Evaluating the Efficacy of Two Regional Pain Management Modalities in Autologous Breast Reconstruction.

At our institution, multimodal opiate-sparing pain management is the cornerstone of our enhanced recovery program for autologous breast reconstruction. The purpose of this study was to compare postoperative outcomes and pain control metrics following implementation of an enhanced recovery program with two different regional analgesia approaches. METHODS: This retrospective cohort study identified 145 women who underwent autologous breast reconstruction from 2015 to 2017. Three groups were included: historical control patients (n = 46) and enhanced recovery patients that received multimodal pain management including a postoperative transversalis abdominis plane block with either a continuous local anesthetic catheter (n = 60) or a single-shot of liposomal bupivacaine (n = 39). The primary outcome was pain scores in the first three postoperative days. Secondary outcomes were opioid consumption in oral morphine equivalents and length of stay. RESULTS: Postoperative pain scores were similar across all three groups until postoperative day 3. Length of stay was significantly shorter in both of the enhanced recovery cohorts (3.0 [3.0, 4.0]) compared with control patients (4.0 [4.0, 5.0], P < 0.001). Likewise, average total oral morphine equivalents consumption was significantly reduced in enhanced recovery patients (continuous catheter 215.9 (95% CI, 165.4-266.3); liposomal bupivacaine 211.0 (95% CI, 154.8-267.2); control 518.4 (95% CI 454.2-582.7), P < 0.001). Neither length of stay (P = 0.953), nor oral morphine equivalents consumption (P = 0.883) differed by type of regional analgesia. CONCLUSION: Compared with control patients, both approaches to regional transversalis abdominis plane block analgesia as part of an opiate-sparing enhanced recovery pain management strategy were successful, but neither superior to the other.

Targeted Muscle Reinnervation following Breast Surgery: A Novel Technique.

Post-mastectomy pain syndrome is a prevalent chronic pain condition that affects numerous patients following breast surgery. The mechanism of this pain has been proposed to be neurogenic in nature. As such, we propose a novel surgical method for the prophylactic management of postsurgical breast pain: targeted muscle reinnervation of the breast. This article serves to review the relevant current literature of post-mastectomy pain syndrome and targeted muscle reinnervation, describe our current surgical technique for this operation, and present an initial cohort of patients to undergo this procedure.

Enhanced Recovery after Surgery Protocols Decrease Outpatient Opioid Use in Patients Undergoing Abdominally Based Microsurgical Breast Reconstruction.

BACKGROUND: Enhanced recovery after surgery (ERAS) protocols have known benefits in the inpatient setting, but little is known about their impact in the subsequent outpatient setting. On discharge, multimodal analgesia has been discontinued, nerve blocks and pain pumps have worn off, and patients enter a substantially different physical environment, potentially resulting in a rebound effect. The objective of this study was to investigate the effect of ERAS protocol implementation on outpatient opioid use and recovery. METHODS: Patients who underwent abdominally based microsurgical breast reconstruction before and after ERAS implementation were reviewed retrospectively. Ohio state law mandates that no more than 7 days of opioids may be prescribed at a time, with the details of all prescriptions recorded in a statewide reporting system, from which opioid use was determined. RESULTS: A total of 105 patients met inclusion criteria, of which 46 (44 percent) were in the pre-ERAS group and 59 (56 percent) were in the ERAS group. Total outpatient morphine milligram equivalents used in the ERAS group were less than in the pre-ERAS group (337.5 morphine milligram equivalents versus 668.8 morphine milligram equivalents, respectively; p =0.016). This difference was specifically significant at postoperative week 1 (p =0.044), with gradual convergence over subsequent weeks. Although opioid use was significantly less in the ERAS group, pain scores in the ERAS group were comparable to those in the pre-ERAS group. CONCLUSIONS: The benefits of ERAS protocols appear to extend into the outpatient setting, further supporting their use to facilitate recovery, and highlighting their potential role in helping to address the prescription opioid abuse problem. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Inframammary Fold Reconstruction in the Previously Reconstructed Breast: A Comprehensive Review.

