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1.
Eur J Cell Biol ; 103(2): 151408, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38583306

RESUMO

BACKGROUND: Therapeutic options for steroid-resistant non-type 2 inflammation in obstructive lung diseases are limited. Bronchial epithelial cells are key in the pathogenesis by releasing the central proinflammatory cytokine interleukine-8 (IL-8). Olfactory receptors (ORs) are expressed in various cell types. This study examined the drug target potential of ORs by investigating their impact on associated pathophysiological processes in lung epithelial cells. METHODS: Experiments were performed in the A549 cell line and in primary human bronchial epithelial cells. OR expression was investigated using RT-PCR, Western blot, and immunocytochemical staining. OR-mediated effects were analyzed by measuring 1) intracellular calcium concentration via calcium imaging, 2) cAMP concentration by luminescence-based assays, 3) wound healing by scratch assays, 4) proliferation by MTS-based assays, 5) cellular vitality by Annexin V/PI-based FACS staining, and 6) the secretion of IL-8 in culture supernatants by ELISA. RESULTS: By screening 100 potential OR agonists, we identified two, Brahmanol and Cinnamaldehyde, that increased intracellular calcium concentrations. The mRNA and proteins of the corresponding receptors OR2AT4 and OR2J3 were detected. Stimulation of OR2J3 with Cinnamaldehyde reduced 1) IL-8 in the absence and presence of bacterial and viral pathogen-associated molecular patterns (PAMPs), 2) proliferation, and 3) wound healing but increased cAMP. In contrast, stimulation of OR2AT4 by Brahmanol increased wound healing but did not affect cAMP and proliferation. Both ORs did not influence cell vitality. CONCLUSION: ORs might be promising drug target candidates for lung diseases with non-type 2 inflammation. Their stimulation might reduce inflammation or prevent tissue remodeling by promoting wound healing.


Assuntos
Brônquios , Células Epiteliais , Receptores Odorantes , Humanos , Células Epiteliais/metabolismo , Receptores Odorantes/metabolismo , Receptores Odorantes/genética , Brônquios/metabolismo , Brônquios/patologia , Células A549 , Interleucina-8/metabolismo , Cálcio/metabolismo , Pneumopatias/metabolismo , Pneumopatias/patologia , Proliferação de Células , Acroleína/análogos & derivados , Acroleína/farmacologia
2.
Front Immunol ; 15: 1296178, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38515755

RESUMO

Background: The neurodegenerative processes leading to glaucoma are complex. In addition to elevated intraocular pressure (IOP), an involvement of immunological mechanisms is most likely. In the new multifactorial glaucoma model, a combination of high IOP and optic nerve antigen (ONA) immunization leads to an enhanced loss of retinal ganglion cells accompanied by a higher number of microglia/macrophages in the inner retina. Here, we aimed to evaluate the immune response in this new model, especially the complement activation and the number of T-cells, for the first time. Further, the microglia/macrophage response was examined in more detail. Methods: Six-week-old wildtype (WT+ONA) and ßB1-connective tissue growth factor high-pressure mice (CTGF+ONA) were immunized with 1 mg ONA. A wildtype control (WT) and a CTGF group (CTGF) received NaCl instead. Six weeks after immunization, retinae from all four groups were processed for immunohistology, RT-qPCR, and flow cytometry, while serum was used for microarray analyses. Results: We noticed elevated numbers of C1q+ cells (classical complement pathway) in CTGF and CTGF+ONA retinae as well as an upregulation of C1qa, C1qb, and C1qc mRNA levels in these groups. While the complement C3 was only increased in CTGF and CTGF+ONA retinae, enhanced numbers of the terminal membrane attack complex were noted in all three glaucoma groups. Flow cytometry and RT-qPCR analyses revealed an enhancement of different microglia/macrophages markers, including CD11b, especially in CTGF and CTGF+ONA retinae. Interestingly, increased retinal mRNA as well as serum levels of the tumor necrosis factor α were found throughout the different glaucoma groups. Lastly, more T-cells could be observed in the ganglion cell layer of the new CTGF+ONA model. Conclusion: These results emphasize an involvement of the complement system, microglia/macrophages, and T-cells in glaucomatous disease. Moreover, in the new multifactorial glaucoma model, increased IOP in combination with autoimmune processes seem to enforce an additional T-cell response, leading to a more persistent pathology. Hence, this new model mimics the pathomechanisms occurring in human glaucoma more accurately and could therefore be a helpful tool to find new therapeutic approaches for patients in the future.


