Identification and analyses of inhibitors targeting apolipoprotein(a) kringle domains KIV-7, KIV-10, and KV provide insight into kringle domain function.

Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for cardiovascular disease. Lp(a) is composed of apolipoprotein(a) (apo(a)) covalently bound to apolipoprotein B of low-density lipoprotein (LDL). Many of apo(a)'s potential pathological properties, such as inhibition of plasmin generation, have been attributed to its main structural domains, the kringles, and have been proposed to be mediated by their lysine-binding sites. However, available small-molecule inhibitors, such as lysine analogs, bind unselectively to kringle domains and are therefore unsuitable for functional characterization of specific kringle domains. Here, we discovered small molecules that specifically bind to the apo(a) kringle domains KIV-7, KIV-10, and KV. Chemical synthesis yielded compound AZ-05, which bound to KIV-10 with a Kd of 0.8 µm and exhibited more than 100-fold selectivity for KIV-10, compared with the other kringle domains tested, including plasminogen kringle 1. To better understand and further improve ligand selectivity, we determined the crystal structures of KIV-7, KIV-10, and KV in complex with small-molecule ligands at 1.6-2.1 Å resolutions. Furthermore, we used these small molecules as chemical probes to characterize the roles of the different apo(a) kringle domains in in vitro assays. These assays revealed the assembly of Lp(a) from apo(a) and LDL, as well as potential pathophysiological mechanisms of Lp(a), including (i) binding to fibrin, (ii) stimulation of smooth-muscle cell proliferation, and (iii) stimulation of LDL uptake into differentiated monocytes. Our results indicate that a small-molecule inhibitor targeting the lysine-binding site of KIV-10 can combat the pathophysiological effects of Lp(a).

Biomarkers of systemic inflammation in farmers with musculoskeletal disorders; a plasma proteomic study.

BACKGROUND: Farmers have an increased risk for musculoskeletal disorders (MSD) such as osteoarthritis of the hip, low back pain, and neck and upper limb complaints. The underlying mechanisms are not fully understood. Work-related exposures and inflammatory responses might be involved. Our objective was to identify plasma proteins that differentiated farmers with MSD from rural referents. METHODS: Plasma samples from 13 farmers with MSD and rural referents were included in the investigation. Gel based proteomics was used for protein analysis and proteins that differed significantly between the groups were identified by mass spectrometry. RESULTS: In total, 15 proteins differed significantly between the groups. The levels of leucine-rich alpha-2-glycoprotein, haptoglobin, complement factor B, serotransferrin, one isoform of kininogen, one isoform of alpha-1-antitrypsin, and two isoforms of hemopexin were higher in farmers with MSD than in referents. On the other hand, the levels of alpha-2-HS-glycoprotein, alpha-1B-glycoprotein, vitamin D- binding protein, apolipoprotein A1, antithrombin, one isoform of kininogen, and one isoform of alpha-1-antitrypsin were lower in farmers than in referents. Many of the identified proteins are known to be involved in inflammation. CONCLUSIONS: Farmers with MSD had altered plasma levels of protein biomarkers compared to the referents, indicating that farmers with MSD may be subject to a more systemic inflammation. It is possible that the identified differences of proteins may give clues to the biochemical changes occurring during the development and progression of MSD in farmers, and that one or several of these protein biomarkers might eventually be used to identify and prevent work-related MSD.

Levels of perfluoroalkyl substances and risk of coronary heart disease: Findings from a population-based longitudinal study.

BACKGROUND: Cross-sectional studies have shown an association between exposure to perfluoroalkyl substances (PFASs) and coronary heart disease (CHD). These findings need to be evaluated in longitudinal settings. OBJECTIVES: To investigate the risk of CHD in relation to PFAS levels in a longitudinal setting among Swedish rural residents. METHODS: In a population-based prospective cohort of male farmers and rural residents recruited in 1990-1991, all men who received a CHD diagnosis between 1992 and 2009 were identified from national registers (n=253). For each CHD case, one control, matched for age, was chosen randomly from the cohort. For all cases and controls, levels of eight PFASs at baseline were measured in stored blood samples. In addition, for a subsample, PFAS levels were also measured in serum samples collected at a follow-up in 2002-2003. RESULTS: There were no statistically significant associations between levels of seven of the eight PFASs at baseline and risk for developing CHD. There was a significant association between perfluoroheptanoic acid (PFHpA) and CHD (OR=2.72; 95% CI: 1.52, 4.84) for the 3rd quartile and (OR=2.45; 95% CI: 1.40, 4.29) for the 4th quartile compared to the lowest quartile. Changes in levels of PFCs between baseline and follow-up did not differ systematically between cases and controls. CONCLUSIONS: This longitudinal study does not lend support to the previously reported cross-sectional relationship between PFAS levels and CHD risk. We found a significant association with PFHpA, but this could be a chance finding, considering its chemical resemblance to other PFASs.

