| 1. |
- Lind, Marcus, et al.
(author)
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Thrombomodulin as a marker for bleeding complications during warfarin treatment
- 2009
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In: Archives of Internal Medicine. - : American Medical Association (AMA). - 0003-9926 .- 1538-3679. ; 169:13, s. 1210-1215
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Journal article (peer-reviewed)abstract
- BACKGROUND: The major adverse effect of warfarin treatment is hemorrhage. Several risk factors for bleeding complications are also risk factors for thromboembolic events, making the clinical decision to initiate or withhold anticoagulant treatment difficult. Specific markers that solely identify patients at high risk of bleeding would have great clinical impact. This study aimed to test if thrombomodulin (TM) concentrations were associated with bleeding complications, cardiovascular events, or mortality in long-term anticoagulant-treated patients. METHODS: In a longitudinal cohort study we followed up 719 patients receiving warfarin treatment for a mean duration of 4.2 years. All bleeding complications causing hospitalization were registered and classified. Soluble TM antigen (sTM) concentration in plasma was measured with an enzyme-linked immunosorbent assay method. RESULTS: During the follow-up time, 113 clinically relevant bleeding events and 73 major bleeding events occurred. Increased concentration of sTM was associated with both clinically relevant bleeding and major bleeding events after adjustment for age. In the multivariable models, hazard ratios for the highest tertiles compared with the lowest were 2.29 (95% confidence interval, 1.35-3.89) and 2.33 (95% confidence interval, 1.21-4.48), respectively. No association between sTM concentration and nonfatal ischemic cardiovascular events or all-cause mortality was found. CONCLUSIONS: Increased levels of sTM are associated with bleeding complications during warfarin treatment but not with cardiovascular events or all-cause mortality. Soluble TM antigen concentration has potential as a new specific marker to identify patients at high risk of bleeding during warfarin treatment.
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| 2. |
- Nilsson, Torbjörn K., et al.
(author)
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Genotyping of the reduced folate carrier-1 c.80G>A polymorphism by pyrosequencing technology : importance of PCR and pre-PCR optimization
- 2008
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In: Scandinavian Journal of Clinical and Laboratory Investigation. - Oslo : Taylor & Francis. - 0036-5513 .- 1502-7686. ; 68:2, s. 166-170
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Journal article (peer-reviewed)abstract
- When developing a genotyping assay by Pyrosequencing™ technology for the RFC1 (SLC19A1) c.80G>A polymorphism (rs1051266), unequal peak heights in the pyrograms were observed, probably due to unequal amplification of the mutated and wild-type alleles. This rarely occurring problem could potentially render assignment of heterozygous genotypes uncertain. When the PCR conditions were studied, it was found that substitution of the dGTP nucleotide in the master mix by dGTP and dITP in proportion 1:1 largely overcame this problem. Heat denaturation of the DNA at 95°C before PCR also counteracted the problem. A combination of these two modifications of the standard pyrosequencing PCR protocol gave the best results. We conclude that, with these modifications, the RFC1 c.80G>A SNP can be reliably assayed by pyrosequencing.
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| 3. |
- Magnusson, Marie, et al.
