Growth factor expression is enhanced, and extracellular matrix proteins are depressed in healing skin wounds in septic patients compared with healthy controls.

Sepsis manifests as a dysregulated immune response to infection, damaging organs. Skin has a critical role in protecting the body. In sepsis, skin wound healing is impaired. The mechanisms behind it have been poorly studied. In this study, suction blister wounds were induced on intact abdominal skin in 15 septic patients. A single blister wound was biopsied from each patient and from 10 healthy controls. Immunohistochemical staining of growth factors and extracellular matrix (ECM) proteins was performed. Significance (p < 0.05) of the differences was calculated. The following growth factors were overexpressed in the skin of septic patients compared with healthy controls: epithelial growth factor (intact epithelium p = 0.007, migrating epithelium p = 0.038), vascular epithelial growth factor (intact epithelium p < 0.001, migrating epithelium p = 0.011) and transforming growth factor beta (migrating epithelium p = 0.002). The expression of syndecan-1 was upregulated in the skin of septic patients compared with healthy controls (intact epithelium p = 0.048, migrating epithelium p = 0.028). The following ECM proteins had lower expression in the epithelium in septic patients than in healthy controls: tenascin-C (migrating epithelium p = 0.03) and laminin-332 (intact epithelium p = 0.036). In sepsis, growth factor and syndecan expression was enhanced, while ECM and basement membrane proteins were mostly depressed.

1H NMR Based Metabolomics in Human Sepsis and Healthy Serum.

Early diagnosis is essential but challenging in severe sepsis. Quantifying and comparing metabolite concentrations in serum has been suggested as a new diagnostic tool. Here we used proton nuclear magnetic resonance spectroscopy (1H NMR) based metabolomics to analyze the possible differences in metabolite concentrations between sera taken from septic patients and healthy controls, as well as between sera of surviving and non-surviving sepsis patients. We took serum samples from 44 sepsis patients when the first sepsis induced organ dysfunction was found. Serum samples were also collected from 14 age and gender matched healthy controls. The samples were analyzed by quantitative 1H NMR spectroscopy for non-lipid metabolites. We found that the serum levels of glucose, glycine, 3-hydroxybutyrate, creatinine and glycoprotein acetyls (mostly alpha-1-acid glycoprotein, AGP) were significantly (p < 0.05) higher in sepsis compared to healthy sera, whereas citrate and histidine were significantly (p < 0.05) lower in sepsis patients compared to healthy controls. We found statistically significantly higher serum lactate and citrate concentrations in non-survivors compared to 30-day survivors. According to our study, 3-hydroxybutyrate, citrate, glycine, histidine, and AGP are candidates for further studies to enable identification of phenotype association in the early stages of sepsis.

Inhibitory effects of serum from sepsis patients on epithelial cell migration in vitro: a case control study.

BACKGROUND: Sepsis delays wound re-epithelialization. In this study we explored the effect of human sepsis sera as well as the effects of cytokines, growth factors and exosomes of sepsis sera treated normal fibroblasts (NF) on keratinocyte migration and proliferation in vitro. METHODS: Serum samples were taken on days 1, 4, and 9 from 44 patients diagnosed with severe sepsis, and from 14 matching healthy controls. We evaluated the effects of sepsis serum with or without TNF-α, EGF, EGF receptor inhibitor or exosomes of sepsis sera treated NF on human keratinocyte (HaCaT) proliferation (BrdU assay), viability (MTT assay), and migration (horizontal wound healing model). Cytokine levels of sepsis and healthy sera were measured by multiplex assay. Comparisons between groups were carried out using SPSS statistics and P < 0.05 was considered significant. RESULTS: Severe-sepsis sera collected on days 1, 4, and 9 reduced keratinocyte proliferation by 6% (P = 0.005), 20% (P = 0.001), and 18% (P = 0.002), respectively, compared to control sera. Cell viability in cultures exposed to sepsis sera from days 4 and 9 was reduced by 38% (P = 0.01) and 58% (P < 0.001), respectively. Open-surface wounds exposed to sepsis sera from days 1 and 4 were larger than those exposed to sera from healthy controls (60 vs. 31%, P = 0.034 and 66 vs. 31%, P = 0.023, respectively). Exosomes of sepsis or healthy sera treated NF inhibited keratinocyte migration. We detected higher serum levels of cytokines TNF-α (5.7 vs. 0.7 pg/ml, P < 0.001), IL-6 (24.8 vs. 3.8 pg/ml, P < 0.001), IL-10 (30.0 vs. 11.9 pg/ml, P = 0.040), and VEGF (177.9 vs. 48.1 pg/ml, P = 0.018) in sepsis sera. Levels of EGF were significantly lower in sepsis sera than in that of healthy controls (6.5 vs. 115.6 pg/ml, P < 0.001). Sepsis serum supplemented with EGF 5 ng/ml and TNF-α in all concentrations improved keratinocyte migration. CONCLUSIONS: Keratinocyte viability, proliferation and migration were reduced in severe sepsis in vitro. Exosomes from NF added in healthy or sepsis serum media inhibited keratinocyte migration. Decreased levels of EGF in sepsis sera may partially explain the delay of wound healing with severe-sepsis patients. Increased levels of TNF-α in sepsis sera do not explain diminished keratinocyte migration.

