[The care of relatives and ICU teams during a crisis]. / Zielgruppengerechte Krisenintervention ­ Angehörige und Team.

Families find themselves in an exceptional situation after the sudden death of someone close. Anxiety, aggression, rage, incomprehension, and distraction are only a few feelings of the concerned people which intensive care staff must take care of. Crisis intervention, developed in the middle of the last century, offers a framework with its concepts for the healthcare staff of how to work with the bereaved people during the first few hours. The BASIS model is a sort of counseling technique that guides nurses and physicians: bonding and urging the acceptance of the facts, providing structure and information, and securing backup support networks. Professionals who offer help need a high level of empathy and compassion for their work. But it is essential to offer help only in situations where advice is possible. Otherwise, physicians and nurses are at high risk to develop compassion fatigue. The right training, advanced education, and supervision are necessary, so that healthcare professionals can support people in crisis.

Gene suppression by tristetraprolin and release by the p38 pathway.

Tristetraprolin (TTP) is a zinc finger protein that has been implicated in the control of tumor necrosis factor (TNF) mRNA stability. We show here that TTP protein has a suppressive effect on promoter elements from TNF-alpha and interleukin-8 and that lipopolysaccharide (LPS) stimulation can release this suppression. The release in LPS-stimulated cells was found to be primarily mediated by the p38 pathway because activation of p38 is sufficient to remove the suppressive effect of TTP. Indeed, TTP seems to be a direct substrate of p38 in vivo since it is an excellent substrate of p38 in vitro, and mutation of potential phosphorylation sites in TTP prevents release of the suppression imposed on TNF transcription. We found TTP protein to be present at low levels in the resting macrophage cell line RAW 264.7 and to be quickly induced after LPS stimulation. The kinetics of TTP induction suggests a potential role of TTP as an important player in switching off LPS-induced genes after induction. In conclusion, TTP plays an important role in maintaining gene quiescence, and this quenching effect on transcription can be released by p38 phosphorylation of TTP.

Phosphorylation of eIF-4E on Ser 209 in response to mitogenic and inflammatory stimuli is faithfully detected by specific antibodies.

Phosphorylation of Ser 209 is thought to modulate the activity of the cap-binding factor eIF-4E which is a crucial component in the initiation complex for cap-dependent translation of mRNA. We report here the full reconstitution of the p38 Map kinase cascade leading to phosphorylation of eIF-4E in vitro and the generation of antibodies specific for phospho-serine 209 in eIF-4E. These antibodies were used to probe the phosphorylation of eIF-4E in mammalian cells stimulated with mitogens and pro-inflammatory cytokines. Treatment of human dermal fibroblasts with FCS led to a transient hyperphosphorylation, followed by hypophosphorylation and return to normal state phosphorylation at 16 h after the initial stimulation. By using a potent small molecular weight inhibitor of Mnk1, the upstream kinase for eIF-4E, we observed a rapid dephosphorylation of eIF-4E within 45 min after addition of the inhibitor, suggesting a high turnover of phosphate on eIF-4E mediated by Mnk1 and a yet unidentified phosphatase.

Independent role of p38 and ERK1/2 mitogen-activated kinases in the upregulation of matrix metalloproteinase-1.

Matrix metalloproteinase-1 (MMP-1) plays an important role in the degradation of extracellular matrix components under several physiological and pathological conditions. The expression of this protease is upregulated by mitogenic growth factors and proinflammatory cytokines, which have been shown to activate different sets of mitogen-activated protein (MAP) kinase pathways. Here we provide evidence that activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) or the p38 MAP kinase pathway is sufficient to induce transcription from the MMP-1 promoter in human primary fibroblasts, whereas modulation of mRNA stability seems to be of minor importance. Upregulation of MMP-1 expression by mitogenic or inflammatory stimuli is blocked by specific small molecular weight inhibitors of the ERK pathway or the p38 pathway, respectively, and constitutively active kinases within the ERK1/2 pathway (MEKK1, MEK1) or the p38 pathway (ASK1, MEKK1, MKK3) are potent activators of the MMP-1 promoter. The current study provides evidence that distinct extracellular signals leading to upregulation of MMP-1 expression in fibroblasts are relayed independently through different MAP kinase pathways and are integrated at the level of the promoter.

Overexpression of activin A in the skin of transgenic mice reveals new activities of activin in epidermal morphogenesis, dermal fibrosis and wound repair.

