Characterization of heme binding to recombinant α1-microglobulin.

Alpha-1-microglobulin (A1M), a small lipocalin protein found in plasma and tissues, has been identified as a heme and radical scavenger that may participate in the mitigation of toxicities caused by degradation of hemoglobin. The objective of this work was to investigate heme interactions with A1M in vitro using various analytical techniques and to optimize analytical methodology suitable for rapid evaluation of the ligand binding properties of recombinant A1M versions

Differential gene expression in pristane-induced arthritis susceptible DA versus resistant E3 rats

Arthritis susceptibility genes were sought by analysis of differential gene expression between pristane-induced arthritis (PIA)-susceptible DA rats and PIA-resistant E3 rats. Inguinal lymph nodes of naïve animals and animals 8 days after pristane injection were analyzed for differential gene expression. mRNA expression was investigated by microarray and real-time PCR, and protein expression was analyzed by flow cytometry or ELISA. Twelve genes were significantly differentially expressed when analyzed by at least two independent methods, and an additional five genes showed a strong a tendency toward differential expression. In naïve DA rats IgE, the bone marrow stromal cell antigen 1 (Bst1) and the MHC class II β-chain (MhcII) were expressed at a higher level, and the immunoglobulin kappa chain (Igκ) was expressed at a lower level. In pristane-treated DA rats the MHC class II β-chain, gelatinase B (Mmp9) and the protein tyrosine phosphatase CL100 (Ptpn16) were expressed at a higher level, whereas immunoglobulins, the CD28 molecule (Cd28), the mast cell specific protease 1 (Mcpt1), the carboxylesterase precursor (Ces2), K-cadherin (Cdh6), cyclin G1 (Ccng1), DNA polymerase IV (Primase) and the tumour associated glycoprotein E4 (Tage) were expressed at a lower level. Finally, the differentially expressed mRNA was confirmed with protein expression for some of the genes. In conclusion, the results show that animal models are well suited for reproducible microarray analysis of candidate genes for arthritis. All genes have functions that are potentially important for arthritis, and nine of the genes are located within genomic regions previously associated with autoimmune disease

Arthritis induced in rats with non-immunogenic adjuvants as models for rheumatoid arthritis

Rat models are useful for studies of the pathogenesis of rheumatoid arthritis (RA) since rats are extraordinarily sensitive to induction of arthritis with adjuvants. Injection of not only the classical complete Freund's adjuvant but also mineral oil without mycobacteria and pure adjuvants such as pristane and squalene, induce severe arthritis in many rat strains. Models like pristane-induced arthritis in rats are optimal models for RA since they fulfill the RA criteria including a chronic relapsing disease course. Arthritogenic adjuvants like pristane, avridine, squalene and mineral oil are not immunogenic since they do not contain major histocompatibility complex (MHC) binding peptides. Nevertheless, the diseases are MHC-associated and dependent on the activation of alphabetaTCR (T-cell receptor)-expressing T cells. However, it has not been possible to link the immune response to joint antigens or other endogenous components although immunization with various cartilage proteins induce arthritis but with different pathogeneses. To unravel the mechanisms behind adjuvant-induced arthritis, a disease-oriented genetic approach is optimal. Several loci that control onset of arthritis, severity and chronicity of the disease have been identified in genetic crosses and most of these have been confirmed in congenic strains. In addition, many of these loci are found in other autoimmune models in the rat as well as associated with arthritis in mice and humans

The radical-binding lipocalin A1M binds to a Complex I subunit and protects mitochondrial structure and function.

Aims: During cell death, energy-consuming cell degradation and recycling programs are performed. Maintenance of energy-delivery during cell death is therefore crucial but the mechanisms to keep the mitochondrial functions intact during these processes are poorly understood. We have investigated the hypothesis that the heme- and radical-binding ubiquitous protein A1M (α1-microglobulin) is involved in protection of the mitochondria against oxidative insult during cell death. Results: Using blood cells, keratinocytes and liver cells, we show that A1M binds with high affinity to apoptosis-induced cells and is localized to mitochondria. The mitochondrial Complex I subunit NDUFAB1 was identified as a major molecular target of the A1M-binding. Furthermore, A1M was shown to inhibit the swelling of mitochondria, and to reverse the severely abrogated ATP-production of mitochondria when exposed to heme and ROS. Innovation: Import of the radical- and heme-binding protein A1M from the extracellular compartment confers protection of mitochondrial structure and function during cellular insult. Conclusion: A1M binds to a subunit of Complex I and has a role in assisting the mitochondria to maintain its energy delivery during cell death. A1M may also, at the same time, counteract and eliminate the ROS generated by the mitochondrial respiration to prevent oxidative damage to surrounding healthy tissue

"Vad fungerar bra? Vad fungerar mindre bra?" : Perspektiv på kartläggning kring elevers stödbehov samt vilket stöd som erbjuds elever.

