Tick-borne encephalitis virus is the cause of much morbidity and sometimes a fatal infection. A vaccine based on formaldehyde inactivated virus is currently the only available way of preventing disease. This vaccine gives a high rate of seroconversion but there are reports of vaccination breakthrough, even in people who have demonstrated a neutralizing antibody response. The T cell response to inactivated TBE vaccine is largely unknown, but could be of importance for the effect of the vaccine. This study characterizes aspects of the T cell response by investigating the expression of two transcription factors, T-bet and GATA-3 with RT-PCR. T-bet is expressed in CD4+ T cells of the Th1 type, while GATA-3 is expressed in CD4+ T cells of the Th2 type. Our data show that vaccination with inactivated TBE vaccine leads to increase in expression of the T-bet gene when cells of vaccinated subjects are cultured with TBE virus. In contrast, the expression of GATA-3 remains unaffected by vaccination. Thus, this study suggests that the inactivated TBE vaccine leads to a Th1-like immune response in humans.

In the demyelinating autoimmune disease multiple sclerosis (MS) there is a great need for validated prognostic biomarkers that can give information about both prognosis and disease course. So far only clinical parameters have been shown to predict future outcome. CXCL13 is a potent B cell chemoattractant that has been suggested to be a potential biomarker candidate. The aim of this study was to investigate the usefulness of CXCL13 as a prognostic biomarker for MS. Clinical, paraclinical, laboratory and MRI data about a large group of MS patients and controls were collected. CXCL13 levels in cerebrospinal fluid (CSF) samples from these patients were determined by standard enzymelinked immunosorbent assay (ELISA). In general CXCL13 were increased in CSF in MS, especially in relapsing-remitting MS during relapses, i.e. with ongoing inflammations in the central nervous system. CXCL13 is a good candidate prognostic marker for MS, since newly diagnosed MS with high CXCL13 levels showed worsened disease course within five years. Most importantly, MS conversion occurred in higher rate in possible MS patients with high concentrations of CXCL13 in CSF, and in a shorter time point. This observation may support an early treatment decision in these patients. In conclusion, this study provides support for an association between CXCL13 levels in the CSF and later development of disease severity in MS.

T lymphocytes can be rendered specific against a wide range of antigens by the genetic transfer of a chimeric receptor, a fusion between the antigen-binding domain of an antibody and the signalling domain of a T cell receptor. The use of such chimeric T lymphocytes has shown promising results for cancer therapy. Previous experiments in our laboratory have shown low rates of gene transfer using retroviral vectors. In this study, investigations have been done to increase the number of genetically modified cells. Different enhancers such as PLL and polybrene have previously been used in combination with retroviral transduction. The optimal retroviral protocol in this study showed to be the use of retrovectors produced with twice the normal concentration of the plasmids encoding env and gag-pol rather than the use of the enhancers. A 6-day pre stimulation of T lymphocytes prior transduction together with a centrifugation step increased the rate of modified cells even further. Alternative approaches of gene transfer were also investigated, including plasmid transfection and adenoviral transduction. While transfection protocols yielded low numbers of modified cells, adenoviral vectors showed the highest rate of gene transfer. / Cancer är den sjukdom som idag, efter hjärt-kärl-sjukdomar, kräver flest dödsfall i i-länder. Som en alternativ behandlingsmetod mot cancer pågår just nu forskning om genetiskt förbättrade immunceller, s.k. chimära T lymfocyter, skulle kunna användas för att döda tumörceller. De chimära cellerna är utrustade med en konstgjord receptor som är en fusion av en antikropp och en signalkedja. Det gör att cellerna kan riktas mot ett brett urval av cancertyper. Att få cellerna att ta upp generna som behövs för den konstgjorda receptorn har visats sig vara problematiskt. Den här studien har därför som mål att förbättra cellernas förmåga att ta upp gener. För detta har vi använt oss av retrovirus- och adenovirus-system tillsammans med försök att få cellerna att spontant ta upp generna, sk. plasmid-transfektion. Studien har visat att de båda virussystemen ger högst antal modifierade celler. Olika substanser som tidigare har visat sig förhöja graden av gentillförsel har testats, men vår studie har visat att tillverkningen av virusvektorerna har större påverkan på resultaten än någon av de olika hjälpmedlen.

