| 1. |
- Barcenilla, Hugo, et al.
(författare)
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Regulatory T-Cell Phenotyping Using CyTOF
- 2023. - 1
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Ingår i: Regulatory T-Cells. - New York : Humana Press. - 9781071626498 - 9781071626474 - 9781071626467 ; , s. 231-242
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Bokkapitel (refereegranskat)abstract
- Regulatory T cells are an important component of the immune system that plays a key role in maintaining homeostasis. Identification of distinct regulatory T cell subsets is essential to understand their function. Mass cytometry or CyTOF is a technology that enables the simultaneous measurement of up to 50 markers in single cells by using antibodies tagged with heavy metals, which are then detected with time-of-flight mass spectrometry. This chapter describes a mass cytometry approach for phenotypic characterization of regulatory T cells and determination of their master transcription factor Foxp3.
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| 2. |
- Bivik Eding, Cecilia, et al.
(författare)
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MTH1 Inhibitors for the Treatment of Psoriasis
- 2021
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Ingår i: Journal of Investigative Dermatology. - : ELSEVIER SCIENCE INC. - 0022-202X .- 1523-1747. ; 141:8, s. 2037-2048
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Tidskriftsartikel (refereegranskat)abstract
- Inflammatory diseases, including psoriasis, are characterized by changes in redox regulation. The MTH1 prevents the incorporation of oxidized nucleotides during DNA replication. Using MTH1 small-molecule inhibitors, we found induced apoptosis through 8-oxodeoxyguanosine triphosphate accumulation and DNA double-strand breaks after oxidative stress in normal and malignant keratinocytes. In psoriasis, we detected increased MTH1 expression in lesional skin and PBMCs compared with that in the controls. Using the imiquimod psoriasis mouse model, we found that MTH1 inhibition diminished psoriatic histological characteristics and normalized the levels of neutrophils and T cells in the skin and skin-draining lymph nodes. The inhibition abolished the expression of T helper type 17-associated cytokines in the skin, which was in line with decreased levels of IL-17-producing gd T cells in lymph nodes. In human keratinocytes, MTH1 inhibition prevented the upregulation of IL-17-downstream genes, which was independent of ROS-induced apoptosis. In conclusion, our data support MTH1 inhibition using small molecules suitable for topical application as a promising therapeutic approach to psoriasis.
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| 3. |
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| 4. |
- Farnebo, Simon, et al.
(författare)
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Continuous assessment of concentrations of cytokines in experimental injuries of the extremity
- 2009
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Ingår i: International Journal of Clinical and Experimental Medicine. - : e-Century Publishing Corporation. - 1940-5901. ; 2:4, s. 354-362
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Tidskriftsartikel (refereegranskat)abstract
- Background. Inflammation plays an important part in the healing process. Little is known about the extent local inflammatory trauma response interacts with the central circulation and inflammation produced by central organs. The aim of the present study was to examine whether high cut-off microdialysis catheters offer potential to in real time assess interstitial cytokines variations in conjunction to markers of metabolism distal to a blunt vascular contusion. Methods. In a standardised contusion trauma model, microdialysis catheters (high MW (100kDa)) were inserted in the gracilis muscle distal to the trauma for the local assessment of IL-6, IL-8, TNF-a, total protein and the metabolic mediators (glycerol, puruvate and lactate). The contra lateral uninjured leg served as control of the centrally mediated inflammation propagated to the extremities. Results. The trauma led to a significant and quantitatively large (8-10 fold) increase in inflammatory cytokines (IL6 and 8) as measured both in the injured and control legs. There was only a minor, and not significant increase in concentrations of cytokines in the injured leg compared to the control leg.. There were no signs of ischemia in either leg. Conclusion. The new finding in this study is that both central, and local, inflammatory responses as well as metabolic mediators may be assessed continuously in skeletal muscle tissue distal to a major injury in an animal model. The findings suggest that the large trauma elicits a generalised inflammatory response to trauma rather than propagating a local one distal to the trauma.
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| 5. |
- Hillman, Jan, 1952-, et al.
(författare)
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A microdialysis technique for routine measurement of macromolecules in the injured human brain
- 2005
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Ingår i: Neurosurgery. - 0148-396X .- 1524-4040. ; 56:6, s. 1264-1268
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: To evaluate a new intracerebral microdialysis catheter with a high-cutoff membrane and its potential for the study of macromolecules in the human brain. METHODS: Paired intracerebral microdialysis catheters were inserted in 10 patients who became comatose after subarachnoid hemorrhage or traumatic brain injury and were then treated in our neurosurgical unit. The only differences from the routine use of microdialysis in our clinic were the length (20 mm) and cutoff properties of the catheter membranes (100 kD) and the perfusion fluids used (standard perfusion fluid, 3.5% albumin, or Ringer-dextran 60). Samples were weighed (for net fluid fluxes) and analyzed at bedside (for routine metabolites) and later in the laboratory (for total protein and interleukin-6). The in vitro recovery of glucose, glutamate, and glycerol were also investigated under different conditions. RESULTS: Even brief perfusion with standard perfusion fluid resulted in a significant loss of volume from the microdialysis system. For albumin and Ringer-dextran 60 fluid, recovery was comparable to standard settings. Interleukin-6 (highest value close to 25,000 pg/ml) was sampled from all catheters, and total protein was analyzed from catheters perfused with Ringer-dextran 60 (average concentration, 234 μg protein/ml). There were detectable patterns of variations in the concentration of interleukin-6, seemingly related to concomitant variations in intracerebral conditions. In the present study, no direct comparison was made with the standard CMA 70 catheter (CMA Microdialysis, Stockholm, Sweden), but in vivo, the measured mean concentrations of glucose, glycerol, lactate, and pyruvate were comparable to those previously reported from standard catheters. In vitro, the recovery of metabolites was better when using Ringer-dextran 60 compared with albumin. CONCLUSION: Microdialysis catheters with high-cutoff membranes can be used in routine clinical practice, allowing for sampling and analysis of cytokines and other macromolecules.
