| 1. |
- Skoglund, Caroline, 1981-
(författare)
-
Platelets in inflammation : Role of complement protein C1q, C-reactive proteinand toll-like receptors
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Platelets are proven essential in haemostasis, however, they are now also increasingly recognized as cells with important immunomodulatory properties, e.g. through interaction with leukocytes and several species of bacteria and by release inflammatory mediators upon activation. Moreover, platelets express receptors involved in immunity and inflammation such as Fcγ‐receptor IIa, complement protein C1q‐receptors (gC1qR, cC1qR, CD93 and α2β1) and toll‐like receptors (TLR‐1, ‐2, ‐4, ‐6 and ‐9). C1q, C‐reactive protein (CRP) and TLRs are all pattern recognition molecules able to recognize non‐self structures and initiate an immune response. Uncontrolled or misdirected activation of platelets and the immune response is involved in the onset and progress of several conditions with an inflammatory component, such as coronary artery disease and autoimmune diseases.Hence, the aims of the present thesis were to investigate the effects and q mechanisms of C1and CRP on platelet activation, and to clarify the intracellular signaling events provoked by TLR‐2 stimulation of platelets. Platelet interaction with immune complexes is poorly understood, however by utilizing well‐characterized model surfaces with adsorbed IgG and microscopy, we show that both C1q and CRP are able to inhibit FcγR‐mediated platelet adhesion and spreading. Using isolated platelets in suspension and flow cytometry, we also found that C1q triggers a rapid, moderate and transient up‐regulation of P‐selectin that is sensitive to blockade of gC1qR and protein kinase C (PKC), but not blockade of α2β1. Additionally, subsequent platelet activation by collagen or collagen‐related peptide (GPVI specific) is inhibited by C1q, suggesting a role for GPVI in C1q‐mediated regulation of collagen‐induced platelet activation. Whole blood studies revealed that C1q inhibits total cell aggregation, formation of platelet‐leukocyte aggregates, and potentiates the production of reactive oxygen species (ROS), all in a platelet‐dependent manner. Furthermore, using the TLR‐2/1 agonist Pam3CSK4 we found that TLR‐2/1‐activation of platelets is mediated via a P2X1‐dependent increase in intracellular free Ca2+, P2Y1 and P2Y12 –receptor ligation, and activation of cyclooxygenase. We also found that platelets express IRAK‐1, however, without being rapidly phosphorylated upon Pam3CSK4 stimulation and thus probably not involved in the early aggregation/secretion response. Furthermore, TLR‐2/6 stimulation does not lead to platelet activation but instead inhibits TLR‐2/1‐provoked activation. Taken together, these findings further strengthen the role of platelets as key players in inflammatory processes.
|
|
| 2. |
- Zhang, Boxi, 1987-
(författare)
-
Modulaton of gene expression in human aortic smooth muscle cells by Porphyromonas gingivalis : a possible association between periodontitis and atherosclerosis
- 2015
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Porphyromonas gingivalis is a gram-negative, rod-shaped, and anaerobic bacterium that is involved in the pathogenesis of periodontitis. P. gingivalis produces a variety of virulence factors including gingipains and fimbriae. These virulence factors not only have a detrimental effect on bacteria adhesion, invasion, and colonization but also affect the host cell inflammatory response. P. gingivalis is also considered to play an role in the development of other diseases, such as atherosclerosis and cancer. Smooth muscle cells are the main components of vascular walls and regulate the width of the blood vessels in the body. To understand the mechanisms underlying the association between periodontitis and atherosclerosis we have, in the studies involved in this thesis, treated human aortic smooth muscle cells (AoSMCs) with wild type, gingipain mutant, and fimbriae mutant strains of P. gingivalis. Using a human whole genome microarray, quanti-tative real time PCR, Western blotting, ELISA, confocal microscopy, and cellular function experiments, we found that P. gingivalis invades AoSMCs, regulates the expression of thousands of genes, and increases cell proliferation by activating the TGFbeta/Notch signaling pathway. The results also show that P. gingivalis increases the ratio of angiopoietin 2 (Angpt2) / angiopoietin 1 (Angpt1) in AoSMCs, which determines the regulatory role of angiopoietins in angiogenesis and their involvement in the development of atherosclerosis. Moreover, we also found that P. gingivalis can induce interleukin-1β (IL-1β) production in AoSMCs, while inhibiting the expression of NLRP3 inflammasome components. Gingipains, especially arginine gingipain, play a fundamental role in P. gingivalis-induced modification of AoSMCs. These findings further support an association between periodontitis and cardiovascular disease.
