Nanoparticles have been used in industry and in medicine due to their properties which give them beneficial uses. This usage of the nanoparticles has risen the question about how harmful they are to the human body, the connection between the exposure to nanoparticles, and many diseases that occur in the body. Methods This study focused on the effect of nanoparticles in a whole human blood loop model. The blood was incubated with Silica, Titanium dioxide and Palladium particles in heparinized loops without any anticoagulants added. The blood’s cell count was analyzed with a cell counter and then complement, and contact system’s markers were analyzed with ELISA to detect a presence of activations in the systems. Experiments one to five were an optimization of test settings. Results An activation of the contact system was initiated in the loops containing the aggregated titanium dioxide nanoparticles. A high platelets consumption up to 73.8 % was observed as well as two visible clots. On top of that, blood smears showed micro-clots in the blood incubated with the aggregated nanoparticles. Conclusion Nanoparticles initiated an activation in the contact system in the aggregated form in comparison with the dispersed form. Further and deeper studies should be executed to observe the importance of the single or the aggregated form in the actual effect on the immune system.

Ovarian cancer is a disease with a high rate of mortality where the need for novel treatments will increase in the near future as older and populous generations reach the age where the cancer is usually diagnosed. Once treated, ovarian cancer tends to recur and display a newfound resistance against the platinum-based chemotherapeutic drugs that are used to treat the disease. Therefore, devising new methods of treatment is of utmost importance. Treatment with oncolytic viruses like LOAd703 offers an alternative treatment option that is more specific, causes immunogenic cell death in tumor cells, can stimulate the patient’s own immune system into fighting the cancer, and also has the potential to induce long term immune memory. In this project, the oncolytic and immunogenic capacity of LOAd703 in three different ovarian cancer cell lines was tested in conjunction with the standard-of-care chemotherapeutical drugs paclitaxel, cisplatin and carboplatin. The chemotherapy did not inhibit the replication, transgene expression or oncolysis of the LOAd703 virus. LOAd703 was able to effectively induce oncolysis in all three cell lines. The oncolytic capacity was generally increased when combined with chemotherapeutics. In cells resistant to chemotherapeutics, combination therapy with LOAd703 increased the killing capacity. While combination therapy proved effective, it did leave behind a small population of tumor cells that appeared to be resistant to both chemotherapy and viral oncolysis but longer culturing times may be tested to evaluate if complete killing will occur or if it is a primary resistance to these treatments in the cell lines. Further, if there is resistance to oncolysis or chemotherapy-mediated killing, employing tumor-immune cell co-cultures or in vivo studies might be necessary in order to assess whether the immunostimulatory effects of LOAd703 will lead to a complete eradication of the remaining tumor cells. The treatments also caused an increase in the expression of certain cell surface markers, like PD-L1 and CD262, which might open the door for future trials combining chemotherapy and LOAd703 with anti-PD-L1 inhibitors or soluble TRAIL.

Human mesenchymal stem cell (MSC) therapy has shown neuroprotective effects and improvement on recovery from neonatal intraventricular hemorrhage (IVH). This study focused on histological evaluations of human amniotic fluid MSC therapy during early prenatal life in a preterm IVH rabbit model. IVH was diagnosed at 24 h of age with ultrasound and animals were randomized into subgroups with confirmed IVH and an IVH negative control group. Animals with confirmed IVH received vehicle only or MSCs at two different doses via intraperitoneal administration. The animals were sacrificed at 48 h post administration. The severity of IVH was histologically analyzed via staining of endogenous peroxidase activity in cryosections and the distribution of red blood cells and cell-free hemoglobin was scored. Primary antibodies targeting human epitopes were validated in IHC assays of frozen MSC pellet. An anti-human nuclear mitotic apparatus (hNuMA) antibody labeled the majority of cells in the MSC pellet and did not cross-react with rabbit NuMA when tested in the nontreated rabbit brain. Significant levels of red blood cells and cell-free