Th2 cells are generated from naive CD4 T cells upon T cell receptor (TCR) recognition of antigen and IL-4 stimulation and play crucial roles in humoral immunity against infectious microorganisms and the pathogenesis of allergic and autoimmune diseases. A tyrosine phosphatase, SHP-1, that contains src homology 2 (SH2) domains is recognized as a negative regulator for various intracellular signal- ing molecules, including those downstream of the TCR and the IL-4 receptor. Here we assessed the role of SHP-1 in Th1/Th2 cell differentiation and in the development of Th2-dependent allergic air- way inflammation by using a natural SHP-1 mutant, the motheaten mouse. CD4 T cells appear to develop normally in the heterozygous motheaten (me/+) thymus even though they express decreased amounts of SHP-1 (about one-third the level of wild-type thymus). The me/+ naive splenic CD4 T cells showed enhanced activation by IL-4 receptor–mediated signaling but only marginal enhancement of TCR-mediated signaling. Interestingly, the generation of Th2 cells was increased and specific cytokine production of mast cells was enhanced in me/+ mice. In an OVA-induced allergic airway inflamma- tion model, eosinophilic inflammation, mucus hyperproduction, and airway hyperresponsiveness were enhanced in me/+ mice. Thus, SHP-1 may have a role as a negative regulator in the development of allergic responses, such as allergic asthma.
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Platelets are critical for normal hemostasis. Their deregulation can lead to bleeding or to arterial thrombosis, a primary cause of heart attack and ischemic stroke. Src homology 2 domain–containing inositol 5-phosphatase 1 (SHIP1) is a 5-phosphatase capable of dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate second messenger into phosphatidylinositol 3,4-bisphosphate. SHIP1 plays a critical role in regulating the level of these 2 lipids in platelets. Using SHIP1-deficient mice, we found that its loss affects platelet aggregation in response to several agonists with minor effects on fibrinogen binding and b 3 integrin tyrosine phosphorylation. Accordingly, SHIP1-null mice showed defects in arterial thrombus formation in response to a localized laser-induced injury. Moreover, these mice had a prolonged tail bleeding time. Upon stimulation, SHIP1-deficient platelets showed large membrane extensions, abnormalities in the open canalicular system, and a dramatic decrease in close cell-cell contacts. Interestingly, SHIP1 appeared to be required for platelet contractility, thrombus organization, and fibrin clot retraction. These data indicate that SHIP1 is an important element of the platelet signaling machinery to support normal hemostasis. To our knowledge, this is the first report unraveling an important function of SHIP1 in the activation of hematopoietic cells, in contrast to its well-documented role in the negative regulation of lymphocytes.
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Physiological angiogenesis depends on the highly coordinated actions of multiple angiogenic regulators. CCN1 is a secreted cysteine-rich and integrin-binding matricellular protein required for proper cardiovascular development. However, our understanding of the cellular origins and activities of this molecule is incomplete. Here, we show that CCN1 is predominantly expressed in angiogenic endothelial cells (ECs) at the leading front of actively growing vessels in the mouse retina. Endothelial deletion of CCN1 in mice using a Cre-Lox system is associated with EC hyperplasia, loss of pericyte coverage and formation of dense retinal vascular networks lacking the normal hierarchical arrangement of arterioles, capillaries and venules. CCN1 is a product of an immediate- early gene that is transcriptionally induced in ECs in response to stimulation by vascular endothelial growth factor (VEGF). We found that CCN1 activity is integrated with VEGF receptor 2 (VEGF-R2) activation and downstream signaling pathways required for tubular network formation. CCN1-integrin binding increased the expression of and association between Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1) and VEGF-R2, which leads to rapid dephosphorylation of VEGF-R2 tyrosine, thus preventing EC hyperproliferation. Predictably, CCN1 further brings receptors/signaling molecules into proximity that are otherwise spatially separated. Furthermore, CCN1 induces integrin-dependent Notch activation in cultured ECs, and its targeted gene inactivation in vivo alters Notch- dependent vascular specification and remodeling, suggesting that functional levels of Notch signaling requires CCN1 activity. These data highlight novel functions of CCN1 as a naturally optimized molecule, fine-controlling key processes in physiological angiogenesis and safeguarding against aberrant angiogenic responses.
