RANTES mRNA Expression Research Articles

E076 To investigate the mechanisms of action of novel compounds ex vivo in cells obtained from the inflammatory synovium of patients with rheumatoid arthritis

Abstract Background/Aims To investigate the ex vivo effect of JAK1/2 inhibitor baricitinib on the expression of pro-inflammatory mediators in normal and RA diseased fibroblasts stimulated with oncostatin M (OSM) as well as in cells derived from RA inflamed synovial membrane cells (RA SMC). Methods RA SMC and fibroblast like synoviocytes (RA FLS) were isolated from synovial membranes of patients undergoing surgery using enzymatic digestion and cell propagation. Normal fibroblast-like synoviocytes (FLS) and normal human skin fibroblasts (HSF) were purchased from commercial sources. All fibroblasts were cultured in media supplemented foetal bovine serum and antibiotics in incubators using established laboratory procedures, and serum-starved prior to stimulation with OSM with or without baricitinib. RA SMC were cultured in the presence of baricitinib without OSM as they spontaneously release pro-inflammatory cytokines. Levels of mRNA and proteins of the various inflammatory cytokines (IL-6, IL-8, MCP-1, CCL5 and IP-10) were determined by qPCR, ELISA and MSD. Western blot was used to measure levels of phospho-JAK specific components. Results qPCR analysis found that baricitinib inhibited OSM induced IP-10 and MCP-1 mRNA expression but not RANTES mRNA expression in normal human skin fibroblasts and RA FLS. In normal FLS however, baricitinib was found to inhibit RANTES and MCP-1 mRNA expression, but not IP-10 mRNA expression. Expression levels of IL-8 were too low for accurate conclusions to be drawn in all cell types apart from the RA SMCs, and in this case baricitinib did not inhibit IL-8 expression. ELISA results showed that baricitinib inhibited OSM induced MCP-1 expression in all cell types investigated. OSM induced IL-6 expression was inhibited in RA FLS but no other cell type. Conclusion Baricitinib inhibits OSM stimulated pro-inflammatory cytokine production through JAK1, 2. It has a varying inhibitory effect on the production of different cytokines: it is shown to particularly inhibit MCP-1. Baricitinib was found to have no effect on the production of spontaneously released pro-inflammatory cytokines in RA mixed cell populations. Disclosure M. Turner: None.

P191 To investigate the mechanisms of action of novel compounds ex vivo in cells obtained from the inflammatory synovium of patients with rheumatoid arthritis

Abstract Background/Aims To investigate the ex vivo effect of JAK1/2 inhibitor baricitinib on the expression of pro-inflammatory mediators in normal and RA diseased fibroblasts stimulated with oncostatin M (OSM) as well as in cells derived from RA inflamed synovial membrane cells (RA SMC). Methods RA SMC and fibroblast like synoviocytes (RA FLS) were isolated from synovial membranes of patients undergoing surgery using enzymatic digestion and cell propagation. Normal fibroblast-like synoviocytes (FLS) and normal human skin fibroblasts (HSF) were purchased from commercial sources. All fibroblasts were cultured in media supplemented foetal bovine serum and antibiotics in incubators using established laboratory procedures, and serum-starved prior to stimulation with OSM with or without baricitinib. RA SMC were cultured in the presence of baricitinib without OSM as they spontaneously release pro-inflammatory cytokines. Levels of mRNA and proteins of the various inflammatory cytokines (IL-6, IL-8, MCP-1, CCL5 and IP-10) were determined by qPCR, ELISA and MSD. Western blot was used to measure levels of phospho-JAK specific components. Results qPCR analysis found that baricitinib inhibited OSM induced IP-10 and MCP-1 mRNA expression but not RANTES mRNA expression in normal human skin fibroblasts and RA FLS. In normal FLS however, baricitinib was found to inhibit RANTES and MCP-1 mRNA expression, but not IP-10 mRNA expression. Expression levels of IL-8 were too low for accurate conclusions to be drawn in all cell types apart from the RA SMCs, and in this case baricitinib did not inhibit IL-8 expression. ELISA results showed that baricitinib inhibited OSM induced MCP-1 expression in all cell types investigated. OSM induced IL-6 expression was inhibited in RA FLS but no other cell type. Conclusion Baricitinib inhibits OSM stimulated pro-inflammatory cytokine production through JAK1, 2. It has a varying inhibitory effect on the production of different cytokines: it is shown to particularly inhibit MCP-1. Baricitinib was found to have no effect on the production of spontaneously released pro-inflammatory cytokines in RA mixed cell populations. Disclosure M.P. Turner: None.

