Cell Therapies for Acute Radiation Syndrome.

The risks of severe ionizing radiation exposure are increasing due to the involvement of nuclear powers in combat operations, the increasing use of nuclear power, and the existence of terrorist threats. Exposure to a whole-body radiation dose above about 0.7 Gy results in H-ARS (hematopoietic acute radiation syndrome), which is characterized by damage to the hematopoietic system; higher doses result in further damage to the gastrointestinal and nervous systems. Only a few medical countermeasures for ARS are currently available and approved for use, although others are in development. Cell therapies (cells or products produced by cells) are complex therapeutics that show promise for the treatment of radiation injury and have been shown to reduce mortality and morbidity in animal models. Since clinical trials for ARS cannot be ethically conducted, animal testing is extremely important. Here, we describe cell therapies that have been tested in animal models. Both cells and cell products appear to promote survival and lessen tissue damage after whole-body irradiation, although the mechanisms are not clear. Because radiation exposure often occurs in conjunction with other traumatic injuries, animal models of combined injury involving radiation and future countermeasure testing for these complex medical problems are also discussed.

Gemcitabine­fucoxanthin combination in human pancreatic cancer cells.

Gemcitabine is a chemotherapeutic agent for pancreatic cancer treatment. It has also been demonstrated to inhibit human pancreatic cancer cell lines, MIA PaCa-2 and PANC-1. The aim of the present study was to investigate the suppressive effect of fucoxanthin, a marine carotenoid, in combination with gemcitabine on pancreatic cancer cells. MTT assays and cell cycle analysis using flow cytometry were performed to study the mechanism of action. The results revealed that combining a low dose of fucoxanthin with gemcitabine enhanced the cell viability of human embryonic kidney cells, 293, while a high dose of fucoxanthin enhanced the inhibitory effect of gemcitabine on the cell viability of this cell line. In addition, the enhanced effect of fucoxanthin on the inhibitory effect of gemcitabine on PANC-1 cells was significant (P<0.01). Fucoxanthin combined with gemcitabine also exerted significant enhancement of the anti-proliferation effect in MIA PaCa-2 cells in a concentration dependent manner (P<0.05), compared with gemcitabine treatment alone. In conclusion, fucoxanthin improved the cytotoxicity of gemcitabine on human pancreatic cancer cells at concentrations that were not cytotoxic to non-cancer cells. Thus, fucoxanthin has the potential to be used as an adjunct in pancreatic cancer treatment.

Intravenous administration of mesenchymal stromal cells leads to a dose-dependent coagulopathy and is unable to attenuate acute traumatic coagulopathy in rats.

BACKGROUND: Mesenchymal stromal cells (MSCs) express surface tissue factor (TF), which may affect hemostasis and detract from therapeutic outcomes of MSCs if administered intravenously. In this study, we determine a safe dose of MSCs for intravenous (IV) administration and further demonstrate the impact of IV-MSC on acute traumatic coagulopathy (ATC) in rats. METHODS: Tissue factor expression of rat bone marrow-derived mesenchymal stromal cell (BMSC) or adipose-derived mesenchymal stromal cell (AMSC) was detected by immunohistochemistry and enzyme-linked immunosorbent assay. The coagulation properties were measured in MSC-treated rat whole blood, and blood samples were collected from rats after IV administration of MSCs. Acute traumatic coagulopathy rats underwent polytrauma and 40% hemorrhage, followed by IV administration of 5 or 10 million/kg BMSCs (BMSC-5, BMSC-10), or vehicle at 1 hour after trauma. RESULTS: Rat MSCs expressed TF, and incubation of rat BMSCs or AMSCs with whole blood in vitro led to a significantly shortened clotting time. However, a dose-dependent prolongation of prothrombin time with reduction in platelet counts and fibrinogen was found in healthy rat treated with IV-MSCs. Bone marrow-derived mesenchymal stromal cells at 5 million/kg or less led to minimal effect on hemostasis. Mesenchymal stromal cells were not found in circulation but in the lungs after IV administration regardless of the dosage. Acute traumatic coagulopathy with prolonged prothrombin time was not significantly affected by 5 or 10 million/kg BMSCs. Intravenous administration of 10 million/kg BMSCs led to significantly lower fibrinogen and platelet counts, while significantly higher levels of lactate, wet/dry weight ratio, and leukocyte infiltration in the lung were present compared with BMSC-5 or vehicle. No differences were seen in immune or inflammatory profiles with BMSC treatment in ATC rats, at least in the acute timeframe. CONCLUSION: Intravenous administration of MSCs leads to a risk of coagulopathy associated with a dose-dependent reduction in platelet counts and fibrinogen and is incapable of restoring hemostasis of rats with ATC after polytrauma and hemorrhagic shock.

