Sensory Neurons Co-opt Classical Immune Signaling Pathways to Mediate Chronic Itch.

Mammals have evolved neurophysiologic reflexes, such as coughing and scratching, to expel invading pathogens and noxious environmental stimuli. It is well established that these responses are also associated with chronic inflammatory diseases, including asthma and atopic dermatitis. However, the mechanisms by which inflammatory pathways promote sensations such as itch remain poorly understood. Here, we show that type 2 cytokines directly activate sensory neurons in both mice and humans. Further, we demonstrate that chronic itch is dependent on neuronal IL-4Rα and JAK1 signaling. We also observe that patients with recalcitrant chronic itch that failed other immunosuppressive therapies markedly improve when treated with JAK inhibitors. Thus, signaling mechanisms previously ascribed to the immune system may represent novel therapeutic targets within the nervous system. Collectively, this study reveals an evolutionarily conserved paradigm in which the sensory nervous system employs classical immune signaling pathways to influence mammalian behavior.

Retinoid X receptor promotes hematopoietic stem cell fitness and quiescence and preserves hematopoietic homeostasis.

Hematopoietic stem cells (HSCs) balance self-renewal and differentiation to maintain hematopoietic fitness throughout life. In steady-state conditions, HSC exhaustion is prevented by the maintenance of most HSCs in a quiescent state, with cells entering the cell cycle only occasionally. HSC quiescence is regulated by retinoid and fatty-acid ligands of transcriptional factors of the nuclear retinoid X receptor (RXR) family. Herein, we show that dual deficiency for hematopoietic RXRα and RXRß induces HSC exhaustion, myeloid cell/megakaryocyte differentiation, and myeloproliferative-like disease. RXRα and RXRß maintain HSC quiescence, survival, and chromatin compaction; moreover, transcriptome changes in RXRα;RXRß-deficient HSCs include premature acquisition of an aging-like HSC signature, MYC pathway upregulation, and RNA intron retention. Fitness loss and associated RNA transcriptome and splicing alterations in RXRα;RXRß-deficient HSCs are prevented by Myc haploinsufficiency. Our study reveals the critical importance of RXRs for the maintenance of HSC fitness and their protection from premature aging.

Application of a New Hemostatic Clip to Prevent Delayed Bleeding After Endoscopic Sphincterotomy: A Propensity Score-matched Analysis.

BACKGROUND AND AIM: Delayed bleeding after endoscopic sphincterotomy (ES) is a serious adverse event of endoscopic retrograde cholangiopancreatography. The aim of this study is to evaluate the effect of prevent delayed bleeding of hemostatic clip (Sureclip) after ES. METHODS: Consecutive patients diagnosed with common bile duct stones with a high risk of delayed bleeding who received ES from January 1, 2013, to July 31, 2022, were analyzed retrospectively. A 1:1 propensity score-matching analysis and logistic regression analysis were used. The patients were allocated into the hemostatic clip and control groups. The rate of delayed bleeding, hyperamylasemia, pancreatitis, and hemostatic clip closing the bile duct or pancreatic duct by mistake were compared between the 2 groups. RESULTS: Overall, 161 and 232 patients were allocated to the control and hemostatic clip groups, respectively, propensity score matching created 120 matched pairs. The rate of delayed bleeding was significantly lower in the hemostatic clip group than in the control group (1.67% vs. 7.5%, P=0.031). After adjusting for confounding factors, logistic regression showed hemostatic clip was associated with decreased odds of delayed bleeding (0.134, 95% CI: 0.025-0.719). No case of hemostatic clip closing the bile duct or pancreatic duct by mistake occurred in the hemostatic clip group. No significant differences were observed in postoperative hyperamylasemia and pancreatitis between the 2 groups. CONCLUSIONS: This study indicated that the prophylactic application of a hemostatic clip is associated with a significantly reduced rate of delayed bleeding after ES in high-risk patients. This approach did not increase the risk of adverse event.

Cytokine exposure mediates transcriptional activation of the orphan nuclear receptor Nur77 in hematopoietic cells.

