@inproceedings{herrmann_2024, title={Minimally invasive sampling of canine skin for investigation of the transcriptome - Tolerability and Feasibility}, booktitle={World Congress of Veterinary Dermatology}, author={Herrmann, I.}, year={2024} }
@misc{herrmann_sanchez_2023, title={Efficacy and Safety of Subcutaneous Allergen-Specific Immuno-Therapy in Horses with Allergic Cutaneous and Respiratory Diseases-A Systematic Review}, volume={10}, ISSN={["2306-7381"]}, url={https://doi.org/10.3390/vetsci10100613}, DOI={10.3390/vetsci10100613}, abstractNote={Allergen-specific immunotherapy (AIT) is the only current intervention that has the ability to modify the immune response toward a tolerogenic state. This study aimed to assess the efficacy and safety of AIT in horses with allergic diseases in a systematic manner. Three databases were searched to identify articles reporting clinical outcomes and adverse events associated with AIT. The articles were evaluated for beneficial responses to AIT, defined as a ≥50% reduction in clinical signs, and clinical remission. Horses with respiratory diseases, urticaria, and pruritic dermatitis receiving insect monotherapy or multi-allergen AIT were included. All adverse events were graded, and analytical and confounding biases were assessed. The results showed that multi-allergen AIT had a beneficial response in 75% of horses with respiratory diseases, 88% with urticaria, and 56% with pruritic dermatitis. However, horses treated solely with insect AIT for pruritic dermatitis had a lower response rate (36%). Self-limiting local reactions were the most common adverse events, with systemic reactions grade II accounting for 11% of reported events. Analytical and confounding biases were identified as major limitations in the available studies. Further research is needed to address these biases and provide stronger evidence on the efficacy and safety of AIT in horses with allergic diseases.}, number={10}, journal={VETERINARY SCIENCES}, author={Herrmann, Ina and Sanchez, Adrianna Jordan}, year={2023}, month={Oct} }
@article{herrmann_mamo_holmes_mohammed_murphy_bizikova_2022, title={Long‐term effects of ciclosporin and oclacitinib on mediators of tolerance, regulatory T‐cells, IL‐10 and TGF‐β, in dogs with atopic dermatitis}, volume={34}, ISSN={0959-4493 1365-3164}, url={http://dx.doi.org/10.1111/vde.13140}, DOI={10.1111/vde.13140}, abstractNote={AbstractBackgroundAtopic dogs often are managed with allergen‐specific immunotherapy (AIT) and concurrent dosages of ciclosporin (CSA) or oclacitinib to alleviate their clinical signs. Both drugs might affect proper tolerance induction by inhibiting regulatory T‐cell (Treg) induction.Hypothesis/ObjectivesWe evaluated Treg cell numbers and serum interleukin (IL)‐10 and transforming growth factor‐beta (TGF‐β)1 levels in dogs diagnosed with atopic dermatitis (AD) and successfully treated with either CSA or oclacitinib for nine or more months.AnimalsWe included 15 dogs receiving oclacitinib, 14 dogs treated with CSA, 15 healthy dogs, 13 dogs with untreated moderate‐to‐severe AD and 15 atopic dogs controlled with AIT.Materials and MethodsPeripheral blood CD4+CD25+FOXP3+ T‐cell percentages were determined using flow cytometry. Serum concentrations of IL‐10 and TGF‐β1 were measured by enzyme‐linked immunosorbent assay.ResultsThe percentage of Treg cells in the CSA group was significantly lower in comparison with the healthy group (p = 0.0003), the nontreated AD group (p = 0.0056) or the AIT group (p = 0.0186). There was no significant difference in Treg cell percentages between the CSA and oclacitinib groups, nor between the oclacitinib and the healthy, nontreated AD or AIT‐treated dogs. No significant differences were detected in IL‐10 and TGF‐β1 serum concentrations between the five groups.Conclusions and Clinical RelevanceLower Treg cell percentages in the CSA‐treated dogs suggest an impact of this drug on this cell population; however, it does not necessarily mean that it diminishes tolerance. Functionality and cytokine production may be more important than the number of Treg cells. Further studies evaluating the treatment outcome of dogs receiving AIT and concurrent drugs are needed to show clinical relevance.}, number={2}, journal={Veterinary Dermatology}, publisher={Wiley}, author={Herrmann, Ina and Mamo, Lisa B. and Holmes, Jenny and Mohammed, Javid P. and Murphy, K. Marcia and Bizikova, Petra}, year={2022}, month={Dec}, pages={107–114} }
@article{herrmann_nagel_luff_traverson_murphy_linder_2022, title={Pathology in Practice}, volume={259}, ISSN={0003-1488}, url={http://dx.doi.org/10.2460/javma.20.12.0688}, DOI={10.2460/javma.20.12.0688}, abstractNote={Abstract
In collaboration with the American College of Veterinary Pathologists}, number={S2}, journal={Journal of the American Veterinary Medical Association}, publisher={American Veterinary Medical Association (AVMA)}, author={Herrmann, Ina and Nagel, Jonathan and Luff, Jennifer and Traverson, Marine and Murphy, K. Marcia and Linder, Keith E.}, year={2022}, month={May}, pages={1–5} }
@inproceedings{herrmann_2021, title={A shortened immunotherapy dose-escalation saves time, but is it safe? a case-control study comparing the rates of adverse reactions between conventional and fast escalation subcutaneous immunotherapy protocols during the induction phase}, booktitle={North American Veterinary Dermatology Forum (NAVDF)}, author={Herrmann, I.}, year={2021} }
@article{herrmann_linder_meurs_friedenberg_cullen_olby_bizikova_2021, title={Canine junctional epidermolysis bullosa due to a novel mutation in LAMA3 with severe upper respiratory involvement}, volume={32}, ISSN={["1365-3164"]}, url={https://doi.org/10.1111/vde.12972}, DOI={10.1111/vde.12972}, abstractNote={BackgroundJunctional epidermolysis bullosa (JEB) is a group of congenital blistering skin diseases characterized by clefting through the lamina lucida of the basement membrane zone.ObjectivesTo characterize the clinical and morphological features of a congenital mechanobullous disease in a litter of puppies with severe upper respiratory involvement, and to identify an associated genetic variant.AnimalsFive of eight puppies in an Australian cattle dog cross‐bred litter showed signs of skin fragility. Three were stillborn and one died at one month of age. The two surviving puppies were presented with blistering skin disease and severe respiratory distress. Additionally, one unaffected sibling was examined and blood was obtained for genetic testing.Methods and materialsPost‐mortem examination, histopathological evaluation and electron microscopy were performed. Whole genome sequencing (WGS) of one affected puppy was compared to a database of 522 dogs of 55 different breeds for variant analysis. Sanger sequencing of one additional affected and one unaffected sibling confirmed the variant.ResultsClinically, severe mucocutaneous ulcers occurred in frictional areas with claw sloughing. Histopathological results revealed subepidermal clefts and electron microscopy confirmed the split in the lamina lucida. Post‐mortem examination documented extensive pharyngeal and laryngeal lesions with granulation tissue and fibrinous exudate obscuring the airway. Moderate tracheal hypoplasia contributed. The WGS revealed a novel missense variant in the laminin α3‐chain XP_537297.2p(Asp2867Val), with an autosomal recessive mode of inheritance.Conclusions and clinical relevanceA novel variant in LAMA3 caused a generalized and severe phenotype of JEB with an unique clinical presentation of upper airway obstruction.}, number={4}, journal={VETERINARY DERMATOLOGY}, author={Herrmann, Ina and Linder, Keith E. and Meurs, Kathryn M. and Friedenberg, Steven G. and Cullen, Jonah and Olby, Natasha and Bizikova, Petra}, year={2021}, month={Aug}, pages={379-+} }
@article{levy_linder_mamo_herrmann_bizikova_2020, title={Cutaneous polyautoimmunity in two unrelated dogs: pemphigus foliaceus and generalized discoid lupus erythematosus}, volume={31}, ISSN={["1365-3164"]}, url={https://doi.org/10.1111/vde.12851}, DOI={10.1111/vde.12851}, abstractNote={BackgroundPolyautoimmunity, the concurrent expression of two or more distinct autoimmune diseases (ADs) in a single individual, is a known phenomenon in humans and has been rarely reported in dogs. To the best of the authors’ knowledge, comorbid pemphigus foliaceus (PF) and generalized discoid lupus erythematosus (GDLE) has not been reported in dogs.Hypothesis/ObjectivesTo describe the clinical, histological and immunological features and treatment outcome of two unrelated dogs with comorbid PF and GDLE.AnimalsOne 10‐year‐old, spayed German shepherd dog and one 8‐year‐old, castrated American Staffordshire terrier presented for evaluation of a symmetrical, facial‐ and/or pedal‐dominant pustular dermatitis with concurrent, truncal scaly plaques.MethodsFor each dog, clinicopathological characterization included physical examination, lesion cytological evaluation, bacterial culture and sensitivity testing, skin histopathological investigation and direct and indirect immunofluorescence testing. Additional diagnostic imaging and haematological testing was performed to exclude extracutaneous disease.ResultsBoth dogs exhibited lesions clinically and histologically compatible with PF and GDLE. Moreover, one dog exhibited generalized leucotrichia and chronic superficial keratitis. Remission was achieved with immunosuppressive dosages of prednisolone [high‐dose pulse (Case 1) or standard immunosuppressive dosage (Case 2)] and ciclosporin (5–6 mg/kg/day). Tissue‐bound antikeratinocyte immunoglobulin (Ig)G and IgM were detected in both dogs. A weak basement membrane zone deposit of C3 was seen in one dog. Circulating antikeratinocyte and anti‐desmocollin‐1 IgG were detected in one dog.Conclusions and clinical importanceCutaneous polyautoimmunity can occur in the dog. Depending on the specific disease combinations, overlapping clinical features may present diagnostic and/or therapeutic challenges. Moreover, these cases should be monitored for development of additional cutaneous or extra‐cutaneous AD(s).}, number={4}, journal={VETERINARY DERMATOLOGY}, author={Levy, Britt J. and Linder, Keith E. and Mamo, Lisa B. and Herrmann, Ina and Bizikova, Petra}, year={2020}, month={Aug}, pages={325-+} }
