Elevated and cross‐responsive CD1a‐reactive T cells in bee and wasp venom allergic individuals

The role of CD1a‐reactive T cells in human allergic disease is unknown. We have previously shown that circulating CD1a‐reactive T cells recognize neolipid antigens generated by bee and wasp venom phospholipase, and here tested the hypothesis that venom‐responsive CD1a‐reactive T cells associate with venom allergy. Circulating T cells from bee and wasp venom allergic individuals, before and during immunotherapy, were exposed to CD1a‐transfected K562 cells in the presence of wasp or bee venom. T‐cell response was evaluated based on IFNγ, GM‐CSF, and IL‐13 cytokine production. Venom allergic individuals showed significantly higher frequencies of IFN‐γ, GM‐CSF, and IL‐13 producing CD1a‐reactive T cells responsive to venom and venom‐derived phospholipase than healthy individuals. Venom‐responsive CD1a‐reactive T cells were cross‐responsive between wasp and bee suggesting shared pathways of allergenicity. Frequencies of CD1a‐reactive T cells were initially induced during subcutaneous immunotherapy, peaking by weeks 5, but then reduced despite escalation of antigen dose. Our current understanding of venom allergy and immunotherapy is largely based on peptide and protein‐specific T cell and antibody responses. Here, we show that lipid antigens and CD1a‐reactive T cells associate with the allergic response. These data have implications for mechanisms of allergy and approaches to immunotherapy.     

Labelling cell structures and tracking cell lineage in zebrafish using SNAP‐tag

We present a method for the specific labelling of fusion proteins with synthetic fluorophores in Zebrafish. The method uses the SNAP‐tag technology and O⁶‐benzylguanine derivatives of various synthetic fluorophores. We demonstrate how the method can be used to label subcellular structures in Zebrafish such as the nucleus, cell membranes, and endosomal membranes. The stability of the synthetic fluorophores makes them attractive choices for long‐term imaging and allows, unlike most of the autofluorescent proteins, the use of acid fixatives such as trichloroacetic acid. Furthermore, the use of O⁶‐benzylguanine derivatives bearing caged fluorescein allows cell lineage tracing through photo‐deprotection of the fluorophore and its detection either through fluorescence microscopy or through immunohistochemistry after fixation using anti‐fluorescein antibodies. Developmental Dynamics 240:820–827, 2011. © 2011 Wiley‐Liss, Inc.     

Rbp-j regulates expansion of pancreatic epithelial cells and their differentiation into exocrine cells during mouse development.

Notch signaling regulates cell fate determination in various tissues. We have reported the generation of mice with a pancreas-specific knockout of Rbp-j using Pdx.cre mice. Those mice exhibited premature endocrine and ductal differentiation. We now generated mice in which the Rbp-j gene was inactivated in Ptf1a-expressing cells using Ptf1a.cre mice. The timing of the Cre-mediated deletion in Rbp-j(f/f) Ptf1a.cre mice is 1 day later than that in Rbp-j(f/f) Pdx.cre mice. In Rbp-j(f/f) Ptf1a.cre mouse pancreases, at E13.5, the reduced Hes1 expression was accompanied by reduced epithelial growth, but premature endocrine cell differentiation was minimal. At E15.5, Pdx1 expression was repressed and acinar cell differentiation was reduced, but an increase in acinar cell proliferation was observed during the perinatal period. Our study indicates that, in addition to its role in preventing premature differentiation of early endocrine cells, Rbp-j regulates epithelial growth, Pdx1 expression, and acinar cell differentiation during mid-pancreatic development.     

Screening of anti‐HPA‐1a antibodies in HPA‐1a‐negative mothers

Results from a study of more than 100 000 pregnant women show that the pathophysiology of neonatal alloimmune thrombocytopenia (NAIT) and haemolytic disease of the newborn (HDN) has many equalities [ 1 ]. It has been reported that NAIT is more than three times more frequent compared with HDN. This makes it tempting to consider a design for screening for HPA 1a negativity and follow up in/after the pregnancy based on the same principles as for RhD‐negative pregnant women. Even without available vaccination, it is strongly indicated that screening and Caesarean section 2–4 weeks prior to term in mothers with the HPA‐1a‐negative platelet type, reduce neonatal morbidity and mortality due to NAIT.     

