Levels Of Related Cytokines Research Articles

Immunological Aspects of Fulminant Type 1 Diabetes in Chinese.

Background. Fulminant type 1 diabetes (FT1D) is a novel subtype of type 1 diabetes characterized by extremely rapid onset and complete deficiency of insulin due to the destruction of pancreatic β cells. However, the precise mechanisms underlying the etiology of this disease remain unclear. Methods. A total of 22 patients with FT1D and 10 healthy subjects were recruited. Serum antibodies to GAD, IA2, and ZnT8 in patients were tested. And peripheral T cell responses to GAD65, insulin B9–23 peptide, or C peptide were determined in 10 FT1D patients and 10 healthy controls. The mRNA levels of several related cytokines and molecules, such as IFN-γ, IL-4, RORC, and IL-17 in PBMCs from FT1D patients were analyzed by qRT-PCR. Result. We found that a certain proportion of Chinese FT1D patients actually have developed islet-related autoantibodies after onset of the disease. The GAD, insulin, or C peptide-reactive T cells were found in some FT1D patients. We also detected a significant increase for IFN-γ expression in FT1D PBMCs as compared with that of healthy controls. Conclusion. Autoimmune responses might be involved in the pathogenesis of Chinese FT1D.

Effects of transcutaneous acupoint electrical stimulation on the imbalance of Th1, Th2, Th17 and Treg cells following thoracotomy of patients with lung cancer.

An imbalance in the various T lymphocytes, including T-helper (Th)1, Th2 and Th17 cells, and regulatory T (Treg) cells, has been associated with immune dysfunction, and may occur following thoracotomy of patients with lung cancer. The use of transcutaneous acupoint electrical stimulation (TAES) has previously been demonstrated to exert immunoregulatory effects; therefore, the present study aimed to evaluate whether TAES was able to attenuate postoperative immune suppression in patients with lung cancer. Thoracic surgical patients with lung cancer (n=27) underwent TAES (frequency, 2/100 Hz; intensity, 4-12 mA) at the bilateral large intestine 4, pericardium 6, small intestine 3 and San Jiao 6 acupuncture points for 30 min, prior to incision, and at 20, 44, 68, 92 and 116 h following thoracotomy. The number of Th1, Th2, Th17 and Treg cells, and the protein and mRNA expression levels of related cytokines were measured by flow cytometry, ELISA and polymerase chain reaction, respectively. The balance of Th1, Th2, Th17 and Treg cells in the peripheral blood of patients with lung cancer was disrupted following thoracotomy. TAES administration increased the percentage of Th1 and Th17 cells, the protein expression levels of interleukin (IL)-2 and interferon-γ, the mRNA expression levels of T-bet and RAR-related orphan receptor-γt, and decreased the percentage of Th2 cells, IL-10 protein expression levels, and GATA binding protein 3 mRNA expression levels. The results of the present study demonstrated that TAES was able to partially attenuate the postoperative immune depression of patients with lung cancer, by regulating the balance of Th1, Th2, Th17 and Treg cells, and the expression levels of related cytokines and transcription factors; therefore, TAES may be considered to be a promising strategy for treating postoperative immune dysfunction in patients with lung cancer.

