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ID PMID Title PublicationDate abstract
14971796 Effect of methotrexate and folinic acid on skeletal growth in mice. 2003 Dec AIM: To investigate whether chronic administration of medium doses of methotrexate (MTX) causes suppression of skeletal growth in young mice and to determine whether folinic acid supplementation could reverse this effect. METHODS: Four equal groups of Balb/c young male mice (6 animals in each group; mean body weight 11.9 +/- 0.25 g, in their rapid growth phase) were subjected to the following drug treatment for a period of 3 wk. Group 1 was given intraperitoneal MTX (3.5 mg kg(-1) body weight) every second day. Group 2 received folinic acid (7.0 mg kg(-1) body weight) intraperitoneally every second day. Group 3 was given both drugs (MTX every second day and folinic acid 8 h post-MTX injection). Group 4 was injected with physiological saline every other day to serve as a control group. Total body weight of the animals in each group was monitored every second day for the entire study period. The animals were sacrificed, the bilateral femurs and tibias of each animal were harvested and X-rays of the bones were taken. The length of each femur and tibia was measured using a micrometer. Measurements from the radiographs were also recorded using image analysis software. The MTX concentrations in the plasma and the folate levels in erythrocytes were determined. The heights of the distal femoral and the proximal tibial growth plate for each animal were measured on histological tissue sections. RESULTS: Mean lengths of both the tibia and femur of animals were compared in the four treatment groups. A significant decrease in the mean lengths (one-way ANOVA, p < 0.005) was observed in the group receiving MTX alone. Similarly, there was a significant decrease (p < 0.001) in the height of the femoral and tibial growth plate in this group when compared with the other groups. The main effect of MTX seemed to be on the hypertrophic proliferative zone of chondrocytes in the growth plate. Furthermore, animals in this MTX-treated group also showed increased levels of MTX in plasma and low levels of erythrocyte folate. CONCLUSION: These data show that chronic administration of MTX induces suppression of skeletal growth in mice, possibly through the inhibition of the pathway of de novo DNA synthesis. Folinic acid treatment following MTX administration appears to reverse this growth inhibition. Based on these observations, children suffering from juvenile rheumatoid arthritis, osteosarcoma or acute lymphoblastic leukaemia and receiving MTX over long periods of time could be at risk of short-term suppression of skeletal growth. If this is the case, it is possible that they could benefit from dietary supplementation with folinic acid.
12954239 Influence of pro-inflammatory (IL-1 alpha, IL-6, TNF-alpha, IFN-gamma) and anti-inflammato 2003 Sep OBJECTIVE: Cytokines produced by inflammatory cells play a pivotal role in synovial inflammation and joint destruction in rheumatoid arthritis. To investigate the influence of pro-inflammatory cytokines (IL-1 alpha, IL-6, TNF-alpha, IFN-gamma) and subsequently the possible beneficial role of an anti-inflammatory cytokine (IL-4) on chondrocyte viability (necrosis/apoptosis), proliferation and nitric oxide (NO) production. METHODS: Primary bovine chondrocytes were cultured until monolayers were obtained. Cells were incubated with cytokines (IL-1 alpha, IFN-gamma, TNF-alpha, IL-4, IL-6) at 0.1, 1, 10 and 100 ng/mL. After 48 h, the viability of the chondrocytes was measured flow cytometrically with propidium iodide. Proliferation was determined by the incorporation of tritiated thymidine. The morphology of the chondrocytes, including presence of apoptotic nuclei, was evaluated by a May-Grünwald-Giemsa staining. In addition, the number of apoptotic chondrocytes was detected flow cytometrically with a TUNEL technique and annexin-V/propidium iodide staining. NO production was evaluated using a spectrophotometric assay, based upon the Griess reaction. RESULTS: The viability and proliferation of bovine chondrocytes decreased after incubation with 100 ng/mL IL-1 alpha, TNF-alpha or IFN-gamma. In contrast, incubation of chondrocytes with IL-4 or IL-6 had no influence on the viability or the proliferation of cells. IL-1 alpha was able to enhance NO production in a dose dependent manner. IFN-gamma and TNF-alpha induced NO production only at the highest concentration (100 ng/mL), whereas IL-4 and IL-6 did not. There was a dose dependent increase in apoptosis of bovine chondrocytes cultured in the presence of IL-1 alpha and TNF-alpha. This effect could not be prevented by preincubation with IL-4. Preincubation with IL-4 diminished IL-1 alpha and TNF-alpha induced NO production and increased proliferation of chondrocytes. In an additional experiment, incubation of human chondrocytes with anti-Fas did not induce apoptosis as measured by annexin-V/propidium iodide staining. CONCLUSIONS: Pro-inflammatory cytokines are able to induce apoptosis, whereas IL-4 as an anti-inflammatory cytokine can inhibit the effect of IL-1 alpha and TNF-alpha on NO production and proliferation of bovine chondrocytes.