Appearance and phenotype of murine follicular dendritic cells expressing VCAM‐1

The architecture of lymphoid follicles is determined by a series of interactions between lymphoid and follicular stromal cells. A cardinal population in the non‐lymphoid compartment is the follicular dendritic cell (FDC), whose communication with resting and activated B cells involves various adhesive interactions. The FDC phenotype variably includes the display of vascular cell adhesion molecule (VCAM‐1). In this report we investigated the appearance and follicular tissue distribution of VCAM‐1 in murine peripheral lymphoid tissues, and compared VCAM‐1 with other FDC markers using immunohistochemistry. Correlating the appearance of VCAM‐1 with other murine FDC‐associated markers (CR1.2 [complement receptor 1.2 or CD35/21] and FDC‐M1) revealed that the display of VCAM‐1 is restricted to a subset of CR1.2‐positive FDCs. We found that the expression of VCAM‐1 antigen in the spleen or peripheral lymph nodes on FDCs requires antigenic stimulus, and that it coincides with germinal center formation. The VCAM‐1 expression is associated with the appearance of mucosal addressin cell adhesion molecule (MAdCAM‐1), with some slight differences in occurrence. The appearance of VCAM‐1 and MAdCAM‐1 antigens on FDCs may serve as indicators of FDC activation. Anat Rec 268:160–168, 2002. © 2002 Wiley‐Liss, Inc.     

Fine‐needle aspiration of cystic pancreatic mucinous tumor: Oncotic cell as an aiding diagnostic feature in paucicellular specimens

In juxtaposition with imaging studies, endoscopic ultrasound‐guided fine‐needle aspiration has gained popularity in the initial evaluation of pancreatic masses, especially cystic lesions of pancreas. Cystic pancreatic mucinous tumors include mucinous cystic neoplasm and intraductal papillary mucinous tumor, both of which have been known to have a low malignant potential and a high rate of association with invasive adenocarcinoma. As such, preoperative diagnosis is of great significance in guiding patient management. Although fine‐needle aspiration cytological diagnosis of pancreatic tumor in cellular specimens has been well described, as with other cystic lesions, the yield of diagnostic cells from needle aspiration of cystic pancreatic mucinous tumors is typically low. Cytological diagnosis from these paucicellular specimens remains challenging. An additional compounding problem is the high frequency of gastrointestinal mucin and epithelial contamination. The diagnostic morphology and criteria in these paucicellular specimens have not been well addressed in the literature. The cytopathologists' ongoing efforts tend to improve the diagnostic accuracy. In this current study, oncotic cells, characterized by cytoplasmic swelling and karyolysis, were analyzed from 17 cases of cystic pancreatic mucinous tumor, of which the diagnosis was either confirmed by surgical resection or supported by cell block and/or increased CEA. Oncotic cells were found in variable amounts in almost all the cystic pancreatic mucinous tumors in this series. None of the five fine‐needle aspirations intended for aspirations of hypoechoic nonlesional pancreas, which yielded either gastrointestinal tract material only or admixture of gastrointestinal and normal pancreatic components, was found to contain oncotic cells, evidencing the utility of oncotic cell as a surrogate morphologic marker in aiding the diagnosis of cystic pancreatic mucinous tumor as well as its differentiation from gastrointestinal contaminant, particularly in paucicellular specimens. Diagn. Cytopathol. 2009. © 2008 Wiley‐Liss, Inc.     

miRNA‐199a‐5p regulates VEGFA in endometrial mesenchymal stem cells and contributes to the pathogenesis of endometriosis

It is believed that endometrial miRNAs contribute to the aetiology of endometriosis in stem cells; however, the mechanisms remain unclear. Here we collected serum samples from patients with or without endometriosis and characterized the miRNA expression profiles of these two groups. MicroRNA‐199a‐5p (miR‐199a‐5p) was dramatically down‐regulated in patients with endometriosis compared with control patients. In addition, we found that the tumour suppressor gene, SMAD4, could elevate miR‐199a‐5p expression in ectopic endometrial mesenchymal stem cells. Up‐regulation of miR‐199a‐5p suppressed cell proliferation, motility and angiogenesis of these ectopic stem cells by targeting the 3′ untranslated region of VEGFA. Furthermore, we established an animal model of endometriosis and found that miR‐199a‐5p could decrease the size of endometriotic lesions in vivo. Taken together, this newly identified miR‐199a‐5p module provides a new avenue to the understanding of the processes of endometriosis development, especially proliferation, motility and angiogenesis, and may facilitate the development of potential therapeutics against endometriosis. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Alkali‐like myosin light chain‐1 (myl1) is an early marker for differentiating fast muscle cells in zebrafish