Recently we demonstrated a strong induction of activin expression after skin injury, suggesting a function of this transforming growth factor-beta family member in wound repair. To test this possibility, we generated transgenic mice that overexpress the activin betaA chain in the epidermis under the control of a keratin 14 promoter. The transgenic mice were significantly smaller than control littermates, and they had smaller ears and shorter tails. In their skin, the fatty tissue was replaced by connective tissue and a severe thickening of the epidermis was found. The spinous cell layer was significantly increased, and the epidermal architecture was highly disorganized. These histological abnormalities seem to result from increased proliferation of the basal keratinocytes and abnormalities in the program of keratinocyte differentiation. After skin injury, a significant enhancement of granulation tissue formation was detected in the activin-overexpressing mice, possibly as a result of premature induction of fibronectin and tenascin-C expression. These data reveal novel activities of activin in the regulation of keratinocyte proliferation and differentiation as well as in dermal fibrosis and cutaneous wound repair.

Different types of ROS-scavenging enzymes are expressed during cutaneous wound repair.

Injury to the skin initiates a series of events including inflammation, new tissue formation, and matrix remodeling. During the early inflammatory phase, polymorphonuclear leukocytes and macrophages infiltrate the wounded tissue. Once activated, they produce large amounts of reactive oxygen species (ROS) as part of their defense mechanism. Although this process is beneficial, increased levels of ROS can inhibit cell migration and proliferation and can even cause severe tissue damage. Therefore, cells must develop strategies for the detoxification of these molecules. To gain insight into the mechanisms which underlie this process, we analyzed the temporal and spatial expression pattern of various ROS-scavenging enzymes during the healing process of full-thickness excisional wounds in mice. Here we demonstrate a strong mRNA expression of two types of superoxide dismutase (SOD), as well as of catalase, and the selenoenzymes glutathione peroxidase (SeGPx) and phospholipid hydroperoxide glutathione peroxidase in normal and wounded skin. Most importantly, mRNA levels of the SODs and of SeGPx increased strongly after skin injury. In situ hybridization and immunofluorescence studies revealed the presence of these transcripts at multiple places in the wound, whereby particularly high expression levels were detected in the hyperproliferative epithelium and the hair follicles at the wound edge. These data suggest an important role of ROS-scavenging enzymes in the detoxification of ROS during cutaneous wound repair.

Connective tissue growth factor: a novel regulator of mucosal repair and fibrosis in inflammatory bowel disease?

Inflammatory bowel disease (IBD) is a multifactorial disorder which is characterized by massive damage of the epithelium and the underlying mesenchyme of the intestine. Due to the potent effect of connective tissue growth factor (CTGF) on fibroblast proliferation and connective tissue deposition we speculated about a possible role of this mitogen in IBD. Here we demonstrate a strikingly increased expression of CTGF mRNA in surgical specimens of patients suffering from two forms of IBD, Crohn's disease and ulcerative colitis. In most specimens, the levels of CTGF mRNA correlated with the degree of inflammation as assessed by histological analysis of adjacent tissue samples and by expression analysis of the pro-inflammatory cytokine interleukin-1 beta. However, areas of little inflammation which were characterized by severe fibrosis also revealed high levels of CTGF mRNA. Expression of transforming growth factor beta-1 (TGF-beta 1), the only known inducer of CTGF so far, as well as of the CTGF target genes collagen I alpha 1, fibronectin and integrin alpha 5 revealed a strong correlation with the expression of CTGF. These data suggest a prominent role of CTGF in the repair of mucosal injury in IBD and in the aberrant deposition of extracellular matrix leading to fibrosis and stenosis, one major complication in IBD, especially in Crohn's disease.

Dexamethasone is a novel potent inducer of connective tissue growth factor expression. Implications for glucocorticoid therapy.

Due to its potent effect on fibroblast proliferation and extracellular matrix deposition, connective tissue growth factor (CTGF) seems to play an important role in the pathogenesis of fibrotic disease. Since glucocorticoids are frequently used for the therapy of these disorders, we determined a potential effect of these steroids on CTGF expression. In cultured fibroblasts, a striking induction of CTGF expression was observed after dexamethasone treatment and occurred in a time- and dose-dependent manner. This effect was obviously not mediated by the CTGF inducer transforming growth factor-beta1, since expression of this factor was down-regulated by the glucocorticoid. Most importantly, CTGF expression levels also increased substantially in various tissues and organs by systemic glucocorticoid treatment of mice. After cutaneous injury, a strong induction of CTGF expression was seen in the wounds of nontreated mice. However, no further increase in the levels of CTGF mRNA occurred in wounded skin compared with unwounded skin of glucocorticoid-treated animals, suggesting the presence of other factors in the wound that might compensate for the effect of the steroids. Tumor necrosis factor-alpha was identified as a possible mediator of this effect because this factor suppressed CTGF expression in cultured fibroblasts and also blocked the glucocorticoid-induced CTGF production by these cells. These findings indicate that glucocorticoids stimulate CTGF expression in normal tissues and organs but not in highly inflamed areas.

