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- Düringer, Caroline, et al.
(författare)
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Agonist-specific patterns of beta(2)-adrenoceptor responses in human airway cells during prolonged exposure.
- 2009
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Ingår i: British Journal of Pharmacology. - : Wiley. - 1476-5381 .- 0007-1188. ; 158, s. 169-179
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Tidskriftsartikel (refereegranskat)abstract
- Background and purpose: beta(2)-Adrenoceptor agonists (beta(2)-agonists) are important bronchodilators used in the treatment of asthma and chronic obstructive pulmonary disease. At the molecular level, beta(2)-adrenergic agonist stimulation induces desensitization of the beta(2)-adrenoceptor. In this study, we have examined the relationships between initial effect and subsequent reduction of responsiveness to restimulation for a panel of beta(2)-agonists in cellular and in vitro tissue models. Experimental approach: beta(2)-Adrenoceptor-induced responses and subsequent loss of receptor responsiveness were studied in primary human airway smooth muscle cells and bronchial epithelial cells by measuring cAMP production. Receptor responsiveness was compared at equi-effective concentrations, either after continuous incubation for 24 h or after a 1 h pulse exposure followed by a 23 h washout. Key findings were confirmed in guinea pig tracheal preparations in vitro. Key results: There were differences in the reduction of receptor responsiveness in human airway cells and in vitro guinea pig trachea by a panel of beta(2)-agonists. When restimulation occurred immediately after continuous incubation, loss of responsiveness correlated with initial effect for all agonists. After the 1 h pulse exposure, differences between agonists emerged, for example isoprenaline and formoterol induced the least reduction of responsiveness. High lipophilicity was, to some extent, predictive of loss of responsiveness, but other factors appeared to be involved in determining the relationships between effect and subsequent loss of responsiveness for individual agonists. Conclusions and implications: There were clear differences in the ability of different beta(2) agonists to induce loss of receptor responsiveness at equi-effective concentrations.
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- Grundström, Gunilla, 1968-
(författare)
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Functional Studies of Collagen-Binding Integrins α2β1 and α11β1 : Interplay between Integrins and Platelet-Derived Growth Factor Receptors
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Integrins are heterodimeric cell surface receptors, composed of an α- and a β-subunit, which mediate cell-extracellular matrix (ECM) interactions. Integrins mediate intracellular signals in response to extracellular stimuli, and cooperate with growth factor and other cytokine receptors. Cells execute their differentiated functions anchored to an ECM. In this thesis functional properties of the two collagen-binding integrins α2β1 and α11β1 were studied. In addition, the impact of β1 cytoplasmic tyrosines in collagen-induced signalling was analyzed.The integrin α11β1 is the latest identified collagen-binding integrin. In this study, tissue distribution of α11 mRNA and protein during embryonal development was explored, and the first α11β1-mediated cellular functions were established. Both α11 protein and mRNA were present in mesenchymal cells in intervertebral discs and around the cartilage of the developing skeleton. α11 protein was also detected in cornea keratinocytes. α11β1 mediated cation-dependent adhesion to collagen types I and IV and localized to focal adhesions. In addition, α11β1 mediated contraction of a collagen lattice and supported cell migration through a collagen substrate. PDGF-BB and FBS both stimulated α11β1-mediated contraction and directed migration.Expression of β1Y783,795F in β1-null cells, prevents activation of FAK in response to fibronectin, and decreases cell migration. In this study, we investigated how this mutation affected α2β1-mediated functions in response to collagen. The β1 mutation impaired collagen gel contraction and prevented activation of FAK, Cas and Src on planar collagen, but not in collagen gels. PDGF-BB stimulated contraction via αvβ3, which also induced activation of Cas in collagen gels. The YY-FF mutation also abolished β1A-dependent downregulation of β3.In the final study integrin-crosstalk during collagen gel contraction was investigated. In cells lacking collagen-binding integrins αvβ3 mediated contraction. Clustering of β1-integrins by antibodies and PDGF-BB stimulated αvβ3-mediated contraction in an ERK-dependent way. Expression of α2β1, but not α11β1, prevented αvβ3-mediated contraction. Contraction by α2β1 and α11β1 was ERK-independent.