Whole blood resuscitation restores intestinal perfusion and influences gut microbiome diversity.

OBJECTIVE: Gut dysbiosis, an imbalance in the gut microbiome, occurs after trauma, which may be ameliorated with transfusion. We hypothesized that gut hypoperfusion following trauma causes dysbiosis and that whole blood (WB) resuscitation mitigates these effects. METHODS: Anesthetized rats underwent sham (S; laparotomy only, n = 6); multiple injuries (T; laparotomy, liver and skeletal muscle crush injuries, and femur fracture, n = 5); multiple injuries and 40% hemorrhage (H; n = 7); and multiple injuries, hemorrhage, and WB resuscitation (R; n = 7), which was given as 20% estimated blood volume from donor rats 1 hour posttrauma. Baseline cecal mesenteric tissue oxygen (O2) concentration was measured following laparotomy and at 1 hour and 2 hours posttrauma. Fecal samples were collected preinjury and at euthanasia (2 hours). 16S rRNA sequencing was performed on purified DNA, and diversity and phylogeny were analyzed with QIIME (Knight Lab, La Jolla, CA; Caporaso Lab, Flagstaff, AZ) using the Greengenes 16S rRNA database (operational taxonomic units; 97% similarity). α and ß diversities were estimated using observed species metrics. Permutational analysis of variance was performed for overall significance. RESULTS: In H rats, an average decline of 36% ± 3.6% was seen in the mesenteric O2 concentration at 1 hour without improvement by 2 hours postinjury, which was reversed following resuscitation at 2 hours postinjury (4.1% ± 3.1% difference from baseline). There was no change in tissue O2 concentration in the S or T rats. ß Diversity differed among groups for all measured indices except Bray-Curtis, with the spatial median of the S and R rats more similar compared with S and H rats (p < 0.05). While there was no difference in α diversity found among the groups, indices were significantly correlated with mesenteric O2 concentration. Members of the family Enterobacteriaceae were significantly enriched in only 2 hours. CONCLUSION: Mesenteric perfusion after trauma and hemorrhage is restored with WB resuscitation, which influences ß diversity of the gut microbiome. Whole blood resuscitation may also mitigate the effects of hemorrhage on intestinal dysbiosis, thereby influencing outcomes.

Extracellular vesicles derived from cardiosphere-derived cells as a potential antishock therapeutic.

BACKGROUND: Extracellular vesicles (EVs) isolated from cardiosphere-derived cells (CDC-EVs) are coming to light as a unique cell-free therapeutic. Because of their novelty, however, there still exist prominent gaps in knowledge regarding their therapeutic potential. Herein the therapeutic potential of CDC-EVs in a rat model of acute traumatic coagulopathy induced by multiple injuries and hemorrhagic shock is outlined. METHODS: Extracellular vesicle surface expression of procoagulant molecules (tissue factor and phosphatidylserine) was evaluated by flow cytometry. Extracellular vesicle thrombogenicity was tested using calibrated thrombogram, and clotting parameters were assessed using a flow-based adhesion model simulating blood flow over a collagen-expressing surface. The therapeutic efficacy of EVs was then determined in a rat model of acute traumatic coagulopathy induced by multiple injuries and hemorrhagic shock. RESULTS: Extracellular vesicles isolated from cardiosphere-derived cells are not functionally procoagulant and do not interfere with platelet function. In a rat model of multiple injuries and hemorrhagic shock, early administration of EVs significantly reduced the elevation of lactate and creatinine and did not significantly enhance coagulopathy in rats with acute traumatic coagulopathy. CONCLUSION: The results of this study are of great relevance to the development of EV products for use in combat casualty care, as our studies show that CDC-EVs have the potential to be an antishock therapeutic if administered early. These results demonstrate that research using CDC-EVs in trauma care needs to be considered and expanded beyond their reported cardioprotective benefits.

Severe Trauma and Hemorrhage Leads to Platelet Dysfunction and Changes in Cyclic Nucleotides in The Rat.

