Lipopolysaccharide-Induced Lysosomal Cell Death Through Reactive Oxygen Species in Rat Liver Cell Clone 9.

In sepsis, bacterial components, particularly lipopolysaccharide (LPS), trigger organ injuries such as liver dysfunction. Although sepsis induces hepatocyte damage, the mechanisms underlying sepsis-related hepatic failure remain unclear. In this study, we demonstrated that the LPS-treated rat hepatocyte cell line Clone 9 not only induced reactive oxygen species (ROS) generation and apoptosis but also increased the expression of the autophagy marker proteins LC3-II and p62, and decreased the expression of intact Lamp2A, a lysosomal membrane protein. Additionally, LPS increased lysosomal membrane permeability and galectin-3 puncta formation, and promoted lysosomal alkalization in Clone 9 cells. Pharmacological inhibition of caspase-8 and cathepsin D (CTSD) suppressed the activation of caspase-3 and rescued the viability of LPS-treated Clone 9 cells. Furthermore, LPS induced CTSD release associated with lysosomal leakage and contributed to caspase-8 activation. Pretreatment with the antioxidant N-acetylcysteine (NAC) not only diminished ROS generation and increased the cell survival rate, but also decreased the expression of activated caspase-8 and caspase-3 and increased the protein level of Lamp2A in LPS-treated Clone 9 cells. These results demonstrate that LPS-induced ROS causes lysosomal membrane permeabilization and lysosomal cell death, which may play a crucial role in hepatic failure in sepsis. Our results may facilitate the development of new strategies for sepsis management.

Using a neonatal rat model to explore the therapeutic potential of coenzyme Q10 in prematurity under hyperoxia.

Hyperoxia, is often used in preterm supportive care, leading to high oxygen exposure in neonates. Coenzyme Q10 (CoQ10) is a free radical scavenger that has been studied in older children but never be investigated for its role in preterm care. We hypothesize that the administration of exogenous CoQ10 would raise serum concentrations of CoQ10 and mitigate the adverse effects of hyperoxia on the organs by reducing oxygen-free radicals and inflammation. The aim of this study was to evaluate the effects of oxidative stress, inflammatory response, and survival in neonatal rats after CoQ10 treatment. Neonatal rats delivered from four pregnant Wistar rats were randomly divided into four groups: (a) control, (b) CoQ10, (c) hyperoxia (O2 group), and (d) treatment (CoQ10 + O2 ) groups. The dose of CoQ10 injected was 30 mg/kg. The CoQ9, CoQ10, cytokines, oxidative stress, and antioxidant enzyme activity were measured. Tissue samples were histologically examined and mortality was monitored for 16 days. The level of CoQ9 significantly increased in the liver, kidney, and plasma, while the level of CoQ10 significantly increased in most organ tissues in the CoQ10 + O2 group. Additionally, CoQ10 decrease oxidative stress in the liver, increase antioxidant enzyme activity in the heart, kidney, and brain, and reverse an inclined level of hematopoietic growth factors. However, CoQ10 had no effect on inflammation, organ damage, or mortality. Therefore, the use of CoQ10 in potential adjuvant therapy for neonatal hyperoxia requires further research.

Correction: Ou-Yang, F. et al. Antiproliferation for Breast Cancer Cells by Ethyl Acetate Extract of Nepenthes thorellii x (ventricosa x maxima). Int. J. Mol. Sci. 2019, 20, 3238.

The authors would like to make corrections to their published paper [...].

M2-like polarization of THP-1 monocyte-derived macrophages under chronic iron overload.

Macrophages are characterized by phenotypical and functional heterogeneity. In different microenvironments, macrophages can polarize into two types: classically activated macrophages (M1) or alternatively activated macrophages (M2). M1 macrophages are a well-known bacteriostatic macrophage, and conversely, M2 macrophages may play an important role in tumor growth and tissue remodeling. M1 macrophages have been reported to have high intracellular iron stores, while M2 macrophages contain lower intracellular iron. It has been well-described that disturbances of iron homeostasis are associated with altered immune function. Thus, it is important to investigate if chronic iron overload is capable of polarizing macrophages. Human monocytic leukemia THP-1 cells were maintained in culture medium that contained 100 µM ferrous sulfate heptahydrate (FeSO4) (I-THP-1) and differentiated into THP-1-derived macrophages (I-TDMs) by induction with phorbol 12-myristate 13-acetate (PMA). We characterized that I-TDMs not only enhanced the surface expression of CD163 and CD206 but also increased arginase and decreased iNOS protein expression. I-TDMs enhanced pSTAT6 expression and decreased pSTAT1 and NF-κB expressions. Furthermore, the gene expression profile of I-TDMs was comparable with M2 macrophages by performing human oligonucleotide DNA microarray analysis. Finally, functional assays demonstrated I-TDMs secreted higher levels of IL-10 but not M1 cytokines. Additionally, the conditional medium of I-TDMs had enhanced migration and increased invasion of A375 melanoma cells which was similar to the characteristics of tumor-associated macrophages. Taken together, we demonstrated that THP-1-derived macrophages polarized to a phenotype of M2-like characteristics when subjected to chronic iron overload.

