@article{mishra_wheeler_pitake_ding_jiang_fukuyama_paps_ralph_coyne_parkington_et al._2020, title={Periostin Activation of Integrin Receptors on Sensory Neurons Induces Allergic Itch}, volume={31}, ISSN={["2211-1247"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85082772179&partnerID=MN8TOARS}, DOI={10.1016/j.celrep.2020.03.036}, abstractNote={Chronic allergic itch is a common symptom affecting millions of people and animals, but its pathogenesis is not fully explained. Herein, we show that periostin, abundantly expressed in the skin of patients with atopic dermatitis (AD), induces itch in mice, dogs, and monkeys. We identify the integrin αVβ3 expressed on a subset of sensory neurons as the periostin receptor. Using pharmacological and genetic approaches, we inhibited the function of neuronal integrin αVβ3, which significantly reduces periostin-induced itch in mice. Furthermore, we show that the cytokine TSLP, the application of AD-causing MC903 (calcipotriol), and house dust mites all induce periostin secretion. Finally, we establish that the JAK/STAT pathway is a key regulator of periostin secretion in keratinocytes. Altogether, our results identify a TSLP-periostin reciprocal activation loop that links the skin to the spinal cord via peripheral sensory neurons, and we characterize the non-canonical functional role of an integrin in itch.}, number={1}, journal={CELL REPORTS}, author={Mishra, Santosh K. and Wheeler, Joshua J. and Pitake, Saumitra and Ding, Huiping and Jiang, Changyu and Fukuyama, Tomoki and Paps, Judy S. and Ralph, Patrick and Coyne, Jacob and Parkington, Michelle and et al.}, year={2020}, month={Apr} }
 @article{ehling_fukuyama_ko_olivry_bäumer_2019, title={Neuromedin B Induces Acute Itch in Mice via the Activation of Peripheral Sensory Neurons}, volume={99}, ISSN={0001-5555}, url={http://dx.doi.org/10.2340/00015555-3143}, DOI={10.2340/00015555-3143}, abstractNote={Neuromedin B is expressed in nociceptive and itch-sensitive dorsal root ganglia neurons, but its peripheral pruritogenic potential is not well described. The potential of neuromedin B as a pruritogen and pro-inflammatory peptide in the skin was tested in vivo in an acute model in mice and monkeys as well as an allergic dermatitis model in mice. To identify the underlying mechanisms in vitro real time PCR analysis for neuromedin B and its receptor expression in murine mast cells and dorsal root ganglia as well as functional calcium imaging in the ganglia was applied. Neuromedin B induces itch when injected intradermally, and the peripheral signal is likely transmitted through the activation of dorsal root ganglia. Thus, neuromedin B could be an interesting new therapeutic target for peripheral processing of itch at the level of sensory neurons.}, number={6}, journal={Acta Dermato Venereologica}, publisher={Acta Dermato-Venereologica}, author={Ehling, S and Fukuyama, T and Ko, M and Olivry, T and Bäumer, W}, year={2019}, pages={587–893} }
 @article{fukuyama_ganchingco_baumer_2017, title={Demonstration of rebound phenomenon following abrupt withdrawal of the JAK1 inhibitor oclacitinib}, volume={794}, ISSN={["1879-0712"]}, DOI={10.1016/j.ejphar.2016.11.020}, abstractNote={The janus kinase-inhibitor oclacitinib is licensed for the control of pruritus associated with allergic skin diseases in dogs. Strikingly, it has been clinically reported that abrupt withdrawal of oclacitinib leads to a rebound pruritus in dogs. Therefore, the primary objective of this study was to mimic the rebound phenomenon of oclacitinib using a chronic pruritic mouse model of allergic contact dermatitis. Chronic allergic contact dermatitis was induced by repetitive toluene-2,4-diisocyanate (TDI) challenge in BALB/c mice. Oclacitinib was orally administered twice daily at 45 mg/kg for 7 days, with concurrent TDI challenge, and then treatment of oclacitinib was abruptly discontinued. Scratching bouts following TDI challenge were evaluated to day 15. Additionally, dorsal root ganglia (DRG) and affected skin were isolated from mice receiving oclacitinib and from mice 24 h after oclacitinib withdrawal and were used to determine pruritogen induced Ca2+ signals in sensory neurons, the number of activated dendritic cells (DCs) within DRG, and the cytokine profiles of affected skin. Mice treated with oclacitinib showed a significant decrease in scratching bouts during treatment, then following abrupt withdrawal scratching bouts were significantly increased. Furthermore, following abrupt withdrawal more DRG neurons were activated by pruritogenic cytokines, TNFα positive DCs were significantly increased, and affected skin revealed a significant increase of TNFα and TSLP. In conclusion, while oclacitinib significantly reduced itch during treatment the abrupt withdrawal led to a rapid rebound phenomenon which can be explained by an increase in pruritogenic cytokines and fast peripheral sensitization.}, journal={EUROPEAN JOURNAL OF PHARMACOLOGY}, author={Fukuyama, Tomoki and Ganchingco, Joy Rachel and Baumer, Wolfgang}, year={2017}, month={Jan}, pages={20–26} }
