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AbstractSince the first discovery of COVID-19 in late 2019, several millions of people have beeninfected worldwide by the novel severe acute respiratory syndrome coronavirus 2 (SARSCoV-2). Towards the end of the following year new mutant variants started emerging,namely 20I/501Y.V1, VOC 202012/01, or B.1.1.7, 20H/501Y.V2 or B.1.351 P.1(descendant of B.1.1.28), which were firstly discovered in United Kingdom, SouthAfrica, and Brazil, respectively. These variants contain several mutations located in thedifferent part of the virus genome. In this work, we focus mainly on commonly sharedmutations in the receptor binding domain (RBD) of the spike protein, E484K and N501Y.There is some evidence indicating that these mutations could increase diseasetransmissibility between humans. Here, we explore a concept of employing a diagnostictool to evaluate the importance of these mutations using short peptide epitopes containingthe mutations. A naturally occurring biologically stable cyclic peptide, sunflower trypsininhibitor 1 (SFTI-1), was used as a molecular scaffold to graft the epitopes. The epitopeswere tested against five serum samples and by using an indirect enzyme-linkedimmunosorbent assay (ELISA) the strength and/or response of the antibody reactivitywas determined. Linear versions of the cyclic peptides and RBD, as well as a longer nativelinear peptide containing both regions of mutation sites were used as controls. Initialresults suggest that short epitopes are insufficient to trigger antibody reactivity and thatepitope regions selection plays an important role. However, the insight presented in thiswork provides substantial information for future development of pharmaceuticalapproaches in COVID-19 therapy.

Abstract   Tripeptidyl-peptidase II (TPPII), is present in most eukaryotic cells. It cuts tripeptides from the N-terminus of peptides and is especially important for degrading peptides longer than 15 amino acids. TPPII also tailors long peptides into suitable substrates for the enzymes which transport and produce the peptides that MHC I present. Increased levels of TPPII have also been found in certain cancer cells, thus it is of interest to determine if TPPII could be used as a tumour marker. The aim of this study was to optimise and evaluate 3 different methods for quantifying TPPII. Western blot, enzyme-linked immunosorbent assay (ELISA) and fluorophore-linked immunosorbent assay (FLISA) protocols were optimised regarding incubation times and antibody dilutions. Sensitivity and linearity were the most important parameters when evaluating the results. The coefficient of determination of western blot was R2=0.98-1 within the range of 1.29-250ng TPPII/well and ELISA had a coefficient of determination of R2=0.96 within the range of 0.03-250ng TPPII/well. Presently western blot is the only one of these methods to yield reliable results with impure samples, but ELISA is superior regarding sensitivity and throughput. Thus further optimisation of ELISA is interesting to pursue.

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.

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.

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.