The amyloidoses are biochemically heterogeneous diseases with patholophysiologic deposits of various proteins. Amyloid deposits can occur either localized to one organ or tissue or as part of a systemic disease with deposits in many different tissue. The clinical course, prognosis and therapy are different for each type of amyloidosis and therefore a type specific diagnosis is demanded as early as possible. We describe a method for typing of the most common systemic amyloidoses based on Western blot analysis combined with specific in- house antibodies, using subcutaneous fat biopsies. We found that the method is reliable and easy to perform and the tissue sample needed is obtained by minor surgery. In the aortic intima amyloid deposits are often associated with atherosclerosis plaques. In our study we also investigated the prevalence of intimal amyloid from 10 patients age 58-94, amyloid deposits were present in 50% of the cases. / Amyloidos är ett sjukdomstillstånd där proteiner som normalt är lösliga i kroppen felveckas och formar långa olösliga fibriller som ansamlas i vävnader och organ såsom t.ex. hjärta, hjärna och lever. Det finns cirka 25 proteiner som kan ge upphov till amyloidos. Man kan skilja på två huvudgrupper av amyloidos, systemisk och lokaliserad. Vid lokal amyloidos kan inlagringar förekomma i specifika vävnader vid framför allt vissa åldersberoende sjukdomar som t.ex. Alzheimers sjukdom. Vid systemisk amyloidos förekommer inlagringar i praktiskt taget alla vävnader. Symtomatologin vid systemisk amyloidos är variabel och sjukdomsbilden kan vara svårtolkad men tidig och specifik diagnostik ger möjlighet till riktad terapi mot den bakomliggande sjukdomen. Syftet med denna studie var att utvärdera en Western blot metod som använts för typning av vanligaste formerna av systemisk amyloidos. De slutsatser som nåtts är att denna metod är snabbt, pålitligt och enkel att utföra. Diagnos erhölls med finnålsbiopsi av bukfettvävnad som är enkel, snabb och billig metod med liten risk för patienternas hälsa. Vi lyckades också med hjälp av immunhistokemisk infärgning titta på prevalens av amyloid i aortas intima.

Group A Streptococcus (GAS) is a globally disseminated pathogen that causes >500,000 deaths yearly and is ranked as ninth leading infectious cause of human mortality by the World Health Organisation. The spectrum of disease ranges from superficial infections of the skin and epithelium to invasive and systemic infections. Although the interaction of GAS with neutrophils has been extensively studied much remains to be discovered about the role of macrophages, which are the first line of defence encountered by invading pathogens. In this study, the aim was to establish a means of deriving macrophages from primary monocytes and to study both the efficiency of macrophage killing of GAS and the macrophage cell death response to GAS infection. Here, we report that monocyte-derived macrophages are able to take up and kill GAS during in vitro infection. Production of reactive oxygen species by macrophages was elicited during infection, but not nearly in as high amounts as produced by neutrophils. Investigating the type of cell death induced by GAS, markers for both apoptosis and necroptosis can be found after 8 hours of infection. These results highlight that macrophages indeed are participating in the clearance of GAS and more studies are needed to understand the roles of macrophages in early control of GAS infection.

The aim of cancer immunotherapy is to eradicate tumours by inducing a tumour-specific immune response. This thesis focuses on how antibodies and peptides can improve antigen presentation and the subsequent tumour-specific T cell response. Tumour recognition by the immune system can be promoted through delivery of antigen in the form of a vaccine. One example is the development of a therapeutic peptide vaccine containing both CD4+ and CD8+ T cell epitopes. So far, peptide vaccinations have shown limited success in clinical trials and further improvements are needed, such as choice of adjuvant and T cell epitopes, as well as targeted delivery of peptides and adjuvants to the same DC. In paper I, we describe the development of a peptide-peptide conjugate (with a tumour T cell epitope) that, via immune complex formation and FcγR binding, enhance antigen uptake and activation of DCs. The conjugate consists of three tetanus toxin-derived linear B cell epitopes (MTTE) that were identified based on specific IgG antibodies in human serum. Three MTTE peptide sequences were conjugated to a synthetic long peptide (SLP) that consists of a T cell epitope derived from the desired target tumour. In paper II, the conjugate was evaluated in a modified Chandler loop model containing human blood, mimicking blood in circulation. The conjugate was internalised by human monocytes in an antibody-dependent manner. A conjugate containing the model CMV-derived T cell epitope pp65NLV generated recall T cell responses dependent on MTTE-specific antibodies and the covalent conjugation of the three MTTE with the SLP. In paper III, a CD40-specific antibody was characterised for local treatment of solid tumours. The antibody eradicated bladder tumours in mice and induced T cell-mediated immunological memory against the tumour. In paper IV, we characterised the Chandler loop model (used in paper II) for its potential use in predicting cytokine release syndrome (CRS) in response to monoclonal antibodies (mAbs). Superagonistic antibodies (e.g., OKT3) induced rapid cytokine release whereas no cytokine release was induced by antibodies (e.g., cetuximab) associated with low incidence of CRS in the clinic. In conclusion, this thesis work demonstrates proof-of-concept of improved strategies for antibody- and peptides-based cancer immunotherapies and their potential use in multiple cancer indications.

