| 1. |
- Atikuzzaman, Mohammad, 1977-
(författare)
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Seminal Influence on the Oviduct : Mating and/or semen components induce gene expression changes in the pre-ovulatory functional sperm reservoir in poultry and pigs
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Internal fertilization occurs in birds and eutherian mammals. Foetal development, however, is either extra- respectively intra-corpore (egg vs uterus). In these animal classes, the female genital tract stores ejaculated spermatozoa into a restricted oviductal segment; the functional pre-ovulatory sperm reservoir, where they survive until ovulation/s occur. Paradoxically, this immunologically foreign sperm suspension in seminal fluid/plasma, often microbiologically contaminated, ought to be promptly eliminated by the female local immune defence which, instead, tolerates its presence. The female immune tolerance is presumably signalled via a biochemical interplay of spermatozoa, as well as the peptides and proteins of the extracellular seminal fluid, with female epithelial and immune cells. Such interplay can result in gene expression shifts in the sperm reservoir in relation to variations in fertility. To further aid our understanding of the underlying mechanisms, this thesis studied the proteome of the seminal fluid (using 2D SDS-PAGE and mass spectrometry) including cytokine content (using Luminex and/or ELISA) of healthy, sexually mature and fertile boars and cocks. As well, gene expression changes (using cDNA microarray) in the oviductal sperm reservoirs of sexually-mature females, mated or artificially infused with homologous sperm-free seminal fluid/plasma were studied. Pigs were of commercial, fertility-selected modern breeds (Landrace), while chicken belonged to the ancestor Red Junglefowl (RJF, low egg laying-capacity), a selected egg-layer White Leghorn (WL) and of their Advanced Intercross Line (AIL). Ejaculates were manually collected as single sample in cocks or as the sperm-rich fraction [SRF] and the post- SRF fraction in boars to harvest seminal fluid/plasma for proteome/cytokine and infusion-studies. Oviducts were retrieved for gene-expression analyses via microarray immediately post-mortem (chicken) or at surgery (pig), 24 h after mating or genital infusion. In pigs, the protein-rich seminal plasma showed the highest amounts of cytokines [interferon-γ, interferon gamma-induced protein 10 (IP-10/CXCL10), macrophage derived chemokine (MDC/CCL22), growth-regulated oncogene (GRO/CXCL1), granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemo-attractant protein-1 (MCP-1/ CCL2), interleukin (IL)-6, IL-8/CXCL8, IL-10, IL-15, IL-17 and transforming growth factor (TGF)-β1-3) in the larger, protein-rich and sperm-poor post-SRF, indicating its main immune signalling influence. Chicken showed also a plethora of seminal fluid proteins with serum albumin and ovotransferrin being conserved through selection/evolution. However, they showed fewer cytokines than pigs, as the anti-inflammatory/immune-modulatory TGF-β2 or the pro-inflammatory CXCL10. The RJF contained fewer immune system process proteins and lacked TGF-β2 compared to WL and AIL, suggesting selection for increased fertility could be associated with higher expression of immune-regulating peptides/proteins. The oviductal sperm reservoir reacted in vivo to semen exposure. In chicken, mating significantly changed the expression of immune-modulatory and pH-regulatory genes in AIL. Moreover, modern fertile pigs (Landrace) and chicken (WL), albeit being taxonomically distant, shared gene functions for preservation of viable sperm in the oviduct. Mating or SP/SF-infusion were able to change the expression of comparable genes involved in pH-regulation (SLC16A2, SLC4A9, SLC13A1, SLC35F1, ATP8B3, ATP13A3) or immune-modulation (IFIT5, IFI16, MMP27, ADAMTS3, MMP3, MMP12). The results of the thesis demonstrate that both mating and components of the sperm-free seminal fluid/plasma elicit gene expression changes in the pre-ovulatory female sperm reservoir of chickens and pigs, some conserved over domestication and fertility-selection.
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| 2. |
- Che, Karlhans Fru
(författare)
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Immunomodulatory Effects of Human ImmunodeficiencyVirus (HIV-1) on Dendritic Cell and T cell Responses : Studies of HIV-1 effects on Dendritic cell functionality reflected in primed T cells
- 2011
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The human immunodeficiency virus (HIV)-1 is the causative agent of acquired immune deficiency syndrome (AIDS) worldwide. Till date there are no vaccines or cure for this infection as the virus has adapted myriad ways to remain persistent in the host where it causes severe damage to the immune system. Both humoral and cellular immune responses are mounted against HIV-1 during the initial phase of infection but fail to control viral replication as these responses are severely depleted during disease progression. Of great importance in HIV-1 research today is the in depth understanding of the types of immune responses elicited, the mechanisms behind their decline and how these responses can be maintained overtime.The focus of this thesis was to examine the possibility of priming HIV-1 specific T cell responses in vitro from whole viral particles and in detail, scrutinize the type of T cell responses and epitope specificities generated. Next was to investigate in vitro the factors responsible for impaired immune responses in HIV-1 infected individuals. We were also interested in understanding the underlying mechanisms through which HIV-1 initiate suppression of T cell functionality.Results showed that using HIV-1 pulsed monocyte derived dendritic cells (DCs), we were able to prime HIV-1 specific CD4+ and CD8+ T cells from naïve T cells in vitro. The epitopes primed in vitro were located within the HIV-1 envelope, gag, and pol proteins and were confirmed ex vivo to exist in acute and chronically infected individuals. We established that many of the novel CD4+ T cell epitopes primed in vitro also existed in vivo in HIV-1 infected individuals during acute infection. These responses declined/disappeared early on, which is in line with HIV-1 preferential infection of HIV-1 specific CD4+ T cells.Besides declining HIV-1 specific T cell responses, many HIV-1 infected individuals also have impaired T cell functionality. We established that one reason behind the decline and impairment in immune responses was the increased expression of inhibitory molecules PD-1, CTLA-4, and TRAIL on HIV-1 primed T cells. These T cells had the capacity to suppress new responses in a cell-cell contact dependent manner. The ability of the HIV-1 primed T cells to proliferate was severely impaired and this condition was reversed after a combined blockade of PD-1, CTLA-4 and TRAIL. Furthermore, more inhibitory molecules TIM-3, LAG-3, CD160, BLIMP-1, and FOXP3 were also found increased at both gene and protein levels on HIV-1 primed T cells. Additionally, we showed decreased levels of functional cytokines IL-2, IFN-γ and TNF-α, and the cytolytic proteins perforin and granzyme in DC T cell priming cocultures containing HIV-1. This could be as a result of the decreased T cell activation or impaired production by T cells. The mechanisms responsible for the elevated levels of inhibitory molecules emanated mainly from the P38MAPK/STAT3 pathways. Blockade of these pathways in both allogeneic and autologous DC-T cell assays significantly suppressed expression of inhibitory molecules and subsequently rescued T cell proliferation.In conclusion, HIV-1 pulsed DCs have the capacity to prime HIV-1 specific responses in vitro that do exist in HIV-1 infected individuals and we found evidence that many of these responses were eliminated rapidly in HIV-1 infected individuals. HIV-1 triggers through P38MAPK/STAT3 pathway the synthesis of inhibitory molecules, namely CTLA-4, PD-1, TRAIL, TIM-3, LAG-3, CD160, and suppression associated transcription factors FOXP3, BLIMP-1 and DTX1. This is followed by decreased T cell proliferation and functionality which are much needed to control viral replication.
