Inflamm. Res. (2017) 66 (Suppl 1):S1–S50 DOI 10.1007/s00011-017-1068-9 Inflammation Research

The First Joint Meeting of the European Research Society and Japanese Histamine Research Society, May 11–14, 2017 Amsterdam, The Netherlands

Editor: G. Sturman

In cooperation with:

K. Barrett (San Diego) D. Bell (Belfast) N. Carruthers (San Diego) P. L. Chazot (Durham) M. Ennis (Belfast) B. Gibbs (Kent) L. Kay (Sheffield) F. L. Pearce (London)

This supplement was not sponsored by outside commercial interests. It was funded entirely by the publishers.

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Meeting Report of the First Joint Meeting of the European and Japanese Histamine Research Societies

G. Sturman

This year’s meeting was in Amsterdam, in the Netherlands positron emission tomography’. Geoff West was a founder at the kind invitation of Professor Rob Leurs and his team. member of the EHRS and was its first General Secretary. This is the second time that histaminologists have met in the At the end of the lecture, Professor Yanai was presented Netherlands; the previous meeting in 2003 was organised by with a copy of Geoff West’s autobiography –‘A Handful of Henk Timmerman and Rob Leurs. This year’s meeting was Luck’. After a coffee break we listened to the first sym- held in the Vrije Universiteit Amsterdam, in the new posium which was on Molecular Pharmacology. Here we research O|2 building (officially opened in 2016). This heard about the various research approaches being used to building houses the research groups of Human Health & study the four different histamine receptors. In the after- Life Sciences. This year there were appropriately 120 people noon, the second symposium was concerned with the registered from many different countries and included a involvement of histamine and its receptors in cancer. Then number from Japan. The Council met as usual late after- each of the poster presenters was given just 2 min to tell noon. Most of the delegates arrived on the Wednesday and the audience about their research findings. Having this time whilst the posters were being put up in the foyer, the constraint worked very well but did cause some hilarity in meeting participants chatted whilst they nibbled on some the process! This was followed by a lively discussion at the snacks. Some regular attendees could not attend and they posters, helped by some alcoholic beverages and followed were missed but a big welcome was made to all the new by a very tasty Indonesian buffet. The remaining evening visitors, who we hope will return to future meetings. was free to explore a little of Amsterdam. Thursday started with the Opening Ceremony: the 45th Friday started with a symposium on ‘ sys- meeting of our society but the first joint meeting with the tems and genetics’ where we heard about some disorders in Japanese Histamine Research Society. We were welcomed which histamine or lack of it has a significant effect. This by our hosts, Professors Henk Timmerman and Rob Leurs, theme continued after coffee but this time centred on digestive who thanked all their helpers. Then the current president of disturbances and the skin. During the lunch time, Madeleine the Japanese Histamine Research Society (JHRS), (Pro- Ennis held a workshop for the ESR people on what helps to fessor Hiroyki Fukui) spoke about the JHRS and how it make a good poster. The first session in the afternoon was on started in 1979 with Professors Kenji Tasaka and Hiroshi Histamine and the Immune System, whilst the following Wada agreeing to have an annual gathering centered on symposium was on the Chemical Biology of the Histamine histamine research. After retirement of these Professors, Receptors. In the evening the organisers had arranged for us to the Japanese histamine researchers have continued these go by 2 canal boats through the various Amsterdam canals. meetings. Their first meeting was organized by Professor Whilst on the boats, the captains told a little of the history of Yamatodani in 1997. Then our EHRS President, Paul Amsterdam as well as something about the more important Chazot gave out the student bursaries certificates and €500 buildings. We had an excellent meal on board and were able to for each of the 7 student members. The El-Sayed Assem chat away to our histaminergic friends. family very kindly sponsored two students while the other In the first session of the following day, we listened to bursary winners were sponsored by our society. This was our younger members (PhD students or not more than followed by the Honorary Membership ceremony where 3 year’s post-doctoral research) who had been selected to Professor Hiroyuki Fukui of Tokushima University, Japan, give presentations for the EHRS Young Investigator Award who is the current President of the Japanese Histamine (YIA). This competition was sponsored by Janssen Phar- Research Society, was presented with his official certificate maceutical Company. It was another difficult task for the beautifully written in Latin and sporting the society’s judges to differentiate between these four excellent pre- official seal. He then thanked the society for this honour. sentations. The winner was Niels Hauwert (Amsterdam, Then we started with the scientific programme with the G. The Netherlands) for his presentation entitled ‘A bi-direc-

B. West Lecture being given by Professor Katzuhiko Yanai tional photo-antagonist toolbox for on ‘The functional significance of the histaminergic neu- photopharmacology’ and the runner-up was Bhanu Priya ronal system: Lessons from gene knockout mice and Ganesh (Houston, Texas, USA) for her presentation

123 The First Joint Meeting of the European Histamine Research Society S3 entitled ‘Histamine and diacylglycerol kinase by Lacto- Dinner in old Amsterdam with the Award Ceremony with bacillus reuteri mediated crosstalk between mammalian certificates and prizes being given out. All YIA finalists intestinal epithelium and immune cell maturation’. The and poster prize winners received a Dutch souvenir from other two finalists were given Highly Commended Cer- the organising committee. Then as usual we had our tificates. The next symposium was concerned with singing session, beginning with ‘‘Anita’s Thank You

‘Histamine H4 antagonists going towards the clinic’ where Song’’ (sung to the tune of ‘Tulips in Amsterdam’) as a big we heard the current situation about these compounds and thank you to Rob and his team for the excellent and their potential therapeutic uses. The final oral session of the memorable meeting. After this we sung our EHRS Anthem, meeting was entitled ‘Histamine and the Nervous System’ before saying ‘au revoir’ to our many ‘histaminergic’ and encompassed 10 talks given on a wide range of topics friends. using multiple species and in vitro and in vivo techniques. Our thanks are given to all of the Dutch and Japanese Over the three days of this meeting, there had been 57 histaminologists for the excellent meeting. The next invited/short talks, plus 4 YIA presentations and 36 posters meeting will be held in Dublin, Ireland (31 May–2 June, displayed and presented. 2018) at the kind invitation of Astrid Sasse. Throughout the meeting the poster committee (Arianna Rosa, Vanina Medina and Mio Mitzunobu) had been working very hard and as usual had a difficult task in identifying winning posters for the poster competition. Eventually first prize was given to Anne Hall et al., from Houston, USA with the poster entitled ‘Histamine pro- duction by Lactobacillus reuteri can be regulated by EriC- family proton/chloride antiporters’, second to Satomi Moriyama et al., from Okayama, Japan with the poster entitled ‘Wide variety expression of vesicular polyamine Paul Chazot, President of the EHRS presenting Professor Katzuhiko Yanoi transporter (VPAT)’ and third prize went to Guadalupe- with a copy of the G. B. West autobiography in thanks for giving the Elide Morales-Figueroa et al., from Mexico with their G. B. West Memorial Lecture. poster entitled ‘Functional interaction between Histamine Department of Pharmacology and Therapeutics, School of H and Adenosine A receptors in rat striato-pallidal nerve 3 2a Biomedical and Health Sciences, Kings College London, terminals’. London, SE1 1UL. E-mail: [email protected] At the end of the final session we held our General Assembly. This was followed by our traditional Farewell

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Honorary Memberships in the European Histamine Research Society

P. L. Chazot, G. Sturman

There are three types of membership in the EHRS: ordi- purification and cloning of the H1 , nary, corporate and affiliated, and honorary (life). The expression topology and signalling, genetic regulation and highest award is that of honorary membership which is pharmacology. Notably he has published over the years only given to very special people. To obtain honorary many papers together with international EHRS colleagues membership, the person has to be elected by over two- across the globe, notably Professors Wada, Hill, Panula, thirds of the ordinary members at the General Assembly. Watanabe and Levi, which highlights his long association There are only 14 current Honorary Members of the with and active support of the EHRS and he rightly Society; Madeleine Ennis, Agnieszka Fogel, Robin deserves being elected an Honorary Member of the Euro- Ganellin, Helmut Haas, Zsuzsanna Husti, Piero Mannaioni, pean Histamine Research Society. Emanuela Masini, Bruno Mondovi, Fred Pearce, Jean- Charles Schwartz, Anita Sydbom, Henk Timmerman, Takehiko Watanabe and Jean West. Former (now sadly deceased) Honorary Members of this society include Sir James Black, Franc Erjavec, Wilfried Lorenz, Czeslaw Maslinski, Wolfgang Schmutzler, Ingrid Olhagen-Uvna¨s, Borje} Uvna¨s and Geoffrey West. At this meeting, the society awarded Honorary Membership to a special person who has contributed significantly to the histamine research over the years and it was very appropriate for this meeting which was the first joint histamine meeting of the EHRS and the Japanese Histamine Research Society, he is Pro- The presentation of Honorary Membership to Professor Hiroyuki Fukui by fessor Hiroyuki Fukui of the Institute of Biomedical the President of the EHRS, Paul Chazot at this Amsterdam meeting. Sciences, Tokushima University Graduate School, Tokushima, Japan. Department of Pharmacology and Therapeutics, School of Professor Hiroyuki Fukui has had a thirty year career in Biomedical and Health Sciences, Kings College London, histamine with over 130 articles published, many in jour- London SE1 1UL. E-mail: [email protected] nals with high impact. His research spans the early days of

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Neurological aspects of histamine

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THE FUNCTIONAL SIGNIFICANCE OF THE are dependent on its concentration: low brain histamine HISTAMINERGIC NEURON SYSTEM: LESSONS causes anxiety and some neurological disorders and high FROM GENE KNOCKOUT MICE AND POSITRON brain concentration causes abnormal sleep-wake cycle and EMISSION TOMOGRAPHY aggressive behaviours. 1. Yoshikawa T, et al. Insufficient Intake of L- K. Yanai, T. Yoshikawa, M. Tashiro reduces brain histamine and causes anxiety-like behaviors in male mice. J. Nutr. 2014; 144:1637–1641 Histamine neurons are exclusively located in the posterior 2. Yanai K, et al. The clinical pharmacology of non- hypothalamus, and send their outputs to almost all regions sedating . Pharmacol. Ther. 2017 Apr 27. In of the brain. They are involved in many functions such as press spontaneous locomotion, arousal, sleep-wake cycle, appe- 3. Yoshikawa T, Yanai K. Histamine clearance through tite control, seizures, learning and memory, behavioral polyspecific transporters in the brain. Handb Exp Phar- sensitization, stress and emotion. We propose that the macol. In press histamine neuron system has a dual effect on CNS, with 4. Naganuma T, et al. Histamine N-methyltransferase both stimulatory and suppressive functions. As a stimula- regulates aggression and the sleep-wake cycle. Submitted. tory function, neuronal histamine is one of the most 5. Kano M, et al. Decreased important systems to maintain and stimulate wakefulness binding in the brain of depressed patients. Eur. J. Neurosci. and cognition. Histamine also functions as a bio-protection 2004; 20: 803–810. system against various noxious and unfavorable stimuli 6. Iwabuchi K, et al. A PET study of histamine H1 such as convulsion, nociception, drug sensitization, and receptors in the schizophrenic patients assessed by [11C]- stress susceptibility. We have clarified the functional roles . Eur Neuropsychopharmacol. 2005; 15:185–191. of histamine neurons using the different methods of his- Department of Pharmacology, Tohoku University Gradu- tamine-related gene knockout mice or human positron ate School of Medicine, Sendai, Japan. E-mail: emission tomography (PET). [email protected] We point out that dietary intake of histidine and car- nosine are important to maintain mental health through the activation of the histaminergic neuron system [1, 2], COMPARISON OF BRAIN- AND NON-BRAIN though the H3 receptors antagonists are clinically available PENETRATING ANTI- AGAINST now. Astrocytes reuptake neuronal histamine via organic MOTION-INDUCED EMESIS IN SUNCUS MURINUS cation transporter 3 (OCT3) and plasma membrane (HOUSE MUSK SHREW) monoamine transporter (PMAT) without recycling in neurons. Then it is metabolized by histamine N-methyl- L. Tu, J. A. Rudd transferase (HNMT) in astrocytes [3]. This is one of the reasons why histidine supplementation is needed to main- Anti-histamines are used in the treatment of motion sick- tain a healthy condition in the brain. We also demonstrated ness but the precise mechanism of action is unknown. In the highly elevated level of brain histamine in histamine the present studies, we used Suncus murinus to investigate N-methyl-transferase (HNMT) gene knockout mice, sug- the anti-emetic potential of the highly selective brain gesting that HNMT is a sole metabolizing enzyme in the penetrating histamine H1 , brain. The high level of brain histamine induced disrupted in comparison to the non-brain penetrating histamine H1 sleep-wake cycle and increased aggressive behaviors receptor antagonist, . The muscarinic receptor through the activation of H1 and H2 receptors, respectively, antagonist scopolamine was used as a positive control. in HNMT-KO mice [4]. Motion-induced (4 cm displacement, 1 Hz, 10 min) emesis We previously examined [11C] doxepin binding in (8.4 ± 0.6 episodes) reduced sniffing activity and depression and schizophrenia [5, 6]. [11C]Doxepin binding increased scratching activity. Mepyramine and scopo- was decreased in the brains of these stress-related psychi- lamine, but not cetirizine, significantly antagonized atric disorders. The decrease of [11C] doxepin binding was motion-induced emesis. The short duration of motion failed proportional to the respective symptom scores. We recently to induce quantifiable c-fos expression in area postrema, 11 observed the similar changes of H1 receptors in the [ C] nucleus tractus solitaries, medial vestibular nucleus and doxepin-PET study of young volunteers with irrita- hypothalamus. However, mepyramine with or without ble bowel syndrome (IBS) because the disorders such as a motion stimulus, but not cetirizine or scopolamine, anxiety, major depression, and chronic fatigue syndrome caused a significant increase in c-fos expression in brain. are common among people with IBS. In conclusion, it is The mepyramine-treated animals were also sedated and suggested that the roles of histamine as a neurotransmitter had fewer episodes of scratching during the motion test.

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Our previous studies using Suncus murinus have shown FAA, expressions of c-Fos mainly in the E2 and E3 that stimuli inducing 6–20 episodes of emesis are associ- subdivisions of TMN were increased in WT in a time ated with increases of c-fos expression in the brain whereas course manner, which was attenuated in Y1KO. Wake- our motion stimulus, which produced ca 10 episodes, did promoting orexinergic neurons in the lateral hypothala- not. The pattern of c-fos activation is paradoxical, being mic area (LHA), that make functional connection with more expected from treatments (e.g. cisplatin) that induce, TMN to control wakefulness, also expressed c-Fos dur- rather than those that inhibit, emesis. Our findings suggest ing the induction of FAA, which was also reduced in that short periods of provocative motion do not transduce Y1KO. These results suggest that NPY activates distinct through c-fos to induce emesis. Conversely, the ability of neurons in TMN subnuclei to induce motivational mepyramine, but not cetirizine, to reduce emesis and arousal before onset of feeding, suggesting a possible scratching and cause sedation appears to involve c-fos and role of NPY in promotion of arousal systems in collab-

H1 receptors located centrally. It is possible that the oration with wake-promoting neurons in the unexpected profile of action of mepyramine relates its hypothalamus. inverse agonist properties opening up new perspectives on Department of Pharmacology, Niigata University Gurad- emesis control and on mechanisms of side-effects involv- uate School of Medical and Dental Sciences, Japan. ing H1 receptors. E-mail: [email protected] School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China. E-mail: [email protected] INFLUENCE OF HISTAMINERGIC CEREBELLAR SYSTEM IN MOTOR LEARNING IN MICE NEUROPEPTIDE Y REGULATES ACTIVATION OF

HISTAMINERGIC SUBGROUPS THROUGH Y1- B. Silva-Marques, A. C. L. Gianlorenc¸o, R. Mattioli RECEPTOR TO INDUCE FOOD-ANTICIPATORY ACTIVITY IN MICE SUBJECTED TO SCHEDULED The central histaminergic nervous system originates FEEDING from the tuberomammillary nucleus of the hypothalamus and widely innervates almost the whole brain including H. Umehara, H. Fukui, H. Higuchi the cerebellum of many species. Experimental evidence indicates that cerebellum is involved in various non- Histaminergic neurons cluster in the tuberomammillary motor as well as motor operations. Since our recent nucleus (TMN), which is morphologically subdivided results have demonstrated the participation of the cere- into 5 groups; E1-E5, and send projections to whole bellar histaminergic system in the consolidation of brain areas to control several functions including arousal emotional memory, this study aims to investigate the and feeding behaviours. Recent evidence suggests that role of the histaminergic neural system in motor learning brain histamine has dissociated roles in appetitive vs. of mice via cerebellar vermis and hereafter establish a consummatory phases of feeding behaviour. Previously possible interaction of this system in processing motor we demonstrated that motivation for food activates his- and non-motor function. First of all, we have conducted taminergic neurons in particular subdivisions of TMN in an experiment to investigate the dose-dependent effects rats that were subjected to scheduled feeding and sug- of histamine microinjected in the cerebellar vermis on gested that histaminergic neurons are composed of motor performance and motor learning in mice. For this, functionally heterogeneous subgroups. Neuropeptide Y we used 50 Swiss albino mice, 10 per dose (weighing (NPY) is an orexigenic neurotransmitter produced in the 25–35 g) and maintained in a thermoregulated environ- arcuate nucleus of hypothalamus (cARC). The orexi- ment. The drugs used were histamine (0.54 nmol, genic action of NPY is mediated mainly through Y1- 1.36 nmol, 2.72 nmol and 4.07 nmol) and saline 0.9%. receptor (Y1) at the paraventricular nucleus of After being anesthetised, a guide cannula was implanted hypothalamus (PVN). Recently we found that TMN into the cerebellar vermis following coordinates from the subdivisions receive NPY-containing axons and express mouse brain atlas of Paxinos and Franklin. The protocol Y1 as well. In this study, we investigated whether NPY was divided in five steps, which were named habituation, activate histaminergic neurons during appetitive phase in microinjection, stage 1, stage 2, and stage 3. At habit- wild type (WT) and Y1-deficient mice (Y1KO) which uation the animals were placed in the rota-rod at speed are subjected to scheduled feeding. WT exhibited of 8–20 rpm until they lost control. Twenty four hours increased locomotor activity before meal time, i.e. food- later, the animals received a microinjection of saline or anticipatory activity (FAA), while Y1KO were less histamine. Five minutes later, the mice were submitted active than WT. Correspondingly with the extent of to stage 1, where they were placed in rota-rod and in the

