Encapsulating peritoneal sclerosis (EPS) is a rare complication of long-term peritoneal dialysis (PD). EPS is associated with extensive thickening and fibrosis of the peritoneum resulting in the formation of a fibrous cocoon encapsulating the bowel leading to intestinal obstruction. The presence of peritoneal thickening, peritoneal calcification and bowel obstruction is considered to be diagnostic of EPS. The current understanding of the pathogenesis of EPS is through the 'two-hit' fibrosis model. This model, however, does not explain the development of peritoneal calcification in patients with EPS. This thesis addresses the hypothesis that altered bone mineral metabolism in ESRF patients together with the mechanical stress of PD influences mesothelial cells to differentiate into osteoblasts promoting calcification in peritoneal tissue. Peritoneal calcification leads to increased tissue stiffness causing progressive fibrosis and the development of EPS. We compared the temporal evolution of the levels of bone mineral markers during PD between patients who developed EPS and control patients on PD. We found that raised serum levels of calcium, phosphate and alkaline phosphatase during PD increased the risk of development of EPS. We compared peritoneum from patients with EPS with that of PD patients without EPS using histological techniques. We found that calcification, organised fibrillary collagen and elastic fibres were significantly more abundant in the EPS peritoneum. Peritoneal calcification was also generalised and distributed not only on the peritoneal surface but also in the sub-mesothelial zone of fibrosis. EPS peritoneum also exhibited osteocalcin, an osteogenic protein, suggesting a cellular mechanism of calcification. Atomic force microscopy of EPS peritoneum showed increased stiffness when compared to control PD peritoneum with the areas of calcification possibly contributing to the increase in tissue stiffness. Human omental cells (HOMCs) were isolated by protease digestion and characterised using a panel of mesothelial markers. HOMCs were cultured in phosphate rich media and phosphate and calcium rich media. HOMCs when cultured with high extracellular levels of calcium showed accelerated mineralisation with upregulation of osteogenic transcription factor runx-2 suggesting osteoblastic transformation. In summary, this thesis indicates that poorly controlled secondary hyperparathyroidism is a risk factor for the development of EPS. On a background of PD related simple sclerosis, uncontrolled secondary hyperparathyroidism can lead to the transformation of mesothelial cells to osteoblasts. This leads to increased matrix deposition and matrix mineralisation causing increased matrix stiffness. Increase in matrix stiffness leads to progressive fibrosis culminating in EPS. Peritoneal calcification can act as the second hit leading to progressive fibrosis and development of EPS.

Post-operative adhesion is a common complication after abdominal surgery, with high impact on patient wellbeing and healthcare costs. The repair of peritoneum is a complex process involving orderly phases which share some common features to normal wound healing. These include coagulation, infiltration of inflammatory cells, cell proliferation, extracellular matrix (ECM) deposition and remodelling, often with overlap between phases. The unique feature of peritoneal repair is that both small and large peritoneal wounds heal in a similar time. The peritoneum is a monolayer of elongated, flattened, squamous-like peritoneal mesothelial cells (PMC). Local mesothelial cell proliferation, centripetal cell migration from the wound edge, as well as incorporation of free-floating mesothelial cells may all contribute to repair of injured peritoneum. To date, the only well-characterised pathologic mechanism underlying post-operative adhesion formation at the molecular level is the