Regulated On Activation, Normal T-cell Expressed And Secreted Expression Research Articles

RANTES and developmental defects of enamel in children: A Brazilian prenatal cohort (BRISA).

Little is known about the effect of maternal immunological factors on the etiology of developmental defects of enamel (DDE). RANTES (Regulated on Activation Normal T Cell Expressed and Secreted) is a chemokine produced by fibroblasts, lymphoid and epithelial mucosa cells in response to various external stimuli. Despite its importance for embryogenesis, RANTES expression has been demonstrated in multiple diseases characterized by inflammation, tumor and immune response, and wound healing. We hypothesized that altered levels of RANTES during pregnancy are associated with the immune and inflammatory response in women, which could lead to the occurrence of DDE in utero (DDE-iu), directly or mediated by preterm birth. Therefore, this study aimed to evaluate the direct and indirect effects of serum levels of RANTES in pregnant women in the occurrence of DDE-iu in children. This is a longitudinal case-control study. The mothers and their children (327) were evaluated in three moments: prenatal care, post childbirth, and when the child was between 12.3 and 36 months of age. The analysis was performed with structural equation modeling, estimating the standardized coefficient (SC), adopting α = 5%. There was a direct and negative effect of RANTES on the outcome (SC = -0.137; p = 0.022). This association was not mediated by preterm birth (SC = 0.007; P = 0.551). When considering the specific types of DDE-iu, RANTES had a direct effect on hypoplasia (SC = -0.190; p = 0.007), but not on opacity (SC = 0.343; p = 0.074). Lower serum levels of RANTES may contribute to a higher number of teeth with DDE-iu, specifically hypoplasia. However, more evidence supported by clinical, laboratory and epidemiological studies is still needed.

CGAS enhances the HTLV-1 reverse transcription intermediate ssDNA90-induced innate immune responses

Objective To investigate the role of cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, in regulating innate immune responses induced by reverse transcription intermediate of human T cell leukemia virus type 1 (HTLV-1). Methods (1)ssDNA90, the reverse transcription intermediate of HTLV-1, was transfected into HeLa cells to observe changes in the expression pattern of cGAS in transfected-HeLa cells with immunoblot assay.(2)HeLa cells were firstly transfected with cGAS-encoding plasmid and then ssDNA90 24 hours later.Real-time PCR was used to measure the expression of interferon (IFN)-β, IFN-gamma-inducible protein 10 (IP-10), regulated on activation, normal T cell expressed and secreted (RANTES) and tumor necrosis factor (TNF)-α.Immunoblot assay was performed to measure phosphorylated interferon regulatory factor 3 (IRF3) and p65.(3)cGAS expression was silenced by siRNA in HeLa and phorbol-12-myristate-13-acetate (PMA)-treated THP1 (PMA-THP1) cells and then ssDNA90 was transfected into these cells 24 hours later.Real-time PCR was used to measure the expression of IFN-β, IP-10, RANTES and TNF-α.Immunoblot assay was performed to measure phosphorylated IRF3 and p65. Results Expression of cGAS was increased in HeLa cells after ssDNA90 transfection.Compared with control cells, cGAS-transfected HeLa cells showed increased expression of IFN-β, IP-10, RANTES and TNF-α and enhanced phosphorylation of IRF3 and p65 after ssDNA90 transfection.Compared with control cells, both HeLa and PMA-THP1 cells with silenced expression of cGAS showed impaired production of IFN-β, IP-10, RANTES and TNF-α after ssDNA90 transfection.Moreover, ssDNA90-induced phosphorylation of IRF3 and p65 were decreased after cGAS gene-knockdown. Conclusion cGAS might promote HTLV-1 RTI ssDNA90-induced innate immune responses. Key words: cGAS; HTLV-1; DNA sensor; Reverse transcription intermediate; Innate immune response

Lactobacillus paracasei strain 06TCa19 suppresses inflammatory chemokine induced by Helicobacter pylori in human gastric epithelial cells.

