CD47 is a cell surface glycoprotein that is expressed by virtually all cells in the body. Binding of CD47 to the macrophage receptor Signal Regulatory Protein alpha (SIRPα) yields an inhibitory signal that counteracts phagocytosis. Red blood cells (RBCs) that lack CD47 are rapidly cleared from the circulation, whereas CD47 expressing cells have a normal turnover rate. CD47 has therefore been proposed to function as a marker of self, enabling the immune system to discriminate between self and foreign. Thus, the studies of the present thesis aimed at further investigating the role of CD47 as a marker of self in regulating phagocytosis of platelets, phagocytosis of viable or senescent RBCs, and the mechanisms involved. CD47 on platelets was found to regulate their turnover in vivo, since platelets from CD47-/- mice transfused into wild type recipients were cleared more rapidly from the circulation than wild type platelets. In addition, CD47-/- mice were found to suffer from a mild spontaneous thrombocytopenia, without any signs of accelerated platelet apoptosis or increased platelet activation. CD47-/- mice were more sensitive to experimental immune thrombocytopenia (ITP), as compared with wild type mice. In vitro phagocytosis experiments proved that platelet CD47 was responsible for this effect, since blocking antibodies to macrophage SIRPα increased phagocytosis of wild type platelets to the levels seen for CD47-/- platelets. When unopsonized platelets or RBCs from CD47+/- mice (expressing about 50 % less CD47 than wild type cells) were transfused into wild type recipients, they were cleared from the circulation at virtually the same rate as wild type cells. However, CD47+/- cells were cleared more rapidly than wild type cells when transfused animals were challenged with an antibody directed against the transfused cell type. In vitro, IgG-opsonized CD47+/- platelets and RBCs were ingested to a higher extent than wild type cells, but less than CD47-/- cells, suggesting that CD47 dose-dependently regulates phagocytosis in macrophages. It was also investigated if inhibitory SIRPα signaling is localized to the site of contact with the cell that is to be ingested, or whether the inhibition of phagocytosis is more general in the whole macrophage. Experiments with a mix of IgG-opsonized wild type and CD47-/- RBCs showed that the effect of inhibitory CD47-SIRPα signaling was local in the macrophage and limited to the site of contact with a specific target cell. Thus, contact with one or several wild type RBCs did not affect the increased phagocytosis of CD47-/- RBCs by the same macrophage. RBC senescence involves oxidation of membrane lipids and proteins, as well as exposure of phosphatidylserine (PS) on the cell surface, and clearance of senescent RBCs is believed to be regulated by several different factors. To investigate the role of CD47 in uptake of experimentally senescent RBCs, RBCs were oxidized with CuSO4/ascorbic acid (Ox-RBCs). Phagocytosis of Ox-RBCs required recognition of PS on the RBCs, recognition by scavenger receptors on the macrophages, and was strongly dependent on serum. CD47 did not inhibit serum-dependent phagocytosis of experimentally senescent unopsonized RBCs, since phagocytosis of senescent wild type or CD47-/- RBCs was virtually similar. The ability of CD47 to cluster in the plasma membrane upon cross-linking with antibodies was reduced in senescent RBCs. Despite this, CD47 inhibited phagocytosis of IgG-opsonized viable or senescent RBCs to the same extent. In summary, CD47 can function as a marker of self on both RBCs and platelets. The phagocytosis-inhibitory effect is dependent on the CD47 expression level, and CD47-SIRPα signaling acts locally in the macrophage at the contact with a target cell. In experimentally senescent RBCs, CD47 does not inhibit serum-dependent phagocytosis in the absence of opsonization, but still inhibits FcγR-mediated phagocytosis. Key words: CD47, SIRPα, platelets, red blood cells, macrophages, phagocytosis, Fcγ receptor, senescence