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| 6. |
- Hillman, Jan, 1952-, et al.
(författare)
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Intracerebral microdialysis in neurosurgical intensive care patients utilising catheters with different molecular cut-off (20 and 100 kD)
- 2006
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Ingår i: Acta Neurochirurgica. - : Springer Science and Business Media LLC. - 0001-6268 .- 0942-0940. ; 148:3, s. 319-324
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Tidskriftsartikel (refereegranskat)abstract
- Objective. To compare the properties of a new intracerebral micro-dialysis catheter with a high cut-off membrane (molecular cut-off 100 kDalton) with a standard catheter (CMA70, molecular cut-off 20 kDalton).Methods. Paired intracerebral microdialysis catheters were inserted in fifteen comatose patients treated in a neurosurgical intensive care unit following subarachnoid haemorrhage or traumatic brain injury. The high-cut-off catheter (D100) differed from the CMA 70 catheter by the length (20 mm) and cut-off properties of the catheter membranes (100 kDalton) and the perfusion fluids used (Ringer-Dextran 60). Samples were collected every 4–6 hours, analyzed bedside (for glucose, glutamate, glycerol, lactate, pyruvate and urea) and later in the laboratory (for total protein).Results. Fluid recovery was similar for the two types of catheters, but significantly more protein was recovered by the D100 catheter. The recovery of glycerol and pyruvate did not differ, while minor differences in recovery of glutamate and glucose were observed. The recovery of lactate was considerably lower in the D100 catheter (p < 0.01), influencing the lactate/pyruvate-ratio. The patterns of concentration changes over time were consistent for all metabolites, and independent of type of catheter.Conclusion. Microdialysis catheters with high cut-off membranes can be used in routine clinical practice in the NSICU, adding the possibility of macro-molecule sampling from the extracellular space during monitoring.
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| 7. |
- Johansson, Joakim, et al.
(författare)
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Dynamics of leukocyte receptors after severe burns: An exploratory study
- 2011
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Ingår i: BURNS. - Oxford : Elsevier Science B.V., Amsterdam.. - 0305-4179 .- 1879-1409. ; 37:2, s. 227-233
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Tidskriftsartikel (refereegranskat)abstract
- Background: Patients with burns are susceptible to organ failure, and there is indirect evidence that leukocytes may contribute to this process. They may change the expression of cell-surface receptors after certain stimuli, for example, the burn. We therefore aimed to assess the changes induced by the burn in the expression of leukocyte cell-surface receptors CD11b, CD14, CD16, and CD62L on the surface of PMNs and monocytes. We also wanted to examine the dynamics of this activation during the first week after the burn, and to relate it to the size of the injury. Methods: Ten patients with burns of andgt;15% (TBSA) were included in the study. Blood samples were collected on arrival and every consecutive morning during the first week. Healthy volunteers acted as controls. Results: PMN CD11b expression was increased. The extent of PMN CD11b expression correlated negatively to the size of the full thickness burn. Monocyte CD14 expression increased initially but there was no relation to the size of the burn. PMN CD16 expression decreased initially during the first days and the decrease was related to burn size. CD62L did not vary depending on the burn in either PMN or monocytes during the first week after the burn. Conclusion: This study showed that specific receptors on the surface of leukocytes (PMN CD11b, monocyte CD14 and PMN CD16) are affected by the burn. Expression of PMN CD11b and CD16 are related to burn size. Burn-induced effects on the expression of PMN receptors, such as PMN CD11b and CD16, may contribute to burn-induced infection susceptibility.
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| 8. |
- Marty, Florence, et al.
(författare)
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Identification of key factors in Accelerated Low Water Corrosion through experimental simulation of tidal conditions : Influence of stimulated indigenous microbiota
- 2014
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Ingår i: Biofouling (Print). - : Informa UK Limited. - 0892-7014 .- 1029-2454. ; 30:3, s. 281-297
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Tidskriftsartikel (refereegranskat)abstract
- Biotic and abiotic factors favoring Accelerated Low Water Corrosion (ALWC) on harbor steel structures remain unclear warranting their study under controlled experimental tidal conditions. Initial stimulation of marine microbial consortia by a pulse of organic matter resulted in localized corrosion and the highest corrosion rates (up to 12-times higher than non-stimulated conditions) in the low water zone, persisting after nine months exposure to natural seawater. Correlations between corrosion severity and the abundance and composition of metabolically active sulfate-reducing bacteria (SRB) indicated the importance and persistence of specific bacterial populations in accelerated corrosion. One phylotype related to the electrogenic SRB Desulfopila corrodens appeared as the major causative agent of the accelerated corrosion. The similarity of bacterial populations related to sulfur and iron cycles, mineral and tuberculation with those identified in ALWC support the relevance of experimental simulation of tidal conditions in the management of steel corrosion exposed to harbor environments.