 @misc{fukuyama_ganchingco_mishra_olivry_rzagalinski_volmer_baeumer_2017, title={Janus kinase inhibitors display broad anti-itch properties: A possible link through the TRPV1 receptor}, volume={140}, ISSN={["1097-6825"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85013487374&partnerID=MN8TOARS}, DOI={10.1016/j.jaci.2016.12.960}, abstractNote={Janus kinase (JAK) inhibitors are being proposed for the treatment of cancer and inflammatory diseases, such as atopic dermatitis. Their mechanism of action, especially that to reduce itch, remains speculative. The JAK inhibitor oclacitinib is currently approved for the treatment of lesions and pruritus in dogs with atopic dermatitis,1Cosgrove S.B. Wren J.A. Cleaver D.M. Martin D.D. Walsh K.F. Harfst J.A. et al.Efficacy and safety of oclacitinib for the control of pruritus and associated skin lesions in dogs with canine allergic dermatitis.Vet Dermatol. 2013; 24 (479-e114)Crossref Scopus (73) Google Scholar, 2Cosgrove S.B. Wren J.A. Cleaver D.M. Walsh K.F. Follis S.I. King V.I. et al.A blinded, randomized, placebo-controlled trial of the efficacy and safety of the Janus kinase inhibitor oclacitinib (Apoquel(R)) in client-owned dogs with atopic dermatitis.Vet Dermatol. 2013; 24 (e141-2): 587-597Crossref PubMed Scopus (93) Google Scholar whereas tofacitinib is under clinical development for the treatment of the homologous human disease (https://www.clinicaltrials.gov/ct2/show/NCT02001181). In rodent and canine models of allergic dermatitis and in human patients with psoriasis and atopic dermatitis, the antipruritic effect of JAK inhibitors is rapidly visible.1Cosgrove S.B. Wren J.A. Cleaver D.M. Martin D.D. Walsh K.F. Harfst J.A. et al.Efficacy and safety of oclacitinib for the control of pruritus and associated skin lesions in dogs with canine allergic dermatitis.Vet Dermatol. 2013; 24 (479-e114)Crossref Scopus (73) Google Scholar, 2Cosgrove S.B. Wren J.A. Cleaver D.M. Walsh K.F. Follis S.I. King V.I. et al.A blinded, randomized, placebo-controlled trial of the efficacy and safety of the Janus kinase inhibitor oclacitinib (Apoquel(R)) in client-owned dogs with atopic dermatitis.Vet Dermatol. 2013; 24 (e141-2): 587-597Crossref PubMed Scopus (93) Google Scholar, 3Fukuyama T. Ehling S. Cook E. Baumer W. Topically administered Janus-kinase inhibitors tofacitinib and oclacitinib display impressive antipruritic and anti-inflammatory responses in a model of allergic dermatitis.J Pharmacol Exp Ther. 2015; 354: 394-405Crossref PubMed Scopus (17) Google Scholar We suspect that the proposed mechanism of action on pruritus (ie, a reduction of signal transduction after binding of pruritogenic cytokines, such as IL-31, to their receptors) might not be the only explanation for the fast onset and sustained antipruritic action of JAK inhibitors. To identify additional mechanisms of action of the JAK inhibitors oclacitinib and tofacitinib, we used both in vitro (dorsal root ganglion [DRG] neuronal culture) and in vivo approaches in itch mouse models that are known to be dependent and independent of JAK signaling pathways. In this study we observed that the JAK inhibitors oclacitinib and tofacitinib exhibit broad anti-itch properties that appear linked to their inhibition of transient receptor potential cation channel subfamily V member 1 (TRPV1), a receptor known to play an important role in the transmission of both pain and itch.4Tóth B.I. Szallasi A. Bíró T. Transient receptor potential channels and itch: how deep should we scratch?.Handb Exp Pharmacol. 2015; 226: 89-133Crossref PubMed Scopus (20) Google Scholar To illustrate the involvement of TRPV1 in the anti-itch properties of JAK inhibitors, we used 3 different approaches: (1) in vitro calcium imaging in DRG neurons; (2) in vivo methods with a mouse model of itch and one of capsaicin-induced pain; and (3) TRPV1-transfected AD293 cells. We used murine primary sensory neurons and compared the responses evoked by several types of pruritogens that are dependent and independent of JAK signaling pathways. We examined the activation of small-diameter DRG neurons (mostly TRPV1-expressing ones) with or without exposure to oclacitinib or tofacitinib using Fura-2 ratiometric Ca2+ imaging. In vitro treatment with these 2 JAK inhibitors resulted in a significant reduction in the response of DRG neurons to the following stimuli: IL-31, TNF-α, histamine, compound 48/80, chloroquine, protease-activated receptor 2 (PAR-2) activating peptide (SLIGRL-NH2), capsaicin, and