The inframammary fold presents a reconstructive challenge once disrupted during total mastectomy or inadequately restored during breast reconstruction. Various methods of recreating the inframammary fold have been proposed, but reports are generally based on small sample sizes and lack long-term analyses and patient-reported outcomes. The authors herein review the literature on inframammary fold anatomy and reconstructive techniques, highlighting the need for more critical analysis of methodology to develop more predictable and durable outcomes.

Novel 3-Dimensional Imaging Analysis of the Ryan Procedure for Inframammary Fold Elevation in the Reconstruction of the Revised Breast.

The inframammary fold (IMF) can be challenging to reconstruct after disruption during mastectomy or breast reconstruction. The Ryan procedure is a previously described technique with little long-term analysis. Our goal is to analyze the long-term results of the Ryan procedure using 3-dimensional (3D) technology, with the hypothesis that 3D measurements will provide quantitative outcomes that add to the qualitative assessment of the reconstruction. We retrospectively reviewed consecutive breast reconstruction patients by a single surgeon from January 1, 2012 to January 31, 2015 to identify patients who underwent the Ryan procedure. Previously obtained 3D photographs were then analyzed to compare breast base diameter, breast projection, and inter-IMF distance pre- and postoperatively. A survey was then given to 15 health professionals in our department to assess the IMF and symmetry pre- and postoperatively. Eight patients were eligible for inclusion. Four patients were unilateral reconstruction and 4 were bilateral. The Ryan procedure resulted in an inter-IMF discrepancy reduction of 39% and a breast projection increase of 18%. Average length of follow-up was 2.82 ± 0.75 years. One patient required a secondary IMF revision. The majority of survey respondents felt that the IMF and IMF symmetry were improved or stable postoperatively. The Ryan procedure seems to be a reliable and durable technique for IMF reconstruction with increased projection, decreased IMF discrepancy, and increased symmetry. Additionally, 3D imaging provides a useful approach in the assessment of breast reconstruction outcomes, adding quantitative outcomes measures to its evaluation.

Insight into the Mechanistic Basis of the Hysteretic-Like Kinetic Behavior of Thioredoxin-Glutathione Reductase (TGR).

A kinetic study of thioredoxin-glutathione reductase (TGR) from Taenia crassiceps metacestode (cysticerci) was carried out. The results obtained from both initial velocity and product inhibition experiments suggest the enzyme follows a two-site ping-pong bi bi kinetic mechanism, in which both substrates and products are bound in rapid equilibrium fashion. The substrate GSSG exerts inhibition at moderate or high concentrations, which is concomitant with the observation of hysteretic-like progress curves. The effect of NADPH on the apparent hysteretic behavior of TGR was also studied. At low concentrations of NADPH in the presence of moderate concentrations of GSSG, atypical time progress curves were observed, consisting of an initial burst-like stage, followed by a lag whose amplitude and duration depended on the concentration of both NADPH and GSSG. Based on all the kinetic and structural evidence available on TGR, a mechanism-based model was developed. The model assumes a noncompetitive mode of inhibition by GSSG in which the disulfide behaves as an affinity label-like reagent through its binding and reduction at an alternative site, leading the enzyme into an inactive state. The critical points of the model are the persistence of residual GSSG reductase activity in the inhibited GSSG-enzyme complexes and the regeneration of the active form of the enzyme by GSH. Hence, the hysteretic-like progress curves of GSSG reduction by TGR are the result of a continuous competition between GSH and GSSG for driving the enzyme into active or inactive states, respectively. By using an arbitrary but consistent set of rate constants, the experimental full progress curves were successfully reproduced in silico.

Advances in addressing full-thickness skin defects: a review of dermal and epidermal substitutes.

full-thickness skin defects remain a reconstructive challenge. Novel regenerative modalities can aid in addressing these defects. A literature review of currently available dermal and epidermal regenerates was performed. The mechanism and application for each skin substitute was analyzed to provide a guide for these modalities. Available epidermal substitutes include autografts and allografts and may be cultured or noncultured. Dermal regenerate templates exist in biologic and synthetic varieties that differ in the source animal and processing. Epidermal and dermal skin substitutes are promising adjunctive tools for addressing certain soft tissue defects and have improved outcomes in reconstructive procedures. The following article provides a comprehensive review of the biologic materials available and the types of complex wounds amenable to their use.