(author)
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A placebo-controlled study of retinal blood flow changes by pentoxifylline and metabolites in humans
- 2006
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In: British Journal of Clinical Pharmacology. - : Wiley. - 0306-5251 .- 1365-2125. ; 61:2, s. 138-147
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Journal article (peer-reviewed)abstract
- AIM: To investigate the possible effects of pentoxifylline metabolites on retinal blood flow in humans. METHODS: A randomized, placebo-controlled, four-period cross-over study that was observer blinded and partly blinded for the eight participants. On one occasion a placebo was given as an intravenous (i.v.) infusion over 100 min. On the other three occasions pentoxifylline was administered as i.v. infusions over 100 min at a rate of 3 mg min(-1). Before two of the pentoxifylline infusions the subjects were pretreated with either ciprofloxacin or rifampicin. Retinal blood flow was measured by scanning laser doppler flowmetry (SLDF) in a selected area of the central temporal retina before, during and until 5 h after the end of infusion. Blood samples for concentration analyses of pentoxifyllin, R-M1, S-M1, M4 and M5 were taken serially and areas under the curves (AUCs) were calculated. Linear mixed models were used for the statistical analyses. RESULTS: Mean AUCs (ng h ml(-1)) were significantly increased for pentoxifylline (1964 vs. 1453) and S-M1 (5804 vs. 4227), but not R-M1 when pentoxifylline was co-administered with ciprofloxacin. The mean AUC for M5 was significantly reduced when subjects were pretreated with rifampicin (2041 vs. 3080). Pentoxifylline with and without pretreatment with rifampicin significantly increased retinal blood flow assessed as mean flow, pulsation (i.e. 1-systole/diastole), and diastolic flow (but not during systole), compared with placebo. The increases over placebo were more pronounced on diastolic flow, 9.7% (95% confidence interval 4.2, 15.5) than on mean flow, 4.6% (1.1, 8.3) after pentoxifylline administration. With pentoxifylline after rifampicin pretreatment the corresponding differences were 11.7% (5.8, 17.9) and 5.1% (1.4, 7.8) over placebo, respectively. After co-administration of pentoxifylline and ciprofloxacin we saw only a nonsignificant trend towards increased flow during diastole, but a significant decrease in pulsation. When AUCs for pentoxifylline and its metabolites were used as regressor variables to retinal mean flow we found that pentoxifylline, R-M1 and M5 had coefficients with a positive sign indicating that they enhanced the retinal blood flow. In contrast, S-M1 and M4 had coefficients with negative sign and thus appeared to decrease the blood flow in subjects treated with pentoxifylline. CONCLUSION: The R-M1 and M5 metabolites of pentoxifylline contributed significantly to the effects of pentoxifylline on retinal blood flow.
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| 4. |
- Mercke Odeberg, Johanna
(author)
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Studies on intrinsic and extrinsic sources of variability in pharmacokinetics and pharmacodynamics
- 2005
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Doctoral thesis (other academic/artistic)abstract
- Variability in pharmacokinetic and pharmacodynamic parameters between individuals can be due to intrinsic and extrinsic factors. In this thesis three different areas regarding sources of variability have been investigated. 1. Formulation properties as an extrinsic source of variability. Lipophilic substances often have poor oral bioavailability and high inter- and intraindividual variability. High fat meals can increase the bioavailability. Two lipophilic substances, astaxanthin and cyclosporine, were incorporated in different lipid based formulations, given orally to healthy volunteers, and shown to alter the bioavailability when compared to reference substances. The combination of different lipid excipients, their ratios and the degree of drug incorporation were factors shown to affect the drug bioavailability. The use of a rational formulation approach in the study with cyclosporine gave successful information in a limited number of trials. 2. Intrinsic and extrinsic sources of variability in the pharmacokinetics and pharmacodynamic of desmopressin. To evaluate factors that can cause variability both the pharmacokinetics and pharmacodynamics have to be investigated. Desmopressin is a synthetic analogue of the antidiuretic hormone vasopressin. We studied in humans if different levels of water hydration had any influence on especially the pharmacokinetics, and found no statistical evidence for that. An indirect response model was described and verified for the concentration-effect relationship. Further, we investigated sex differences and the impact of concomitant medication with piroxicam , and we found a difference in the pharmacodynamics of desmopressin, but not in the pharmacokinetics. 3. Genetic polymorphism as an intrinsic source of variability in drug metabolism and drug transport. We investigated, in a Swedish cohort (248 individuals) and in a human diversity panel (HDP) (DNA from 14 ethnicities), the prevalence of different polymorphisms and haplotypes in the UGT1A- and MDR1-genes, both being associated with variations in pharmacokinetics. Also, to estimate UGT1A genotype-dependent glucuronidation efficiency, we used the endogenous substrate bilirubin as an indicator. We found that several of the genetic variants investigated in the UGT1A-gene and also the SNP (exon 26, C3435T) in the MDR1 gene are common in the cohort. The HDP indicated differences in prevalence of the allelic variants due to ethnicity. Three major haplotypes of the UGT1A-gene constitute 84% of the allelic variants. Tools for optimisation, prediction and individualisation of drug treatment are important for attaining the aim of explaining and reducing variability in drug response.
