A placebo-controlled randomised trial to assess the effect of TGF-ß1-expressing chondrocytes in patients with arthritis of the knee.

The aim of this study was to assess the effect of injecting genetically engineered chondrocytes expressing transforming growth factor beta 1 (TGF-ß1) into the knees of patients with osteoarthritis. We assessed the resultant function, pain and quality of life. A total of 54 patients (20 men, 34 women) who had a mean age of 58 years (50 to 66) were blinded and randomised (1:1) to receive a single injection of the active treatment or a placebo. We assessed post-treatment function, pain severity, physical function, quality of life and the incidence of treatment-associated adverse events. Patients were followed at four, 12 and 24 weeks after injection. At final follow-up the treatment group had a significantly greater improvement in the mean International Knee Documentation Committee score than the placebo group (16 points; -18 to 49, vs 8 points; -4 to 37, respectively; p = 0.03). The treatment group also had a significantly improved mean visual analogue score at final follow-up (-25; -85 to 34, vs -11 points; -51 to 25, respectively; p = 0.032). Both cohorts showed an improvement in Western Ontario and McMaster Osteoarthritis Index and Knee Injury and Osteoarthritis Outcome Scores, but these differences were not statistically significant. One patient had an anaphylactic reaction to the preservation medium, but recovered within 24 hours. All other adverse events were localised and resolved without further action. This technique may result in improved clinical outcomes, with the aim of slowing the degenerative process, leading to improvements in pain and function. However, imaging and direct observational studies are needed to verify cartilage regeneration. Nevertheless, this study provided a sufficient basis to proceed to further clinical testing.

Immune response against 2,4-dinitrofluorobenzene-induced atopic dermatitis-like clinical manifestation is suppressed by spermidine in NC/Nga mice.

Of the biogenic polyamines, spermidine is a natural constituent of living cells and organisms. Spermidine is associated with regulation of cell growth, proliferation and differentiation, and with the suppression of oxidation and inflammation. Atopic dermatitis (AD) is a chronic inflammatory skin disease that has a complex and multiple pathogenesis, which includes genetic abnormality, modified or abnormal immune response and the production of nitric oxide and reactive oxygen species. We investigated whether spermidine can relieve AD-like clinical manifestation induced by the continual application of 2,4-dinitrofluorobenzene (DNFB) in NC/Nga mice. Spermidine at concentrations of 1 or 10 mg/kg reduced increasing ear swelling and attenuated oedema, haemorrhage and hyperkeratosis in AD-like skin lesions. Repetitive application of DNFB induced inflammatory cell infiltration to skin lesions, whereas intraperitoneal injection of spermidine inhibited DNFB-evoked infiltration of eosinophils, mast cells and T lymphocytes. Furthermore, spermidine suppressed mast cell degranulation and production of interferon-gamma by activated CD4(+) T cells in AD-like skin lesions. Spermidine may be a potential therapeutic agent for treatment of AD.

Bone regeneration at dental implant sites with suspended stem cells.

