Calcium/Calmodulin-Dependent Protein Kinase is Involved in the Release of High Mobility Group Box 1 Via the Interferon-beta Signaling Pathway

Previously, we have reported that high mobility group box 1 (HMGB1), a proinflammatory mediator in sepsis, is released via the IFN-beta-mediated JAK/STAT pathway. However, detailed mechanisms are still unclear. In this study, we dissected upstream signaling pathways of HMGB1 release using various molecular biology methods. Here, we found that calcium/calmodulin-dependent protein kinase (CaM kinase, CaMK) is involved in HMGB1 release by regulating IFN-beta production. CaMK inhibitor, STO609, treatment inhibits LPS-induced IFN-beta production, which is correlated with the phosphorylation of interferon regulatory factor 3 (IRF3). Additionally, we show that CaMK-I plays a major role in IFN-beta production although other CaMK members also seem to contribute to this event. Furthermore, the CaMK inhibitor treatment reduced IFN-beta production in a murine endotoxemia. Our results suggest CaMKs contribute to HMGB1 release by enhancing IFN-beta production in sepsis.

Expression Pattern of Immunoproteasome Subunits in Human Thymus

The expression pattern of immunoproteasomes in human thymus has not been analyzed but may have important consequences during thymic selection. Here we examined the expression patterns of immunoproteasome subunits in fetal and adult thymic tissues by immunohistochemistry and found that all three subunits are expressed in both cortical and medullary stromal cells. These data suggest that thymic selection in human can be affected by peptide repertoires generated by immunoproteasomes.

Sepsis Mortality in CIITA Deficient Mice is Associated with Excessive Release of High-mobility Group Box 1

BACKGROUND: Down regulation of major histocompatibility complex class II transactivator (CIITA) has been identified as a major factor of immunosuppression in sepsis and the level of CIITA expression inversely correlates with the degree of severity. However, it has not been fully elucidated whether the lower expression of CIITA is a cause of disease process or a just associated sign. Here we determined whether the CIITA deficiency decreased survival rate using murine sepsis model. METHODS: Major histocompatibility complex class II (MHC-II) deficient, CIITA deficient and wild type B6 mice were subjected to cecal ligation puncture (CLP) surgery. CIITA and recombination activation gene (RAG)-1 double deficient mice were generated to test the role of lymphocytes in CIITA-associated sepsis progression. RESULTS: Sepsis mortality was enhanced in CIITA deficient mice, not by impaired bacterial clearance resulted from CD4 T cell depletion, but hyper-inflammatory response such as excessive release of a pro-inflammatory cytokine, high-mobility group box 1 (HMGB1). CONCLUSION: Our results demonstrate that CIITA deficiency affects the course of sepsis via the unexpected function of CIITA, regulation of cytokine release.

CD99 activates T cells via a costimulatory function that promotes raft association of TCR complex and tyrosine phosphorylation of TCR zeta

We investigated the co-stimulatory role of a cell-surface protein, CD99. Co-ligation of CD99 and suboptimal CD3 induced T-cell activation to a level comparable to that obtained with optimal CD3 or CD3+CD28. We also noted concomitant enhancement of the earliest T-cell receptor (TCR) signaling events. In addition, co-ligation of CD99 and CD3 led to translocation of TCR complexes into the lipid raft, without concomitant migration of CD99 to the raft, and consequent enhancement of TCR zeta-mediated signal 1. These data demonstrate the unique properties of CD99 co-stimulation that distinguish this molecule from CD28 and other raft-resident co-stimulatory factors.

Effect of Erythromycin on Pro-inflammatory Signalings by Particles

Purpose: In periprosthetic osteolysis, cytokines, which are secreted from macrophages by the stimulation of particles, up-regulate the signaling for osteoclast activation through RANKL (Receptor activator of Nuclear Factor Kappa-B Ligand). This study compared the reaction to the particles and RANKL in the macrophages by examining the changes in the pro-inflammatory signals. In addition, because erythromycin has an anti-inflammatory effect, the effect of erythromycin on the pro-inflammatory signals by particles and RANKL was also analyzed to clarify the mechanism for the anti-resorptive effect with particles. Materials and Methods: The Raw 264.7 cell line (murine macrophage cell line) was used for these experiments. The particles were made from PMMA (poly-methyl-meth-acrylate) and UHMWPE (ultra high molecular weight polyethylene) to enhance their stimulatory effects. Under the same culture conditions used for macrophages, the cells were treated with either particles or RANKL. The differences in the production of TNF-α, activities of MAP kinase, I-κB and reactive oxygen species (ROS) between the particle and RANKL treated macrophages were examined. The influence of erythromycin on these models was also observed. Results: Erythromycin inhibited ERK and p38 phosphorylation in both models, and suppressed the increase in H2O2 production in the particle-treated macrophages. However, erythromycin inhibited neither the production of TNF- in both models nor the production of H2O2 in the RANKL-treated macrophages. In addition, erythromycin reversed the suppression of I-κB by the particles. Conclusion: For the response of macrophages, erythromycin mainly suppresses the particle induced ROS and NF-κB activation compared with RANKL-induced osteoclastogenesis signaling. Erythromycin might suppress particle-induced osteolysis through these anti-inflammatory effects. Therefore, further studies on the downstream signals of osteoclastogenesis will be needed.

