Induction of the endogenous sialoglycan ligand for eosinophil death receptor Siglec-8 in chronic rhinosinusitis with hyperplastic nasal polyposis.

Siglec-8, an immune-inhibitory sialoglycan binding lectin (S8), is expressed on the surface of eosinophils and mast cells, which are potent mediators of allergic inflammation. When S8 engages endogenous sialoglycan ligands, eosinophils undergo apoptosis and mast cell mediator release is inhibited. In the human airway, Siglec-8 ligands (S8L) are sialylated keratan sulfate chains carried on isoforms of the protein Deleted in Malignant Brain Tumors-1 (DMBT1), an immunoregulatory protein that we recently identified as the endogenous ligand for S8, DMBT1S8. We herein report that S8L is overexpressed in chronic rhinosinusitis with nasal polyposis (CRSwNP), a prevalent eosinophilic laden airway disease. Quantification and comparison of the degree to which DMBT1 carries the S8L by immunoblot analysis and lectin blot overlay, respectively, from nasal lavage showed that the S8L/DMBT1 ratio was significantly increased in CRSwNP vs. control or CRS patients. We identified the histological sites of S8L and DMBT1 expression in fresh surgically resected human nasal polyps. Histochemistry of diseased polyps and adjacent nondiseased middle turbinate (MT) tissue from CRSwNP demonstrated colocalization of S8L and DMBT1 with highest staining in submucosal glands >> epithelium > stoma. S8L expression was specifically elevated in the submucosal glands and epithelium of polyp tissue compared to MT. We hypothesize that expression of the isoform of DMBT1 carrying the Siglec-8 binding sialoglycan, DMBT1S8, is induced in polyps of CRSwNP specifically at the site of disease, is produced in the submucosal glands of polyps and secreted into the lumen of the sinonasal cavity as a host response to mitigate eosinophil-mediated inflammation.

Isolation, identification, and characterization of the human airway ligand for the eosinophil and mast cell immunoinhibitory receptor Siglec-8.

BACKGROUND: The immunoinhibitory receptor Siglec-8 on the surface of human eosinophils and mast cells binds to sialic acid-containing ligands in the local milieu, resulting in eosinophil apoptosis, inhibition of mast cell degranulation, and suppression of inflammation. Siglec-8 ligands were found on postmortem human trachea and bronchi and on upper airways in 2 compartments, cartilage and submucosal glands, but they were surprisingly absent from the epithelium. We hypothesized that Siglec-8 ligands in submucosal glands and ducts are normally transported to the airway mucus layer, which is lost during tissue preparation. OBJECTIVE: Our aim was to identify the major Siglec-8 sialoglycan ligand on the mucus layer of human airways. METHODS: Human upper airway mucus layer proteins were recovered during presurgical nasal lavage of patients at a sinus clinic. Proteins were resolved by gel electrophoresis and blotted, and Siglec-8 ligands detected. Ligands were purified by size exclusion and affinity chromatography, identified by proteomic mass spectrometry, and validated by electrophoretic and histochemical colocalization. The affinity of Siglec-8 binding to purified human airway ligand was determined by inhibition of glycan binding. RESULTS: A Siglec-8-ligand with a molecular weight of approximately 1000 kDa was found in all patient nasal lavage samples. Purification and identification revealed deleted in malignant brain tumors 1 (DMBT1) (also known by the aliases GP340 and SALSA), a large glycoprotein with multiple O-glycosylation repeats. Immunoblotting, immunohistochemistry, and enzyme treatments confirmed that Siglec-8 ligand on the human airway mucus layer is an isoform of DMBT1 carrying O-linked sialylated keratan sulfate chains (DMBT1S8). Quantitative inhibition revealed that DMBT1S8 has picomolar affinity for Siglec-8. CONCLUSION: A distinct DMBT1 isoform, DMBT1S8, is the major high-avidity ligand for Siglec-8 on human airways.

Application of computer-aided diagnosis in breast ultrasound interpretation: improvements in diagnostic performance according to reader experience.

