1.
Wilms, Torben, 1973-
(författare)
Squamous cell carcinoma of the head and neck, focusing on Epstein-Barr-virus, programmed cell death ligand 1 and serum lipoproteins
2021
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Background: Squamous cell carcinoma of the head and neck (SCCHN)comprises a large group of tumours including the oral cavity and nasopharyngealarea, and typically affects older males in association with alcohol/tobacco usage.Within the oral cavity, the mobile tongue is the most common site for tumourdevelopment. The incidence of squamous cell carcinoma of the oral tongue(SCCOT) is increasing in younger people, which has been suggested to associatewith other than the traditional risk factors for this disease. Two common humanoncogenic viruses, human papillomavirus (HPV) and Epstein-Barr virus (EBV)are connected to certain types of SCCHN, in oropharynx and nasopharynxrespectively. The receptor programmed cell death 1 (PD)-1 and its ligandprogrammed cell death ligand 1 (PD-L1) are particularly relevant in immunecheckpoint control, and elevated levels have been seen in various cancer types. Alink between hyperlipidemia and cancer risk has previously been suggested. Theaim of this thesis was to investigate risk factors and prognostic features forSCCHN, by focusing on EBV, PD-L1 and serum lipoproteins.Materials and methods: Ninety-eight cases of SCCOT and 15 cases of tonsillarsquamous cell carcinoma were examined for the presence of EBV-encodedribonucleic acids (EBERs), EBV deoxyribonucleic acid (DNA) and the proteinEBV-encoded nuclear antigen-1 (EBNA-1), using in situ hybridisation,polymerase chain reaction (PCR) and immunohistochemistry respectively. Onehundred and one cases of SCCOT were examined for expression of PD-L1 intumour and surrounding immune cells using Ventana SP263immunohistochemistry assay and a QuickScore (QS) method. An estimation oftumour-infiltrating immune cells was also performed in 25 of the patients.Circulating levels of PD-L1 were measured using an electrochemiluminescenceassay platform in serum from 30 patients. Finally, serum samples from 106patients and 28 healthy controls were investigated for levels of total cholesterol,low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides andlipoprotein(a).Results: In the first study, using an in situ hybridisation kit no EBER transcriptswere detected. No EBV DNA was identified with PCR analysis, andimmunohistochemistry for EBNA-1 was also negative. In the second study, highertumour cell PD-L1 levels were found in females than males (p = 0.019). Forpatients with low PD-L1 in tumour cells, better survival was shown in males thanfemales (overall survival p = 0.021, disease-free survival p = 0.020). Tumourinfiltrating natural killer (NK) T cells, immature dendritic cells (DCs) and M1macrophages correlated positively with tumour cell PD-L1 (p < 0.05). In the laststudy, the only lipoprotein showing significant difference in concentration iiibetween healthy controls and patients was HDL (p = 0.012). Kaplan-Meiersurvival curves showed that patients with high levels of total cholesterol or LDLhad better survival than patients with normal levels (p = 0.028 and p = 0.007respectively). Adjusting for the effects of age at diagnosis, TNM stage and weightchange, multivariate Cox regression models showed LDL to be an independentprognostic factor for both overall (p = 0.010) and disease-free survival (p =0.018).Conclusion: We excluded EBV as a potential player in SCCOT in both old andyoung patients and highlight the importance of appropriate controls for EBVencoded RNA in-situ hybridization (EBER-ISH) when investigating EBV inhuman diseases. Regarding PD-L1, our data supported the significance of genderon tumour cell PD-L1 expression and demonstrated combined effects of genderand PD-L1 levels on clinical outcome in patients with SCCOT. Data also indicatedthe involvement of specific immune cell types in PD-L1-regulated immuneevasion. Looking at serum lipoproteins, we found high LDL levels to be beneficialfor survival outcome in patients with SCCHN. Furthermore, the use of cholesterollowering medicine for prevention or management of SCCHN needs to be carefullyevaluated.
