Coupling of a Major Allergen to the Surface of Immune Cells for Use in Prophylactic Cell Therapy for the Prevention of IgE-Mediated Allergy.

Up to a third of the world's population suffers from allergies, yet the effectiveness of available preventative measures remains, at large, poor. Consequently, the development of successful prophylactic strategies for the induction of tolerance against allergens is crucial. In proof-of-concept studies, our laboratory has previously shown that the transfer of autologous hematopoietic stem cells (HSC) or autologous B cells expressing a major grass pollen allergen, Phl p 5, induces robust tolerance in mice. However, eventual clinical translation would require safe allergen expression without the need for retroviral transduction. Therefore, we aimed to chemically couple Phl p 5 to the surface of leukocytes and tested their ability to induce tolerance. Phl p 5 was coupled by two separate techniques, either by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or by linkage via a lipophilic anchor, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-maleimide (DSPE-PEG-Mal). The effectiveness was assessed in fresh and cultured Phl p 5-coupled cells by flow cytometry, image cytometry, and immunofluorescence microscopy. Chemical coupling of Phl p 5 using EDC was robust but was followed by rapid apoptosis. DSPE-PEG-Mal-mediated linkage was also strong, but antigen levels declined due to antigen internalization. Cells coupled with Phl p 5 by either method were transferred into autologous mice. While administration of EDC-coupled splenocytes together with short course immunosuppression initially reduced Phl p 5-specific antibody levels to a moderate degree, both methods did not induce sustained tolerance towards Phl p 5 upon several subcutaneous immunizations with the allergen. Overall, our results demonstrate the successful chemical linkage of an allergen to leukocytes using two separate techniques, eliminating the risks of genetic modifications. More durable surface expression still needs to be achieved for use in prophylactic cell therapy protocols.

Deep immune profiling of chronic rhinosinusitis in allergic and non-allergic cohorts using mass cytometry.

Chronic rhinosinusitis (CRS) is a persistent nasal and paranasal sinus mucosa inflammation comprising two phenotypes, namely CRS with nasal polyps (CRSwNP) and without (CRSsNP). CRSwNP can be associated with asthma and hypersensitivity to non-steroidal anti-inflammatory drug (NSAID) in a syndrome known as NSAID-exacerbated respiratory disease (N-ERD). Furthermore, CRS frequently intertwines with respiratory allergies. This study investigated levels of 33 different nasal and serum cytokines and phenotypic characteristics of peripheral blood mononuclear cells (PBMCs) within cohorts of CRS patients (n = 24), additionally examining the influence of comorbid respiratory allergies by mass cytometry. N-ERD patients showed heightened type 2 nasal cytokine levels. Mass cytometry revealed increased activated naive B cell levels in CRSwNP and N-ERD, while resting naive B cells were higher in CRSsNP. Th2a cell levels were significantly elevated in allergic subjects, but not in CRS groups. In conclusion, there are distinct immunological features in PBMCs of CRS phenotypes and allergy.

Platelet-to-lymphocyte ratio might predict the response to dupilumab treatment for patients with nasal polyposis.

BACKGROUND: Dupilumab is a monoclonal antibody against interleukin 4 receptor alpha and has proven to be clinically effective in treating patients with chronic rhinosinusitis with nasal polyps (CRSwNP). However, a certain number of patients are non- or partial responders. This study aims to investigate the relevance of inflammatory markers with regard to therapy response to dupilumab in CRSwNP patients. METHODS: All patients with CRSwNP treated with dupilumab at a tertiary healthcare center with available pretreatment inflammatory markers were included. The values of pretreatment neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) were associated with the outcome. Patients were stratified according to the respective median value (> median was considered high). The binary logistic regression was performed with regard to total treatment response (post-treatment total nasal polyp score (NPS) 0). RESULTS: A total of 65 CRSwNP patients with available pretreatment peripheral blood values were included in the study. The mean pre- and post-treatment total NPS values were 4.3 ± 1.9 and 1.2 ± 1.6, respectively. High PLR (> 131.2) was independently associated with a 3.9-fold higher probability of reaching the NPS value of 0 in the multivariable analysis. On the other hand, High NLR (> 1.9) did not significantly associate with the outcome. CONCLUSIONS: The current study provides insights into the potential positive predictive value of the high PLR (> 131.2) in CRSwNP patients regarding treatment with dupilumab. There is a need for further prospective studies for validation of these results, especially in cohorts of patients with severe CRSwNP.

