Non-lymphoid Cells Research Articles

Homeostatic NF-κB Signaling in Steady-State Migratory Dendritic Cells Regulates Immune Homeostasis and Tolerance

Migratory non-lymphoid tissue dendritic cells (NLT-DCs) transport antigens to lymph nodes (LNs) and are required for protective immune responses in the context of inflammation and to promote tolerance to self-antigens in steady-state. However, the molecular mechanisms that elicit steady-state NLT-DC maturation and migration are unknown. By comparing the transcriptome of NLT-DCs in the skin with their migratory counterparts in draining LNs, we have identified a novel NF-κB-regulated gene network specific to migratory DCs. We show that targeted deletion of IKKβ in DCs, a major activator of NF-κB, prevents NLT-DC accumulation in LNs and compromises regulatory T cell conversion in vivo. This was associated with impaired tolerance and autoimmunity. NF-κB is generally considered the prototypical pro-inflammatory transcription factor, but this study describes a role for NF-κB signaling in DCs for immune homeostasis and tolerance that could have implications in autoimmune diseases and immunity.

The NADPH oxidase NOX5 protects against apoptosis in ALK-positive anaplastic large-cell lymphoma cell lines

Reactive oxygen species (ROS) are key modulators of apoptosis and carcinogenesis. One of the important sources of ROS is NADPH oxidases (NOXs). The isoform NOX5 is highly expressed in lymphoid tissues, but it has not been detected in any common Hodgkin or non-Hodgkin lymphoma cell lines. In diverse, nonlymphoid malignant cells NOX5 exerts an antiapoptotic effect. Apoptosis suppression is the hallmark feature of a rare type of lymphoma, termed anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL), and a major factor in the therapy resistance and relapse of ALK+ ALCL tumors. We applied RT-PCR and Western blot analysis to detect NOX5 expression in three ALK+ ALCL cell lines (Karpas-299, SR-786, SUP-M2). We investigated the role of NOX5 in apoptosis by small-interfering RNA (siRNA)-mediated gene silencing and chemical inhibition of NOX5 using FACS analysis and examining caspase 3 cleavage in Karpas-299 cells. We used immunohistochemistry to detect NOX5 in ALK+ ALCL pediatric tumors. NOX5 mRNA was uniquely detected in ALK+ ALCL cells, whereas cell lines of other lymphoma classes were devoid of NOX5. Transfection of NOX5-specific siRNA and chemical inhibition of NOX5 abrogated calcium-induced superoxide production and increased caspase 3-mediated apoptosis in Karpas-299 cells. Immunohistochemistry revealed focal NOX5 reactivity in pediatric ALK+ ALCL tumor cells. These results indicate that NOX5-derived ROS contribute to apoptosis blockage in ALK+ ALCL cell lines and suggest NOX5 as a potential pharmaceutical target to enhance apoptosis and thus to suppress tumor progression and prevent relapse in pediatric ALK+ ALCL patients that resist classical therapeutic approaches.

Cytologic mimics of non-Hodgkin lymphoma in the head and neck

Non-lymphoid small round blue cell tumors of the head and neck are particularly difficult to diagnose when metastatic to a lymph node. The cytopathologist or surgical pathologist evaluating these lesions has to be aware of the various non-hematopoetic neoplasms that present in the head and neck area that can mimic non-Hodgkin lymphoma. Presented here are the various lesions commonly seen which needs to be entertained depending on where in the head and neck the lesion is located. More recently, a plethora of HPV related head and neck tumors has been described and these lesions add to the mix in this challenging milieu of cases.

Atrazine induces endoplasmic reticulum stress-mediated apoptosis of T lymphocytes via the caspase-8-dependent pathway.

