TLR2 deficiency promotes IgE and inhibits IgG1 class-switching following ovalbumin sensitization

2020 ◽  
Author(s):  
Yuqin Li ◽  
Qiu Chen ◽  
Wei Ji ◽  
Yujie Fan ◽  
Li Huang ◽  
...  
Keyword(s):  
Enzyme Linked Immunosorbent Assay ◽  
Lung Pathology ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Rt Pcr ◽  
Class Switching ◽  
Th2 Cytokine ◽  
Stat3 Phosphorylation ◽  
Hematoxylin And Eosin

Abstract Background: To explore the roles of Toll-like receptor (TLR)2 in Th2 cytokine production and immunoglobulin (Ig) class switching following ovalbumin (OVA) sensitization.Methods: TLR2-/- and wild-type C57BL/6 mice were sensitized by intraperitoneal injection with OVA. Lung pathology was assessed by hematoxylin and eosin staining. Abundance of interleukin (IL)4, IL5, IL13, and IL21 transcripts in the lungs was quantified by RT-PCR. OVA-specific IgG1, IgG2a, IgG2b, IgE and IgM were quantified by enzyme-linked immunosorbent assay. Phosphorylated signal transducer and activator of transcription (STAT)3 in lung tissue was detected by immunohistochemistry staining and nuclear factor (NF) κB activation was measured by immunofluorescence staining. STAT3 activation was inhibited using cryptotanshinone (CPT) treatment. Germline transcripts (Iμ-Cμ, Iγ-Cγ, Iα-Cα or Iε-Cε), post-recombination transcripts (Iμ-Cγ, Iμ-Cα or Iμ- Cε) and mature transcripts (VHDJH-Cγ, VHDJH-Cα or VHDJH-Cε) were analyzed from splenic B cells of OVA-sensitized wild-type mice (with or without CPT treatment) and TLR2-/- mice (with or without IL21 treatment). Results: The lungs of TLR2-/- mice showed a lesser degree of inflammation than wild-type mice after OVA sensitization. Following OVA sensitization, levels of IL4, IL13, and IL21, but not IL5, were significantly lower in TLR2-/- compared with wild-type mice. Moreover, OVA-specific IgG1 and IgE titers were markedly lower and higher, respectively, in TLR2-/- mice. TLR2 deficiency inhibited STAT3 activation but not NF-κB p65 activation. CPT treatment reduced IgG1 titers via inhibition of Stat3 phosphorylation. Both TLR2 knockout and CPT treatment reduced the frequencies of Iγ1-Cγ1, Iγ3-Cγ3 and Iα-Cα transcripts, but IL21 treatment compensated for the effects of TLR2 deficiency.Conclusion: These results suggest a role of TLR2 in restricting OVA-sensitized lung inflammation via promotion of IgG1 and inhibition of IgE class switching regulated by IL21 and STAT3.

scholarly journals TLR2 deficiency promotes IgE and inhibits IgG1 class-switching following ovalbumin sensitization

2021 ◽  
Vol 47 (1) ◽  
Author(s):  
Yuqin Li ◽  
Qiu Chen ◽  
Wei Ji ◽  
Yujie Fan ◽  
Li Huang ◽  
...  
Keyword(s):  
Enzyme Linked Immunosorbent Assay ◽  
Lung Pathology ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Rt Pcr ◽  
Class Switching ◽  
Th2 Cytokine ◽  
Stat3 Phosphorylation ◽  
Hematoxylin And Eosin

