Blood samples (5 mL) to determine sST2 concentration were collected upon admission to the hospital using stan- dard collection techniques into vacuum tubes containing a clot activator. The samples were centrifuged for 5 min at 3000 rpm after the formation of a clot, and the super- natant (serum) was immediately separated and frozen at -76°C. Then, after thawing the serum, the soluble ST2 level was measured by quantitative assay using a sand- wich ELISA kit (Medical & Biological Laboratories Co., Ltd, Nagoya, Japan) validated for use with human serum. This kit uses 2 monoclonal antibodies against 2 differ- ent epitopes of human sST2. The serum samples were incubated in microwells coated with the first antihuman sST2 monoclonal antibody. After the washing stage, the second incubation with the peroxidase conjugated anti- human sST2 monoclonal antibody was conducted. After further washing, the peroxidase substrate was added to each well and the optical density was measured at 450 nm using a microplate reader.
Hospital, Gwagwalada, Abuja. The project was supported by Award Number D43TW008330 from the Fogarty Interna- tional Center. The content is solely the responsibility of the authors and does not necessarily represent the official view of the Fogarty International Center or the National Insti- tutes of Health. The authors would like to acknowledge the funding source for the biomarker assays: National Research Foundation South Africa, SERVIER and the Maurice Hat- ter Foundation. The abstract of this paper was presented at the World Congress of Cardiology, June 2016, in Mexico City, Mexico, as a poster presentation. We found that in a cohort of hypertensive HF subjects, soluble ST2 correlated significantly with RVSP, RVDD and right atrial. The poster’s abstract was published in Journal of Global Health 2016.
sST2, soluble ST2; WC, waist circumference; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HR, heart rate; Total-C, total cholesterol; TG, triglycerides; LDL-C, low-density lipoprotein-cholester- ol; HDL-C, high-density lipoprotein-cholesterol; AST, aspartate transaminase; ALT, alanine transaminase; BUN, blood urea nitrogen; GGT, gamma glutamyl- transferase; WBC, white blood cell count; CACS, coronary artery calcium score; hsCRP, high-sensitivity C-reactive protein.
ronchial asthma is thought to be T helper 2 (Th2) cell-mediated immune diseases. Th2 cells produce cytokines, such as interleukin (IL)-33 which is also a chemoattractant for human Th2 cells. IL-33 is produced by mast cells after immunoglobulin (Ig) E-mediated activation and is able to trigger mast cells to release proinflammatory cytokines in vitro  . IL-33 is a member of the IL-1 family of cytokines and binds to two receptors: ST2 (IL-1R1) and IL-1 receptor accessory protein (IL-1RAP). There are two isoforms of ST2 proteins: ST2L, a transmembrane form, and soluble ST2 (sST2), a secreted form that can serve as a decoy
Growth stimulation expressed gene 2 (ST2) is a mem- ber of the Toll-like/interleukin-1 receptor superfamily [14, 15]. It has two isoforms: transmembrane ST2 expressed on the cell surface and soluble ST2 (sST2) in the serum . sST2 expression is elevated in several in- flammatory diseases, including atopic individuals with allergic symptoms or exacerbation of asthma [17, 18], atopic dermatitis , rheumatoid arthritis [20, 21], ul- cerative colitis, Crohn’s disease , and systemic lupus erythematosus . In liver diseases, sST2 was reported to positively correlates with alanine aminotransferase (ALT) levels in chronic hepatitis patients . And it has been proved to be a promising prognostic biomarker in HBV-related acute-on-chronic liver failure .