|
|
| 3. |
- Jayaprakash, Kartheyaene, 1983-
(författare)
-
Monocyte and Neutrophil Inflammatory Responses to the Periodontopathogen Porphyromonas gingivalis
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Periodontitis is one of the most common adult infections. Duing bacteremia in healthy individuals or patients with chronic periodontitis, a number of oral bacteria such as Porphyromonas gingivalis encounter inflammatory cells in the blood eg. platelets, neutrophils and monocytes. Although several studies have suggested an association between periodontitis and cardiovascular diseases, the infection and inflammatory mechanisms are poorly understood. Hence, the aim of this thesis was to elucidate the mechanisms that are involved in P. gingivalis interaction with blood leukocytes, in order to further understand the molecular pathogenesis that renders periodontitis as a risk factor for several systemic conditions. We have demonstrated that P. gingivalis induces ROS production in neutrophils, THP1 cells and in whole blood, through activation of pattern recognition receptors, such as toll-like receptors, nuclear oligomerizing domains and protease- activated receptors. Besides, we have also shown that monocytes secrete IL-1β and CXCL8 in response to P. gingivalis. Both these cytokines prime neutrophils, endothelial cells and other vascular cells in an autocrine and paracrine manner. P. gingivalis has a plethora of virulence factors of which gingipains are very unique. In addition to activating inflammatory signalling pathways in cells, gingipains also regulate CXCL8 and IL-1β, thereby curtailing the host defence strategies. We demonstrated that oxidized LDL, but not native LDL, induces IL-1β release and CD36 expression on THP1 cells. Furthermore, LDL mildly modifies P. gingivalis-induced inflammatory responses as well as CD36 expression in THP1 cells. We also observed that P. gingivalis is eliminated mainly by phagocytosis in neutrophils. In summary, these studies clarify the mechanisms of interaction between P. gingivalis and leukocytes, which can increase the understanding of the pathogenesis of periodontitis and associated systemic disorders.
|
|
| 4. |
- Koskela von Sydow, Anita, 1979-
(författare)
-
Regulation of fibroblast activity by keratinocytes, TGF-β and IL-1α : studies in two- and three dimensional in vitro models
- 2016
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Dysregulated wound healing is commonly associated with excessive fibrosis. Connective tissue growth factor (CTGF/CCN2) is characteristically overexpressed in fibrotic diseases and stimulated by transforming growth factor-β (TGF-β) in dermal fibroblasts. Reepithelialisation and epidermal wound coverage counteract excessive scar formation. We have previously shown that interleukin-1α (IL-1α) derived from keratinocytes conteracts TGF-β-stimulated CTGF-expression. The aim of this thesis was to further explore the effects of keratinocytes and IL-1α on gene and protein expression, as well as pathways, in TGF-β stimulated fibroblasts. Fibroblasts were studied in vitro by conventional two dimensional cell culture models and in a three dimensional keratinocyte-fibroblast organotypic skin culture model.The results showed that IL-1 suppresses basal and TGF-β-induced CTGF mRNA and protein, involving a possible TAK1 mechanism. Keratinocytes regulate the expression of fibroblast genes important for the turnover of the extracellular matrix. Most of the genes analysed (11/13) were regulated by TGF-β and counter regulated by keratinocytes. The overall results support a view that keratinocytes regulate fibroblasts to act catabolically (anti-fibrotic) on the extracellular matrix.Transcriptional microarray and gene set enrichment analysis showed that antagonizing effects of IL-1α on TGF-β were much more prominent than the synergistic effects. The most confident of these pathways was the interferon signaling, which were inhibited by TGF-β and activated by IL-1α. A proteomics study confirmed that IL-1α preferentially conteracts TGF-β effects. Six new fibroblast proteins involved in synthesis/ regulation were identified, being regulated by TGF-β and antagonized by IL-1α. Pathway analysis confirmed counter-regulation of interferon signaling by the two cytokines. These findings have implications for understanding the role of fibroblasts for inflammatory responses and development of fibrosis in the skin.
|
|
| 5. |
- Lönn, Johanna, 1982-
(författare)
-
The role of peridontitis and hepatocyte growth factor in systemic inflammation
- 2013
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- An essential goal in addressing inflammation is the return of tissue to homeostasis. Persistent infections often cause prolonged response and accumulation of immune cells, inducing imbalance in pro- and anti-inflammatory mediators, tissue destruction, and chronic inflammation. In periodontal disease, bacteria of the dental plaque are the primary aetiologic agents. Coronary artery disease (CAD) and chronic renal failure (CRF) are associated with periodontitis and involve systemic inflammation with atherosclerotic and fibrotic processes. The aims of this thesis were to study the effect of the bacterium Porphyromonas gingivalis and the anti-inflammatory mediator lipoxin A4 (LXA4) on blood cells in vitro, as well as to measure the expression of hepatocyte growth factor (HGF) in patients with periodontitis, CAD, and CRF. We found that LXA4 inhibits P. gingivalis–induced leukocyte platelet aggregation and reactive oxygen species (ROS) production in whole blood, by antagonizing the upregulation of CD11b/CD18 on leukocytes. The serum concentration of HGF was elevated in patients with periodontitis, CAD and CRF, indicating a systemic inflammation. However, the biological activity of HGF was reduced in serum from CRF patients and in saliva and gingival crevicular fluid of patients with periodontitis. This finding correlated with reduced growth of gingival epithelial cells incubated with saliva from patients with periodontitis. Neutrophil proteases reduced the biological activity of HGF in patients with CRF, and HGF expression in patients with periodontitis was associated with higher concentration and numbers of species of periodontal bacteria. In conclusion, these studies suggest that systemic spreading of periodontal bacteria, leukocyte-platelet activation and disturbed HGF-expression are crucial components involved in tissue degradation and progression of chronic inflammation.