hemoglobin were found in the IVH confirmed group, whereas the control group showed no hemorrhage. The MSC therapy groups showed similar scoring results as the IVH-vehicle group. Anti-hNuMA immunolabeling did not detect any cells in the brain of MSC treated rabbits. However, extracellular (nonnuclear) immunolabeling was detected, located in the midbrain of the animals that received MSCs, indicating the presence of MSC nuclear debris. The preterm rabbit model was proven successful for inducing IVH, whereas MSC treatment did not affect the degree of hemorrhage. To increase the possibility to detect the MSCs post administration, future studies should include prelabeling of the MSCs with a suitable cell tracker and analyses at time points closer to the administration of MSCs. / Human mesenkymal stamcellsterapi har visat neuroprotektiva effekter samt förbättring av återhämtning efter neonatal intraventrikulär blödning (IVH). Denna studie fokuserade på histologiska utvärderingar av stamcellsterapi med humana mesenkymala stamceller (MSC) från fostervatten under tidigt prenatalt liv i en prematur IVH-kaninmodell. IVH diagnostiserades med ultraljud vid 24 tim. ålder och djuren delades slumpmässigt in i undergrupper med konfirmerad IVH och en IVH-negativ kontrollgrupp. Djur med konfirmerad IVH mottog endast bärmedel eller MSC i två olika doser via intraperitoneal administration. Djuren avlivades 48 tim. post administration. Graden av IVH analyserades histologiskt genom färgning av peroxidasaktivitet i kryosnitt av hjärna och distributionen av erytrocyter samt fritt hemoglobin graderades. Primärantikroppar riktade mot humana epitop validerades i immunhistokemiska analyser av fryst MSC-pellet. En anti-human nukleär mitotisk apparat (hNuMA) antikropp märkte majoriteten av cellerna i MSC-pelleten och korsreagerade inte med kanin-NuMA under försök i obehandlad kaninhjärna. Signifikanta nivåer av erytrocyter och fritt hemoglobin påvisades i gruppen med konfirmerad IVH, medan kontrollgruppen inte visade någon blödning. Samtliga IVH-grupper; IVH-vehicle och de som mottog MSC- terapi, visade liknande blödningsgrad. Anti-hNuMA-immuninmärkning kunde inte detektera några celler i de MSC-behandlade kaninhjärnorna. Dock detekterades extracellulär (icke-nukleär) immuninmärkning lokaliserad i mitthjärnan hos de djur som mottog MSC, vilket indikerar närvaro av nukleär MSC-debris. Den prematura kaninmodellen bevisades vara framgångsrik för induktion av IVH, men MSC-terapi påverkade inte blödningsgraden. För att öka sannolikheten att detektera MSC efter administration, borde framtida studier inkludera förmärkning av MSC med en lämplig cellmarkör samt analyser vid tidpunkter närmare administrationen av MSC.

Alteration of gut microbiota has been associated with development of immune mediated diseases, such as allergy. In part, this could be due to the influence of microbes in shaping the immune response. In paper I, we investigated the association of early-life gut colonization with bacteria, and numbers of IL-4, IL-10 and IFN-γ producing cells at two years of age in response to PBMC stimulation with phytohemagglutinin (PHA) in vitro. Early Staphylococcus (S) aureus colonization was directly proportional to increased numbers of IL-4 and IL-10 secreting cells, while early co-colonization with lactobacilli and S. aureus associated with a decrease in IL-4, IL-10 and IFN-γ secreting cells compared to S. aureus alone. This was also confirmed in in vitro stimulations of PBMC with Lactobacillus and/or S. aureus strains, where S. aureus-induced IFN-γ production by Th cells was down regulated by co-stimulation with Lactobacillus. In paper II, we investigated the effects of UV-killed and/or culture supernatant (sn) of Lactobacillus strains and S. aureus strains on IEC and immune cell responses. IEC exposed to S. aureus-sn produced CXCL-1/GRO-α and CXCL-8/IL-8, while UV-killed bacteria had no effect. Further, PBMC from healthy donors exposed to Lactobacillus-sn and S. aureus-sn were able to produce a plethora of cytokines, but only S. aureus induced the T-cell associated cytokines: IL-2, IL-17, IFN-γ and TNF-α; which were down regulated by co-stimulation with any of the different Lactobacillus strains. Intracellular staining verified S. aureus-induced IFN-γ and IL-17 production by Th cells, and increased CTLA-4 expression and IL-10 production by T reg cells. In conclusion, we show that colonization with gut microbiota at early age modulates the cytokine response in infancy. In addition, bacterial species influence cytokine response in a species-specific manner and we demonstrate that lactobacilli modulate S. aureus-induced immune response away from an inflammatory phenotype.