a low-pH solution (Fig 1, A). However, the response of DRG neurons to serotonin (5-HT) and allyl isothiocyanate (AITC) was not significantly changed compared with that in the vehicle-only control group after treatment with these JAK inhibitors (Fig 1, A and B). To examine whether the JAK inhibitors also reduced the itch response in vivo, we induced scratching responses in mice with all the compounds previously tested on the primary DRG sensory neurons. We confirmed in vivo a similar inhibitory pattern after oral administration of the JAK inhibitors compared with the vehicle control group, whereas there was no change in itch behavior after 5-HT (Fig 1, C). It has been shown above that these 2 JAK inhibitors reduced the itch induced by JAK-dependent cytokines involved in sensory perception, such as IL-31.3Fukuyama T. Ehling S. Cook E. Baumer W. Topically administered Janus-kinase inhibitors tofacitinib and oclacitinib display impressive antipruritic and anti-inflammatory responses in a model of allergic dermatitis.J Pharmacol Exp Ther. 2015; 354: 394-405Crossref PubMed Scopus (17) Google Scholar However, our results indicated a broader action than previously demonstrated with the reduction of itch induced by JAK-independent compounds that included TNF-α, histamine, compound 48/80, chloroquine, and SLIGRL-NH2. Similarly, we also observed a reduction of pain (manifested by wiping behavior) induced by capsaicin, the latter being a specific agonist for TRPV1. In support of this proposed novel mechanism of JAK inhibitors, a strikingly similar pattern of reduced scratching behavior to histamine, compound 48/80, and PAR-2 has similarly been shown by a TRPV1 antagonist.5Yun J.W. Seo J.A. Jang W.H. Koh H.J. Bae I.H. Park Y.H. et al.Antipruritic effects of TRPV1 antagonist in murine atopic dermatitis and itching models.J Invest Dermatol. 2011; 131: 1576-1579Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Chloroquine interacts not only with TRPV1 but also with the other TRP ion channels.6Than J.Y. Li L. Hasan R. Zhang X. Excitation and modulation of TRPA1, TRPV1, and TRPM8 channel-expressing sensory neurons by the pruritogen chloroquine.J Biol Chem. 2013; 288: 12818-12827Crossref PubMed Scopus (68) Google Scholar Additionally, IL-31–induced itch is known to be dependent on both TRPV1- and transient receptor potential cation channel subfamily A member 1 (TRPA1)-expressing neurons.7Cevikbas F. Wang X. Akiyama T. Kempkes C. Savinko T. Antal A. et al.A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: involvement of TRPV1 and TRPA1.J Allergy Clin Immunol. 2014; 133: 448-460Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar Interestingly, we did not see any effect of the tested JAK inhibitors on TRPA1-associated sensory perception induced by AITC and 5-HT (Fig 1, A and C).8Liu B. Escalera J. Balakrishna S. Fan L. Caceres A.I. Robinson E. et al.TRPA1 controls inflammation and pruritogen responses in allergic contact dermatitis.FASEB J. 2013; 27: 3549-3563Crossref PubMed Scopus (165) Google Scholar After these observations, we hypothesized that the broad antipruritic effect of oclacitinib and tofacitinib could be related to their direct inhibition of TRPV1 rather than only JAK inhibition. To confirm the direct association between TRPV1 and JAK inhibitors, we examined the in vitro and in vivo responses to IL-31, TNF-α, histamine, compound 48/80, chloroquine, SLIGRL-NH2, and capsaicin in TRPV1-knockout mice. Our inhibitory results are similar to those previously published,5Yun J.W. Seo J.A. Jang W.H. Koh H.J. Bae I.H. Park Y.H. et al.Antipruritic effects of TRPV1 antagonist in murine atopic dermatitis and itching models.J Invest Dermatol. 2011; 131: 1576-1579Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 6Than J.Y. Li L. Hasan R. Zhang X. Excitation and modulation of TRPA1, TRPV1, and TRPM8 channel-expressing sensory neurons by the pruritogen chloroquine.J Biol Chem. 2013; 288: 12818-12827Crossref PubMed Scopus (68) Google Scholar, 7Cevikbas F. Wang X. Akiyama T. Kempkes C. Savinko T. Antal A. et al.A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: involvement of TRPV1 and TRPA1.J Allergy Clin Immunol. 