Human diamine oxidase cellular binding and internalization in vitro and rapid clearance in vivo are not mediated by N-glycans but by heparan sulfate proteoglycan interactions.

Human diamine oxidase (hDAO) rapidly inactivates histamine by deamination. No pharmacokinetic data are available to better understand its potential as a new therapeutic modality for diseases with excess local and systemic histamine, like anaphylaxis, urticaria or mastocytosis. After intravenous administration of recombinant hDAO to rats and mice, more than 90% of the dose disappeared from the plasma pool within 10 min. Human DAO did not only bind to various endothelial and epithelial cell lines in vitro, but was also unexpectedly internalized and visible in granule-like structures. The uptake of rhDAO into cells was dependent on neither the asialoglycoprotein-receptor (ASGP-R) nor the mannose receptor (MR) recognizing terminal galactose or mannose residues, respectively. Competition experiments with ASGP-R and MR ligands did not block internalization in vitro or rapid clearance in vivo. The lack of involvement of N-glycans was confirmed by testing various glycosylation mutants. High but not low molecular weight heparin strongly reduced the internalization of rhDAO in HepG2 cells and HUVECs. Human DAO was readily internalized by CHO-K1 cells, but not by the glycosaminoglycan- and heparan sulfate-deficient CHO cell lines pgsA-745 and pgsD-677, respectively. A docked heparin hexasaccharide interacted well with the predicted heparin binding site 568RFKRKLPK575. These results strongly imply that rhDAO clearance in vivo and cellular uptake in vitro is independent of N-glycan interactions with the classical clearance receptors ASGP-R and MR, but is mediated by binding to heparan sulfate proteoglycans followed by internalization via an unknown receptor.

Histone deacetylase 1 (HDAC1): A key player of T cell-mediated arthritis.

Rheumatoid Arthritis (RA) represents a chronic T cell-mediated inflammatory autoimmune disease. Studies have shown that epigenetic mechanisms contribute to the pathogenesis of RA. Histone deacetylases (HDACs) represent one important group of epigenetic regulators. However, the role of individual HDAC members for the pathogenesis of arthritis is still unknown. In this study we demonstrate that mice with a T cell-specific deletion of HDAC1 (HDAC1-cKO) are resistant to the development of Collagen-induced arthritis (CIA), whereas the antibody response to collagen type II was undisturbed, indicating an unaltered T cell-mediated B cell activation. The inflammatory cytokines IL-17 and IL-6 were significantly decreased in sera of HDAC1-cKO mice. IL-6 treated HDAC1-deficient CD4+ T cells showed an impaired upregulation of CCR6. Selective inhibition of class I HDACs with the HDAC inhibitor MS-275 under Th17-skewing conditions inhibited the upregulation of chemokine receptor 6 (CCR6) in mouse and human CD4+ T cells. Accordingly, analysis of human RNA-sequencing (RNA-seq) data and histological analysis of synovial tissue samples from human RA patients revealed the existence of CD4+CCR6+ cells with enhanced HDAC1 expression. Our data indicate a key role for HDAC1 for the pathogenesis of CIA and suggest that HDAC1 and other class I HDACs might be promising targets of selective HDAC inhibitors (HDACi) for the treatment of RA.

Impact of infrared radiation on UVB-induced skin tumourigenesis in wild type C57BL/6 mice.

Although infrared radiation (IR) represents more than 50% of the solar radiation reaching the Earth's surface, this waveband has been hardly investigated in terms of tumourigenesis. The objective of the present study was to investigate the influence of IR on ultraviolet B (UVB)-induced carcinogenesis in male and female wild type mice. For this purpose, male and female C57BL/6N mice were subjected to a long-term irradiation protocol. Mice were irradiated once neonatally and from the age of eight weeks for 36 weeks with a cumulative dose of 576 kJ m-2 UVB and/or 78 895 kJ m-2 IR. In order to resemble natural sun irradiation, exposure to physiological doses of UVB and IR was performed simultaneously. Mice were screened for arising lesions twice a week. Lesions were excised and histologically diagnosed. Kaplan-Meier analyses were carried out and lesion counts and cumulated hazard rates for the development of lesions in the UVB and IR + UVB-exposed groups in male and female mice were compared. We found that IR-exposure did not change the number of epithelial malignant tumours in UVB-exposed wild type mice. In combination with IR there was a tendency of more tumours with increased malignancy: 23 vs. seven spindle cell shaped sarcomas and seven vs. two MelanA+/S100+ tumours in groups of 35 C57BL/6 mice. IR did not influence UVB-induced carcinogenesis differently in male and female mice. However, comparing UVB and sham irradiated animals irrespective of IR exposure, UVB-induced non-epithelial tumours arose significantly earlier in male mice than in female mice.