Abstract P083: Fetuin-a Induces Vascular Dysfunction Through Toll-like Receptor 4 And Nox1/4 Activation

Although studies demonstrate an important role for fetuin-A (FetA) in the inhibition of vascular calcification, convincing evidence suggests that fetuin-A is also involved in insulin resistance, inflammation and cardiovascular damage. The present study seeks to unravel FetA vascular effects and associated molecular mechanisms, focusing on oxidative stress and toll-like receptor 4 (TLR4). Vascular function studies were performed in mesenteric resistance arteries from WKY rats, wild-type, Nox1 KO, Nox4 KO and Ang II-dependent hypertensive mice (LinA3) and rat aortic endothelial cells (RAEC). ROS production (chemiluminescence, Amplex Red, ELISA) and pro-inflammatory markers expression (RT-PCR) were measured in VSMCs from WKY rats and RAEC. FetA impaired endothelium-dependent (LogEC50 7.320±0.08 M vs control 8.025±0.06) and endothelium-independent vasorelaxation (LogEC50 6.48±0.19 M vs control 7.38±0.12), p<0.05; effects blocked by tempol (superoxide dismutase mimetic), Nox1 inhibitor, ML171, and TLR4 inhibitor, CLI095. We did not observe any changes in contraction. FetA increased ROS production (62%) and peroxynitrite levels (158%) in VSMCs; while in RAEC, FetA increased ROS production (105%) followed by a decrease in H2O2 (62%) levels (p<0.05 vs control). FetA-induced effects on ROS were inhibited by ML171 and GKT137831 (Nox1/Nox4 inhibitor), as well as CLI095. Vascular dysfunction in arteries from Nox1 and Nox4 KO mice was unaffected by FetA. Activation of the FetA/TLR4/Nox axis led to an increase in IL-1β (190%), Il-6 (124%) and RANTES mRNA expression(116%) in RAEC, p<0.05 vs control. FetA enhanced vascular dysfunctionin LinA3 mice. Together, these results suggest that FetA through TLR4/Nox1 and 4-derived ROS leads to vascular dysfunction and inflammation, which may play an important role in the development of vascular injury during hypertension.

Toll-Like Receptor Agonists Modulate Wound Regeneration in Airway Epithelial Cells.

Background: Impaired regeneration of airway epithelium may lead to persistence of inflammation and remodelling. Regeneration of injured epithelium is a complex phenomenon and the role of toll-like receptors (TLRs) in the stimulation of respiratory virus products in this process has not been established. Objective: This study was undertaken to test the hypothesis that the wound repair process in airway epithelium is modulated by microbial products via toll-like receptors. Methods: Injured and not-injured bronchial epithelial cells (ECs) (BEAS-2B line) were incubated with the TLR agonists poly(I:C), lipopolisacharide (LPS), allergen Der p1, and supernatants from virus-infected epithelial cells, either alone or in combination with TLR inhibitors. Regeneration and immune response in injured and not-injured cells were studied. Results: Addition of either poly(I:C) or LPS to ECs induced a marked inhibition of wound repair. Supernatants from RV1b-infected cells also decreased regeneration. Preincubation of injured and not-injured ECs with TLR inhibitors decreased LPS and poly(I:C)-induced repair inhibition. TGF-β and RANTES mRNA expression was higher in injured ECs and IFN-α, IFN-β, IL-8, and VEGF mRNA expression was lower in damaged epithelium as compared to not-injured. Stimulation with poly(I:C) increased IFN-α and IFN-β mRNA expression in injured cells, and LPS stimulation decreased interferons mRNA expression both in not-injured and injured ECs. Conclusion: Regeneration of the airway epithelium is modulated by microbial products via toll-like receptors.

Rosmarinic acid suppresses adipogenesis, lipolysis in 3T3-L1 adipocytes, lipopolysaccharide-stimulated tumor necrosis factor-α secretion in macrophages, and inflammatory mediators in 3T3-L1 adipocytes