12882648 Oxysterol and 9-cis-retinoic acid stimulate the group IIA secretory phospholipase A2 gene 2003 Dec 1 The inflammation that occurs during rheumatoid arthritis or atherosclerosis is characterized by the release of large amounts of sPLA(2) (group IIA secretory phospholipase A(2)). We have shown previously that the sPLA(2) promoter in SMC (smooth-muscle cells) is activated by interleukin-1beta and cAMP-signalling pathways, through the interplay of multiple transcription factors [Antonio, Brouillet, Janvier, Monne, Bereziat, Andreani, and Raymondjean (2002) Biochem. J. 368, 415-424]. In the present study, we have investigated the regulation of sPLA(2) gene expression in rat aortic SMCs by oxysterols. We found that oxysterol ligands that bind to the LXR (liver X receptor), including 25-HC (25-hydroxycholesterol) and 22( R )-HC, cause the accumulation of sPLA(2) mRNA and an increased enzyme activity. Transient transfection experiments demonstrated that the sPLA(2) promoter is synergistically activated by 22( R )-HC in combination with 9- cis -retinoic acid, a ligand for the LXR heterodimeric partner RXR (retinoid X receptor). Promoter activity was also increased in a sterol-responsive fashion when cells were co-transfected with LXRalpha/RXRalpha or LXRbeta/RXRalpha. Mutagenesis studies and gel mobility-shift assays revealed that LXR/RXR heterodimers regulate sPLA(2) transcription directly, by interacting with a degenerated LXRE (LXR response element) at position [-421/-406] of the sPLA(2) promoter. Chromatin immunoprecipitation revealed the in vivo occupancy of LXR on the sPLA(2) promoter. In addition, the orphan nuclear receptor LRH-1 (liver receptor homologue-1) potentiated the sterol-dependent regulation of the sPLA(2) promoter by binding to an identified promoter element (TCAAGGCTG). Finally, we have demonstrated that oxysterols act independent of interleukin-1beta and cAMP pathways to activate the sPLA(2) promoter. In the present study, we have identified a new pathway activating sPLA(2) gene expression in SMCs.
23720862 (99m)Tc-Type II tumor necrosis receptor-Fc-interlukin-1 receptor antagonist fusion protein 2004 Tumor necrosis factor (TNF) is a proinflammatory cytokine produced by macrophages, and it plays an important role in inflammation and immune response (1). There are two types of TNF (TNF-α, TNF-β). TNF-α is mainly responsible for initiating inflammatory responses by induction of several proinflammatory cytokines, chemokines, matrix metalloproteinases, and vascular endothelial adhesion molecules that attract leukocytes known to promote inflammation. There are two types of TNF receptors: TNFR1 and TNFR2. Soluble forms of TNF receptors are released upon proteolytic cleavage of the membrane-bound TNF receptors. Soluble TNF receptors participate in limiting the availability of TNF to bind to its receptors (2, 3). Several anti-TNF monoclonal antibodies (TNF blockers) to further reduce circulating TNF have been approved by the United States Food and Drug Administration (FDA) for the treatment of a variety of inflammatory diseases (4, 5). The interleukin-1 family consists of two proinflammatory cytokines, IL-1α and IL-1β, which bind to two IL-1 receptors (IL-1R1 and IL-1R2), and an IL-1R antagonist (IL-1ra), which is mainly produced by activated macrophages and tissue macrophages (6). IL-1α and IL-1β are important mediators of the inflammatory response and hematopoiesis, and they are involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis. IL-1 is involved in chronic inflammatory diseases and in neuropathological conditions (7, 8). The balancing act of IL-1 and IL-1ra plays an important role in the regulation of inflammation and immune responses (9). IL-1ra has been shown to be effective as an anti-inflammatory treatment in several chronic inflammatory diseases and stroke (10, 11). A human recombinant, non-glycosylated form of the human IL-1ra (rhIL-1ra, Anakinra) has been approved by the FDA for the treatment of rheumatoid arthritis (12). IL-1β and TNF-α exhibit additive or synergistic effects in promoting pathophysiological processes observed in many inflammatory diseases (13). A dual domain TNFR2-Fc-IL-1ra fusion protein was constructed by joining TNFR2 and IL-1ra cDNA to the Fc fragment of human IgG(1) cDNA in an expression plasmid (14). The amino-terminal segment binds to TNF, and the carboxyl-terminal sequence binds to the IL-1R. Liu et al. (14) radiolabeled TNFR2-Fc-IL-1ra with (99m)Tc via 2-iminothiolane reduction to produce (99m)Tc-TNFR2-Fc-IL-1ra for use with single-photon emission computed tomography (SPECT) imaging of inflammation in mice.
23193618 (64)Cu-1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-Arg-rich Cys knot scaffold 2004 Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell–cell and cell–matrix interactions (1). Integrins consist of an α and a β subunit and are important for cell adhesion and signal transduction. α(v)β(3) integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (2-7). Expression of α(v)β(3) integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. α(v)β(3) antagonists are being studied as antitumor and antiangiogenic agents, and the agonists are being studied as angiogenic agents for coronary angiogenesis (6, 8, 9). A tripeptide sequence consisting of Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including α(v)β(3) and α(v)β(6). Various radiolabeled antagonists have been introduced for imaging of tumors and tumor angiogenesis (10). Integrin α(v)β(6) plays an important role in the development of epithelial cells and is nearly undetectable on adult normal tissues. However, the levels of α(v)β(6) integrin can be upregulated during tissue remodeling and wound healing (11). On the other hand, α(v)β(6) integrin is strongly expressed on tumor cells of the oral cavity, pancreas, breast, ovary, colon, and stomach (12-14). α(v)β(6) integrin affects tumor growth, tumor invasiveness, and metastasis (13). α(v)β(6) binds to the RGD motif in fibronectin, tenascin, and the viral protein 1 (VP1) of foot-and-mouth disease virus (FMDV) (15). FMDV binds to cells through the RGD motif of the GH loop of the VP1. A consensus α(v)β(6)-binding motif DLXXL was identified by using phage display screening with minimal binding to α(v)β(3), α(IIb)β(3), and α(v)β(5) (16). Engineered cysteine knot peptides (knots) comprise a rigid molecular scaffold of ~4 kDa with three disulfide bonds and a centrally located β sheet. Kimura et al. (17) grafted a α(v)β(6)-binding peptide (RSLARTDLDHLRGR) into the loop 1 of an Arg-rich knot to produce the knot known as R(0)2, which was radiolabeled with (64)Cu via 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugation to form (64)Cu-DOTA-R(0)2 as a positron emission tomography (PET) probe for in vivo imaging of α(v)β(6) integrin in tumor-bearing nude mice.