2.
Heyman, Lovisa
(författare)
Berries in Prevention of Metabolic Disease – focus on obesity, diabetes and gut microbiota
2015
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
The increasing prevalence of obesity is a worldwide health problem closely linked to diet and lifestyle factors. Obesity is associated with increased risk of several metabolic disorders including insulin resistance, nonalcoholic fatty liver disease and type 2 diabetes. Hence, there is a great need to identify dietary strategies for the prevention of obesity and related diseases. This thesis investigates the potential of different berries to mediate beneficial health effects in a mouse model of diet-induced obesity and prediabetes. We found that supplementation with lingonberries, blackcurrants and bilberries reduced body weight gain, insulin resistance, low-grade inflammation and hepatic lipid accumulation in C57BL/6J mice fed a high-fat diet. Supplementation with raspberries, crowberries, blackberries or prunes had no or small effects, whereas açai berries promoted development of obesity and fatty liver compared to the control group receiving high-fat diet without berries. Global hepatic gene expression analysis revealed that the phenotype in the lingonberry and bilberry groups was coupled to an anti-inflammatory effect, including downregulation of acute-phase proteins and inflammatory mediators. Mice receiving açai displayed an upregulation of steatosis markers and genes related to lipid synthesis, in line with the exacerbation of high-fat-induced fatty liver in these mice. The HELP-tagging assay was used to identify differentially methylated CpG sites in the lingonberry group compared to the high-fat control group. Lingonberries induced genome-wide and specific alterations of DNA methylation, however the significance of these findings remains to be established. Furthermore, different batches of lingonberries were found to have different capacity to prevent obesity. However lingonberries prevented low-grade inflammation, metabolic endotoxemia and modified the gut microbiota of high-fat fed mice, including increasing the Firmicutes/Bacteroidetes ratio. These findings were independent of effects on body weight gain and achieved regardless of the source of berries. The capacity of lingonberries to counteract negative outcomes of an unhealthy diet should be further evaluated in humans, including assessment of anti-inflammation and microbiota modulation. The generated knowledge about berries and their effects on metabolism may be useful in designing future dietary strategies aimed at preventing metabolic disease.
3.
Chenna Narendra, Sudeep
(författare)
Systemic and local regulation of experimental arthritis by IFN-α, dendritic cells and uridine
2017
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
In this thesis, we have studied the immunological processes of joint inflammation that may be targets for future treatment of patients with arthritis. We focus on the immune-modulating properties of interferon-α (IFN-α) and uridine in experimental arthritis. The nucleoside uridine, which is regarded a safe treatment has anti-inflammatory properties notably by inhibiting tumor necrosis factor (TNF) release. Because the inflamed synovium in rheumatoid arthritis (RA) is characterised by pathogenic TNF-production, uridine could potentially be away to ameliorate arthritis. Systemic administration of uridine had no effect on antigeninduced arthritis (AIA), which is a T-cell dependent model where animals are immunized twice (sensitization) with bovine serum albumin (mBSA), before local triggering of arthritis by intra-articular antigen (mBSA) re-challenge. In contrast, intra-articular administration of uridine clearly down modulated development of AIA in a dose dependent manner and inhibited the expression of synovial adhesion molecules, influx of inflammatory leukocytes and synovial expression of TNF and interleukin 6, but did not affect systemic levels of proinflammatory cytokines or antigen-specific T-cell responses. Local administration of uridine may thus be a viable therapeutic option for treatment of arthritis in the future.Viral double-stranded deoxyribonucleic acid (dsRNA), a common nucleic acid found in most viruses, can be found in the joints of RA patients and local deposition of such viral dsRNA induces arthritis by activating IFN-α. Here we show that arthritis induced by dsRNA can be mediated by