ABSTRACTBackground: Rosmarinic acid (RA) is a natural phenol carboxylic acid with many promising biological effects. It may be a suitable candidate for improving obesity-related adipose tissue dysfunction.Objective: We aimed to investigate the therapeutic use of RA as an anti-obesity agent by measuring its effects on adipogenesis, lipolysis, and messenger RNA (mRNA) expression of major adipokines in 3T3-L1 adipocytes; and its effects on lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) secretion in macrophages and inflammatory mediators in 3T3-L1 adipocytes incubated with macrophage-conditioned medium (MCM).Methods: 3T3-L1 preadipocytes were used to explore how RA affects adipogenesis, as well as the involvement of phosphorylated extracellular signal-regulated kinase-1/2 (p-ERK1/2) and mothers against decapentaplegic homolog 3 (p-Smad3). 3T3-L1 preadipocytes were also differentiated into mature adipocytes to explore how RA affects basal and isoproterenol- and forskolin-stimulated lipolysis; and how RA affects key adipokines’ mRNA expression. RAW 264.7 macrophages were stimulated with LPS in the absence or presence of RA to explore RA’s effects on TNF-α secretion. MCM was collected and 3T3-L1 adipocytes were incubated with MCM to explore RA’s effects on interleukin-6 (IL-6), IL-1β, monocyte chemoattractant protein-1 (MCP-1), and RANTES mRNA expression.Results: During the preadipocyte differentiation process, RA suppressed peroxisome proliferator-activated receptor-γ and CCAAT/enhancer binding protein-α, and activated p-ERK1/2 and p-Smad3; inhibition of adipogenesis by RA was partially restored following treatment with p-ERK1/2 and p-Smad3 inhibitors. In mature adipocytes, RA inhibited basal lipolysis; phosphodiesterase-3 inhibitor reversed this. RA also inhibited isoproterenol- and forskolin-stimulated glycerol and free fatty acid release, and the phosphorylation of hormone-sensitive lipase and perilipin. RA had no effects on leptin, adiponectin, resistin, or visfatin mRNA expression. RA suppressed TNF-α mRNA expression and secretion in LPS-stimulated RAW 264.7 macrophages; and reduced LPS-MCM-induced IL-6, IL-1β, MCP-1, and RANTES mRNA expression in 3T3-L1 adipocytes.Conclusions: RA exerts inhibitory effects on adipogenesis, lipolysis, and inflammation. RA could be a promising natural product for improving adipose mobilization in obesity.

The selective biomarker IL-8 identifies IFTA after kidney transplantation in blood cells

Cellular and antibody-mediated rejection processes and also interstitial fibrosis/tubular atrophy (IFTA) lead to allograft dysfunction and loss. The search for accurate, specific and non-invasive diagnostic tools is still ongoing and essential for successful treatment of renal transplanted patients. Molecular markers in blood cells and serum may serve as diagnostic tools but studies with high patient numbers and differential groups are rare. We validated the potential value of several markers on mRNA level in blood cells and serum protein level in 166 samples from kidney transplanted patients under standard immunosuppressive therapy (steroids±mycophenolic acid±calcineurin inhibitor) with stable graft function, urinary tract infection (UTI), IFTA, antibody-mediated rejection (ABMR), and T-cell-mediated rejection (TCMR) applying RT-PCR and ELISA. The mRNA expression of RANTES, granulysin, granzyme-B, IP-10, Mic-A and Interferon-γ in blood cells did not distinguish specifically between the different pathologies. We furthermore discovered that the mRNA expression of the chemokine IL-8 is significantly lower in samples from IFTA patients than in samples from patients with stable graft function (p<0.001), ABMR (p<0.001), Borderline (BL) TCMR (p<0.001), tubulo-interstitial TCMR (p<0.001) and vascular TCMR (p<0.01), but not with UTI. Serum protein concentrations of granzyme-B, Interferon-γ and IL-8 did not differ between the patient groups, RANTES concentration was significantly different when comparing UTI and ABMR (p<0.01), whereas granulysin, Mic-A and IP-10 measurement differentiated ongoing rejection or IFTA processes from stable graft function but not from each other.The measurement of IL-8 mRNA in blood cells distinguishes clearly between IFTA and other complication after kidney transplantation and could easily be used as diagnostic tool in the clinic.

COMPARISON OF THE DIAGNOSTIC INFORMATIVE VALUE OF DETERMINATION OF MRNA OF SOME HOMEOSTATIC AND PROINFLAMMATORY CYTOKINES IN THE SYNOVIAL MEMBRANE OF PATIENTS WITH RHEUMATOID ARTHRITIS