Skin collagen synthesis is depressed in patients with severe sepsis.

BACKGROUND: Skin is an essential barrier in maintaining a stable internal environment. Adequate regenerative capacity is crucial to overcome the homeostatic challenges caused by a septic insult. In sepsis, coagulation and inflammation are activated to restore homeostasis, but it is not known whether sepsis also alters tissue regeneration processes such as skin collagen synthesis. METHODS: In this prospective observational study, we measured aminoterminal propeptides of collagens I and III (PINP, PIIINP) from blister fluid of sepsis patients. Blister fluid was obtained from experimental blisters induced on intact abdominal skin 4 times: within the first 48 hours from the first organ failure, on the fifth day, and at 3 and 6 months thereafter. Forty-four patients with severe sepsis were enrolled. The median age was 63 years (25th-75th percentile, 53-71 years). The median Acute Physiology and Chronic Health Evaluation II score on admission was 26 (22-30). Thirty-day mortality was 25%. Fifteen healthy adults were used as controls. RESULTS: Median PIIINP and PINP levels in septic patients were lower in comparison with controls in the first blister (40.8 microg/L [25th-75th percentile, 22.2-77.1 microg/L], P = 0.028 and 69.9 microg/L [32.4-112.7 microg/L], P < 0.001, respectively) and in the blister induced on day 5 (38.8 microg/L [19.9-68.5 microg/L], P < 0.001 and 90.0 [35.1-138.8 microg/L], P < 0.001, respectively). The survivors revealed an overexpression at 3 months, whereas normal values of PIIINP and PINP were reestablished at 6 months. CONCLUSIONS: Skin collagen synthesis is depressed during severe sepsis and is followed by a compensatory response 3 and 6 months after the onset of sepsis.

Matrix-metalloproteinase-2, -8 and -9 in serum and skin blister fluid in patients with severe sepsis.

INTRODUCTION: Matrix metalloproteinases (MMPs) have various roles in inflammatory states. They seem to be able to modulate endothelial barriers and regulate the activity of chemokines and cytokines. The timely development of the levels during severe sepsis and thereafter have not been investigated. In addition it was of interest to study alterations of MMP-levels in intact skin, as the skin is the largest barrier against external pathogens and MMPs have not been studied at organ level in human sepsis. The aim of this study was to investigate the timely development of serum and skin MMP-2, -8 and -9 levels in human severe sepsis and their association with disease severity and mortality. METHODS: Forty-four patients with severe sepsis and fifteen healthy controls were included in this prospective longitudinal study. The amounts of MMP-2, -8 and -9 were analyzed from serum at days 1, 4, 6, 8, and 10, and from skin suction blister fluid at days 1 and 5 from the beginning of severe sepsis. Additionally, samples from the survivors were obtained after three and six months. RESULTS: The levels of MMP-2 and -8 were up-regulated in severe sepsis in comparison to healthy controls in skin blister fluid and serum. Compared to the controls MMP-9 levels were lower in sepsis from the fourth day on in serum and both the first and fifth day in skin blister fluid. Active forms of MMP-2 and -9 were present only in severe sepsis. The non-survivors had higher pro- and active MMP-2 levels than the survivors in skin blister fluid samples. Furthermore, MMP-2 levels were more pronounced in blister fluid and serum samples in patients with more severe organ failures. In the survivors at 3 and 6 month follow-up the MMP levels had returned to normal. CONCLUSIONS: MMP-2 and -8 are elevated in serum and blister fluid in severe sepsis, implying that they may play a significant role in the pathogenesis of severe sepsis and organ dysfunctions. Active forms of MMP-2 and 9 were only present in patients with severe sepsis, and higher MMP-2 levels in skin blister and serum were associated with more severe organ dysfunctions.