@article{gotovina_bianchini_fazekas‐singer_herrmann_pellizzari_haidl_hufnagl_karagiannis_marshall_jensen‐jarolim_2020, title={Epinephrine drives human M2a allergic macrophages to a regulatory phenotype reducing mast cell degranulation in vitro}, volume={75}, ISSN={0105-4538 1398-9995}, url={http://dx.doi.org/10.1111/all.14299}, DOI={10.1111/all.14299}, abstractNote={To the Editor, As the prevalence of allergies rises, the impact of social factors such as physiological stress has gained much attention. While stress is suggested to exacerbate allergic conditions, including asthma and atopic dermatitis, less is known about the effect of acute stress mediator epinephrine on allergic M2a macrophages in a Th2 environment. This study aimed to investigate whether human M2a macrophages express adrenergic receptors to respond to epinephrine and what effect epinephrine could exhibit on M2a macrophages in an in vitro Th2 environment. We further assessed whether epinephrine-treated M2a macrophages could affect IgE-mediated degranulation in human mast cells in vitro. To study the effect of epinephrine on human M2a macrophages, we isolated monocytes from healthy donors and matured them in the presence of M-CSF according to a standard protocol1 into monocyte-derived macrophages (M0). M0 were subsequently treated with IL-4 and IL-13 to differentiate them into M2a phenotype, which showed higher expression of CD206 marker and IL-10 production. Detailed information on this study is available in this article's online supplementary information. The presence of the β2-adrenergic receptor (β2-AR) was confirmed in the M2a subtype, but no expression of α2A-AR, β1-AR, and β3-AR was detected (online repository; Figure S2D-E). The 16-hour treatment of M2a macrophages with 1 µmol/L epinephrine led to a significant upregulation of the cytokines IL-10 (P = .0131), TNF (P = .0012) and IL-6 (P = .0001), while no M1 marker IL-12 was detected (Figure 1A-D). This effect was not observed in the supernatants of M2a macrophages treated with the vehicle (negative control). Also, CD86 surface marker expression was significantly upregulated (P = .0313) (Figure 1G, Figure S3), indicating an antigen presentation capacity of this phenotype. Since epinephrine can induce cytokine production already after a few hours, we also observed the mRNA production of IL-10, IL-6, TNF, IL-1β , and CCL1 after 2 hours. Other M2 markers, including CCL2, CCL22, CCL18, and TGF-β, were less affected, and expression of IFN-γ was not detected after epinephrine treatment (Figure 2A). The production of anti-inflammatory IL-10 cytokine alongside IL-6, TNF, and IL-1β and upregulation of CD86 suggest that epinephrine can drive M2a macrophages towards an immunoregulatory M2b phenotype in vitro. Since the M2b phenotype is commonly induced by exposure to immune complexes and TLR ligands, which was not the case in our study, and we did not observe CCL1 production in the supernatants of epinephrine-treated M2a macrophages,1, 2 we termed this immunoregulatory phenotype "M2b-like." It is important to note that the immunoregulatory function of this phenotype was confirmed in vitro on human cord blood–derived mast cells (CBMCs), where treatment with supernatants from epinephrine-treated M2b-like macrophages significantly reduced the IgE-mediated β-hexosaminidase degranulation (P = .0013). Interestingly, this effect was significantly pronounced compared to treatment with epinephrine alone (P ≤ .05) (Figure 2B). To the best of our knowledge, this is the first report about the presence of the β2-AR receptor on the M2a macrophage phenotype, which is an important player in allergy. We, however, acknowledge that our study has its limitations. Although µM epinephrine in mouse cells can induce regulatory macrophages3 and dose-dependent studies of epinephrine on human monocytes revealed the strongest effect on chemokine/cytokine production in 1-10 µmol/L concentration range, often used to stimulate human monocytes in vitro,4, 5 our results do not necessarily translate into real human setting. However, there is reason to believe that during stress the local epinephrine concentrations at the immunological synapse are higher than in circulation due to sympathetic neuronal discharge and local catecholamine production from neighbouring immune cells (previously termed "diffusely expressed adrenergic organ"6). Another limitation of results (Figure 2A) is the normalization against a single housekeeping gene. We acknowledge that under given conditions, using a second gene for normalization had been advisable. This was a study on epinephrine effect on in vitro Th2 inflammation. To translate these data and develop targeted therapies in the future, it would be important to obtain the information on the exact signalling pathway that epinephrine might have activated on M2a human macrophages and drive the M2b-like phenotype. FcR signalling known to induce the M2b phenotype by activating phosphoinositide 3-kinase (PI3K)2 , may be a possible pathway induced by epinephrine in our study; furthermore, catecholamine activation of a β2-AR noncanonical pathway through phosphoinositol 3-kinase (PI3K) induced regulatory macrophages in mice.3 Even though M2b-like macrophages retain the ability to produce many pro-inflammatory cytokines including IL-6, TNF, and IL-1β, the upregulation of IL-10 (IL-10high/IL-12low) is certainly a central part of this phenotype and in the range reported in the previous studies.1, 7 Future studies should address the involvement of IL-10, but also IL-6, TNF, and IL-1β in the observed reduction of β-hexosaminidase production by CBMCs, as this was beyond the scope of this work. However, IL-10 could be a possible target, since it was shown to suppress the FcεRI signalling pathway and reduce histamine release in CBMCs8 or to directly affect the FcεRI expression and reduce degranulation in human skin mast cells.9 Although mast cells are known to express β2-AR and can respond to epinephrine stimulation (control treatment; Figure 2B), the observed effect on degranulation of CBMCs with supernatants from M2b-like macrophages was significantly higher than the impact of epinephrine alone. Due to its short half-life and its instability under supernatant storage conditions (−20°C), epinephrine is not expected to be present in the supernatants of M2b-like macrophages. In conclusion, the treatment of human allergic M2a macrophages with epinephrine led to a phenotypic switch to a macrophage subtype, which we term "M2b-like." In vitro data suggest that the M2b-like phenotype suppresses the IgE-dependent release of inflammatory mediators from mast cells. In allergic patients, acute stress may drive the plasticity of macrophages towards a regulatory M2b phenotype and reduce allergic symptoms, but further studies are needed to translate the results of this in vitro study into real life. However, as recently demonstrated in a clinical study in which the effects of acute stress on skin prick testing greatly varied among individuals,10 the net outcome of short-term acute stress in patients seems to be more complex and also depends on coping mechanisms. Together, our findings support further studies on the role of acute stress mediators in allergies. We would like to thank Prof. Regina Sommer, Head of Water Hygiene Unit, Inst. for Hygiene and Applied Immunology, for her kind assistance in the quantification of LPS amounts in our samples. Dr Gotovina, Dr Bianchini, Dr Singer, Dr Herrmann, Dr Pellizzari, Dr Haidl, Dr Hufnagl and Dr Marshall have nothing to disclose. Dr Karagiannis reports grants from NIHR Biomedical Research Centre at Guy's and St Thomas's Hospitals NHS Trust and King's College London; Medical Research Council; Breast Cancer Now; CR UK/NIHR in England/DoH for Scotland, Wales and Northern Ireland Experimental Cancer Medicine Centre; Cancer Research UK; Guy's and St Thomas's Charity, during the conduct of the study; and IGEM Therapeutics Ltd., outside the submitted work; in addition, Dr Karagiannis has a patent (IgE antibody technology) issued to IGEM Therapeutics Ltd. Dr Jensen-Jarolim reports grants and other from Biomedical International R + D GmbH, Vienna, and Bencard Allergie GmbH, Germany, and other from Allergy Therapeutics Ltd., UK, outside the submitted work. This study was supported by the Austrian Science Fund grants W1205-B09 (CCHD) and SFB F4606-B28 to EJJ. The authors acknowledge support by the Medical Research Council (MR/L023091/1) (SNK); the Academy of Medical Sciences (SNK); CR UK//NIHR in England/DoH for Scotland, Wales and Northern Ireland Experimental Cancer Medicine Centre (C10355/A15587) (SNK); Cancer Research UK (C30122/A11527; C30122/A15774) (SNK, GP); and Breast Cancer Now (147), working in partnership with Walk the Walk (SNK). The research was supported by the National Institute for Health Research (NIHR) Biomedical Research Centre (BRC) based at Guy's and St. Thomas' NHS Foundation Trust and King's College London (IS-BRC-1215-20006) (SNK). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.}, number={11}, journal={Allergy}, publisher={Wiley}, author={Gotovina, Jelena and Bianchini, Rodolfo and Fazekas‐Singer, Judit and Herrmann, Ina and Pellizzari, Giulia and Haidl, Ian D. and Hufnagl, Karin and Karagiannis, Sophia N. and Marshall, Jean S. and Jensen‐Jarolim, Erika}, year={2020}, month={Apr}, pages={2939–2942} }