Delayed allograft rejection by the suppression of class II transactivator

We examined the effect of class II transactivator (CIITA) down-modulation on allograft rejection. To inhibit the function of CIITA, we constructed a series of CIITA mutants and found one exhibiting the dominant-negative effect on the regulation of major histocompatibility complex (MHC) class II expression. To test whether the CIITA dominant-negative mutant reduces immunogenecity, CIITA-transfected melanoma cells were injected into allogeneic host and assessed for immune evading activity against host immune cells. We demonstrated that the CIITA dominant-negative mutant allowed tumor nodules to develop earlier in the lung than control by this tumor challenge study. Furthermore, skin grafts deficient for CIITA also survived longer than wild-type in allogeneic hosts. Both the tumor challenge and skin graft studies suggest the inhibition of CIITA molecules in donor tissue would be beneficial to the control of allo-response.

The Limited Immune Modulatory Effect of Early Escharectomy on Adhesion Molecules in Major Burn Patients / 대한마취과학회지

BACKGROUND: Early escharectomy has been shown to improve the survival rates and the treatment outcomes of major burn patients. However, its exact mechanism, especially in terms of the human immune system, has not been fully elucidated. This observational study, which placed a focus on adhesion molecules, was conducted to assess changes of soluble intercelluar adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), and E-selectin in major burn patients undergoing early eschar excision. METHODS: Seventeen ASA physical status II or III adult major burn patients, admitted for plastic and reconstructive surgery for burn wound care, were initially recruited. When early escharectomy was scheduled, a series of blood samples were obtained four times at 72 and 24 hours preop and 24 and 72 hours postop, respectively. Changing levels of sICAM-1, sVCAM-1, and E-selectin were measured using quantitative sandwich immunoassay techniques. RESULTS: All patients suffered from major burns. Early escharectomy does not appear to have any significant impact on the levels of sICAM-1 and sVCAM-1. On the other hand, E-selectin levels showed a significant decrease after escharectomy. CONCLUSIONS: Major burn injury certainly induces a systemic inflammatory response. Adhesion molecules behave in such a way that escharectomy has a limited immunomodulatory effect in major burns. This is probably related to the timing and extent of surgery, and the complex nature of burn related inflammation.

Western Blot Analysis of Alterations of Skeletal Muscle Nicotinic Acetylcholine Receptor Subunits in the Major Burns: Burn vs. Nonburn Sites / 대한마취과학회지

BACKGROUND: Major burn injury causes aberrant responses to neuromuscular blocking agents, and an increase in fetal type nicotinic acetylcholine receptors (nAchR) has been speculated to contribute to the altered response. This study was conducted to test the hypothesis that the increases in nAchR are due to generalized systemic effects, not limited to area of burn in humans. Using the rectus abdominis muscle of the burned area and quadriceps muscle of nonburned area from the single major burn patient, the changes of nAchR subunits of alpha1, gamma and alpha7 were compared. METHODS: Twelve adults (M:F = 11:1) with total body surface area (TBSA) of 48.8 +/- 19.3% major burns, undergoing burn related elective surgery, were included at 30.8 +/- 16.4 days after the burn injury. Only those with burns in abdominal area and no burns in the lower extremity, were selected. Under general anesthesia, muscle biopsy was taken from the rectus abdominis muscle under the burned abdominal area as well as from the quadriceps muscle under the unburned thigh, at least 30 cm away from the closest burn wound margin. The nAchR changes in the muscles were assayed by western blot using subtype specific mouse antibody for alpha1, gamma and alpha7. RESULTS: The nAchR subunit quantitation (volume% compared to nonburn controls) of alpha1, gamma and alpha7 in the rectus and quadriceps by densitometry were 335.4 +/- 45.4 vs. 321.7 +/- 76.2, 298.3 +/- 234.3 vs. 290.0 +/- 202.3, 149.7 +/- 22.6 vs. 141.6 +/- 35.6, respectively, showing no significant statistical differences between burned and nonburned area (P > 0.05). However, large coefficients of variation were noted in both muscles of gamma group, which may suggest indirect evidence of proliferation of fetal type nAchR in the major burns. TBSA and days after the burn injury were poorly correlated with the amount of expression of subtypes of acetylcholine receptors tested. CONCLUSIONS: Our results confirm that systemic effects of thermal injury include an increase in nAchR at sites distant from the thermal injury, which may account for the skeletal muscle dysfunction and aberrant responses to neuromuscular relaxation. Further studies need to address the importance of burn size and its effects on quantitative and qualitative changes in receptor.