During the maintenance of bone marrow-derived mesenchymal stem cells (BMMSCs), suspended cells are discarded normally. We noted the osteogenic potential of these cells to be like that of anchorage-dependent BMMSCs. Therefore, we characterized suspended BMMSCs from rabbit bone marrow by bioengineering and applied the suspended BMMSCs to double-canaled dental implants inserted into rabbits. After primary isolation of BMMSCs, we collected the suspended cells during primary culture on the third day. The cells were transferred and maintained on an extracellular-matrix-coated culture plate. The cells were characterized and compared with BMMSCs by colony-forming-unit fibroblast (CFU-f) and cell proliferation assay, fluorescence-activated cell sorter (FACS), in vitro multipotency, and reverse transcription polymerase chain reaction (RT-PCR). We also analyzed the osteogenic potential of cells mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) and transplanted into immunocompromised mice. We compared the viability and proliferation of the suspended BMMSCs and BMMSCs on the titanium implant surface and observed cell morphology. Then, the cells mixed with HA/TCP were applied to the double-canaled implants during installation into rabbit tibia. Four weeks later, we analyzed bone formation inside the canal by histomorphometry. The suspended cells showed higher CFU-f on the extracellular matrix (ECM)-coated culture plate and similar results of proliferation capacity compared with BMMSCs. The cells also showed osteogenic, adipogenic, and chondrogenic ability. The suspended cells showed levels of attachment survival and proliferation on the surfaces of titanium implant discs to be higher than or similar to those of BMMSCs. The suspended cells as well as BMMSCs showed stronger bone formation ability in both upper and lower canals of the implants compared with controls on double-canaled implants inserted into rabbit tibia. In this study, we showed that suspended cells after primary BMMSC isolation have bone regeneration capacity like that of BMMSCs, not only in vitro but also in vivo. ECM was valuable for propagation of MSCs for cell-based bone regeneration. Therefore, the suspended cells could also be useful tools for bone regeneration after implant surgery.

Comparability and utility of body composition measurement vs. anthropometric measurement for assessing obesity related health risks in Korean men.

BACKGROUND: Obesity is commonly assessed by body mass index (BMI) of which limitations come from an inability to distinguish body fat mass from lean mass. Several anthropometric measurements, including BMI, waist circumference, waist-to-height ratio and waist-to-hip ratio have been used to predict metabolic syndrome. The purpose of this study was to evaluate the utility of FMI or BF% combined with previous known anthropometric indices to assess the risk of metabolic syndrome in clinical practice. METHODS: In 5534 men visiting a hospital for health check-ups, blood tests, anthropometric measurements and body composition analysis using BIA were performed. Logistic regression analysis was performed to compare the odds ratios for metabolic syndrome and each component of metabolic syndrome among BMI, waist-to-height ratio, waist-to-hip ratio, FMI and BF%. The area under the curve (AUC) of the receiver operating characteristic curve (ROC) for metabolic syndrome was compared between several measurements. The net reclassification improvement with integrated discrimination improvement was used for assessing value of body composition measurement. RESULTS: The adjusted odds ratios of metabolic syndrome was 1.80 (95% CI, 1.71-1.89) for FMI and 1.15 (95% CI, 1.13-1.17) for BF%. Odds ratio of each metabolic component was highest for FMI among several anthropometric and body composition measurements. AUCs using the ROC curve for metabolic syndrome was highest for waist-to-height ratio, 0.823 (95% CI, 0.808-0.837) by National Cholesterol Education Program criteria. FMI caused a mild increase in integrated discrimination improvement when combined with waist-to-height ratio. CONCLUSIONS: Waist-to-height ratio seems to be the best screening tool for evaluating metabolic syndrome in Korean men, and adding FMI could result in a modest increase in integrated discrimination improvement.

Effect of immature dendritic cell injection before heterotropic cardiac allograft.

Although dendritic cells (DCs) are unrivaled for initiation of immune responses, the immunomodulatory capacity of chemically fixed DC has not been thoroughly evaluated. We monitored the tolerogenic capacity of chemically fixed DCs using allogeneic heart transplantations. Bone marrow progenitors were differentiated into immature DCs which were then chemically fixed and injected intravenously into recipient mice at 14 days before allogeneic heart transplantation. Chemically fixed DCs markedly prolonged graft survival in the major histocompatibility complex (MHC) I/II mismatch cardiac transplantation (B6 --> B10.A; median survival time [MST] 12.5 days vs >70 days). T cells that encountered chemically fixed DCs showed attenuated apoptotic cell death and inactivated phenotypes after allogeneic heterotropic heart transplantation. Furthermore, when DCs from interleukin (IL)-10-/- mice were treated, the in vitro T-cell response was greater than that from IL-12-/- mice. We have suggested that the chemically fixed DCs may mediate peripheral T-cell tolerance, with therapeutic potential for allogeneic transplantation.