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| 3. |
- Tjomsland, Veronica
(författare)
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Complement activation - good or evil in HIV-1 infection? : interaction of free and complement opsonized HIV-1 with monocyte derived dendritic cells and immune cells in the cervical mucosa
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Worldwide, the heterosexual route is the most common mode of sexual transmission of HIV-1 and women are particularly susceptible to this infection. After penetration of the mucosal epithelium HIV-1 interacts with potential target cells, i.e. dendritic cells (DCs) and CD4+ T cells. The complement system, a key component of the innate immune system, is immediately activated by HIV-1 in vivo. However, HIV-1 can resist complement mediated lysis and become coated with complement fragments and this opsonization influences the viral interaction with immune cells. The DCs are the most potent antigen presenting cell. This cell effectively links the innate recognition of viruses to the generation of an adaptive immune response. However, HIV-1 exploits the function of the DCs to facilitate viral spread and infection. HIV-1 interacts with a range of receptors expressed by the DCs including C-type lectins, integrins and complement receptors (CRs). The uptake of virions by DCs leads to their activation and migration to the lymph nodes. At this site DCs present HIV-1 derived antigen on MHC class I and II molecules and trigger an HIV-1 specific T cell response. The interplay between the virus and the DCs is complex and the initial receptor binding may affect antigen uptake, infection, and antigen presentation.The fundamental questions of this thesis are the following: How is free and opsonized HIV-1 internalized, processed, and presented on MHC class I and II molecules by DCs and how do free and opsonized HIV-1 particles interact with immune cells in the cervical mucosa?Our results indicate that opsonization of HIV-1 plays a critical role in the interaction with immune cells. Complement opsonization of HIV-1 (C-HIV) significantly enhanced the internalization by the DCs compared to free HIV (F-HIV). Both C-HIV and F-HIV interacted with the CD4 receptor, C-type lectins and integrins. In addition, opsonization of HIV-1 favored an MHC class I presentation by DCs compared to F-HIV. However, the endocytic receptors macrophage mannose receptor, β7 integrin, and CR3 guided the antigens to different compartments with distinct properties and efficiencies for degradation and MHC class I and II presentation of viral antigens. MHC class I presentation of F-HIV and C-HIV was dependent of viral fusion in a CD4/coreceptor dependent manner. Moreover, MHC class II presentation of antigens derived from HIV-1 required endocytosis and proteolysis in acidified compartments. HIV-1 infection of cervical mucosa immune cells and tissue was assessed in a cervical tissue explant model. C-HIV significantly enhanced infection of DCs compared to F-HIV, whereas C-HIV decreased the infection of CD4+ T cells. Blocking the viral use of integrins in the cervical tissue explants significantly decreased the HIV-1 infection of both emigrating DCs and CD4+ T cells and the establishment of founder populations in these tissues. This thesis work has brought forward new facts that can be used to facilitate the development of an effective vaccine or microbicide.
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| 4. |
- Carlsson, Anders, 1980-
(författare)
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Role of mast cells and probiotics in the regulation of intestinal barrier function
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The intestinal mucosa is the largest contact area and one of the most important barriers to the outside environment. It is highly specialized in aiding us digest and absorb nutrients. It is daily exposed to several potentially dangerous substances and microorganisms, which if they were allowed to pass into the body, could give rise to diseases. Throughout the small intestine certain sites specialized in antigen sampling are found. These sites are named Peyer’s patches and are lymphoid follicles. The epithelium covering the Peyer’s patches is called follicle-associated epithelium and is specialized in antigen sampling and uptake. The special epithelium enables presentation of luminal antigen to immune cells in the underlying follicle.Persistent life stress and stressful life events affect the course of irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) through largely unknown mechanisms. Regulation of epithelial permeability to antigens is crucial for the balance between inflammation and immune-surveillance, and increased intestinal permeability has been shown in patients with ulcerative colitis and Crohns disease. Vasoactive intestinal polypeptide (VIP) and corticotropin-releasing factor have been implicated as important mediators of stress-induced abnormalities in intestinal mucosal functions in animal models. Both of these mediators have been reported to regulate bowel ion secretion in humans during stress and uptake of horseradish peroxidase in rodents. Probiotics have been shown to ameliorate the deleterious effects of stress on intestinal function, but mechanisms remain to be elucidated.The aim of this thesis was to elucidate whether mast cells play an important role in intestinal barrier function during stress and inflammation. Moreover, we wanted to determine whether probiotics can ameliorate the mucosal barrier integrity during stress and inflammation.To study the function of mast cells we conducted in vitro experiments on cell lines and ex vivo experiments in Ussing chambers on mouse, rat and human intestinal tissue. The Ussing chamber technique measures electrophysiological properties of the tissue and also gives the possibility to study transcellular and paracellular passage of markers and bacteria. Immunohistology and confocal microscopy have been used to identify mast cells and receptors of interest.Our results show that stress affects the follicle-associated epithelium barrier by mechanisms involving VIP and mast cells. These findings were corroborated by the localization of VIP receptors on mucosal mast cells. Furthermore, pretreatment with probiotics was effective in protecting the gut against stress-induced intestinal barrier dysfunction and mucosal inflammation. This protection appeared to involve a mast cell and peroxisome proliferatoractivated receptor-γ dependent mechanism.
