Thesis (PhD (Pharmaceutics))--PU for CHE, 1999.

There is an increased risk for cardiovascular (CV) comorbidity among patients with rheumatoid arthritis (RA), with premature atherosclerosis, and a higher incidence of CV events, compared with the general population. Disease related factors add to the CV risk, and interact with the traditional CV risk factors. The underlying mechanism for this is not completely understood. In active RA there is a loss of muscle mass and an increase in body fat content. Production of cytokines, i.e., adipokines, in the adipose tissue could link the inflammation with the CV system. Control of the inflammation has been suggested to modify the CV risk in RA, and the recently introduced biological drugs, such as the tumor necrosis factor inhibitors (TNFi), have opened up new treatment opportunities. The aim of this thesis was to evaluate aspects of the interaction between inflammation and CV comorbidity in RA using biochemical and epidemiological methods. Methods In the first two studies, patients with established RA were examined for clinical disease activity, and blood samples were analysed for cytokines and adipokines using ELISAs and multiplex technology. In Study I (n RA=23) anthropometric measurements were assessed and in Study II (n RA=51) measurements of intima-media thickness (IMT), and endothelial function (FMD). From a subgroup of patients (Study II, n RA=13) samples of abdominal subcutaneous adipose tissue (SAT) were analysed for content of adipokines. In study III and IV associations between treatment with TNFi and acute coronary syndromes (ACS) were analysed using data from the Swedish Rheumatology Register; in Study III regarding early RA (n TNFi exposed=1,271, n bionaïve RA=4,729), and in Study IV comprising patients with RA of all stages (n TNFi exposed=7,213, n bionaïve RA=17,769) and with a matched general population comparator cohort (n=32,161). Associations between response to TNFi therapy and risk for ACS in the early RA cohort were evaluated in a nested case-control design (cases n=24, controls n=81). Results Serum levels of the cytokines/adipokines interleukin-1 receptor antagonist (IL-1Ra), IL-6, osteopontin, visfatin and TNF were increased in patients compared with controls (p≤0.001-0.036). The amount of TNF receptor II extracted from SAT was greater in patients (p=0.006). The serum (s-) levels of IL-1Ra correlated with s-leptin (r=0.71, p≤0.001) and s-haptoglobin in RA patients (r=0.56, p≤0.01). The result from a factor analysis indicated IL-1Ra to be associated with both adipose tissue and inflammation. Levels of s-visfatin (p=0.019) and s-IL-1Ra (p=0.023), respectively, were positively associated with IMT independently of inflammatory activity and CV risk factors. PAI-1 and MCP-1 extracted from SAT showed inverse associations with IMT. Patients with RA, whether exposed to TNFi or bio-naïve, had a doubled risk for ACS compared with the general population; HR 2.09 (95%CI 1.58-2.76) and 1.80 (1.49-2.17), respectively. No significant associations between risk for ACS and TNFi exposure were detected after adjustments; HR 0.80 (0.52-1.24) in early RA and HR 1.08 (0.82-1.41) in RA of any duration. Furthermore, no association between the risk for ACS and response to TNFi treatment in patients with early RA was observed, OR 1.5 (0.3-6.9). Conclusions The results indicate that cytokines/adipokines may have a role in the development of atherosclerosis in RA patients. A continuing increase in the risk of ACS in RA compared with the general population, despite modern therapeutic strategies, was noted. Neither exposure nor response to treatment with TNFi was associated with any modification of the risk for ACS.