Effects of Paeonia lactiflora root extracts on the secretions of monocyte chemotactic protein-1 and -3 in human nasal fibroblasts.

In the field of Oriental medicine, the root of Paeonia lactiflora (Paeoniae Radix) has been prescribed usually to treat the common cold, and it was thought to alleviate upper respiratory infection or nasal inflammation. Monocyte chemotactic proteins (MCP)-1 and MCP-3 are known as the most potent chemokines to mediate allergic inflammation. The object of the study was to investigate the effect of Paeoniae Radix on the release of the chemokines such as MCP-1 and MCP-3. To detect the secretion of MCPs, an enzyme-linked immunosorbent assay was performed for human nasal mucosal fibroblasts after stimulation with several cytokines such as tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and interleukin-1beta (IL-1beta). The secretion of MCP-1 was increased after stimulation with TNF-alpha or IL-1beta treatment. Co-treatment of TNF-alpha and IFN-gamma, or IL-1beta and IFN-gamma increased the secretion of MCP-1 and MCP-3. Paeoniae Radix was tested with inflammatory cytokines, and the results showed that the Paeoniae Radix significantly decreased the secretion of MCP-1 and MCP-3. In our study, Paeoniae Radix may play an important role in nasal inflammation with a modulation of MCP-1 and MCP-3.

Roles of protein kinase A and C in the opioid potentiation of the GABAA response in rat periaqueductal gray neuron.

The periaqueductal gray (PAG) is the main target site of the opioid-induced analgesia. The present study was designed to examine the roles of protein kinase A (PKA) and C (PKC) in the opioid-induced modulation of the currents activated by an inhibitory neurotransmitter, gamma-aminobutyric acid (GABA). The PAG neurons were acutely isolated and voltage-clamped under the nystatin-perforated patch-clamp mode. The GABA-activated current was sensitively blocked by a GABA(A) receptor antagonist, bicuculline, and selectively carried by chloride ions. The GABA(A) receptor-activated Cl(-) current was potentiated by a mu-opioid receptor agonist, [D-Ala(2),N-MePhe(4),Gly(5)-ol]-enkephalin acetate (DAMGO). The GABA response was also potentiated by phorbol-12-myristate-13-acetate (PMA). Pretreatment with PMA occluded the DAMGO potentiation. However, both chelerythrine and 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X) also potentiated the GABA response. Pretreatment with chelerythrine or GF109203X also occluded the DAMGO potentiation. Meanwhile, the GABA response was potentiated by N-(2-[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide (H-89), while not altered by forskolin. Pretreatment with H-89 occluded the potentiation effect of DAMGO on the GABA response. In addition, the DAMGO effect was completely blocked by pretreatment with forskolin. From the result, it can be suggested that activation of mu-opioid receptor potentiates the GABA(A) response through the mediation of PKA inhibition, and that PKC is not directly involved in the action mechanism of DAMGO.

Antifibrotic effect of silymarin in rat secondary biliary fibrosis is mediated by downregulation of procollagen alpha1(I) and TIMP-1.

BACKGROUND/AIMS: Silymarin reduces hepatic collagen accumulation by 35% in rats with secondary biliary cirrhosis. The aim of the present study was to explore its antifibrotic mechanism. METHODS: Thirty female adult Wistar rats were allocated to (1) bile duct occlusion, (2) bile duct occlusion and oral silymarin at 50 mg/kg per day, and (3) sham operation and oral silymarin at 50 mg/kg per day. Steady-state mRNA levels for procollagen alpha1(I), tissue inhibitor of metalloproteinases-1 (TIMP-1), and transforming growth factor (TGF) beta1 were determined by multi-probe ribonuclease protection assay. RESULTS: After 6 weeks of bile duct occlusion, liver collagen content was increased 12-fold, when compared with the sham-operated controls. These animals displayed 17-, 6.5- and 16-fold higher transcript levels for procollagen alpha1(I), TIMP-1 and TGFbeta1 (P < 0.01). Silymarin downregulated elevated procollagen alpha1(I), TIMP-1 and TGFbeta1 mRNA levels by 40-60% (P < 0.01). These lowered hepatic profibrogenic transcript levels correlated with decreased serum levels of the aminoterminal propeptide of procollagen type III. CONCLUSIONS: Silymarin suppresses expression of profibrogenic procollagen alpha1(I) and TIMP-1 most likely via downregulation of TGFbeta1 mRNA in rats with biliary fibrosis. The serum procollagen type III propeptide level mirrors profibrogenic mRNA expression in the liver.