IFN-producing dendritic cells in the joint and this may thus explain why viral infections are sometimes associated with arthritis.Earlier, to study the effect of dsRNA and IFN-α in an arthritis model, that like RA, is dependent on adaptive immunity, dsRNA and IFN-α were administered individually during the development of AIA. Both molecules clearly protected against AIA in a type I IFN receptor-dependent manner but were only effective if administered in the sensitization phase of AIA. Here we show that the anti-inflammatory effect of IFN-α is critically dependent on signalling via transforming growth factor β (TGF-β) and the enzymatic activity of indoleamine 2,3 dioxygenase 1 (IDO). The IDO enzyme is produced by plasmacytoid DC and this cell type was critically required both during antigen sensitization and in the arthritis phase of AIA for the protective effect of IFN-α against AIA. In contrast, TGF-β and the enzymatic activity of IDO were only required during sensitization, which indicate that they are involved in initial steps of tolerogenic antigen sensitization. In this scenario, IFN- α first activates the enzymatic activity of IDO in pDC, which converts Tryptophan to Kynurenine, which thereafter activates TGF-β. Common for IDO-expressing pDC, Kyn and TGF-β is their ability to induce development of regulatory T cells (Tregs). We found that Tregs were crucial for IFN-α-mediated protection against AIA, but only in the arthritis phase. In line with this, adoptive transfer of Tregs isolated from IFN-α treated mice to recipient animals in the arthritis phase clearly protected against AIA. The numbers of Tregs were not significantly altered by IFN-α but IFN-α increased the suppressive capacity of Tregs against antigen-induced proliferation. This enhanced suppressive activity of Tregs in the arthritis phase was dependent on the earlier activated enzyme IDO1 during the sensitization phase of AIA. Thus, presence of IFN-α at the time of antigen sensitization activates the enzymatic activity of IDO, which generates Tregs with enhanced suppressive capacity that upon antigen re-challenge prevents inflammation. We have thus identified one example of how immune tolerance can be developed, that may be a future way to combat autoimmunity.
4.
Jönsson, Göran
(författare)
Studies on hereditary C2 deficiency: Frequent occurrence of severe infections, atherosclerosis and rheumatological manifestations
2007
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
The complement system is a part of the innate immunity and is essential in the defence against microorganisms. Hereditary C2 deficiency (C2D) is one of the most common complement deficiency states with an estimated prevalence of 1:20,000 in persons of Western descent. In the present investigation, the identification of more than 40 C2D persons at a single centre combined with long observation periods provided a unique basis for assessment of C2D-associated manifestations and diseases. The predominant clinical manifestation was severe bacterial infections. The infections were mainly caused by Streptococcus pneumoniae. Repeated infections occurred primarily during infancy and childhood. On the other hand, about 25-30 % of the C2D persons remained healthy during the observation period. Immunological factors as IgG subclass levels, GM allotypes, complement proteins, and Fc receptors were assessed to explain this difference. Homozygosity for the G2M*n allele was strongly associated with protection against severe infections (p<0.001). This indicated that an efficient antibody response to polysaccharide antigens is of great importance in C2D. Mannan-binding lectin deficiency also contributed to the susceptibility to infection. The association between C2D and systemic lupus erythematosus (SLE) was confirmed, but notably the severity of SLE in patients with C2D was similar to that of other SLE patients. Another novel finding was a high occurrence of anti-cardiolipin antibodies (aCL) and antibodies to the collagen-like region of C1q. Both autoantibodies have a pro-atherosclerotic effect that might explain the high occurrence of cardiovascular disease found in the cohort. Interestingly, anti-phospholipid syndrome was not observed despite the high occurrence of aCL. Vaccination in 25 C2D persons resulted in antibody responses which show that C2D persons benefit from vaccination against infections caused by encapsulated bacteria such as pneumococci.
5.
