Capsule endoscopy in the diagnosis of small bowel disease

Capsule endoscopy (CE) is a method for examining the small bowel by means of an ingested encapsulated video camera, propelled by peristalsis, to continuously take images during its passage through the gastrointestinal tract. The method has been in clinical use in Sweden since 2002 and is considered user-friendly and well tolerable by patients. CE is used to diagnose obscure small-bowel bleeding, Crohn´s disease (CD) and suspected small-bowel tumors. It is known for having a high sensitivity but a lower specificity. In study I CE was performed in 18 patients with chronic intestinal dysmotility (CID), in which a high frequency of mucosal breaks (89%) was observed. There were signs of motility disturbances but the small-bowel transit time did not differ significantly between the two types of CID or to a control group. This was the first study to use CE in CID patients. CE was shown to be feasible for the examination of small bowel mucosa in patients with CID. When CE is used to find a bleeding source in the small bowel, the most common finding are vascular malformations; angioectasias. These can also be found in non-bleeding patients but what triggers bleeding in some patients is not fully understood. In study II a group of 25 patients with bleeding from gastrointestinal angioectasias were tested for bleeding disorders with special focus on acquired von Willebrand syndrome (AVWS), a condition previously identified as a possible explanation for bleeding. Compared to a control group, no significant differences between groups were found in coagulation parameters, bleeding time or von Willebrand multimer levels. These results did not support the need for routine bleeding tests in cases of bleeding from angioectasias and do not demonstrate an overall increased risk of AVWS among these patients. Inflammatory lesions in the small bowel showed by CE may be due to CD but also to other conditions. Since biopsies from the small bowel might be difficult to obtain the relevance of the lesions may remain unclear. In study III 30 patients with small bowel lesions were tested for inflammatory markers in blood (CRP) and faeces (calprotectin). Harvey-Bradshaw Index (HBI) was used to grade patient symptoms. The patients were followed up after nine months with a second capsule endoscopy, CRP, calprotectin and HBI. A significant correlation was found between endoscopic inflammation and calprotectin that persisted over time. A correlation between endoscopic inflammation and CRP was found at inclusion but did not persist at follow up. Symptoms did not correlate with endoscopic findings of inflammation at any time. Study IV aimed to evaluate complications of capsule endoscopy, specifically incomplete examinations and capsule retention and to determine the risk factors for these. In this consecutive study 2300 CE examinations - performed at four different hospitals in Stockholm, Sweden from 2003 to 2009 - were included. The frequency of incomplete examinations was 20%. Older age, male gender and suspected or known CD were risk factors for an incomplete examination. Capsule retention occurred in 1.3%. Risk factors for capsule retention were known CD and a suspected tumor. CE was concluded to be an overall safe procedure, although obstructive symptoms and serious complications due to capsule retention can be found in large patient series

Cell derived microparticles: method development, and clinical and experimental studies

Background Cell derived microparticles (MP) are released from the cell membrane upon activation or apoptosis. They resemble their parent cell by exposing similar proteins or surface receptors. This enables identification of their cellular origin. MP are considered to facilitate cross-talk between cells, and to be involved in coagulation, inflammation and vascular function. Elevated circulating MP have been shown in previous studies. Assessment of MP is, however, difficult due to methodological issues. Aims To evaluate pre-analytical procedures and a flow cytometric method for detection of microparticles. To study the effects of statin treatment and inflammation on phenotype and functional properties of microparticles. Methods and Results In Paper I we describe a flow cytometric method for measurements of platelet derived microparticles (PMP) exposing CD62P or CD142. Mean fluorescence intensity measurements were more reproducible than concentration measurements. The presence of platelet fragments could be detected with the peptide phalloidin. This approach can be used as a quality control of samples. Samples frozen and stored as platelet-free plasma generated lowest number of platelet fragments upon flow cytometric analysis. Using our flow cytometric protocol we found two times higher exposure of CD62P and CD142 on PMP in plasma from type 1-diabetes patients compared to healthy controls. In Paper II and III we investigated the effects of atorvastatin on MP. Nineteen patients with atherothrombotic disease were randomized to treatment with atorvastatin or placebo in a cross-over fashion. Thrombin generation and exposure of CD61, CD62P, CD142 and phosphatidylserine (PS) were assessed on PMP (Paper II). Endothelial derived MP (EMP) were assessed by CD144 or CD144+ CD142+ exposure (Paper III). During atorvastatin treatment both thrombin generation and exposure of CD61, CD62P, and CD142 on PMP decreased. No effect was seen on PS exposure. Furthermore, we demonstrated that MP enhanced thrombin generation through PS and CD142 exposure. Unexpectedly, circulating EMP measured as CD144 or CD144+ CD142+increased significantly during atorvastatin treatment. In Paper IV we investigated and characterized in vivo release of MP from 15 healthy volunteers administered lipopolysaccharide (LPS) in the presence of hydrocortisone with or without inhaled nitric oxide. MP from platelets (CD42a or CD41), endothelial cells (CD144 or CD62E) and monocytes (CD14) were studied. Nuclear content in MP was assessed (SYTO 13 binding) as well as HMGB1 exposure. Irrespective of treatment, LPS led to an increase in numbers of all MP, as well as the number of PMP and monocyte MP positive for anti-HMGB1 and SYTO 13. Conclusions We describe a flow cytometric method to measure MP in plasma, and we demonstrate that MP from platelets and endothelial cells respond differently to statin treatment, reflecting the complexity of MP formation. Furthermore, we show that experimental inflammation leads to elevated circulating MP, and that MP may be a source of extracellular HMGB1. MP may be used as biomarkers, an idea that deserves to be investigated more extensively in future studies