The problem of the early diagnosis of rheumatoid arthritis (RA) remains relevant, which is associated with the limited potential of available biomarkers and the heterogeneity of the disease. Chemokines and cytokines produced by the synovial membrane of the patients play a leading role in the pathogenesis of the disease, suggesting that they may be used as promising biomarkers. Objective: to comparatively investigate the relative mRNA expression of the homeostatic chemokines: stromal cellderived factor 1 (SDF-1/CXCL12), B-cell-attracting chemokine-1 (BCA-1/CXCL13), their receptors CXCR4 and CXCR5, the proinflammatory chemokines: macrophage chemotactic protein-1 (MCP-1/CCL2), T cell expressed and secreted chemokine on activation (CCL5/RANTES), interleukin-8 (IL-8) (IL-8/CXCL8), IL-17, and vascular endothelial growth factor (VEGF) in the synovial membrane biopsy specimens from patients with RA or osteoarthritis (OA) and from healthy individuals and to estimate the diagnostic informative value of these biomarkers. Subjects and methods. The expression of mRNA was estimated using a real-time polymerase chain reaction assay. The RA group included 28 patients, their median age was 47 [35; 54] years; disease duration – 8 [4; 12] years; DAS28 – 4.9 [3.9; 5.5]; 16 and 14 patients were seropositive for rheumatoid factor and anti-cyclic citrulinated peptide antibodies, respectively. The OA group comprised 22 patients whose median age was 70.5 [61; 74] years. Fifteen clinically healthy individuals with a median age of 45 [28; 64] years were also examined. Results and discussion. The RA group was noted to have significantly higher RANTES, IL-8, IL-17, VEGF, BCA-1, and CXCR5 mRNA levels than the OA group and healthy individuals. In the patients with OA, the level of the homeostatic chemokines BCA-1 and SDF-1, which had B-cell chemoattractant activities, was higher than that in the control group, which may suggest that they are implicated in the pathogenesis of the disease. The determination of RANTES mRNA expression was most informative in diagnosing RA: the area under the curve (AUC), 0.91 [95% confidence interval (CI), 0.84–0.99]; diagnostic sensitivity, 72.97% (95% CI, 55.88–86.21), diagnostic specificity, 96.15% (95% CI, 80.36–99.90); positive likelihood ratio (LR+), 18.95; negative likelihood ratio (LR-), 0.28 at a diagnostic threshold of 0.0350, so was CBC1 mRNA. The AUC for BCA-1 was 0.78 (95% CI, 0.66–0.89); diagnostic sensitivity, 56.25% (95% CI, 37.66–73.64), diagnostic specificity, 92.59% (95% CI, 75.71–99.09); LR+, 7.59; LR-, 0.47 at a diagnostic threshold of 0.0184.

The Role of Microenvironment Expression of Pd-1 Ligands and Chemokines in Clinical Outcome of Hodgkin'S Lymphoma

ABSTRACT Aim: Classical Hodgkin's lymphoma (cHL) is characterized by only few malignant cells and an abundance of inflammatory cells. cHL infiltrating cells produce cytokines and growth factors that provide essential stimulatory signals for survival and proliferation of Hodgkin and Reed–Sternberg (HRS) cells. Moreover, clinical behaviour of cHL may be directly regulated by the cross-talk between HRS cells and other cells in their microenvironment. The aim of our study was to estimate the role of microenvironment expression of PD-1 ligands and chemokines in clinical outcome of cHL. Methods: The case group comprised 49 patients with cHL (stage IIA: 23, IIB: 3, III-IV: 23). The patients received chemotherapy regimens (ABVD or BEACOPP 14/esc) and radiotherapy by indications. cHL specimens were obtained from lymph node biopsies of patients at diagnosis. PD-L1, PD-L2, MIP-1a, RANTES mRNA expression levels were analyzed in fresh tissue specimens using real-time RT-PCR. Results: We observed that 26.5 % (13/49) of cHL cases were PD-L1 negative, 16.3 % (8/49) - RANTES negative and 14.3 % (7/49) – both PD-L1 and RANTES negative. MIP-1a and RANTES expression levels were slightly higher in mixed cellularity cHL (p = 0.15), whereas PD-L1 – in nodular sclerosis cHL and advanced stages of disease (p = 0.19). PD-L2 expression level was independent of histological cHL variant or disease stage. All cases with the absence of both PD-L1 and RANTES markers had a complete response to the therapy and long-term remission. For the patients with increasing of RANTES/MIP-1a rate, a trend for a higher risk of relapse was observed (P = 0.09). ROC analysis revealed that PD-L1 expression level in tumor is an important marker which is associated with clinical outcome of cHL patients (Se = 87.5 %; Sp = 64.5 %; AUC = 0.75; p = 0.002). High PD-L1 expression was associated with reduced progression-free survival (PFS) in cHL patients. The 2-year PFS rate for cHL patients with high PD-L1 expression was 47 % compared to 95 % for low or absent of PD-L1 expression (p = 0.04). Conclusions: Our results suggest that tumor microenvironment play an important role in clinical behavior of cHL. PD-L1 expression level can be used as a marker of prognosis in patients with cHL and represent an attractive target for a cHL immunotherapy in patients with poor outcome. Disclosure: All authors have declared no conflicts of interest.