Abstract Background To explore the roles of Toll-like receptor (TLR)2 in Th2 cytokine production and immunoglobulin (Ig) class switching following ovalbumin (OVA) sensitization. Methods TLR2−/− and wild-type C57BL/6 mice were sensitized by intraperitoneal injection with OVA. Lung pathology was assessed by hematoxylin and eosin staining. Abundance of interleukin (IL)4, IL5, IL13, and IL21 transcripts in the lungs was quantified by RT-PCR. OVA-specific IgG1, IgG2a, IgG2b, IgE and IgM were quantified by enzyme-linked immunosorbent assay. Phosphorylated signal transducer and activator of transcription (STAT)3 in lung tissue was detected by immunohistochemistry staining and nuclear factor (NF) κB activation was measured by immunofluorescence staining. STAT3 activation was inhibited using cryptotanshinone (CPT) treatment. Germline transcripts (Iμ-Cμ, Iγ-Cγ, Iα-Cα or Iε-Cε), post-recombination transcripts (Iμ-Cγ, Iμ-Cα or Iμ- Cε) and mature transcripts (VHDJH-Cγ, VHDJH-Cα or VHDJH-Cε) were analyzed from splenic B cells of OVA-sensitized wild-type mice (with or without CPT treatment) and TLR2−/− mice (with or without IL21 treatment). Results The lungs of TLR2−/− mice showed a lesser degree of inflammation than wild-type mice after OVA sensitization. Following OVA sensitization, levels of IL4, IL13, and IL21, but not IL5, were significantly lower in TLR2−/− compared with wild-type mice. Moreover, OVA-specific IgG1 and IgE titers were markedly lower and higher, respectively, in TLR2−/− mice. TLR2 deficiency inhibited STAT3 activation but not NF-κB p65 activation. CPT treatment reduced IgG1 titers via inhibition of Stat3 phosphorylation. Both TLR2 knockout and CPT treatment reduced the frequencies of Iγ1-Cγ1, Iγ3-Cγ3 and Iα-Cα transcripts, but IL21 treatment compensated for the effects of TLR2 deficiency. Conclusion These results suggest a role of TLR2 in restricting OVA-sensitized lung inflammation via promotion of IgG1 and inhibition of IgE class switching regulated by IL21 and STAT3.

TLR2 deficiency promotes IgE and inhibits IgG1 class-switching following ovalbumin sensitization

2020 ◽  
Author(s):  
Yuqin Li ◽  
Qiu Chen ◽  
Wei Ji ◽  
Yujie Fan ◽  
Li Huang ◽  
...  
Keyword(s):  
Enzyme Linked Immunosorbent Assay ◽  
Lung Pathology ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Rt Pcr ◽  
Class Switching ◽  
Th2 Cytokine ◽  
Stat3 Phosphorylation ◽  
Hematoxylin And Eosin

Abstract Background: To explore the roles of Toll-like receptor (TLR)2 in Th2 cytokine production and immunoglobulin (Ig) class switching following ovalbumin (OVA) sensitization. Methods: TLR2-/- and wild-type C57BL/6 mice were sensitized by intraperitoneal injection with OVA. Lung pathology was assessed by hematoxylin and eosin staining. Abundance of interleukin (IL)4, IL5, IL13, and IL21 transcripts in the lungs was quantified by RT-PCR. OVA-specific IgG1, IgG2a, IgG2b, IgE and IgM were quantified by enzyme-linked immunosorbent assay. Phosphorylated signal transducer and activator of transcription (STAT)3 in lung tissue was detected by immunohistochemistry staining and nuclear factor (NF) κB activation was measured by immunofluorescence staining. STAT3 activation was inhibited using cryptotanshinone (CPT) treatment. Germline transcripts (Iμ-Cμ, Iγ-Cγ, Iα-Cα or Iε-Cε), post-recombination transcripts (Iμ-Cγ, Iμ-Cα or Iμ- Cε) and mature transcripts (VHDJH-Cγ, VHDJH-Cα or VHDJH-Cε) were analyzed from splenic B cells of OVA-sensitized wild-type mice (with or without CPT treatment) and TLR2-/- mice (with or without IL21 treatment). Results: The lungs of TLR2-/- mice showed a lesser degree of inflammation than wild-type mice after OVA sensitization. Following OVA sensitization, levels of IL4, IL13, and IL21, but not IL5, were significantly lower in TLR2-/- compared with wild-type mice. Moreover, OVA-specific IgG1 and IgE titers were markedly lower and higher, respectively, in TLR2-/- mice. TLR2 deficiency inhibited STAT3 activation but not NF-κB p65 activation. CPT treatment reduced IgG1 titers via inhibition of Stat3 phosphorylation. Both TLR2 knockout and CPT treatment reduced the frequencies of Iγ1-Cγ1, Iγ3-Cγ3 and Iα-Cα transcripts, but IL21 treatment compensated for the effects of TLR2 deficiency. Conclusion: These results suggest a role of TLR2 in restricting OVA-sensitized lung inflammation via promotion of IgG1 and inhibition of IgE class switching regulated by IL21 and STAT3.

scholarly journals TLR9–IL-2 axis exacerbates allergic asthma by preventing IL-17A hyperproduction