PURPOSE: The purpose of this study was to evaluate the usefulness of applying computer-aided diagnosis (CAD) to breast ultrasound (US), depending on the operator's experience with breast imaging. METHODS: Between October 2015 and January 2016, two experienced readers obtained and analyzed the grayscale US images of 200 cases according to the Breast Imaging Reporting and Data System (BI-RADS) lexicon and categories. They additionally applied CAD (S-Detect) to analyze the lesions and made a diagnostic decision subjectively, based on grayscale US with CAD. For the same cases, two inexperienced readers analyzed the grayscale US images using the BI-RADS lexicon and categories, added CAD, and came to a subjective diagnostic conclusion. We then compared the diagnostic performance depending on the operator's experience with breast imaging. RESULTS: The sensitivity values for the experienced readers, inexperienced readers, and CAD (for experienced and inexperienced readers) were 91.7%, 75%, 75%, and 66.7%, respectively. The specificity values for the experienced readers, inexperienced readers, and CAD (for experienced and inexperienced readers) were 76.6%, 71.8%, 78.2%, and 76.1%, respectively. When diagnoses were made subjectively in combination with CAD, the specificity significantly improved (76.6% to 80.3%) without a change in the sensitivity (91.7%) in the experienced readers. After subjective combination with CAD, the sensitivity and specificity improved in the inexperienced readers (75% to 83.3% and 71.8% to 77.1%). In addition, the area under the curve improved for both the experienced and inexperienced readers (0.84 to 0.86 and 0.73 to 0.8) after the addition of CAD. CONCLUSION: CAD is more useful for less experienced readers. Combining CAD with breast US led to improved specificity for both experienced and inexperienced readers.

Diagnostic Performance of and Breast Tissue Changes at Early Breast MR Imaging Surveillance in Women after Breast Conservation Therapy.

Purpose To investigate the diagnostic performance and tissue changes in early (1 year or less) breast magnetic resonance (MR) imaging surveillance in women who underwent breast conservation therapy for breast cancer. Materials and Methods This prospective study was approved by the institutional review board, and written informed consent was obtained. Between April 2014 and June 2016, 414 women (mean age, 51.5 years; range, 21-81 years) who underwent 422 early surveillance breast MR imaging examinations (median, 6.0 months; range, 2-12 months) after breast conservation therapy were studied. The cancer detection rate, positive predictive value of biopsy, sensitivity, specificity, accuracy, and area under the curve of surveillance MR imaging, mammography, and ultrasonography (US) were assessed. Follow-up was also obtained in 95 women by using positron emission tomography (PET)/computed tomography (CT). Background parenchymal enhancement (BPE) changes in the contralateral breast were assessed according to adjuvant therapy by using the McNemar test. Results Of 11 detected cancers, six were seen at MR imaging only, one was seen at MR imaging and mammography, two were seen at MR imaging and US, one was seen at mammography only, and one was seen at PET/CT only. Three MR imaging-depicted cancers were observed at the original tumor bed, and two MR imaging-depicted cancers were observed adjacent to the original tumor. Among two false-negative MR imaging diagnoses (two cases of ductal carcinoma in situ), one cancer had manifested as calcifications at mammography without differentiated enhancement at MR imaging, and the other cancer was detected at PET/CT, but MR imaging results were negative because of marked BPE, which resulted in focal lesion masking. The positive predictive value of biopsy and the sensitivity, specificity, accuracy, and area under the curve for MR imaging were 32.1% (nine of 28), 81.8% (nine of 11), 95.1% (391 of 411), 94.7% (400 of 422), and 0.88, respectively. The sensitivity of surveillance MR imaging (81.8%; 95% confidence interval [CI]: 48.2%, 97.7%) was higher than that of mammography (18.2%; 95% CI: 2.3%, 51.8%) and US (18.2%; 95% CI: 2.3%, 51.8%), with an overlap in CIs. The BPE showed a significant decrease in the group of patients who received adjuvant chemotherapy (43 BPE decreases and four BPE increases) and the group of patients who received hormone therapy (55 BPE decreases and two BPE increases) (P < .0001 for both). Conclusion Early MR imaging surveillance after breast conservation therapy can be useful in patients who have breast cancer, with superior sensitivity compared with that of mammography and US. The BPE tends to be decreased at short-term follow-up MR imaging in patients who receive adjuvant therapy. © RSNA, 2017.

