| 11. |
- Birzniece, Vita
(författare)
-
Neuroactive steroids and rat CNS
- 2004
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Several studies suggest profound effects on mood and cognition by neuroactive steroids. Estrogen alone or in combination with antidepressant drugs affecting the serotonin system has been used to treat mood disorders. On the other hand, progesterone is related to negative effects on mood and memory. A major part of the progesterone effects on the brain can be mediated by its metabolite allopregnanolone, which is also de novo synthesized in the brain, and affects the GABAA receptors. It would be of great importance to find a substance that antagonize allopregnanolone adverse effects. To investigate how long term supplementation of estradiol and progesterone, resembling postmenopausal hormone replacement therapy, affects serotonin receptors in different brain areas important for mood and memory functions, we used ovariectomized female rats. After 2 weeks of supplementation with 17β-estradiol alone or in combination with progesterone, or placebo pellets, estradiol alone decreases but estradiol supplemented together with progesterone increases 5HT1A mRNA expression in the hippocampus. Estradiol decreases the 5HT2C receptor gene expression, while estradiol in combination with progesterone increases the 5HT2A mRNA expression in the ventral hippocampus. Thus, estradiol alone has opposite effects compared to the estradiol/progesterone combination. To detect if acute tolerance develops to allopregnanolone, an EEG method was used where male rats by continuous allopregnanolone infusion were kept on anesthesia level of the silent second (SS). After different time intervals (first SS, 30 min or 90 min of anesthesia) several GABAA receptor subunit mRNAs were measured for detecting if changed expression of any GABAA receptor subunits is involved in development of acute tolerance. There is development of acute tolerance to allopregnanolone and brain regions of importance are hippocampus, thalamus and hypothalamus. The GABAA receptor alpha4 subunit in thalamus and alpha2 subunit in the dorsal hippocampus are related to development of acute tolerance. For assessing allopregnanolone behavioral effects, we studied how this neurosteroid affects spatial learning in the Morris water maze task Allopregnanolone inhibits spatial learning short after the injection and shows a specific behavioral pattern with swimming close to the pool wall. The steroid UC1011 can inhibit the increase in chloride ion uptake induced by allopregnanolone. UC1011 decreases allopregnanoloneinduced impairment of spatial learning in the water maze, as well as the specific behavioral swim pattern. In conclusion, the present work demonstrates that neuroactive steroids affect the 5HT and GABA systems in a brain region specific way. GABAA receptor subunit changes in hippocampus and thalamus are related to acute allopregnanolone tolerance. Allopregnanolone induces cognitive deficits, like spatial learning impairment and UC1011 can inhibit allopregnanolone-induced effects in vitro and in vivo. Key words: Estradiol, progesterone, HRT, allopregnanolone, UC1011, serotonin receptor, GABAA receptor, mRNA, Morris water maze, silent second, tolerance.
|
|
| 12. |
- Birznieks, Ingvars
(författare)
-
Tactile Sensory Control of Dexterous Manipulation in Humans
- 2003
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- During dexterous manipulation with the fingertips, forces are applied to objects' surfaces. To achieve grasp stability, these forces must be appropriate given the properties of the objects and the skin of the fingertips, and the nature of the task. It has been demonstrated that tactile sensors in the fingertips provide crucial information about both object properties and mechanical events critical for the control of fingertip forces, while in certain tasks vision may also contribute to predictions of required fingertip actions. This thesis focuses on two specific aspects of the sensory control of manipulation: (i) how individual fingers are controlled for grasp stability when people restrain objects subjected to unpredictable forces tangential to the grasped surfaces, and (ii) how tactile sensors in the fingertips encode direction of fingertip forces and shape of surfaces contacted by the fingertips. When restraining objects with two fingers, subjects adjust the fingertip forces to the local friction at each digit-object interface for grasp stability. This is accomplished primarily by partitioning the tangential force between the digits in a way that reflects the local friction whereas the normal forces at the involved digits are scaled by the average friction and the total load. The neural control mechanisms in this task rely on tactile information pertaining to both the friction at each digit-object interface and the development of tangential load. Moreover, these mechanisms controlled the force application at individual digits while at the same time integrating sensory inputs from all digits involved in the task. Microneurographical recordings in awake humans shows that most SA-I, SA-II and FA-I sensors in the distal phalanx are excited when forces similar to those observed during actual manipulation are applied to the fingertip. Moreover, the direction of the fingertip force influences the impulse rates in most afferents and their responses are broadly tuned to a preferred direction. The preferred direction varies among the afferents and, accordingly, ensembles of afferents can encode the direction of fingertip forces. The local curvature of the object in contact with the fingertip also influenced the impulse rates in most afferents, providing a curvature contrast signals within the afferent populations. Marked interactions were observed in the afferents' responses to object curvature and force direction. Similar findings were obtained for the onset latency in individual afferents. Accordingly, for ensembles of afferents, the order by which individual afferents initially discharge to fingertip events effectively represents parameters of fingertip stimulation. This neural code probably represents the fastest possible code for transmission of parameters of fingertip stimuli to the CNS.