Assuntos
Glaucoma , Humanos , Camundongos , Animais , Retina/patologia , Células Ganglionares da Retina , Imunidade , Antígenos/metabolismo , Complexo Antígeno-Anticorpo/metabolismo , RNA Mensageiro/metabolismo
3.
Cells ; 12(13)2023 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-37443808

RESUMO

BACKGROUND: A total of 262 million people worldwide suffer from asthma and 461000 people died from it in 2019. Asthma is a disease with different endotypes defined by the granulocytes found in the asthmatic lung. In allergic asthma, the eosinophilic endotype is present, driven by a TH2 response. A TH17 immune response leads to the neutrophil endotype. This often causes uncontrolled asthma and is triggered by pollutants, microbes, and oxidative stress. It has been described that a significant number of patients with eosinophilic asthma develop mixed granulocytic asthma over time. The severity of asthma in the mixed endotype is related to the proportion of neutrophils in the lungs. PURPOSE: In this report, we address the question of how a TH2 response interacts with IL-17A in allergic asthma. METHODS: To this end, we used a mouse model to induce allergic asthma followed by an aerosol challenge with ovalbumin. To investigate the role of IL-17A, we administered IL-17A intranasally during the challenge phase. RESULTS: IL-17A alone did not elicit an immune response, whereas in combination with allergic asthma, it resulted in a shift of the asthmatic endotype from eosinophilic to neutrophilic. TGFß1 was increased in these lungs compared to asthmatic lungs without IL-17A, as was the expression of the IL-17A receptor subunits IL-17RA and IL-17RC. In cultures with human cells, we also found that IL-17A increased the expression of its receptors only in combination with IL-13. We also found this effect for IL-8, which attracts neutrophils in humans. CONCLUSIONS: The TH2 response increased the sensitivity to IL-17A in a mouse asthma model as well as in human cell lines.


Assuntos
Asma , Interleucina-17 , Camundongos , Animais , Humanos , Interleucina-17/metabolismo , Pulmão/metabolismo , Granulócitos/metabolismo , Inflamação/metabolismo
4.
Inflamm Res ; 72(3): 577-588, 2023 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-36658268

RESUMO

INTRODUCTION: Chronic inflammatory lung diseases are a common cause of suffering and death. Chronic obstructive pulmonary disease (COPD) is the reason for 6% of all deaths worldwide. A total of 262 million people are affected by asthma and 461,000 people died in 2019. Idiopathic pulmonary fibrosis (IPF) is diagnosed in 3 million people worldwide, with an onset over the age of 50 with a mean survival of only 24-30 months. These three diseases have in common that remodeling of the lung tissue takes place, which is responsible for an irreversible decline of lung function. Pathological lung remodeling is mediated by a complex interaction of different, often misguided, repair processes regulated by a variety of mediators. One group of these, as has recently become known, are the Wnt ligands. In addition to their well-characterized role in embryogenesis, this group of glycoproteins is also involved in immunological and structural repair processes. Depending on the combination of the Wnt ligand with its receptors and co-receptors, canonical and noncanonical signaling cascades can be induced. Wnt5A is a mediator that is described mainly in noncanonical Wnt signaling and has been shown to play an important role in different inflammatory diseases and malignancies. OBJECTIVES: In this review, we summarize the literature available regarding the role of Wnt5A as an immune modulator and its role in the development of asthma, COPD and IPF. We will focus specifically on what is known about Wnt5A concerning its role in the remodeling processes involved in the chronification of the diseases. CONCLUSION: Wnt5A has been shown to be involved in all three inflammatory lung diseases. Since the ligand affects both structural and immunological processes, it is an interesting target for the treatment of lung diseases whose pathology involves a restructuring of the lung tissue triggered in part by an inflammatory immune response.