Parkinson’s disease (PD) is a progressive neurodegenerative disease and characterized by the loss of dopaminergic (DA) neurons in the substantia nigra in the midbrain. The causes of the disease are still unknown. The most commonly used treatment is administration of L-DOPA, however, another possible treatment strategy is to transplant DA neurons to the striatum of PD patients to substitute the loss of neurons. Clinical trials have demonstrated beneficial effects from transplantation, but one obstacle with the grafting trials has been the variable outcome, where limited graft reinnervation of the host brain is one important issue to solve. To improve and control the graft DA nerve fiber outgrowth organotypic tissue cultures can be utilized. Cultures of fetal ventral mesencephalon (VM) have been used to investigate astrocytic migration and dopamine nerve fiber formations at different time points and under varying conditions to study how to control nerve fiber formation. The early appearing DA nerve fibers as revealed by tyrosine hydroxylase (TH) –immunoreactivity, form their fibers in the absence of glial cell bodies, are not persistent over time, and is called non-glial-associated TH-positive nerve fiber outgrowth. A monolayer of astrocytes guides a second persistent subpopulation of nerve fibers, the glial-associated TH-positive nerve fiber formation. Investigations of the interactions between the astrocytic migration and nerve fiber formations were made. In embryonic (E) day 14 VM cultures the mitosis of the astrocytes was inhibited with the antimitotic agent β-D-arabinofuranoside. The results revealed decreased astrocytic migration, reduced glial-associated TH-positive outgrowth, and enhanced presence of the non-glial-associated TH-positive outgrowth in the cultures. Thus, astrocytes affect both the non-glial- and the glial-associated growths by either its absence or presence, respectively. The astrocytes synthesize proteoglycans. Therefore the nerve fiber formation was studied in VM or spinal cord cultures treated with the proteoglycan blockers chondroitinase ABC (ChABC), which degrades the proteoglycans, or methyl-umbelliferyl-β-D-xyloside (β-xyloside), which blocks the proteoglycan synthesis. β-xyloside inhibited the migration of the astrocytes and the outgrowth of the glial-associated TH-positive nerve fibers in both VM and spinal cord cultures, whereas ChABC treatment had no effect in E14 VM or spinal cord cultures. E18 VM and spinal cord cultures were evaluated to investigate how the different developmental stages influence astrocytes and the two nerve fiber formations after 14 DIV. No nerve fiber formation was found in E18 VM cultures, while the non-glial-associated nerve fiber outgrowth was obvious as long and robust fibers in E18 spinal cord cultures. The astrocytic migration was similar in VM and spinal cord cultures. β-xyloside and ChABC did not affect nerve fiber growth but astrocytic migration in E18 VM cultures, while no effects was found in the spinal cord cultures. However, the neuronal migration found in control cultures was abolished in both VM and spinal cord cultures after both ChABC and β-xyloside. Neuroinflammation plays a critical role in the development of PD. Increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) are observed in postmortem PD brains and the levels of TNFα receptors on circulating T-lymphocytes in cerebrospinal fluid of PD patients are increased. The effects of TNFα were studied on E14 VM cultures. The outgrowth of the non-glial-associated TH-positive nerve fibers was inhibited while it stimulated astrocytic migration and glial-associated TH-positive nerve fiber outgrowth at an early treatment time point. Furthermore, blocking the endogenous levels of TNFα resulted in cell death of the TH-positive neurons. Furthermore, cultures of E14 mice with gene deletion for the protein CD47 were investigated. CD47 is expressed in all tissues and serves as a ligand for the signal regulatory protein (SIRP) α, which promotes e.g migration and synaptogenesis. CD47-/- cultures displayed massive and long non-glial-associated TH-positive nerve fiber outgrowth despite a normal astrocytic migration and the presence of glial-associated TH-positive nerve fiber outgrowth. For the first time, it was observed that the non-glial-guided TH-positive nerve fiber outgrowth did not degenerate after 14 DIV. Taken together, there is an interaction between astrocytes and TH-positive nerve fiber formations. Both nerve fiber formations seem to have their task during the development of the DA system.

