| 51. |
- Jonasson, Jenni, 1971-
(författare)
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Genetic and Molecular analysis of the Spinocerebellar ataxia type 7 (SCA7) disease gene
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Spinocerebellar ataxia type 7 (SCA7) is a hereditary neurodegenerative disorder affecting the cerebellum, pons and retina. SCA7 patients present with gait ataxia and visual impairment as the main symptoms. Anticipation, commonly observed in SCA7 families, is a phenomenon where an earlier age at onset and a more severe progression of disease is seen in successive generations. In order to identify the gene responsible for SCA7, we performed linkage analysis on a Swedish SCA7 kindred. Evidence for linkage of the SCA7 disease locus to a 32 cM region on chromosome 3p12-21.1, between markers D3S1547 and D3S1274, was established. A number of neurodegenerative disorders associated with anticipation are caused by expanded (CAG)n repeats in their respective disease genes. In order to isolate the SCA7 disease gene we, therefore, screened a human infant brain stem cDNA library for CAG repeat containing clones, mapping to chromosome 3. Four candidate clones were isolated and analysed, but could all be excluded as the SCA7 disease gene. In 1997, the SCA7 disease gene was identified and, as expected, shown to harbour a CAG repeat, expanded in SCA7 patients. Analysis of the SCA7 CAG repeat region in Swedish SCA7 patients demonstrated that CAG repeat size was negatively correlated to age at onset of disease. Furthermore, patients with larger repeats presented with visual impairment, whereas patients with smaller repeats presented with ataxia as the initial symptom. SCA7 is the most common autosomal dominant cerebellar ataxia in Sweden and Finland, but rare in other populations. In order to investigate if the relatively high frequency of SCA7 in these countries is the result of a founder effect in the region, a haplotype analysis was performed on all SCA7 families available. All 7 families shared a common haplotype of at least 1.9 cM surrounding the SCA7 locus. In addition, strong linkage disequilibrium was demonstrated for marker D3S1287 closely linked to the SCA7 gene, suggesting a founder effect for the SCA7 mutation in Sweden and Finland. The function of the SCA7 protein, ataxin-7, is not known and it does not show significant homologies to any previously known proteins. In order to gain insight into the function of ataxin-7 we analysed the expression of ataxin-7 in brain and peripheral tissue from SCA7 patients and controls. In brain, expression was found to be mainly neuronal with a nuclear subcellular localisation. Ataxin-7 expression was found throughout the CNS, not restricted to sites of pathology. We also confirmed previously reported findings of neuronal intranuclear inclusions (NIls) in the brains of SCA7 patients. Based on our findings, we conclude that the cell type specific neurodegeneration in SCA7 is not due to differences in expression pattern in affected and non-affected tissue or the distribution pattern of aggregated protein.
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| 52. |
- Kalezic, Ivana
(författare)
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Experimental studies of spinal mechanisms associated with muscle fatigue
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Muscle fatigue is ubiquitous in every day life.Muscle fatigue might be considered as an altered state of motor behaviour, which impairs motor performance. By contrast, muscle fatigue could also be considered a positive phenomenon, which protects muscle tissue from damage that might be incurred to it by overuse. The principal aim of the thesis was to explore some of the mechanisms of muscle fatigue at the spinal level in animal models.The activation of multiple motor units of a single calf muscle may influence contractile properties of its neighbouring, otherwise inactive units, providing evidence for spatial spreading of fatigue between different muscle parts. The release of metabolites, their action on inactive muscle units and the effects of local hypoxia are the most likely causes. Fatigue-induced metabolite shift in the interstitium provokes excitation and/or sensitisation of high-threshold afferent fibers, with complex effects on the spinal premotoneuronal network involved in the modulation of motoneuronal output. This was examined by studing the intrasegmental lamellar distribution of the lumbar spinal interneurons following fatiguing contractions of the triceps surae muscle. Furthermore, fatigue of calf muscles enhanced the activity of fusimotor neurons to these muscles irrespective of the regime of muscle activity (isometric vs. lengthening) in conditions that simulate locomotion. Altered fusimotor activity, through increased or maintained muscle spindle afferent responsiveness may be advantageous, providing support to the skeletomotor activity and enhanced information about muscle periphery to higher nervous centres. The particular effects of interneuronal network at motor input (presynaptic inhibition system) and output (recurrent inhibition system) stages were then addressed. Fatigue of triceps surae muscle induced a suppression of the monosynaptic reflex. The intensity of presynaptic inhibition increased, while the intensity of recurrent inhibition decreased. Post fatigue-evoked changes in monosynaptic reflexes and presynaptic inhibition indicate the possibility that high-threshold afferents inhibit group Ia terminals pre-synaptically, which would allow fatigue-induced signals from the muscle to reduce the relevance of proprioceptive feedback. Besides intrasegmental, intersegmental spreading of nociceptive signals was explored. Activation of sensory afferents from dorsal neck muscles by capsaicin induces powerful activation of interneurons located in the cervical spinal cord, as well as a widespread activation of cells in lumbar spinal cord segments. The results confirm the pivotal role of small diameter muscle afferents in the orchestration of segmental responses to fatigue and show complex interactions that may lead to limited accuracy of motor output. They also depict processes that may be related to, and even become precursors of chronic muscle pain.