The orphan nuclear receptor Nur77 is an immediate-early response gene that based on tissue and cell context is implicated in a plethora of cellular processes, including proliferation, differentiation, apoptosis, metabolism, and inflammation. Nur77 has a ligand-binding pocket that is obstructed by hydrophobic side groups. Naturally occurring, cell-endogenous ligands have not been identified, and Nur77 transcriptional activity is thought to be regulated through posttranslational modification and modulation of protein levels. To determine whether Nur77 is transcriptionally active in hematopoietic cells in vivo, we used an upstream activating sequence (UAS)-GFP transgenic reporter. We found that Nur77 is transcriptionally inactive in vivo in hematopoietic cells under basal conditions, but that activation occurs following cytokine exposure by G-CSF or IL-3. We also identified a series of serine residues required for cytokine-dependent transactivation of Nur77. Moreover, a kinase inhibitor library screen and proximity labeling-based mass spectrometry identified overlapping kinase pathways that physically interacted with Nur77 and whose inhibition abrogated cytokine-induced activation of Nur77. We determined that transcriptional activation of Nur77 by G-CSF or IL-3 requires functional JAK and mTor signaling since their inhibition leads to Nur77 transcriptional inactivation. Thus, intracellular cytokine signaling networks appear to regulate Nur77 transcriptional activity in mouse hematopoietic cells.

Endogenous and combination retinoids are active in myelomonocytic leukemias.

Retinoid therapy transformed response and survival outcomes in acute promyelocytic leukemia (APL), but has demonstrated only modest activity in non-APL forms of acute myeloid leukemia (AML). The presence of natural retinoids in vivo could influence the efficacy of pharmacologic agonists and antagonists. We found that natural RXRA ligands, but not RARA ligands, were present in murine MLL-AF9-derived myelomonocytic leukemias in vivo and that the concurrent presence of receptors and ligands acted as tumor suppressors. Pharmacologic retinoid responses could be optimized by concurrent targeting RXR ligands (e.g. bexarotene) and RARA ligands (e.g. all-trans retinoic acid, ATRA), which induced either leukemic maturation or apoptosis depending on cell culture conditions. Co-repressor release from the RARA:RXRA heterodimer occurred with RARA activation, but not RXRA activation, providing an explanation for the combination synergy. Combination synergy could be replicated in additional, but not all, AML cell lines and primary samples, and was associated with improved survival in vivo, although tolerability of bexarotene administration in mice remained an issue. These data provide insight into the basal presence of natural retinoids in leukemias in vivo and a potential strategy for clinical retinoid combination regimens in leukemias beyond acute promyelocytic leukemia.

The F-box protein COI1 functions upstream of MYB305 to regulate primary carbohydrate metabolism in tobacco (Nicotiana tabacum L. cv. TN90).

Jasmonate (JA) plays an important role in regulating plant male fertility and secondary metabolism, but its role in regulating primary metabolism remains unclear. The F-box protein CORONATINE INSENSITIVE 1 (COI1) is a critical component of the JA receptor, and mediates JA-signalling by targeting JASMONATE ZIM-domain (JAZ) proteins for proteasomal degradation in response to JA perception. Here, we found that RNA interference-mediated knockdown of NtCOI1 in tobacco (Nicotiana tabacum L. cv. TN90) recapitulated many previously observed phenotypes in coi1 mutants, including male sterility, JA insensitivity, and loss of floral anthocyanin production. It also affected starch metabolism in the pollen, anther wall, and floral nectary, leading to pollen abortion and loss of floral nectar. Transcript levels of genes encoding starch metabolism enzymes were significantly altered in the pollen, anther wall, and floral nectary of NtCOI1-silenced tobacco. Changes in leaf primary metabolism were also observed in the NtCOI1-silenced tobacco. The expression of NtMYB305, an orthologue of MYB305 previously identified as a flavonoid metabolic regulator in Antirrhinum majus flowers and as a floral-nectar regulator mediating starch synthesis in ornamental tobacco, was extremely downregulated in NtCOI1-silenced tobacco. These findings suggest that NtCOI1 functions upstream of NtMYB305 and plays a fundamental role in coordinating plant primary carbohydrate metabolism and correlative physiological processes.

A phase I dose escalation study of oral bexarotene in combination with intravenous decitabine in patients with AML.

The response rate of non-M3 acute myeloid leukemia (AML) to all trans retinoic acid has been limited. Using Affymetrix expression arrays, we found that in diverse AML blasts RXRA was expressed at higher levels than RARA and that mouse Ctsg-PML-RARA leukemia responded to bexarotene, a ligand for RXRA. We therefore performed a phase I study of combination bexarotene and decitabine in elderly and relapsed AML patients. We found that this combination was well tolerated, although outcomes were modest (1 CRi, and 3 PR among 19 patients). Correlative studies found that patients with clinical response had increased differentiation to bexarotene both in vivo and ex vivo, suggesting that pre-treatment analysis might identify a more susceptible subgroup of patients.