INTRODUCTION: Rats subjected to polytrauma and hemorrhage develop a coagulopathy that is similar to acute coagulopathy of trauma in humans, and is associated with a rise in prothrombin time and a fall in clot strength. Because platelet aggregation accounts for a major proportion of clot strength, we set out to characterize the effects of polytrauma on platelet function. METHODS: Sprague-Dawley rats were anesthetized with isoflurane. Polytrauma included laparotomy and damage to 10 cm of the small intestines, right and medial liver lobes, right leg skeletal muscle, femur fracture, and hemorrhage (40% of blood volume). No resuscitation was given. Blood samples were taken before and after trauma for the measurement of impedance electrode aggregometry, and intracellular levels of cyclic adenosine and guanosine monophosphate (cAMP, cGMP), inositol trisphosphate (IP3), and adenosine and guanosine triphosphates (ATP, GTP). RESULTS: Polytrauma significantly increased the response of collagen (24%) and thrombin (12%) to stimulate platelet aggregation. However, aggregation to adenosine diphosphate (ADP) or arachidonic acid (AA) was significantly decreased at 2 (52% and 46%, respectively) and 4 h (45% and 39%). Polytrauma and hemorrhage also led to a significant early rise in cAMP (101 ±â€Š11 to 202 ±â€Š29 pg/mL per 1,000 platelets), mirrored by a decrease in cGMP (7.8 ±â€Š0.9 to 0.6 ±â€Š0.5). In addition, there was a late fall in ATP (8.1 ±â€Š0.7 to 2.2 ±â€Š0.6 ng/mL per 1,000 platelets) and GTP (1.5 ±â€Š0.2 to 0.3 ±â€Š0.1). IP3 rose initially, and then fell back to baseline. CONCLUSIONS: Polytrauma and hemorrhage led to a deficit in the platelet aggregation response to ADP and AA after trauma, likely due to the early rise in cAMP, and a later fall in energy substrates, and may explain the decrease in clot strength and impaired hemostasis observed after severe trauma.

Effect of tranexamic acid administration on acute traumatic coagulopathy in rats with polytrauma and hemorrhage.

Trauma and hemorrhagic shock can lead to acute traumatic coagulopathy (ATC) that is not fully reversed by prehospital resuscitation as simulated with a limited volume of fresh whole blood (FWB) in a rat model. Tranexamic Acid (TXA) is used as an anti-fibrinolytic agent to reduce surgical bleeding if administered prior to or during surgery, and to improve survival in trauma if given early after trauma. It is not clear from the existing clinical literature whether TXA has the same mechanism of action in both settings. This study sought to explore the molecular mechanisms of TXA activity in trauma and determine whether administration of TXA as a supplement to FWB resuscitation could attenuate the established ATC in a rat model simulating prehospital resuscitation of polytrauma and hemorrhagic shock. In a parallel in-vitro study, the effects on clotting assays of adding plasmin at varying doses along with either simultaneous addition of TXA or pre-incubation with TXA were measured, and the results suggested that maximum anti-fibrinolytic effect of TXA on plasmin-induced fibrinolysis required pre-incubation of TXA and plasmin prior to clot initiation. In the rat model, ATC was induced by polytrauma followed by 40% hemorrhage. One hour after trauma, the rats were resuscitated with FWB collected from donor rats. Vehicle or TXA (10mg/kg) was given as bolus either before trauma (TXA-BT), or 45min after trauma prior to resuscitation (TXA-AT). The TXA-BT group was included to contrast the coagulation effects of TXA when used as it is in elective surgery vs. what is actually feasible in real trauma patients (TXA-AT group). A single dose of TXA prior to trauma significantly delayed the onset of ATC from 30min to 120min after trauma as measured by a rise in prothrombin time (PT). The plasma d-dimer as well as plasminogen/fibrinogen ratio in traumatized liver of TXA-BT were significantly lower as compared to vehicle and TXA-AT. Wet/dry weight ratio and leukocytes infiltration of lungs were significantly decreased only if TXA was administrated later, prior to resuscitation (TXA-AT). In conclusion: Limited prehospital trauma resuscitation that includes FWB and TXA may not correct established systemic ATC, but rather may improve overall outcomes of resuscitation by attenuation of acute lung injury. By contrast, TXA given prior to trauma reduced levels of fibrinolysis at the site of tissue injury and circulatory d-dimer, and delayed development of coagulopathy independent of reduction of fibrinogen levels following trauma. These findings highlight the importance of early administration of TXA in trauma, and suggest that further optimization of dosing protocols in trauma to exploit TXA's various sites and modes of action may further improve patient outcomes.