|
|
| 13. |
- Björklund, Martin, 1961-
(författare)
-
Effects of repetitive work on proprioception and of stretching on sensory mechanisms : implications for work-related neuromuscular disorders
- 2004
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aims of the thesis were (i) to investigate the impact of repetitive low-intensity work exposure on proprioception and (ii) to examine effects of muscle stretching (especially sensory effects and effects on muscle nociception) and to relate its application to the prevention, alleviation and/or treatment of work-related neuromuscular disorders. The effects of low-intensity repetitive work on the shoulder proprioception were tested in healthy subjects. The effect of working time on the retention of subjective fatigue and their relation to changes in proprioception, and the immediate effect of stretching on shoulder proprioception were investigated. A new method to test the stretchability of the rectus femoris muscle was investigated for reliability and validity and used to assess the effects of a two-week stretching regimen on range of motion and on subjective stretch sensation. Finally, the interactions between innocuous muscle stretch and nociceptive chemical stimulation on discharge behavior of nociceptive dorsal horn neurons in the feline spinal cord were explored. The main findings were as follows: 1) The repetitive low-intensity work to fatigue diminished the shoulder proprioception; the working time as well as the retention of subjective fatigue were partly related to the extent of changed proprioception. 2) There was no effect of acute muscle stretching on the proprioception. 3) The new method for testing muscle stretchability proved valid and reliable. A two-week stretching regimen increased the tolerance to stretch torque, but the range of motion remained unchanged. 4) Half of the nociceptive dorsal horn neurons that responded to close arterial injections of bradykinin were modulated by muscle stretching applied directly after the injections. Altogether, the results give credence to the hypothesis of an involvement of sensory information distortion due to repetitive low-intensity work exposure in the development of work-related neuromuscular disorders. Increased tolerance to stretch torque may be an important mechanism in explaining improvements following stretch treatment. The spinal interactions between innocuous stretch and nociceptive muscle afferent inputs indicate a possible mechanism involved in stretching-induced pain alleviation.