2014; 133: 448-460Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar and the inhibition pattern mimics that of the JAK inhibitors (Fig 2, A and B). To test the effects of the JAK inhibitors in a more clinically relevant chronic itch setting, we used the toluene-2,4,-diisocyanate (TDI)–induced mouse model of chronic allergic itch. First, we observed a significant increase in expression of TRPV1 receptor–positive neurons in the DRG neurons of chronically challenged mice (Fig 2, C and D). Second, TDI-challenged mice topically treated with tofacitinib during the challenge phase showed a significant decrease in itch behavior (Fig 2, E). To further demonstrate a direct interaction of JAK inhibitors with TRPV1, we performed Ca2+ imaging on TRPV1-transfected AD293 cells and studied the effects of oclacitinib and tofacitinib on capsaicin-induced calcium influx. The 2 JAK inhibitors significantly reduced the response in TRPV1-transfected cells to capsaicin stimulation (see Fig E1, A, in this article's Online Repository at www.jacionline.org). Finally, we conducted matrix-assisted laser desorption/ionization (MALDI)–ultra-high-resolution mass spectrometric experiments on TRPV1 immunoprecipitates isolated from the TRPV1-transfected AD293 cells (see Fig E1, B). We detected higher concentrations of JAK inhibitors in TRPV1-transfected AD293 cells compared with those in vehicle-treated and mock-transfected AD293 cells (see Fig E1, C and D), although there were still some traces of JAK inhibitors in mock-transfected gel (see Fig E1, C). Because a small Ca2+ influx to a higher concentration of capsaicin was also found in mock-transfected cells, a low level of TRPV1 expression was likely present in wild-type cells. Future studies will include radioligand experiments to demonstrate direct binding to TRPV1. The inhibition of itch by JAK inhibitors has been reported to be both fast and sustained in dogs and human patients with psoriasis.1Cosgrove S.B. Wren J.A. Cleaver D.M. Martin D.D. Walsh K.F. Harfst J.A. et al.Efficacy and safety of oclacitinib for the control of pruritus and associated skin lesions in dogs with canine allergic dermatitis.Vet Dermatol. 2013; 24 (479-e114)Crossref Scopus (73) Google Scholar, 2Cosgrove S.B. Wren J.A. Cleaver D.M. Walsh K.F. Follis S.I. King V.I. et al.A blinded, randomized, placebo-controlled trial of the efficacy and safety of the Janus kinase inhibitor oclacitinib (Apoquel(R)) in client-owned dogs with atopic dermatitis.Vet Dermatol. 2013; 24 (e141-2): 587-597Crossref PubMed Scopus (93) Google Scholar, 9Bushmakin A.G. Mamolo C. Cappelleri J.C. Stewart M. The relationship between pruritus and the clinical signs of psoriasis in patients receiving tofacitinib.J Dermatolog Treat. 2015; 26: 19-22Crossref PubMed Scopus (31) Google Scholar However, this is difficult to explain by just the reduction of itch signaling by a few cytokines, such as IL-31. This direct inhibitory action on TRPV1 is likely the missing link, explaining the broad inhibitory action of the JAK inhibitors oclacitinib and tofacitinib. In summary, our results indicate a much more comprehensive relief of itch by JAK inhibitors, which is mediated through TRPV1 receptors and not just by inhibiting the signal transduction of itch-inducing cytokines. In addition to itch inhibition, the reduced response to capsaicin by JAK inhibitors indicates an extension of possible benefit in attenuating pain transduction through TRPV1, as already documented in patients with rheumatoid arthritis. Our study provides a novel mechanism for the antipruritic role of JAK inhibitors through TRPV1 receptor channels. We appreciate the technical support provided by Jenny Wilzopolski. Tofacitinib (CP-690550, C16H20N6O.C6H8O7) and 5-HT were purchased from Tocris (Minneapolis, Minn), and oclacitinib (PF-03394197, C15H23N5O2S) was purchased from Adooq Bioscience (Irvine, Calif) or Zoetis (Florham Park, NJ). Capsaicin, chloroquine, compound 48/80, histamine dihydrochloride, potassium chloride, poly-l-lysine, laminin, and TDI were obtained from Sigma (St Louis, Mo). AITC, collagenase, Dynabeads Protein G, Fura-2–acetoxymethyl ester, methylcellulose, PBS, NuPAGE LDS Sample Buffer, NuPAGE Sample Reducing Agent, Tris/Glycine/SDS buffer, PAR-2–activating peptide (SLIGRL-NH2), Tween 20, and Tween 80 were from Thermo Fisher Scientific (Waltham, Mass). Dulbecco modified Eagle medium, dispase, and Ca2+- and Mg2+-free HBSS were from Mediatech (Manassas, Va). Recombinant mouse IL-31 and TNF-α were obtained from PeproTech (Rocky Hill, NJ). FuGENE HD Transfection Reagent was from Promega (Madison, Wis). Mini-PROTEAN TGX Stain-Free Precast Gels were from Bio-Rad Laboratories (Richmond, Calif). VR1 antibody (P-19) was obtained from Santa Cruz Biotechnology (Santa Cruz, Calif). Rabbit TRPV1 antibody and mouse TUJ-1 antibody were obtained from Chemicon (Billerica, Mass). Female BALB/cAnN mice (7 weeks old) were purchased from Charles River Laboratories (Raleigh, NC). Female C57BL/6 wild-type and TRPV1 KO mice (age 6 weeks) were purchased from Jackson Laboratory (Bar Harbor, Me). They were housed in groups of 4 mice per cage under controlled lighting (12-hour light-dark cycle), temperature (22°C ± 3°C), humidity (55% ± 15%), and ventilation (at least 10 complete fresh-air changes per hour). Standard rodent chow and water were available ad libitum. All aspects of the current study