Platelets derived from fresh and cold-stored whole blood participate in clot formation in rats with acute traumatic coagulopathy.

The in vitro haemostatic functions of fresh whole blood (FWB) are well preserved after cold storage. This study aimed to determine whether platelets derived from FWB and stored whole blood (SWB) contribute to clot formation in tissue injury after transfusion into coagulopathic rats with polytrauma/haemorrhage (T/H). The rats were resuscitated 1 h after trauma with FWB or SWB collected from green fluorescence protein (GFP) transgenic rats. After transfusion, a liver incision was made and the tissue was collected 10 min after injury to identify GFP+ platelets by immunohistochemistry. In comparison to FWB, platelet aggregation to adenosine diphosphate and protease-activated receptor-4 was reduced by 35% and 20%, and clotting time was shortened by 25% in SWB. After transfusion, SWB led to a significant increase in platelet activation as measured by an elevation of CD62P and phosphatidylserine expression. The platelets from SWB were in a higher activation state, and showed higher clearance rate and formation of platelet-leucocyte aggregates than those from FWB after transfusion. Platelets from both FWB and SWB were equivalently incorporated into the clot at the incisional site, as determined by co-localization of CD61 and GFP. This study suggests that SWB contributes to haemostatic function and is an effective alternative resource to treat trauma patients.

Acute traumatic coagulopathy: The elephant in a room of blind scientists.

Acute traumatic coagulopathy (ATC) is the failure of coagulation homeostasis that can rapidly arise following traumatic injury, hemorrhage, and shock; it is associated with higher injury severity, coagulation abnormalities, and increased blood transfusions. Acute traumatic coagulopathy has historically been defined by a prolonged prothrombin time, although newer, more informative measurements of hemostatic function have been used to improve diagnosis and support clinical decision making. The underlying biochemical mechanisms of and best practice therapeutics for ATC remain under active investigation because of its significant correlation to poor outcomes. The wide range of hypothesized mechanisms for ATC results from the large number of symptoms, phenotypes, and altered states in these patients as observed by multiple research groups. Much like the ancient fable of blind men describing an elephant from their limited perspectives, the limited nature of clinical and laboratory tools used to diagnose coagulopathy or evaluate hemostatic function has made finding causation difficult. The prolonged prothrombin time, degree of fibrinolysis, depletion of coagulation factors and inhibitors, and general failure of the blood have all been identified as being primary indicators for ATC. Therapeutic interventions including recombinant coagulation factors, antifibrinolytics, and blood products have been used with varying degrees of success as they are used to address specific symptoms. To truly understand the causes of ATC, research efforts must recognize the complexity of the hemostatic system and get to the heart of the matter by answering the question: "Is ATC a pathological condition that develops from the observed deficiencies in coagulation, fibrinolysis, and autoregulation, or is ATC an adaptive response generated as the body attempts to restore perfusion and avoid massive organ failure?" Because patient management must proceed without definitive answers regarding the entire causative chain, the current therapeutic focus should be on using what knowledge has been gained to the patient's advantage: control hemorrhage, maintain appropriate homeostatic balances of coagulation proteins, and restore oxygen perfusion.

Severe Acinetobacter baumannii sepsis is associated with elevation of pentraxin 3.

