Introduction: Chronic hepatitis B (CHB) is a viral infection that can lead to development of fibrosis and hepatocellular carcinoma (HCC). Several factors affecting disease progression have been reported, such as sex and region of origin. Liver stiffness and fibrosis can be evaluated using transient elastography. The degree of fibrosis is an important parameter when deciding if treatment and HCC surveillance is indicated. Aim1) To compare patients with CHB according to sex and region of origin regarding the parameters liver stiffness, presence of significant fibrosis, hepatitis B e antigen (HBeAg) positivity, frequency of elevated alanine aminotransferase (ALT) levels and HCC surveillance.2) To identify factors associated with significant liver fibrosis. Methods: 410 patients with a registered doctor’s visit 2015–2018 at the Department of Infectious Diseases at Örebro University Hospital were included. A systematic review of medical records was performed and groups (women-men, regions of origin) were compared. Multivariate logistic regression was used to identify factors associated with significant fibrosis. Results: Men had significantly higher liver stiffness values, higher presence of significant fibrosis, and were more frequently under HCC surveillance compared to women. No other significant differences were found regarding the studied parameters, neither related to sex, nor to region of origin. Factors associated with significant fibrosis were: male sex, elevated ALT levels and hepatitis D virus (HDV) co-infection. Conclusions: Men had a higher frequency of significant fibrosis compared to women. Factors associated with significant fibrosis were male sex, elevated ALT values and HDV co-infection.

Aim: The aim of this thesis is to describe the process by which the inherited disease, cystic fibrosis, (CF) was recognised as an important clinical entity in the United Arab Emirates (UAE) and the Sultanate of Oman (Oman). It examines the clinical presentation of the first patients and assesses their degree of severity. Further, it describes the first studies carried out to determine the underlying CF mutations associated with the disease in the UAE and Oman. An estimate is offered of the birth frequency of the condition. Overall, the cultural, geographical and historical aspect of the societies in which the disease occurs is stressed. Methods: An initial literature search was carried out using Medline of any literature pertaining to the Arab World and CF. this was read and classified into the relevance to Arabs in general, the Middle East and then specifically the Arab (Persian) Gulf societies. Thereafter, a clinic was established at Tawam Hospital, Al Ain, UAE, for children presenting With chronic respiratory disease that could serve as a national referral centre. It was run by the Author as a service of the Paediatric Department of the UAE University Medical School. I sent a letter to every Paediatrician working in the UAE informing them of our clinic and offering our services for the diagnosis and management of chronic respiratory disease in children. This was based on the author's experience as a respiratory paediatrician in Australia and New Zealand and as the Professor of Paediatrics in the UAE. No such service then existed in the UAE. Funding was sought to establish a research programme and develop a molecular genetics laboratory in the UAE Medical School. A series of successful research applications provided the grants to commence the investigations. Once a small number of children had been identified as having CF from those referred to the respiratory clinic, the initial project was to assess and report their clinical presentation. Following this an early start was made on the identification of the mutations responsible. Once these were established an attempt was made to estimate the frequency of the condition at birth. Additional clinical studies revolved around assessing the severity of the condition that was associated with the main mutations that were identified. A clinical comparison was made with those with the mutation AF508 and the other main mutation, despite the obvious limitation of small numbers then available. Radiological assessment was made to evaluate the progression of the disease. The final aspect of the study was to assess patients from Oman and compare their findings and mutations with the neighbouring UAE. Based on information gained hypotheses are proposed regarding the spread of the gene mutation by population drift. Thesis outline: A literature review is presented in the form of a critique on the disease and a resume of the relevant aspects of the genetics of CF. Additionally, facts about the two countries' geography and history are presented. Finally, knowledge about CF mutations and population origins from other areas is presented. The second main section deals with the clinical features of the disorder as it presents in the UAE. Molecular findings are then presented and details of the common mutation found in Bedouin Arabs. Hypotheses are then presented based on the information gathered. Results: CF is not a rare disease in the Arab children of the UAE and Oman. These findings refute previous reports of CF being a rare or non-existent disease in Arabs. The condition presents with a severe clinical picture, with early colonisation of the respiratory tract with staphylococcus, haemophilus and pseudomonas organisms, even with conventional CF management practices in place. The CF mutation S549R is prevalent in Arabs of Bedouin stock, while AF508 is found in those of Baluch origin. The former may be descendants of Arabs who left southern Arabia and travelled to the Trucial Coast at the time of the destruction of the great dam at Marib. The origins of this mutation may lie in the area that corresponds to the modern Republic of Yemen. The latter groups are descendants of those who came originally from Baluchistan. It is hypothesised also that the ancestral home of the AF508 mutation may be in the geographical area now known as Baluchistan, that spans three separate modern political territories. The evidence presented supports the concept that the S549R mutation may be associated with a severe, if not the severest, clinical pattern recognised. It equates with that seen with the homozygous AF508 genotype. The absence of an additional mutation in the promoter region accounts for the different clinical pattern seen in previously described patients. Conclusions: There needs to be a major awareness of the presence of CF as a severe clinical disease in the children of the Gulf States. The clinical presentation and findings support the concept of under recognition of the disease. Climatic conditions put the children at special risk of hyponatraemia and electrolyte imbalance. The absence of surviving adults with the disease suggests premature deaths have occurred, but the high fertility rates have maintained the gene pool for this recessive disorder.