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| 5. |
- Malm-Erjefält, Monika
(author)
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Assessment of eosinophil degranulation in allergic diseases and experimental models
- 2004
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Doctoral thesis (other academic/artistic)abstract
- In allergic disorders, the eosinophilic granulocytes migrate to affected tissues and release granule proteins with cytotoxic, immunoregulatory, and remodeling-promoting properties. Given that degranulation is reflected by a loss in eosinophil granule density of living cells, or by cell membrane rupture and release of intact granules, these morphological changes may represent a biomarker of the allergic disease. In this thesis, eosinophil structural changes were quantified by transmission electron microscopy. The main objective was to reveal the degranulation status of blood eosinophils during allergic disease, and to determine the eosinophil morphology in common experimental cell- and animal models used for studying eosinophil activation and its pathogenic consequences. First, the eosinophil ultrastructure in common mouse models of allergic airway inflammation was determined and the relevance of these models to human disease was assessed. Both in vivo and in vitro studies revealed that eosinophil degranulation occurred in human but not in mouse eosinophils. Thus, eosinophil-driven pathologic events, reflecting human allergic disease, should not unconditionally be expected in current mouse models. Furthermore, the granule structure of isolated human blood eosinophils was studied and compared with the baseline morphology in blood to examine whether the transformation from an intact eosinophil to a degranulating phenotype can be accurately studied in vitro. The standard procedures of erythrocyte lysis were shown to induce artefactual eosinophil degranulation that also increased the susceptibility of cells to further treatment. Hence, caution should be taken when assessing data and concepts generated in the many previous studies on isolated human blood eosinophils. However, by a novel protocol described herein, eosinophils with minimal granule abnormalities can now be recovered and used for studies on the events regulating the early degranulation. Finally, using a novel approach to assess the ultrastructure of blood eosinophils the degranulation status of circulating eosinophils in a variety of allergic diseases was determined. The results showed that in symptomatic allergic disease, eosinophils retain their granule contents until they have reached their target organ. Hence, eosinophil degranulation in the circulation cannot be used as a biomarker of allergic disease. This finding also suggests that eosinophils in different body compartments have different effector functions and may have distinct susceptibility to therapeutic interventions. Altogether, this thesis underlines the general importance of validating samples and applied methodology when exploring eosinophil degranulation in vitro or in allergic disease.
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| 6. |
- Pesonen, Erkki, et al.
(author)
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Elevated infection parameters and infection symptoms predict an acute coronary event.
- 2008
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In: Therapeutic Advances in Cardiovascular Disease. - : SAGE Publications. - 1753-9447 .- 1753-9455. ; 2:6, s. 419-424
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Journal article (peer-reviewed)abstract
- BACKGROUND: The etiology and significance of flu-like symptoms often appearing before myocardial infarction should be clarified. METHODS: In a case-control study of 323 matched controls and a random sample of 110 out of 351 cases the presence of infection symptoms during the preceding four weeks before admission were asked and blood samples taken. RESULTS: Enterovirus (EV), herpes simplex virus (HSV), and Chlamydia pneumoniae IgA titers were significantly higher in cases than in controls (p<0.001, 0.008 and 0.046, respectively). Flu-like symptoms appeared significantly more often in patients than in controls the most common one being fatigue (p<0.001). In controls with fatigue, EV and HSV titers showed a trend to be higher (1.50 vs 1.45 and 4.29 vs 3.73) than in controls without fatigue but only HSV titers were statistically significantly higher (3.47 vs 3.96, p = 0.02). Even CRP and amyloid A concentrations (3.49 vs 2.08, p<0.0001 and 5.70 vs 3.77 mg/l, p = 0.003, respectively) as well as C4 (0.40 vs 0.44, p = 0.02) were higher in controls with fatigue. CONCLUSIONS: Odds ratios for a coronary event in a logistic regression model were 4.79 for fatigue and 2.72 for EV antibody levels in their fourth quartile. A linear-by-linear association test showed increasing number of single symptoms with higher EV titer quartiles (p = 0.004).