12102627 Inhibition of cathepsin K with lysosomotropic macromolecular inhibitors. 2002 Jul 16 Cathepsin K is the major enzyme responsible for the degradation of the protein matrix of bone and probably for the destruction of articular cartilage in rheumatoid arthritis joints. These processes occur mainly in the resorption lacuna and within the lysosomal compartment. Here, we have designed, synthesized, and evaluated new lysosomotropic (water-soluble) polymer-cathepsin K inhibitor conjugates. In particular, we characterized the relationship between conjugate structures and their activity to inhibit cathepsins K, B, L, and papain. A potent selective cathepsin K inhibitor, 1,5-bis(N-benzyloxycarbonylleucyl)carbohydrazide, was modified to 1-(N-benzyloxycarbonylleucyl)-5-(phenylalanylleucyl)carbohydrazide (I) to facilitate polymer conjugation. It was conjugated to the polymer chain termini of two water-soluble polymers [alpha-methoxy poly(ethylene glycol), abbreviated as mPEG-I; semitelechelic poly[N-(2-hydroxypropyl)methacrylamide], abbreviated as ST-PHPMA-I]. The conjugation of inhibitor I to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer side chains was accomplished via either a Gly-Gly spacer (PHPMA-GG-I) or with no spacer between I and the copolymer backbone (PHPMA-I). Kinetic analysis revealed that free inhibitor I possessed an apparent second-order rate constant against cathepsin K (k(obs)/[I] = 1.3 x 10(6) M(-1) s(-1)) similar to that of unmodified 1,5-bis(Cbz-Leu) carbohydrazide, while I conjugated to the chain termini of mPEG and ST-PHPMA-COOH had slightly lower values (about 5 x 10(5) M(-1) s(-1)). The k(obs)/[I] values for I attached to the side chains of HPMA copolymers (PHPMA-GG-I and PHPMA-I) were about 3 x 10(4) M(-1) s(-1). When tested against cathepsin L, inhibitor I and all its polymer conjugates produced k(obs)/[I] values 1-2 orders of magnitude less than those determined for cathepsin K, while for cathepsin B and papain, the values were 2-4 orders of magnitude lower. The ability of mPEG-I and ST-PHPMA-I to inhibit cathepsin K activity in synovial fibroblasts was also evaluated. Both polymer-bound inhibitors were internalized by endocytosis and were ultimately trafficked to the lysosomal compartment. ST-PHPMA-I was internalized faster than mPEG-I. The inhibitory activity in the synovial fibroblast assay correlated with the rate of internalization.
15680249 NSAIDs increase GM-CSF release by human synoviocytes: comparison with nitric oxide-donatin 2005 Jan 31 Non-steroidal anti-inflammatory drugs (NSAIDs) are used to treat the condition of rheumatoid arthritis, where levels of prostaglandin E2 (PGE2) and granulocyte macrophage-colony stimulating factor (GM-CSF) are elevated in the synovial fluid. NO-NSAIDs are a new class of cyclooxygenase (COX)-inhibitors developed by coupling a nitric oxide (NO)-donating moiety to conventional NSAIDs. We show that, in cytokine-treated synoviocytes (from non-rheumatic patients), NO-naproxen and NO-flurbiprofen like their parent compounds concentration-dependently reduce the levels of PGE2 (an index of COX-2 activity), with a corresponding rise in the release of GM-CSF. Unlike acetylsalicylic acid (ASA), NO-ASA reduces the levels of PGE2, without increasing GM-CSF release, although cell viability is reduced at the highest concentration (1 mM). The effects of NSAIDs and NO-NSAIDs on GM-CSF release were attributable to the PGE2 mediated cyclic (c) AMP pathway because PGE2 reversed the effects of COX blockade. Second, phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX) and Ro-201724 (both of which elevate cAMP levels) decreased GM-CSF release, in the presence of PGE2. Finally, neither sodium nitroprusside nor zaprinast (both of which elevate cGMP levels) affected GM-CSF or PGE2 release. Our findings demonstrate that GM-CSF is regulated by NSAIDs and NO-NSAIDs via inhibition of COX and appears to be mediated via the cAMP pathway. NO-ASA is the exception, because it does not increase GM-CSF release, although at millimolar concentrations cell viability is reduced.