Assuntos
Asma , Fibrose Pulmonar Idiopática , Doença Pulmonar Obstrutiva Crônica , Humanos , Pré-Escolar , Ligantes , Fibrose Pulmonar Idiopática/patologia , Pulmão/patologia , Asma/patologia , Doença Crônica , Proteína Wnt-5a
5.
Mol Med ; 28(1): 150, 2022 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-36503361

RESUMO

BACKGROUND: Therapeutic options for steroid-resistant non-type 2 inflammation in obstructive lung diseases are lacking. Alveolar macrophages are central in the progression of these diseases by releasing proinflammatory cytokines, making them promising targets for new therapeutic approaches. Extra nasal expressed olfactory receptors (ORs) mediate various cellular processes, but clinical data are lacking. This work investigates whether ORs in human primary alveolar macrophages could impact pathophysiological processes and could be considered as therapeutic targets. METHODS: Human primary alveolar macrophages were isolated from bronchoalveolar lavages of 50 patients with pulmonary diseases. The expression of ORs was validated using RT-PCR, immunocytochemical staining, and Western blot. Changes in intracellular calcium levels were analyzed in real-time by calcium imaging. A luminescent assay was used to measure the cAMP concentration after OR stimulation. Cytokine secretion was measured in cell supernatants 24 h after stimulation by ELISA. Phagocytic ability was measured by the uptake of fluorescent-labeled beads by flow cytometry. RESULTS: We demonstrated the expression of functional OR2AT4 and OR1A2 on mRNA and protein levels. Both ORs were primarily located in the plasma membrane. Stimulation with Sandalore, the ligand of OR2AT4, and Citronellal, the ligand of OR1A2, triggered a transient increase of intracellular calcium and cAMP. In the case of Sandalore, this calcium increase was based on a cAMP-dependent signaling pathway. Stimulation of alveolar macrophages with Sandalore and Citronellal reduced phagocytic capacity and release of proinflammatory cytokines. CONCLUSION: These are the first indications for utilizing olfactory receptors as therapeutic target molecules in treating steroid-resistant lung diseases with non-type 2 inflammation.


Assuntos
Pneumopatias , Receptores Odorantes , Humanos , Cálcio/metabolismo , Citocinas/metabolismo , Inflamação/metabolismo , Ligantes , Pneumopatias/metabolismo , Macrófagos Alveolares/metabolismo , Receptores Odorantes/genética , Receptores Odorantes/metabolismo , Esteroides
6.
Cells ; 11(19)2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-36230982

RESUMO

Clinical success of Toll-Like receptor-4 (TLR-4) antagonists in sepsis therapy has thus far been lacking. As inhibition of a receptor can only be useful if the receptor is active, stratification of patients with active TLR-4 would be desirable. Our aim was to establish an assay to quantify phosphorylated TLR-4 using the proximity ligation assay (PLA). HEK293 TLR4/MD2/CD14 as well as THP-1 cells were stimulated with LPS and the activation of TLR-4 was measured using the PLA. Furthermore, peripheral blood mononuclear cells (PBMCs) from 25 sepsis patients were used to show the feasibility of this assay in clinical material. Activation of TLR-4 in these samples was compared to the PBMCs of 11 healthy individuals. We could show a transient activation of TLR-4 in both cell lines. Five min after the LPS stimulation, the signal increased 6.7-fold in the HEK293 cells and 4.3-fold in the THP-1 cells. The assay also worked well in the PBMCs of septic patients. Phosphorylation of TLR-4 at study inclusion was 2.9 times higher in septic patients compared to healthy volunteers. To conclude, we established a diagnostic assay that is able to quantify the phosphorylation of TLR-4 in cell culture and in clinical samples of sepsis patients. This makes large-scale stratification of sepsis patients for their TLR-4 activation status possible.