123 The First Joint Meeting of the European Histamine Research Society S9 balance beam, each for 3 times, with 5 min of rest N-terminal region of the huntingtin protein. In the between each time. The protocol was repeated 4 h later hypothalamus, the aggregation of this protein was more for stage 2 and repeated again 24 h later for stage 3. strongly in the TMN. Moreover, we found that the HDC- Statistical analysis included the homogeneity test and mRNA levels were increased as was the HMT expres- multi-factor analysis of variance (ANOVA) followed by sion in other brain areas. In addition, alterations of the Duncan’s Multiple Range test. A p value of B0.05 was histaminergic system in depression or schizophrenia was required for significance. The results showed a possible discussed. Potential clinical implications of our findings facilitation of histamine at the highest dose in the are that the histamine H3 receptor antagonists were evaluation of learning and motor performance in the found to have positive effects in AD patients in several rota-rod. This suggests that cerebellar histaminergic ongoing phase II clinical trials. In AD, HDC-mRNA projections are involved in motor learning and make a levels in the TMN were unchanged, in spite of TMN modulating role in the cerebellar circuit to ensure that neuronal loss. In the course of AD only females had movements are performed efficiently. increased prefrontal cortex expression of H3R- and Department of Physiotherapy, UFSCar, Sa˜o Carlos, Brazil. HMT-mRNA. Regarding the possibility the already E-mail: [email protected] higher activity of the remaining TMN neurons in AD

patients, the applications of these H3R antagonists should be well controlled. THE HUMAN HISTAMINEGIC SYSTEM IN Netherlands Institute for Neuroscience, an Institute of the HEALTH AND NEUROPSYCHIATRIC Royal Netherlands Academy of Arts and Sciences, Ams- DISORDERS: A POSTMORTEM STUDY terdam, the Netherlands. E-mail: [email protected]

L. Shan, A.-M. Bao, D. F. Swaab HISTAMINE RELEASE FROM THE The hypothalamic tuberomammillary nucleus (TMN) is HYPOTHALAMUS TARGETS SPECIFIC the exclusive source of neuronal histamine. The his- NEURONAL POPULATIONS IN THE taminergic system is crucially involved in a number of PREFRONTAL CORTEX basic physiological brain functions. We performed quantitative radioactive in situ hybridization of histidine D. Lucaci, C. Houston, X. Yu, W. Wisden, S. G. Brickley decarboxylase (HDC), the rate limiting enzyme for his- tamine production, to study changes in histamine The tuberomamillary nucleus promotes arousal by acti- production. Moreover, expression changes of the his- vating histamine receptors throughout the brain. We have taminergic receptors and of the inactivating enzyme recently demonstrated that in addition to this histamine histamine methyltransferase (HMT) were studied by release, co transmission of GABA from this projection will qPCR in specific projection areas in these disorders. A also lead to activation of extrasynaptic GABA-A receptors diurnal fluctuation in HDC-mRNA expression was found with the potential to dampen the excitability produced by for the first time in controls while a disorder of this histamine receptor activation. However, it is not clear to diurnal fluctuation was observed in neurodegenerative what extent the GABA and histamine systems target dif- diseases including Parkinson’s (PD), Huntington’s (HD) ferent neuronal populations or whether co transmission is a or Alzheimer’s (AD) disease. Earlier studies in an ani- feature of release from the tuberomamillary nucleus mal PD model showed that inhibition of histamine throughout the brain. In this study, we have focused our production in the early stage would rescue dopaminergic attention on the actions of GABA and histamine in the neurons from degenerating in the substantia nigra and prefrontal cortex; a brain region associated with working improve locomotion. So the question was whether in PD memory, decision making and executive function. We have the TMN was activated. In PD patients there was an used whole-cell recording techniques in acute slice prepa- abundant presence of Lewy bodies and Lewy neurites in rations of the adult prefrontal cortex to monitor the impact the TMN, which were presumed to indicate a strong of optogenetic stimulation of axonal fibres originating from degeneration of this nucleus. In spite of that, HDC- the tuberomamillary nucleus. We have observed excitatory mRNA in the TMN remained unaffected in either early actions of histamine on fast spiking interneurons and or late stage of PD. A higher histaminergic receptor inhibitory actions of GABA on neocortical pyramidal cells. expression and HMT was found in the substantia nigra We are currently incorporating these observations into a and putamen, which may play a protective role against model of decision making within the prefrontal cortex. augmentation of histamine in these areas of PD. HD Department of Life Sciences, Imeprial College London, results from an abnormal polyglutamine extension in the London, UK. E-mail: [email protected]

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LACK OF HISTAMINE INCREASES LEVO-DOPA the direct DA agonist apomorphine tended to induce INDUCED AXIAL DYSKINESIAS IN MICE more contralateral rotations in HDC KO mice than in wildtype mice. These results suggest that the lack of S. Rannanpa¨a¨, S. Nousiainen, S. Leino, P. Panula, histamine could have a protective effect against the O. Salminen lesion while at the same time the DA receptors might be more sensitised. To study LID, daily L-DOPA injections The dopamine replacement therapy with L-DOPA is the were given and dyskinesias recorded and analysed. The most effective treatment for motor symptoms of HDC KO mice displayed more more severe axial dysk- Parkinson’s disease (PD). A disadvantage of L-DOPA inesia than wild type mice (L-DOPA dose 24 mg/kg s.c.; treatment is abnormal involuntary movements (AIMs, *p \ 0.05 and 48 mg/kg **p \ 0.01, Mann–Whitney also called L-DOPA-induced dyskinesia, LID) develop test. N = 5WT,N= 4HDCKO)butnodifferencesin in most patients 4–6 years after the initiation of the orolingual or forelimb dyskinesia between genotypes treatment. In addition to the loss of brain dopamine were observed. When comparing total dyskinesia scores (DA) neurons, brain histaminergic system is affected in between moderately and severely dyskinetic animals, a PD and HA neurons can take up L-DOPA and convert it clear tendency of HDC KO mice to develop more severe to DA and thereby affecting PD treatment. Here we dyskinesia was observed (L-DOPA dose 24 mg/kg s.c.; studied the role of HA in LID in a 6-hydroxydopamine p = 0.07, Mann–Whitney test. N = 5WT,N= 4HDC (6-OHDA) lesioned knockout KO). In conclusion, these results suggest that when the (HDC KO) mice. HDC KO mice do not synthesize brain histaminergic system is genetically dampened, the histamine whereas the histamine neurons are not mor- mice develop more severe LID than control animals. The phologically altered. Female HDC KO mice and their underlying mechanism could be that the histamine neu- littermate wild type mice underwent a stereotaxic sur- rons lacking the histamine synthesizing enzyme can gery where the dorsal striatal DA neurones were replace their histamine synthesis with dopamine syn- unilaterally lesioned by two injections of 6-OHDA thesis as a compensatory mechanism and cause an (2 9 6 lg). To study the interactions between pharma- imbalance during the dopamine replacement therapy and cological agents and the absence of histamine induce more severe dyskinesia. This work was supported amphetamine- and apomorphine-induced rotation tests by the Academy of Finland (grant #12677612). were performed. Injection of the indirect DA agonist Division of Pharmacology and Pharmacotherapy, Faculty amphetamine tended to induce more ipsilateral rotations of Pharmacy, University of Helsinki, Finland. E- in wildtype mice than HDC KO mice and injection of mail:saara.rannanpaa@helsinki.fi

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Immunological aspects of histamine

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HUMAN BASOPHILS PRODUCE AND ARE EFFECTS OF DEXAMETHASONE ON MURINE DIFFERENTIALLY ACTIVATED BY IL-31 MATURE MAST CELLS

B. F. Gibbs, M. Gehring, S. Kleiner, B. Eiz-Vesper, S. Tanaka A. Kapp, U. Raap Steroidal anti-inflammatory drugs have been widely Basophils crucially contribute to the symptoms of used in chronic inflammatory diseases such as atopic allergic inflammation including itch, which is thought to dermatitis. Because they have multiple targets, it be attributable to the release of histamine from these remains to be clarified how they modulate the func- cells as well as mast cells. However, recent evidence tions of tissue mast cells. We previously established a suggests that IL-31 also plays a major role in causing murine mast cell culture system that mimics the pro- itch, especially in chronic urticaria which is associated cess of cutaneous mast cell maturation and with increased IL-31 serum levels and basophil activa- demonstrated that histamine synthesis is required for tion. We therefore investigated the potential association granule maturation. Here, we investigated the effects between IL-31 and basophils by assessing the biological of dexamethasone on the maturation of murine mast effects of IL-31 on basophils and whether these cells cells using this model, where IL-3-dependent bone are a source for this important proinflammatory marrow-derived cultured mast cells (BMMCs) were co- cytokine. cultured with Swiss 3T3 fibroblasts in the presence of IL-31 protein and receptor expressions were analyzed on stem cell factor. The presence of 1 lM dexamethasone healthy donor basophils by Western blotting and flow (Dex) during the co-culture period (16 days) sup- cytometry, respectively. Basophil responses to IL-31 were pressed the mast cell proliferation but did not affect examined for chemotaxis, CD63 and CD203c expressions, the staining profiles with Safranin O. The prolonged release of histamine and Th2-type cytokines as well as in treatment with Dex significantly suppressed the storage terms of regulation of intracellular signalling. of chymase and tryptase but substantially up-regulated Both the IL-31 receptor and protein were expressed in that of histamine and b-hexosaminidase. Dex signifi- basophils and protein expressions of the cytokine were cantly suppressed degranulation induced by compound increased after stimulation with either IL-3 or anti-IgE 48/80 and substance P but had no effects on that and strikingly enhanced following fMLP stimulation. IL- induced by IgE-mediated antigen stimulation. Dex was

31 induced basophil chemotaxis and the release of IL-3, found to suppress the expression of Gbi1,whichwas IL-4, IL-6 and IL-13. It also moderately induced the up-regulated during the co-culture period. The number phosphorylation of ERK1/2 an increased expression of of cutaneous mast cells were not changed after 6 days HIF-1alpha, an important regulator of basophil cytokine of topical application of Dex (0.05%–0.2%) whereas synthesis. Surprisingly, and in stark contrast to priming the content of tissue histamine was significantly cytokines such as IL-3 and IL-33, IL-31 did not poten- increased. Dex might impair the functions of cutaneous tiate IgE-dependent basophil activation or histamine endothelial cells because vascular responses were release and had no effect on CD63 and CD203c down-regulated even in the presence of histamine. We expressions. Additionally, IL-31 was strongly expressed then measured the number of degranulated cutaneous in basophils present in the skin from patients with mast cells upon treatment with compound 48/80 as chronic spontaneous urticaria. well as IgE-mediated passive cutaneous anaphylaxis Together, our study clearly demonstrates that basophils and found that prolonged treatment with Dex signifi- are a source of and are differentially activated by IL-31 cantly suppressed it under both conditions. We which stimulates basophils to release proinflammatory conclude that prolonged treatment with dexamethasone cytokines and induces chemotaxis but does not affect might considerably change the granule contents and degranulation. The basophil is therefore a potential major suppress IgE-independent degranulation of cutaneous contributor to itch in urticaria and other basophil-related mast cells. diseases due to the effects of not only histamine but also Department of Immunobiology, Graduate School of Medi- IL-31. cine, Dentistry, and Pharmaceutical Sciences, Okayama University of Kent, Medway School of Pharmacy, United University, Japan. E-mail: [email protected] Kingdom. E-mail: [email protected]

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MAST CELL-DERIVED HISTAMINE REGULATES Unit of Drug Discovery and Development, Italian Institute LIVER KETONE BODY PRODUCTION VIA of Technology, Genoa, Italy. OLEOYLETHANOLAMIDE-MEDIATED E-mail: [email protected] SIGNALLING

A. Misto, A. Basit, G. Provensi, V. Vozella, M. B. Passani, REGULATION OF ANAPHYLATOXIN C3A D. Piomelli RECEPTOR EXPRESSION ON HUMAN M2 MACROPHAGES BY IL-4 AND HISTAMINE Oleoylethanolamide (OEA) is a lipid mediator that regu- lates feeding and stimulates lipolysis and fatty acid S. Mommert, D. Aslan, H. Stark, R. Gutzmer, T. Werfel oxidation in adipocytes and hepatocytes through activation of nuclear peroxisome proliferator-activated receptor type- Complement activation culminates in a strong amplifica- a (PPAR-a). Fasting is accompanied by a substantial ele- tion of the immune response. The anaphylatoxin C3a vation in liver OEA levels, but the biochemical mechanism triggers inflammation by binding to its specific G-protein and physiological implications of this effect are unknown. coupled C3a receptor (C3aR). Histamine plays an important role in the control of feeding Since the number of C3aR expressed on the cell surface behavior and contributes to the anorexic effects of sys- of immune cells modulates the cell responsiveness to C3a, temically administered OEA. we intended to investigate the expression levels of C3aR on In the present study, we examined whether histamine- human M2 macrophages in allergic situations where high mediated signalling might contribute to OEA mobilization levels of the Th2 cytokine IL-4 and also histamine are in response to changes in feeding status. present in a local microenvironment. Histidine decarboxylase-null (HDC-/-)mice,mast For this purpose we differentiated human monocytes cell-deficient (C57BL/6 J-KitW-v/J) mice, NAPE- in vitro in the presence of macrophage colony stimulating PLD-/- mice and their wild-type littermates (all males) factor (M-CSF) to M2 macrophages. The expression of the were subjected to three different conditions: a) free- different histamine receptors was analyzed on fully dif- feeding (FF); b) 12 h food-deprivation (FD) and c) 1 h ferentiated M2 macrophages compared to IL-4 activated refeeding after 12 h FD (RF). A dose of 50 lgor100lg M2a macrophages by quantitative PCR. of histamine (volume of injection 0.1 mL) was admin- Expression of the C3aR was measured by quantitative istered by a single intrahepatic (i.h.) injection for PCR and by flow cytometry: (i) On macrophages which 30 min. Histamine receptor antagonists ( were differentiated in the presence of histamine or the and , each to 10 mg/kg and ciproxifan, 3 mg/ (H4R) agonist ST-1006, (ii) on fully kg) were administered by intraperitoneal (i.p.) injection, differentiated M2 macrophages stimulated with different while alpha-fluoromethylhistidine (a-FMH), a suicide histamine receptor agonists, (iii) on IL-4 activated M2a inhibitor of HDC, was administered by intracere- macrophages. broventricular (i.c.v.) infusion (1 lg/ll). Endogenous Fully differentiated M2 macrophages expressed H1R, OEA, N-oleoyl-PE, oleic acid and histamine levels were H2R and H4R at mRNA level. Expression of the H3R was quantified by liquid chromatography/mass spectrometry not detected. Importantly, the H2R and H4R mRNA (LC/MS) and ketone body production was quantified by expression were significantly up-regulated by IL-4. colorimetric assay. The presence of ST-1006 during the differentiation We found that 12-hour fasting stimulates visceral mast process of monocytes into M2 macrophages significantly cells to release histamine, which enhances N-oleoyl-PE down-regulated C3aR expression. The stimulation of fully biosynthesis and NAPE-PLD-dependent OEA production differentiated M2 macrophages with histamine or ST-1006 in liver tissue through activation of H1 receptors. In mutant down-regulated C3aR expression in a time dependent mice in which this signalling process is defective— manner. Also the activation of fully differentiated M2 HDC-/- mice, mast cell-deficient mice and NAPE-PLD-/- macrophages to M2a macrophages by IL-4 led to down- mice—ketone body production induced by food depriva- regulation of C3aR expression. Moreover, ST-1006 tion is markedly diminished. The results reveal an showed an additive effect on the IL-4 induced down-reg- unexpected role for mast cells in the regulation of liver ulation of C3aR surface expression. ketone body production during fasting.