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| 9. |
- Mellergard, Pekka, et al.
(författare)
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Release of VEGF and FGF in the extracellular space following severe subarachnoidal haemorrhage or traumatic head injury in humans
- 2010
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Ingår i: British Journal of Neurosurgery. - : Informa Healthcare. - 0268-8697 .- 1360-046X. ; 24:3, s. 261-267
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Tidskriftsartikel (refereegranskat)abstract
- Microdialysate fluid from 145 severely injured NSICU-patients, 88 with subarachnoidal haemorrage (SAH), and 57 with traumatic brain injury (TBI), was collected by microdialysis during the first 7 days following impact, and levels of the neurotrophins fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) were analysed. The study illustrates both similarities and differences in the reaction patterns of the 2 inflammatory proteins. The highest concentrations of both FGF2 and VEGF were measured on Day 2 (mean (+/- SE) values being 47.1 +/- 15.33 and 116.9 +/- 41.85 pg/ml, respectively, in the pooled patient material). The VEGF concentration was significantly higher in TBI-patients, while the FGF2 showed a tendency to be higher in SAH-patients. This is the first report presenting in some detail the human cerebral response of FGF2 and VEGF following SAH and TBI. Apart from increasing the understanding of the post-impact inflammatory response of the human brain, the study identifies potential threshold values for these chemokines that may serve as monitoring indicators in the NSICU.
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| 10. |
- Mellergard, Pekka, et al.
(författare)
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The Cerebral Extracellular Release of Glycerol, Glutamate, and FGF2 Is Increased in Older Patients following Severe Traumatic Brain Injury
- 2012
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Ingår i: Journal of Neurotrauma. - : Mary Ann Liebert. - 0897-7151 .- 1557-9042. ; 29:1, s. 112-118
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Tidskriftsartikel (refereegranskat)abstract
- Old age is associated with a poor recovery from traumatic brain injury (TBI). In a retrospective study we investigated if the biochemical response following TBI is age dependent. Extracellular fluids were continuously sampled by microdialysis in 69 patients admitted to our NSICU following severe TBI. The concentrations of glycerol, glutamate, lactate, pyruvate, and eight different cytokines (IL-1 beta, IL-6, IL-10, IL-8, MIP-1 beta, RANTES, FGF2, and VEGF) were determined by fluorescence multiplex bead technology. Patients in the oldest age group (andgt;= 65 years) had significantly higher microdialysate concentrations of glycerol and glutamate compared to younger patients: the mean microdialysate concentration of glycerol increased from 55.9 mu mol/L (25-44 year) to 252 mu mol/L (andgt;= 65 years; p andlt; 0.0001); similarly glutamate increased from 15.8 mmol/L to 92.2 mmol/L (p andlt; 0.0001). The lactate-pyruvate ratio was also significantly higher in the patients andgt;= 65 years of age (63.9) compared with all the other age groups. The patterns of cytokine responses varied. For some cytokines (IL-1b, IL-10, and IL-8) there were no differences between age groups, while for others (MIP-1b, RANTES, VEGF, and IL-6) some differences were observed, but with no clear correlation with increasing age. For FGF2 the mean microdialysate concentration was 43 pg/mL in patients andgt;= 65 years old, significantly higher compared to all other age groups (p andlt; 0.0001). Increased concentrations of glycerol and glutamate would indicate more extensive damaging processes in the elderly. An increase in concentration of FGF2 could serve a protective function, but could also be related to a dysregulation of the timing in the cellular response in elderly patients.
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| 11. |
- Mellergård, Pekke, et al.
(författare)
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Changes in Extracellular Concentrations of Some Cytokines, Chemokines, and Neurotrophic Factors After Insertion of Intracerebral Microdialysis Catheters in Neurosurgical Patients
- 2008
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Ingår i: Neurosurgery. - 0148-396X .- 1524-4040. ; 62:1, s. 151-157
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: The extracellular levels of eight different inflammatory agents were analyzed during the initial 36 hours after insertion of microdialysis catheters in patients. METHODS: Cerebral extracellular fluid from 38 patients who were treated in a neurosurgical intensive care unit for severe brain injury was collected every 6 hours for 36 hours. The concentration of interleukin (IL)-1ß, IL-6, IL-8, macrophage inflammatory protein-1ß, regulated on activation, normal T-cell expressed and secreted (RANTES), fibroblast growth factor-2, and vascular endothelial growth factor was determined by a multiplex assay, and IL-10 was determined by enzyme-linked immunosorbent assay. RESULTS: This is the first report regarding the presence of IL-10, IL-8, macrophage inflammatory protein-1ß, regulated on activation, T-cell expressed and secreted, vascular endothelial growth factor, and fibroblast growth factor-2 in the tissue level proper of the living human brain. The study also provides new information regarding the response of IL-1ß and IL-6 after insertion of a microdialysis catheter. The study confirms that the intriguing patterns of interplay between different components of the inflammatory response studied in laboratory settings are present in the human brain. This was most clearly observed in the variations in response between the three different chemokines investigated, as well as in the rapid and transient response of fibroblast growth factor-2. CONCLUSION: The data presented illustrate the opportunity to monitor biochemical events of possible importance in the human brain and indicate the potential of such monitoring in neurosurgical intensive care. The study also underlines that any analysis of events in the brain involving mechanical invasiveness needs to take into account biochemical changes that are directly related to the manipulation of brain tissue.