Background: The COVID-19 pandemic is caused by the SARS-CoV-2 virus, which enter the host cells through interactions between the receptor-binding domain (RBD) on the S-protein and the ACE-2 receptor on the host cell. A novel type of vaccine strategy is peptide vaccines, with great potential as a faster and more selective approach to conventional vaccine development. This study focuses on the possibility of generating an antibody response through synthetic peptides harboring B cell epitopes.  Aim: This project aims to investigate the potential of immunogenic peptides to generate an antibody response when used as synthetically produced peptides. As proof-of-concept, the project studies the interactions between previously identified monoclonal antibodies with defined B cell epitopes and the corresponding peptide sequences.  Method: The interactions are evaluated by different ELISA experiments. The candidate peptides are additionally investigated on their binding to polyclonal serum with established S reactive antibodies. Furthermore, the project includes synthesis of one peptide by solid phase peptide synthesis. Results: The ELISA experiments presented no interaction between the synthetic peptides and the monoclonal antibodies or human sera.  Conclusion: The project fulfilled its aim to study the interaction between the B cell epitopes and the monoclonal antibodies. However, no binding was observed. Despite the many advantages in production and stability, development of B cell epitope vaccines come with many challenges. Future will entail if synthetic peptides harboring B cell epitopes can be used as vaccines, or if peptide vaccines will be a focus when a T cell response is to be induced.

Upon encounter of an antigen, epidermis-resident Langerhans cells (LCs) become activated and present the processed antigen to T cells of the draining lymph nodes, resulting in tolerogenic or inflammatory responses. In psoriasis plaques, skin homeostasis is disrupted and replaced by an inflammatory dermatitis. Topical application of the anti-viral compound, imiquimod, induces a psoriasiform inflammatory condition, partly driven by LC production of pro-inflammatory cytokines. Differentiation of the myeloid progenitor cell line, MUTZ-3, produces MUTZ-3 derived Langerhans cells (MUTZ-LCs) which can be used as an in vitro model of LCs. This project aimed to investigate the phenotype and function of imiquimod-treated MUTZ-LCs in monolayer cultures, co-culture with T cells and inserted into a 3D skin model. LC-related surface markers (HLA-DR, CD1a, CD207, CCR7) were upregulated in MUTZ-LCs after 7 days of differentiation with 40 ng/ml GM-CSF, 10 ng/ml TGF-β and 2.5 ng/ml TNF-α. Supernatants of imiquimod-treated monolayer cultures of MUTZ-LCs showed subtle concentrations of IL-6 and TNF-α, but not IL-23. mRNA expression showed no significant upregulation of IL-6, IL-23 or TNF-α after 24 h treatment with imiquimod. The presence of MUTZ-LCs in T cell co-cultures greatly increased the production of IL-2, but did not affect expression of CD25. After 16 h exposure to imiquimod, IL-6, IL-23 and TNF-α could not be detected in culture supernatants of a 3D model consisting of fibroblasts, keratinocytes and MUTZ-LCs. The model was devoid of fibroblasts after 19 days of culture, most likely compromising the immunocompetence, as LC migration in response to activation could not be detected. Further studies could refine and optimize the imiquimod-3D skin model, which has potential as a possible substitute for animal models in psoriasis research.

