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  • 1.
    Farnebo, Simon
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of Plastic Surgery, Hand surgery UHL.
    Karlander, Lars-Erik
    Linköping University, Department of Clinical and Experimental Medicine, Surgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of Plastic Surgery, Hand surgery UHL.
    Steinwall, Ingrid
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of Plastic Surgery, Hand surgery UHL.
    Sjögren, Florence
    Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology . Linköping University, Faculty of Health Sciences.
    Sjöberg, Folke
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of Plastic Surgery, Hand surgery UHL.
    Continuous assessment of concentrations of cytokines in experimental injuries of the extremity2009In: International Journal of Clinical and Experimental Medicine, ISSN 1940-5901, Vol. 2, no 4, p. 354-362Article in journal (Refereed)
    Abstract [en]

    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.

  • 2.
    Hillman, Jan
    et al.
    Linköping University, Department of Neuroscience and Locomotion, Neurosurgery. Linköping University, Faculty of Health Sciences.
    Milos, Peter
    Linköping University, Department of Neuroscience and Locomotion, Neurosurgery. Linköping University, Faculty of Health Sciences.
    Zhengquan, Yu
    Linköping University, Department of Neuroscience and Locomotion, Neurosurgery. Linköping University, Faculty of Health Sciences.
    Sjögren, Florence
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Anderson, Chris
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Mellergård, Pekka
    Linköping University, Department of Neuroscience and Locomotion, Neurosurgery. Linköping University, Faculty of Health Sciences.
    Intracerebral microdialysis in neurosurgical intensive care patients utilising catheters with different molecular cut-off (20 and 100 kD)2006In: Acta Neurochirurgica, ISSN 0001-6268, E-ISSN 0942-0940, Vol. 148, no 3, p. 319-324Article in journal (Refereed)
    Abstract [en]

    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.

  • 3.
    Johansson, Joakim
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine.
    Sjögren, Florence
    Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Dermatology and Venerology in Östergötland.
    Bodelsson, Mikael
    Lund University.
    Sjöberg, Folke
    Linköping University, Department of Clinical and Experimental Medicine, Burn Center. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Plastic Surgery, Hand surgery UHL. Östergötlands Läns Landsting, Sinnescentrum, Department of Anaesthesiology and Surgery UHL.
    Dynamics of leukocyte receptors after severe burns: An exploratory study2011In: BURNS, ISSN 0305-4179, Vol. 37, no 2, p. 227-233Article in journal (Refereed)
    Abstract [en]

    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.

  • 4.
    Mellergard, Pekka
    et al.
    Östergötlands Läns Landsting, Reconstruction Centre, Department of Neurosurgery UHL.
    Sjögren, Florence
    Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Dermatology and Venerology in Östergötland.
    Hillman, Jan
    Linköping University, Department of Clinical and Experimental Medicine, Neurosurgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of Neurosurgery UHL.
    Release of VEGF and FGF in the extracellular space following severe subarachnoidal haemorrhage or traumatic head injury in humans2010In: British Journal of Neurosurgery, ISSN 0268-8697, E-ISSN 1360-046X, Vol. 24, no 3, p. 261-267Article in journal (Refereed)
    Abstract [en]

    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.

  • 5.
    Mellergard, Pekka
    et al.
    Östergötlands Läns Landsting, Sinnescentrum, Department of Neurosurgery UHL.
    Sjögren, Florence
    Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Dermatology and Venerology in Östergötland.
    Hillman, Jan
    Linköping University, Department of Clinical and Experimental Medicine, Neurosurgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Neurosurgery UHL.
    The Cerebral Extracellular Release of Glycerol, Glutamate, and FGF2 Is Increased in Older Patients following Severe Traumatic Brain Injury2012In: Journal of Neurotrauma, ISSN 0897-7151, E-ISSN 1557-9042, Vol. 29, no 1, p. 112-118Article in journal (Refereed)
    Abstract [en]

    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.