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| 53. |
- Kallin, Kristina
(författare)
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Falls in older people in geriatric care settings : predisposing and precipitating factors
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Falls and their consequences are a major health problem in the older population, increasing their immobility, morbidity and mortality. This thesis focuses on older people living in geriatric care settings, frail older people who are most prone to suffer falls. The aim was to study predisposing and precipitating factors associated with falls in older people with or without cognitive impairment. In a cross-sectional study with a one-year prospective follow-up for falls 63% of the 83 residents suffered 163 falls and 65% of the fallers fell more than once. The antidepressants selective serotonine reuptake inhibitors (SSRIs), impaired vision and being unable to use stairs independently were the factors most strongly associated with sustaining falls. Acute diseases were judged to have precipitated 32 % of the falls and drug side effects 9%. In another cross-sectional study with a one-year follow-up for falls, including 199 residents, previous falls and treatment with antidepressants (mainly SSRIs) were found to be the most important predisposing factor for falls. Acute disease was judged to be the precipitating factor alone or in combination, in 39% of the falls, medical drugs in 8%, external factors such as obstacles in 8% and other conditions both related to the individual and the environment, such as misinterpretation, misuse of roller walkers or mistakes made by the staff were judged to have precipitated 17% of the falls. In a population-based cross-sectional study including 3604 residents in geriatric care settings more than 8% sustained a fall at least once during the preceding week. A history of falls, the ability to get up from a chair, the need for a helper when walking, pain, cognitive impairment, use of neuroleptics and use of antidepressants were all associated with falls in multivariate analyses. In the subgroup of people with cognitive impairment (2008 residents) more than 9% had sustained a fall at least once during the preceding week. As for the whole population, being able to get up from a chair, previous falls, needing a helper when walking with the addition of hyperactive symptoms were the factors independently associated with falls. In a study with a one-year prospective follow up for falls, including 439 residents in residential care facilities, 63% sustained 1354 falls, corresponding to an incidence rate of 3.5 falls / person year. Thirty-three percent of the falls and 37% of the injurious falls occurred during the night (9pm-6am). There were significantly higher fall rates in the evening and in January, April, May, November and December. There were no associations between fall rates and any of the weather parameters studied. In conclusion falls and fall-related injuries in older people in geriatric care settings are common. Both predisposing and precipitating factors contribute to the risk of falling. Addressing precipitating factors for falls seems to be important in an individualised preventive strategy among older people in geriatric care settings.