Angiotensin II signals cardiovascular disease through metalloproteinases including MMP-2, MMP-7 and ADAM-17/TACE. We hypothesized that these metalloproteinases regulate each other at the transcriptional level. Further, MMP-2, being a major gelatinase in cardiac and vascular tissue, could mediate angiotensin II-induced cardiovascular disease. We studied the development of hypertension (by tail cuff plethysmography), cardiac hypertrophy (by M-mode echocardiography and qRT-PCR analysis of hypertrophy marker genes) and fibrosis (by collagen staining and qRT-PCR analysis of fibrosis marker genes) in mice receiving angiotensin II. Angiotensin II induced hypertension, cardiac hypertrophy and fibrosis which correlated with an upregulation of MMP-2. Downregulation of MMP-2 by pharmacological inhibition and RNA interference attenuated hypertension but not cardiac hypertrophy or fibrosis. Downregulation of MMP-7 or ADAM-17/TACE by RNA interference attenuated angiotensin II-induced MMP-2 upregulation as well as hypertension, cardiac hypertrophy and fibrosis. We conclude that MMP-2 selectively mediates angiotensin II-induced hypertension under the transcriptional control of MMP-7 and ADAM-17/TACE.

Fibrosis in the bone marrow is usually denominated myelofibrosis and may contribute to impaired hematopoiesis. Myelofibrosis is seen both in malignant and non-malignant diseases. The normal microenvironment in the bone marrow consists of a heterogenous population of hematopoietic and non-hematopoietic stromal cells, their extracellular products and hematopoietic cytokines. The stromal cells produce a complex array of molecules, among others collagens and glycosaminoglycans (GAGs) of which hyaluronan (HYA) is the most abundant. Marrow fibrosis results from an increased deposition of collagens, which are polypeptides. Staining for reticulin, mostly composed of collagen type III, is the common way of visualizing myelofibrosis. HYA, like the collagens, is widely distributed in connective tissues. Little is known about the distribution of HYA in bone marrow. The aims of this thesis have been to determine how HYA is distributed in normal and malignant bone marrow, compared to reticulin staining, and to follow patients with chronic myeloproliferative diseases (CMPD) during two years treatment with anagrelide considering development of cellularity and fibrosis. In bone marrow biopsies from healthy volunteers, the controls, HYA was found in a pattern that was concordant with the reticulin staining. Comparing patients with different malignant diseases with and without bone marrow involvemen, HYA staining was found to be significantly stronger in both groups compared to the controls. The HYA scores were also significantly higher in the bone marrow of patients with de novo acute myeloid leukemia (AML), compared to the controls. There was a correlation between HYA and reticulin in the patients with de novo AML, and in the patients with different malignant diseases with and without bone marrow involvement as in the controls. Increase of HYA, reticulin and cellularity in the bone marrow of patients with CMPD after two years of treatment with anagrelide indicated progression of fibrosis. Anagrelide is a valuable drug for reduction of platelets but seems unable to stop progression of fibrosis and hypercellularity. HYA is an interesting molecule with properties not only contributing to the structure of extracellular matrix but also to cell signaling and behaviour, although the understanding of the detailed mechanisms is still incomplete.