The "real life" efficacy of dupilumab is independent of initial polyp size and concomitant steroids in CRSwNP.

BACKGROUND: Dupilumab significantly improves symptom control in chronic rhinosinusitis with nasal polyps (CRSwNP). Patients with large polyps at the initiation of treatment (total polyp score (TPS) ≥ 5) have been the focus in published studies. Patients with significant burden of disease but small polyps (TPS ≤ 4) have not yet been evaluated for clinical response. This study set out to evaluate the benefit of dupilumab treatment on cohorts of small (TPS ≤ 4) compared to large polyps (TPS ≥ 5). Furthermore, benefit of concomitant oral and/or nasal steroid therapy has been evaluated. METHODS: 97 patients with CRSwNP, who were begun on dupilumab between January 2020 and October 2021, were included. All patients were followed-up for 6 months. At each visit they underwent nasal endoscopy, smell identification tests and filled out validated patient questionnaires. RESULTS: Significant drops in TPS were seen in both patient groups after 6 months of therapy, dropping from a median score of 3 to 0 and from 6 to 2 in patients with small and large polyps respectively. Furthermore, a linear mixed model calculated a drop of 22% and 24% in TPS per month in patients with small and large polyps respectively with no significant difference in rate of decline. Finally the model showed that neither oral nor nasal steroids influenced the rate of response to dupilumab therapy. CONCLUSIONS: Polyp size at the initiation of dupilumab therapy and whether patients continue to take steroid therapy does not appear to influence effectiveness of dupilumab treatment.

Nasal IL-13 production identifies patients with late-phase allergic responses.

BACKGROUND: There is limited knowledge on how local cytokine secretion patterns after nasal allergen challenge correlate with clinical symptoms especially with regard to the "late allergic response," which occurs in approximately 40% to 50% of patients with allergy. OBJECTIVE: We sought to characterize the immunologic and clinical nasal responses to birch pollen allergen challenge with a special focus on the late allergic response. METHODS: In this randomized, double-blind, placebo-controlled trial, birch pollen-allergic participants were challenged with birch pollen extract (n = 20) or placebo (n = 10) on 3 consecutive days. On days 1 and 3, nasal secretions were collected at selected time points over a 24-hour time course for the measurement of 33 inflammatory mediators. Clinical responses were determined through subjective symptom scores and objective nasal airflow measurements. RESULTS: Provoked participants had significantly greater clinical responses and showed significant increases in tryptase and the soluble IL-33 receptor serum stimulation 2 (sST2) in nasal secretions within minutes compared with the placebo group. Eight of 20 provoked participants displayed high IL-13 levels 2 to 8 hours after allergen provocation. This group also showed significant changes in clinical parameters, with a secondary drop in nasal airflow measured by peak nasal inspiratory flow and increased symptoms of nasal obstruction, which significantly differed from IL-13 nonresponders after 6 hours. CONCLUSIONS: IL-13 response status correlates with clinical responses and type 2 cytokine responses in the late phase after allergen provocation.

The nasal microbiome in patients suffering from non-steroidal anti-inflammatory drugs-exacerbated respiratory disease in absence of corticosteroids.