Multidrug-resistant Acinetobacter baumannii is among the most prevalent bacterial pathogens associated with trauma-related wound and bloodstream infections. Although septic shock and disseminated intravascular coagulation have been reported following fulminant A. baumannii sepsis, little is known about the protective host immune response to this pathogen. In this study, we examined the role of PTX3, a soluble pattern recognition receptor with reported antimicrobial properties and stored within neutrophil granules. PTX3 production by murine J774a.1 macrophages was assessed following challenge with A. baumannii strains ATCC 19606 and clinical isolates (CI) 77, 78, 79, 80, and 86. Interestingly, only CI strains 79, 80, and 86 induced PTX3 synthesis in murine J774a.1 macrophages, with greatest production observed following CI 79 and 86 challenge. Subsequently, C57BL/6 mice were challenged intraperitoneally with CI 77 and 79 to assess the role of PTX3 in vivo. A. baumannii strain CI 79 exhibited significantly (P < 0.0005) increased mortality, with an approximate 50% lethal dose (LD50) of 10(5) CFU, while an equivalent dose of CI 77 exhibited no mortality. Plasma leukocyte chemokines (KC, MCP-1, and RANTES) and myeloperoxidase activity were also significantly elevated following challenge with CI 79, indicating neutrophil recruitment/activation associated with significant elevation in serum PTX3 levels. Furthermore, 10-fold-greater PTX3 levels were observed in mouse serum 12 h postchallenge, comparing CI 79 to CI 77 (1,561 ng/ml versus 145 ng/ml), with concomitant severe pathology (liver and spleen) and coagulopathy. Together, these results suggest that elevation of PTX3 is associated with fulminant disease during A. baumannii sepsis.

Insulin effects on glucose tolerance, hypermetabolic response, and circadian-metabolic protein expression in a rat burn and disuse model.

Insulin controls hyperglycemia after severe burns, and its use opposes the hypermetabolic response. The underlying molecular mechanisms are poorly understood, and previous research in this area has been limited because of the inadequacy of animal models to mimic the physiological effects seen in humans with burns. Using a recently published rat model that combines both burn and disuse components, we compare the effects of insulin treatment vs. vehicle on glucose tolerance, hypermetabolic response, muscle loss, and circadian-metabolic protein expression after burns. Male Sprague-Dawley rats were assigned to three groups: cage controls (n = 6); vehicle-treated burn and hindlimb unloading (VBH; n = 11), and insulin-treated burn and hindlimb unloading (IBH; n = 9). With the exception of cage controls, rats underwent a 40% total body surface area burn with hindlimb unloading, then IBH rats received 12 days of subcutaneous insulin injections (5 units·kg(-1)·day(-1)), and VBH rats received an equivalent dose of vehicle. Glucose tolerance testing was performed on day 14, after which blood and tissues were collected for analysis. Body mass loss was attenuated by insulin treatment (VBH = 265 ± 17 g vs. IBH = 283 ± 14 g, P = 0.016), and glucose clearance capacity was increased. Soleus and gastrocnemius muscle loss was decreased in the IBH group. Insulin receptor substrate-1, AKT, FOXO-1, caspase-3, and PER1 phosphorylation was altered by injury and disuse, with levels restored by insulin treatment in almost all cases. Insulin treatment after burn and during disuse attenuated the hypermetabolic response, increased glucose clearance, and normalized circadian-metabolic protein expression patterns. Therapies aimed at targeting downstream effectors may provide the beneficial effects of insulin without hypoglycemic risk.

A novel significance score for gene selection and ranking.

MOTIVATION: When identifying differentially expressed (DE) genes from high-throughput gene expression measurements, we would like to take both statistical significance (such as P-value) and biological relevance (such as fold change) into consideration. In gene set enrichment analysis (GSEA), a score that can combine fold change and P-value together is needed for better gene ranking. RESULTS: We defined a gene significance score π-value by combining expression fold change and statistical significance (P-value), and explored its statistical properties. When compared to various existing methods, π-value based approach is more robust in selecting DE genes, with the largest area under curve in its receiver operating characteristic curve. We applied π-value to GSEA and found it comparable to P-value and t-statistic based methods, with added protection against false discovery in certain situations. Finally, in a gene functional study of breast cancer profiles, we showed that using π-value helps elucidating otherwise overlooked important biological functions. AVAILABILITY: http://gccri.uthscsa.edu/Pi_Value_Supplementary.asp CONTACT: xy@ieee.org, cheny8@uthscsa.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The promotion of a functional fibrosis in skeletal muscle with volumetric muscle loss injury following the transplantation of muscle-ECM.