Thulin Hedberg, Sara
(författare)
Antibiotic susceptibility and resistance in Neisseria meningitidis : phenotypic and genotypic characteristics
2009
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Neisseria meningitidis, also known as the meningococcus, is a globally spread obligate human bacterium causing meningitis and/or septicaemia. It is responsible for epidemics in both developed and developing countries. Untreated invasive meningococcal disease is often fatal, and despite modern intensive care units, the mortality is still remarkably high (approximately 10%). The continuously increasing antibiotic resistance in many bacterial pathogens is a serious public health threat worldwide and there have been numerous reports of emerging resistance in meningococci during the past decades. In paper I, the gene linked to reduced susceptibility to penicillins, the penA gene, was examined. The totally reported variation in all published penA genes was described. The penA gene was highly variable (in total 130 variants were identified). By examination of clinical meningococcal isolates, the association between penA gene sequences and penicillin susceptibility could be determined. Isolates with reduced susceptibility displayed mosaic structures in the penA gene. Two closely positioned nucleotide polymorphisms were identified in all isolates with reduced penicillin susceptibility and mosaic structured penA genes. These alterations were absent in all susceptible isolates and were successfully used to detect reduced penicillin susceptibility by real-time PCR and pyrosequencing in paper II. In papers III and IV, antibiotic susceptibility and characteristics of Swedish and African meningitis belt meningococcal isolates were comprehensively described. Although both populations were mainly susceptible to the antibiotics used for treatment and prophylaxis, the proportion of meningococci with reduced penicillin susceptibility was slightly higher in Sweden. A large proportion of the African isolates was resistant to tetracycline and erythromycin. In paper V, the gene linked to rifampicin resistance, the rpoB gene, was examined in meningococci from 12 mainly European countries. Alterations of three amino acids in the RpoB protein were found to always and directly lead to rifampicin resistance. A new breakpoint for rifampicin resistance in meningococci was suggested. The biological cost of the RpoB alterations was investigated in mice. The pathogenicity/virulence was significantly lower in rifampicin resistant mutants as compared with susceptible wild-type bacteria.
6.
de Oliveira, Ana Henriques
(författare)
RsbX and stress response in Listeria monocytogenes
2021
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Listeria monocytogenes is a ubiquitous foodborne Gram-positive bacterium. Despite being mainly a soil bacterium, it can reach the food processing environment and contaminate food destined for human consumption, causing outbreaks. Because of its pathogenicity, it poses a danger for certain high-risk groups, including children, elderly, and immune-compromised people, as well as pregnant women, being capable of causing a life-threatening systemic infection known as listeriosis.All bacteria require an efficient transcriptional response and its fine-tuned modulation in order to survive the different stresses it encounters. This is especially true for L. monocytogenes, which presents an impressive range of stress adaptions that allows it survival in certain extreme conditions such as low temperature, low pH and high osmolarity. The alternative Sigma factor B, SigB, is responsible for the expression of the general stress response of this bacterium and plays a key role in the survival and adaption to new environments. The activation of SigB requires an intricate system of partner switching mechanisms, involving anti-sigma and anti-anti-sigma factors, triggered by a number of phosphorylation and dephosphorylation events that culminates with SigB being available to interact with RNA polymerase and lead the transcription of the general stress response regulon. At the top of this signal transduction pathway lies a large multi-protein complex, known as the stressosome. It is formed by RsbR (and its paralogs), RsbS and RsbT and is believed to function as a sensory hub for environmental stimuli. After signal detection, the stressosome proteins are phosphorylated and the complex goes through conformational changes that will ultimately allow for SigB activation. The reset of the stressosome to its pre-stress conformation, is hypothesized to be exerted by a putative phosphatase, RsbX, which most likely dephosphorylates the stressosome proteins post-stress.The role of RsbX in modulating the activity and conformation of the stressosome as well as in subsequent regulation of SigB activity was investigated. RsbX was shown to be required for maintaining SigB levels and activity low in non-stressed conditions as well as for proper SigB mediated stress adaptation. A ΔrsbX mutant strain was shown to have a very slight growth defect, but it also exhibited impaired motility, reduced biofilm formation, as well as a more acid resistant phenotype. The absence of RsbX was shown to alter the composition of the stressosome without drastically affecting its phosphorylation pattern. In general, RsbX was shown to play a crucial role in modulating the signal transduction pathways by preventing SigB activation under