Conclusion:Medical treatment may have a role in platelet count after transfusion. Since the TACSI platelets passed the quality requirements, and the vast majority of patients platelet count increased after TACSI platelet transfusion, the TACSI platelets will replace the old method to produce platelets at the Uppsala University hospital.     Methods: A new approach that pools 8 buffy coats (TACSI platelets) that were separated into 2 units instead of 4-6 buffy coats pooled to 1 unit was investigated in this study. After the platelets were extracted from the buffy coats their quality was controlled and subsequently the platelet product was evaluated in 96 patients.   Results: The results showed that 80 % of the platelet units passed the European quality requirements. Further, the platelet count was increased in most patients that received TACSI platelets. Conclusion: Medical treatment may have a role in platelet count after transfusion. Since the TACSI platelets passed the quality requirements, and the vast majority of patients platelet count increased after TACSI platelet transfusion, the TACSI platelets will replace the old method to produce platelets at the Uppsala University hospital.

Background The proliferation signal inhibitors (PSIs) sirolimus (SRL) and everolimus (ERL) are the potent immunosuppressive drugs using in organ transplantation and has been used successfully in renal transplant recipients (RTX) as well. PSIs are the key factors to overcome the allograft rejections after successful organ transplantation since the immune system starts to react against the graft. SRL and ERL prevents the action of immune system b inhibits the proliferation of T- and B-cells by inhibiting the intracellular signaling of interleukin-2. The presence of excess amount of serum proteins including albumin in the urine is considered as proteinuria, which reflects the loss of kidney function. The occurrence of proteinuria can be the result of abnormal glomerular filtration and/or impaired tubular endocytic function of renal proximal tubular epithelial cells (PTECs). Megalin and cubulin are two scavenger receptors present on epical surface of PTECs and involved in reabsorption of proteins after glomerular ultrafiltration process in the kidney. Proteinuria appears too high in renal transplanted patients during ongoing   treatment with PSIs. Aim Our study aimed to investigate and correlate the expression level of megalin and cubilin and albumin uptake in PTEC of renal transplanted patients before and after conversion to PSI. Methods To retrieve the maximal expression of our interest molecules in renal PTECs, we optimized antigen retrieval (AR) method and primary antibody dilution for each molecule separately. An optimization experiment was performed on 3 different normal patients renal biopsies were used. Later, human renal biopsy specimens originated from 4 different renal transplanted patients were used in this study. From all the 4 patients biopsy specimens were taken before and ongoing administration of PSIs (SRL, ERL). The expression of megalin, cubilin and albumin uptake in PTEC of renal transplant patients was determined by immunohistochemical staining. Results Based on the optimization experiments, we selected the AR method and primary antibody dilution for the expression of megalin, cubilin and albumin uptake. In 4 renal transplanted patients following administration of PSIs results in patients 1, 2, 3 expression of megalin, cubilin and albumin uptake during ongoing PSI treatment was not comparable or even more intense than before PSIs introduction. The expression of megalin, cubilin and albumin uptake was reduced in patient 4 during ongoing PSI treatment. Conclusion Our findings suggest that the renal transplant patient 4 developed proteinuria during PSI medication. The expression of megalin, cubilin and albumin uptake was markedly decreased during ongoing PSI treatment in patient 4. We concluded that there is a direct link between PSI medication and tubular dysfunction, which might cause proteinuria

Type I interferons (IFNs), perhaps the most important of cytokines in fighting viral infections, have been target for detailed research only the past few decades and much is yet to be investigated. Hidden in the mysteries of IFNs might be powerful anti-viral and anti-tumor therapies, alongside greatly increased understanding of vertebrate immunology. This project aims at investigating IFN expression of in vitro stimulated equine cell lines and studies of IFN expression in horses, both healthy and a number of horses diagnosed with inflammatory bowel disease (IBD). Amongst equine diseases, IBD is of increasing concern and scientific progressions within the project are, in several aspects, also applicable for human medicine.

The tolerance towards the semi-allogenic foetus is obtained through both systemic and local changes in the maternal immune response. Locally, in the decidua, the cell composition differs from that found in the blood; natural killer (NK) cells and macrophages being the major cell types. Decidual macrophages (dMØ), which are alternatively activated, and trophoblasts, placental cells of foetal origin, are believed to participate in the foetal tolerance at the foetal-maternal interface. To test the recruitment ability of macrophages and trophoblasts, and to test if these cells are responsible for the special cell composition in the decidua, a migration assay was established. In this migration assay peripheral blood mononuclear cells (PBMC) were allowed to migrate through Matrigel-coated transwell inserts into lower wells containing a recruiting stimulus. After testing several conditions, a protocol was established for further use. The results showed that in vitro alternatively activated macrophages, which display many of the surface markers as dMØ, hold a recruiting ability and recruit monocytes. Further there was an indication that trophoblasts also hold a recruiting ability. Neither cell types were shown to recruit NK cells. In conclusion, this study presents a suitable protocol for assessing chemotactic factors and different cell type’s ability to recruit cells from blood. Although the experiments need to be repeated and extended and the recruitment ability of dMØ needs to be evaluated in detail before a final conclusion can be drawn, the preliminary data indicated that macrophages and trophoblasts can recruit monocytes.