Tissue engineering strategies that primarily use biological extracellular matrices (ECMs) with or without the inclusion of a stem or progenitor cell source are under development for the treatment of trauma resulting in the loss of a large volume of skeletal muscle (i.e., volumetric muscle loss; VML). The explicit goal is to restore functional capacity to the injured tissue by promoting generation of muscle fibers. In the current study, a syngeneic muscle-derived ECM (mECM) was transplanted in a rat tibialis anterior (TA) muscle VML model. Instead of muscle fiber generation a large fibrotic mass was produced by mECM transplantation out to six months post-injury. Surprisingly, recovery of one-third of the original functional deficit was still achieved by two months post-injury following mECM transplantation. These counterintuitive findings may be due, at least in part, to the ability of mECM to attenuate muscle damage in the remaining muscle as compared to non-repaired muscle. These findings point to a novel role of biological ECMs for the treatment of VML, wherein the remaining muscle mass is protected from prolonged overload injury.

Proximal gut mucosal epithelial homeostasis in aged IL-1 type I receptor knockout mice after starvation.

BACKGROUND: Previous studies have shown that starvation induces small bowel atrophy, and that atrophy diminishes with aging. In this experiment, we assessed whether starvation-induced atrophy of proximal gut mucosa is associated with the Interleukin-1 receptor (IL-1R) signaling pathway in aged mice. MATERIALS AND METHODS: Thirty 26-month-old IL-1R knockout mice and age-matched wild-type C57BL/6 mice were randomly divided into two groups: ad libitum fed and fasted. Mice were euthanized 12 or 48 hours after starvation. The proximal small bowel was harvested for morphologic analysis. Gut epithelial cell proliferation was detected using immunohistochemical staining for proliferating cell nuclear antigen (PCNA), and apoptosis was identified using terminal deoxyuridine nick-end labeling (TUNEL) staining. RESULTS: Aged IL-1R knockout mice were larger than aged-matched wild-type mice (P < 0.05). Proximal gut mucosal height and mucosal cell number were not different between aged IL-1R knockout and wild-type groups. The apoptosis index in gut epithelial cells was higher in fed IL-1R knockout versus wild-type mice (P < 0.05), while there was no significant difference in cell proliferation between both groups. Mucosal atrophy was induced in both aged IL-1R knockout and wild-type groups by starvation (P < 0.05), however, aged IL-1R knockout mice experienced greater loss in proximal gut weight, mucosal length, and corresponding cell number than did wild-type mice at the 12-h time point (P < 0.05). The apoptosis index in gut epithelial cells significantly increased in both groups after starvation (P < 0.05). Starvation decreased cell proliferation in IL-1R knockout mice (P < 0.05), but not in wild-type mice. CONCLUSIONS: The response in aged IL-1R knockout mice differs from wild-type mice in that starvation increases atrophy and is associated with decreased cell proliferation rather than increased apoptosis.

Second hit post burn increased proximal gut mucosa epithelial cells damage.

Secondary infections after burn are common and are a major contributor to morbidity and mortality. We previously showed that burn disrupted proximal gut mucosal homeostasis through increased epithelial cell apoptosis. In the present study, we sought to determine whether proximal gut mucosal disruption is additively affected by secondary endotoxemia after a severe burn. C57BL/6 mice received 30% total body surface area full-thickness scald burns and were randomized to receive saline or LPS 1 mg/kg body weight given intraperitoneally 72 h after burn. Proximal small bowel was harvested 12 h after LPS injection. Mucosal height and epithelial cell number were assessed on hematoxylin-eosin sections, intestinal epithelial cell apoptosis was identified by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and cell proliferation by immunohistochemical staining for proliferating cell nuclear antigen. Results showed that proximal gut mucosa impairment occurred 12 h after injury, including significantly decreased proximal gut wet weight, gut mucosal height, and epithelial cell number associated with increased proximal gut epithelial apoptosis (P < 0.05). This impairment diminished 72 h after burn. Second-hit endotoxemia caused additional proximal gut mucosa damage with decreased proximal gut weight, cell number, and mucosal height (P < 0.05) and significantly increased small intestinal epithelial apoptosis and mucosal atrophy, even after the first event, indicating a second detrimental effect of endotoxemia after the initial injury.

Insulin decreases hepatic acute phase protein levels in severely burned children.