Chronic rhinosinusitis (CRS) is a chronic inflammatory disease phenotypically classified by the absence (CRSsNP) or presence of nasal polyps (CRSwNP). The latter may also be associated with asthma and hypersensitivity towards non-steroidal anti-inflammatory drugs (NSAID) as a triad termed NSAID-exacerbated respiratory disease (N-ERD). The role of the microbiome in these different disease entities with regard to the underlying inflammatory process and disease burden is yet not fully understood. To address this question, we measured clinical parameters and collected nasal samples (nasal mucosal fluids, microbiome swabs from middle meatus and anterior naris) of patients suffering from CRSsNP (n=20), CRSwNP (n=20) or N-ERD (n=20) as well as from patients without CRS (=disease controls, n=20). Importantly, all subjects refrained from taking local or systemic corticosteroids or immunosuppressants for at least two weeks prior to sampling. The nasal microbiome was analyzed using 16S rRNA gene amplicon sequencing, and levels of 33 inflammatory cytokines were determined in nasal mucosal fluids using the MSD platform. Patients suffering from N-ERD and CRSwNP showed significantly worse smell perception and significantly higher levels of type 2 associated cytokines IL-5, IL-9, Eotaxin and CCL17. Across all 4 patient groups, Corynebacteria and Staphylococci showed the highest relative abundances. Although no significant difference in alpha and beta diversity was observed between the control and the CRS groups, pairwise testing revealed a higher relative abundance of Staphylococci in the middle meatus in N-ERD patients as compared to CRSwNP (p<0.001), CRSsNP (p<0.01) and disease controls (p<0.05) and of Lawsonella in patients suffering from CRSwNP in middle meatus and anterior naris in comparison to CRSsNP (p<0.0001 for both locations) and disease controls (p<0.01 and p<0.0001). Furthermore, we observed a positive correlation of Staphylococci with IL-5 (Pearson r=0.548) and a negative correlation for Corynebacteria and Eotaxin-3 (r=-0.540). Thus, in patients refraining from oral and nasal corticosteroid therapy for at least two weeks known to alter microbiome composition, we did not observe differences in microbiome alpha or beta diversity between various CRS entities and disease controls. However, our data suggest a close association between increased bacterial colonization with Staphylococci and decreased colonization by Corynebacteria as well as increased type 2 inflammation.

Dupilumab increases aspirin tolerance in NSAID-exacerbated respiratory disease.

BACKGROUND: Nonsteroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (N-ERD) comprises the triad of chronic rhinosinusitis with nasal polyps, asthma and intolerance to NSAIDs. Dupilumab treatment, targeting the interleukin-4 (IL-4) receptor α, significantly reduces polyp burden as well as asthma symptoms. Here we aimed to investigate the effect of dupilumab on aspirin intolerance, burden of disease and nasal cytokine profiles in patients with N-ERD. METHODS: In this open-label trial, adult patients with confirmed N-ERD were treated with dupilumab for 6 months. Clinical parameters (e.g. total polyp scores, quality of life questionnaires, smell test, spirometry), oral aspirin provocation testing and blood, nasal and urine sampling were monitored at regular intervals for up to 6 months after starting dupilumab therapy. RESULTS: Of the 31 patients included in the study, 30 completed both aspirin provocation tests. After 6 months of treatment with dupilumab, 23% of patients (n=7 of 30) developed complete aspirin tolerance and an additional 33% of patients (n=10 of 30) tolerated higher doses. Polyp burden was significantly reduced (total polyp score: -2.68±1.84, p<0.001), while pulmonary symptoms (asthma control test: +2.34±3.67, p<0.001) and olfactory performance improved (University of Pennsylvania Smell Identification Test: +11.16±9.54, p<0.001) in all patients after therapy. Patients with increased aspirin tolerance showed a significant decrease in urinary leukotriene E4 levels and their improvement in clinical parameters was associated with a reduction of eotaxin-1, C-C motif chemokine ligand 17, IL-5, IL-17A and IL-6. CONCLUSION: In this study, 57% of N-ERD patients tolerated higher doses of aspirin under dupilumab therapy.

Using mass cytometry for the analysis of samples of the human airways.

