Mechanisms of delayed wound healing by commonly used antiseptics.

BACKGROUND: The cytotoxic effects of antiseptics on pivotal cell types of the healing process have been well documented. The purpose of our investigation was to explore the ability of subcytotoxic levels of antiseptics to interfere with fibroblast function. METHODS: Cell proliferation assays were performed by culturing fibroblasts in the presence of commonly used antiseptics. Migration was evaluated using scratch assays in which monolayers were "wounded" and cellular movement was monitored by digital photography. Matrix metalloproteinase (MMP) release was analyzed by zymography. RESULTS: H2O2 and povidone-iodine reduced both migration and proliferation of fibroblasts in a dose-dependent fashion. Treatment with silver-containing antiseptics and chlorhexidine exhibited reductions in proliferation at high concentrations, but enhanced growth at lower doses. Silver-containing compounds and chlorhexidine also proved to be the least detrimental to migration in these assays. metalloproteinase release from the cells was differently affected depending on the dosage and class of antiseptic applied. CONCLUSIONS: When debridement of the wound bed is not sufficient to reduce bacterial loads, the application of broad-spectrum antiseptics maybe indicated. Our data would suggest that H2O2 and iodine are poor choices, potentially retarding the contribution of fibroblasts to the healing process. Silver sulfadiazine and chlorhexidine, at levels still proven to be bactericidal, had fewer detrimental effects on fibroblast activity in these assays. The silver-containing antiseptics may even increase the proliferative potential of these cells in culture.

A diketopiperazine fragment of human serum albumin modulates T-lymphocyte cytokine production through rap1.

BACKGROUND: Aspartyl-alanyl- diketopiperazine (DA-DKP) is generated by cleavage and cyclization from the N-terminus of human albumin during the preparation of commercial serum albumin product. Antigen-stimulated human T lymphocytes produce significantly lower quantities of interferon-gamma and tumor necrosis factor-alpha after stimulation in vitro in the presence of DA-DKP. METHODS: T lymphocytes activated in the presence of DA-DKP were analyzed by pull-down western blot assay for the activation of the guanosine triphosphatase Rap1 and by quantitative immunoassay for the phosphorylated transcription factors ATF-2 (activating transcription factor-2) and c-jun, which regulate the production of interferon-gamma and tumor necrosis factor-alpha. RESULTS: Exposure of human T lymphocytes to DA-DKP resulted in increased levels of active Rap1 and decreased activation factors relevant to the T-cell receptor signal transduction pathway and subsequently, decreased phosphorylated ATF-2 and c-jun expression. CONCLUSION: The cyclized N- terminal fragment of human serum albumin, DA-DKP, can modulate the inflammatory immune response through a molecular pathway implicated in T- lymphocyte anergy.

Commercial human albumin preparations for clinical use are immunosuppressive in vitro.

OBJECTIVE: We previously reported significant variations in oxidation status and molecular length among sources and lots of human serum albumin (HSA) commercial preparations intended for clinical use. In this report, we investigated what effect the presence of HSA products have on the immune response in vitro. DESIGN: Laboratory study. SETTING: Trauma research basic science laboratory. SUBJECTS: Activated human peripheral blood mononuclear cells. INTERVENTIONS: Six commercial HSA preparations were tested for their effect on cytokine release from activated human peripheral blood mononuclear cells (PBMCs) and T-lymphocytes. Mass spectrometry analysis of aspartyl-alanyl diketopiperazine (DA-DKP) content of HSA and percentage of HSA having lost its amino terminal dipeptide aspartyl alanyl (HSA-DA) were correlated. MEASUREMENTS AND MAIN RESULTS: Human PBMCs were cultured in the presence of six commercial HSA preparations and activated via the T-cell receptor complex. A cloned T-lymphocyte cell line, activated with specific antigen, was also cultured with both synthetic DA-DKP and small molecular weight extracts from the commercial HSA tested. Supernatants were quantified by enzyme-linked immunosorbent assay for interferon-gamma and tumor necrosis factor-alpha content. DA-DKP was extracted from HSA by centrifugal filters and quantified by anion exchange liquid chromatography coupled to negative electrospray ionization mass spectrometry. HSA species were determined by reverse phase liquid chromatography coupled to positive electrospray ionization, time of flight mass spectrometry. All HSA preparations significantly inhibited the in vitro production of interferon-gamma and tumor necrosis factor-alpha by activated PBMCs. DA-DKP was detected in all HSA sources at concentrations ranging between 42.0 and 79.6 microM. A synthetic form of DA-DKP possessed similar immunosuppressive qualities in a dose-dependent manner on T lymphocytes. CONCLUSIONS: DA-DKP was present in significant concentrations in all HSA sources tested and was partially responsible for the immunosuppressive effects of HSA on activated PBMCs and T-lymphocytes in vitro. In view of these findings, administering HSA to immunocompromised critically ill patients might be reevaluated.