14. Chapuis J, Hot D, Hansmannel F, Kerdraon O, Ferreira S, Hubans C, Maurage CA, Huot L, Bensemain F, Laumet G, Ayral AM, Fievet N, Hauw JJ, DeKosky ST, Lemoine Y, Iwatsubo T, Wavrant-Devrièze F, Dartigues JF, Tzourio C, Buée L, Pasquier F, Berr C, Mann D, Lendon C, Alpérovitch A, Kamboh MI, Amouyel P, Lambert JC. Transcriptomic and genetic studies identify IL-33 as a candidate gene for Alzheimer ’ s disease. Mol Psychiatry. 2009;14:1004 – 16. 15. Díaz-Jiménez D, Núñez LE, Beltrán CJ, Candia E, Suazo C, Alvarez-Lobos M, González M-J, Hermoso MA, Quera R. Soluble ST2: a new and promising activity marker in ulcerative colitis. World J Gastroenterol. 2011;17:2181 – 90. 16. Dignass A, Van Assche G, Lindsay JO, Lémann M, Söderholm J, Colombel JF,
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reduced adiposity with reduced fasting glucose and improved glucose/insulin tolerance in ob/ob mice, and reduced cardiac hypertrophy and fibrosis in a murine model of pressure overload [8,9,10]. Furthermore, ApoE 2/2 mice treated with sST2 developed significantly larger atherosclerotic plaques in the aortic sinus compared to control mice, and sST2 reversed the anti-hypertrophic effects of IL-33 on cultured cardiomyocytes  . Thus, the soluble form of ST2 may play an important role in negatively regulating a putative protective role for IL-33 in the cardiovascular system. In humans, elevated circulating levels of sST2 are associated with adverse prognosis in patients with acute myocardial infarction (AMI) [11,12] and heart failure [13,14]. Recently, some studies have shown that sST2 levels are higher in patients with prevalent CVD who also have diabetes [12,15,16,17]. However, so far the association between sST2 levels with cardiometabolic markers has not been carefully studied in any general population cohort.
How these genetic variants differentially relate to these previ- ously described immune and inflammatory diseases compared with cardiovascular disease is unknown. We found 2 variants with suggestive associations with clinical phenotypes. The directional- ity suggests that variants with lower sST2 concentrations may be associated with higher C-reactive protein and adverse outcomes, but these data should be considered hypothesis generating. This association is counter to clinical observations, in which higher sST2 concentrations were associated with adverse outcomes (19). Experimental data support an overall protective effect of IL33/ST2 signaling; activation of membrane-bound ST2L by IL33 leads to reduced hypertrophy and fibrosis (9). The genetic variants studied in our manuscript are located in membrane-bound ST2L, and we show that genetic variants in membrane-bound ST2L enhance both sST2 expression and also IL-33 responsiveness of ST2L itself. Thus, genetic variants likely influence both soluble and membrane-bound ST2. It may be that increased expression of ST2 reflects a compen- satory response to cardiac stress, as seen with other molecules (such as BNP), in which the association of genetic variants with outcomes has the opposite directionality from circulating levels (39). We found strong evidence linking IL1RL1 genotypes with soluble ST2; on the other hand, the associations with clinical outcomes warrant further confirmation. Furthermore, because variants in this region are in high linkage disequilibrium, it is not possible to determine whether the effect is due solely to variants in IL1RL1 or neighboring loci within the linkage disequilibrium block, and identification of other causal variants requires additional study.
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Novel biomarkers such as soluble ST2 (sST2), Interleukin-34 (IL34), some traditional biomarkers as BNP, and TNF-α, could be used in diagnosis and prognosis of patients with chronic heart failure (CHF). This study aimed at evaluating the clinical effect and economic impact values of these above-mentioned biomarkers in CHF patients. 41 CHF stage III patients with LVEF ≤ 40 and thirty healthy control volunteers were recruited and accessed via clinical examination, echocardiography, lipid profile, serum electrolytes, and serum biomarkers. Data showed that, serum levels of the four studied biomarkers were significantly elevated in CHF patient as compared to the control group (P< 0.05). A significant negative correlation was observed between serum levels of these biomarkers and LVEF. A significant positive correlation was observed between CRI-I and CRI-II and total cholesterol, LDL-C, sST2, and IL-34 (P< 0.05). It was found that, sST2 is the most prominent predictor in providing independent and additive prognostic information of mortality (P= 0.043) and the most cost-effective biomarker as compared to EF, IL-34, BNP and TNF-α. Elevated serum levels of these novel and traditional biomarkers may be used as indicator for risk stratification and can also, be applied as independent predictors for the mortality among CHF patients. Using specific and cost-effective biomarker may reduce the numbers of patients requiring echocardiography and may help in early diagnosis that, may help in selecting the evidence-based therapy that, in turn, may modify patient’s outcomes, and useful to follow the effectiveness of the treatment.