|
|
| 14. |
- Bukachi, Frederick, 1960-
(författare)
-
Ventricular Long Axis Function: Amplitudes and Timings : Echocardiographic Studies in Health and Disease
- 2004
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: The ageing process not only increases the risk of coronary artery disease (CAD) but also complicates its diagnosis and treatment. It is therefore important to understand the newer concepts of cardiovascular ageing physiology as well as methods of predicting the outcomes of therapeutic options available for the elderly with severe CAD. Studies of atrioventricular (AV) ring or plane motion have attracted considerable interest in the last few years as a means of assessing ventricular and atrial function. As the displacement of AV rings towards the ventricular apex is a direct reflection of longitudinal fibre contraction, its measurement by echocardiography provides additional information regarding global and regional systolic and diastolic function. Left ventricular (LV) long axis amplitude of motion, referred to as mitral valve annular (MA) motion, is reduced in CAD and to some extent in the elderly as part of the normal ageing process. Objectives & Methods: The aim of the present study was two-fold. First, to investigate the relationship between the timing of MA motion and transmitral and pulmonary venous flow in healthy subjects, and to define the physiological significance of that relationship including its potential diagnostic utility. Second, to investigate the relationship between the clinical outcome and the behaviour of long axis function in patients with severe ischaemic LV dysfunction (SLVD) after percutaneous coronary angioplasty (PTCA). Transmitral early (E) and late (A) filling, and pulmonary venous flow reversal (Ar) were studied by Doppler echocardiography, while at the left lateral AV ring, the MA motion in early (Em) and late (Am) diastole were recorded by Doppler tissue imaging (DTI) and M-mode echocardiography. Results: Healthy subjects – In early diastole the onsets of LV filling (E) and relaxation (Em) were simultaneous, and peak Em preceded peak E by 26 msec in all age groups, constituting a time interval referred to as early diastolic temporal discordance (EDTD). Similarly, the onsets of Am, A and Ar were simultaneous at onset and began approximately 84 msec after the electrocardiographic P wave. Peak Am preceded peak A by 23 msec in the young and by 13 msec in the elderly, a time interval referred to as late diastolic temporal discordance (LDTD). Peak Ar, on the other hand, coincided with peak Am in all age groups. With increasing age and sequential prolongation of isovolumic relaxation time, the peaks of Am, Ar and A converged. This point of convergence is described as atrial mechanical alignment (AMA). Patients – MA total amplitude of motion, rates of shortening and lengthening were all reduced in patients with SLVD. At mid-term, 3-6 months after PTCA, there was improvement in all these variables. A pre-procedure long axis cut off value of ≥5 mm was associated with favourable symptomatic outcome. Overall angiographic success was 95.2%, and event-free survival was 78.4% at one month and declined steadily to 62.3% at one year with 2.5% mortality. Conclusions: EDTD, which reflects ventricular restoring forces (suction) is age independent while the narrowing of LDTD leading to AMA provides a novel method to identify healthy subjects at increased dependency on left atrial contraction for late diastolic filling. Peak atrial contraction (Am) coincides with peak Ar, thus the timing of regional atrial contraction by DTI can be used to estimate corresponding measurements of Ar, which is often difficult to image by transthoracic echocardiography. In patients with SLVD long axis total amplitude of at least 5 mm at the left MA suggests a significant potential for segmental function recovery after PTCA. Keywords: Echocardiography, Doppler tissue imaging, ageing, coronary disease, left ventricular dysfunction, atrial contraction, electromechanical function, coronary angioplasty.
|
|
| 15. |
- Burén, Jonas, 1972-
(författare)
-
Glucose and lipid metabolism in insulin resistance : an experimental study in fat cells
- 2003
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Type 2 diabetes is usually caused by a combination of pancreatic β-cell failure and insulin resistance in target tissues like liver, muscle and fat. Insulin resistance is characterised by an impaired effect of insulin to reduce hepatic glucose production and to promote glucose uptake in peripheral tissues. The focus of this study was to further elucidate cellular mechanisms for insulin resistance that may be of relevance for type 2 diabetes in humans. We used rat and human adipocytes as an established model of insulin’s target cells. Glucocorticoids, e.g. cortisol, can induce insulin resistance in vivo. In the present study, pretreatment of rat adipocytes in vitro for 24 h with the cortisol analogue dexamethasone produced a downregulation of glucose uptake capacity as well as a marked depletion of cellular insulin receptor substrate 1 (IRS-1) and protein kinase B (PKB), two proteins suggested to play a critical role in the intracellular signal transduction pathway of insulin. The amount of phosphorylated PKB in response to acute insulin treatment was decreased in parallel to total PKB content. The basal rate of lipolysis was enhanced, but insulin’s