Mass cytometry (MC) is a powerful method for mapping complex cellular systems at single-cell levels, based on the detection of cellular proteins. Numerous studies have been performed using human blood, but there is a lack of protocols describing the processing and labeling of bronchoalveolar lavage fluid (BALF) and nasal polyps (NP) for acquisition by MC. These specimens are essential in the investigation of immune cell characteristics in airway diseases such as asthma and chronic rhinosinusitis with NP (CRSwNP). Here we optimized a workflow for processing, labeling, and acquisition of BALF and NP cells by MC. Among three methods tested for NP digestion, combined enzymatic/mechanical processing yielded maximum cell recovery, viability and labeling patterns compared to the other methods. Treatment with DNAse improved sample acquisition by MC. In a final step, we performed a comparison of blood, BALF and NP cell composition using a 31-marker MC antibody panel, revealing expected differences between the different tissue but also heterogeneity among the BALF and NP samples. We here introduce an optimized workflow for the MC analysis of human NP and BALF, which enables comparative analysis of different samples in larger cohorts. A deeper understanding of immune cell characteristics in these samples may guide future researchers and clinicians to a better disease management.

Comprehensive Analysis of Nasal Polyps Reveals a More Pronounced Type 2 Transcriptomic Profile of Epithelial Cells and Mast Cells in Aspirin-Exacerbated Respiratory Disease.

Chronic rhinosinusitis with nasal polyps is affecting up to 3% of Western populations. About 10% of patients with nasal polyps also suffer from asthma and intolerance to aspirin, a syndrome called aspirin-exacerbated respiratory disease. Although eosinophilic inflammation is predominant in polyps of both diseases, phenotypic differences in the tissue-derived microenvironment, elucidating disease-specific characteristics, have not yet been identified. We sought to obtain detailed information about phenotypic and transcriptional differences in epithelial and immune cells in polyps of aspirin-tolerant and intolerant patients. Cytokine profiles in nasal secretions and serum of patients suffering from aspirin-exacerbated respiratory disease (n = 10) or chronic rhinosinusitis with nasal polyps (n = 9) were assessed using a multiplex mesoscale discovery assay. After enrichment for immune cell subsets by flow cytometry, we performed transcriptomic profiling by employing single-cell RNA sequencing. Aspirin-intolerant patients displayed significantly elevated IL-5 and CCL17 levels in nasal secretions corresponding to a more pronounced eosinophilic type 2 inflammation. Transcriptomic profiling revealed that epithelial and mast cells not only complement one another in terms of gene expression associated with the 15-lipoxygenase pathway but also show a clear type 2-associated inflammatory phenotype as identified by the upregulation of POSTN, CCL26, and IL13 in patients with aspirin-exacerbated respiratory disease. Interestingly, we also observed cellular stress responses indicated by an increase of MTRNR2L12, MTRNR2L8, and NEAT1 across all immune cell subsets in this disease entity. In conclusion, our findings support the hypothesis that epithelial and mast cells act in concert as potential drivers of the pathogenesis of the aspirin-exacerbated respiratory disease.

Zerumbone acts as a radiosensitizer in head and neck squamous cell carcinoma.

INTRODUCTION: Zerumbone is a phytochemical compound of the ginger plant Zingiber zerumbet with cytotoxic effects in various cancer cell lines. To date, zerumbone has shown an antiproliferative effect in oral squamous cell carcinoma cells lines. However, the effect of combination with radiation or cisplatin in head and neck squamous cell carcinoma (HNSCC) is unclear. The aim of this study was to investigate the effect of zerumbone alone, and in combination with irradiation and cisplatin on HNSCC cell lines. METHODS: The three HNSCC cell lines SCC25, Cal27 and FaDu were treated with zerumbone, radiation and/or cisplatin. Cell viability and clonogenic assays were performed. The interaction between zerumbone and radiation or cisplatin was evaluated using the combination index. Apoptosis was measured by flow cytometry and cell migration was assessed using a wound healing assay. RESULTS: Treatment with zerumbone resulted in a dose dependent induction of cytotoxicity and apoptosis in all three cell lines. The combination with cisplatin revealed a synergistic to additive effect in Cal27. The clonogenic assay showed a significant radiosensitizing effect in all three cell lines. The wound healing assay showed a reduction of cell migration in Cal27. CONCLUSION: The natural compound zerumbone shows a cytotoxic and proapoptotic effect on HNSCC cell lines. Furthermore, zerumbone enhances the radiation effect in all three cell lines and thus may be a suitable candidate for combination therapy in HNSCC.