15158604 Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and auto 2004 May 15 Commensal microflora (normal microflora, indigenous microbiota) consists of those micro-organisms, which are present on body surfaces covered by epithelial cells and are exposed to the external environment (gastrointestinal and respiratory tract, vagina, skin, etc.). The number of bacteria colonising mucosal and skin surfaces exceeds the number of cells forming human body. Commensal bacteria co-evolved with their hosts, however, under specific conditions they are able to overcome protective host responses and exert pathologic effects. Resident bacteria form complex ecosystems, whose diversity is enormous. The most abundant microflora is present in the distal parts of the gut; the majority of the intestinal bacteria are Gram-negative anaerobes. More than 50% of intestinal bacteria cannot be cultured by conventional microbiological techniques. Molecular biological methods help in analysing the structural and functional complexity of the microflora and in identifying its components. Resident microflora contains a number of components able to activate innate and adaptive immunity. Unlimited immune activation in response to signals from commensal bacteria could pose the risk of inflammation; immune responses to mucosal microbiota therefore require a precise regulatory control. The mucosal immune system has developed specialised regulatory, anti-inflammatory mechanisms for eliminating or tolerating non-dangerous, food and airborne antigens and commensal micro-organisms (oral, mucosal tolerance). However, at the same time the mucosal immune system must provide local defense mechanisms against environmental threats (e.g. invading pathogens). This important requirement is fulfilled by several mechanisms of mucosal immunity: strongly developed innate defense mechanisms ensuring appropriate function of the mucosal barrier, existence of unique types of lymphocytes and their products, transport of polymeric immunoglobulins through epithelial cells into secretions (sIgA) and migration and homing of cells originating from the mucosal organised tissues in mucosae and exocrine glands. The important role of commensal bacteria in development of optimally functioning mucosal immune system was demonstrated in germ-free animals (using gnotobiological techniques). Involvement of commensal microflora and its components with strong immunoactivating properties (e.g. LPS, peptidoglycans, superantigens, bacterial DNA, Hsp) in etiopathogenetic mechanism of various complex, multifactorial and multigenic diseases, including inflammatory bowel diseases, periodontal disease, rheumatoid arthritis, atherosclerosis, allergy, multiorgan failure, colon cancer has been recently suggested. Animal models of human diseases reared in defined gnotobiotic conditions are helping to elucidate the aetiology of these frequent disorders. An improved understanding of commensal bacteria-host interactions employing germ-free animal models with selective colonisation strategies combined with modern molecular techniques could bring new insights into the mechanisms of mucosal immunity and also into pathogenetic mechanisms of several infectious, inflammatory, autoimmune and neoplastic diseases. Regulation of microflora composition (e.g. by probiotics and prebiotics) offers the possibility to influence the development of mucosal and systemic immunity but it can play a role also in prevention and treatment of some diseases.
15059481 [Effect of melatonin on the expression of nuclear factor-kappa B and airway inflammation i 2004 Feb OBJECTIVE: Nuclear factor-kappa B (NF-kappaB) is a critical transcription factor governing the expression of many cytokines that are involved in the pathogenesis of inflammatory diseases, such as asthma and rheumatoid arthritis. Melatonin (MT), a relatively safe and potent antioxidant which has shown efficacy in several chronic inflammatory models, may inhibit the expression of NF-kappaB and therefore might have a therapeutic use in asthma. This study aimed at observing the effect of MT on the expression of NF-kappaB and airway inflammation in a rat model of bronchial asthma. METHODS: Twenty-four male Sprague-Dawley (SD) rats weighing 120 g to 170 g were randomly divided into three experimental groups (8 in each): (1) Asthmatic group: Rats were immunized on day 1 by intraperitoneal injection of 100 mg ovalbumin (OVA) in 1 ml of saline with 100 mg of alu minum hydroxide. From day 15 the animals were challenged with aerosolized OVA (1% in saline) for 20 minutes per day for 7 consecutive days. (2) MT group: OVA-sensitized rats were injected intraperitoneally with 10 mg/kg MT 30 minutes before each OVA challenge. (3) CONTROL GROUP: OVA for inhalation and MT for intraperitoneal injection was replaced with normal saline (NS). Airway responsiveness to aerosolized acetylcholine of 24 rats was detected six hours after the last challenge. Then the rats were lavaged and total and differentiated leukocytes counts in bronchoalveolar lavage fluid (BALF) were performed after staining with Wright-Giemsa staining. At the same time, levels of nitric oxide (NO) in BALF, inducible nitric oxide synthesis (iNOS) and constitute nitric oxide synthesis (cNOS) in the lung tissues were assessed with the use of nitrate reductase and chemical colorimetry, respectively. The expression of NF-kappaB in the lung tissues was observed by means of immunohistochemical staining. RESULTS: (1) After OVA challenge, there was a significant decrease in airway responsiveness, lymphocytes and eosinophils in BALF in MT group compared with asthmatic group (P < 0.01 respectively); (2) There was a significant decrease in amounts of NO(2)(-)/NO(3)(-) in the BALF and levels of iNOS in the lung tissues in MT group comparing with asthmatic group (P < 0.01 respectively); and the levels of iNOS in the lung tissues was correlated positively with NO(2)(-)/NO(3)(-) in the BALF (P < 0.01), but there were no significant differences in activity of cNOS in any of the groups analyzed. (3) There was a significant increase in expression of NF-kappaB in lung tissues in asthmatic group compared with the other groups (P < 0.01), and so was in MT group compared with control group (P < 0.05). CONCLUSIONS: MT could partially inhibit the expression of NF-kappaB and down-regulate the activity of iNOS in lung tissue, decrease the production of NO in BALF. These data suggest that the inhibitory effect of MT probably play a role in decreasing airway hyperresponsiveness and airway inflammation of asthmatic rats model.