Restoring full-thickness defects with spray skin in conjunction with dermal regenerate template and split-thickness skin grafting: a pilot study.

Complex, full-thickness soft tissue defects secondary to large burns, trauma and war-related injuries continue to challenge reconstructive surgeons. To achieve positive surgical outcomes in these patient populations, novel approaches are needed to restore the functional, protective and aesthetic properties of skin. Herein, we provide the first report describing the staged use of a dermal regenerate template (DRT) with a spray-on epidermal regenerative modality (spray skin) in addition to autologous split-thickness skin grafting (STSG) in non-burn trauma and compare these results with those of patients treated with DRT and STSG alone. A pilot study was performed to evaluate whether the use of spray skin technology (ReCell, Avita Medical) as an adjunct to DRT (Integra, Integra Lifesciences) and autologous skin grafting in the treatment of patients with large full-thickness soft tissue losses impacts donor site burden as well as recipient and donor site re-epithelialization. In this retrospective study, two patients who were treated with DRT and STSG alone (control group) were compared with two patients who were treated with DRT and spray skin/STSG in combination (experimental group). The mechanisms of injury, total defect and treatment sizes, time to complete re-epithelialization, lengths of follow-up, outcomes and complications were reviewed. Our group observed that using a DRT in conjunction with spray skin/STSG can reduce donor site burden and decrease time to complete healing. It can also permit greater or larger meshing ratios, while aiding in improved re-pigmentation when compared with similar wounds treated with a DRT and autologous skin grafting alone. Copyright © 2017 John Wiley & Sons, Ltd.

A Case Report of the First Nonburn-related Military Trauma Victim Treated with Spray Skin Regenerative Therapy in Combination with a Dermal Regenerate Template.

Massive soft tissue and skin loss secondary to war-related traumas are among the most frequently encountered challenges in the care of wounded warriors. This case report outlines the first military nonburn-related trauma patient treated by a combination of regenerative modalities. Our case employs spray skin technology to an established dermal regenerate matrix. Our patient, a 29-year-old active duty male, suffered a combat blast trauma in 2010 while deployed. The patient's treatment course was complicated by a severe necrotizing fasciitis infection requiring over 100 surgical procedures for disease control and reconstruction. In secondary delayed reconstruction procedures, this triple-limb amputee underwent successful staged ventral hernia repair via a component separation technique with biologic mesh underlay although this resulted in a skin deficit of more than 600 cm2. A dermal regenerate template was applied to the abdominal wound to aid in establishing a "neodermis." Three weeks after dermal regenerate application, spray skin was applied to the defect in conjunction with a 6:1 meshed split thickness skin graft. The dermal regenerate template allowed for optimization of the wound bed for skin grafting. The use of spray skin allowed for a 6:1 mesh ratio, thus minimizing the donor-site size and morbidity. Together, this approach resulted in complete healing of a large full-thickness wound. The patient is now able to perform activities of daily living, walk without a cane, and engage in various physical activities. Overall, our case highlights the potential that combining regenerative therapies can achieve in treating severe war-related and civilian traumatic injuries.

Intestine immune homeostasis after alcohol and burn injury.

Traumatic injury remains one of the most prevalent reasons for patients to be hospitalized. Burn injury accounts for 40,000 hospitalizations in the United States annually, resulting in a large burden on both the health and economic system and costing millions of dollars every year. The complications associated with postburn care can quickly cause life-threatening conditions including sepsis and multiple organ dysfunction and failure. In addition, alcohol intoxication at the time of burn injury has been shown to exacerbate these problems. One of the biggest reasons for the onset of these complications is the global suppression of the host immune system and increased susceptibility to infection. It has been hypothesized that infections after burn and other traumatic injury may stem from pathogenic bacteria from within the host's gastrointestinal tract. The intestine is the major reservoir of bacteria within the host, and many studies have demonstrated perturbations of the intestinal barrier after burn injury. This article reviews the findings of these studies as they pertain to changes in the intestinal immune system after alcohol and burn injury.