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These data suggest that down-regulation of C3aR process. However, these first results already indicate a expression by mediators present in allergic situations such possible role for histamine in modulating AMPs in as IL-4 and histamine has an anti-inflammatory impact by inflammatory skin diseases. reducing the sensitivity to C3a-induced down-stream sig- Department of Dermatology and Allergy, Hannover Med- naling and thereby inhibiting an overwhelming immune ical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, response. Germany. E-mail: [email protected] Division of Immunodermatology and Allergy Research, Department of Dermatology and Allergy, Hannover Med- ical School, D-30625 Hannover, Germany. E-mail: ANTI-INFLAMMATORY DRUGS WITH DUAL [email protected] INHIBITORY EFFECTS ON CAIX AND COX-2

L. Lucarini, M. Durante, C. Lanzi, A. Pini, F. Carta, HISTAMINE MODULATES THE PRODUCTION OF C. T. Supuran, E. Masini ANTIMICROBIAL PEPTIDES BY HUMAN KERATINOCYTES The carbonic anhydrase (CA) family includes 16 catalyti- cally active zinc metallo-enzymes that catalyze the K. Schaper-Gerhardt, J. S. Trampenau, S. Mommert, reversible interconversion of carbon dioxide and water to T. Werfel, R. Gutzmer bicarbonate and protons. Sulphonamide CA inhibitors, besides antitumour activity, exhibit anti-inflammatory Histamine and antimicrobial peptides (AMPs) are both effects in rats with permanent middle cerebral artery important immunomodulatory mediators in the pathogen- occlusion and in LPS-stimulated RAW 264.7 macrophages. esis and maintenance of inflammatory skin diseases like Macrophages are involved in inflammation, and histamine atopic dermatitis and psoriasis. AMPs are a multiple group up-regulates the production of pro-inflammatory cytokines of polypeptides with antimicrobial cytotoxic activity stimulated by LPS. Fibrosis of lung tissue is characterized against bacteria, fungi and virus. They are divided in by chronic inflammation that determines a pathological cathelecidine and members of the RNase, which are remodelling of lung parenchyma. The animal model expressed constitutive in the upper layers of the epidermis, obtained by intra-tracheal administration of bleomycin in in defensine and the S100 family, which are upregulated C57BL/6 mice is one of the most validated murine models. through pathogens infiltrating the skin. Besides their direct This study investigated the effects of compound 1E, a antimicrobial effects, AMPs also have the capability to novel nonsteroidal anti-inflammatory compound and car- modulate the immune response by attracting immune cells bonic anhydrase inhibitor hybrid (NSAID-CAI) in the and induce cytokine production. However, the interaction modulation of inflammation. between histamine and AMPs has not been investigated in We administered the compound 1E on COX-2 inhibition detail, yet. Our aim was to explore the impact of histamine in a murine model of bleomycin-induced lung fibrosis. on the production of AMPs in keratinocytes. In first studies, C57BL/6 mice were treated with bleomycin (0.05 IU) or we found that stimulation with histamine alone was able to saline intratracheally to induce lung fibrosis. Immediately enhance hBD-2, LL-37 and RNase7 production signifi- after, mice were treated with vehicle, compound 1e (1 mg/ cantly at mRNA-level in human neonatal keratinocytes kg b.wt.), ibuprofen (0.5 mg/kg b.wt.) or acetazolamide (Wilcoxon Signed Rank Test). To mimic the inflammatory (0.5 mg/kg b.wt.) at equimolar doses, sub-cutaneously conditions of Th1 and Th2 cytokine-mediated skin dis- released by micro-osmotic pumps for 21 days. We assayed eases, we stimulated keratinocytes with IL-4 and with a airway resistance to inflation and lung samples were pro- combination of IFNc and IL-17. While IL-4 reduced the cessed to measure 8OHdG, a marker of oxidative stress, production of hBD-2 and RNase7, pre-incubation with COX-2 expression, pro-inflammatory cytokines production histamine reversed this decrease. In contrast to IL-4, and apoptosis. Fibrosis and airway remodeling were eval- stimulation with IFNc and IL-17 enhanced the production uated by measuring TGF-b, the percentage of positive of hBD-2, RNAse 7 and LL-37, which could be further Goblet cells, and smooth muscle layer thickness slightly potentiated by pre-incubation with histamine. First determination. results with immunohistochemical staining against RNase Our preliminary results indicate that the compound 1E 7 on cultivated keratinocytes also revealed an impact of decreased oxidative stress marker and inflammation mark- histamine at protein level. In further experiments we will ers, as shown by the reduction of 8-OHdG (9.5 ± 1.1 vs extend our studies on protein level and additionally per- 14.04 ± 1.3 ng/lg of DNA), and inflammatory (IL-1b, form 3D skin cultures models. Additionally, we want to 40.1 ± 3 vs 58.2 ± 2 pg/lg of protein; TNFa, 0.6 ± 0.1 vs elucidate which histamine receptors are involved in this 0.9 ± 0.2 pg/lg of protein) and pro-fibrotic (TGF-b,

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110.5 ± 13 vs 146.04 ± 11 pg/lg of protein) cytokine total IgE concentration in mouse serum (37.2 ± 3.0 ng/ml production compared to the control mice. The treatment in normal serum (n = 13), 74.9 ± 8.8 ng/ml in L-ASP- reduces the thickness of smooth muscle layer (4.9 ± 2.4 vs sensitized (n = 14)) and CY, at 150 mg/kg and 300 mg/kg, 9.33 ± 4.3 lm) and the goblet cell relative number (2 ± 0.5 also augmented the increase in IgE in the sera vs 7 ± 3), both markers of bronchial remodeling, and the (230.3 ± 30.9 ng/mL (n = 7) and 213.1 ± 52.3 ng/mL collagen deposition, a functional parameter of fibrosis. (n = 7), respectively). Anti-IgE treatment decreased total The results here reported demonstrate that these com- IgE concentration in L-ASP-sensitized and 150 mg/kg CY- pounds, endowed with a dual COX-2 and CAIX inhibition, treated L-ASP-sensitized mice serum to 15.8 ± 4.2 ng/mL are interesting new anti-inflammatory drugs and have a (n = 8) and 12.6 ± 2.4 ng/mL (n = 8), respectively. beneficial effect in a model of lung fibrosis in mice, thus When RBL-2H3 cells were sensitized by anti-L-ASP sera indicating that these compounds could be a novel thera- in vitro, L-asp challenge induced beta-hexosaminidase peutic strategy for lung inflammatory diseases. (Hex) release. Anti-L-ASP serum of CY-treated mice Department of Neuroscience, Psychology, Drug Research induced higher Hex release than normal anti-L-ASP serum and Child Health (NEUROFARBA), University of Flor- (spontaneous release 1.8 ± 0.1% (n = 29), L-ASP-sensi- ence, Viale G. Pieraccini 6, 50139 Florence, Italy. E-mail: tized serum 3.5 ± 0.3% (n = 27), CY (150 mg/kg)-treated laura.lucarini@unifi.it L-ASP-sensitized serum 15.3 ± 2.7%). Anti-IgE Ab treatment ex vivo inhibited L-ASP-induced Hex release in L-ASP sensitized serum and CY-treated L-ASP-sensitized L-ASPARAGINASE-INDUCED ALLERGY IN MICE: serum to 2.3 ± 0.2% (n = 13) and 1.9 ± 0.1% (n = 13), IN VIVO SENSITIZATION AND IN VITRO respectively. ACTIVATION OF RBL-2H3 CELLS From the present results, L-ASP sensitization induced IgE production in vivo and this serum induced Hex release M. Mio, A. Nogami-Hara, K. Yabuki, K. Suwaki, A. Handa, from RBL-2H3 cells. C. Mitsuhata, K. Kajiyama, A. Shimada Laboratory of Pharmacology, Shujitsu University, School of Pharmacy, 703-8516, Japan. E-mail: mio@ L-Asparaginase (L-ASP) is one of the essential drugs for shujitsu.ac.jp acute lymphoblastic leukemia, Since L-ASP proteins used Account number: 620058103818 clinically are derived from bacteria, they often cause allergies, including anaphylaxis. We have established mouse model of L-ASP allergy and observed that a neu- H2R AGONISM AND H4R ANTAGONISM tralizing antibody (Ab) of IgE is effective to inhibit L-ASP- AMELIORATES INFLAMMATION IN A MOUSE induced allergic reaction. In the present study, we used MODEL OF PSORIASIS RBL-2H3 cells to establish in vitro model of L-ASP allergy. K. Rossbach, K. Wahle, K. Schaper, W. Ba¨umer, Male BALB/c mice (7 week old) were sensitized with M. Kietzmann L-ASP (100 units) with Al(OH)3 gel (1 mg) on days 0 and 14. The right ears of the mice were locally sensitized on Psoriasis is a chronic inflammatory skin disorder that is days 17, 20, 23 by i.d. injection of L-ASP (10 units). characterized by hyperproliferative keratinocytes and Antigen challenge was carried out on day 26 by i.d. immune cell infiltration into the skin. Manifestations of injection (10 units). Antisera of L-ASP were prepared from psoriasis are diverse, but one hallmark is scaling, which the blood obtained by cardiac puncture under anesthesia. results from the hyperproliferative epidermis. Histamine Cyclophosphamide (CY) was i.p. administrated at day –2 might play a pathogenetic role in psoriasis since histamine and day 12. Total IgE was determined using an ELISA kit increases the proliferation of keratinocytes and elevated (Yamasa). Anti-IgE treatment (100 lg, i.p.) was carried out histamine levels can be found in psoriatic skin. Aim of this on the day before L-ASP challenge. study was to test the role of histamine receptors (HxR) in L-ASP challenge-induced ear edema was inhibited by the AldaraTM-induced psoriasis-like skin inflammation pretreatment with anti-IgE Ab (BD Bioscience, clone R35- mouse model. BALB/c mice (n = 6–7 per group) were

92). CY, at 150 mg/kg, enhanced L-ASP-induced ear treated intraperitoneally with 20 mg/kg mepyramine (H1R edema. L-ASP-induced CY-enhanced ear edema was also inverse agonist), (H2R agonist), JNJ-39758979 inhibited by anti-IgE Ab. L-ASP sensitization increased (H4R antagonist) or vehicle twice daily for 17 days. Drug

123 The First Joint Meeting of the European Histamine Research Society S17 administration started one week prior to application of HISTAMINE AS A POTENTIAL MEDIATOR AldaraTM. AldaraTM cream (contains 5% imiquimod) was TAHT ACTIVELY PROMOTES applied once daily onto the back skin for 10 consecutive THE DEVELOPMENT OF ORGAN INJURY -/- days. In addition, wildtype and H4R mice were treated IN SEPSIS with AldaraTM for 10 days. Statistical significances were assessed by Mann–Whitney U-Test. Data in the text are Y. Hattori given as means ± SD. Stimulation of the H2R and block- ade of the H4R clearly reduced the scaling response (score Sepsis, a syndrome that occurs when microbial invasion (0–4) on day 10: Vehicle: 2 ± 0, amthamine: 1.3 ± 0.5; induces systemic illness, is one of the most common rea- vehicle: 2.4 ± 0.8, JNJ-39758979: 1.2 ± 0.4; wildtype: sons for critically ill patients to be admitted to an intensive -/- 2.5 ± 0.5, H4R : 1.3 ± 0.5), diminished epidermal care unit and remains the major cause of death from thickening (vehicle: 53 ± 9 lm, amthamine: 41 ± 4 lm, microbial infection, despite recent progress in our under- p \ 0.01; vehicle: 58 ± 10 lm, JNJ-39758979: standing of the disease mechanisms and modern advances -/- 46 ± 2 lm, p \ 0.05; wildtype: 58 ± 10 lm, H4R : in hemodynamic, antibiotic, and ventilator clinical support. 45 ± 12 lm) and lowered the influx of inflammatory cells The development of a failure of one or more organs poses a into the skin (score (none—++++): Vehicle: ++++ , major threat to the survival of patients with sepsis, but the amthamine: +++; vehicle: +++, JNJ-39758979: ++; wild- pathological process in the development of sepsis-induced -/- type: ++++ , H4R : +++). The concentration of pro- multiple organ dysfunction to death remains incompletely inflammatory Th1 cytokine IL-33 (vehicle: 136 ± 32 pg/ understood. In the very recent past, the third international lg, amthamine: 77 ± 36 pg/lg, p \ 0.05; vehicle: consensus definitions have been advocated for sepsis and 13 ± 2 pg/lg, JNJ-39758979: 8 ± 1 pg/lg, p \ 0.05; septic shock, and sepsis is now redefined as life-threatening -/- wildtype: 31 ± 13 pg/lg, H4R :22± 14 pg/lg) as well organ dysfunction due to a dysregulated host response to as the Th17 cytokines IL-17A (vehicle: 30 ± 9 pg/lg, infection. Histamine assumes a critical role as a major amthamine: 18 ± 6 pg/lg, p \ 0.05; vehicle: 3 ± 1 pg/ mediator of many disorders with inflammation and immune lg, JNJ-39758979: 2 ± 1 pg/lg; wildtype: 17 ± 4 pg/lg, reactions. Since a long time ago, it has been reported that -/- H4R :11± 1 pg/lg; p \ 0.01) and IL-23 (vehicle: plasma histamine levels are elevated in animal models of 33 ± 10 pg/lg, amthamine: 20 ± 7 pg/lg, p \ 0.05; sepsis as well as in patients with septic shock. To date, vehicle: 1 ± 0.3 pg/lg, JNJ-39758979: 0.6 ± 0.2 pg/lg, however, little is known about the role of histamine in the -/- p \ 0.05; wildtype: 21 ± 4 pg/lg, H4R :14± 3 pg/lg, pathogenesis of sepsis. When polymicrobial sepsis was p \ 0.01) were reduced at lesional skin sites in mice induced by cecal ligation and puncture (CLP) in mice, we -/- treated with amthamine or JNJ- 39758979 and in H4R found that tissue histamine levels were elevated in asso- mice. These cytokines play key roles in the pathogenesis of ciation with histidine decarboxylase (HDC) expression. In psoriasis. Furthermore, Aldara increased the number of HDC knockout mice rendered septic by CLP, tissue levels skin-draining lymph node cells. This increase was lower in of pro-inflammatory cytokines, such as TNF-a, IL-1b, IL- -/- -/- H4R mice (wildtype: 3 ± 2 million (mio); H4R : 6, and MCP-1, were less marked than their littermate wild- 2 ± 1, p \ 0.09) and in mice treated with JNJ-39758979 type (WT) mice. Blood biochemical and histopathological (vehicle 6 ± 1 mio, JNJ-39758979 3 ± 1 mio, p \ 0.01) examinations showed that sepsis-induced multiple organ but not in mice treated with the H2R agonist (vehicle 3 ± 1 injury (lung, liver, and kidney) was significantly reduced in mio, amthamine: 3 ± 1 mio). The H1R inverse agonist HDC knockout mice as compared with WT controls. His- mepyramine did not show anti-inflammatory effects in the tamine H1- and H2-receptor gene-double knockout mice Aldara model. The inflammatory response was reduced in apparently behaved similar to HDC knockout mice in terms amthamine- and JNJ-39758979-treated mice as well as in of reductions in blood biochemical and histopathological -/- H4R mice. Thus, H2R agonist and H4R antagonists changes after CLP. Pharmacological interventions with H1- could represent a new strategy for the treatment of psori- and H2-receptor antagonists (d-chlorpheniramine and asis. A combined treatment with H2R agonists and H4R famotidine) indicated that both H1- and H2-receptors are antagonists might provide synergistic anti-inflammatory involved in septic lung and liver injury, while only H2- effects and will be tested in future studies. receptors can contribute to septic kidney injury. Several University of Veterinary Medicine Hannover, Institute for transcription factors play key roles in the transcription of Pharmacology, Toxicology and Pharmacy, Hannover, inducible genes involved in immune and inflammatory Germany. E-mail: [email protected] responses in sepsis. Most notably, nuclear factor-jB (NF-

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jB) is a pivotal participant in modulating the transcription We establish that endogenous histamine acting on H1- of the immunoregulatory mediators contributing to the and H2-receptors is identified as an aggravating mediator to sepsis pathology. NF-jB is a family of key transcription contribute to the development of major end-organ injury in factors that exist in virtually all cell types and its respon- sepsis. We thus shed light on the new role of histamine in sive site, jB site, has been characterized in the promoters the pathophysiology of sepsis disorder. Our results are also and enhancers of numerous pro-inflammatory genes. The in favor of H1- and H2-receptor antagonists being safe histamine-mediated development of major end-organ in critically ill patients. injury is likely to be associated with an increase in the Department of Molecular and Medical Pharmacology, nuclear factor-jB signaling pathway. Indeed, phosphory- Graduate School of Medicine and Pharmaceutical Sci- lation of IjBa, an inhibitor of jB, and nuclear ences, University of Toyama, Toyama, Japan. E-mail: translocation of NF-jB were diminished in lungs of HDC [email protected] knockout mice following sepsis induction.