A Polymeric Nanomedicine Diminishes Inflammatory Events in Renal Tubular Cells

The polyglutamic acid/peptoid 1 (QM56) nanoconjugate inhibits apoptosis by interfering with Apaf-1 binding to procaspase-9. We now describe anti-inflammatory properties of QM56 in mouse kidney and renal cell models.In cultured murine tubular cells, QM56 inhibited the inflammatory response to Tweak, a non-apoptotic stimulus. Tweak induced MCP-1 and Rantes synthesis through JAK2 kinase and NF-κB activation. Similar to JAK2 kinase inhibitors, QM56 inhibited Tweak-induced NF-κB transcriptional activity and chemokine expression, despite failing to inhibit NF-κB-p65 nuclear translocation and NF-κB DNA binding. QM56 prevented JAK2 activation and NF-κB-p65(Ser536) phosphorylation. The anti-inflammatory effect and JAK2 inhibition by QM56 were observed in Apaf-1−/− cells. In murine acute kidney injury, QM56 decreased tubular cell apoptosis and kidney inflammation as measured by down-modulations of MCP-1 and Rantes mRNA expression, immune cell infiltration and activation of the JAK2-dependent inflammatory pathway.In conclusion, QM56 has an anti-inflammatory activity which is independent from its role as inhibitor of Apaf-1 and apoptosis and may have potential therapeutic relevance.

Distinct lines of chickens express different splenic cytokine profiles in response to Salmonella Enteritidis challenge

Chicken meat and eggs contaminated with Salmonella result in economic losses in the poultry industry and potential human infection. Intestinal parasites have been shown to lead to a reduction in the utilization of nutrients and performance in poultry. This study provides insight into the immune responses used by hens of 3 genetically distinct chicken lines (broiler, Leghorn, and Fayoumi) in the presence and absence of Salmonella Enteritidis infection. Understanding the range of immune responses used by different lines in response to Salmonella Enteritidis may help the poultry industry genetically select birds that are more pathogen resistant. The splenic mRNA levels of several immune-related genes [IL-6, IL-8, IL-10, IL-18, macrophage inflammatory protein 1β, interferon (IFN)-γ, transforming growth factor β1, and regulated upon activation, normal T cell expressed, and secreted (RANTES)] were analyzed by quantitative PCR. Line, challenge, and their interaction were considered fixed effects. Line had a significant effect on the mRNA expression of RANTES (P < 0.02) and IFN-γ (P < 0.03). Broilers expressed significantly more splenic RANTES mRNA than Fayoumis, and significantly more splenic IFN-γ mRNA than Leghorns (P < 0.05). There was a significant interaction of genetic line and challenge on IL-18 (P < 0.02) and IL-6 (P < 0.01) mRNA expression. Although there was a significant interaction of genetic line and challenge for IL-18, Tukey's test analysis only showed differences at a suggestive level (P < 0.1). Bacterial challenge had a significant effect on IL-6 mRNA expression only within the Fayoumi line. Challenged Fayoumis expressed significantly less IL-6 mRNA than nonchallenged Fayoumis (P < 0.05). The observed differences in mRNA expression of selected cytokines support the concept that these distinct genetic lines utilize different immune responses at homeostasis and in response to Salmonella Enteritidis infection.

The value of regulated upon activation normal T cell expressed and secreted in the pathogenesis of varicose ulcer of lower limbs

To study the effectiveness of RANTES in the pathogenesis of venous ulceration. 40 Chronic venous insufficiency (CVI) individuals were enrolled in the study and separated into the ulceration group (n=20) and non-ulceration group (n=20), according to CVI with or without ulceration, 10 healthy individuals were enrolled as control group. The expression of RANTES mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) in blood. Immunohistochemical staining of RANTES were carried in normal skin, in the tissue at the brim of ulceration and in the tissue of cured or curing ulceration. In ulceration group, the expression of RANTES mRNA in blood increased obviously. Compared with the other two groups, they were significant different (P < 0.05). The difference was also significance in the other two groups (P < 0.05). The average number of RANTES positive expression in normal skin is 28 +/- 13, in the tissue at the brim of ulceration is 107 +/- 44, and in the tissue of cured or curing ulceration is 35 +/- 20. There are significant difference between normal skin and the tissue at the brim of ulceration (P < 0.05). The average number of RANTES positive expression in the tissue of cured or curing ulceration is lower than that's in the tissue at the brim of ulceration. The high expression of RANTES may be one of the important mechanisms of varicose ulcer.

Rotavirus and coxsackievirus infection activated different profiles of toll-like receptors and chemokines in intestinal epithelial cells

To understand the inflammatory-immune response in intestinal epithelial cells after infection of rotavirus and coxsackievirus B3. We examined by quantitative PCR the expression profiles of genes encoding five toll-like receptors (TLR) and levels of three chemokines in response to rotavirus and coxsackievirus B3 infection in a human intestinal epithelial cell line (HT-29 cells). We demonstrated that rotavirus induced significantly increased levels of mRNA expression for TLR2, TLR3, TLR7 and TLR8 in HT-29 cells in a time-dependent manner. In contrast, coxsackievirus B3 did not stimulate mRNA expression for TLR3. Rotavirus and coxsackievirus B3 also induced higher levels of mRNA expression for RANTES, IP-10 and IL-8 during the period of infection in a different manner. Finally, significantly elevated levels of RANTES, IP-10 and IL-8 were detected by ELISA in rotavirus-infected cells from 24 to 48 h. Our findings suggest that different patterns of TLRs and chemokines were induced in the initiation and modulation of immune response to rotavirus and coxsackievirus B3 infection.