Lung Injury Induced by Major Burns does not Affect Fentanyl Clearance / 대한마취과학회지

BACKGROUND: Major burns can alter the pharmacokinetics of opiate analgesics, which are commonly used perioperatively. Fentanyl undergoes a significant amount of pulmonary pharmacokinetic transition. This study was conducted to compare the pharmacokinetics of fentanyl in major burns, with and without lung injury, during the subacute hyperdynamic phase of recovery. METHODS: Twelve adults, with total body surface area (TBSA) 51.0 +/- 11.8% burns, aged 34.9 +/- 9.6 years, with a lung injury related to the burn, were studied at 15.4 +/- 9.4 days after the injury. Another 8 patients, aged 39.8 +/- 10.5 years, with TBSA 46.3 +/- 19.4%, at 19.3 +/- 10.9 days, without lung injury, served as controls. Fentanyl 200microgram was given intravenously over 10 seconds. Blood samples (n = 20) were collected at predetermined intervals. A two-compartment model was used for pharmacokinetic analyses of the fentanyl concentrations, as determined by LC/MS. The cardiac index (CI) was also measured using an esophageal Doppler monitor. RESULTS: There were no differences in the patient characteristics between the two groups. Those with burns had a significantly higher cardiac index (4.1 +/- 2.4 L/min/m2), clearance (Cl), central (V1) and total volume of distribution (Vd), but there were no differences between those with and without lung injury (30.2 +/- 14.3 vs. 30.1 +/- 5.8 ml/min/kg, 0.8 +/- 0.3 vs. 0.6 +/- 0.2 L/kg, 5.8 +/- 1.7 vs. 5.2 +/- 2.1 L/kg, respectively). Prolonged distribution (t1/2alpha) and elimination half-lives (t1/2beta) were noted in those with burns, but there were no differences between the two groups (3.2 +/- 1.3 vs. 3.3 +/- 1.3 minutes, 2.6 +/- 1.4 vs. 2.0 +/- 0.7 hours, respectively). CONCLUSIONS: The increased Cl of fentanyl in those with burns is primarily dependent upon the resultant increased hepatic blood flow. The pulmonary kinetics is a saturable process, which is not affected by a single bolus of fentanyl. The lung injury induced by major burns would have no influence on the elimination kinetics of fentanyl.

CD24 Expression in Gastric Adenocarcinoma Is Associated with Tumor Invasiveness

BACKGROUND: CD24, also referred to as the heat stable antigen in mice, is a glycosyl phosphatidylinositol- linked glycoprotein expressed by thymocytes, B cells, neutrophils and immature neuronal cells. It has been recently observed in a variety of human malignancy. Here, we demonstrated the expression of CD24 in gastric adenocarcinomas. METHODS: A total of 40 gastric adenocarcinomas and 20 tubular adenomas were immunohistochemically examined for the expression of CD24 and matrix metalloproteinase-2 (MMP-2) proteins. The immunoreactivity of CD24 was semiquantitatively scored (0, 1+, 2+) and compared with clinicopathologic variables and MMP-2 expression in tumor cells. RESULTS: CD24 was rarely expressed in normal gastric tissue and not expressed in tubular adenoma. In contrast, a moderate/strong expression (2+) of CD24 was observed in 25% of gastric adenocarcinomas, and 30% cases showed a weak CD24 staining (1+). Moreover, CD24 expression was significantly correlated with the depth of tumor invasion and MMP-2 expression. CONCLUSION: These results suggest that the aberrant expression of CD24 in gastric adenocarcinomas might be associated with tumor progression and invasiveness.

Effect of Atorvastatin, a HMG-CoA Reductase Inhibitor, in Experimental Colitis in Mice

BACKGROUND: The statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, are approved for cholesterol reduction, and may also be beneficial in the treatment of inflammatory disease. In this study, atorvastatin was tested in experimental colitis, a disease model of inflammatory bowel disease. METHODS: To induce colitis, dextran sodium sulfate (DSS) or trinitrobenzene sulfonic acid (TNBS) were administrated to C57BL/6 or BALB/c mice. Mice were monitored daily for loss of body weight and survival for indicated days. Colon length and histology were examined after sacrifice. RESULTS: The administration of DSS induced marked colonic inflammation and shortening, and resulted in a loss of body weight. DSSinduced colitis was not affected by atorvastatin treatment, but in contrast, the administration of atorvastatin relieved TNBS-induced colitis with a resultant rapid recovery of weight loss and a reduction in colonic length shortening. Histologically, inflammatory cell infiltration in the colonic wall, mucosal ulceration and crypt disruption were also suppressed in atorvastatin treated mice. CONCLUSION: These results suggest that atorvastatin preserves intestinal integrity in colitis, probably via the modulation of Th cell-mediated immune response, in a manner independent of innate immunity.

Homophilic Interaction of CD99 via Its Extracellular Domain / 대한면역학회지

No abstract available.