Richard D. Klein

An Essential Role for Lipopolysaccharide-binding Protein in Pulmonary Innate Immune Responses

Lipopolysaccharide (LPS)-binding protein (LBP) greatly facilitates LPS activation of monocytic cells through the CD14 receptor, triggering activation of innate immune responses. An acute phase protein, LBP is produced predominantly by the liver; however, we and others have shown that LBP is produced extrahepatically in multiple locations, including the lung. The importance of LBP in the lung has remained unclear. LBP may make the host more acutely sensitive to LPS and development of septic complications; alternatively, it may be protective, aiding in detection, opsonization, and killing of bacteria. Our objective was to determine the role LBP plays in local pulmonary immune defenses to bacterial challenge. LBP knockout mice and age-matched C57BL/6 wild-type controls were challenged with direct intratracheal inoculation of Klebsiella pneumoniae. We observed a significant increase in mortality, earlier onset of bacteremia, and greater pulmonary bacterial loads in LBP knockout mice compared with controls. Total lung myeloperoxidase (MPO) activity, neutrophil recruitment to the alveolar space, and levels of KC—a chemokine involved in neutrophil recruitment—in bronchoalveolar lavage (BAL) fluid and lung homogenates were found to be significantly diminished in knockout mice compared with controls. Together, our findings suggest that LBP is essential in local pulmonary innate immune responses against bacteria.

Can the clearance of tumor necrosis factor alpha and interleukin 6 be enhanced using an albumin dialysate hemodiafiltration system

Patients with acute hepatic failure (AHF) have elevated levels of inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6). Recently, we have shown selective hemodiafiltration with albumin dialysis, as an extracorporeal liver support device (ECLVS), to be effective in the clearance of multiple toxins that are elevated in AHF. Our objective was to evaluate whether ECLVS would be effective in the clearance of TNF-alpha and IL-6. An in vitro continuous hemodiafiltration circuit was used with single pass counter-current dialysis. A known amount of recombinant rat TNF-alpha and IL-6 was added to heparinized bovine blood and filtered across a polyalkyl sulfone hemofilter using matched filtration and dialysate flow rates. During 4 hours, the serial TNF-alpha and IL-6 concentrations were measured in the circulating blood, and the content of each cytokine was calculated using mass balance. For each cytokine, clearance was determined for two dialysate groups at constant temperature and pH (group 1: dialysate = 0.9 normal saline, n = 5; group 2: dialysate = albumin 2 gm/dl, n = 5). Analysis of data was performed using ANOVA and Students t-test. There was improved clearance of TNF-alpha and IL-6 when albumin was used in the dialysate (81+/-0.09% of the initial TNF-alpha and 77+/-0.04% of the IL-6 quantities) compared with when 0.9 normal saline was used as the dialysate (58+/-0.14% of the initial TNF-alpha and 56+/-0.18% of the IL-6 quantities); p < 0.03. An ECLVS utilizing hemodiafiltration with albumin dialysis is more effective than conventional hemofiltration in the clearance of TNF-alpha and IL-6 and, therefore, may benefit patients with acute hepatic failure.

Protegrin-1 enhances bacterial killing in thermally injured skin.

ObjectiveSeptic complications and the emergence of drug-resistant microbes represent serious risks to patients. Recently, naturally occurring peptides have been discovered that possess potent and broad-spectrum antimicrobial activity. Protegrin-1 is particularly attractive for clinical use in human wounds because, unlike defensins, protegrin-1 retains broad antimicrobial and antifungal activity at physiologic salt concentration and in the presence of serum. The objective of this study was to examine the efficacy of protegrin-1 in killing multiple drug-resistant microbes isolated from human burn patients. DesignFor the in vitro experiment, bilayer radial diffusion was performed comparing standard antibiotics with protegrin-1 on multiple-drug-resistant microbial organisms isolated from infected burn wounds. In vivo, rats received a 20% total body surface area partial-thickness burn by immersion in 60°C water for 20 secs followed by wound seeding with 106 colony forming units of Silvadene-resistant Pseudomonas aeruginosa. SettingUniversity of Michigan research laboratory. SubjectsAdult, male Sprague-Dawley rats. InterventionsRats were randomized into three groups: those receiving synthetic protegrin-1, acetic acid (carrier), or gentamicin (positive control). Protegrin-1 was administered by topical application or intradermal injection. Wound tissues were harvested aseptically at different time points for quantitative bacterial counts. Measurements and Main Results In vivo and in vitro experiments revealed rapid and significant decreases in bacterial counts for protegrin-1-treated groups compared with controls. ConclusionsThis study shows that protegrin-1 potentially may be used as an alternative or adjunct therapy to standard agents used to treat wound infections.

Feasibility of biolistic gene therapy in burns.

Skin is an especially attractive target for genetic manipulation because it is readily accessible and easily monitored for both the presence and the expression of inserted genes. This study was designed to assess the feasibility of particle mediated gene transfer to burned skin and to compare the transfection efficiency, anatomic distribution, and duration of transgene expression achievable in normal versus burned skin. Two days following scald injury of varying depths in 60 degrees C water (10 s: superficial partial; 20 s: deep partial; 40 s: full thickness) reporter gene (beta-galactosidase) constructs were delivered using a gene gun at various helium pressures (200-600 psi) to normal and burned skin. A time course study was performed to examine the kinetics of transgene expression. Animals received a superficial partial thickness burn and were sacrificed 12 h, 1, 3, 5, 7, 14, or 21 days after gene transfer. India Ink injection and immunohistochemistry were used to assess the depth of the scald injury. Transfection efficiency was measured in skin homogenates 24 h after gene transfer by morphometric and chemoluminescent assays. We found that the extent of tissue damage was directly related to the duration of heat source exposure. Reporter gene activity was significantly higher in superficial partial thickness burns compared to normal controls and gradually declined with increasing tissue injury. No activity was seen in the full thickness burn group. Beta-galactosidase activity reached a maximum level 12 h after gene transfer in both normal and superficial partial thickness burned skin with no levels seen after 5 days post-transfection. These findings indicate that particle-mediated gene transfer in thermally injured skin is feasible and may provide a means of introducing biologic agents into injured tissue capable of enhancing bacterial clearance and improving wound healing.