Tracing Human IgE B Cell Antigen Receptor-Bearing Cells With a Monoclonal Anti-Human IgE Antibody That Specifically Recognizes Non-Receptor-Bound IgE.

Up to 30% of the population suffers from immunoglobulin E (IgE)-mediated allergies. Despite current stepwise gating approaches, the unambiguous identification of human IgE-producing cells by flow cytometry and immunohistology remains challenging. This is mainly due to the scarcity of these cells and the fact that IgE is not only expressed in a membrane-bound form on the surface of IgE-producing cells in form of the B cell antigen receptor (BCR), but is more frequently found on various cell types bound to the low and high affinity receptors, CD23 and FcϵRI, respectively. Here we sought to develop a sequential gating strategy for unambiguous detection of cells bearing the IgE BCR on their surface. To that aim we first tested the monoclonal anti-IgE antibody omalizumab for its ability to discriminate between IgE BCR and receptor-bound IgE using cells producing IgE or bearing IgE bound to CD23 as well as basophils exhibiting FcϵRI receptor-bound IgE. Using flow cytometry, we demonstrated that omalizumab recognized IgE producing cells with a high sensitivity of up to 1 IgE+ cell in 1000 human peripheral blood mononuclear cells (PBMCs). These results were confirmed by confocal microscopy both in cell suspensions as well as in nasal polyp tissue sections. Finally, we established a consecutive gating strategy allowing the clear identification of class-switched, allergen-specific IgE+ memory B cells and plasmablasts/plasma cells in human PBMCs. Birch pollen specific IgE+ memory B cells represented on average 0.734% of total CD19+ B cells in allergic patients after allergen exposure. Thus, we developed a new protocol for exclusive staining of non-receptor bound allergen-specific IgE+ B cell subsets in human samples.

Differences in men and women suffering from CRSwNP and AERD in quality of life.

PURPOSE: While the overall impact of chronic rhinosinusitis (CRS) on patients' health is diverse, many affected individuals have a substantially impaired quality of life (QoL). The aim of this study was to evaluate the impact of sex-associated differences specifically in the subgroups of CRS with nasal polyps (CRSwNP) and aspirin-exacerbated respiratory disease (AERD) by assessing QoL parameters in women and men separately. METHODS: In a retrospective single-center study, 59 patients with CRSwNP (39 males and 20 females) and 46 patients with AERD (18 males and 28 females) were included. Patient-reported outcome measures (PROM) evaluating QoL via the Sino-Nasal Outcome Test-20 German Adapted Version (SNOT-20 GAV) as well as the total polyp score (TPS) were analysed. RESULTS: There was no significant difference in TPS (p = 0.5550) and total SNOT-20 GAV scores (p = 0.0726) between male or female patients with CRSwNP or AERD. Furthermore, no significant sex differences were found within disease groups regarding the subcategories of the SNOT-20 GAV items. CONCLUSION: Thus, quality of life is severely impaired in patients suffering from various forms of CRS regardless of their sex.

Associations between the Quality of Life and Nasal Polyp Size in Patients Suffering from Chronic Rhinosinusitis without Nasal Polyps, with Nasal Polyps or Aspirin-Exacerbated Respiratory Disease.

Chronic rhinosinusitis (CRS) is a common disease that substantially impairs the quality of life (QoL). Here, we aimed to assess patients' QoL in different subtypes of CRS and correlated this with nasal polyp size to improve the clinical understanding of the burden of disease. In this retrospective single-center study, 107 patients with the following diagnoses were analyzed: CRS without nasal polyps (CRSsNP), CRS with nasal polyps (CRSwNP), or aspirin-exacerbated respiratory disease (AERD). Sino-Nasal Outcome Test-20 German Adapted Version (SNOT-20 GAV) scores and their correlation with endoscopic Total Polyp Scores (TPS) were evaluated. The mean SNOT-20 GAV scores were highest in patients with AERD (AERD = 43.4, CRSwNP = 36.3, CRSsNP = 30.9). A statistically significant correlation of total SNOT-20 GAV score with TPS was observed in CRSwNP patients (r = 0.3398, p = 0.0195), but not in AERD patients (r = 0.2341, p = 0.1407). When analyzing single SNOT-20 parameters, a strong correlation with TPS was observed for blockage/congestion of the nose, particularly in AERD patients (r = 0.65, p < 0.0001). The impact of nasal polyp size on the QoL differs amongst the subgroups of CRS. Nasal symptoms have the greatest impact on QoL in patients suffering from AERD. CRSwNP and AERD patients should be separately analyzed in clinical investigations and interpretations due to significant differences in QoL.