Assuntos
Sepse , Receptor 4 Toll-Like/metabolismo , Células HEK293 , Humanos , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia
7.
Am J Physiol Lung Cell Mol Physiol ; 323(4): L450-L463, 2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-35972838

RESUMO

The enzyme, nitric oxide-sensitive guanylyl cyclase (NO-GC), is activated by binding NO to its prosthetic heme group and catalyzes the formation of cGMP. The NO-GC is primarily known to mediate vascular smooth muscle relaxation in the lung, and inhaled NO has been successfully used as a selective pulmonary vasodilator. In comparison, NO-GC's impact on the regulation of airway tone is less acknowledged and, most importantly, little is known about the issue that NO-GC signaling is accomplished by two isoforms: NO-GC1 and NO-GC2, implying the existence of distinct "cGMP pools." Herein, we investigated the functional role of the NO-GC isoforms in respiration by measuring lung function parameters of isoform-specific knockout (KO) mice using noninvasive and invasive techniques. Our data revealed the participation and ongoing influence of NO-GC1-derived cGMP in the regulation of airway tone by showing that respiratory resistance was enhanced in NO-GC1-KOs and increased more pronouncedly after the challenge with the bronchoconstrictor methacholine. The tissue resistance and stiffness of NO-GC1-KOs were also higher because of narrowed airways that cause tissue distortion. Contrariwise, NO-GC2-KOs displayed reduced tissue elasticity, elastic recoil, and airway reactivity to methacholine, which did not even increase in an ovalbumin model of asthma that induced hyperresponsiveness in NO-GC1-KOs. In addition, conscious NO-GC2-KOs showed a higher breathing rate with a shorter duration of inspiration and expiration time, which remained faster even in the presence of bronchoconstrictors that slow down breathing. Thus, we provide evidence of two distinct NO/cGMP pathways in airways, accomplished by either NO-GC1 or NO-GC2, adjusting differentially the airway reactivity.


Assuntos
Broncoconstritores , Guanilato Ciclase , Animais , GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Heme , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Knockout , Óxido Nítrico/metabolismo , Ovalbumina , Isoformas de Proteínas/metabolismo , Guanilil Ciclase Solúvel/metabolismo , Vasodilatadores
8.
Nat Commun ; 13(1): 4009, 2022 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-35817801

RESUMO

Diphthamide, a post-translationally modified histidine residue of eukaryotic TRANSLATION ELONGATION FACTOR2 (eEF2), is the human host cell-sensitizing target of diphtheria toxin. Diphthamide biosynthesis depends on the 4Fe-4S-cluster protein Dph1 catalyzing the first committed step, as well as Dph2 to Dph7, in yeast and mammals. Here we show that diphthamide modification of eEF2 is conserved in Arabidopsis thaliana and requires AtDPH1. Ribosomal -1 frameshifting-error rates are increased in Arabidopsis dph1 mutants, similar to yeast and mice. Compared to the wild type, shorter roots and smaller rosettes of dph1 mutants result from fewer formed cells. TARGET OF RAPAMYCIN (TOR) kinase activity is attenuated, and autophagy is activated, in dph1 mutants. Under abiotic stress diphthamide-unmodified eEF2 accumulates in wild-type seedlings, most strongly upon heavy metal excess, which is conserved in human cells. In summary, our results suggest that diphthamide contributes to the functionality of the translational machinery monitored by plants to regulate growth.


Assuntos
Arabidopsis , Proteínas de Saccharomyces cerevisiae , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Histidina/análogos & derivados , Histidina/metabolismo , Humanos , Mamíferos/metabolismo , Camundongos , Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Int Arch Allergy Immunol ; 183(7): 714-725, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35193134