Roles of purinergic receptor P2Y, G protein-coupled 12 in the development of atherosclerosis in apolipoprotein E-deficient mice.

OBJECTIVE: The aim of the study was to evaluate the role of purinergic receptor P2Y, G protein-coupled 12 (P2Y12), an ADP receptor, in the development of atherosclerotic lesions. METHODS AND RESULTS: Apolipoprotein E-null mice were crossed with P2y12(-/-) mice to generate double knockout mice. The double knockout mice and the control apolipoprotein E-null mice were fed a high-fat diet for 20 weeks. Assessment of the atherosclerotic lesions in the control and double knockout mice demonstrated that P2Y12 deficiency caused a diminished lesion area, an increased fibrous content at the plaque site, and decreased monocyte/macrophage infiltration of the lesions. Polymerase chain reaction studies revealed that white blood cells do not express significant levels of P2Y12. Bone marrow transplantation experiments confirmed that P2Y12 expressed on platelets is a key factor responsible for atherosclerosis, but do not exclude a role of smooth muscle cell P2Y12. Supernatant fluid from activated P2y12(+/+) but not P2y12(-/-) platelets was capable of causing monocyte migration. In vitro studies showed that platelet P2Y12 deficiency suppressed platelet factor 4 secretion and P-selectin expression. Further work demonstrated that platelet P2Y12, through inhibition of the cAMP/protein kinase A pathway, critically regulates the release of platelet factor 4, and thereby affects monocyte recruitment and infiltration. CONCLUSIONS: These results demonstrate that P2Y12 modulates atherogenesis, at least in part by augmenting inflammatory cell recruitment via regulation of platelet α-granule release.

Blood natural killer cell deficiency reveals an immunotherapy strategy for atopic dermatitis.

Atopic dermatitis (AD) is a widespread, chronic skin disease associated with aberrant allergic inflammation. Current treatments involve either broad or targeted immunosuppression strategies. However, enhancing the immune system to control disease remains untested. We demonstrate that patients with AD harbor a blood natural killer (NK) cell deficiency that both has diagnostic value and improves with therapy. Multidimensional protein and RNA profiling revealed subset-level changes associated with enhanced NK cell death. Murine NK cell deficiency was associated with enhanced type 2 inflammation in the skin, suggesting that NK cells play a critical immunoregulatory role in this context. On the basis of these findings, we used an NK cell-boosting interleukin-15 (IL-15) superagonist and observed marked improvement in AD-like disease in mice. These findings reveal a previously unrecognized application of IL-15 superagonism, currently in development for cancer immunotherapy, as an immunotherapeutic strategy for AD.

Jasmonate-Sensitivity-Assisted Screening and Characterization of Nicotine Synthetic Mutants from Activation-Tagged Population of Tobacco (Nicotiana tabacum L.).

Nicotine is a secondary metabolite that is important to the defense system and commercial quality of tobacco (Nicotiana tabacum L.). Jasmonate and its derivatives (JAs) are phytohormone regulators of nicotine formation; however, the underlying molecular mechanism of this process remains largely unclear. Owing to the amphitetraploid origin of N. tabacum, research on screening and identification of nicotine-synthetic mutants is relatively scarce. Here, we describe a method based on JA-sensitivity for screening nicotine mutants from an activation-tagged population of tobacco. In this approach, the mutants were first screened for abnormal JA responses in seed germination and root elongation, and then the levels of nicotine synthesis and expression of nicotine synthetic genes in the mutants with altered JA-response were measured to determine the nicotine-synthetic mutants. We successfully obtained five mutants that maintained stable nicotine contents and JA responses for three generations. This method is simple, effective and low-cost, and the finding of transcriptional changes of nicotine synthetic genes in the mutants shows potentials for identifying novel regulators involved in JA-regulated nicotine biosynthesis.

Endogenous retinoid X receptor ligands in mouse hematopoietic cells.

