Pattern-recognition receptors in airway inflammation

Airway inflammation is a defining feature of allergic rhinitis and asthma. Bacterial and viral infections are known to cause exacerbations of both diseases, but knowledge about the mechanisms involved is limited. The pattern-recognition receptors (PRRs) comprise several receptor families which recognize microbial structures or host-derived danger signals, and trigger an immune response. The Toll-like receptors (TLRs) are the best characterized set of receptors, but another family of PRRs, the Nod-like receptors (NLRs), has recently been described. The TLRs and NLRs are found both in leukocytes and structural cells throughout the airways and are becoming increasingly implicated in airway inflammation. This thesis characterizes the presence and functional response of various members of the TLR and NLR families. Lipopolysaccharide (LPS) is found in the cell wall of Gram-negative bacteria and exerts its effects through TLR4. In the first three papers nasal LPS challenge was used as a method to study neutrophil inflammatory responses in the nose. In the first paper we explored if the upper airways could be used as a model for inflammatory events in the lung. LPS-induced neutrophil inflammation in the nose was inhibited and the results compared with findings in a study of similar design on the lower airways. The nasal model was found to mimic the responses seen in the lower airway study, with no signs of systemic activation or adverse effects. This suggests the nasal LPS model to be a safe and convenient method for studying neutrophilic airway inflammation. The nasal model was subsequently used in the second paper to analyze the role of macrophage inflammatory protein (MIP)-1α in LPS-induced local inflammation. The LPS challenge resulted in a neutrophil-mediated secretion of MIP-1α, dependent on the nuclear factor (NF)-κB, protein kinase C (PKC) and p38 mitogen activated protein kinase (MAPK) pathways. LPS also delayed neutrophil apoptosis in vitro, suggesting that the secretion of MIP-1α may be boosted by a prolonged neutrophil survival. This indicates that MIP- 1α plays a role in neutrophilic airway disease. In the third paper we investigated whether symptomatic allergic rhinitis affected the expression of TLR4 in nasal lavage, blood and bone marrow. Provocation with LPS in a milieu of allergic inflammation, caused by allergen challenge, resulted in a release of cytokines like interleukin (IL)-4, IL-5, IL-10, IL-13, Interferon (IFN)-γ and tumor necrosis factor (TNF)-α. No such cytokine release was seen with either allergen or LPS alone, nor when LPS preceded allergen. The systemic up-regulation of TLR4 seen during on-going allergic rhinitis might contribute to the presently seen increase in LPS response. It is therefore tempting to extrapolate these findings to a clinical situation in which a local infection can cause exacerbation or aggravation of allergic symptoms. The systemic up-regulation of TLR4 seen during symptomatic allergic rhinitis might contribute to the observed increase in response when LPS was applied following allergen challenge. The results strengthen the suggestion that a local infection may exacerbate allergic inflammation. The fourth study aimed to visualize the effects of allergen on two major populations of dendritic cells, the myeloid (mDCs) and the plasmacytoid dendritic cells (pDCs), in the nose of patients with allergic rhinitis. Allergen challenge increased the number of pDCsin the nasal sub-epithelium. In vitro studies of pDCs revealed that they could be activated by TNF-α, IL-4 and CpG stimulation, and that TNF-α caused a higher activation among atopic than non-atopic subjects. The data support the notion of mDCs and pDCs as distinct populations with different roles in the allergic process. Further, it suggests that the pDCs observed upon allergen challenge might be of an activated phenotype and play a role in the course of allergic rhinitis. The fifth study focused on the effects of TLR7 stimulation on airway smooth muscle contraction. Guinea pig tracheas were stimulated with the TLR7 agonists R837 and R848 for three days, and the contractile response along with the underlying signal pathways were investigated in a myograph model. TLR7 stimulation was found to reduce airway smooth muscle contraction in an epithelium-independent manner, dependent on p38 MAPK and NF-κB pathways. This indicates that TLR7 stimulation can be part of a protective mechanism against virus infection and that TLR7 deficiency might be a cause of airway disease. Further, it suggests that TLR7 ligands might be a future option for treatment of airway hyperresponsiveness. The sixth study characterized the expression and function of NLRs in neutrophils, with focus on Nucleotide-binding oligomerization domain (NOD)1, NOD2 and NACHTLRR- PYD binding protein (NLRP)3. An expression of NOD2 and NLRP3 protein and mRNA was found in isolated neutrophils. NOD2 activation resulted in IL-8 secretion and a change in neutrophil phenotype, while activation of NLRP3 caused secretion of IL-1β. Both receptors caused an increased neutrophil migration. The findings might reflect a previously unknown pathway for activation of these cells