Helicobacter (H.) pylori infection is an important risk factor for gastric cancer that causes gastric inflammation. Inflammatory chemokines such as interleukin (IL)-8 and regulated on activation normal T cell expressed and secreted (RANTES) are elevated in the gastric mucosa by H. pylori. This study aimed to investigate the effects of Lactobacillus paracasei strain 06TCa19, a probiotic strain, on IL-8 and RANTES expression and production induced by H. pylori using human gastric epithelial cell lines. Strain 06TCa19 was shown to suppress H. pylori-mediated elevation of gene expression related to these chemokines in MKN45 cells. The strain also suppressed the increase in IL-8 and RANTES products induced by H. pylori in AGS cells as well as in MKN45 cells. In MKN45 cells inoculated with H. pylori, strain 06TCa19 was shown to downregulate the activation of NF-κB and p38 MAPK signaling pathways. Additionally, the level of the CagA virulence protein of H. pylori in the MKN45 cells and the number of viable H. pylori adhering to MKN45 cells decreased with the addition of strain 06TCa19. Moreover, the strain 06TCa19 notably increased lactic acid in the supernatant of MKN45 cells. Thus, lactic acid released from strain 06TCa19 might have inhibited the adhesion of H. pylori to MKN45 cells and prevented the insertion of H. pylori CagA into the cells, and elevation of IL-8 and RANTES genes and proteins might be suppressed by downregulating the NF-κB and p38 MAPK pathways. Therefore, use of strain 06TCa19 may prevent H. pylori-associated gastric inflammation.

RANTES and SDF-1 Are Keys in Cell-based Therapy of TMJ Osteoarthritis

The present study aimed to investigate the therapeutic effect of injections of local bone marrow mesenchymal stem cells (BMSCs) on osteoarthritis (OA) of the temporomandibular joint (TMJ) and to explore the role of stromal cell–derived factor 1 (SDF-1) and regulated on activation, normal T-cell expressed and secreted (RANTES) in this effect. Fundamentally, OA of the TMJ was induced by unilateral anterior crossbite in mice. Exogenous green fluorescent protein–labeled BMSCs (GFP-BMSCs) were weekly injected into the TMJ region for 4, 8, and 12 wk. The reparative effects of exogenous GFP-BMSCs were investigated by morphological observation and micro–computed tomography. The differentiation of GFP-BMSCs in the cartilage was examined by double immunofluorescence of GFPs with type II collagen, and the expression of related factors in the condylar cartilage was quantified by real-time polymerase chain reaction. The role of RANTES and SDF-1 in the therapeutic effect of exogenous BMSCs was examined by both in vitro and in vivo studies. The OA cartilage of the TMJ displays a synchronous increase in SDF-1 and RANTES expression and a higher capability of attracting the migration of GFP-BMSCs. The implanted GFP-BMSCs differentiated into type II collagen–positive cells and reversed cartilage degradation and subchondral bone loss in mice with OA of the TMJ. The migration of GFP-BMSCs towards OA cartilage and the rescuing effect of GFP-BMSC injections were impaired by the inhibitors of C-X-C chemokine receptor type 4 (CXCR4) and C-C chemokine receptor type 1 (CCR1), which are the receptors of SDF-1 and RANTES, respectively. Our data indicated that SDF-1/CXCR4 and RANTES/CCR1 signals are pivotal and function synergistically in the recruitment of GFP-BMSCs towards degraded cartilage in mice OA of the TMJ.

Beneficial Effects of Hormone Replacement Therapy on Periodontitis Are Vitamin D Associated

Possible synergism between female sex hormones and vitamin D on periodontitis pathology has not been assessed. Here, the authors investigate effects of estrogen, progesterone, and vitamin D on periodontitis in a population-based sample and use cell studies to explore mechanistic explanations of the population-based findings. The epidemiologic analysis uses cross-sectional data from the continuous National Health and Nutrition Examination Survey 2001 to 2004. The cross sections include 1,230 women aged 40 to 85 years who received a periodontal examination, responded to questions regarding hormone replacement therapy (HRT), and provided a blood sample for serum vitamin D assessments. For mechanistic cell culture studies, human monocytes were cultured with or without lipopolysaccharide (LPS), estradiol, progesterone, and/or 1,25-dihydroxyvitamin D3; and transcriptional activity of interleukin (IL)-6, IL-1β, B lymphocyte chemoattractant (BLC), and regulated on activation normal T-cell expressed and secreted (RANTES) was assessed. HRT use (versus none) was associated with higher attachment levels and more teeth only among participants who were vitamin D sufficient (>20 ng/mL). The odds ratio for having moderate/severe periodontitis among users of HRT versus participants who did not use HRT was 0.69 among participants who were vitamin D sufficient and 1.19 in participants who were vitamin D deficient. LPS-induced IL-6, IL-1β, and BLC expression was attenuated in human monocytes treated with estrogen and progesterone. Downregulation of IL-6 expression by estrogen and progesterone was potentiated when vitamin D was included. LPS-induced IL-6 and RANTES expression was decreased, and BLC expression was totally reversed, by vitamin D treatment. The association between HRT and clinical periodontal measures was strongest among women with high vitamin D levels. This association is plausibly mediated via an anti-inflammatory transcriptional mechanism.