Epidermal wound healing in severe sepsis and septic shock in humans.

INTRODUCTION: The effect of sepsis on epidermal wound healing has not been previously studied. It was hypothesised that epidermal wound healing is disturbed in severe sepsis. METHODS: Blister wounds were induced in 35 patients with severe sepsis and in 15 healthy controls. The healing of the wounds was followed up by measuring transepidermal water loss and blood flow in the wound, reflecting the restoration of the epidermal barrier function and inflammation, respectively. The first set of suction blisters (early wound) was made within 48 hours of the first sepsis-induced organ failure and the second set (late wound) four days after the first wound. In addition, measurements were made on the intact skin. RESULTS: The average age of the whole study population was 62 years (standard deviation [SD] 12). The mean Acute Physiology and Chronic Health Evaluation II (APACHE II) score on admission was 25 (SD 8). The two most common causes of infections were peritonitis and pneumonia. Sixty-six percent of the patients developed multiple organ failure. The decrease in water evaporation from the wound during the first four days was lower in septic patients than in the control subjects (56 g/m2 per hour versus 124 g/m2 per hour, P = 0.004). On the fourth day, septic patients had significantly higher blood flow in the wound compared with the control subjects (septic patients 110 units versus control subjects 47 units, P = 0.001). No difference in transepidermal water loss from the intact skin was found between septic patients and controls. Septic patients had higher blood flow in the intact skin on the fourth and on the eighth day of study compared with the controls. CONCLUSIONS: The restoration of the epidermal barrier function is delayed and wound blood flow is increased in patients with severe sepsis.

Markers of collagen synthesis and degradation are increased in serum in severe sepsis: a longitudinal study of 44 patients.

INTRODUCTION: Sepsis-related multiple organ dysfunction is a common cause of death in the intensive care unit. The effect of sepsis on markers of tissue repair is only partly understood. The aim of this study was to measure markers of collagen synthesis and degradation during sepsis and investigate the association with disease severity and outcome. METHODS: Forty-four patients with severe sepsis participated in the study and 15 volunteers acted as controls. Blood samples were collected for 10 days after the first sepsis-induced organ dysfunction and after three and six months. Procollagen type I and III aminoterminal propeptides (PINP and PIIINP) and cross-linked telopeptides of type I collagen (ICTP) were measured. RESULTS: The PIIINP concentration was elevated in the septic patients (8.8 microg/L, 25th to 75th percentile = 6.8 to 26.0) when compared with controls (3.0 microg/L, 25th to 75th percentile = 2.7 to 3.3; P < 0.001) on day one. Maximum serum PIIINP concentrations during sepsis were higher in non-survivors compared with survivors (26.1 microg/L, 25th to 75th percentile = 18.7 to 84.3; vs. 15.1 microg/L, 25th to 75th percentile = 9.6 to 25.5; P = 0.033) and in multiple organ failure (MOF) compared with multiple organ dysfunction syndrome (MODS) (24.2 ug/L, 25th to 75th percentile = 13.4 to 48.2; vs. 8.9 microg/L, 25th to 75th percentile = 7.4 to 19.4; P = 0.002). Although the PINP values of the septic patients remained within the laboratory reference values, patients with MOF had higher values than patients with MODS (79.8, 25th to 75th percentile = 44.1 to 150.0; vs.40.4, 25th to 75th percentile = 23.6 to 99.3; P = 0.007). Day one ICTP levels were elevated in septic patients compared with the controls (19.4 microg/L, 25th to 75th percentile = 12.0 to 29.8; vs. 4.1 microg/L, 25th to 75th percentile = 3.4 to 5.0; P < 0.001). CONCLUSIONS: Markers of collagen metabolism are increased in patients with severe sepsis and can be investigated further as markers of disease severity and outcome.