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| 5. |
- Vicente Carrillo, Alejandro, 1989-
(författare)
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Sperm Membrane Channels, Receptors and Kinematics : Using boar spermatozoa for drug toxicity screening
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Internal fertilization usually implies that a spermatozoon, with intact attributes for zygote formation, passes all hurdles during its transport through the female genitalia and reaches the oocyte. During this journey, millions to billions of other spermatozoa perish. Spermatozoa are highly differentiated motile cells without synthetic capabilities. They generate energy via glycolysis and oxidative phosphorylation to sustain motility and to maintain the stability and functionality of their plasma membrane. In vivo, they spend their short lifespan bathing in female genital tract fluids of different origins, or are in vitro exposed to defined media during diverse sperm handling i.e. extension, cryopreservation, in vitro fertilization, etc. Being excitable cells, spermatozoa respond in vivo to various stimuli during pre-fertilization (capacitation, hyperactivation, oocyte location) and fertilization (acrosome reaction, interaction with the oocyte) events, mediated via diverse membrane ion-conducting channels and ligand-gated receptors. The present Thesis has mapped the presence and reactivity (sperm intactness and kinematics) of selected receptors, water and ion channels in ejaculated boar spermatozoa. The final aim was to find a relevant alternative cell type for in vitro bioassays that could ease the early scrutiny of candidate drugs as well as decreasing our needs for experimental animals according to the 3R principles. Spermatozoa are often extended, cooled and thawed to warrant their availability as fertile gametes for breeding or in vitro testing. Such manipulations stress the cells via osmotic variations and hence spermatozoa need to maintain membrane intactness by controlling the exchange of water and the common cryoprotectant glycerol, via aquaporins (AQPs). Both AQPs-7 and -9 were studied for membrane domain changes in cauda- and ejaculated spermatozoa (un-processed, extended, chilled or frozen-thawed). While AQP-9 maintained location through source and handling, thawing of ejaculated spermatozoa clearly relocated the labelling of AQP-7, thus appearing as a relevant marker for non-empirical studies of sperm cryopreservation. Alongside water, spermatozoa interact with calcium (Ca2+) via the main Ca2+ sperm channel CatSper. Increments in intracellular Ca2+ initiate motility hyperactivation and the acrosome reaction. The four subunits of the CatSper channel were present in boar spermatozoa, mediating changes in sperm motility under in vitro capacitation-inducing conditions (increased extracellular Ca2+ availability and bicarbonate) or challenge by the CatSper antagonists mibefradil and NNC 55-0396. Uterine and oviduct fluids are richest in endogenous opioids as β-endorphins during mating and ovulation. Both μ- and δ- opioid receptors were present in boar spermatozoa modulating sperm motility, as in vitro challenge with known agonists (μ: morphine; δ: DPDPE and κ: U 50488) and antagonists (μ: naloxone; δ: naltrindole and κ: nor-binaltrorphimine) showed that the μ-opioid receptor maintained or increased motility while the δ-opioid receptor mediated decreased motility over time. Finally, boar spermatozoa depicted dose-response effects on sperm kinematics and mitochondrial potential following in vitro challenge with 130 pharmacological drugs and toxic compounds as well as with eight known mito-toxic compounds. In conclusion, boar spermatozoa expressing functional water (AQPs-7 and -9) and ion (CatSper 1-4) channels as well as μ- and δ-opioid receptors are able to adapt to stressful environmental variations, capacitation and pharmacological compounds and drug components. Ejaculated sperm suspensions are easily and painlessly obtained from breeding boars, and are suitable biosensors for in vitro drug-induced testing, complying with the 3R principles of reduction and replacement of experimental animals, during early toxicology screening.
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| 6. |
- Drobni, Mirva, 1976-
(författare)
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Adhesion-related interactions of Actinomyces and Streptococcus biofilm bacteria
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Adhesion of bacteria is a key event in biofilm formation and is mediated by bacterial adhesins recognising host or bacterial partner receptors. In oral biofilm formation, primary Actinomyces and Streptococcus colonizers adhere to salivary pellicle proteins such as proline-rich proteins (PRPs) as well as to mucosal surfaces. Subsequently, Actinomyces and Streptococcus strains and other bacteria, such as Veillonella, Fusobacterium and Porphyromonas, adhere to each other. The nature of this community is highly important for the health or disease status, although specific pathogenic species may also have been implicated. The aim of this thesis was to study key players in early oral colonisation, Actinomyces and Streptococcus species, and more specifically the nature of their adhesins and ligands. A further aim was to study the function of the salivary PRP proteins and an innate peptide derived thereof on bacterial adhesion, proliferation and regulation of pH, i.e. key factors in biofilm formation. In paper I and II, adhesion, proliferation and pH affecting features of the RGRPQ (arginine-glycine-arginine-proline-glutamine) peptide, derived from PRP-1, were demonstrated. By use of an alanine-scan (I), motifs for adhesion inhibition and desorption of Actinomyces naeslundii, and proliferation stimulation, ammonia production and inhibition of sucrose induced pH drop by Streptococcus gordonii were indicated. The RGRPQ peptide also stimulated S. gordonii colonisation in vivo. In paper II, a more sophisticated quantitative structure-activity relationship (QSAR) study, using statistical molecular design (SMD) and multivariate modelling (partial least squares projections to latent structures, PLS), further narrowed down the RGRPQ peptide motifs. The R and Q amino acids were crucial for activity. For proliferation a hydrophobic and large size third position amino acid was crucial, while adhesion inhibition and desorption needed a small hydrophilic second position amino acid. All functions depended on a low polarity hydrophobic fourth position. Accordingly, activities could be optimized separately, with decreased function in the others. In paper III and IV, focus was on the bacterial adhesins and their binding epitopes. The genes for FimA major subunit proteins of type-2 fimbriae were sequenced from A. naeslundii genospecies 1 and 2 and Actinomyces odontolyticus, each with unique carbohydrate binding specificities (III). Three major subtypes of FimA proteins were found that correlated with binding specificity, including a novel fimA gene in A. odontolyticus. All subtypes contained a pilin, LPXTG and E box motif. In paper IV, multiple PRP binding patterns for Actinomyces and Streptococcus strains were mapped using a hybrid peptide construct. The two most deviating binding groups deviated in type-1 fimbriae mediated binding to milk and saliva protein ligands. In conclusion, differences in bacterial adhesins and their ability to utilise salivary proteins may render bacteria tropism for different niches. Peptides derived from protein receptors, such as RGRPQ, may be important modulators of biofilm formation, giving commensal bacteria a competitive edge in the bacterial community.