32. Wenisch, C., S. Varijonantoa, S. Looareesuwan, W. Graninger, R. Pichler, and W. H. Wernsdorfer. 1994. Soluble intercellular adhesion molecule-1 (ICAM-1), endothelial leukocyte adhesion molecule-1 (ELAM-1) and tumor necrosis factor receptor (55kDa TNF-R) in patients with acute Plasmodium falciparum malaria. Clin. Immunol. Immunopathol. 71:344–348.
, significantly higher than that of the control treatment (11.92 mg.L -1 ). However, at the concentrations of 1 and 2% NaCl, IAA productions produced by ST2-1 were not significantly different with each other, but these two IAA productions were significantly higher than that of the treatment with 3% NaCl. Thus, this result showed that the maximum IAA (28.65-29.55 mg.L -1 ) productions were found at 1 and 2% NaCl and this strain can tolerant the environmental stress to 3% NaCl. In the case of IAA production, IAA is an auxin required by most plant cells for division and root initiation . Egamberdiyeva (2009)  reported that IAA-producing bacteria significantly increased plant growth under salt stress. In addition, Nakbanpote et al., (2014)  also reported that Pseudomonas sp. PDMZnCd2003 isolated from from a Zn/Cd contaminated soil was classified as salt-tolerant bacteria. This bacteria had indole-3-acetic acids (IAA) production, nitrogen fixation, and phosphate solubilization, under 8% (w/v) NaCl condition, moreover, this strain stimulated the germination and seedlings of Oryza sativa L.cv. RD6 under a salinity of 0–16 dS.m -1 . Therefore, the isolate of ST2-1 which was halotolerant bacteria is considered as plant growth promoting bacteria.
pipe is a highly conserved gene in the insects. In insects other than the cyclorrhaphan (true) flies, only a single Pipe isoform is normally present. Moreover, the individual Pipe isoforms that are predicted to be expressed in mosquito (Anopheles gambiae), silkworm (Bombyx mori), honeybee (Apis mellifera) and flour beetle (Tribolium casteneum) exhibit greatest sequence similarity to the Pipe-ST2 isoform, among the multiple isoforms expressed by Drosophila melanogaster. By contrast, the genomes of all Drosophila species that have been sequenced (D. ananassae, D. erecta, D. grimshawi, D. mojavensis, D. persimilis, D. pseudoobscura, D. sechellia, D. simulans, D. virilis, D. willistoni and D. yakuba) predict multiple Pipe protein isoforms. Taken together, these observations suggest that an insect ancestral to the holometabolous Diptera, Coleoptera, Hymenoptera and Leptidoptera expressed a single Pipe isoform, the role of which was to regulate the formation of the embryonic DV axis. Pipe protein may also regulate DV axis formation in more basal genera of insects and in other arthropods, as the genome of the water flea (Daphnia pulex), a crustacean, apparently encodes an orthologous protein that is more similar to Pipe-ST2 than to HS2ST or D/C2ST (http:/wfleabase.org).