Atrazine (ATR) is one of the most commonly applied broad-spectrum herbicides. Although ATR is well known to be a biologically hazardous molecule with potential toxicity in the immune system, the molecular mechanisms responsible for ATR-induced immunotoxicity remain unclear. In this study, we found that the immunotoxic properties of ATR were mediated through the induction of apoptotic changes in T lymphocytes. Mice exposed to ATR for 4 weeks exhibited a significant decrease in the number of spleen CD3(+) T lymphocytes, while CD19(+) B lymphocytes and nonlymphoid cells were unaffected. ATR exposure also led to inhibition of cell growth and induction of apoptosis in human Jurkat T-cells. Importantly, ATR triggered the activation of caspase-3 and the cleavage of caspase-8 and PARP, whereas it did not affect the release of cytochrome c from the mitochondria in Jurkat T-cells. In addition, ATR activated the unfolded protein response signaling pathway, as indicated by eIF2α phosphorylation and CHOP induction. Our results demonstrate that ATR elicited an immunotoxic effect by inducing ER stress-induced apoptosis in T-cells, therefore providing evidence for the molecular mechanism by which ATR induces dysregulation of the immune system. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 998-1008, 2016.

Short Communication: Diversity of TNF-α region in Chinese domestic goats.

Tumor necrosis factor-α is a cytokine with a wide range of effects on both lymphoid and non-lymphoid cells. In this study, we identified polymorphisms in major histocompatibility complex class III gene in the 4th exon and the 3' untranslated region of tumor necrosis factor-α to evaluate the immunogenetic diversity of Chinese south indigenous goat. Three single-nucleotide polymorphisms were identified and showed similar frequencies in different except MI loci. These data suggest that the high immunodiversity of the tumor necrosis factor-α region within these breeds can be used for strengthening variety improvement and promoting animal husbandry development in Chinese indigenous goats.

Abstract 4531: INCB40093 is a selective PI3Kδ inhibitor with potent antiproliferative activity against human B-cell tumors

Abstract Phosphatidylinositol 3-kinases (PI3Ks) belong to a family of lipid signaling kinases that phosphorylate phosphatidylinositol-4,5-bisphosphate, giving rise to phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 functions as a second messenger that controls a number of cellular processes, including growth, survival, adhesion and migration. The delta isoform of PI3K (PI3Kδ) plays an essential role in B-cell development and functions by mediating the signaling of key receptors on B cells including BCR, BAFF-R, IL-4R, CD40 and TLRs. In addition, increased malignant B cell proliferation and survival has been associated with aberrant activation of PI3Kδ, and data from recent clinical studies using idelalisib, a PI3Kδ specific inhibitor, indicate that the enzyme is a therapeutic target for the treatment of B cell lymphomas. INCB40093 is a novel, potent small molecule inhibitor of PI3Kδ with 74 to > 900-fold selectivity over the other PI3K family members. It also does not inhibit (< 30% inhibition) a broad panel of 196 other kinases when tested at a concentration of 1 μM. INCB40093 blocks the proliferation of primary B cells induced by the activation of PI3Kδ-mediated signaling through several key receptors (e.g. BCR, CD40). More importantly, INCB40093 is potent (IC50 values of ∼10-100 nM) in cell-based assays relevant to the pathogenesis of B cell malignancies, such as PI3Kδ-mediated signaling and growth of human B cell lines. This effect is not due to general cytotoxicity because 10 μM INCB40093 had no significant effect on the growth of non-lymphoid cell lines. Compared to the inhibition of primary B cell proliferation, INCB40093 is at least 50 times less potent in assays that measure human T cell or NK cell proliferation, and neutrophil and macrophage function, suggesting that the impact of INCB40093 on the human immune system will likely be restricted to B cells. In vivo, the effects of INCB40093 were evaluated in two human hematological cancer xenograft models. In the Pfeiffer model of non-Hodgkin's lymphoma, oral administration of INCB40093 inhibited tumor growth as a single agent. INCB40093 also potentiated the anti-tumor effects of the clinically relevant chemotherapeutic agent, bendamustine. The tumor growth inhibitory effects of INCB40093 were also demonstrated in a myeloma tumor xenograft model, INA-6.Tu1. Taken together, pharmacological data obtained in both in vitro and in vivo model systems support the potential utility of orally administered INCB40093 in the treatment of B cell malignancies. Citation Format: Niu Shin, Kathy Wang, Leslie Hall, Qian Wang, Gengjie Yang, Yanlong Li, Yun-Long Li, Maryanne Covington, Jordan Fridman, Robert Newton, Peggy Scherle. INCB40093 is a selective PI3Kδ inhibitor with potent antiproliferative activity against human B-cell tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4531. doi:10.1158/1538-7445.AM2014-4531

Ikaros and RAG-2-mediated antisense transcription are responsible for lymphocyte-specific inactivation of NWC promoter.