Nanoparticles are used more and more extensively in today's society, especially in the industry sector. Humans get exposed to nanoparticles daily but the effect is a topic that has not been fully explored yet and its effect on humans is still unknown.The purpose of this project was to investigate whether the size of nanoparticles is a factor that influences their effect on humans, mainly the effect on blood activation. In order to study this, nanopaticles of polystyrene with three different sizes (75, 120 and 260 nm) were selected and incubated in a human whole blood model, the Chandler loop. The samples from the Chandler loop experiments were analysed with three different ELISA's: C3a, terminal complement complex (TCC, sC5b-9) and thrombin-antithrombincomplexes (TAT).The results in this study indicate that the smallest nanoparticle has a higher potential for activating the coagulation system than the larger ones. The complement system did not seem to be significantly activated from the nanoparticles. More experiments needs to be done in order to get a better statistic value but just as it is the results look promising and there is a tendency for a higher activation of the coagulation system with the 75 nm nanoparticles.

Njurtransplantation är det enda botande behandlingsalternativet för patienter som befinner sig i kronisk njursviktsstadium 5. Dagens mest använda immunsuppressiva kombinationsbehandling i klinisk praxis består av calcineurinhämmaren takrolimus, mykofenolatmofetil och kortikosteroider i form av prednisolon. Belatacept (Nulojix®) är ett fusionsprotein som introducerades på marknaden 2011 och ska ses som ett alternativ för primär immunsuppressiv behandling. Verkningsmekanismen för belatacept är att hämma aktiveringen av T-celler genom blockera co-stimulatoriska signaler från antigenpresenterande celler. Teorin bakom belatacept är att man genom en mer specifik immunsuppression ska kunna undvika de nefrotoxiska biverkningar som calcineurinhämmarna takrolimus(Prograf®) och ciklosporin(Sandimmun®) ofta ger. Nefrotoxicitet kan på långsikt leda till en försämring av njurfunktion vilket på sikt kan leda till förlust av transplantatet. Syftet med detta arbete var att undersöka effektiviteten av belatacept jämfört med calcineurinhämmare med avseende på graftöverlevnad, njurfunktion och förekomsten av akuta rejektioner. Detta arbete är en litteraturstudie som gjorts genom att utvärdera fem studier som hittades på sökdatabasen PubMed. Samtliga studier som utvärderades i detta arbete påvisade inga signifikanta skillnader i graftöverlevnad mellan de patienter som behandlades med belatacept och de som behandlades med en calcineurinhämmare. Samtliga studier påvisade en signifikant högre njurfunktion mätt i cGFR, Calculated Glomerular Filtration Rate, hos patienter som behandlades med belatacept. Hos dessa patienter ökade njurfunktionen över tid vilket bekräftar teorin bakom belatacept som säger att man genom att undvika nefrotoxicitet ska kunna behålla en stabil nivå i njurfunktion.  I samtliga studier förutom i studie 2 så har patientgrupperna som mottagit belatacept drabbats av en högre incidens av akuta rejektioner där nästan alla skedde inom de första sex månader efter transplantation vilka oftast ger lindriga komplikationer. Slutsatserna som kan dras är att belatacept ger en högre njurfunktion på lång sikt vilket gör det mycket fördelaktigt framför calcineurinhämmare. Belatacept är förenat med ökad förekomst av akuta rejektioner men fördelen med den höga njurfunktionen kan anses väga tyngre då akuta rejektioner oftast ger lindriga komplikationer. Då belatacept är ett nytt läkemedel så kommer det behövas längre studier framöver för att påvisa en högre graftöverlevnad. / Kidney transplant is the only curing treatment for patients who have chronic kidney disease stage 5. Today’s most used immunosuppressive treatment after kidney transplant in Sweden and worldwide is the combination of the calcineurin inhibitor tacrolimus, mycophenolate mofetil and corticosteroids. Belatacept (Nulojix®) is a fusion protein which was introduced on the pharmaceutical market 2011 and should be viewed as an alternative for primary immunosuppressive treatment after kidney transplant. The mechanism of action for belatacept is to inhibit the activation of T-cells by blocking co-stimulatory signals provided by antigen-presenting cells. The theory behind belatacept is to avoid the nephrotoxic adverse events through a more specific immunosuppression. Nephrotoxicity is often seen with the calcineurin inhibitors tacrolimus (Prograf®) and cyclosporine (Sandimmune®). The consequence of nephrotoxicity is a deterioration in renal function