Blocking Stat3 to Regulate the Balance of Th17/Treg in Cgvhd Therapy

Background: Our preliminary research found that STAT3, IL-17A, and IL-21 expressed in cGVHD patients. So, we provide the blocking STAT3 signal to the induction of Treg cells differentiation, and to provide experimental basis on new targets of cGVHD immunotherapy.Methods:1. Mice spleen CD4+ CD62L+ naiveT cells were separated by immune magnetic bead and then activated for 72 h. After 96 h infection with STAT3-shRNA and negative control lentivirus, the Th subgroup proportion were measured by flow cytometry. Th related cytokines levels test by Luminex. Real time quantitative PCR was to detect STAT3 and Th subgroup related transcription factor mRNA levels. CD117+ mouse bone marrow stem progenitor cells were sorted by flow cytometry, and transfected by STAT3-shRNA. Inhibition of STAT3 gene in mRNA level was measured at 96 h. Cell proliferation activity was test with CCK8 kit, and cell apoptosis rate determined by flow cytometry. Differentiation of CD117+ cells was induced by 2.2% of methyl cellulose and different cytokines.2. BALB/c female mice, after the linear accelerator 700cGy of whole body irradiation, accepted miHA mismatched male B10. D2 mice bone marrow cells and spleen cells (8 x 106, 1:1). Randomly assigned 6 mice of cGVHD clinical score of 0.6 or above to each group. After STAT3-shRNA or negative control lentivirus treatment, the observe end point was 58th day after transplantation. The clinical and pathologic scores compared. Th17 and Treg cells measured by flow cytometry. Th related cytokines measured by Luminex. Purpose genes in blood and protein expression levels in target organs were found by Q-PCR and western blot test, respectively.Results:1. The Th17 / Treg ratio of shRNA group was significantly decreased than that in the NC group (P < 0.05). Except for the Foxp3 gene, other purpose genes, including T-bet, Gata3, RORγt, TGFβ, Notch1, and Jagged2 mRNA levelsin interference group were cut. GM-CSF, IFN-gamma, beta, IL- 3, IL-2, IL-4, IL-6, TNF alpha, IL-17, IL-22a, IL-27, and IL-9 factor expression levels were significant difference between shRNA and negative control group (P < 0.05). There was no significant difference of cell proliferation activity, early apoptosis rate, and differentiation ability in STAT3-shRNA treated CD117+ bone marrow, compared with negative control group and blank control group (P > 0.05).2. After 50th day, shRNA treatment group appear hair recovery, energy recovery, weight gain, shortness of breath better, mean of cGVHD score decreased. At the 58th day, clinical scores of cGVHD between shRNA treatment group and the negative control group overall mean difference was statistically significant (P < 0.05). cGVHD pathological score of lungs in shRNA treat group reduced (P < 0.05). STAT3mRNA levels in peripheral blood, phosphorylated STAT3, and STAT3 expression level of lung declined than control groups. The proportion of Th17 / Treg cells of spleen was significant reduced in shRNA group, compared with negative control group (P < 0.05).Conclusion:1. STAT3 knocking down in naïve CD4+ Th cells induced the increased Treg cells, and the decreased Th17 cells. IL-2 confirmed to promote the growth of Treg cells. It speculated that blocking STAT3 might bring Th9 cells differentiation. STAT3 blocking in CD117+ stem progenitor cells have no significant effect on the proliferation, apoptosis and differentiation, validation the safety of STAT3-shRNA.2. STAT3-shRNA treatment cGVHD mice in vivo achieved curative effect. The main target organs was the lung, which might be closely related to the fall in the proportion of Th17 /Treg. STAT3 may be used as a new target for immunotherapy of cGVHD.AcknowledgmentThe project was sponsored by grants from National Natural Science Foundation of China (No. 30972790; No.81270648; No.81370665; No.81300446), Provincial Natural Science Foundation of Guangdong (No. S2012010009560), Provincial Science and Technology Planning Project of Guangdong (No.2013B021800186; No.2013B021800201), and Science and Technology Planning Project of Guangzhou (No. 201400000003-4, 201400000003-1). DisclosuresNo relevant conflicts of interest to declare.

Evaluation of the circulating levels of IL-12 and IL-33 in patients with breast cancer: influences of the tumor stages and cytokine gene polymorphisms.

IL-12 as an anti-tumor cytokine and IL-33 a novel identified cytokine with both pro- or anti-tumor activities, play important roles in response against tumor cells. Our aim was to evaluate the IL-12 and IL-33 levels and single nucleotide polymorphisms (SNP) in their genes in patients with breast cancer. Blood samples were collected from 100 patients with breast cancer, and 100 healthy women were controls. The serum IL-12 and IL-33 levels were measured by ELISA. The SNP rs3212227 (in IL-12 gene) and rs1929992 (in IL-33 gene) were determined using PCR-RFLP. The IL-12 levels similarly expressed in patients and controls. IL-12 levels in patients at stage I were significantly lower than in the healthy group (P<0.05). IL-33 levels and the IL-33/IL-12 ratio were significantly higher in patients than the control group (P<0.001). The IL-33 levels and IL-33/IL-12 ratio in stage IV patients were significantly higher than other stages and controls (P<0.0001 and P<0.001, respectively). There were no significant differences in the frequencies of genotypes in rs3212227 and rs1929992 between patients and the control group. No significant differences were observed between subjects with various genotypes at rs3212227 and rs1929992 with respect to related cytokine levels. These results indicate that the diminished IL-12 production may contribute to the tumor establishment. The higher IL-33 levels and IL-33/IL-12 ratio in patients also indicate an imbalance in Th1/Th2 responses that may contribute to tumor development. Thus, correcting the imbalance of Th1/Th2 could be an important strategy for cancer immunotherapy.