Association of the G403A polymorphism in the RANTES gene with coronary artery disease: a meta-analysis

The G403A polymorphism in the RANTES (regulated on activation normal T cell expressed and secreted) gene has a key role in the expression of RANTES, which has been detected in a range of cells in atherosclerotic plaque. However, the association of this polymorphism with the risk of coronary artery disease (CAD) remains controversial. A meta-analysis was performed to assess the association of the G403A polymorphism in the RANTES gene with the risk of CAD. A comprehensive search was conducted to identify all studies published on the association of the RANTES gene G403A polymorphism with CAD risk. The fixed or random-effect pooled measure was adopted based on a heterogeneity test among studies, which was evaluated using I(2). Potential sources of between-study heterogeneity were explored using meta-regression analysis. Publication bias was estimated with Begg's rank correlation method. Eight articles were included in this meta-analysis, with 4601 CAD cases and 2522 controls. No significant association of RANTES gene G403A polymorphism with CAD was identified in any of the codominant, dominant, recessive, homozygote, or heterozygote inheritance models. No evidence of publication bias was detected. The meta-analysis suggested that the A allele of the G403A polymorphism in the RANTES gene has no effect on the risk of CAD. This relationship needs to be confirmed by further studies.

Increased upper airway cytokines and oxidative stress in severe obstructive sleep apnoea

Inflammation may contribute to upper airway pathophysiology in obstructive sleep apnoea (OSA). Our objective was to compare upper airway pro-inflammatory cytokine expression, oxidative stress and connective tissue deposition in severe (n = 25) versus mild (n = 17) OSA patients. Upper airway surgical specimens were separated by predominance of either mucosal or muscle tissue. Expression levels of interleukin (IL)-1α, IL-6, interferon-γ, RANTES (regulated on activation, normal T-cell expressed and secreted), transforming growth factor (TGF)-β and l-selectin were measured by ribonuclease protection assay. Oxidative stress was assessed via protein carbonyl group detection by immunoblotting. Histochemistry was employed for immunolocalisation of selected cytokines and connective tissue morphometry. In the severe OSA group, expression of IL-1α, IL-6 and TGF-β was significantly higher in mucosa-predominant tissues, whereas in muscle-predominant specimens, RANTES expression was greater in severe OSA. Increased protein carbonylation was observed in severe OSA within both mucosal and muscle compartments. Immunohistochemistry localised TGF-β to submucosal and perimuscular inflammatory cells, while IL-6 was primarily localised to myocytes. Consistent with the pro-fibrotic cytokine profile observed in mucosa-predominant tissue, morphometric analysis revealed greater submucosal and perimuscular connective tissue in severe OSA subjects. There is increased pro-inflammatory and pro-fibrotic cytokine expression, oxidative stress, and connective tissue deposition in upper airway tissues from severe versus mild OSA patients.

Expression of TNF-α and RANTES in drug-induced human gingival overgrowth

Objectives:Regulated on activation, normal T cell expressed and secreted (RANTES) is a chemokine that is produced by fibroblasts, lymphoid and epithelial cells of the mucosa in response to various external stimuli. RANTES expression has been demonstrated in a variety of diseases characterized by inflammation, including asthma, transplantationassociated accelerated atherosclerosis, endometriosis and fibrosis. RANTES mRNA is quickly up-regulated by tumor necrosis factor (TNF)-α stimulation. Cyclosporine A (CsA) is widely used in organ transplant patients, often causing various side-effects including gingival overgrowth, which is fibrotic in nature. This study was carried out to assess the mRNA expression of TNF-α and RANTES in healthy individual, chronic periodontitis and CsAinduced gingival overgrowth tissues.Materials and Methods:Gingival tissue samples were collected from chronic periodontitis, CsAinduced gingival overgrowth patients and healthy individuals. Total RNA was isolated and reverse transcription polymerase chain reaction (RT-PCR) was performed for TNF-α and RANTES expression.Results:The results suggest that CsAinduced gingival overgrowth tissues expressed significantly increased TNF-α and RANTES compared to control and chronic periodontitis.Conclusion:The findings of the present study suggest that CsA can modify the expression of TNF-α and RANTES in drug-induced human gingival overgrowth.