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yusuke Murakami ◽  
Takashi Ishii ◽  
Hiroki Nunokawa ◽  
Keigo Kurata ◽  
Tomoya Narita ◽  
...  
Keyword(s):  
Allergic Asthma ◽  
Allergic Diseases ◽  
Innate Immune ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Inhibitory Antibody ◽  
Th2 Cytokine ◽  
Dust Mite ◽  
The Relationship

Abstract Allergic asthma is one of most famous allergic diseases, which develops lung and airway inflammation. Recent studies have revealed the relationship between the pathology of allergic asthma and the increase of host-derived DNA in inflamed lung, but the role of the DNA-recognizing innate immune receptor for the inflammation is unknown well. Here we investigated the role of Toll-Like Receptor 9 in the pathogenesis of allergic asthma without synthesized CpG-ODNs. To examine that, we analyzed the pathology and immunology of house-dust-mite (HDM)-induced allergic asthma in Tlr9–/– mice and TLR9-inhibitory-antibody-treated mice. In Tlr9–/– mice, airway hyperresponsiveness (AHR) and the number of eosinophils decreased, and production of the Th2 cytokines IL-13, IL-5, and IL-4 was suppressed, compared with in wild-type mice. Interestingly, unlike Th2 cytokine production, IL-17A production was increased in Tlr9–/– mice. Furthermore, production of IL-2, which decreases IL-17A production, was reduced in Tlr9–/– mice. Blockade of TLR9 by treatment with TLR9-inhibitory-antibody, NaR9, effectively suppressed the development of allergic asthma pathology. IL-17A production in NaR9-treated mice was enhanced, which is comparable to Tlr9-/- mice. These results suggest that the TLR9–IL-2 axis plays an important role in Th2 inflammation by modulating IL-17A production in HDM-induced allergic asthma and that targeting of TLR9 might be a novel therapeutic method for allergic asthma.

scholarly journals Spontaneous Abortion and Chikungunya Infection: Pathological Findings

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 554
Author(s):  
Natália Salomão ◽  
Michelle Brendolin ◽  
Kíssila Rabelo ◽  
Mayumi Wakimoto ◽  
Ana Maria de Filippis ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Spontaneous Abortion ◽  
Ultrastructural Changes ◽  
Rt Pcr ◽  
Chorionic Villi ◽  
Decidual Cells ◽  
Hofbauer Cells ◽  
Hematoxylin And Eosin ◽  
Maternal Fetal Transmission

Intrauterine transmission of the Chikungunya virus (CHIKV) during early pregnancy has rarely been reported, although vertical transmission has been observed in newborns. Here, we report four cases of spontaneous abortion in women who became infected with CHIKV between the 11th and 17th weeks of pregnancy. Laboratorial confirmation of the infection was conducted by RT-PCR on a urine sample for one case, and the other three were by detection of IgM anti-CHIKV antibodies. Hematoxylin and eosin (H&E) staining and an electron microscopy assay allowed us to find histopathological, such as inflammatory infiltrate in the decidua and chorionic villi, as well as areas of calcification, edema and the deposition of fibrinoid material, and ultrastructural changes, such as mitochondria with fewer cristae and ruptured membranes, endoplasmic reticulum with dilated cisterns, dispersed chromatin in the nuclei and the presence of an apoptotic body in case 1. In addition, by immunohistochemistry (IHC), we found a positivity for the anti-CHIKV antibody in cells of the endometrial glands, decidual cells, syncytiotrophoblasts, cytotrophoblasts, Hofbauer cells and decidual macrophages. Electron microscopy also helped in identifying virus-like particles in the aborted material with a diameter of 40–50 nm, which was consistent with the size of CHIKV particles in the literature. Our findings in this study suggest early maternal fetal transmission, adding more evidence on the role of CHIKV in fetal death.

scholarly journals The Na+/H+ exchanger isoform 3 is required for active paracellular and transcellular Ca2+ transport across murine cecum

2013 ◽  
Vol 305 (4) ◽  
pp. G303-G313 ◽  
Author(s):  
Juraj Rievaj ◽  
Wanling Pan ◽  
Emmanuelle Cordat ◽  
R. Todd Alexander
Keyword(s):  
Water Movement ◽  
Water Flux ◽  
Wild Type Mouse ◽  
Wild Type ◽  
Rt Pcr ◽  
Ussing Chambers ◽  
Voltage Clamps ◽  
Paracellular Absorption ◽  
Mouse Intestine