CDNA for an immune response gene encoding low molecular weight polypeptide from flounder, Paralichthys olivaceus.

The cDNA for an immune response gene encoding the low molecular weight polypeptide (LMP7) was cloned and sequenced from a flounder (Paralichthys olivaceus) leukocyte cDNA library. The cDNA clone was 1,160 bp, and composed of an open reading frame of 822 bp that corresponded to a protein of 273 amino acid residues with a calculated mass of 30.5 kDa. The ScanProsite search indicated that the deduced amino acid sequence from the flounder LMP7 contains a proteasome beta-type subunit signature, which is well conserved during evolution. The sequence shares a high degree of identity with other LMP7 sequences varying from a 66% identity with zebra fish (Danio renio) to a 57% identity with the African clawed frog (Xenopus laevis), which was confirmed from a phylogenetic tree. A reverse transcription-polymerase chain reaction (RT-PCR) was used to determine tissue specificity, and the expression of LMP7 was detected from the liver, kidney, leukocyte, and spleen of the flounder.

Anti-elastase and anti-hyaluronidase of phenolic substance from Areca catechu as a new anti-ageing agent.

We have previously screened 150 medicinal plants for the inhibition of elastase and found significant inhibitory effects of the extracts of Areca catechu L. on the ageing and inflammation of skin tissues. To isolate and identify the compounds having biological activity, they were further purified by each fraction of solvents, silica gel column chromatography, preparative TLC and reversed-phase HPLC. The peak in HPLC, which coincided with the inhibitory activity against elastase, was identified as a phenolic substance by using various colorimetric methods, UV and IR. IC(50) values of this phenolic substance were 26.9 mug mL(-1) for porcine pancreatic elastase (PPE) and 60.8 mug mL(-1) for human neutrophil elastase (HNE). This phenolic substance showed more potent activity than that of reference compounds, oleanolic acid (76.5 mug mL(-1) for PPE, 219.2 mug mL(-1) for HNE) and ursolic acid (31.0 mug mL(-1) for PPE, 118.6 mug mL(-1) for HNE). According to the Lineweaver-Burk plots, the inhibition against both PPE and HNE by this phenolic substance was competitive inhibition with the substrate. The phenolic substance from A. catechu effectively inhibited hyaluronidase activity (IC(50) : 210 mug mL(-1) ). These results suggest that the phenolic substance purified from A. catechu has an anti-ageing effect by protecting connective tissue proteins.

Activation of the hepatic endothelin-system in rats with biliary liver fibrosis.

Circulating plasma endothelin-1 (ET-1) is elevated in liver cirrhosis, in a disease-stage-dependent manner. However, ET-1 exerts its effects mainly via paracrine and autocrine pathways. Therefore, the aim of the present study was to analyze the hepatic endothelin (ET) system in liver cirrhosis resulting from bile duct obstruction (BDO). Wistar rats were subjected for 6 weeks to either sham operation (control) or BDO. Thereafter, hepatic ET-1 concentrations were elevated 7.2-fold in BDO compared to control (p <0.001), whereas big ET-1 was unchanged. The density of both ET receptor subtypes was upregulated in BDO (ETA: 7.4-fold and ETB: 4.9-fold vs control, p < 0.001, respectively). The affinity of both receptor subtypes was significantly reduced in BDO. In conclusion, our data demonstrated for the first time that the hepatic ET system in liver cirrhosis is characterized by a simultaneous upregulation of both ET-1 tissue concentration as well as the density of hepatic ETA- and ETB-receptors, suggesting a synergistic activation of the hepatic ET system in rats with BDO. The increased ET-1 tissue concentration is not a result of an altered big ET-1 synthesis in biliary liver fibrosis, suggesting an increased activity of endothelin-converting enzyme (ECE) in liver cirrhosis.