Th17/Treg balance in children with obstructive sleep apnea syndrome and the relationship with allergic rhinitis

ObjectiveThis study aims to explore the role of the Th17 to Treg cell ratio in children with OSA and its relationship with allergic rhinitis. MethodsThe study included 127 children diagnosed with OSA by polysomnography (PSG) testing and 29 children without OSA. The 127 children with OSA were divided into the following groups: OSA with moderate adenoidal hypertrophy (n=47), OSA with severe adenoidal hypertrophy (n=49), and OSA complicated by allergic rhinitis (AR) (n=31). The adenoids of the 29 children without OSA were mildly hypertrophic. We measured the number of Th17 and Treg cells, the levels of related serum cytokines in cellular secretions, and the expression of key transcription factors in both the peripheral blood and adenoid tissue. The Th17/Treg ratio was calculated and analyzed between groups. The numbers of Th17 and Treg cells were measured by flow cytometry; the secreted IL-17, IL-10, and TGF-β were measured by ELISA; and the expression levels of RORγt and Foxp3 were measured by RT-PCR. ResultsCompared with the control group, OSA children exhibited a significant increase in the number of peripheral Th17 cells, Th17-related cytokine secretion (IL-17), and RORγt mRNA levels, whereas they exhibited a decrease in the number of Treg cells, Treg-related cytokine secretions (IL-10, TGF-β) and Foxp3 mRNA levels. The Th17/Treg ratio was higher (p<0.05) in the OSA groups than in the control group. The Th17/Treg ratio was correlated with the size of the adenoids. We also found that the Th17/Treg balance in OSA patients was complicated by allergic rhinitis; the increase was significantly larger in the AR group (p<0.05, p=0.021) than in OSA groups without AR. These results were observed in both the peripheral blood and local adenoid tissue. ConclusionThe Th17/Treg imbalance may increase the risk of developing OSA, and AR may promote the development of the disease. These results provide an alternative explanation for OSA pathogenesis that warrants additional research and presents new directions for the prevention and treatment of OSA in children.

The Roles of T Helper Type 17/Regulatory T Cells in Acute Rejection After Liver Transplantation in Rats

Liver transplantation is a widely accepted treatment for end-stage liver disease. The aim of our study was to investigate the imbalance between T helper type 17 (Th17) and CD4(+) CD25(+) Foxp3(+) regulatory T (Treg) cells during hepatic allograft rejection in a rodent grafts model because their roles in liver transplantation is not fully understood. We compared median survival time (MST) survivals, Th17/Treg cells and related cytokines levels (interleukin [IL]-17, IL-6, IL-10, and transforming growth factor-β1), as well as serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (T-Bili) after liver transplantation. In addition, the allograft groups were injected with IL-17 neutralizing antibody to evaluate the role of IL-17 in acute rejection. Th17 cells and related cytokines (IL-17 and IL-6) were obviously increased in the allograft group (all P < 0.01), whereas Treg cells and related cytokines (IL-10 and transforming growth factor-β1) were markedly lower than those of isograft and control groups (all P < 0.01). In addition, liver grafts in allograft groups rejected acutely (MST, 8.0 ± 1.2 days) accompanied by impaired liver function (AST, ALT, and T-Bili), while isograft groups survived long term (MST, 50.0 ± 3.8 days, all P < 0.01 vs. allograft group). Finally, neutralization of IL-17 in allograft groups significantly prolonged the survival (MST, 32.0 ± 4.8 days, P < 0.01), improved the liver function (AST, ALT, and T-Bili), and increased Treg cells in the liver (all P < 0.01). Th17 cells and secreted IL-17 play critical roles in acute rejection after liver grafts. The reduction of IL-17 may be 1 mechanism of immune tolerance after liver transplantation.

Differential Cytokine Changes in Patients with Myasthenia Gravis with Antibodies against AChR and MuSK.

Neuromuscular transmission failure in myasthenia gravis (MG) is most commonly elicited by autoantibodies (ab) to the acetylcholine receptor or the muscle-specific kinase, constituting AChR-MG and MuSK-MG. It is controversial whether these MG subtypes arise through different T helper (Th) 1, Th2 or Th17 polarized immune reactions and how these reactions are blunted by immunosuppression. To address these questions, plasma levels of cytokines related to various Th subtypes were determined in patients with AChR-MG, MuSK-MG and healthy controls (CON). Peripheral blood mononuclear cells (PBMC) were activated in vitro by anti-CD3, and cytokines were quantified in supernatants. In purified blood CD4+ T cells, RNA of various cytokines, Th subtype specific transcription factors and the co-stimulatory molecule, CD40L, were quantified by qRT-PCR. Plasma levels of Th1, Th2 and Th17 related cytokines were overall not significantly different between MG subtypes and CON. By contrast, in vitro stimulated PBMC from MuSK-MG but not AChR-MG patients showed significantly increased secretion of the Th1, Th17 and T follicular helper cell related cytokines, IFN-γ, IL-17A and IL-21. Stimulated expression of IL-4, IL-6, IL-10 and IL-13 was not significantly different. At the RNA level, expression of CD40L by CD4+ T cells was reduced in both AChR-MG and MuSK-MG patients while expression of Th subset related cytokines and transcription factors were normal. Immunosuppression treatment had two effects: First, it reduced levels of IL12p40 in the plasma of AChR-MG and MuSK-MG patients, leaving other cytokine levels unchanged; second, it reduced spontaneous secretion of IFN-γ and increased secretion of IL-6 and IL-10 by cultured PBMC from AChR-MG, but not MuSK-MG patients. We conclude that Th1 and Th17 immune reactions play a role in MuSK-MG. Immunosuppression attenuates the Th1 response in AChR-MG and MuSK-MG, but otherwise modulates immune responses in AChR-MG and MuSK-MG patients differentially.