Gallstas är ett patologiskt tillstånd där antingen produktionen eller flödet av galla är hämmat. Orsaken kan vara mekaniskt stopp och kallas då för extrahepatisk eller annan underliggande kronisk leversjukdom och kallas då för intrahepatisk. Vid gallstas samlas kopparassocierat protein (KAP) i levern. Dessa proteiner kan synliggöras med orcein. Orcein är en histokemisk färg som färgar KAP, och elastiska fibrer. Syftet med arbetet är att utifrån etablerade orceinprotokoll utföra avvikelser för att se hur dessa påverkar orceins förmåga att synliggöra elastiska fibrer. Metoden ska valideras med positivt kontrollmaterial för att på sikt användas inom diagnostik för att påvisa KAP i levervävnad vid avdelningen för Klinisk patologi i Region Kalmar Län. Som kontrollmaterial användes tunntarm och extransplanterad cirrotisk lever. Initialt testades etablerade protokoll och utifrån resultaten gjordes fortsatta tester. Fortsatta tester inkluderade (i) varierande inkubationstider i orcein vid rumstemperatur samt vid 60°C, (ii) varierande koncentration av surgjord kaliumpermanganat samt varierande inkubationstid i orcein i rumstemperatur, (iii) jämförelse mellan orcein från två olika tillverkare (HistoLab och RAL-diagnostic), samt (iv) validering av metod med positivt kontrollmaterial, vid varierande inkubationstid i värme (60°C) och med orcein från annan tillverkare (HistoLab). Resultaten visade att elastiska fibrer och bakgrund färgades mer intensivt vid längre inkubationstid samt att värme minskar bakgrunden i tunntarm. Högre koncentrationer kaliumpermanganat påverkade inte resultatet märkbart förutom vid längre inkubationstid där bakgrunden blev svagare. Stor skillnad upptäcktes mellan olika tillverkare av orcein. Vid infärgning av lever syntes KAP först efter infärgning över natt med RAL-diagnostics orcein och efter enbart 30 minuter med HistoLabs orcein. Bakgrunden i lever är allmänt högre än i tunntarm. Den slutsats som drogs av arbetet är att elastiska fibrer lämpligast synliggörs vid lång inkubationstid i 60°C. KAP synliggörs med båda varianterna av orcein, vidare tester är däremot nödvändigt. / Cholestasis is a pathological condition where bile flow from the liver to the intestines is somehow obstructed. Obstruction can result from mechanical blockage of the ducts (called extrahepatic) or metabolic inhibition (called intrahepatic). During cholestasis copper associated protein accumulate in the liver. These proteins can be made visible with the use of orcein. Orcein is a histochemical stain that stain copper associated proteins and elastic fibres. The purpose of this thesis was to use established protocols to stain elastic fibres and to make know deviations from these protocols to studied orceins ability to stain these structures. Validation of the method shall be performed with known copper associated protein material, so that the method can be used in the long run for diagnostic use at the department of Clinical Pathology, Region Kalmar County. Initial tests were conducted to compare the different protocols. Based on the results further testing was done. These tests included variation in temperature, variation in incubation time in orcein, variation in potassium permanganate concentration and testing the outcome of orcein from different manufacturers (RAL-diagnostic and HistoLab).  A validation was also conducted. The results showed that elastic fibres gained increased visibility with longer incubation time, higher temperatures increased visibility of elastic fibres even further and reduced background. Higher concentrations of potassium permanganate had limited effect, though it seemed to reduce background staining at longer incubations. Orcein from different manufacturers was also showed to preform very differently, with HistoLab giving best results. Copper associated proteins was visualised in liver with both types of orcein. The results showed a faster staning with orcein from HistoLab (30 min) than orcein from RAL-diagnostic (overnight). The conclusion is that elastic fibres is best visualised with orcein in 60°C and with longer incubation time. Visualisation of copper associated proteins can be done with orcein from both manufacturers, although further testing is required.