Intestinal calcium (Ca2+) absorption occurs via paracellular and transcellular pathways. Although the transcellular route has been extensively studied, mechanisms mediating paracellular absorption are largely unexplored. Unlike passive diffusion, secondarily active paracellular Ca2+ uptake occurs against an electrochemical gradient with water flux providing the driving force. Water movement is dictated by concentration differences that are largely determined by Na+ fluxes. Consequently, we hypothesized that Na+ absorption mediates Ca2+ flux. NHE3 is central to intestinal Na+ absorption. NHE3 knockout mice (NHE3−/−) display impaired intestinal Na+, water, and Ca2+ absorption. However, the mechanism mediating this latter abnormality is not clear. To investigate this, we used Ussing chambers to measure net Ca2+ absorption across different segments of wild-type mouse intestine. The cecum was the only segment with net Ca2+ absorption. Quantitative RT-PCR measurements revealed cecal expression of all genes implicated in intestinal Ca2+ absorption, including NHE3. We therefore employed this segment for further studies. Inhibition of NHE3 with 100 μM 5-( N-ethyl- N-isopropyl) amiloride decreased luminal-to-serosal and increased serosal-to-luminal Ca2+ flux. NHE3−/− mice had a >60% decrease in luminal-to-serosal Ca2+ flux. Ussing chambers experiments under altered voltage clamps (−25, 0, +25 mV) showed decreased transcellular and secondarily active paracellular Ca2+ absorption in NHE3−/− mice relative to wild-type animals. Consistent with this, cecal Trpv6 expression was diminished in NHE3−/− mice. Together these results implicate NHE3 in intestinal Ca2+ absorption and support the theory that this is, at least partially, due to the role of NHE3 in Na+ and water absorption.

scholarly journals GPR110 (ADGRF1) mediates anti-inflammatory effects of N-docosahexaenoylethanolamine

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Taeyeop Park ◽  
Huazhen Chen ◽  
Hee-Yong Kim
Keyword(s):  
Inflammatory Cytokine ◽  
Inflammatory Responses ◽  
Cytokine Expression ◽  
Regulatory Function ◽  
Enzyme Linked Immunosorbent Assay ◽  
Inflammatory Condition ◽  
Wild Type ◽  
Knock Out ◽  
The Brain

Abstract Background Neuroinflammation is a widely accepted underlying condition for various pathological processes in the brain. In a recent study, synaptamide, an endogenous metabolite derived from docosahexaenoic acid (DHA, 22:6n-3), was identified as a specific ligand to orphan adhesion G-protein-coupled receptor 110 (GPR110, ADGRF1). Synaptamide has been shown to suppress lipopolysaccharide (LPS)-induced neuroinflammation in mice, but involvement of GPR110 in this process has not been established. In this study, we investigated the possible immune regulatory role of GPR110 in mediating the anti-neuroinflammatory effects of synaptamide under a systemic inflammatory condition. Methods For in vitro studies, we assessed the role of GPR110 in synaptamide effects on LPS-induced inflammatory responses in adult primary mouse microglia, immortalized murine microglial cells (BV2), primary neutrophil, and peritoneal macrophage by using quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA) as well as neutrophil migration and ROS production assays. To evaluate in vivo effects, wild-type (WT) and GPR110 knock-out (KO) mice were injected with LPS intraperitoneally (i.p.) or TNF intravenously (i.v.) followed by synaptamide (i.p.), and expression of proinflammatory mediators was measured by qPCR, ELISA, and western blot analysis. Activated microglia in the brain and NF-kB activation in cells were examined microscopically after immunostaining for Iba-1 and RelA, respectively. Results Intraperitoneal (i.p.) administration of LPS increased TNF and IL-1β in the blood and induced pro-inflammatory cytokine expression in the brain. Subsequent i.p. injection of the GPR110 ligand synaptamide significantly reduced LPS-induced inflammatory responses in wild-type (WT) but not in GPR110 knock-out (KO) mice. In cultured microglia, synaptamide increased cAMP and inhibited LPS-induced proinflammatory cytokine expression by inhibiting the translocation of NF-κB subunit RelA into the nucleus. These effects were abolished by blocking synaptamide binding to GPR110 using an N-terminal targeting antibody. GPR110 expression was found to be high in neutrophils and macrophages where synaptamide also caused a GPR110-dependent increase in cAMP and inhibition of LPS-induced pro-inflammatory mediator expression. Intravenous injection of TNF, a pro-inflammatory cytokine that increases in the circulation after LPS treatment, elicited inflammatory responses in the brain which were dampened by the subsequent injection (i.p.) of synaptamide in a GPR110-dependent manner. Conclusion Our study demonstrates the immune-regulatory function of GPR110 in both brain and periphery, collectively contributing to the anti-neuroinflammatory effects of synaptamide under a systemic inflammatory condition. We suggest GPR110 activation as a novel therapeutic strategy to ameliorate inflammation in the brain as well as periphery.

