The interaction between neuropeptides and the innate immune system in the nasal mucosa of healthy individuals and subjects with allergic rhinitis

Allergic rhinitis is a highly common disease prevalent worldwide. The symptoms include rhinorrhea, itchy eyes and swelling. It is driven by Immunoglobulin E and a type 2 T cell response resulting in inflammation in the upper airways. Allergic rhinitis is often under diagnosed and has no cure, only treatments with varying efficiency. The role of the peripheral nervous system has classically not been thought of as part of the immune system. Recent studies have however, been successful in demonstrating its involvement. One of its most studied mediators, substance P (SP), seems to have a role in priming the innate immune system for incoming dangers. SP and Toll-like Receptors (TLRs) have been shown to regulate one another’s expression in healthy individuals. This project aimed to compare this cross-talk in healthy and allergic subjects. Analysis was made for additional receptors CD10, NK1R and VIPR2 and will also involve the cytokine interleukin 6 (IL-6) which is an important pro-inflammatory cytokine expressed following TLR ligand binding. Healthy and allergic individuals volunteered to leave nasal epithelial cells for culturing. These samples were stimulated with either SP or with R837, the synthetic ligand for TLR7. The project included three major methods; Enzyme-linked immunosorbent assay (ELISA), Realtime quantitative PCR and fluorescent activated cell sorting (FACS). Stimulation with R837 did not give any significant differences in the transcription of genes coding for SP, tac1, and its receptor of choice, tacr1, between 7 healthy and 9 allergic subjects. Real-time qPCR for genes tlr4 and tlr9 showed that transcription of tlr4 was significantly elevated in allergic subjects and that the same was true for transcription of tlr9 but in healthy individuals, following SP exposure. Competitive-ELISA for SP proved that allergic individuals have a slower response to R837 stimulation, secreting significantly less of SP after 15 minutes compared to healthy individuals. The FACS results were hard to interpret and are unusable due to heavily split cells and persistent infections that resulted in a too small patient group for the allergic subjects (n=1). Future projects could include more patients for FACS, more extensive analysis for CD10, incorporating it in the qPCR analysis and to perform an ELISA measuring the antiinflammatory neuropeptide Vasoactive Intestinal Peptide (VIP) as it has antagonistic properties towards SP

The interaction between neuropeptides and the innate immune system in the nasal mucosa of healthy individuals and subjects with allergic rhinitis

Allergic rhinitis is a highly common disease prevalent worldwide. The symptoms include rhinorrhea, itchy eyes and swelling. It is driven by Immunoglobulin E and a type 2 T cell response resulting in inflammation in the upper airways. Allergic rhinitis is often under diagnosed and has no cure, only treatments with varying efficiency. The role of the peripheral nervous system has classically not been thought of as part of the immune system. Recent studies have however, been successful in demonstrating its involvement. One of its most studied mediators, substance P (SP), seems to have a role in priming the innate immune system for incoming dangers. SP and Toll-like Receptors (TLRs) have been shown to regulate one another’s expression in healthy individuals. This project aimed to compare this cross-talk in healthy and allergic subjects. Analysis was made for additional receptors CD10, NK1R and VIPR2 and will also involve the cytokine interleukin 6 (IL-6) which is an important pro-inflammatory cytokine expressed following TLR ligand binding. Healthy and allergic individuals volunteered to leave nasal epithelial cells for culturing. These samples were stimulated with either SP or with R837, the synthetic ligand for TLR7. The project included three major methods; Enzyme-linked immunosorbent assay (ELISA), Realtime quantitative PCR and fluorescent activated cell sorting (FACS). Stimulation with R837 did not give any significant differences in the transcription of genes coding for SP, tac1, and its receptor of choice, tacr1, between 7 healthy and 9 allergic subjects. Real-time qPCR for genes tlr4 and tlr9 showed that transcription of tlr4 was significantly elevated in allergic subjects and that the same was true for transcription of tlr9 but in healthy individuals, following SP exposure. Competitive-ELISA for SP proved that allergic individuals have a slower response to R837 stimulation, secreting significantly less of SP after 15 minutes compared to healthy individuals. The FACS results were hard to interpret and are unusable due to heavily split cells and persistent infections that resulted in a too small patient group for the allergic subjects (n=1). Future projects could include more patients for FACS, more extensive analysis for CD10, incorporating it in the qPCR analysis and to perform an ELISA measuring the antiinflammatory neuropeptide Vasoactive Intestinal Peptide (VIP) as it has antagonistic properties towards SP

The interaction between neuropeptides and the innate immune system in the nasal mucosa of healthy individuals and subjects with allergic rhinitis