Hjärt-kärlsjukdomar är de vanligaste dödsorsakerna i Sverige och under 2016 stod de för 35 % av alla dödsfall i Sverige. Vid behandling av hjärtinfarkt är målet att återställa blodflödet till hjärtat. Trots att återställandet av blodflödet (reperfusion) till hjärtat leder till förbättrat kort- och långsiktigt hälsotillstånd så kan reperfusionen leda till irreversibla skador såsom kardiell kontraktionsbandsnekros. Luxol-fast-blue är en specialfärgning som används för att undersöka myelin samt visats färga in myofibrilla degenereringar såsom kontraktionsbandsnekros. Syftet med detta examensarbete var att göra det möjligt att påvisa akut hjärtinfarkt med luxor-fast-blue, en mer specifik färgmetod istället för rutindiagnostisk klassisk hematoxylin och eosin-färgning. Arbetet utfördes genom att vävnad från hjärta med känd hjärtinfarkt förbereddes och färgades med luxor-fast-blue. Metoden för infärgning med luxor-fast-blue modifierades för att uppnå optimal infärgning av kontraktionsbandsnekros vid hjärtinfarkt. De olika modifikationerna som gjordes var ändrad tid vid infärgning, differentiering och kärninfärgning. Resultatet av arbetet visade att vävnaden, med hjärtinfarkt, som färgades med luxor-fast-blue över natt, differentieringen under 50 sekunder och kärninfärgning med nuclear-fast-red under en minut var den mest optimala infärgningen av kontraktionsbandsnekros vid hjärtinfarkt. Där kunde blå infärgade kontraktionsband visualiseras i hjärtinfarktsbiopsin medan resterande celler var infärgade rött.

Neutrophil granulocytes play an important role in the host defence against invading microorganisms and constitute the frontline of defence within the innate immune system and are among the first cells to arrive at the site of inflammation. Effective phagocytosis and killing of invading pathogens by neutrophils is of significant importance for successful resistance to infectious diseases. An important complication in diabetes mellitus is an increased sensitivity to infections and increased tissue damage, leading to many secondary diseases. This may in part be explained by an impaired function of neutrophil granulocytes. Since the exact mechanisms underlying defective neutrophil function in diabetes mellitus are not fully understood, the aim of the present study was to investigate the effects of elevated glucose and insulin concentrations on phagocytosis of opsonized yeast and on production of reactive oxygen metabolites (ROS) in normal human neutrophils. Elevated D-glucose concentrations (15-25 mM) inhibited the phagocytosis of C3bi- or IgG-opsonized yeast particles, which was neither an osmotic effect nor an effect due to reduced binding of opsonized yeast particles to the neutrophils. Inhibition of protein kinase C (PKC) by GF109203X or Go6976 could completely reverse the inhibitory effect of 25 mM D-glucose on phagocytosis. Diacylglycerol (DAG) dose-dependently inhibited phagocytosis and suboptimal inhibitory concentrations of DAG and glucose showed an additive inhibitory effect. Elevated concentrations of insulin (80-160 μU/ml) also inhibited neutrophil phagocytosis, an effect shown in part to be due to a delayed phagocytosis process. Insulin was found to increase the accumulation of cortical F-actin, without affecting the total cellular F-actin content. The PKCalpha/beta inhibitor, Go6976, abolished the insulin-mediated increase in cortical F-actin content and both Go6976 and the PKCalpha/beta/delta/epsilon-specific inhibitor GF109203X reversed the inhibitory effects of insulin on phagocytosis. The inhibition of phagocytosis by either glucose or insulin resulted in an expected reduction of intracellular respiratory burst. However, the extracellular release of ROS during phagocytosis was increased by insulin, but inhibited by glucose. The ability of insulin to enhance ROS production was found to be F-actin dependent. Data suggests that glucose inhibited intracellular respiratory burst activation by interfering with intracellular signaling downstream of PKC activation, whereas extracellular release of ROS was inhibited by glucose upstream of PKC signaling. Taken together these results suggest that both hyperglycemia and hyperinsulinemia inhibit complement receptor and Fc receptor-mediated phagocytosis in human neutrophils. Insulin, but not glucose, also induced an enhanced extracellular release of ROS during phagocytosis. The combination of reduced phagocytosis and alterations in ROS production may possibly explain both the increased sensitivity to infections and tissue damage seen in type 2 diabetes.