Cystic fibrosis (CF) is an inherited disease with symptoms mainly in the respiratory tract. The airway epithelium is covered with a thin layer of fluid, the airway surface liquid (ASL). The volume and composition of ASL are important in the pathogenesis of cystic fibrosis. The composition of ASL was determined. Firstly, pig airways were analyzed by X-ray microanalysis in the frozen-hydrated state. Secondly, small Sephadex beads were left to absorb the ASL in situ and were analyzed by X-ray microanalysis. The Na and Cl concentrations in the ASL of the pig were close to those of these ions in serum. Rat tracheal ASL was hypotonic. However, rat nasal fluid was hypertonic with an extremely high concentration of K. The composition of the ASL could be influenced by pharmacological stimulation. The development of transgenic mouse models for CF may help to develop therapies for the disease. The composition of mouse ASL was investigated using different collection techniques. (1) beads mounted on filter paper, (2) beads randomly spread over the airway epithelium, and (3) beads spread over the epithelium with a syringe. No significant difference could be detected between these techniques, and mouse ASL was hypotonic. Calibration curves had to be made for each element of interest. Nasal fluid from healthy human volunteers was collected with: (1) a pipette, (2) filter paper, (3) cotton wool, or (4) Sephadex beads. Collection on filter paper and equilibration with Sephadex beads gave reliable results. The Na and Cl concentrations in nasal fluid of control subjects were about the same as in serum, but the K concentration was higher. Rhinitis or primary ciliary dyskinesia patients and CF heterozygotes had abnormally high concentrations of Na and Cl in their nasal fluid (probably due to inflammation of the nasal epithelium), and CF homozygotes had even higher concentrations of Na and Cl.

Fibrosis is a sequel of chronic inflammation and is defined as an excessive deposition of collagen that ultimately leads to organ dysfunction. To date there are no effective treatments for fibrosis. The main cell type involved in collagen deposition and organization is the myofibroblast. In the first study we examined how myofibroblasts differentiate in human fibrotic conditions and in experimental animal models. Human tissues were stained with antibodies that recognize integrin receptors and in addition we also stained for α-SMA, a myofibroblast marker. We found a co-localization between these two markers in stromal cells and hypothesized that integrin α1 is important for the acquisition of the myofibroblast phenotype. To tests this hypothesis we used knockout animals for this integrin subunit. These animals showed a reduction of α-SMA positive fibroblasts, indicating that the α1 integrin subunit is required for proper myofibroblast differentiation. In the second study we used a neuroblastoma tumor model to study tumour growth when a drug targeting the synthesis of cellular NAD was administered. In treated animals an expansion of the nonvascular stroma was observed compared to controls. Normalization of the vasculature was observed in treated tumors together with a decrease in hypoxia. Moreover, this was followed by a decrease in stromal PDGF-B and VEGF expression, suggesting a deactivation of the stroma. In the third study the effects of over-expression of the two pro-fibrotic growth factors TGF-β and PDGF-B in skin was evaluated. We observed that both growth factors induced fibrosis. Over time, a decrease in blood vessel density was observed in both treatment groups. Both factors also stimulated an expansion of the connective tissue cell population originating from the microvascular pericyte, but the phenotype of these cells differed in the different treatments with regards to expression of markers. Furthermore, in tissue over-expressing PDGF-B but not TGF-β, the fibrotic process was partially reversible.