Systemic lupus erythematosus (SLE) is a systemic inflammatory disease. Clinically, the distinction between ongoing inflammation attributed to SLE, and organ damage due to medication or co-morbidities remains challenging. In addition, SLE is a heterogeneous disease where the various disease phenotypes complicate the search for biomarkers that adequately reflect disease activity and/or signs of increasing organ damage. The aim of the thesis was to investigate and evaluate potential new biomarkers of disease activity and/or organ damage in SLE patients. High mobility group box protein-1 (HMGB1) is a nuclear non-histone protein that can shuttle to the cytoplasm, become secreted extracellularly, and participate in systemic inflammation. Administration of monoclonal anti-HMGB1 antibodies has been reported both to attenuate and intensify disease in animal models of arthritis and lupus. In Paper I of the thesis, circulating anti-HMGB1 was found in 23% of the SLE patients and correlated with disease activity variables. The biological role of these autoantibodies remains to be elucidated. As a consequence of massive circulating levels of cellular debris and immune complexes, SLE patients have insufficient capacity to remove such material via the reticuloendothelial system. Pentraxin 3 (PTX3) may possibly protect against lupus flares due to classical complement activation, opsonization of apoptotic cells, and cytokine induction. In Paper II, circulating PTX3 was found to be inhibited or exhausted by interferon (IFN)-α, a key cytokine of SLE pathogenesis, and serum levels of PTX3 in SLE patients were inversely related to IFN-α levels. Suppressed PTX3 levels may contribute to a vicious circle resulting in impaired waste clearance, autoantigen exposure and autoantibody production, and sustained disease activity. Osteopontin (OPN), a protein known to influence cell signaling and apoptosis, has been proposed as a marker of organ damage in pediatric lupus. In a Swedish cross-sectional study, circulating OPN levels were found to be raised in SLE (Paper III). In patients with recent-onset disease, OPN reflected disease activity, while in established disease, OPN appeared to mirror damage accrual and cardiovascular damage. In Paper IV, OPN was instead analyzed in an international longitudinal multi-center study based on patients with recent-onset SLE and follow-up data. OPN turned out to be a poor predictor of organ damage, but significant associations were observed between OPN and disease activity both at disease onset, as well as over 5 years of follow-up. In conclusion, increased anti-HMGB1 antibody and decreased PTX3 levels could potentially sustain the impaired waste-disposal. Of the molecules analyzed in this thesis, OPN seems to be the best marker of disease activity. Further studies of these proteins may help to better understand SLE pathogenesis and to optimize treatment of patients.

Prostatacancer är den vanligaste cancerformen hos män i Sverige, och det är även den cancerform som flest män dör utav. Detta till trots så är mekanismerna bakom canceretiologin till stor del okända. Uppskattningsvis 20% av alla canceruppkomster i människor tros vara länkade till infektioner från patogener och de inflammationer som de orsakar. Tidigare studier har visat indikationer på att kronisk inflammation av prostatan kan hjälpa tumörer att undvika immunförsvaret. Bakterien Cutibacterium acnes förekommer i stor utsträckning i prostatavävnad hos patienter som lider av prostatacancer vilket kan peka på en länk mellan mikroorganismen och cancerformen. Tumörassocierade makrofager kan producera immunhämmande proteiner, däribland liganden PD-L1, ett protein som kan hjälpa tumörcellerna att undgå immunförsvaret. Studien ämnade därför att undersöka huruvida makrofager som infekterats med typ I eller typ II C. acnes producerar en högre mängd av PD-L1 än obehandlade makrofager och i detta syfte utfördes ett ELISA-test som bestämde koncentrationen av PD-L1 i odlingsmediumet. Genom en statistisk analys konstaterades att makrofager som infekterats med bakterien ökade produktionen av PD-L1 jämfört med obehandlade makrofager. Resultatet av studien visar indikationer på att C. acnes kan spela en roll i spridningen av tumörceller i kroppen, dock krävs vidare studier för att bekräfta hypotesen. / Prostate cancer is the most common form of cancer among men in Sweden, and the form of cancer that most men die from. In spite of this, the mechanisms behind the etiology of the cancer are largely unknown. Approximately 20% of all cancer etiologies are believed to be linked to infection by pathogens, and the inflammatory response they cause within the body. Earlier studies have shown indications that chronic inflammation of the prostate might create conditions that are favourable for the tumour cells. The bacterium Cutibacterium acnes is prevalent in prostate tissue from patients suffering from prostate cancer, which might indicate a link between the microorganism and this form of cancer. Tumour associated macrophages produce immunosuppressive proteins, such as the ligand PD-L1, a protein that might provide immunoevasive qualities for the tumour cells. The aim of the study was to examine whether macrophages that have been infected with either type I or type II C. acnes would produce a greater amount of PD-L1 than untreated macrophages. For this purpose, an ELISA test was performed to determine the concentrations of PD-L1 in the culture media. I found that macrophages that had been infected with the bacterium produced greater amounts of PD-L1 than untreated macrophages. The result of this study shows some indication that C. acnes could play a role in the proliferation of tumour cells and thus the spread of cancer through immunosuppresive mechanisms, although further studies are needed to confirm this hypothesis.