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| 5. |
- Grundström, Gunilla, et al.
(författare)
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Integrin αvβ3 mediates platelet-derived growth factor-BB-stimulated collagen gel contraction in cells expressing signaling deficient integrin α2β1
- 2003
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Ingår i: Experimental Cell Research. - : Elsevier BV. - 0014-4827 .- 1090-2422. ; 291:2, s. 463-473
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Tidskriftsartikel (refereegranskat)abstract
- The interplay between the collagen-binding integrin, α2β1, and platelet-derived growth factor (PDGF) receptors in the context of functional interactions with collagen was studied. We expressed either wild-type α2β1 (α2β1A) or α2β1 with a Y783/795F mutation in the cytoplasmic tail of the β1 subunit (α2β1Amut) in the β1-null fibroblastic cell line, GD25. GD25 cells lack endogenous expression of the α1 and α2 integrin subunits and do not adhere to collagen even after transfection with β1A. Cells expressing α2β1Amut contracted three-dimensional collagen lattices less efficiently than those expressing α2β1A. PDGF-BB significantly stimulated lattice contraction by GD25-α2β1Amut cells. Both cell types responded chemotactically to PDGF-BB. Focal adhesion kinase (FAK) and p130Cas were phosphorylated when GD25-α2β1A cells, but not GD25-α2β1Amut cells were seeded on collagen-coated dishes. Subsequent treatment with PDGF-BB further increased phosphorylation of FAK and p130Cas only in GD25-α2β1A cells. However, when cultured within collagen lattices, FAK and p130Cas phosphorylation were stimulated in both α2β1A- and α2β1Amut-expressing cells but further phosphorylation, in response to subsequent treatment with PDGF-BB, was seen only in GD25-α2β1A cells. We show that the stimulatory effects of PDGF-BB on collagen gel contraction and chemotaxis by GD25-α2β1Amut cells were mediated by the αvβ3 integrin. Phosphorylation of p130Cas, but not FAK, in GD25-α2β1Amut cells seeded in collagen lattices also depended on αvβ3. Our results show that PDGF-BB stimulation of fibroblast–collagen interactions is mediated by the αvβ3 integrin when β1 integrin function is impaired.
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| 6. |
- Grundström, Gunilla, et al.
(författare)
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Skin Sensitization Testing-What's Next?
- 2019
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1422-0067. ; 20:3
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Tidskriftsartikel (refereegranskat)abstract
- There is an increasing demand for alternative in vitro methods to replace animal testing, and, to succeed, new methods are required to be at least as accurate as existing in vivo tests. However, skin sensitization is a complex process requiring coordinated and tightly regulated interactions between a variety of cells and molecules. Consequently, there is considerable difficulty in reproducing this level of biological complexity in vitro, and as a result the development of non-animal methods has posed a major challenge. However, with the use of a relevant biological system, the high information content of whole genome expression, and comprehensive bioinformatics, assays for most complex biological processes can be achieved. We propose that the Genomic Allergen Rapid Detection (GARD™) assay, developed to create a holistic data-driven in vitro model with high informational content, could be such an example. Based on the genomic expression of a mature human dendritic cell line and state-of-the-art machine learning techniques, GARD™ can today accurately predict skin sensitizers and correctly categorize skin sensitizing potency. Consequently, by utilizing advanced processing tools in combination with high information genomic or proteomic data, we can take the next step toward alternative methods with the same predictive accuracy as today's in vivo methods-and beyond.
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| 7. |
- Huang, Shan, et al.