|
|
| 6. |
|
|
| 7. |
- Bengtsson, Sara, 1978-
(författare)
-
Stress steroids as accelerators of Alzheimer's disease. : Effects of chronically elevated levels of allopregnanolone in transgenic AD models.
- 2013
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background Alzheimer’s disease (AD) and dementia are devastating conditions not only for the affected patients but also for their families. The economical costs for the society are tremendous. Mid-life psychological stress, psychosocial stress and post-traumatic stress disorder cause cognitive dysfunction and lead to increased risk for dementia. However, the mechanisms behind stress-induced AD and dementia are not known. AD is characterized by solid amyloid plaques in the CNS. However, over the last decade it has been concluded that the levels of soluble beta-amyloid (Aβ) correlate to cognitive performance while plaques often do not. The soluble Aβ accumulate intracellularly and disturb the synaptic function. Interestingly, the levels of intracellular Aβ depend on neuronal activity. Previous studies have shown that decreased neuronal activity cause increased intracellular levels of Aβ and cognitive decline. Stress steroids produced in the brain, e.g. allopregnanolone, enhance the activity of the GABAergic system, i.e. the main inhibitory system of the brain. Consequently, allopregnanolone affects neuronal activity. Therefore, it is possible that elevated levels of allopregnanolone (due to e.g. stress) cause increased intracellular levels of Aβ. This could be a mechanism behind stress-induced AD. The purpose of this thesis was to investigate if elevation of allopregnanolone is a possible link in the mechanism behind stress-induced AD by investigating the effects of chronically elevated levels of allopregnanolone in transgenic mouse models for AD.Methods Swe/PS1 and Swe/Arc mice (transgenic models for AD) were treated chronically with elevated allopregnanolone levels, comparable to those at mild stress. After an interval of no treatment, the mice were tested for learning and memory performance in the Morris water maze. The brain tissue of the mice was then analyzed for disease markers, i.e. soluble and insoluble Aβ40 and Aβ42 using enzyme-linked immunosorbent assay, and amyloid plaques using immunohistochemistry and Congo red staining technique. The brain tissue was also analyzed for a marker of synaptic function, i.e. synaptophysin.Results Chronic treatment of allopregnanolone caused impaired learning performance in both the Swe/PS1 and the Swe/Arc mouse models. The Swe/PS1 mice had increased levels of soluble Aβ in both hippocampus and cortex. Interestingly, the levels of soluble Aβ were unchanged in the Swe/Arc mice. Three months of allopregnanolone treatment in the Swe/PS1 mouse model caused decreased plaque size, predominantly in hippocampus. It may be concluded that chronic allopregnanolone elevation caused smaller but more abundant congophilic plaques as both total plaque area and number of plaques were increased in mice with poor learning ability. Additional spots for accumulation of Aβ, predominantly the more toxic Aβ42, and thus additional starting points for plaque production could be a part of the mechanism behind stress-induced Alzheimer’s disease.Conclusions The conclusion of this thesis is that chronic elevation of allopregnanolon accelerated the development of Alzheimer’s disease in the Swe/PS1 and the Swe/Arc transgenic mouse models. Allopregnanolone may be an important link in the mechanism behind stress-induced AD. However, further studies are required to grasp the extent of its pathological influence.