14989423 The IL-1 family and inflammatory diseases. 2002 Sep IL-1 and its related family member IL-18 are primarily proinflammatory cytokines by their ability to stimulate the expression of genes associated with inflammation and autoimmune diseases. For IL-1 (IL-1alpha and IL-1beta), the most salient and relevant properties are the initiation of cyclooxygenase type 2 (COX-2), type 2 phospholipase A and inducible nitric oxide synthase (iNOS). This accounts for the large amount of prostaglandin-E2 (PGE2), platelet activating factor and nitric oxide (NO) produced by cells exposed to IL-1 or in animals or humans injected with IL-1. Another important member of the proinflammatory IL-1 family is IL-18. IL-18 is also an important player in autoimmune disease because of its ability to induce IFNgamma, particularly in combination with IL-12 or IL-15. Both IL-1 and IL-18 increase the expression of adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) on mesenchymal cells and vascular-cell adhesion molecule-1 (VCAM-1) on endothelial cells. This latter property promotes the infiltration of inflammatory and immunocompetent cells into the extravascular space. IL-1 and IL-18 are also an angiogenic factors by increasing the expression of vascular endothelial growth factor; IL-1 and IL-18 thus play a role in pannus formation and blood vessel supply. The strongest case for the importance of IL-1 in disease processes come from the administration of the IL-1 receptor antagonist, also a member of the IL-1 family and IL-18 binding protein (IL-18BP), a constitutively expressed and secreted protein that binds and neutralizes IL-18. Data from the human genome project have revealed other members of the IL-1 family. However, these appear to be antagonists rather than agonists. IL-1 also acts as an adjuvant during antibody production and stimulates bone marrow stem cells for differentiation in the myeloid series. IL-1 is distinct from tumor necrosis factor (TNF); IL-1 and TNFalpha share several biological properties but the salient difference is that TNF receptor signaling induces programmed cell death whereas IL-1 receptor signaling does not. In fact, IL-1 is a hematopoietic growth factor and IL-1 was administered to humans to reduce the nadir of white blood cells and platelets in patients during bone-marrow transplantation. This property, of IL-1 is not observed in the responses to TNFalpha. Furthermore, in animal models of destructive rheumatoid arthritis, IL-1 is necessary but TNFalpha is not.
12960348 IL-6 induces NF-kappa B activation in the intestinal epithelia. 2003 Sep 15 IL-6 is a potent proinflammatory cytokine that has been shown to play an important role in the pathogenesis of inflammatory bowel disease (IBD). It is classically known to activate gene expression via the STAT-3 pathway. Given the crucial role of IL-6 in the pathogenesis of chronic intestinal inflammation, it is not known whether IL-6 activates NF-kappaB, a central mediator of intestinal inflammation. The model intestinal epithelial cell line, Caco2-BBE, was used to study IL-6 signaling and to analyze whether suppressor of cytokine signaling 3 (SOCS-3) proteins play a role in the negative regulation of IL-6 signaling. We show that IL-6 receptors are present in intestinal epithelia in a polarized fashion. Basolateral IL-6 and, to a lesser extent, apical IL-6 induces the activation of the NF-kappaB pathway. Basolateral IL-6 stimulation results in a maximal induction of NF-kappaB activation and NF-kappaB nuclear translocation at 2 h. IL-6 induces polarized expression of ICAM-1, an adhesion molecule shown to be important in the neutrophil-epithelial interactions in IBD. Using various deletion constructs of ICAM-1 promoter, we show that ICAM-1 induction by IL-6 requires the activation of NF-kappaB. We also demonstrate that overexpression of SOCS-3, a protein known to inhibit STAT activation in response to IL-6, down-regulates IL-6-induced NF-kappaB activation and ICAM-1 expression. In summary, we demonstrate the activation of NF-kappaB by IL-6 in intestinal epithelia and the down-regulation of NF-kappaB induction by SOCS-3. These data may have mechanistic and therapeutic implications in diseases such as IBD and rheumatoid arthritis in which IL-6 plays an important role in the pathogenesis.
12820370 TNF alpha down-regulates CD105 expression in vascular endothelial cells: a comparative stu 2003 Mar The vascular endothelium participates in angiogenesis, inflammation and the immune response, which are modulated by vasoactive cytokines such as tumour necrosis factor-alpha (TNF alpha) and transforming growth factor-beta 1 (TGF beta 1). CD105 is a component of the TGF beta receptor complex and is abundantly expressed in activated/injured endothelium where it is implicated in multiple cellular processes. Up-regulation of CD105 in synovial cells of rheumatoid arthritis and psoriatic lesions implies a possible role in the pathogenesis of such inflammatory disorders. The pro-inflammatory cytokine, TNF alpha, and anti-inflammatory cytokine, TGF beta 1, regulate multiple cellular processes such as proliferation, differentiation and apoptosis. Our hypothesis is that CD105 gene expression in endothelial cells is regulated by the multifunctional cytokines TNF alpha and TGF beta 1. By using human dermal microvascular endothelial cells the present study has shown that long-term treatment with TNF alpha (0.1-5 ng/ml) elicited a concentration- and time-dependent significant suppression (over 50% reduction) in CD105 protein levels. The observations that no significant alterations in the CD105 mRNA levels or in the CD105 promoter activity were found and that the potent inhibitor of NF kappa B, PDTC, did not affect the TNF alpha action suggest that CD105 down-regulation by TNF alpha is not at the transcriptional level. In contrast to TNF alpha, TGF beta 1 significantly elevated CD105 protein and mRNA expression (approximately 2-fold increase) through activation of its promoter activity. From these data we conclude that TNF alpha and TGF beta 1 exert opposing effects on CD105 expression in human vascular endothelial cells and that CD105 is enmeshed in the network of signal pathways modulating multiple cellular functions.
