A retrospective analysis of mortality risk and immunosuppressive therapy for Stevens-Johnson Syndrome and toxic epidermal necrolysis syndrome using the TriNetX research network.

Stevens-Johnson Syndrome (SJS) and Toxic Epidermal Necrolysis (TEN) exist on a spectrum of autoimmune conditions which cause epidermal detachment and keratinocyte necrosis. Due to the rare incidence of these conditions, a dramatic heterogeneity in treatment algorithms exists. To better appreciate pharmacologic immunosuppressive therapies' impact on survival, the authors queried a multi-institutional data network. Data for this study was extracted from TriNetX Research Network, a platform that contains ICD-9/ICD-10 coding data from a consortium of international healthcare organizations. Seventy-one institutions were queried to identify adult patients diagnosed with SJS, TEN or SJS-TEN Overlap. Cohorts were created based on the therapy received: systemic steroids (SS), diphenhydramine (DH), cyclosporine (CS), intravenous immunoglobulin (IVIG), tumor necrosis factor alpha inhibitors (TNFαi), or a combination of treatments. Cohorts were then propensity matched with patients who received supportive care. Patients who only received one of the above treatments showed no significant reduction in 90-day mortality. Patients who received CS or IVIG as part of their multitherapy showed a significantly increased risk of death when compared to supportive care (CS: RR = 1.583, 95% CI [1.119, 2.240]; IVIG: RR = 2.132, 95% CI [1.485, 3.059]). Despite their frequent utilization, this study's analysis suggests that none of these therapies confer significant 90-day mortality survival over supportive care alone. These results highlight the heterogeneity of therapies and emphasize the need for critical prospective appraisal of their outcomes in SJS and TEN.

Pre-existing skin diseases as predictors of post-burn pruritus.

BACKGROUND: Post-burn pruritus (PBP) has been shown to adversely affect burn patients' quality of life. However, the predictors of PBP are not known. We hypothesize a pre-existing pruritic skin diagnosis is associated with an increased risk of adverse outcomes following a burn injury. METHODS: This retrospective study utilized data from the TriNetX electronic health record. Burn patients with a history of a pruritic skin disorder were compared to patients without a diagnosed skin disorder and the occurrence of pruritus was compared between the two cohorts. RESULTS: Patients with pre-existing skin conditions were more likely to develop PBP. The risk of PBP was highest 1 year after injury. Stratification by percent TBSA burned, gender, race, and age showed an increased risk of PBP for females, Caucasians, older patients, and those with large burns. CONCLUSION: A pre-existing pruritic skin diagnosis is highly associated with developing pruritus following a burn injury.

Impact of pre-burn statin use on metabolic and cardiovascular disorders.

INTRODUCTION: Statins are among the most widely prescribed medications with proven effectiveness in patients with hyperlipidemia and atherosclerotic cardiovascular diseases. We investigated the relationship between statin use, metabolic and cardiovascular outcomes after burn. METHODS: We utilized data from the TriNetX electronic health database. Burn patients with prior statin use were compared to patients without prior use and analyzed the occurrence of metabolic and cardiovascular disorders. RESULTS: Prior statin use burn patients were 1.33 times as likely to develop hyperglycemia, 1.20 times for cardiac arrhythmia, 1.70 times for coronary artery disease (CAD), 1.10 times for sepsis, and 0.80 times for death. High percent TBSA burn, male sex, and lipophilic statin use were associated with higher odds of outcome development. CONCLUSION: Prior statin use in severely burned patients is associated with an increased risk of developing hyperglycemia, arrhythmias, and CAD, with higher odds in males, higher TBSA burn, and lipophilic statin users.

Higher risk of acute kidney injury and death with rhabdomyolysis in severely burned patients.

BACKGROUND: Rhabdomyolysis is a severe condition responsible for up to 10% of acute kidney injury (AKI) cases. Severely burned patients are susceptible to developing both rhabdomyolysis and acute kidney injury, but whether burned patients with rhabdomyolysis are at greater risk of acute kidney injury compared to non-burned patients with rhabdomyolysis is unclear. METHODS: TriNetX, a clinical research platform, was used to obtain electronic medical record information in 3 patient cohorts over the recent 20-year period. Cohort 1 included burn patients who developed rhabdomyolysis, cohort 2 were those who developed rhabdomyolysis due to non-burn causes, and cohort 3 included burn patients who did not develop rhabdomyolysis. Matching was performed to balance demographics and comorbidities for each cohort. Cohorts were then compared for the development of acute kidney injury and death. RESULTS: We found 84 of 111 patients in cohort 1 (75.676%), 56 of 111 patients in cohort 2 (50.45%), and 18 of 111 patients in cohort 3 (16.216%) developed acute kidney injury within 5 days. The odds ratio for developing acute kidney injury for burned patients compared to non-burned patients with rhabdomyolysis was 3.056 with a 95% confidence interval of 1.726 to 5.41 (P < .05), and between burned patients with and without rhabdomyolysis was 16.074 with a 95% confidence interval of 8.263 to 31.268 (P < .05). The incidence of death within 1 year was also significantly higher in cohort 1 (48%) than the other cohorts (9% and 14%, respectively) (P < .05). CONCLUSION: The risk of acute kidney injury and mortality in severely burned patients with rhabdomyolysis is significantly increased compared to matched non-burn patients with rhabdomyolysis or burned patients without rhabdomyolysis.