RESUMO

Different endotypes of asthma were described in human. Atopic asthma is a T-helper 2 (Th2)-mediated disease consisting mainly of an eosinophilic inflammation in the airways. Other endotypes show neutrophilic inflammation of the airways that is probably based on a Th17 response. There are several mouse models described in the literature to study the Th2 polarized eosinophilic disease, however, only a few models are available which characterize the neutrophilic endotype. The aim of this study was to compare both endotypes in relation to the severity of the allergen-induced inflammation. Groups of either Balb/c or DO11.10 mice were sensitized with ovalbumin (OVA) adsorbed to aluminum hydroxide. Mice were subsequently challenged with OVA for different periods of time. They were evaluated for airway hyperreactivity (AHR), cytokine production, airway inflammation, and remodeling of the airways. As expected, Balb/c mice developed a Th2 response with AHR, eosinophilic airway inflammation, and allergen-specific IgE and IgG1. By contrast DO11.10 mice showed a mixed Th1/Th17 response with strong neutrophilic airway inflammation, IgG2a, but only limited induction of AHR. While Balb/c mice showed remodeling of the airways with subepithelial fibrosis and goblet cell metaplasia, airway remodeling in DO11.10 mice was marginal. Both airway inflammation and remodeling resolved after prolonged periods of challenge in both models. In conclusion, strong allergen-induced airway remodeling in mice seems to be triggered by the specific conditions arising from infiltration with eosinophilic granulocytes in the lung. A Th1/Th17 response leading to neutrophilic inflammation does not seem to be sufficient to induce pronounced airway remodeling.


Assuntos
Remodelação das Vias Aéreas , Asma , Alérgenos/efeitos adversos , Animais , Asma/induzido quimicamente , Modelos Animais de Doenças , Inflamação , Pulmão , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/efeitos adversos , Células Th2
10.
CRISPR J ; 5(1): 53-65, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-35099270

RESUMO

Cerebral organoids are a promising model to study human brain function and disease, although the high inter-organoid variability is still challenging. To overcome this limitation, we introduce the method of labeled mixed organoids generated from two different human induced pluripotent stem cell (hiPSC) lines, which enables the identification of cells from different origin within a single organoid. The method combining gene editing and organoid differentiation offers a unique tool to study gene function in a complex human three-dimensional model. Using a CRISPR-Cas9 gene-editing approach, different fluorescent proteins were fused to ß-actin or lamin B1 in hiPSCs, and mixtures of differently edited cells were seeded to induce cerebral organoid differentiation. Consequently, the development of the organoids was detectable by live confocal fluorescence microscopy of whole organoids and immunofluorescence staining in fixed samples. We demonstrate that a direct comparison of the individual cells is possible by having the edited and the control (or the two differentially labeled) cells within the same organoid, thus overcoming the inter-organoid inhomogeneity limitations. Furthermore, the approach enables mosaic analysis of mutant clones in a wild-type three-dimensional cellular environment. It paves the way for the reliable analysis of human genetic disorders using organoids and the gain of fundamental understanding of the molecular mechanisms underlying pathological conditions.


Assuntos
Células-Tronco Pluripotentes Induzidas , Organoides , Sistemas CRISPR-Cas/genética , Diferenciação Celular/genética , Edição de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Organoides/metabolismo
11.
Pharmaceuticals (Basel) ; 14(8)2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34451852

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by extensive fibrosis of the lung tissue. Wnt5a expression was observed to be upregulated in IPF and suggested to be involved in the progression of the disease. Interestingly, smooth muscle cells (SMC) are a major source of Wnt5a in IPF patients. However, no study has been conducted until now to investigate the precise role of smooth muscle-derived Wnt5a in IPF. Here, we used the bleomycin-induced lung fibrosis model in a conditional gene-deficient mouse, where the Wnt5a gene was excised from SMC. We show here that the excision of the Wnt5a gene in SMC led to significantly improved health conditions with minimized weight loss and improved lung function. This improvement was based on a significantly lower deposition of collagen in the lung with a reduced number of fibrotic foci in lung parenchyma. Furthermore, the bleomycin-induced cellular infiltration into the airways was not altered in the gene-deficient mice compared with wild-type mice. Thus, we demonstrate that the Wnt5a expression of SMC of the airways leads to aggravated fibrosis of the lung with poor clinical conditions. This aggravation was not an influence in the bleomycin-induced inflammatory processes but on the development of fibrotic foci in lung parenchyma and the deposition of collagen.