Antiproliferation for Breast Cancer Cells by Ethyl Acetate Extract of Nepenthes thorellii x (ventricosa x maxima).

Extracts from the Nepenthes plant have anti-microorganism and anti-inflammation effects. However, the anticancer effect of the Nepenthes plant is rarely reported, especially for breast cancer cells. Here, we evaluate the antitumor effects of the ethyl acetate extract of Nepenthes thorellii x (ventricosa x maxima) (EANT) against breast cancer cells. Cell viability and flow cytometric analyses were used to analyze apoptosis, oxidative stress, and DNA damage. EANT exhibits a higher antiproliferation ability to two breast cancer cell lines (MCF7 and SKBR3) as compared to normal breast cells (M10). A mechanistic study demonstrates that EANT induces apoptosis in breast cancer cells with evidence of subG1 accumulation and annexin V increment. EANT also induces glutathione (GSH) depletion, resulting in dramatic accumulations of reactive oxygen species (ROS) and mitochondrial superoxide (MitoSOX), as well as the depletion of mitochondrial membrane potential (MMP). These oxidative stresses attack DNA, respectively leading to DNA double strand breaks and oxidative DNA damage in γH2AX and 8-oxo-2'deoxyguanosine (8-oxodG) assays. Overall these findings clearly revealed that EANT induced changes were suppressed by the ROS inhibitor. In conclusion, our results have shown that the ROS-modulating natural product (EANT) has antiproliferation activity against breast cancer cells through apoptosis, oxidative stress, and DNA damage.

Heat-shock pretreatment reduces expression and release of TSLP from keratinocytes under Th2 environment.

BACKGROUND: Atopic dermatitis is a chronic, relapsing inflammatory disease of the skin. Current therapy is not curative, and recalcitrant disease is a big stress and challenge for parents and physicians. This study explored the potential role of heat-shock protein 70 (HSP-70) and its anti-inflammatory effects on keratinocyte under TH2 environment. METHODS: Human keratinocyte cell line (HaCa T) was stimulated with IL-4, IL-13, and TNF-α to synthesize and secrete thymic stromal lymphopoietin (TSLP), an important cytokine of immunopathogenesis in atopic dermatitis. Heat shock was performed by immersing the cell-contained flash into a water bath of 45°C for 20 min. Cell viability, TSLP expression, and secretion of HaCa T cells were measured and compared. Possible regulatory mechanisms influencing the expression of TSLP, such as the STAT6 and NF-κB signal pathways, were investigated. RESULTS: Heat-shock treatment induced intracellular HSP-70 expression in HaCa T cells without affecting cell viability. The induced expression and secretion of TSLP in HaCa T cells were suppressed by heat shock. The NF-κB signal pathway was inhibited by heat shock, leading to decreased TSLP expression and secretion. CONCLUSION: Heat stress-induced HSPs can significantly reduce the production and secretion of TSLP from HaCaT cells under Th2 environment. Thus, the evidence highlights the potential role of HSP-70 for atopic dermatitis in the future.

Low-dose imiquimod induces melanogenesis in melanoma cells through an ROS-mediated pathway.