Parkinson’s disease is a progressive neurodegenerative disorder, characterized by a depletion of the dopaminergic neurons in the substantia nigra. The cause of the disease is yet unknown but age, oxidative stress, and neuroinflammation are some of the features involved in the degeneration. In addition, substantial cell death of noradrenergic neurons occurs in the locus coeruleus (LC). Noradrenaline has been suggested to protect the dopamine neurons from oxidative stress and neuroinflammation. The main treatment of Parkinson’s disease is Levo-dopa, although severe side effects arise from this therapy. Hence, grafting fetal ventral mesencephalic (VM) tissue into the adult striatum has been evaluated as an alternative treatment for Parkinsons’s disease. However, the survival of the grafted neurons is limited, and the dopamine-denervated striatum does not become fully reinnervated. Therefore, elucidating factors that enhance dopamine nerve fiber formation and/or survival of the grafted neurons is of utmost importance. To investigate dopamine nerve fiber formation and the interactions with glial cells, organotypic VM tissue cultures were utilized. Two morphologically different nerve fiber outgrowths from the tissue slice were observed. Nerve fibers were initially formed in the absence of migrating astrocytes, although thin vimentin-positive astrocytic processes were detected within the same area. A second, persistent nerve fiber outgrowth was observed associated with migrating astrocytes. Hence, both of these nerve fiber outgrowths were to some extent dependent on astrocytes, and appeared as a general feature since this phenomenon was demonstrated in β-tubulin, tyrosine hydroxylase (TH), and aldehyde dehydrogenase A1 (ALDH1)-positive nerve fibers. Neither oligodendrocytes (NG2-positive cells), nor microglia (Iba-1-positive cells) exerted any effect on these two neuronal growths. Since astrocytes appeared to influence the nerve fiber formation, the role of proteoglycans, i.e. extracellular matrix molecules produced by astrocytes, was investigated. β-xyloside was added to the cultures to inhibit proteoglycan synthesis. The results revealed a hampered astrocytic migration and proliferation, as well as a reduction of the glia-associated TH-positive nerve fiber outgrowth. Interestingly, the number of cultures displaying the non-glia-mediated TH-positive nerve fibers increased after β-xyloside treatment, although the amount of TH-protein was not altered. Thus, proteoglycans produced by astrocytes appeared to be important in affecting the dopamine nerve fiber formation. The noradrenaline neurons in LC have been suggested to protect dopamine neurons from damage. Therefore, the interaction between VM and LC was evaluated. Using the intraocular grafting method, fetal VM and LC were grafted either as single grafts or as VM+LC co-grafts. Additionally, the recipient animals received 2% blueberry-enriched diet. The direct contact of LC promoted graft volume and survival of TH-positive neurons in the VM grafts. The number of dopamine neurons, derived preferably from the A9 (ALDH1/TH-positive) was increased, whereas the dopamine neurons from the A10 (calbindin/TH-positive) were not affected. A dense dopamine-β-hydroxylase (DBH)-positive innervation was correlated to the improved survival. Blueberry-enriched diet enhanced the number of TH-positive neurons in VM, although the graft size was not altered. The combination of blueberries and the presence of LC did not yield additive effects on the survival of VM grafts. The attachment of VM or the addition of blueberries did not affect the survival of TH-positive neurons in LC grafts. The number of Iba-1-positive microglia was decreased in co-grafted VM compared to single VM transplants. The addition of blueberries reduced the number of Iba-1-positive microglia in single VM transplants. Hence, the direct contact of LC or the addition of blueberries enhanced the survival of VM grafts. Taken together, these data demonstrate novel findings regarding the importance of astrocytes for the nerve fiber formation of dopamine neurons. Further, both the direct attachment of LC or antioxidant-enriched diet promote the survival of fetal VM grafts, while LC is not affected.