Hard Tissue Augmentation of Aged Bone by Means of a Tin-Free PLLA-PCL Co-Polymer Exhibiting in vivo Anergy and Long-Term Structural Stability.

BACKGROUND: Due to aging, tissue regeneration gradually declines. Contemporary strategies to promote tissue-specific regeneration, in particular in elderly patients, often include synthetic material apt for implantation primarily aiming at upholding body functions and regaining appropriate anatomical and functional integrity. OBJECTIVE: Biomaterials suitable for complex reconstruction surgical procedures have to exert high physicochemical stability and biocompatibility. METHOD: A polymer made of poly-L-lactic acid and poly-ε-caprolactone was synthesized by means of a novel tin-free catalytic process. The material was tested in a bioreactor-assisted perfusion culture and implanted in a sheep model for lateral augmentation of the mandible. Histological and volumetric evaluation was performed 3 and 6 months post-implantation. RESULTS: After synthesis the material could be further refined by cryogrinding and sintering, thus yielding differently porous scaffolds that exhibited a firm and stable appearance. In perfusion culture, no disintegration was observed for extended periods of up to 7 weeks, while mesenchymal stromal cells readily attached to the material, steadily proliferated, and deposited extracellular calcium. The material was tested in vivo together with autologous bone marrow-derived stromal cells. Up to 6 months post-implantation, the material hardly changed in shape with composition also refraining from foreign body reactions. CONCLUSION: Given the long-term shape stability in vivo, featuring imperceptible degradation and little scarring as well as exerting good compatibility to cells and surrounding tissues, this novel biomaterial is suitable as a space filler in large anatomical defects.

Bioavailability, metabolism, and excretion of a complex Alternaria culture extract versus altertoxin II: a comparative study in rats.

Despite the frequent infection of agricultural crops by Alternaria spp., their toxic secondary metabolites and potential food contaminants lack comprehensive metabolic characterization. In this study, we investigated their bioavailability, metabolism, and excretion in vivo. A complex Alternaria culture extract (50 mg/kg body weight) containing 11 known toxins and the isolated lead toxin altertoxin II (0.7 mg/kg body weight) were administered per gavage to groups of 14 Sprague Dawley rats each. After 3 h and 24 h, plasma, urine and feces were collected to determine toxin recoveries. For reliable quantitation, an LC-MS/MS method for the simultaneous detection of 20 Alternaria toxins and metabolites was developed and optimized for either biological matrix. The obtained results demonstrated efficient excretion of alternariol (AOH) and its monomethyl ether (AME) via feces (> 89%) and urine (> 2.6%) after 24 h, while the majority of tenuazonic acid was recovered in urine (20 and 87% after 3 and 24 h, respectively). Moreover, modified forms of AOH and AME were identified in urine and fecal samples confirming both, mammalian phase-I (4-hydroxy-AOH) and phase-II (sulfates) biotransformation in vivo. Despite the comparably high doses, perylene quinones were recovered only at very low levels (altertoxin I, alterperylenol, < 0.06% in urine and plasma, < 5% in feces) or not at all (highly genotoxic, epoxide-holding altertoxin II, stemphyltoxin III). Interestingly, altertoxin I was detected in all matrices of rats receiving altertoxin II and suggests enzymatic de-epoxidation in vivo. In conclusion, the present study contributes valuable information to advance our understanding of the emerging Alternaria mycotoxins and their relevance on food safety.

Pervasion of beta-tricalcium phosphate with nanodiamond particles yields efficient and safe bone replacement material amenable for biofunctionalization and application in large-size osseous defect healing.