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Broadly speaking, the SH2 consensus binding motifs identified from interactions observed using addressable arrays of physiological peptides are remarkably similar to the motifs described using peptide library approaches (Table 3). Yet binding specificities observed for physio- logical phosphotyrosine peptide ligands may in some cases represent more than the specificity of the isolated SH2 domain. The EDSM position weighted matrices noted in Additional file 2: Figure S5 reveal a number of cases in which the residues outside of the conventional window of residues at positions +1 to +4 appear to influ- ence binding. Longer contact regions have been noted for certain SH2 domains in the past, though these are generally exceptions to the rule. For instance, the SH2 domain of SH2D1A/SAP binds to an extended peptide in the SLAM receptor comprised of residues − 2 to +3 and shows a diminished dependence on phosphorylation of the tyrosine for binding . Physiological peptide ligands co-evolve to allow recognition by their cognate SH2 domain partner, while also acting as competent substrates for their cognate kinases. In some cases, the observed specificity for physiological peptide ligands may therefore represent an amalgam of SH2 specificity, kinase recognition, and other factors. This may, for ex- ample, explain the apparent observed preference of the Crk SH2 domain for an Asp residue at the −2 position. The presence of an aspartic acid residue at the −2 pos- ition does not appear to contribute to Crk SH2 domain binding (Figure 4B), however, this may instead reveal a signature for a distinct event such as kinase recognition for a specific subset of physiological peptides. Indeed, a large number of tyrosine kinases have reported prefer- ence for acidic residues preceding the target tyrosine residue [80,81]. Not surprisingly, acidic residues are commonly observed in the EDSM logos for the SH2 domains (Additional file 2: Figure S5). In addition to act- ing as kinase substrates and SH2 domain binding sites, the peptide motif must also presumably be surface exposed, and potentially disordered prior to binding, and these factors may also contribute to the overall physio- logical peptide motif. Combining multiple motifs in computational searches has been shown to markedly in- crease predictive accuracy , suggesting that the in- clusion of indirect components such as kinase specificity
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region . The presence of numerous low copy repeats results in instability and can trigger frequent segmental loss by unequal crossover or end-joining events; there- fore, such unstable CNV-rich subtelomeric regions are predisposed to deletion or duplication events . Note- worthily, locus-specific mutation rates for CNVs or structural rearrangements are between 10 -6 and 10 -4 , at least 2 to 4 orders of magnitude (100- to 10,000-fold) greater than those for point mutations or SNPs . Considering the causes of sporadic disease such as MSA, the new high mutation rate of CNVs is fascinat- ing, and should be studied while searching for gene(s) related to such disorders . CNVs in the distal 350-kb region of the 19p13.3 subtelomere have not been described for other neurologic diseases. For instance, our preliminary CNV beadchip analysis did not detect specific copy number loss in patients with multiple sclerosis (unpublished data).