Methodological studies applying new techniques for evaluation of arterial function

Atherosclerotic cardiovascular disease is the most common cause of death in the Western world today. Atherosclerosis in the arterial wall is related to aging and the inflammatory and calcification processes, and is influenced by risk factors such as smoking, male gender, diabetes, hypertension, obesity, stress, inactivity, hyperlipidemia, and genetic factors. Adequate diagnostic tools are needed for early detection and evaluation of treatment effects. The general aim of this thesis was to evaluate new and more established noninvasive techniques for measurement of arterial structure and function. Methods: Study I measured carotid intima–media thickness (cIMT) in ultrasound recordings from 99 participants without known cardiovascular disease and compared the results between two semiautomated techniques: AMS and GE. In Study II aortic pulse wave velocity (PWVao) and aortic augmentation index (AIxao) were measured with Arteriograph and SphygmoCor in 63 (20–69 years old) healthy participants and compared the results between the newer technique and the more established technique. Study III evaluated the effects of vitamin D, parathyroid hormone, and calcium levels on vascular structure and function measured by different noninvasive techniques in 48 patients with mild primary hyperparathyroidism and 48 controls. In Study IV we investigated the effects of gender, insulin resistance, and low-intensity physical activity on vascular stiffness, structure, and function in 201 participants. Results: The cIMT measured by GE was slightly but significantly higher than by AMS, but these correlated well in subjects without known cardiovascular disease (Study I). The Arteriograph gave higher PWVao values than the SphygmoCor, especially in women, while the correlation for AIxao between the methods was excellent. Arterial stiffness measured with both methods was related to age, cIMT and serum cholesterol level (Study II). PWVao, AIxao, cIMT, and radial artery IMT were related to systolic blood pressure but not to vitamin D level. (Study III). Greater arterial stiffness in women was indicated by AIxao, which was related to systemic vascular resistance and exercise capacity. However, a 4-month program of low-intensity physical activity did not improve vascular variables, and there were no gender differences in the responses to the exercise intervention (Study IV). Conclusion: In follow-up studies and when evaluating treatment effects, it seems favourable not to change the techniques used to measure cIMT and to estimate stiffness. Neither vitamin D level nor low-intensity physical activity training influenced vascular morphology and function as evaluated by the applied noninvasive techniques