Radiation dose affected by mammographic composition and breast size: first application of a radiation dose management system for full-field digital mammography in Korean women.

BACKGROUND: Relative to Western women, Korean women show several differences in breast-related characteristics, including higher rates of dense breasts and small breasts. We investigated how mammographic composition and breast size affect the glandular dose during full-field digital mammography (FFDM) in Korean women using a radiation dose management system. METHODS: From June 1 to June 30, 2015, 2120 FFDM images from 560 patients were acquired and mammographic breast composition and breast size were assessed. We analyzed the correlations of patient age, peak kilovoltage (kVp), current (mAs), compressed breast thickness, compression force, mammographic breast composition, and mammographic breast size with the mean glandular dose (MGD) of the breast using a radiation dose management system. The causes of increased radiation were investigated, among patients with radiation doses above the diagnostic reference level (4th quartile, ≥75%). RESULTS: The MGD per view of 2120 images was 1.81 ± 0.70 mGy. In multivariate linear regression analysis, age was negatively associated with MGD (p < 0.05). The mAs, kVp, compressed breast thickness, and mammographic breast size were positively associated with MGD (p < 0.05). The "dense" group had a significantly higher MGD than the "non-dense" group (p < 0.05). Patients with radiation dose values above the diagnostic reference value had large breasts of dense composition. CONCLUSIONS: Among Korean women, patients with large and dense breasts should be more carefully managed to ensure that a constant radiation dose is maintained.

USPIO enhanced lymph node MRI using 3D multi-echo GRE in a rabbit model.

Ultrasmallsuperparamagnetic iron oxide (USPIO) has been suggested to be a negative MR contrast agent to detect metastatic lymph nodes. Previously reported studies have evaluated the diagnostic performance of USPIO-enhanced MR lymph node imaging based on signal intensity. In this study, we investigate the specific performance of three different parametric approaches (normalized signal intensity, R2 * and susceptibility) using 3D multi-echo gradient echo to quantify the USPIO particles in lymph nodes. Nine rabbits with VX2 tumor implants were scanned before and after USPIO injection. From 3D multi-echo GRE magnitude and phase data, we generated multi-echo combined T2 *-weighted images, an R2 * map, and a quantitative susceptibility map. Eighteen lymph nodes (nine reactive and nine metastatic) were evaluated and showed remarkable signal drops in the area of USPIO accumulation. On parametric analysis, the R2 * difference before and after USPIO injection was significantly different (p < 0.05) between reactive and metastatic lymph nodes; in contrast, the normalized signal intensity and susceptibility were not significantly different between the nodes. Our study showed the potential utility of USPIO-enhanced MRI using R2* mapping from 3D multi-echo GRE for the detection of lymph node metastasis and parametric analysis of lymph node status in a rabbit model. Copyright © 2016 John Wiley & Sons, Ltd.

Perfusion Parameters in Dynamic Contrast-enhanced MRI and Apparent Diffusion Coefficient Value in Diffusion-weighted MRI:: Association with Prognostic Factors in Breast Cancer.

RATIONALE AND OBJECTIVES: To evaluate the association of prognostic factors and subtypes of breast cancer with perfusion parameters in dynamic contrast-enhanced magnetic resonance imaging and apparent diffusion coefficient (ADC) values in diffusion-weighted magnetic resonance imaging. MATERIALS AND METHODS: Quantitative perfusion parameters (constant of transfer from plasma to interstitium, constant of transfer from the interstitium to the plasma, extravascular/extracellular volume per unit of volume of tissue [ve], and initial area under the concentration curve [iAUC]) and ADC values in the entire tumor volume of 52 invasive ductal carcinomas were obtained using histogram analysis. Four measures (25th percentile, mean, median, 75th percentile) were calculated for each parameter and the ADC value. Associations of perfusion parameters and ADC values with prognostic factors and tumor subtypes were analyzed. RESULTS: Among perfusion parameters, iAUCmean and iAUCmedian were greater in tumors larger than 2 cm (8.23 ± 2.33, 8.64 ± 2.67 × 10(4)) than in those smaller than 2 cm (6.99 ± 1.92, 7.04 ± 2.15 × 10(4); P = 0.046, 0.023). Ve median was higher in tumors with progesterone receptor (PR) positivity (0.54 ± 0.18) than in those with PR negativity (0.44 ± 0.1, P = 0.041). There were higher ADCmean and ADCmedian in tumors with human epidermal growth factor receptor 2 (HER2) positivity (1.306 and 1.278 × 10(-3)mm(2)/s) than in those with HER2 negativity (1.078 and 1.053 × 10(-3)mm(2)/s; P = 0.012 and 0.020). Higher ADCmean and ADCmedian were observed in HER2-enriched type (1.404 and 1.378 × 10(-3)mm(2)/s) than in luminal type (1.096 and 1.073 × 10(-3)mm(2)/s; P = 0.030 and 0.045). CONCLUSIONS: Among perfusion parameters, iAUC was associated with tumor size and ve median was associated with PR positivity. Mean and median ADC values showed positive correlation with HER2-positive and HER2-enriched tumors.