Fouchet är specialiserad färgningsmetod för att påvisa gallpigment i leverpreparat. Fouchet-reagens innehåller järnklorid som oxiderar gult bilirubin till grönt biliverdin i närvaro av triklorättiksyra och ger en klar grön färg på leverns gallpigment. Ökad mängd gallpigment kan ses vid sjukdom som exempelvis vid hepatocellulär cancer. Syftet med arbetet var att utifrån befintliga protokoll optimera infärgning av bindväv och gallpigment i levervävnad med hjälp av manuell histopatologisk infärgning med Fouchet. För att diagnostisera leversjukdomar där ökat gallpigment förekommer nyttjas infärgning med Fouchet vid avdelningen för Klinisk patologi i Region Kalmar län och infärgningen var i behov av optimering. Vävnad från tarm och lever användes. Metoden optimerades genom förändringar av inkubationstiden med Fouchet-reagens och van Gieson-lösning samt genom tillsats av ren pikrinsyra. Bedömningskriterierna bestod av infärgning av bindväv, muskler och gallpigment samt utvärdering av kontrast mellan bindväv, muskler och gallpigment. Bedömningen av den infärgade vävnaden gjordes av legitimerad patolog. Resultatet visade att den mest optimala färgningsmetoden var infärgning i elva minuter Fouchet följt av tre minuter ren pikrinsyra samt två minuter van Gieson. Infärgningen gav bäst bakgrundsfärg och klarast infärgning av grönt gallpigment. Slutsatsen var att den nya manuella infärgningen med Fouchet samt extra pikrinsyra utan sköljning i destillerat vatten visade ett så pass bra resultat att metoden har potential att ersätta det nuvarande infärgningssprotokollet (ursprungligt färgningsprotokoll) vid avdelningen för Klinisk patologi i Region Kalmar län. / Fouchet dye specializes in detecting bile pigments in the liver. Fouchet-reagent contains iron chloride, which oxidizes yellow bilirubin to green biliverdin in the presence of trichloroacetic acid and gives a clear green color to the liver bile pigment. Increased amounts of bile pigments can be obtained in diseases such as hepatocellular cancers. The work aimed to optimize the staining of connective tissue and bile pigments in liver tissue based on existing protocols with the help of manual histopathological staining with Fouchet. Staining with Fouchet is used at the Clinical Pathology in Kalmar to diagnose liver diseases with bile pigment. Tissue was taken from the intestine and liver. The method was optimized by changes of incubation times of tissues stained with Fouchet reagent, van Gieson solution, and the addition of pure picric acid. Assessment criteria consisted of staining of connective tissue, muscles, and bile pigments and evaluation of contrast between connective tissue, muscles, and bile pigments. The assessment of the stained tissue was made by a licensed pathologist. Optimal results were obtained with eleven minutes Fouchet followed by three minutes picric acid and two minutes van Gieson. The best-stained liver tissue was based on a consistent background color and evidence of green bile pigment staining. The conclusion was that the new manual staining with Fouchet and extra picric acid without rinsing in distilled water showed such good results that the method can potentially replace the current staining protocol at Clinical Pathology in Kalmar.