12806611 Infliximab but not etanercept induces apoptosis in lamina propria T-lymphocytes from patie 2003 Jun BACKGROUND & AIMS: Steroid-refractory Crohn's disease responds to therapy with the chimeric anti-tumor necrosis factor (TNF)-alpha antibody infliximab. Etanercept, a recombinant TNF receptor/immunoglobulin G fusion protein, is highly effective in rheumatoid arthritis but not in Crohn's disease. Because both infliximab and etanercept are TNF-alpha-neutralizing drugs, we investigated the differences in TNF-alpha-neutralizing capacity and human lymphocyte binding and apoptosis-inducing capacity of both molecules. METHODS: We used a nuclear factor kappaB reporter assay and a cytotoxicity bioassay to study TNF-alpha neutralization by infliximab and etanercept. Lymphocyte binding and apoptosis-inducing capacity was investigated using fluorescence-activated cell sorter analysis, annexin V staining, and cleaved caspase-3 immunoblotting using mixed lymphocyte reaction-stimulated peripheral blood lymphocytes (PBL) from healthy volunteers and lamina propria T cells from patients with Crohn's disease. RESULTS: Both infliximab and etanercept neutralized TNF-alpha effectively. Infliximab bound to activated PBL and lamina propria T cells, whereas binding of etanercept was equal to a nonspecific control antibody. Infliximab but not etanercept induced peripheral and lamina propria lymphocyte apoptosis when compared with a control antibody. Infliximab activated caspase 3 in a time-dependent manner, whereas etanercept did not. CONCLUSIONS: Although both infliximab and etanercept showed powerful TNF-alpha neutralization, only infliximab was able to bind to PBL and lamina propria T cells and subsequently to induce apoptosis of activated lymphocytes. These data may provide a biological basis for the difference in efficacy of the 2 TNF-alpha-neutralizing drugs.
12598651 IL-1 is required for tumor invasiveness and angiogenesis. 2003 Mar 4 Here, we describe that microenvironmental IL-1 beta and, to a lesser extent, IL-1 alpha are required for in vivo angiogenesis and invasiveness of different tumor cells. In IL-1 beta knockout (KO) mice, local tumor or lung metastases of B16 melanoma cells were not observed compared with WT mice. Angiogenesis was assessed by the recruitment of blood vessel networks into Matrigel plugs containing B16 melanoma cells; vascularization of the plugs was present in WT mice, but was absent in IL-1 beta KO mice. The addition of exogenous IL-1 into B16-containing Matrigel plugs in IL-1 beta KO mice partially restored the angiogenic response. Moreover, the incorporation of IL-1 receptor antagonist to B16-containing plugs in WT mice inhibited the ingrowth of blood vessel networks into Matrigel plugs. In IL-1 alpha KO mice, local tumor development and induction of an angiogenic response in Matrigel plugs was less pronounced than in WT mice, but significantly higher than in IL-1 beta KO mice. These effects of host-derived IL-1 alpha and IL-1 beta were not restricted to the melanoma model, but were also observed in DA/3 mammary and prostate cancer cell models. In addition to the in vivo findings, IL-1 contributed to the production of vascular endothelial cell growth factor and tumor necrosis factor in cocultures of peritoneal macrophages and tumor cells. Host-derived IL-1 seems to control tumor angiogenesis and invasiveness. Furthermore, the anti-angiogenic effects of IL-1 receptor antagonist, shown here, suggest a possible therapeutic role in cancer, in addition to its current use in rheumatoid arthritis.
12560232 Genetic evidence for convergence of c-Kit- and alpha4 integrin-mediated signals on class I 2003 Jun 15 Mast cells play a critical role in host defense against a number of pathogens. Increased mast cell infiltration has been described in allergic asthma, in rheumatoid arthritis, and during helminthes infection. Despite the importance of mast cells in allergic disease and defense against infection, little is known about the mechanisms by which mast cells migrate to various tissues under steady state conditions or during infection or inflammation. Here, we show that activation of c-Kit by its ligand, stem cell factor (SCF), cooperates with alpha4 integrin in inducing directed migration of mast cells on fibronectin. A reduction in migration and activation of a small G protein, Rac, was observed in mast cells derived from class IA phosphoinositide-3 kinase (PI-3kinase)-deficient mice in response to SCF stimulation and in mast cells expressing the dominant-negative Rac (RacN17), as well as in mast cells deficient in the hematopoietic-specific small G protein, Rac2. In addition, a PI-3kinase inhibitor inhibited alpha4- as well as SCF-induced migration in a dose-dependent fashion. In contrast, a mitogen-activated protein kinase (MAPK) inhibitor had little effect. Consistent with the pharmacologic results, abrogating the binding of the p85alpha subunit of class IA PI-3kinase to c-Kit also resulted in inhibition of SCF-induced migration on fibronectin. These genetic and biochemical data demonstrate that both c-Kit and alpha4 integrin signaling are linked to class IA PI-3kinase and Rac pathways and regulate integrin-directed (haptotactic) migration in mast cells.