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| 12. |
- Mellergård, Pekka, et al.
(författare)
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Differences in Cerebral Extracellular Response of Interleukin-1 beta, Interleukin-6, and Interleukin-10 After Subarachnoid Hemorrhage or Severe Head Trauma in Humans
- 2011
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Ingår i: NEUROSURGERY. - : Williams and Wilkins. - 0148-396X. ; 68:1, s. 12-19
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Microdialysis has become a routine method for biochemical surveillance of patients in neurosurgical intensive care units. OBJECTIVE: To analyze the intracerebral extracellular levels of 3 interleukins (ILs) during the 7 days after major subarachnoid hemorrhage or traumatic brain injury). METHODS: Microdialysate from 145 severely injured neurosurgical intensive care unit patients (88 with subarachnoid hemorrhage, 57 with traumatic brain injury) was collected every 6 hours for 7 days. The concentrations of IL-1 beta and IL-6 were determined by fluorescence multiplex bead technology, and IL-10 was determined by enzyme-linked immunosorbent assay. RESULTS: Presented are the response patterns of 3 ILs during the first week after 2 different types of major brain injury. These patterns are different for each IL and also differ with respect to the kind of pathological impact. For both IL-1 beta and IL-6, the initial peaks (mean values for all patients at day 2 being 26.9 +/- 4.5 and 4399 +/- 848 pg/mL, respectively) were followed by a gradual decline, with IL-6 values remaining 100-fold higher compared with IL-1 beta. Female patients showed a stronger and more sustained response. The response of IL-10 was different, with mean values less than 23 pg/mL and with no significant variation between any of the postimpact days. For all 3 ILs, the responses were stronger in subarachnoid hemorrhage patients. The study also indicates that under normal conditions, IL-1 beta, IL-6, and IL-10 are present only at very low concentrations or not at all in the extracellular space of the human brain. CONCLUSION: This is the first report presenting in some detail the human cerebral response of IL-1 beta, IL-6, and IL-10 after subarachnoid hemorrhage and traumatic brain injury. The 3 ILs have different reaction patterns, with the response of IL-1 beta and IL-6 being related to the type of cerebral damage sustained, whereas the IL-10 response was less varied.
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| 13. |
- Seifert, Oliver, et al.
(författare)
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Gene expression profiling of macrophages: implications for an immunosuppressive effect of dissolucytotic gold ions
- 2012
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Ingår i: Journal of Inflammation. - : BioMed Central. - 1476-9255. ; 9:43
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Tidskriftsartikel (refereegranskat)abstract
- Background: Gold salts has previously been used in the treatment of rheumatoid arthritis but have been replaced by biologicals such as TNF-alpha inhibitors. The mechanisms behind the anti-inflammatory effect of metallic gold ions are still unknown, however, recent data showed that charged gold atoms are released from pure metallic gold implants by macrophages via a dissolucytosis membrane, and that gold ions are taken up by local macrophages, mast cells and to some extent fibroblasts. These findings open the question of possible immunomodulatory effects of metallic gold and motivate efforts on a deeper understanding of the effect of metallic gold on key inflammatory cells as macrophages. less thanbrgreater than less thanbrgreater thanMethods: Human macrophage cells (cell line THP-1) were grown on gold foils and intracellular uptake was analysed by autometallography. The impact of phagocytised gold ions on viability of THP-1 cells was investigated by trypan blue staining and TUNEL assay. The global gene expression profile of THP-1 cells after incorporation of gold ions was studied using microarray analysis comprising approximately 20,000 genes. The gene expression data was confirmed by measurement of secreted proteins. less thanbrgreater than less thanbrgreater thanResults: Autometallography showed intracellular uptake of gold ions into THP-1 cells. No significant effect on viability of THP-1 cells was demonstrated. Our data revealed a unique gene expression signature of dissolucytotic THP-1 cells that had taken up gold ions. A large number of regulated genes were functionally related to immunomodulation. Gold ion uptake induced downregulation of genes involved in rheumatoid arthritis such as hepatocyte growth factor, tenascin-C, inhibitor of DNA binding 1 and 3 and matrix metalloproteinase 13. less thanbrgreater than less thanbrgreater thanConclusion: The data obtained in this study offer new insights into the mode of action of gold ions and suggest for the investigation of effects on other key cells and a possible future role of metallic gold as implants in rheumatoid arthritis or other inflammatory conditions.
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| 14. |
- Sjögren, Florence, 1950-, et al.
(författare)
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Acetone has anti-inflammatory effects on experimental contact reactions
- 1999
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Ingår i: Contact Dermatitis. - : Wiley. - 0105-1873 .- 1600-0536. ; 41:1, s. 22-29
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Tidskriftsartikel (refereegranskat)abstract
- The effects of a topically applied corticosteroid and its acetone vehicle on experimental allergic, toxic and irritant reactions are presented. The corticosteroid budesonide in acetone or acetone alone was applied to reactions immediately after and at different time intervals within the 1st h after provocation. Classical naked eye observation was performed and the dermal cellular infiltrate was differentiated and counted using a previously well-characterized method. “Treatment”, whether with the steroid in acetone or acetone alone, had anti-inflammatory effects. For all reaction types, erythema and oedema diminished and a significant decrease in mononuclear cells was seen, when application occurred within the first 5 min after provocation. The effects were most marked for the toxic reaction to croton oil, the steroid and the vehicle being anti-inflammatory to the same extent. Application up to 60 min after provocation had anti-inflammatory effects for this reaction type. The mechanisms of acetone's anti-inflammatory effects are at present unclear. One possible explanation is that intercellular lipid organisation and, by extension, cellular membrane lipid organisation, are altered, influencing membrane receptor function. Possible anti-inflammatory effects of acetone should be considered in experimental and perhaps even clinical situations. Further investigation of the therapeutic possibilities of the finding seems warranted.