The overall aim was to improve the knowledge and understanding of eating habits of older men with somatic diseases, and the men's perceptions about managing food-related habits, such as grocery shopping and cooking. A total of 67 men between 64 and 89 years of age were visited in their homes on two occasions with 1-2 weeks in between. The participants were diagnosed with one of the three diseases Parkinson’s disease, rheumatoid arthritis, or stroke. A food survey, with repeated 24-h recall, was used to assess food intake and meal patterns. Interviews with 18 participants were conducted with open-ended questions. The interviews were further analysed with a thematic framework approach.The findings showed that eating events were distributed over a 24-h period.Further, co-living men had a significantly larger number of eating events over the day (p=0.001). No differences in daily energy intake were observed between co-living and single-living men. Co-living men’s hot eating events were compared with those of single-living men more often cooked from fresh ingredients (p=0.001), including a greater mix of vegetables/roots (p=0.003).Thematic analysis revealed three different approaches to food-related activities(FRA), namely ‘Cooking as a pleasure’, describing joy in cooking; ‘Cooking as a need’, indicating no habits or skills in cooking; and ‘Food is served’, that is, being served meals by a partner. The men's approaches to FRA were affected in particular by gender-related roles, but also by changed life circumstances, activity limitations, personal interests, and a wish to maintain continuity and independence. Further adaptive strategies were used among the men in attempts to maintain continuity and independence in FRA. In conclusion, single-living older men, especially those with activity limitations, were identified as being a vulnerable group from a nutritional perspective. Further, health care efforts in promoting FRA should preferably be individualised with respect to the older man’s approach to these activities.

Predisposition to autoimmune diseases such as, rheumatoid arthritis, diabetes, and multiple sclerosis, is caused by the effect of multiple genes and a strong influence from the environment. In this study, I have investigated genetic factors that confer susceptibility to rheumatoid arthritis in a rat model. This work has led to the identification of several chromosomal regions, containing uncharacterized genes that directly or indirectly are associated to the arthritis development in these rats. We have observed that timing, gender, and genetic interactions are features that play a part in the effect that these genetic factors exert. Unarguably, animal models for human disorders display differences to the human form of disease. An important fact is however that the same chromosomal regions are identified in both rodent and human studies, which suggests that there are genetic factors that we have in common, which are involved directly or indirectly with an autoimmune response. Focusing the interest on these similarities, and on the possibility to apply a wide set of genetic tools, make animal models an invaluable, and probably necessary, instrument to dissect the genetic component of complex disorders. To fully comprehend the genetic basis for a complex disorder like this, will require understanding of how multiple genes interact with each other to cause disease. We have been able to demonstrate that chronic arthritis, in a rat model for rheumatoid arthritis, is regulated by several genes and that these act during different temporal phases of the disease. These findings will hopefully contribute to our understanding of the etiology and progression of rheumatoid arthritis.

In the present studies, aspects of some disease activity measures in rheumatoid arthritis (RA) have been investigated, including the effect of glucocorticoids on this activity. In RA, serum interleukin(IL)-6 levels were elevated and were shown to have a circadian rhythm, with peak levels in the morning, declining towards low or normal levels in the afternoon and evening. In contrast, serum levels of tumour necrosis factor(TNF) alpha were low and stable. In other connective tissue diseases, serum TNF alpha levels were elevated but without circadian variation, while IL-6 levels were low and stable. Nocturnal administration (at 2:00 a.m.) of low-dose prednisolone a few hours before the early morning peak of IL-6 was shown to be significantly more effective in reducing clinical symptoms of disease activity and serum IL-6 levels than the traditional morning administration (at 7:30 a.m.) of the same dose of prednisolone. Circulating monocytes are activated in RA, expressing receptors related to adhesion and phagocytosis. Treatment with glucocorticoids suppressed the expression of these receptors on monocytes, and this may be one mechanism of the beneficial effect of glucocorticoids in RA. Endogenous levels of cortisol seem to play a minor role in expression of monocyte receptors. The different acute phase markers used to assess disease activity in RA showed good corrrelations with each other and with serum IL-6 levels. There were especially strong corrrelations between C-reactive protein (CRP) and Serum amyloid protein A (SAA), and between fibrinogen and erythrocyte sedimentation rate (ESR). Fibrinogen and CRP showed stronger correlation than ESR with the Modified Health Assessment Questionnaire (MHAQ) score and with the neutrophil count. Four simple objective function tests were each compared with the HAQ score a with a radiological joint damage score (Larsen score). The objective function tests correlated with the MHAQ score, and each of these two methods of assessing physical disability correlated with pain, CRP and ESR. In addition, most of the objective function tests correlated significantly with radiological joint damage, while the MHAQ score did not.

Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. We developed a novel multiplexed method for SNP genotyping based on four-color fluorophore tag-microarray minisequencing. This method allows simultaneous genotyping of 80 samples and up to 200 SNPs in any allele combination. In study I we set up the method for a panel of SNPs from genes in the type I interferon system, and applied it in study III. In study II we used the technique to genotype SNPs from the coding region of the mitochondrial genome. A panel of 150 SNPs was genotyped in 265 individuals representing nine different populations. We demonstrated that the multiplexed SNP genotyping method for mitochondrial DNA increases the power of forensic identification in combination with sequencing of the hypervariable region of mitochondrial DNA. In study III we performed a genetic association study of SNPs in genes related to the type I Interferon system in Systemic Lupus Erythematosus (SLE). SLE is a chronic autoimmune inflammatory disease with a complex etiology. The SNPs were genotyped in DNA samples from Swedish, Finnish, and Icelandic patients with SLE, unaffected family members, and unrelated controls. The analysis identified SNPs in two genes, the tyrosine kinase 2 (TYK2) and interferon regulatory factor 5 (IRF5) genes that are highly associated with SLE with p-values <10-7 for joint linkage and association. Study IV describes the analysis of the TYK2 and IRF5 SNPs in a large Rheumatoid Arthritis (RA) sample cohort. We found that SNPs in the IRF5 gene were significantly associated with RA with a p-value = 0.00008. In contrast, we did not detect an association with SNPs in the TYK2 gene. These findings demonstrate that SLE and RA may have a common genetic background in the case of IRF5, while the TYK2 variants appear to be unique for SLE.

Neuropeptides, such as substance P (SP) and bombesin/gastrin-releasing peptide (BN/GRP), and neurotrophins are involved in neuro-immunomodulatory processes and have marked trophic, growth-promoting and inflammation-modulating properties. The impact of these modulators in rheumatoid arthritis (RA) is, however, unclear. An involvement of the innervation, including the peptidergic innervation, is frequently proposed as an important factor for arthritic disease. Many patients with RA, but not all, benefit from treatment with anti-TNF medications. The studies presented here aimed to investigate the roles of neuropeptides, with an emphasis on BN/GRP and SP, and neurotrophins, especially with attention to brain-derived neurotrophic factor (BDNF), in human and murine knee joint tissue. The expression patterns of these substances and their receptors in synovial tissue from patients with either RA or osteoarthritis (OA) were studied in parallel with the levels of these factors in blood and synovial fluid from patients with RA and from healthy controls. Correlation studies were also performed comparing the levels of neuropeptides with those of pro-inflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6)]. Furthermore, the impact of anti-TNF treatment on the levels of BDNF in blood was investigated. In a murine model of RA, the expression of these substances on articular chondrocytes along with their expression in synovial tissue was investigated. The expression of BN/GRP in human synovial tissue was confined to fibroblast-like and mononuclear-like cells whereas SP was detected in nerve-related structures. Receptors for these neuropeptides (GRP-R and NK-1R) were frequently present in blood vessel walls, and on fibroblast-like and mononuclear-like cells. The expression of BDNF and its receptors, p75 neurotrophin receptor and TrkB, was mainly confined to nerve structures. The levels of SP, and particularly those of BN/GRP, in synovial fluid and peripheral blood correlated with the levels of pro-inflammatory cytokines. There were clearly more correlations between SP-BN/GRP and inflammatory parameters than between BDNF and these factors. Plasma levels of BDNF were decreased following anti-TNF-treatment. In the joints of the murine model, there was a marked expression of neurotrophins, neurotrophin receptors and NK-1R/GRP-R in the articular chondrocytes. The expression was down-regulated in the arthritic animals. A neurotrophin system was found to develop in the inflammatory infiltrates of the synovium in the arthritic mice. The results presented suggest that there is a local, and not nerve-related, supply of BN/GRP in the human synovial tissue. Furthermore, BN/GRP and SP have marked effects in the synovial tissue of patients with RA, i.e., there were abundant receptor expressions, and these neuropeptides are, together with cytokines, likely to be involved in the neuro-immunomodulation that occurs in arthritis. The observations do on the whole suggest that the neuropeptides, rather than BDNF, are related to inflammatory processes in the human knee joint. A new effect of anti-TNF treatment; i.e., lowering plasma levels of BDNF, was observed. Severe arthritis, as in the murine model, lead to a decrease in the levels of neurotrophin, and neurotrophin and neuropeptide receptor expressions in the articular cartilage. This fact might be a drawback for the function of the chondrocytes. Certain differences between the expression patterns in the synovial tissue of the murine model and those of human arthritic synovial tissue were noted. It is obvious that local productions in the synovial tissue, nerve-related supply in this tissue and productions in chondrocytes to different extents occur for the investigated substances.