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| 7. |
- Holm, Angelika
(författare)
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Aquaporins in Infection and Inflammation
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The ability of eukaryotic cells to change their shape and to migrate directionally is highly dependent on active volume regulation in cells building up tissues as well as in individual cells. Transmembrane fluxes of water via specialized water channels, called aquaporins (AQPs), facilitate the changes of volume and shape, which additionally require a complex interplay between the plasma membrane and the cytoskeleton. AQPs have been shown to be involved in the development of inflammatory processes and diseases. The aims of the studies underlying this thesis were to further elucidate the expression and function of AQPs in both bacterial and viral infections as well as in the inflammatory disease, microscopic colitis. For this, molecular techniques qPCR, immunoblotting and live, holographic, confocal and super-resolution imaging were used.When cells of the innate immune system encounter pathogens they need to respond and prepare for migration and phagocytosis and do so through volume regulatory processes. The Gramnegative bacterium Pseudomonas aeruginosa utilizes a small molecule-based communication system, called quorum sensing (QS) to control the production of its virulence factors and biofilms. We found that P. aeruginosa with a complete QS system elicits a stronger phagocytic response in human blood-derived macrophages compared to its lasI-/rhlI- mutant lacking the production of the QS molecules N-butyryl-L-homoserine lactone (C4-HSL) and N-3-oxododecanoyl-L-homoserine lactone (3O-C12-HSL). Infection with P. aeruginosa further increases the expression of AQP9 and induces re-localisation of AQP9 to the front and trailing ends of macrophages. Moreover, the 3O-C12-HSL alone elevates the expression of AQP9, redistribute the water channel to the front and rear ends and increases the cell area and volume of macrophages. Both infection with the wild type P. aeruginosa and the treatment with 3OC12-HSL change the nano-structural architecture of the AQP9 distribution in macrophages.Viruses use the intracellular machinery of the invaded cells to produce and assemble new viral bodies. Intracellular AQPs are localised in a membranes of cellular organelles to regulate their function and morphology. C3H10T1/2 fibroblasts transiently expressing green fluorescent protein (GFP)-AQP6 show a reduced expression of AQP6 after Hazara virus infection and an increased cell area. Overexpressing AQP6 in C3H10T1/2 cells reduces the infectivity of Hazara virus indicating that AQP6 expression has a protective role in virus infections.Ion and water channels in the epithelial cell lining tightly regulate the water homeostasis. In microscopic colitis (MC), patients suffer from severe watery diarrhoeas. For the first time, we have shown that the expression of AQP1, 8 and 11 and the sodium/hydrogen exchanger NHE1 are reduced in colonic biopsies from MC patients compared to healthy control individuals. Following treatment with the glucocorticoid budesonide the patients experienced a rapid recovery and we observed a restored or increased expression of the AQPs and NHE1 during treatment, suggesting a role for AQPs in the diarrhoeal mechanisms in MC.Taken together, this thesis provides new evidence on the importance of water homeostasis regulation through AQPs during infections and inflammation and opens up a door for further investigations of roles for AQPs in inflammatory processes.
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| 8. |
- Karlsson, Thommie, 1983-
(författare)
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Water Fluxes and Cell Migration : How Aquaporin 9 Controls Cell Shape and Motility
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Prerequisites for all modes of cell migration are cell-substratum interactions that require a sophisticated interplay of membrane dynamics and cytoskeletal rearrangement. Generally, a migrating cell is polarized with a distinct rear and front, from which it extends a wide and thin membrane protrusion- lamellipodium, small fingerlike projections- filopodia, and membrane blisters- blebs. The development of these structures is primarily driven by cytoskeletal contractions and actin polymerization, which are under regulation of several actin-binding proteins and the small GTPases Cdc42, Rac and Rho. Lamellipodia and filopodia are assumed to arise from polymerizing actin, pushing the membrane forward through a Brownian-ratchet mechanism. However, other models based on shifts in the local hydrostatic pressure have also been suggested since blebs are initially void of actin. Recently, fluxes of water through membrane-anchored water channels, aquaporins (AQPs), have been implicated in cell motility, while they appeared to localize to lamellipodia and facilitate cell locomotion. Indeed, expression of AQP9 was shown to induce filopodia in fibroblasts. Here, we have focused on the effects of AQP9 on cell morphology and motility. By using primarily live cell imaging of GFP-AQP9 and other cytoskeletal components we found that AQP9: (i) enhances cell polarization and migration in a Rac1 and serine11 phosphorylation-dependent manner in neutrophils, (ii) induces and accumulates in filopodia, before actin polymerization, (iii) locally deforms the membrane upon rapid reductions osmolarity, (iv) accumulates in the cell membrane underlying bleb development, (v) induces multiple protrusions and thereby impairs the intrinsic directionality, and (vi) facilitates epithelial wound closure through a mechanism involving swelling and expansion of the monolayer. Based on these findings, we have presented models for how water fluxes through AQPs aids actin polymerization in the formation of membrane protrusions. In summary, these models rely on localized accumulation of ion and water channels that control the influx of water and thereby the buildup of a hydrostatic pressure between the membrane and the cytoskeleton. Upon reaching a critical pressure, it will dislocate the membrane from the cytoskeleton and force it to protrude outwards. Moreover, this will promote a local cytoplasmic gel-to-sol transformation, which facilitates diffusion of cytoskeletal reactants. Hereby, we can furthermore assign to filopodia a role as osmo-sensors, protecting the cell from different osmotic loads. In addition, we have postulated a novel model for wound healing involving force generation by cell swelling. Taken together, this thesis provides the field of cell migration with solid evidence for pivotal roles of water fluxes through AQP9 in particular, but most likely AQPs in general, during cell locomotion and localized volume control.