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| 15. |
- Sjögren, Florence, et al.
(författare)
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Are cutaneous microdialysis cytokine findings supported by end-point biopsy immunohistochemistry findings?
- 2010
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Ingår i: AAPS Journal. - : Springer Science and Business Media LLC. - 1550-7416. ; 12:4, s. 741-749
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Tidskriftsartikel (refereegranskat)abstract
- Insertion of a cutaneous microdialysis catheter into normal dermis has been shown to induce the production of IL1b, IL6 and IL8 in an innate response to minimal trauma. In the present study, skin biopsy for immunohistochemistry has been performed at the site of the microdialysis catheter to compare the findings with that of the microdialysis findings 24 h after insertion. Of the three named cytokines, concordance between the two investigated technologies was highest for IL8 (100%) followed by IL6 (70%) and IL1b (50%). For seven other pro-inflammatory and T cell-relevant cytokines studied, concordance ranged between 50% and 80%. The total number of positive (microdialysis or immunofluorescence) findings was similar between the two methodologies. Technical and biological phenomenon can explain the differences. We conclude that both methodologies illustrate important features of tissue biology and that a combination of the two methods in clinical research can provide the chronology of soluble mediator participation and the more classic, but also more invasive, biopsy-based methodology at a point which constitutes the end of the observation period. We conclude further that at the 24-h time period here studied, microdialysis catheters are still functional and thus capable of producing relevant data which can be corroborated and extended by the “end point biopsy”.
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| 16. |
- Sjögren, Florence, et al.
(författare)
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Cutaneous Microdialysis : Cytokine Evidence for Altered Innate Reactivity in the Skin of Psoriasis Patients?
- 2012
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Ingår i: AAPS Journal. - : American Association of Pharmaceutical Scientists. - 1550-7416. ; 14:2, s. 187-195
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Tidskriftsartikel (refereegranskat)abstract
- Cutaneous microdialysis demonstrates cytokine production in living human skin. In the present study, microdialysis samples taken from uninvolved and lesional skin in three test subjects with psoriasis over 24 h have been investigated for cytokine content with a bead-based multiplex immunoassay from Luminex. Concentration curves for a set of Th1/Th2 and pro-inflammatory cytokines measured differed from a reference group of ten subjects without psoriasis. The time to return to near baseline values after innate insertion reactivity is between 9 and 16 h. Post-equilibration levels (17-24 h) for the three main cytokines elevated in the reference group were differentially elevated outside the range of the reference group for interleukin-1 beta (IL1 beta) and IL8 but not so for IL6. Two further cytokines, granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha not generally elevated in the reference group, showed elevated values in the test subjects. Multivariate time series analysis (chemometry) showed that cytokine patterns for the individual test subjects often fell outside the 99% confidence intervals of a model generated from the reference group. In a clinical research situation, cutaneous microdialysis is feasible, gives generally higher cytokine levels than in the blood and generates interpretable data on an individuals reactivity compared with a reference group. This may well prove useful in delineation of pathogenetic issues, selection of appropriate therapy and monitoring of subsequent response in inflammatory dermatoses such as psoriasis.
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| 17. |
- Sjögren, Florence, et al.
(författare)
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Cytokine suspension array analysis of microdialysate samples from normal human skin during 24 hours after 100 kiloDalton cut of membrane catheter insertion
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background Cytokines play important roles in steering homeostatic and inflammtory activities in human tissues not the least skin. In vivo, human, large pore membrane microdialysis technique can be used to measure cytokines in human tissues such as skin, muscle and brain. The new analytical technique of flow cytometry based analysis of polystyrene bead conjugated antibodies (suspension array) allows the analysis of multiple cytokines or other proteins on the typically small sample volumes (10 - 15 ul) provided by microdialysis. Another challenge for microdialysis is the differentiation of observations due to the pathological process being studied from those caused by the insertion and presence in the tissue of the catheter itself.Objectives The objective of the present study was to use suspension array analysis of 10 cytokines to illustrate the chronology of the response of normal living human skin to the introduction of a microdialysis probe in-vivo.Methods CE-marked, commercially manufactured microdialysis catheters equipped with a 100 kiloDalton cut-off polyethersulphone membrane were introduced into the normal skin of the ventral forearm in 10 volunteers. Probes were perfused with Ringer Dextran Braun at a speed of 0.3 ul min-I. Samples were collected at 1 hour intervals for the first 7-8 hours, for a period of 9- 14 hours during the evening and during a 15-21 hours night period. Hourly sampling was again done the following morning until at least 24 hours after catheter insertion. Total protein was analysed by a protein assay kit. A cytokine suspension array was used to estimate the levels ofinterlenkin (IL) 1beta (b), 2, 4, 5, 6, 8, 10, granulocyte monocyte colony stimulating factor (GM-CSF), interferon gamma (IFNg) and turnor necrosis factor alpha (TNFa).Results All probes were in place as planned for 24-28 hours with a small and acceptable degree of discomfort to the subjects. Total protein levels confirmed that probes were functioning with high initial levels followed by a fall. The cytokines IL6, IL8 and IL-1 beta were detected in all patients at all time points after the first hour. IL-6 levels reached a peak at 5-8 hours, falling to levels between 25 - 820 pg ul-1 at 24 hours. IL-8 and IL-1b showed similar time courses. IL-2, GM-CSF and TNFalpha were detected at levels above the lowest standard for the technique at some time points in some subjects. IL-10, IL-5, and IL-4 were only detected in isolated samples in a few individuals. IFNg was not detected at any time point, in any of the ten subjects.Conclusions The previously observed elevation of IL-6 immediately after microdialysis catheter insertion into normal skin was confirmed with levels peaking at 5-8 hours and then falling to lower levels by 24 hours. IL-8 and IL-1b showed a similar time course though absolute levels at peak were lower. Other cytokines showed variable outcomes from similarly high levels, to variably low levels as well as absent levels. The cytokine spectrum was pro-inflammatory with a profile of non-Th2 character according to the Th1/Th2 paradigm. Suspension array represents a significant analytical advance in the applicability of in-vivo microdialysis whether it be experimental or clinical since most analytical capture molecules can be conjugated to the beads. The levels of cytokines seen after probe insertion provide a basis for normal biology and chronology of the cytokines as well as the interpretation of levels in the study of pathological reactions.