Clinical Features and Prognosis of Invasive Pulmonary Aspergillosis in Korean Children with Hematologic/Oncologic Diseases.

Invasive pulmonary aspergillosis (IPA) is the most frequent form of invasive fungal diseases in immunocompromised patients. However, there are only a few studies on IPA in immunocompromised children in Korea. This study was designed to characterize IPA in Korean children with hematologic/oncologic diseases. Medical records of children with hematologic/oncologic diseases receiving antifungal therapy were reviewed. The enrolled children were divided into the IPA group (proven and probable IPA) and non-IPA group, and the clinical characteristics and prognosis were compared between the two groups. During the study period, 265 courses of antifungal therapy were administered to 166 children. Among them, two (0.8%) episodes of proven IPA, 35 (13.2%) of probable IPA, and 52 (19.6%) of possible IPA were diagnosed. More children in the IPA group suffered from neutropenia lasting for more than two weeks (51.4% vs. 21.9%, P<0.001) and showed halo signs on the chest computed tomography (78.4% vs. 40.7%, P<0.001) than in the non-IPA group. No other clinical factors showed significant differences between the two groups. Amphotericin B deoxycholate was administered as a first line antifungal agent in 33 (89.2%) IPA group episodes, and eventually voriconazole was administered in 27 (73.0%) episodes. Ten (27.0%) children in the IPA group died within 12 weeks of antifungal therapy. In conclusion, early use of chest computed tomography to identify halo signs in immunocompromised children who are expected to have prolonged neutropenia can be helpful for early diagnosis of IPA and improving prognosis of children with IPA.

Serum galactomannan index for early prediction of mortality in immunocompromised children with invasive pulmonary aspergillosis.

BACKGROUND: Invasive pulmonary aspergillosis (IPA) is the most common invasive fungal disease in immunocompromised patients, and it has a 30 % mortality rate despite appropriate antifungal therapy. This retrospective study was performed to determine risk factors for mortality in immunocompromised children with IPA. METHODS: Medical records of 45 probable/proven IPA cases diagnosed in children with hematologic/oncologic diseases were reviewed. Selected cases were divided into the survival (n = 30) and fatality (n = 15) groups based on survival at 12 weeks after antifungal therapy. Clinical characteristics and serum galactomannan indices (GMIs) were compared between the two groups. RESULTS: Significantly more children in the fatality group were male (p = 0.044), not in complete remission of the underlying malignancies (p = 0.016), and had received re-induction/salvage or palliative chemotherapy (p = 0.035) than those in the survival group. However, none of these factors was significantly associated with mortality in a multivariate analysis. Serum GMIs were higher in the fatality group than in the survival group during the entire period of antifungal therapy, and serum GMI at 1 week after antifungal therapy was most significantly associated with mortality. A serum GMI > 1.50 at 1 week after antifungal therapy exhibited a sensitivity and specificity of 61.5 % and 89.3 %, respectively, in predicting mortality within 12 weeks after antifungal therapy. CONCLUSIONS: Higher serum GMI in the early phase of antifungal therapy was associated with mortality in immunocompromised children with IPA. These children should receive more intensive care for IPA than others.