T cell IFN-γ suppression following alcohol and burn injury is independent of miRNA155.

miRNA155 has been implicated in normal T cell function and their differentiations into the Th1 subtype. We have shown that acute alcohol (ethanol) intoxication combined with burn injury suppresses T cell IFN-γ release. Herein, we examined whether the decrease in IFN-γ is resulted from altered expression of miRNA155 and transcription factors--NFAT, Tbx21, Jun and Fos--in T cells following ethanol and burn injury. Mice received ethanol (∼3 g/Kg) 4 hours prior to ∼12.5% total body surface area sham or burn injury and were sacrificed one day after injury. Splenic T cells were harvested and cultured with anti-CD3 (2 µg/ml) in the presence or absence of rIL-12 (10 ng/ml) or PMA (10 ng/ml) plus ionomycin (50 ng/ml) for 48 hours. We observed a significant decrease in miRNA155, NFAT, Tbx21, Jun and Fos expression as well as IFN-γ release in T cells cultured with anti-CD3 following ethanol and burn injury compared with shams. The co-treatment of T cells with rIL-12 prevented the decrease in IFN-γ and NFAT, Tbx21, Jun and Fos, but not miRNA155. In contrast, the co-treatment with PMA plus ionomycin normalized the expression of NFAT. It did not prevent the decrease in IFN-γ, Tbx21, Jun, Fos and miRNA155. Finally, results obtained in miRNA155-/- mice did not show any change in T cell release of IFN-γ or expression of nuclear factors compared to wildtype mice. Together, these findings suggest that while ethanol and burn injury decreases the expression of miRNA155, it may not be involved in decreased IFN-γ under those conditions.

The role of aryl hydrocarbon receptor in interleukin-23-dependent restoration of interleukin-22 following ethanol exposure and burn injury.

OBJECTIVE: T-helper (Th)-17 lymphocytes play a crucial role in maintenance and regulation of gut immunity. Our laboratory has demonstrated that acute ethanol (EtOH) exposure before burn injury results in intestinal T cell suppression and enhanced bacterial translocation. BACKGROUND: To extend these studies, we examined the effects of EtOH exposure and burn injury on Th17 responses within intestinal lymphoid Peyer's patches (PP). We further investigated whether restitution of interleukin (IL)-23 enhances PP cell IL-17 and IL-22 after EtOH and burn injury. METHODS: Male mice, approximately 25 g, were gavaged with EtOH (2.9 mg/kg) before receiving an approximately 12.5% total body surface area full thickness burn. One day postinjury, PP mixed cells were cultured in the presence of plate-bound anti-CD3/soluble anti-CD28 in the presence or absence of IL-23 for 48 hours. Supernatants were harvested for IL-17 and IL-22 levels. RESULTS: When combined with EtOH intoxication, burn injury significantly decreased IL-17 and IL-22, as compared with sham injury. IL-23 treatment successfully increased levels of IL-22 but not IL-17. This restoration was prevented when PP cells were treated with CH-223191, an aryl hydrocarbon receptor inhibitor. To further delineate the mechanism of differential IL-17 and IL-22 suppression, PP cells were treated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, which signal via protein kinase C (PKC) and calcium flux. Treatment with PMA and ionomycin significantly prevented the decrease in IL-17 but not IL-22 after EtOH exposure and burn injury. CONCLUSIONS: These findings suggest that IL-23-mediated restoration of IL-22 is aryl hydrocarbon receptor dependent, whereas IL-17 requires activation of protein kinase C and intracellular calcium signaling.

Noninvasive measurement of intestinal inflammation after burn injury.