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Histamine H3 receptor

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HETEROCOMPLEXES OF DOPAMINE D1- A2ARs on GP nerve terminals was confirmed by radioli- HISTAMINE H3 RECEPTORS REPRESENT gand binding (1327 and 454 fmol/mg protein, respectively; A NOVEL THERAPEUTIC TARGET FOR 4–5 determinations). A2AR activation with the selective HUNTINGTON’S DISEASE agonist CGS-21680 significantly enhanced K+-evoked [3H]-GABA release from perfused GP synaptosomes D. Moreno-Delgado, M. Puigdellı´vol, E. Moreno, (153 ± 20% of control values at 3 nM; n = 5, p [ 0.05, J. Botta, M. Rodrı´guez-Ruiz, P. Gasperini, A. Cordomı´, one-way ANOVA and Dunnett’s test), and this effect was

L. A. Howell, M. Scarselli, G. Navarro, V. Casado´, reduced by the H3R selective agonist (EC50 E. Canela, S. Ferre´, M. Guzma´n, L. Pardo, J. Alberch, 5.2 nM; n = 5). The inhibitory effect of 100 nM immepip C. Lluı´s, S. Gine´s, P. J. McCormick was in turn antagonized by (3 lM; n = 4;

p [ 0.05, one-way ANOVA and Tukey’s test). The A2AR- Early Huntington’s disease (HD) includes over-activation mediated facilitation of GABA release has been reported to of dopamine D1 receptors (D1R) producing an imbalance in involve the cAMP/PKA pathway, and both forskolin dopaminergic neurotransmission and cell death. Employing (10 lM; n = 5) and 8-Bromo-cAMP (0.5 mM) mimicked + 3 a new strategy to reduce D1R over-activation by targeting the effect of A2ARs on K -evoked [ H]-GABA release complexes of D1R and histamine H3 receptors (H3R), we from GP synaptosomes (n = 5). In GP slices A2AR acti- block D1R-mediated cell death. D1R-H3R heteromers are vation increased cAMP accumulation (295% of basal, EC50 expressed and function in an HD cell model and in dif- 3.2 nM; n = 5) and H3R activation by immepip reduced by ferent brain areas of HD mice in early but not late disease 50% this effect (IC50 5 nM; n = 5). A2AR activation did + 2+ stages. We found D1R-H3R expressed in human control not increase K -evoked Ca entry in GP synaptosomes subjects and in low-grade HD patients but not high-grade suggesting an action on the exocytotic machinery and not 2+ HD patients. We demonstrate that H3R ligands allosteri- on voltage-activated Ca channels. The interaction cally modulate D1R via the D1R-H3R heteromer interface, between H3Rs and A2ARs may also involve the formation and show this can be disrupted using synthetic peptides. of heteromers. In GP synaptosomal membranes the H3R Treatment of HD mice with an H3R antagonist prevents agonist immepip reduced the A2AR affinity for CGS- cognitive and motor learning deficits and the loss of het- 21680, whereas the A2AR agonist increased the H3R eromer expression. Our results indicate that D1R-H3R affinity for immepip (n = 4, p [ 0.05, Student’s t test). heteromers play a pivotal role in controlling dopamine These data support that H3R/A2AR dimerization does take signaling and are targets for treating HD. place, although the changes (*2 fold) in affinities appear University of Surrey, School of Veterinary Medicine, Dept. modest to explain the functional effects observed. Our of Pre-Clinical Sciences, Guildford, Surrey, GU2 7AL, UK. results indicate that H3Rs modulate GABA release from E-mail: [email protected] striato-pallidal neurons by selectively opposing the A2AR- induced stimulatory effect, most likely by inhibiting A2AR- induced cAMP formation and PKA activation. FUNCTIONAL INTERACTION BETWEEN Departamento de Neurociencias, Centro de Investigacio´n y HISTAMINE H3 AND ADENOSINE A2A de Estudios Avanzados (Cinvestav) del IPN, 07360 Ciudad RECEPTORS IN RAT STRIATO-PALLIDAL NERVE de Me´xico, Me´xico. E-mail: mofi[email protected] TERMINALS

G.-E. Morales-Figueroa, R. Gonza´lez-Pantoja, NOVEL PERIPHERAL HISTAMINE H3 RECEPTOR J. Escamilla-Sa´nchez, J. A. Arias-Montan˜o ANTAGONIST ZPL-868087 ATTENUATES MECHANICAL HYPERSENSITIVITY IN The globus pallidus (GP) belongs to the basal ganglia and NEUROPATHIC PAIN IN MICE is therefore involved in the control of motor behavior. Striato-pallidal neurons express high levels of adenosine E. Battell, A. Rosa, P. L. Chazot, A. Pini, W. L. S. Liu, A2A receptors (A2ARs) and histamine H3 receptors (H3Rs). I. Obara A2ARs couple to Gas proteins and their activation facili- tates depolarization-evoked GABA release from rat GP Despite increasing evidence supporting the histamine H3 slices. H3Rs couple to Gai/o proteins, and in slices from receptor (H3R) expression in nociceptive pathways, including striatum and substantia nigra pars reticulata their activation our earlier studies showing H3R on a population of A delta + 3 decreases K -evoked [ H]-GABA release by opposing the fibres, and a role for H3R in the modulation of mechanical stimulatory action of dopamine D1 receptors, coupled to pathological pain, many of the findings reporting the func- Gas proteins. In this work the high expression of H3Rs and tional implication of H3R in chronic pain have been somewhat 123 S22 The First Joint Meeting of the European Histamine Research Society contradictory. Recent development of a selective and Neuropathic pain is a chronic condition following nerve peripherally acting/centrally-sparing H3R ligand ZPL-868087 damage and degeneration induced by various pathologies has provided an interesting tool for further investigation of the such as diabetes, infection, autoimmune diseases and role of H3R in nociception and validation of peripheral H3Ras malignancies. Allodynia, a painful abnormal sensation a potential target for therapeutic intervention in chronic pain, caused by innocuouc stimuli, is a major symptom of particularly in peripheral neuropathies. We therefore evalu- neuropathic pain, but currently there is no effective ated the antinociceptive efficacy of ZPL-868087 in peripheral clinical treatments. A previous study indicated that a H3 neuropathic pain induced by chronic constriction injury (CCI) antagonist attenuated allodynia in rat neuropathic noci- of the sciatic nerve and in a model of severe hyperglycaemia- ception models. The site of action for H3 antagonist in induced hypersensitivity developed after a single streptozo- CNS, however, is not totally understood. In this study, tocin (STZ, 200 mg/kg i.p.) injection in adult male C57BL/6 J we examined the central effects of H3 antagonists in the mice (n = 6/group). The effect of ZPL-868087 on mechani- rat neuropathic nociception model with L5 spinal root cal and heat hypersensitivity at the plantar surface of the hind ligation. We injected a JNJ- paw was assessed using von Frey filaments and Hargreaves 101814572 (JNJ) (1, 3, 10 and 30 mg/kg) to evaluate its test in the CCI model and it was observed that 3 and 10 mg/kg effect on allodynia quantified by von Frey filament (4 i.p. injections every 24 h for 4 days) resulted in alleviation (n = 8). The expressions of histamine receptor in the of mechanical, but not heat hypersensitivity, while 1 mg/kg spinothalamic nociception tract were determined by was ineffective compared to controls (area under the curve quantitative RT-PCR (n = 8). The histamine concentra- (AUC) for mechanical hypersensitivity—10 mg: tions in ventral posterolateral nucleus of thalamus 7.14 ± 0.97 g, 3 mg: 6.79 ± 0.91 g, 1 mg: 2.38 ± 0.44 g, (VPL), periaqueductal gray matter (PAG), locus ceruleus vehicle: 2.92 ± 0.64 g, F(5,31) = 5.85, P = 0.0006). In the (LC) and spinal cord (SC) before and after JNJ i.p. hyperglycaemia-induced hypersensitivity model ZPL- injection (10 mg/kg) were measured (n = 8). JNJ 868087 (10, 30 and 100 mg/kg p.o.; 6 treatments every 24 h (10 nmol, 1 lL) was administered focally to VPL, PAG, for 6 days) on mechanical and cold hypersensitivity was LC and SC (intrathecal) to evaluate the effects to reduce assessed using von Frey filaments and the acetone test. Here, allodynia (n = 8). We found that H3 receptor was pre- all tested doses inhibited cold hypersensitivity (AUC— dominantlyexpressedinVPL,PAGandSC.JNJ 100 mg: 35.95 ± 3.06, 30 mg: 42.64 ± 4.47, 10 mg: peritoneal injection decreased allodynia (50% PAW in

45.04 ± 4.08, STZ: 99.55 ± 7.97, F(5,30) = 27.07, 10 mg/kg 5.09 v.s. 1.98 g, p \ 0.05). We found that the P \ 0.0001), but only 100 mg/kg inhibited mechanical increase of histamine concentration in the VPL, PAG hypersensitivity (AUC—100 mg: 10.91 ± 1.13 g vs. STZ: and LC after the injection of JNJ (p \ 0.05), indicating

3.66 ± 0.59 g, F(5,30) = 20.52, P \ 0.0001). In contrast, that thalamus and descending inhibitory pathways might none of the ZPL-868087 doses affected nociceptive thresh- play importatnt roles. Finally, direct focal injection of olds in control sham animals. In addition, our immunoblotting JNJ to PAG, LC and SC ameliorated allodynia and immunohistochemistry studies indicated that ZPL- (p \ 0.05). These data demonstrated that VPL and SC as

868087 antinociceptive/analgesic effects may be mediated key regions in allodynia relief achieved by H3R through the extracellular signal-regulated kinases pathways antagonism. involved in pain-induced sensitization. Together, this study Department of Pharmacology, Tohoku University Gradu- emphasizes the importance of the H3R in the modulation of ate School of Medicine, Sendai, Japan. E-mail: chronic pain and in alleviation of, in particular, peripheral [email protected] neuropathies. School of Medicine, Pharmacy and Health, Durham University, UK. E-mail: [email protected] DIHYDROBENZOFURANYL-PIPERAZINES AS MOLECULAR SCAFFOLD FOR DESIGNING

SELECTIVE H3R ANTAGONISTS: INITIAL SAR THE EFFECT OF H3 RECEPTOR ANTAGONISM DATA ON RAT NEUROPATHIC NOCICEPTION M. F. Correˆa, A´ .J. R. Barbosa, D. A. Duarte, T. Nakamura, T. Matsuzawa, M. Mogilevskaya, A. Mogi, L. B. Teixeira, M. Bouvier, C. M. Costa-Neto, T. Yoshikawa, F. Naganuma, N. Okamura, K. Yanai J. P. S. Fernandes

123 The First Joint Meeting of the European Histamine Research Society S23

Histamine is one of the most important chemical trans- pharmacological treatments is 25% reduction of individual mitters mainly involved in allergic, inflammatory and CNS baseline IOP. We previously demonstrated that H3R processes, which acts activating the histamine receptors antagonists such as ABT239, GSK189254 as well as DL-76

(H1R-H4R). The H3R is mainly expressed in the CNS as a and ciproxifan, at 1% concentration, reduced IOP in rabbit presynaptic auto/heteroreceptor, controlling the release of glaucoma models displaying features similar to a reference several neurotransmitters and histamine as well. The H4R anti-glaucomatous drug. is mainly found in inflammatory cells. Herein, we present a The aim of the present research was to study the role of novel molecular scaffold, the dihydrobenzofuranyl-piper- the histaminergic system in the eye by targeting trabecular azines (LINS01 series), to design ligands of H3R/H4R meshwork (TM) cells derived from the ciliary body. using ABT-239 and JNJ-7777120 as prototypes. A set of 9 Trabecular meshwork was isolated under sterile condi- compounds with different substituents in the piperazine N tions to provide a primary TM cell culture. These cells and in 5-position of the dihydrobenzofuran nucleus was were characterized by immunostaining showing positivity synthesized and evaluated for their affinity and functional to alpha-SMA, histaminergic H1,H3 and H4 receptors and activity on H3R and H4R. The preparation of the com- negativity to myosin heavy chain, vimentine and his- pounds was done through classical synthetic methods, taminergic H2 receptors. The expression of aquaporin 1 which resulted in final compounds with good yields. The tested by Western blot analysis and myocillin expression in binding assays in both receptors were performed by com- response to 5 days treatment with 100 nM dexamethasone petition assays using [3H]-histamine. The results show that confirmed that these cells are TM cells. When we tested the the compounds present moderate and low affinities for H3R response of these cells to ciproxifan using a calcium and H4R, with up to 10-fold H3R selectivity. The chloro imaging technique, we observed a modest increase in derivative LINS01007 shows no significant selectivity for intracellular calcium concentration suggesting a direct either H3RorH4R(pKi 6.07 and 6.06 respectively), effect of ciproxifan in IOP regulation targeting these cells. although it displays the highest affinity for H4R in the The peripheral nervous system could therefore repre- series, indicating the importance of the chlorine for H4R sent a new interesting target for H3 receptor antagonists, affinity. Regarding H3R, we conclude that 5-substitution as it was for the treatment of Me´nı`ere’s disease by improves the binding affinity. In addition, the presence of . Histaminergic H3R antagonists represent an the N-allyl group (LINS01004) increases the affinity for interesting new therapeutic option for the treatment of

H3R(pKi 6.40). Functional activity was assessed by Bio- glaucoma. luminescence Resonance Energy Transfer (BRET) assays Department of NEUROFARBA, Section of Pharmacology, to directly evaluate activation of G-protein. All compounds University of Florence, Italy. E-mail: cecilia.lanzi@unifi.it showed no Gi activation and blocked histamine activity, thus acting as antagonists on both H3R and H4R. In sum- mary, our work presents a novel scaffold that can be NON-IMIDAZOLE HISTAMINE H3 RECEPTOR improved to achieve up to 10-fold receptor subtype LIGANDS–IN VITRO SAFETY EVALUATION selectivity. Department of Pharmaceutical Sciences, Federal Univer- G. Latacz, E. Honkisz, M. Kaleta, T. Karcz, A. Siwek, sity of Sa˜o Paulo, Diadema-SP, 09913-030, Brazil. E-mail A. Olejarz, D. Łazewska,_ H. Stark, K. Kiec´-Kononowicz [email protected] Modern drug discovery and drug development process requires not only the pharmacological evaluation, but also A POSSIBLE ROLE FOR CALCIUM IN HISTAMINE evaluation of the ADME-Tox properties of new molecules

H3 RECEPTOR ANTAGONISM ON RABBIT at the earliest stage possible. Toxicity testing is an essential TRABECULAR MESHWORK CELLS part of new chemical entities examination, because insuf- ficient safety profile is one of the main reasons C. Lanzi, L. Lucarini, M. Durante, F. Resta, H. Stark, disqualifying potential drug candidates during preclinical E. Masini and clinical trials. Therefore, evaluation of safety profiles should be performed simultaneously with determination of Glaucoma is the second most prevalent eye condition the pharmacological properties. In our search for histamine known to cause blindness, affecting about 66.8 million H3 receptor ligands among non-imidazole derivatives many people worldwide. Elevated intraocular pressure (IOP) is compounds with nanomolar histamine H3R affinity were the major risk factor for the development of glaucoma; obtained (e.g. 2, 3). For the present study we selected three therefore the reduction of IOP is considered the main- compounds from a new series: E-162, E-163 and E-165 stream of glaucoma therapy. The target for the and examined them in vitro for safety profile. MTS-assay

123 S24 The First Joint Meeting of the European Histamine Research Society was used for the antiproliferative studies against normal be also achieved through the combination of histamine cells HEK-293, hepatoma HepG2 and neuroblastoma IMR- H3R antagonism with e.g. AChE inhibition in a single 32. To predict potential drug–drug interactions (DDI), the molecule). Recently, we have reported inhibitory

H3R ligands were examined to determine their influence on activity against acetylcholinesterase (AChE) and cytochrome CYP3A4. Additional study for evaluation butyrylcholinesterase (BuChE) of histamine H3R hERG channel inhibition potency was performed using a ligands—chlorophenoxy derivatives of homo- or (un)- patch clamp assay. The antiproliferative assays showed no substituted piperidines. These compounds displayed

H3R ligands’ toxicity against HEK-293 cells (IC50 from high to moderate histamine H3R affinity. As a [50 lM), weak neurotoxicity and hepatotoxicity of E-163 continuation of those previous studies we have tested and E-165 (IMR-32 cells IC50 values: 32.32 and selected of our histamine H3R ligands (H3R Ki 35.73 lM; HepG2 cells IC50 values: 38.65 and 25.32 lM, C40 nM) for inhibitory potency against AChE and respectively). The examined H3R ligands also showed very BuChE. Some of evaluated compounds showed inhi- weak CYP3A4 inhibition activity, reaching around 50% of bitory effect on both enzymes in a low micromolar and its activity only at the highest used concentration 25 lM. submicomolar range. Most of them strongly inhibited Regarding hERG channel inhibition study compound E- BuChE than AChE. The most potent in this group was 165 showed similar inhibition potency (IC50 = 1.57 lM) 1-(6-(4-phenylphenoxy)hexyl)homopiperidine inhibiting to the recently approved as a drug H3R ligand the both enzymes (EeAChE IC50 = 2.76 lMand (IC50 = 1.70 lM). EqBuChE IC50 = 0.60 lM). Acknowledgement: Partly supported by National Science Acknowledgement: Partly supported by National Science Center, on the basis of decision No DEC-2011/02/A/NZ4/ Center, on the basis of decision No DEC-2011/02/A/NZ4/ 00031. 00031. Department of Technology and Biotechnology of Drugs, Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical College, Medyczna Street Jagiellonian University Medical College, Medyczna Street 9, 30-688 Krakow, Poland. E-mail: mfkonono@cyf- 9, 30-688 Krakow, Poland. E-mail: dlazewska@cm- kr.edu.pl uj.krakow.pl

ACETYL- AND BUTYRYLCHOLINESTERASE BEHAVIORAL AND NEUROCHEMICAL EFFECTS

INHIBITORY ACTIVITY OF HISTAMINE H3 OF HISTAMINE RECEPTOR 3 KNOCKOUT IN THE RECEPTOR LIGANDS LARVAL ZEBRAFISH

D. Łazewska,_ M. Bajda, S. Hagenow, E. Stawarska, H. A. J. Puttonen, M. Sundvik, Y. Shirai, S. Semenova, P. Zare˛ba, H. Stark, B. Malawska, K. Kiec´-Kononowicz Y.-C. Chen, P. Panula