Glycogen synthase kinase‐3 negatively regulates anti‐inflammatory interleukin‐10 for lipopolysaccharide‐induced iNOS/NO biosynthesis and RANTES production in microglial cells

The inflammatory effects of glycogen synthase kinase-3 (GSK-3) have been identified; however, the potential mechanism is still controversial. In this study, we investigated the effects of GSK-3-mediated interleukin-10 (IL-10) inhibition on lipopolysaccharide (LPS)-induced inflammation. Treatment with GSK-3 inhibitor significantly blocked LPS-induced nitric oxide (NO) production as well as inducible NO synthase (iNOS) expression in BV2 murine microglial cells and primary rat microglia-enriched cultures. Using an antibody array and enzyme-linked immunosorbent assay, we found that GSK-3-inhibitor treatment blocked LPS-induced upregulation of regulated on activation normal T-cell expressed and secreted (RANTES) and increased IL-10 expression. The time kinetics and dose-response relations were confirmed. Reverse transcription-polymerase chain reaction showed changes on the messenger RNA level as well. Inhibiting GSK-3 using short-interference RNA, and transfecting cells with dominant-negative GSK-3beta, blocked LPS-elicited NO and RANTES expression but increased IL-10 expression. In contrast, GSK-3beta overexpression upregulated NO and RANTES but downregulated IL-10 in LPS-stimulated cells. Treating cells with anti-IL-10 neutralizing antibodies to prevent GSK-3 from downregulating NO and RANTES showed that the anti-inflammatory effects are, at least in part, IL-10-dependent. The involvement of Akt, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase and nuclear factor-kappaB that positively regulated IL-10 was demonstrated. Furthermore, inhibiting GSK-3 increased the nuclear translocation of transcription factors, that all important for IL-10 expression, including CCAAT/enhancer-binding protein beat (C/EBPbeta), C/EBPdelta, cAMP response binding element protein and NF-kappaB. Taken together, these findings reveal that LPS induces iNOS/NO biosynthesis and RANTES production through a mechanism involving GSK-3-mediated IL-10 downregulation.

NF-κB Decoy Oligonucleotides Suppress RANTES Expression and Monocyte Chemotactic Activity via NF-κB Inactivation in Stromal Cells of Ectopic Endometrium

The nuclear factor kappa B (NF-kappaB) pathway is a critical mediator of regulated on activation, normal T cell expressed and secreted (RANTES) gene regulation and therefore represents a potential target for therapy of endometriosis-associated symptoms. The objective of this study was to investigate the effect of NF-kappaB decoy oligonucleotides (ODNs) on NF-kappaB activation, RANTES expression, and monocyte chemotactic activity in ectopic endometrial stromal cells in vitro. A specific sandwich enzyme-linked immunosorbent assay (ELISA) was used to quantify RANTES expression in ectopic and normal endometrial stromal cells stimulated by interleukin (IL)-1beta. Four hours after transfection of NF-kappaB decoy ODNs, 10 ng/ml IL-1beta was added to induce the ectopic endometrial stromal cells to secrete RANTES. The NF-kappaB activation, RANTES expression, and monocyte chemotactic activity in ectopic endometrial stromal cells were respectively evaluated by electrophoretic mobility shift assay, ELISA, and Boyden chambers. IL-1beta induced significantly higher levels (P < 0.05) of RANTES expression in a time-dependent manner in ectopic endometrial stromal cells compared with IL-1beta-untreated ectopic and normal endometrial stromal cells. The RANTES accounts for the majority (68%) of the monocyte chemotactic activity in conditioned media of ectopic endometrial stromal cells. In vitro transfection of NF-kappaB decoy ODNs dramatically decreased (P < 0.05) the NF-kappaB activation, RANTES expression, and monocyte chemotactic activity in IL-1beta-induced ectopic endometrial stromal cells. NF-kappaB decoy ODNs may exert anti-inflammatory effects in ectopic endometrial stromal cells via the suppression of NF-kappaB activation, RANTES expression, and monocyte chemotactic activity.