During myogenesis, muscle precursors become divided into either fast‐ or slow‐twitch fibres, which in the zebrafish occupy distinct domains in the embryo. Genes encoding sarcomeric proteins specific for fast or slow fibres are frequently used as lineage markers. In an attempt to identify and evaluate early definitive markers for cells in the fast‐twitch pathway, we analysed genes encoding proteins contributing to the fast sarcomeric structures. The previously uncharacterized zebrafish alkali‐like myosin light chain gene (myl1) was found to be expressed exclusively in cells in the fast‐twitch pathway initiated at an early stage of fast fibre differentiation. Myl1 was expressed earlier, and in a more fibre type restricted manner, than any of the previously described and frequently used fast myosin light and heavy chain and troponin muscle markers mylz2, mylz3, tnni2, tnnt3a, fMyHC1.3. In summary, this study introduces a novel marker for early differentiating fast muscle cells. Developmental Dynamics 240:1856–1863, 2011. © 2011 Wiley‐Liss, Inc.     

Liver sinusoidal endothelial cells induce immunosuppressive IL‐10‐producing Th1 cells via the Notch pathway

Under homeostasis, liver sinusoidal endothelial cells (LSECs) shift intrahepatic T‐cell responses towards tolerance. However, the role of LSECs in the regulation of T‐cell‐induced liver inflammation is less clear. Here, we studied the capacity of LSECs to modulate pro‐inflammatory Th1‐cell differentiation in mice. Using in vitro co‐culture systems and subsequent cytokine analysis, we showed that LSECs induced high amounts of the anti‐inflammatory cytokine IL‐10 in developing Th1 cells. These LSEC‐stimulated Th1 cells had no pro‐inflammatory capacity in vivo but instead actively suppressed an inflammatory Th1‐cell‐induced delayed‐type hypersensitivity reaction. Blockage of IL‐10 signaling in vivo inhibited immunosuppressive activity of LSEC‐stimulated Th1 cells. We identified the Notch pathway as a mechanism how LSECs trigger IL‐10 expression in Th1 cells. LSECs expressed high levels of the Delta‐like and Jagged family of Notch ligands and induced expression of the Notch target genes hes‐1 and deltex‐1 in Th1 cells. Blockade of Notch signaling selectively inhibited IL‐10 induction in Th1 cells by LSECs. Our findings suggest that LSEC‐induced IL‐10 expression in Th1 cells via the Notch pathway may contribute to the control of hepatic inflammatory immune responses by induction of a self‐regulatory mechanism in pro‐inflammatory Th1 cells.     

Genotoxicity of 1‐methylpyrene and 1‐hydroxymethylpyrene in chinese hamster V79‐derived cells expressing both human CYP2E1 and SULT1A1

1‐Methylpyrene (1‐MP) is a widespread pollutant that is carcinogenic in animals following metabolic activation. Previous studies have shown that benzylic hydroxylation of 1‐MP, catalyzed by multiple CYP isoforms, gives rise to 1‐hydroxymethylpyrene (1‐HMP), which becomes bioreactive following further metabolism by various sulfotransferase (SULT) isoforms. However, the mutagenic and chromosome damaging effects of 1‐MP and 1‐HMP in mammalian cells have not been investigated. In this study a Chinese hamster V79‐derived cell line expressing both human CYP2E1 and human SULT1A1 was used to investigate the ability of 1‐MP and 1‐HMP to induce cytotoxicity (using the CCK‐8 assay), micronuclei and Hprt gene mutations. The role of each enzyme was investigated through co‐exposure in the presence of an enzyme inhibitor. We found that at concentrations of 0.5–4 μM and 5–20 μM, under conditions where no reduction in cell viability/growth occurred, 1‐HMP and 1‐MP induced micronuclei in V79‐hCYP2E1‐hSULT1A1 cells in a concentration‐dependent manner; however, both compounds were inactive in V79 cells. Similarly, they both caused an increase in Hprt mutant frequency in V79‐hCYP2E1‐hSULT1A1 cells in these concentration ranges, with 1‐MP impairing cell viability/growth at 10 μM and above in the mutagenicity assay. The compounds were again both inactive in V79 cells. The effects of 1‐HMP in V79‐hCYP2E1‐hSULT1A1 cells were blocked or reduced by addition of pentachlorophenol (PCP), a SULT1 inhibitor; the genotoxicity of 1‐MP was significantly reduced by either 1‐aminobenotrazole, a CYP2E1 inhibitor, or PCP. The results suggest that human CYP2E1 and SULT1A1 cooperate to activate 1‐MP and cause genotoxicity in mammalian cells. Environ. Mol. Mutagen. 56:404–411, 2015. © 2014 Wiley Periodicals, Inc.     