formation of the fibrin layer and regulation of peritoneal fibrinolytic capacity. However, the contributions of collagen deposition and ECM remodelling to the peritoneal repair mechanism are not well understood. This thesis focuses on the role of PMC in the regulation of ECM deposition and remodelling in response to inflammatory stimuli in both in vivo and in vitro models, aiming to identify other key pro-fibrotic factors involved in the development of post-operative adhesion. We first identified that lysyl oxidase (LOX) played a key role in the progression of peritoneal fibrosis by regulating collagen cross-linking and deposition in vivo. The inhibition of LOX enzyme activity prevented the formation of fibrotic tissue by reducing collagen deposition. Meanwhile, dexamethasone (DEX) treatment also minimized the fibrotic response. Furthermore, in vitro studies showed that the induction of collagen deposition factors in PMC, including LOX and pro-collagen I, required both IL-1 and TGF-β signalling pathways. Thus, the combination of IL-1 + TGF-β was adopted in an in vitro model to mimic the inflammatory environment during peritoneal repair. Treatment of PMC with IL-1+TGF-β caused an epithelial-to-mesenchymal transition (EMT). These transformed PMC had enhanced cell motility and were more adherent to fibronectin. Finally, a real-time quantitative PCR-based microarray was used for genomic analysis of ECM-adhesion-related PMC genes in response to IL-1 and TGF-β treatment. The results showed that IL-1 was more involved in regulating ECM degradation by inducing expression of matrix metalloproteinase (MMP) genes, whereas TGF-β mainly affected genes involved in ECM deposition, including collagens and other ECM components. However, both cytokines were shown to regulate some key genes involved in the development of adhesion, including COL16A1, COL7A1, FN1, ITGA5, and TGFB1. Moreover, IL-1 was shown to reduce ITGA4 and ITGB6 expression affecting adherence of PMC to basement membrane, while TGF-β increased MMP14 and MMP16 expression, which could facilitate invasion of EMT-transformed PMC to the site of tissue repair. In summary, this thesis indicates that LOX plays an important role in peritoneal fibrosis. Secondly, a combination of IL-1 and TGF-β1 treatment demonstrates how these factors can act in concert to orchestrate tissue remodelling during peritoneal repair. Finally, genomic analysis of ECM-adhesion genes increases our understanding of aspects of the pathology of post-operative adhesion and identifies novel potential therapeutic targets to prevent adhesion formation.

Peritoneal dialysis (PD) is a form of renal replacement therapy using the peritoneum as a dialysis membrane. PD solutions employed to remove nitrogen metabolites and excess plasma fluid, and to restore electrolyte and acid-base balance are being developed to minimize local and systemic inflammatory responses while maintaining peritoneal homeostasis and host defense. The effect of chronic action of PD solutions on the peritoneum results in its remodeling and, possibly, eventual loss of peritoneal ultrafiltration capacity. Factors most responsible for late complications and peritoneal remodeling include high glucose levels in PD solutions, and the presence and formation of glucose degradation products (GDP) and advanced glycation end - products (AGEs) in the peritoneal cavity. The aim of our study described in this dissertation was to test various PD solutions with different glucose content and GDP and, using AGEs receptor ligands, to define their systemic effects and identify PD solutions with highest biocompatibility. This part of the dissertation characterizes conventional glucose - based solutions, low - glucose and GDP load solutions as well as glucose polymer (icodextrin) - based PD solutions while determining the plasma and dialysate levels of soluble receptor for AGEs (s - RAGE) and its...