Activin A: a novel player and inflammatory marker in inflammatory bowel disease?

Recently, we demonstrated a strong induction of activin expression after cutaneous injury. We speculated, therefore, that activin may be overexpressed during inflammatory processes in other tissues characterized by mesenchymal/epithelial structure. Herein, we show a strikingly increased expression of the activin beta A-subunit in surgical specimens from the gut of patients suffering from ulcerative colitis and Crohn's disease, whereas no activin beta A mRNA could be detected in the normal human digestive tract. The levels of activin beta A expression showed an outstanding correlation with the degree of inflammation as assessed by histologic analysis of adjacent tissue and expression analysis of the proinflammatory cytokine interleukin-1 beta. In situ hybridization studies revealed the highest levels of activin mRNA in the mucosa and submucosa of highly inflamed areas, particularly where the intestinal epithelium was damaged, but not in control tissue. In contrast, activin beta B mRNA levels in most specimens from inflamed areas were only slightly higher compared to control tissue. The strong overexpression of activin beta A in inflammatory bowel disease suggests a novel and important role of this growth and differentiation factor during inflammatory processes of the gut.

Regulation of the expression of stromelysin-2 by growth factors in keratinocytes: implications for normal and impaired wound healing.

Keratinocyte growth factor (KGF) has been implicated in wound re-epithelialization and branching morphogenesis of several organs. To determine whether KGF induces these effects via induction of matrix metalloproteinase expression we have analysed the effect of KGF on the expression of stromelysin-2 in cultured HaCaT keratinocytes. Here we show a strong induction of stromelysin-2 mRNA within 5-8 h of stimulation of these cells with KGF. The degree of induction was similar to that achieved by treatment with epidermal growth factor or tumour necrosis factor alpha, whereas the stimulatory effect of transforming growth factor beta 1 was even stronger. To determine whether the induction of stromelysin-2 expression by growth factors and cytokines might be important for wound healing, we analysed the expression of this gene during the healing process of full-thickness excisional wounds in mice. Whereas stromelysin-2 mRNA could hardly be detected in unwounded skin, a biphasic induction was seen after injury and highest levels were found at days 1 and 5 after wounding. Hybridization in situ revealed the presence of stromelysin-2 mRNA in basal keratinocytes at the wound edge but not in the underlying mesenchymal tissue. During impaired wound healing as seen in glucocorticoid-treated mice, stromelysin-2 expression was significantly increased compared with untreated control mice. Taken together, these results suggest that correct regulation of this broad-spectrum metalloproteinase might be important for normal repair.

Ultraviolet B and H2O2 are potent inducers of vascular endothelial growth factor expression in cultured keratinocytes.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is strongly expressed by epidermal keratinocytes during wound healing, in psoriasis, and in bullous diseases such as erythema multiforme and bullous pemphigoid. All of these disorders are characterized by increased microvascular permeability and angiogenesis. Since the development of erythema as a result of hyperpermeable blood vessels is also a common feature after excess sun exposure, we speculated about an up-regulation of VEGF expression by ultraviolet (UV) light. To test this hypothesis, we analyzed the effect of UVB irradiation on VEGF expression in cultured keratinocytes. Thereby we found a large increase in VEGF mRNA and protein levels upon irradiation of quiescent keratinocytes with sublethal and physiologically relevant doses of UVB. Although H2O2 was also a potent inducer of VEGF expression, the effect of UVB irradiation is unlikely to be mediated by reactive oxygen species as determined by the use of antioxidants. Further experiments revealed that the UVB-induced overexpression of VEGF is dependent on de novo protein synthesis and might occur via release of soluble mediators, which subsequently turn on VEGF expression. In summary, our results suggest a novel role of VEGF in the induction of erythema after excess sun exposure.

Keratinocyte growth factor is highly overexpressed in inflammatory bowel disease.

