Initial Characterization of Transgenic Mice Overexpressing Human Histamine H2 Receptors.

In an integrative approach, we studied the role of histamine H2 receptors in the mouse heart. We noted that histamine, added cumulatively to the organ bath, failed to affect the force of contraction in left atrial preparations and did not change spontaneous heart rate in right atrial preparations from wild-type mice. By contrast, in the same preparations from mice that overexpressed the human H2 receptor in a cardiac-specific way, histamine exerted concentration- and time-dependent positive inotropic and positive chronotropic effects. Messenger RNA of the human H2 receptor was only detected in transgenic mice. Likewise, immunohistology and autoradiography only gave signals in transgenic but not in wild-type cardiac preparations. Similarly, a positive inotropic and positive chronotropic effect was observed with histamine in echocardiography of living transgenic mice and isolated perfused hearts (Langendorff preparation). Phosphorylation of phospholamban was increased in atrial and ventricular preparations from transgenic mice, but not in wild-type animals. The effects of histamine were mimicked by dimaprit and amthamine and antagonized by cimetidine. In summary, we generated a new model to study the physiologic and pathophysiologic cardiac role of the human H2 receptor.

The negative effect of unloading exceeds the bone-sparing effect of alkaline supplementation: a bed rest study.

Potassium bicarbonate was administrated to an already alkaline diet in seven male subjects during a 21-day bed rest study and was able to decrease bed rest induced increased calcium excretion but failed to prevent bed rest-induced bone resorption. INTRODUCTION: Supplementation with alkali salts appears to positively influence calcium and bone metabolism and, thus, could be a countermeasure for population groups with an increased risk for bone loss. However, the extent to which alkalization counteracts acid-induced bone resorption or whether it merely has a calcium and bone maintenance effect is still not completely understood. In the present study, we hypothesized that additional alkalization to an already alkaline diet can further counteract bed rest-induced bone loss. METHODS: Seven healthy male subjects completed two parts of a crossover designed 21-day bed rest study: bed rest only (control) and bed rest supplemented with 90 mmol potassium bicarbonate (KHCO3) daily. RESULTS: KHCO3supplementation during bed rest resulted in a more alkaline status compared to the control intervention, demonstrated by the increase in pH and buffer capacity level (pH p = 0.023, HCO3p = 0.02, ABE p = 0.03). Urinary calcium excretion was decreased during KHCO3 supplementation (control 6.05 ± 2.74 mmol/24 h; KHCO3 4.87 ± 2.21 mmol/24 h, p = 0.03); whereas, bone formation was not affected by additional alkalization (bAP p = 0.58; PINP p = 0.60). Bone resorption marker UCTX tended to be lower during alkaline supplementation (UCTX p = 0.16). CONCLUSIONS: The more alkaline acid-base status, achieved by KHCO3 supplementation, reduced renal calcium excretion during bed rest, but was not able to prevent immobilization-induced bone resorption. However, advantages of alkaline salts on bone metabolism may occur under acidic metabolic conditions or with respect to the positive effect of reduced calcium excretion within a longer time frame. TRIAL REGISTRATION: Trial number: NCT01509456.

Label-free analysis of GPCR-stimulation: The critical impact of cell adhesion.

Label-free cell-based assays have been attracting growing attention in drug research. Optical approaches based on evanescent electric fields (e.g. EPIC, RWG/DMR, SPR) and electrochemical impedance analysis (ECIS, xCELLigence) are by far the most widespread techniques for such purposes. We compared three label-free approaches (ECIS, RWG/DMR and SPR) with respect to the activation of the human histamine H1 receptor (H1R) expressed by U-373 MG glioblastoma and genetically engineered HEK 293T cells. HEK 293T cells were either expressing the hH1R alone or in combination with the adhesion protein hMSR1. The ß2-adrenergic receptor (ß2-AR) expressed by bovine aortic endothelial cells (BAEC) served as a second cell model. Reduced cell adhesion to the surface of the sensing devices affected both, the optical and the impedance-based readout, but became much more obvious in case of RWG- or SPR-based assays. By contrast, the co-expression of hH1R and hMSR1 in HEK 293T cells strongly enhanced the signal compared to hH1R expression alone. As the sensitivity of the optical readouts is confined to a distance of 100-200nm from the surface, depending on the wavelength of the incident light, this observation is in accordance with tighter adhesion of the co-transfectants, inducing a shorter distance between the cell membrane and the substrate. Combining ECIS and SPR, allowing for simultaneous registration of both signals for a single cell population, provided a direct correlation of both readouts, when H1R or ß2-AR stimulation was investigated for the same cell populations. Cell adhesion was found to have a critical impact on the results of label-free cell monitoring, in particular when techniques based on evanescent electric fields are applied.