Adipokine gene expression in peripheral blood of adult and juvenile dermatomyositis patients and their relation to clinical parameters and disease activity measures.

ObjectiveRecently adipokines have been implicated in the regulation of immune and inflammatory responses in autoimmune disease. To investigate the role of adipokines in adult and pediatric patients with newly diagnosed dermatomyositis (DM), we analyzed peripheral blood and skeletal muscle gene expression of four adipokines: visfatin, leptin, adiponectin and resistin.MethodsPeripheral blood mononuclear cells (PBMCs) were collected for 21 adult DM, 26 juvenile DM, 5 non-disease adult controls, and 6 non-disease pediatric controls at two time points: baseline and 6 months. Muscle biopsies from 5 adult DM patients and 5 non-disease adult controls were collected at baseline. Similarly, muscle biopsies from 7 juvenile DM patients and 5 non-disease pediatric controls were collected at baseline. The gene expression levels of leptin, adiponectin, resistin, visfatin and related inflammatory cytokines, IL-6, TNF- α, and housekeeping genes GAPDH, B2M, and ACTB were generated using a custom RT2 Profiler PCR Array.ResultsVisfatin gene expression levels in peripheral blood were significantly higher in newly diagnosed adult DM cases compared to non-disease controls (P = 0.004) and these levels correlated with baseline clinical parameters such as age (r = 0.34, P = 0.020), male sex (r = −0.35, P = 0.017), prednisone use (r = −0.42, P = 0.006), and DMARD use (r = 0.35, P = 0.025). No significant association was found between change in visfatin gene expression levels and change in disease activity measures. While visfatin gene expression was significantly up-regulated in muscle tissue of juvenile DM patients (P = 0.028), in adult DM patients only a trend towards significance was observed (P = 0.08). Also, muscle gene expression levels of resistin were significantly elevated in both adult and juvenile DM patients compared respectively to non-disease adult and pediatric controls. Furthermore, an association between peripheral blood resistin gene expression and DM disease activity, including global, muscle, and extra-skeletal disease activity was also observed.ConclusionPeripheral blood visfatin gene expression and muscle resistin gene expression are significantly increased in newly diagnosed adult DM patients. Further longitudinal studies should explore the possibility of using gene expression levels of adipokines such as visfatin and resistin as novel clinical diagnostic biomarkers in DM.

Lysophosphatidic acid alters the expression profiles of angiogenic factors, cytokines, and chemokines in mouse liver sinusoidal endothelial cells.

Background and AimsLysophosphatidic acid (LPA) is a multi-function glycerophospholipid. LPA affects the proliferation of hepatocytes and stellate cells in vitro, and in a partial hepatectomy induced liver regeneration model, the circulating LPA levels and LPA receptor (LPAR) expression levels in liver tissue are significantly changed. Liver sinusoidal endothelial cells (Lsecs) play an important role during liver regeneration. However, the effects of LPA on Lsecs are not well known. Thus, we investigated the effects of LPA on the expression profiles of angiogenic factors, cytokines, and chemokines in Lsecs.MethodsMouse Lsecs were isolated using CD31-coated magnetic beads. The mRNA expression levels of LPAR’s and other target genes were determined by quantitative RT-PCR. The protein levels of angiogenesis factors, cytokines, and chemokines were determined using protein arrays and enzyme immunoassay (EIA). Critical LPAR related signal transduction was verified by using an appropriate chemical inhibitor.ResultsLPAR1 and LPAR3 mRNA’s were expressed in mouse LPA-treated Lsecs. Treating Lsecs with a physiological level of LPA significantly enhanced the protein levels of angiogenesis related proteins (cyr61 and TIMP-1), cytokines (C5/C5a, M-CSF, and SDF-1), and chemokines (MCP-5, gp130, CCL28, and CXCL16). The LPAR1 and LPAR3 antagonist ki16425 significantly inhibited the LPA-enhanced expression of cyr61, TIMP-1, SDF-1, MCP-5, gp130, CCL28, and CXCL16, but not that of C5/C5a or M-CSF. LPA-induced C5/C5a and M-CSF expression may have been through an indirect regulation mechanism.ConclusionLPA regulated the expression profiles of angiogenic factors, cytokines, and chemokines in Lsecs that was mediated via LPAR1 and LPAR3 signaling. Most of the factors that were enhanced by LPA have been found to play critical roles during liver regeneration. Thus, these results may prove useful for manipulating LPA effects on liver regeneration.