Syftet med den här studien är att undersöka speciallärares och specialpedagogers erfarenheter av hur elevers behov av stödinsatser i grundskolan kartläggs, samt vilka insatser de erfar att kartläggningen kan leda till. Våra två frågeställningar blev: Hur bedrivs arbetet enligt våra informanter för att ta reda på vilka stödinsatser som en elev behöver för att nå minsta kunskapskrav? Vilket stöd erbjuds elever som riskerar att inte nå kunskapskraven? Vår sociokulturella teori är som Vygotskijuttrycker att samspelet med andra människor är viktigt vid all socialutveckling och lärande.  För att få svar på vårt syfte och frågeställningar intervjuade vi 22 specialpedagoger och speciallärare vid grundskolor utspridda i Sverige. Vi använde oss av en kvalitativ forskningsmetod. I studiens resultat framkom att det finns olika vägar för informationen att nåfram till specialpedagog och speciallärare samt till elevhälsoteamet. Fleraskolor screenar kontinuerligt eleverna i varje årskurs för att upptäcka elever i behov av stöd på olika sätt. Kartläggningsprocessen och stödinsatserna såg olika ut mellan informanternas skolor. Stödet kunde röra sig om att medverka i bildandet av grupper, olika digitala material, intensivkurser för att på kort tid komplettera kunskapsluckor, stöd utanför klassrummet och mindre grupper. Vår slutsats är att det finns en stor variation i vårt lands skolor på hur kartläggningen går till och vilken form av stödåtgärder i och utanför klassrummet som ges till elever som inte når målen

"Vad fungerar bra? Vad fungerar mindre bra?" : Perspektiv på kartläggning kring elevers stödbehov samt vilket stöd som erbjuds elever.

Syftet med den här studien är att undersöka speciallärares och specialpedagogers erfarenheter av hur elevers behov av stödinsatser i grundskolan kartläggs, samt vilka insatser de erfar att kartläggningen kan leda till. Våra två frågeställningar blev: Hur bedrivs arbetet enligt våra informanter för att ta reda på vilka stödinsatser som en elev behöver för att nå minsta kunskapskrav? Vilket stöd erbjuds elever som riskerar att inte nå kunskapskraven? Vår sociokulturella teori är som Vygotskijuttrycker att samspelet med andra människor är viktigt vid all socialutveckling och lärande.  För att få svar på vårt syfte och frågeställningar intervjuade vi 22 specialpedagoger och speciallärare vid grundskolor utspridda i Sverige. Vi använde oss av en kvalitativ forskningsmetod. I studiens resultat framkom att det finns olika vägar för informationen att nåfram till specialpedagog och speciallärare samt till elevhälsoteamet. Fleraskolor screenar kontinuerligt eleverna i varje årskurs för att upptäcka elever i behov av stöd på olika sätt. Kartläggningsprocessen och stödinsatserna såg olika ut mellan informanternas skolor. Stödet kunde röra sig om att medverka i bildandet av grupper, olika digitala material, intensivkurser för att på kort tid komplettera kunskapsluckor, stöd utanför klassrummet och mindre grupper. Vår slutsats är att det finns en stor variation i vårt lands skolor på hur kartläggningen går till och vilken form av stödåtgärder i och utanför klassrummet som ges till elever som inte når målen

Chronicity of pristane-induced arthritis in rats is controlled by genes on chromosome 14

To, address; he possibility, that genes specifically control the chronic phase of arthritis we have isolated a congenic fragment from the resistant E3; rat cm the, susceptible DA rat background. The isolated fragment covers the Pia6 quantitative trait locus on chromosome 14, which previously has been identified to be linked to chronic pristane induced arthritis (PIA) in gene segregation experiments; of an (E3 x DA) F-2-cross. Heterozygous. Pia6 congenic rats (DA.Pia6) were protected from chronic active arthritis specifically, as determined by macroscopic scoring, histopathology and release of cartilage oligomeric matrix protein (-reflecting ongoing cartilage destruction). The difference was seen only during the chronic active phase of arthritis starting approximately 5 weeks after onset of arthritis. Interestingly, the plasma concentration of the lipocalins alpha(1)-acid glycoprotein and alpha(1)-microglobulin was found significantly altered in naive congenic rats compared to the DA rat. The concentration of alpha(1)-microglobulin was found to be significantly higher throughout the disease course, while alpha(1)-acid glycoprotein had a lower concentration in naive rats, which was highly significant in the chronic phase. The altered concentrations of these proteins already before development of chronic arthritis may provide a clue to the pathogenic process controlled by the Pia6 genes. We conclude that the active relapsing chronic arthritis is under a unique genetic control that is different from the control of acute arthritis and postulate that the liver plays an important role in this relagation. (C) 2003 Elsevier Ltd. All rights reserved.