BACKGROUND: Melanogenesis is the process of melanin maturation which not only protects skin from UV radiation but also plays an important role in antigenicity of melanomas. Imiquimod (IMQ) is a toll-like receptor 7 (TLR7) agonist that exhibits antiviral and anticancer activity. OBJECTIVE: To explore whether IMQ could induce melanogenesis in melanoma cells. METHODS: The mouse melanoma cell line B16F10, the mouse immortalized melanocyte Melan-A, and human melanoma cell lines MNT-1, C32 and A375 were utilized in this study. The pigmented level was observed by the centrifuged cell pellet. The intracellular and extracellular melanin levels were examined in the absorbance in NaOH-extracted cell lysate and cell-cultured medium, respectively. The expression of melanogenesis related proteins was examined by immunoblotting. The intracellular cyclic AMP amount was evaluated by the cAMP Glo assay kit. The activity of phosphodiesterase 4B (PDE4B) was investigated by CREB reporter assay with overexpressed PDE4B or not. RESULTS: We demonstrated that a low dose of IMQ could trigger melanogenesis in B16F10 cells. IMQ induced microphthalmia-associated transcription factor (MITF) nuclear translocation, upregulated the expression of melanogenesis-related proteins, increased tyrosinase (TYR) activity, and led to pigmentation in B16F10 cells. Next, we found that IMQ-induced melanogenesis was activated by excessive intracellular cAMP accumulation, which was regulated through IMQ-mediated PDE4B inhibition. Finally, IMQ-induced ROS production was found to be involved in melanogenesis by its control of PDE4B activity. CONCLUSIONS: Low dose of IMQ could activate melanogenesis through the ROS/PDE4B/PKA pathway in melanoma cells.

p53 modulates the AMPK inhibitor compound C induced apoptosis in human skin cancer cells.

Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53 status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status.

Heat shock protein 70 protects the lungs from hyperoxic injury in a neonatal rat model of bronchopulmonary dysplasia.

Hyperoxia plays a significant role in the pathogenesis of lung injury, such as bronchopulmonary dysplasia (BPD), in premature infants or newborns. BPD management aims to minimize further injury, provide an optimal environment to support growth and recovery. In clinic neonatal care, we need a new therapy for BPD. Heat shock protein 70 (Hsp70) inhibit cell apoptosis and promote cell repair allowing cells to survive lethal injury. We hypothesized that Hsp70 could be used to prevent hyperoxia related BPD in the neonatal rat model through its anti-apoptotic and anti-inflammatory effects. In this study, we explored the effect of Hsp70 on hyperoxia-induced lung injury using neonatal rats. Neonatal Wistar rats were delivered naturally at full term of gestation and were then pooled and randomly assigned to several groups to receive heat stimulation (41°C for 20 min) or room temperature conditions. The Hsp70 group received recombinant Hsp70 intraperitoneally (200 µg/kg, daily). All newborn rats were placed under hyperoxic conditions (85% oxygen) for 21 days. Survival rates in both heat-hyperoxia and Hsp70-hyperoxia groups were higher than those in the hyperoxia group (p < 0.05). Both endogenous and exogenous Hsp70 could reduce early apoptosis of alveolar cells under hyperoxia. Additionally, there were less macrophage infiltration in the lung of the Hsp70 groups (p < 0.05). Heat stress, heat shock proteins, and exogenous recombinant Hsp70 significantly increased the survival rate and reduced pathological hyperoxia induced lung injuries in the development of BPD. These results suggest that treating hyperoxia-induced lung injury with Hsp70 may reduce the risk of developing BPD.

Advanced Detection Method for Dengue NS1 Protein Using Ultrasensitive ELISA with Thio-NAD Cycling.

Dengue fever, a mosquito-borne disease in tropical and subtropical climates caused by the dengue virus (DENV), has become a major social and economic burden in recent years. However, current primary detection methods are inadequate for early diagnosis of DENV because they are either time-consuming, expensive, or require training. Non-structural protein 1 (NS1) is secreted during DENV infection and is thus considered a suitable biomarker for the development of an early detection method. In the present study, we developed a detection method for the NS1 protein based on a previously reported thio-NAD cycling ELISA (i.e., ultrasensitive ELISA) and successfully achieved a LOD of 1.152 pg/mL. The clinical diagnosis potential of the detection system was also evaluated by using 85 patient specimens, inclusive of 60 DENV-positive and 25 DENV-negative specimens confirmed by the NAAT method. The results revealed 98.3% (59/60) sensitivity and 100% (25/25) specificity, which was in almost perfect agreement with the NAAT data with a kappa coefficient of 0.972. The present study demonstrates the diagnostic potential of using an ultrasensitive ELISA as a low-cost, easy-to-use method for the detection of DENV compared with NAAT and could be of great benefit in low-income countries.

Perception of medical care systems and stress responses in preschoolers' caregivers at a pediatric emergency department in Taiwan.