Skeletal muscle wasting after a severe burn is a consequence of cachexia and sarcopenia.

Muscle wasting is common and persistent in severely burned patients, worsened by immobilization during treatment. In this review, we posit two major phenotypes of muscle wasting after severe burn, cachexia and sarcopenia, each with distinguishing characteristics to result in muscle atrophy; these characteristics are also likely present in other critically ill populations. An online search was conducted from the PubMed database and other available online resources and we manually extracted published articles in a systematic mini review. We describe the current definitions and characteristics of cachexia and sarcopenia and relate these to muscle wasting after severe burn. We then discuss these putative mechanisms of muscle atrophy in this condition. Severe burn and immobilization have distinctive patterns in mediating muscle wasting and muscle atrophy. In considering these two pathological phenotypes (cachexia and sarcopenia), we propose two independent principal causes and mechanisms of muscle mass loss after burns: (1) inflammation-induced cachexia, leading to proteolysis and protein degradation, and (2) sarcopenia/immobility that signals inhibition of expected increases in protein synthesis in response to protein loss. Because both are present following severe burn, these should be considered independently in devising treatments. Discussing cachexia and sarcopenia as independent mechanisms of severe burn-initiated muscle wasting is explored. Recognition of these associated mechanisms will likely improve outcomes.

Burn Serum Stimulates Myoblast Cell Death Associated with IL-6-Induced Mitochondrial Fragmentation.

BACKGROUND: Burn patients suffer muscle mass loss associated with hyperinflammation and hypercatabolism. The mitochondria are affected by this metabolic alteration. Mitochondrial fission activates a caspase cascade that ultimately leads to cell death. We postulate that burn-induced muscle loss is associated with increased mitochondrial fission and subsequent functional impairment. Further, we investigated whether the cytokine IL-6 plays a major role in mitochondrial fission-associated cell death after burn. METHODS: Murine myoblast C2C12 cells were treated with 10% serum isolated either from control rats or 40% total body surface area burned rats. Mitochondria were labeled with MitoTracker Green for live cell images. Mitochondrial function was assessed with an Enzo Mito-ID membrane potential cytotoxicity kit. Protein signals were detected by Western blot analysis. Moreover, recombinant IL-6 was applied to stimulate C2C12 to differentiate the role of cytokine IL-6; lastly, we treated burn serum-stimulated cells with IL-6 antibodies. RESULTS: Caspase 3 activity increased in C2C12 cells with burn serum stimulation, suggesting increased cell death in skeletal muscle after burn. Mitochondrial morphology shortened and mitochondrial membrane potential decreased in cells treated with burn serum. Western blot data showed that mitofusion-1 expression significantly decreased in burn serum-treated cells, supporting the morphologic observation of mitochondrial fission. Mitochondrial fragmentation increased with IL-6 stimulation, and IL-6 antibody decreased caspase 3 activity and mitochondrial membrane potential improved in burn serum-stimulated cells. CONCLUSION: Burn serum caused muscle cell death associated with increased mitochondrial fission and functional impairment. This alteration was alleviated with IL-6 antibody treatment, suggesting the cytokine plays a role in mitochondrial changes in muscle after systemic injury.

Umbilical cord blood-derived non-hematopoietic stem cells retrieved and expanded on bone marrow-derived extracellular matrix display pluripotent characteristics.