VALIDERING AV MAY-GRÜNWALD GIEMSA VID FÄRGNING AV CYTOLOGIPROVEROPTIMERING AV IN VITRO-DIAGNOSTIK, MED ANLEDNING AV NYA EU-KRAVKARIN SVANTESSONSvantesson, K. Validering av May-Grünewald Giemsa vid färgning av cytologiprover. Optimering av in vitro-diagnostik, med anledning av nya EU-krav. Examensarbete i biomedicinsk laboratorievetenskap, 15 högskolepoäng. Malmö Universitet: Fakulteten för hälsa och samhälle, institutionen för Biomedicinsk vetenskap, 2023.Cancer uppstår vid onormal celldelning, men uppkommer även vid kronisk inflammation. För att kunna konstatera om patienten har cancer krävs olika typer av diagnostik, där cytologidiagnostik ingår. Med hjälp av olika färglösningar kan cancerceller i serösa vätskor färgas för att upptäckas, således kan den cancerdrabbade patienten behandlas. May-Grünwald Giemsa (MGG) ingår i Romanowsky-färgningsteknikerna och har använts sedan 1800-talet.Färglösningen ger en högkvalitativ visualisering av cellernas morfologi. Färgen används inom histologi-, hematologi- och cytologidiagnostiken. Laboratorier som arbetar med in vitro-diagnostik (IVD) måste arbeta med märkta produkter som är reglerade för IVD. År 2017 fastställdes att inom fem år skall alla laboratorier i Europeiska unionen (EU) som utför IVD, arbeta med in vitro-diagnostik reglerade (IVDR) produkter för att uppnå EU-direktiven. Syftet med studien var att optimera en ny färgblandning av MGG vid färgning av cytologiprover, så att IVDR-kraven uppfylls på Patologen i Skövde. Studien omfattade flera försök för att fastställa vilket av färgprotokollen som gav bästa kvalité på cellerna i cytologiska preparat. Under studiens gång har det även visats att olika faktorer, exempelvis färskheten av provet kan påverka infärgningens kvalité av preparatet. Gammalt eller felhanterat provmaterial kan leda till försämrad infärgning av cellerna. Resultat från studien visade att färgprotokollet Histo Lab (HL) gav goda resultat efter en modifiering av sammansättningen och tiden vid infärgning. / VALIDATION OF MAY-GRÜNWALD GIEMSA IN THE STAINING OF CYTOLOGY SAMPLESOPTIMIZATION OF IN VITRO-DIAGNOSTICS, DUE TO NEW EU REQUIREMENTSKARIN SVANTESSONSvantesson, K. Validation of May-Grünwald Giemsa in the staining of cytology samples. Optimization of in vitro-diagnostics, due to new EU requirements. Degree project in biomedical laboratory science, 15 higher education credits. Malmö University: Faculty of Health and Society, Department of Biomedical Sciences, 2023.Cancer is caused by abnormal cell division, but also occurs by chronic inflammation. In order to determine whether the patient has cancer, different types of diagnostics are required, where cytology diagnostics are included. With thehelp of different dye solutions, cancer cells in serous fluids can be stained and detected, allowing for diagnosis and treatment. May-Grünwald Giemsa (MGG) is part of the Romanowsky staining techniques and has been used since the 19th century. The dye solution provides a high-quality visualization of the cells' morphology and is used in histology-, hematology- and cytology diagnostics.Laboratories that work with in-vitro diagnostics (IVD) must use products that are in vitro-diagnostics regulated (IVDR). In 2017 it was determined that within five years all laboratories in the European Union (EU) performing IVD must work with IVDR labeled products to achieve EU directives. The aim of the study was to optimize a new dye stain of MGG for cytology samples, so that the IVDR requirements are achieved at the pathology laboratory in Skövde. The study included several attempts to determine which of the staining protocols produced the best quality of the cells in cytology preparations. During the study, it has also been shown that various factors can negatively affect the results. If sample material is too old or mishandled, it can lead to poor staining of the cells. Results from the study showed that the Histo Lab (HL) staining protocol gave goodresults after modification of the composition and time of staining.

Peritoneal dialysis (PD) is a form of renal replacement therapy using the peritoneum as a dialysis membrane. PD solutions employed to remove nitrogen metabolites and excess plasma fluid, and to restore electrolyte and acid-base balance are being developed to minimize local and systemic inflammatory responses while maintaining peritoneal homeostasis and host defense. The effect of chronic action of PD solutions on the peritoneum results in its remodeling and, possibly, eventual loss of peritoneal ultrafiltration capacity. Factors most responsible for late complications and peritoneal remodeling include high glucose levels in PD solutions, and the presence and formation of glucose degradation products (GDP) and advanced glycation end - products (AGEs) in the peritoneal cavity. The aim of our study described in this dissertation was to test various PD solutions with different glucose content and GDP and, using AGEs receptor ligands, to define their systemic effects and identify PD solutions with highest biocompatibility. This part of the dissertation characterizes conventional glucose - based solutions, low - glucose and GDP load solutions as well as glucose polymer (icodextrin) - based PD solutions while determining the plasma and dialysate levels of soluble receptor for AGEs (s - RAGE) and its...