antilipolytic effect was not consistently altered following dexamethasone pretreatment. Alterations in blood glucose as well as insulin levels may be of great importance for cellular as well as whole-body insulin resistance. High glucose (≥15 mM) for 24 h induced a decrease in glucose uptake capacity in rat adipocytes and IRS-1 content was reduced whereas IRS-2 was increased. Long-term pretreatment with a high insulin concentration downregulated insulin binding capacity and when combined with high glucose, it produced a pronounced reduction of cellular IRS-1 and 2 content together with insensitivity to insulin’s effect to activate PKB and a decrease in glucose uptake capacity. A common denominator for a decrease in glucose uptake capacity in our rat adipocyte studies seems to be a decrease in IRS-1 content. Adipocytes from type 2 diabetes patients are insulin-resistant, but in our work the insulin resistance could be reversed by incubation of the cells at a physiological glucose level for 24 h. Insulin resistance in fresh adipocytes from type 2 diabetes patients was associated with in vivo insulin resistance and glycemic level and with adipocyte cell size and waist-hip ratio (WHR). As a potential mechanism for postprandial dyslipidemia in type 2 diabetes, we examined the nutritional regulation of subcutaneous adipose tissue lipoprotein lipase (LPL) activity. It was upregulated by ~40-50 % after a standardised lipid-enriched meal and this was very similar in type 2 diabetes patients and control subjects, suggesting that the postprandial hypertriglyceridemia found in type 2 diabetes is not explained by an altered nutritional regulation of LPL in subcutaneous fat. In conclusion, the present work provides evidence for novel interactions between glucocorticoids and insulin in the regulation of glucose metabolism that may potentially contribute to the development of insulin resistance. High levels of glucose and insulin produce perturbations in the insulin signalling pathway that may be of relevance for human type 2 diabetes. Cellular insulin resistance may be secondary to the diabetic state in vivo, e.g. via glucotoxicity. This is supported by our finding that insulin resistance in adipocytes from type 2 diabetes patients can be reversed after incubation at a physiological glucose level. Key words: adipocyte, insulin resistance, type 2 diabetes, insulin signalling, glucose uptake, insulin, glucose, dexamethasone, insulin receptor substrate, protein kinase B, GLUT4, lipoprotein lipase.
|
|
| 16. |
- Burstedt, Marie, 1964-
(författare)
-
Bothnia dystrophy, a clinical, genetical and electrophysiological study
- 2003
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- A high frequency of retinitis pigmentosa (RP) is found in Northern Sweden. In an inventory of autosomal recessive RP patients in Västerbotten County, a great number of cases with a unique phenotype was noticed, denoted Bothnia Dystrophy (BD). The aim of the study was to describe the phenotype, to determine the chromosomal location, and to identify the gene. Patients typically show night blindness from early childhood. Symptoms of defect macular function with a decrease of visual acuity can appear in early adulthood. The retinal fundus shows irregular white spots in a central, and parafoveal pattern along the arcades. Centrally areolar maculopathy develops and round circular atrophies are observed in the periphery. The disease was shown to be associated with a missense mutation in the RLBP1 gene resulting in an amino acid substitution (R234W) in the cellular retinaldehyde-binding protein (CRALBP). The R234W mutation was found in a homozygous state in 61 patients affected with BD. Ten patients were heterozygous for the R234W mutation, and presented a similar phenotype. No additional mutations in the coding sequence or exon-intron junctions were found. CRALBP is localised in retinal pigment epithelium (RPE), and Müller cells of the retina. In the RPE, CRALBP functions as a carrier protein for endogenous retinoids. Dark adaptometry and electrophysiologic testing showed an initial loss of rod function followed by a progressive reduction of the cone responses in older ages. A compromised rod function, dysfunction of the Müller cells, and indications of a disturbed function of the inner retina were found. With prolonged dark adaptation, a gradual increase in retinal sensitivity to light and an improvement of the ERG components occurred. The findings indicate a prolonged synthesis of photopigments, retardation of the visual process in the retinal pigment epithelium and a loss of retinal cells probably starting at a relative early age in BD. To evaluate the subjective visual function in BD patients, a battery of objective tests of visual function and composite score of the 25-item NEI-Visual Function Questionnaire (VFQ-25) were analyzed. We found that weighted distance logMAR visual acuity (WVA), had the strongest association with subjective visual function, and that there was a considerable loss of subjective and objective visual function with increasing age in BD patients. The prevalence of BD is as high as 1:3600 in Västerbotten County. The possibility that recycling of retinoids localized in the RPE might be impaired in BD might give future therapeutic possibilities. Due to the large and clinically well-characterized set of patients with this disease, they constitute a suitable study group.