Regulation of the hepatic endothelin system in advanced biliary fibrosis in rats.

The aim of the present study was to analyze the hepatic endothelin system and its regulation in liver cirrhosis due to bile duct obstruction. Wistar rats were subjected for 6 weeks to: 1) sham operation; 2) bile duct obstruction; 3) bile duct obstruction and the selective oral endothelin A receptor antagonist LU 135252; 4) bile duct obstruction and oral silymarin, a hepatoprotective and antifibrotic compound. We determined tissue concentrations of endothelin-1 and big-endothelin-1 by ELISA and the density of both endothelin receptor subtypes in plasma membrane fractions by Scatchard analysis. The hepatic endothelin system in liver cirrhosis due to chronic bile duct obstruction is characterized by a simultaneous up-regulation of both endothelin-1 tissue concentration (7.2 fold compared to sham operation; p<0.001) as well as the density of both endothelin receptor subtypes (ET(A) 7.4-fold, ET(B) 4.9-fold, p<0.001, respectively) suggesting a synergistic activation of the hepatic endothelin system in this rat model of non-inflammatory cirrhosis. Treatment with proven antifibrotic agents such as silymarin or a selective endothelin-A-receptor blocker (LU 135252) did not reduce the activity of the hepatic endothelin system, suggesting that the hepatic endothelin system is not activated by the fibrotic process itself.

An oral endothelin-A receptor antagonist blocks collagen synthesis and deposition in advanced rat liver fibrosis.

BACKGROUND & AIMS: Endothelin 1 induces contraction, proliferation, and collagen synthesis of hepatic stellate cells in vitro, which may be mediated via the endothelin A receptor. It is unknown if specific blockade of the endothelin A receptor inhibits hepatic fibrosis in vivo. METHODS: Groups of 10-20 rats with bile duct occlusion were treated with the nonpeptide endothelin-A receptor antagonist LU 135252 at 80 mg. kg(-1). day(-1) from week 1-6 or from week 4-6, or with LU at 10 mg. kg(-1). day(-1) from week 1-6. Animals with bile duct occlusion alone and sham-operated rats without or with LU at 80 mg. kg(-1). day(-1) over 6 weeks served as controls. After 6 weeks, parameters of fibrogenesis were determined. RESULTS: LU treatment led to improved histology, paralleled by a dose-dependence up to 60% reduction of liver collagen, even when administered at an advanced fibrosis stage. This was accompanied by a decreased messenger RNA of hepatic procollagen alpha1(I) and tissue inhibitor of metalloproteinase 1, 2 major effectors of fibrosis, and of serum procollagen type III, a surrogate marker of liver fibrogenesis. CONCLUSIONS: Selective endothelin-A receptor blockade can dramatically reduce collagen accumulation in rat secondary biliary fibrosis, a model refractory to most potential antifibrotic agents. Endothelin-A receptor antagonists are promising antifibrotic agents in chronic liver disease.

Inhibitory Effects of 150 Plant Extracts on Elastase Activity, and Their Anti-inflammatory Effects.