Intestinal inflammation has been linked with multiorgan failure in patients with burn and other traumatic injuries. We hypothesized that markers of intestinal inflammation are detectible noninvasively. Fecal samples were collected from seven severely burned patients and 15 control patients for the measurement of inflammatory cytokines using a multiplex assay kit. In addition, fecal levels of myeloperoxidase (MPO) and elastase were measured using standard procedures. Compared with a control group, levels of inflammatory cytokines were significantly increased in the burn group. Interleukin (IL)-6 increased to a mean (± SEM) of 2.16 ± 0.61 to 3.81 ± 0.49 pg/mg (P < .05), as did IL-8 (3.32 ± 0.76 to 20.51 ± 6.65 pg/mg; P < .05), IL-12 (6.23±0.98 to 8.11±0.95pg/mg; P=0.01), IL-13 (3.86 ± 0.32 to 11.83 ± 1.47 pg/mg; P < .05), monocyte chemoattractant protein-1 (2.78 ± 2.61 to 6.5 ± 3.97 pg/mg; P < .05), MPO (13.41 ± 1.40 to 24.52 ± 4.31 units/mg protein; P < .05), and elastase (2.46 ± 0.38 to 5.08 ± 0.72 pg/mL; P < .05). Our results suggest that markers of intestinal inflammation are measurable by noninvasive means and are increased after burn injury compared with controls. Of note, increased IL-8 correlated with increased MPO and elastase activity, suggesting a role for neutrophil activation in burn-mediated intestinal inflammation. Thus, these inflammatory cytokine profiles may be valuable biomarkers of intestinal inflammation after burn injury.

Reduced neutrophil chemotaxis and infiltration contributes to delayed resolution of cutaneous wound infection with advanced age.

Advanced age is associated with alterations in innate and adaptive immune responses, which contribute to an increased risk of infection in elderly patients. Coupled with this immune dysfunction, elderly patients demonstrate impaired wound healing with elevated rates of wound dehiscence and chronic wounds. To evaluate how advanced age alters the host immune response to cutaneous wound infection, we developed a murine model of cutaneous Staphylococcus aureus wound infection in young (3-4 mo) and aged (18-20 mo) BALB/c mice. Aged mice exhibit increased bacterial colonization and delayed wound closure over time compared with young mice. These differences were not attributed to alterations in wound neutrophil or macrophage TLR2 or FcγRIII expression, or age-related changes in phagocytic potential and bactericidal activity. To evaluate the role of chemotaxis in our model, we first examined in vivo chemotaxis in the absence of wound injury to KC, a neutrophil chemokine. In response to a s.c. injection of KC, aged mice recruited fewer neutrophils at increasing doses of KC compared with young mice. This paralleled our model of wound infection, where diminished neutrophil and macrophage recruitment was observed in aged mice relative to young mice despite equivalent levels of KC, MIP-2, and MCP-1 chemokine levels at the wound site. This reduced leukocyte accumulation was also associated with lower levels of ICAM-1 in wounds from aged mice at early time points. These age-mediated defects in early neutrophil recruitment may alter the dynamics of the inflammatory phase of wound healing, impacting macrophage recruitment, bacterial clearance, and wound closure.

Interleukin-22 modulates gut epithelial and immune barrier functions following acute alcohol exposure and burn injury.

Interleukin-22 (IL-22) maintains gut epithelial integrity and expression of antimicrobial peptides Reg3ß and Reg3γ. Our laboratory has shown that acute alcohol/ethanol (EtOH) exposure before burn injury results in increased gut permeability, intestinal T-cell suppression, and enhanced bacterial translocation. Herein, we determined the effect of combined EtOH intoxication and burn injury on intestinal levels of IL-22 as well as Reg3ß and Reg3γ expression. We further examined whether in vivo restitution of IL-22 restores gut permeability, Reg3ß and Reg3γ levels, and bacterial load (e.g., gut bacterial growth) within the intestine after EtOH and burn injury. Male mice, ∼25g, were gavaged with EtOH (2.9 mg/kg) before receiving a ∼12.5% total-body-surface-area, full-thickness burn. Mice were immediately treated with saline control or IL-22 (1 mg/kg) by i.p. injection. One day after injury, there was a significant decrease in intestinal IL-22, Reg3ß, and Reg3γ expression along with an increase in intestinal permeability and gut bacterial load after EtOH combined with burn injury, as compared with sham injury. Treatment with IL-22 normalized Reg3ß and Reg3γ expression and attenuated the increase in intestinal permeability after EtOH and burn injury. Qualitatively, IL-22 treatment reduced the bacterial load in nearly half of mice receiving EtOH combined with burn injury. Our data indicate that IL-22 maintains gut epithelial and immune barrier integrity after EtOH and burn injury; thus, the IL-22/antimicrobial peptide pathway may provide a therapeutic target for the treatment of patients who sustain burn injury under the influence of EtOH.