Discovery of the Fibrinolysis Inhibitor AZD6564, Acting via Interference of a Protein-Protein Interaction.

A class of novel oral fibrinolysis inhibitors has been discovered, which are lysine mimetics containing an isoxazolone as a carboxylic acid isostere. As evidenced by X-ray crystallography the inhibitors bind to the lysine binding site in plasmin thus preventing plasmin from binding to fibrin, hence blocking the protein-protein interaction. Optimization of the series, focusing on potency in human buffer and plasma clotlysis assays, permeability, and GABAa selectivity, led to the discovery of AZD6564 (19) displaying an in vitro human plasma clot lysis IC50 of 0.44 µM, no detectable activity against GABAa, and with DMPK properties leading to a predicted dose of 340 mg twice a day oral dosing in humans.

Type 2 diabetes among farmers and rural and urban referents: cumulative incidence over 20 years and risk factors in a prospective cohort study.

BACKGROUND: Few studies have examined the risk of type 2 diabetes in various occupational groups. Farmers in Sweden have a low risk of coronary heart disease, but less is known about diabetes. OBJECTIVE: To analyze the cumulative incidence and relative risk of type 2 diabetes among farmers and referents taking lifestyle factors and components of the metabolic syndrome into account. METHODS: In a longitudinal observational cohort study we followed 1,220 farmers, 1,130 rural non-farmer referents and 1,219 urban referents over 20 years. Outcomes were generated from national registers and from two surveys 12 years apart. Baseline data were assessed at the first survey conducted in 1990-91. RESULTS: Farmers had a significantly lower risk of all diabetes compared with urban and rural referents (p<0.05). A total of 91 farmers (8.4%) and 102 non-farming rural referents (11.5%) were identified with type 2 diabetes over the 20 year study period (OR=0.70; 95% CI 0.52-0.95). Fractional analyses of lifestyle factors and components of the metabolic syndrome showed that the low risk of type 2 diabetes among farmers was explained in terms of physical activity and meal quality. Farmers had significantly higher physical capacity (p<0.001) and scored higher in a meal quality index than rural referents (p<0.001). CONCLUSIONS: The prevalence of type 2 diabetes was significantly lower among farmers. The low relative risk was explained by high physical activity and better meal quality, indicating that farmers' lifestyles and their work environment are health-promoting.

Potent fibrinolysis inhibitor discovered by shape and electrostatic complementarity to the drug tranexamic acid.

Protein-protein interfaces provide an important class of drug targets currently receiving increased attention. The typical design strategy to inhibit protein-protein interactions usually involves large molecules such as peptides and macrocycles. One exception is tranexamic acid (TXA), which, as a lysine mimetic, inhibits binding of plasminogen to fibrin. However, the daily dose of TXA is high due to its modest potency and pharmacokinetic properties. In this study, we report a computational approach, where the focus was on finding electrostatic potential similarities to TXA. Coupling this computational technique with a high-quality low-throughput screen identified 5-(4-piperidyl)-3-isoxazolol (4-PIOL) as a potent plasminogen binding inhibitor with the potential for the treatment of various bleeding disorders. Remarkably, 4-PIOL was found to be more than four times as potent as the drug TXA.

High dairy fat intake related to less central obesity: a male cohort study with 12 years' follow-up.

OBJECTIVE: To study associations between dairy fat intake and development of central obesity. DESIGN: A prospective population-based cohort study with two surveys 12 years apart. SETTING: Nine municipalities selected from different parts of Sweden representing the rural areas in the country. SUBJECTS: 1782 men (farmers and non-farmers) aged 40-60 years at baseline participated in a baseline survey (participation rate 76%) and 1589 men participated at the follow-up. 116 men with central obesity at baseline were excluded from the analyses. MAIN OUTCOME MEASURES: Central obesity at follow-up defined as waist hip ratio ≥ 1. RESULTS: 197 men (15%) developed central obesity during follow-up. A low intake of dairy fat at baseline (no butter and low fat milk and seldom/never whipping cream) was associated with a higher risk of developing central obesity (OR 1.53, 95% CI 1.05-2.24) and a high intake of dairy fat (butter as spread and high fat milk and whipping cream) was associated with a lower risk of central obesity (OR 0.52, 95% CI 0.33-0.83) as compared with medium intake (all other combinations of spread, milk, and cream) after adjustment for intake of fruit and vegetables, smoking, alcohol consumption, physical activity, age, education, and profession. The associations between dairy fat intake and central obesity were consistent across body mass index categories at baseline. CONCLUSION: A high intake of dairy fat was associated with a lower risk of central obesity and a low dairy fat intake was associated with a higher risk of central obesity.