Imaging features of bilateral breast plasmacytoma as unusual initial presentation of multiple myeloma: case report and literature review.

Breast plasmacytoma (BP) is an extremely rare extramedullary manifestation of multiple myeloma (MM). We report the imaging findings of an unusual case in which BP was the initial presentation of MM. A 53-year-old woman with no contributory medical history underwent chest computed tomography to evaluate intermittent nocturnal anterior chest pain, and bilateral multiple breast masses were found. Following an ultrasound-guided core needle biopsy, these lesions were confirmed to be BP.

Constitutive expression of vascular endothelial cell growth factor (VEGF) gene family ligand and receptors on human upper and lower airway epithelial cells.

BACKGROUND: We previously reported that vascular endothelial cell growth factor (VEGF) is abundantly expressed by primary human nasal epithelial cells (PNECs) and functions to promote cell hyperplasia in polyposis. Therefore, we aimed to examine the full expression profile of other members of the VEGF gene family of ligands and receptors, which may play a role in cell growth and the development of chronic rhinosinusitis with nasal polyposis (CRSwNP). METHODS: Messenger RNA (mRNA) and protein expression of VEGF genes, receptors, and co-receptors was examined from cultured PNECs (n = 4) and compared to that from primary human bronchial epithelial cells (PBECs; n = 4) and the BEAS2B cell line (n = 4) by real-time polymerase chain reaction (PCR) and flow cytometry. RESULTS: We report abundant expression of VEGFA, VEGFB, and VEGFC, detected by mRNA and flow cytometric analysis on PNECs. We herein report the novel finding that there is significant expression of VEGFR1, VEGFR2, VEGFR3, and both neuropilin co-receptors, NP1 and NP2, at baseline conditions on PNECs. Lower airway PBECs and BEAS2B cells displayed similar patterns of expression. CONCLUSION: PNECs express high constitutive levels of the VEGF gene family homolog of ligands and receptors. Expression of multiple VEGF ligand-receptor combinations may function as redundant pathways to promote upper and lower airway epithelial cell growth during inflammation.

Cigarette smoke inhibits nasal airway epithelial cell growth and survival.

BACKGROUND: Cigarette smoke (CS) exposure has been shown to be associated with chronic rhinosinusitis (CRS). We hypothesized that that CS exposure results in impairment nasal epithelial cell growth and survival necessary for normal cell function. Furthermore, we hypothesized that normal nasal epithelial cell growth is dependent on vascular endothelial growth factor (VEGF) and that CS inhibits normal nasal epithelial cell growth and survival through VEGF-dependent mechanisms. METHODS: To examine whether nasal epithelial cell growth is dependent upon VEGF, we exposed in vitro cultures of human primary nasal epithelial cells (PNECs) from normal subjects to blocking antibodies against the VEGF co-receptor, neuropilin-1 (NP1), and measured cell growth using a proliferation assay. To study whether CS inhibits cell growth, we exposed PNECs to cigarette smoke extract (CSE). We also examined the ability of CSE exposure of PNECs to induce apoptosis. RESULTS: Exposure of PNECs from normal subjects to VEGF receptor anti-NP1 antibody resulted in inhibition of constitutive cell growth. CSE exposure resulted in dose-dependent inhibition of constitutive cell growth. Addition of anti-NP1 antibody to CSE-exposed cells resulted in no further inhibition of cell growth. CSE exposure also resulted in induction of apoptosis in a dose-dependent manner, comparable to that seen with VEGF blockade using anti-NP1 antibody. CONCLUSION: PNECs from normal healthy control subjects display VEGF-dependent constitutive cell growth. CSE impairs cell growth and promotes apoptosis of normal healthy nasal epithelial cells. The effect of CSE may occur in part through VEGF-dependent mechanisms.

Vascular endothelial growth factor drives autocrine epithelial cell proliferation and survival in chronic rhinosinusitis with nasal polyposis.