12.
Front Immunol ; 12: 635411, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33995354

RESUMO

More than fifty c-type lectin receptors (CLR) are known and have been identified so far. Moreover, we know the group of galectins and sialic acid-binding immunoglobulin-type lectins that also belong to the carbohydrate-binding receptors of the immune system. Thus, the lectin receptors form the largest receptor family among the pathogen recognition receptors. Similar to the toll-like receptors (TLRs), the CLR do not only recognize foreign but also endogenous molecules. In contrast to TLRs, which have a predominantly activating effect on the immune system, lectin receptors also mediate inhibitory signals. They play an important role in innate and adaptive immunity for the induction, regulation and shaping of the immune response. The hygiene hypothesis links enhanced infection to protection from allergic disease. Yet, the microbial substances that are responsible for mediating this allergy-protective activity still have to be identified. Microbes contain both ligands binding to TLRs and carbohydrates that are recognized by CLR and other lectin receptors. In the current literature, the CLR are often recognized as the 'bad guys' in allergic inflammation, because some glycoepitopes of allergens have been shown to bind to CLR, facilitating their uptake and presentation. On the other hand, there are many reports revealing that sugar moieties are involved in immune regulation. In this review, we will summarize what is known about the role of carbohydrate interaction with c-type lectins and other sugar-recognizing receptors in anti-inflammation, with a special focus on the regulation of the allergic immune response.


Assuntos
Hipersensibilidade/metabolismo , Inflamação/metabolismo , Lectinas Tipo C/metabolismo , Animais , Humanos , Hipersensibilidade/imunologia , Inflamação/imunologia , Lectinas Tipo C/imunologia , Ligantes , Transdução de Sinais
13.
Clin Exp Allergy ; 51(11): 1471-1481, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33550702

RESUMO

BACKGROUND: Asthma is an inflammatory disease of the respiratory system, and a major factor of increasing health care costs worldwide. The molecular actors leading to the development of chronic asthma are not fully understood and require further investigation. OBJECTIVE: The aim of this study was to monitor the proteome dynamics during asthma development from early inflammatory to late fibrotic stages. METHODS: A mouse asthma model was used to analyse the lung proteome at four time points during asthma development (0 weeks = control, 5, 8 and 12 weeks of treatment, n = 6 each). The model was analysed using lung function tests, immune cell counting and histology. Furthermore, a multi-fraction mass spectrometry-based proteome analysis was performed to achieve a comprehensive coverage and quantification of the lung proteome. RESULTS: At early stages, the mice showed predominant eosinophilic inflammation of the airways, which disappeared at later stages and was replaced by marked airway hyper-reactivity and fibrosis of the airways. 3325 proteins were quantified with 435 proteins found to be significantly differentially abundant between the experimental groups (ANOVA p-value ≤.05, maximum fold change ≥1.5). We applied hierarchical clustering to identify common protein abundance profiles along the asthma development and analysed these clusters using gene ontology annotation and enrichment analysis. We demonstrate the correlation of protein clusters with the course of asthma development, that is eosinophilic inflammation and fibrotic remodelling of the airways. CONCLUSIONS AND CLINICAL RELEVANCE: Proteome analysis revealed proteins that were previously described to be important during asthma chronification. Moreover, we identified additional proteins previously not described in the context of asthma. We provide a comprehensive data set of a long-term mouse model of asthma that may contribute to a better understanding and allow new insights into the progression and development of chronic asthma. Data are available via ProteomeXchange with identifier PXD011159.


Assuntos
Asma , Proteoma , Animais , Modelos Animais de Doenças , Ontologia Genética , Humanos , Pulmão , Camundongos
14.
Front Allergy ; 2: 777545, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35386998

