Severe Traumatic Brain Injury (TBI) and aneurysmal Subarachnoid Hemorrhage (SAH) are severe cerebral emergencies. They are common reasons for extensive morbidity and mortality in young people and adults in the western world. This thesis, based on five clinical studies in patients with severe TBI (I-IV) and SAH (V), is concentrated on examination of pathophysiological developments and of evaluation of therapeutic approaches in order to improve outcome after cerebral emergency. The treatment for severe TBI patients at Umeå University Hospital, Sweden is an intracranial pressure (ICP)-targeted therapy according to “the Lund-concept”. This therapy is based on physiological principles for cerebral volume regulation, in order to preserve a normal cerebral microcirculation and a normal ICP. The main goal is to avoid development of secondary brain injuries, thus avoiding brain oedema and worsened microcirculation. Study I is evaluating retrospectively 41 children with severe TBI, from 1993 to 2002. The boundaries of the ICP-targeted protocol were obtained in 90%. Survival rate was 93%, and favourable outcome (Glasgow Outcome Scale, score 4+5) was 80%. Study II is retrospectively analysing fluid administration and fluid balance in 93 adult patients with severe TBI, from 1998 to 2001.The ICP-targeted therapy used, have defined fluid strategies. The total fluid balance was positive day one to three, and negative day four to ten. Colloids constituted 40-60% of total fluids given/day. Severe organ failure was evident for respiratory insufficiency and observed in 29%. Mortality within 28 days was 11%. Study III is a prospective, randomised, double-blind, placebo-controlled clinical trial in 48 patients with severe TBI. In order to improve microcirculation and prevent oedema formation, prostacyclin treatment was added to the ICP-targeted therapy. Prostacyclin is endogenously produced, by the vascular endothelium, and has the ability to decrease capillary permeability and vasodilate cerebral capillaries. Prostacyclin is an inhibitor of leukocyte adhesion and platelet aggregation. There was no significant difference between prostacyclin or placebo groups in clinical outcome or in cerebral microdialysis markers such as lactatepyruvate ratio and brain glucose levels. Study IV is part of the third trial and focus on the systemic release of pro-inflammatory mediators that are rapidly activated by trauma. The systemically released pro-inflammatory mediators, interleukin-6 and CRP were significantly decreased in the prostacyclin group versus the placebo group. Study V is a prospective pilot study which analyses asymmetric dimethylarginine (ADMA) concentrations in serum from SAH patients. Acute SAH patients have cerebral vascular, systemic circulatory and inflammatory complications. ADMA is a marker in vascular diseases which is correlated to endothelial dysfunction. ADMA concentrations in serum were significantly elevated seven days after the SAH compared to admission and were still elevated at the three months follow-up. Our results show overall low mortality and high favourable outcome compared to international reports on outcome in severe TBI patients. Prostacyclin administration does not improve cerebral metabolism or outcome but significantly decreases the levels of pro-inflammatory mediators. SAH seems to induce long-lasting elevations of ADMA in serum, which indicates persistent endothelial dysfunction. Endothelial dysfunction may influence outcome after severe cerebral emergencies.