  • 6.
    Mellergård, Pekka
    et al.
    Östergötlands Läns Landsting, Reconstruction Centre, Department of Neurosurgery UHL.
    Åneman, Oscar
    Linköping University, Department of Clinical and Experimental Medicine, Neurosurgery. Linköping University, Faculty of Health Sciences.
    Sjögren, Florence
    Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Dermatology and Venerology in Östergötland.
    Säberg, Carina
    Östergötlands Läns Landsting, Sinnescentrum, Department of Neurosurgery UHL.
    Hillman, Jan
    Linköping University, Department of Clinical and Experimental Medicine, Neurosurgery. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of Neurosurgery UHL.
    Differences in Cerebral Extracellular Response of Interleukin-1 beta, Interleukin-6, and Interleukin-10 After Subarachnoid Hemorrhage or Severe Head Trauma in Humans2011In: NEUROSURGERY, ISSN 0148-396X, Vol. 68, no 1, p. 12-19Article in journal (Refereed)
    Abstract [en]

    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.

  • 7.
    Mellergård, Pekke
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Neurosurgery . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Reconstruction Centre, Department of Neurosurgery UHL.
    Åneman, Oscar
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Neuroscience and Locomotion.
    Sjögren, Florence
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology .
    Pettersson, P.
    Linköping University, Department of Clinical and Experimental Medicine, Neurosurgery . Linköping University, Faculty of Health Sciences.
    Hillman, Jan
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Neurosurgery . Östergötlands Läns Landsting, Reconstruction Centre, Department of Neurosurgery UHL.
    Changes in Extracellular Concentrations of Some Cytokines, Chemokines, and Neurotrophic Factors After Insertion of Intracerebral Microdialysis Catheters in Neurosurgical Patients2008In: Neurosurgery, ISSN 0148-396X, E-ISSN 1524-4040, Vol. 62, no 1, p. 151-157Article in journal (Refereed)
    Abstract [en]

    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.

  • 8.
    Seifert, Oliver
    et al.
    Ryhov Hospital, Sweden .
    Matussek, Andreas
    Ryhov Hospital, Sweden .
    Sjögren, Florence
    Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology. Linköping University, Faculty of Health Sciences.
    Geffers, Robert
    Helmholtz Centre Infect Research, Germany .
    Anderson, Chris
    Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Center, Department of Dermatology and Venerology.
    Gene expression profiling of macrophages: implications for an immunosuppressive effect of dissolucytotic gold ions2012In: Journal of Inflammation, ISSN 1476-9255, E-ISSN 1476-9255, Vol. 9, no 43Article in journal (Refereed)
    Abstract [en]

    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.

  • 9.
    Sjögren, Florence
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Dermal cell trafficking: from microscopy to microdialysis2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    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.

    List of papers
    1. The cellular dermal infiltrate in experimental immediate type cutaneous hypersensitivity
    Open this publication in new window or tab >>The cellular dermal infiltrate in experimental immediate type cutaneous hypersensitivity
    1995 (English)In: Acta Dermato-Venereologica, ISSN 0001-5555, E-ISSN 1651-2057, Vol. 75, no 6, p. 417-421Article in journal (Refereed) Published
    Abstract [en]

    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.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-84289 (URN)8651014 (PubMedID)
    Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2017-12-07Bibliographically approved
    2. The spectrum of inflammatory cell response to dimethyl sulfoxide
    Open this publication in new window or tab >>The spectrum of inflammatory cell response to dimethyl sulfoxide
    2000 (English)In: Contact Dermatitis, ISSN 0105-1873, E-ISSN 1600-0536, Vol. 42, no 4, p. 216-221Article in journal (Refereed) Published
    Abstract [en]