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ST2 is an IL-1 receptor family member with transmembrane (ST2L) and soluble (sST2) isoforms. sST2 is a mechanically induced cardiomyocyte protein, and serum sST2 levels predict outcome in patients with acute myocardial infarction or chronic heart failure. Recently, IL-33 was identified as a functional ligand of ST2L, allowing exploration of the role of ST2 in myocardium. We found that IL-33 was a biomechanically induced protein predominantly synthesized by cardiac fibroblasts. IL-33 markedly antagonized angiotensin II– and phenylephrine-induced cardiomyocyte hypertrophy. Although IL-33 activated NF-κB, it inhibited angio- tensin II– and phenylephrine-induced phosphorylation of inhibitor of NF-κBα (IκBα) and NF-κB nuclear binding activity. sST2 blocked antihypertrophic effects of IL-33, indicating that sST2 functions in myo- cardium as a soluble decoy receptor. Following pressure overload by transverse aortic constriction (TAC), ST2 –/– mice had more left ventricular hypertrophy, more chamber dilation, reduced fractional shortening,
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Materials—Human embryonic kidney 293 and EL4 murine thymoma cells were obtained from the European Collection of Animal Cell Cul- tures (Salisbury, UK). Cell culture media and serum were obtained from Invitrogen. The murine mastocytoma cell line P815 was a gift from Dr. T. Kamradt (Deutches Rheumaforchungszentrum, Berlin, Germany). The IL1RAcP-deficient cell line EL4 D6/76 was a kind gift from Prof. Werner Falk (Universita¨t Regensburg, Regensburg, Germa- ny). Human recombinant IL-1 ␣ was a gift from the NCI, National Institutes of Health, Biological Resources Branch (Rockville, MD). The 22-bp oligonucleotide, 5 ⬘ -AGT TGA GGG GAC TTT CCC AGG C-3 ⬘ , containing the NF-B consensus sequence (underlined), and the T4 polynucleotide kinase were obtained from Promega (Madison, WI). PhosphoPlus™ SAPK/JNK (Thr-183/Tyr-185) and p42/p44 MAP kinase (Thr-202/Tyr-204) antibody kits and the phospho-c-Jun (Ser-63) II and c-Jun antibodies were obtained from New England Biolabs, Ltd. (Hitchen, UK). The monoclonal anti-T1/ST2 antibody 3E10 has been described previously (9). The anti-FLAG M2 antibody was obtained from Sigma. The I B antibody was a kind gift from Prof. R. Hay (University of St. Andrews, St. Andrews, UK). The JNK inhibitor SP600125 was purchased from Calbiochem (CN Biosciences, Notts, UK).
The work presented in this dissertation investigates the interaction of activated CD8 + T cells arising in the context of systemic inflammation with their surrounding environment, both in their response to antigen-independent cues and their contribution to the cytokine storm. We have shown that the IL-33/ST2 pathway plays a key role in amplifying inflammation above the threshold for fatal disease in FHL2 mice. ST2 intrinsically promotes LCMV-specific CD8 + and CD4 + T cell proliferation and potentiates IFNγ production, thereby raising systemic quantities of IFNγ to lethal levels. Blockade of ST2 signaling provides therapeutic benefit in murine FHL by acutely dampening the antiviral T cell response, which prolongs the survival of the mice and thus enables CD8 + T cell exhaustion to arise and mediate long-term protection from morbidity. In a different model of hemophagocytic syndrome, TLR9-MAS, a unique population of effector-like IL- 10-producing CD8 + T cells arises independently of antigen stimulation and accumulates within damaged liver. These studies provide new insight into the role of CD8 + T cells in hemophagocytic syndromes, but several important questions remain.
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In summary, this report demonstrates the occurrence, persis- tence, and potential spread of an MDR genotype of S. epidermidis causing health care-associated infections in an Australian teaching hospital. This genotype (ST2) accounted for 85% of isolates tested, was indistinguishable from an MDRSE genotype identified FIG 1 Cluster analysis of the genetic similarity of 27 isolates of multidrug-resistant Staphylococcus epidermidis using pulsed-field gel electrophoresis (PFGE). The horizontal upper bar represents genetic similarity (percent). The dotted lines in the center represent digitalized transformation of the PFGE-DNA pattern. Source of culture, PFGE type, sequence type (ST), ward, and year of isolation are described in the columns to the right. a BMTU, bone marrow transplant unit.