@article{herrmann_kradischnig_skor_pakozdy_panakova_2021, title={Higher prevalence of seizure activity in a small population of atopic dogs: a retrospective breed- and age-matched study}, volume={32}, ISSN={["1365-3164"]}, url={https://doi.org/10.1111/vde.12913}, DOI={10.1111/vde.12913}, abstractNote={Background – Atopic dermatitis (AD) is considered to be a systemic disease in people shown to have an association with epilepsy. However, so far, no data about the association of epilepsy and atopy have been reported in dogs. Objectives – Given the homology between human and canine AD, and the increased incidence of epilepsy in atopic people, we investigated the association between AD and seizure‐associated activity in a small canine population. Conclusion and clinical importance – In our small retrospective study, we observed an increased prevalence of seizure activity in the atopic dog population. Further larger and prospective studies are needed to confirm these results.}, number={2}, journal={VETERINARY DERMATOLOGY}, publisher={Wiley}, author={Herrmann, Ina and Kradischnig, Clarissa and Skor, Ondrej and Pakozdy, Akos and Panakova, Lucia}, year={2021}, month={Apr}, pages={126-+} }
@article{high_linder_mamo_levy_herrmann_bizikova_2020, title={Rapid response of hyperkeratotic erythema multiforme to oclacitinib in two dogs}, volume={31}, ISSN={["1365-3164"]}, url={https://doi.org/10.1111/vde.12852}, DOI={10.1111/vde.12852}, abstractNote={BackgroundHyperkeratotic erythema multiforme (HKEM) is a clinically distinct dermatosis and poorly characterized syndrome, comprised of hyperkeratotic plaques with variable symmetry and apoptosis similar to “classic” erosive canine EM. Hyperkeratotic EM has a protracted clinical course and, although treatments with glucocorticoids, azathioprine and/or ciclosporin have been tried, rates of remission are low.ObjectivesTo describe successful treatment of HKEM in two dogs using oclacitinib.AnimalsA 7‐year‐old, spayed Havanese dog (Case 1) and a 1‐year‐old, intact cryptorchid Dachshund dog (Case 2).MethodsCase characterization and clinical diagnoses were based on lesion character, surgical biopsy, cytological evaluation, culture, direct immunofluorescence (DIF) and expected responses to treatments.ResultsBoth cases exhibited multifocal, often symmetrical hyperkeratotic plaques with adherent scale. Histological findings revealed prominent epidermal hyperplasia, parakeratotic hyperkeratosis, lymphocytic dermatitis and transepidermal apoptosis with lymphocytic satellitosis. DIF revealed fine, patchy IgG, IgM and IgA basement membrane deposits (Case 2). Both dogs exhibited rapid improvement with oral oclacitinib (0.6–0.9 mg/kg twice daily) with a complete remission of clinical signs observed in 12 and seven weeks in cases 1 and 2, respectively.Conclusion and Clinical ImportanceOclacitinib could be considered as a fast‐acting and effective treatment option for HKEM in dogs.}, number={4}, journal={VETERINARY DERMATOLOGY}, author={High, Endya J. and Linder, Keith E. and Mamo, Lisa B. and Levy, Britt J. and Herrmann, Ina and Bizikova, Petra}, year={2020}, month={Aug}, pages={330-+} }
@article{herrmann_loft_olivry_2020, title={Shortened immunotherapy dose‐escalation saves time, but is it safe? A case‐control study comparing the rates of adverse reactions between conventional and fast‐escalation subcutaneous immunotherapy protocols during the induction phase}, url={https://doi.org/10.1111/vde.12892}, DOI={10.1111/vde.12892}, abstractNote={BackgroundAllergen immunotherapy (AIT) is the only intervention believed to change the course of atopic diseases. As dogs appear to have fewer severe adverse events (AEs) compared to people receiving AIT, a prolonged dose‐escalation induction phase might not be needed.ObjectivesTo report the incidence and characteristics of AEs induced by a fast‐escalation subcutaneous immunotherapy (f‐SCIT) protocol compared to a conventional (c‐SCIT) regimen.AnimalsOne hundred dogs treated with either f‐ SCIT (Centre 1, 50 dogs) or c‐SCIT (Centre 2, 50 dogs).Methods and materialsA case‐control study retrospectively evaluating AEs during the induction of AIT. We determined the incidence and type of AEs in each SCIT group; we also assessed factors such as self‐limitation and the need for AE‐associated protocol changes.ResultsTwelve of 100 dogs (12%) developed a SCIT‐attributable AE during the induction phase, with one dog having a local and 11 having systemic reactions (nine Grade I, two Grade II, none of grades III or IV). Dogs treated with the f‐SCIT had a significantly higher rate of AEs (11 of 50; 22%) compared to those receiving the c‐SCIT (one of 50; 2%). Most of the AEs (10 of 11; 91%) in the f‐SCIT group were mild and self‐limiting.Conclusions and clinical importanceThe induction phase of f‐SCIT is simpler, and the maintenance phase is reached faster than that of the c‐SCIT. Despite its higher rate of AEs than with the c‐SCIT regimen, the majority of f‐SCIT‐associated AEs were mild and self‐limiting. Whether or not this f‐SCIT protocol leads to a faster time‐to‐efficacy needs to be determined.}, journal={Veterinary Dermatology}, author={Herrmann, Ina and Loft, Klaus E. and Olivry, Thierry}, year={2020}, month={Dec} }
@article{fazekas-singer_singer_ilieva_matz_herrmann_spillner_karagiannis_jensen-jarolim_2018, title={AllergoOncology: Generating a canine anticancer IgE against the epidermal growth factor receptor}, volume={142}, ISSN={0091-6749}, url={http://dx.doi.org/10.1016/j.jaci.2018.04.021}, DOI={10.1016/j.jaci.2018.04.021}, abstractNote={Cancer immunotherapy with antibodies has revolutionized clinical oncology, giving hope for patients with cancer. Among these, cetuximab, a mouse-human chimeric antibody, targeting the human epidermal growth factor receptor (EGFR),1Ferris R.L. Jaffee E.M. Ferrone S. Tumor antigen-targeted, monoclonal antibody-based immunotherapy: clinical response, cellular immunity, and immunoescape.J Clin Oncol. 2010; 28: 4390-4399Crossref PubMed Scopus (249) Google Scholar inhibits growth signaling and mediates antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP)2Pozzi C. Cuomo A. Spadoni I. Magni E. Silvola A. Conte A. et al.The EGFR-specific antibody cetuximab combined with chemotherapy triggers immunogenic cell death.Nat Med. 2016; 22: 624-631Crossref PubMed Scopus (169) Google Scholar, E5Singer J. Weichselbaumer M. Stockner T. Mechtcheriakova D. Sobanov Y. Bajna E. et al.Comparative oncology: ErbB-1 and ErbB-2 homologues in canine cancer are susceptible to cetuximab and trastuzumab targeting.Mol Immunol. 2012; 50: 200-209Crossref PubMed Scopus (71) Google Scholar of tumor cells. Harnessing TH2 immune responses against cancer is a subject in the emerging field of "AllergoOncology," taking advantage of the high capacity of IgE to trigger immune reactions against cancer.3Jensen-Jarolim E. Bax H.J. Bianchini R. Capron M. Corrigan C. Castells M. et al.AllergoOncology—the impact of allergy in oncology: EAACI position paper.Allergy. 2017; 72: 866-887Crossref PubMed Scopus (56) Google Scholar Unlike Fcγ-receptors, all Fcε-receptors are activating.3Jensen-Jarolim E. Bax H.J. Bianchini R. Capron M. Corrigan C. Castells M. et al.AllergoOncology—the impact of allergy in oncology: EAACI position paper.Allergy. 2017; 72: 866-887Crossref PubMed Scopus (56) Google Scholar Because fewer than 1 in 10 anticancer drugs translate from murine preclinical studies to human clinical trials,4Fulkerson C.M. Dhawan D. Ratliff T.L. Hahn N.M. Knapp D.W. Naturally occurring canine invasive urinary bladder cancer: a complementary animal model to improve the success rate in human clinical trials of new cancer drugs.Int J Genomics. 2017; 2017: 6589529Crossref PubMed Scopus (29) Google Scholar we focused on dogs (Canis lupus familiaris) with spontaneously occurring cancer as potential efficacy models. Mammary carcinomas and osteosarcomas in dogs and humans frequently overexpress EGFR.4Fulkerson C.M. Dhawan D. Ratliff T.L. Hahn N.M. Knapp D.W. Naturally occurring canine invasive urinary bladder cancer: a complementary animal model to improve the success rate in human clinical trials of new cancer drugs.Int J Genomics. 2017; 2017: 6589529Crossref PubMed Scopus (29) Google Scholar, 5Mantovani F.B. Morrison J.A. Mutsaers A.J. Effects of epidermal growth factor receptor kinase inhibition on radiation response in canine osteosarcoma cells.BMC Veter Res. 2016; 12: 82Crossref PubMed Scopus (18) Google Scholar Also, human and canine immune systems are highly similar,3Jensen-Jarolim E. Bax H.J. Bianchini R. Capron M. Corrigan C. Castells M. et al.AllergoOncology—the impact of allergy in oncology: EAACI position paper.Allergy. 2017; 72: 866-887Crossref PubMed Scopus (56) Google Scholar rendering canine clinical trials a promising alternative to rodent experiments. We previously developed a caninized cetuximab IgG, "can225IgG,"6Singer J. Fazekas J. Wang W. Weichselbaumer M. Matz M. Mader A. et al.Generation of a canine anti-EGFR (ErbB-1) antibody for passive immunotherapy in dog cancer patients.Mol Cancer Therapeut. 2014; 13: 1777-1790Crossref PubMed Scopus (23) Google Scholar which here we engineered into cetuximab IgE, "can225IgE-λ," retaining the exact epitope specificity, but with the canine λ- and ε-constant domains. Can225IgE-λ was cloned using a ligation-free cloning techniqueE13Ilieva K.M. Fazekas-Singer J. Achkova D.Y. Dodev T.S. Mele S. Crescioli S. et al.Functionally active Fc mutant antibodies recognizing cancer antigens generated rapidly at high yields.Front Immunol. 