Il processo infiammatorio è un meccanismo di difesa aspecifico innato, che costituisce una risposta protettiva dell’organismo a vari tipi di insulto (infezione, danno tissutale, trauma, stress, malattie autoimmune,). Esso comporta il rilascio in circolo di mediatori pro-infiammatori (es. citochine) and anti-infiammatori (es lipossine ed alcune citochine). Le citochine pro-infiammatorie inducono effetti infiammatori (es. anoressia e febbre) e stimolano la risposta di fase acuta (APR). Invece, le lipossine e le citochine anti-infiammatorie tendono ad attenuare l’infiammazione. Gli scopi di questa ricerca erano due: distinguere i soggetti in base al grado di severità della APR dopo il parto, e dopo stimolazione intramammaria con lipopolisaccaride (LPS) in bovine da latte sottoposte a diversi stress metabolici (NaCl, BHB, EuG e IpoG). I soggetti EuG e BHB hanno mostrato una APR più severa rispetto a IpoG e NaCl. Un ulteriore scopo è stato proposto un indice composto da alcune proteine di fase acuta al fine di stimare i processi infiammatori e le conseguenze epatiche (PICE). Le bovine con PICE più basso prima del parto, avevano più alti livelli plasmatici di citochine pro-infiammatorie e lipossine prima del parto (e mostravano una APR più severa dopo il parto), anche in assenza di sintomi clinici. / Inflammation is the innate, non-specific response of the host to disturbances in his homeostasis caused by infection, tissue injury, stress, trauma, neoplastic growth, immunological disorders. It involves pro- (e.g. cytokines) and anti-inflammatory mediators (e.g. lipoxins, some cytokines). The pro-inflammatory cytokines induce inflammatory effects (e.g. anorexia, fever) and play key roles in the stimulation of acute phase response (APR). The lipoxins and anti-inflammatory cytokines tend to mitigate the inflammation. Two were the aims of this research: to investigate in dairy cows the severity of APR at calving time as well as after intramammary lypopolysaccharide (LPS) administration in cows challenged with hyperinsulinemic hypoglycemic (HypoG, n=4), hyperinsulinemic euglycemic (EuG, n=5), hyperketonemic (BHB, n=4) and control (NaCl, n=6) clamps. Plasma samples were assayed for a wide metabolic and inflammatory profile. With respect to HypoG and NaCl animals, more severe APR was observed in EuG and BHB. A further aim was the proposal of an Index, composed by several acute phase proteins, to estimate Inflammatory Processes and Hepatic Consequences (IPHC). The dairy cows with lower IPHC after calving, had higher plasma levels of pro-inflammatory cytokines and of lipoxins before calving (and showed a stronger APR after calving); this was seen also in absence of clinical symptoms.

Il periodo di transizione (TP) delle vacche da latte è caratterizzata da disfunzione del sistema immunitario e dalla comparsa di fenomeni infiammatori. La tesi ha presentato una vasta revisione della letteratura seguita da 3 articoli sperimentali. Nel capitolo II sono stati investigati i cambiamenti delle citochine pro-infiammatorie (PIC) nel TP. I livelli di PIC hanno mostrato una elevata variabilità in tarda gravidanza, ma i livelli più alti hanno mostrato un’associazione con i problemi di salute e le prestazioni dopo il parto. Nel capitolo III, l'attività immunitaria di vacche in transizione è stata valutata utilizzando un test ex vivo di stimolazione del sangue con lipopolisaccaridi (WBA) e un test cutaneo alla carragenina. I risultati hanno rivelato che il sistema immunitario è molto sensibile in prossimità del parto. Entrambi i test descrivono i cambiamenti del sistema immunitario durante il TP. Nel capitolo IV, è stata valutata l’espressione genica dei leucociti durante il TP con la tecnica dell’ RNA-Seq. Confrontando i geni differenzialmente espressi con i risultati del capitolo II e III sono stati resi noti i cambiamenti funzionali dei leucociti. Complessivamente, queste ricerche contribuiscono a definire meglio la fisiologia della fase di transizione della vacche da latte. / The transition period of dairy cows is characterized by immune dysfunction and inflammatory like conditions. The thesis presented a wide review literature followed by 3 research papers. Chapter II investigated the pattern of changes of pro-inflammatory cytokines (PIC) around parturition and discovered an association with periparturient health status. PIC levels showed a high variability in late pregnancy but the highest levels demonstrated a good relationship with health troubles and performance after calving. In Chapter III, immune activity of transition cows were evaluated using: an ex vivo whole blood stimulation assay (WBA) with lipopolysaccharides and a carrageenan skin test. Results revealed that immune system is very sensitive around calving in respect to both tests, with a significant increase of pro-inflammatory cytokines and a reduction of the skin thickness after carrageenan challenge. Thus, both tests are able to describe the complex changes of the immune system combined to conventional metabolic and immune parameters. In Chapter IV, changes of leukocyte gene expression were evaluated from 20 days before to 7 days after calving using RNA-seq technique. Comparing the differentially expressed genes with the results of Chapter II and III were disclosed fundamental functional changes in leukocytes. Overall, these researches contribute to define better the physiology of the most vulnerable phase of dairy cows.