Alzheimer’s disease (AD) is one of the most common The histamine receptor H3 (hrh3) has been implicated in forms of dementia affecting about 10% of people older many brain processes, such as cognition, feeding behavior than 65 in our society. The etiology of AD is complex and the regulation of sleep. In this study, we first describe and many factors can influence its course and devel- the distribution of hrh3 expression by fluorescent in situ opment. The loss of cognitive function in patients with hybridization in the zebrafish brain. The strongest expres- AD is related mainly to the constant and progressive sion was found in the dorsal pallium, the area analogous to damage of cholinergic transduction in the brain and the mammalian neocortex, where hrh3 receptor was lowering the levels of acetylcholine. Histamine H3 expressed mainly in glutamatergic neurons. Hrh3 expres- receptors (H3R) are predominantly found inthe central sion was also found in gabaergic and histaminergic neurons nervous system, where they inhibit release of certain in the diencephalon, however, no coexpression was neurotransmitters including acetylcholine. Many pre- observed with other populations. Secondly, clinical and clinical studies confirm the efficacy of we created a hrh3 knockout zebrafish using the CRISPR/ certain ligands of these receptors in the treatment of Cas9 system. Hrh3 knockout zebrafish showed a shorter cognitive impairments, which are associated with neu- period of hyperactivity after onset of darkness, indicating a rodegenerative disorders. The effectiveness of several faster adaptation to darkness (p \ 0.05, n = 30–32 per compounds has been investigated in clinical studies in group). The histamine dependent dark-induced flash patients with AD e.g. ABT-288 (Abbott), GSK-239512 response was intact, suggesting that this response is not (GlaxoSmithKline) or MK-3134 (Merck). Enhanced mediated by hrh3. Neurochemically, the hrh3 knockout cholinergic neurotransmission in the brain cortex can zebrafish had decreased levels of serotonin and dopamine

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(p \ 0.05 and p \ 0.01, respectively, n = 4–5), with evidence of the relevance of hrh3 signalling in the regu- noradrenaline levels being unchanged. The morphology of lation of monoaminergic neurotransmission. As serotonin the dopaminergic and serotonergic networks in the brain is involved in the regulation of dark-avoidance behavior in was unchanged. As no changes were seen in the expression the zebrafish, the faster adaptation to darkness might be of key genes in monoaminergic neurotransmission, the caused by decreased levels of serotonin. decreased neurotransmitter levels most likely reflect post- Department of Anatomy, POB 63 (Haartmaninkatu 8), transcriptional functional changes in the monoaminergic 00014 University of Helsinki, Finland E-mail: henri. neurons following hrh3 knockout, providing further puttonen@helsinki.fi

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Histamine H4 receptor

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APPROACHES TO UNDERSTANDING BIAS COMPARISON OF PHARMACOLOGICAL

SIGNALLING OF THE H4-HISTAMINE RECEPTOR ACTIVITY OF HYDROCHLORIDE SALT AND FREE BASE OF PUTATIVE HISTAMINE H4 A. B. Tobin, S. Riaz LIGANDS

It is now clear that ligands to GPCRs can have the M. Adami, C. Micheloni, D. Grandi, H. Stark, D. Łazewska,_ capacity to drive signalling preferentially down one K. Kiec´-Kononowicz signalling pathway in preference to others. These so called biased ligands hold the promise that novel drugs The histamine H4 receptor (H4R) is an increasing attractive can be developed that target signalling pathways with drug target for inflammatory disorders and within the therapeutic value in preference to signalling that drive growing number of H4R ligands there is a large group of adverse responses. A significant barrier to exploiting the azine derivatives. In the present study, the putative anti- full potential of biased ligands is the fact that we cur- inflammatory activity of base JN-35 (4-(4-methylpiperazin- rently do not know which GPCR-pathways are 1-yl)-1,3,5-triazine derivative), its hydrochloride salt (JN- responsible for clinical efficacy and which are mediators 35xHCl) and the hydrochloride H4R antagonist of adverse responses. We have addressed this issue by JNJ7777120xHCl, as well as their effect on gastric ulcer- generating genetically engineered mice that express ation, were evaluated for possible differences in receptors that are mutated so that they are uncoupled pharmacological activity. Compounds were tested in the from one signalling pathway but remained normally mouse ear edema assay after challenge with local appli- coupled to others. Such mice can be used to generate a cation of 2.5% croton oil (CO). Ear weight and histological map of the physiological responses that lay downstream tissue damage were investigated 2 h after CO application; of particular signalling pathways. We have developed CO-induced itch was evaluated 1 h afterwards. CO-in- this approach by conducting studies on the prototypical duced ear edema was significantly reduced by JN-35 acetylcholine M3 muscarinic receptor (M3-receptor). We (50 mg/kg subcutaneously, sc), JN-35xHCl (50 mg/kg sc) firstly identified the sites of receptor phosphorylation or JNJ7777120xHCl (30 mg/kg sc) (D mg/2 h: which were then mutated to alanine residues. This gen- 29.18 ± 2.12, 4.74 ± 2.39, 8.55 ± 1.58 and 4.88 ± 1.63 erated a receptor that was uncoupled from receptor for vehicle, JN-35, JN-35xHCl and JNJ7777120xHCl, phosphorylation/arrestin-dependent signalling but that respectively, P \ 0.01). The anti-inflammatory effect of remained normally coupled to Gq/11-calcium mobilisa- JN-35 was confirmed by histological evaluation of ear tion. By engineering a mouse strain expressing the tissues. At anti-inflammatory doses, both JN-35 and JN- phosphorylation deficient mutant of the M3-receptor we 35xHCl displayed anti-pruritic effect (D scratching bouts/ have been able for the first time to define a map of M3- 1 h: 152.80 ± 18.95, 14.40 ± 5.34 and 61.14 ± 19.73 for receptor mediated physiological responses that are vehicle, JN-35 and JN-35xHCl, respectively, P \ 0.01); by downstream of receptor phosphorylation/arrestin sig- contrast, JNJ7777120xHCl was ineffective. Gastric lesions nalling and those downstream of Gq/11 dependent were evaluated in mice 4 h after indomethacin signalling. We are now extending this approach to other (IND) + bethanechol (BET). Conversely from JN-35xHCl receptor classes including the histamine H4 receptor. In and JNJ7777120xHCl, base JN-35 (50 mg/kg sc) inhibited this regard we have conducted the first mass spectrom- IND + BET-induced gastric damage (lesion index: etry based analysis of the phosphorylation sites on the 68.07 ± 12.53 and 33.57 ± 7.02 mm for vehicle and JN-

H4 receptor identifying at least 13 serine/threonine sites 35, respectively, P \ 0.05). In conclusion, present data of phosphorylation. Using both non-biased (histamine) show that JN-35, its hydrochloride salt and the and supposedly biased (JNJ7777120) agonists to H4 hydrochloride H4R antagonist JNJ7777120xHCl displayed receptors are in the process of assessing the changes in anti-inflammatory activity. However, differently from phosphorylation of the H4 receptor and relating these JNJ7777120xHCl, only JN-35 and JN-35xHCl inhibited changes to signalling responses in a programme aimed at CO-induced itch. The anti-inflammatory and anti-pruritic the determination of the relevance of biased signalling to activities of JN-35, combined with its gastroprotective

H4 receptor function. effect, would be of the utmost importance when consid- The Centre for Translation Pharmacology, University of ering that most of the therapeutically available anti- Glasdgow, Glasgow G12 8QQ, UK. E-mail: Andrew. inflammatory drugs are still endowed with gastrointestinal [email protected] toxicity. This study was partly supported by National

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Science Center, based on decision No DEC-2011/02/A/ DIGESTING THE HISTAMINE H4 RECEPTOR IN NZ4/00031. INTESTINAL DISEASES Department of Medicine and Surgery, University of Parma, 43125 Parma, Italy. E-mail: [email protected] A. D. Kraneveld, P. J. Koelink, G. Folkerts, J. Garssen

Substantial evidence implicates histamine in the patho-

INTRA-VERMIS H4 RECEPTOR AGONIST genesis of intestinal disorders ranging from inflammatory IMPAIRS EMOTIONAL MEMORY bowel disease (IBD), irritable bowel syndrome (IBS), and CONSOLIDATION IN ANXIETY- AND food allergy to intestinal cancer. Besides histaminergic FEAR-MEDIATED MODELS neurons, basophils and the microbiota, the intestinal mast cell is a prominent source of histamine. Histamine exerts C. E. M. Fernandes, K. R. Serafim, A. C. L. Gianlorenco, its effects on various cell types through four known R. Mattioli G-protein coupled receptors (H1R, H2R, H3R and H4R). H4R was the most recently-discovered histamine receptor Neural histaminergic system modulates cognitive perfor- (in 2000) and has been, since its discovery, one of the most mance in various animal models. However, little is known actively-investigated targets in chronic inflammatory dis- regarding the effects of H4 histamine receptors in the eases. The deregulated mucosal immune response, in central nervous system. The purpose of this study was to which both genetic and environmental factors play a role, investigate the effect of histamine H4 agonist VUF-8430 is believed to play an important role in the above-men- microinjection into cerebellar vermis in the consolidation tioned disorders. The histamine H4 receptor is expressed on of emotional memory in mice submitted to the elevated cells of the immune system (such as mast cells, eosino- plus maze (EPM) and to the inhibitory avoidance task phils, basophils, dendritic cells and T cells) but also on (IAT). On the fourth day after surgical recovery, the ani- endocrine cells and enteric neurons. Activation of the mals received saline (SAL) or VUF (0.15 nmol; 0.49 nmol; histamine H4 receptor induces chemotaxis of mast cells and 1.48 nmol/0.1 ll) administered post-exposure. All experi- granulocytes and activation of lymphocytes and neurons. ments were performed on two consecutive days: Exposure Functional studies using selective and non-selective his-

(T1 and D1) and 24 h after, the re-exposure (T2 and D2). tamine H4 receptor ligands have rapidly increased the Experiment 1 was held in the EPM and the animals were knowledge of H4 receptor involvement in intestinal pro- free to explore the maze for 5 min; in T1, immediately cesses in models of intestinal diseases. Evidence points after exposure, the pharmacological treatment occurred; in towards a role for histamine H4 receptors in the modulation T2, there were only re-exposure to EPM. Experiment 2 was of immune-mediated responses and visceral pain in held on the IAT and the pharmacological treatment was intestinal inflammatory disorders. Moreover, histamine H4 post-D1; in D2, the animals were only re-exposed to the receptors are implicated in gastrointestinal carcinogenesis.

IAT. The data were analyzed using two-way repeated Taken together, the histamine H4 receptor is an interesting measures ANOVA. Differences indicated by significant F target for pharmacological modulation of a disturbed mucosal values were further verified by post hoc Duncan’s multiple immune system, associated inflammation, visceral pain and range tests. In all cases, p \ 0.05 was considered to be malignancies in a broad range of intestinal disorders. significant. In Experiment 1, an increased open arm Division of Pharmacology, Utrecht Institute for Pharma- exploration (% open arms time) for VUF 1.48 nmol was ceutical Sciences, Faculty of Sciences, Utrecht University, determined in T2 compared to T1. In Experiment 2, a The Netherlands. E-mail: [email protected] significant decrease of consolidation latency in relation to D2 for the group VUF 1.48 nmol compared to SAL group was revealed. These results indicate that VUF 1.48 nmol PRECLINICAL MODELS TRANSLATIONALLY microinjection into the cerebellar vermis impairs emotional PREDICT CLINICAL PK/PD RELATIONSHIPS memory consolidation in both models, one to anxiety- OF THE ANTI-VERTIGO DRUG SENS-111 (EPM) and the other to fear-mediated (IAT). Laboratory of Neuroscience, Physiotherapy Department, J. Dyhrfjeld-Johnsen, E. Wersinger, M. Petremann, Federal University of Sao Carlos, Brazil. E-mail: V. Brieuc, D. Challuau, C. Gueguen, S. Bressieux, [email protected] C. Tran Van Ba, P. Attali

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The treatment of vertigo and dizziness of peripheral We have previously demonstrated that histamine induces vestibular origin is currently limited to a few older the epithelial-mesenchymal transition (EMT) program by vestibular suppressants, and off-label use of sedatives and stimulating the histamine H4 receptor (H4R) in the human anti-emetics potentially interfering with long-term recov- mammary carcinoma cell line MDA-MB-231. The H4R ery. The recent discovery of significant reduction of ligands increase the expression of the mesenchymal marker peripheral vestibular neuronal activity in vitro by H4R vimentin and the nuclear expression of b-catenin and Slug antagonists suggests their potential therapeutic use for transcription factor, and also cell migration. All these EMT symptomatic treatment of peripheral vestibular imbalance. related events were prevented by Src inhibitors. Src is a Our mechanism based rat model of vestibular injury, non receptor tyrosine kinase, which is tightly involved in centred on excitotoxic peripheral insult, generates a num- cell migration. In addition, TGF-b 1 is a recognized ber of established, vertigo associated symptoms such as inducer of EMT, a critical process for invasion and spontaneous nystagmus, postural deviations and gastric metastasis. paresis. Using a combination of preclinical and clinical The aim of this work was to investigate whether the pharmacodynamic and pharmacokinetic approaches, we EMT induced by H4R ligands could be mediated by TGF-b demonstrate a clear correspondence between preclinical type I receptor activation. prediction of effective drug concentration and clinical All experiments were performed in RPMI medium outcome for the H4 R receptor antagonist, SENS-111, without fetal bovine serum. MDA-MB-231 cells treated analyzed using PK/PD modeling. with H4R ligands and/or the TGF-b type I receptor kinase Preclinical experiments using iv SENS-111 administra- inhibitor A83-01 (10 lM) were incubated for 24 h. Indirect tion (10/20/40 mg/kg) 1 h after induction of acute vertigo immunofluorescence and Western blot studies confirmed demonstrated a dose/efficacy relationship with an optimal the presence of cytoplasmic TGF-b 1 in cells treated with dose of 10 mg/kg corresponding to symptom reduction of 10 lM of the H4R ligands VUF8430 or clobenpropit. TGF- 20–30%, while doses of 20–40 mg/kg resulted in a loss of b 1 levels were not increased when cells received A83- efficacy. For the effective dose of 10 mg/kg SENS-111, 01 + H4R ligands. blood plasma concentrations did not exceed 500 ng/mL The H4R ligand-mediated increase in vimentin and Slug while the non-effective dose of 20 mg/kg reached levels as expression was also blocked by pre-treatment with A83-01 high as 1000 ng/mL. (p \ 0.05). In agreement, immunofluorescence studies did In a phase 1b multiple ascending dose study, the effect not reveal nuclear localization of b-catenin and Slug in of SENS-111 on vertigo parameters following modified cells in the presence of the combined treatment. Migration caloric irrigation was determined in healthy volunteers in studies carried out by filming cells in transwell inserts parallel with pharmacokinetic parameters. PK/PD model- showed an enhancement in migratory capacity induced by ing of drug exposure levels and pharmacodynamic effects clobenpropit or VUF8430, which was blocked in the on vertigo measures determined that positive treatment presence of A83-01 (p \ 0.05). effects of 20–30% were also restricted to exposures Src phosphorylation, evaluated by Western blotting, was B500 ng/mL while exposures [500 ng/mL led to a loss of increased in H4R ligand-treated cells as previously reported. efficacy on vertigo outcome measures. Interestingly this rise was prevented by A83-01 (p \ 0.05). The observed clinical and preclinical correspondence of Our results suggest that EMT induced by H4R ligands in PK/PD relationships strengthens the rationale for dose MDA-MB-231 breast cancer cells may be mediated by selection and clinical trial design in upcoming phase 2/3 signaling through the TGF-b type I receptor that involves trials. Src phosphorylation. Sensorion, Montpellier, France. E-mail: jonas.dyhrfjeld- Universidad de Buenos Aires, Facultad de Farmacia y [email protected] Bioquı´mica, Ca´tedra de Fı´sica, Laboratorio de Radioiso´topos, Argentina. E-mail: [email protected]

TGFb TYPE I RECEPTOR AS A POSSIBLE MEDIATOR OF THE EPITHELIAL TO 2,4-DIAMINOPYRIMIDINES: TOWARDS MESENCHYMAL TRANSITION INDUCED BY MOLECULAR TOOLS FOR INVESTIGATIONS

HISTAMINE H4 RECEPTOR LIGANDS IN MDA- ON HUMAN AND RODENT HISTAMINE H4 MB-231 BREAST CANCER CELLS RECEPTORS

M. A. Ta´quez Delgado, T. E. Galarza, N. A. Mohamad, E. Bartole, T. Littmann, G. Bernhardt, T. Ozawa, G. M. Vedoya, V. A. Medina, G. A. Martı´n, G. P. Cricco A. Buschauer