Label-free versus conventional cellular assays: Functional investigations on the human histamine H1 receptor.

A set of histamine H1 receptor (H1R) agonists and antagonists was characterized in functional assays, using dynamic mass redistribution (DMR), electric cell-substrate impedance sensing (ECIS) and various signaling pathway specific readouts (Fura-2 and aequorin calcium assays, arrestin recruitment (luciferase fragment complementation) assay, luciferase gene reporter assay). Data were gained from genetically engineered HEK293T cells and compared with reference data from GTPase assays and radioligand binding. Histamine and the other H1R agonists gave different assay-related pEC50 values, however, the order of potency was maintained. In the luciferase fragment complementation assay, the H1R preferred ß-arrestin2 over ß-arrestin1. The calcium and the impedimetric assay depended on Gq coupling of the H1R, as demonstrated by complete inhibition of the histamine-induced signals in the presence of the Gq inhibitor FR900359 (UBO-QIC). Whereas partial inhibition by FR900359 was observed in DMR and the gene reporter assay, pertussis toxin substantially decreased the response in DMR, but increased the luciferase signal, reflecting the contribution of both, Gq and Gi, to signaling in these assays. For antagonists, the results from DMR were essentially compatible with those from conventional readouts, whereas the impedance-based data revealed a trend towards higher pKb values. ECIS and calcium assays apparently only reflect Gq signaling, whereas DMR and gene reporter assays appear to integrate both, Gq and Gi mediated signaling. The results confirm the value of the label-free methods, DMR and ECIS, for the characterization of H1R ligands. Both noninvasive techniques are complementary to each other, but cannot fully replace reductionist signaling pathway focused assays.

Safety and pharmacodynamic dose response of short-term prednisone in healthy adult subjects: a dose ranging, randomized, placebo-controlled, crossover study.

BACKGROUND: Glucocorticoids (GCs), such as prednisone, are the standard of care for several inflammatory and immunologically mediated diseases, but their chronic systemic administration is severely limited by serious adverse effects that are both dose and time dependent. Short-term treatment (7-14 days) with oral prednisone is used for many acute inflammatory and allergic conditions. This study was conducted to characterize the safety and pharmacodynamic (PD) dose-response of a 7-day course of oral prednisone on biomarkers of GC receptor agonism. METHODS: In this randomized, single-blind, placebo-controlled, crossover study (A9001309), 37 healthy subjects received placebo or a prednisone dose from 2.5-60 mg daily over 7 days in each of three treatment periods. White blood cell counts and plasma samples for measuring cortisol, osteocalcin and procollagen type 1 N-propeptide (P1NP) were obtained at 2, 4, 8, and 12 h post-dose on Day 1, immediately prior to dosing on Days 1, 2, and 4, and at nominal dosing time on Days 0 and 8. Urine samples for urinary N-terminal cross-linked telopeptide of type 1 collagen (uNTX) were collected on Days 0, 1, 2, 4, and 8. Serum samples for adiponectin were obtained prior to dosing on days 0, 1, 4 and 8. RESULTS: Daily doses of prednisone up to 60 mg resulted in dose- and time-dependent decreases in plasma osteocalcin, plasma P1NP, serum cortisol, and absolute blood eosinophil counts. Absolute blood neutrophil counts increased, while blood lymphocyte counts rebounded to an increased level following an initial rapid decrease after dosing. An increase was observed for uNTX and adiponectin. The incidence of adverse effects with prednisone was not dose related, and nervous system disorders, mainly headache, were the most frequently reported adverse effects. CONCLUSIONS: This characterization provides important and relevant information on safety and PD responses of short-term prednisone dosing over the commonly-used clinical dose range, and also provides a reference for early clinical development of dissociated agents targeting a differentiated PD profile. TRIAL REGISTRATION NUMBER: NCT02767089 (retrospectively registered: 21 April 2016).