RATIONALE: The pathogenesis of nasal polyps in chronic rhinosinusitis is poorly understood. OBJECTIVES: These studies seek to implicate a functional role for vascular endothelial growth factor (VEGF) in perpetuating primary nasal epithelial cell overgrowth, a key feature of hyperplastic polyps. METHODS: Comparison of VEGF and receptor expression was assessed by ELISA of nasal lavage, immunohistochemistry of sinus tissue, flow cytometry of nasal epithelial cells, and ELISA of supernatants. VEGF-dependent cell growth and apoptosis were assessed with blocking antibodies to VEGF, their receptors, or small interfering RNA knockdown of neuropilin-1 by cell proliferation assays and flow cytometric binding of annexin V. MEASUREMENTS AND MAIN RESULTS: VEGF protein was sevenfold higher in nasal lavage from patients with polyposis compared with control subjects (P < 0.001). We also report elevated expression of VEGF (P < 0.012), receptors VEGFR2 and phospho-VEGFR2 (both P < 0.04), and identification of VEGF coreceptor neuropilin-1 in these tissues. Nasal epithelial cells from patients with polyps demonstrated faster growth rates (P < 0.005). Exposure of cells to blocking antibodies against VEGF resulted in inhibition of cell growth (P < 0.05). VEGF receptor blockade required blockade of neuropilin-1 (P < 0.05) and resulted in increased apoptosis (P < 0.001) and inhibition of autocrine epithelial VEGF production (P < 0.05). CONCLUSIONS: These data demonstrate that VEGF is a novel biomarker for chronic rhinosinusitis with hyperplastic sinonasal polyposis that functions in an autocrine feed-forward manner to promote nasal epithelial cell growth and to inhibit apoptosis. These findings implicate a previously unrecognized and novel role of VEGF functioning through neuropilin-1 on nonneoplastic primary human airway epithelial cells, to amplify cell growth, contributing to exuberant hyperplastic polyposis.

FcepsilonRI-mediated mast cell migration: signaling pathways and dependence on cytosolic free Ca2+ concentration.

IgE-sensitized rat basophilic leukemia (RBL)-2H3 mast cells have been shown to migrate towards antigen. In the present study we tried to identify the mechanism by which antigen causes mast cell migration. Antigen caused migration of RBL-2H3 cells at the concentration ranges of 1000-fold lower than those required for degranulation and the dose response was biphasic. This suggests that mast cells can detect very low concentration gradients of antigen (pg/ml ranges), which initiate migration until they degranulate near the origin of antigen, of which concentration is in the ng/ml ranges. Similar phenomenon was observed in human mast cells (HMCs) derived from CD34(+) progenitors. As one mechanism of mast cell migration, we tested the involvement of sphingosine 1-phosphate (S1P). Fc epsilon RI-mediated cell migration was dependent on the production of S1P but independent of a S1P receptor or its signaling pathways as determined with S1P receptor antagonist VPC23019 and Gi protein inhibitor pertussis toxin (PTX). This indicated that the site of action of S1P produced by antigen stimulation was intracellular. However, S1P-induced mast cell migration was dependent on S1P receptor activation and inhibited by both VPC23019 and PTX. Cell migration towards antigen or extracellular S1P was dependent on the activation of the phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways, while only migration towards antigen was inhibited by the inhibitors of sphingosine kinase and phospholipase C (PLC) and intracellular calcium chelator BAPTA. In summary, our data suggest that the high affinity receptor for IgE (Fc epsilon RI)-mediated mast cell migration is dependent on the production of S1P but independent of S1P receptors. Cell migration mediated by either Fc epsilon RI or S1P receptors involves activation of both PI3K and MAPK.

Cross-linking of FcepsilonRI causes Ca2+ mobilization via a sphingosine kinase pathway in a clathrin-dependent manner.