Expression of a functional proteinase inhibitor capable of accepting xylose: bikunin

Bikunin is a Kunitz-type proteinase inhibitor, which is cross-linked to heavy chains via a chondroitin sulfate chain, forming inter-alpha-inhibitor and related molecules. Rat bikunin was produced by baculovirus-infected insect cells. The protein could be purified with a total yield of 20 mg/liter medium. Unlike naturally occuring bikunin the recombinant protein had no galactosaminoglycan chain. Endoglycosidase digestion also suggested that the recombinant form lacked N-linked oligosaccharides. Bikunin is translated as a part of a precursor, alpha1-microglobulin/bikunin, but the functional significance of the cotranslation is unknown. Our results indicate that the proteinase inhibitory function of bikunin is not regulated by the alpha1-microglobulin-part of the alpha1-microglobulin/bikunin precursor since recombinant bikunin had the same trypsin inhibitory activity as the recombinant precursor. Both free bikunin and the precursor were also functional as a substrate in an in vitro xylosylation system. This demonstrates that the alpha1-microglobulin-part is not necessary for the first step of galactosaminoglycan assembly

Older people with swallowing dysfunction and poor oral health are at greater risk of early death

Objectives: We investigated the associations between swallowing dysfunction, poor oral health and mortality among older people in intermediate care in Sweden. Methods: This prospective cohort study investigated 391 older people in 36 intermediate care units (clusters). Swallowing function was assessed with the timed water swallow test (TWST), and oral health with the revised oral assessment guide (ROAG) at baseline. Data were collected on age, sex, education level, multimorbidity, cognitive impairment, care dependency and body mass index (BMI). Time to mortality was recorded during the following year. The mixed effects Cox regression model with cluster as a random factor was used to estimate hazards ratios (HR) with 95% confidence intervals (CI). Results: The median age of the participants was 84 years (interquartile range [IQR]: 11), and 53.3% were females. Mortality within one year was 25.1%. In the adjusted model, swallowing dysfunction and poor oral health were both independently associated with mortality (adjusted HR [aHR]: 1.67, 95% CI 1.02‐2.75; P = .041 and aHR: 1.98, 95% CI 1.07‐3.65; P = .029, respectively). Participants with combined swallowing dysfunction and poor oral health showed the highest mortality (35.0%) and 2.6 (95% CI 1.15‐5.89; P = .022) times higher mortality risk than those with normal swallowing function and good oral health (13.0%). Conclusions: Swallowing dysfunction and poor oral health were identified as independent risk factors for mortality in older people in intermediate care. Although further studies are required to verify these findings, they suggest that systematic assessment of swallowing function and oral health status should be performed for care considerations.Originally included in thesis in manuscript form</p

Carbohydrate groups of alpha1-microglobulin are important for secretion and tissue localization but not for immunological properties

The role of the carbohydrates for the structure and functions of the plasma and tissue protein alpha1-microglobulin (alpha1m) was investigated by deletion of the sites for N-glycosylation by site-directed mutagenesis (N17,96-->Q). The mutated cDNA was expressed in a baculovirus-insect cell system resulting in a nonglycosylated protein. The biochemical properties of N17,96Q-alpha1m were compared to nonmutated alpha1m, which carries two short non-sialylated N-linked oligosaccharides when expressed in the same system. Both proteins carried a yellow-brown chromophore and were heterogeneous in charge. Circular dichroism spectra and antibody binding indicated a similar overall structure. However, the secretion of N17,96Q-alpha1m was significantly reduced and approximately 75% of the protein were found accumulated intracellularly. The in vitro immunological effects of recombinant nonmutated alpha1m and N17,96Q-alpha1m were compared to the effects of alpha1m isolated from plasma, which is sialylated and carries an additional O-linked oligosaccharide. All three alpha1m variants bound to human peripheral lymphocytes and mouse T cell hybridomas to the same extent. They also inhibited the antigen-stimulated proliferation of peripheral lymphocytes and antigen-stimulated interleukin 2-secretion of T cell hybridomas in a similar manner. After injection of rats intravenously, the blood clearance of recombinant nonmutated and N17,96Q-alpha1m was faster than that of plasma alpha1m. Nonmutated alpha1m was located primarily to the liver, most likely via binding to asialoglycoprotein receptors, and N17,96Q-alpha1m was located mainly to the kidneys. It is concluded that the carbohydrates of alpha1m are important for the secretion and the in vivo turnover of the protein, but not for the structure or immunological properties