Signaling Pathways Involved In Dengue-2 Virus Infection Induced RANTES Overexpression

Dengue viruses participate in liver inflammation by inducting the expression of various chemokines including Regulated on Activation Normal T-cell Expressed and Secreted (RANTES). However, the underlying signaling remains unknown. Here, we reveal that Ras, Raf-1 and three mitogen-activated protein kinases (MAPKs) p38, extracellular signal-regulated kinase (Erk), and c-jun-NH2-terminal kinase (JNK) can be activated or phosphorylated in dengue-2 virus infected hepatocyte and epithelial cells by western blotting and confirmed by dominant negative mutants of ras, raf-1, p38, Erk, and JNK. The Tet-off inducible plasmids harboring dengue-2 virus prM, core, E or NS1 gene were utilized to reveal their role in RANTES activation. However, no effect was detected among the genes tested indicating that they are either dispensable or not sufficient for RANTES activation. Taken-together, Ras, Raf-1, JNK, Erk and p38 related signaling pathways are essential for the activation of RANTES by dengue-2 virus. The knowledge gathered will shed light on developing a novel therapeutic approach to block inflammatory infiltrates through decreasing RANTES expression.

Expression and Secretion of RANTES (CCL5) in Granulomatous Calcified Tissue before and after Lipopolysaccharide Treatment In Vivo

RANTES (regulated on activation, normal T cell-expressed and secreted) is a CC chemokine appearing to be involved in the recruitment of leukocytes at inflammation sites. RANTES is produced by CD8(+) T cells, epithelial cells, fibroblasts, and platelets. It acts in vitro in leukocyte activation and human immunodeficiency virus suppression, but its role in vivo is still uncertain. In our study, we established the involvement of RANTES in an in vivo model of chronic inflammation induced by potassium permanganate, leading to calcified granulomas. In our rat model, RANTES expression (mRNA and protein) was significantly upregulated in granulomatous tissue; RANTES expression was further increased upon i.p. injection of lipopolysaccharide (LPS), while it was kept at basal levels by dexamethasone (Dex) given 18 hours before sacrifice. LPS and Dex increased and decreased, respectively, the recruitment of mononuclear cells in granulomatous tissue compared with control granulomas from phosphate-buffered saline (PBS)-treated animals. In granuloma tissue, levels of RANTES were higher in LPS-treated rats and lower in the Dex group compared to controls. RANTES was also found in the conditioned medium of granuloma tissue from treated (LPS or Dex) and untreated (PBS) rats. When LPS was added in vitro for 18 hours, RANTES was further increased, except in the Dex group (P > 0.05). On serum analysis, RANTES levels were higher in the LPS group and lower in the Dex group compared to controls. This study shows for the first time that RANTES is produced in vivo in chronic, experimental inflammatory states, an effect increased by LPS and inhibited by Dex.

Interferon Regulatory Factor 1 Is an Essential and Direct Transcriptional Activator for Interferon γ-induced RANTES/CCl5 Expression in Macrophages

Interferon regulatory factor 1 (IRF-1) is an important transcription factor in interferon gamma (IFNgamma)-mediated signaling in the development and function of NK cells and cytotoxic T lymphocytes. RANTES (regulated on activation normal T cell expressed and secreted; CCL5) is a member of the CC chemokine family of proteins, which is strongly chemoattractant for several important immune cell types in host defense against infectious agents and cancer. However, the role of IFNgamma and IRF-1 in the regulation of RANTES gene expression and their operative mechanisms in macrophages have not been established. We report here that RANTES expression in IRF-1-null mice, primarily in macrophages, in response to carcinogenic stimulation in vivo and in vitro and to IFNgamma but not to lipopolysaccharide in vitro, was markedly decreased. As a result, RANTES-mediated chemoattraction of CCR5(+) target cells was also severely impaired. Adenovirus-mediated gene transduction of IRF-1 in primary macrophages resulted in enhanced RANTES expression. The IFNgamma and IRF1 response element was localized to a TTTTC motif at -147 to -143 of the mouse RANTES promoter, to which endogenous or recombinant IRF-1 can physically bind in vitro and in vivo. This study uncovers a novel IFNgamma-induced pathway in RANTES expression mediated by IRF-1 in macrophages and elucidates an important host defense mechanism against neoplastic transformation.