Observer variability in the assessment of the acromiohumeral interval using anteroposterior shoulder radiographs.

BACKGROUND: An acromiohumeral interval narrower than 6 mm measured on AP shoulder radiographs has been considered pathological and suggestive of rotator cuff tears. This prospective study was conducted to assess inter- and intraobserver variation in the radiographic assessment of the acromiohumeral interval and its critical value on routinely taken AP shoulder radiographs off-study use to evaluate the accuracy of this measurement method. METHODS: The acromiohumeral distance from the inferior anterior acromial aspect to the humeral head was measured in millimeters. Thirty blinded, anteroposterior shoulder radiographs were independently reviewed by five board-certified orthopedic shoulder surgeons at two time points in random order. RESULTS: The results of three investigators showed significant intraobserver variation. Five investigator pairs showed significant interobserver variation at both examination time points. The maximum interobserver difference for the same radiograph was 8 mm (range 0 to 8 mm). CONCLUSION: Our results indicate that the assessment of the acromiohumeral interval using non-standardized anteroposterior radiographs off-study use cannot be seen as a reproducible and reliable method of measurement.

Functional reconstitution of human neuropeptide Y (NPY) Y(2) and Y(4) receptors in Sf9 insect cells.

The four functionally expressed human neuropeptide Y receptor subtypes (hY(1)R, hY(2)R, hY(4)R, hY(5)R) belong to class A of the G-protein-coupled receptors (GPCRs) and interact with pertussis toxin-sensitive G(i/o)-proteins. The number of small molecules described as ligands for hY(1)R and hY(5)R exceeds by far those for hY(2)R. Potent non-peptidergic ligands for the hY(4)R are not available so far. Here, we report on the functional reconstitution of the hY(2)R and the hY(4)R in Sf9 insect cells using the baculovirus system. Sf9 cells were genetically engineered by infection with up to four different baculoviruses, combining the receptors with G-proteins of the G(i/o) family and regulators of G-protein signaling (RGS) proteins to improve signal-to-noise ratio. In steady-state GTPase assays, using pNPY (Y(2)) and hPP (Y(4)), the GPCRs coupled to various G(i)/G(o)-proteins and both, RGS4 and GAIP, enhanced the signals. Co-expression systems hY(2)R + G?(i2) and hY(4)R + G?(i2)/G?(o) + RGS4, combined with G?(1)?(2), yielded best signal-to-noise ratios. hY(2)R function was validated using both agonistic peptides (NPY, PYY, NPY(13?36)) and selective non-peptidergic antagonists (BIIE0246 and derivatives), whereas the hY(4)R model was characterized with peptidergic agonists (PP, NPY, GW1229, and BW1911U90). Tunicamycin inhibited receptor N-glycosylation diminished NPY signals at hY(2)R and abolished hY(4)R function. Investigations with monovalent salts showed sensitivity of hY(4)R toward Na(+), revealing moderate constitutive activity. After validation, an acylguanidine (UR-PI284) was identified as a weak non-peptide Y(4)R antagonist. In summary, the established steady-state GTPase assays provide sensitive test systems for the characterization of Y(2) and Y(4) receptor ligands.

Author Correction: Fibrinogen - A Practical and Cost Efficient Biomarker for Detecting Periprosthetic Joint Infection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Increasing the throughput of label-free cell assays to study the activation of G-protein-coupled receptors by using a serial agonist exposure protocol.