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| 54. |
- Karlsson, Berndt
(författare)
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Metabolic disturbances in shift workers
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- An increased risk for coronary heart disease among shift workers is earlier shown in the epidemiological literature. The aim of this thesis has been to penetrate metabolic disturbances and obesity among shift workers compared to day workers, and to compare if there are differences in total mortality or cause specific mortality of coronary heart disease (CHD), diabetes or ischaemic stroke in between the two groups. In an intervention study on female nurses (N=11), on night schedules in Umeå hospital, the highest peak value of glucose and insulin after meal ingestion was seen in the late evening (23:30). The post-prandial area under curve (AUC) of glucose and insulin was correspondingly largest after meal ingestion the same clock hour compared to meal ingestion other clock times. In two different cross-sectional studies Västerbotten Inventory Study (VIP) (N= 27,485) and in a subset of Work, Lipids and Fibrinogen Study (WOLF) (N= 1,324) metabolic differences in between shift and day workers has been evaluated. In both studies have obesity, high triglycerides and low HDL-cholesterol been more prevalent among the shift-working group compared to the day-working group. After adjustments for age and socio-economic factors in the VIP-study obesity and high triglycerides remained as risk factors in shift workers in both men and women. After directly age standardisation, a clustering effect, simultaneously, of two or more metabolic risk factors (obesity, hypertension, and high triglycerides) was seen in both genders among the shift workers compared to the day workers. Correspondingly, in the Wolf study low HDL-cholesterol and high triglycerides remained as significant risk factors after adjustments of confounders as age, socio-economic group, physical activity, current smoking, low social support and high job strain. In a cohort study from one company (MoDo) with two plants in the pulp and paper industry 2,354 male shift workers and 3,088 male day workers were followed from January 1, 1952 to December 31, 2001 regarding total and cause specific mortality due to CHD, diabetes and ischaemic stroke. Groups of workers defined by different duration of shift exposure were compared with day workers by calculating standardised relative rates (SRR). No increased risk of total mortality was seen among shift workers compared to day workers. Higher duration of shift work was associated with increased risk for CHD, and shift workers with 30 years or more had the highest risk. Diabetes was more common with increasing number of shift year exposure. Compared to day workers shift workers had also an increased risk to die because of ischaemic stroke, with the highest relative difference in the least shift exposed group (< 5 years).
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| 55. |
- Kavianipour, Mohammad, 1973-
(författare)
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Myocardial energy metabolism in ischemic preconditioning, role of adenosine catabolism
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Brief episodes of ischemia and reperfusion render the myocardium more resistant to necrosis from a subsequent, otherwise lethal ischemic insult. This phenomenon is called ischemic preconditioning(IP). Today, much is known about the signalling pathways involved in IP; however, the details of the final steps leading to cardioprotection, remain elusive. Adenosine (a catabolite of ATP) plays a major role in the signalling pathways of IP. Following IP there is an unexplained discrepancy between an increased adenosine production (evidenced by increased 5’-nucleotidase activity) and the successively lower adenosine levels observed in the interstitial space. We propose that this discrepancy in adenosine production vs. availability may be due to an increased metabolic utilisation of adenosine by the IP myocardium. According to our hypothesis, IP induces/activates a metabolic pathway involving deamination of adenosine to inosine. Inosine is further catalysed (in presence of Pi) to hypoxanthine and ribose-1-phosphate. Ribose-1-phosphate can be converted to ribose-5-phosphate in a phosphoribomutase reaction. Ribose-5-phosphate is an intermediate of the hexose monophosphate pathway also operative under anaerobic conditions. Hence the ribose moiety of adenosine can be utilised to generate pyruvate and ultimately ATP (via lactate formation) n.b. without any initial ATP investment. Such cost-effective adenosine utilisation may at least partly explain the cardioprotective effect of IP. Objectives & Methods: In the current studies we investigated the role of adenosine metabolism according to the suggested metabolic pathway by addition of adenosine and inhibition of its metabolism during IP as well as by comparing