Adrenal gland insufficiency – the clinical manifestation of deficient production or action of adrenal steroids – is a life-threatening disorder. Among many factors which can predispose to primary adrenal failure, an autoimmune adrenalitis and infectious agents play a major role. The initial host defense against bacterial infections is executed primarily by the pattern recognition receptors, e.g. Toll-like receptors (TLRs), expressed in cells from the innate immune system. Upon activation, TLRs have been found to regulate various levels of innate and adaptive immunity as well as control tissue inflammation. TLRs are implicated in adrenal cell turnover and steroidogenesis during inflammation. Therefore, TLRs play a crucial role in the activation of adrenal inflammation mediating adrenal gland dysfunction during septicemia. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Adrenal gland insufficiency – the clinical manifestation of deficient production or action of adrenal steroids – is a life-threatening disorder. Among many factors which can predispose to primary adrenal failure, an autoimmune adrenalitis and infectious agents play a major role. The initial host defense against bacterial infections is executed primarily by the pattern recognition receptors, e.g. Toll-like receptors (TLRs), expressed in cells from the innate immune system. Upon activation, TLRs have been found to regulate various levels of innate and adaptive immunity as well as control tissue inflammation. TLRs are implicated in adrenal cell turnover and steroidogenesis during inflammation. Therefore, TLRs play a crucial role in the activation of adrenal inflammation mediating adrenal gland dysfunction during septicemia. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Over the past few years the NAD-related compounds nicotinamide (NAM), nicotinamide riboside (NR) and 1-methylnicotinamide (MNA) have been established as important molecules in signalling pathways that contribute to metabolic functions of many cells, including those of the immune system. Among immune cells, monocytes/macrophages, which are the major players of inflammatory processes, are especially susceptible to the anti-inflammatory action of NAM. Here we asked whether NAM and the two other compounds have the potential to regulate differentiation and LPS-induced biological answers of the monocytic cell line THP-1. We show that treatment of THP-1 cells with NAM, NR and MNA resulted in growth retardation accompanied by enrichment of cells in the G0/G1-phase independent of p21 and p53. NAM and NR caused an increase in intracellular NAD concentrations and SIRT1 and PARP1 mRNA expression was found to be enhanced. The compounds failed to up-regulate the expression of the cell surface differentiation markers CD38, CD11b and CD14. They modulated the reactive oxygen species production and primed the cells to respond less effectively to the LPS induced TNF-a production. Our data show that the NAD metabolites interfere with early events associated with differentiation of THP-1 cells along the monocytic path and that they affect LPS-induced biological responses of the cell line.

No description available.

Upon encounter of an antigen, epidermis-resident Langerhans cells (LCs) become activated and present the processed antigen to T cells of the draining lymph nodes, resulting in tolerogenic or inflammatory responses. In psoriasis plaques, skin homeostasis is disrupted and replaced by an inflammatory dermatitis. Topical application of the anti-viral compound, imiquimod, induces a psoriasiform inflammatory condition, partly driven by LC production of pro-inflammatory cytokines. Differentiation of the myeloid progenitor cell line, MUTZ-3, produces MUTZ-3 derived Langerhans cells (MUTZ-LCs) which can be used as an in vitro model of LCs. This project aimed to investigate the phenotype and function of imiquimod-treated MUTZ-LCs in monolayer cultures, co-culture with T cells and inserted into a 3D skin model. LC-related surface markers (HLA-DR, CD1a, CD207, CCR7) were upregulated in MUTZ-LCs after 7 days of differentiation with 40 ng/ml GM-CSF, 10 ng/ml TGF-β and 2.5 ng/ml TNF-α. Supernatants of imiquimod-treated monolayer cultures of MUTZ-LCs showed subtle concentrations of IL-6 and TNF-α, but not IL-23. mRNA expression showed no significant upregulation of IL-6, IL-23 or TNF-α after 24 h treatment with imiquimod. The presence of MUTZ-LCs in T cell co-cultures greatly increased the production of IL-2, but did not affect expression of CD25. After 16 h exposure to imiquimod, IL-6, IL-23 and TNF-α could not be detected in culture supernatants of a 3D model consisting of fibroblasts, keratinocytes and MUTZ-LCs. The model was devoid of fibroblasts after 19 days of culture, most likely compromising the immunocompetence, as LC migration in response to activation could not be detected. Further studies could refine and optimize the imiquimod-3D skin model, which has potential as a possible substitute for animal models in psoriasis research.