2017; 8: 1112Crossref PubMed Scopus (12) Google Scholar and expressed in Expi293F cells on the basis of our previously published method7Ilieva K.M. Fazekas-Singer J. Achkova D.Y. Dodev T.S. Mele S. Crescioli S. et al.Functionally active Fc mutant antibodies recognizing cancer antigens generated rapidly at high yields.Front Immunol. 2017; 8: 1112Crossref PubMed Scopus (2) Google Scholar (Fig 1, A; see this article's Methods section in the Online Repository at www.jacionline.org). The affinity-purified can225IgE-λ had an apparent molecular mass of 235 kDa (Fig 1, B) and was correctly assembled (Fig 1, C and D) with low amounts of free chains. We confirmed antibody specificity to recombinant human EGFR by Western blot (Fig 1, E), to native EGFR on human A431 breast cancer cells (Fig 1, F), to P114 canine mammary carcinoma (Fig 1, G) and D17 canine osteosarcoma (Fig 1, H) cells, but no binding to EGFR-negative CHO K1 (control) cells (Fig 1, I). Next, we assessed FcεRI expression on effector cell lines. Both human promonocytic U937 and canine monocytic/macrophage-like DH82 cells showed moderate to high FcεRI expression (Fig 1, J and K). Furthermore, canine MPT-1 mast cells showed relatively lower FcεRI expression (Fig 1, L) compared with RBL-SX38 rat basophilic leukemia cells transfected with human tetrameric FcεRI (Fig 1, M). Because no cross-reactive antibody for canine FcεRII (CD23) was available, we evaluated CD23 expression on canine effector cell lines by RT-PCR. We observed a more than 1000-fold higher relative CD23 expression by MPT-1, compared with DH82 cells (Fig 1, N). The can225IgE-λ antibody bound to Fcε receptors on U937 cells (Fig 2, A), DH82 cells (Fig 2, B), and MPT-1 (Fig 2, C) cells. Having shown tumor target specificity and recognition of immune effector cells by can225IgE-λ, we next used a tumor cell killing assayE4Bracher M. Gould H.J. Sutton B.J. Dombrowicz D. Karagiannis S.N. Three-colour flow cytometric method to measure antibody-dependent tumour cell killing by cytotoxicity and phagocytosis.J Immunol Methods. 2007; 323: 160-171Crossref PubMed Scopus (41) Google Scholar to quantify immune-mediated tumor cell killing by can225IgE-λ via flow cytometry. Tumor cells killed by cytotoxicity (ADCC) and phagocytosis (ADCP) were counted separately (for gating strategy, see Fig E1, A, in this article's Online Repository at www.jacionline.org). We first used human A431 as cancer target cells, U937 as effector cells, and human and dog cetuximab IgGE5Singer J. Weichselbaumer M. Stockner T. Mechtcheriakova D. Sobanov Y. Bajna E. et al.Comparative oncology: ErbB-1 and ErbB-2 homologues in canine cancer are susceptible to cetuximab and trastuzumab targeting.Mol Immunol. 2012; 50: 200-209Crossref PubMed Scopus (71) Google Scholar and IgE versions. Both cetuximab IgG and IgE showed high tumor cell killing potential, mostly via ADCC (Fig 2, D) mediated by effector cells. Canine IgE shows a 33 times weaker binding strength to the human FcεRI compared with human IgE. Accordingly, can225IgE-λ–triggered tumor killing with human effector cells was significantly lower than that triggered by human cetuximab-IgE (Fig 2, E). None of the antibodies had a significant effect on the ADCP of tumor cells (see Tables E1 and E3 in this article's Online Repository at www.jacionline.org). We furthermore evaluated whether can225IgE-λ could activate canine effector cells (Fig 2, F and G; Table E1). In a time-dependent manner (Fig E1, K), can225IgE-λ triggered significantly higher levels of ADCC by canine DH82 macrophages compared with can225IgG (Fig 2, F). Conversely, and in agreement with previous studies,8Karagiannis P. Singer J. Hunt J. Gan S.K. Rudman S.M. Mechtcheriakova D. et al.Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells.Cancer Immunol Immunother. 2009; 58: 915-930Crossref PubMed Scopus (83) Google Scholar, E7Spillner E. Plum M. Blank S. Miehe M. Singer J. Braren I. Recombinant IgE antibody engineering to target EGFR.Cancer Immunol Immunother. 2012; 61: 1565-1573Crossref PubMed Scopus (19) Google Scholar can225IgG was significantly superior to IgE with regard to triggering tumor cell ADCP (see Table E4 in this article's Online Repository at www.jacionline.org). In addition, we investigated the effects of an approach using 50:50 can225IgG and can225IgE-λ at the same total antibody amount as samples treated with individual antibodies. Combinational treatment performed comparable tumor cell ADCC to that by can225IgE-λ alone, but significantly better ADCC than that by can225IgG alone. ADCP was lower than that triggered by can225IgG, but higher than in can225IgE-λ monotherapy (Fig 2, F). Overall, can225IgE-λ mediated higher levels of total tumor cell killing compared with those mediated by can225IgG alone. Moreover, combining can225IgG and can225IgE-λ was significantly superior to each isotype alone (Fig 2, G; see Table E2 and Table E4 in this article's Online Repository at www.jacionline.org). Similarly, we evaluated the cytotoxic killing potency of can225IgE-λ by MPT-1 canine mast cells against A431 and measured specific tumor cell killing (Fig 2, H). Although ADCP assessment by flow cytometry was not possible in this experimental setting due to cell clumping, we could indeed confirm mast cell–mediated ADCC by fluorescence microscopy (Fig 2, I). Such effector functions may be due to can225IgE-λ specifically binding to IgE receptors (FcεRI on RBL-SX38 cells, Fig 2, J) and canine CD23, expressed on transfected CHO K1 cells (Fig 2, K). Therefore, we established an in vitro system for quantification and side-by-side comparison of anticancer ADCC/ADCP triggered by cetuximab IgG, IgE, and their canine counterparts. Unlike in humans where κ:λ light chains are expressed in a ratio of 2:1, the average κ:λ ratio in dogs is 1:9.E11Arun S.S. Breuer W. Hermanns W. Immunohistochemical examination of light-chain expression (lambda/kappa ratio) in canine, feline, equine, bovine and porcine plasma cells.Zentralblatt fur Veterinarmedizin Reihe A. 1996; 43: 573-576Crossref PubMed Scopus (86) Google Scholar Hence, we engineered can225IgE-λ. Because canine IgE is able to bind human FcεRI, we could demonstrate dose-dependent binding of can225IgE-λ on human U937 (Fig 2, A) in addition to the expected binding to canine FcεRI on DH82 and MPT-1 cells (Fig 2, B and C). Furthermore, we showed that DH82 macrophages express low levels of CD23 (Fig 1, N), but high levels of functional FcεRI (Fig 1, K), which is likely responsible for triggering IgE-mediated tumor cell killing (Fig 2, F and G), comparable to that mediated by human U937 monocytic effector cells. Using the canine DH82 macrophages, we were able to generate a species-relevant in vitro model system with which we confirmed the tumoricidal potencies of our canine antibodies, can225IgG or can225IgE-λ, alone, or combined. It has been observed that human IgG triggers considerable levels of ADCP, whereas IgE mainly elicits ADCC by human monocytes/macrophages.8Karagiannis P. Singer J. Hunt J. Gan S.K. Rudman S.M. Mechtcheriakova D. et al.Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells.Cancer Immunol Immunother. 2009; 58: 915-930Crossref PubMed Scopus (83) Google Scholar, E7Spillner E. Plum M. Blank S. Miehe M. Singer J. Braren I. Recombinant IgE antibody engineering to target EGFR.Cancer Immunol Immunother. 2012; 61: 1565-1573Crossref PubMed Scopus (19) Google Scholar We observed the same in the dog system and we also report significantly higher total tumor cell killing induced by IgE. We furthermore anticipated that IgE could act complementary to IgG, through using different Fc-receptors on effector cells. Coincubation with can225IgG and can225IgE-λ exerted higher tumor cell death, than did IgG or IgE treatment alone (Fig 2, F). Finally, we also demonstrate that can225IgE-λ is able to trigger specific ADCC against EGFR+ tumor cells by canine MPT-1 mast cells (Fig 2, H and I). Additional studies are needed to further evaluate these promising results with other IgG or IgE effector cells.3Jensen-Jarolim E. Bax H.J. Bianchini R. Capron M. Corrigan C. Castells M. et al.AllergoOncology—the impact of allergy in oncology: EAACI position paper.Allergy. 2017; 72: 866-887Crossref PubMed Scopus (56) Google Scholar, 9Singer J. Jensen-Jarolim E. IgE-based immunotherapy of cancer: challenges and chances.Allergy. 2014; 69: 137-149Crossref PubMed Scopus (25) Google Scholar This study presents the first in vitro functionally active canine anticancer IgE antibody for future clinical studies in dog cancer patients that may offer new options in cancer immunotherapy.9Singer J. Jensen-Jarolim E. IgE-based immunotherapy of cancer: challenges and chances.Allergy. 2014; 69: 137-149Crossref PubMed Scopus (25) Google Scholar In contrast to previous reports,E1Peng Z. Estelle F. Simons R. Becker A.B. Differential binding properties of protein A and protein G for dog immunoglobulins.J Immunol Method. 1991; 145: 255-258Crossref PubMed Scopus (22) Google Scholar we did not succeed in purifying can225IgE-λ by Protein A affinity columns. This might be possibly due to 2 IgE isoforms in dogs, IgE1 having a high affinity toward Staphylococcal Protein A and IgE2 not at all binding to protein A.E2Peng Z. Arthur G. Rector E.S. Kierek-Jaszczuk D. Simons F.R. Becker A.B. Heterogeneity of polyclonal IgE characterized by differential charge, affinity to protein A, and antigenicity.J Allergy Clin Immunol. 