BACKGROUND: Severe burn induces the hepatic acute phase response. In this study, we wondered whether continuous insulin treatment decreases acute phase protein levels in the severely burned. METHODS: Eighteen children aged 2 to 17 years with burns >40% of total body surface area were randomized to receive either insulin (n=9) or no treatment (n=9) within 72 hours after injury until the wounds were 95% healed. Insulin was given at a continuous rate of > or =1.5 microU/kg/min to maintain euglycemia (serum glucose 100-140 microg/dL). Plasma was examined at days 7, 14, 21, and 28 for acute phase protein levels including C-reactive protein, C3 complement, alpha1-acid glycoprotein, haptoglobin, alpha2-macroglobulin, prealbumin, transferrin, and retinol-binding protein. Statistical analysis was by ANOVA and t test. RESULTS: With insulin treatment, alpha1-acid glycoprotein, C3 complement, alpha2-macroglobulin, and haptoglobin levels decreased (P<.05) after a severe burn compared with control, especially at days 21 and 28. Additionally, the hepatic constitutive proteins (prealbumin, transferrin, and retinol-binding protein) were lower in the insulin-treatment group than those of the control group at day 21 (P<.05). CONCLUSIONS: Continuous insulin treatment decreases acute phase protein levels after a severe burn. The results suggest insulin downregulation of the hepatic acute phase response to injury.

Burn-induced gut mucosal homeostasis in TCR delta receptor-deficient mice.

Gammadelta T lymphocytes make up approximately 50% of lymphocytes in the intestine. These cells have been shown to prime macrophages for TNF-alpha production after burn. We previously showed that neutralizing anti-TNF-alpha antibodies reduce mucosal atrophy by decreasing gut epithelial apoptosis after severe burn. We hypothesized that burn-induced mucosal turnover is diminished in T cell receptor delta gene knockout (TCR delta-/-) mice through diminished TNF-alpha activity. Forty-two wild-type and 42 TCR delta-/- mice (C57-BL6) were randomly assigned to burn and sham burn groups. The burn group underwent a 25% total body surface area (TBSA) scald burn. The proximal small intestine was harvested at 2, 12, and 48 h. To assess mucosal atrophy, mucosal height and cell numbers in the villi and crypts were determined on hematoxylin and eosin-stained tissue sections. Apoptotic gut epithelium was identified by terminal deoxyuridine nick-end labeling (TUNEL) staining, and cell proliferation was detected by immunostaining for proliferative cell nuclear antigen (PCNA). TNF-alpha mRNA expression was measured by RT-PCR. Caspase-8 activity was measured by colorimetric assay. Statistical analysis was performed with two-way analysis of variance and t testing. Significance was accepted at P < 0.05. Data are expressed as means +/- SEM. TNF-alpha mRNA expression was significantly decreased in TCR delta-/- mice at 2 h after burn. Gut epithelial apoptosis and proliferation in both wild-type and TCR delta-/- mice were significantly increased after burn, but TCR delta-/- mice had a significantly lower levels of apoptosis (P < 0.01) and proliferation (P < 0.05) when compared with wild-type mice. Burn-induced mucosal atrophy was identified in groups by decreasing villus height, crypt depth, and villus and crypt cell number (P < 0.001) compared with sham, but no difference was found between wild-type and TCR delta-/- mice. Caspase-8 activity was significantly diminished in TCR delta-/- mice compared with wild-type mice. Gammadelta T cells are associated with increased TNF-alpha expression and gut epithelial turnover in the small bowel after severe burn. However, absence of delta T cell receptor did not inhibit mucosal atrophy after severe burn. This study suggests that gut mucosal atrophy after severe burn is a multifactorial process associated with increased TNF-alpha activity.

Decreased lymphocyte apoptosis by anti-tumor necrosis factor antibody in Peyer's patches after severe burn.

Severe burn results in immunosuppression, with increased lymphocyte apoptosis in both the central and peripheral immune system. As atrophy of the small intestine has been described in mouse models and intestinal lymphocytes have been implicated in the burn inflammatory response, we examined the effects of burn and tumor necrosis factor (TNF)-alpha on lymphocytes in intestinal Peyer's patches. Anesthetized C57BL6 mice received a 30% full-thickness scald burn on the upper back. Sham-burned animals served as controls. Anti-TNF or control immunoglobulin (Ig) G antibody (200 microg) was given immediately after the burn. The animals were initially resuscitated with 2 mL of normal saline, and were then sacrificed 12 h postburn. Terminal deoxyuridine nick-end labeling (TUNEL) and proliferative cell nuclear antigen (PCNA) staining was performed. Apoptosis was quantified as apoptotic lymphocytes/high-powered field (hpf). Results, expressed as mean +/- SEM, were compared using analysis of variance (ANOVA) and the Student-Newman-Keuls test. All mice survived the burn. An initial time-course experiment demonstrated maximal Peyer's patch apoptosis 12 h after the burn. Sham mice had 25 +/- 7 TUNEL-stained cells/hpf in Peyer's patches, whereas burned mice had 93 +/- 18 cells/hpf (P < 0.05). In contrast, burned mice receiving anti-TNF antibody had 28 +/- 8 TUNEL-stained cells/hpf (P < 0.05 vs. burn), whereas sham mice receiving anti-TNF antibody had 20 +/- 4 cells/hpf. There were no significant differences in PCNA staining between the groups. Scald burn results in lymphocyte apoptosis in Peyer's patches. This apoptosis can be abrogated by the addition of anti-TNF antibody. Apoptotic changes may lead to the failure of the intestinal immunological barrier and increased risk of sepsis.