Primary Nasal Epithelial Cells From Allergic and Non-allergic Individuals Show Comparable Barrier Function.

Previous reports suggested that ex vivo cultured primary nasal epithelial cells from allergic patients differ from those from non-allergic individuals by genuinely reduced barrier function. By contrast, we found that primary nasal epithelial cells from allergic and non-allergic individuals showed comparable barrier function and secretion of cytokines.

Allergen-specific IgE levels and the ability of IgE-allergen complexes to cross-link determine the extent of CD23-mediated T-cell activation.

BACKGROUND: CD23 mediates IgE-facilitated allergen presentation and subsequent allergen-specific T-cell activation in allergic patients. OBJECTIVE: We sought to investigate key factors regulating IgE-facilitated allergen presentation through CD23 and subsequent T-cell activation. METHODS: To study T-cell activation by free allergens and different types of IgE-Bet v 1 complexes, we used a molecular model based on monoclonal human Bet v 1-specific IgE, monomeric and oligomeric Bet v 1 allergen, an MHC-matched CD23-expressing B-cell line, and a T-cell line expressing a human Bet v 1-specific T-cell receptor. The ability to cross-link Fcε receptors of complexes consisting of either IgE and monomeric Bet v 1 or IgE and oligomeric Bet v 1 was studied in human FcεRI-expressing basophils. T-cell proliferation by monomeric or oligomeric Bet v 1, which cross-links Fcε receptors to a different extent, was studied in allergic patients' PBMCs with and without CD23-expressing B cells. RESULTS: In our model non-cross-linking IgE-Bet v 1 monomer complexes, as well as cross-linking IgE-Bet v 1 oligomer complexes, induced T-cell activation, which was dependent on the concentration of specific IgE. However, T-cell activation by cross-linking IgE-Bet v 1 oligomer complexes was approximately 125-fold more efficient. Relevant T-cell proliferation occurred in allergic patients' PBMCs only in the presence of B cells, and its magnitude depended on the ability of IgE-Bet v 1 complexes to cross-link CD23. CONCLUSION: The extent of CD23-mediated T-cell activation depends on the concentration of allergen-specific IgE and the cross-linking ability of IgE-allergen complexes.

Two years of treatment with the recombinant grass pollen allergy vaccine BM32 induces a continuously increasing allergen-specific IgG4 response.

BACKGROUND: BM32, a grass pollen allergy vaccine containing four recombinant fusion proteins consisting of hepatitis B-derived PreS and hypoallergenic peptides from the major timothy grass pollen allergens adsorbed on aluminium hydroxide has been shown to be safe and to improve clinical symptoms of grass pollen allergy upon allergen-specific immunotherapy (AIT). We have investigated the immune responses in patients from a two years double-blind, placebo-controlled AIT field trial with BM32. METHODS: Blood samples from patients treated with BM32 (n = 27) or placebo (Aluminium hydroxide) (n = 13) were obtained to study the effects of vaccination and natural allergen exposure on allergen-specific antibody, T cell and cytokine responses. Allergen-specific IgE, IgG, IgG1 and IgG4 levels were determined by ImmunoCAP and ELISA, respectively. Allergen-specific lymphocyte proliferation by 3H thymidine incorporation and multiple cytokine responses with a human 17-plex cytokine assay were studied in cultured peripheral blood mononuclear cells (PBMCs). FINDINGS: Two years AIT comprising two courses of 3 pre-seasonal injections of BM32 and a single booster after the first pollen season induced a continuously increasing (year 2 > year 1) allergen-specific IgG4 response without boosting allergen-specific IgE responses. Specific IgG4 responses were accompanied by low stimulation of allergen-specific PBMC responses. Increases of allergen-specific pro-inflammatory cytokine responses were absent. The rise of allergen-specific IgE induced by seasonal grass pollen exposure was partially blunted in BM32-treated patients. INTERPRETATION: AIT with BM32 is characterised by the induction of a non-inflammatory, continuously increasing allergen-specific IgG4 response (year 2 > year1) which may explain that clinical efficacy was higher in year 2 than in year 1. The good safety profile of BM32 may be explained by lack of IgE reactivity and low stimulation of allergen-specific T cell and cytokine responses. FUNDINGS: Grants F4605, F4613 and DK 1248-B13 of the Austrian Science Fund (FWF).