RESUMO

Background: The use of ovalbumin as a model allergen in murine models of allergic asthma is controversially discussed since it is not an aeroallergen and sensitization can only be achieved by using strong Th2-inducing adjuvants. Therefore, in this study, a murine model of asthma has been established in which sensitization against the major grass pollen allergen Phl p5b was performed without using aluminum hydroxide (alum). We used this model for specific immunotherapy. Methods: Female, 5-6-week-old mice were sensitized by six subcutaneous (s.c.) injections of 20 µg Phl p5b followed by four provocations to induce allergic airway inflammation. For desensitization, 1 mg of Phl p5b was injected subcutaneously during allergen challenge for one to a maximum of four times. Three days after the last challenge, the allergic immune response was analyzed. Results: Sensitized and challenged animals showed a significant infiltration of eosinophils into the airways, and the production of interleukin-5 (IL-5) by in vitro re-stimulated splenocytes could be detected. Furthermore, hyper-responsiveness of the airways was verified by invasive measurement of airway resistance in methacholine-challenged animals. Desensitized animals showed a significant reduction of all parameters. Conclusion: In this study, a murine model of asthma has successfully been established by sensitization against the clinically relevant allergen Phl p5b without using alum. S.c. injection of allergen dose dependently led to desensitization of sensitized mice. We suggest that this model is useful to study adjuvant effects of immune modulatory substances on immunotherapy without the interference of alum.

15.
Chemistry ; 27(3): 928-933, 2021 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-32579239

RESUMO

Arabinogalactan, a microheterogeneous polysaccharide occurring in plants, is known for its allergy-protective activity, which could potentially be used for preventive allergy treatment. New treatment options are highly desirable, especially in a preventive manner, due to the constant rise of atopic diseases worldwide. The structural origin of the allergy-protective activity of arabinogalactan is, however, still unclear and isolation of the polysaccharide is not feasible for pharmaceutical applications due to a variation of the activity of the natural product and contaminations with endotoxins. Therefore, a pentasaccharide partial structure was selected for total synthesis and subsequently coupled to a carrier protein to form a neoglycoconjugate. The allergy-protective activity of arabinogalactan could be reproduced with the partial structure in subsequent in vivo experiments. This is the first example of a successful simplification of arabinogalactan with a single partial structure while retaining its allergy-preventive potential.

16.
Psychiatry Res ; 289: 112979, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32438208

RESUMO

Early detection markers for substance use disorders are urgently needed. Recently, an association between the methylation of Ganglioside-induced differentiation-associated protein 1 (GDAP1) and alcohol addiction was found in a US and German population. In this study, we investigate whether GDAP1 expression might be affected by cigarette smoke as well and thus might be a marker of substance addiction in general. 11 adult female C57BL/6 J mice (6 wildtype and 5 lacking the NO-sensitive guanylyl cyclase1 (NO-GC1 KO)) were exposed to cigarette smoke over a period of 5 weeks, their brains immunohistochemically stained and compared to 11 non exposed mice (5 WT and 6 KO). The deletion of NO-GC1 results in a complete loss of synaptic plasticity, therefore, addiction-related alterations might become more obvious. Co-staining of anti-GDAP1 and DAPI revealed protein in the stratum granulare of the hippocampus. Three randomized frames for dentate gyrus (DG) and three for Cornu Ammonis region 1 (CA1) were used to count GDAP1. Cigarette smoke exposure significantly influenced GDAP1 expression depending on the hippocampal region but was not influenced by guanyl cyclase. In conclusion, cigarette smoke exposure alone had an effect on GDAP1 amount in both regions. Therewith, GDAP1might be a biomarker for substance addiction in general.


Assuntos
Região CA1 Hipocampal/metabolismo , Giro Denteado/metabolismo , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/efeitos dos fármacos , Poluição por Fumaça de Tabaco/efeitos adversos , Animais , Fumar Cigarros , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal , Receptores Nicotínicos
18.
Interv Neuroradiol ; 25(6): 619-627, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31248313