scholarly journals Toll-Like Receptor 9 Modulates Immune Responses to Aspergillus fumigatus Conidia in Immunodeficient and Allergic Mice

2008 ◽  
Vol 77 (1) ◽  
pp. 108-119 ◽  
Author(s):  
Hemanth Ramaprakash ◽  
Toshihiro Ito ◽  
Theodore J. Standiford ◽  
Steven L. Kunkel ◽  
Cory M. Hogaboam
Keyword(s):  
Immune Responses ◽  
Aspergillus Fumigatus ◽  
Inflammatory Responses ◽  
Fungal Growth ◽  
Lower Airway ◽  
Interleukin 17 ◽  
Toll Like Receptor ◽  
Wild Type ◽  
Immunodeficient Mice

ABSTRACT The role of Toll-like receptor 9 (TLR9) in antifungal responses in the immunodeficient and allergic host is unclear. We investigated the role of TLR9 in murine models of invasive aspergillosis and fungal asthma. Neutrophil-depleted TLR9 wild-type (TLR9+/+) and TLR9-deficient (TLR9−/−) mice were challenged with resting or swollen Aspergillus fumigatus conidia and monitored for survival and lung inflammatory responses. The absence of TLR9 delayed, but did not prevent, mortality in immunodeficient mice challenged with resting or swollen conidia compared to TLR9+/+ mice. In a fungal asthma model, TLR9+/+ and TLR9−/− mice were sensitized to soluble A. fumigatus antigens and challenged with resting or swollen A. fumigatus conidia, and both groups of mice were analyzed prior to and at days 7, 14, and 28 after the conidium challenge. When challenged with resting conidia, TLR9−/− mice exhibited significantly lower airway hyper-responsiveness compared to the TLR9+/+ groups. In contrast, A. fumigatus-sensitized TLR9−/− mice exhibited pulmonary fungal growth at days 14 and 28 after challenge with swollen conidia, a finding never observed in their allergic wild-type counterparts. Increased fungal growth in allergic TLR9−/− mice correlated with markedly decreased dectin-1 expression in whole lung samples and isolated dendritic cell populations. Further, whole lung levels of interleukin-17 were lower in allergic TLR9−/− mice compared to similar TLR9+/+ mice. Together, these data suggest that TLR9 modulates pulmonary antifungal immune responses to swollen conidia, possibly through the regulation of dectin-1 expression.

The Critical Role of Peptidyl-Prolyl cis/trans Isomerase, Pin1 in Acute Myeloid Leukemia with C/EBPalpha Mutations.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2213-2213
Author(s):  
J. Pulikkan ◽  
A. Peer Zada ◽  
M. Geletu ◽  
V. Dengler ◽  
Daniel G. Tenen ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Dominant Negative ◽  
Pcr Analysis ◽  
Wild Type ◽  
Rt Pcr ◽  
Promoter Assay ◽  
Type C ◽  
Acute Myeloid