Nerve growth factor receptors in equine synovial membranes vary with osteoarthritic disease severity

Nerve growth factor (NGF) is a neurotrophin that has been implicated in pain signaling, apoptosis, inflammation and proliferation. The resultant effects depend on interaction with two different receptors; tyrosine kinase A (TrkA) and p75(NTR). NGF increases in synovial fluid from osteoarthritic joints, and monoclonal antibody therapy is trialed to treat osteoarthritis (OA)-related pain. Investigation of the complex and somewhat contradictory signaling pathways of NGF is conducted in neural research, but has not followed through to orthopaedic studies. The objectives of this study were to compare the expression of NGF receptors and the downstream regulator BAX in synovial membranes from joints in various stages of OA. The horse was used as a model. Synovial membranes were harvested from five healthy horses postmortem and from clinical cases with spontaneous OA undergoing arthroscopic surgery for lameness. Four horses with synovitis without gross cartilage changes, four horses with synovitis and cartilage damage, and four horses with synovitis and intracarpal fractures were included. Samples were investigated by immunohistochemistry and results showed that nuclear staining of TrkA, p75(NTR) and BAX increases in OA-associated synovitis. TrkA expression increased in early disease stages whereas increases in p75(NTR) were most prominent in later disease stages with cartilage damage and fibrosis. Clinical significance: Suppression of NGF may result in varied effects depending on different stages of the osteoarthritic disease process

Bovine B cells : B lymphopoiesis and the Generation of Primary Immunoglobulin Repertoire in Fetal Cattle