Experimental autoimmune encephalomyelitis (EAE) mice were administered with murine anti-CD52 antibody to investigate its therapeutic effect and whether the treatment modulates IL-33 and ST2 expression. EAE severity and central nervous system (CNS) inflammation were reduced following the treatment, which was accompanied by peripheral T and B lymphocyte depletion and reduced production of various cytokines including IL-33, while sST2 was increased. In spinal cords of EAE mice, while the number of IL-33 + cells remained unchanged, the extracellular level of IL-33 protein was significantly reduced in anti-CD52 antibody treated mice compared with controls. Furthermore the number of ST2 + cells in the spinal cord of treated EAE mice was downregulated due to decreased inflammation and immune cell infiltration in the CNS. These results suggest that treatment with anti-CD52 antibody differentially alters expression of IL-33 and ST2, both systemically and within the CNS, which may indicate IL-33/ST2 axis is involved in the action of the antibody in inhibiting EAE.
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The immune cells expressing ST2 are as fol- lows: Th2 lymphocytes, NK cells, NKT cells, mast cells, macrophages, monocytes, dendritic cells, epithelial cells, neutrophils and so forth [10, 14, 17, 18]. IL-33 binds to ST2 and acti- vates downstream signals: the adaptors (e.g. MyD88, MAL or TIRAP, TRIF or TICAM1, TRAM or TIICAM2, SARM, et al.) interact with the recep- tor intracellular domain and IRAK; recruits TR- AF6 ; activates MEKKK which activates p38MAPK, NIK1 which activates IκB, and TA- K1 which activates NF-κB, JNK or p38MAPK. Besides, IL-33 indirectly regulates TNFα and IL-1β through binding to IL-1RAcP [20, 21], induces the expression of INF-γ, these demon- strates that in addition to mediate Th2 inflam- matory response via ST2 receptor, IL-33 also exerts other functions not through ST2 rece- ptor.
Figure 3. The failure of IL-33 to induce mast cell degranulation is associated with lack of intracellular mobilisation of calcium. PDMCs (a,c) and BMMCs (b,d) were sensitized with murine anti-DNP IgE (0.5 μ g/ ml) or not (a,b) and then stimulated with DNP-HSA (0.5 µg/ml) to induce cross-linking of Fc ε RI (XL), IL-33 (10 ng/ml) or PMA plus Ionomycin (both 1 μ M) for 30 mins at 37 °C. Degranulation was determined as the % β -hexosaminidase released relative to the total activity of the cells and data are presented from a single experiment (a,b) or mean ± SEM values from the combined results of five (c) or three (d; IL-33, n = 2) independent experiments where statistical analysis was by one-way ANOVA with Bonferroni’s post test and *p < 0.05, **p < 0.01 and ***p < 0.001. WT or ST2 −/− PDMCs (e–g) and BMMCs (h,i) loaded with Fura-2/
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Experimental studies — role of IL-33/ST2 in endotoxemia The role of the IL-33-ST2 axis has been extensively studied in experimental endotoxemia. Even before the identification of IL-33, it was demonstrated that the ST2 receptor func- tions as a negative regulator of TLR4 signaling and main- tains LPS tolerance . In these studies, ST2-deficient mice did not develop endotoxin tolerance . Specifically, Liu et al.  found that ST2 also negatively regulates TLR2 signaling but is not required for bacterial lipoprotein- induced tolerance. A plausible explanation for these differ- ences may lie in the unique signaling transduction and mo- lecular mechanisms of TLR4-mediated tolerance (LPS tolerance) vs TLR2-mediated tolerance (BLP tolerance). Despite the implicated roles of ST2 in endotoxin tolerance, IL-33 triggering of ST2 failed to induce LPS desensitization but instead enhanced the LPS-induced proinflammatory cytokine production (IL-6, TNF-α and IL-1β) in mouse mac- rophages . This effect is ST2 dependent, as it was not observed in ST2 knockout mice . IL-33 treatment in- creases macrophage expression of the MD2/TLR-4 compo- nents of the LPS receptor as well as levels of the soluble form of CD14, and preferentially affects the MyD88- dependent pathway downstream of TLR-4 and TLR-2, which may explain the enhanced LPS responses of macro- phages . These conflicting results indicate distinct roles for IL-33 and ST2 in the pathogenesis of LPS responses.