Immunity to islet grafts transduced with adenovirus vectors does not inhibit long-term islet function

Adenoviral gene transfer is a potential ex vivo gene therapy for islet transplantation. However, the immunogenicity of adenoviral vectors can potentially impair vector efficacy in transplants where long-term gene expression is required. We investigated the effect of this antiviral immunity on vector expression and islet graft function. Syngeneic murine islets transduced with adenovirus encoding β-galactosidase (AdCMVnt-βgal) were transplanted under the renal capsule. At different time points after transplant, blood glucose and glucose tolerance, intragraft cellular infiltration and IgG, IgM productions, and expression of transgene, cytokines and chemokines/receptors were assessed. Splenocytes and sera were analyzed to evaluate the systemic anti-adenoviral immune response. Diabetes was reversed in 1 day in recipients of a marginal amount (200) of untransduced islets, while AdCMVnt-βgal-transduced islets failed to reverse diabetes until 10 days post-transplant ( p ≤ 0.048). A profound and progressive infiltration with CD4 +, CD8 + cells, natural killer cells, and macrophages was identified in adenovirus transduced grafts, which paralleled a decline in β-gal expression. However, this did not extinguish transgene and insulin expression, which persisted for over 3 months. Glucose tolerance was impaired in transduced grafts at 25 days compared with non-transduced grafts ( p = 0.008), but was equivalent at 3 months ( p = 0.275) post-transplant. Intragraft interferon (IFN)-γ and RANTES mRNA expression was not different between transduced and untransduced islet grafts, despite an increased expression of CC chemokine receptor 5 (CCR5). No significant splenocyte IFN-γ and interleukin (IL)-4 production or serum anti-adenoviral antibodies were discovered albeit IgM antibody was detected in transduced grafts. Transduction of islets with adenoviral vectors results in overt local inflammation of islet grafts and inhibits early graft function. However, long-term graft function is preserved, and transgene expression persists. Thus, antivector immunity leads to neither loss of islet graft nor eradication of vector.

Human platelets can activate peripheral blood B cells and increase production of immunoglobulins

Blood platelets represent a link between hemostasis, inflammation, and tissue repair. Their role in immune responses and inflammation mainly involves many molecules, among which Toll-like receptor, major histocompatibility complex class I, CD40 and CD154/CD40 ligand (CD40L). As platelets are the major purveyor of soluble CD40L (sCD40L), we sought to determine their involvement in CD40/CD40L-dependent immune responses and to understand the interactions between platelets and peripheral B lymphocytes. We examined the capacity of platelets to bind nonstimulated B cells, and phenotypic changes by flow cytometry and confocal scanning laser microscopy. Modulation of cytokines/chemokines and total levels of immunoglobulin (Ig) A, IgG, IgM, and IgG subclasses in supernatants of coculture, platelets, and B lymphocytes was performed by sandwich enzyme-linked immunosorbent assay and differential production of cytokine mRNA as determined by reverse transcriptase polymerase chain reaction. In coculture, platelets and B lymphocytes were mutually activated, as demonstrated by the increased expression of platelet CD62p and B-cell CD86. Platelet/B-cell interactions were accompanied by changes in membrane expression of CD40 and CD40L by both platelets and B lymphocytes. IL12p70 and IL8 gene transcription were significantly reduced, which was attributable to B cells. Conversely, there was a significant, platelet-dependent reduction of sCD40L and RANTES mRNA expression. After a 3-day incubation with platelets, differentiated B cells increased their in vitro production of IgG1, IgG2, and IgG3, but not IgG4, IgA, or IgM. These data emphasize the potentially important role of platelets in the adaptive immune response. Platelets have an immunoregulatory role that might be applied clinically in multitransfused patients (e.g., hematopoietic stem cell transplantation).