Predictors of sick leave owing to neck or low back pain: a 12-year longitudinal cohort study in a rural male population.

Back pain is a common cause of sick leave. We analyzed how individual, work-related and lifestyle factors predicted sick leave owing to neck or low back pain over a 12-year period. In this prospective cohort study, 1,405 rural middle-aged farmers and non-farmers were surveyed in 1990-1991 (participation rate 76%) and followed up 12 years later (participation rate 68%). The 836 men who reported having experienced unspecific neck or low back pain the year prior to survey 1 were followed up for self-reported sick leave owing to neck or low back problems. Individual, occupational and lifestyle factors and data on acquired specific neck or back diagnosis were included in multiple logistic regression models. Seven percent reported neck or low back related sick leave during the 12 year period. Self-employment was associated with a lower risk of sick leave while sedentary leisure time, snuff use and a specific neck or back diagnosis was associated with a higher risk. Age, education, physical workload, marital status, sense of coherence, smoking, and alcohol consumption were not independently associated with sick leave. The low risk of sick leave among the self-employed is notable from a societal and public health perspective.

Farmers and retirement: a longitudinal cohort study.

BACKGROUND: Studies report low prevalence of several health problems among farmers. Health status and psychosocial factors are generally assumed to impact on retirement age and the ability to stay in work. OBJECTIVES: To study retirement in a male rural population and to test the hypothesis that farmers retire later than nonfarmers thanks to better health status and advantageous psychosocial factors. METHOD: The study design was a longitudinal cohort study on farmers and rural nonfarmers with data assessment at two surveys 12 years apart. Analyses were performed with multiple logistic regression models. RESULTS: Among subjects 64 years or younger at survey 2, 93% of the farmers were still working (full or part time) as compared with 76% of the employed nonfarmers. Among those 65 years or older, 64% of the farmers, 33% of nonfarming entrepreneurs, and 6% of employed nonfarmers were still occupationally active. Significant differences in reported diseases and psychosocial factors were found between farmers and nonfarmers, but in the fully adjusted model these variables did not modify the low odds ratios of retirement found for farmers. The farmers adjusted risk (OR) for premature retirement (64 or younger) was 0.30 (95% CI 0.16-0.55) and for standard retirement (65 or older) 0.01 (95% CI 0.00-0.05). CONCLUSIONS: Farmers continue to work full or part time around retirement age to a much larger extent than employees. The employment status of farmers may only partly explain this. Health status and psychosocial factors did not impact on the results.

Mortality and morbidity among farmers, nonfarming rural men, and urban referents: a prospective population-based study.

A cohort of 1,220 farmers, 1,130 nonfarming rural men, and 1,087 urban referents from Sweden were monitored for 12 years. Farmers had lower mortality than urban referents for all causes of death (hazard ratio [HR] = 0.51; 95% confidence interval [CI], 0.37-0.71), cancer (HR = 0.44; 95% CI, 0.24-0.78) and cardiovascular diseases (HR = 0.60; 95% CI, 0.36-0.99). Nonfarming rural men had lower mortality than urban referents for all causes of deaths (HR = 0.81; 95% CI, 0.70-0.94). Farmers and nonfarming rural men had significantly lower morbidity risks of cancer and of psychiatric disorders than urban referents. Farmers had significantly lower risk of endocrine disorders, cardiovascular disorders, and respiratory disorders. In general, morbidity was lower among nonfarming rural men compared with urban referents and was even lower among farmers. Urban referents had, however, significantly less musculoskeletal disorder morbidity. An urban-rural factor and a farming occupational or lifestyle factor results in lower mortality and morbidity rates except concerning musculoskeletal disorders.

Food choices and coronary heart disease: a population based cohort study of rural Swedish men with 12 years of follow-up.

Coronary heart disease is associated with diet. Nutritional recommendations are frequently provided, but few long term studies on the effect of food choices on heart disease are available. We followed coronary heart disease morbidity and mortality in a cohort of rural men (N = 1,752) participating in a prospective observational study. Dietary choices were assessed at baseline with a 15-item food questionnaire. 138 men were hospitalized or deceased owing to coronary heart disease during the 12 year follow-up. Daily intake of fruit and vegetables was associated with a lower risk of coronary heart disease when combined with a high dairy fat consumption (odds ratio 0.39, 95% CI 0.21-0.73), but not when combined with a low dairy fat consumption (odds ratio 1.70, 95% CI 0.97-2.98). Choosing wholemeal bread or eating fish at least twice a week showed no association with the outcome.