1997; 100: 87-95Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar The EGFR-negative CHO K1 cell line was used as control target cells. These cells were not recognized by can225IgE-λ (Fig 1, I). Consequently, CHO K1 cells were also not attacked by the human monocytic U937 cells (Fig E1, C and D) or the canine monocytic/macrophage-like DH82 cells (Fig E1, E and F). DH82 cells appeared as single, round cells in culture and showed only 1 distinct population in flow cytometry scatter plots (Fig E1, B); 77.4% of DH82 cells were positive for CD16 (ΔMFI, 713), 99.9% for CD14 (ΔMFI, 12922), and 72.2% for CD80 (ΔMFI, 2797) (Fig E1, G-I). DH82 cells transform over time from a monocyte- to a macrophage-like phenotype, associated with changes in CD14 and CD80 surface marker expression.E3Heinrich F. Contioso V.B. Stein V.M. Carlson R. Tipold A. Ulrich R. et al.Passage-dependent morphological and phenotypical changes of a canine histiocytic sarcoma cell line (DH82 cells).Veter Immunol Immunopathol. 2015; 163: 86-92Crossref PubMed Scopus (18) Google Scholar However, both CD14 and CD80 expression levels, as well as morphology and the uniform population in flow-cytometric scatter plots, identified a monocytic phenotype,E3Heinrich F. Contioso V.B. Stein V.M. Carlson R. Tipold A. Ulrich R. et al.Passage-dependent morphological and phenotypical changes of a canine histiocytic sarcoma cell line (DH82 cells).Veter Immunol Immunopathol. 2015; 163: 86-92Crossref PubMed Scopus (18) Google Scholar which was advantageous because the 3-color flow-cytometric assay had been established for monocytes.E4Bracher M. Gould H.J. Sutton B.J. Dombrowicz D. Karagiannis S.N. Three-colour flow cytometric method to measure antibody-dependent tumour cell killing by cytotoxicity and phagocytosis.J Immunol Methods. 2007; 323: 160-171Crossref PubMed Scopus (41) Google Scholar For proof-of-concept studies in the field of comparative immuno-oncology and AllergoOncology, the recombinant canine IgE antibody can225IgE-λ was developed against EGFR, which has 95% amino acid sequence homology and 92% identity among humans and dogs.E5Singer J. Weichselbaumer M. Stockner T. Mechtcheriakova D. Sobanov Y. Bajna E. et al.Comparative oncology: ErbB-1 and ErbB-2 homologues in canine cancer are susceptible to cetuximab and trastuzumab targeting.Mol Immunol. 2012; 50: 200-209Crossref PubMed Scopus (71) Google Scholar The antibody was expressed in a transient expression system and characterized with respect to correct assembly, EGFR specificity, and binding to IgE receptors FcεRI and CD23. In addition, the functionality of can225IgE-λ was evaluated in a flow-cytometric ADCC and ADCP assay using human and canine monocyte-like cells, as well as canine mast cells as effectors against EGFR-overexpressing tumor and EGFR-negative control cells, as described below. Human monocytic cell line U937 (CRL-1593.2), human epidermoid carcinoma cell line A431 (CRL-1555), canine osteosarcoma cell line D17 (CCL183), canine monocyte/macrophage-like cell line DH82 (CRL-10389), and Chinese hamster ovary cell line CHO K1 (CCL-61) were purchased from the American Type Culture Collection (Manassas, Va). Canine mammary carcinoma cell line P114 was a kind gift of G. Rutteman, University of Utrecht (Utrecht, The Netherlands). Canine mast cell line MPT-1E6Amagai Y. Tanaka A. Ohmori K. Matsuda H. Establishment of a novel high-affinity IgE receptor-positive canine mast cell line with wild-type c-kit receptors.Biochemic Biophys Res Commun. 2008; 366: 857-861Crossref PubMed Scopus (11) Google Scholar was a kind gift of Prof. H. Matsuda, Tokyo University of Agriculture and Technology (Tokyo, Japan). U937 was cultured in RPMI 1640 GlutaMAX medium (Gibco, Thermo Fisher Scientific, Waltham, Mass) supplemented with 10% (v/v) FBS (Gibco, Thermo Fisher Scientific) and 100 U/mL penicillin and 100 μg/mL Streptomycin (Gibco, Thermo Fisher Scientific). A431 was grown in high glucose (4.5 g/L) Dulbecco modified Eagle medium (DMEM) GlutaMAX medium (Gibco, Thermo Fisher Scientific), supplemented with 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin. D17 was grown in Minimum Essential Medium (MEM) GlutaMAX (Gibco, Thermo Fisher Scientific), augmented with 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin. P114 was cultured in DMEM/F-12 GlutaMAX (Gibco, Thermo Fisher Scientific) media supplemented with 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin. DH82 was cultured in low glucose (1 g/L) DMEM GlutaMAX medium (Gibco, Thermo Fisher Scientific), supplemented with 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin. MPT-1 was grown in alpha-MEM GlutaMAX medium (Gibco, Thermo Fisher Scientific), supplemented with 10% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin. CHO K1 was grown in Ham's F-12 Nutrient Mix GlutaMAX (Gibco, Thermo Fisher Scientific), augmented with 0% FBS and 100 U/mL penicillin and 100 μg/mL streptomycin. All cells were maintained in a humidified incubator at 37°C under a 5% CO2 atmosphere. Cetuximab (Erbitux) was purchased from Merck (Darmstadt, Germany). Rituximab (MabThera) was used as human IgG isotype control and was purchased from Roche (Basel, Switzerland). Cetuximab IgEE7Spillner E. Plum M. Blank S. Miehe M. Singer J. Braren I. Recombinant IgE antibody engineering to target EGFR.Cancer Immunol Immunother. 2012; 61: 1565-1573Crossref PubMed Scopus (19) Google Scholar was provided by Dr E. Spillner, Aarhus University (Aarhus, Denmark). MOv18-IgEE8Gould H.J. Mackay G.A. Karagiannis S.N. O'Toole C.M. Marsh P.J. Daniel B.E. et al.Comparison of IgE and IgG antibody-dependent cytotoxicity in vitro and in a SCID mouse xenograft model of ovarian carcinoma.Eur J Immunol. 1999; 29: 3527-3537Crossref PubMed Scopus (88) Google Scholar—used as human IgE isotype control—was provided by Dr S.N. Karagiannis and Prof. H.J. Gould, King's College London (London, UK). Purified canine IgG (cat# IR-DG-GF, Innovative Research, Novi, Mich) was used as canine IgG isotype control and purified canine IgE (cat# P115, Bethyl Laboratories, Montgomery, Tex) as canine IgE isotype control. Can225IgG was produced as described by Singer et al.E9Singer J. Fazekas J. Wang W. Weichselbaumer M. Matz M. Mader A. et al.Generation of a canine anti-EGFR (ErbB-1) antibody for passive immunotherapy in dog cancer patients.Mol Cancer Therapeut. 2014; 13: 1777-1790Crossref PubMed Scopus (40) Google Scholar Recombinant human EGFR (extracellular domain) was purchased from ACROBiosystems (cat# EGR-H5222). Recombinant human epidermal growth factor receptor 2 (extracellular domain) was produced in-house by Lec-1 cells,E10Cho H.S. Mason K. Ramyar K.X. Stanley A.M. Gabelli S.B. Denney Jr., D.W. et al.Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab.Nature. 2003; 421: 756-760Crossref PubMed Scopus (1222) Google Scholar which were a kind gift of D. Leahy from Johns Hopkins University School of Medicine (Baltimore, Md). Cetuximab heavy-chain variable region sequence was fused with the canine immunoglobulin epsilon constant sequence (NCBI GenBank: AAA56797.1). Cetuximab light-chain variable region sequence was fused with the canine lambda (because of its higher prevalence in dogs than in humans).E11Arun S.S. Breuer W. Hermanns W. Immunohistochemical examination of light-chain expression (lambda/kappa ratio) in canine, feline, equine, bovine and porcine plasma cells.Zentralblatt fur Veterinarmedizin Reihe A. 1996; 43: 573-576Crossref PubMed Scopus (86) Google Scholar The light-chain constant sequence (NCBI Ref Seq: XP_013963655.1) can be found among the coding sequences in Table E5. The final sequences were synthesized by GeneArt in Strings DNA format (Thermo Fisher Scientific) and cloned into pCR-Blunt vector using Zero Blunt PCR Cloning Kit (Thermo Fisher Scientific). Coding sequences were recloned into pVitro1-hygro (Invivogen, San Diego, Calif) using a ligation-free polymerase incomplete primer extension cloning method adapted from Dodev et al.E12Dodev T.S. Karagiannis P. Gilbert A.E. Josephs D.H. Bowen H. James L.K. et al.A tool kit for rapid cloning and expression of recombinant antibodies.Sci Rep. 2014; 4: 5885Crossref PubMed Scopus (62) Google Scholar All primers used for polymerase incomplete primer extension cloning are listed in Table E6. The pVitro-hygro-can225IgE-λ construct was then transformed into Escherichia coli Top10 (Thermo Fisher Scientific). Correct clones were verified using Sanger sequencing. Can225IgE-λ was produced according to Ilieva et alE13Ilieva K.M. Fazekas-Singer J. Achkova D.Y. Dodev T.S. Mele S. Crescioli S. et al.Functionally active Fc mutant antibodies recognizing cancer antigens generated rapidly at high yields.Front Immunol. 2017; 8: 1112Crossref PubMed Scopus (12) Google Scholar in human Expi293F cells using the pVitro1 vector (Fig 1, A) and purified with a custom affinity column using the ÄKTAFPLC system from GE Healthcare Life Sciences (Chicago, Ill). Custom anti-dog IgE column was made by crosslinking 1 mg goat anti-dog IgE (cat# NB7343, Novus Biologicals, Littleton, Colo) to HiTrap Protein G HP column (GE Healthcare Life Sciences) in the presence of 50-fold molar excess disuccinimidylsuberate (Sigma-Aldrich, St Louis, Mo). PBS, pH 7.4, was used as binding and washing buffer. Bound can225IgE-λ was then eluted with 0.1 M glycine (pH 2.5) followed by immediate pH neutralization using 1 M tris-HCl, pH 8.0. PAGE was run using tris-glycine–based 4% to 20% Mini-PROTEAN TGX gels (Bio-Rad Laboratories, Hercules, Calif), Laemmli loading buffer, and molecular weight markers Spectra Multicolor High Range Protein Ladder (Fig 1, B-D) and PageRuler Plus Prestained Protein Ladder (Fig 2, A) from Thermo Fisher. The gels were loaded with 0.5 μg can225IgE-λ/canine IgE isotype or 1 μg recombinant human EGFR/recombinant human epidermal growth factor receptor 2/BSA and the proteins were subsequently detected either by silver staining or by immunoblot. Blocking and antibody dilutions were performed with 5% (w/v) skimmed milk powder dissolved in tris buffered saline, pH 7.2, + 0.1% (v/v) tween-20. Canine IgE was detected using horseradish peroxidase–labeled goat anti-dog IgE (cat# 7346, Novus Biologicals) at 1:5000 for 1 hour, or