Over the past few years the NAD-related compounds nicotinamide (NAM), nicotinamide riboside (NR) and 1-methylnicotinamide (MNA) have been established as important molecules in signalling pathways that contribute to metabolic functions of many cells, including those of the immune system. Among immune cells, monocytes/macrophages, which are the major players of inflammatory processes, are especially susceptible to the anti-inflammatory action of NAM. Here we asked whether NAM and the two other compounds have the potential to regulate differentiation and LPS-induced biological answers of the monocytic cell line THP-1. We show that treatment of THP-1 cells with NAM, NR and MNA resulted in growth retardation accompanied by enrichment of cells in the G0/G1-phase independent of p21 and p53. NAM and NR caused an increase in intracellular NAD concentrations and SIRT1 and PARP1 mRNA expression was found to be enhanced. The compounds failed to up-regulate the expression of the cell surface differentiation markers CD38, CD11b and CD14. They modulated the reactive oxygen species production and primed the cells to respond less effectively to the LPS induced TNF-α production. Our data show that the NAD metabolites interfere with early events associated with differentiation of THP-1 cells along the monocytic path and that they affect LPS-induced biological responses of the cell line.

Les neutrophiles sont généralement considérés résistants aux glucocorticoïdes. Cependant, peu d’études comparant l’effet de ces drogues sur les neutrophiles et les autres leucocytes sanguins (monocytes, lymphocytes et éosinophiles) ont été rapportées. Dans notre étude, nous avons évalué la réponse aux glucocorticoïdes de ces deux populations cellulaires chez le cheval et l’homme. Les cellules, préalablement isolées du sang de 6 chevaux et 4 sujets humains sains, ont été incubées pendant 5 h en présence de lipopolysaccharide (LPS; 100 ng/mL) seul ou combiné avec de l’hydrocortisone, de la prednisolone ou de la dexaméthasone (10-8M et 10-6M). L’expression d’ARNm pour l’IL-1β, le TNF-α, l’IL-8, la glutamine synthétase et le récepteur α des glucocorticoïdes (GR-α) a été quantifiée par qPCR. Les neutrophiles équins ont également été incubés pendant 20 h en présence de ces 3 glucocorticoïdes et la survie cellulaire a été évaluée par cytométrie de flux et microscopie optique. Nous avons démontré que les glucocorticoïdes inhibaient l’expression des gènes pro-inflammatoires induite par le LPS pour les deux populations cellulaires chez les deux espèces étudiées. L’expression de la glutamine synthétase était également significativement augmentée par les glucocorticoïdes chez les neutrophiles et les autres leucocytes sanguins équins. De manière générale, l’intensité de la réponse aux glucocorticoïdes s’est avérée similaire dans les 2 populations leucocytaires et chez les deux espèces. Les glucocorticoïdes augmentaient également la survie des neutrophiles équins, phénomène également rapporté dans d’autres espèces. Ainsi, les glucococorticoïdes exercent des effets d’intensité comparable sur les neutrophiles et les autres leucocytes sanguins. Nous spéculons que la faible réponse à la corticothérapie observée lors de maladies inflammatoires chroniques neutrophiliques comme l’asthme sévère ou la Maladie Pulmonaire Obstructive Chronique (MPOC) ne s’explique pas par une corticorésistance intrinsèque des neutrophiles. / Neutrophils are generally considered resistant to glucocorticoids compared to other inflammatory cells. However, there are few studies comparing the effects of glucocorticoids in neutrophils and those of other blood leukocytes (monocytes, lymphocytes and eosinophils). In our study, we assessed glucocorticoid-responsiveness in equine and human peripheral blood neutrophils and in neutrophil-depleted leukocytes. Cells were isolated from 6 healthy horses and 4 human healthy subjects. They were incubated for 5 h with or without lipopolysaccharide (LPS; 100 ng/mL) alone or combined with hydrocortisone, prednisolone or dexamethasone (10-8M and 10-6M). IL-1β, TNF-α, IL-8, glutamine synthetase and Glucocorticoid Receptor α (GR-α) mRNA expression was quantified by qPCR. Equine neutrophils were also incubated for 20 h with or without the three glucocorticoids and cell survival was assessed by flow cytometry and light microscopy. We found that glucocorticoids down-regulated LPS-induced pro-inflammatory mRNA expression in both cell populations and species. These drugs also significantly increased glutamine synthetase gene expression in both equine cell populations. The magnitude of glucocorticoid response was generally similar in both cell populations and species. As reported in other species, glucocorticoids significantly increase the survival in equine neutrophils. Based on these results, it appears that glucocorticoids exert effects of similar magnitude on neutrophils and on other blood leukocytes. We speculate that the poor response to glucocorticoids observed in some chronic neutrophilic human diseases such as severe asthma or Chronic Obstructive Pulmonary Disease (COPD) is not explained by an inherent attenuated response of neutrophils to these drugs.