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| 18. |
- Sjögren, Florence, 1950-
(författare)
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Dermal cell trafficking : from microscopy to microdialysis
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The term dermal cell trafficking has been used to describe the dynamic nature of cell movement in the dermis reflecting the skin's role as an immunological organ. A light microscopic experimental model for qualitative and quantitative counting of the dermal inflammatory cell infiltrate in allergic, acute irritant and cumulative irritant contact reactions has been developed In human studies use of microdialysis technique has enabled observation of biochemical events in the skin, in vivo over a period of time. This method might allow measurement of cytokines and other inflammatory mediators in the intercellular space of the dermis without the need of multiple biopsies. For confirmation that cytokines detected/not detected in the dialysate are present /absent in the dermis, and to determine the possible cellular source of the cytokines, a return to biopsy technique at time points of special interest would be required.The general aims of this thesis have been to extend the experimental studies on skin reactions to immediate reaction types and to develop the use of microdialysis technique for the measurement of cytokines.Plastic embedding, thin sectioning and optimal staining were the basis for inflammatory cell counting. The immediate hypersensitivity reaction to ovalbumin and the non immunological immediate contact reaction to dimethyl sulfoxide were studied. The effect of topical glucocorticosteroid on delayed contact reaction types was also studied. A polyethersulfone membrane, with a cut-off value of 100,000 Daltons was used. Reliable sample volumes and high analyte recovery was achieved either by push pull pumping or standard pumping using a perfusate consisting of Ringer Dextran 60. ELISA and flow cytometry based analysis of polystyrene bead conjugated antibodies (suspension array) was used to estimate the levels of interleukins 1 beta, 2, 4, 5, 6, 8, 10, granulocyte monocyte colony stimulating factor, interferon gamma and tumor necrosis factor alpha in normal skin for up to 24 -28 hours. Tissue sections from the area of the microdialysis membrane were examined with double immunofluorescence for cytokines and resident dermal cells.The immediate Type 1 hypersensitivity reaction to ovalbumin showed an early phase with basophil granulocytes and a late lymphocytic phase. 100% DMSO gave a basophil rich non immunological immediate reaction while repeated applications of 12% DMSO gave a reaction most like the cumulative irritant reaction. Topical glucocorticosteroid and its acetone vehicle showed anti inflammatory effects most pronounced on the acute irritant reaction. Microdialysis showed IL6, IL8 and IL1b in response to insertion with a slow equilibration period. Other cytokines were detected less frequently and in smaller amounts. The biopsies revealed intracellular cytokines in general concordance with the microdialysis fmdings. Confocal microscopy using double immunofluorescence allowed demonstration of cytokines and cellular markers in the same preparation.The experimental model illustrates differences in dermal inflammatory histological patterns in various common reaction types. Findings are relevant for discussion of pathogenetic mechanisms and as background information for continued clinical studies. Microdialysis is well suited to chronological studies of cytokine patterns in vivo. Suspension array technique allows measurement of multiple cytokines and other analytes, the results of which need interpretation against background knowledge of the particular analyte. End point biopsy for immunofluorescence studies enable intracellular localization of cytokines and even speculation about cellular origin.
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| 19. |
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| 20. |
- Sjögren, Florence, et al.