were conducted in accordance with the Animal Care and Use Program of the North Carolina State University (IACUC protocol no. 13-111-B). Mice were killed by means of CO2 asphyxiation, and DRGs along the whole vertebral column were excised. Isolated DRGs were enzymatically digested in dispase (2.5 U/mL) and collagenase (2.5 mg/mL) dissolved in HBSS at 37°C. Neurons were dissociated by means of mechanical trituration with fire-polished Pasteur pipettes. Cells were washed in DRG medium (Dulbecco modified Eagle medium with l-glutamine, 10% heat-inactivated FCS, and 1% penicillin-streptomycin) and resuspended in 200 to 300 μL of DRG medium, and 20 μL was placed onto poly-l-lysine (0.1 mg/mL)– and laminin (0.1 mg/mL)–coated coverslips. Cells were incubated at 37°C in a 5% CO2 atmosphere for 2 hours and then flooded with 1 mL of DRG medium and further incubated at 37°C in a 5% CO2 atmosphere. Measurements were performed within the next 24 hours. FuGENE HD Transfection Reagent was used to transiently transfect AD293 cells with the TRPV1-pEAK8 plasmid. The functionality of TRPV1-transfected AD293 cells was validated by measuring capsaicin-evoked cytosolic calcium mobilization 48 hours after transfection. Calcium imaging was performed on cultured mouse DRG neurons and TRPV1-transfected AD293 cells, as described previously,E1Rossbach K. Nassenstein C. Gschwandtner M. Schnell D. Sander K. Seifert R. et al.Histamine H1, H3 and H4 receptors are involved in pruritus.Neuroscience. 2011; 190: 89-102Crossref PubMed Scopus (117) Google Scholar with minor modifications. Cells were first loaded with 2 μmol/L Fura-2–acetoxymethyl ester for 30 minutes at 37°C in a 5% CO2 atmosphere protected from light. The coverslips were mounted on a perfusion block and viewed through an inverted fluorescence microscope (TE200; Nikon, Melville, NY). Fluorescence was excited by UV light at 340 and 380 nm alternately, and the emitted light was collected every 100 ms by using a camera attached to a Lambda LS lamp and a Lambda optical filter changer. Cells were incubated in Locke buffer (pH 7.4; 136 mmol/L NaCl, 5.4 mmol/L KCl, 2.9 mmol/L CaCl2, 2.5 mmol/L MgCl2, 10.9 mmol/L D-glucose, 0.6 mmol/L NaHPO4, and 14.3 mmol/L NaHCO3) during imaging. Solutions were delivered through a temperature-controlled perfusion system at a flow rate of 3 mL/min. Recombinant mouse IL-31 (1 μg/mL), recombinant mouse TNF-α (1 μg/mL), histamine dihydrochloride (1 mmol/L), compound 48/80 (1 mmol/L), the PAR-2 agonist SLIGRL-NH2 (1 mmol/L), chloroquine (100 μmol/L), 5-HT (1 mmol/L), AITC (10 μmol/L), capsaicin (1 μmol/L), and low-pH solution (pH 4.0) were delivered. At the end of the experiment, the viability of the neurons was confirmed by an increase in Ca2+ evoked by an application of 150 mmol/L potassium chloride. Cells were judged to be responsive if the ratio value increased by greater than 10% of the resting level after chemical application. DRG neurons and TRPV1-transfected AD293 cells were exposed to 0.1% dimethyl sulfoxide (vehicle) and each JAK inhibitor (oclacitinib or tofacitinib at 1 μmol/L) in Locke buffer just before (approximately 1 minute) the stimulus exposure. The selected concentrations were adapted from previous reports.E2Fukuyama T. Ehling S. Cook E. Baumer W. Topically administered Janus-kinase inhibitors tofacitinib and oclacitinib display impressive antipruritic and anti-inflammatory responses in a model of allergic dermatitis.J Pharmacol Exp Ther. 2015; 354: 394-405Crossref PubMed Scopus (69) Google Scholar, E3Heine A. Held S.A. Daecke S.N. Wallner S. Yajnanarayana S.P. Kurts C. et al.The JAK-inhibitor ruxolitinib impairs dendritic cell function in vitro and in vivo.Blood. 2013; 122: 1192-1202Crossref PubMed Scopus (255) Google Scholar, E4Kubo S. Yamaoka K. Kondo M. Yamagata K. Zhao J. Iwata S. et al.The JAK inhibitor, tofacitinib, reduces the T cell stimulatory capacity of human monocyte-derived dendritic cells.Ann Rheum Dis. 2014; 73: 2192-2198Crossref PubMed Scopus (110) Google Scholar This concentration (1 μmol/L) did not induce any cell toxicity. The hair on the thoracic region of the backs and cheeks of the mice was shaved with a hair clipper a day before the experiment. The JAK inhibitors oclacitinib or tofacitinib were administered orally 30 minutes before injection of the pruritogen. Each JAK inhibitor was diluted in a 0.5% methylcellulose/0.25% Tween 20 solution to doses: tofacitinib, 30 mg/kg; oclacitinib, 45 mg/kg. The oral doses of tofacitinib and oclacitinib used in this study were selected based on our previously published study.E2Fukuyama T. Ehling S. Cook E. Baumer W. Topically administered Janus-kinase inhibitors tofacitinib and oclacitinib display impressive antipruritic and anti-inflammatory responses in a model of allergic dermatitis.J Pharmacol Exp Ther. 2015; 354: 394-405Crossref PubMed Scopus (69) Google Scholar Recombinant mouse IL-31, 1 μg per injection site; recombinant mouse TNF-α, 1 μg per injection site; histamine dihydrochloride, 50 μg per injection site; compound 48/80, 20 μg per injection site; and chloroquine, 50 μg or 200 μg per injection site (note the higher concentration corresponds to former studies in which no difference between wild-type and TRPV1−/− mice was observedE5Kim S. Barry D.M. Liu X.Y. Yin S. Munanairi A. Meng Q.T. et al.Facilitation of TRPV4 by TRPV1 is required for itch transmission in some sensory neuron populations.Sci Signal. 