The inhibitory effects of 150 medicinal plants on elastase activity were investigated. Among the 150 plants, six plant extracts (final concentration 1 mg/ml in methanol) exhibited more than 65% of inhibition of elastase activity. The inhibitory effects of six active plants, including Areca catechu (IC50, 42.4 mug/ml), Cinnamonum cassia (IC50, 208.7 mug/ml), Myristica fragrans (IC50, 284.1 mug/ml), Curcuma longa (IC50, 398.4 mug/ml), Alpinia katsumadai (IC50, 465.7 mug/ml) and Dryopteris cassirrhizoma (IC50, 714.4 mug/ml) on the activity of human leukocyte elastase, hyaluronidase and lipid peroxidation were examined. In the lipid peroxidation assay, using the TBA method, three of the six plants, including Curcuma longa (IC50, 45.5 mug/ml), Areca catechu (IC50, 51.0 mug/ml) and Alpinia katsumadai (IC50, 116.3 mug/ml) exhibited more than 70% inhibition at the concentration of 1 mug/ml, but only one plant, Areca catechu (IC50, 563 mug/ml) showed high inhibitory effect on hyaluronidase activity. The results suggest that medicinal plants showing several biological activities may be potent inhibitors of the anti-ageing process in skin. This property might be useful for application in cosmetics.

Enzyme-linked immunosorbent assay for serum procollagen type III peptide in rats with hepatic fibrosis.

The process of hepatic fibrosis, and the changes in contents of hepatic hyproxyproline (HYP) and serum procollagen type III peptide (PIIINP) were examined in two rat models for hepatic fibrosis, i.e. bile duct ligation/scission (BDL/s)- and dimethylnitrosamine (DMN)-induced models. In addition, an expression of type III collagen mRNA in the liver of BDL/s model was also examined. In BDL/s model, hepatic fibrosis started at 2 weeks after operation (WAO) and cirrhosis with prominent bile duct hyperplasia was detected at and after 5 WAO. Serum PIIINP content measured using a modified double armed inhibition enzyme-linked immunosorbent assay (ELISA) method proposed by us started to increase at 1 WAO and continued to increase thereafter. Hepatic HYP content measured colorimetrically started to increase at 3 WAO and it continued to increase until 7 WAO. An expression of type III collagen mRNA in the liver was enhanced at and after 2 WAO, especially at 4 and 5 WAO. In DMN model, marked hepatic fibrosis was detected at 1 week after the last DMN administration (WAA), and the degree of fibrosis was apparently reduced at 4 WAA. Serum PIIINP content prominently increased at 1 WAA and decreased at and after 3 WAA. Hepatic HYP content showed a marked increase at 1 WAA and decreased thereafter. The present results indicated that the sequences of hepatic fibrosis, hepatic HYP content and serum PIIINP content were well correlated with each other in both BDL/s and DMN models. In conclusion, ELISA system for the detection of serum PIINP content is considered to be reliable method for assessment of cirrhotic liver, and the present two rat models for liver fibrosis/cirrhosis seems to be a good tool for researching antifibrotic agents.

Artemisinin derivatives in the treatment of falciparum malaria in pregnancy.

An artemisinin derivative (artesunate or artemether) was used for the treatment of multidrug-resistant Plasmodium falciparum malaria in 83 Karen pregnant women in Thailand; 55 women were treated for recrudescent infection following quinine or mefloquine, 12 for uncomplicated hyperparasitaemic episodes, and 16 had not declared their pregnancy when treated. The women were followed weekly until delivery. Artesunate and artemether were well tolerated and there was no drug-related adverse effect. Recrudescence within 42 d occurred in 16% of the treated episodes. Overall 73 pregnancies (88%) resulted in live births, 3 (4%) in abortions and 2 (3%) in still births, and 5 women were lost to follow-up before delivery. There was no congenital abnormality in any of the newborn children, and the 46 children followed for more than one year all developed normally.

Identification and characterization of the inducible murine mast cell gene, imc-415.

