing radiation, elevated serum endostatin levels significantly enhanced the time for tumors to grow from 200 to 1,000 mm(3) (radiation, 34 days; endostatin plus radiation, 50 days, P<0.05). CONCLUSION: The delivery of endostatin via rAAV vectors may provide an effective means of enhancing the anti-tumor efficacy of radiation therapy.

Animals↗