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| 7. |
- Andersson, David
(author)
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Vasodilator actions of EDHF and anandamide
- 2001
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Doctoral thesis (other academic/artistic)abstract
- Vasodilator responses to endothelium-derived hyperpolarizing factor (EDHF) and anandamide in isolated arteries were investigated. A combination of the potassium channel blockers charybdotoxin and apamin inhibits EDHF-mediated relaxations. Experiments in which charybdotoxin or apamin was substituted with other potassium channel inhibitors indicated that small- and intermediate-conductance calcium-activated potassium channels contribute to EDHF-mediated relaxations. The possibility that EDHF is potassium ions was also examined. Potassium ions failed to mimic the action of EDHF and no evidence was found for involvement of Na+/K+ ATPase and inwardly rectifying potassium channels in EDHF-mediated responses. In the presence of the Na+/K+ ATPase inhibitor ouabain, acetylcholine and KCl stimulated release of CGRP from sensory nerves. Relaxant responses to the endogenous cannabinoid anandamide were examined in isolated rat and guinea pig arteries. Cannabinoid receptors did not mediate anandamide-induced relaxations. Capsaicin pretreatment, the CGRP receptor antagonist CGRP 8-37 and the vanilloid receptor antagonist capsazepine inhibited anandamide-induced relaxations, indicating that vanilloid receptors on perivascular sensory nerves mediate the effects of anandamide. Anandamide also activated the cloned rat vanilloid receptor (VR1). Effects of anandamide and related vanilloid receptor ligands in guinea-pig mesenteric arteries and main bronchi were compared. Capsaicin was equally potent in the two tissues, while anandamide, resiniferatoxin and olvanil were more potent vasodilators than bronchoconstrictors. Schild plots for capsazepine yielded similar pA2 values in both tissues. Data simulations suggested that the differences in effects of the vanilloid receptor agonists might be explained by differences in agonist efficacies and receptor densities rather than by receptor heterogeneity.
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| 8. |
- Johansson, Rebecka
(author)
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Regulation of inducible nitric oxide synthase - Consequences in experimental models of bladder disease
- 2003
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Doctoral thesis (other academic/artistic)abstract
- Inducible nitric oxide synthase (iNOS) expression is associated with various pathophysiological conditions in the lower urinary tract. The aims of this thesis have been to investigate the regulation of iNOS in bladder smooth muscle cells and the consequences on neuromuscular regulation, cell growth and differentiation. Primary cultures of rat bladder smooth muscle cells (BSMC) expressed iNOS mRNA and protein upon stimulation with the cytokines IL-1β and TNF-alpha in combination. No constitutive isoforms of NOS were detected at the mRNA level. BSMC were put into different stages of differentiation. The capacity of BSMC to express iNOS was negatively correlated to differentiation status measured as smooth muscle myosin-heavy chain expression. iNOS was shown to be preferentially expressed in immature dedifferentiated BSMC. Actin cytoskeletal dynamics and Rho signalling were involved in regulation of cytokine-induced iNOS expression in BSMC. Phenotypic changes and impairment in actin cytoskeletal formation may potentiate cytokine activation and in turn increase NO production during bladder disease. The consequences of iNOS induction on bladder neuromuscular function were investigated by incubating isolated rat bladder strips with cytokines and recording the mechanical activity in organ baths. iNOS protein was observed in the smooth muscle layer in cytokine stimulated bladder strips. Nerve-mediated, but not acetylcholine-mediated, contraction of the bladder was impaired in bladder strips exposed to cytokines. This impairment was restored by the iNOS inhibitor aminoguanidine and partially by the neurotrophic factor BDNF. iNOS induction affected bladder function and bladder nerves were more sensitive to NO exposure than smooth muscle. The activity and expression of different NOS isoforms were investigated in an in vivo model of bladder hypertrophy induced by urethral obstruction in rats. During obstruction the nNOS activity decreased whereas iNOS activity increased initially. The total NOS activity was decreased. iNOS was expressed in smooth muscle, urothelium and inflammatory cells. Exogenous NO exposure decreased DNA- and protein-synthesis and stimulated differentiation in cultured BSMC. The results indicate that the decreased NO production during obstruction may stimulate a growth response and phenotypic changes in the bladders smooth muscle. The results in this thesis show that the smooth muscle in the bladder should be considered as a source of iNOS expression and NO production during inflammation. Neuronally derived NO is suggested to be involved in bladder smooth muscle physiology by maintaining and stabilizing the smooth muscle phenotype.