Recombination activating gene-2 (RAG-2) and NWC are strongly evolutionarily conserved overlapping genes which are convergently transcribed. In non-lymphoid cells the NWC promoter is active whereas in lymphocytes it is inactive due to the DNA methylation. Analysing the mechanism responsible for lymphocyte-specific methylation and inactivation of NWC promoter we found that Ikaros, a lymphocyte-specific transcription factor, acts as a repressor of NWC promoter - thus identifying a new Ikaros target - but is insufficient for inducing its methylation which depends on the antisense transcription driven by RAG-2 promoter. Possible implications of these observations for understanding evolutionary mechanisms leading to lymphocyte specific expression of RAG genes are discussed.

Fish thymic non-lymphoid cells and the problem of the blood-thymus barrier

This review is devoted to a comparative histological analysis of non-lymphoid thymic cell populations in fish and mammals. The structures and functions of these cells, as well as their role in the formation of the blood-thymus barrier, are discussed. Data that point to the involvement of the thymus in the immune response of fish are given.

El receptor NKG2D en la frontera de la inmunovigilancia y la carcinogénesis

El sistema inmune es capaz de realizar la detección y eliminación de células transformadas por un mecanismo fisiológico conocido como inmunovigilancia. En este proceso participa el receptor activador NKG2D presente en linfocitos T y células NK, ambos de suma relevancia en la inmunovigilancia contra el cáncer. Al reconocer el receptor NKG2D a sus ligandos (NKG2DLs) en las células que experimentan neotransformación se desencadena la respuesta lítica específica de las células linfoides citotóxicas. Asimismo, se ha descrito en diversos tipos de cáncer formas solubles de NKG2DLs que se ha demostrado son utilizadas para la evasión tumoral al saturar los receptores NKG2D presentes en las células efectoras linfoides evitando de esta manera ser reconocidas y eliminadas y, con ello escapando de la inmunovigilancia. Aunque este fenómeno de evasión inmune, donde participan algunos NKG2DLs, ha sido ya descrito y corroborado clínicamente no se ha estudiado si el receptor NKG2D está presente en las células tumorales per se ya que también podría estar implicado en subvertir la inmunovigilancia. En este trabajo se analizan evidencias recientes de que la expresión del receptor NKG2D no es exclusiva de linfocitos T y NK ya es expresado por células epiteliales tumorales tanto in vitro como in vivo. Las consecuencias de esta anómala expresión en células no linfoides tiene amplias implicaciones en la carcinogénesis que serán revisadas. También se analizan estudios clínicos recientes donde se comprueba la participación del receptor NKG2D en diferentes patologías tumorales.

Extracellular adenosine regulates colitis through effects on lymphoid and nonlymphoid cells.

Adenosine is a purine metabolite that can mediate anti-inflammatory responses in the digestive tract through the A(2A) adenosine receptor (A(2A)AR). We examined the role of this receptor in the control of inflammation in the adoptive transfer model of colitis. Infection of A(2A)AR(-/-) mice with Helicobacter hepaticus increased colonic inflammation scores compared with uninfected A(2A)AR controls. Comparison of T cell subsets in wild-type and A(2A)AR(-/-) mice revealed differences in markers associated with activated helper T (Th) cells and regulatory T (Treg) cells. Previous studies showed that expression of A(2A)AR on CD45RB(HI) and CD45RB(LO) Th cells is essential for the proper regulation of colonic inflammation. Adoptive transfer of CD45RB(HI) with CD45RB(LO) from wild-type mice into RAG1(-/-)/A(2A)AR(-/-) mice induced severe disease within 3 wk, although transfer of the same subsets into RAG1(-/-) mice does not induce colitis. This suggests that the presence of A(2A)AR on recipient cells is also important for controlling colitis. To investigate the role of A(2A)AR in myeloid cells, chimeric recipients were generated by injection of bone marrow from RAG1(-/-) or RAG1(-/-)/A(2A)AR(-/-) mice into irradiated RAG1(-/-) mice. After adoptive transfer, these recipients did not develop colitis, regardless of A(2A)AR expression by the donor. Together, our results suggest that the control of inflammation in vivo is dependent on A(2A)AR signaling through multiple cell types that collaborate in the regulation of colitis by responding to extracellular adenosine.