Label-free, holistic assays, monitoring, for example, the impedance of cells on electrodes, are gaining increasing popularity in the evaluation of G-protein-coupled receptor (GPCR) ligands. It is the strength of these approaches to provide the integrated cellular response non-invasively, highly automated and with a device-dependent time resolution down to several milliseconds. With an increasing number of samples to be studied in parallel, the available time resolution is, however, reduced and the cost for the disposable sensor arrays may become limiting. Inspired by protocols from organ pharmacology, we investigated a simple serial agonist addition assay that circumvents these limitations in impedance-based cellular assays. Using a serial addition of increasing concentrations of a GPCR agonist while continuously monitoring the sample's impedance, we were able to establish a full concentration-response curve for the endogenous agonist histamine on a single layer of U-373 MG cells endogenously expressing the histamine 1 receptor (H1R). This approach is validated with respect to conventional, parallel agonist addition protocols and studies using H1R antagonists such as mepyramine. Applicability of the serial agonist addition assay was shown for other GPCRs known for their signaling via one of the canonical G-protein pathways, Gq, Gi/0 or Gs as well. The serial agonist addition protocol has the potential to further strengthen the output of label-free analysis of GPCR activation.

Influence of body mass index on health-related quality of life after surgical treatment of intra-articular distal radius fractures. A retrospective 7-year follow-up study.

Morbid obesity is associated with reduced health-related quality of life (HRQOL), increased morbidity and mortality. Little is known about the correlation between obesity and complex distal radius fractures (DRF). The purpose of this study was to examine the effect of being overweight on postoperative HRQOL after surgically treated intra-articular DRF. Fifty-three patients were included in this retrospective study with 7 years' mean follow-up (mean 7.2±0.4, range 6.4-7.9 years) after volar plating of an intra-articular DRF (AO-type C). All patients were categorized by their body mass index (BMI) into two study groups: group 1 (normal weight) with a BMI<25 (n=24); group 2 (obese) with a BMI≥25 (n=29). HRQOL and functional outcomes were assessed through range of motion (ROM) and four different scores - the 36-item short form health survey (SF-36), the disability of arm and shoulder score (DASH), the Gartland and Werley score and the Castaing score - along with X-rays to measure volar tilt, radial inclination, radial length and articular congruity. All HRQOL assessments and clinical outcomes were correlated to BMI by comparing group 1 versus group 2. There was no difference in terms of postoperative ROM. The group of normal weight patients achieved slightly better but non-significant results for the Gartland and Werley score. No differences were seen in the DASH score or SF-36. There were also no differences regarding the Castaing score. Overall, normal and obese patients had no significant differences their HRQOL and functionality after volar plating of DRF.

Fibrinogen - A Practical and Cost Efficient Biomarker for Detecting Periprosthetic Joint Infection.

The early and accurate diagnosis of periprosthetic joint infection (PJI) can be challenging. Fibrinogen plays an important role in mediating inflammation of bacterial infections and therefore could be a valuable biomarker for PJI. The purpose of this study was to investigate the sensitivity and specificity of serum levels of fibrinogen in detecting PJI, and to compare the results with the established PJI biomarkers C-reactive protein (CRP) and leukocyte count. Eighty-four patients (124 surgeries) were prospectively included. The preoperatively analyzed parameters were fibrinogen, CRP and leukocyte count. The sensitivity and specificity of the biomarkers were calculated and compared. Fibrinogen (p < 0.001), CRP (p < 0.001) and leukocyte count (p < 0.001) had a statistically significant correlation with the criteria defining the presence of PJI. For fibrinogen, the value of 519 mg/dl had a sensitivity of 0.90 and a specificity of 0.34. The CRP cut-off point of 11.00 mg/dl had a sensitivity of 0.90 and a specificity of 0.74. The leukocyte count of 5.68 G/l had a sensitivity of 0.90 and a specificity of 0.39. Our results indicated that fibrinogen is a significant biomarker for detecting a bacterial PJI. It has shown to be a cost-efficient diagnostic support with high sensitivity and specificity.