Functioning in neck and low back pain from a 12-year perspective: a prospective population-based study.

OBJECTIVE: The aim of this study was to evaluate the effects of unspecific neck pain and low back pain at a given time (1990-91) with respect to physical and social functioning and role limitations due to emotional problems 12 years later. METHODS: A rural male study population (2351 individuals) was established in 1989 and a first survey conducted in 1990-91. A follow-up survey was performed in 2002-03. A total of 1405 persons participated in both surveys. Functioning and role limitations in 2002-03 were evaluated using the SF-36 instrument. Several possible confounders were included in the analyses. RESULTS: Unspecific neck pain or low back pain in 1990-91 was shown in a multivariate longitudinal regression model to be significantly related to limited physical (odds ratio (OR)=2.08; 95% confidence interval (CI) 1.51-2.87) and social (OR=1.92; 95% CI 1.33-2.75) functioning 12 years later. The effects were only slightly modified by the confounders analysed. However, higher education independently and significantly predicted a low risk for functional limitations. CONCLUSION: Non-specific neck pain and low back pain at a given time impacted on the risk of limited physical and social functioning many years later. Current symptoms of depression and anxiety at the time for the second survey had a high impact on functional limitations.

Nasal complaints and signs of disease in farmers--a methodological study.

CONCLUSION: The methods used in this study are suitable for field studies that involve examinations of groups of workers. For individual examinations, there is no gold standard method that can discriminate work-related discomfort from other causes of rhinitis. OBJECTIVES: Studies of the effects of occupation on farmers' health have mainly focused on lower airways; few studies have examined effects on upper airways. This study investigated nasal functions in three groups of farmers (swine, milk and grain producers) and a control group using different methods, suitable for field studies. SUBJECTS AND METHODS: Health-related complaints were examined and several functional tests, such as expirogram, olfactory threshold test, acoustic rhinometry, nasal lavage with biomarkers of inflammation (eosinophilic cationic proteins (ECP), myeloperoxidase (MPO), tryptase, albumin) and allergy tests were performed. The different tests were correlated to nasal complaints and to each other. RESULTS: Nasal blockage complaints were more common among farmers; overall, nasal polyps were more frequent in grain producers. Objective parameters showed more pronounced mucosal swelling in farmers and higher concentrations of ECP in nasal lavage compared with controls. Lung function, olfactory threshold, atopy frequency and allergen-specific IgE to the storage mite Lepidoglyphus destructor did not differ between farmers and controls. Mucosal swelling measured with acoustic rhinometry was more pronounced in subjects with nasal complaints, hypersensitivity, nasal polyps and symptoms from lower airways. There was a correlation between biomarkers in nasal lavage (MPO, albumin and ECP).

Hip osteoarthritis in a rural male population: A prospective population-based register study.

OBJECTIVES: A cohort of rural men with urban referents was followed over 13 years to study the risk of developing osteoarthritis of the hip joint. METHODS: A group of 1,220 farmers, 1,130 matched rural non-farming men, and 1,087 urban men were identified in 1989 and followed over time. Information on hospital care and surgery was obtained from the national Swedish register of hospital care and surgery. RESULTS: More farmers than referents had been hospitalized for osteoarthritis and had undergone surgery for osteoarthritis of the hip joint. Hazard ratio for osteoarthritis of the hip joint was 3.0 (95% CI: 1.7-5.3) for farmers versus urban controls. Non-farming rural men had no increased risk as compared with urban referents. CONCLUSION: Farmers but not non-farming rural men had a significantly increased risk of developing osteoarthritis of the hip joint as compared with urban referents.

Primary care consultation, hospital admission, sick leave and disability pension owing to neck and low back pain: a 12-year prospective cohort study in a rural population.