non-stressed conditions.Strains that acquire sigB operon mutations have been shown to have a growth advantage under certain mild stress conditions recurrent in a laboratory set. These strains were shown to outcompete the wild-type strain when grown in these conditions, demonstrating how a deficient SigB activity poses an advantage to the cell. On the other hand, and the ΔrsbX mutant strain was shown to have a growth disadvantage, since it was outcompeted by the wild-type strain when co-cultured. The data highlights the significant cost stress protection presents to this pathogen, since deploying the general stress response is a burden on cellular resources, and in its absence the cell can redirect energy for growth. In contrast, in the presence of a lethal stress (low pH) the strains with impaired SigB activity showed a reduced survival and an overall increased sensitivity to the stress. Hence demonstrating that in a more stressful condition the high cost of the general stress response regulon is outweighed by the protection benefits it confers to the cell. The importance of RsbX, which prevents unnecessary SigB activation, is even more evident. RsbX is not only critical to shut down the general stress response post-stress and subsequent recovery of homeostasis, but it also keeps SigB activity to low levels in non-stressed conditions, avoiding unwarranted gene expression and contributing to important energy saving. SigB also plays an important role in the transition of L. monocytogenes from a saprophytic to a pathogenic lifestyle. Even though most of the virulence factors are under the control of PrfA, the master regulator of virulence, SigB is fundamental in the survival of the bacteria inside the host’s gastro-intestinal tract (e.g., stomach high acidity and bile salt release in the duodenum), as well as in the early stages of infection, such as internalization into not phagocytic cells. Because of the importance of SigB for virulence, we speculated if RsbX, by controlling activity of SigB, would also impact the virulence of the bacteria. The data showed somewhat contradicting results, but in general it suggests that even though the expression of the virulence genes responsible for the uptake of the bacteria are increased in a strain lacking RsbX compared with the wild-type strain, the effect on the general infectivity of this strain was either minimal or not existent at all. A reason for this could be the suggested growth defect caused by the absence of RsbX, which could also jeopardize the bacteria’s ability to efficiently grow within infected cells or organisms.Overall, RsbX seems to play a crucial role for L. monocytogenes, since it is responsible to maintain a very important, but extremely costly, stress protection mechanism in an inactive mode in absence of stress. Its functions span from alteration of stressosome conformation and subsequent modulation of stress response, to homeostasis recovery, motility, biofilm formation, stress survival, and even to indirect impact in the bacteria’s infectivity. This shows the diversified, but impactful range of effects RsbX seems to have for the bacterial cell.
7.
Bratanis, Eleni
(författare)
Bacterial antibody hydrolyzing enzymes – as bacterial virulence factors and biotechnological tools
2019
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Antibodies are an essential part of the human immune system, and antibody mediated immunity has been an area of interest for many researchers for almost a century. An accumulation of knowledge regarding antibody structure, glycosylation and receptor interactions has contributed to the current understanding of antibody mediated immunity. It has more recently become evident how bacteria and other microorganisms evade host recognition and eradication through specific antibody degradation or modification. The importance of antibody glycosylation and how glycan modification can fine-tune the elicited immune response has also contributed to the development of antibody-based drugs with improved clinical efficacy. In turn these insights have paved the way and created a need for the development of biotechnological methods and tools to specifically engineer antibodies with defined properties, for analysis to ensure quality and safety, and for improved antibody purification.This thesis highlights the importance of glycosylation for antibody function and presents different aspects and applications of antibody modifications by bacteria. We show, for the first time, activity of the IgG-specific Streptococcal endoglycosidase EndoS during Streptococcus pyogenes infection, clearly demonstrating that EndoS contributes to S. pyogenes pathogenesis and bacterial survival in the context of adaptive immunity. Further this thesis presents the use of bacterial enzymes as antibody modifying tools and their potential as binding reagents for selective antibody purification. The identification and characterization of two novel proteases, BspK and BspE exhibiting unique IgG and IgA cleavage profiles respectively, from Bdellovibrio bacteriovorus highlights the potential of using Bdellovibrio as a source for the identification of novel enzymes with biotechnological applications. Finally, I present the development of a novel method for selective antibody purification, using the inactive variants of the bacterial enzymes EndoS and EndoS2, ensuring the purification of native, correctly folded and modified antibodies.