Clathrin-coated pits are now recognized to be involved in cell signaling in addition to receptor down-regulation. Here we tried to identify signaling pathways that might be dependent on clathrin. Our initial data with pharmacological inhibitors of formation of clathrin-coated pits or lipid-rafts indicated that Ca(2+) response evoked by cross-linking of the high affinity receptors for IgE (FcepsilonRI) was dependent on clathrin. To confirm this finding, we created clathrin-knockdown cells by transfecting the mast cell line RBL-2H3 with a shRNA-clathrin heavy chain construct. In these cells, the FcepsilonRI-mediated Ca(2+) response was almost completely abolished, which was accompanied by the inhibition of sphingosine 1-phosphate (S1P) production with no changes in inositol 1,4,5-trisphosphate (IP(3)) production. This suggests that the Ca(2+) signaling pathway via a sphingosine kinase (SK) is dependent on clathrin. Furthermore, antigen-induced tyrosine phosphorylation of p85 and p110 subunits of PI3K was almost completely inhibited in clathrin-knockdown cells. In contrast, antigen-induced tyrosine phosphorylation of phospholipase Cgamma was not affected by clathrin-knockdown and tyrosine phosphorylation of Syk and degranulation were partially inhibited in clathrin-knockdown cells. The present study identifies the SK/Ca(2+) pathway to be dependent on clathrin.

Inhibition of FcepsilonRI-dependent mediator release and calcium flux from human mast cells by sialic acid-binding immunoglobulin-like lectin 8 engagement.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of glycan-binding inhibitory receptors, and among them, Siglec-8 is selectively expressed on human eosinophils, basophils, and mast cells. On eosinophils, Siglec-8 engagement induces apoptosis, but its function on mast cells is unknown. OBJECTIVE: We sought to study the effect of Siglec-8 engagement on human mast cell survival and mediator release responses. METHODS: Human mast cells were generated from CD34+ precursors. Apoptosis was studied by using flow cytometry. Mast cell mediator release or human lung airway smooth muscle contraction was initiated by FcepsilonRI cross-linking with or without preincubation with Siglec-8 or control antibodies, and release of mediators was analyzed along with Ca++ flux. RBL-2H3 cells transfected with normal and mutated forms of Siglec-8 were used to study how Siglec-8 engagement alters mediator release. RESULTS: Siglec-8 engagement failed to induce human mast cell apoptosis. However, preincubation with Siglec-8 mAbs significantly (P < .05) inhibited FcepsilonRI-dependent histamine and prostaglandin D(2) release, Ca++ flux, and anti-IgE-evoked contractions of human bronchial rings. In contrast, release of IL-8 was not inhibited. Siglec-8 ligation was also shown to inhibit beta-hexosaminidase release and Ca++ flux triggered through FcepsilonRI in RBL-2H3 cells transfected with full-length human Siglec-8 but not in cells transfected with Siglec-8 containing a tyrosine to phenylalanine point mutation in the membrane-proximal immunoreceptor tyrosine-based inhibitory motif domain. CONCLUSION: These data represent the first reported inhibitory effects of Siglec engagement on human mast cells.

Cloning and characterization of rat sphingosine kinase 1 with an N-terminal extension.

Sphingosine kinase (SK) is an enzyme that converts sphingosine to sphingosine 1-phosphate, a lysophospholipid with various cellular functions. Here, we report cloning and characterization of a novel isoform of rat SK1 (rSK1) with an N-terminal extension (long-rSK1). Long-rSK1 is 458 amino acid-long and has a calculated molecular weight of 49.8kDa. Deduced amino acid sequences of rat and human long-SK1 have high homology (71.3% identity and 78.9% similarity). Long-rSK1 mRNA is widely expressed throughout the tissues including brain, heart, lung, liver, kidney, and spleen, whereas mRNA encoding rSK1 has been shown not to be expressed in heart or liver. When the long-rSK1 was transfected in COS-7 cells, SK activity was 53-fold increased. Substrate specificity and dependency on divalent cations of long-rSK1 were similar to those of rSK1. Taken together, the fact that long-rSK1 with similar enzymatic characteristics to rSK1 displays differential tissue distribution may suggest an additional role of long-rSK1.

Identification of Jmjd1a as a STAT3 downstream gene in mES cells.

Mouse embryonic stem (mES) cells can be maintained in undifferentiated state in the presence of a cytokine, leukemia inhibitory factor (LIF). Many investigators found that STAT3 activation is important for the maintenance of pluripotency by LIF. However, the downstream pathways of STAT3 activation are still unknown. To look for STAT3-downstream target genes, we performed DD-RT PCR in the presence or absence of LIF. Through further confirmation, we finally selected 8 genes whose expressions were significantly dependent upon the presence of LIF. Among them, Jmjd1a was down-regulated after LIF withdrawal, and it was selected for further investigation. Its expression started to decrease 1 day after the removal of LIF, and disappeared on day 3. It was also shown that STAT3 could bind to the promoter region of Jmjd1a gene. These data demonstrate that Jmjd1a might be a critical signaling molecule underlying the maintenance of pluripotency in mES cells.