    Dimethyl sulfoxide (DMSO), depending upon the concentration and mode of application to the skin, can induce either a non-immunological immediate contact urticaria or an irritant reaction. The dermal cellular infiltrate after open application of varying concentrations of DMSO has been studied in an experimental guinea pig model. The composition of the dermal cellular infiltrate showed a spectrum dependent on the concentration and number of applications of DMSO. The immediate reaction infiltrate 3 h after application of 100% DMSO consisted of 50% granulocytes, basophils being predominant. On the other hand, 12% DMSO applied 3 × daily for 3 days (cumulative insult) caused histologically a cellular reaction in which 80% of the infiltrate consisted of mononuclear cells. The present findings are compared to the microscopic findings in 3 other cutaneous reactions previously studied in this animal model, namely, the Type I immediate hypersensitivity reaction, the Type IV delayed hypersensitivity reaction, and the irritant reaction. Differing cellular infiltrate patterns are discernible at the same time points. The study illustrates the spectrum of inflammatory reactions seen in the skin and provides background information for future clinical studies, for instance, on the role of the basophil granulocyte in immediate contact reactions.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-24852 (URN)10.1034/j.1600-0536.2000.042004216.x (DOI)9251 (Local ID)9251 (Archive number)9251 (OAI)
    Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
    3. Acetone has anti-inflammatory effects on experimental contact reactions
    Open this publication in new window or tab >>Acetone has anti-inflammatory effects on experimental contact reactions
    1999 (English)In: Contact Dermatitis, ISSN 0105-1873, E-ISSN 1600-0536, Vol. 41, no 1, p. 22-29Article in journal (Refereed) Published
    Abstract [en]

    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.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-24846 (URN)10.1111/j.1600-0536.1999.tb06203.x (DOI)9244 (Local ID)9244 (Archive number)9244 (OAI)
    Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
    4. Technical prerequisites for in vivo microdialysis determination of interleukin-6 in human dermis
    Open this publication in new window or tab >>Technical prerequisites for in vivo microdialysis determination of interleukin-6 in human dermis
    2002 (English)In: British Journal of Dermatology, ISSN 0007-0963, E-ISSN 1365-2133, Vol. 146, no 3, p. 375-382Article in journal (Refereed) Published
    Abstract [en]

    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.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-24850 (URN)10.1046/j.1365-2133.2001.144003650_146_3.x (DOI)9249 (Local ID)9249 (Archive number)9249 (OAI)
    Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
    5. Cytokine suspension array analysis of microdialysate samples from normal human skin during 24 hours after 100 kiloDalton cut of membrane catheter insertion
    Open this publication in new window or tab >>Cytokine suspension array analysis of microdialysate samples from normal human skin during 24 hours after 100 kiloDalton cut of membrane catheter insertion
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    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.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-84290 (URN)
    Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2012-10-03Bibliographically approved
    6. Immunofluorescence staining for cytokine and cellular localisation in 24 hour biopsies from the site of cutaneous microdialisys in normal human skin
    Open this publication in new window or tab >>Immunofluorescence staining for cytokine and cellular localisation in 24 hour biopsies from the site of cutaneous microdialisys in normal human skin
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    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.

    Keywords
    Immunostaining, cytokines, cutaneous microdialysis, confocal microscopy normal skin
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-84291 (URN)
    Available from: 2012-10-03 Created: 2012-10-03 Last updated: 2012-10-03Bibliographically approved
  • 10.
    Sjögren, Florence
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Light microscopic assessment of the dermal inflammatory cell infiltrate in experimental inflammatory reactions1999Licentiate thesis, comprehensive summary (Other academic)
    Abstract [en]

    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.

  • 11.
    Sjögren, Florence
    et al.
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Anderson, C.
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Cytokine suspension array analysis of microdialysate samples from normal human skin during 24 hours after 100 kiloDalton cut of membrane catheter insertionManuscript (preprint) (Other academic)
    Abstract [en]

    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.

  • 12.
    Sjögren, Florence
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology . Linköping University, Faculty of Health Sciences.
    Anderson, Chris
    Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology . Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Medicine, Department of Dermatology and Venerology in Östergötland.
    Sterile trauma to normal human dermis invariably induces IL1beta, IL6 and IL8 in an innate response to "danger".2009In: Acta Dermato-Venereologica, ISSN 0001-5555, E-ISSN 1651-2057, Vol. 89, no 5, p. 459-465Article in journal (Refereed)
    Abstract [en]

    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.