TRIM21 is a cytosolic ubiquitin ligase and an antibody receptor that providesa last line of defense against invading pathogens. By utilizing the diversity ofantibody repertoire to identify pathogens, TRIM21 serves as a link betweenintrinsic cellular defense and adaptive immunity. A variety of diseases havebeen linked to mutations of the TRIM family, including cancer, inflammatorydiseases, and autoimmune diseases. In this project, TRIM21 was producedand purified from Escherichia coli, (E.coli). Protein characterization wasperformed with SDS-PAGE, size exclusion chromatography and cryo-electronmicroscopy (cryo-EM). Previously TRIM21 has been shown to form a dimerwhen produced in SF9. Results from size exclusion chromatography show thatTRIM21 form a larger complex when expressed in E.coli. Cryo-EM resultsshow that the complex structure is more globular than previously thought.Purified TRIM21 was bound to the antibody IC100. SDS-PAGE and sizeexclusion chromatography results show much lower affinity to antibodies thanexpected. / TRIM21 är en cytosolisk ubiquitinligas- och antikroppsreceptor som ger ensista försvarslinje mot invaderande virus. Genom att använda mångfalden avantikroppsrepertoar för att identifiera patogener, fungerar TRIM21 som enlänk mellan inre cellulärt försvar och adaptiv immunitet. En mängd olikasjukdomar har kopplats till mutationer i TRIM-familjen, inklusive cancer,inflammatoriska sjukdomar och autoimmuna sjukdomar. I detta projekt produceradesoch renades TRIM21 från Escherichia coli, (E.coli). Proteinkarakteriseringutfördes med SDS-PAGE, gelfiltreringskromatografi och kryo-elektronmikroskopi(cryo-EM). Tidigare har TRIM21 visat sig bilda en dimer när den producerasi SF9. Resultat från gelfiltrering visar att TRIM21 bildar ett större komplexnär det uttrycks i E.coli. Cryo-EM-resultat visar att den komplexa strukturenär mer klotformig än man tidigare trott. Renad TRIM21 bands till antikroppenIC100. SDS-PAGE och gel-filtrerings resultat visar mycket lägre affinitet tillantikroppar än förväntat.

Introduktion: Alzheimers sjukdom är en neurodegenerativ sjukdom som orsakas av ansamling av amyloid-β (Aβ) i hjärnan. Prevalensen av Alzheimers sjukdom ökar, och symtom inkluderar minnesförlust, ångest, depression, förvirring, nedsatt omdöme och desorientering. Ålder och genetiska varianter är två riskfaktorer för att utveckla Alzheimers sjukdom. Det finns två modeller som förklarar hur sjukdomen kan uppstå, 1) den amyloida hypotesen som beskriver hur deposition av Aβ leder till ökad aggregation av proteinet tau vilket orsakar celldöd och neurodegeneration, och 2) den kolinerga hypotesen vilken beskriver att Aβ-plack minskar produktionen av acetylkolin vilket leder minskad aktivitet i kolinerga nerver. Det är främst entorhinala cortex och hippocampus som drabbas. Diagnostisering görs genom medicinska och neurologiska undersökningar och genom standardiserade test/instrument. I dagläget kan symtom av Alzheimers sjukdom behandlas med acetylkolinesterasinhibitorer och memantin. Ett annat behandlingssätt är att använda monoklonala antikroppar som riktats mot Aβ för att minska belastningen av Aβ i hjärnan. För att bedöma effekt av sådana läkemedel används bedömningsmetoder baserade på kognitiva och funktionella tester.  Syfte: Syftet med detta arbete var att undersöka effektivitet och säkerhet av anti-Aβ antikroppar för behandling av Alzheimers sjukdom, vilket gjordes genom att analysera kognitiv förmåga, biomarkörer och biverkningar.  Metod: Detta arbete är en litteraturstudie som baserades på fem läkemedelsstudier vilka erhölls från databasen PubMed. Sökord som användes vid litteratursökning var ”aducanumab”, ”lecanemab”, ”donanemab”, ”crenezumab” och ”bapineuzumab”.  Resultat: Hög dos aducanumab i studien EMERGE och lecanemab visade statistiskt signifikant förändring på alla utfallsvariabler, och analys av biomarkörer visade minskad amyloid-belastning i hjärnan. Donanemab visade statistiskt signifikant skillnad på den primära utfallsvariabeln och på analys av biomarkörer, men resultat på sekundära utfallsvariabler var inte statistiskt signifikanta. Crenezumab visade endast statistiskt signifikant förändring på den primära utfallsvariabeln i CREAD2. Bapineuzumab visade ingen statistiskt signifikant skillnad på någon utfallsvariabel eller på förändringar i biomarkörer. De resultat som var statistiskt signifikanta indikerade minskad kognitiv försämring hos patienterna. ARIA var en vanlig biverkning hos alla läkemedel utom crenezumab, men förekomsten av ARIA var i de flesta fall mild till måttlig. Andra vanliga biverkningar inkluderade infusionsrelaterade reaktioner, huvudvärk och fall.  