Immunological research is dominated by studies on man and mouse, however, only some aspects of this field are universal among vertebrates. Whilst the production of T cells is universal, B cell production cannot be extrapolated from one species to another. As such, ruminant B cell biology has distinctive features not similar to the conventional man or mouse based models. Cattle are large long-lived ruminants, of major significance globally. The bovine ileal Peyer s patch, an organ of B cell follicles along the gut, is where V(D)J immunoglobulin gene rearranged B cells proliferate, however it is not known where and how the DNA rearrangements take place before the B cells enter these follicles. Furthermore, it is unclear whether post-recombinatorial modifications, such as somatic hypermutation or gene conversion, facilitate the generation of more antibody specificities of the immunoglobulin genes in these follicles. Herein, the bovine immunoglobulin light chain genomic locus was characterized and only a moderate number of functional gene segments that cause low combinatorial antibody diversity were found. The lambda locus is the larger of the two light chain loci, containing 25 functional variable gene segments, compared to the kappa locus, which contains only eight. Functional genes comprise less than half of all the variable genes in both loci, the remainder representing unfunctional pseudogenes. The immunoglobulin genes of the fetal ruminant ileal Peyer s patch can possibly be further modified. Accordingly, the expression of activation-induced cytidine deaminase (AID), a mutator protein, was demonstrated here in fetal cattle ileal Peyer s patch. Sequencing of expressed heavy chain variable genes in these follicles showed ongoing hypermutation. The mutations were concentrated on the complementarity determining regions (CDR) of the variable genes, and on the hotspot target sequences of AID. AID-dependent mutations have usually been ascribed to antigen dependent affinity maturation, but this work demonstrates mutations in fetal immunoglobulin genes before exposure to external antigen. Bovine B lymphopoiesis is studied here, its localization in the fetal cattle in particular. By analyzing the expression of RAG1 and RAG2, which take part in the rearrangement of immunoglobulin genes, active B lymphopoiesis was demonstrated in fetal bone marrow and lymph node. The expression of surrogate light chain genes VPREB1 and IGLL1 was also shown in these same tissues. The expression of these genes implicated that a pre-B cell stage exists in cattle. This was further confirmed by the presence of a phenotypic pre-B cell population in fetal bone marrow and lymph node. VPREB2 and VPREB3 were expressed differently from the other surrogate light chain genes, which indicate that their function in cattle might not be related to pre-B cells. Overall, B lymphopoiesis was shown to take place in fetal, but not in adult, bovine tissues. Pre-B cell related genes RAGs, VPREB1 and IGLL1 were not expressed in adult tissues. Further, adult bone marrow cells were not able to differentiate into B lineage cells in cell culture. These results suggest that no new immunoglobulin rearrangements are generated during bovine adult life. Thus it is likely that the animal manages its whole life with the peripheral B cell pool produced during the fetal and neonatal period.Immunologinen tietämys perustuu lähinnä ihmisen ja hiiren immunologiaan, vaikka kaikki immunologinen tieto ei sinällään ole yleistettävissä muihin eläinlajeihin. Esimerkiksi T-solujen tuotanto on hyvin samankaltainen lajista riippumatta, mutta B-solujen tuotannossa erot ovat merkittäviä. Näitä B-solubiologian eroja on havaittu esimerkiksi märehtijöillä, joista tässä väitöskirjassa keskitytään nautaan, pitkäikäiseen ja maailmanlaajuisesti tärkeään märehtijään. Naudan suolistossa olevat ileumin Peyerin levyt ovat solusaarekkeita, joissa B-solut jakautuvat ja lisääntyvät. Tiedetään, että ennen kuin nämä solusaarekkeet alkavat muodostua, ovat B-solut uudelleenjärjestäneet vasta-ainegeeninsä. Uudelleenjärjestäytyminen aikaa ja paikkaa ei tiedetä kuten ei sitäkään, tapahtuuko Peyerin levyssä tämän jälkeistä vasta-ainegeenien muokkausta hypermutaation tai geenikonversion keinoin. Tässä väitöskirjassa naudan vasta-aineiden kevyen ketjun geenilokus karakterisoitiin, ja siitä löydettiin vain kohtalainen määrä toimivia geenisegmenttejä, mistä johtuen segmenttien yhdistelmien tarjoama monimuotoisuus on alhainen. Lambda-lokus sisältää 25 toimivaa variaabelia geenisegmenttiä, kappa-lokuksessa niitä on vain 8. Molemmissa lokuksissa alle puolet geeneistä on toimivia, ja loput ovat toimimattomia pseudogeenejä. On mahdollista, että märehtijöiden sikiön Peyerin levyissä vasta-ainegeenejä mutatoidaan vielä uudelleenjärjestäytymisen jälkeen. Tässä väitöskirjassa osoitettiin tunnetun mutaattori-proteiinin, aktivaatio-indusoidun sytidiini deaminaasin (AID) ekspressio naudan sikiön Peyerin levyissä. Vasta-ainegeenien sekvensoinnin perusteella mutaatiot keskittyivät erityisesti geenien vaihteleville alueille, AID-proteiinin tunnetuille kohdesekvensseille. AID:n on aiemmin tiedetty mutatoivan vasta-ainegeenejä syntymän jälkeen ulkoisen antigeenin kohtaamisen seurauksena, mutta tässä työssä osoitettiin AID:n toiminta jo sikiöaikana. Tämän väitöskirjan eräs osatavoite oli kuvata naudan B-solujen syntyä eli B-lymfopoieesia. Sen osoitettiin tapahtuvan sikiön luuytimessä ja imusolmukkeessa, sillä näissä kudoksissa vasta-ainegeenien uudelleenjärjestäytymiseen liittyvät RAG-proteiinit ekspressoituivat. Myös B-solun erään kehitysvaiheen, preB-solun, olemassaolo todistettiin naudalla. PreB-solun tunnusmerkki on preB-solureseptori, jonka osia koodaavien geenien, VPREB1:n ja IGLL1:n, todettiin olevan aktiivisia B-lymfopoieettisissa kudoksissa. VPREB1:n rinnakkaiset muodot VPREB2 ja VPREB3 ekspressoituivat poikkeavasti, joten niiden funktio ei välttämättä liity preB-soluihin. Väitöskirjassa todistettiin, että naudalla B-lymfopoieesia tapahtuu sikiöaikana, muttei enää aikuisena. PreB-soluihin liittyvää ekspressiota RAG-, VPREB1- ja IGLL1-geeneistä ei havaittu aikuisen kudoksissa. Aikuisen luuydinsolut eivät myöskään pystyneet erilaistumaan B-solulinjan soluiksi viljelyolosuhteissa. Näiden tulosten perusteella vaikuttaa todennäköiseltä, että B-soluerilaistumista ei tapahdu enää aikuisella eläimellä. Tällöin eläimen tulee pärjätä koko elämänsä niillä B-soluilla, jotka ovat syntyneet sikiö- tai nuoruusiällä