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For investigations on the (patho)physiological role of the stably expressing the human H4 receptor, the mouse H4 histamine H4 receptor, high affinity ligands, antagonists as receptor or the rat H4 receptor. Additionally, the compounds well as agonists, are required. Comparative in vitro studies were investigated in luciferase reporter gene and b-arrestin on human, mouse and rat histamine H4 receptors are ham- recruitment assays, using HEK293T cells stably expressing pered by the lack of universal molecular tools, in particular, the respective H4 receptor orthologues. Binding affinities radiolabeled and fluorescent ligands, binding with similar were in the one- to three digit nM range at the human H4 affinities to the receptor orthologues across species. In view receptor. The spinaceamine derivative was a partial to full of the need for a high affinity radioligand addressing the agonist with potencies in the subnanomolar range across the rodent H4Rs, we explored the structure–activity- and struc- orthologues in the reporter gene assay. The introduction of ture-selectivity relationship of a set of 2,4- (R)-N,N-dimethylpyrrolidin-3-amine gave the ‘‘cold’’ form diaminopyrimidines on the human and rodent H4 receptors. of a potential radioligand with partial to full agonistic Inspired by a report on (R)-4-(3-aminopyrrolidin-1-yl)-N- potencies in the one- to two-digit nM range at the H4 neopentylpyrimidin-2-amine as a partial to full agonist, receptor orthologues (proximal readout: [35S]GTPcS, b-ar- equipotent at human, rat and mouse H4 receptors, we restin assay). Therefore, radioligands for H4 receptor binding explored structural variations in position 4, focusing on studies across species should be accessible from an opti- aliphatic amines and their guanidinylated and methylated mized diaminopyrimidine. derivatives (e.g. (R)-N,N-dimethylpyrrolidin-3-amine) as Department of Medicinal Chemistry II, University of well as substructures derived from histamine (e.g. spina- Regensburg, Germany. E-mail: [email protected] ceamine). Radioligand binding and [35S]GTPcS assays were regensburg.de performed, using membrane preparations of Sf9 insect cells

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Molecular, genetic and chemical aspects of histamine, its receptors and enzymes

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NEW BIOIMAGING APPROACHES TO STUDY TOOLS FOR THE GENETIC ANALYSIS OF

HISTAMINE H1-RECEPTOR PHARMACOLOGY HISTAMINE RECEPTORS

S. J. Hill, L. Stoddart, M. Bouzo Lorenzo, S. J. Briddon A. Sasse, S. Micallef

A full understanding of the kinetics of ligand–protein inter- Genetic analysis identifies the genetic make-up of a par- actions is important in the design of new drugs and in ticular area of the genome. Variations in the genomic DNA delineating their mode of action in vivo. For G protein- may lead to inherited diseases, as certain proteins may be coupled receptors the techniques that are often used to up- or downregulated, may have higher or lower activity; determine ligand binding kinetics use cell membranes or or functional variants may not be expressed. There are remove the receptor from its native membrane environment many potential variations going beyond simple SNPs entirely. It is not known, however, if kinetic parameters (single-nucleotide polymorphisms), for example VNTR measured in membranes are the same as those measured in a (variable number of tandem repeats), TRP (tri-nucleotide whole cell environment. To address this we have designed repeats), SSR (short-sequence repeats), microsatellites and

fluorescent histamine H1-receptor (H1R) ligands to investi- CNV (copy-number variations). DNA fingerprinting is a gate ligand binding in living cells. Furthermore the use of versatile method for analysing a person’s genome. The these fluorescent ligands in combination with a novel bio- method is based on variations found in minisatellites, luminescent protein (NanoLuc) can provide a novel which are 10–60 bp long regions of genomic DNA that bioluminescence resonance energy transfer (BRET) tech- have a particularly high variability. Pharmacogenomics is nique (NanoBRET; 1). The technique uses Nanoluc attached the study of how genes affect a person’s response to drugs to the N-terminus of the H1R as the energy donor and a and there are many examples from current clinical practice. fluorescently tagged ligand as the acceptor. Using the his- Personalised treatment or ‘Precision Medicine’ is very tamine H1R as a model system we have determined kinetic much a focal point of translational pharmacologists, pro- parameters for labelled and unlabelled ligands in living cells. viding a ‘bench to bedside and back to be bench’ approach

cDNA encoding the human histamine H1 receptor was in drug discovery. Methodologies in genetic analysis will amplified and fused in frame into a vector containing Nluc to be discussed in this presentation, such as HapMap and generate Nluc-H1 and a HEK293 cell line stably expressing 1000genomes, as well as bioinformatic approaches such as this construct generated. For kinetic studies, cells grown to LD (linkage disequilibrium) and GWAS (genome–wide confluence or cell membranes were incubated with the Nluc association studies). Pharmacogenetics of the histamine substrate (10 lM furimazine) for 10 min prior to the addi- receptors, both at the bench and at the bedside, will be used tion of the fluorescent ligand in the presence or absence of to illustrate these valuable tools of analyses. unlabelled ligands. Non-specific binding was determined in School of Pharmacy and Pharmaceutical Sciences, Trinity the presence of 10 lM doxepin. Luminescence and the Biomedical Sciences Institute, Trinity College Dublin, resulting BRET was measured every minute for 60 min University of Dublin, Dublin 2, Ireland. E-mail: using the PHERAstar FS plate reader (BMG labtech). Fil- [email protected]. tered light emissions were measured at 460 nm (80 nm bandpass) and [610 nm (longpass). Using the NanoBRET assay, the kinetic parameters of the fluorescent antagonist HISTAMINE DYSREGULATION IN mepyramine-alanine-alanine-BODIPY630/650 (mep-AA- TOURETTE SYNDROME AND RELATED

BY630) at the H1R was determined in whole cells with low NEUROPSYCHIATRIC CONDITIONS: STUDIES levels of non-specific binding. When using membranes, both IN MICE AND MEN a physiological buffer (HEPES-buffered saline solution) and a traditional membrane binding buffer was used and there C. Pittenger was no difference in the kinetic and affinity values obtained.

During this presentation we described how fluorescent H1R While the normal functions of histamine (HA) in the central ligands can be used to investigate the pharmacology of the nervous system have gradually come into focus over the past

H1R in living cells. 30 years, the relationship of abnormalities in neurotransmitter This work has received support from the EU/EFPIA HA to human disease has been slower to emerge. New insight Innovative Medicines Initiative Joint Undertaking, K4DD came with the 2010 description of a rare nonsense mutation in grant n° 115366. the biosynthetic enzyme histidine decarboxylase (Hdc) that Cell Signalling Research Group. School of Life Science, was associated with Tourette syndrome (TS) and related University of Nottingham, Nottingham, UK. E-mail: conditions in a single family pedigree. Subsequent genetic [email protected]. work has provided further support for abnormalities of HA

123 The First Joint Meeting of the European Histamine Research Society S35 signaling in sporadic TS. As a result of this genetic work, Hdc is involved in. The interrogation of our network models for knockout mice, which were generated more than 15 years the molecular details of the implication of histamine in a ago, have been re-examined as a model of the pathophysiol- complex disease such as diabetes, suggests hypotheses ogy of TS and related conditions. Parallel work in these KO about the role of histamine and glycation end products in mice and in human carriers of the Hdc mutation has revealed diabetic complications, and the relationship between dia- abnormalities in the basal ganglia system and its modulation betes and cancer. by dopamine (DA) and has confirmed the etiologic, face, and Department of Molecular Biology and Biochemistry. predictive validity of the model. The Hdc-KO model thus Universidad de Ma´laga, 29071 Ma´laga Spain. E-mail: serves as a unique platform to probe the pathophysiology of [email protected] TS and related conditions, and to generate specific hypotheses for subsequent testing in humans. This presentation will summarize the development and validation of this model and SPLIT-LUCIFERASE COMPLEMENTATION recent and ongoing work using it to further investigate ENABLES QUANTIFICATION OF Gaq pathophysiological changes that may contribute to these ACTIVATION AND LIVE CELL IMAGING disorders. Departments of Psychiatry and Psychology, Child Study T. Littmann, T. Ozawa, C. Hoffmann, G. Bernhardt, Center, and Interdepartmental Neurosciecne Program, A. Buschauer Yale University School of Medicine, New Haven, CT, USA. E-mail: [email protected] Aiming at the combined detection of alternate signal transduction pathways by optical methods and spatiotem- poral analyses, here we report on the application of a NETWORK PERSPECTIVE OF HISTAMINE technique that uses split-luciferase complementation to RELATED DISEASES quantify the activation of Gaq proteins, for example via the histamine H1 receptor (H1R), in whole cells. A red light- A. A. Moya-Garcia, R. Rodrı´guez-Lo´pez, M. A´ . Medina, emitting luciferase from the click beetle Pyrophorus pla- F. Sa´nchez-Jime´nez giophthalamus (CBR) was dissected into two fragments, capable of combining to form a catalytically active Histamine is the most pleiotropic biogenic amine. Pro- enzyme. On the one hand, the C-terminal fragment of CBR duced and stored by a limited set of cells—histaminergic was utilized to create several different fusion proteins with neurons, enterochromaffin-like cells, and mast cells—it Gaq. On the other hand, the N-terminal part of CBR was broadcasts intercellular communication signals to a wide fused to the phospholipase C-b3. Two Gaq variants with variety of cell types through its tissue-specific receptors. CBR tags inserted in loop regions showed highest signal- The many molecular interactions of these receptors and to-background ratios, when stimulated via the H1R. Fur- other mediators result in complex cellular networks whose thermore, both variants allow for the quantification of alteration result in disease. Therefore, complex diseases agonistic and antagonistic activities not only at the H1R, map to modules of these cellular networks in the but also at the muscarinic M3 receptor, yielding pharma- diseasomes. cological values in good agreement with published data In this communication, we survey the histamine cellular obtained in different assays. In addition, we demonstrated networks to map the histamine diseasome, presenting a that the interaction of the two fusion proteins is fully network view of the pleiotropy of histamine and its role in reversible. several complex diseases. Furthermore, one of the variants showed exceptionally We found 87 genes modulated by histamine through high signal-to-background ratios, enabling microscopic literature mining queries with the tools CTDBASE and live cell imaging. PhenUMA. These genes form a connected subnetwork in The presented approach to the quantification of Gaq the human interactome extracted from the STRING data- activation via split-luciferase complementation is a first base, i.e. histamine functional network. step towards spatiotemporal investigations of Gaq signal- The histamine functional network map to more than 200 ing and harbors a potential with regard to multiparametric diseases as defined in the diseasome developed by Menche analyses of cellular signaling networks. and Barabasi. These diseases group in 12 categories with Department of Medicinal Chemistry II, University of similar aetiologies and underlying molecular causes, which Regensburg, Regensburg, Germany. E-mail: provide a richer, expanded view of the diseases histamine [email protected]

123 S36 The First Joint Meeting of the European Histamine Research Society

(-)MAACKIAIN, AN ACTIVE COMPOUND FROM laboratory comprises expression of target transporters in KUJIN, ALLEVIATES BOTH HISTAMINE H1 insect cells or Escherichia coli, purification and recon- RECEPTOR AND INTETLEUKIN-33 GENE stitution into liposomes. The resultant proteoliposomes EXPRESSIONS enable us to quantify the transport activity upon imposing any type of artificial driving forces. Using this H. Fukui, H. Mizuguchi, Y. Kitamura, H. Nemoto, approach, we found that SLC18B1 protein transports not Y. Kashiwada, N. Takeda. only monoamines but also polyamines such as spermine (SPM) and spermidine (SPD) through H+ substrate Antihistamines are major therapeutics for pollinosis and antiport at the expense of an electrochemical H+ gra- other allergic diseases. However, they are not potent to dient established by vacuolar H+-ATPase. SLC18B1 alleviate chronic symptoms, and the development of novel protein is associated with neuronal synaptic vesicles and therapeutics is highly expected. Eosinophils play an secretory granules in neuroendocrine cells and is important role in chronic symptoms. There are pollinosis involved in vesicular storage and release of polyamines. patients with eosinophilia. We discovered the levels of Thus, SLC18B1 protein is regarded as a vesicular eosinophils were correlatively elevated with nasal mucosal polyamine transporter (VPAT). Presence of VPAT interleukin (IL)-33 mRNA levels in pollinosis patients, defines polyaminergic chemical transmission and suggesting that the activation of IL-33 gene expression explains the mechanism how SPM and SPD are released participates in eosinophilia. We satisfactorily observed that through exocytosis to get access to the extracellular- IL-33 gene expression was activated with protein kinase C exposing specific binding site(s) on the NMDA or (PKC)-activating phorbol ester (PMA), and the activation AMPA-type receptors, modulating glutamatergic chem- was suppressed with inhibitors of PKC-delta isoform. ical transmission. VPAT is also expressed in various Previously, we demonstrated that histamine H1 receptor polyaminergic cells. In mast cells, VPAT is associated (H1R) gene expression is suppressed with inhibitors of with a new polulation of secretory granules and involves PKC-delta isoform. Then PKC-delta inhibiting herbal vesicular release of SPM and SPD to regulate secretion medicines are suggested to alleviate not only H1R gene of histamine. It is concluded that SLC18 members may expression-dependent but also IL-33 gene expression/ fulfill vesicular storage of essentially all ranges of bio- eosinophil-dependent allergic symptoms. genic amines and acetylcholine, and the physiological Institute of Biomedical Sciences, Tokushima University relevance of SLC18 family is wider and more complex Graduate School, Tokuchima, Japan. E-mail: hfukui@ than that so far understood. tokushima-u.ac.jp Departments of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmacetical Sciences, and of Biochemistry, Matsumoto VESICULAR AMINE TRANSPORTERS AND Dental University, Japan. E-mail: moriya-y@okayama- HISTAMINE ACTION u.ac.jp

Y. Moriyama WIDE VARIETY EXPRESSION OF VESICULAR Solute carrier type 18 transporter family (SLC18) con- POLYAMINE TRANSPORTER (VPAT) tains three members, A1, A2 and A3 that encodes vesicular monoamine transporter 1 (VMAT1), vesicular S. Moriyama, Y. Harada, T. Takeuchi, N. Ko, S. Tanaka, monoamine transporter 2 (VMAT2) and vesicular Y. Moriyama, M. Hiasa acetylcholine transporter (VAchT), respectively. VMATs and VAchT transport a wide variety of Vesicular polyamine transporter, the fourth member of monoamines and acetylcholine, and are responsible for solute carrier type 18 transporter family (SLC18), is vesicular storage and subsequent release of monoamines responsible for vesicular storage of polyamines (sper- and acetylcholine as chemical transmitters. Recent midine and spermine) at the expense of an electrochemical studies identified two additional SLC18 members, gradient of protons across the membranes, which is SLC18A4, an insect specific isoform, and SLC18B1, an established by vacuolar proton ATPase. In brain, VPAT is isoform ubiquitously present in almost all animals. A present in neuronal synaptic vesicles and secretory vesicles clean biochemical approach established in my in astrocytes, and responsible for vesicular storage and

123 The First Joint Meeting of the European Histamine Research Society S37 release of polyamines. Consequently, the polyamines may recombineering was used to generate mutants with pre- act as neurotransmitters and gliotransmitters to modulate dicted functional effects in EriC and EriC2. In a histamine the glutamatergic chemical transmission. Because VPAT production assay, 8 of 9 mutant strains produced approxi- mRNA distributes through the body, in the present study, mately half as much histamine as the wild type (WT) we investigated expression and functions of VPAT in strain. Expression of hdcA was unchanged, or slightly various organs of rodents. We found that VPAT is highly increased relative to WT in mutant strains. Membrane expressed and associated with a population of secretory potential was evaluated in each mutant using a fluorescent granules other than histamine granules in mast cells. RNAi dye aggregation assay. Certain mutants carried weaker of SLC18B1 gene resulted in decreased vesicular uptake of membrane potentials than the WT strain. Differences in polyamines followed by release of polyamines from mast intracellular pH were observed across EriC/EriC2 mutants cells. In the retina, VPAT is present in photoreceptor cells, using a pH-sensitive fluorophore. In the WT strain, we bipolar cells, astrocytes and Mueller cells. VPAT is also observed histamine production varying directly with expressed in, indicating vesicular storage and release of extracellular acidity, with the most histamine being pro- spermines from mouse must cells. In mouse retina, VPAT duced at the lowest pH over the range pH 4.6–7.6. These is expressed in the type II alveolar epithelial cells in lung, data establish a regulatory role for EriC-family proton/ collecting ducts and glomerulus in kidney and bile chloride antiporters in the expression and function of his- canaliculi. The highest expression was observed in the tamine production machinery. Mechanistically, these bladder epithelium. Thus, VPAT is expressed in wide transporters can affect membrane potential and may alter variety of cells, and may be involved in vesicular storage the internal pH of the bacteria. In the gut, pH and chloride and release of polyamines in these cells. gradients may alter histamine production, and thus the Department of Membrane Biochemistry, Okayama efficacy of L. reuteri as an anti-inflammatory probiotic. University Graduate School of Medicine, Dentistry and Department of Molecular Virology and Microbiology, Pharmaceutical Sciences, Okayama, Japan. E-mail: Baylor College of Medicine, Houston, TX, USA, 2Depart- [email protected] ment of Pathology, Texas Children’s Hospital, Houston, TX, USA. E-mail: [email protected]

HISTAMINE PRODUCTION BY LACTOBACILLUS REUTERI CAN BE REGULATED BY ERIC-FAMILY SURFACTANT-INDUCED INTRAEPIDERMAL PROTON/CHLORIDE ANTIPORTERS NERVE FIBER ELONGATION IS ATTENUATED BY HISTIDINE DECARBOXYLASE DEFICIENCY A. Hall, J. Versalovic Y. Inami, A. Sato, H. Ohtsu, Y. Mano, Y. Kuraishi, T. Andoh Histamine produced by the human commensal Lacto- bacillus reuteri ATCC PTA 6475 reduces inflammation Sodium dodecyl sulphate (SDS) is a synthetic anionic and tissue damage in models of inflammatory bowel dis- surfactant commonly found in laundry detergents, dish ease (IBD). Histamine generation and secretion by soaps and shampoos. Anionic surfactants are frequently histidine decarboxylase (HdcA) and its coupled histidine/ used as the main components of detergents because of their histamine exchanger (HdcP) consumes protons, alkaliniz- high detergency. In daily life, the repeated use of deter- ing the cell interior and generating an inside-negative gents is possible to cause cutaneous sensory membrane potential. Neutral-to-alkaline pH and highly hypersensitivity reactions characterized by itching and negative membrane potential have been shown to suppress eczema on the skin and scalp. However, the mechanisms the activities of HdcA and HdcP in other lactic acid bac- underlying cutaneous sensory hypersensitivity induced by teria. However, it is unknown how L. reuteri balances its detergents remains unclear. Our previous report has shown internal ion environment in order to sustain histamine that repeated applications of SDS to mouse skin increase production. Two proton/chloride antiporters, EriC and the density of epidermal nerve fibers in accompany with EriC2, have been identified in L. reuteri as potential reg- the increase of the expression of histamine-synthesizing ulators of intracellular pH and membrane potential, and enzyme histidine decarboxylase (HDC) and the concen- thus histamine synthesis. To investigate how EriC-family tration of histamine in the epidermis. These findings ion transport might alter expression and function of the suggest that histamine may contribute to the increase of histamine production machinery in L. reuteri, single-strand epidermal nerve fibers. In this study, we used ICR mice