Pericytes are cells of mesenchymal origin located on the abluminal side, juxtapositioned to the endothelial cells in capillaries, venules and small arterioles. They are important for maintaining vessel integrity in resting tissues as well as the formation and stabilization of new vessels. They have been suggested to function as mesenchymal stem cells thereby contributing to the connective tissue cell population in reactive tissues. In this thesis the role of pericytes as progenitors for fibroblasts was further defined both in vitro and in vivo. In the first study connective tissue cells of mesenchymal origin were investigated based on their marker expression and relation to the microvasculature. The expression of alpha smooth muscle actin (α-SMA), a marker for myofibroblasts, was compared to the expression of certain integrins in three reactive conditions in human tissues. There was a co-localization of α-SMA and α1β1 integrins, indicating that α1 integrin was important for acquiring the α-SMA myofibroblast phenotype. To further investigate this, two animal models for carcinoma growth and wound healing using α1 deficient mice were employed. Reduction/lack of α-SMA expressing myofibroblasts substantiated or findings in human tissues, strengthening the hypothesis that the α1 integrin is important for the differentiation of α-SMA expressing myofibroblasts. In study two the effects of the HDAC inhibitor valproic acid (VPA) on pericyte function in vitro was investigated. This revealed that VPA had an inhibitory effect on pericyte proliferation, migration and differentiation into collagen type I producing fibroblasts. In addition qPCR array studies on angiogenesis related gene expression identified an up-regulation of genes involved in vessel stabilization in VPA treated pericytes. This suggests that VPA promotes a pericyte phenotype favoring vessel stability. In study three the differentiation from early mesenchymal stem cell like pericyte to fully differentiated fibroblast was further defined by flow cytometry marker analysis. By isolating pericytes from human placenta with a phenotype resembling the in vivo phenotype the differentiation pathway could be defined in five consecutive steps. The five steps were defined by their marker expression and their ability to give rise to the other cell populations in the differentiation lineage, as well as their slow cycling characteristics. A better understanding of how connective tissue cells are derived in fibrotic conditions may be beneficial in trying to modulate the outcome of the healing process towards optimal tissue regeneration with minimal fibrosis.

Angiotensin-(1-7) [Ang-(1-7)] is a heptapeptide component of the renin angiotensin system and the endogenous ligand for the Mas receptor. Ang-(1-7) is generated mainly via angiotensin converting enzyme 2 (ACE2)-dependent cleavage of Angiotensin (Ang) II. Studies suggest Ang-(1-7) may protect against progression of renal injury in experimental models of chronic kidney disease, although the responses may be dose dependent. The role of Ang-(1-7) in the progression of renal fibrosis in unilateral ureteral obstruction (UUO) remains unclear. We tested the hypothesis that endogenous Ang-(1-7) and low dose exogenous Ang-(1-7) would protect against renal injury in the UUO model, while high dose Ang-(1-7) would exacerbate renal injury. Male C57Bl/6 mice underwent UUO and received vehicle, the Ang-(1-7) antagonist A779, or one of three doses of Ang-(1-7) for 10 days. Treatment with A779 exacerbated renal injury as seen by increased fibronectin, transforming growth factor-β (TGF-β), and α-smooth muscle actin (α-SMA) expression, increased tubulointerstitial fibrosis scores, macrophage infiltration, apoptosis, and NADPH oxidase activity in obstructed kidneys. Paradoxically, delivery of exogenous Ang-(1-7) was associated with increased renal injury regardless of dose. Taken together, these data indicate the Mas receptor may be sensitive to concentrations of Ang-(1-7) within the obstructed kidney and that exogenous Ang-(1-7) stimulates pro-fibrotic and pro-inflammatory signalling through unclear pathways.

Rationale: Ventilation inhomogeneity (VI) may be an early sign of obstructive airway disease. The lung clearance index (LCI) has been suggested as a sensitive marker of VI, although it has not been well characterized in young children in health and in those with CF and asthma. Objective: To determine if LCI can detect VI in asymptomatic infants and preschool-age subjects with CF or wheeze/asthma compared to healthy controls. Methods: Sulphur hexafluoride (SF6) multiple breath washout (MBW) testing was completed in all subjects. Results: LCI was found to be dependent on age in a large healthy cohort. Accounting for age, LCI was significantly elevated in disease groups compared to healthy controls in early childhood, illustrating early presence of VI in wheezy infants and the progression of disease in CF. Furthermore, the effects of breathing pattern and the variability of MBW parameters showed positive associations with age and VI.

Rationale: Ventilation inhomogeneity (VI) may be an early sign of obstructive airway disease. The lung clearance index (LCI) has been suggested as a sensitive marker of VI, although it has not been well characterized in young children in health and in those with CF and asthma. Objective: To determine if LCI can detect VI in asymptomatic infants and preschool-age subjects with CF or wheeze/asthma compared to healthy controls. Methods: Sulphur hexafluoride (SF6) multiple breath washout (MBW) testing was completed in all subjects. Results: LCI was found to be dependent on age in a large healthy cohort. Accounting for age, LCI was significantly elevated in disease groups compared to healthy controls in early childhood, illustrating early presence of VI in wheezy infants and the progression of disease in CF. Furthermore, the effects of breathing pattern and the variability of MBW parameters showed positive associations with age and VI.