Characteristics as unknown cause, complicated pathophysiology and a great amount of complexity are describing systemic lupus erythematosus (SLE) more than well. It’s an autoimmune disease that is almost exclusive for women in their reproductive years and are believed to correlate with both genetics and environmental factors. Risk factors like stress, usage of cigarettes or birth controls with estrogen and infections are believed to trigger the progression of SLE. The spectra of therapeutic drugs are narrow due to the complexity and requirement of financial resources for scientific causes. Treatment is mainly symptomatic. The alternative splicing (AS) is a highly complex mechanism that is essential and are able to generate a great diversity of proteins encoded by the same gene are referred to as isoforms. Splicing occurs after the transcription that generates pre-mRNA because the exons need to fuse together and excision of introns. In patients with cancer diagnosis, they have observed that progression of disease and AS are correlated by the means of isoforms and splicing regulators. In studies, the relevance of alternative splicing events in SLE has been shown for both splicing regulators like SRSF1 and different isoforms for example CD44 and CD45. The aim of this study was to evaluate the potential biomarker AS in SLE.  This study of literature started with looking for clinical trials within databases like PubMed and Web of Science, that matched the aim of the study. Usage of terms like ‘SLE and biomarker’ och ‘SLE and alternative splicing’ etcetera. After inclusion of six scientific articles the author started the work with this literature of study.  Results gave strong indications that usage of alternative splicing as biomarker do have strong potential. Although the need of more goal-oriented scientific studies is required. Results from all six studies can be summarized by the line of argument that AS, in different ways, are somehow involved in the pathogenesis and progression of SLE. Both spliceosome, isoforms, splicing factors, other proteins and is also a possible, in the future, therapeutic target for example monoclonal antibodies. Other therapeutic targets maybe against phosphatases and kinases. New strategies are going to bring hope for the patients that are suffering from SLE, especially when the disease is active for 2–3 years. Being able to individualize treatment are going to generate a better quality of life for many SLE patients and usage of AS as a biomarker for disease severity.

With increasing incidence rates aligned with poor prognosis; sepsis represents one of the biggest challenges in modern health care. It is a multifactorial syndrome defined as organ dysfunction caused by disturbed systemic response to an infection. Most of the inpatient sepsis are caused by Gram positive bacteria and one of its major constituents of the cell envelope: lipoteichoic acid (LTA). An adjuvant treatment that has gained prominence recently is extracorporeal blood removal therapies i.e., hemoperfusions. The concept is to remove the bacterial virulence factors that triggers immune responses and therefor stabilize the hemodynamic parameters of the patient. The dominating research of this method centres around adsorption of the Gram negative bacterias’ endotoxin lipopolysaccharide (LPS) but not LTA, whose biochemical and physiological properties resembles each other. The aim of this study was to determine whether LTA can be adsorbed using immobilized truncated derivates of antimicrobial peptides (AMPs). LTA was quantified using ELISA comparing before and after passage through columns with immobilized peptides. Further, the absorption abilities of LTA from two different solid phases with distinctive surfaces were investigated. This was of interest to elucidate the nature of the mechanisms behind LTA extractions. All results generated inconclusive data, except for one trial which demonstrated that peptide KEF-19 adsorbed most LTA and that the electrostatic force had the greatest influence of the adsorption. Future studies should however be carried out to validate these statements as well as feasibility and safety estimations for KEF-19 as the sorbent in hemoperfusions for Gram positive bacteria and LTA.