Role of RIG-I, MDA-5, and PKR on the Expression of Inflammatory Chemokines Induced by Synthetic dsRNA in Airway Epithelial Cells

Background: We hypothesized that synthetic double-stranded (ds)RNA may mimic viral infection and reported that dsRNA stimulates expression of inflammatory chemokines through a receptor of dsRNA Toll-like receptor (TLR) 3 in airway epithelial cells. In this study, we focused our study on the role of other receptors for dsRNA, such as retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), and double-stranded RNA-dependent protein kinase (PKR). Methods: Airway epithelial cell BEAS-2B was cultured in vitro. Expression of target RNA and protein were analyzed by PCR and ELISA. To analyze the role of receptors for dsRNA, knockdown of theses genes was performed with short interfering RNA (siRNA). Results: We first investigated the effects of chloroquine, an inhibitor of lysosomal acidification, on the expression of chemokines. Preincubation with 100 µM chloroquine significantly inhibited the expression of mRNA for RANTES, IP-10, and IL-8, stimulated by poly I:C, indicating that poly I:C may react with a receptor expressed inside the cells. RIG-I, MDA-5, and PKR are supposed to be expressed inside the airway epithelial cells. However, the expression of chemokines stimulated with poly I:C was not significantly inhibited for these putative receptors in the cells which were transfected with siRNA. Conclusions: Synthetic dsRNA poly I:C stimulates the expression of inflammatory chemokines in airway epithelial cells, but the putative receptors for dsRNA such as RIG-I, MDA-5, or PKR may not play pivotal roles in this process. TLR3 may play a major role as reported previously.

Simvastatin Down Regulates mRNA Expression of RANTES and CCR5 in Posttransplant Renal Recipients With Hyperlipidemia

Chemokines and hyperlipidemia are involved in the mechanism of chronic allograft nephropathy (CAN). In this study, the mRNA expression of RANTES and its receptor CCR5 on peripheral blood mononuclear cells were measured in renal transplant recipients with hyperlipidemia, and the effect of simvastatin treatment observed to investigate the mechanism and prevention of CAN. Sixty recipients selected from 167 renal transplant recipients were divided into two groups: group A without hyperlipidemia ( n = 30) and group B with hyperlipidemia ( n = 30). The control group consisted of 30 healthy volunteers. The recipients in group B were treated with simvastatin for 3 months. We estimated serum lipid levels and mRNA expressions of RANTES and CCR5. The mRNA expressions of RANTES and CCR5 were significantly higher in renal transplant recipients compared with controls. The expressions were much higher in group B than in group A patients. In group B patients, serum lipid levels decreased dramatically after simvastatin treatment. Meanwhile, the mRNA expressions of RANTES and CCR5 were reduced significantly after 1.5 months of simvastatin treatment to a level significantly lower than that in group A after 3 months of treatment. The increased expressions of RANTES and CCR5 mRNAs in renal transplant recipients with hyperlipidemia might be involved in CAN due to hyperlipidemia. Simvastatin seemed to reduce the chemokine transcripts in renal recipients with hyperlipidemia.

Erhöhte CCR3- und Eotaxin-mRNA-Expression in eosinophilen Nasenpolypen

Background: Recent cytokine and chemokine research has rapidly expanded our understanding of nasal polyposis. Specific cytokines and chemokines that appear to play a central role in the pathogenesis of nasal polyposis, have now been identified. The objective of the present study was to investigate the mRNA expression of the chemokine receptor CCR3 and his ligands eotaxin and RANTES in nasal polyps in a precisely characterized patient population, with special attention to etiological factors and to compare the results with turbinate nasal mucosa of control subjects and within the patient group. Methods: Eosinophilic nasal polyps from 30 patients (comparable CT score and eosinophilia) and tissue samples taken from the inferior turbinate of 10 control subjects were obtained during endonasal surgery. Histologic examination was performed to determine the extent of eosinophilia. Realtime TaqMan PCR was performed to analyze and quantify CCR-3n eotaxin―, RANTES mRNA expression. The results were analyzed using the Mann-Whitney U test and the Spearman test. Results: In nasal polyps, CCR3― (p < 0,001) and eotaxin (p = 0,001) mRNA expression were significantly higher than in controls. There significantly correlation between CCR3― and eotaxin mRNA expression (p = 0,01). No correlation was detected between the CCR3 and RANTES mRNA values and the RANTES and eotaxin mRNA values. In nasal polyps of patients with ASS sensitivity and allergy, CCR3 mRNA expression was significantly evelated, compared with the other patients. There was no difference in RANTES mRNA expression between patients and controls, but within the patient group, non-allergic patients (p = 0,012) and allergic patients (p = 0,029) had increased RANT'ES mRNA values compared with patients suffering from ASS sensitivity and allergy. Conclusions: Our results suggest that the CCR3 eotaxin system plays a key role in the selective recruitment of eosinophils and in the development of nasal polyps. The differences in RANTES mRNA expression within the patient group indicate a disease-specific pathomechanism in patients with different etiological factors. Data may lead to the assumption that ASS sensitivity and also allergy are important etiological factors in the multifactorial genesis of polyposis nasi.