which in a long-term can lead to graft loss. The aim of this study is to evaluate the efficacy of belatacept in comparison to calcineurininhibitors regarding graft survival, renal function and the occurrence of acute rejections. This literature study was conducted by evaluating five studies found in the PubMed database. None of the studies that were evaluated in this study showed any significant differences in graft survival of the patients treated with belatacept compared to calcineurin inhibitors. All studies demonstrated a significantly higher renal function measured in cGFR among patients treated with belatacept. The renal function increased over time which confirms the theory behind belatacept, i.e., that you can keep a more stable renal function over time by avoiding nephrotoxicity. All studies except study 2 demonstrated a higher incidence of acute rejection among patients who received belatacept as treatment. Almost all acute rejections in each study occurred within the first 6 months of the study which most of the time give minor complications.The conclusion that can be drawn from this literature study is that treatment with belatacept results in a higher renal function which makes it favorable to calcineurin inhibitors. Treatment with belatacept also results in a higher incidence of acute rejections but the benefit of a higher renal function can be considered to outweigh the risk of acute rejection.4In order to observe a significant difference in graft survival between patients receiving belatacept and those receiving calcineurin inhibitors several and longer studies, including more patients, need to be conducted.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) constitute a group of vasculitides characterized by neutrophil-rich necrotizing inflammation of small vessels and the presence of ANCA in the circulation. Dying neutrophils surrounding the walls of small vessels are a histological hallmark of AAV. Traditionally it has been assumed that these neutrophils die by necrosis, but neutrophil extracellular traps (NETs) have recently been visualized at the sites of vasculitic lesions. NETs were first described to be involved in capture and elimination of pathogens but dysregulated production and/or clearance of NETs are thought to contribute to vessel inflammation in AAV; directly by damaging endothelial cells and indirectly by acting as a link between the innate and adaptive immune system through the generation of pathogenic PR3-ANCA and MPO-ANCA that can activate neutrophils. ANCA can, however, be found in all individuals and are therefore suggested to belong to the repertoire of natural antibodies produced by innate-like B cells, implying that not all ANCA are pathogenic.  In paper I, we found neutrophils in patients to be more prone to undergo NETosis/necrosis spontaneously compared with neutrophils in healthy controls (HC), as well as that active patients possessed elevated levels of NETs in the circulation. Our results also suggest that ANCA during remission could contribute to the clearance of NETs as we observed an inverse relation between ANCA and NETs. In paper II, we observed neutrophils in patients to be more easily activated upon ANCA stimulation as they produced more ROS than neutrophils in HC. In paper III, we showed for the first time that cells of adaptive immunity (B and T cells) in addition to cells of innate immunity can release ET-like structures, in this case consisting of mitochondrial (mt) DNA. mtDNA can act as a damage-associated pattern molecule (DAMP) and promote inflammation, and increased levels of mtDNA has been observed in AAV. Our finding broadens our perspective of the possible roles of T and B cells in immunological responses, and should be further investigated in AAV. In paper IV, we observed reduced frequencies of MZ-like B cells, considered to be innate-like B cells that produce natural antibodies, and of the proposed regulatory B (Breg) cell populations CD24highCD27+ and CD25+CD27+ B cells in patients, particularly in those with active disease. We also observed the phenotypes of these different Breg cell populations to be different from the corresponding cells in HC. We hypothesize that the increased activation potential by neutrophils in AAV to produce ROS and undergo NETosis/necrosis contribute to the excessive inflammation as well as an increased antigen load of PR3 and MPO, and that this in combination with dysregulation of innate-like B cells and Breg cells could lead to break of tolerance to these antigens and production of pathogenic autoantibodies. ANCA can in turn activate neutrophils to release NETs, suggesting a vicious circle in disease development.