Biofunctionalization of scaffold materials can enable the healing of large bone defects. In case of minimally invasive guided-bone regeneration (GBR), limitations are however hard-to-control side effects related to the potential release of biofactors into the systemic environment. Biofactors can be stably bound to nanodiamond particles (ND) through physisorption. We therefore tested the biological and clinical effects of refining beta-tricalcium phosphate (ßTCP) with ND in vitro and in vivo. In vitro, ßTCP carrying 4% ND resulted in enhanced attachment of mesenchymal stem cells. When assessing GBR after lateral augmentation of the mandible in sheep showed that ND in ßTCP resulted in a consistently steady bone formation when compared to pure ßTCP, demonstrating the biological inert behavior and the potential clinical safety of ND.

CCR6 controls autoimmune but not innate immunity-driven experimental arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease, characterized by synovial infiltration of various inflammatory cells. Chemokines are involved in controlling the recruitment of different cell types into the synovial membrane. The role of CCR6 in the development of arthritis so far remains unclear. In this study, we investigated the role of CCR6 in the pathogenesis of arthritis using three different murine arthritis models. Compared to WT animals, CCR6-/- mice developed less clinical signs of arthritis in the collagen-induced arthritis model but not in the K/BxN serum transfer arthritis model and in the human tumour necrosis factor transgenic arthritis model, suggesting a defect in adaptive effector functions but intact innate effector functions in the development of arthritis in CCR6-/- animals. In line with this, anti-collagen antibody levels were significantly reduced in CCR6-/- mice compared with WT mice. Moreover, we demonstrate enhanced osteoclastogenesis in vitro in CCR6-/- mice compared with WT mice. However, we did not detect differences in bone mass under steady state conditions in vivo between WT and CCR6-deficient mice. These data suggest that CCR6 is crucially involved in adaptive but not in innate immunity-driven arthritis. CCR6 or its chemokine ligand CCL20 might represent a possible new target for the treatment of RA.

Differential effects of purified low molecular weight Poly(I:C) in the maternal immune activation model depend on the laboratory environment.

The Poly (I:C) (polyriboinosinic-polyribocytidilic acid) paradigm of maternal immune activation (MIA) is most widely used as experimental model for the evaluation of the effects of gestational infection on the brain and behavior of the progeny. We have previously reported significant batch-to-batch variability in the effects of Poly (I:C), purchased from the same supplier (Sigma-Aldrich), on maternal and fetal immune responses and found these differences to be dependent on the relative amount of synthetic double-stranded RNA fragments in the high versus low molecular weight (LMW) range contained in the compound. We here resorted to Poly (I:C) purified for LMW dsRNA fragments to establish a MIA paradigm with increased reproducibility and enhanced standardization in an effort to refine the MIA paradigm and characterize its effect on offspring behavior. We found that the parallel application of LMW Poly (I:C) in two different MIA-experienced laboratories (Vienna and Zurich) yielded differential outcomes in terms of maternal immune responses and behavioral phenotypes in the offspring generation. In both experimental sites, administration of LMW Poly (I:C) induced a significant sickness response and cytokine induction in the pregnant dam and fetal brains, while the expected deficit in sociability as one main behavioral outcome parameter in the MIA progeny, was only present in the Zurich, but not the Vienna cohort. We conclude that although using Poly (I:C) purified for a defined molecular weight range reduces batch-to-batch variability, it does not make the MIA model more reliable and robust. The differential response in behavioral phenotypes of the MIA offspring between the two laboratories illustrates the highly complex interaction between prenatal and postnatal milieus - including the laboratory environment - that determine offspring phenotypic outcomes after MIA. Consequently, establishing a new MIA protocol or implementing the MIA model firstly under new or changed environmental conditions must include the assessment of offspring behavior to ensure solid and reproducible experimental outcomes.

Interaction of the pre- and postnatal environment in the maternal immune activation model.