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| 9. |
- Norlén, Per
(author)
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Histamine and chromogranin A-derived peptides in rat stomach ECL cells
- 2000
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Doctoral thesis (other academic/artistic)abstract
- The ECL cells constitute the predominant endocrine cell population of the stomach. They secrete histamine which is a major stimulator of acid secretion. Chormogranin A (CgA)-derived peptides, such as pancreastatin, are co-secreted with histamine. In this thesis it was demonstrated that pancreastatin may serve as a reliable marker for ECL cell secretory activity in the rat. The function of pancreastatin and other CgA-derived peptides is however obscure, and therefore, attempts were made to develop a method for continuous monitoring of ECL-cell histamine in conscious rats. By using microdialysis it was demonstrated that histamine is secreted from ECL cells in response to gastrin and food and to a number of neuropeptides, such as PACAP, VIP and adrenaline. It could also be demonstrated that anaesthesia greatly impairs ECL-cell histamine secretion.
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| 10. |
- Nylén, Anders
(author)
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Nitric oxide in central nervous structures associated with water balance and micturition
- 2001
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Doctoral thesis (other academic/artistic)abstract
- The role of nitric oxide (NO) in the central control of water balance and micturition has not yet been established. In the present thesis, where female rats were used, neuronal NO synthase (nNOS) distribution was studied in structures of the central nervous system known to be associated with the above mentioned control. Moreover, the concert action of a NOS inhibitor and oxytocin (OT), intrathecally (i.t.) administered, on cystometry was studied. In rostral circumventricular organs, nNOS-immunoreactive (IR) neurons were observed. In some of these cell bodies, 8-arginine vasopressin (AVP)-immunoreactivity was also found. In the supraoptic nucleus (SON) seven different populations of neuronal cell bodies containing all combinations of nNOS, AVP and OT were demonstrated. The populations in the SON containing either nNOS, but lacking AVP and OT, or nNOS together with AVP and OT, constituted each 2-3% of all observed cell bodies. In the hypothalamic paraventricular nucleus (PVN), about 30% of all nNOS-IR cell bodies were OT-IR. In contrast, around 3% of all nNOS-IR cell bodies were AVP-IR. Further, in the PVN, about 10% of all nNOS-IR neuronal cell bodies were labelled by the retrograde tracer True Blue (TB) injected in the spinal cord. About 40% of these neurons were magnocellular. TB/nNOS cell bodies were most abundant in the caudal PVN, with a clear ipsilateral dominance. OT were found to mediate bladder contractions both in vitro and in vivo. However, when a NOS inhibitor, inactive per se, was injected i.t. prior to OT, the effect of OT was reduced or completely prevented. Moreover, all rats developed overflow incontinence and hindlimb paralysis. The above data suggest a possible interaction between NO and AVP/OT at hypothalamic/suprapontine and spinal levels in the control of water balance and micturition.
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