(författare)
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Low concentrations of citrate reduce complement and granulocyte activation in vitro in human blood
- 2015
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Ingår i: Clinical Kidney Journal. - : Oxford University Press (OUP). - 2048-8505 .- 2048-8513. ; 8:1, s. 31-37
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND:The use of acetate in haemodialysis fluids may induce negative effects in patients including nausea and increased inflammation. Therefore, haemodialysis fluids where acetate is substituted with citrate have recently been developed. In this study, we investigated the biocompatibility of citrate employing concentrations used in haemodialysis.METHODS:The effects of citrate and acetate were investigated in human whole blood in vitro under conditions promoting biomaterial-induced activation. Complement activation was measured as generation of C3a, C5a and the sC5b-9 complex, and granulocyte activation as up-regulation of CD11b expression. For the experimental set-up, a mathematical model was created to calculate the concentrations of acetate and citrate attained during haemodialysis.RESULTS:Citrate reduced granulocyte activation and did not induce higher complement activation compared with acetate at concentrations attained during haemodialysis. Investigating different citrate concentrations clearly showed that citrate is a potent complement inhibitor already at low concentrations, i.e. 0.25 mM, which is comparable with concentrations detected in the blood of patients during dialysis with citrate-containing fluids. Increased citrate concentration up to 6 mM further reduced the activation of C3a, C5a and sC5b-9, as well as the expression of CD11b.CONCLUSIONS:Our results suggest that citrate is a promising substitute for acetate for a more biocompatible dialysis, most likely resulting in less adverse effects for the patients.
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| 8. |
- Meijer, P J, et al.
(författare)
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Directed evolution of a type I antifreeze protein expressed in Escherichia coli with sodium chloride as selective pressure and its effect on antifreeze tolerance
- 1996
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Ingår i: Protein Engineering. - : Oxford University Press (OUP). - 0269-2139. ; 9:11, s. 4-1051
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Tidskriftsartikel (refereegranskat)abstract
- Both freezing tolerance and NaCl tolerance are improved when antifreeze proteins are expressed as fusion proteins with two domains of staphylococcal protein A (SPA) in Escherichia coli. To characterize these properties further we created a randomly mutated expression library in E. coli, based on the winter flounder antifreeze protein HPLC-8 component gene. Low-fidelity PCR products of this gene were fused to the spa gene encoding two domains of the SPA. The library was screened for enhanced NaCl tolerance and four clones were selected. The freezing tolerance of each of the selected clones was enhanced to varying extents. DNA sequencing of the isolated mutants revealed that the amphiphilic properties of the native antifreeze protein were essentially conserved. Furthermore, by studying the primary sequence of the randomly mutated clones, in comparison with the degree of freezing tolerance, we have identified clues which help in understanding the relationship between salt and freezing tolerance.
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| 9. |
- Pihl-Karlsson, Gunilla, et al.
(författare)
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Ozonmätnätet i södra Sverige. Marknära ozon i bakgrundsmiljön i södra Sverige med hänsyn till ozonets variation i landskapet. Resultat 2009
- 2010
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Rapport (övrigt vetenskapligt/konstnärligt)abstract