Comparative Genomic Profiling of Synovium Versus Skin Lesions in Psoriatic Arthritis

ObjectiveTo our knowledge, there is no broad genomic analysis comparing skin and synovium in psoriatic arthritis (PsA). Also, there is little understanding of the relative levels of cytokines and chemokines in skin and synovium. The purpose of this study was to better define inflammatory pathways in paired lesional skin and affected synovial tissue in patients with PsA.MethodsWe conducted a comprehensive analysis of cytokine and chemokine activation and genes representative of the inflammatory processes in PsA. Paired PsA synovial tissue and skin samples were obtained from 12 patients on the same day. Gene expression studies were performed using Affymetrix HGU133 Plus 2.0 arrays. Confirmatory quantitative real-time polymerase chain reaction (PCR) was performed on selected transcripts. Cell populations were assessed by immunohistochemistry and immunofluorescence.ResultsGlobally, gene expression in PsA synovium was more closely related to gene expression in PsA skin than to gene expression in synovium in other forms of arthritis. However, PsA gene expression patterns in skin and synovium were clearly distinct, showing a stronger interleukin-17 (IL-17) gene signature in skin than in synovium and more equivalent tumor necrosis factor (TNF) and interferon-γ gene signatures in both tissues. These results were confirmed with real-time PCR.ConclusionThis is the first comprehensive molecular comparison of paired lesional skin and affected synovial tissue samples in PsA. Our results support clinical trial data showing that PsA skin and joint disease are similarly responsive to TNF antagonists, while IL-17 antagonists have better results in PsA skin than in PsA joints. Genes selectively expressed in PsA synovium might direct future therapies for PsA.

Protective effect of Th22 cells and intrahepatic IL-22 in drug induced hepatocellular injury

Th22 cells regulate host immunity against pathogenic invasion, including protecting host against chronic hepatitis B; however, the relationship between drug induced liver injury (DILI) and Th22/Th17 cells is still unclear. We investigated the role of Th22 cells in DILI development. The frequencies of peripheral Th22/Th17/Th1 cells and intrahepatic IL-22/IL-17 production from DILI, non-DILI liver diseases, and healthy controls were examined. Plasma IL-22/IL-17 and the related cytokines were determined in DILI patients at week 0 (defined as the occurrence of liver injury within 7days), 4 and 24. Multivariable stepwise logistic regression was applied to explore the associations between various factors and recovery of DILI. The frequencies of Th22/Th17 cells were significantly higher in DILI onset patients than HC. Significant increase of Th22 cells and the related cytokines levels was observed in DILI with hepatocellular injury type. There was a positive correlation between intrahepatic IL-22 level and liver regeneration. Plasma IL-22 level was higher in DILI patients with improved liver function than unimproved function. Multivariable analysis showed that the odds ratio (OR) of plasma IL-22 at 4weeks was 1.054 [95% confidence interval (CI), 1.012, 1.124]. Increased peripheral and intrahepatic IL-22-secreting cells are detected in DILI. Th22 and its related cytokines might be hepato-protective, which might provide new perspective for understanding the immunopathogenesis of DILI. Plasma IL-22 might be a reliable indicator to evaluate the prognosis of DILI and provide a novel therapeutic target for DILI treatment.