Allergic rhinitis is a highly common disease prevalent worldwide. The symptoms include rhinorrhea, itchy eyes and swelling. It is driven by Immunoglobulin E and a type 2 T cell response resulting in inflammation in the upper airways. Allergic rhinitis is often under diagnosed and has no cure, only treatments with varying efficiency. The role of the peripheral nervous system has classically not been thought of as part of the immune system. Recent studies have however, been successful in demonstrating its involvement. One of its most studied mediators, substance P (SP), seems to have a role in priming the innate immune system for incoming dangers. SP and Toll-like Receptors (TLRs) have been shown to regulate one another’s expression in healthy individuals. This project aimed to compare this cross-talk in healthy and allergic subjects. Analysis was made for additional receptors CD10, NK1R and VIPR2 and will also involve the cytokine interleukin 6 (IL-6) which is an important pro-inflammatory cytokine expressed following TLR ligand binding. Healthy and allergic individuals volunteered to leave nasal epithelial cells for culturing. These samples were stimulated with either SP or with R837, the synthetic ligand for TLR7. The project included three major methods; Enzyme-linked immunosorbent assay (ELISA), Realtime quantitative PCR and fluorescent activated cell sorting (FACS). Stimulation with R837 did not give any significant differences in the transcription of genes coding for SP, tac1, and its receptor of choice, tacr1, between 7 healthy and 9 allergic subjects. Real-time qPCR for genes tlr4 and tlr9 showed that transcription of tlr4 was significantly elevated in allergic subjects and that the same was true for transcription of tlr9 but in healthy individuals, following SP exposure. Competitive-ELISA for SP proved that allergic individuals have a slower response to R837 stimulation, secreting significantly less of SP after 15 minutes compared to healthy individuals. The FACS results were hard to interpret and are unusable due to heavily split cells and persistent infections that resulted in a too small patient group for the allergic subjects (n=1). Future projects could include more patients for FACS, more extensive analysis for CD10, incorporating it in the qPCR analysis and to perform an ELISA measuring the antiinflammatory neuropeptide Vasoactive Intestinal Peptide (VIP) as it has antagonistic properties towards SP

Myší modely pro Angelmanův syndrom: generace a charakterizace

Angelman syndrome (AS) is a neurodevelopmental disease found in 1 to 10,000 to 40,000 births, exhibiting an equal gender ratio. Key characteristics of the disease include an ataxic gait with tremor, severe mental retardation, profound speech impairment and seizures. Behavioral deficits such as increased anxiety and autism spectrum disorder features is found in affected individuals as well. The disease stems from the imprinted region 15q11.2-13q where genes are either maternally or paternally expressed as a result of parent-of-origin specific expression of the alleles. There are four main genetic etiologies causing AS namely, i) a large deletion ranging from 4-6 Mb on the maternally inherited allele including imprinted and bi-allelically expressed genes, ii) maternal deletion of the Ubitiquin ligase E3 (UBE3A) gene, iii) paternal uniparental disomy and iv) imprinting defect leading to inappropriate methylation of the locus. So far, there is no cure for AS rather the symptoms are ameliorated using a multidisciplinary approach. The goal of the doctoral study was to further decipher the role of Ube3a and Gabra5 using two mouse models to gain more knowledge about the involvement of these two genes for future therapeutic interventions in for Angelman syndrome. One model generated was a full gene deletion...Angelmanův syndrom (AS) je neurovývojové onemocnění s celosvětovou incidencí 1:10 000, vyskytuje se tedy u 40 000 porodů ročně, poměr mezi pohlavími je stejný. Mezi klíčové charakteristiky onemocnění patří ataktická chůze s třesem, těžká mentální retardace, těžké poruchy řeči a záchvaty. U postižených jedinců se také vyskytují behaviorální deficity, jako je zvýšená úzkost a poruchy autistického spektra. Onemocnění pochází z imprintované oblasti 15q11.2-13q, kde jsou geny exprimovány buď maternálně, nebo paternálně jako výsledek exprese alel specifické pro rodiče. Existují čtyři hlavní genetické mutace způsobující AS, a to i) velká delece v rozsahu 4- 6 Mb na alele zděděné maternálně včetně imprintovaných a bialelicky exprimovaných genů, ii) delece maternálního původu v genu Ubitiquin ligázy E3 (UBE3A), iii) paternální uniparentální disomie a iv) defekt imprintingu vedoucí k nevhodné metylaci lokusu. Dosud neexistuje žádný lék na AS, spíše se symptomy zmírňují pomocí multidisciplinárního přístupu. Cílem doktorského studia bylo dále dešifrovat roli UBE 3A a GABRA5 pomocí dvou myších modelů s cílem získat více znalostí o zapojení těchto dvou genů do generování modelů pro budoucí terapeutické intervence. Jedním vytvořeným modelem byla úplná genová delece Ube3a z 5'UTR do 3'UTR zahrnující přibližně 76...Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyFaculty of SciencePřírodovědecká fakult

Mouse models for Angelman syndrome: generation and characterization

Angelman syndrome (AS) is a neurodevelopmental disease found in 1 to 10,000 to 40,000 births, exhibiting an equal gender ratio. Key characteristics of the disease include an ataxic gait with tremor, severe mental retardation, profound speech impairment and seizures. Behavioral deficits such as increased anxiety and autism spectrum disorder features is found in affected individuals as well. The disease stems from the imprinted region 15q11.2-13q where genes are either maternally or paternally expressed as a result of parent-of-origin specific expression of the alleles. There are four main genetic etiologies causing AS namely, i) a large deletion ranging from 4-6 Mb on the maternally inherited allele including imprinted and bi-allelically expressed genes, ii) maternal deletion of the Ubitiquin ligase E3 (UBE3A) gene, iii) paternal uniparental disomy and iv) imprinting defect leading to inappropriate methylation of the locus. So far, there is no cure for AS rather the symptoms are ameliorated using a multidisciplinary approach. The goal of the doctoral study was to further decipher the role of Ube3a and Gabra5 using two mouse models to gain more knowledge about the involvement of these two genes for future therapeutic interventions in for Angelman syndrome. One model generated was a full gene deletion..