Influenza infected vitamin C deficient mice have increased lung pathology and an altered immune response to influenza virus infection

This study was designed to determine the effects of ascorbic acid deficiency on the immune response to influenza virus infection. Previous work in our laboratory has demonstrated that the antioxidant nutrients selenium and vitamin E are important mediators of the immune response to infection. Because one of the functions of vitamin C is as an antioxidant, we tested whether vitamin C could impact the immune response and/or lung pathology following influenza virus infection. L-gulono-γ-lactone oxidase gene inactivated mice (gulo −/− mice) require L-ascorbic acid supplementation in their drinking water for survival. Five-week old male and female gulo−/ − mice were provided with water or water containing 330 mg/mL ascorbic acid for three weeks prior to inoculation with influenza A/Bangkok/1/79. There were no differences in influenza lung titers between supplemented and unsupplemented groups, however lung pathology in the vitamin C deficient mice was decreased on days 1 and 3 post infection, but increased at day 7 compared with vitamin C sufficient mice. There was a gender difference on the effect of ascorbic acid on the immune response: male vitamin C deficient mice had a decreased expression of mRNA for RANTES, IL-1β and TNF-α in the lungs at 24 hours post-infection. However, 3 days post infection vitamin C deficient male mice had an increased expression of RANTES, MCP-1 and IL-12. None of these differences were observed in female vitamin C deficient mice. These data suggest that ascorbic acid is required for limiting lung pathology post infection and can influence the immune response. Supported by NIH grant R01 AI055050

Retinoic acid‐inducible gene‐I is induced by interleukin‐1β in cultured human gingival fibroblasts

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH box family protein, and details of its biological function are not known. We have studied the mechanism of the interleukin-1beta (IL-1beta)-induced RIG-I expression in human gingival fibroblasts in culture. We also addressed the possibility of enhanced expression of COX-2, RANTES and galectin-9 in fibroblasts overexpressed RIG-I. We stimulated cultured human gingival fibroblasts with IL-1beta and examined the expression of RIG-I mRNA and protein by reverse transcriptase-mediated polymerase chain reaction and Western blot analysis. The effect of cycloheximide, a protein synthesis inhibitor, on the IL-1beta-induced expression of RIG-I was examined. The expression of COX-2, RANTES, galectin-9 and monocyte chemoattractant protein-1 in gingival fibroblasts transfected with RIG-I cDNA was also examined. IL-1beta stimulated the expressions of mRNA and protein for RIG-I, in cultured fibroblasts, in a time- and concentration-dependent manner. Cycloheximide did not suppress the IL-1beta-induced RIG-I expression. Introduction of RIG-I cDNA into fibroblasts resulted in enhanced expression of COX-2 mRNA, and slightly enhanced the expression of mRNA for RANTES and galectin-9. In contrast, RIG-I overexpression did not alter the level of mRNA for monocyte chemoattractant protein-1. We conclude that IL-1beta stimulates RIG-I expression in human gingival fibroblasts.

Inhibition of HIV-1 replication by GB virus C infection through increases in RANTES, MlP-lα, MIP-1β, and SDF-1

Background People coinfected with HIV and GB virus C (GBV-C) have lower mortality than HIV-positive individuals without GBV-C infection. HIV uses either of the chemokine receptors CCR5 and CXCR4 for entry into CD4-positive cells. Longer survival in HIV-positive individuals is associated with high serum concentrations of ligands for CCR5 (RANTES [regulated on activation, normal T-cell expressed and secreted] and macrophage inflammatory proteins [MIP] 1alpha and 1beta) and CXCR4 (stromal-derived factor [SDF-1]), and with decreased expression of CCR5 on lymphocytes. Methods Peripheral-blood mononuclear cells were coinfected with GBV-C and HIV, and HIV replication was monitored by measuring infectivity and HIV p24 antigen production. Chemokine secretion was measured by ELISA, chemokine-receptor expression by flow cytometry, and cellular chemokine mRNA expression by differential hybridisation. Findings GBV-C infection of peripheral-blood mononuclear cells resulted in decreased replication of both clinical and laboratory HIV strains that use either CCR5 or CXCR4 as their coreceptor. Inhibition was related to the dose and timing of the GBV-C infection. Expression of mRNA for RANTES, MIP-1alpha, MIP-1beta, and SDF-1 and secretion of the chemokines into culture supernatants were higher in GBV-C-infected cells than in mock-infected cells. The inhibitory effect of GBV-C on HIV replication was blocked by incubation with neutralising antibodies against the relevant chemokines, and surface expression of CCR5 was significantly lower in GBV-C-infected cells than in mock-infected cells. Interpretation GBV-C induces HIV-inhibitory chemokines and reduces expression of the HIV coreceptor CCR5 in vitro. This study provides insight into the epidemiological association between GBV-C infection and longer survival in HIV-infected individuals.