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changes in JEV-induced neuroinflammation. SHIP1 knockdown by siRNA in BV-2 cells resulted in higher expression of inflamma- tory cytokines than in untreated cells. Further, cotransfection of BV-2 cells with SHIP1 siRNA and anti-miR-155 resulted in higher expression of proinflammatory cytokines than with their individ- ual transfections (see Fig. S3 in the supplemental material). After JEV infection of BV-2 cells, SHIP1 knockdown blunted the bene- ficial effects of anti-miR-155 transfection (see Fig. S3 in the sup- plemental material). These sets of observations provide evidence for an antineuroinflammatory role of SHIP1 (see Fig. S3 in the supplemental material). As shown in Fig. 3A, we observed a sig- nificant decrease in the SHIP1 protein level by transfection with a miR-155 mimic. This observation, along with the previously men- tioned inverse relationship between miR-155 and SHIP1 expres- sion post-JEV infection, suggests that the expression level of SHIP1 is negatively regulated by miR-155. Previous reports have identified SHIP1 as a potential target of miR-155 (20). To deter- mine whether miR-155 binds to the 3= UTR of SHIP1, we per- formed a luciferase reporter assay. A pMIR-REPORT construct encompassing a firefly luciferase reporter followed by the full- length 3= UTR of SHIP1, pMIR-REPORT SHIP1, was used. miR- 155 and miR-29b mimic (as a negative/nonspecific miRNA con- trol) oligonucleotides and an empty vector (pMIR-REPORT) were cotransfected with pMIR-REPORT SHIP1. Luciferase activ- ity was diminished in the reporter containing the 3= UTR of SHIP1 treated with miR-155 compared with pMIR-REPORT SHIP1 alone. In addition, when pMIR-REPORT SHIP1was cotransfected with an irrelevant miR-29b mimic, the luciferase activity did not FIG 4 miR-155 regulates JEV-induced infection in microglia. Expression of total and phosphorylated TBK-1, IRF3, IRF7, and NF- B proteins in JEV-infected BV-2 cells. BV-2 cells were transfected with anti-miR-155 for 24 h, followed by JEV infection for 24 h. Subsequently, the cells were lysed, protein was extracted, and the levels of total and phosphorylated TBK-1, IRF3, IRF7, and NF- B were determined by immunoblotting. The graphs represent the densitometric analysis of the immunoblots. The light parts of the graphs denote the phosphorylated fractions, and the dark parts represent the total protein. ␤ -Tubulin was used as a loading control. CON, noninfected BV-2 cells. The data represent means ⫾ SEM from 3 independent experiments. *,P ⬍ 0.05 compared to uninfected BV-2 cells, and #, P ⬍ 0.05 compared to JEV-infected BV-2 cells.
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Age-related changes in T cell subset composition in- volve a reduced frequency/number of naïve T cells, in- creased number of memory T cells and increased proportion of CD28 - T cells, especially in the CD8 + sub- population . A decrease in CD28 expression would affect the efficiency of the co-stimulatory pathway and this situation has been suggested to be in part respon- sible for impaired T cell activation with aging . On the other hand, data from our laboratories have provided evidence for a direct correlation between the state of ac- tivation of Lck and LAT and their association/recruit- ment with lipid rafts of CD4 + and CD8 + T cells . In relation with these reports, recent data have provided evidence that, in addition to being a required component of TCR/CD3 signaling, Lck is an obligatory link for T cell activation through the CD28 co-stimulatory path- way. Kong et al.  have put forward a model whereby activated Lck associates with pY207 of the C-terminal portion of CD28 through its SH2 domain. This associ- ation retains Lck in lipid rafts and allows its concomitant interaction with GLK-dependent phosphorylated PKCθ through its SH3 domain. The model predicts that Lck therefore provides an essential molecular bridge between CD28 and PKCθ. Our data suggest that interfering with the negative regulatory effect of SHP-1 would have a beneficial effect on signals 1 and 2 of T cell activation and would help to restore T cell proliferation in aging, as shown here (Figure 7). Furthermore, it has been recently reported that impairment of early signalling events in ac- tivated T cells allows prediction of the levels of expres- sion of the co-stimulatory molecules CD28 and CD27. This situation further predicts the number of population divisions in culture from a limited subset of signalling molecules such as Lck .
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Figure 1.—(A) Phylogram of PLC-␥ homologs. The number of changes between each sequence is represented by the length of each branch, and the single best tree is shown. Numbers at each node indicate bootstrap values. (B) Alignment of vertebrate and invertebrate PLC-␥ proteins in the region where tyrosine phosphorylation occurs in mammalian PLC-␥. The amino acid sequence between the C-terminal SH2 domain and the SH3 domain was aligned using ClustalW 1.4 and improved by eye. Dark- gray shading indicates a position where at least half the proteins have same amino acid; light-gray shading indicates a position where at least half the proteins have an amino acid with similar biochemical properties. The tyrosines known to be phosphorylated in human PLC-␥1 and PLC-␥2 are indicated by a circled P above the alignment.