8.
Strand, Mårten, 1982-
(författare)
The discovery of antiviral compounds targeting adenovirus and herpes simplex virus : assessment of synthetic compounds and natural products
2014
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
There is a need for new antiviral drugs. Especially for the treatment of adenovirus infections, since no approved anti-adenoviral drugs are available. Adenovirus infections in healthy persons are most often associated with respiratory disease, diarrhea and infections of the eye. These infections can be severe, but are most often self-limiting. However, in immunocompromised patients, adenovirus infections are associated with morbidity and high mortality rates. These patients are mainly stem cell or bone marrow transplantation recipients, however solid organ transplantation recipients or AIDS patients may be at risk as well. In addition, children are at higher risk to develop disseminated disease.Due to the need for effective anti-adenoviral drugs, we have developed a cell based screening assay, using a replication-competent GFP expressing adenovirus vector based on adenovirus type 11 (RCAd11GFP). This assay facilitates the screening of chemical libraries for antiviral activity. Using this assay, we have screened 9800 small molecules for anti-adenoviral activity with low toxicity. One compound, designated Benzavir-1, was identified with activity against representative types of all adenovirus species. In addition, Benzavir-1 was more potent than cidofovir, which is the antiviral drug used for treatment of adenovirus disease. By structure-activity relationships analysis (SAR), the potency of Benzavir-1 was improved. Hence, the improved compound is designated Benzavir-2. To assess the antiviral specificity, the activity of Benzavir-1 and -2 on both types of herpes simplex virus (HSV) was evaluated. Benzavir-2 displayed better efficacy than Benzavir-1 and had an activity comparable to acyclovir, which is the original antiviral drug used for therapy of herpes virus infections. In addition, Benzavir-2 was active against acyclovir-resistant clinical isolates of both HSV types.To expand our search for compounds with antiviral activity, we turned to the natural products. An ethyl acetate extract library was established, with extracts derived from actinobacteria isolated from sediments of the Arctic Sea. Using our screening assay, several extracts with anti-adenoviral activity and low toxicity were identified. By activity-guided fractionation of the extracts, the active compounds could be isolated. However, several compounds had previously been characterized with antiviral activity. Nonetheless, one compound had uncharacterized antiviral activity and this compound was identified as a butenolide. Additional butenolide analogues were found and we proposed a biosynthetic pathway for the production of these compounds. The antiviral activity was characterized and substantial differences in their toxic potential were observed. One of the most potent butenolide analogues had minimal toxicity and is an attractive starting point for further optimization of the anti-adenoviral activity.This thesis describes the discovery of novel antiviral compounds that targets adenovirus and HSV infections, with the emphasis on adenovirus infections. The discoveries in this thesis may lead to the development of new antiviral drugs for clinical use.
9.