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| 9. |
- Kowalewski, Jacob, 1978-
(författare)
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Modeling and Data Analysis in Cellular Biophysics
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cellular biophysics deals with the physical aspects of cell biology. This thesis presents a number of studies where mathematical models and data analysis can increase our understanding of this field. During recent years development in experimental methods and mathematical modeling have driven the amount of data and complexity in our understanding of cellular biology to a new level. This development has made it possible to describe cellular systems quantitatively where only qualitative descriptions were previously possible. To deal with the complex data and models that arise in this kind of research a combination of tools from physics and cell biology has to be applied; this constitutes a field we call cellular biophysics. The aim of this doctoral thesis is to develop novel approaches in this field. I present eight studies where quantitative modeling and analysis are involved. The first two studies concern cells interacting with their surrounding environment in the kidney. These cells sense fluid flow and respond with calcium (Ca2+) signals. The interaction between fluid and cells in renal tubular epithelium can be described by biomechanical models. This thesis describes a mathematical model of flow sensing by cilia with focus on the flow frequency response and time delay between the mechanical stress and the Ca2+ signaling response. Intracellular Ca2+ is kept at a very low level compared to the extracellular environment, while several intracellular compartments have higher Ca2+ concentration than the cytoplasm. This makes Ca2+ an efficient messenger for intracellular signaling, the process whereby signals are transduced from an extracellular stimulus to an intracellular activity such as gene expression. An important type of Ca2+ signaling is oscillations in intracellular Ca2+ concentration which occur due to the concerted interplay between different transport mechanisms within a cell. A study in this thesis examines ways to explain these mechanisms in terms of a mathematical model. Another study in the thesis reports that erythropoietin can regulate the water permeability of astrocytes and that it alters the pattern of Ca2+ oscillations in astrocytes. In this thesis the analysis of this Ca2+ signaling is described. Simulations described in one of the studies show how different geometries can affect the fluorescence recovery and that geometrically constrained reactions can trap diffusing receptors in dendritic spines. When separate time scales are present in a fluorescence revovery after photobleaching (FRAP) experiment the reaction and diffusion components can be studied separately. Applying single particle tracking methods to the migration trajectories of natural killer cells shows that there is a correlation between the formation of conjugates and transient confinement zones (TCZs) in these trajectories in vitro. TCZs are also present in in vivo experiments where they show strong similarities with the in vitro situation. This approach is a novel concept in data analysis methods for tracking immune cells.
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| 10. |
- Stenberg, Åsa, 1973-
(författare)
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Studies of SIRPα-mediated regulation of neutrophil functions
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Neutrophil granulocytes constitute the front line of defense in the innate immune response to invading microorganisms, but can also contribute to development of inflammatory disease and tissue destruction following e.g. myocardial infarction or stroke. During inflammatory activation, neutrophils leave the blood, interact with extracellular matrix proteins, and migrate into tissues in response to chemotactic factors to phagocytose and kill infectious agents by using toxic granule contents and reactive oxygen metabolites. The functional neutrophil response relies on exocytosis of cytoplasmic granules, each containing membrane proteins, which are thereby mobilized to the plasma membrane. Specific programmed cell death (apoptotic) pathways regulate neutrophil homeostasis, where an inflammatory milieu can prolong the life span of neutrophils to several days, whereas non-activated neutrophils are committed to constitutive/spontaneous apoptosis within hours.Signal regulatory protein alpha (SIRPα) is a surface glycoprotein with two intracellular immunoreceptor-tyrosine-based inhibitory motifs (ITIMs), which is highly expressed in neutrophils and other myeloid cells. In other cell types, SIRPα has been shown to regulate cellular functions such as cell migration and phagocytosis. The aim of the present thesis was to investigate neutrophil SIRPα expression in response to inflammatory activation or apoptosis, and how this receptor can regulate neutrophil adhesion and cell migration.Neutrophils contain several subcellular granule compartments, including primary (azurophilic), secondary (specific), tertiary (gelatinase) granules, and a fourth compartment called secretory vesicles. In resting neutrophils, SIRPα was found to be present in the plasma membrane and in all types of granules except for the azurophilic granules. Stimulation with the bacterial peptide fMLF in vitro, or inflammatory activation in vivo, was found to rapidly mobilize SIRPα to the neutrophil cell surface. In mice expressing a mutated form of SIRPα, where the cytoplasmic signaling domain was deleted, we found an enhanced accumulation of neutrophils in the peritoneal cavity in a peritonitis model. These findings therefore suggest that an increased amount of SIRPα on the surface of activated neutrophils could serve to negatively fine-tune neutrophil accumulation in inflammation.Neutrophil priming means that the cell becomes partially activated, in a way that facilitates subsequent full activation. One part of the priming process is a moderate exocytosis of granules, mostly the secretory vesicles, which increases the density of certain receptors on the cell surface. It also involves the activation of adhesion receptors called integrins. We found that TNFα-induced priming involved an increased accumulation of SIRPα on the cell surface. When comparing wild-type and SIRPα-mutant neutrophils, we found a strongly reduced TNFα-stimulated and β2 integrin-dependent adhesion of mutant neutrophils to type I collagen or fibrinogen. This adhesion defect resulted in a reduced adhesion-dependent activation of the respiratory burst and an increased chemotactic response of SIRPα-mutant neutrophils in vitro.During neutrophil apoptosis, several receptors are known to be shed from the cell surface (e.g. CD16 and CD43). We found that also SIRPα is shed from the surface during spontaneous as well as Fas-induced apoptosis. The shedding mechanism was found to involve matrix metalloproteinase (MMP) activity, mostly that of MMP-3 and MMP-8.In conclusion, neutrophil cell surface SIRPα expression is regulated during neutrophil activation and seems to play an important role in stimulating β2-integrin-dependent adhesion. This way, SIRPα can negatively fine-tune neutrophil migration and accumulation in inflammation. During apoptosis, SIRPα is shed from the cell surface, which may be one mechanism contributing to the well-known down-regulation in the adhesiveness of apoptotic neutrophils.