by 0.5 μg/mL can225IgE-λ for 2 hours at room temperature. Blots were developed with Clarity Western enhanced chemiluminescence substrate (Bio-Rad) using the VersaDoc Imaging System (Bio-Rad). Light chains were detected using 0.5 μg/mL anti-dog light-chain antibody (cat# A40-124A, Bethyl Laboratories) followed by alkaline phosphatase-labeled donkey anti-goat IgG detection antibody (cat# A16008, Thermo Fisher) at 1:2000, and subsequently visualized using 1-Step NBT/BCIP substrate solution (Thermo Fisher). The apparent molecular mass of can225IgE-λ on silver-stained PAGE was determined using GelAnalyzer 2010a software (Istvan Lazar and Dr Istvan Lazar). Spectra Multicolor High Range Protein Ladder (Thermo Fisher Scientific) was used as a reference. A431, D17, P114, and CHO K1 cells were detached using Accutase solution (Sigma-Aldrich). A total of 3 × 105 cells/tube were stained with 10 μg/mL can225IgE-λ (or isotype control) for 30 minutes on ice followed by 10 μg/mL fluorescein isothiocyanate (FITC)-labeled goat anti-dog IgE (cat# NB7345, Novus Biologicals) for 30 minutes on ice in a light-protected environment. The cells were analyzed on a 3-laser FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ). DH82 and MPT-1 (3 × 105 per test) were stained with 10 μg/mL phycoerythrin-labeled anti-mouse FcεRI (clone MAR-1) (cat# 12-5898-83, eBioscience, Santa Clara, Calif) or appropriate isotype controls. U937 and RBL-SX38 cells (3 × 105 per test) were stained with 10 μg/mL anti-human FcεRI (clone CRA-1) (cat# 14-5899-82, eBioscience, Santa Clara, Calif), or mouse IgG2b isotype control, detected by 10 μg/mL FITC-labeled goat anti-mouse IgG (cat# F0479, DAKO, Agilent Technologies, Santa Clara, Calif). U937, DH82, and MPT-1 cells (3 × 105 per test) were incubated with can225IgE-λ at different concentrations (1, 2.5, 5, and 10 μg/mL) for 30 minutes at 37°C. Bound can225IgE-λ was detected with 10 μg/mL FITC-labeled goat anti-dog IgE. RBL-SX38 or canine CD23-transfected CHO K1 cells (3 × 105 per test) were incubated with 10 μg/mL can225IgE-λ for 30 minutes. Following a washing step, bound can225IgE-λ was detected with 10 μg/mL FITC-labeled goat anti-dog IgE. DH82 cells (passages 16-27, 3 × 105 per test) were stained with APC-labeled anti-human CD14 clone Tük4 (cat# MHCD1405, Thermo Fisher)/FITC-labeled anti-human CD16 (clone LNK16) (cat# MA1-19611, Thermo Fisher)/phycoerythrin-labeled anti-mouse CD80 (clone 16-10A1) (cat# 12-0801-82, eBioscience), or the respective controls. Cells were incubated with antibodies at concentrations according to the manufacturer's instructions for 30 minutes and washed before acquisition. Total RNA of 5 × 106 DH82 and MPT-1 was isolated using the RNeasy Mini Kit (cat# 74104, Qiagen, Venlo, The Netherlands) and transcribed using the SuperScript II reverse transcriptase (cat# 18064022, Thermo Fisher) along with oligo(dT)12-18 primers (cat# 18418012, Thermo Fisher) according to the manufacturer's instructions. Real-time PCR was performed using SYBR Green PCR Master Mix (cat# 4309155, Applied Biosystems, Thermo Fisher). Primers for canine CD23: forward: CCCAGAGCTTGAACGAGAGAA, reverse: TCCTCGCCGAAGTAGTAGCAC, as described by Amagai et al.E6Amagai Y. Tanaka A. Ohmori K. Matsuda H. Establishment of a novel high-affinity IgE receptor-positive canine mast cell line with wild-type c-kit receptors.Biochemic Biophys Res Commun. 2008; 366: 857-861Crossref PubMed Scopus (11) Google Scholar Primers for beta-actin: forward: ATTGCCGACAGGATGCAGAA, reverse: GCTGATCCACATCTGCTGAA. RT-PCR analysis was performed using 3 biological replicates (and 3 technical replicates of each biological replicate). Flow-cytometric ADCC/ADCP assays were performed using the method described by Bracher et al.E4Bracher M. Gould H.J. Sutton B.J. Dombrowicz D. Karagiannis S.N. Three-colour flow cytometric method to measure antibody-dependent tumour cell killing by cytotoxicity and phagocytosis.J Immunol Methods. 2007; 323: 160-171Crossref PubMed Scopus (41) Google Scholar A431 tumor cells were prestained with 5 μM of the green fluorescent dye carboxyfluoresceinsuccinimidyl ester for 10 minutes, 18 hours before the experiment. The tumor cells (70000/test) were incubated with 5 μg/mL antibody solution in assay buffer (RPMI 1640 GlutaMAX +2% (v/v) FBS) at 37°C for 30 minutes. Next, 210,000 U937 cells/tube were coincubated with the tumor cells at 37°C for 2.5 hours. Following the incubation, U937 were labeled with APC-conjugated anti-human CD89 antibody (cat# 354106, BioLegend, San Diego, Calif). Dead cells were detected using the blue fluorescent DNA dye 4′,6-diamidino-2-phenylindole (cat# D1306, Thermo Fisher), few seconds before acquisition on the FACSCanto II. When using DH82 or MPT-1 as effector cells, both tumor and effector cells were stained with cell-tracking dyes 18 hours in advance to the treatment antibodies and coincubation: Tumor cells were prestained with carboxyfluoresceinsuccinimidyl ester (as described earlier), whereas DH82 and MPT-1 were prestained with 1 μM CellTrace Far Red (Thermo Fisher) for 10 minutes. Because effector cells were already prelabeled in this case, no CD89 staining was needed to tag the cells after the coincubation of the tumor and effector cells. All assays were performed at least 3 times using 3 technical replicates per condition. Data were analyzed using FlowJo V10.0.7 (Flow Jo LLC, Ashland, Ore). Samples were prepared as described in the 3-color flow-cytometric ADCC and ADCP assay section using MPT-1 canine mast cells and A431 tumor cells. Fifteen microliter of the nonfixed cell suspension was mounted on a glass slide immediately after 4′,6-diamidino-2-phenylindole staining, and fluorescence microscopy pictures were recorded 20 minutes within mounting using an Axioplan 2 fluorescent microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) with a 20× air objective. CHO K1 cells were transfected with pcDNA3.3-TOPO (Thermo Fisher) containing the coding sequence of canine CD23 (back-translated from the amino acid sequence disclosed under GenBank accession number AAN20273.1) using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer's instructions. Forty-eight hours after transfection, 1 mg/mL G418 (cat# CP11.3, Carl Roth) was added and selection pressure was maintained over 3 weeks before proceeding to IgE-binding assays. Statistical analyses were carried out using GraphPad Prism v5.00 for Windows (GraphPad Software, Inc, La Jolla, Calif). Fig 2, A-C, shows mean values + SEM, and Fig 2, D-H, and Fig E1, C-F, depict mean + SD. To calculate the statistical significance of induced killing between the different treatment groups (Table E3 and Table E4), we used 2-way ANOVA with Bonferroni posttest (phagocytosis and cytotoxicity). To compare the total killing induced by the antibodies (Fig 2, E and G, and Fig E1, D and F), we used 1-way ANOVA followed by Tukey multiple comparison test. Statistical significances are defined as *P < .05, **P < .01, and ***P < .001.Table E1ADCC and ADCP triggered by different antibody treatmentsU937 as effector cellsKilling mechanismPBSHuman IgG ISOCetuximab IgGHuman IgE ISOCetuximab IgEDog IgE ISOcan225IgE-λCytotoxicity20% ± 6.0%18% ± 5.0%56% ± 8.6%18% ± 5.2%68% ± 4.7%18% ± 4.0%55 % ± 14%Phagocytosis4.0% ± 2.3%3.3% ± 2.4%5.3% ± 4.3%4.4% ± 4.0%4.2% ± 3.3%3.7% ± 2.9%3.6% ± 3.4%DH82 as effector cellsKilling mechanismPBSDog IgG ISOcan225IgGDog IgE ISOcan225IgE-λDog IgG + dog IgE ISOcan225IgG + can225IgE-λCytotoxicity15% ± 2.3%13% ± 1.7%40% ± 4.9%14% ± 2.4%64% ± 3.4%15% ± 2.8%63% ± 5.1%Phagocytosis18% ± 2.3%19% ± 2.9%28% ± 2.6%19% ± 2.5%11% ± 1.3%19% ± 1.1%18% ± 2.5%Depicted are mean values ± SDs in percent of the total number of tumor cells in the sample. n = 9 in all groups except combinational treatment with dog IgG and IgE isotype control and can225IgG+IgE, where n = 6.ISO, Isotype control antibody; PBS, phosphate buffered saline, mock-treated control. Open table in a new tab Table E2Total tumor cell killing triggered by different antibody treatmentsU937 as effector cellsTreatmentPBSHuman IgG ISOCetuximab IgGHuman IgE ISOCetuximab IgEDog IgE ISOcan225IgE-λTotal killing24% ± 6.0%21% ± 5.2%61% ± 12%22% ± 6.1%72% ± 7.5%21% ± 5.0%58% ± 16%DH82 as effector cellsTreatmentPBSDog IgG ISOcan225IgGDog IgE ISOcan225IgEDog IgG + dog IgE ISOcan225IgG + can225IgE-λTotal killing32% ± 2.9%32% ± 3.6%67% ± 3.8%32% ± 1.6%74% ± 2.6%34% ± 1.9%82% ± 3.1%Depicted are mean values ± SDs in percent of the total number of tumor cells in the sample. n = 9 in all groups except combinational treatment with dog IgG and IgE isotype control and can225IgG+IgE, where n = 6.ISO, Isotype control antibody; PBS, phosphate buffered saline, mock-treated control. Open table in a new tab Table E3Detailed statistical analysis of antibody treatments in ADCC and ADCP assays using the human U937 as effector cellsPhagocytosis ↓Cytotoxicity →PBSHuman IgG ISOCetuximab IgGHuman IgE ISOCetuximab IgEDog IgE ISOcan225IgEPBSns***P < .001.ns***P < .001.ns***P < .05.Human IgG ISOns***P < .001.ns***P < .001.ns***P < .001.Cetuximab IgGnsns***P < .001.***P < .001.∗P < .05.nsHuman IgE ISOnsnsns***P < .001.ns***P < .001.Cetuximab IgEnsnsnsns***P < .001.***P < .001.Dog IgE ISOnsnsnsnsns***P < .001.can225IgEnsnsnsnsnsnsStatistical analysis was calculated by 2-way ANOVA and Bonferroni posttest. Significance is determined as following: ns, nonsignificant (P > .05).∗ P < .05.∗∗∗ P < .001. Open table in a new tab Table E4Detailed statistical analysis of antibody treatments in ADCC and ADCP assays using the canine DH82 as effector cellsPhagocytosis ↓Cytotoxicity →PBSDog IgG ISOcan225IgGDog IgE ISOcan225IgEDog IgG + dog IgE ISOcan225IgG + can225IgEPBSns***P < .001.ns***P < .001.ns***P < .001.Dog IgG ISOns***P < .001.ns***P < .001.ns***P < .001.can225IgG***P < .001.