12490655 IL-7 induces bone loss in vivo by induction of receptor activator of nuclear factor kappa 2003 Jan 7 IL-7, a powerful lymphopoietic cytokine, is elevated in rheumatoid arthritis (RA) and known to induce bone loss when administered in vivo. IL-7 has been suggested to induce bone loss, in part, by stimulating the proliferation of B220(+) cells, a population capable of acting as early osteoclast (OC) precursors. However, the mechanism by which IL-7 leads to differentiation of precursors into mature OCs remains unknown. We previously reported that, in vitro, IL-7 up-regulated T cell cytokines including receptor activator of nuclear factor kappaB ligand (RANKL). To demonstrate the importance of T cells to the bone-wasting effect of IL-7 in vivo, we have now examined IL-7-induced bone loss in T cell-deficient nude mice. We show that T cell-replete mice undergo significant osteoclastic bone loss after IL-7 administration, concurrent with induction of RANKL and tumor necrosis factor alpha (TNF-alpha) secretion by splenic T cells. In contrast, nude mice were resistant to IL-7-induced bone loss and showed no detectable increase in either RANKL or TNF-alpha, despite an up-regulation of B220(+) cells. Importantly, T cell adoptive transfer into nude mice restored IL-7-induced bone loss, and RANKL and TNF-alpha secretion, demonstrating that T cells are essential mediators of IL-7-induced bone loss in vivo.
11918541 A pilot study of the recombinant soluble human tumour necrosis factor receptor (p75)-Fc fu 2002 Apr Laboratory observations suggest that, in some myelodysplastic syndromes (MDS), immune mechanisms may contribute to the impaired blood cell production. Tumor necrosis factor alpha (TNF-alpha), a potent inhibitor of haematopoiesis, has been hypothesized to mediate suppressive effects in MDS: TNF-alpha levels are elevated and correlated with marrow apoptosis and cytopenia. Inhibition of TNF-alpha production using the soluble TNF receptor (Enbrel) has been successful in rheumatoid arthritis, and we have now applied the same principle to MDS. We determined spontaneous TNF-alpha production by marrow cells in MDS; TNF-alpha production was elevated (> mean + 2 x SD of controls) in > 1/3 of patients, but did not correlate with clinical parameters. Sixteen patients participated in a 3-month pilot study of Enbrel. The drug was well tolerated and 15 patients were evaluable. Of these, one became temporarily (14 weeks) transfusion independent. In another patient, absolute neutrophil count (ANC) rose from 0.5 x 10(9)/l to 0.84 x 10(9)/l. Serious infections were seen in two out of six neutropenic patients. Progression to refractory anaemia with excess blasts in transformation (RAEBt) or leukaemia was observed in three patients. When the effects of Enbrel on haematopoietic colony formation were studied, no significant increase was seen in MDS and there was no correlation with TNF-alpha levels. Although anti-TNF therapy with Enbrel was well tolerated at the dosages used in MDS, its efficacy as a single agent appears low.
23720860 (99m)Tc-Interleukin-18-binding protein-Fc-interlukin-1 receptor antagonist. 2004 Interleukin-18 (IL-18) is a proinflammatory cytokine produced by macrophages, epithelial cells, and activated T cells (1, 2), and it plays an important role in inflammation and immune response (3, 4). IL-18 induces production of tumor necrosis factor and IL-1 in mononuclear cells. A variety of normal and malignant cells can produce and respond to IL-18 through its receptor (IL-18R). A soluble secreted IL-18 binding protein (IL-18bp) was found to bind to IL-18 with high affinity (dissociation constant (K(d)) = 0.4 nM) and to neutralize the biological effects of IL-18 by blocking its interaction with IL-18R (5, 6). IL-18bp-Fc is a 40-kDa glycoprotein with an immunoglobulin (Ig) domain. The interleukin-1 family of two proinflammatory cytokines, IL-1α and IL-1β, which bind to two IL-1 receptors (IL-1R1 and IL-1R2), and an IL-1R antagonist (IL-1ra), is mainly produced by activated macrophages and tissue macrophages (7). IL-1α and IL-1β are important mediators of the inflammatory response and hematopoiesis, and they are involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis. IL-1 is involved in chronic inflammatory diseases and in neuropathological conditions (8, 9). The balancing action of IL-1 and IL-1ra plays an important role in the regulation of inflammation and immune responses (10). IL-1ra has been shown to be effective as an anti-inflammatory treatment in several chronic inflammatory diseases and stroke (11, 12). A human recombinant, non-glycosylated form of the human IL-1ra (rhIL-1ra, Anakinra) has been approved by the United States Food and Drug Administration for the treatment of rheumatoid arthritis (13). IL-1 and IL-18 exhibit additive or synergistic effects in promoting pathophysiological processes observed in many inflammatory diseases (14). A dual domain IL-18bp-Fc-IL-1ra fusion protein was constructed by joining IL-18bp and IL-1ra cDNA to the Fc fragment of human IgG(1) cDNA in an expression plasmid (15). The amino-terminal segment binds to IL-18, and the carboxyl-terminal sequence binds to the IL-1R. Liu et al. (15) radiolabeled IL-18bp-Fc-IL-1ra with (99m)Tc via 2-iminothiolane reduction to produce (99m)Tc-IL-18bp-Fc-IL-1ra for use with single-photon emission computed tomography (SPECT) imaging of inflammation in mice.