The murine pan T cell marker CD96 is an adhesion receptor for CD155 and nectin-1.

The CD155 ligand CD96 is an immunoglobulin-like protein tentatively allocated to the repertoire of human NK receptors. We report here that the CD96/CD155-interaction is preserved between man and mouse although both receptors are only moderately conserved in amino acid sequence. Moreover, murine CD96 (mCD96) binds to nectin-1, a receptor related to CD155. Applying newly generated monoclonal antibodies specifically recognizing mCD96, an expression profile is revealed resembling closely that of human CD96 (hCD96) on cells of hematopoietic origin. A panel of anti-mCD96 but also recently established anti-mCD155 antibodies effectively prevents formation of CD96/CD155-complexes. This was exploited to demonstrate that the only available receptor for mCD96 present on thymocytes is mCD155. Moreover, T cell adhesion to insect cells expressing mCD155 is blocked by these antibodies depending on the T cell subtype. These results suggest a function of the CD96/CD155-adhesion system in T cell biology.

[The Effect of Intraoperative Screw Monitoring (Root Monitoring) with the INS-1 System (NUVASIVE) on the Radiological Outcome of Dorsal Instrumentation of the Lumbar Spine]. / Der Einfluss des intraoperativen Nervenwurzelmonitorings mit dem INS-1-System (NuVasive) auf das radiologische Outcome bei dorsaler transpedikulärer Instrumentation der Lendenwirbelsäule.

INTRODUCTION: Transpedicular screw fixation of spinal segments has been described for a variety of surgical indications and is a key element in spinal surgery. The aim of transpedicular screw fixation is to achieve maximal stability. Screw malposition should be obviated to avoid neurological complications. There are published methods of applying evoked EMG to control screw position in relation to neural structures. These studies demonstrated that an intact bony pedicle wall acts as an electrical isolator between the screw and spinal nerve root. The aim of our study was to evaluate the impact of intraoperative pedicle screw monitoring on screw positioning. MATERIAL AND METHODS: We enrolled 22 patients in this prospective randomised study, who underwent spinal instrumentation after being split into two equal groups. In the first group, dorsal instrumentation was supplemented with intraoperative nerve root monitoring using the INS-1-System (NuVasive, San Diego USA). In the second group, screws were inserted without additional pedicle monitoring. All patients underwent monosegmental instrumentation with "free hand implanted" pedicle screws. 44 screws were inserted in each group. The screw position was evaluated postoperatively using CT scans. The position of the screws in relation to the pedicle was measured in three different planes: sagittal, axial and coronal. The accuracy of the screw position was described using the Berlemann classification system. Screw position is classified in three groups: type 1 correct screw position, type 2 encroachment on the inner cortical wall, type 3 pedicle cortical perforation. Screw angulation and secondary operative criteria were also evaluated. RESULTS: The use of neuromonitoring did not influence the distance between the centre of the screws and the pedicle wall. Distances only depended on the implantation side (right and left) and the height of implantation (caudal or cranial screw). Because of the low number of cases, no conclusion could be reached about the influence of root monitoring on the correct positioning of the screws. There was at least a non-significant trend towards more frequent perforation of the pedicle in the monitor group. In the present study, we showed that root monitoring had a significant effect on the scattering of transversal angles. These were increased compared to the control group. Otherwise, the implantation angle was not shown to depend on the use of neuromonitoring. Neuromonitoring did not influence blood loss or operative time. DISCUSSION: The data did not permit any conclusion as to whether this technique can minimise the frequency of pedicle screw malposition. The four coronal plane distances did not depend on the use of neuromonitoring. The inclination angle was also unaffected by neuromonitoring. The only parameter for which we found any effect was the transverse angle. The mean values were similar in both groups, but the variances were not equal. The effect of monitoring on the only parameter which could not be evaluated by fluoroscopy is thus rather unfavourable.