tissue and interstitial levels of key energy-metabolites following different protocols of IP. Furthermore, we studied the importance of the IP protocol with regard to the number of ischemia and reperfusion cycles for the cardioprotective effect of IP. In addition, the validity of the microdialysis technique for experimental in vivo studies of myocardial energy metabolism was evaluated. For these purposes the microdialysis technique, tissue biopsies, and planimetric infarct size estimation in an open chest porcine heart-model was used. Results: Addition of adenosine via microdialysis probes enhanced the interstitial release of inosine, hypoxanthine and lactate in the myocardium of IP-subjects during prolonged ischemia. This finding did not occur in non-preconditioned subjects. Similar addition of deoxyadenosine a non-metabolizable adenosine receptor-agonist, did not evoke the same metabolic response. Purine nucleoside phosphorylase (PNP) is responsible for the conversion of inosine to hypoxanthine being a key enzyme in the above mentioned metabolic pathway. Inclusion of 8' aminoguanosine (a competitive inhibitor of PNP) decreased interstitial hypoxanthine release (as a token of PNP inhibition) and increased the release of taurine (marker of cellular injury) in the ischemic IP myocardium. Addition of inosine (a natural substrate of PNP) reverted these changes. Four IP cycles protected the heart more than one IP cycle as evidenced by morphometric and energy-metabolic data.Proportionally more hypoxanthine was found in the myocardium of IP subjects during prolonged ischemia. The ratio of tissue levels of inosine/hypoxanthine (used as an indicator of PNP activity) was significantly smaller in the IP groups. In addition, myocardial interstitial levels of energy-related metabolites (lactate, adenosine, inosine, and hypoxanthine) obtained by the microdialysis technique correlated with tissue biopsy levels of corresponding metabolites. Conclusions: IP activated a metabolic pathway favouring metabolism of exogenous adenosine to inosine, hypoxanthine and eventually lactate. Inhibition of adenosine metabolism following IP (via inhibition of PNP-activity resulted in enhanced cellular injury.PNP-activity is proportionally higher in IP-myocardium. Metabolic utilisation of adenosine in IP-myocardium (as outlined above) may represent a costeffective way to produce ATP and at least partly explain the cardioprotective effect of IP. IP protects the myocardium in a graded fashion. Furthermore, we confirmed the validity of the microdialysis technique (in the current setting) for studying dynamic changes of myocardial energy metabolism.
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| 56. |
- Kjellgren, Daniel, 1963-
(författare)
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Human extraocular muscles : molecular diversity of a unique muscle allotype
- 2004
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Introduction: The extraocular muscles (EOMs) are considered a separate class of skeletal muscle, allotype. Myosin is the major contractile protein in muscle. The myosin heavy chain (MyHC) isoforms are the best molecular markers of functional heterogeneity of muscle fibers. The relaxation rate, reflects the rate at which Ca2+ is transported back into the sarcoplasmic reticulum (SR) mostly by SR Ca2+ATPase (SERCA). Myosin binding protein C (MyBP-C), plays a physiological role in regulating contraction. The laminins (Ln) are the major non-collagenous components of the basement membrane (BM) surrounding muscle fibers and are important for muscle fiber integrity. Methods: Adult human EOMs were studied with SDS-PAGE, immunoblots and immunocytochemistry, the latter with antibodies against six MyHC, 2 SERCA, 2 MyBP-C and 8 laminin chain isoforms. The capillary density was also determined. Results: Most fibers contained a mixture of MyHC isoforms. Three major groups of fibers could be distinguished. Fast fibers that stained with anti-MyHCIIa, slow fibers that stained with anti-MyHCI and MyHCeompos/MyHCIIaneg-fibers that stained with neither of these antibodies but with anti-MyHCI+IIa+eom and anti-MyHCeom. A majority of the fibers contained both SERCA1 and 2 whereas 1% were unstained with both antibodies. Biochemically SERCA2 was more abundant than SERCA1. MyBP-Cfast was not present in the EOMs and MyBP-Cslow was only detected immunocytochemically. The extrasynaptical BM of the EOM muscle fibers contained Lna2, b1, b2, g1, a4 and a5 chains. The capillary density in the EOMs was very high (1050 +/-190 capillaries/mm2) and significantly (p<0.05) higher in the orbital than in the global layer. Conclusions: The co-existence of complex mixtures of several crucial protein isoforms provide the human EOMs with a unique molecular portfolio that a) allows a highly specific fine-tuning regime of contraction and relaxation, and b) imparts structural properties that are likely to contribute to protection against certain neuromuscular diseases.