23193616 (111)In-Diethylenetriamine pentaacetic acid-NAVPNLRGDLQVLAQKVART. 2004 Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell–cell and cell–matrix interactions (1). Integrins consist of an α and a β subunit and are important for cell adhesion and signal transduction. α(v)β(3) integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (2-7). Expression of α(v)β(3) integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. α(v)β(3) antagonists are being studied as antitumor and antiangiogenic agents, and the agonists are being studied as angiogenic agents for coronary angiogenesis (6, 8, 9). A tripeptide sequence consisting of Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including α(v)β(3) and α(v)β(6). Various radiolabeled antagonists have been introduced for imaging of tumors and tumor angiogenesis (10). The α(v)β(6) integrin plays an important role in the development of epithelial cells and is nearly undetectable on normal adult tissues. However, the levels of α(v)β(6) integrin can be upregulated during tissue remodeling and wound healing (11). On the other hand, α(v)β(6) integrin is strongly expressed on tumor cells of the oral cavity, pancreas, breast, ovary, colon, and stomach (12-14). α(v)β(6) integrin affects tumor growth, tumor invasiveness, and metastasis (13). α(v)β(6) binds to the RGD motif in fibronectin, tenascin, and the viral protein 1 (VP1) of the foot-and-mouth disease virus (FMDV) (15). FMDV binds to cells through the RGD motif of the GH loop of the VP1. The consensus α(v)β(6)-binding motif DLXXL was identified with the use of phage display screening, with minimal binding to α(v)β(3), α(IIb)β(3), and α(v)β(5) (16). A 20-amino acid peptide, NAVPNLRGDLQVLAQKVART (A20FMDV2), was identified as a ligand binding to α(v)β(6) integrin (15). A20FMDV2 was radiolabeled with N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]SFB) to study in vivo biodistribution of the tracer in tumor-bearing mice (17). [(18)F]FB-A20FMDV2 was found to have specific accumulation in α(v)β(6)-positive tumors. For single-photon emission computed tomography (SPECT), Saha et al. (18) prepared (111)In-diethylenetriamine pentaacetic acid-A20FMDV2 ((111)In-DTPA-A20FMDV2) for in vivo imaging of α(v)β(6)-positive tumors.
23166958 (99m)Tc-Hydrazinonicotinyl(tricine)(TPPTS)-Glu-c(RGDyK)-bombesin[7-14]NH(2). 2004 The amphibian bombesin (BBN or BN, a peptide of 14 amino acids) is an analog of human gastrin-releasing peptide (GRP, a peptide of 27 amino acids) that binds to GRP receptors (GRPR) with high affinity and specificity (1, 2). Both GRP and BBN share an amidated C-terminus sequence homology of seven amino acids, Trp-Ala-Val-Gly-His-Leu-Met-NH(2). BBN-Like peptides have been shown to induce various biological responses in diverse tissues, including the central nervous system (CNS) and the gastrointestinal (GI) system. They also act as potential growth factors for both normal and neoplastic tissues (3). Specific BBN receptors (BBN-Rs) have been identified on CNS and GI tissues and on a number of tumor cell lines (4). The BBN-R superfamily includes at least four different subtypes, namely the GRPR subtype (BB2), the neuromedin B receptor subtype (BB1), the BB3 subtype, and the BB4 subtype. The findings of GRPR overexpression in various human tumors, such as breast, prostate, lung, colon, ovarian, and pancreatic cancers, provide opportunities for tumor imaging by designing specific molecular imaging agents to target the GRPR (5, 6). Integrins are a family of heterodimeric glycoproteins on cell surfaces that mediate diverse biological events involving cell–cell and cell–matrix interactions (7). Integrins consist of an α and a β subunit and are important for cell adhesion and signal transduction. The α(v)β(3) integrin is the most prominent receptor affecting tumor growth, tumor invasiveness, metastasis, tumor-induced angiogenesis, inflammation, osteoporosis, and rheumatoid arthritis (8-13). Expression of the α(v)β(3) integrin is strong on tumor cells and activated endothelial cells, whereas expression is weak on resting endothelial cells and most normal tissues. A peptide sequence consisting of Arg-Gly-Asp (RGD) has been identified as a recognition motif used by extracellular matrix proteins (vitronectin, fibrinogen, laminin, and collagen) to bind to a variety of integrins, including α(v)β(3). Various ligands have been introduced for imaging of tumors and tumor angiogenesis (14). Because breast and prostate cancers express both GRPR and α(v)β(3), Liu et al. (15) designed an RGD-BBN heterodimer in which BBN[7-14]NH(2) and c(RGDyK) are connected with a glutamate linker (BBN on the Glu side chain γ-carboxylate group and RGD on the Glu side chain α-carboxylate group). 1,4,7-triazacyclononane-1,4-7-triacetic acid (NOTA) was used as a bifunctional chelator for labeling RGD-BBN to form (64)Cu-NOTA-RGD-BBN for use in positron emission tomography (PET) imaging of α(v)β(3) and GRPR in nude mice bearing human tumors. For single-photon emission computed tomography (SPECT), Liu et al. (16) conjugated Glu-RGD-BBN with 6-hydrazinonicotinyl (HYNIC) and labeled the product with (99m)Tc, using tricine and trisodium triphenylphosphine-trisulfonate (TPPTS) as the coligands. (99m)Tc-HYNIC-RGD-BBN was evaluated in C57/BL6 mice bearing mouse Lewis lung carcinomas (LLC).