(författare)
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Immunofluorescence staining for cytokine and cellular localisation in 24 hour biopsies from the site of cutaneous microdialisys in normal human skin
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Background Classically, participation of cytokines in a tissue process is demonstrated by biopsy, immunostaining and fluorescence microscopy or by PCR technique. If the chronology of a reaction is to be studied, multiple biopsies are required. This can pose practical and ethical problems. Cutaneous microdialysis is a technique which allows the qualitative and quantitative, chronological study of endogenous molecules including cytokines in living, human skin. For confumation that cytokines detected/not detected in the dialysate are present /absent in the dermis, and to determine the possible cellular source of the cytokines, a return to biopsy technique at time points of special interest is required.Objectives The first objective of the study was to use a single immunofluorescence staining technique to demonstrate the presence or absence of ten pro-inflammatory and lymphocyte regulatory cytokines in biopsies from the site of a microdialysis catheter. Findings were to be compared with analysis of the same cytokines in microdialysates from the same subjects immediately prior to the biopsy. A further objective was to investigate use of double immunostaining, confocal microscopy to compare the localisation of individual cytokines with cellular markers for four major candidate cell types -fibroblasts, endothelial cells, mast cells and dendritic/Langerhans cells.Methods In 10 volunteers studied by dermal microdialysis in normal skin for 24 -28 hours, a biopsy was taken from the area of the microdialysis membrane at the tip of the catheter. Biopsies were frozen in liquid nitrogen. Single immunostaining for interleukin (IL) I beta (b), 2, 4, 5, 6, 8, 10, granulocyte monocyte colony stimulating factor (GM-CSF), interferon gamma (IFNg) and tumor necrosis factor alpha (TNFa), was performed and assessed semiquantitatively. Results were then compared with the microdialysis cytokine levels in the same subject innnediately prior to biopsy. Double immunostaining confocal microscopy for the cytokine and markers of the four main cell types in the dermis was performed on biopsies positive for the cytokines.Results Negative controls contained no fluorescence and there was reproducibility of results in multiple sections. Cellular localisation of cytokines was noted to varying degrees: IL8 was seen in all subjects; IL6 was seen in 70% of the subjects; TNFa in 50%; IL1b, IL2, GM-CSF and IFNg were seen in 30-40% of subjects; IL4, IL5 and IL10 were seen least often, 10 - 20% of subjects. At an individual subject level, concordance between positive or negative results for fluorescence microscopy and microdialysis was highest for IL8 (100%), lowest for TNFa (50%) with the remaining cytokines distributed between these two values. Double immunofluorescence and confocal microscopy enabled study of cytokine and cellular markers in the same section. Apparent co-localisation of the two markers was seen to varying degrees.Conclusions Fluorescence microscopy and microdialysis illustrate different aspects of cytokine presence in tissue reactions. Though individual variability can be seen in both methods and concordance of results was not seen in all subjects, the methodology was useful for better interpretation of microdialysis data. The individual concordance for the three main cytokines, IL8, IL6 and IL1b, seen after probe insertion were 100%, 80% and 60% respectively. Microdialysis fmdings in the 24 hours up to the biopsy bad suggested a non-Th2 cytokine pattern according to the Th1/Th2 paradigm. The immunofluorescence findings in the present paper indicate an even higher degree of positivity for TNFa and IFNg. Two important Th2 cytokines, IL4 and IL5 found in 1 of 10 subjects in microdialysis were not detected more frequently with immunofluorescence. The microdialysis and fluorescence findings together support a conclusion that dermal stimulation as reflected by catheter insertion creates a Th1 cytokine environment. The use of end point biopsy in the experimental design of microdialysis studies seems capable of producing worthwhlle data in regard to expression of cytokines and possible even cellular origin of cytokines.
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| 21. |
- Sjögren, Florence, 1950-
(författare)
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Light microscopic assessment of the dermal inflammatory cell infiltrate in experimental inflammatory reactions
- 1999
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In vivo experimental models have been used traditionally for the study of inflammatory contact reactions in the skin. The naked eye appearance and the histology of the skin reaction is in principle similar to that seen in man. More easily than in human experimentation, a cutaneous inflammatory reaction can, by the use of adequate group size, be studied histologically at many different time points. Plastic embedding and staining with May-GrUnewald Giemsa stains allow microscopic assessment of inflammatory cells infiltrating from the blood. Standardized, predetermined view-field selection and cellular criteria are necessary. Having previously studied the dermal infiltrate in contact reactions of delayed onset, the immediate Type I hypersensitivity reaction to ovalbumin and the non immunological immediate contact reaction to dimethyl sulfoxide (DMSO) were studied. Using the same experimental method, the effect of local application of the glucocorticosteroid, budesonide, on the delayed contact inflammatory reactions was also studied. The immediate Type I hypersensitivity reaction showed a dermal cellular infiltrate that was not uniformly distributed nor limited to the subepidermal area. The chronological study showed an influx of neutrophil and eosinophil granulocytes in the early phase and basophil granulocytes and mononuclear cells (chiefly lymphocytes) in the late phase. The non immunological immediate reaction to 100% DMSO showed the majority of the early infiltrating granulocytes to be basophils. One application of DMSO 12% gave no visible or microscopic reaction but repeated applications resulted in a delayed onset reaction with a predominant mononuclear cell infiltrate. Comparison of the two early reactions and the three previously studied reaction types showed differences and similarities at various time points in the dermal inflammatory cell infiltrate. Topically applied budesonide and its acetone vehicle showed anti inflammatory effects most pronounced on the toxic reaction to croton oil.The present model illustrates differences in dermal inflammatory histological patterns in various common model reaction types. The findings are relevant for the discussion of pathogenetic mechanisms and constitute background information for continued clinical studies.
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| 22. |
- Sjögren, Florence, et al.
(författare)
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Sterile trauma to normal human dermis invariably induces IL1beta, IL6 and IL8 in an innate response to "danger".