***P < .001.***P < .001.***P < .001.***P < .001.***P < .001.Dog IgE ISOnsns***P < .001.∗P < .05.ns***P < .001.can225IgE***P < .001.***P < .001.***P < .001.***P < .001.***P < .001.nsDog IgG + dog IgE ISOnsns***P < .001.ns***P < .001.***P < .001.can225IgG +can225IgEnsns***P < .001.ns***P < .001.nsStatistical analysis was calculated by 2-way ANOVA and Bonferroni posttest. Significance is determined as following: ns, nonsignificant (P > .05).∗ P < .05.∗∗∗ P < .001. Open table in a new tab Table E5Amino acid and coding DNA sequence of can225IgE-λSequence descriptionSequenceCan225IgE-λ EPSILON chain amino acid sequence Legend:Bold, dog immunoglobulin heavy-chain variable region leader (ACI25521.1)Underlined, 225 variable regionRegular, dog immunoglobulin epsilon constant regionMESVLGWVFLVAILQGVQGQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAATSQDLSVFPLASCCKDNIASTSVTLGCLVTGYLPMSTTVTWDTGSLNKNVTTFPTTFHETYGLHSIVSQVTASGKWAKQRFTCSVAHAESTAINKTFSACALNFIPPTVKLFHSSCNPVGDTHTTIQLLCLISGYVPGDMEVIWLVDGQKATNIFPYTAPGTKEGNVTSTHSELNITQGEWVSQKTYTCQVTYQGFTFKDEARKCSESDPRGVTSYLSPPSPLDLYVHKAPKITCLVVDLATMEGMNLTWYRESKEPVNPGPLNKKDHFNGTITVTSTLPVNTNDWIEGETYYCRVTHPHLPKDIVRSIAKAPGKRAPPDVYLFLPPEEEQGTKDRVTLTCLIQNFFPADISVQWLRNDSPIQTDQYTTTGPHKVSGSRPAFFIFSRLEVSRVDWEQKNKFTCQVVHEALSGSRILQKWVSKTPGKCan225IgE-λ EPSILON chainDNA sequence(HEK293-optimized)Legend:Bold, STOP codonATGGAAAGCGTGCTGGGCTGGGTGTTCCTGGTGGCCATTCTGCAGGGCGTGCAGGGACAGGTGCAGCTGAAGCAGTCTGGCCCTGGACTGGTGCAGCCTAGCCAGAGCCTGAGCATCACCTGTACCGTGTCCGGCTTCAGCCTGACCAACTACGGCGTGCACTGGGTGCGCCAGAGCCCTGGAAAAGGCCTGGAATGGCTGGGAGTGATTTGGAGCGGCGGCAACACCGACTACAACACCCCCTTCACCAGCAGACTGTCCATCAACAAGGACAACAGCAAGAGCCAGGTGTTCTTCAAGATGAACAGCCTGCAGAGCAACGACACCGCCATCTACTACTGCGCTCGGGCCCTGACCTACTATGACTACGAGTTCGCCTACTGGGGCCAGGGCACACTCGTGACAGTGTCTGCCGCCACCAGCCAGGACCTGAGCGTGTTCCCTCTGGCCAGCTGCTGCAAGGACAATATCGCCAGCACCAGCGTGACCCTGGGCTGCCTCGTGACCGGCTACCTGCCTATGAGCACCACAGTGACCTGGGACACCGGCAGCCTGAACAAGAACGTGACCACCTTCCCCACCACCTTTCACGAGACATACGGCCTGCACAGCATCGTGTCTCAAGTGACCGCCAGCGGCAAGTGGGCCAAGCAGAGATTCACATGCAGCGTGGCCCACGCCGAGAGCACCGCCATTAACAAGACCTTCAGCGCCTGCGCCCTGAACTTCATCCCTCCCACCGTGAAGCTGTTCCACAGCAGCTGCAACCCCGTGGGCGATACCCACACCACCATTCAGCTGCTGTGCCTGATCAGCGGCTACGTGCCCGGCGACATGGAAGTGATCTGGCTGGTGGATGGCCAGAAGGCTACCAACATCTTCCCCTACACCGCCCCTGGCACCAAAGAGGGCAATGTGACCAGCACCCACTCCGAGCTGAACATCACCCAGGGCGAGTGGGTGTCACAGAAAACCTACACCTGTCAAGTGACATACCAGGGCTTCACCTTCAAGGACGAGGCCCGGAAGTGCAGCGAGAGCGATCCTAGAGGCGTGACCTCCTACCTGAGCCCCCCTAGCCCTCTGGACCTGTACGTGCACAAGGCCCCCAAGATCACCTGTCTGGTGGTGGACCTGGCCACAATGGAAGGCATGAACCTGACCTGGTACAGGGAAAGCAAAGAACCCGTGAACCCAGGCCCTCTGAACAAAAAGGACCACTTCAACGGCACCATCACCGTGACAAGCACCCTGCCCGTGAACACCAACGACTGGATCGAGGGCGAGACTTACTACTGCAGAGTGACCCACCCTCATCTGCCCAAGGACATCGTGCGGTCTATCGCCAAGGCCCCAGGCAAAAGGGCCCCTCCCGATGTGTACCTGTTCCTGCCTCCCGAGGAAGAACAGGGCACCAAGGACAGAGTGACACTGACCTGCCTGATCCAGAACTTCTTCCCCGCCGACATCTCCGTGCAGTGGCTGAGAAACGACAGCCCCATCCAGACCGACCAGTACACCACCACCGGCCCTCACAAGGTGTCCGGAAGCAGACCCGCCTTCTTCATCTTCAGCAGGCTGGAAGTGTCCCGGGTGGACTGGGAGCAGAAGAACAAGTTCACATGCCAGGTGGTGCACGAGGCCCTGAGCGGCTCTCGGATCCTGCAGAAATGGGTGTCCAAGACCCCCGGCAAGTGACan225IgE-λ LAMBDA chain amino acid sequence Legend:Bold, dog immunoglobulin lambda light-chain variable region leader (XP_013963655.1)Underlined, 225 light-chain variable regionRegular, dog immunoglobulin lambda constant regionMTSTMAWSSFLLTLLAHFTGSWADILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAQPKASPSVTLFPPSSEELGANKATLVCLISDFYPSGVTVAWKADGSPVTQGVETTKPSKQSNNKYAASSYLSLTPDKWKSHSSFSCLVTHEGSTVEKKVAPAECSCan225IgE-λLAMBDA light-chainDNA sequence(HEK293-optimized)Legend:Bold, STOP codonATGACCAGCACAATGGCCTGGTCCAGCTTCCTGCTGACCCTGCTGGCCCACTTTACAGGCAGCTGGGCCGACATTCTGCTGACACAGAGCCCCGTGATCCTGAGCGTGTCCCCTGGCGAGAGAGTGTCCTTCAGCTGCAGAGCCAGCCAGAGCATCGGCACCAACATCCACTGGTATCAGCAGCGGACCAACGGCAGCCCCAGACTGCTGATTAAGTACGCCAGCGAGTCCATCAGCGGCATCCCCAGCAGATTTTCCGGCAGCGGCTCCGGCACCGACTTCACCCTGAGCATCAACAGCGTGGAAAGCGAGGATATCGCCGACTACTACTGCCAGCAGAACAACAACTGGCCCACCACCTTCGGAGCCGGCACCAAGCTGGAACTGAAGAGAACCGTGGCCCAGCCCAAGGCCAGCCCTAGCGTGACACTGTTCCCTCCAAGCAGCGAGGAACTGGGCGCCAACAAGGCCACACTCGTGTGCCTGATCAGCGACTTCTACCCCAGCGGCGTGACCGTGGCCTGGAAGGCTGATGGCTCTCCTGTGACCCAGGGCGTGGAAACCACCAAGCCCAGCAAGCAGTCCAACAACAAATACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGATAAGTGGAAGTCCCACAGCAGCTTCAGCTGTCTCGTGACCCACGAGGGCAGCACCGTGGAAAAGAAAGTGGCCCCTGCCGAGTGCAGCTGATAG Open table in a new tab Table E6PIPE primers used for ligation-free cloning can225IgE-λ heavy- and light- chain sequences into pVitro-1 hygroPrimer nameSequencecan225IgE-λ epsilon forwardTACAGCTAGCTGGCCAGACATGATAAGATACATTGATGAGcan225IgE-λ epsilon reverseTCCGGATTGCTTTGAATTAGCGGTGGCTTTCACAACACCTcan225IgE-λ lambda forwardAGGGATCCCGTACGCCTAGGAGCAGGTTTCCCCAATGACAcan225IgE-λ lambda reverseACCGGTTGCTTTGAATTAGCGGTGGTTTTCACAACACCTAPIPE, Polymerase incomplete primer extension. Open table in a new tab Depicted are mean values ± SDs in percent of the total number of tumor cells in the sample. n = 9 in all groups except combinational treatment with dog IgG and IgE isotype control and can225IgG+IgE, where n = 6. ISO, Isotype control antibody; PBS, phosphate buffered saline, mock-treated control. Depicted are mean values ± SDs in percent of the total number of tumor cells in the sample. n = 9 in all groups except combinational treatment with dog IgG and IgE isotype control and can225IgG+IgE, where n = 6. ISO, Isotype control antibody; PBS, phosphate buffered saline, mock-treated control. Statistical analysis was calculated by 2-way ANOVA and Bonferroni posttest. Significance is determined as following: ns, nonsignificant (P > .05). Statistical analysis was calculated by 2-way ANOVA and Bonferroni posttest. Significance is determined as following: ns, nonsignificant (P > .05). PIPE, Polymerase incomplete primer extension.}, number={3}, journal={Journal of Allergy and Clinical Immunology}, publisher={Elsevier BV}, author={Fazekas-Singer, Judit and Singer, Josef and Ilieva, Kristina M. and Matz, Miroslawa and Herrmann, Ina and Spillner, Edzard and Karagiannis, Sophia N. and Jensen-Jarolim, Erika}, year={2018}, month={Sep}, pages={973–976.e11} }
@article{carvalho_bianchini_fazekas-singer_herrmann_flickinger_thalhammer_pires_jensen-jarolim_queiroga_2018, title={Bidirectional Regulation of COX-2 Expression Between Cancer Cells and Macrophages}, volume={38}, ISSN={0250-7005 1791-7530}, url={http://dx.doi.org/10.21873/anticanres.12525}, DOI={10.21873/anticanres.12525}, abstractNote={Our aim was to investigate the crosstalk between tumor and immune cells (M2 macrophages) and its effects on cyclo-oxygenase-2 (COX2) regulation in canine mammary tumors (CMT).Sh1b CMT cells and human BT474 mammary or HT29 colon cancer cells were co-cultured with canine peripheral blood mononuclear cells (PBMCs) or with macrophage-like differentiated THP1 monocytes (dTHP1). Intracellular COX2 expression by PBMCs, dTHP1 and cancer cells was evaluated by flow cytometry.Co-culturing of Sh1b and canine PBMCs induced COX2 overexpression in CMT cells. In turn, COX2 expression by PBMCs, mostly CD68+ macrophages, was attenuated by co-culture with Sh1b (p=0.0001). In accordance, co-culture with dTHP1 prompted intracellular production of COX2 in both Sh1b CMT cells and HT29 human colon cancer cells and reduced production of COX2 in BT474 human mammary cancer cells. The intracellular COX2 expression from dTHP1 decreased when treated with conditioned medium from cultured Sh1b and HT29 cancer cells.Bidirectional COX2 regulation between cancer and monocytes/macrophages might shape a tolerogenic tumor microenvironment in CMT.}, number={5}, journal={Anticancer Research}, publisher={Anticancer Research USA Inc.}, author={Carvalho, M. and Bianchini, R. and Fazekas-Singer, J. and Herrmann, I. and Flickinger, I. and Thalhammer, J. and Pires, I. and Jensen-Jarolim, E. and Queiroga, F.}, year={2018}, month={May} }
@article{herrmann_gotovina_fazekas-singer_fischer_hufnagl_bianchini_jensen-jarolim_2018, title={Canine macrophages can like human macrophages be in vitro activated toward the M2a subtype relevant in allergy}, volume={82}, ISSN={0145-305X}, url={http://dx.doi.org/10.1016/j.dci.2018.01.005}, DOI={10.1016/j.dci.2018.01.005}, abstractNote={The M2a subtype of macrophages plays an important role in human immunoglobulin E (IgE-mediated allergies) and other Th2 type immune reactions. In contrast, very little is known about these cells in the dog. Here we describe an in vitro method to activate canine histiocytic DH82 cells and primary canine monocyte-derived macrophages (MDMs) toward the M2a macrophages using human cytokines. For a side-by-side comparison, we compared the canine cells to human MDMs, and the human monocytic cell line U937 activated towards M1 and M2a cells on the cellular and molecular level. In analogy to activated human M2a cells, canine M2a, differentiated from both DH82 and MDMs, showed an increase in CD206 surface receptor expression compared to M1. Interestingly, canine M2a, but not M1 derived from MDM, upregulated the high-affinity IgE receptor (FcεRI). Transcription levels of M2a-associated genes (IL10, CCL22, TGFβ, CD163) showed a diverse pattern between the human and dog species, whereas M1 genes (IDO1, CXCL11, IL6, TNF-α) were similarly upregulated in canine and human M1 cells (cell lines and MDMs). We suggest that our novel in vitro method will be suitable in comparative allergology studies focussing on macrophages.}, journal={Developmental & Comparative Immunology}, publisher={Elsevier BV}, author={Herrmann, Ina and Gotovina, Jelena and Fazekas-Singer, Judit and Fischer, Michael B. and Hufnagl, Karin and Bianchini, Rodolfo and Jensen-Jarolim, Erika}, year={2018}, month={May}, pages={118–127} }