Abstract CCAAT enhancer binding protein alpha (C/EBPα) is a myeloid specific transcription factor that coordinates cellular differentiation and cell cycle arrest. Loss of C/EBPα expression or function in leukemic blasts contributes to a block in myeloid cell differentiation. C/EBPα is mutated in around 9% of acute myeloid leukemia (AML). The mutations reported in C/EBPα are frame shift mutations and point mutations at basic region Leucine zipper. The mutant form of C/EBPα ie C/EBPα-p30 exhibits dominant negative function over the wild type protein. The role of peptidyl-prolyl cis/trans isomerase, Pin1 in tumorogenesis and its overexpression in many cancers led us to investigate its role in acute myeloid leukemia with C/EBPα mutation. Here we show that Pin1 is upregulated in patients with acute myeloid leukemia by affymetrix analysis. By quantitative Real-Time RT-PCR analysis, we show C/EBPα-p30 could induce Pin1 transcription, while the wild type C/EBPα downregulates Pin1 expression. Luciferase promoter assay for the Pin1 promoter shows that wild type C/EBPα is able to block Pin1 promoter activity. Mean while, C/EBPα-p30 couldn’t block Pin1 promotor activity. By silencing Pin1 by RNA Interference as well as with inhibitor against Pin1 (PiB) we could show myeloid differentiation in human CD34+ cord blood cells as well as in Kasumi-6 cells as assessed by FACS analysis with granulocytic markers. We investigated the mechanism underlying the dominant negative action of C/EBPα-p30 over the wild type protein. We report that Pin1 increases the transcriptional activity of the oncogene c-jun. We also show that c-jun blocks the DNA binding and transactivation of C/EBPα protein as assessed by gel shift assay and promoter assay respectively. We have previously shown that c-jun expression is high in AML patients with C/EBPα mutation and c-jun could block C/EBPα function by protein-protein interaction. Quantitative Real-Time RT-PCR analysis shows that inhibition of Pin1 by the inhibitor PiB downregulates c-jun mRNA expression. In conclusion, inhibition of Pin1 leads to granulocytic differentiation. Our results show Pin1 as a novel target in treating AML patients with C/EBPα mutation.

PAX5/TEL Exhibits a Dominant-Negative Role by Inhibiting the Expression of Genes Involved in BCR Signalling and Suppressing the Binding of Wild Type PAX5 to Its Direct Target Gene CD79A (mb-1) in a human Pre-B ALL Cell Line.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3455-3455
Author(s):  
Gabriela B. Iwanski ◽  
Nils Heinrich Thoennissen ◽  
PohYeen Lor ◽  
Norihiko Kawamata ◽  
Daniel Nowak ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Fusion Gene ◽  
Cell Receptor ◽  
Dominant Negative ◽  
Fusion Product ◽  
Wild Type ◽  
Rt Pcr ◽  
Downstream Target

Abstract Abstract 3455 Poster Board III-343 Acute lymphoblastic leukemia (ALL), one of the most common malignancies in childhood, is a heterogeneous disease with individual leukemia subtypes differing in their response to chemotherapy. Recent findings suggest that disruptions of B cell receptor (BCR) signalling pathways may be involved in the development of ALL. The transcription factor PAX5 is essential for the commitment of lymphoid progenitors to the B-lymphocytic lineage. In 30% of childhood B-ALL cases, PAX5 is a frequent target of aberrancies, showing monoallelic loss, point mutations, or chromosomal translocations, whereas the role of these aberrancies is still poorly understood. Using high resolution SNP-chip analysis, we have recently identified several candidate partner genes fused to PAX5 in pediatric ALL, ETV6 (TEL), FOXP1, AUTS2, C20orf112, which bind to PAX5 recognition sequences as strongly as wild-type PAX5 (wt PAX5) suppressing its transcriptional activity in a dominant-negative fashion. In order to study the role of PAX5/TEL in leukemic evolution of B-ALL, we transfected the leukemic BCP cell line Nalm 6, which endogenously expresses PAX5, with a retroviral vector encoding PAX5/TEL and confirmed its expression by Western blotting and RT-PCR. Previously, the fusion gene PAX5/TEL has been cloned into the retroviral vector pMSCV-IRES-GFP (MIGR) from a patient diagnosed with B-cell precursor ALL (BCP) with t(9;12)(q11;p13). This fusion product consists of the 5′-end NH2 terminal region of the PAX5 gene and the almost whole sequence of the TEL gene. PAX5/TEL-MIGR expressing cells were sorted for GFP and analyzed by gene expression profiling on Affymetrix HG-U133 plus 2.0 Array in comparison to cells transfected with vector control (MIGR) and a MIGR vector encoding wt PAX5 (wtPAX5/MIGR). The probes were normalized with the Affymetrix MAS5.0 software. Probes were considered to be differentially expressed with a fold change ≤ 2 or ≥ 2, respectively. We identified a set of about 200 genes that were differentially expressed in the PAX5/TEL expressing cells, most of which were downregulated, compared to the controls. A subset of these genes encodes proteins important for BCR signalling: RAG1, one of two key mediators in the process of V(D)J recombination, VPREB3, which is involved in the early phase of pre-BCR assembly, the Runt domain transcription factor Runx1 (AML1) and FOXP1. The latter two genes are fusion partners of PAX5 in pediatric B-ALL and loss of FOXP1 leads to impaired DH–JH and VH–DJH rearrangement. Additionally, we found BACH2, which plays an important role during B-cell development, as well as protein kinase C-epsilon (PKCe) to be downregulated. PKCe is highly expressed in B cells linking the BCR to the activation of mitogen-activated protein kinases (MAPK). We confirmed the downregulation of the affected genes by RT-PCR. Strikingly, VPREB3 expression showed a significant downregulation of up to 170-fold, and RAG1 up to 90-fold. Loss of the RAG1/2 locus has been found in four precursor B-cell ALL cases, which indicates that defects in this process might contribute to leukemogenesis. We also detected a significant decrease in the expression of wt PAX5 as well as its direct downstream target CD79A (mb-1). CD79A (mb-1) encodes the B cell receptor component Ig-a and its early B cell-specific mb-1 promoter is a target for regulation by early B cell-specific transcription factors like E2A, early B cell factor (EBF), and PAX5. The latter is important for the activation of the mb-1 promoter by recruiting Ets proteins through protein-protein interactions. We investigated the binding efficiency of wt PAX5 to the promoter region of CD79A by chromatin-immunoprecipitation (ChIP). For the ChIP assay, we used a PAX5 antibody detecting the C-terminal region of PAX5 so that the antibody can bind the wt PAX5 but not the fusion product PAX5/TEL of which the C-terminal side is fused to TEL. Binding of wt PAX5 to the promoter region of CD79A was diminished by expression of the PAX5/TEL-fusion protein compared to the controls, leading to repression of CD79A, which we also confirmed by RT-PCR. In conclusion, we show that the expression of PAX5/TEL in a leukemic cell line has a repressor function on the expression of wt PAX5 as well as other genes important in BCR signalling. Also, we demonstrated that PAX5/TEL has a negative impact on the binding affinity of one of the direct downstream target genes of wt PAX5. Our results indicate a repressor role of the fusion gene PAX5/TEL including BCR signalling and point towards its contribution to leukemic transformation. Disclosures No relevant conflicts of interest to declare.