Bos taurus genome sequence reveals the assortment of immunoglobulin and surrogate light chain genes in domestic cattle

<p>Abstract</p> <p>Background</p> <p>The assortment of cattle immunoglobulin and surrogate light chain genes has been extracted from the version 3.1 of <it>Bos taurus </it>genome sequence as a part of an international effort to sequence and annotate the bovine genome.</p> <p>Results</p> <p>63 variable lambda chain and 22 variable kappa chain genes were identified and phylogenetically assigned to 8 and 4 subgroups, respectively. The specified phylogenetic relationships are compatible with the established ruminant light chain variable gene families or subgroups. Because of gaps and uncertainties in the assembled genome sequence, the number of genes might change in the future versions of the genome sequence. In addition, three bovine surrogate light chain genes were identified. The corresponding cDNAs were cloned and the expression of the surrogate light chain genes was demonstrated from fetal material.</p> <p>Conclusion</p> <p>The bovine kappa gene locus is compact and simple which may reflect the preferential use of the lambda chain in cattle. The relative orientation of variable and joining genes in both loci are consistent with a deletion mechanism in VJ joining. The orientation of some variable genes cannot be determined from the data available. The number of functional variable genes is moderate when compared to man or mouse. Thus, post-recombinatorial mechanisms might contribute to the generation of the bovine pre-immune antibody repertoire. The heavy chains probably contribute more to recombinational immunoglobulin repertoire diversity than the light chains but the heavy chain locus could not be annotated from the version 3.1 of <it>Bos taurus </it>genome.</p

Identification of major cell types in paraffin sections of bovine tissues

BACKGROUND: Identification of cell types in bovine tissue sections is complicated by the limited availability of anti-bovine antibodies, and by antigen retrieval treatments required for formalin-fixed tissue samples. We have evaluated an antibody and lectin panel for identifying major cell types in paraffin-embedded bovine tissue sections, and report optimized pretreatments for these markers. RESULTS: We selected 31 useful antibodies and lectins which can be used to identify cell types of epithelia, connective tissue, muscle, and nervous tissue, as well as cell proliferation and apoptosis. CONCLUSION: The panel of markers allows the identification of all major cell types in paraffin-embedded cattle tissue sections by immunohistochemistry or lectin histochemistry. Heat-induced epitope retrieval methods are required for most antibodies

Responsiveness of a modified version of the postural assessment scale for stroke patients and longitudinal change in postural control after stroke- Postural Stroke Study in Gothenburg (POSTGOT) -