Ethanol exposure suppresses bone marrow-derived dendritic cell inflammatory responses independent of TLR4 expression.

Acute alcohol (ethanol) exposure is linked with increased susceptibility to infection and increased mortality in trauma and burn patients. Dendritic cells (DCs) are central mediators in innate and adaptive immune responses, and they play a role in the presentation of pathogens to adaptive immune cells. We investigated the effects of acute ethanol exposure on bone marrow-derived DC (BM-DC) responses. Total bone marrow cells, obtained from 8 to 10 week old C57BL/6 male mice, were cultured in the presence of granulocyte/monocyte-colony stimulating factor and interleukin (IL)-4 for 7 days. BM-DCs were harvested and treated with increasing doses of ethanol (50, 100, and 250 mM) at the time of, or 3 h before, lipopolysaccharide (LPS). After LPS, supernatants were collected for cytokine measurement, and cells were harvested for flow cytometry. Concurrent acute ethanol exposure and LPS treatment resulted in a dose-dependent suppression of IL-6, IL-12p40, IL-23, and IL-10. In addition, ethanol exposure before LPS dysregulated the IL-12p40/IL-23 balance and more profoundly suppressed IL-6 and IL-10 secretion by BM-DCs, as compared with cells concurrently treated with ethanol and LPS. Ethanol treatment did not affect either toll-like receptor (TLR)4 or TLR2 expression. In summary, our study demonstrates that acute ethanol exposure suppresses BM-DC LPS-induced responses, irrespective of affecting TLR4 or TLR2 expression.

Th17 cells: critical mediators of host responses to burn injury and sepsis.

Th cells have long been recognized as vital components of the adaptive immune system. Until recently, CD3(+)CD4(+) Th cells were divided into cell-mediated Th1 or humoral Th2 responses. However, the Th1-Th2 hypothesis failed to accommodate the more recently described Th17 cells. Today, the major Th cell subsets include Th1, Th2, Th9, Th17, Th22, and Tregs, each of which produce specific effector cytokines under unique transcriptional regulation. Specifically, Th17 cells produce effector cytokines IL-17, IL-21, and IL-22 under the regulation of ROR-γt. Th17 lymphocytes were first described as orchestrators of neutrophil recruitment and activation and as key players in chronic inflammation and autoimmunity. More recent evidence suggest that Th17 lymphocytes and their effector cytokines play a crucial role in maintaining mucosal immunity and barrier integrity, including the skin, lung, and gut. Burn injury induces global changes to the systemic immune response, including suppressed immune function and increased susceptibility to infection. Moreover, burn trauma is associated with remote organ injury. This relationship between burn and remote organ injury supports the hypothesis that immune suppression may facilitate the development of sepsis, systemic inflammatory response syndrome, and multiple organ dysfunction syndrome in critically ill burn patients. Herein, we discuss this emerging adaptive cell subset in critical care settings, including burn injury and clinical sepsis, and highlight the potential therapeutic role of IL-22.

Activation of toll-like receptor 2 prevents suppression of T-cell interferon γ production by modulating p38/extracellular signal-regulated kinase pathways following alcohol and burn injury.