Olika typer av blodcancer är några av de vanligast förekommande cancerformerna i världen. Ju tidigare sjukdomen upptäcks och beroende på vilken typ av blodcancer någon drabbats av ser prognosen i många fall god ut då dagens behandlingsmetoder under flera år effektiviserats. Dock finns det fall där patienter inte svarar på traditionella terapier som cytostatika och stålning eller gånger där sjukdomen kommer tillbaka i aggressivare former. I dessa fall krävs andra metoder för att försöka behandla och bota cancern.   En metod som på senare tid fått mycket uppmärksamhet i forskarvärlden är användandet av kroppens egna immunceller som behandlingsform mot blodcancer. Genom att rena fram en patients egna T-celler från ett blodprov, kan dessa sedan genetiskt modifieras till att känna igen specifika tumörassocierade antigener (TAA) som bara vissa typer av cancerceller uttrycker. Metoden går ut på att T-cellerna får chimära antigen-receptorer (CAR), som uttrycks på cellytan, där CAR-T cellerna sedan injiceras tillbaka till patienten. CAR-T cellerna känner igen cancercellerna och attackerar sedan, med målet att patienten efter behandlingen inte ska ha några cancerceller kvar i kroppen.   Kliniska försök gjorda på patienter med olika typer av blodcancer har visat lovande resultat, särskilt gällande patienter som fått återfall av blodcancertypen Akut Lymfatisk Leukemi (ALL). De som fått delta i studierna har haft mycket dåliga prognoser och har innan blivit behandlade med de konventionella behandlingsterapierna, men utan eller med mycket dåligt resultat. Förhoppningar som väckts från dessa forskningsresultat har lett till diskussion att CAR-T celler kan komma att förändra cancervården och i framtiden kanske vara en lika vanlig behandlingsmetod som strålning eller kemoterapi. Dock kvarstår många problem som forskarna måste lyckas lösa innan CAR-T celler kan räknas som konventionell. Bland annat finns stora risker att patienter vid behandling kan drabbas av cytokinfrisläppningssyndrom (CRS, eng. cytokine release syndrome), där immunförsvaret kan attackera kroppens egna organ som i värsta fall kan leda till döden. Cancercellerna kan även komma tillbaka efterbehandling, då de har utvecklat en resistens mot CAR-T cellerna. Metoder för att undvika dessa toxiska responser och göra T-cellerna mer effektiva är bara några av de problem som kvarstår.

Benign prostatic hyperplasia (BPH) and chronic prostatitis (CP) are common diseases in aging men. Though medications are available to alleviate these conditions, problems of possible side-effects of first-line synthetic drugs for prostatic conditions have allowed patients to switch to a safer plant-based medication. CernitinTM, a pollen extract, is used to alleviate these conditions. A recent in vitro study showed that CernitinTM inhibits cell proliferation and induce a regulatory effect on inflammatory parameters. To validate those results, the inter-batch variability of CernitinTM was assessed using the active ingredients CernitinTM T60 and CernitinTM GBX on the human prostatic cell lines BPH‐1 and WPMY‐ 1 and on human peripheral blood mononuclear cells (hPBMCs) in vitro. Cell proliferation assay was performed in prostatic cell lines, while inflammatory parameters were analyzed in hPBMCs. Results revealed that both CernitinTM active ingredients, regardless of batch production, significantly inhibited the proliferation of both prostatic cell lines after 48 and 72 hours, respectively (p < 0.05 to p < 0.001). Among the batches, there were no significant differences observed. Notably, the GBX batches 14164, 14548 and 14160 had a more pronounced effect on cell proliferation right after 48 hours on both cell lines. Whilst, T60 batches 11539 and 14144 had a pronounced effect right after 48 hours on BPH cells. In hPBMC, the production of the anti-inflammatory cytokine interleukin (IL)- 10 and its receptor IL-10 receptor subunit beta (RB), as well as pro-inflammatory cytokine IL-6 was significantly increased after treatment with the T60 formulation regardless of the batch, but not after treatment with the GBX batch. Moreover, IL-10 receptor subunit alpha (RA) and tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) expression increased after the use of both formulations (p < 0.05 to p < 0.001). The pro-inflammatory cytokine IL-8 and chemokine CXCL-10 was significantly decreased using both batches of T60 (p < 0.05 to p < 0.001). Collectively, these results support the claim of the role of CernitinTM as an anti-proliferative agent and as a cytokine regulator.

Recombinant adeno-associated virus vectors are one of the most promising gene delivery tools for applications within gene- and cell therapy. The high level of wild-type adeno-associated virus infections in humans is a limitation due to the pre-existing immunity against the vector or its transgene product. An important tool to develop effective and safe therapies is the ability to measure the pre-existing immune responses against the virus capsids in humans. This master thesis at Gyros Protein Technologies aimed to investigate if the Gyrolab immunoassay system can be used to screen for pre-existing anti-capsid immunity in human sera by optimizing and evaluate three different assay formats: an indirect assay, a generic anti-AAV adsorption assay and a bridging assay. The evaluation focused on immunity against adeno-associated virus serotype 2. All immunoassay formats performed well and depending on application, the different formats offers different advantages. The generic anti-AAV adsorption assay offers the ability to easily screen for several viral serotypes without having to label the capsid, and the bridging assay provides high sensitivity. When screening 31 individual human sera, 58% were positive using the indirect assay and the generic anti-AAV adsorption assay and 65% using the bridging assay format. Provided, is automated and high throughout immunoassays where 16 individuals can be screened in one-two hours. It is shown that all three immunoassay formats can be used to screen for anti-adeno-associated virus antibodies, even though further optimization, cut off development and a larger data set is needed to obtain a fully sophisticated screening tool.