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(naı¨ve mice), HDC knockout mice and HDC inhibitor used. Histamine increased the total length and the maxi- (histidine methyl ester dihydrochloride, HME) to investi- mum length of each neurite in small-sized, but not large- gate the role of histamine on SDS-induced nerve fiber sized, DRG neurons. Therefore, it is suggested that his- elongation into the epidermis. The repeated application of tamine plays an important role in intraepidermal nerve fiber SDS increased the density of epidermal nerve fibers in wild elongation in murine skin treated repeatedly with SDS. type mice and ICR mice, which was significantly decreased Department of Applied Pharmacology, University of by HDC deficiency and HME, respectively. To determine Toyama, Toyama, Japan. E-mail: [email protected] the effect of histamine on neurite outgrowth, primary cul- toyama.ac.jp tures of mouse dorsal root ganglion (DRG) neurons were

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Histamine in the cardiovascular and gastro-intestinal systems, plus growth

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HISTAMINE AND DIACYLGLYCEROL KINASE BY the intestinal mucosa. Interestingly, mild inflammation in the LACTOBACILLUS REUTERI MEDIATED intestine by 1% DSS reduced the CD11b+ MHCIIhiCD45lo CROSSTALK BETWEEN MAMMALIAN activated microglia in brain and this was reversed by INTESTINAL EPITHELIUM AND IMMUNE CELL administration of WT L. reuteri in mice compared to mutant MATURATION L. reuteri treated mice. These findings provide a deeper mechanistic understanding of intestinal immunomodulatory B. P. Ganesh, A. Hall, S. Ayyaswamy, R. Fultz, J. Nelson, probiotics and microbiome: host interactions. A. Major, M. Whary, J. Fox, J. Versalovic The presenter of this abstract was the runner-up of the Young Investigator Award (2017). The probiotic bacterium, Lactobacillus reuteri ATCC PTA Department of Pathology and Immunology, Baylor College 6475, with an intact hdc gene cluster is known to synthesize of Medicin and Department of Pathology, Texas Children’s and secrete histamine and can suppress inflammation in Hospital, Houston, Texas, USA. E-mail: Bhanu. mammalian systems via specific activation of the type 2 [email protected] histamine receptor (H2R). Gut microbes such as L. reuteri 6475 promote H2R signaling and may suppress H1R pro- inflammatory signaling pathways in parallel by unknown BRAIN HISTAMINE AND THE GUT-BRAIN AXIS mechanisms. It is unclear if L. reuteri-derived histamine also interacts with H1R in the intestinal epithelium. In this work, M. B. Passani we identified a soluble secreted isoform of diacylglycerol kinase (DGK) from L. reuteri 6475. DGKs belong to a dis- The central nervous system and viscera constitute a func- tinct, conserved family of intracellular lipid kinases that tional ensemble, the gut-brain axis, that allows phosphorylate diacylglycerol (DAG), catalyzing conversion bidirectional information flow that contributes to the con- of DAG to phosphatidic acid (PA). This reaction may trol of feeding behaviour, appetite, memory, and the diminish DAG quantities in the cell membrane, possibly response to stress and pain. Recent research on the gut- modifying host intracellular signaling downstream of DAG. brain axis has revealed the contributions of extensive Histamine binding to H1R can cause phosphorylation of neuronal networks and chemical factors, amongst which is PKC via DAG activation. We found that L. reuteri 6475 a lipid compound synthesised in the intestine upon inges- suppressed basal levels of the pro-inflammatory cytokines tion of dietary fat, the anandamide monounsatured IL-6, IL-1, Eotaxin (eosinophilic chemoattractant proteins) analogue, oleoylethanolamide (OEA). OEA affects home- and G-CSF in the luminal mucosa and in blood plasma. L. ostatic and cognitive function mainly via activation of the reuteri lacking DGK could not suppress the aforementioned vagus nerve. We recently found that the central neuro- pro-inflammatory biomarkers. In addition, we demonstrated transmitter systems recruited by peripheral OEA to inhibit that histamine synthesized by L. reuteri 6475 activates both food intake include histaminergic neurons. Using different

H1 and H2 receptors but DGK synthesized by the bacterium behavioural settings, we observed that OEA induced a suppresses H1R downstream signaling. Inhibition of sig- hypophagic effect that was significantly attenuated in mice naling downstream of H1R was supported by diminished lacking the gene encoding for histidine decarboxylase or PKC phosphorylation in the intestines of wild-type (WT) L. acutely depleted of histamine through i.c.v. infusions of the reuteri treated but not in DdgkA mutant treated germ-free HDC blocker a-fluoromethylhistidine. OEA improves (GF) mice. In addition, we also report suppression of memory retention in several tasks, presumably to optimize CD11b+Gr1+ Ly6Ghi immature myeloid cells (IMCs) after food searching and the ability to remember the context WT L. reuteri treatment. The proportion was consistent with associated with food availability. We found that OEA in vivo experiments in our mouse model. PKC phosphory- induced an exaggerated emotional response in an aver- lation was reduced in human epithelial cells after treating the sively-motivated task—the contextual fear conditioning cells with L. reuteri derived conditioned media. L. reuteri paradigm. This effect was abrogated by the inhibition of suppressed immune responses by direct effects of the histaminergic neurotransmission or the local blockade of metabolite, histamine, and a secreted bacterial enzyme, either H1 or H2 receptors in the basolateral amygdala. Our DGK, which converts a membrane lipid signal to an inactive findings are relevant to recent studies on patients suffering form. Additionally, conventional BALB/c mice treated with from post-traumatic stress disorder In this regard, markers 109 cells of WT L. reuteri before and after 1% DSS induced of histaminergic dysregulation are found in several neu- mild colitis showed reduced proinflammatory CD11b+F4/ ropsychiatric disorders characterized by repetitive 80+MHCIIhi M/ compared to the mice that received DdgkA behaviours, thoughts and stereotypic behaviours. There- L. reuteri in the intestinal mucosa. Also, CD4+CD25+Foxp3+ fore, we analyzed the effect of acute brain histamine T-reg cells were increased with WT L. reuteri treatment in depletion on the temporal organization of motor sequences

123 S42 The First Joint Meeting of the European Histamine Research Society during behaviours of CD1 mice in an open-field test, using These results look promising and warrant further dedicated software. We found that histamine deficiency experimental, clinical and translational studies of the use of was associated with a general enhancement of behavioral agents targeting H1R, H4R or a combination of H1 and H4 pattern complexity, suggesting a putative involvement of receptors in the treatment of visceral pain and symptoma- histamine in the pathophysiology of tics and related dis- tology in IBS populations. orders. Systemic OEA reversed the effects of histamine University of Antwerp, Laboratory of Experimental Medi- depletion, suggesting a potential role in the treatment of cine and Pediatrics, Universiteitsplein 1, 2610 Antwerp, such diseases. Belgium. E-mail: [email protected] In conclusion, we are beginning to unravel unsuspected functions of the brain histaminergic system as part of the complex gut-brain axis. HISTAMINE AND DIABETES: WHERE WE ARE Dept of Health Sciences, University of Florence, VIale AND WHERE ARE WE GOING Pieraccini 6 50139 Florence, Italy. E-mail: beatrice. passani@unifi.it A. C. Rosa, A. Pini, I. Obara, A. Sasse, F. Sanchez- Jimenez, H. Stark, E. Masini, R. Leurs, P. L. Chazot

THERAPEUTIC OPTIONS FOR INHIBITION OF H1 Almost 60 years have passed by since the first study on AND H4 RECEPTORS IN THE GI TRACT histamine and diabetes; over most of that time period additional controversial evidence supportive of a role for B. Y. De Winter histamine in diabetes has confined this amine to a marginal role. However, in the last decade, through the identification

Irritable bowel syndrome (IBS) is a functional bowel dis- of the newer members of the histamine receptor family, H3 order characterised by motility disturbances such as and H4 receptors, several lines of evidence support the diarrhea and/or constipation, and sensitivity disturbances contribution of histamine in the diabetic milieu resulting such as abdominal pain. Inflammation and stress are two from persistent hyperglycaemia, providing scope for new well-known triggers in the pathogenesis of IBS resulting in insights into the role of histamine in diabetes and its the activation of epithelial cells, immune cells and mast complications. Therefore, we are increasingly convinced cells. Each of these can release numerous mediators cap- that it is time to re-evaluate the functional role of histamine able of affecting neighbouring afferent neurons and smooth in diabetes and its complications. Under this aim, some muscle cells. Histamine is one of the mediators released effort has been dedicated to understand the histamine-in- and has recently been studied for its role in visceral sulin loop in the pancreas. Indeed, diabetic rats showed an hypersensitivity. Experimental data from animal models increase in histamine plasma content up to 77.6% due to a support a role for H1 and H4 receptors in the occurrence of neosynthesis by histidine decarboxylase (HDC). Insulin visceral hypersensitivity, with evidence for a functional induced a 22.07% reduction of histamine plasma content. interplay between both receptor subtypes (Deiteren et al., Interestingly, similar results were obtained in different Gut, 2014: 63:1873–82). The exact mechanism of action of tissues such as the retina and kidney. The first evidence for

H1R and H4R activation as well as the level of activation a contribution of the histamine H4 receptor in diabetic (either peripherally in the gut wall at the level of the dorsal nephropathy has also been provided. However, many other root ganglia, or centrally in the brain) need further study, complications, such as diabetic neuropathy and retinopa- notwithstanding the potential involvement of these recep- thy, still require a dedicated investigation. All these issues tors on inflammatory cells. Currently there is evidence that will be addressed in future studies which will include a the transient potential receptor (TRP) family is involved in focus on all the microvascular complications of diabetes the sensitising effects of histamine. Histamine is able to (nephropathy, neuropathy and retinopathy) through a sensitise the transient receptor potential vanilloid (TRPV) 1 multidisciplinary programme including epidemiology, receptors at the level of the dorsal root ganglia, and bioinformatics, genetics, medicinal chemistry, pharmacol- supernatants of biopsies from IBS patients can sensitise ogy and histology. This goal will be realized with the human submucosal neurons and mouse serosal afferent constitution and action of an ad hoc consortium (the neurons (Wouters et al., Gastroenterology, 2016, HAdDoCk consortium), which currently comnsists of 6 150:875–87). In a proof-of-concept clinical trial, , different European countries. an H1 receptor antagonist, had beneficial effects on rectal Department of Scienza e Tecnologia del Farmaco, visceral hypersensitivity as well as on associated symptoms University of Turin, Italy. E-mail: ariannacarolina. and abdominal pain in IBS patients. [email protected]

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HEMODYNAMIC AND BIOCHEMICAL EFFECTS TRANSGENIC MICE AS MODEL TO STUDY THE

OF IMMUNOLOGICAL STRESS IN MICE WITH ROLE OF THE HUMAN H2 RECEPTOR IN THE CARDIAC SPECIFIC OVEREXPRESSION OF THE HEART

HUMAN H2 RECEPTOR U. Gergs, K. Griethe, N. Mißlinger, J. Neumann J. Neumann, M. Mahnkopf, H. Edler, F. Bergmann, U. Gergs In the human heart, histamine H2 receptors are expressed and can mediate positive inotropic, chronotropic and

Epidemiological studies indicate that H2-receptor proarrhythmic effects of histamine. In the mouse heart, we antagonists might be beneficial in patients for reducing could not detect functional H2 receptors. Therefore, we the incidence and severity of heart failure suggesting have generated transgenic mice (TG) that overexpress the that activation of H2 receptors is detrimental for cardiac human H2 receptor specifically in cardiomyocytes. In left function. To study this hypothesis in an animal model, atrial preparations of TG mice, but not of wild type mice we have generated transgenic mice (TG) which overex- (WT), histamine (logEC50 =-7.47) or amthamine press the human H2 receptor specifically in (logEC50 =-7.99), a selective H2 receptor agonist, cardiomyocytes via the alpha myosin heavy chain pro- induced a positive inotropic effect (PIE). To further char- moter. In these transgenic mice (TG) but not in wild acterize this TG model, we studied some aspects of the type litter mates (WT), histamine induced positive ino- signal transduction of the PIE in isolated electrically driven tropic effects (PIE) and chronotropic effects (PCE) in (1 Hz) left atria and, moreover, to investigate whether the isolated left or right atrial preparations and in vivo as H2 receptor has any impact on cardiac function during studied via echocardiography. This mouse model was ischemia and/or reperfusion, we perfused isolated hearts used to study the relevance of histamine in a model of (Langendorff mode, 2 ml/min flow) from TG and WT cardiac failure. Therefore, LPS (lipopolysaccharides) mice. Firstly, using partially depolarized left atrial prepa- were intraperitoneally injected at 30 lg/g (or isotonic rations (44 mM KCl in the organ bath decreased force of sodium chloride: NaCl) body weight in TG and WT. contraction to 27 ± 4.7% in WT, n = 7, and to 51 ± 7.4% Thereafter, the time course (1, 3, 7 h) of LPS-action was in TG, n = 7; p \ 0.05 vs. Ctr and WT), histamine followed by echocardiography in isoflurane-anaes- (100 lM) induced a PIE only in TG atria (by 31.2%, thetized mice. LPS led to a time-dependent reduction in p \ 0.05) that was abolished by (10 lM), a H2 ejection fraction and arterial and venous flow parameters receptor antagonist, whereas isoproterenol (10 lM) whereas heart rate was increased (n = 6–9). Of note, increased force in both WT (by 15.3%) and TG (by 17.4%). cardiac output was less in TG than in WT at 7 h after Furthermore, we tested whether analgesics known to release LPS injection (n = 8). After 7 h, hearts were obtained histamine were inotropically active in TG. However, mor- and Western blotting or RNA isolation and reverse phine (10 lM) was unable to affect contractility in TG or transcription—quantitative polymerase reaction (RT- WT, whereas ketamine (100 lM) and fentanyl (10 and PCR) were performed for genes of interest. LPS- 30 lM) increased left atrial contractility in both TG and WT induced increases in IL-6 and IkBalpha were less pro- (n = 4, p \ 0.05 vs. Ctr). Secondly, using perfused heart nouncedinTGthaninWT.Moreover,inNaCl-treated preparations, global ischemia was simulated by interrupting TG, levels of IL-1b were much smaller than in LPS- the perfusion for 20 min followed by 30 min of reperfusion. treated TG. Finally, LPS increased CD14-levels only in In our experimental setup, ischemia did not cause a per- TG but not in WT. Using RT-PCR, we detected a manent damage as after reperfusion, force and heart rate of reduction in density of endogenous mouse H2 receptors both, TG and WT hearts, reached pre-ischemic values again. in WT and less so in TG. Interestingly, the density of But during ischemia, the time to 50% decline of force (F‘) overexpressed human H2,whichwasveryhighinTG, was reduced in TG (to 34.7 ± 8.4 s versus 60.5 ± 17.8 s in was hardly measurable in TG after LPS treatment. It is WT, n = 6) and during reperfusion, the time to return to suggested that LPS reduces the stability of human H2 sinus rhythm was increased in TG (71.0 ± 7.6 s) compared mRNA in TG. Hence, TG are more sensitive to stress to WT (47.7 ± 6.9 s, n = 6, p \ 0.05). Taken together, we

(induced by LPS), which explains why H2 antagonists demonstrated, firstly, an involvement of the L-type calcium might play a beneficial role in human heart failure. channel current in the H2 receptor-mediated PIE in TG atria. Institute for Pharmacology and Toxicology, Medical Fac- Secondly, we failed to release inotropically active histamine ulty, Martin-Luther-University Halle-Wittenberg, 06097 using classical analgesics, arguing that a direct effect also in Halle (Saale), Germany. E-mail: joachim.neumann@ the human heart is unlikely to occur. Thirdly, we conclude medizin.uni-halle.de that during cardiac infarction/reperfusion, H2 receptors may

123 S44 The First Joint Meeting of the European Histamine Research Society be in part responsible for further deterioration of the situa- HISTAMINE RECEPTORS IN CANCER tion in patients. Institute for Pharmacology and Toxicology, Medical Fac- V. Medina ulty, Martin Luther University Halle-Wittenberg, 06097 Halle (Saale), Germany. E-mail: ulrich.gergs@medizin. Cancer is one of the leading causes of morbidity and mor- uni-halle.de tality worldwide. Substantial evidence demonstrates that histamine modulates biological responses related to tumor progression with different effects depending on the his-