Potential relationship and clinical significance of miRNAs and Th17 related cytokines in patients with multiple myeloma

目的探讨miRNA和Th17相关细胞因子在多发性骨髓瘤(MM)患者发病中的作用机制。方法收集27例MM患者骨髓标本，以8名健康人为正常对照，应用荧光实时定量PCR(qRT-PCR)法检测其miR-15a/16、-34a、-194-2-192簇、-181a/b的表达情况，采用ELISA法检测患者血清Th17相关细胞因子IL-17、IL-21、IL-22、IL-23、IL-27的表达水平。分析miRNA与Th17相关细胞因子表达的相关性。结果与对照组比较，MM患者组miR-15a/16、-34a、-194-2-192簇表达水平降低，而miR-181a/b表达水平升高，差异均有统计学意义（P值均<0.05）；MM患者血清IL-17、IL-21、IL-27的表达水平升高，IL-22表达水平降低，差异均有统计学意义（P值均<0.05）；IL-23表达水平与对照组相比差异无统计学意义（P>0.05)。ISS分期Ⅲ期患者的miR-181a/b和IL-17、IL-21、IL-23、IL-27表达水平高于Ⅰ期患者，而miR-15a/16、-34a、-194和IL-22的表达水平则低于后者，差异均有统计学意义（P值均< 0.05）。miR-34a与IL-21，miR-181a与IL-23，miR-192与IL-21、IL-27，miR-194与IL-27的表达水平均呈正相关，而miR-192与IL-21呈负相关（P值均<0.05）。结论miRNA和Th17相关细胞因子在MM患者中均存在异常表达，其表达水平与MM患者的ISS分期有关，同时两者之间也存在密切的联系，提示失调的miRNA可能影响Th17细胞的分化进而参与MM的发生、发展。

Inhibition of Janus kinase-2 signalling pathway ameliorates portal hypertensive syndrome in partial portal hypertensive and liver cirrhosis rats

Background and aimsJAK2/STAT3 signalling promotes fibrosis, angiogenesis and inflammation in many diseases; however, the role of this pathway in portal hypertension remains obscure. This study aimed to explore the function of JAK2/STAT3 signalling in portal hypertension and estimate the potential therapeutic effect of treatment with the specific inhibitor AG490. MethodsRats induced by partial portal vein ligation and common bile duct ligation were treated with AG490 for two weeks. Haemodynamic parameters were assessed. The levels of phospho-STAT3 protein and related cytokines were detected by western blotting of splanchnic organs. Liver, spleen and intestine characterization was performed using histological analyses. Peripheral blood cell counts were also detected. ResultsHigh levels of phospho-STAT3 protein were detected in portal hypertensive rats. AG490 effectively inhibited JAK2/STAT3 signalling and its downstream cytokines and provided protective effects by decreasing splanchnic neovascularization and inflammation and by attenuating portal pressure and hyperdynamic splanchnic circulation. In cirrhosis rats, AG490 inhibited intrahepatic fibrosis, angiogenesis and inflammation. AG490 improved the peripheral blood cell counts and the splenomegaly observed in these rats. ConclusionsJAK2/STAT3 signalling is essential in portal hypertension, and targeting JAK2/STAT3 may be a promising therapy to treat this condition.

Murine β-defensin-2 may regulate the effect of bacillus Calmette-Guérin (BCG) in normal mouse bladder

PurposeWe investigated whether bacillus Calmette-Guérin (BCG)–induced secretion of murine β-defensin-2 (mBD2) and determined whether mBD2 regulated BCG effects in the normal mouse bladder. Materials and methodsA total of 140 C57BL/6 female mice were divided into 28 groups, and the experiment was performed over 3 steps. In the first step (20 groups), mice bladders were stimulated with different doses of BCG (multiplicity of infection [MOI] 0, 1, 10, 30, and 100) and histological analysis was conducted in bladder specimens isolated at different times (0, 4, 8, and 24h after instillation) to determine optimal dose and time point of BCG internalization and urine mBD2 and cytokine concentration. In the second step (4 groups), BCG internalization and urine cytokine levels were measured after pretreatment of different recombinant mBD2 (rmBD2) (0, 1, 2.5, and 5ng/ml) at optimal dose and time point. In the third step (4 groups), BCG internalization and urine cytokine levels were compared between pretreatment conditions (control, rmBD2, anti-mBD2 Ab, and rmBD2+anti-mBD2 Ab). Urine was collected for estimating mBD2 levels and a multiplex analysis for 9 cytokines. Real-time polymerase chain reaction assay was used for estimating the relative BCG cell number in mice bladder tissue. ResultsBladder edema was induced by BCG (MOI 30 and 100), which progressed to an inflammatory infiltrate composed primarily of neutrophils and increased mBD2 secretion at 4 hours after instillation. Relative BCG cell number and urinary cytokine levels (interferon-γ and interleukins [IL]-2, -4, -6, and -10) response pattern was characterized by a peak at 4 hours after instillation followed by rapid decline. The levels of interferon-γ, and IL-1β, -2, -4, -6, and -10 and relative BCG cell numbers decreased in a dose-dependent manner according to pretreatment with rmBD2 protein, and the responses were potentiated in the anti-mBD2 pretreatment group at 4 hours after BCG (MOI 30) instillation. ConclusionThe present results suggest that the mouse urothelium produces mBD2 in response to intravesicular BCG as a defense mechanism against BCG, and blocking mBD2 by an anti-mBD2 antibody increased the effectiveness of BCG.