- I maj 2009 startade IVL Svenska Miljöinstitutet AB länsbaserade undersökningar av marknära ozon inom 'Ozonmätnätet i södra Sverige' i Skåne, Blekinge, Halland, Kronoberg, Kalmar, Gotland, Jönköping, Västra Götaland, Östergötland samt Västmanland på uppdrag av Länsstyrelser och Luftvårdsförbund. Grundtanken med detta Gemensamma Delprogram är att ge en mer detaljerad heltäckande bild av ozonbelastningen i bakgrundsmiljö i södra Sverige. Målet är även att kartlägga eventuella överskridanden av olika målvärden för ozon, både miljökvalitetsnormerna för utomhusluft och miljökvalitetsmålet Frisk Luft. Inriktningen ligger i första hand på det ozonindex som beskriver inverkan av ozon på växtligheten (AOT40). Ett mätår omfattar perioden 1 mars till den 30 september. Området som omfattas av Ozonmätnätet ligger huvudsakligen söder om den biologiska norrlandsgränsen, vilket kan vara viktigt för sambandet mellan ozondynamiken och temperaturdynamiken. Urbana och periurbana områden omfattas inte av mätprogrammet. Eftersom ozon är en gränsöverskridande luftförorening är mätstationerna inom Ozonmätnätet i södra Sverige indelade i zoner som baseras på klimatologi och ej efter länsgränser. De fem zonerna är en kustzon, en central zon som domineras av småländska höglandet, en västlig zon, en ostlig zon och en nordlig zon. Övervakningen baseras på en metodik att uppskatta viktiga ozonindex utifrån enkla ozonmätningar med diffusionsprovtagare på månadsbasis i kombination med lufttemperaturmätningar på timbasis. Dessutom används förekommande ozonmätningar med instrument på timbasis. Metoden bygger på att det finns ett samband mellan variationen hos timvisa värden av ozonhalter inom en mätperiod och variationen hos luft-temperaturerna under samma period. Resultaten från 2009 års mätningar bekräftade att denna metodik fungerar. Sommarhalvåret 2009 karakteriserades i hög grad av lågtrycksbetonat väder och därmed även låga ozonhalter. När denna vädertyp råder brukar skillnaderna mellan olika mätplatser vad gäller temperatur och ozonhalter att bli förhållandevis små. Årets resultat inom Ozonmätnätet bekräftar detta, med relativt små skillnader av de genomsnittliga dygnsvariationerna i temperatur mellan kustlokaler samt högt och lågt belägna lokaler i inlandet. De kustnära lokalerna hade som väntat minst dygnsvariation i temperatur, främst beroende på att nattemperaturerna var högre än vid andra typer av lokaler. Lågt belägna lokaler hade i genomsnitt lägre temperaturer nattetid än övriga. Generellt var ozonförekomsten under 2009 högst under april och maj, vilket är ett vanligt förekommande mönster. Under de följande månaderna sjönk ozonhalterna betydligt, men var dock i genomsnitt högre i juni än under juli, augusti och september. Kustlokalerna hade högst ozonkoncentrationer, följt av högt belägna lokaler i inlandet. Sannolikt hade dessa skillnader, liksom skillnaderna mellan olika lokaler och kategorier i temperatur, varit större under en sommar med mer högtrycksbetonat väder. Inom kategorin låglänta platser fanns det en tendens till lägre ozonhalter i nordlig-, ostlig- och västlig zon jämfört med den centrala zonen och kustzonen men variationen är relativt stor inom zonerna. Generationsmålet för ozon, som gäller från 2020 är satt till 50 µg m-3 (april-september), överskreds vid samtliga kategorier i samtliga zoner. Överskridandet var dock litet för låglänta platser i den nordliga och ostliga zonen. Variationen mellan mätlokaler gör att på enstaka låglänta lokaler i den ostliga zonen samt i den nordliga zonen kan medelhalten april-september ha varit strax under målvärdet. Det finns ett förslag till miljömål för skydd av växligheten på AOT40 20 000 µg m-3 timmar för sommarhalvåret som föreslås gälla från 2015. Resultaten från 2009 visar att detta målvärde sannolikt inte överskreds på någon plats i södra Sverige. Miljökvalitetsnormen för ozon som ej får överskridas 2010 - 2019 med mer än 18 000 µg m-3 timmar beräknat som AOT 40 (maj-juli), bestämt som ett glidande 5-årsmedelvärde. Från 2020 sänks målärdet till 6 000 µg m-3 timmar som ej får överskridas något år. Årets resultat visar att för 2009 låg de beräknade AOT40-värdena klart under miljökvalitetsnormen vid samtliga kategorier i samtliga zoner. Även om den nya strängare normen, som skall gälla från och med 2020 hade gällt idag hade normen endast överskridits vid en kustnära lokal i kustzonen och vid en låglänt lokal i den centrala zonen. Sammanfattningsvis kan sägas att under sommaren 2009 överskreds inga föreslagna miljömål eller gällande miljökvalitetsnormer som gäller AOT40 inom något område i södra Sverige. Endast generationsmålet inom Frisk Luft på 50 µg m-3 som sommarhalvårsmedelvärde, överskreds under 2009 i hela södra Sverige. Avsaknaden av överskridanden kan förklaras av de låga ozonhalter som uppmättes under 2009. Sannolikt låg ozonhalterna under 80 µg m-3 den mesta tiden, vilket medförde att det ej bidrog till AOT40 värdena
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