Adverse influences during pregnancy are associated with a range of unfavorable outcomes for the developing offspring. Maternal psychosocial stress, exposure to infections and nutritional imbalances are known risk factors for neurodevelopmental derangements and according psychiatric and neurological manifestations later in offspring life. In this context, the maternal immune activation (MIA) model has been extensively used in preclinical research to study how stimulation of the maternal immune system during gestation derails the tightly coordinated sequence of fetal neurodevelopment. The ensuing consequence of MIA for offspring brain structure and function are majorly manifested in behavioral and cognitive abnormalities, phenotypically presenting during the periods of adolescence and adulthood. These observations have been interpreted within the framework of the "double-hit-hypothesis" suggesting that an elevated risk for neurodevelopmental disorders results from an individual being subjected to two adverse environmental influences at distinct periods of life, jointly leading to the emergence of pathology. The early postnatal period, during which the caregiving parent is the major determinant of the newborn´s environment, constitutes a window of vulnerability to external stimuli. Considering that MIA not only affects the developing fetus, but also impinges on the mother´s brain, which is in a state of heightened malleability during pregnancy, the impact of MIA on maternal brain function and behavior postpartum may importantly contribute to the detrimental consequences for her progeny. Here we review current information on the interaction between the prenatal and postnatal maternal environments in the modulation of offspring development and their relevance for the pathophysiology of the MIA model.

Exposure to soiled bedding reduces abnormal repetitive behaviors in mice.

Hygiene management protocols in laboratory mouse husbandries worldwide most commonly employ soiled bedding-exposed sentinel mice to monitor the occurrence of infections in mouse colonies. Using this approach, sentinel mice repeatedly receive a mixture of used bedding, supplied by a variety of cages of a defined hygienic unit for a period of several months. Hereby, microorganisms shed in the used bedding can infect the sentinel animals and can be detected in subsequent health monitoring procedures. However, murine excrements carry more than only microorganisms. Mouse feces and urine also contain a multitude of olfactory molecules, which the animals use to code information about social status and context. However, if and how the persistent and repeated experience with these odor cues affects the behavior of sentinel mice, has not yet been explored. To address this question, we conducted a longitudinal study for neurochemical output parameters related to an organism's responsiveness to challenging conditions, and for the exploratory assessment of a panel of home cage behaviors in soiled bedding and control female C57BL/6J mice. We found that the number of mice showing abnormal repetitive behaviors, including barbering and bar mouthing, was lower in the soiled bedding group. While neutrophil/lymphocyte ratios and fecal corticosterone metabolites did not differ between groups, the within-group variance of the neutrophil/lymphocyte ratio was reduced in the soiled bedding group. These results show that the occurrence of abnormal repetitive behaviors is lower in sentinel than in control mice and suggest a beneficial effect of soiled bedding on the welfare of laboratory mice and on outcome variability.

Markers for DNA damage are induced in the rat colon by the Alternaria toxin altertoxin-II, but not a complex extract of cultured Alternaria alternata.

Mycotoxins produced by Alternaria spp. act genotoxic in cell-based studies, but data on their toxicity in vivo is scarce and urgently required for risk assessment. Thus, male Sprague-Dawley rats received single doses of a complex Alternaria toxin extract (CE; 50 mg/kg bw), altertoxin II (ATX-II; 0.21 mg/kg bw) or vehicle by gavage, one of the most genotoxic metabolites in vitro and were sacrificed after 3 or 24 h, respectively. Using SDS-PAGE/Western Blot, a significant increase of histone 2a.X phosphorylation and depletion of the native protein was observed for rats that were exposed to ATX-II for 24 h. Applying RT-PCR array technology we identified genes of interest for qRT-PCR testing, which in turn confirmed an induction of Rnf8 transcription in the colon of rats treated with ATX-II for 3 h and CE for 24 h. A decrease of Cdkn1a transcription was observed in rats exposed to ATX-II for 24 h, possibly indicating tissue repair after chemical injury. In contrast to the observed response in the colon, no markers for genotoxicity were induced in the liver of treated animals. We hereby provide the first report of ATX-II as a genotoxicant in vivo. Deviating results for similar concentrations of ATX-II in a natural Alternaria toxin mixture argue for substantial mixture effects.