An unexpected role of Smad4 to control autoimmunity and T cell function (BA8P.125)

Abstract Smad-dependent signaling pathway is known to mediate TGF-beta signal in non-lymphoid cells. How this pathway controls T cell function remains poorly understood. In particular, the function of Smad4 (co-Smad) in T cells is perplexing. On the contrary to the prediction that Smad4-deficient mice will pheno-copy TGF-betaR-deficient mice and develop aggressive autoimmunity, Smad4-deficient mice are phenotypically grossly normal. Two explanations may account for such observations. One is that Smad4 is dispensable for TGF-beta function in T cells. The other is that, while Smad4 is important for TGF-beta function in T cells, it is also critical to promote T cell function to offset its TGF-beta-dependent function. To test these possibilities, we generated DKO mice, where both TGF-betaR and Smad4 are deficient in T cells. Much to our surprise, Smad4 deletion rescued the lethal autoimmunity of TGF-betaR-deficient mice. DKO mice phenocopy Smad4-deficient mice. These findings suggest that the second explanation, albeit paradoxical, is correct. Further investigation revealed that, Smad4 is indeed required for TGF-beta function in T cells and, unexpectedly, it is also important to promote T cell function. These findings therefore identify a novel function of Smad4 critical for T cell function and autoimmunity. Here, we will discuss in detail these new findings and the potential underlying mechanisms.

Role of innate immune response in non-alcoholic Fatty liver disease: metabolic complications and therapeutic tools.

Non-alcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide, both in adults and children. It is characterized by an aberrant lipid storage in hepatocytes, named hepatic steatosis. Simple steatosis remains a benign process in most affected patients, while some of them develop superimposed necroinflammatory activity with a non-specific inflammatory infiltrate and a progression to non-alcoholic steatohepatitis with or without fibrosis. Deep similarity and interconnections between innate immune cells and those of liver parenchyma have been highlighted and showed to play a key role in the development of chronic liver disease. The liver can be considered as an “immune organ” because it hosts non-lymphoid cells, such as macrophage Kupffer cells, stellate and dendritic cells, and lymphoid cells. Many of these cells are components of the classic innate immune system, enabling the liver to play a major role in response to pathogens. Although the liver provides a “tolerogenic” environment, aberrant activation of innate immune signaling may trigger “harmful” inflammation that contributes to tissue injury, fibrosis, and carcinogenesis. Pathogen recognition receptors, such as toll-like receptors and nucleotide oligomerization domain-like receptors, are responsible for the recognition of immunogenic signals, and represent the major conduit for sensing hepatic and non-hepatic noxious stimuli. A pivotal role in liver inflammation is also played by cytokines, which can initiate or have a part in immune response, triggering hepatic intracellular signaling pathways. The sum of inflammatory signals and deranged substrate handling induce most of the metabolic alteration traits: insulin resistance, obesity, diabetes, hyperlipidemia, and their compounded combined effects. In this review, we discuss the relevant role of innate immune cell activation in relation to NAFLD, the metabolic complications associated to this pathology, and the possible pharmacological tools.

A 46-year-old Chinese woman presenting with retroperitoneal follicular dendritic cell sarcoma: a case report

IntroductionFollicular dendritic cells are non-phagocytic, non-lymphoid cells of the immune system that are necessary for antigen presentation and the regulation of reactions in the germinal centers of the lymph nodes. Follicular dendritic cell sarcoma is an unusual cancer, particularly in the intra-abdominal region. In the present report we describe an unusual case of retroperitoneal follicular dendritic cell sarcoma that emphasizes the difficulty of diagnosing and treating this tumor. Retroperitoneal follicular dendritic cell sarcoma has only been rarely reported in the literature to date.Case presentationA 46-year-old Chinese woman of Han ethnicity presented with chronic right lower quadrant abdominal pain over the preceding 4 weeks. The tumor was resected and submitted to histopathological examination. The case was verified as retroperitoneal follicular dendritic cell sarcoma by microscopic examination and immunohistochemical analysis. After diagnosis, she received postoperative radiotherapy and chemotherapy. She has survived 3 years postoperatively, although she has a pulmonary metastasis.ConclusionsRetroperitoneal follicular dendritic cell sarcoma may demonstrate aggressive potential. This study indicated that postoperative adjuvant radiotherapy and chemotherapy could extend the survival of a patient with retroperitoneal follicular dendritic cell sarcoma.