Abstract Background Responsiveness data certify that a change in a measurement output represents a real change, not a measurement error or biological variability. The objective was to evaluate the responsiveness of the modified version of the Postural Assessment Scale for Stroke Patients (SwePASS) in patients with a first event of stroke. An additional aim was to estimate the change in postural control during the first 12 months after stroke onset. Methods The SwePASS assessments were conducted during the first week and 3, 6 and 12 months after stroke in 90 patients. Svensson’s method, Relative Position (RP), Relative Concentration (RC) and Relative Rank Variance (RV), were used to estimate the scale’s responsiveness and the patients’ change in postural control over time. Results From the first week to 3 months after stroke, the patients improved in terms of postural control with 2 to 12 times larger systematic changes in Relative Position (RP), for which 9 items and the total score showed a significant responsiveness to change when compared to the intrarater reliability measurement error of the SwePASS reported in a previous study. When SwePASS was used to assess change in postural control between the first week and 3 months, 74% of the patients received higher scores while 10% received lower scores, RP 0.31 (95% CI 0.219-0.402). The corresponding figures between 3 and 6 and between 6 and 12 months were 37% and 16%, RP 0.09 (95% CI 0.030-0.152), and 18% and 26%, RP −0.07 (95% CI −0.134- (−0.010)), respectively. Conclusions The SwePASS is responsive to change. Postural control evaluated using the SwePASS showed an improvement during the first 6 months after stroke. The measurement property, in the form of responsiveness, shows that the SwePASS scoring method can be considered for use in rehabilitation when assessing postural control in patients after stroke, especially during the first 3 months.</p

Att välja grundläggande karttjänst : utveckling av jämförelsemodell och testverktyg för utvärdering

Sammanfattning: Många verksamheter använder karttjänster för att lösa problem och underlätta beslutsfattande. Allt fler upptäcker fördelarna med detta och integrerar karttjänster i verksamhetssystem. Dessa tjänster utvecklas från grundläggande karttjänster som kan ha betydande skillnader mellan sig vilket gör valet av tjänst till en viktig fråga. Eftersom karttjänsterna är olika och uppbyggda av en mängd mindre beståndsdelar blir jämförelsen av karttjänsterna tidsödande om alla komponenter ska utvärderas mot varandra. Som stöd vid val av karttjänst utvecklades inom examensarbetet en jämförelsemodell. Modellen kan användas för att underlätta urvalsarbetet genom att strukturera de beståndsdelar som bygger upp en karttjänst för att tydliggöra skillnader mellan dem. Under litteraturstudier upptäcktes hur olika och komplexa verksamheters kravbilder på en kartjänst kan vara. En karttjänst är uppbyggd av en mängd beståndsdelar, olika parametrar är viktiga för olika verksamheter. För att inte proritera bort parametrar som i vissa situationer kan vara av värde strukturerades de under sju kategorier för att öka överblicken. För att spegla att verksamheter har olika behov kan användare justera vikter efter en kravspecifikation så att tyngdpunkten läggs på viktiga kategorier. Modellen anpassas efter användarens krav och användarbarheten ökar. För att underlätta användningen av jämförelsemodellen utvecklades ett testverktyg anpassat efter modellen. I verktyget kan användare ställa in vikter för att anpassa verktyget efter de egna behoven. Det finns test för de flesta kategorierna där användaren får en uppfattning om karttjänsternas för- och nackdelar då de utvärderas sida vid sida. Användaren betygsätter sedan respektive kategori. När utvärderingen är klar visas ett resultat där vikter och betyg presenteras. För att förbättra modell samt testverktyg utfördes användartester och två större pilotfall. I pilotstudien konstruerades verklighetstrogna testfall vilka användes för att utvärdera modell och verktyg. Den slutsats som framkommit är att ett användningsområde för en generell jämförelsemodell samt testverktyg finns. De prototyper som framtagits under examensarbetet behöver dock utvecklas för att fungera framgångsrikt.Summary: Many organizations use map services in their daily work to solve problems and enhance decision making processes. The number of organizations who enjoy the benefits of map services and integrate them into their system is increasing. To choose a certain map service is an important decision since the differences between them can be significant. The service is dependent on many minor components which all affect the outcome of the integrated system. To simplify the procedure of choosing a fundamental map service, a general model was defined. The benchmarking model is used to ease the selection process by structuring the features that constitute a map service and clarifying the differences between them. An existing structure was sought to inspire and help define the benchmarking model. Studies of GIS related organizations and map services were also conducted, and the knowledge was used for choosing the components of the model. Requirement specifications of map services for organizations can be complex and varied. Parameters for map services are measured differently depending on needs and business focus. Therefore, parameters in the model were structured under seven categories to create an overview. The benchmarking model can be adjusted with weights for each category to put emphasis on the most important requirements. This way the model can be kept general while being specific for its users. A testing tool was implemented in the form of a web application, designed to evaluate and complement the model. The tool is an implementation of the benchmarking model and gives the user a deeper knowledge and understanding of the model. While using the testing tool, a user may configure the tool to better suit the individual priorities. This is done by adjusting weights for each category. Some of the categories have hands on tests where both map services are presented together and commands chosen by the user are carried out simultaneously. These tests help the user grade the different categories which are finally presented along with the weights in a result page. User tests and a pilot study helped to detect minor faults in the model and the application during the working process. For the pilot study two users cases were designed to test the model in different situations close to reality. The conclusion of the master thesis is that a benchmarking model in collaboration with a testing tool can be useful in the decision making process of integrating a map service. However, the prototypes that have been developed necessitates improvement to operate successfully