Case control studies have shown that a high consumption of fish, olive oil, and cooked vegetables is associated with a decreased risk of developing rheumatoid arthritis (RA). These foods have a central position in the traditional Cretan Mediterranean diet, and it has been suggested that dietary factors contribute to the low prevalence of RA in Mediterranean countries. The overall aim of this thesis was to examine whether a modified Cretan Mediterranean diet can reduce signs and symptoms of RA. This was investigated in a three-month dietary intervention trial in which 51 patients with well controlled, although active RA of at least two years duration took part. A further aim was to study the compliance with the experimental and control diets used in the study, and to validate the diet history interview method used to assess the dietary intake. The validation was carried out by means of biological markers of dietary intake. From baseline to the end of the study the group that had adopted the Cretan Mediterranean diet (MD group; n=26) obtained a reduction in disease activity, improved physical function, and improved vitality, while no changes was seen in the control diet group (CD group; n=25). According to the dietary assessments, the intake frequencies of antioxidant-rich food items increased in the MD group. This group also had a significantly higher intake of vitamin E, vitamin C and selenium compared to the CD group. Despite the reported increase in the consumption frequencies of antioxidant-rich foods, the plasma levels of carotenoids, vitamin C, lipid adjusted tocopherols, uric acid and urine malondialdehyde, a marker of oxidative stress, were unchanged at the end of the study. The plasma levels of retinol, vitamin C and uric acid were, however, correlated to indices of disease activity. Changes in the reported consumption of food groups with relevance to the fat intake were also observed in the MD group, including an increased intake of fish, shellfish and poultry, and a decreased intake of meat and high fat dairy products. As a result, the total fat intake was lower in the MD group compared to the CD group. Furthermore, in the MD group a slightly higher percentage of the energy intake was derived from polyunsaturated fatty acids and a lower percentage from saturated fatty acids. This group also had a lower ratio of n-6:n-3 fatty acids. A corresponding change in the relation between n-6 and n-3 fatty acids was also observed in s-phospholipids. The validation of the diet history interview method showed that the diet history interview could capture the dietary intake fairly well. The validity of the reported dietary intake did not differ between the MD and the CD group, which indicates that the dietary assessment was not biased by the dietary intervention.