Expression profile of Toll-like receptor mRNA in pigs co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2

Field and experimental studies have shown that co-infection of pigs with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) increases the severity of the disease. The present study investigates the mRNA expression profile of Toll-like receptors (TLRs) in pigs co-infected with PRRSV and PCV2. SPF pigs were infected with PRRSV, PCV2 or in a combination of both. The mRNA expression levels of TLRs and related cytokines in peripheral blood mononuclear cells (PBMCs) of pigs were determined by quantitative real-time RT-PCR. The mRNA expression profiles of TLRs by PBMCs from pigs co-infected with PRRSV and PCV2 displayed two distinct patterns: an increased expression profile for TLRs2, 4 and 8, and a decreased expression profile for TLRs3, 7 and 9. An up-regulated expression of IL-1β and IL-10 mRNA and a down-regulated expression of INF-α and TNF-α mRNA in PBMCs of co-infected pigs were also observed.

Endoplasmic reticulum stress preconditioning antagonizes low-density lipoprotein-induced inflammation in human mesangial cells through upregulation of XBP1 and suppression of the IRE1α/IKK/NF-κB pathway.

Elevated plasma low‑density lipoprotein (LDL) is associated with systemic inflammation, and is an important factor in the pathogenesis of chronic kidney disease. The aim of the present study was to investigate the effects of endoplasmic reticulum (ER) stress preconditioning on LDL‑induced inflammatory responses, in human mesangial cells (HMCs). HMCs were exposed to LDL (200 nm), with or without pretreatment with tunicamycin, an ER stress inducer, and tested for changes to gene expression levels. Small interfering RNA technology was used to knockdown the expression of inositol‑requiring enzyme‑1α (IRE1α) and X‑box‑binding protein‑1 (XBP‑1), in order to determine their effects on LDL‑treated HMCs. LDL treatment resulted in a significant, and time‑dependent, increase in the relative mRNA expression levels of proinflammatory cytokines and CD40, which was coupled with enhanced phosphorylation of IRE1α, IκB kinase (IKK), and nuclear factor (NF)‑κB p65 and p65 nuclear translocation. The LDL‑induced inflammatory responses were significantly reduced in the IRE1α‑depleted HMCs. Furthermore, pretreatment with tunicamycin significantly attenuated the induction of proinflammatory cytokines and CD40, by LDL. Whereas, silencing XBP1 expression significantly restored the production of proinflammatory cytokines, in the LDL‑treated HMCs with ER stress preconditioning. The phosphorylation levels of IRE1α, IKK, and NF‑κB p65 were markedly increased in the XBP1‑depleted HMCs. Conversely, overexpression of XBP1 blocked LDL‑induced inflammation in the HMCs. The results of the present study demonstrate that ER stress preconditioning antagonizes LDL‑induced inflammatory responses in HMCs, which may be mediated through upregulation of XBP1, and subsequent inactivation of the IRE1α/IKK/NF‑κB pathway.

Allergenicity of two Anisakis simplex allergens evaluated in vivo using an experimental mouse model

Anisakis (Anisakidae) is one of the most important causes of helminth-induced allergic reactions and elicits clinical responses that include urticaria, rhinitis, bronco-constriction, cough, and/or gastrointestinal symptoms. More than 13 reactive allergens have been identified in the serum of Anisakis allergy patients, but the allergenicity of only a few of these have been evaluated in vivo using a mouse model. To evaluate the allergenicity of two important allergens, Ani s 1 and Ani s 9, we induced experimental allergic airway inflammation in a mouse model by repeated intranasal administration of the allergens. Both recombinant proteins (rAni s 1 and rAni s 9) elicited increased airway hyperresponsivity, airway infiltration by inflammatory cells (especially eosinophils), bronchial epithelial cell hyperplasia, all of which are characteristic of allergic airway inflammation. These allergens significantly increased the levels of Th2-related cytokines (IL-4, IL-5, IL-13, and IL-25) and Th17 related cytokines (IL-6 and IL-17) in both splenocytes and airway (except IL-17 in airway by rAni s 9). OVA-specific IgE and total IgE were increased in rAni s 1 and rAni s 9 treated mice as compared with controls treated with OVA alone. In addition, these two allergens induced gene expression of thymic stromal lymphopoietin (TSLP) and IL-25 (initiators of the Th2 response), as well as CXCL1 (initiator of the Th17 response) in mouse lung epithelial cells. In conclusion, repeated intranasal treatments with rAni s 1 and rAni s 9 induced airway inflammation in mice by elevating of Th2 and Th17 responses in the lung.