Slutsats: Baserat på resultaten från detta arbete dras slutsatsen att aducanumab, lecanemab och donanemab var de läkemedel med högst effektivitet. Framtiden ser mest lovande ut för aducanumab och lecanemab med anledning av positiva resultat på primära och sekundära utfallsvariabler och biomarkörer samt FDAs godkännande av läkemedlen i USA. Förekomsten av ARIA påverkar säkerheten av läkemedlen och därför behöver fler studier genomföras för att undersöka deras säkerhet ytterligare. / Background: Alzheimer’s disease is a neurodegenerative disease that is caused by accumulation of amyloid-β (Aβ) in the brain. The prevalence of Alzheimer’s disease is increasing, and symptoms of the disease include memory loss, anxiety, depression, confusion, impaired judgment and disorientation. Age and genetic variants are the two main risk factors for developing Alzheimer’s disease. There are two models which describe the development of the disease, 1) the amyloid hypothesis which describes how the deposition of Aβ leads to increased aggregation of the protein tau, which causes neuronal cell death and neurodegeneration, and 2) the cholinergic hypothesis which describes that Aβ plaques decrease the production of acetylcholine, this causes less activity in cholinergic neurons. The two areas in the brain which are mainly affected by neurodegeneration are the entorhinal cortex and the hippocampus. Diagnosing Alzheimer’s disease is done by medicinal and neurological assessments and by using standardized tests/instruments. Currently only symptomatic treatments for Alzheimer’s disease are available; acetylcholine esterase inhibitors and memantine. Another treatment method is using monoclonal antibodies against Aβ to decrease the Aβ load in the brain. To assess the effectiveness of these drugs assessment methods based on cognitive and functional tests can be used.  Aim: This study aimed to analyse the efficacy and safety of anti-Aβ antibodies as a treatment for Alzheimer’s disease, which was done by analysing cognitive ability, biomarkers and adverse events.  Method: This literature study was based on 5 clinical randomized controlled trials which were obtained from the PubMed database. Keywords that were used in the searches were “aducanumab”, “lecanemab”, “donanemab”, “crenezumab” and “bapineuzumab”.  Results: High-dose aducanumab in the study EMERGE and lecanemab showed statistically significant differences on all endpoints, and analysis of biomarkers showed a decrease in amyloid load in the brain. Donanemab showed statistically significant differences on the primary endpoint and analysis of biomarkers but results on secondary endpoints were not statistically significant. Crenezumab only showed statistically significant change on the primary endpoint in CREAD2. Bapineuzumab did not show statistically significant differences on any endpoint or on changes in levels of biomarkers. Statistically significant results on primary and secondary endpoints indicated decreased cognitive impairment among the patients. ARIA was a common adverse event in all drugs, with exception of crenezumab, but the occurrence of ARIA was in most cases mild to moderate. Other common adverse events were infusion-related reactions, headaches and falls.  Conclusion: With consideration of the results of this paper a conclusion can be drawn that aducanumab, lecanemab and donanemab have been shown to be effective on primary endpoints and analysis of biomarkers. The drugs that seem the most promising are aducanumab and lecanemab, mainly because they showed efficacy on both primary and secondary endpoints, and biomarkers and because of the FDA’s recent approval of both drugs in the US. The occurrence of ARIA is something that affects the safety of these drugs and because of this more studies are needed to further assess their safety.

Coronavirus eller SARS-CoV-2 (severe acute respiratory syndrom coronavirus 2) som fick utbrott i december 2019 har väckt en stor uppmärksamhet och påverkat mänskligheten världen runt och inte minst påverkat den socioekonomiska balansen. Viruset som har ursprung i staden Wuhan i Kina sprider sig snabbt mellan människorna genom frekvent rekombination av det genetiska materialet. Redan efter ett år från utbrottet beräknades antal fall till 98 miljoner och dödsfallen till 2 miljoner globalt. Förutom lunginflammation leder infektionen till högre halter av proinflammatoriska markörer som CRP och höga nivåer av cytokiner som IL-6 som i slutändan kan resultera i en cytokinstorm. Därför anses en blockering av IL6 produktionen och/eller blockering av receptorbindningen vara en terapeutisk lösning för att begränsa patogenicitet. Tocilizumab som är den första immunmodulator som introducerades och testades mot covid-19 pneumoni har i flera studier gett upphov till heterogent resultat angående dess effekt på sjukdomen. Syftet med detta litteraturabete är att studera tocilizumabs effekt på covid-19 sjuka patienter med pneumoni och med måttlig till svår sjukdom. Genom sökning på databasen PubMed hittades sex randomiserade kliniska studier som valdes för att undersökas i detta arbete. Studierna jämförde effekten av standardbehandling med och utan tocilizumab. Resultatet blev heterogent men en signifikant förbättring observerades för tocilizumab vad gäller överlevnad, sjukdomsprogression och hälsostatus. Trots att tocilizumabs fördelar överväger dess nackdelar krävs det dock mer forskning kring läkemedlet för att kunna dra bättre samband mellan tocilizumab och dess effekter.