Role of GLP-1 receptor agonists in endothelial function

BACKGROUND: The leading cause of death for patients suffering from type 2 diabetes is macrovascular disease. Endothelial dysfunction is one of the earliest events identified in the pathogenesis of atherosclerosis. Hence, there is a need for finding glucose-lowering agents that cause direct positive effects on vasculature in diabetic patients. The aim of this work was to evaluate the putative role and potential effects of incretins i.e. GLP-1 and exendin-4 on the vasculature and to elucidate the mechanisms behind these effects. STUDY I: We investigated whether incretins influence proliferation of human coronary artery endothelial cells (HCAECs) in vitro and studied the molecular mechanisms behind such effects. Exendin-4, GLP-1 (7-36) and GLP-1 (9-36) elicited dose-dependent increases in DNA synthesis and increased cell number. This mitogenic effect was associated with increased eNOS and Akt activity, which along with the augmented cell proliferation were blocked by PKA-, PI3K-, Aktand eNOS-inhibitors and by a GLP-1 receptor antagonist, exendin (9-39). STUDY II: We studied the role of exendin-4 on apoptosis of HCAECs under lipotoxic conditions in vitro. Palmitate provoked apoptosis, an effect that was inhibited by exendin-4 or GLP-1 (7-36). In contrast, palmitate-induced apoptosis was not affected by GLP-1 (9-36). Palmitate alone resulted in increased eNOS, p-38 MAPK and JNK phosphorylation, which were neutralized by exendin- 4. The protective effect of exendin-4 on apoptosis was prevented after treatment of the cells with specific inhibitors for PKA, PI3K, eNOS, p38 MAPK or for JNK. The effect of exendin-4 on lipoapoptosis was blocked by the GLP-1 receptor antagonist, exendin (9-39). STUDY III: In this study, we investigated the long-term in vitro effect of palmitate or high glucose, and the role of exendin-4, on gene expression in HCAECs. Our data show that the expression of eNOS was up-regulated by exendin-4 in the presence of either palmitate or high glucose, as demonstrated by both microarray and Western blotting analyses. However, microarray analysis showed a suppressed eNOS expression by palmitate, which was not observed in Western blot. The expression of tyrosine kinase receptor Tie-2 and its ligand Ang-1 was up-regulated in the presence of exendin-4. Moreover, exendin-4 increased the expression of tissue plasminogen activator (TPA) and cell adhesion molecules involved in angiogenesis, such as platelet endothelial cell adhesion molecule (PECAM), cadherin-5 and extracellular matrix protein fibronectin. Angiotensin І-converting enzyme (ACE) expression was up-regulated by high glucose, whereas exendin-4 inhibited expression of this gene at high glucose. STUDY IV: The aim of this study was to investigate whether exendin-4 could protect against endothelial dysfunction induced by a triglyceride-rich fat emulsion, and if there were any differences in vasorelaxant capacity between GLP-1 (7-36), GLP-1 (9-36) and exendin-4 in rat femoral arterial rings from non-diabetic rats ex vivo. Exendin-4 did not protect against lipotoxicity, whereas GLP-1 (7-36) and GLP-1 (9-36) exerted vasorelaxation. CONCLUSIONS: GLP-1 receptor agonists stimulate the proliferation of HCAECs, protect them from lipoapoptosis and improve endothelial function in part through regulating expression of genes involved in angiogenesis, inflammation and thrombogenesis by reversing glucolipotoxic gene regulation. Improvement of endothelial dysfunction may translate into beneficial effects on many cardiovascular risk factors and may thus have important clinical implications in preventing and treating macroangiopathy in type 2 diabetes

The glucocorticoid receptor as a regulator of cortisol responses in cortisol resistant patients and healthy subjects

Glucocorticoids are essential for life, and are involved in growth, reproduction, intermediary metabolism, immune and inflammatory reactions as well as central nervous system and cardiovascular functions. Glucocorticoids are also used as treatment of many diseases. Resistance to exogenous glucocorticoids is sometimes seen in patients treated with glucocorticoids. Resistance to endogenous glucocorticoid is seen in some patients causing a syndrome called primary generalized glucocorticoid resistance. Glucocorticoids exert their effect through the glucocorticoid receptor, which belongs to the nuclear hormone receptor superfamily. The receptor consists of three functional domains, the N-terminal, the DNA binding domain and the ligand binding domain. The overall aim of this thesis was to study the glucocorticoid receptor in patients with primary generalized resistance to glucocorticoids i.e. resistance to endogenous glucocorticoids. In 12 unrelated patients with primary generalized glucocorticoid resistance we identified two novel mutations in the glucocorticoid receptor gene in two different patients, R477H and G679S respectively, situated in the DNA binding domain and in the ligand binding domain of the receptor. The R477H mutation is the only mutation described in the DNA binding domain of the human glucocorticoid receptor. We characterized these two mutations in vitro in terms of ligand binding, DNA binding, transactivation and transrepression as well as studies of crosstalking with the inflammatory transcription factor NFκB. We could demonstrate that the phenotype of the two patients expressing these two mutations correlated to the in vitro findings. We further demonstrated that the R477H and G679S were true mutations and not present as polymorphisms among healthy individuals. Glucocorticoid sensitivity among healthy individuals was also compared between two groups characterized as low and high secretors of urinary free cortisol studied with respect to their responses to a low dose of exogenous glucocorticoid. We concluded that individuals with a low cortisol profile, though still in the normal range, seems to be more sensitive to exogenous cortisol than those with high profile. This could have impact on the response to treatment with exogenous glucocorticoids and the prediction of therapeutic effect and adverse side effects