2016; 9: ra71Crossref PubMed Scopus (54) Google Scholar), SLIGRL-NH2 (50 μg per injection site) and 5-HT (10 μg per injection site) were injected once intradermally in a volume of 50 μL into the shaved part of the back 30 minutes after administration of the JAK inhibitor. Capsaicin (10 μg per injection site) was injected intradermally in a volume of 10 μL into the shaved part of the cheek 30 minutes after administration of the JAK inhibitor. Scratching or wiping behavior was evaluated for 30 minutes immediately after injection of the pruritogen or capsaicin. A TDI-induced itch model was generated based on the method described previously,E2Fukuyama T. Ehling S. Cook E. Baumer W. Topically administered Janus-kinase inhibitors tofacitinib and oclacitinib display impressive antipruritic and anti-inflammatory responses in a model of allergic dermatitis.J Pharmacol Exp Ther. 2015; 354: 394-405Crossref PubMed Scopus (69) Google Scholar with the difference that the allergic itch was chronic. Hair was removed from the abdominal regions of female BALB/c mice with a depilatory cream the day before the first sensitization (day 1). On day 1, the shaved abdominal region was stripped 10 times with adhesive tape just before sensitization with 50 μL of 5% TDI. The same sensitization without tape stripping was performed on days 2 and 3. To induce a chronic model, the allergic reaction was boosted repeatedly for the next 6 weeks through application of 50 μL of 0.5% TDI onto the shaved neck (once weekly). A week after the last TDI boost, either vehicle (1:7 acetone/dimethyl sulfoxide) or tofacitinib (0.5%) was topically administered 30 minutes before TDI challenge to neck skin (n = 6 for each group). The tofacitinib concentration was selected according to a former study performed by our group.E2Fukuyama T. Ehling S. Cook E. Baumer W. Topically administered Janus-kinase inhibitors tofacitinib and oclacitinib display impressive antipruritic and anti-inflammatory responses in a model of allergic dermatitis.J Pharmacol Exp Ther. 2015; 354: 394-405Crossref PubMed Scopus (69) Google Scholar Scratching bouts were monitored for 1 hour immediately after 0.5% TDI challenge. Immunohistochemistry was performed on DRG sections (12 μm) from untreated and TDI-treated mice. Rabbit TRPV1 (1:500 dilution) and mouse TUJ-1 (1:500 dilution) antibodies were used. Total numbers of neurons expressing the TRPV1 receptor were counted for untreated and treated samples and were normalized to the TUJ-1 as a neuron marker. Either mock- or TRPV1-transfected AD293 cells were incubated for 10 minutes with oclacitinib (10 μmol/L) or tofacitinib (10 μmol/L) and then thoroughly washed with PBS, solubilized, and immunoprecipitated with Dynabeads Protein G and VR1 antibody (P-19). SDS-PAGE of the eluted sample was performed with NuPAGE LDS Sample Buffer, NuPAGE Sample Reducing Agent, and gel electrophoresis on Mini-PROTEAN TGX Stain-Free Precast 10% Gel. Precipitates from the gel were analyzed with MALDI–ultra-high-resolution Fourier-transform ion cyclotron resonance (FTICR) mass spectrometry using α-cyanohydroxycinnamic acid as a matrix substance after cutting the respective bands from the gel and extracting them with ethyl acetate to visualize the presence of tofacitinib or oclacitinib interacting with TRPV1.E6Fukuyama T. Tschernig T. Qi Y. Volmer D.A. Baumer W. Aggression behaviour induced by oral administration of the Janus-kinase inhibitor tofacitinib, but not oclacitinib, under stressful conditions.Eur J Pharmacol. 2015; 764: 278-282Crossref PubMed Scopus (19) Google Scholar, E7Qi Y. Hainz N. Tschernig T. Meier C. Volmer D.A. Differential distribution of probenecid as detected by on-tissue mass spectrometry.Cell Tissue Res. 2015; 360: 427-429Crossref PubMed Scopus (5) Google Scholar Signals for the drugs were acquired with mass accuracies of less than 1 ppm, and results were confirmed with tandem mass spectrometry by using authentic standards. All data were expressed as means ± 1 SDs. Statistical significance of the difference between the vehicle control and treated groups were estimated at the 5% and 1% levels of probability. The Student t test was used to test the significance of differences between mean values between 2 groups. Comparisons for more than 3 groups were carried out by using a 1-way ANOVA, followed by the Dunnett multiple comparison test. Comparisons of proportions were made with the Fisher exact test. Data were analyzed with Prism 4 software (GraphPad Software, San Diego, Calif).}, number={1}, journal={JOURNAL OF ALLERGY AND CLINICAL IMMUNOLOGY}, author={Fukuyama, Tomoki and Ganchingco, Joy Rachel and Mishra, Santosh K. and Olivry, Thierry and Rzagalinski, Ignacy and Volmer, Dietrich A. and Baeumer, Wolfgang}, year={2017}, month={Jul}, pages={306-+} }