"Ett förändrat samhälle" - en kvantitativ studie av föräldrars syn på att hantera och förebygga stress hos barn

It´s a general opinion that stress among children is something that has increased (Stressforskningsinstitutet, 2015). This study aims to investigate opinions among parents about their knowledge in the subject. The study also aims to look at strategies parents use in their everyday life to prevent stress in the family, and how competent parents feel to protect their children against stress. A questinnaire was handed out to parents of children from six to seven years old, in a small comunity in the South of Sweden (n=166). The empirical material shows that a majority of parents, 65.7%, think they have a good knowledge about stress among children and 69.9% use strategies in their everyday life. 42.8% think they can greatly protect children against stress and 53% think they can protect to a lower extent. We found a significant relationship between parents who think they have good knowledge about stress in children, and the use of strategies. We also found a relationship between the use of strategies and the trust in ability to protect the children against stress. Women are more likely to use strategies in everyday life than men. Keywords: stress in children, parents, coping, gender, knowledge, strategies, protectio

Psychosocial resources predict frequent pain differently for men and women: A prospective cohort study

Introduction Psychosocial resources, psychological and social factors like self-efficacy and social support have been suggested as important assets for individuals with chronic pain, but the importance of psychosocial resources for the development of pain is sparsely examined, especially sex and gender differences. The aim of this study was to investigate associations between psychosocial resources and sex on the development of frequent pain in a general population sample, and to deepen the knowledge about sex and gender patterns. Methods A sample from the Swedish Health Assets Project, a longitudinal cohort study, included self-reported data from 2263 participants, 53% women, with no frequent pain at baseline. The outcome variable was frequent pain at 18–months follow-up. Psychosocial resources studied were general self-efficacy, instrumental and emotional social support. Log binomial regressions in a generalised linear model were used to calculate risk ratios (RRs), comparing all combinations of men with high psychosocial resources, men with low psychosocial resources, women with high psychosocial resources and women with low psychosocial resources. Results Women with low psychosocial resources had higher risk of frequent pain at follow-up compared to men with high resources: general self-efficacy RR 1.82, instrumental social support RR 2.33 and emotional social support RR 1.94. Instrumental social support was the most important protective resource for women, emotional social support was the most important one for men. Results were discussed in terms of gender norms. Conclusions The psychosocial resources general self-efficacy, instrumental and emotional support predicted the risk of developing frequent pain differently among and between men and women in a general population sample. The results showed the importance of studying sex and gender differences in psychological and not least social predictors for pain.publishedVersio