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| 11. |
- Tafazoli, Farideh, 1955-
(författare)
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Perturbation of the epithelial barrier by enteric pathogens
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Gastrointestinal infections in humans have been associated with a number of diseased condition, including stomach ulcers, gastroenteritis, Crohn's disease, and rheumatic arthritis. Such infections often cause altered intestinal permeability through perturbation of the tight junctions that hold epithelial cells together. The objective of the present studies was to detennine whether the enteric pathogens Salmonella, Yersinia, and Rotavirus can disrupt the integrity of the epithelial barrier, and, if so, how this is achieved. Another aim was to elucidate regulation of the epithelial batrier in relation to the structure of the cytoskeleton.To accomplish these goals, we assessed the mechanism of enhanced cytotoxicity of Yersinia YopE and the response to this protein by its target in the epithelial bartier, both of which require contact between the bacteria and the eukaryotic cells. YopK appeared to control Yop effector delivery by regulating the size of the translocation pore, and enhanced translocation was accompanied by decreased transepithelial resistance and disruption of barrier function. We also examined the interaction of Yersinia with polarized MDCK cells to detemrine the target of these bacteria. We found that wild-type Yersinia adhered apically to the tight junction areas, and, in adjacent cells, these contact points displayed ß1 integrins and tight junction proteins that allowed localized invasin-mediated binding and translocation of cytotoxins. Studying signal transduction pathways involved in the disruption of barrier function by Salmonella typhimurium, we found that infection with the wild-type strain increased the level of activated. Rac1 and Cdc42 small G-proteins and caused them to accumulate apically in MDCK cells, and this was prevented by appropriate inhibitors. Activation of these proteins was a prerequisite of disruption of barrier integrity by S. typhimurium. We also considered specific effects of the rota virus non-structural protein NSP4 on the function of tight junctions. NSP4 has been desctibed as the first viral enterotoxin, and we found that incubation of noncontluent MDCK-1 cells with NSP4 prevented development of the permeability barrier, as well as lateral targeting of the tight junction-associated zonula occludence-1 protein.In conclusion, our results provide strong evidence that the studied pathogens perturb the epithelial barrier by binding to specific cell receptors to deliver cytotoxins (Yesinia); by interfering with cell signaling pathways (Salmonella); and by impairing normal formation of tight junctions (NSP4).
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| 12. |
- Borutinskaite, Veronika Viktorija, 1977-
(författare)
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Characterization of proteins involved in differentiation and apoptosis of human leukemia and epithelial cancer cells
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Today, cancer is understood as an epigenetic as well as a genetic disease. The main epigenetic hallmarks of the cancer cell are DNA methylation and histone modifications. The latter changes may be an optimal target for novel anticancer agents. The main goal of using histone deacetylase inhibitors (HDACIs) would be restoration of gene expression of those tumor-suppressor genes that have been transcriptionally silenced by promoter-associated histone deacetylation. However, HDACIs have pleiotropic effects that we are only just starting to understand. These may also be responsible for the induction of differentiation, cell-cycle arrest and pro-apoptotic effects.There are now so many HDACIs available, with such different chemical structures and biological and biochemical properties, that it is hopeful that at least some of them will succeed, probably in combination with other agents or therapies.In our studies we focussed ourselves on studies some new HDACIs, that can be useful for treating cancers, including leukemia and epithelial cancer. To do that, we used novel HDACIs, like BML-210, and their combination with the differentiation inducer all-trans retinoic acid (ATRA). Cell differentiation and proliferation in general, and specific gene expression require de novo protein synthesis and/or post-translational protein modifications. So, we tried to identify proteins in general and specifically the proteins that could be important for the cell differentiation process, and when and where in the cell the proteins appear.We delineated that HDACIs inhibited leukemia (NB4 and HL-60) cell growth in a time- and dose-dependent way. Moreover, BML-210 blocked HeLa cell growth and promoted apoptosis in a time-dependent way. Combining of BML-210 with ATRA induced a differentiation process in leukemia cell lines that lead to apoptosis. This correlated with cell cycle arrest in G0/G1 stage and changes in expression of cell cycle proteins (p21, p53), transcription factors (NF-κB, Sp1) and their binding activity to consensus or specific promoter sequences. We also assessed histone modifications, i.e. H3 phosphorylation and H4 hyperacetylation due to HDACI, leading to chromatin remodeling and changes in gene transcriptions.We have also studied changes in protein maps caused by HDACIs and differentiation agents, identifying differences for a few proteins due to growth inhibition and induction of differentiation in NB4 cells using BML-210 alone or in combination with ATRA. These proteins are involved in cell proliferation and signal transduction, like Rab, actin and calpain. One of them was alpha-dystrobrevin (α-DB). To further study possible roles of the latter, we determined changes of α-DB protein isoform expression that correlated with induction of differentiation. We thus identified a novel ensemble of α-DB interacting proteins in promyelocytic leukemia cells, including tropomyosin 3, actin, tubulin, RIBA, STAT and others, being important in cytoskeleton reorganization and signal transduction. Using confocal microscopy, we determined that α-DB co-localizes with HSP90 and F-actin in NB4 and HeLa cells. We also revealed that it changes sub-cellular compartment after treatment with ATRA and/or BML-210. α-DB silencing affected F-actin expression in HeLa cells, further supporting the idea that α-DB is involved in cytoskeleton reorganization in cells. Altogether, our results suggest that α−DB may work as a structural protein during proliferation and differentiation processes of human cancer cells.Based on our findings, we suggest that HDACIs, like BML-210, can be promising anticancer agents, especially in leukemia treatment, by inducing apoptosis and regulating proliferation and differentiation through the modulation of histone acetylations and gene expression.
