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| 2. |
- Andersson, Sandra, et al.
(författare)
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Insufficient antibody validation challenges oestrogen receptor beta research
- 2017
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Ingår i: Nature Communications. - : NATURE PUBLISHING GROUP. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- The discovery of oestrogen receptor beta (ER beta/ESR2) was a landmark discovery. Its reported expression and homology with breast cancer pharmacological target ER alpha (ESR1) raised hopes for improved endocrine therapies. After 20 years of intense research, this has not materialized. We here perform a rigorous validation of 13 anti-ER beta antibodies, using well-characterized controls and a panel of validation methods. We conclude that only one antibody, the rarely used monoclonal PPZ0506, specifically targets ER beta in immunohistochemistry. Applying this antibody for protein expression profiling in 44 normal and 21 malignant human tissues, we detect ER beta protein in testis, ovary, lymphoid cells, granulosa cell tumours, and a subset of malignant melanoma and thyroid cancers. We do not find evidence of expression in normal or cancerous human breast. This expression pattern aligns well with RNA-seq data, but contradicts a multitude of studies. Our study highlights how inadequately validated antibodies can lead an exciting field astray.
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| 3. |
- Clausson, Carl-Magnus, 1985-, et al.
(författare)
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Compaction of rolling circle amplification products increases signal integrity and signal–to–noise ratio
- 2015
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 5, s. 12317:1-10
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Tidskriftsartikel (refereegranskat)abstract
- Rolling circle amplification (RCA) for generation of distinct fluorescent signals in situ relies upon the self-collapsing properties of single-stranded DNA in commonly used RCA-based methods. By introducing a cross-hybridizing DNA oligonucleotide during rolling circle amplification, we demonstrate that the fluorophore-labeled RCA products (RCPs) become smaller. The reduced size of RCPs increases the local concentration of fluorophores and as a result, the signal intensity increases together with the signal-to-noise ratio. Furthermore, we have found that RCPs sometimes tend to disintegrate and may be recorded as several RCPs, a trait that is prevented with our cross-hybridizing DNA oligonucleotide. These effects generated by compaction of RCPs improve accuracy of visual as well as automated in situ analysis for RCA based methods, such as proximity ligation assays (PLA) and padlock probes.
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| 4. |
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| 5. |
- Clausson, Carl-Magnus, et al.
(författare)
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Increasing the dynamic range of in situ PLA
- 2011
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 8:11, s. 892-893
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Tidskriftsartikel (refereegranskat)
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| 6. |
- Clausson, Carl-Magnus, 1985-
(författare)
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Making Visible the Proximity Between Proteins
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Genomic DNA is the template of life - the entity which is characterized by a self-sustaining anatomical development, regulated signaling processes, the ability to reproduce and to respond to stimuli. Through what is classically known as the central dogma, the genome is transcribed into mRNA, which in turn is translated into proteins. The proteins take part in most, if not all, cellular processes, and it is by unraveling these processes that we can begin to understand life and disease-causing mechanisms.In vitro and in vivo assays are two levels at which protein communication may be studied, and which permit manipulation and control over the proteins under investigation. But in order to retrieve a representation of the processes as close to reality as possible, in situ analysis may instead be applied as a complement to the other two levels of study. In situ PLA offers the ability to survey protein activity in tissue samples and primary cell lines, at a single cell level, detecting single targets in their natural unperturbed environment. In this thesis new developments of the in situ PLA are described, along with a new technique offering in situ enzyme-free detection of proximity between biomolecules.The dynamic range of in situ PLA has now been increased by several orders of magnitude to cover analogous ranges of protein expression; the output signals have been modified to offer a greater signal-to-noise ratio and to limit false-positive-rates while also extending the dynamic range further; simultaneous detection of multiple protein complexes is now possible; proximity-HCR is presented as a robust and inexpensive enzyme-free assay for protein complex detection.The thesis also covers descriptions on how the techniques may be simultaneously applied, also together with other techniques, for the multiple data-point acquisition required by the emerging realm of systems biology. A future perspective is presented for how much more information may be simultaneously acquired from tissue samples to describe biomolecular interactions in a new manner. This will allow new types of biomarkers and drugs to be discovered, and a new holistic understanding of life.
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| 7. |
- Clausson, Carl-Magnus, et al.
(författare)
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Methods for analysis of the cancer microenvironment and their potential for disease prediction, monitoring and personalized treatments
- 2012
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Ingår i: The EPMA journal. - : Springer Science and Business Media LLC. - 1878-5085 .- 1878-5077. ; 3:7
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Forskningsöversikt (refereegranskat)abstract
- A tumor does not consist of a homogenous population of cancer cells. Therefore, to understand cancer, the tumor microenvironment and the interplay between the different cell types present in the tumor has to be taken into account, and how this regulates the growth and survival of the cancer cells. To achieve a full picture of this complex interplay, analysis of tumor tissue should ideally be performed with cellular resolution, providing activity status of individual cells in this heterogeneous population of different cell-types. In addition, in situ analysis provides information on the architecture of the tissue wherein the cancer cells thrive, providing information of the identity of neighboring cells that can be used to understand cell-cell communication. Herein we describe how padlock probes and in situ PLA can be used for visualization of nucleic acids and protein activity, respectively, directly in tissue sections, and their potential future role in personalized medicine.
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| 8. |
- Erjefält, Jonas S., et al.