|
|
| 8. |
|
|
| 9. |
- Kälvegren, Hanna, 1978-
(författare)
-
The Role of Chlamydia pneumoniae-induced Platelet Activation in Cardiovascular Disease : In vitro and In vivo studies
- 2007
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The common risk factors for atherosclerosis, such as obesity, high cholesterol levels, sedentary lifestyle, diabetes and high alcohol intake, only explain approximately 50% of cardiovascular disease events. It is thereby important to identify new mechanisms that can stimulate the process of atherosclerosis. During the past decades, a wide range of investigations have demonstrated connections between infections by the respiratory bacterium Chlamydia pneumoniae and atherosclerosis. Earlier studies have focused on the interaction between C. pneumoniae and monocytes/macrophages, T-lymphocytes, smooth muscle cells and endothelial cells, which are present in the atherosclerotic plaque. However, another important player in atherosclerosis and which is also present in the plaques is the platelet. Activation of platelets can stimulate both initiation and progression of atherosclerosis and thrombosis, which is the ultimate endpoint of the disease. The aim of the present thesis was to investigate the capacity of C. pneumoniae to activate platelets and its role in atherosclerosis.The results show that C. pneumoniae at low concentrations binds to platelets and stimulates platelet aggregation, secretion, reactive oxygen species (ROS) production and oxidation of low-density lipoproteins (LDL), and that these effects are mediated by lipopolysaccharide (LPS). Activation of protein kinase C, nitric oxide synthase and 12-lipoxygenase (12-LOX) was required for platelet ROS production, whereas platelet aggregation was dependent on activation of GpIIb/IIIa. Pharmacological studies showed that the C. pneumoniae-induced platelet activation is prevented by inhibitors against 12-LOX, platelet activating factor (PAF) and the purinergic P2Y1 and P2Y12 receptors, but not against cyclooxygenase (COX). These findings were completely opposite to the effects of these inhibitors on collagen-stimulated platelets. We also present data from a clinical study indicating that percutaneous coronary intervention (PCI or balloon dilatation) leads to release of C. pneumoniae into the circulation, which causes platelet activation and LDL oxidation.In conclusion, these data support a role for C. pneumoniae-induced platelet activation in the process of atherosclerosis. Stimulation of platelets by C. pneumoniae leads to release of growth factors and cytokines, oxidation of LDL and platelet aggregation, which are processes that can stimulate both atherosclerosis and thrombosis. Development of novel drugs that prevent C. pneumoniae-platelet interaction by inhibiting 12-LOX and/or PAF, may be important in the future treatment of cardiovascular disease.
|
|
| 10. |
- Musa, Amani, 1983-
(författare)
-
Plantaricins as a novel group of antibacterial compounds and enhancers of antibiotics
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibiotics have revolutionized medicine, however, the rapid development of an-tibiotic resistance among bacteria is diminishing their efficacy. Antimicrobial pep-tides produced by Lactobacillus plantarum, i.e., plantaricins, are considered prom-ising alternatives to antibiotics against infections. In this thesis, the antimicrobial activities of different plantaricins (Pln A, Pln EF, Pln JK, and PLNC8 αβ) were investigated against antibiotic-resistant and susceptible strains of Staphylococcus spp, biofilm-forming strains, as well as clinical isolates of ESKAPE pathogens, and Escherichia coli. Moreover, the stability, cytotoxicity, and immunomodulatory effects of PLNC8 αβ were characterized. The results show that Pln EF and Pln JK have potent antimicrobial activity against Staphylococcus epidermidis and effectively enhance the effects of various antibiotics. Furthermore, PLNC8 αβ shows potent antibacterial effects against different Gram-positive and Gram-negative bacteria, including vancomycin- and methicillin-resistant strains. The antibacterial effects and stability following peptide truncation and D-amino acid substitution were investigated. D-amino acid substitution did not change the antimicrobial activity of PLNC8 αβ, however, it increased the stability of the peptide as it was more resistant to proteolysis by trypsin compared to the native L-enantiomer. Moreover, among the truncated peptides, α1–22, β7–34, and β1–20 retained bacteriostatic effects without displaying bactericidal activity. L-PLNC8 αβ peptides were tested for their antibiofilm properties and displayed rapid disruption of surface-associated S. epidermidis. Electron microscopy shows that PLNC8 αβ targets bacterial cell membranes, ultimately resulting in rapid permeabilization and altered homeostasis, including ATP release. PLNC8 αβ does not show any cytotoxic or hemolytic effects on human cells in vitro. Furthermore, PLNC8 αβ counteracted the cytotoxic effects and expression of inflammatory mediators that were induced by S. aureus, including MMPs and growth factors that are essential in cell regeneration. Pathogen recognition receptors (TLR2, TLR4, and PAR2), intracellular signaling events (c-Jun, c-Fos), and inflammatory mediators (IL-1β, IL-6, CXCL-8), that facilitate pathogen recognition, cell survival, and cellular communication, were all enhanced by the peptides. At sub-MIC concentrations, PLNC8 αβ enhanced the activity of various antibiotics against both Gram-positive and Gram-negative ESKAPE bacteria. In conclusion, plantaricins efficiently impede bacterial pathogens and enhance the activity of antibiotics and thereby constitute a therapeutic option to counter the threatening situation with severe antibiotic-resistant infections.
|
|