BACKGROUND: Umbilical cord blood (UCB) not only contains hematopoietic stem cells (HSCs), but also non-hematopoietic stem cells (NHSCs) that are able to differentiate into a number of distinct cell types. Based on studies published to date, the frequency of NHSCs in UCB is believed to be very low. However, the isolation of these cells is primarily based on their adhesion to tissue culture plastic surfaces. METHODS AND RESULTS: In the current study, we demonstrate that this approach overlooks some of the extremely immature NHSCs because they lack the ability to adhere to plastic. Using a native extracellular matrix (ECM), produced by bone marrow (BM) stromal cells, the majority of the UCB-NHSCs attached within 4 h. The colony-forming unit fibroblast frequency of these cells was 1.5 × 104/108 mononuclear cells, which is at least 4000-fold greater than previously reported for UCB-NHSCs. The phenotype of these cells was fibroblast-like and different from those obtained by plastic adhesion; they formed embryonic body-like clusters that were OCT4-positive and expressed other human embryonic stem cell-related markers. Importantly, when implanted subcutaneously for 8 weeks into immunocompromised mice, these ECM-adherent and expanded NHSCs generated three germ layer-derived human tissues including muscle, fat, blood vessel, bone, gland, and nerve. Moreover, injection of these cells into muscle damaged by cryoinjury significantly accelerated muscle regeneration. CONCLUSIONS: These results indicate that UCB may be a virtually unlimited source of NHSCs when combined with isolation and expansion on ECM. NHSCs may be a practical alternative to embryonic stem cells for a number of therapeutic applications.

Skeletal Muscle Loss is Associated with TNF Mediated Insufficient Skeletal Myogenic Activation After Burn.

Muscle loss accompanies severe burn; in this hyper-catabolic state, muscle undergoes atrophy through protein degradation and disuse. Muscle volume is related to the relative rates of cellular degradation and myogenesis. We hypothesize that muscle atrophy after injury is in part because of insufficient myogenesis associated with the hyper-inflammatory response. The aim of this study was to investigate the role of skeletal myogenesis and muscle cell homeostasis in response to severe burn. Twenty-eight male C57BL6 mice received 25% TBSA scald. Gluteus muscle from these animals was analyzed at days 1, 3, 7, and 14 after injury. Six additional animals without burn served as controls. We showed muscle wet weight and protein content decreased at days 3 and 7 after burn, with elevated tumor necrosis factor (TNF) mRNA expression (P < 0.05). Increased cell death was observed through TUNEL staining, and cleaved caspase-3 levels reached a peak in muscle lysate at day 3 (P < 0.05). The cell proliferation marker proliferating cell nuclear antigen (PCNA) significantly increased after burn, associated with increased gene and protein expression of myogenesis markers Pax7 and myogenin. Desmin mRNA expression and the ratio of desmin to PCNA protein expression, however, significantly decreased at day 7 (P < 0.05). In vitro, the ratio of desmin to PCNA protein expression significantly decreased in C2C12 murine myoblasts after TNF-α stimulation for 24 h. We showed that severe burn induces both increased cell death and proliferation. Myogenesis, however, does not counterbalance increased cell death after burn. Data suggest insufficient myogenesis might be associated with pro-inflammatory mediator TNF activity.

Hepatic autophagy after severe burn in response to endoplasmic reticulum stress.

BACKGROUND: Previous studies showed that liver dysfunction develops soon after severe burn, and this is associated with activation of endoplasmic reticulum (ER) stress. Autophagy is a catabolic process to maintain cellular organelle balance; ER stress is associated with autophagy signaling cascades. We thus sought to determine whether autophagy signals were associated with damage in the liver after burn, and further whether burn-associated ER stress activates autophagy signals in hepatocytes. METHODS: C57BL/6 male mice received a 25% total body surface area full-thickness scald burn, and liver was harvested at 24 h after burn. HepG2 cells were stimulated with an ER stress inducer thapsigargin (TG) for 24 h to mimic ER stress in vitro. Terminal deoxyuridine nick-end labeling staining was performed on histologic sections of liver. Autophagy was assessed by immunoblotting. Statistical analysis was performed using the Student t-test and significance was accepted at P < 0.05. RESULTS: Terminal deoxyuridine nick-end labeling positive-stained hepatocytes increased in burned animals with a significant elevation of caspase 3 activity (P < 0.05). Hepatic autophagy-related (ATG) protein 3, ATG5 and light chain (LC) 3B elevated significantly in burn animals as well (P < 0.05). Expression of Beclin-1, LC3A, and LC3B increased in HepG2 cells in response to TG, similar to the response seen in vivo. Cytosolic adenosine triphosphate dropped significantly, and adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) were phosphorylated as well in response to TG (P < 0.05). CONCLUSIONS: ER stress, which occurs in hepatocytes after severe injury, is associated with autophagy and liver damage after severe burn. In response to ER stress, activated autophagy is associated with adenosine monophosphate-activated protein kinase and mammalian target target of rapamycin pathway.