Feline Immunodeficiency Virus Envelope Glycoproteins Antagonize Tetherin through a Distinctive Mechanism That Requires Virion Incorporation

BST2/tetherin inhibits the release of enveloped viruses from cells. Primate lentiviruses have evolved specific antagonists (Vpu, Nef, and Env). Here we characterized tetherin proteins of species representing both branches of the order Carnivora. Comparison of tiger and cat (Feliformia) to dog and ferret (Caniformia) genes demonstrated that the tiger and cat share a start codon mutation that truncated most of the tetherin cytoplasmic tail early in the Feliformia lineage (19 of 27 amino acids, including the dual tyrosine motif). Alpha interferon (IFN-α) induced tetherin and blocked feline immunodeficiency virus (FIV) replication in lymphoid and nonlymphoid feline cells. Budding of bald FIV and HIV particles was blocked by carnivore tetherins. However, infectious FIV particles were resistant, and spreading FIV replication was uninhibited. Antagonism mapped to the envelope glycoprotein (Env), which rescued FIV from carnivore tetherin restriction when expressed in trans but, in contrast to known antagonists, did not rescue noncognate particles. Also unlike the primate lentiviral antagonists, but similar to the Ebola virus glycoprotein, FIV Env did not reduce intracellular or cell surface tetherin levels. Furthermore, FIV-enveloped FIV particles actually required tetherin for optimal release from cells. The results show that FIV Envs mediate a distinctive tetherin evasion. Well adapted to a phylogenetically ancient tetherin tail truncation in the Felidae, it requires functional virion incorporation of Env, and it shields the budding particle without downregulating plasma membrane tetherin. Moreover, FIV has evolved dependence on this protein: particles containing FIV Env need tetherin for optimal release from the cell, while Env(-) particles do not. HIV-1 antagonizes the restriction factor tetherin with the accessory protein Vpu, while HIV-2 and the filovirus Ebola use their envelope (Env) glycoproteins for this purpose. It turns out that the FIV tetherin antagonist is also its Env protein, but the mechanism is distinctive. Unlike other tetherin antagonists, FIV Env cannot act in trans to rescue vpu-deficient HIV-1. It must be incorporated specifically into FIV virions to be active. Also unlike other retroviral antagonists, but similar to Ebola virus Env, it does not act by downregulating or degrading tetherin. FIV Env might exclude tetherin locally or direct assembly to tetherin-negative membrane domains. Other distinctive features are apparent, including evidence that this virus evolved an equilibrium in which tetherin is both restriction factor and cofactor, as FIV requires tetherin for optimal particle release.

S1P lyase in thymic perivascular spaces promotes egress of mature thymocytes via up-regulation of S1P receptor 1

Sphingosine 1-phosphate (S1P) and S1P receptor 1 (S1P1) play an important role in the egress of mature CD4 or CD8 single-positive (SP) thymocytes from the thymus. Fingolimod hydrochloride (FTY720), an S1P1 functional antagonist, induced significant accumulation of CD62L(high)CD69(low) mature SP thymocytes in the thymic medulla. Immunohistochemical staining using anti-S1P1 antibody revealed that S1P1 is predominantly expressed on thymocytes in the thymic medulla and is strongly down-regulated even at 3h after FTY720 administration. 2-Acetyl-4-tetrahydroxybutylimidazole (THI), an S1P lyase inhibitor, also induced accumulation of mature SP thymocytes in the thymic medulla with an enlargement of the perivascular spaces (PVS). At 6h after THI administration, S1P1-expressing thymocytes reduced partially as if to form clusters and hardly existed in the proximity of CD31-expressing blood vessels in the thymic medulla, suggesting S1P lyase expression in the cells constructing thymic medullary PVS. To determine the cells expressing S1P lyase in the thymus, we newly established a mAb (YK19-2) specific for mouse S1P lyase. Immunohistochemical staining with YK19-2 revealed that S1P lyase is predominantly expressed in non-lymphoid thymic stromal cells in the thymic medulla. In the thymic medullary PVS, S1P lyase was expressed in ER-TR7-positive cells (reticular fibroblasts and pericytes) and CD31-positive vascular endothelial cells. Our findings suggest that S1P lyase expressed in the thymic medullary PVS keeps the tissue S1P concentration low around the vessels and promotes thymic egress via up-regulation of S1P1.