(författare)
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Diffuse alveolar damage patterns reflect the immunological and molecular heterogeneity in fatal COVID-19
- 2022
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Ingår i: EBioMedicine. - : Elsevier BV. - 2352-3964. ; 83
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Tidskriftsartikel (refereegranskat)abstract
- Background: Severe COVID-19 lung disease exhibits a high degree of spatial and temporal heterogeneity, with different histological features coexisting within a single individual. It is important to capture the disease complexity to support patient management and treatment strategies. We provide spatially decoded analyses on the immunopathology of diffuse alveolar damage (DAD) patterns and factors that modulate immune and structural changes in fatal COVID-19. Methods: We spatially quantified the immune and structural cells in exudative, intermediate, and advanced DAD through multiplex immunohistochemistry in autopsy lung tissue of 18 COVID-19 patients. Cytokine profiling, viral, bacteria, and fungi detection, and transcriptome analyses were performed. Findings: Spatial DAD progression was associated with expansion of immune cells, macrophages, CD8+ T cells, fibroblasts, and (lymph)angiogenesis. Viral load correlated positively with exudative DAD and negatively with disease/hospital length. In all cases, enteric bacteria were isolated, and Candida parapsilosis in eight cases. Cytokines correlated mainly with macrophages and CD8+T cells. Pro-coagulation and acute repair were enriched pathways in exudative DAD whereas intermediate/advanced DAD had a molecular profile of elevated humoral and innate immune responses and extracellular matrix production. Interpretation: Unraveling the spatial and molecular immunopathology of COVID-19 cases exposes the responses to SARS-CoV-2-induced exudative DAD and subsequent immune-modulatory and remodeling changes in proliferative/advanced DAD that occur side-by-side together with secondary infections in the lungs. These complex features have important implications for disease management and the development of novel treatments. Funding: CNPq, Bill and Melinda Gates Foundation, HC-Convida, FAPESP, Regeneron Pharmaceuticals, and the Swedish Heart & Lung Foundation.
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| 9. |
- Hvidtfeldt, Morten, et al.
(författare)
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Airway hyperresponsiveness reflects corticosteroid-sensitive mast cell involvement across asthma phenotypes
- 2023
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Ingår i: Journal of Allergy and Clinical Immunology. - : Elsevier BV. - 0091-6749. ; 152:1, s. 4-116
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Tidskriftsartikel (refereegranskat)abstract
- Background: Airway hyperresponsiveness is a hallmark of asthma across asthma phenotypes. Airway hyperresponsiveness to mannitol specifically relates to mast cell infiltration of the airways, suggesting inhaled corticosteroids to be effective in reducing the response to mannitol, despite low levels of type 2 inflammation. Objective: We sought to investigate the relationship between airway hyperresponsiveness and infiltrating mast cells, and the response to inhaled corticosteroid treatment. Methods: In 50 corticosteroid-free patients with airway hyperresponsiveness to mannitol, mucosal cryobiopsies were obtained before and after 6 weeks of daily treatment with 1600 μg of budesonide. Patients were stratified according to baseline fractional exhaled nitric oxide (FENO) with a cutoff of 25 parts per billion. Results: Airway hyperresponsiveness was comparable at baseline and improved equally with treatment in both patients with FENO-high and FENO-low asthma: doubling dose, 3.98 (95% CI, 2.49-6.38; P < .001) and 3.85 (95% CI, 2.51-5.91; P < .001), respectively. However, phenotypes and distribution of mast cells differed between the 2 groups. In patients with FENO-high asthma, airway hyperresponsiveness correlated with the density of chymase-high mast cells infiltrating the epithelial layer (ρ, −0.42; P = .04), and in those with FENO-low asthma, it correlated with the density in the airway smooth muscle (ρ, −0.51; P = .02). The improvement in airway hyperresponsiveness after inhaled corticosteroid treatment correlated with a reduction in mast cells, as well as in airway thymic stromal lymphopoietin and IL-33. Conclusions: Airway hyperresponsiveness to mannitol is related to mast cell infiltration across asthma phenotypes, correlating with epithelial mast cells in patients with FENO-high asthma and with airway smooth muscle mast cells in patients with FENO-low asthma. Treatment with inhaled corticosteroids was effective in reducing airway hyperresponsiveness in both groups.
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| 10. |
- Hvidtfeldt, Morten, et al.
(författare)
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Mucosal cryobiopsies : a new method for studying airway pathology in asthma
- 2022
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Ingår i: ERJ open research. - : European Respiratory Society (ERS). - 2312-0541. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- Background In vivo studies of airway pathology in obstructive lung disease are limited by poor quality of specimens obtained with forceps. Obtainment of cryobiopsies has increased diagnostic yield in cancer and interstitial lung disease but has not been used in patients with asthma. In a recent pilot study, we found mucosal cryobiopsies to be larger and more intact than conventional forceps biopsies. The aim of the present study was to compare quality and safety of mucosal cryobiopsies versus conventional forceps biopsies in patients with asthma. Methods Endobronchial biopsies were obtained with forceps and cryoprobe from patients with asthma not currently treated with inhaled steroids and evaluated histologically. Results A total of 240 cryobiopsies and 288 forceps biopsies were obtained from 48 patients. Bleeding from the biopsy site was common but self-limiting. No major complications related to the procedure were seen. Cryobiopsy cross areas were four times larger compared with forceps. Stretches of intact epithelium were detected in all cryobiopsies compared to 33% in forceps biopsies. Further, the length of intact epithelium was on average four times longer in the cryobiopsies. Importantly, there was a good preservation of both antigens and mRNA in the cryobiopsies ensuring a suitability and robustness for immunohistochemistry and in situ hybridisation. Conclusion Obtainment of mucosal cryobiopsies in patients with asthma is safe and yields biopsies that are significantly larger and morphologically better preserved compared with traditional forceps biopsies. The cryotechnique thus seems to be a promising tool for future in vivo studies of airway pathology.
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