RESUMO

BACKGROUND: Stent-assisted coiling and extra-saccular flow diversion require dual anti-platelet therapy due to the thrombogenic properties of the implants. While both methods are widely accepted, thromboembolic complications and the detrimental effects of dual anti-platelet therapy remain a concern. Anti-thrombogenic surface coatings aim to solve both of these issues. Current developments are discussed within the framework of an actual clinical case. CASE DESCRIPTION: A 33-year-old male patient lost consciousness while doing sport and was administered 500 mg acetylsalicylic acid on site. Computed tomography revealed a massive subarachnoid haemorrhage, and digital subtraction angiography showed an aneurysm of the right middle cerebral artery. Stent-assisted coiling using a neck bridging device with a hydrophilic coating (pCONUS_HPC) was considered as an appropriate approach. Another 500 mg acetylsalicylic acid IV was given. After the single anti-platelet therapy was seen to be effective, a pCONUS_HPC was implanted, and the aneurysm sac subsequently fully occluded using coils. No thrombus formation was encountered. During the following days, 2 × 500 mg acetylsalicylic acid IV daily were required to maintain single anti-platelet therapy, monitored by frequent response testing. Follow-up digital subtraction angiography after 13 days confirmed the occlusion of the aneurysm and the patency of the middle cerebral artery. CONCLUSION: A variety of ways to reduce the thrombogenicity of neurovascular stents is discussed. Hydrophilic surface coatings are a valid concept to improve the haemocompatibility of neurovascular implants while avoiding the use of dual anti-platelet therapy. Phosphorylcholine and phenox hydrophilic polymer coating are currently the most promising candidates. This concept is supported by anecdotal experience. However, formalised registries and randomised trials are currently being established.


Assuntos
Aneurisma Intracraniano/terapia , Stents , Hemorragia Subaracnóidea/terapia , Adulto , Angiografia Digital , Aspirina/uso terapêutico , Angiografia Cerebral , Materiais Revestidos Biocompatíveis , Diagnóstico Diferencial , Embolização Terapêutica , Humanos , Aneurisma Intracraniano/diagnóstico por imagem , Aneurisma Intracraniano/tratamento farmacológico , Masculino , Inibidores da Agregação Plaquetária/uso terapêutico , Hemorragia Subaracnóidea/diagnóstico por imagem , Hemorragia Subaracnóidea/tratamento farmacológico , Tomografia Computadorizada por Raios X
20.
J Mol Med (Berl) ; 97(6): 817-828, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30929031

RESUMO

COPD patients have an increased susceptibility to bacterial airway infections that can induce exacerbations. In response to infections, circulating monocytes become recruited to the infected tissue and secrete cytokines. We hypothesized that this cytokine response is reduced in COPD. Cultured peripheral blood monocytes of never smokers (NS) and smokers without (S) and with COPD (3 study populations, n = 36-37) were stimulated with extracts of Haemophilus influenzae, Staphylococcus aureus, or Streptococcus pneumoniae or with four different pathogen-associated molecular patterns (PAMPs). Four cytokines and 9 PAMP-related signaling molecules were measured and compared between the groups. Granulocyte-macrophage-colony-stimulating-factor responses to all stimulants were reduced in S and COPD compared to NS. Tumor-necrosis-factor-α responses to all bacterial extracts, peptidoglycan, and lipopolysaccharide were reduced in S and/or COPD. Interleukin-10 responses to S. aureus and lipoteichoic acid were increased in COPD. Correlations to pack-years and lung function were found. The peptidoglycan-receptor NOD2 and the mRNA of the lipopolysaccharide-receptor TLR4 were reduced in S and COPD. Cytokine responses of monocytes to bacteria are suppressed by smoking and in COPD possibly due to NOD2 and TLR4 reduction and/or interleukin-10 increase. This might help to explain the increased susceptibility to bacterial infections. These systemic molecular pathologies might be targets for therapeutic strategies to prevent infection-induced exacerbations. KEY MESSAGES: COPD subjects have an increased susceptibility to bacterial infections. This implies defects in the immune response to bacteria and is critical for disease progression. The cytokine response of monocytes to bacteria is reduced in COPD. This might be due to a reduced NOD2 and TLR4 and an increased IL-10 expression. This can explain the increased susceptibility to infections and help to identify drug targets.


Assuntos
Bactérias/imunologia , Monócitos/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/microbiologia , Fumar/efeitos adversos , Anticorpos/farmacologia , Feminino , Volume Expiratório Forçado , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Haemophilus influenzae/fisiologia , Humanos , Lipopolissacarídeos , Masculino , Pessoa de Meia-Idade , Proteína Adaptadora de Sinalização NOD2/metabolismo , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
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