Interleukin-16 expression in the peripheral blood and CD8 T lymphocytes after traumatic injury.

BACKGROUND: Expressed in several pathologic conditions, interleukin (IL-16) can induce chemotaxis and regulate the activation of CD4-positive leukocytes. This study investigated the expression of IL-16 in trauma patient plasma and peripheral blood leukocytes to determine its involvement in the physiologic response to injury. METHODS: In this study, 25 consecutive patients requiring trauma team activation and 15 noninjured subjects were evaluated for plasma IL-16 by enzyme-linked immunosorbent assay and peripheral blood leukocyte expression of intracellular cytokine by flow cytometry. RESULTS: Trauma patient plasma IL-16 was transiently increased after injury in comparison with levels in noninjured control subjects. In patients with worse outcome, both peripheral blood T lymphocyte intracellular IL-16 levels and CD4/CD8 lymphocyte ratios were lower than those for less severely injured patients and control subjects. CONCLUSION: Posttraumatic changes in IL-16 expression were found to be associated with worse patient outcome, suggesting an innate immune mechanism with a role in regulation of the T lymphocyte response to injury.

Copper stimulates the synthesis and release of interleukin-8 in human endothelial cells: a possible early role in systemic inflammatory responses.

Endogenous copper can play an important role in postischemic reperfusion injury, a condition associated with endothelial cell activation and increased interleukin 8 (IL-8) production. Excessive endothelial IL-8 secreted during trauma, major surgery, and sepsis may contribute to the development of systemic inflammatory response syndrome (SIRS), adult respiratory distress syndrome (ARDS), and multiple organ failure (MOF). No previous reports have indicated that copper has a direct role in stimulating human endothelial IL-8 secretion. Increased IL-8 in the culture medium of human umbilical vein (HUVEC), lung microvascular, and iliac artery endothelial cells was observed 24 h after the addition of 10 to 50 microM CuCl2 (cupric ions). HUVEC IL-8 induction by copper was higher than by 50 pg/mL tumor necrosis factor-alpha, whereas 50 pg/mL IL-1beta and 1 ng/mL platelet-activating factor did not stimulate IL-8 production or release. HUVEC IL-8 mRNA increased 3 h after CuCl2 stimulation and remained elevated after 24 h, implying sustained transcriptional activation. Copper did not stimulate HUVECs to secrete other cytokines. Cu(II) appeared to be the primary copper ion responsible for the observed increase in IL-8 because a specific high-affinity Cu(II)-binding peptide, d-Asp-d-Ala-d-His-d-Lys (d-DAHK), completely abolished this effect in a dose-dependent manner. These results suggest that Cu(II) may induce endothelial IL-8 by a mechanism independent of known Cu(I) generation of reactive oxygen species. Furthermore, in vivo studies are warranted to determine if copper is involved in the pathogenesis of systemic inflammation and if Cu(II) chelation can reduce this IL-8-induced endothelial inflammatory response.

Copper inhibits activated protein C: protective effect of human albumin and an analogue of its high-affinity copper-binding site, d-DAHK.

Activated protein C (APC) is useful in the treatment of sepsis. Ischemia and acidosis, which often accompany sepsis, cause the release of copper from loosely bound sites. We investigated (i) whether physiological concentrations of copper inhibit APC anticoagulant activity and (ii) if any copper-induced APC inhibition is reversible by human serum albumin (HSA) or a high-affinity copper-binding analogue of the human albumin N-terminus, d-Asp-d-Ala-d-His-d-Lys (d-DAHK). APC activity after 30 min of incubation with CuCl2 (10 microM) was decreased 26% below baseline. HSA, both alone and when combined with various ratios of CuCl2, increased APC activity significantly above baseline. d-DAHK alone and 2:1 and 4:1 ratios of d-DAHK:CuCl2 also increased APC activity. APC contained 1.4 microM copper, which helps explain the increased APC activity with HSA and d-DAHK alone. These in vitro results indicate that copper inhibits APC activity and that albumin and d-DAHK reverse the copper-induced APC deactivation.