Multiple endocrine neoplasia type 1‐associated cystic pancreatic endocrine neoplasia and multifocal cholesterol granulomas

A novel combination of tumors was found in a 68 year‐old female with Multiple Endocrine Neoplasia type‐1 (MEN 1) that included a cystic pancreatic endocrine neoplasm (CPEN), a pituitary adenoma, and multifocal cholesterol granulomas (MCGs) in the breast, pleura, and the extremities. The pancreatic tumor displayed a single central locule surrounded by a thin rim of neoplastic parenchyma. The tumor showed heterogeneity in the architecture that included glandular, trabecular and solid patterns. The tumor cells of the pancreas were immunohistochemically positive for both endocrine and pancreatic acinar markers including chromogranin A, synaptophysin, glucagon, lipase, and reg protein. Electron microscopy revealed that there were numerous smaller dense‐cored neurosecretory granules, larger zymogen‐like granules and microvilli on the apical side of the tumor cells. The pancreatic tumor was diagnosed as CPEN with acinar cell features. Analysis of the DNA extracted from the tissues revealed that there is a MEN1 germline mutation in exon 10 codon 527, and somatic mutation in exon 2 codon 32 in the pancreatic tumor, and one base pair deletion in exon 2 codon 79 in the pituitary adenoma. Here, we report the case and discuss possible pathogenesis of CPEN and MCGs in a patient with MEN 1.     

Severe functional impairment and elevated PD‐1 expression in CD1d‐restricted NKT cells retained during chronic HIV‐1 infection

Invariant CD1d‐restricted NKT cells play important roles in regulating both innate and adaptive immunity. They are targeted by HIV‐1 infection and severely reduced in number or even lost in many infected subjects. Here, we have investigated the characteristics of NKT cells retained by some patients despite chronic HIV‐1 infection. NKT cells preserved under these circumstances displayed an impaired ability to proliferate and produce IFN‐γ in response to CD1d‐restricted lipid antigen as compared with cells from uninfected control subjects. HIV‐1 infection was associated with an elevated expression of the inhibitory programmed death‐1 (PD‐1) receptor (CD279) on the CD4^? subset of NKT cells. However, blocking experiments indicated that the functional defects in NKT cells were largely PD‐1‐independent. Furthermore, the elevated PD‐1 expression and the functional defects were not restored by anti‐retroviral treatment, and the NKT cell numbers in blood did not recover significantly in response to treatment. The functional phenotype of NKT cells in these patients suggests an irreversible immune exhaustion due to chronic activation in vivo. The data demonstrate a severe functional impairment in the remaining NKT‐cell compartment in HIV‐1‐infected patients, which limits the prospects to mobilize these cells in immunotherapy approaches in patients.     

Sphingosine‐1‐phosphate receptor 1 signalling in T cells: trafficking and beyond

Sphingosine-1-phosphate (S1P) is a lipid second messenger that signals via five G protein-coupled receptors (S1P_1–5). S1P receptor (S1PR) signalling is associated with a wide variety of physiological processes including lymphocyte biology, their recirculation and determination of T-cell phenotypes. The effect of FTY720 (Fingolimod, Gilenya™) to regulate lymphocyte egress and to ameliorate paralysis in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis led to the use of FTY720 as a first-line oral agent for treatment of relapsing–remitting multiple sclerosis. However, a significant body of research suggests that S1P signalling may participate in diverse immune regulatory functions other than lymphocyte trafficking. This review article discusses the current knowledge of S1P signalling in the fate and function of T regulatory, T helper type 17 and memory T cells in health and disease.     