  • 13.
    Sjögren, Florence
    et al.
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Anderson, Chris
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    The spectrum of inflammatory cell response to dimethyl sulfoxide2000In: Contact Dermatitis, ISSN 0105-1873, E-ISSN 1600-0536, Vol. 42, no 4, p. 216-221Article in journal (Refereed)
    Abstract [en]

    Dimethyl sulfoxide (DMSO), depending upon the concentration and mode of application to the skin, can induce either a non-immunological immediate contact urticaria or an irritant reaction. The dermal cellular infiltrate after open application of varying concentrations of DMSO has been studied in an experimental guinea pig model. The composition of the dermal cellular infiltrate showed a spectrum dependent on the concentration and number of applications of DMSO. The immediate reaction infiltrate 3 h after application of 100% DMSO consisted of 50% granulocytes, basophils being predominant. On the other hand, 12% DMSO applied 3 × daily for 3 days (cumulative insult) caused histologically a cellular reaction in which 80% of the infiltrate consisted of mononuclear cells. The present findings are compared to the microscopic findings in 3 other cutaneous reactions previously studied in this animal model, namely, the Type I immediate hypersensitivity reaction, the Type IV delayed hypersensitivity reaction, and the irritant reaction. Differing cellular infiltrate patterns are discernible at the same time points. The study illustrates the spectrum of inflammatory reactions seen in the skin and provides background information for future clinical studies, for instance, on the role of the basophil granulocyte in immediate contact reactions.

  • 14.
    Sjögren, Florence
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology. Linköping University, Faculty of Health Sciences.
    Anderson, Chris D
    Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Dermatology and Venerology in Östergötland.
    Are cutaneous microdialysis cytokine findings supported by end-point biopsy immunohistochemistry findings?2010In: AAPS Journal, ISSN 1550-7416, E-ISSN 1550-7416, Vol. 12, no 4, p. 741-749Article in journal (Refereed)
    Abstract [en]

    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”.

  • 15.
    Sjögren, Florence
    et al.
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Andersson, C.
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Immunofluorescence staining for cytokine and cellular localisation in 24 hour biopsies from the site of cutaneous microdialisys in normal human skinManuscript (preprint) (Other academic)
    Abstract [en]

    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.

  • 16.
    Sjögren, Florence
    et al.
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Andersson, C.
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Groth, O.
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    The cellular dermal infiltrate in experimental immediate type cutaneous hypersensitivity1995In: Acta Dermato-Venereologica, ISSN 0001-5555, E-ISSN 1651-2057, Vol. 75, no 6, p. 417-421Article in journal (Refereed)
    Abstract [en]

    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.

  • 17.
    Sjögren, Florence
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology. Linköping University, Faculty of Health Sciences.
    Davidsson, Karin
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Sjöström, Michael
    Umeå University.
    Anderson, Chris
    Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Dermatology and Venerology in Östergötland.
    Cutaneous Microdialysis: Cytokine Evidence for Altered Innate Reactivity in the Skin of Psoriasis Patients?2012In: AAPS Journal, ISSN 1550-7416, E-ISSN 1550-7416, Vol. 14, no 2, p. 187-195Article in journal (Refereed)
    Abstract [en]

    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.

  • 18.
    Sjögren, Florence
    et al.
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Groth, O.
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Anderson, Chris
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Acetone has anti-inflammatory effects on experimental contact reactions1999In: Contact Dermatitis, ISSN 0105-1873, E-ISSN 1600-0536, Vol. 41, no 1, p. 22-29Article in journal (Refereed)
    Abstract [en]

    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.

  • 19.
    Sjögren, Florence
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Dermatology.
    Ljunghusen, O
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Baas, A
    Coble, Britt-Inger
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Dermatology. Östergötlands Läns Landsting, Centre for Medicine, Department of Dermatology and Venerology in Östergötland.
    Stendahl, Olle
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Expression and function of beta-2 integrein CD11B/CD18 on leukocytes from patients with psoriasis. 1999In: Acta Dermato-Venereologica, ISSN 0001-5555, E-ISSN 1651-2057, Vol. 79, p. 105-110Article in journal (Refereed)
  • 20.
    Sjögren, Florence
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Biomedicine and Surgery, Dermatology.
    Stendahl, Olle
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    Ljunghusen, Olof
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology.
    The influence of retinoic acid and retinoic acid derivatives on Beta2 integrins and L-selectin expression in HL-60 cells in vitro2000In: Inflammation, ISSN 0360-3997, E-ISSN 1573-2576, Vol. 24, no 1, p. 21-32Article in journal (Refereed)
    Abstract [en]

    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.