BACKGROUND: Neck and low back pain are common musculoskeletal complaints generating large societal costs in Western populations. In this study we evaluate the magnitude of long-term health outcomes for neck and low back pain, taking possible confounders into account. METHOD: A cohort of 2,351 Swedish male farmers and rural non-farmers (40-60 years old) was established in 1989. In the first survey, conducted in 1990-91, 1,782 men participated. A 12-year follow-up survey was made in 2002-03 and 1,405 men participated at both times. After exclusion of 58 individuals reporting a specific back diagnosis in 1990-91, the study cohort encompassed 1,347 men. The health outcomes primary care consultation, hospital admission, sick leave and disability pension were assessed in structured interviews in 2002-03 (survey 2). Symptoms and potential confounders were assessed at survey 1, with the exception of rating of depression and anxiety, which was assessed at survey 2. Multiple logistic regression generating odds ratios (OR) with 95% confidence intervals (95% CI) was performed to adjust the associations between reported symptoms and health outcomes for potential confounders (age, farming, workload, education, demand and control at work, body mass index, smoking, snuff use, alcohol consumption, psychiatric symptoms and specific back diagnoses during follow up). RESULTS: Of the 836 men reporting current neck and/or low back pain at survey 1, 21% had had at least one primary care consultation for neck or low back problems, 7% had been on sick leave and 4% had disability pension owing to the condition during the 12 year follow up. Current neck and/or low back pain at survey 1 predicted primary care consultations (OR = 4.10, 95% CI 2.24-7.49) and sick leave (OR = 3.22, 95% CI 1.13-9.22) after potential confounders were considered. Lower education and more psychiatric symptoms were independently related to sick leave. Lower education and snuff use independently predicted disability pension. CONCLUSION: Few individuals with neck or low back pain were on sick leave or were granted a disability pension owing to neck or low back problems during 12 years of follow up. Symptoms at baseline independently predicted health outcomes. Educational level and symptoms of depression/anxiety were important modifiers.

Unexpected high prevalence of IgG-antibodies to hepatitis E virus in Swedish pig farmers and controls.

Hepatitis E virus (HEV) infections are responsible for large waterborne outbreaks in developing countries. Sporadic cases in the developed world are mainly imported via immigrants and travellers from endemic areas. HEV has been suggested to be a zoonotic infection where pigs may be an important reservoir for the disease and specific swine strains of HEV have been identified which can infect also humans. The aim of this study was to analyse if Swedish pig farmers are more exposed to HEV than persons with other occupations. A total of 115 male pig farmers aged 40-60 y and 108 age- and geographically- matched control subjects were tested for IgG anti-HEV antibodies. No statistical difference in anti-HEV prevalence was noted between pig farmers (13.0%) and control subjects (9.3%). The prevalence of anti-HEV antibodies in the pig farmers and controls was higher than that previously reported among other populations in Europe (<1-9%). Further studies are needed to elucidate the routes for infection of indigenous HEV and if sub-clinical infections with pig associated HEV strains occur in Sweden.

Gene expression analysis suggests that EBF-1 and PPARgamma2 induce adipogenesis of NIH-3T3 cells with similar efficiency and kinetics.

Differentiation of multipotent mesenchymal stem cells into lipid-accumulating adipocytes is a physiological process induced by transcription factors in combination with hormonal stimulation. We have used Affymetrix microarrays to compare the adipogenic differentiation pathways of NIH-3T3 fibroblasts induced to undergo in vitro differentiation by ectopic expression of early B cell factor (EBF)-1 or peroxisome proliferator-activated receptor (PPAR)gamma2. These experiments revealed that commitment to the adipogenic pathway in the NIH-3T3 cells was not reflected in gene expression until 4 days after induction of differentiation. Furthermore, gene expression patterns at the earlier time points after stimulation indicated that EBF-1 and PPARgamma2 induced different sets of genes, while the similarities increased upon differentiation, and that several genes linked to adipocyte differentiation were also transiently induced in the vector-transduced cells. These data suggest that the initial activation of genes associated with adipocyte development is independent of commitment to the adipogenic pathway and that EBF-1 and PPARgamma2 induce adipocyte differentiation with comparable kinetics and efficiency.

Low back pain comorbidity among male farmers and rural referents: a population-based study.

Farmers report more low back pain (LBP) than rural referents. We have previously demonstrated that the difference in reporting rate cannot be fully explained by known risk factors such as physical work exposures, psychosocial factors and lifestyle. Other etiological factors must be involved. In this cross-sectional population-based study, we investigate LBP comorbidity in terms of coexistent symptoms. A total of 1,013 male farmers, 40-60 years old, and 769 matched rural referents participated in an extensive health survey. Information on causes of primary health care and hospital admissions, symptoms, lifestyle factors, physical work exposures and psychosocial factors were gathered through standardized interviews and questionnaires. In the combined farmer-referent group, the prevalence of LBP was associated with musculoskeletal symptoms other than LBP, chest discomfort, dyspepsia, symptoms from eyes, nose and throat mucous membranes, skin problems, work-related fever attacks, and primary care appointments due to digestive disorders. The associations were independent of age, educational level, smoking habits, body mass index, physical work exposures and psychosocial factors. Presence of both respiratory and digestive disorders doubled the LBP prevalence. Significant associations between LBP and digestive and respiratory disorders were revealed, indicating that LBP and these disease entities may have etiological factors in common.