- 2009
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Ingår i: Acta Dermato-Venereologica. - : Medical Journals Sweden AB. - 0001-5555 .- 1651-2057. ; 89:5, s. 459-465
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Tidskriftsartikel (refereegranskat)abstract
- Microdialysis allows the study of the local production and temporal resolution of cytokines in living skin. Samples were taken from the normal skin of 10 healthy subjects for 24-28 h after insertion of a concentric microdialysis catheter, and analysed with a Luminex bead-based assay. Interleukin-1 beta (IL1b), IL6 and IL8 were seen in all subjects at all time-points after the first hour. Levels peaked at 5-8 h, equilibrating to lower levels at 24 h. Immunohistological double staining for human leukocyte antigen (HLA)-DR and intracellular cytokines on biopsies taken after catheter removal showed many stained cells in the dermis, in contrast to the few cells stained in the epidermis. This study demonstrates the reactive capability of the dermis when provoked separately from the epidermis. The production of IL1b, IL6 and IL8 occurs invariably in what can be termed an innate, dermal response to "danger"; in this case in the form of sterile needle trauma.
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| 23. |
- Sjögren, Florence, 1950-, et al.
(författare)
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Technical prerequisites for in vivo microdialysis determination of interleukin-6 in human dermis
- 2002
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Ingår i: British Journal of Dermatology. - : Oxford University Press (OUP). - 0007-0963 .- 1365-2133. ; 146:3, s. 375-382
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Tidskriftsartikel (refereegranskat)abstract
- Background Cutaneous microdialysis in vivoin human skin is demonstrably of use in the study of skin metabolism, percutaneous absorption and skin inflammation. A promising area for cutaneous microdialysis is the measurement of cytokines. This requires catheters equipped with membranes permeable to molecules of high molecular weight.Objectives To address technical problems of poor sample volume retrieval and analysis sensitivity in the simplest model of provocation, namely the insertion of the catheter itself in vivo into human dermis.Methods Use of a polyethylenesulphone membrane, with a cut-off value of 100 000 Da, allowed measurement of target molecules of large molecular weight. Using an adaptation of a commercially available high sensitivity enzyme-linked immunosorbent assay, the ubiquitous proinflammatory cytokine interleukin (IL)-6 was measured in the normal skin of six healthy volunteers after insertion of the microdialysis catheter.Results Reliable sample volumes and high analyte recovery were achieved either by push–pull pumping or by standard pumping using a perfusate consisting of Ringers Dextran 60 Braun. No IL-6 was detected in 25 of 26 samples taken during the first 100 min after catheter insertion. The IL-6 concentration then increased and remained elevated for the duration of the experiments.Conclusions Technical and analytical modifications in the microdialysis technique have allowed the measurement of IL-6 in vivo in human dermis. It is suggested that the cytokine production is the result of the dermal trauma caused by catheter insertion, but the cellular source of the IL-6 is at present unknown.
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| 24. |
- Sjögren, Florence, et al.
(författare)
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The cellular dermal infiltrate in experimental immediate type cutaneous hypersensitivity
- 1995
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Ingår i: Acta Dermato-Venereologica. - 0001-5555 .- 1651-2057. ; 75:6, s. 417-421
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Tidskriftsartikel (refereegranskat)abstract
- A previously developed guinea pig model for the study of the dermal inflammatory cell infiltrate of allergic, toxic, and irritant reactions was adapted to the study of the immediate intradermal reaction to ovalbumin, Comparison of qualitative and quantitative counts of infiltrating cells at three levels in the dermis showed that counting 20 subepidermal fields starting from the injection point of the allergen gave reliable figures, The reaction showed microscopically two phases. The first was of rapid onset and characterized by a high proportion of neutrophils, unlike the picture seen in the previously studied (allergic and toxic) reaction types. In the second phase, which can be termed 'late phase' reaction, mononuclear cells and basophil granulocytes predominated. The late phase of the reaction bears similarities to the delayed allergic contact reaction at the same timepoint in that the response was rich in basophils. There were, however, other differences; e.g, eosinophils and neutrophils were more common.
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| 25. |
- Sjögren, Florence, 1950-, et al.
(författare)
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The influence of retinoic acid and retinoic acid derivatives on Beta2 integrins and L-selectin expression in HL-60 cells in vitro
- 2000
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Ingår i: Inflammation. - 0360-3997 .- 1573-2576. ; 24:1, s. 21-32
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Tidskriftsartikel (refereegranskat)abstract
- A decreased expression of the ▀2-integrin CD11b molecules on peripheral neutrophils from patients with pustular psoriasis occurred during treatment with retinoid compounds. Since this effect could not be mimicked in vitro with isolated peripheral neutrophils, the effect of retinoid compounds on cell differentiation was investigated. The promyelocytic cell line, HL60, was used to study what effect different retinoid compounds had on the cell surface expression of CD11b and L-selectin (CD62L) molecules, complement- mediated phagocytosis, adhesion and the oxidative burst. Retinoid- differentiated cells showed a significantly lower expression of CD11b and CD62L, and a decreased phagocytosis and oxidative burst compared to DMSO- differentiated HL60 cells or peripheral blood neutrophils. The diminished expression of ▀2-integrins or L-selectin did not affect their adhesion to non-activated or lipopolysaccharide-activated endothelial cells in vitro but may however affect adhesion to vascular endothelium under shear forces during blood flow. These results suggest that retinoid treatment could affect several early steps in the inflammatory process.
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