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To better understand the function of TSAd, we used an algorithm for identification of SH2 domain-ligand pairs (SMALI) to identify possible binding partners for the TSAd phosphotyrosines. SMALI pointed to a pos- sible interaction between TSAd and the adaptor Nck. Nck is known to regulate the actin cytoskeleton. It con- sists of one C-terminal Src homology 2 (SH2) domain and three N-terminal SH3 domains which allows for multiple protein-protein interactions. More than 60 interaction partners for Nck have been identified [9, 10]. Nck interacts constitutively with the guanine nucleotide exchange factor Vav1 . Upon TCR-triggering, Nck and Vav1 interacts with SLP-76, leading to the activation of the actin rearrangement at the T-cell APC interface. Thus, Nck is a key adaptor in T cell activation-dependent actin filament formation through its interactions with compo- nents of the TCR/CD3 complex and cytoskeletal regula- tors including Vav1 and SLP-76 [9, 12–14]. Nck plays a universal role in regulation of the signaling networks critical for organizing the actin cytoskeleton; including formation of the IS following TCR engagement, cell prolif- eration and cell migration [9, 15, 16].
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HCC. Overexpression of miR-365 in the HCC cell line HepG2 could inhibit cell proliferation and cell migration, which mimics the clinical phenomenon in which patients with higher miR- 365 expression tend to be in the early stage of the disease or are unlikely to progress, and vice versa. Importantly, in the clinic, HCC cases with a higher level of miR-365 were not likely to suf- fer metastasis, most likely due to the suppres- sive effect of miR-365 on cell migration, as wit- nessed by the in vitro tests . Nevertheless, to date, the in-depth molecular mechanism of miR-365 in HCC remains largely undetermined. No study has unveiled the target genes of miR- 365 in HCC. However, the targets of miR-365 have been reported in several cancers. For example, miR-365 has been shown to target adaptor protein Src homology 2 domain-con- taining 1 (SHC1) and apoptosis-promoting pro- tein BAX in pancreatic cancer cells , neuro- pilin 1 (NRP1) in malignant melanoma , thyroid transcription factor 1 (TTF-1) in non- small cell lung cancer (NSCLC) , ADAMTS-1 in breast cancer , nuclear factor I/B (NFIB) in cutaneous squamous cell carcinoma , PAX6 in retinoblastoma , and NKX2-1 in lung cancer . To gain a deeper insight into the prospective target genes of miR-365 in HCC, we combined the genes predicted by online software and those key genes closely related to HCC from the NLP and TCGA an- alysis with in silico methods. Eventually, 238 overlapping genes, which were the most signifi- cant target genes of miR-365 in HCC, were identified to be enriched in several classical signaling pathways related to the progression and metastasis of HCC, especially in the regula- tion of cell proliferation and regulation of pro- grammed cell death from the GO analysis, the Ras Pathway from the PANTHER analysis, and the ‘Pathways in Cancer’ from the KEGG analy- sis [42-47]. However, dual-luciferase reporter assays are still needed to ascertain the rela- tionship between miR-365 and these prospec- tive target genes in the future.