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| 57. |
- Kotova, Irina, 1972-
(författare)
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Purification of general RNA polymerase II transcription factors from mouse for studies of proliferation-specific transcription
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Accurate initiation of transcription by RNA polymerase II depends on general transcription factors (GTFs), which include the TATA-binding protein (TBP) and the transcription factors (TF) IIB, IIF, IIE and IIH. In order to reconstitute mouse transcription in vitro, we cloned the genes encoding mouse TFIIB, and both subunits of TFIIE and TFIIF from a mouse cDNA library. TBP and TFIIB were expressed in E.coli, while both subunits of TFIIE and the two subunits of TFIIF were expressed in a baculovirus system. All these factors were purified to > 90% homogeneity. The more complex transcription factors, TFIIH and RNA polymerase II, were purified more than 1000-fold and to near homogeneity, respectively, from tissue cultured mouse ascites cells. We have shown that the purified mouse transcription factors are active in a reconstituted RNA polymerase II in vitro transcription assay. The transcription reaction was inhibited by α-amanitine, and dependent on the addition of all the GTFs.Ribonucleotide reductase is a key enzyme in deoxyribonucleotide synthesis. It consists of two subunits, R1 and R2, which are both required for the enzyme activity. Transcription of the R1 and R2 genes is restiricted to the S-phase of the cell cycle, but the mechanisms that control this coordinated expression remain to be identified. We have studied initiation of transcription from the mouse R2 gene using a combination of in vivo reporter gene assays and in vitro transcription assays with crude nuclear extracts or with purified transcription factors. This promoter has an atypical TATA-box and a CCAAT-box that binds the transcription factor NF-Y.We found that a mutation in the R2 CCAAT-box had no effect on the transcription level in in vitro transcription assays reconstituted with pure transcription factors. However, it significantly decreased the level of transcription in similar experiments using crude nuclear extract. We also found that the sequence downstream from the R2 transcription start site (5´-UTR) (from +1 to +17 base pairs relative to transcription start site) is essential for initiation of transcription from this promoter. The presence of the wild type 5´-UTR made the R2 TATA-box redundant. On the other hand, the R2 5´-UTR had a repressing effect on transcription from the mouse R2 promoter. This region contains a palindrome sequence that covers 10 base pairs, and it is partially conserved in the human R2 promoter. Gel shift assays and in vitro transcription experiments using antibodies against mouse TAF4 (=TAF135) demonstrate that TAF4 is a component of the protein complex that interacts with this palindrome region, and suggest involvement of this component of the TFIID complex in negative regulation of the R2 promoter.The Adenovirus Major Late (AdML) promoter is commonly used as a model for studies of transcription initiation and regulation. It is a TATA-box dependent promoter, which also contains an initiator (Inr) element, a CCAAT-box interacting with transcription factor NF-Y, and an E-box binding the upstream stimulatory factor (USF). Using gel shift assays with recombinant NF-Y, USF, and immunopurified human TFIID, we show that binding of USF1 and NF-Y to DNA is not cooperative and that both factors independently facilitate binding of TFIID to the core promoter. The activation domains of NF-Y are expendable for this effect. Negative cofactor (NC2) comprises two subunits, which have a histone-fold structure similar to NF-Y, and represses transcription through formation of an inhibitory complex with TBP. Using an in vitro transcription system based on crude nuclear extracts, we show that NC2 has a negative effect on transcription in the presence of NF-Y or USF1, indicating that the two activators do not act as antirepressors. In vitro transcription using highly purified transcription factors efficiently reproduces repression of transcription by NC2. However, USF1 was inactive and NF-Y had a repressing effect in this system, which suggests that the activator functions of USF and NF-Y depend on cofactors.