Detection of HIV-1 Neutralizing Antibodies in a Human CD4+/CXCR4+/CCR5+ T-Lymphoblastoid Cell Assay System

Sensitive assays are needed to meaningfully assess low levels of neutralizing antibodies (NAbs) that may be important for protection against the acquisition of HIV-1 infection in vaccine recipients. The current assay of choice uses a non-lymphoid cell line (TZM-bl) that may lack sensitivity owing to over expression of CD4 and CCR5. We used transfection of a human CD4+/CXCR4+/α4β7+ T-lymphoblastoid cell line (A3.01) with a CMV IE promoter-driven CCR5neo vector to stably express CCR5. The resulting line, designated A3R5, is permissive to a wide range of CCR5-tropic circulating strains of HIV-1, including HIV-1 molecular clones containing a Tat-inducible Renilla luciferase reporter gene and expressing multiple Env subtypes. Flow cytometric analysis found CCR5 surface expression on A3R5 cells to be markedly less than TZM-bl but similar to CD3.8 stimulated PBMC. More importantly, neutralization mediated by a diverse panel of monoclonal antibodies, HIV-1 positive polyclonal sera and sCD4 was consistently greater in A3R5 compared to TZM-bl cells. The A3R5 cell line provides a novel approach to guide the development and qualification of promising new HIV-1 vaccine immunogens.

Sustained Alterations In Bone Marrow Stromal Cells From Patients With Myeloproliferative Neoplasms (MPN) Contribute To Remodelling Of The Bone Marrow Microenvironment Prior To The Manifestation Of Myelofibrosis