Recent studies indicate that toll-like receptors (TLRs) are expressed on T cells and that these receptors directly or indirectly activate the adaptive immune system. We have shown previously that acute alcohol/ethanol (EtOH) intoxication combined with burn injury suppresses mesenteric lymph node (MLN) T-cell interleukin-2 (IL-2) and interferon γ (IFN-γ) production. We examined whether direct stimulation of T cells with TLR2, 4, 5 and 7 agonists modulates CD3-mediated T-cell IL-2/IFN-γ release following EtOH and burn injury. Male mice were gavaged with EtOH (2.9 gm/kg) 4 h prior to receiving an ~12.5% total body surface area sham or full-thickness burn injury. Animals were killed on d 1 after injury and T cells were purified from MLN and spleens. T cells were cultured with plate-bound anti-CD3 in the presence or absence of various TLR ligands. Although TLR2, 4 and 5 agonists potentiate anti-CD3-dependent IFN-γ by T cells, the TLR2 agonist alone induced IFN-γ production independent of CD3 stimulation. Furthermore, T cells were treated with inhibitors of myeloid differentiation primary response protein 88 (MyD88), TIR domain-containing adaptor protein (TIRAP), p38 and/or extracellular signal-regulated kinase (ERK) to determine the mechanism by which TLR2 mediates IL-2/IFN-γ production. IL-2 was not influenced by TLR agonists. MyD88 and TIRAP inhibitory peptides dose-dependently diminished the ability of T cells to release IFN-γ. p38 and ERK inhibitors also abolished TLR2-mediated T-cell IFN-γ. Together, our findings suggest that TLR2 directly modulates T-cell IFN-γ production following EtOH and burn injury, independent of antigen-presenting cells. Furthermore, we demonstrated that MyD88/TIRAP-dependent p38/ERK activation is critical to TLR2-mediated T-cell IFN-γ release following EtOH and burn injury.

Hysteresis in thioredoxin-glutathione reductase (TGR) from the adult stage of the liver fluke Fasciola hepatica.

Thioredoxin-glutathione reductase (TGR) was purified from the adult stage of the liver fluke Fasciola hepatica. At 38° C and pH 7.8, specific activity values were 10.2U mg(-1) and 64.5U mg(-1), with DTNB or GSSG as substrates, respectively. Under the same conditions, apparent Km values were 46±8 µM (DTNB) and 30 ± 5 µM (GSSG). The enzyme was also able to catalyze thiol/disulfide exchange reactions. A subunit Mr of 61,000 was obtained. Like the homologous enzyme from the tapeworms, a lag time was observed in the enzyme assays at moderate or high concentrations of the substrate GSSG. The hysteretic behavior was reverted in the presence of GSH and was notably dependent on pH, such that the magnitude of the lag time increased with the acidity of the medium. These results strongly suggest that a hysteretic kinetic is a common feature of TGR from any parasitic flatworm. A sequence comparison revealed the structural cysteine residues proposed to be in the origin of the peculiar kinetic behavior of TGR are absent from the F. hepatica enzyme. Based on these observations, the model proposed recently to explain the GSSG-dependent hysteretic kinetic of TGR, which assumes the covalent modification of specific cysteine residues through glutathionylation [Bonilla M. et al. (2008) J Biol Chem 283: 17898] needs to be reevaluated.

Mitochondrial Thioredoxin-Glutathione Reductase from Larval Taenia crassiceps (Cysticerci).

Mitochondrial thioredoxin-glutathione reductase was purified from larval Taenia crassiceps (cysticerci). The preparation showed NADPH-dependent reductase activity with either thioredoxin or GSSG, and was able to perform thiol/disulfide exchange reactions. At 25 degrees C specific activities were 437 +/- 27 mU mg(-1) and 840 +/- 49 mU mg(-1) with thioredoxin and GSSG, respectively. Apparent K(m) values were 0.87 +/- 0.04 muM, 41 +/- 6 muM and 19 +/- 10 muM for thioredoxin, GSSG and NADPH, respectively. Thioredoxin from eukaryotic sources was accepted as substrate. The enzyme reduced H(2)O(2) in a NADPH-dependent manner, although with low catalytic efficiency. In the presence of thioredoxin, mitochondrial TGR showed a thioredoxin peroxidase-like activity. All disulfide reductase activities were inhibited by auranofin, suggesting mTGR is dependent on selenocysteine. The reductase activity with GSSG showed a higher dependence on temperature as compared with the DTNB reductase activity. The variation of the GSSG- and DTNB reductase activities on pH was dependent on the disulfide substrate. Like the cytosolic isoform, mTGR showed a hysteretic kinetic behavior at moderate or high GSSG concentrations, but it was less sensitive to calcium. The enzyme was able to protect glutamine synthetase from oxidative inactivation, suggesting that mTGR is competent to contend with oxidative stress.