MCP-1 Is Induced by Receptor Activator of Nuclear Factor-κB Ligand, Promotes Human Osteoclast Fusion, and Rescues Granulocyte Macrophage Colony-stimulating Factor Suppression of Osteoclast Formation

Human osteoclast formation from monocyte precursors under the action of receptor activator of nuclear factor-kappaB ligand (RANKL) was suppressed by granulocyte macrophage colony-stimulating factor (GM-CSF), with down-regulation of critical osteoclast-related nuclear factors. GM-CSF in the presence of RANKL and macrophage colony-stimulating factor resulted in mononuclear cells that were negative for tartrate-resistant acid phosphatase (TRAP) and negative for bone resorption. CD1a, a dendritic cell marker, was expressed in GM-CSF, RANKL, and macrophage colony-stimulating factor-treated cells and absent in osteoclasts. Microarray showed that the CC chemokine, monocyte chemotactic protein 1 (MCP-1), was profoundly repressed by GM-CSF. Addition of MCP-1 reversed GM-CSF suppression of osteoclast formation, recovering the bone resorption phenotype. MCP-1 and chemokine RANTES (regulated on activation normal T cell expressed and secreted) permitted formation of TRAP-positive multinuclear cells in the absence of RANKL. However, these cells were negative for bone resorption. In the presence of RANKL, MCP-1 significantly increased the number of TRAP-positive multinuclear bone-resorbing osteoclasts (p = 0.008). When RANKL signaling through NFATc1 was blocked with cyclosporin A, both MCP-1 and RANTES expression was down-regulated. Furthermore, addition of MCP-1 and RANTES reversed the effects of cyclosporin A and recovered the TRAP-positive multinuclear cell phenotype. Our model suggests that RANKL-induced chemokines are involved in osteoclast differentiation at the stage of multinucleation of osteoclast precursors and provides a rationale for increased osteoclast activity in inflammatory conditions where chemokines are abundant.

The Role of RANTES in a Murine Model of Food Allergy

Food allergy is an important and common health issue, and there is a need to identify and characterize the sensitizing mechanisms. One of the common causes of food allergy is ovalbumin (OVA), a dietary antigen from eggs. We hypothesized that OVA‐induced food allergy in the gut involves the activation of the chemokine regulated on activation, normal T cell expressed and secreted (RANTES), which then recruits eosinophils to lesioned tissue. The purpose of this study was to clarify whether RANTES expression correlates with eosinophil infiltration in the gut of OVA‐sensitized BALB/c mice in response to oral OVA challenge. BALB/c mice were immunized with OVA 1 µg and sensitized after 2 weeks by intragastric administration of OVA. Sensitization to the oral OVA challenge was analyzed by examining eosinophil infiltration into the gut tissue (immunohistochemistry), mucosal eosinophil cationic protein (ECP) concentration, and RANTES mRNA expression (reverse‐transcriptase polymerase chain reaction and Southern blotting) at 3, 6, 12, and 24 h after the challenge. There was marked edema of the intestinal villi, and eosinophil infiltration to the lamina propria peaked at 6 h in OVA‐sensitized mice. RANTES mRNA expression peaked at 3 h and 6 h and declined thereafter. The expression of RANTES mRNA in the allergic mice was much higher than in the nonallergic, normal, or unsensitized control mice. Tissue eosinophilia and intestinal ECP levels were significantly correlated with the RANTES mRNA level. We conclude that RANTES may play a central role in the pathogenesis of food‐mediated gastrointestinal allergy.