|
|
| 17. |
- Bylund, Sonya H, 1953-
(författare)
-
Hand-arm vibration and working women : Consequences and affecting factors
- 2004
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The use of hand-held vibrating tools may lead to hand-arm vibration syndrome (HAVS), a condition with vascular, neurological and musculoskeletal symptoms. Vibrating tools are used in several occupations in which women can be found, e.g. by metal- and wood workers, drivers, and dental personnel. The risk of women developing HAVS is hard to estimate, as little research has been done on women exposed to hand-arm vibration. The overall aim of this thesis has been to fill this gap of knowledge. It is based upon one questionnaire study and one interview study on women who have reported an occupational injury related to hand-arm vibration. The thesis also comprises two laboratory studies of female and male subjects exposed to hand-arm vibration from a handle. The questionnaire and the interview study showed that the women had a high prevalence of symptoms, such as numbness, weakness, pain and white fingers. Neurological symptoms were more common and developed after shorter time of exposure compared to vascular symptoms. The symptoms had a considerable impact on all domains of the women’s lives, not only on their physical functioning, such as the ability to work, to participate in leisure activities and to do household activities, but also on their relationships and identity. Forty per cent of the women had retired or retrained due to the injury. Dental personnel had the highest relative risk of vibration injuries. In one of the laboratory studies 12 female and 12 male subjects were exposed to vibration in two vibration directions, (Xh and Zh) and at two vibration levels. The absorbed power was higher in the Zh direction and at the higher vibration level. The volumes of the subjects’ arms affected the power absorption in the Zh direction. There were no indications of a gender difference in the absorption of power. In the other laboratory study, the effect of handle size, vibration level, anthropometric measures and maximal grip force on the ability to perform a precision task was studied in 20 female and 20 male subjects. Ratings of difficulty and discomfort were made after each test round. The results indicate that the male subjects performed better in all the tests, but no gender difference was seen in the ratings. The higher vibration level resulted in higher ratings of discomfort. In the female subjects, the handle size, the anthropometric measures and maximal grip force affected both the performance and the ratings. In conclusion, the studies indicate that vibration injuries are severely disabling and influence many parts of the sufferer’s life. Vibration injuries are preventable, and the extensive consequences found underscore the importance of preventive action. This can be done by informing employees about the risks, and by giving them the opportunity to choose suitable machines and to practice work tasks when starting a new job.
|
|
| 18. |
- Caldera Aburto, José Trinidad
(författare)
-
Mental health in Nicaragua : with special reference to psychological trauma and suicidal behaviour
- 2004
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis explores mental health problems relating to war and natural disaster and suicidal behaviour in the Nicaraguan population. The more specific aims of the study were to assess the prevalence and sociodemographic correlates of mental disorder in a community-based study during time of war (Paper I), to assess the mental health impact of Hurricane Mitch in 1998 (Paper II), to assess the incidence of hospitalized parasuicide cases and groups at risk (Paper III), and to examine suicide intent among attempters relating to gender, suicide method and sociodemographic factors and identify predictors for repetition of an attempt (Paper IV).Method: Based on 4453 family food ration books for families living in an urban area of León, Subtiava, 219 families including 746 adults were selected through a systematic sampling procedure. The study was conducted in 1987 during the war. We were able to reach 584 adults for interview according to the Present State Examination for ICD-9 diagnoses and Self-Report Questionnaire (Paper I). In Paper II, 496 adult primary health care attendees were interviewed six months after Hurricane Mitch according to the Harvard Trauma Questionnaire and were diagnosed for post-traumatic stress disorder (PTSD) according to DSM-IV. In Papers III and IV, all cases from León city admitted to HEODRA Hospital for a suicide attempt over a three-year period (n=233) were interviewed regarding sociodemographic factors and method, time and place of the suicide attempt. A subgroup of 204 cases was interviewed using the Suicide Intent Scale (SIS). Out of those 106 cases were followed-up regarding repetition of attempt or completed suicide after a mean period of 1172 days.Results: In the Paper I study, the one-month prevalence of any mental disorder was 28.8% for men and 30.8% for women. Among men, alcoholism was