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| 13. |
- 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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| 14. |
- Hájková, Lucie, 1967-
(författare)
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The dynamic microfilament system : cellular organization of actin and profilin and their association with cell signalling
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Active shape changes and movement of whole cells, and translocations within cells are essential to life. For instance, during embryogenesis cell migration is a central theme. Wound healing depends on increased and directed motile activity of several cell types. Platelets change their shape dramatically during blood clotting and restoration of blood flow. Cells of the immune system extravasate (leave blood vessels) and migrate to sites of infection, where they carry out their defense reactions etc. Furthermore, increased motile activity is one of the hallmarks of malignant tumor cells.All these movements are carried out by a system of muscle proteins. They are present in all our cells, in fact in all eukaryotic cells. The proteins actin and myosin play central roles in this system. They form superstructures inside the cell, converting chemical energy into movements. In non-muscle cells, they are part of a highly dynamic system called the microfilament system, where the basic component are filaments formed from actin. Microfilaments are particularly concentrated beneath the plasma membrane, where they are linked, directly or indirectly, to transmembrane proteins - receptors, adhesion proteins, and ion channels. The dynamic activity of the microfilament system is based on the ability the cell has to change the system, both with respect to organization and activity, and it does so in response to the interactions that it has with the immediate surroundings.The protein profilin is one of the crucial components in the regulation of the microfilament system. It binds to actin and controls its polymerizability. In addition to that, it appears to be an important link between the transmembrane signalling and the mechanisms that polymerize actin into filaments.This thesis deals with the role of profilin in vivo. It reports on the localization of profilin in cells and describes the effects of microinjecting normal and mutant profilins, and a chemically crosslinked profilin:actin complex into cells. The results strengthen the view that profilin plays a role in the activation of the microfilament system, and provides evidence that the profilin:actin complex is the precursor for filament formation and that dissociation of the complex is a crucial step in the polymerization reaction. The thesis also describes effects on the microfilament system by microinjection of antibodies against two isoforms of PI3-kinase, an enzyme that is involved in the metabolism of a particular type of phospholipid present in the plasma membrane and linked to the control of the microfilament system.
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| 15. |
- Hollén, Elisabet, 1966-
(författare)
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Coeliac Disease in Childhood : On the Intestinal Mucosa and the Use of Oats
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Celiaki, eller glutenintolerans, är en av våra vanligaste kroniska sjukdomar i barnaåren. Sjukdomen orsakar en kraftig inflammation i tunntarmens slemhinna efter intag av glutenhaltig föda hos personer med ärftlig benägenhet att utveckla celiaki. En frisk tarm är kraftigt veckad för att öka ytan för upptag av näringsämnen. Ytan består dessutom av åtskilliga fingerliknande utskott, s.k. villi, och mellan villi finns kryptorna där celldelning och celldifferentiering sker. Villi och kryptor kantas av epitelceller, enterocyter, vilkas uppgift är att ta upp näring från tarminnehållet samt att utgöra en selektiv barriär mellan den yttre och inre miljön i tarmen. Den typiska tarmskadan vid celiaki karakteriseras av avsaknad av villi och kraftigt förlängda kryptor, och både näringsupptaget och barriärfunktionen är dessutom störda. Den enda behandling som finns att tillgå vid celiaki är en livslång glutenfri diet. De skadliga proteinerna i vetegluten kallas gliadin, och det finns liknande proteiner i råg, korn, och havre. I havre kallas proteinet avenin. Möjligheten att använda havre vid celiaki har diskuterats flitigt, men numera anses det riskfritt för majoriteten av både barn och vuxna att använda havre i den glutenfria dieten.Målet med den här avhandlingen var att undersöka hur barn med celiaki reagerar på havre i kosten. Detta studerades med avseende på antikroppar mot avenin samt med en metod som mäter halten av kväveoxid- (NO-) produkter i urinen. Ett andra mål var att studera tunntarmens struktur vid olika stadier av celiaki.I den första studien undersökte vi om celiakibarn har antikroppar i serum mot avenin. Vi fann att så var fallet och att nivåerna var signifikant högre än hos friska kontrollbarn. När barnen sattes på glutenfri kost sjönk antikroppsnivåerna, för att öka igen när gluten återinfördes i kosten. Blodproverna till den här studien togs innan debatten om havre kom igång, vilket gör att vi tror att de olika dieterna även speglar ett sant intag av havre. Studien visade också att det inte var någon korsreaktion mellan antikroppar mot avenin och gliadin.Vi använde sedan vår metod för att mäta antikroppar mot avenin i en randomiserad studie där havre gavs till barn med nydiagnostiserad celiaki. Barnen fick antingen en vanlig glutenfri diet eller en med tillsats av specialhavre. Antikroppsnivåerna sjönk markant redan efter tre månader i båda grupperna, och vid studietidens slut, efter ca ett år, hade alla utom ett par patienter återfått normala nivåer. Samma barn studerades även med avseende på NO-produkter i urinen. NO är en kortlivad molekyl som fungerar som budbärare i och mellan celler, och produktionen av den ökar markant vid en inflammation. Tidigare studier har visat att barn med obehandlad celiaki har extremt höga halter av NO-produkter i urinen. I vår studie sjönk även dessa värden signifikant efter tre månader, och det var ingen skillnad mellan grupperna. Efter ett år hade dock fyra barn i havregruppen och ett barn i den grupp som fick vanlig glutenfri kost, fortfarande extremt höga nivåer av NO-produkter.Dessa båda studier styrker den kliniska uppfattningen att de flesta barn med celiaki kan tåla havre, men de visar också att man bör följa upp de celiakibarn som kompletterar sin glutenfria kost med havre eftersom vissa barn verkar ha kvarstående tecken på inflammation i tarmen.I tarmbiopsier från barn med olika stadier av celiaki studerades förekomst och lokalisering av occludin och claudiner, proteiner som är viktiga för att upprätthålla barriärfunktionen i tarmen. Vi fann ett ökat uttryck av occludin vid obehandlad celiaki, vilket vi tror speglar den ökade celldelning och de förändrade barriäregenskaper som man ser vid aktiv celiaki. Resultaten tyder även på att uttrycket av claudin 1-5 inte tycks påverkas av kosten hos barn med celiaki.