HISTAMINE H3 RECEPTOR BLOCKAGE- tamine receptor subtype involved and the cell type. INDUCED RESUSCITATING EFFECT IN Expression of histamine H4 receptors (H4R) has been HAEMORRHAGE-SHOCKED RATS—NOT ONLY described in different tumor subtypes, with these receptors CARDIOVASCULAR EFFECTS playing a key role in histamine-mediated effects. Thus, his- tamine and H4R agonists reportedly inhibit proliferation of J. Jochem, Z. Czuba, K. Jasikowska, A. Mitera human breast cancer and melanoma cells in vitro and in vivo, augmenting cell death, differentiation and senescence. There are two phases of cardiovascular response to acute Chemotherapy and radiotherapy are major treatments of blood loss—an initial sympathoexcitatory phase, with an medical oncology. However, their related toxicities fre- increase in total peripheral resistance and heart rate (HR), quently outweigh their clinical benefits and worsen a followed by a sympathoinhibitory phase, with vasorelax- patient’s quality of life. In this regard, histamine increases ation and bradycardia. Prolonged tissue hypoperfusion and the antitumor efficacy of a chemotherapeutic agent or ion- subsequent reperfusion in haemorrhagic shock induces izing radiation in experimental models of human breast immunological changes which may lead to multiorgan cancer and melanoma developed in immunodeficient mice, dysfunction. Since histamine H3 receptors are present on by modulating DNA damage, oxidative stress and cell death. postganglionic endings of the sympathetic neurons and are Recent studies in murine breast cancer developed in able to regulate the synthesis and release of postganglionic immunocompetent mice allowed an examination of the neurotransmitters and thus the neurogenic vasopressor effect of histamine not only on metastatic potential, but tone, the aim of the study was to examine cardiovascular also on the immune system and inflammatory processes, and immunological effects of H3 receptor ligands in the since these latter processes play a fundamental role in sympathoinhibitory phase of haemorrhagic shock. Experi- regulating tumor development and progression. Modula- ments were carried out in ketamine (100 mg/kg)/xylazine tion of tumor immunity was found to be involved in the (10 mg/kg)-anaesthetised adult male Wistar rats, divided effects of histamine on tumor development. into groups of 6 and subjected to haemorrhagic hypoten- The latest evidence regarding the involvement of his- sion, with mean arterial pressure (MAP) stabilized at tamine and H4R in breast cancer and melanoma was 30–35 mmHg. In the control group, all animals survived discussed in my presentation. The findings presented sup- 2 h with MAP 40–50 mmHg, renal, mesenteric and port exploitation of the H4R as a molecular target for hindquarters blood flows decreased to 35–50%, and cancer drug development. increased plasma concentrations of INF-alpha, IL-1alpha, Institute for Biomedical Research (BIOMED CONICET- IL-1beta, IL-5, IL-6, IL-10, IL-12, IL-13 and TNF-alpha. UCA), School of Medical Sciences, Pontifical Catholic

H3 receptor inverse agonist/antagonist BF 2649 (10 and University of Argentina (UCA), Buenos Aires, Argentina. 50 mg/kg) and antagonist ROS 234 (5 and 15 mg/kg) given E-mail: [email protected] intravenously at 5 min of hypotension induced long-lasting increases in MAP, HR and peripheral blood flows. Addi- tionally, BF 2649 (50 mg/kg) and ROS 234 (15 mg/kg) LACK OF HISTAMINE H4 RECEPTOR IN TUMOR reduced increases in measured immunological parameters. MICROENVIRONMENT RESULTS IN REDUCED

In conclusion, blockage of histamine H3 receptors in the AGRESSIVENESS IN A MODEL OF BREAST sympathoinhibitory phase of haemorrhagic shock in rats CANCER. ROLE OF ANTITUMOR IMMUNITY induces a long-lasting pressor effect and influences— probably due to the improvement in tissue perfusion— M. Nicoud, H. Sterle, N. Massari, M. V. Herrero Ducloux, hypoxia/reperfusion-induced immunological changes. D. Martinel Lamas, G. Cremaschi, V. Medina Departments of Physiology and of Microbiology and Immunology, School of Medicine with the Division of The expression of histamine H4 receptor (H4R) and his- Dentistry in Zabrze, Poland. E-mail: jjochem@ tamine-induced modulation of proliferation in different poczta.onet.pl types of tumors has been previously reported. However, the

123 The First Joint Meeting of the European Histamine Research Society S45 role of H4R in tumor microenvironment, including immune was observed. The lack of H4R is associated with an cells, was not yet studied. Present work aims to investigate increase in the percentage of CD8 + (32.0 ± 2.5 vs. the effect on breast cancer development and progression of 16.1 ± 3.1, P \ 0.05) and CD19 + (27.2 ± 4.8 vs. the lack of H4R in tumor microenvironment. We evaluated 6.5 ± 1.5, P \ 0.01) tumor infiltrating lymphocytes and a growth parameters, histological characteristics of tumors decrease in CD4 + lymphocytes (11.9 ± 3.4 vs. and the composition of tumoral immune subsets in a syn- 19.4 ± 1.2, P \ 0.05). Similar differences in immune geneic model of breast cancer developed orthotopically subsets are observed in the spleen. Finally, H4R-deficient with 4T1 cells in H4R knockout (H4R-KO) and wild-type mice showed non-significant changes in lung metastasis. (WT) mice. Mice lacking H4R show reduced tumor size These results suggest an interplay between H4R expressing and weight and exhibit a more differentiated histopatho- cells in tumor microenvironment and cancer cells, which logical pattern (grade 2) compared with WT mice (grade 3, has implications in breast cancer progression. highly aggressive potential). Tumors of H4R-KO mice Institute for Biomedical Research (BIOMED CONICET- display significant decrease in neutrophils (number of UCA), School of Medical Sciences, Pontifical Catholic neutrophils per field: 3.2 ± 0.9 vs. 9.0 ± 2.1, University of Argentina (UCA), Buenos Aires, Argentina. P\0.05) while non-significant differences in angiogenesis E-mail: [email protected]

123 S46 The First Joint Meeting of the European Histamine Research Society

Clinical aspects of histamine

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123 S48 The First Joint Meeting of the European Histamine Research Society

HISTAMINE RESPONSE AND GENETICS IN THE LONG AND WINDING ROAD—CLINICAL

ASTHMA DEVELOPMENT OF H4R ANTAGONISTS

B. L. Jones, C. Sherwin, X. Liu, H. Dai, C. A. Vyhlidal R. L. Thurmond

There is growing knowledge of the wide ranging effects The histamine H4 receptor (H4R) is a high affinity receptor for of histamine throughout the body therefore it is impor- histamine and was discovered via a genomics method about tant to better understand the effects of this amine in 17 years ago. Since that time numerous ligands, both antag- patients with asthma. We have demonstrated that his- onists and agonists, have been described for the receptor. The tamine pharmacodynamic (PD) response varies among identification of these selective ligands have been crucial in children and adults and that PD response phenoytpes are uncovering the functions of the receptor. Most of this work has able to be identified utilizing the surrogate endpoint, been in the preclinical space, but over the past few years Histamine Iontophoresis with Laser Doppler minoriting clinical data have been reported furthering our understanding (HILD). These differences may be important in pre- for the role of the receptor in human physiology and patho- dicting therapeutic response to anti-histamine/anti- physiology. One of the first selective H4Rantagonists allergic agents in the treatment of asthma. We have also discovered was JNJ 7777120 that has become one of the observed that genes within the histamine pathway differ standard ligands for the H4R. The use of this ligand, along with between children with allergic vs. non-allergic asthma. studies in H4R –deficient mice, has pointed to a role for the Therefore, we aimed to explore the association between receptor in a number of human diseases including pruritus, histamine pharmacodynamic (PD) response and genetic asthma and atopic dermatitis. However, the short in vivo half- variation in the histamine pathway in children with life of the compound along with the induction of hypoad- asthma. In this study HILD was performed in children renocorticism toxicity in rats and dogs prevented it from with asthma and estimates for area under the effect curve progressing into clinical studies. Subsequent antagonists such (AUEC), maximal response over baseline (Emax), and as JNJ 28307474 and JNJ 39758979 were used to prove that time of Emax (Tmax) were calculated using non-com- the adrenal effects were not related to H4R antagonism. JNJ partmental analysis and non-linear mixed-effects model 39758979 advanced into clinical testing and was shown to be with a linked effect PK/PD model. DNA isolation and efficacious in reducing histamine-induced pruritus and pru- genotyping were performed among participants to detect ritus associated with atopic dermatitis, providing clinical known single nucleotide polymorphisms (SNPs) evidence for the first time for the potential of H4Rantagonists (n = 10) among genes (HDC, HNMT, ABP1, HRH1, for the treatment of human diseases. However, clinical testing HRH4) within the histamine pathway. General linear with JNJ 39758979 had to be terminated since it was shown to model was used to identify associations between his- cause drug-induced agranulocytosis. Once again this did not tamine related genetic variants and measured histamine appear to be related to antagonism of the H4R as a second H4R, PD response parameters. Genotyping and HILD response toreforant, had also advanced into clinical studies and did not profiles were completed for 163 children. SNPS in show this side-effect. In all the experience with the develop-

HNMT and ABP1, were associated with Emax (ABP1 47 ment of H4R antagonists demonstrates the difficulties in C/T, CC 167.21 vs. CT/TT 139.20, p = 0.04; ABP1 finding ligands with all of the right properties needed to 4107 C/G, CC 141.72 vs. CG/GG 156.09, p = 0.005; become a successful human therapeutic. Despite some of the

HNMT-1639 C/T, CC 132.62 vs. CT/TT 155.3, failures, the clinical efficacy showen to date with H4R p = 0.02). In a stratified analysis among African antagonists point to their potential as future therapeutics for American children only, ABP1 and HNMT SNPs were the treatment of atopic dermatitis, pruritus and perhaps other also associated with PD response; HRH4 413 CC indications. genotype was associated with lower Emax, p = 0.009. Janssen Research & Development, LLC San Diego, CA We show for the first time that histamine pathway 92107, USA. E-mail: [email protected] genetic variation is associated with measureable changes in histamine response in children with asthma. The variability in histamine response and impact of histamine THE IMPACT OF H4R BLOCKADE IN ATOPIC pathway genotype is important to further explore in DERMATITIS AND PSORIASIS patients with asthma so as to improve disease pheno- typing leading to more personalized treatments. T. Werfel, R. Gutzmer, M. Kietzmann, S. Mommert, Department of Pediatrics University of Missouri-Kansas K. Rossbach, K. Schaper-Gerhardt City, 64108 Missouri, USA.. E-mail: [email protected]

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Histamine-induced inflammation is largely mediated exercise or passive warming. The underlying causes and through the H4 receptor (H4R). T lymphocytes and ker- pathophysiological mechanisms remain to be identified and atinocytes from patients with atopic dermatitis (AD) characterized in detail. In this study, we sought to analyze if express high levels of H4R which may be due to genetic histamine, as a wheal forming agent in human sweat, could variations or effects of Th2 cytokines on H4R expression. play a role in CholU symptom elicitation. To this end, we first High H4R expression may be associated with a more determined the histamine content in the sweat of 35 CholU pronounced Th2 cytokine polarization driving atopic der- patients and 38 healthy controls (HC). Next, we analyzed the matitis and histamine-induced proliferation. skin reactivity to intracutaneous (i.c.) injections to autolo- Two studies have so far been described with blockers of gous sweat, histamine (50 ll, 100 lg/ml, positive control) the H4R in atopic dermatitis. In one, the H4R-antagonist and saline (negative control) in both populations. JNJ-39758979 was given to adult Japanese patients with Exposure to 80 °C for 15 min resulted in sweating in moderate atopic dermatitis. Eighty-eight of 105 planned both CholU patients and HCs (CholU 97 ml ± 66, HC patients were randomized before the study was stopped and 105 ml ± 60, p = n.s.). The sweat of CholU patients and unblinded for safety reasons (two cases of neutropenia). HCs showed similar levels of histamine as determined via The study did not meet the primary end-points but HistareaderÒ (CholU 98.4 ng/ml ± 117.2, HC 101.7 ng/ decreases in the median severity score of eczema for JNJ- ml ± 121.5, p = n.s.). Injection of 1:100 diluted autolo- 39758979 versus placebo were observed at week 6. Sig- gous sweat resulted in wheal formation in 74% of the nificant improvements on pruritus were also observed. CholU patients (5.4 mm ± 4.2) but not in HC (p \ 0.001). In another randomised, double-blind, placebo-controlled, In response to i.c. injections of histamine, CholU patients parallel group study, the selective H4R antagonist ZPL-389 had more than 2-fold bigger wheals than HC (CholU was studied in 98 adult subjects with moderate to severe atopic 14.9 mm ± 8.7 vs. HC 5.9 mm ± 4.6, p \ 0.001). dermatitis. Following 8 weeks treatment with oral ZPL-389, In summary, CholU patients exhibit normal sweat vol- results showed a 50% reduction in the eczema score EASI ume and histamine levels, but markedly increased skin compared to 27% for the placebo (p = 0.01). ZPL-389 was reactivity to sweat and histamine. This suggests, that well tolerated and, particularly, no cases of neutropenia were CholU may be caused by cutaneous hypersensitivity to observed in this study. The results showed for the first time histamine. Detailed studies of the underlying mechanisms that an H4R antagonist has a clinically significant anti-in- are needed and ongoing. flammatory effect in moderate to severe AD in humans. Department of Dermatology and Allergology, Charite´— The H4R is also functionally expressed on Th17 cells Universita¨tsmedizin D-10117 Berlin, Germany E-mail: and plasmacytoid dendritic cells (pDCs) which play a [email protected] prominent role in the pathogenesis of psoriasis. On pDCs, a higher basal expression level of the H4R in psoriasis patients compared to healthy controls has been detected. In THE ENIGMA OF H1-ANTIHISTAMINES IN a recent study we described genetic variations within the URTICARIA H4R gene which might be associated with special clinical features of psoriasis (severity, palmoplantar involvement). M. K Church, E. Alarbeed, K. Krause, M. Maurer These findings lend weight to the therapeutic potential of H4R antagonists in psoriasis and are the rational for Urticaria is hypothesised to be is mast cell driven disease in ongoing studies in this inflammatory skin disease. which the symptoms are mediated by the actions of his- Dept. Dermatology and Allergy, Hannover Medical tamine on H1-receptors located on vascular smooth muscle School, Hannover, Germany. E-mail: Werfel.thomas@mh- (vasodilatation) endothelial cells (the wheal) and on sen- hannover.de sory nerves (neurogenic flare and pruritus). This is supported by efficacy of H1-antihistamines in the treatment of urticaria. But is this conclusion based on real evidence? CHOLINERGIC URTICARIA IS ASSOCIATED To test this hypothesis, cold urticaria was provoked in WITH A HIGHER SKIN REACTIVITY TO SWEAT 20 patients following 7 days treatment with placebo and AND HISTAMINE 80 mg. Clinical effectiveness was assessed by measurement of critical temperature thresholds (CTT) for S. Altrichter, O. Alraboni, P. Schumacher, M. K. Church, wheal formation. A reduction of CTT to\4 °C by bilastine M. Maurer was considered to be completely effective. Histamine was measured in the provocation sites by microdialysis. Patients with cholinergic urticaria (CholU) develop itchy Bilastine was completely effective in reducing symp- wheal and flare type skin reactions in response to physical toms in 12 patients, partially effective in 7 patients and

123 S50 The First Joint Meeting of the European Histamine Research Society ineffective in only one person. Concentrations of histamine up to the anaphylaxis and administration of adrenaline will recovered by microdialysis, which were not reduced by be described in this patient who had no prior history of IgE bilastine, were extremely variable up to 230 nM. In 5 mediated allergy or anaphylaxis. patients, there were no increases histamine concentrations had been taken for one year, at a dose of above baseline, 3–5 nM, and yet in these individuals 300 mg at night, to manage reflux related to Ehlers- bilastine was fully effective. This suggests that the effects Danlos Syndrome Hypermobility Type (EDS-HT). This of other mediators are being blocked H1- was increased to 300 mg twice daily six weeks prior the therapy. Possible candidates are bradykinin and platelet anaphylactoid event, to reduce symptoms of nausea, activating factor (PAF), the ability of both to produce vomiting, bloating and diarrhoea related to mast cell wheal and flare responses has been shown previously to be activation (MCA). The patient also suffers from postural prevented by H1-antihistamine in the absence of histamine orthostatic tachycardia syndrome (POTS). This concurs release. What is the explanation for this? Is it receptor with the proposed association between MCA, POTS and cooperation or dimerization? This apparent enigma EDS-HT. requires further investigation. A mechanism for the reaction is proposed, requiring Charite´-Universita¨tsmedizin Berlin, D-10117 Berlin, critique, in the context of ranitidine and a rebound effect/ Germany. E-mail: [email protected] reverse agonist rather than an antagonist/upregulation explanation. Recognition of this event may have implica- tions for patients with MCA disorders and or EDS-HT who A CASE OF ANAPHYLAXIS FOLLOWING are prescribed ranitidine and other pharmacologically CESSATION OF HIGH DOSE RANTIDINE: similar drugs. There are potential policy and patient guid- MECHANISM? ance implications for primary and secondary care with respect to stopping these medications. It is proposed that P. L. Chazot, S. Allen research in this area is lacking and of benefit. Department of Biosciences, Durham University, Durham, The anaphylactoid reaction reported here followed cessa- UK. E-mail: [email protected] tion of a high dose of the antagonist, ranitidine, in a 19 year old female patient. Events leading

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