A Phase 1 Dose-Escalation Study of ASP2409, a Selective T-Cell Costimulation Inhibitor, in Stable Rheumatoid Arthritis Patients on Methotrexate Therapy.

ASP2409 represents a new class of CTLA4-Ig molecules with higher binding avidity and selectivity to CD86. This first-in-human study was to assess the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics of ASP2409 in stable rheumatoid arthritis patients on methotrexate therapy with a randomized, double-blind, placebo-controlled dose-escalation study design. Patients were enrolled and randomized in each of 8 dose-escalation cohorts ranging from 0.001 to 3.0 mg/kg to receive either ASP2409 or placebo in a sequential manner. Escalation to higher dose levels occurred in the absence of dose-limiting toxicity. A total of 57 patients completed the study. ASP2409 showed nonlinear PK over the dose range of 0.01 to 3.0 mg/kg following a single intravenous administration, indicating target-mediated drug disposition. Area under the concentration-time curve (AUC) and maximum concentration (Cmax ) increased at a greater than dose-proportional rate. The half-life of ASP2409 increased dose dependently and ranged from 1.57 to 6.68 days. ASP2409 showed a dose-dependent increase in the extent and duration of CD86 receptor occupancy. There were no clinically relevant safety issues up to a single dose of 3.0 mg/kg. No maximum tolerated dose was reached. The incidence and duration of antidrug antibodies did not correlate with adverse events. ClinicalTrials.gov identifier: NCT02171143.

Pharmacokinetics, Pharmacodynamics, Safety, and Tolerability of ASP2408, a Potent Selective T-Cell Costimulation Modulator After Single and Multiple Ascending Doses in Healthy Volunteers and RA Patients.

ASP2408 is a next-generation anti-cytotoxic T lymphocyte antigen-4 fusion protein engineered for improved CD86 binding affinity as a treatment for rheumatoid arthritis (RA). In 72 healthy subjects (n = 6/treatment), ASP2408 was administered as single ascending doses intravenously at 0.003 to 10.0 mg/kg or subcutaneously at 0.3 to 3.0 mg/kg. It showed decreased clearance and prolonged half-life with increasing doses, consistent with target-mediated disposition. The apparent bioavailability was 36.3%-56.7% across single subcutaneous doses. Sixteen RA patients (n = 8/treatment) on stable methotrexate received 3 × 3.0 mg/kg subcutaneously every 4 weeks or every 2 weeks. Similar to single-dose treatment, ASP2408 concentrations peaked 2 to 3 days postdose, with a median t1/2 of approximately 8 days. Using CD86 receptor occupancy (RO) as a mechanistic biomarker, ASP2408 demonstrated dose-dependent binding to its target. ASP2408 3.0 mg/kg subcutaneously every 4 weeks and every 2 weeks led to a mean %CD86 RO ≥ 74.7% and ≥ 81.5%, respectively, within each dosing interval. ASP2408 was well tolerated across studies with no evidence of dose-limiting toxicity or clinically significant changes in clinical laboratory test results, vital signs, or 12-lead electrocardiograms. ASP2408 elicited antidrug antibodies in the majority of patients, but with no clinical sequelae.

Trans-activation of human MYC: the second promoter is target for the stimulation by adenovirus E1a proteins.

The detailed mechanisms leading to transcriptional activation of the human MYC oncogene in general as well as in certain tumor cells are poorly understood. In view of the ability of a number of viral oncogenes to stimulate transcription in trans, the identification of cellular target genes could contribute to the understanding of components of the transformation process. It is demonstrated that the human MYC promoter is such an efficient target for the trans-acting activity mediated by E1a proteins of adenoviruses (Ad). Using the chloramphenicol acetyltransferase (CAT) gene as a marker for promoter activity, co-transfection of constructs containing both MYC promoters and most of the untranslated first exon with plasmids expressing the E1a gene of different adenoviruses stimulates CAT activity up to 24-fold. Trans-activation depends upon the presence of the second promoter (P2), and transcription is initiated at the authentic cap site of P2. This observation is confirmed by the behaviour of stably transformed cell lines carrying single or multiple copies of MYC-cat constructs, which were transfected either with E1a-expressing plasmids, infected with Ad5, or fused with 293 cells constitutively expressing E1a protein. These results suggest that E1a proteins can lead to an imbalance of the regulation of the human MYC gene, which might be a sufficient prerequisite for initiation and progression of transformation.