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| 16. |
- Lidén, Åsa, 1975-
(författare)
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Integrin αVβ3-Directed Contraction by Connective Tissue Cells : Role in Control of Interstitial Fluid Pressure and Modulation by Bacterial Proteins
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis aimed at studying mechanisms involved in control of tissue fluid homeostasis during inflammation.The interstitial fluid pressure (PIF) is of importance for control of tissue fluid balance. A lowering of PIF in vivo will result in a transport of fluid from the circulation into the tissue, leading to edema. Loose connective tissues that surround blood vessels have an intrinsic ability to take up fluid and swell. The connective tissue cells exert a tension on the fibrous network of the tissues, thereby preventing the tissues from swelling. Under normal homeostasis, the interactions between the cells and the fibrous network are mediated by β1 integrins. Connective tissue cells are in this way actively controlling PIF.Here we show a previously unrecognized function for the integrin αVβ3, namely in the control of PIF. During inflammation the β1 integrin function is disturbed and the connective tissue cells release their tension on the fibrous network resulting in a lowering of PIF. Such a lowering can be restored by platelet-derived growth factor (PDGF) -BB. We demonstrated that PDGF-BB restored PIF through a mechanism that was dependent on integrin αVβ3. This was shown by the inability of PDGF-BB to restore a lowered PIF in the presence of anti-integrin β3 IgG or a peptide inhibitor of integrin αVβ3. PDGF-BB was in addition unable to normalize a lowered PIF in β3 null mice. Furthermore, we demonstrated that extracellular proteins from Streptococcus equi modulated αVβ3-mediated collagen gel contraction. Because of the established concordance between collagen gel contraction in vitro and control of PIF in vivo, a potential role for these proteins in control of tissue fluid homeostasis during inflammation could be assumed. Sepsis and septic shock are severe, and sometimes lethal, conditions. Knowledge of how bacterial components influence PIF and the mechanisms for tissue fluid control during inflammatory reactions is likely to be of clinical importance in treating sepsis and septic shock.
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| 17. |
- Nilsson, Anna, 1978-
(författare)
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Mechanisms involved in macrophage phagocytosis of apoptotic cells
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Efficient removal of apoptotic cells is critical for development, tissue remodelling, maintenance of homeostasis, and response to injury. Phagocytosis of apoptotic cells is mediated by many phagocytic receptors, soluble bridging molecules, and pro-phagocytic ligands on the surface of apoptotic cells. Macrophage phagocytosis in general is controlled by stimulatory and inhibitory mechanisms. An example of the latter mechanism is that mediated by the cell surface glycoprotein CD47, which by binding to the inhibitory receptor Signal Regulatory Protein alpha (SIRPα) on macrophages, is known to inhibit phagocytosis of viable host cells. The studies of the present thesis aimed at investigating possible changes to CD47 on apoptotic cells, which could influence their elimination by macrophages. The endoplasmatic protein calreticulin (CRT), in conjunction with Low density lipoprotein Receptorrelated Protein 1 (LRP1) on the phagocyte, can act as a receptor for collectin family members and mediate uptake of apoptotic cells. However, CRT itself was found to also be expressed on the surface of many viable cell types, and the CRT expression increased on apoptotic cells. By using antibodies to LRP1 or receptor‐associated protein (RAP), an antagonist blocking LRP1 ligand binding, we found that CRT on target cells could interact in trans with LRP1 on a phagocyte and stimulate phagocytosis. CD47 on the target cell inhibited LRP1‐mediated phagocytosis of viable cells (e.g. lymphocytes or erythtocytes), but not that of apoptotic cells. The inability of CD47 on apoptotic cells to inhibit LRP1‐ mediated phagocytosis could be explained in two ways: 1) Some apoptotic cell types (fibroblasts and neutrophils, but not Jurkat T cells) lost CD47 from the cell surface, or 2) CD47 is evenly distributed on the surface of viable cells, while it was redistributed into patches on apoptotic cells, segregated away from areas of the plasma membrane where the pro‐phagocytic ligands CRT and phoaphatidylserine (PS) were concentrated. Apoptotic murine thymocytes also showed a patched distribution of CD47, but no significant loss of the receptor. However, both PS‐independent and PS‐dependent macrophage phagocytosis of apoptotic CD47‐/‐ thymocytes was less efficient than uptake of apoptotic wild‐type (wt) thymocytes. This contradictory finding was explained by the fact that CD47 on apoptotic thymocytes did no longer inhibit phagocytosis, but rather mediated binding of the apoptotic cell to the macrophage. These effects could in part be dependent on the apoptotic cell type, since uptake of experimentally senescent PS+ wt or CD47‐/‐ erythrocytes by macrophage in vitro, or by dendritic cells (DC) in vivo, were the same. In vivo, PS+ erythrocytes were predominantly trapped by marginal zone macrophages and by CD8+ CD207+ DCs in the splenic marginal zone. DCs which had taken up PS+ erythrocytes showed a slight increase in expression levels of CD40, CD86 and MHC class II. These findings suggest that PS+ erythrocytes may be recognized by splenic macrophages and DCs in ways similar to that reported for apoptotic T cells. Uptake of senescent erythrocytes by DCs may serve as an important mechanism to maintain self‐tolerance to erythrocyte antigens, and defects in this function may facilitate development of AIHA. Glucocorticoids are used to treat inflammatory conditions and can enhance macrophage uptake of apoptotic cells. We found that the glucocorticoid dexamethasone time‐ and dose‐dependently stimulated macrophage cell surface LRP1 expression. Dexamethasone‐stimulated macrophages also showed enhanced phagocytosis of apoptotic thymocytes and unopsonized viable CD47‐/‐ erythrocytes. In summary, LRP1 can mediate phagocytosis of both viable and apoptotic cells by binding CRT on the target cell. Macrophage expression of LRP1 is increased by glucocorticoids, which could be one explanation for the anti‐inflammatory role of glucocorticoids. While CD47 on viable cells efficiently inhibits phagocytosis in macrophages, CD47 on apoptotic cells does not and can sometimes even promote their removal.
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