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- Carlsson, Hanna, 1978-
(författare)
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Laboratory methods for investigation of the immunological orchestra in response to pathogens
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Laboratory methods used for investigation of immune response often involve collection of whole blood and analysis of different biomarkers in blood components or generated from pathogen stimulation of whole blood or peripheral blood mononuclear cells (PBMC). Methods used to measure biomarkers are for example enzyme-linked immunosorbent assay (ELISA) which measures one biomarker at a time or multiplex assays for example x-unknown, multi-analyte profiling (xMAP) by Luminex or proximity extension assay (PEA), which can measure up to just over 3000 biomarkers at a time. Analysis of one biomarker at a time are time consuming, costly, and dependent of a large sample size to enable repeated measurements of different analytes. Therefore, multiplex assays that are time saving, more cost effective and measures multiple bi-omarkers at once in a small sample can be applied. The aim of this thesis was to evaluate multiplex laboratory methods for investigation of the immunological orchestra in response to Borrelia infection and influenza immunisation and if possible, further characterize individuals with different clinical outcomes or serological response, respectively. In our studies (paper I-III) we included 1113 blood donors of which 66 were found to previously have had a subclinical borreliosis (defined as presence of Borrelia-specific antibodies without recall of previous Lyme borreliosis), of the 66 individuals 60 were available for participation. We also included 22 patients previously diagnosed with Lyme neuroborreliosis (LNB). In paper IV we included in total 73 individuals consisting of healthcare workers and patients attending seasonal influenza vaccination. We applied whole blood, PBMC and plasma stimulations and measured a range of cytokines, chemokines and complement factors with ELISA, nephelometry, xMAP and PEA. Our results show that subclinical Lyme borreliosis (SB) individuals display the following pattern, low age, male sex, low amount of secreted interleukin (IL)-17, CCL20 and higher secretion of IL-10 by PBMCs stimulated three days with Borrelia garinii compared to patients with previous Lyme neuroborreliosis (LNB). The subclinical individuals also show higher activation of the complement system in response to Borrelia afzelii. We performed multiplex analysis of complement factors in attempt to further characterize our SB individuals and LNB patient but found the results to deviate largely from reference values retrieved with other standardized methods. This highlights the importance of critical review of generated results from all form of assays. To investigate immune responses after influenza immunisation and further characterize serological responders and nonresponders we included measurement of influenza-specific antibodies and total immunoglobulins (Ig) in blood serum, influenza-specific mucosal IgA (nasal-swabs) and cell-mediated immune response in supernatants from PBMCs stimulated with influenza vaccine using PEA. We found the serological responders to be characterised by lower levels of total IgM, Granzyme B (GZMB) and IL-12 together with higher levels of CXCL13 compared with nonresponders. To conclude, xMAP and PEA are two valuable methods that can be applied together with multivariate statistical methods in the investigation of both innate and adaptive immunity characteristics and association to clinical outcome or serological response after Borrelia infection and influenza immunisation, respectively.
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| 2. |
- Svanberg, Cecilia, 1991-
(författare)
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HIV-1 Exploitation of Dendritic Cell Functionality and Initial Responses in Mucosal Tissues : Elucidation of Influence of HIV-1 Complement Opsonization, and HIV-1-HSV-2 Co-infection
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Human immunodeficiency virus (HIV)-1 is transmitted between individuals via sexual intercourse or via blood products. To date there are 38 million people living with chronic HIV infection and around 1.5 million yearly acquire a new infection. HIV is known to have detrimental effects on the immune system and its cell function and leads to the development of acquired immune deficiency syndrome (AIDS). AIDS is characterized by opportunistic infections often fatal to the individual. Mucosal tissues, which are the main site of HIV-1 infection, consist of a complex network of different cell types building up a barrier against the outside world. In the mucosa there are multiple immune cells and one of them is the dendritic cells (DCs), a professional antigen presenting cell needed for priming of adaptive T cell responses. DCs are rarely productively infected with HIV-1 but the virus can modulate the DC phenotype and function by mere exposure to the virus. There are many factors that influence viral transmission including prevalent inflammatory conditions or infections in the genital tract. For instance, a newly acquired herpes simplex virus-2 (HSV-2) infection increases the risk of transmission at HIV-1 exposure by four times. A vital part of the innate immune defense is the complement system, which consists of proteins found in all body fluids. Normally during complement activation, it leads to lysis of pathogens, but HIV-1 can evade this process. This is achieved by the incorporated complement regulatory proteins in the viral plasma membrane leading to accumulation of opsonizing complement fragments on the HIV-1 particles. Our aim with these studies was to deepen the understanding of the HIV-1 exploitation of DC function and elucidate the initial effects exposure and establishment of HIV-1 infection has on the mucosal tissues and immune cells. In addition, we aimed to elucidate the effects HSV-2 exerts on the HIV-1-infection of DCs and how viral complement opsonization affects the viral infection and activation of immune responses. In Paper I we found that coinfection of monocyte derived DCs (MDDCs) with HIV-1 and HSV-2 decreases the amount of the HIV-1 restriction factors SAMHD1 and TREX1 in MDDCs, leading to increased productive HIV-1 infection. In Paper II and III we found that HIV-1 utilizes the complement system to induce higher productive infection of the colorectal and cervical mucosal DCs. The different forms of HIV, free or complement opsonized, had distinct effects on the immune responses and T cell phenotypes in the tissues, all in favor of HIV-1 establishment and productive infection. In Paper IV, HIV-1 exposed DCs triggered after crosstalk with suppressive T cells a prolonged type I interferon response and an upregulation of coinhibitory molecules on the DCs. The findings in this thesis add to the knowledge of HIV-1 early transmission events, how co-infection modulates DC function, and how the presence of HIV-1 affects the priming of adaptive immune responses.
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