Inhibition of RANTES expression by indirubin in influenza virus-infected human bronchial epithelial cells

The human bronchial epithelial cells are the primary sites of influenza virus infection. In this study, the effect of indirubin on the expression of the chemokine regulated on activation, normal T cell expressed and secreted (RANTES) by the influenza virus-infected H292 human epithelial cell line was examined. The expression of RANTES mRNA was analyzed using reverse transcription polymerase chain reaction and the concentration of RANTES production was determined by the enzyme-linked immunosorbent assay. At the non-cytotoxic concentrations, indirubin was found to reduce both the expression and production of RANTES in influenza A/NWS/33-infected H292 cells. Inhibition was also observed in influenza virus B/Lee-infected cells. Significant reduction of the expression of IL-8 was not observed after the infection. Indirubin-3′-oxime, a recently developed derivative with kinase inhibitory activity, also mediates a potent inhibitory effect on the expression of RANTES. The influenza virus infection-induced phosphorylation of the nuclear transcription NF-kB regulatory molecule IkBα and the p38 MAP kinase were also found to be inhibited by indirubin-3′-oxime. This finding suggests that indirubin is one of the components in the Chinese medicinal herbs Isatis indigotica and Strobilanthes cusia with immunomodulatory activity on the expression of RANTES.

Double genetic modification of adenovirus fiber with RGD polylysine motifs significantly enhances gene transfer to isolated human pancreatic islets.

New strategies for improving durable functional islet mass will be instrumental in facilitating islet transplantation as a cure for type 1 diabetes mellitus. The ability to transfer immunoregulatory or cytoprotective genes into pancreatic islets may enhance survival. Adenoviral vectors (Ad5) have been used widely to deliver therapeutic genes to different tissues. Limitations associated with the use of Ad5 for gene therapy are related to the reliance of the virus on the presence of its primary receptor, the transient nature of the transgene expression, and the immediate inflammatory and immune response elicited by the infection. Because the arginine-glycine-aspartame (RGD) and polylysine (pK7) motifs have been shown to enhance Ad5 infection through an Ad5 receptor-independent pathway, we hypothesized that they could act additively to improve infectivity and reduce toxicity to isolated human pancreatic islets (IHPI). Hand-picked IHPI were infected with nonmodified Ad5, single-modified Ad5 with RGD (Ad5RGD) or pK7 (ad5pK7), and Ad5RGDpK7. Transfection efficiency was evaluated by green fluorescent protein and luciferase expression. Apoptosis was assessed using a quantitative assay, activation of caspase 3 by a colorimetric assay, nuclear factor (NF)-kappaB nuclear translocation using a promoter-luciferase NF-kappaB responsive construct, regulated on activation normal T-cell expressed and secreted (RANTES) by enzyme-linked immunosorbent assay. In vivo functionality was evaluated after transplantation into diabetic nonobese diabetic severe combined immunodeficiency mice. Compared with unmodified and singly-modified Ad5 vectors, Ad5RGDpK7 demonstrated the highest infectivity. After the infection of IHPI with adenoviral vectors using the minimal dose required to infect greater than 80% of the islet cells (Ad5, 500 viral particles [VP]/cell; Ad5RGD and Ad5pK7, 10 VP/cell; Ad5RGDpK7, 0.1 VP/cell), islets infected with Ad5RGDpK7 presented a significant reduction in apoptosis, NF-kappaB nuclear translocation, RANTES expression, and higher glucose disposal rate; reduced Ad5-driven specific Th1 and antibody response were also observed. Ad5RGDpK7 exhibited higher transfection efficiency, allowing a significant reduction in the viral dose required to infect greater than 80% of the islet cells. The reduction in the viral dose was associated with reduced toxicity, inflammation, and immune responses related to Ad5 infection. This strategy may thus be used to successfully modify isolated pancreatic islets.

Exacerbations of Bronchitis: bronchial eosinophilia and gene expression for interleukin-4, interleukin-5, and eosinophil chemoattractants.