 @article{cruse_yin_fukuyama_desai_arthur_baumer_beaven_metcalfe_2016, title={Exon skipping of Fc epsilon RI beta eliminates expression of the high-affinity IgE receptor in mast cells with therapeutic potential for allergy}, volume={113}, ISSN={["0027-8424"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85002784626&partnerID=MN8TOARS}, DOI={10.1073/pnas.1608520113}, abstractNote={Significance We identified an innovative use for the technique of antisense oligonucleotide-mediated exon skipping to specifically target and down-regulate IgE receptor expression in mast cells. Exon skipping is typically used as part of personalized medicine, where a mutant exon is skipped after sequencing the patients’ affected genes. Our approach, however, targets a nonmutated gene and an exon that is critical for surface IgE receptor expression. It does not require a personalized approach with genetic sequencing or multiple iterations of oligonucleotides that would require clinical trials. Furthermore, the diseases to be treated with this technology are ideal for local delivery of the oligonucleotides by aerosols or topical cream formulations. Allergic diseases are driven by activation of mast cells and release of mediators in response to IgE-directed antigens. However, there are no drugs currently available that can specifically down-regulate mast cell function in vivo when chronically administered. Here, we describe an innovative approach for targeting mast cells in vitro and in vivo using antisense oligonucleotide-mediated exon skipping of the β-subunit of the high-affinity IgE receptor (FcεRIβ) to eliminate surface high-affinity IgE receptor (FcεRI) expression and function, rendering mast cells unresponsive to IgE-mediated activation. As FcεRIβ expression is restricted to mast cells and basophils, this approach would selectively target these cell types. Given the success of exon skipping in clinical trials to treat genetic diseases such as Duchenne muscular dystrophy, we propose that exon skipping of FcεRIβ is a potential approach for mast cell-specific treatment of allergic diseases.}, number={49}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Cruse, Glenn and Yin, Yuzhi and Fukuyama, Tomoki and Desai, Avanti and Arthur, Greer K. and Baumer, Wolfgang and Beaven, Michael A. and Metcalfe, Dean D.}, year={2016}, month={Dec}, pages={14115–14120} }
 @article{stover_fukuyama_young_daniele_oberley_crapo_baeumer_2016, title={Topically applied manganese-porphyrins BMX-001 and BMX-010 display a significant anti-inflammatory response in a mouse model of allergic dermatitis}, volume={308}, ISSN={["1432-069X"]}, DOI={10.1007/s00403-016-1693-0}, abstractNote={In this study, we topically administered two antioxidant compounds, the manganese-porphyrin-derivatives BMX-001 and BMX-010, in a mouse model of allergic dermatitis and compared the efficacy for reduction of itch and inflammation. In vitro effects of BMX-001 and BMX-010 on keratinocytes, bone marrow derived dendritic cells (BMDCs) and T-cells were initially analysed. For assessment of scratching behaviour, BMX-001 and BMX-010 (0.01 and 0.1 %) were topically applied 16 h and/or 1 h before compound 48/80 or toluene-2,4,-diisocyanate (TDI) challenge in a TDI induced mouse dermatitis model. Additionally, assessment of allergic skin inflammation was performed in a similar manner in the TDI model. Post-treatment ear thickness was measured 24 h after TDI challenge and compared to basal values. The mice were sacrificed and the ear auricle was removed for further analysis. In vitro, both BMX substances significantly inhibited cytokine production of keratinocytes as well as of BMDC and T-cell proliferation. Topical treatment with BMX cream resulted in a significant decrease in scratching behaviour in the compound 48/80 model, but not in the TDI model. Mice treated with BMX-001 and BMX-010 showed a moderate dose dependent decrease in ear thickness, and interestingly, the concentration of the cytokines IL-1β and IL-4 in inflamed skin was reduced by 80–90 % by all treatment options. These first results suggest the potential benefit of a BMX-001 and BMX-010 cream for the treatment of allergic-inflammatory skin diseases.}, number={10}, journal={ARCHIVES OF DERMATOLOGICAL RESEARCH}, author={Stover, Kelsey and Fukuyama, Tomoki and Young, Ashlyn T. and Daniele, Michael A. and Oberley, Rebecca and Crapo, James D. and Baeumer, Wolfgang}, year={2016}, month={Dec}, pages={711–721} }