Construction, affinity maturation, and biological characterization of an anti-tumor-associated glycoprotein-72 humanized antibody.

The tumor-associated glycoprotein (TAG)-72 is expressed in the majority of human adenocarcinomas but is rarely expressed in most normal tissues, which makes it a potential target for the diagnosis and therapy of a variety of human cancers. Here we describe the construction, affinity maturation, and biological characterization of an anti-TAG-72 humanized antibody with minimum potential immunogenicity. The humanized antibody was constructed by grafting only the specificity-determining residues (SDRs) within the complementarity-determining regions (CDRs) onto homologous human immunoglobulin germ line segments while retaining two mouse heavy chain framework residues that support the conformation of the CDRs. The resulting humanized antibody (AKA) showed only about 2-fold lower affinity compared with the original murine monoclonal antibody CC49 and 27-fold lower reactivity to patient serum compared with the humanized antibody HuCC49 that was constructed by CDR grafting. The affinity of AKA was improved by random mutagenesis of the heavy chain CDR3 (HCDR3). The highest affinity variant (3E8) showed 22-fold higher affinity compared with AKA and retained the original epitope specificity. Mutational analysis of the HCDR3 residues revealed that the replacement of Asn(97) by isoleucine or valine was critical for the affinity maturation. The 3E8 labeled with (125)I or (131)I showed efficient tumor targeting or therapeutic effects, respectively, in athymic mice with human colon carcinoma xenografts, suggesting that 3E8 may be beneficial for the diagnosis and therapy of tumors expressing TAG-72.

Antigen-induced Ca2+ mobilization in RBL-2H3 cells: role of I(1,4,5)P3 and S1P and necessity of I(1,4,5)P3 production.

Inositol 1,4,5-trisphosphate (IP3) has long been recognized as a second messenger for intracellular Ca2+ mobilization. Recently, sphingosine 1-phosphate (S1P) has been shown to be involved in Ca2+ release from the endoplasmic reticulum (ER). Here, we investigated the role of S1P and IP3 in antigen (Ag)-induced intracellular Ca2+ mobilization in RBL-2H3 mast cells. Antigen-induced intracellular Ca2+ mobilization was only partially inhibited by the sphingosine kinase inhibitor dl-threo-dihydrosphingosine (DHS) or the IP3 receptor inhibitor 2-aminoethoxydiphenyl borate (2-APB), whereas preincubation with both inhibitors led to complete inhibition. In contrast, stimulation of A3 adenosine receptors with N5-ethylcarboxamidoadenosine (NECA) caused intracellular Ca2+ mobilization that was completely abolished by 2-APB but not by DHS, suggesting that NECA required only the IP3 pathway, while antigen used both the IP3 and S1P pathways. Interestingly, however, inhibition of IP3 production with the phospholipase C inhibitor U73122 completely abolished Ca2+ release from the ER induced by either stimulant. This suggested that S1P alone, without concomitant production of IP3, would not cause intracellular Ca2+ mobilization. This was further demonstrated in some clones of RBL-2H3 cells excessively overexpressing a beta isoform of Class II phosphatidylinositol 3-kinase (PI3KC2beta). In such clones including clone 5A4C, PI3KC2beta was overexpressed throughout the cell, although endogenous PI3KC2beta was normally expressed only in the ER. Overexpression of PI3KC2beta in the cytosol and the PM led to depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), resulting in a marked reduction in IP3 production. This could explain the abolishment of intracellular Ca2+ mobilization in clone 5A4C. Supporting this hypothesis, the Ca2+ mobilization was reconstituted by the addition of exogenous PI(4,5)P2 in these cells. Our results suggest that both IP3 and S1P contribute to FcvarepsilonRI-induced Ca2+ release from the ER and production of IP3 is necessary for S1P to cause Ca2+ mobilization from the ER.