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| 58. |
- Kottorp, Anders, 1965-
(författare)
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Occupation-based evaluation and intervention : validity of the assessment of motor and process skills when used with persons with mental retardation
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The ability to perform everyday life occupations is a critical component in both evaluation and intervention for persons with mental retardation (MR). While the ability to perform personal and instrumental activities of daily living (ADL) has always been important for occupational therapy (OT) practice, there is an absence in OT literature and research with a focus on ADL and persons with MR. The overall aim of this thesis was to evaluate the validity of the Assessment of Motor and Process Skills (AMPS) for evaluation and intervention of ADL ability for persons with MR.In order to evaluate the evidence of validity of the AMPS ability measures based on relation to level of MR, two groups of participants with MR were evaluated with the AMPS (,#=22; #= 39). The results indicated expected moderate relationships between ADL motor and ADL process ability measures and level of MR, despite different methods used for evaluating level of MR. The results also indicated that the results of the AMPS evaluation could be used to directly describe and measure the consequences in performance of ADL tasks for persons with different levels of MR.The evidence of validity of the AMPS was further examined in a study including participants with different types of developmental disabilities (e.g., MR, cerebral palsy, spina bifida) (#=1724). An application of many-faceted Rasch analysis was used to examine goodness-of-fit of the responses for the tasks, skill items, and participants included in the study. All tasks and all items except one demonstrated acceptable goodness-of-fit to the model on the ADL motor and ADL process scales. An expected proportion of participants demonstrated acceptable goodness-of-fit on the ADL motor scale. On the ADL process scale, a slightly lower proportion of participants than expected demonstrated acceptable goodness-of-fit. The results indicated further that persons with more severe levels of MR and persons with more limited ADL process abilities demonstrated different response patterns across tasks and possibly items.The evidence of validity of the internal structure of the AMPS scales was also evaluated between persons with mild and moderate MR (#=178; #=170). Group specific ADL motor and ADL process skill item hierarchies were generated using many-faceted Rasch analyses and compared. The hierarchies of ADL motor and ADL process skill items remained stable across groups, indicating evidence of validity of the AMPS scales when used to evaluate persons with MR. The results also indicated that although participants with moderate MR demonstrated overall lower mean ADL motor and ADL process ability, they did perform some specific ADL motor and ADL process skills at a similar level as persons with mild MR.Finally, the utility of the AMPS ability measures for detecting change were examined in an intervention study including three female participants with moderate MR. The study was based on a single case design and evaluated the effectiveness of a structured occupational therapy intervention program. Improvements were found for the participants in relation to the implementation of the program, but the pattern of changes were different between the participants and across the dependent variables. ADL process ability was the only variable that improved across all participants. The results supported the ADL process abilities as sensitive measures for detecting changes in ADL ability of persons with MR.In conclusion, the results of these studies contribute to the evidence of validity of the AMPS ability measures and scales, specifically in relation to the evaluation of persons with MR. The finding that an OT program resulted in improved ADL process ability also suggest that the results of the AMPS can be used to plan as well as evaluate outcomes of OT practice. Further research is also suggested in order to improve validity evidence and utility of the AMPS when used with persons with MR.
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| 59. |
- Kozlenkov, Alexey, 1973-
(författare)
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Structural / functional studies on human alkaline phosphatases
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The recent elucidation of the three-dimensional structure of human placental alkaline phosphatase (PLAP) has enabled me to perform structural studies aimed at characterizing the properties of human PLAP and tissue-nonspecific AP (TNAP) as paradigms for mammalian APs in general, using site-directed mutagenesis, protein expression, kinetic analysis and computer modeling. In Paper I, we found that a single critical E429G substitution explains the difference in stability and kinetics between the common allelic variants of PLAP and the D allozyme. In Paper II, we demonstrated the role of residue E429 in PLAP in stabilizing the active site metals, elucidated the distinct roles of residues H153 and H317 in catalysis, and the relative importance of five Cys residues in PLAP. We also discovered the significance of Y367, a unique feature of mammalian APs, for enzyme stability and specific inhibition by amino acids. Paper III focused on the identification and mutagenesis analysis of a novel, non-catalytic peripheral binding site of PLAP that appears to mediate a mitogenic effect of PLAP. This site provides indications that PLAP may function as a fetal growth factor. The last two papers focus on the TNAP isozyme as paradigm. A deficiency in TNAP activity is the cause of the human disease hypophosphatasia, characterized by rickets, osteomalacia and occasionally epileptic seizures. Paper IV has been able to partially explain the variable expressivity of hypophosphatasia traits by examining site-directed mutants of TNAP and performing kinetic analysis using natural substrates PPi and PLP. Finally, Paper V has clarified the mechanism of inhibition of TNAP by uncompetitive inhibitors L-homoarginine, levamisole and theophylline. We identified residues that confer to TNAP its distinct inhibitory properties. These data have significance for future drug design of specific TNAP inhibitors to therapeutically target TNAP as a way of elevating PPi extracellular level and alleviating pathological bone hypermineralization conditions.