The retinoid X receptor α (RXRA) has been implicated in diverse hematological processes. To identify natural ligands of RXRA that are present in hematopoietic cells, we adapted an upstream activation sequence-green fluorescent protein (UAS-GFP) reporter mouse to detect natural RXRA ligands in vivo. We observed reporter activity in diverse types of hematopoietic cells in vivo. Reporter activity increased during granulocyte colony-stimulating factor (G-CSF)-induced granulopoiesis and after phenylhydrazine (PHZ)-induced anemia, suggesting the presence of dynamically regulated natural RXRA ligands in hematopoietic cells. Mouse plasma activated Gal4-UAS reporter cells in vitro, and plasma from mice treated with G-CSF or PHZ recapitulated the patterns of reporter activation that we observed in vivo. Plasma from mice with dietary vitamin A deficiency only mildly reduced RXRA reporter activity, whereas plasma from mice on a fatty acid restriction diet reduced reporter activity, implicating fatty acids as plasma RXRA ligands. Through differential extraction coupled with mass spectrometry, we identified the long-chain fatty acid C24:5 as a natural RXRA ligand that was greatly increased in abundance in response to hematopoietic stress. Together, these data suggest that natural RXRA ligands are present and dynamically increased in abundance in mouse hematopoietic cells in vivo.

Soft-template synthesis of single-crystalline CdS dendrites.

The single-crystalline CdS dendrites have been fabricated from the reaction of CdCl2 and thiourea at 180 degrees C, in which glycine was employed as a soft template. The obtained products were explored by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, and selected area electronic diffraction. The optical properties of CdS dendrites have been investigated by ultraviolet and visible light (UV-vis) and photoluminescence techniques. The investigations indicated that the dendrites were grown due to the anisotropic properties enhanced by the use of Glycine in the route.

Pathways of retinoid synthesis in mouse macrophages and bone marrow cells.

In vivo pathways of natural retinoid metabolism and elimination have not been well characterized in primary myeloid cells, even though retinoids and retinoid receptors have been strongly implicated in regulating myeloid maturation. With the use of a upstream activation sequence-GFP reporter transgene and retrovirally expressed Gal4-retinoic acid receptor α in primary mouse bone marrow cells, we identified 2 distinct enzymatic pathways used by mouse myeloid cells ex vivo to synthesize retinoic acid receptor α ligands from free vitamin A metabolites (retinyl acetate, retinol, and retinal). Bulk Kit(+) bone marrow progenitor cells use diethylaminobenzaldehyde-sensitive enzymes, whereas bone marrow-derived macrophages use diethylaminobenzaldehyde-insensitive enzymes to synthesize natural retinoic acid receptor α-activating retinoids (all-trans retinoic acid). Bone marrow-derived macrophages do not express the diethylaminobenzaldehyde-sensitive enzymes Aldh1a1, Aldh1a2, or Aldh1a3 but instead, express Aldh3b1, which we found is capable of diethylaminobenzaldehyde-insensitive synthesis of all trans-retinoic acid. However, under steady-state and stimulated conditions in vivo, diverse bone marrow cells and peritoneal macrophages showed no evidence of intracellular retinoic acid receptor α-activating retinoids, despite expression of these enzymes and a vitamin A-sufficient diet, suggesting that the enzymatic conversion of retinal is not the rate-limiting step in the synthesis of intracellular retinoic acid receptor α-activating retinoids in myeloid bone marrow cells and that retinoic acid receptor α remains in an unliganded configuration during adult hematopoiesis.

[Research on the mechanism of fluorescence quenching of HRP by NaN3].

The fluorescence and synchronous fluorescence spectra were employed to study the binding between sodium azide (NaN3) and horseradish peroxidase (HRP), which was affected by the molecular conformation and the microenvironment of the fluorescence residues. The mechanism of the fluorescence quenching of HRP by NaN3 was discussed and the binding constant and the number of binding sites of non-covalent bond between them were calculated.

Peptide LSARLAF induces integrin ß3 dependent outside-in signaling in platelets.