Skoog, Emma C, 1983
(författare)
Helicobacter spp. interactions with mucins: adhesion and mucin regulation of pathogen proliferation and gene expression
2014
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Helicobacter pylori colonizes the gastric mucosa of approximately half of the world’s population and is a risk factor for gastritis, peptic ulcers and gastric cancer. H. pylori is surrounded by, and adheres to, the heavily glycosylated mucins that build up the mucus layer. The carbohydrate structures on the mucins that act as ligands for H. pylori vary between individuals and change during disease. In this thesis, we investigated how H. pylori interacts with differently glycosylated mucins by analyzing adhesion, proliferation, gene expression and the resulting effect on virulence to host cells. We found that mucins can interfere with H. pylori proliferation, partly dependent on binding to the mucins and the presence of known antimicrobial structures, but also observed an inhibition of H. pylori proliferation independent of these two factors. The gene expression of H. pylori varied greatly in the response to differently glycosylated mucins. Expression of the virulence factor CagA increased in response to some mucins, presumably by Fur-dependent regulation as a result of binding via the SabA adhesin. The varying interaction of H. pylori and mucins resulted in alterations in the response of infected gastric epithelial cells in vitro. There are several Helicobacter species that commonly infects other animals, but can also infect and cause disease in humans. Their modes of interaction with mucins are unclear. We examined the adhesion of two non-H. pylori Helicobacter species to differently glycosylated gastric mucins and mucosal tissue from a range of animals. Our results demonstrated that they can adhere to mucins and gastric tissue via specific glycan structures that change during infection, although the binding ability to human mucins are lower than that of H. pylori. In addition, there are other bacteria in the stomach that may interfere with mucin interactions of Helicobacter spp. We showed that Lactobacillus species isolated from the same stomachs as H. pylori did not compete for the same mucin ligands and did not markedly change how co-isolated H. pylori interact with the mucins. In summary, H. pylori adhesion to human mucins differs from that of other Helicobacter spp. and is not affected by co-colonizing Lactobacillus spp. The interactions of H. pylori to mucins affect proliferation and expression of virulence factors that may influence the colonization ability, virulence and host response and ultimately play a role in the development of symptoms displayed in the host.
10.
Wrighton, Sebastian
(författare)
Exploring Monoclonal Antibody Action Against the Group A Streptococcal M Protein
2023
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Group A Streptococcus (GAS) is a significant human pathogen that has developed multipleimmune evasion mechanisms to counter the host immune response. One of these mechanisms involvesthe production of the M protein, which, amongst other things, acts as an anti-phagocytic factor and canbind host proteins. Another is the ability of M protein to bind a human protein called fibronectin (Fn). Thisprotein plays a key role in a number of physiological processes and can be used by GAS to evade theimmune system. In this PhD dissertation, we aimed to assess the binding efficacy and function ofmonoclonal antibodies targeting the GAS M protein.In the first paper we start by developing a robust method to assess phagocytosis. This method highlightsthe importance of factors such as volume, time, and the ratio of phagocyte to prey on the phagocyticprocess. It has allowed us to, henceforth, attain precise, high quality phagocytosis data and has been amajor driving force for other projects within the lab – especially the three other papers included in thisthesis.In the second paper we discovered a novel form of antibody binding whereby a monoclonal binds theGAS M protein in a bivalent dual-Fab cis mode. This means that both Fab arms of the Ab bind to distinctepitopes on the target molecule simultaneously. Even so this antibody bound to a region of the M proteinassociated with non-opsonic antibodies we found that this Ab could enhance phagocytosis suggestingthat this novel binding form can circumvent the M protein's anti-phagocytic properties.In the third paper we investigated the M protein’s ability to bind fibronectin. While this function wasdescribed in previous studies, we found it could only do so with very low affinity. We found that the bindingof antibodies from the blood of donors who had recently recovered from a severe GAS infection couldgreatly enhance this fibronectin binding. We show that same occurs with certain anti-M monoclonals andthat this mechanism leads to a reduction in opsonophagocytosis. Moreover we find that Ab flexibility mayplay a role and that Ab Fc domains are a crucial factor in mechanism.In the fourth paper we further explore this anti-phagocytic effect. Here we assess the effects of varyingconcentrations of Fn since this can differ greatly within the human body. We found that both very low andhigh concentrations of Fn, corresponding with the nasopharyngeal niche and blood respectively, led to asubstantial reduction in phagocytosis. We moreover found that this reduction in phagocytosis is likelylinked to a modulation of integrins. Overall, this work provides insights into immune evasion mechanismsdeveloped by GAS and highlights how this remarkable pathogen always seems to be one step ahead ofus.