Pancreatic ß-cells are heterogeneous in their secretory responsiveness, glucose sensitivity and metabolic rate. A diminished and delayed first-phase insulin release is an early sign of failing ß-cells in diabetes. Mechanisms controlling functional characteristics, such as lag time for insulin release or magnitude of the response in each individual cell are unknown. To find out whether the heterogeneity represents a random phenomenon in ß-cell or is a manifestation of reproducible characteristics, we compared parameters of Ca2+ response in Fura-2 labelled ob/ob mouse ß-cells during two consecutive stimulations with glucose. Lag times, as well as peak heights and nadirs of initial lowering showed a strong correlation between the first and second stimulation. Thus, timing and magnitude of the early Ca2+ response were specific for each cell. ß-Cells from lean mice, diabetic db/db mice and rats also showed cell-specific responses characteristics. This indicates that a cell-specific Ca2+ response to glucose is common in rodent ß-cells, both normal and diabetic. Another question was whether aggregated ß-cells show cell-specific responses. Using the same protocol as for dispersed ß-cells, we analysed Ca2+ responses in clusters of different size and in intact islets from ob/ob and lean mice. Correlations were found between the first and second stimulation for timing and magnitude of [Ca2+]i rise, and for the initial lowering. Next, we tested if the ß-cell response is cell-specific, when induced at different steps of the stimulus-secretion coupling. The glycolytic intermediate glyceraldehyde, the mitochondrial substrate KIC, the KATP-channel blocker tolbutamide and arginine were used as tools. [Ca2+]i changes were studied in dispersed ß-cells from lean, ob/ob and db/db mice. NADH responses to glucose and KIC were analyzed as a measure of metabolic flux. The correlation between Ca2+ and insulin response from individual ß-cells was tested using Fluo-3 and Fluozin-3. Both timing and magnitude of calcium responses were cell-specific in lean mouse ß-cells with all tested secretagogues. ß-Cells from ob/ob and db/db mice showed cell-specific timing of Ca2+ responses to glyceraldehyde but not to KIC, tolbutamide or arginine. However, ob/ob mouse ß-cells within intact islets showed cell-specific timing of tolbutamide-induced response. NADH responses to glucose were cell-specific in all three mouse models, but the timing of NADH responses to KIC was cell-specific only in lean mice. Thus, a cell-specific response can be induced in normal ß-cells at several steps of stimulus-secretion coupling for nutrient-stimulated insulin release. Cell-specific properties of ß-cell ion channels and the mitochondrial metabolism are affected in db/db and ob/ob mice. The relation between mitochondrial mass and parameters of Ca2+ responses were investigated in Mitotracker Red and Fluo-3 labelled ß-cells using confocal microscopy. Data show that ß-cell mitochondrial state may play an important role in determining the timing of [Ca2+]i changes. In summary, the early Ca2+ response pattern in ß-cells, including the lag time, the nadir of initial lowering and the height of the first peak response is cell-specific. Isolated and functionally coupled ß-cells show cell-specific timing of Ca2+ responses when stimulated with metabolic and non-metabolic agents. This may be a robust mechanism of importance for the adequate function of ß-cells and a basis for the pacemaker function of some cells. A disturbed cell specificity of the mitochondrial metabolism and ion channel function appears to be a marker of ß-cell dysfunction in hyperglycemia and diabetes and may explain the delayed insulin release in ß-cells from diabetic subjects.

Akut hjärtinfarkt är globalt sett en vanlig dödsorsak. Hjärtinfarkter behöver inte ge tydliga symptom och dödsorsaken kan därmed vara okänd fram till en klinisk obduktion och att histopatolgiska studier av hjärtmuskelvävnad genomförs. Fosforwolframsyra-hematoxylin (PTAH) är en färgningsmetod som kan användas vid visualisering av hjärtinfarkt genom att färga myokardceller, fibrin, kontraktionsband och cellkärnor blå medan kollagen färgas rosa till rödbrunt. Syftet med examensarbetet var att optimera PTAH-färgningsprotokoll för att underlätta diagnosticering av hjärtinfarkt. Tre olika färgningsprotokoll, två med PTAH med Mallory Bleach (#1 och #2) och ett med PTAH med refixering i Bouins lösning (#3), jämfördes vid inkubering över natt i rumstemperatur samt 3-4 h i värmeskåp vid 56 ºC. Vävnader som färgades in var från paraffininbäddade klossar vilka tillhörde utsvarade fall med konstaterad hjärtinfarkt. Dessa snittades och värmdes fast på oladdade samt laddade objektglas. Efter