INTRODUCTION: Peptide LSARLAF (LSA) can bind and activate integrin αIIbß3 in the absence of 'inside-out' signal. The active αIIbß3 mediates 'outside-in' signaling that elicits platelet aggregation, granule secretion and TxA2 production. Here we identify the membrane glycoproteins which mediate LSA-induced platelet activation other than αIIbß3, and determine the roles of Src, PLCγ2, FcRγ-chain, and SLP-76 in LSA-induced platelet activation. METHOD: Ligand-receptor binding assay was performed to study the effect of peptide LSA or its control peptide FRALASL (FRA) on integrins binding to their ligands. Spreading of CHO cells expressing αIIbß3 or αVß3 on immobilized fibrinogen was measured in the presence of LSA or FRA. Washed ß3, Src, FcRγ-chain, LAT and SLP-76 deficient platelets aggregation and secretion were tested in response to LSA. RESULTS: Ligand-receptor binding assay indicated that LSA promoted the binding of multiple ligands to αIIbß3 or αVß3. LSA also enhanced CHO cells with αIIbß3 or αVß3 expression spreading on immobilized fibrinogen. ß3 deficient platelets failed to aggregate and secrete in response to LSA. The phosphorylation of PLCγ2 and Syk was also ß3 dependent. Src, FcRγ-chain, LAT and SLP-76 deficient platelets did not aggregate, secrete ATP or produce TxA2 in response to LSA. CONCLUSION: LSA-induced platelet activation is ß3 dependent, and signaling molecules Src, FcRγ-chain, SLP-76 and LAT play crucial roles in LSA-induced ß3 mediated signaling.

Integrin αIIb-mediated PI3K/Akt activation in platelets.

Integrin αIIbß3 mediated bidirectional signaling plays a critical role in thrombosis and haemostasis. Signaling mediated by the ß3 subunit has been extensively studied, but αIIb mediated signaling has not been characterized. Previously, we reported that platelet granule secretion and TxA2 production induced by αIIb mediated outside-in signaling is negatively regulated by the ß3 cytoplasmic domain residues R(724)KEFAKFEEER(734). In this study, we identified part of the signaling pathway utilized by αIIb mediated outside-in signaling. Platelets from humans and gene deficient mice, and genetically modified CHO cells as well as a variety of kinase inhibitors were used for this work. We found that aggregation of TxA2 production and granule secretion by ß3Δ724 human platelets initiated by αIIb mediated outside-in signaling was inhibited by the Src family kinase inhibitor PP2 and the PI3K inhibitor wortmannin, respectively, but not by the MAPK inhibitor U0126. Also, PP2 and wortmannin, and the palmitoylated ß3 peptide R(724)KEFAKFEEER(734), each inhibited the phosphorylation of Akt residue Ser473 and prevented TxA2 production and storage granule secretion. Similarly, Akt phosphorylation in mouse platelets stimulated by the PAR4 agonist peptide AYPGKF was αIIbß3-dependent, and blocked by PP2, wortmannin and the palmitoylated peptide p-RKEFAKFEEER. Akt was also phosphorylated in response to mAb D3 plus Fg treatment of CHO cells in suspension expressing αIIbß3-Δ724 or αIIbß3E(724)AERKFERKFE(734), but not in cells expressing wild type αIIbß3. In summary, SFK(s) and PI3K/Akt signaling is utilized by αIIb-mediated outside-in signaling to activate platelets even in the absence of all but 8 membrane proximal residues of the ß3 cytoplasmic domain. Our results provide new insight into the signaling pathway used by αIIb-mediated outside-in signaling in platelets.

BF0801, a novel adenine derivative, inhibits platelet activation via phosphodiesterase inhibition and P2Y12 antagonism.

Though antiplatelet drugs are proven beneficial to patients with coronary heart disease and stroke, more effective and safer antiplatelet drugs are still needed. In this study we report the antiplatelet effects and mechanism of BF0801, a novel adenine derivative. BF0801 dramatically inhibited platelet aggregation and ATP release induced by ADP, 2MeSADP, AYPGKF, SFLLRN or convulxin without affecting shape change in vitro . It also potentiated the inhibitory effects of adenosine-based P2Y12 antagonist AR-C69931MX or phosphodiesterase (PDE) inhibitor IBMX on platelet aggregation. The cAMP levels in both resting and forskolin-stimulated platelets were increased by BF0801 suggesting its PDE inhibitor activity, which is further confirmed by the concentration-dependent suppression of BF0801 on the native and recombinant PDE. Similar to AR-C69931MX, BF0801 drastically inhibited 2MeSADP- induced adenylyl cyclase inhibition in platelets indicating its P2Y12 antagonism activity, which is substantiated by the inhibition of BF0801 on the interaction between ADP and P2Y12 receptor expressed in CHO-K1 cells measured by atomic force microscopy. Moreover, we confirmed the antiplatelet effects of BF0801 using platelets from rats intravenously given BF0801. In summary, for the first time we developed a novel adenine derivative bearing dual activities of PDE inhibition and P2Y12 antagonism, which may have therapeutic advantage as a potential antithrombotic drug.