Myeloproliferative neoplasms (MPN) are characterized by the loss of normal hematopoiesis and the excessive production and accumulation of non-lymphoid cells and platelets in the BM. Clonal hematopoiesis in MPN is assumed to generate factors which induce profound changes in the non-clonal BM microenvironment. Alterations include massive deposition of extracellular matrix proteins (ECM), progression to bone marrow (BM)-fibrosis and osteosclerosis. We hypothesize that particularly in MPN, alterations in the cross talk between hematopoietic and bone marrow stromal cells (BMSC) play a critical role for an impaired bone marrow microenvironment long before overt signs of myelofibrosis can be detected by conventional methods.To dissect the hematopoiesis supporting capacity and extracellular matrix remodelling of BMSC from patients with MPN, we isolated BMSC from BM of patients with essential thrombocytemia (ET, n=5), polycythemia vera (PV, n=5), chronic myeloid leukemia (CML, n=5) and control BM (n=6). BMSC isolates were taken only from pre-fibrotic MPN patients (bone marrow trephine biopsy reticulin staining graded 0 or 1) and the resulting expansion cultures fullfilled MSC criteria according to the common consensus (Dominici et al., Cytotherapy, 2006; 8(4):315-317). When subjected to myeloid colony forming unit assays, MPN-BMSC conditioned supernatants showed a significantly reduced capacity to stimulate a CFU-GM/G/M growth of non-malignant hematopoietic stem and progenitor cells as compared to control BMSC (control (n=6) vs CML (n=5): p= 0.0032; control vs PV (n=5): p=0.016, control vs ET (n=4) not significant; student«s T- test ). BMSC-dependent matrix remodelling was analysed in a previously established robust matrix remodelling assay in vitro. MPN-BMSC displayed a pronounced increased matrix remodelling capacity compared to control BMSC. Interestingly, among the different MPN subtypes, this effect was highly significant in BMSC derived from patients with ET (Control (n=6) vs. ET (n=5): p<0.001). Furthermore, in vitro ECM production by MPN-BMSC was paralleled by ECM changes observed in matched bone marrow punches as shown by fibronectin immunohistochemistry. Co-expression of the stroma marker CD271 and fibronectin -as shown by confocal microscopy- points towards stroma-mediated ECM production in vivo. As upregulation of fibronectin expression was also detected in reticulin 0 graded BM punches, we hypothesized that fibronectin staining might be a potential marker for pre-fibrotic ECM changes in –so far- reticulin-negative MPN biopsies. To validate this hypothesis, we stained fibronectin in a tissue microarray (TMA), containing primary BM biopsies from patients with ET (n=14), PV (n=14), CML (n=14), MF (n=11) and controls (Non-Hodgkin's lymphoma without bone marrow involvement, n=17). Interestingly, within the reticulin-negative subcohort of pre-fibrotic MPN (n=34), fibronectin stained positive (grade 1 or higher) in 5/7 cases with ET (71%), 6/9 cases with PV (66%) and in 14/14 cases with CML (100%) as well as in all cases with PMF (100%). Furthermore, fibronectin staining correlated significantly with patients' decreased haemoglobin levels as shown by ANOVA analysis of routine clinical parameters (F=5.71; Prob> F 0.0037).We conclude that prior to the manifestation of fibrosis in MPN, BMSC loose their capacity to support non-malignant hematopoiesis and increase their matrix remodelling potential. As to our surprise, these effects are stably maintained in the absence of the malignant hematopoietic clone and since BMSC cultures develop over numerous population doublings in vitro from proliferating mesenchymal precursors, we hypothesize that cell intrinsic effects in BMSC from patients with MPN contribute to the development of myelofibrosis in MPN. [Display omitted] Disclosures:Bruemmendorf: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria.

Correlation of increased PARP14 and CCL26 expression in biopsies from children with eosinophilic esophagitis

Correlation of increased PARP14 and CCL26 expression in biopsies from children with eosinophilic esophagitis

Transcriptional regulation of teleost Aicda genes. Part 1 – Suppressors of promiscuous promoters

In order to better understand antibody affinity maturation in fishes we sought to identify gene regulatory elements that could drive expression of activated B-cell specific fluorescent reporter transgenes in zebrafish. Specifically the promoter and several non-coding regions of the channel catfish (Ictalurus punctatus) and zebrafish (Danio rerio) were tested for transcriptional activity using a dual luciferase reporter system in transfected fish leukocytes and two mammalian cell lines that constitutively express Aicda (activation-induced cytidine deaminase). The promoters of both fish Aicda genes were as transcriptionally active as an SV40 promoter control in all cell lines tested, regardless of the cells ability to express Aicda. Coupling of a putative intron 1 enhancer or a region 10 kb upstream of the zebrafish promoter effectively silenced transcription from the fish Aicda promoter. Paradoxically these suppressor elements enhanced transcription when they were coupled to the mouse Aicda intron 1 enhancer. The results are considered in context of similar observations for Aicda transcriptional regulation in mice and in light of recent evidence that Aicda is utilized for epigenetic reprogramming of several non-lymphoid cell types.

The Invasive Chytrid Fungus of Amphibians Paralyzes Lymphocyte Responses

The chytrid fungus, Batrachochytrium dendrobatidis, causes chytridiomycosis and is a major contributor to global amphibian declines. Although amphibians have robust immune defenses, clearance of this pathogen is impaired. Because inhibition of host immunity is a common survival strategy of pathogenic fungi, we hypothesized that B. dendrobatidis evades clearance by inhibiting immune functions. We found that B. dendrobatidis cells and supernatants impaired lymphocyte proliferation and induced apoptosis; however, fungal recognition and phagocytosis by macrophages and neutrophils was not impaired. Fungal inhibitory factors were resistant to heat, acid, and protease. Their production was absent in zoospores and reduced by nikkomycin Z, suggesting that they may be components of the cell wall. Evasion of host immunity may explain why this pathogen has devastated amphibian populations worldwide.