Generation and Characterization of a Novel Angelman Syndrome Mouse Model with a Full Deletion of the Ube3a Gene

Angelman syndrome (AS) is a neurodevelopmental disorder caused by deficits in maternally inherited UBE3A. The disease is characterized by intellectual disability, impaired motor skills, and behavioral deficits, including increased anxiety and autism spectrum disorder features. The mouse models used so far in AS research recapitulate most of the cardinal AS characteristics. However, they do not mimic the situation found in the majority of AS patients who have a large deletion spanning 4–6 Mb. There is also a large variability in phenotypes reported in the available models, which altogether limits development of therapeutics. Therefore, we have generated a mouse model in which the Ube3a gene is deleted entirely from the 5′ UTR to the 3′ UTR of mouse Ube3a isoform 2, resulting in a deletion of 76 kb. To investigate its phenotypic suitability as a model for AS, we employed a battery of behavioral tests directed to reveal AS pathology and to find out whether this model better mirrors AS development compared to other available models. We found that the maternally inherited Ube3a-deficient line exhibits robust motor dysfunction, as seen in the rotarod and DigiGait tests, and displays abnormalities in additional behavioral paradigms, including reduced nest building and hypoactivity, although no apparent cognitive phenotype was observed in the Barnes maze and novel object recognition tests. The AS mice did, however, underperform in more complex cognition tasks, such as place reversal in the IntelliCage system, and exhibited a different circadian rhythm activity pattern. We show that the novel UBE3A-deficient model, based on a whole-gene deletion, is suitable for AS research, as it recapitulates important phenotypes characteristic of AS. This new mouse model provides complementary possibilities to study the Ube3a gene and its function in health and disease as well as possible therapeutic interventions to restore function

Ablation of <i>Gabra5</i> Influences Corticosterone Levels and Anxiety-like Behavior in Mice

Stress responses are activated by the hypothalamic-pituitary-adrenal axis (HPA axis), culminating in the release of glucocorticoids. During prolonged periods of secretion of glucocorticoids or inappropriate behavioral responses to a stressor, pathologic conditions may occur. Increased glucocorticoid concentration is linked to generalized anxiety, and there are knowledge gaps regarding its regulation. It is known that the HPA axis is under GABAergic control, but the contribution of the individual subunits of the GABA receptor is largely unknown. In this study, we investigated the relationship between the α5 subunit and corticosterone levels in a new mouse model deficient for Gabra5, which is known to be linked to anxiety disorders in humans and phenologs observed in mice. We observed decreased rearing behavior, suggesting lower anxiety in the Gabra5−/− animals; however, such a phenotype was absent in the open field and elevated plus maze tests. In addition to decreased rearing behavior, we also found decreased levels of fecal corticosterone metabolites in Gabra5−/− mice indicating a lowered stress response. Moreover, based on the electrophysiological recordings where we observed a hyperpolarized state of hippocampal neurons, we hypothesize that the constitutive ablation of the Gabra5 gene leads to functional compensation with other channels or GABA receptor subunits in this model

Ablation of Gabra5 Influences Corticosterone Levels and Anxiety-Like Behavior in Mice

Stress responses are activated by the hypothalamic-pituitary-adrenal axis (HPA axis), culminating in the release of glucocorticoids. During prolonged periods of secretion of glucocorticoids or inappropriate behavioral responses to a stressor, pathologic conditions may occur. Increased glucocorticoid concentration is linked to generalized anxiety, and there are knowledge gaps regarding its regulation. It is known that the HPA axis is under GABAergic control, but the contribution of the individual subunits of the GABA receptor is largely unknown. In this study, we investigated the relationship between the &alpha;5 subunit and corticosterone levels in a new mouse model deficient for Gabra5, which is known to be linked to anxiety disorders in humans and phenologs observed in mice. We observed decreased rearing behavior, suggesting lower anxiety in the Gabra5&minus;/&minus; animals; however, such a phenotype was absent in the open field and elevated plus maze tests. In addition to decreased rearing behavior, we also found decreased levels of fecal corticosterone metabolites in Gabra5&minus;/&minus; mice indicating a lowered stress response. Moreover, based on the electrophysiological recordings where we observed a hyperpolarized state of hippocampal neurons, we hypothesize that the constitutive ablation of the Gabra5 gene leads to functional compensation with other channels or GABA receptor subunits in this model