Plasmin has been suggested to be involved in degradation of extracellular matrix (ECM) and tissue remodeling during a number of physiological and pathological processes. The aims of this thesis were to study the functional roles of plasmin during pathological inflammation in autoimmune and nonautoimmune disease models of rheumatoid arthritis (RA), multiple sclerosis (MS), wound healing and infection. In order to explain the obtained results in our functional studies as well as some previous results on the functional roles of plasmin during different tissue remodeling processes, I propose that there is a functional correlation between absence of plasmin and an inability to activate complement. The role of plasminogen during autoimmune collagen type II-induced arthritis (CIA) was studied first. The data revealed that whereas 83% of wild-type (plg+/+) mice developed CIA, none of the plasminogendeficient (plg-/-) mice got arthritis within a 40-day period. When plg+/+ mice were injected with a mixture of monoclonal antibodies against collagen type II they developed arthritis within a 5-day period, whereas no arthritis could be seen in plg-/- mice, although these mice had normal binding of antibody to the cartilage surface. These data suggest that plasmin plays an essential role in the step between antibody binding and inflammatory cell infiltration during CIA, probably during the step of complement activation. When plg+/+ and plg-/- mice were injected intra-articularly with collagen type II or 0.9% NaCl following CIA induction, plg-/- mice developed typical CIA, but the disease was less severe than in the plg+/+ mice and restricted to the injected joints. Sustained tissue necrosis was found only in the plg-/- mice after the local injection. When the antigen-induced arthritis (AIA) model was used, plg-/- mice developed a much more severe arthritis than the plg+/+ mice. These results indicate that different forms of pathogenesis exist for CIA and AIA, and further emphasize the importance of trauma in the induction of CIA in plg-/- mice. We further investigated the role of plasmin in experimental autoimmune encephalomyelitis (EAE), which is an autoimmune disease model for MS. During a 2-month period, the severity, incidence, mean onset day, mean maximal score and mean accumulative score of EAE were essentially identical in plg-/- and plg+/+ mice of B10.Q background. Histopathological studies revealed similar levels of inflammation and demyelination in plg-/- and plg+/+ mice. These data indicate that plasmin does not play an essential role in the development of EAE. The findings that plasmin is essential for the development of CIA but not needed for the development of EAE suggest that plasmin may play a pivotal role in autoimmune diseases where complement activation is critically involved in the pathogenesis. The role of plasmin was also studied in a tympanic membrane (TM) wound healing model. After TM perforations were performed, the plg+/+ TMs had all healed by day 11, whereas TM healing was completely arrested in plg-/- mice even as late as day 143. Immunohistochemical studies revealed a disturbed inflammation and tissue remodeling pattern in plg-/- mice. These data indicate that plasmin plays a central role in the healing of TM perforations. The involvement of plasminogen in ear infections was also investigated in plg-/- mice. During an 18-week experimental period, spontaneous otitis media (OM) was essentially developed in all of the plg-/- mice, whereas all of the plg+/+ mice kept a normal TM status. Positive bacterial growth was found in 5 out of 6 plg-/- mice, but only in 1 out of 6 plg+/+ mice. Immunohistochemical studies showed an accumulation of inflammatory cells, fibrin and also other extracellular matrix in the middle-ear cavity and the external-ear canal of plg-/- mice. These results show a spontaneous development of OM in plg-/- mice, but not in plg+/+ controls, suggesting that plasmin plays a critical role in the defense mechanisms during ear infections. Taken together, plasmin appears to play essential roles during autoimmune and non-autoimmune diseases in which complement activation is critical in the pathogenesis.

This thesis comprised patients with chronic rheumatic diseases. The studies aimed to elucidate food sensitivity by measuring mucosal inflammatory reactivity and thereby a possible link between the gut and joints. In all the studies, the mucosal path technique was used to evaluate the rectal mucosal response to rectal challenge with gluten and/or cow’s milk protein (CM). In some patients with primary Sjögren’s syndrome (pSS) and the genetic susceptibility genes HLA DQ2, mucosal reactivity measured with nitric oxide (NO) was found after rectal gluten challenge without detectable serum antibodies to gluten or transglutaminase. This gluten sensitivity was not linked to coeliac disease. After rectal CM challenge, a rectal mucosal inflammatory response measured with NO and myeloperoxidase (MPO) was detected in 38% of pSS patients, all of whom fulfilled the criteria for irritable bowel syndrome. In a questionnaire study of self-experienced adverse reactions to food, 27% of patients with rheumatoid arthritis (RA) reported intolerance to various foods and CM in particular. After rectal CM challenge performed in RA patients (n=27), strong mucosal reactivity to CM was observed in a few patients and a moderate increase in 23%. After gluten challenge, a moderate increase in mucosal reactivity was found in 35% of patients. No correlation to self-perceived intolerance and mucosal reactivity measured with NO and MPO was seen. Inflammation of the gut is a prominent feature of spondyloarthropathies (SpA). After rectal challenges with CM protein and gluten, an increase in rectal NO production was seen in 26% and 19% respectively (p<0.001). An increase in the mucosal release of MPO as a sign of neutrophil activation was seen in the CM- and gluten-sensitive patients. NO production in SpA patients was more enhanced compared with RA and pSS patients and could contribute to the increased barrier permeability described in SpA patients.