Stabilities of neutral and basic esters of bendamustine in plasma compared to the parent compound: kinetic investigations by HPLC.

Esters of the cytostatic bendamustine (1), previously demonstrated to be much more potent than the parent compound as antiproliferative agents in vitro, were investigated for stability in buffer and plasma, as well as against porcine liver esterase in the presence of different amounts of albumin using a validated RP-HPLC method with fluorescence detection. The hydrolysis of the nitrogen mustard moiety was retarded (for 1: approximately 130 vs. 11 min) in the presence of plasma proteins. For the derivatives, both cleavage of ester and nitrogen mustard moieties were analyzed. Enzymatic hydrolysis was very fast in the case of 2-pyrrolidino-, 2-piperidino- and 2-(4-methylpiperazino)-ethyl esters, whereas methyl, ethyl, morpholinoethyl and branched 2-pyrrolidinoethyl esters were considerably more stable (half-lives between 41 and 116 min, compared to <5 min). Inhibition by physostigmine indicated unspecific cholinesterases to be involved in the rapid ester cleavage. Due to lower protein content and higher enzymatic activity in murine compared to human plasma, reduced stability of all investigated esters in mouse plasma (t½<2 min) has to be taken into account with respect to the design of animal studies.

Molecular determinants for the high constitutive activity of the human histamine H4 receptor: functional studies on orthologues and mutants.

BACKGROUND AND PURPOSE: Some histamine H4 receptor ligands act as inverse agonists at the human H4 receptor (hH4 R), a receptor with exceptionally high constitutive activity, but as neutral antagonists or partial agonists at the constitutively inactive mouse H4 receptor (mH4 R) and rat H4 receptor (rH4 R). To study molecular determinants of constitutive activity, H4 receptor reciprocal mutants were constructed: single mutants: hH4 R-F169V, mH4 R-V171F, hH4 R-S179A, hH4 R-S179M; double mutants: hH4 R-F169V+S179A, hH4 R-F169V+S179M and mH4 R-V171F+M181S. EXPERIMENTAL APPROACH: Site-directed mutagenesis with pVL1392 plasmids containing hH4 or mH4 receptors were performed. Wild-type or mutant receptors were co-expressed with Gαi2 and Gß1 γ2 in Sf9 cells. Membranes were studied in saturation and competition binding assays ([(3) H]-histamine), and in functional [(35) S]-GTPγS assays with inverse, partial and full agonists of the hH4 receptor. KEY RESULTS: Constitutive activity decreased from the hH4 receptor via the hH4 R-F169V mutant to the hH4 R-F169V+S179A and hH4 R-F169V+S179M double mutants. F169 alone or in concert with S179 plays a major role in stabilizing a ligand-free active state of the hH4 receptor. Partial inverse hH4 receptor agonists like JNJ7777120 behaved as neutral antagonists or partial agonists at species orthologues with lower or no constitutive activity. Some partial and full hH4 receptor agonists showed decreased maximal effects and potencies at hH4 R-F169V and double mutants. However, the mutation of S179 in the hH4 receptor to M as in mH4 receptor or A as in rH4 receptor did not significantly reduce constitutive activity. CONCLUSIONS AND IMPLICATIONS: F169 and S179 are key amino acids for the high constitutive activity of hH4 receptors and may also be of relevance for other constitutively active GPCRs. LINKED ARTICLES: This article is part of a themed issue on Histamine Pharmacology Update published in volume 170 issue 1. To view the other articles in this issue visit http://onlinelibrary.wiley.com/doi/10.1111/bph.2013.170.issue-1/issuetoc.