@inbook{jensen-jarolim_herrmann_panakova_janda_2017, title={Allergic and Atopic Eczema in Humans and Their Animals}, ISBN={9783319470054 9783319470078}, url={http://dx.doi.org/10.1007/978-3-319-47007-8_9}, DOI={10.1007/978-3-319-47007-8_9}, abstractNote={The encounter of high levels of allergens via the skin, such as from house dust mite, may induce allergic dermatitis. Atopic individuals, both human and animal, are genetically predisposed for a deficient skin barrier function and have an inborn higher jeopardy for percutaneous allergy and infections. The atopic phenotype also has typically a higher risk for environmental allergies which is in humans termed the atopic march. Atopic dermatitis (AD) or eczema is clinically associated with chronic or recurrent, often persistent skin inflammation at typical body sites: head and face, neck, intertrigo areas, and bend and hollow sites of arms and legs. It often occurs from early age, may persist lifelong, and is complexed by the associated itch. The clinics in humans and animals are comparable in terms of the pruritic inflammation, and scratching bears a high risk for superinfections. Further symptoms may be thickening of the keratocyte layer associated with overall atrophy of the skin (lichenification); more rarely severe systemic courses may take place. The diagnostic criteria are the eczematous skin appearance, elevated total IgE levels, and occurrence of allergen-specific IgE associated with hay fever, asthma, and food allergies. Treatments of AD are based on skin repair and anti-inflammatory and immunomodulatory drugs, mostly local, in severe cases systemically. All treatments today are symptomatic. The great homology of human and veterinary AD should be recognized to speed up the understanding of the underlying pathophysiology and result in development of an improved generation of drugs with true healing potency.}, booktitle={Comparative Medicine}, publisher={Springer International Publishing}, author={Jensen-Jarolim, Erika and Herrmann, Ina and Panakova, Lucia and Janda, Jozef}, year={2017}, pages={131–150} }
@inbook{pali-schöll_herrmann_jensen-jarolim_iben_2017, title={Allergies, with Focus on Food Allergies, in Humans and Their Animals}, ISBN={9783319470054 9783319470078}, url={http://dx.doi.org/10.1007/978-3-319-47007-8_8}, DOI={10.1007/978-3-319-47007-8_8}, abstractNote={Hypersensitivity reactions to respiratory, ingested, percutaneously encountered, or injected allergens are classified according to different pathophysiological mechanisms. In the case that food causes the adverse reactions, most typically symptoms along the digestive route (oral allergy syndrome, angioedema, stomachache, vomiting, diarrhea) but also systemic reactions (urticaria/hives, asthma, up to life-threatening anaphylaxis) may occur. On the contrary, food intolerance reactions are disagreeable but do not elicit dangerous systemic reactions. Therefore, it is important to diagnostically differentiate between immune-mediated hypersensitivities and the more harmless food intolerances. Principally, food adverse reactions may occur in all mammalian species. To single out the suspected food in children and animal patients the allergist is much dependent on collaboration with parents or owners, respectively. For diagnosis of food allergies in humans and animals, evaluation of the allergen-specific serum IgE levels, skin tests, and sometimes elimination diets and oral provocation tests are performed. Intolerances are diagnosed via hydrogen breath test or blood glucose test, in addition to elimination diets. The offending food allergen must be avoided. Clinical tolerization strategies and experimental immunotherapies have shown promising results. Symptomatic treatment may include the prescription of emergency self-medication in patients at risk for anaphylaxis. Whereas mostly murine models are used for developing more effective diagnostic and treatment options for food allergies, we propose the systematic inclusion of companion animals as spontaneous food allergy models in examination and diagnosis of allergy.}, booktitle={Comparative Medicine}, publisher={Springer International Publishing}, author={Pali-Schöll, Isabella and Herrmann, Ina and Jensen-Jarolim, Erika and Iben, Christine}, year={2017}, pages={109–129} }
@article{herrmann_einhorn_panakova_2017, title={Gender aspects in allergies of pets – A secondary publication and update}, volume={10}, ISSN={1939-4551}, url={http://dx.doi.org/10.1186/s40413-017-0172-1}, DOI={10.1186/s40413-017-0172-1}, abstractNote={Allergies need not only affect humans; this multifactorial and complex disease can also affect animals. Comparative allergology investigates the many similarities between the pathogenesis, clinics, diagnosis, and therapy of the disorders in humans and pet animals. In contrast to human allergy research, the veterinary field lacks access to a central database, which means there are no cohort studies published. This limits not only the research on breed and regional differences in allergies, but also further studies on the impact of gender in allergies of domestic animals. Moreover, domestic cats, dogs and male horses are castrated in most cases, which neutralises any effects of sexual hormones. In this review article a few interesting findings regarding gender aspects in companion animals were extracted from current literature. In summary, there is a lack of data on gender effects on allergies in cats, dogs or horses.}, journal={World Allergy Organization Journal}, publisher={Elsevier BV}, author={Herrmann, Ina and Einhorn, Lukas and Panakova, Lucia}, year={2017}, pages={42} }
@article{herrmann_panakova_2017, title={Genderaspekte bei den Allergien unserer Haustiere}, volume={40}, ISSN={0344-5062}, url={http://dx.doi.org/10.5414/alx01912}, DOI={10.5414/alx01912}, number={03}, journal={Allergologie}, publisher={Dustri-Verlgag Dr. Karl Feistle}, author={Herrmann, I. and Panakova, L.}, year={2017}, month={Mar}, pages={128–131} }
@article{oida_einhorn_herrmann_panakova_resch_vrtala_hofstetter_tanaka_matsuda_jensen-jarolim_2017, title={Innate function of house dust mite allergens: robust enzymatic degradation of extracellular matrix at elevated pH}, volume={10}, ISSN={1939-4551}, url={http://dx.doi.org/10.1186/s40413-017-0154-3}, DOI={10.1186/s40413-017-0154-3}, abstractNote={Background Exposure to the house dust mite Dermatophagoides pteronyssinus (D.p.) increases the risk for developing allergic diseases in humans and their best friends, the dogs. Here, we explored whether this allergenic mite via its enzymes may impact the cutaneous extracellular matrix (ECM), which critically determines epithelial barrier integrity both structurally and functionally.Methods Two extracts obtained from either dust-purified or cultured D.p. bodies were used in the present study. To assess the potential impact of D.p. on protein components of the ECM, proteolytic activity of the D.p. extracts were determined by casein and gelatin gel zymography, and their N-acetyl-β-hexosaminidase activity determined colorimetrically. In addition, IgE-dependent and innate degranulation potential of D.p. was examined in canine MPT-1 mast cells and neurite outgrowth assay using rat pheochromocytoma PC-12 cells.Results In gel zymography, both extracts digested the substrates casein and gelatin in a dose-dependent manner, especially at alkaline pH, and effective in a wide range of temperatures (30 °C−42 °C). In particular, a 25-kDa band corresponding to Der p 1, the major D.p. allergen for humans, was found enzymatically active in both casein and gelatin gels regardless of the presence of metal ions and of alkaline conditions. Besides protease activity, N-acetyl-β-hexosaminidase activity was detected in both extracts, suggesting that D.p. affects the cutaneous ECM through deteriorating both proteins and glycosaminoglycans. While both D.p. extracts induced IgE-dependent mast cell degranulation, much less innate effects on mast- and neuronal cells were observed.Conclusions Our data highlight that D.p. is a robust source of several distinct enzymes with protease- and N-acetyl-β-hexosaminidase activities. In alkaline milieu they can degrade components of the ECM. Therefore, D.p. may contribute to epithelial barrier disruption especially when the skin surface pH is elevated.}, journal={World Allergy Organization Journal}, publisher={Elsevier BV}, author={Oida, Kumiko and Einhorn, Lukas and Herrmann, Ina and Panakova, Lucia and Resch, Yvonne and Vrtala, Susanne and Hofstetter, Gerlinde and Tanaka, Akane and Matsuda, Hiroshi and Jensen-Jarolim, Erika}, year={2017}, pages={23} }
@inproceedings{herrmann_2016, title={Characterization of circulating monocytes/macrophages and polarization of M2a macrophage subtype in healthy and atopic dogs}, booktitle={8th World Congress of Veterinary Dermatology}, author={Herrmann, I.}, year={2016} }
@inproceedings{herrmann_2015, title={House dust mites provide enzymes that disrupt epithelial integrity}, booktitle={28th Annual Congress of European Society of Veterinary Dermatology (ESVD)}, author={Herrmann, I.}, year={2015} }
@article{jensen‐jarolim_einhorn_herrmann_thalhammer_panakova_2015, title={Pollen Allergies in Humans and their Dogs, Cats and Horses: Differences and Similarities}, volume={5}, ISSN={2045-7022 2045-7022}, url={http://dx.doi.org/10.1186/s13601-015-0059-6}, DOI={10.1186/s13601-015-0059-6}, number={1}, journal={Clinical and Translational Allergy}, publisher={Wiley}, author={Jensen‐Jarolim, Erika and Einhorn, Lukas and Herrmann, Ina and Thalhammer, Johann G and Panakova, Lucia}, year={2015}, month={Jan} }
@inproceedings{herrmann_2014, title={Positive effects of videootoscopy and otic flushing in dogs with chronic and recurrent otitis externa, a retrospective study of 62 diseased ears}, booktitle={29th Annual Meeting of the Vereinigung Österreichischer Kleintiermediziner}, author={Herrmann, I.}, year={2014} }