Quantitative proteome analysis of Merkel cell carcinoma cell lines using SILAC.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive neuroendocrine tumour of the skin with growing incidence. To better understand the biology of this malignant disease, immortalized cell lines are used in research for in vitro experiments. However, a comprehensive quantitative proteome analysis of these cell lines has not been performed so far. METHODS: Stable isotope labelling by amino acids in cell culture (SILAC) was applied to six MCC cell lines (BroLi, MKL-1, MKL-2, PeTa, WaGa, and MCC13). Following tryptic digest of labelled proteins, peptides were analysed by mass spectrometry. Proteome patterns of MCC cell lines were compared to the proteome profile of an immortalized keratinocyte cell line (HaCaT). RESULTS: In total, 142 proteins were upregulated and 43 proteins were downregulated. Altered proteins included mitoferrin-1, histone H2A type 1-H, protein-arginine deiminase type-6, heterogeneous nuclear ribonucleoproteins A2/B1, protein SLX4IP and clathrin light chain B. Furthermore, several proteins of the histone family and their variants were highly abundant in MCC cell lines. CONCLUSIONS: The results of this study present a new protein map of MCC and provide deeper insights in the biology of MCC. Data are available via ProteomeXchange with identifier PXD008181.

Growth hormone resistance exacerbates cholestasis-induced murine liver fibrosis.

UNLABELLED: Growth hormone (GH) resistance has been associated with liver cirrhosis in humans but its contribution to the disease remains controversial. In order to elucidate whether GH resistance plays a causal role in the establishment and development of liver fibrosis, or rather represents a major consequence thereof, we challenged mice lacking the GH receptor gene (Ghr(-/-), a model for GH resistance) by crossing them with Mdr2 knockout mice (Mdr2(-/-)), a mouse model of inflammatory cholestasis and liver fibrosis. Ghr(-/-);Mdr2(-/-) mice showed elevated serum markers associated with liver damage and cholestasis, extensive bile duct proliferation, and increased collagen deposition relative to Mdr2(-/-) mice, thus suggesting a more severe liver fibrosis phenotype. Additionally, Ghr(-/-);Mdr2(-/-) mice had a pronounced down-regulation of hepatoprotective genes Hnf6, Egfr, and Igf-1, and significantly increased levels of reactive oxygen species (ROS) and apoptosis in hepatocytes, compared to control mice. Moreover, single knockout mice (Ghr(-/-)) fed with a diet containing 1% cholic acid displayed an increase in hepatocyte ROS production, hepatocyte apoptosis, and bile infarcts compared to their wild-type littermates, indicating that loss of Ghr renders hepatocytes more susceptible to toxic bile acid accumulation. Surprisingly, and despite their severe fibrotic phenotype, Ghr(-/-);Mdr2(-/-) mice displayed a significant decrease in tumor incidence compared to Mdr2(-/-) mice, indicating that loss of Ghr signaling may slow the progression from fibrosis/cirrhosis to cancer in the liver. CONCLUSION: GH resistance dramatically exacerbates liver fibrosis in a mouse model of inflammatory cholestasis, therefore suggesting that GH resistance plays a causal role in the disease and provides a novel target for the development of liver fibrosis treatments.