the most common diagnosis, whereas neurosis, crisis reaction and depression were dominant among women. Alcoholism was scored as the second most severe disorder after psychosis in terms of functional level. In the Mitch study six months after the hurricane, traumatic events were common and 39% reported death or serious injury of a close relative as a result of the hurricane. The prevalence of PTSD ranged from 4.5% in the least damaged area to 9.0% in the worst damaged area. At the prolonged follow-up six months later, half of the cases still retained their diagnosis. Trauma-related symptoms were common and death of a relative, destroyed house, female sex, illiteracy and previous mental health problems were associated with a higher level of symptoms. Suicidal ideation was reported among 8.5% and was significantly associated with previous mental health problems and illiteracy. The studies regarding hospitalized parasuicides showed the highest rate among girls aged 15–19 years (302 attempts per 100 000 inhabitants and year). After drug intoxication, pesticide was the second most common method and most often used by men (23%). Half of the women had recent contact with health care services before attempting suicide. There were significant peaks regarding time of attempt in terms of seasonal and diurnal distribution. Overall scores regarding seriousness of the intent (SIS) were equal between the sexes, but the pattern of SIS items showed significant gender differences in terms of relation to background factors and method used. For women, having a child was one factor associated with higher seriousness. Factor analysis of SIS items revealed a four-factor solution, explaining 59% of the variance. Risk for fatal repetition was 3.2% after three years and for non-fatal repetition 4.8%. During follow-up, three men (11%) had completed suicide but no women. We failed to identify any predictors for repetition from background factors or SIS.Conclusion: The studies have identified different groups at risk for mental health problems relating to war and disasters. Parasuicide rates equalled those from European countries. Whereas young girls dominated, attempts among men were more severe in terms of the methods used and completed suicide at follow-up. SIS seemed to give a meaningful pattern among women but not for men. In our study, seriousness of attempt in terms of method or suicide intent did not predict repetition. Overall non-fatal repetition rate was very low as compared to other studies.
|
|
| 19. |
- Campino, Susana, 1975-
(författare)
-
Genetic analysis of murine malaria
- 2003
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Malaria, an infectious disease caused by Plasmodium parasites, is one of the major world-scale health problems. Despite the efforts aimed at finding an effective way to control the disease, the success has been thwarted by the emergence of parasite drug resistance and mosquito resistance to insecticides. This thesis focuses on the genetic analysis of resistance to murine malaria induced by the lethal Plasmodium berghei ANKA using a wild-derived-inbred strain (WDIS). The aim of this thesis was to exploit the genetic diversity represented among WDIS for identifying loci contributing to resistance/susceptibility to murine malaria. The work included a genome-wide polymorphism survey using microsatellite markers performed on 10 WDIS. Comparisons of these strains to laboratory inbred strains confirmed a higher rate of polymorphism among the WDIS. We conclude that these WDIS represent repositories of unique naturally occurring genetic variability that may prove to be invaluable for the study of complex phenotypes. Next, we used the WDIS to search for novel phenotypes related to malaria pathogenesis. Whereas most laboratory strains were susceptible to experimental cerebral malaria (ECM) after infection with P. berghei ANKA, several WDIS were found to be resistant. To study the genetic inheritance of resistant/susceptibility to P. berghei ANKA infection we analysed backcross and F2 cohorts derived from crossing the WLA wild-derived strain with a laboratory mouse strain (C57BL/6). A novel phenotype represented by the cure of infection, clearance of parasitaemia and establishment of immunological memory was observed in the F2 progeny. The backcross progeny was used to genetically map one locus on chromosome 1 (Berr1) and one locus on chromosome 11 (Berr2) that mediate control of resistance to ECM induced by P. berghei ANKA. Genetic mapping using the F2 progeny showed that a locus on chromosome 1 (Berr1) and a locus on chromosome 9 (Berr3) were contributing to control survival time after infection with lethal Plasmodium. Finally, we identified, a locus on chromosome 4 (Berr4) that appears to control time of death due to hyperparasitaemia. This thesis underlines the value of using WDIS to reveal genetic factors involved in the aetiology of disease phenotypes. The characterisation of the genetic factors represented by the malaria resistance loci identified here are expected to provide a better understanding of the malaria pathology.