Activation of mast cells results in the generation and release of bioactive mediators which in turn initiate allergic inflammation. Mast cell function is enhanced following stimulation in part because of the induction of specific genes and their products. To identify additional genes induced in mast cells that support this process, we thus constructed an activation-specific mast cell subtraction library. To date, we have isolated 26 novel inducible murine mast cell (imc) cDNA clones. Among them, a full-coding region of the murine gene imc-415 was found to have a greater than 90% nucleotide sequence homology and a 97.5% amino acid sequence homology to both a human beta4 integrin-binding protein (p27(BBP)) and a human translation initiation factor 6 (eIF6), which in turn are identical. In vitro translation of the imc-415 gene yielded a band of an approximately 26 kDa. This is the same as the calculated molecular weight of murine IMC-415 protein based on the predicted amino acid sequence and is the molecular weight of p27(BBP)/eIF6. Murine imc-415 message was also induced in inflamed lung tissues in a mouse model of asthma. These results suggest a role for murine imc-415 in allergic inflammation where it may enhance protein synthesis. Human eIF6/p27(BBP) may also play a role in allergic diseases based on the similarities in sequence and in gene expression patterns.

Identification and categorization of inducible mast cell genes in a subtraction library.

Mast cells play an important role in allergic inflammation by releasing inducible proinflammatory cytokines. While many inducible genes have been identified, we hypothesized that a significant number remain to be identified. We thus constructed an activation-specific mast cell subtraction library to establish a profile of induced genes in mast cells following allergic stimulation. To date, we have sequenced 150 cDNA clones. Among them, we have isolated 22 known genes whose expression has not been reported in mast cells, and an additional 26 cDNA clones which do not have significant homology to known genes in the Genbank database. We next selected 10 cDNA clones with strong signals by differential plaque hybridization. Of these cDNA clones, five genes were induced in mast cells upon Fc epsilon RI-mediated stimulation. They are cofilin, annexinVI, interferon (IFN)-beta, serglycin, and a novel inducible mast cell (IMC) gene, IMC-415. Characterization and relevant studies of this novel gene and other inducible known genes in mast cells will provide insight into the functions of mast cells in mammalian biology.

Two different negative regulatory elements control the transcription of T-cell activation gene 3 in activated mast cells.

T-cell activation gene 3 (TCA3) encodes a beta-chemokine that is transcriptionally regulated in mast cells; the gene has a functional NF-kappaB element at positions -194 to -185. The 5'-flanking region of this gene is also known to have a negative regulatory region between -2057 and -1342. To characterize the negative regulatory elements (NREs), this region was sequenced and then digested by HindIII enzyme into two fragments, NRE-1 (-2057 to -1493) and NRE-2 (-1492 to -1342). Both NRE-1 and NRE-2 in the 5'-3' orientation inhibited chloramphenicol acetyltransferase (CAT)-protein synthesis by a TCA3-CAT construct transfected into mast cells that were then activated. Only NRE-1 inhibited CAT-protein synthesis in the 3'-5' orientation. Further deletion of the 5' region of NRE-1 partially abolished the inhibitory activity. Both NRE-1 and NRE-2 inhibited the activity of a CD20-CAT construct independent of cell activation. Electrophoretic mobility shift assays showed DNA-protein complex formation with subsequences (CCCCCATTCT) of NRE-1 (NRE-1a) and (CCATGA) of NRE-2 (NRE-2b). NRE-1a appears to be novel. NRE-2b is identical with a putative silencer motif in the alphaIIb integrin gene. Site-directed mutagenesis demonstrated that both NRE-1a and NRE-2b are important in the negative regulation of TCA3 promoter activity. In vivo ligation-mediated PCR footprinting of the NRE-2 region revealed protection between -1372 and -1354, which contains NRE-2b. The data thus demonstrate identity of a silencer motif, here termed NRE-2b, in both the alphaIIb integrin gene and the TCA3, and that this silencer region in mast cells is functional both in vivo and in vitro. Further, evidence is presented that the promoter for TCA3 contains a novel silencer motif, termed NRE-1a, characterized by a CT-rich sequence.