Loss of natural killer T cells promotes pancreatic cancer in LSL‐KrasG12D/+ mice

The role of the unique T‐cell population, natural killer T (NKT) cells, which have similar functions to NK cells in pancreatic cancer (PC), is not yet evaluated. To address the regulatory roles of NKT cells on tumour progression through tumour‐associated macrophages (TAM) and their production of microsomal prostaglandin E synthase‐1 (mPGES‐1) and 5‐lipoxygenase (5‐LOX) in (Kras)‐driven pancreatic tumour (KPT) progression, we crossed CD1d^−/− mice deficient in both invariant and variant NKT cells with the Kras^G12D mice. Loss of NKT cells significantly increased pancreatic intraepithelial neoplasia (PanIN) lesions and also increased 5‐LOX and mPGES‐1 expression in M2‐type macrophages and cancer stem‐like cells in pancreatic tumours. Pharmacological inhibition of mPGES‐1 and 5‐LOX in M2 macrophages with specific inhibitor YS‐121 in KPT‐CD1d^−/− mice decreased PanIN lesions and suppressed tumour growth in association with elevated levels of active CD8a cells. Hence, NKT cells regulate PC by modulating TAMs (M2) through mPGES‐1 and 5‐LOX; and the absence of NKT cells leads to aggressive development of PC.     

Two distinct types of theca cells in the medaka gonad: Germ cell‐dependent maintenance of cyp19a1‐expressing theca cells

Aromatase is a steroidogenic enzyme catalyzing the production of estrogens and is important for the proper development and function of the reproductive system. The lineage of cyp19a1 (ovarian‐type aromatase)‐expressing cells in the developing gonad was analyzed using a transgenic medaka (Oryzias latipes) that recapitulates endogenous cyp19a1 expression with EGFP fluorescence. Our results show that cyp19a1‐expressing cells arise in the ventral stromal cells of the developing female gonad, then expand anteriorly as the gonadal region extends anteriorly. These cells become located close to the developing follicles, and are distinguishable from the P450c17‐I‐expressing theca cells. In the adult ovary, the expression of P450c17‐I and cyp19a1 are mutually exclusive in the outer theca‐cell layer. Cyp19a1 expression in the granulosa cells is found only in the population of large follicles. These observations demonstrate two types of theca cells in the medaka ovary. We also show that the maintenance of cyp19a1‐expressing cells depends on germ cells. Developmental Dynamics 238:2652–2657, 2009. © 2009 Wiley‐Liss, Inc.     

Notch1 modulates mesenchymal stem cells mediated regulatory T‐cell induction

Notch1 signaling is involved in regulatory T (Treg)‐cell differentiation. We previously demonstrated that, when cocultured with CD3⁺ cells, mesenchymal stem cells (MSCs) induced a T‐cell population with a regulatory phenotype. Here, we investigated the molecular mechanism underlying MSC induction of human Treg cells. We show that the Notch1 pathway is activated in CD4⁺ T cells cocultured with MSCs. Inhibition of Notch1 signaling through GSI‐I or the Notch1 neutralizing antibody reduced expression of HES1 (the Notch1 downstream target) and the percentage of MSC‐induced CD4⁺CD25^highFOXP3⁺ cells in vitro. Moreover, we demonstrate that FOXP3 is a downstream target of Notch signaling in human cells. No crosstalk between Notch1 and TGF‐β signaling pathways was observed in our experimental system. Together, these findings indicate that activation of the Notch1 pathway is a novel mechanism in the human Treg‐cell induction mediated by MSCs.     

MicroRNA‐664a‐5p promotes neuronal differentiation of SH‐SY5Y cells

MicroRNAs (miRNAs) belong to a class of small noncoding RNAs that play important roles in the translational regulation of gene expression. A number of miRNAs are known to act as key regulators of diverse processes such as neuronal differentiation. In this study, we have attempted to identify novel miRNAs related to neuronal differentiation via microarray analysis in the human neuronal differentiation model neuroblastoma SH‐SY5Y cells. We identified 15 up‐regulated and eight down‐regulated miRNAs in SH‐SY5Y cells treated with all‐trans retinoic acid to induce differentiation. We further showed that one of the up‐regulated miRNAs, miR‐664a‐5p, promoted neuronal differentiation of SH‐SY5Y cells. These findings enhance our understanding of the miRNAs involved in the process of neurogenesis and, in particular, highlight an important role of miR‐664a‐5p in SH‐SY5Y cell neuronal differentiation. Further studies will be required to confirm the function of miR‐664‐5p in neuronal development and disease and to identify its relevant target genes.