Recently we demonstrated an important function of keratinocyte growth factor (KGF) in wound re-epithelialization. As KGF is mitogenic for various epithelial cells, we speculated about a role of KGF in epithelial repair processes of other organs as seen in a variety of inflammatory diseases. Here we demonstrate a strikingly increased expression of KGF in surgical specimens from patients suffering from Crohn's disease and ulcerative colitis. The levels of KGF expression strongly correlated with the degree of inflammation as assessed by histological analysis of adjacent tissue and expression analysis of the pro-inflammatory cytokine interleukin-1 beta. The highest levels of KGF mRNA and protein were found in mesenchymal cells of the lamina propria, particularly in highly inflamed areas. As the KGF receptor is expressed in intestinal epithelial cells, KGF seems to act in a paracrine manner to stimulate proliferation of these cells. These data suggest a crucial role of KGF in epithelial repair after injury caused by inflammatory processes.

Differential regulation of pro-inflammatory cytokines during wound healing in normal and glucocorticoid-treated mice.

It has long been speculated that pro-inflammatory cytokines play an important role in wound repair. However, little is known about the temporal and spatial expression pattern of these cytokines during normal and impaired wound healing. In this study we show a strong and early induction of interleukins 1 alpha and beta (IL-alpha and beta) and of tumour necrosis factor alpha (TNF-alpha) expression after cutaneous injury. Highest levels of these cytokines were seen as early as 12-24 h after wounding. After completion of the proliferative phase of wound healing, mRNA levels of these cytokines returned to the basal level. During the early phase of wound repair, proinflammatory cytokines were predominantly expressed in polymorphonuclear leukocytes, suggesting a novel function of these cells in the initiation of wound healing. At later stages of the repair process, expression of IL-1 alpha, IL-1 beta and TNF-alpha was also seen in macrophages. Furthermore, TNF-alpha was detected in the hyperproliferative epithelium at the wound edge and IL-1 alpha was found in keratinocytes of the hair follicles. Induction of these cytokines after injury was significantly reduced during wound repair in healing-impaired glucocorticoid-treated mice. This finding demonstrates that wound healing defects are associated with impaired cytokine expression and suggests that the early induction of these genes is important for normal repair.

Suppression of keratinocyte growth factor expression by glucocorticoids in vitro and during wound healing.

We have recently demonstrated an important function of keratinocyte growth factor (KGF) in morphogenesis of epithelium and wound re-epithelialization. Furthermore, abnormalities in KGF expression or responsiveness are associated with wound-healing defects. In this study we have analyzed the regulation of KGF expression during wound repair in glucocorticoid-treated mice that are characterized by severe wound healing abnormalities. Induction of KGF mRNA expression after skin injury was significantly reduced in these mice, whereas KGF receptor mRNA levels were only affected to a minor extent by glucocorticoid treatment. The reduced KGF expression during wound healing in steroid-treated animals is at least partially due to a direct effect of glucocorticoids on the KGF expressing mesenchymal cells, because treatment of cultured fibroblasts with dexamethasone reduced KGF mRNA levels in a time- and concentration-dependent manner. The inhibitory effect of glucocorticoids on KGF expression was compensated for by high levels of serum growth factors or pro-inflammatory cytokines, demonstrating that KGF expression is subject to positive and negative regulation. Thus it seems likely that a fine balance of various KGF-regulating factors is important for normal wound healing.

Large induction of keratinocyte growth factor expression by serum growth factors and pro-inflammatory cytokines in cultured fibroblasts.

We have recently demonstrated a large induction of keratinocyte growth factor expression in dermal fibroblasts during wound healing. To identify possible mediators of KGF induction, we have now analysed the regulation of KGF expression in vitro in cultured murine and human fibroblasts. Here we demonstrate that KGF mRNA and protein expression is low in quiescent fibroblasts but is strongly induced upon serum stimulation. This induction can be mediated by at least two different intracellular pathways involving protein kinase C or cAMP-dependent kinases. Our finding that induction of KGF expression by serum is independent of de novo protein synthesis demonstrates, that KGF is the product of a primary response gene. The stimulatory effect of serum on KGF expression is likely to be a combinatorial effect of different mitogens, since several purified serum growth factors also stimulated KGF expression but to a lesser extent compared to serum. Furthermore, we also found a strong KGF induction by interleukin-1 beta, tumor necrosis factor-alpha and interleukin-6, cytokines which are released by polymorphonuclear leukocytes and activated macrophages during wound healing. These data suggest that serum which is released upon hemorrhage as well as pro-inflammatory cytokines might be responsible for the KGF induction in vivo during skin repair.