Effect of Slc26a6 deletion on apical Cl−/HCO3− exchanger activity and cAMP-stimulated bicarbonate secretion in pancreatic duct

2007 ◽  
Vol 292 (1) ◽  
pp. G447-G455 ◽  
Author(s):  
Hiroshi Ishiguro ◽  
Wan Namkung ◽  
Akiko Yamamoto ◽  
Zhaohui Wang ◽  
Roger T. Worrell ◽  
...  
Keyword(s):  
Pancreatic Duct ◽  
Apical Membrane ◽  
Pancreatic Ducts ◽  
Bicarbonate Secretion ◽  
Wild Type ◽  
Rt Pcr ◽  
Secretory Rate ◽  
Transport Fluid ◽  
Exchange Activity

The role of Slc26a6 (PAT1) on apical Cl−/HCO3− exchange and bicarbonate secretion in pancreatic duct cells was investigated using Slc26a6 null and wild-type (WT) mice. Apical Cl−/HCO3− exchange activity was measured with the pH-sensitive dye BCECF in microperfused interlobular ducts. The HCO3−-influx mode of apical [Cl−]i/[HCO3−]o exchange (where brackets denote concentration and subscripts i and o denote intra- and extracellular, respectively) was dramatically upregulated in Slc26a6 null mice ( P < 0.01 vs. WT), whereas the HCO3−-efflux mode of apical [Cl−]o/[HCO3−]i exchange was decreased in Slc26a6 null mice ( P < 0.05 vs. WT), suggesting the unidirectionality of the Slc26a6-mediated HCO3− transport. Fluid secretory rate in interlobular ducts were comparable in WT and Slc26a6 null mice ( P > 0.05). In addition, when pancreatic juice was collected from whole animal in basal and secretin-stimulated conditions, neither juice volume nor its pH showed differences between WT and Slc26a6 null mice. Semiquantitative RT-PCR demonstrated more than fivefold upregulation in Slc26a3 (DRA) expression in Slc26a6 knockout pancreas. In conclusion, these results point to the role of Slc26a6 in HCO3− efflux at the apical membrane and also suggest the presence of a robust Slc26a3 compensatory upregulation, which can replace the function of Slc26a6 in pancreatic ducts.

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