The combination of tumour necrosis factor-α -308A and interleukin-10 -1082G gene polymorphisms and increased serum levels of related cytokines: susceptibility to vitiligo.

The aetiopathogenesis of vitiligo is still under investigation. To assess the role of single nucleotide polymorphisms (SNPs) of the genes for tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10, as well as the serum levels of these three cytokines in the pathogenesis of vitiligo. The study enrolled 105 patients with vitiligo, and 211 age- and sex-matched controls. TNF-α (-308), IL-6 (-174) and IL-10 (-1082) promoter polymorphisms were investigated by LightSNiP assay and analysed by χ(2) test. Subsequently, the serum cytokine levels were assessed by ELISA and evaluated by Mann-Whitney U-test and Kruskal-Wallis test. The frequency of the GG genotype of the IL-10 -1082 polymorphism was significantly higher in the vitiligo group compared with the healthy control group (P=0.02). Further investigations using combinations of these variant alleles detected a significant risk for vitiligo for individuals carrying both the IL-10 -1082G and TNF-α -308A alleles (OR=12.57, 95% CI 1.44-110.0, P<0.01). Serum IL-10 and TNF-α levels were higher in the vitiligo group (P=0.001). In addition, TNF-α levels in patients with active disease were significantly higher than in patients with stable disease (P<0.02). The concomitant presence of IL-10 -1082G and TNF-α -308A alleles significantly raises the risk for vitiligo. Furthermore, in accordance with these findings, serum IL-10 and TNF-α were also increased in this study, confirming the role of these cytokines in the pathogenesis of vitiligo.

Downregulation of Th17 cells and the related cytokines with treatment in Kawasaki disease

Given the inflammatory nature of Kawasaki disease (KD) and the pro-inflammatory properties of Th17, this study aimed to determine the frequency of Th17 cells and the levels of corresponding cytokines in acute phase of KD and to evaluate their alterations one and eight weeks after treatment. Th17 and the related cytokine levels were measured in 21 KD patients and 42 positive and negative controls, using flow cytometry and ELISA, respectively. Th17, IL-17, IL-22 and IL-23 were significantly higher (P<0.05) in patients compared with negative controls, but no significant differences (P>0.05) with the positive controls. Furthermore, Th17, IL-17, IL-22 and IL-23 were significantly higher in patients before treatment than those one and eight weeks after. Considering the downregulation of Th17 and its related cytokines with aspirin and intravenous immunoglobulin therapy implies the probable role of Th17 in KD pathogenesis.

530P - Angiogenic Switch As Predictor of Response to Chemotherapy+ Bevacizumab in Patients with Metastatic Colorectal Cancer

ABSTRACT Aim: There is a need to develop biomarkers to predict the outcome of patients with metastatic colorectal cancer (CRC) treated with chemotherapy (Ch) plus Bevacizumab (B). It has been reported that during exposure to B there is a switch in the plasma levels of angiogenesis growth factors and related cytokines called angiogenic switch (AS). It is not known if changes in these circulating factors affect the response of patients with CRC to Ch + B. The aim of this study is to determine the influence of AS in the PFS in pts with metastatic CRC treated with Ch + B. Methods: Treatment naive, ECOG 0-1, metastatic CRC pts were eligible. Patients received induction treatment with XELOX-B or XELIRI-B at standard doses x 6 cycles followed by maintenance treatment with B plus Capecitabine until progression. Angiogenic related cytokines (HGF, PIGF, MCP-3, MM-9, Eotaxin, bFGF and IL-18) were prospectively analyzed at baseline and at the time of each CT evaluation (every eight weeks). AS + was defined by A) doubling of PIGF compared to baseline or B) PIGF elevation with a simultaneous elevation of any two of bFGF, HGF, MCP-3, IL-18 or MMP-9 compared to baseline. Results: Of 62 patients enrolled, 24 (36.7%) have progressed. Median PFS is 15.6 (95% CI: 8.3 to 16.4) months for the entire population. Twenty-five pts (40.3%) were AS + , out of which 23 were detected at first on-study evaluation (week 8). 18 were classified as AS+ based on both criteria, 4 based on criteria A) and 3 based on criteria B). One year (95% CI) PFS for AS+ pts was 82.6% (53.0% to 94.4%) versus 32.9% (13.9% to 53.5%) for AS- pts (p Conclusions: There is a statistically significant improved PFS amongst metastatic CRC AS+ pts treated with first line Ch + B combination compared with AS–pts. Disclosure: All authors have declared no conflicts of interest.