Characterizing vasopressin and other vasoactive mediators released during resuscitation of trauma patients.

BACKGROUND: We sought to perform the first characterization of vasopressin and other vasoactive mediators released during resuscitation of hypotensive trauma patients. METHODS: This institutional review board-approved study was conducted under waiver of consent. Adults with clinical evidence of acute traumatic injury and systolic blood pressure less than or equal to 90 mm Hg within 1 hour of arrival were evaluated at our Level I trauma center. Two hundred three patients were screened with 50 enrolled from February 2010 to February 2011. Demographic information was also collected. Blood samples were obtained at 0, 30, 60, 90, 120, and 240 minutes after arrival, and assays were performed for vasopressin, angiotensin II, epinephrine, and cortisol. We assessed the significance of variation in these vasoactive mediators with injury and transfusion of more than 600 mL, with adjustment for time using repeated-measures linear models in log units. RESULTS: We found that vasopressin (p = 0.005) and epinephrine (p = 0.01) increased significantly with injury, while angiotensin (p = 0.60) and cortisol (p = 0.46) did not and that vasopressin (p < 0.001) and epinephrine (p = 0.004) increased significantly in patients requiring transfusion of more than 600 mL but angiotensin II (p = 0.11) and cortisol (p = 0.90) did not. Relatively low levels of vasopressin (<30 pg/mL) were observed at least once during the first 2 hours in 88% of trauma patients, and abnormally low epinephrine levels (<100 pg/mL) were observed at least once during the first 2 hours in 18% of trauma patients. CONCLUSION: This is the first clinical trial to serially evaluate vasopressin and other vasoactive mediators following trauma during the resuscitation phase. Vasopressin, in particular, and epinephrine seem to be the key mediators produced in the human response to severe injury. A deficiency of vasopressin may contribute to intractable shock after trauma. LEVEL OF EVIDENCE: Prognostic/epidemiologic study, level III.

Propranolol improves impaired hepatic phosphatidylinositol 3-kinase/akt signaling after burn injury.

Severe burn injury is associated with induction of the hepatic endoplasmic reticulum (ER) stress response. ER stress leads to activation of c-Jun N-terminal kinase (JNK), suppression of insulin receptor signaling via phosphorylation of insulin receptor substrate 1 and subsequent insulin resistance. Marked and sustained increases in catecholamines are prominent after a burn. Here, we show that administration of propranolol, a nonselective ß1/2 adrenergic receptor antagonist, attenuates ER stress and JNK activation. Attenuation of ER stress by propranolol results in increased insulin sensitivity, as determined by activation of hepatic phosphatidylinositol 3-kinase and Akt. We conclude that catecholamine release is responsible for the ER stress response and impaired insulin receptor signaling after burn injury.

Measurement of hepatic protein fractional synthetic rate with stable isotope labeling technique in thapsigargin stressed HepG2 cells.

Severe burn-induced liver damage and dysfunction is associated with endoplasmic reticulum (ER) stress. ER stress has been shown to regulate global protein synthesis. In the current study, we induced ER stress in vitro and estimated the effect of ER stress on hepatic protein synthesis. The aim was two-fold: (1) to establish an in vitro model to isotopically measure hepatic protein synthesis and (2) to evaluate protein fractional synthetic rate (FSR) in response to ER stress. Human hepatocellular carcinoma cells (HepG2) were cultured in medium supplemented with stable isotopes 1,2-(13)C(2)-glycine and L-[ring-(13)C(6)]phenylalanine. ER stress was induced by exposing the cells to 100 nM of thapsigargin (TG). Cell content was collected from day 0 to 14. Alterations in cytosolic calcium were measured by calcium imaging and ER stress markers were confirmed by Western blotting. The precursor and product enrichments were detected by GC-MS analysis for FSR calculation. We found that the hepatic protein FSR were 0.97 ± 0.02 and 0.99 ± 0.05%/hr calculated from 1,2-(13)C(2)-glycine and L-[ring-(13)C(6)]phenylalanine, respectively. TG depleted ER calcium stores and induced ER stress by upregulating p-IRE-1 and Bip. FSR dramatically decreased to 0.68 ± 0.03 and 0.60 ± 0.06%/hr in the TG treatment group (p<0.05, vs. control). TG-induced ER stress inhibited hepatic protein synthesis. The stable isotope tracer incorporation technique is a useful method for studying the effects of ER stress on hepatic protein synthesis.

Severe injury is associated with insulin resistance, endoplasmic reticulum stress response, and unfolded protein response.