POPULAR SCIENTIFIC SUMMARY Systemic lupus erythematosus (SLE) is not your average disorder. It behaves like a mischievous troublemaker, wreaking havoc throughout the body, causing inflammation that affects multiple organs. SLE presents a puzzle that keeps health care professionals worldwide intrigued, searching for answers amidst its complex of immunologic manifestations and clinical symptoms. While we’ve made progress in understanding SLE, its specific cause remains a mystery. What we do know is that SLE triggers a fascinating interplay between genetic, hormonal, and environmental factors in susceptible individuals. Macrophages, specialized white blood cells, can be likened to moody actors on a stage wearing different masks and wielding functional props. Among them are M1 macrophages, fiery troublemakers who provoke pro-inflammatory responses, and M2 macrophages, peacemakers striving for balance by generating anti-inflammatory responses. Then there is NRF2, the vigilante, normally held by its captor, KEAP1. However, when cells stress NRF2 manages to break free from KEAP1 and spring into action, embarking on a crucial journey into the cell nucleus where DNA is stored. Once inside, NRF2 binds specific regions of the DNA, promoting genes associated with protective activities, including antioxidative responses and detoxification processes, thereby shielding cells from further harm. Now, let us envision a therapeutic strategy that utilizes this; if we can deliberately unleashNRF2 on command, triggering a powerful cascade of antioxidative responses throughout the body,such a treatment would offer tremendous promise and serve as a paradigm for patients sufferingfrom chronic inflammation. But the question remains: Is it possible? In this study, we investigated the effects of certain chemicals on macrophages in a controlledlab environment. Our goal was to explore their potential for therapeutic purposes. Excitingly, wediscovered that these chemicals can indeed influence macrophages to produce a stronger antiinflammatory and antioxidant response. These findings could be promising for developing futuretreatments, especially in patients diagnosed with conditions such as SLE. / ABSTRACT Systemic lupus erythematosus (SLE) is a multifaceted, chronic autoimmune disorder that leads to inflammation and affects various organs. A wide range of immunologic manifestations and clinical symptoms characterizes SLE. While the specific cause remains unknown, it is thought to result from a combination of genetic susceptibility and the intricate interplay between environmental and hormonal factors. A significant subset of SLE patients also experience renal manifestation, lupus nephritis (LN), characterized by distinct inflammatory responses in which macrophages play a role. Macrophages exhibit different functional characteristics depending on their environment, and generally display two contrasting phenotypes; M1, which elicits proinflammatory responses, and M2, which generates anti-inflammatory responses Homeostasis is vital, yet environmental stress is inevitable. NRF2, a transcription factor known for its involvement in oxidative stress response, plays a pivotal role. Under basal conditions, NRF2 resides in the cytoplasm and is targeted for degradation by the protein KEAP1. However, during cellular stress, the NRF2-KEAP1 complex dissociates, allowing NRF2 to translocate into the nucleus where it binds specific regulatory regions of genes that promote cytoprotective activities. The NRF2 pathway has gained attention as a potential target for therapeutic strategies in inflammatory conditions, including SLE. This study aimed to assess the effects of certain chemical NRF2 activators and a KEAP1 inhibitor on an in vitro model of M1 and M2 macrophage polarization. The objective was to investigate whether these compounds could enhance antioxidative response. To evaluate this, key genes and proteins involved in antioxidative pathways were analyzed. Gene expression was assessed using quantitative real-time PCR (qPCR), and protein presence was determined through immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). The findings of this study indicate that stimulation of macrophage subgroups with the selected compounds promotes a shift towards anti-inflammatory and antioxidative response. / <p>Rektor tilldelade Leo Svahn stipendie Österby för <em>välartade obemedlade studier</em>.</p>