|
|
| 20. |
- Carlsson, Lennart, 1970-
(författare)
-
Aspects of interferon alpha signalling in hematopoetic cells
- 2004
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The type I interferons (IFN) are a family of cytokines with pleiothropic activities that include inhibition of viral replication, cell proliferation and activation of the immune system. These properties give the IFNs important physiological and pathological roles in infection and cancer and have led to their therapeutic use for many clinical conditions. In humans, the type I IFNs consist of 12 different IFNa subtypes as well as single IFNb, w and k subtypes. They all compete for binding to a common receptor, consisting of two subunits, IFNAR1 and IFNAR2. In almost all cell types proliferation is inhibited by IFNs as a consequence of the antiviral properties. However, previous studies on human peripheral B-lymphocytes have shown increased survival as well as proliferation upon IFN treatment.We established a purification system for extraction of B-lymphocytes from buffy-coat, utilizing density centrifugation in combination with anti-CD19 magnetic beads. In an attempt to identify the molecular mechanisms of increased survival, the expression and/or activation pattern of different signaling proteins were analysed by Western blot. It was previously reported that phosphatidylinositol 3’-kinase (PI3K) physically interacts with the IFNAR complex, via adaptor proteins. Activated PI3K indirectly activates Akt/PKB, a kinase involved in a pathway leading to both survival and proliferation signals. We were able to show a novel signaling pathway - IFN treatment activated Akt/PKB as well as a downstream effector, one member of the Forkhead family (FKHR) was inactivated by phosphorylation and as a consequence p27/Kip1 expression was downregulated. Activation of this pathway resulted in increased survival as measured by TUNEL assay, an effect efficiently counteracted by the the synthetic PI3K inhibitor, LY294002.In additional experiments we investigated the molecular mechanisms of proliferation. Activation of B-cells was ensured by using limiting concentrations of anti-IgM antibodies, mimicing natural activation. Using thymidine incorporation, we discovered that IFN treatment increased the sensitivity to anti-IgM stimulation. As a consequence, more cells proliferated as measured by CFSE staining. However, on its own, IFN was unable to induce proliferation. IFN turned out to be as efficient as IL-2, a classical B-lymphocyte growth factor. In order to distinguish proliferation from increased survival, Rb phoshorylation was analysed by Western blot. Phosphorylation induced by anti-IgM was further enhanced by IFN. As we determined earlier, p27/Kip1 expression was downregulated, releasing the cell cycle block. However, p21/Cip1 expression was upregulated but almost exclusively localised to the cytoplasm, therefore unable to perform the classical growth inhibitory functions. We conclude that type I interferons contribute to increased survival as well as proliferation of human primary B-lymphocytes.The IFN receptor subunits was studied in a human myeloma cell line (U266), using a variant of which that are totally resistant towards the anti-proliferative properties of IFN. The reason for resistance in clinical situations is seldom elucidated, but is often believed to be due to development of antibodies against interferon. The resistant cells were unable to bind radio-labelled IFN, and through Southern Blot we could determine that the IFNAR1 gene was not functional. Also the IFNAR2 gene was affected, since Northern blot and sequencing detected an aberrant transcript not present in the wild type cells. Karyotyping showed that the cells had 3-4 copies of chromosome 21, but Southern blot did not detect any cytoplasmic region of IFNAR2. The IFN receptors are close to each other on the genome, and a deletion affecting one receptor gene is likely to affect the other as well. We conclude that the IFN resistance in U266Res cells is due to lack of functional receptor subunits.
|
|