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this question, the pGTR4 reporter was cotransfected into BJAB cells along with either an empty expression vector (Fig. 1C, lanes 1 and 2) or the LANA expression construct pCDNA3:73 (lanes 3 and 4) or pCMN:73 (lanes 5 and 6). While pCDNA3:73 and pCMN:73 contain the same functional elements (Fig. 1A), pCMN:73 differs from pCDNA3:73 in the absence of noncoding sequences upstream of ORF73 and the presence of a Kozak-optimized start codon for ORF73, result- ing in at least 10-fold-higher expression levels for LANA, as judged by Western blot analysis (data not shown). Both LANA expression constructs enabled replication of the cotransfected pGTR4 reporter plasmid, with efficiencies of 4 (pCDNA3:73) and 8% (pCMN:73). The lower percentage of replicated plas- mids obtained when LANA is provided in trans is likely to be a result of reduced levels of LANA compared to those gener- ated by plasmids in which the TRs are linked to LANA in cis. (Unlike pGTR4:73, pCDNA3:73 and pCMN:73 do not contain TR units and are therefore not replicated, resulting in declin- ing plasmid levels over the 72-h period.) In another set of experiments, either pGFP or pGTR4 was transfected into BJAB:73 cells, a cell line stably expressing LANA. The BJAB:73 cell line was generated by stable transduction of BJAB cells with a retrovirus expressing ORF73 as well as a puromycin resistance gene. Approximately 60% of these pu- romycin-resistant mass cultures expressed LANA, as judged by immunofluorescence analysis (data not shown). As shown in Fig. 1D, only pGTR4, not pGFP, is replicated in BJAB:73 cells, indicating that replication is strictly dependent on the presence of the TRs. Fourteen percent of pGTR4 episomes recovered from BJAB:73 cells were DpnI resistant. This per- centage is comparable to the values obtained with pGTR4:73 in BJAB cells when the proportion of LANA-negative cells (40%) in the BJAB:73 cultures is considered.
b) However, in odd number of crossing we always receive a knot; this confirms that at highest homological grading there exists a space against the quantum grading 3n th . Moreover, at ( n + 2 ) th quantum grading one space should appear with positive coefficient in the Equation (4). Thus, a space actually appears at the position ( n + 2, n − 1 ) . The homology of 1
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Bone marrow transduction and transplantation assay The pMIG-p210bcr/abl vector was used to produce retro- viruses directing the expression of BCR-ABL-GFP as de- scribed previously . Balb/c Shb wild type or knockout mice 8-10 weeks old were treated with 5 –fluorouracil (5- FU) (Sigma-Aldrich, St. Lois, MO) at a dose of 150 mg/kg body weight 6 days prior to bone marrow isolation, in order to enrich for HSCs. Isolated donor bone marrow was stimulated in RPMI 1640 (Sigma Aldrich) supple- mented with 10% FCS (Sigma Aldrich), 2 mM L-glutam- ine, streptomycin (0.1 mg/ml), penicillin (100 U/ml) (All from Gibco, Paisley, UK), IL-3 (10 ng/ml), stem cell factor (SCF) (10 ng/ml) and IL-6 (10 ng/ml) (all cytokines were purchased from PeproTech, Rocky Hill, NJ) for 24 hours. The cells were subsequently subjected to two rounds of spin infections over the following 48 hours as described
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The present study examines the role of Pyk2 in acid regulation of sodium/hydrogen exchanger 3 (NHE3) activity in OKP cells, a kidney proximal tubule epithelial cell line. Incubation of OKP cells in acid media caused a tran- sient increase in Pyk2 phosphorylation that peaked at 30 seconds and increased Pyk2/c-Src binding at 90 sec- onds. Pyk2 isolated by immunoprecipitation and studied in a cell-free system was activated and phosphorylated at acidic pH. Acid activation of Pyk2 (a) was specific for Pyk2 in that acid did not activate focal adhesion kinase, (b) required calcium, and (c) was associated with increased affinity for ATP. Transfection of OKP cells with dominant-negative pyk2 K457A or small interfering pyk2 duplex RNA blocked acid activation of NHE3, while nei-
Fig.2: WMJ-S-001 inhibited angiogenesis and tumor growth in a mouse xenograft model. Rat aortic rings were placed in Matrigel and treated with VEGF-A (20 ng/ml) in the presence or absence of WMJ-S-001 at indicated concentrations. The effect of WMJ-S-001 on formation of vessel sprout from various aorta samples was examined on day 8. Bar graphs show compiled data of average microvessels area (n=4). *p < 0.05, compared with the group treated with VEGF-A alone. (B) Matrigel mixed with VEGF-A was injected subcutaneously into the right flank of nude mice. After implantation, animals were treated intraperitoneally with vehicle or WMJ-S-001 (20 mg/kg/day) for 10 days. Matrigel plugs removed from the mice administered intraperitoneally with vehicle or WMJ-S-001 were shown. Hemoglobin levels in the Matrigel plug were quantified. Each column represents the mean ± S.E.M. of four plugs in each group ( * p < 0.05