Eosinophilia has been reported during exacerbations of bronchitis, but the mechanisms of tissue recruitment of eosinophils are unclear. We quantified eosinophils and the concurrent expression of cytokines and chemokines probably responsible for the tissue eosinophilia in bronchial biopsies obtained from three groups of nonatopic subjects: (1) healthy nonsmokers (n = 7; FEV1 % predicted = 108 +/- 4 [mean +/- SEM]); (2) nonasthmatic smokers with chronic bronchitis (CB) in a stable phase of their disease (n = 11; FEV1 % predicted: 75 +/- 5); and (3) nonasthmatic subjects with CB who sought medical advice for an exacerbation of their condition (n = 9; FEV(1) % predicted: 61 +/- 8). We applied anti-EG2 antibody and immunostaining to detect and count eosinophils. We performed in situ hybridization to visualize and enumerate cells expressing the genes for interleukin (IL)-4 and IL-5 and the eosinophil chemokines eotaxin, monocyte chemoattractant protein (MCP)-4, or regulated on activation, normal T-cell expressed and secreted (RANTES). We confirmed an increase in EG2-positive eosinophils in patients with CB in exacerbation. We found messenger RNA (mRNA) positivity for IL-4 and IL-5 in CB, but the between-group differences were not statistically significant. However, the numbers of lymphomononuclear cells expressing eotaxin mRNA were significantly greater in the smokers with CB than in the healthy nonsmokers without CB (p < 0.01). Following an exacerbation, RANTES expression was upregulated and this chemokine was strongly expressed in both the surface epithelium and in subepithelial lymphomononuclear cells: only RANTES showed a significant positive correlation with the increasing number of EG2-positive cells (r = 0.51; p < 0.03). In conclusion, an allergic profile of inflammation can also occur in CB: the marked upregulation of RANTES in the epithelium and subepithelium most likely accounts for the increased eosinophilia associated with an exacerbation of bronchitis.

Analysis of local cytokine gene expression in patients with allergic rhinitis treated with CO2 laser surgery.

Laser surgery of the inferior turbinates has become a popular surgical treatment for patients with allergic rhinitis, particularly for those who have persistent nasal obstruction and do not respond well to pharmacological therapy. The aim of this study was to investigate the effects of the laser surgery on local cytokine gene expression at the mucosal surface in relation to the improvement of nasal symptoms. A prospective analysis of 25 patients with allergic rhinitis caused by the house dust mite who underwent laser surgery twice with a 1-month interval on an outpatient basis. Fifteen healthy volunteers served as normal control subjects. Improvement of the nasal symptoms was evaluated on a graded scale. Nasal mucosal cells were obtained by brushing from the inferior turbinate at each visit. The expression of messenger RNA (mRNA) encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-6, IL-8, RANTES (regulated on activation, normal T-cell expressed and secreted), and eotaxin was semiquantitatively analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Two months after treatment, the nasal symptom scores significantly decreased from baseline. The decrease was most apparent in nasal obstruction. RT-PCR analysis revealed that a significant decrease in IL-8 and RANTES expression (P < .001 and P = .012, respectively) was observed after successive laser treatment, and the reduction in these cytokines was significantly correlated. On the other hand, mRNA expression of GM-CSF, IL-6, and eotaxin remained unchanged. This study provided evidence that the expression of local inflammatory cytokines can be attenuated in part by CO2 laser treatment, which may be closely related to the clinical effectiveness of this procedure.

Quantitative analysis of eotaxin and RANTES messenger RNA in nasal polyps: association of tissue and nasal eosinophils.

Nasal polyps develop in the ethmoidal and middle turbinate area, often in relation to inflammatory conditions. Their exact etiology and pathogenesis are still under debate. Histologically, the polyps are infiltrated by a number of inflammatory cells, with eosinophil predominating in most specimens. This finding suggests that the nasal polyp is an inflammatory growth that is controlled by the local environment. The chemokines eotaxin and RANTES (regulated on activation normal T cell expressed and secreted) have been postulated to be involved in the recruitment and activation of eosinophils to certain inflamed tissues. The purpose of this study was to investigate eotaxin and RANTES mRNA expression in nasal polyps and its effect on tissue and nasal eosinophils. Nasal polyps (917 allergic and 30 nonallergic cases) were obtained from endoscopic sinus surgery, and 15 normal inferior turbinates also were taken. Immunohistochemical staining for eosinophils and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) tests for eotaxin and RANTES mRNA expression were performed, and the concentration of nasal eosinophil cationic protein (ECP) was measured. The amounts of eotaxin mRNA in the allergic nasal polyps were 11.4 times higher and the levels in the nonallergic polyps were 6.4 times higher than in the normal inferior turbinate. However, the RANTES mRNA expression did not show any differences among the three groups. Tissue eosinophilia and nasal ECP levels were significantly correlated with eotaxin mRNA level but not with RANTES mRNA expression. Nasal polyp eosinophilic infiltration and activation correlate mainly with increased eotaxin gene expression rather than with RANTES expression.