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| 60. |
- Kroca, Michal, 1970-
(författare)
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Expansion of circulatory Vγ9Vδ2 T cells in tularemia and Pontiac fever, two intracellular bacterial diseases with widely different clinical expression
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Although well established that human Vγ9Vδ2 T cells may expand in circulation during intracellular bacterial infections, most underlying studies included only a few cases and only some diseases had been studied so far. In tularemia, a severe invasive disease, only one patient had been described. Legionellosis, including the mild flue-like Pontiac disease caused by Legionella micdadei, had not been studied at all. The aim of the present thesis was to study the circulatory Vγ9Vδ2-T cell response in these two intracellular bacterial diseases. The number of cases included was large enough to draw general conclusions. At various intervals, Vγ9Vδ2-T-cell counts and the capability of the cells to produce proinflammatory cytokines were assayed. Finally, the nature of the stimulating antigens was determined.In the acute phase of tularemia, we showed a marked increase of circulatory Vγ9Vδ2 T cells. When 181 samples from 108 patients with ulceroglandular tularemia were assayed, the percentage of Vγ9Vδ2 T cells was found to increase from ~5 to > 20% after the first week of disease. During the ensuing 24 months, levels were normalized. Vaccination with the live attenuated vaccine strain Francisella tularensis LVS, on the other hand, did not cause an increase in numbers of Vγ9Vδ2 T cells.Within an outbreak of Pontiac fever, 14 cases were well defined with regard to incubation time and onset of disease. In samples obtained 4 to 6 days after onset of disease, the mean percentage of Vγ9Vδ2 T cells was ~ 1%, i.e., 20% of normal values. Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset of disease, values were ~ 15%. Later, values slowly decreased. In both tularemia and Pontiac fever, the capacity of Vγ9Vδ2 T cells to produce TNF-α in response to phorbol myristate acetate in vitro was transiently decreased, in tularemia up to 6 weeks after onset of disease and in Pontiac fever in samples obtained 5-7 weeks after onset of disease.Nonpeptidic pyrophosphorylated molecules, referred to as phosphoantigens, are powerful stimuli for Vγ9Vδ2 T cells. Various strains of F. tularensis, including LVS, and a strain of L. micdadei were shown to produce Vγ9Vδ2 T-cell stimulating phosphoantigen. Notably, stimulation with an extract from each agent caused a similar degree of expansion of cells from subjects infected with the homologous and heterologous agent and also of cells from healthy subjects. Thus no immunospecific memory was detected in the Vγ9Vδ2-T cell response.Since it had been suggested that homologs of the conserved heat shock protein, chaperon-60, may be recognized by human Vγ9Vδ2 T cells, we determined the subpopulation of T cells responding to this protein as well as to DnaK, another heat-shock protein. Under in vitro conditions allowing a vigorous expansion of Vγ9Vδ2 T in response to a phosphoantigen, no expansion of γδ T cells in response to Cpn60 or DnaK of F. tularensis occurred. αβ T cells of tularemia-primed subjects, on the other hand, responded vigorously to the heat-shock proteins.In conclusion, two intracellular bacterial diseases with widely varying clinical expression were both associated with expansion of circulating Vγ9Vδ2 T cells. The expansion was prominent, long-lasting, and consistent within large numbers of individuals tested. In Pontiac fever, the expansion of Vγ9Vδ2 T cells was preceded by a depletion of the cells in circulation, implicating a possible extravasal migration into an infected site before the occurrence of rapid expansion and reentrance to blood. Both in tularemia and Pontiac fever, a modulation of the cytokine expression of Vγ9Vδ2 T cells was demonstrated in vitro, suggesting the presence of modulation of the inflammatory response. In extracts from in vitro culture of F. tularensis and L. micdadei, Vγ9Vδ2 T-cell stimulating phosphoantigens were identified and according to cross stimulation experiments, they induced expansion in vitro of Vγ9Vδ2 T cells without regard to immunospecific memory.
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