färgning graderades infärgning efter kategorierna vävnadsdifferentiering och cellkomponenter med en poängskala från 0, ej bedömbar, till 3, optimal. Det protokoll med högst poäng optimerades för att hitta bästa inkuberingstid i PTAH-lösning, därefter implementerades protokollet genom att bekräfta korrekt infärgning vid upprepade infärgningar av hjärtmuskelvävnad. Protokoll #3 visade på högst poäng efter infärgning vid jämförelsen med protokoll #1 och #2 och under optimering framgick inkubering under 4 h i värmeskåp vid 56 ºC som mest gynnsam, då denna inkuberingstid även visade på mindre känslighet vid dehydrering. Slutsatsen blev att PTAH-färgning efter protokoll #3 med refixering i Bouins lösning med 4 h i värmeskåp gav bäst resultat, men färgnyansen varierar med tid mellan dödsfallet och obduktionen. / Acute myocardial infarction (MI) is a common cause of death globally. MI without symptoms or with atypical symptoms is usually first detected at clinical autopsy with histological analyses of the myocardium. Phosphtungstic acid haematoxylin (PTAH) is a method to visualize MI by staining myocytes, fibrin, contraction bands and nuclei blue while collagen stains reddish brown. The aim of this degree project is to optimize PTAH staining protocol for MI diagnosis. Three different staining protocols, two protocols with PTAH staining and Mallory Bleach solution and one protocol with PTAH staining with refixation in Bouin’s solution (#1-3), where compared along with incubation overnight in room temperature and at different hours in heat (56 ºC). Paraffin embedded tissues from myocardium from different autopsy cases diagnosed with MI were cut before mounting and heating on non-charged as well as charged slides. After staining, results were evaluated using scores 0-3 for two parameters; differences in tissues and cell components. Highest evaluated protocol where optimized to find the ultimate incubation in PTAH staining solution. For implementations, additional myocardial tissues from other cases were stained to confirm repeated results. Protocol #3 was evaluated higher in comparison with #1-2 and optimized to 4 h incubation in PTAH-solution in heating with staining results less sensitive to dehydration. In conclusion, staining protocol #3 with 4 h heating was optimal, however, vulnerable to prolonged morgue storage time losing the insensitivity of colour.

Grocott metoden är den känsligaste metoden för infärgning av svampstrukturer i histologisk vävnad i jämförelse med till exempel Periodic Acid-Schiff (PAS). Handhavandet av det cancerogena oxidationsmedlet kromsyra samt tidsaspekten för Grocott är signifikanta problem. Syftet med studien var att undersöka om den automatiserade färgningsmetoden för Grocott ger likvärdiga resultat som vid den manuella färgningsmetoden. Tolv preparat färgades med både den manuella och den automatiserade metoden för Grocott. Den automatiserade färgningsmetoden använder ett färdigt kit med alla ingående lösningar. Visuell bedömning av preparaten utfördes med hjälp av legitimerade biomedicinska analytiker samt en patolog. Svampstrukturer i åtta av ela preparat samt kontroll bedömdes vara tydligare infärgade med den automatiserade metoden i färhållande till den manuella. Svampstrukturer i endast ett preparat bedömdes vara tydligare infärgade med den manuella metoden. Resterande två preparat visade inga svampstrukturer. Resultaten tyder på att den automatiserade färgningsmetoden för Grocott ger likvärdiga, alternativt tydligare infrägning av svampstrukturer i jämförelse med den manuella metoden. / The Grocott method is the most sensitive staining method for fungal structures in histological tissues in comparison to for example Periodic Acid-Schiff (PAS). Usage of the cancerogenic chromic acid and the time aspect for Grocott are significant problems. The aim of this study was to examine if the automated staining method for Grocott provides equivalent results in comparison to the manual staining method. Twelve tissue sections were stained with both the automated and the manual method. The automated method contains a kit with all solutions. Visual evaluation was conducted with authorized biomedical scientists and a pathologist. Fungi structures in eight of eleven tissue sections and a control were evaluated as more distinct in the automated method in comparison to the manual method. Fungal structures in one of the tissue sections were evaluated more distinct in the manual method. In the remaining two tissue sections, no fungal structures were identified. Results from this study indicates that the automated staining method for Grocott provides equally, alternatively more distinct fungal staining in comparison to the manual method.