OBJECTIVE: We determined whether postburn hyperglycemia and insulin resistance are associated with endoplasmic reticulum (ER) stress/unfolded protein response (UPR) activation leading to impaired insulin receptor signaling. BACKGROUND: Inflammation and cellular stress, hallmarks of severely burned and critically ill patients, have been causally linked to insulin resistance in type 2 diabetes via induction of ER stress and the UPR. METHODS: Twenty severely burned pediatric patients were compared with 36 nonburned children. Clinical markers, protein, and GeneChip analysis were used to identify transcriptional changes in ER stress and UPR and insulin resistance-related signaling cascades in peripheral blood leukocytes, fat, and muscle at admission and up to 466 days postburn. RESULTS: Burn-induced inflammatory and stress responses are accompanied by profound insulin resistance and hyperglycemia. Genomic and protein analysis revealed that burn injury was associated with alterations in the signaling pathways that affect insulin resistance, ER/sarcoplasmic reticulum stress, inflammation, and cell growth/apoptosis up to 466 days postburn. CONCLUSION: Burn-induced insulin resistance is associated with persistent ER/sarcoplasmic reticulum stress/UPR and subsequent suppressed insulin receptor signaling over a prolonged period of time.

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.

Characterization of the inflammatory response during acute and post-acute phases after severe burn.

Severe burn causes a pronounced hypermetabolic response characterized by catabolism and extensive protein wasting. We recently found that this hypermetabolic state is driven by a severe inflammatory response. We characterized in detail the kinetics of serum levels of a panel of cytokines in a rat model, which may serve as reference for the development of therapeutic interventions applicable to humans. Male Sprague-Dawley rats (n = 8) received a full-thickness burn of 60% total body surface area. Serum was harvested 1, 3, 6, 12, 24, 48, 96, and 168 h after burn. Eight serum cytokines commonly used to assess the inflammatory response in humans, such as IL-1beta, IL-6, IL-10, TNF, vascular endothelial growth factor, and monocyte chemotactic protein 1, and the rat-specific cytokines cytokine-induced neutrophil chemoattractant (CINC) 1, CINC-2, and CINC-3 were measured by enzyme-linked immunosorbent assay technique and were compared with controls (n = 4). Statistical analysis was conducted using the t test, with P < 0.05 considered as significantly different. Thermal injury resulted in significantly increased serum levels of IL-1beta, IL-6, IL-10, monocyte chemotactic protein 1, CINC-1, CINC-2, and CINC-3 when compared with the concentrations detected in nonburned rats (P < 0.05). Serum levels of TNF-alpha and vascular endothelial growth factor in burned rats were not found to be significantly different to controls. Burn causes a profound inflammatory response in rats. Specific cytokines known to increase in humans postburn such as IL-1 beta, IL-6, IL-10, MCP-1, and IL-8 (CINC-1, CINC-2, and CINC-3 in the rat) were also observed in our rat burn model, which now allows us to study new anti-inflammatory treatment options.

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.

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.

Immunomodulation of hepatic ischemic injury via increased Bcl-X(L) and decreased Bcl-X(S).

BACKGROUND: Although classic ischemia-reperfusion injury is mediated by reactive oxygen intermediaries, increasing evidence implicates a role for immune-mediated apoptosis during ischemic injury in transplantation. Herein, we report the effects of polyclonal rabbit anti-thymocyte globulin (rATG) on mediators of hepatic apoptosis during cold storage. METHODS: Three-month-old male Lewis rats were placed under halothane anesthesia and the portal vein cannulated. University of Wisconsin (UW) solution (35 ml) with (n = 5) and without (n = 5) 20 mg/kg anti-rat rATG was infused before hepatectomy. The liver was stored in UW solution +/- rATG (143 ng/ml) at 4 degrees C for various times up to 24 h. Specimens were terminal deoxyuridine nick end labeling-stained for apoptosis. Tissue lysates were analyzed by Western blotting and densitometry. RESULTS: Compared to UW alone, significantly fewer apoptotic cells were present in UW + rATG perfused and stored livers. There were early and sustained significant increases in Bcl-X(L) and decreases in Bcl-X(S) with rATG. There was an initial, but not sustained, significant decrease in Bax with rATG. Moreover, there was a significant one-third decrease in caspase-9 production with rATG at 0, 6, 12, and 18 h. CONCLUSION: Decreased proapoptotic Bcl-X(S) and increased antiapoptotic Bcl-X(L), as well as decreased downstream proapoptotic caspase-9 expression, during liver ischemia after treatment with rATG, all favor cell survival. Because apoptotic ischemic injury results in allograft dysfunction, preservation strategies that ameliorate such immunological effects may improve organ function.

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.