Thermal injury induces expression of CD14 in human skin

BACKGROUND Skin is equipped with an array of immune mediators aimed at fighting invading microbes. CD14 has been shown to play a key role in modulating the activation of cells by LPS. Since LPS levels within burn wounds are often found to be elevated, we sought to examine the expression of CD14 within human skin following thermal injury. METHODS Patients who sustained partial thickness burns, were recruited into the study (n=57). Total RNA was isolated from both burn and normal (control) skin. Northern blot analysis and TaqMan RT-PCR were used to determine skin CD14 mRNA levels. Immunohistochemistry was used to localize CD14 expression in burned and normal skin. RESULTS Quantitative PCR showed significantly increased CD14 expression levels in the immediate post-burn period (P<0.05 burn versus non-burn). Immunohistochemistry revealed more pronounced CD14 staining 24 h after the injury, reaching normal levels approximately 5-7 days post-burn. CONCLUSION CD14 expression peaks within the first week post-burn before declining, reaching normal levels after 14 days. This loss of supranormal CD14 expression locally within the wound may contribute to a weakened host defense response 5-6 days after injury, when patients become especially vulnerable to infection.

Skin lipopolysaccharide-binding protein and IL-1β production after thermal injury

In response to a burn injury, skin can have an inflammatory response characterized by the production of inflammatory cytokines, recruitment of immune cells, containment of invading organisms, and clearance of noxious substances from the wound. Lipopolysaccharide-binding protein (LBP) is a molecule that is capable of coordinating all 4 functions; we previously found evidence that suggested that LBP is produced within surgical wounds. Because of the central role of LBP in the response to bacterial infection, as well as in the high rate of infection after burn injuries, we sought to determine whether a thermal injury could affect wound LBP production and thereby affect host responses against bacterial infection. Rats were given either a burn or a sham burn and were killed 24, 48, and 72 hours after the injuries. Wound specimens were assayed for bacterial counts and for the presence of LBP, messenger (m)RNA, and interleukin (IL)-1beta mRNA. Wound LBP mRNA was significantly upregulated at 24 hours in the group with burn injuries (P < .05; burn vs sham burn); this was followed by decreases at 48 and 72 hours. Immunohistochemistry showed LBP protein in the epidermis of animals with burns. Bacterial counts increased in the group with burn injuries (P < .05; burn vs sham burn) and continued to rise for 72 hours. IL-1beta mRNA levels were elevated at all time points in the group with burn injuries (P < .05). These results suggest an inverse correlation between burn wound LBP expression and bacterial wound counts. This failure to maintain local LBP production after severe thermal injury despite localized inflammation shown by high IL-1beta levels may predispose local wounds to bacterial invasion.

Tu1772 The Evolving Landscape of Esophageal Cancer: A Four Decade Analysis

The incidence of esophageal cancer in the United States seems to have significantly increased since the 1970s. In undertaking this study, we sought to describe changes in the incidence, histologic type, and presenting stage of esophageal cancer over the past four decades. With Institutional Review Board approval, the Surveillance, Epidemiology, and End Results database of the National Cancer Institute was queried. Regression analysis was used to analyze data, and significance was accepted with 95 per cent probability. Forty-two thousand seven hundred thirty-nine patients had squamous cell carcinoma or adenocarcinoma located in their upper, middle, and/or lower esophagus from 1973 through 2010, reflecting a 7.5-fold annual increase from 1973 through 2010. Squamous cell carcinoma increased annually 2.5-fold (P < 0.001) and esophageal adenocarcinoma increased annually 57-fold from 1973 through 2010 (P < 0.001), whereas the overall population in the United States increased only 43 per cent (215,092,900 to 308,745,538) in the same period. From 1973 through 2010, there was a significant increase in the incidence of esophageal cancer in the United States. This increase was much greater than the increase in the population in the United States. The incidence of adenocarcinoma increased much more than that of squamous cell carcinoma of the esophagus from 1973 through 2010.

Su1783 What Are the Financial Implications of Centers for Regional Healthcare

Introduction: Financial implications on regionalization of healthcare and programmatic development are not often considered. We undertook this study to evaluate and compare hospital cost of care and income with a common operation (laparoscopic cholecystectomy) versus an operation often associated with HPB programmatic development and healthcare regionalization (pancreaticoduodenectomy). Methods and Procedures: The charges and reimbursements of all laparoscopic cholecystectomies (n=201) and pancreaticoduodenectomies (n=44) at one hospital undertaken from June 2012 to June 2013 were determined. Comparisons were undertaken using ANOVA with significance accepted at p ≤ 0.05. Data are reported as median data or as median (mean ± SD). Results: Pancreaticoduodenectomy, relative to laparoscopic cholecystectomy, had greater time in the operating room (283 min vs. 93 min), hospital charges (

Kupffer cell activation by lipopolysaccharide in rats: Role for lipopolysaccharide binding protein and toll‐like receptor 4

108,040.87 vs.

CD14 and lipopolysaccharide binding protein expression in a rat model of alcoholic liver disease.

25,055.85), and hospital costs (