Internetized obesity treatment : from recruitment to practice

Background: The prevalence of obesity is reaching alarming altitudes worldwide, and the current healthcare systems face challenges to treat everyone. Thus, new strategies are of high priority. The Internet, for instance, has the potential to reach individuals irrespective of geographical location. Consequently, the overall aim of this thesis was to gain further understanding of the Internet’s applicability in obesity treatment, targeting lifestyle-related health aspects. Methods: This thesis has been built on four studies (I, II, III & IV). Study I used a prospective cohort design, examining members’ program performance in an Internet-based weight loss club during six months of participation (n=23,233, 20% males), focusing on “active members” (n= 4,440, 18% males). Study II characterized a randomized intervention study (n= 3,876, 67% males) in which the participants received either Internet-based counseling (personalized automated health feedback) with or without added telephone counseling, compared with no counseling, on health behavior improvements. Study III was a validation study evaluating the ability of our newly designed Internet-based virtual food plate (with pictures of food items) to assess food intake (lunch meal). We compared the results with participants’ (n=55, 100% males) composed meal of real food items on a real food plate. Lastly, study IV was a descriptive study of the effects of reminders (emails, flyers, oral presentations etc.) on overall participation in study II. Results: The findings from study I suggest that older members (≥ 65 years) performed equally well or better than younger members (< 65 years). They logged-in and recorded their health more frequently, and reported a higher total weight loss (women: 5.6kg, 6.8%; men: 6.4kg, 6.8%). The results from study II indicate an overall health improvement from the intervention per se, rather than for specific interventions. However, those participants who received personalized automated feedback enhanced their motivation to change health habits at follow-up. Study III supports the validity of our Internet-based virtual food plate, with Spearman and concordance correlations ranging between 0.58-0.70 and 0.59-0.81 for energy intake and nutritional components, respectively. A slight overestimation using our virtual food plate was found (+310kJ), but less among overweight participants (+147 kJ). Finally, study IV completes this thesis concluding that a high number of reminders were effective on response rate, predominantly for those with high Internet availability. The participants’ characteristics (age, BMI, motivation etc.), nonetheless, did not influence when they participated. Conclusions: The Internet possesses unique potentials in obesity treatment. This thesis presents valuable effects on health improvements primarily on middle-aged or older, overweight men – a subgroup known to be challenging to include in health promoting activities. This work is only one of the building blocks in the investigation of the Internet’s applicability in medicine. Future research is urged to continue searching the needs and preferences of Internet-based strategies targeting obesity

Venous thromboembolism in women : an assessment of hormonal, genetic and other risk factors

Background Venous thromboembolism (VTE) occurs in 1-2 in 1000 individuals per year. VTE is found in both sexes, but women have a higher incidence at younger ages, particularly during the childbearing years. Although several acquired and genetic risk factors for venous thrombosis have been identified, the modes and consequences of combinations of these risk factors are not fully understood. Aim: The overall aim of this thesis was to clarify risk factors for VTE in women. Methods: The ThromboEmbolismHormoneStudy (TEHS) is a nation-wide populationbased case-control study that included 1470 cases and 1590 controls. All participants were recruited prospectively in Sweden from 2003 to 2009. Reports on acquired risk factors for thrombosis were collected through telephone interviews of the participants and genetic risk factors were identified by DNA analyses on blood samples. Results: In Study I we found that risks associated with recognized acquired and genetic risk factors for VTE generally were of similar magnitude in pre-and postmenopausal women. The acquired, transient risk factors were stronger than the genetic factors and the combination of surgery and plaster cast yielded a 50-fold increased risk for VTE in both pre- and postmenopausal women. In study II current use of combined hormonal contraception (CHC) was associated with a five-fold increased risk of VTE, adjusted odds ratio (ORadj)=5.3, 95% confidence interval (CI)=4.0-6.9. In adjusted analyses combinations with desogestrel had the highest risk (OR=11.4, 95% CI=6.0-22.0) followed by drospirenone, etonogestrel, norgestimate, levonorgestrel and norethisterone (OR=2.0, 95% CI=1.1-3.8). Current use of progestogen-only contraception (POC) was not associated with increased risks of VTE (ORadj=0.9, 95% CI=0.7-1.2). In stratified analyses (by dose) current users of “high dose” POC had an increased risk of VTE (ORadj=2.2, 95% CI=1.3-4.0). In study III for the group of propionic acid derivatives, most women used ibuprofen (92%); of the women who used acetic acid derivatives, almost all used diclofenac (97%). In adjusted analyses overall use of NSAIDs was not associated with increased risks of VTE (ORadj=1.0, 95% CI=0.8–1.2). The adjusted OR was 0.9 for propionic acid derivatives (95% CI=0.72–1.10), 1.2 for acetic acid derivatives (95% CI=0.8–1.7) and 1.8 for coxibs (95% CI=0.7–4.3). For users of acetic acid derivatives and coxibs, the adjusted ORs increased by cumulative dose, suggesting a dose–effect relationship for these drugs. Conclusion: Menopausal status has only a minor impact on the risk levels with regard to recognized risk factors for VTE. The risk of VTE associated with the use of CHC varies depending on the type of progestogen used, even after adjustment for individual factors such as smoking and body mass index (BMI). Except for high-dose preparations, POC seems to be a safer alternative to CHC, with no obvious increased risks for VTE. There is no apparent risk of VTE associated with the use of propionic acid derivatives in young and middle-aged women. Key words venous thromboembolism, combined oral contraception, progestogen-only contraception, NSAID, premenopausal, postmenopausal, risk factor, SN