Growth hormone down-regulation of Interleukin-1beta and Interleukin-6 induced acute phase protein gene expression is associated with increased gene expression of suppressor of cytokine signal-3.

Severe burn induces the hepatic acute phase response. We previously showed that recombinant human growth hormone (GH) treatment after burn down-regulated acute phase protein (APP) production and gene expression in vivo. In this study, we hypothesized that the inhibitory effect of GH on the hepatic acute phase response was due to increased suppressor of cytokine signaling (SOCS) gene expression. HepG2 cells were treated with Interleukin-1beta (IL-1beta; 2 ng/mL) and interleukin 6 (IL-6; 20 ng/mL) alone or combined with GH (2 microg/mL) for 15 and 30 min, and 1, 2, and 4 h. The levels of gene expression for alpha1-acid glycoprotein (AGP), alpha1-antitrypsin (ATT), and SOCS (CIS, SOCS-1, 2, and 3) were measured by reverse transcript-polymerase chain reaction (RT-PCR). APP levels in the supernatant were determined by enzyme-linked immunosorbent sandwich assay (ELISA). The gene expression of AGP and ATT were also measured in HepG2 cells transfected with pEF-Flag-l/mSOCS-3 plasmid after IL-1beta or IL-6 treatment. Data are expressed as means +/- SEM, and statistical analysis was performed by one- or two-way analysis of variance. IL-1beta and IL-6 induced AGP and ATT gene expression and protein production, respectively, which was down-regulated by GH treatment. SOCS-3 but not CIS, SOCS-1, or SOCS-2 gene expression was significantly increased by GH treatment. APP gene expression was significantly decreased in cells transfected with plasmid over expressing SOCS-3 after IL-6 and IL-1beta treatment. GH attenuates IL-1beta or IL-6 induced APP gene expression, which is associated with increased expression of SOCS-3. This study suggests that SOCS-3 plays an important role in the suppression of cytokine signaling by GH in down-regulating the acute phase response after injury.

Effect of bombesin on gut mucosal impairment after severe burn.

Severe cutaneous bum alters gut epithelial homeostasis. In previous studies, treatment with bombesin decreased mucosal atrophy and improved maintenance of gut mucosal integrity after severe burn. Our current hypothesis is that bombesin reduces burn-induced gut impairment by decreasing gut epithelial cell death. Fifty-four adult male Fisher-344 rats were randomly assigned to three groups: control, sham burn (I), burn (II), and burn + bombesin (III). Animals in groups II and III received a 60% total body surface area full thickness scald burn, and the treatment group (III) received bombesin subcutaneously (10 microg/kg, every 8 h) beginning immediately before the experiment. The proximal small bowel was harvested at 12 and 72 h after burn with measurement of wet and dry weight, mucosal weight, and protein content, and a 1-cm length of proximal end was excised and fixed in fomalin for histological and immunohistochemical observation. Data are expressed as means +/- SEM. Statistical analysis was by done by analysis of variance (significance at P < 0.05). Bombesin treatment attenuated mucosal atrophy demonstrated by restoration of the mucosal weight, mucosal protein content, and maintenance of mucosal height and total mucosal epithelial cell count. Gut epithelial cell apoptosis was, at least in part, inhibited by bombesin compared with a significant increase of gut cell apoptosis at 12 h after burn. Gut epithelial proliferation was not affected. Bombesin diminished burn-induced gut mucosal atrophy and gut epithelial cell apoptosis, suggesting that bombesin treatment may play an important role in the recovery of gut impairment after severe burn.