 @article{fukuyama_tschernig_qi_volmer_baumer_2015, title={Aggression behaviour induced by oral administration of the Janus-kinase inhibitor tofacitinib, but not oclacitinib, under stressful conditions}, volume={764}, DOI={10.1016/j.ejphar.2015.06.060}, abstractNote={Janus kinase (JAK) inhibitors have recently been developed for allergic diseases. We focused on the 2 different JAK inhibitors, tofacitinib (selective for JAK3) and oclacitinib (selective for JAK1 and 2), to clarify the mechanism of anti-inflammatory and anti-itching potency of these drugs. In the process of detecting anti-itching potency, we observed that tofacitinib treated mice showed aggression behaviour. The objective of the study reported here was to investigate the aggressive behaviour induced by tofacitinib by using a mouse model of allergic dermatitis and the resident-intruder test. For the allergic dermatitis model, female BALB/c mice were sensitised and challenged topically with toluene-2,4-diisocyanate (TDI). Vehicle, tofacitinib or oclacitinib, was administered orally 30 min before TDI challenge. Scratching, aggression and standing behaviours were monitored in the 60 min period immediately following challenge of TDI. Another group of male BALB/c mice treated with vehicle, tofacitinib or oclacitinib was evaluated in the resident-intruder test and brains were obtained to determine blood brain barrier penetration. In the allergic dermatitis model, a significant increase in aggression and standing behaviour was only obvious in the tofacitinib treatment group. There was no effect in non-sensitised mice, but similar aggression was also induced by tofacitinib in male resident-intruder test. Penetration of blood-brain barrier was observed both in tofacitinib and oclacitinib treated mice. These results suggest that aggression was induced by tofacitinib under some kind of stressful environment. This study indicates a possible role of the JAK-STAT pathway in modulation of aggression behaviour.}, journal={European Journal of Pharmacology}, author={Fukuyama, T. and Tschernig, T. and Qi, Y. L. and Volmer, D. A. and Baumer, W.}, year={2015}, pages={278–282} }
 @article{fukuyama_ehling_cook_baeumer_2015, title={Topically Administered Janus-Kinase Inhibitors Tofacitinib and Oclacitinib Display Impressive Antipruritic and Anti-Inflammatory Responses in a Model of Allergic Dermatitis}, volume={354}, ISSN={["1521-0103"]}, DOI={10.1124/jpet.115.223784}, abstractNote={The prevalence of allergic skin disorders has increased rapidly, and development of therapeutic agents to alleviate the symptoms are still needed. In this study, we orally or topically administered the Janus kinase (JAK) inhibitors, tofacitinib and oclacitinib, in a mouse model of dermatitis, and compared the efficacy to reduce the itch and inflammatory response. In vitro effects of JAK inhibitors on bone marrow–derived dendritic cells (BMDCs) were analyzed. For the allergic dermatitis model, female BALB/c mice were sensitized and challenged with toluene-2,4-diisocyanate (TDI). Each JAK inhibitor was orally or topically applied 30 minutes before and 4 hours after TDI challenge. After scratching bouts and ear thickness were measured, cytokines were determined in challenged skin and the cells of the draining lymph node were analyzed by means of flow cytometry. In vitro, both JAK inhibitors significantly inhibited cytokine production, migration, and maturation of BMDCs. Mice treated orally with JAK inhibitors showed a significant decrease in scratching behavior; however, ear thickness was not significantly reduced. In contrast, both scratching behavior and ear thickness in the topical treatment group were significantly reduced compared with the vehicle treatment group. However, cytokine production was differentially regulated by the JAK inhibitors, with some cytokines being significantly decreased and some being significantly increased. In conclusion, oral treatment with JAK inhibitors reduced itch behavior dramatically but had only little effect on the inflammatory response, whereas topical treatment improved both itch and inflammatory response. Although the JAK-inhibitory profile differs between both JAK inhibitors in vitro as well as in vivo, the effects have been comparable.}, number={3}, journal={JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS}, author={Fukuyama, Tomoki and Ehling, Sarah and Cook, Elizabeth and Baeumer, Wolfgang}, year={2015}, month={Sep}, pages={394–405} }