Heparin-binding motif mutations of human diamine oxidase allow the development of a first-in-class histamine-degrading biopharmaceutical.

Background: Excessive plasma histamine concentrations cause symptoms in mast cell activation syndrome, mastocytosis, or anaphylaxis. Anti-histamines are often insufficiently efficacious. Human diamine oxidase (hDAO) can rapidly degrade histamine and therefore represents a promising new treatment strategy for conditions with pathological histamine concentrations. Methods: Positively charged amino acids of the heparin-binding motif of hDAO were replaced with polar serine or threonine residues. Binding to heparin and heparan sulfate, cellular internalization and clearance in rodents were examined. Results: Recombinant hDAO is rapidly cleared from the circulation in rats and mice. After mutation of the heparin-binding motif, binding to heparin and heparan sulfate was strongly reduced. The double mutant rhDAO-R568S/R571T showed minimal cellular uptake. The short α-distribution half-life of the wildtype protein was eliminated, and the clearance was significantly reduced in rodents. Conclusions: The successful decrease in plasma clearance of rhDAO by mutations of the heparin-binding motif with unchanged histamine-degrading activity represents the first step towards the development of rhDAO as a first-in-class biopharmaceutical to effectively treat diseases characterized by excessive histamine concentrations in plasma and tissues. Funding: Austrian Science Fund (FWF) Hertha Firnberg program grant T1135 (EG); Sigrid Juselius Foundation, Medicinska Understödsförening Liv och Hälsa rft (TAS and SeV).

Beneficial Metabolic Effects of TREM2 in Obesity Are Uncoupled From Its Expression on Macrophages.

Obesity-induced white adipose tissue (WAT) hypertrophy is associated with elevated adipose tissue macrophage (ATM) content. Overexpression of the triggering receptor expressed on myeloid cells 2 (TREM2) reportedly increases adiposity, worsening health. Paradoxically, using insulin resistance, elevated fat mass, and hypercholesterolemia as hallmarks of unhealthy obesity, a recent report demonstrated that ATM-expressed TREM2 promoted health. Here, we identified that in mice, TREM2 deficiency aggravated diet-induced insulin resistance and hepatic steatosis independently of fat and cholesterol levels. Metabolomics linked TREM2 deficiency with elevated obesity-instigated serum ceramides that correlated with impaired insulin sensitivity. Remarkably, while inhibiting ceramide synthesis exerted no influences on TREM2-dependent ATM remodeling, inflammation, or lipid load, it restored insulin tolerance, reversing adipose hypertrophy and secondary hepatic steatosis of TREM2-deficient animals. Bone marrow transplantation experiments revealed unremarkable influences of immune cell-expressed TREM2 on health, instead demonstrating that WAT-intrinsic mechanisms impinging on sphingolipid metabolism dominate in the systemic protective effects of TREM2 on metabolic health.

First insights into Alternaria multi-toxin in vivo metabolism.

Alternaria mycotoxins frequently contaminate agricultural crops and may impact animal and human health. However, data on mammalian metabolism and potential biomarkers of exposure for human biomonitoring (HBM) are scarce. Here, we report the preliminary investigation with respect to metabolism and excretion of Alternaria toxins in Sprague Dawley rats. Four animals were housed in metabolic cages for 24 h after gavage administration of an Alternaria alternata culture extract containing ten known toxins. LC-MS/MS analysis of 17 Alternaria toxins in urine and fecal samples allowed to gain first insights regarding xenobiotic metabolism and excretion rates. Alternariol (6-10%), alternariol monomethyl ether (AME, 6-7%) and tenuazonic acid (up to 55%) were recovered in urine and fecal samples (9%, 87%, 0.3%, respectively), while perylene quinones administered at comparatively high levels, were either determined at very low levels (up to 0.5% altertoxin I in urine and 15% in feces; 0.2% alterperylenol in urine and 3% in feces) or not at all (altertoxin II, stemphyltoxin III). AME-3-sulfate, which was not present in the administered extract, was determined in urine, representing up to 23% of the AME intake. Critical evaluation of the applied sample preparation protocol and LC-MS/MS analysis revealed interesting preliminary results and information crucial for improving follow-up experiments.