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Plant based compounds serve as curing agents of various disease for decades. Virtual screening using computational tools provide a wide platform of plant based bioactivity prediction. The current study provides molecular mechanism of compound isolated from roots of Piper betle. This work comprises of target prediction and molecular mechanism of Aristolactam A II (ARL), 4-allyl resorcinol (ARL) and Stigmast-4-en-3, 6-dione (STM), compounds isolated from P. betle root. The target validation showed proto-oncogene tyrosine protein kinase Src (c-Src) and cyclin dependent kinases (CDK) as the top- ranked targets. Molecular docking of ARL showed good score against c-Src (-17.88 Kcal/mol) and CDK2 (-16.96 Kcal/mol) using Biosolveit LeadIT. Autodock 4.2 validation also showed similar amino acid pattern. Comparison with known c-Src inhibitor Bosutinib (BST) (-27.70 Kcal/mol) and CDK2 inhibitor Staurosporine (STP) (-35.20 Kcal/mol) showed similarity in binding pattern. Toxicity study showed ARL was non-toxic whereas Bosutinib (BST) was reported with high side-effect and toxicity. This concludes that these compounds works as some of the factors to support the ethnomedicinal claim of anticancer activity of P. betle.
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Before PRCC knockdown, we examined baseline PRCC expres- sion levels in four lung cancer cell lines (NCI-H23, NCI-H358, NCI-H460 and A549) by Western blot analysis. In three of the four cell lines, protein expression levels of PRCC were pro- foundly increased, compared with normal lung cells (CCD25- LU) (Fig. 2). Among them, NCI-H358 and A549 showed the highest levels of baseline PRCC expression (4.51 and 3.87 folds, respectively); therefore, both cells were selected for the fol- lowing experiments. To assess the biological effects of PRCC overexpression on lung carcinogenesis, we transfected three PRCC-specific siRNA constructs (siPRCC-1, -2, and -3) and siNEG into the NCI-H358 and A549 cell lines. Of the three siPRCCs, siPRCC-1 showed best knockdown performance in
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The homology model of the Protein HER 2 was modeled using the Modeller software. The structure was predicted with the active site identification. The validation of the protein by Procheck and Rampage were promising. Hence this protein can be an alternative for the Researchers further studies.
tools for finding novel cancer antigens  and has been applied on a nationwide basis to target many cancers, in- cluding gliblastoma [6-8]. However, the specific and cru- cial changes in the protein expression in low-grade gliomas have not been identified yet. In contrast, it is well known that activation of the receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) is the most frequent molecular aberration found in high-grade gliomas . The receptor tyrosine kinases make the ras pathway activation through a protein-protein interaction of the adaptor protein called GRB2 with Son of Sevenless (Sos) protein through src-homology 3 (SH3) domain [10,11]. The connection of the adaptor protein and Sos is a key step toward activating the ras-mediated oncogenic pathways in the downstream of receptor tyrosine kinases.
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Basophils also appear to be involved in the pathogene- sis of CSU. Peripheral blood basopenia is seen in patients with high disease activity and may be explained by the recruitment of basophils from the blood into skinlesions . CSU basophils also show functional abnormalities during active disease that revert during disease remi- sion. Greaves was first to show the hyporesponsiveness of basophils of patients with CSU to anti-IgE . Reduced basophil responsiveness to anti-IgE and altered signal transduction are reportedly seen in at least half of the patients [24–28]. Vonakis and coworkers reported baso- phil hypo-responsiveness in about half of the patients with chronic urticaria that is linked to excessive activity of the negative regulator Src homology inositol phos- phatase (SHIP). SHIP dephosphorylates kinases such as spleen tyrosine kinase (Syk) and consequently decreases cell responsiveness. Reversal of anti-IgE hypo-respon- siveness was observed upon disease remission, which suggests a relationship with the disease pathogenesis .
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