  • 21.
    Sjögren, Florence
    et al.
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Svensson, C.
    Linköping University, Department of Biomedicine and Surgery, Dermatology. Linköping University, Faculty of Health Sciences.
    Anderson, Chris
    Department of Dermatology, Liverpool Health Service, Australia.
    Technical prerequisites for in vivo microdialysis determination of interleukin-6 in human dermis2002In: British Journal of Dermatology, ISSN 0007-0963, E-ISSN 1365-2133, Vol. 146, no 3, p. 375-382Article in journal (Refereed)
    Abstract [en]

    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.

  • 22.
    Sjöwall, Johanna
    et al.
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Infectious Diseases.
    Fryland, Linda
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Clinical Immunology.
    Nordberg, Marika
    Linköping University, Department of Clinical and Experimental Medicine, Infectious Diseases. Linköping University, Faculty of Health Sciences.
    Sjögren, Florence
    Linköping University, Department of Clinical and Experimental Medicine, Dermatology and Venerology. Linköping University, Faculty of Health Sciences.
    Garpmo, Ulf
    Kalmar Hospital.
    Jansson, Christian
    Aland Borrelia Grp.
    Carlsson, Sten-Anders
    Aland Borrelia Grp.
    Bergstrom, Sven
    Umea University.
    Ernerudh, Jan
    Linköping University, Department of Clinical and Experimental Medicine, Clinical Immunology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Immunology and Transfusion Medicine.
    Nyman, Dag
    Aland Borrelia Grp.
    Forsberg, Pia
    Linköping University, Department of Clinical and Experimental Medicine, Infectious Diseases. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Medicine, Department of Infectious Diseases in Östergötland.
    Ekerfelt, Christina
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Clinical and Experimental Medicine, Clinical Immunology.
    Decreased Th1-Type Inflammatory Cytokine Expression in the Skin Is Associated with Persisting Symptoms after Treatment of Erythema Migrans2011In: PLOS ONE, ISSN 1932-6203, Vol. 6, no 3, p. 0018220-Article in journal (Refereed)
    Abstract [en]

    Background: Despite the good prognosis of erythema migrans (EM), some patients have persisting symptoms of various character and duration post-treatment. Several factors may affect the clinical outcome of EM, e. g. the early interaction between Borrelia (B.) burgdorferi and the host immune response, the B. burgdorferi genotype, antibiotic treatment as well as other clinical circumstances. Our study was designed to determine whether early cytokine expression in the skin and in peripheral blood in patients with EM is associated with the clinical outcome. Methods: A prospective follow-up study of 109 patients with EM was conducted at the A land Islands, Finland. Symptoms were evaluated at 3, 6, 12 and 24 months post-treatment. Skin biopsies from the EM and healthy skin were immunohistochemically analysed for expression of interleukin (IL)-4, IL-10, IL-12p70 and interferon (IFN)-gamma, as well as for B. burgdorferi DNA. Blood samples were analysed for B. burgdorferi antibodies, allergic predisposition and levels of systemic cytokines. Findings: None of the patients developed late manifestations of Lyme borreliosis. However, at the 6-month follow-up, 7 of 88 patients reported persisting symptoms of diverse character. Compared to asymptomatic patients, these 7 patients showed decreased expression of the Th1-associated cytokine IFN-gamma in the EM biopsies (p = 0.003). B. afzelii DNA was found in 48%, B. garinii in 15% and B. burgdorferi sensu stricto in 1% of the EM biopsies, and species distribution was the same in patients with and without post-treatment symptoms. The two groups did not differ regarding baseline patient characteristics, B. burgdorferi antibodies, allergic predisposition or systemic cytokine levels. Conclusion: Patients with persisting symptoms following an EM show a decreased Th1-type inflammatory response in infected skin early during the infection, which might reflect a dysregulation of the early immune response. This finding supports the importance of an early, local Th1-type response for optimal resolution of LB.

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