OBJECTIVES: This study was undertaken to understand caregivers' perception of medical care systems and their stress responses throughout their stay with preschoolers at a pediatric emergency department (ED). METHODS: Overall, 201 preschoolers' caregivers in pediatric ED were recruited in this study. They were asked to answer self-made questionnaires regarding the perception of medical care systems and their stress responses immediately before preschoolers' release. RESULTS: The results show that caregivers with a low education or low income level were prone to exhibit greater appreciation for the efficiency of medical care systems and greater degree of anxiety for their lack of treatment and care knowledge than those of caregivers with a high education or high income level. Interestingly, caregivers older than 38 years reported greater emotional responses and physical exhaustion than did younger caregivers. Immigrant caregivers had higher emotional reaction and lower concentration than did Taiwanese caregivers. Paradoxically, caregivers undergoing over 3-time pediatric ED visits for observation expressed stronger stress reactions as compared with caregivers with less observation experiences. Not surprisingly, caregivers reported the highest emotional responses when their preschoolers were diagnosed as having very urgent degree in triage classification. Finally, caregivers' perception of "lack of family support" and "lack of treatment and care knowledge" correlated positively with all aspects of the stress responses. CONCLUSIONS: These results indicate that attention should be paid to the specific psychological weakness and need for the caregivers with certain demographic characteristics by the medical team in pediatric ED.

Overlap syndrome in a 12-year-old girl with systemic lupus erythematosus and anti-oj antibody-positive polymyositis: a case report.

BACKGROUND: The peculiar presentation of overlap syndrome in children makes precise diagnosis difficult. Children with overlap syndrome may or may not have specific antibodies. We present the case of a 12-year-old girl diagnosed with overlap syndrome of systemic lupus erythematosus (SLE) and juvenile polymyositis (JPM) who tested positive for anti-OJ antibodies. CASE PRESENTATION: We describe the case of a 12-year-old girl diagnosed with SLE at the age of 7 and presented with fever with malar rash, periungual erythema, generalized weakness, and multiple joint pain at admission. The patient had persistent joint pain and weakness after intravenous methylprednisolone administration and complained of an inability to walk with a positive test for Gower's sign one week after admission, accompanied by elevated alanine aminotransferase (ALT) and creatine-phospho-kinase (CPK) levels. The results of nerve conduction velocity test were normal. Electromyography revealed abundant spontaneous activity and myopathic motor unit action potentials in the right deltoid, biceps, and iliopsoas, in addition to fibrillation and mild myopathic motor unit action potentials in the right rectus femoris muscle. Magnetic resonance imaging revealed diffusely increased signal intensities in the myofascial planes of the bilateral iliopsoas, gluteus, obturator, pectineus, and hamstring muscles. Anti-nuclear antibody, anti-RNP, and rheumatoid factor IgG tests were positive, and inflammatory myopathy autoantibodies revealed anti-OJ antibody positivity, which strongly indicated autoimmune myositis. High-resolution computed tomography of the lung revealed mild pericardial effusion without any evidence of interstitial lung disease. We initiated intravenous pulses of methylprednisolone treatment, followed by cyclosporine, mycophenolate mofetil, and oral steroids. Clinical improvement with a delayed, slowly reduced CPK level after the above treatment and she was discharged after the 18th day of hospitalization. CONCLUSION: Overlap syndrome with inflammatory myositis can occur years later in pediatric SLE cases. We should be alert when patients with SLE develop a new presentation characterized by decreased SLE-specific autoantibody titers, positive anti-RNP antibodies, and elevated CPK. Treatment of the overlap syndrome of SLE and JPM is individualized, and the course differs between pediatric and adult patients.

Imiquimod-induced ROS production causes lysosomal membrane permeabilization and activates caspase-8-mediated apoptosis in skin cancer cells.

BACKGROUND: Lysosomal cell death is induced by lysosomal membrane permeabilization (LMP) and the subsequent release of lysosomal proteolytic enzymes, including cathepsins (CTSs), which results in mitochondrial dysfunction and apoptosis. Imiquimod (IMQ), a synthetic TLR7 ligand, has both antiviral and antitumor activity against various skin malignancies in clinical treatment. Previously, we demonstrated IMQ not only caused lysosomal dysfunction but also triggered lysosome biogenesis to achieve lysosomal adaptation in cancer cells. OBJECTIVE: To determine whether lysosomes are involved in IMQ-induced apoptosis. METHODS: The human skin cancer cell lines BCC, A375 and mouse melanoma cell line B16F10 were used in all experiments. Cell death was determined by the Cell Counting Kit-8 (CCK-8) assay and DNA content assay. Protein expression was determined by immunoblotting. Caspase-8 activity was assessed using a fluorescence caspase-8 kit and determined by flow cytometry and confocal microscopy. RESULTS: IMQ not only induced lysosome damage but also abrogated lysosome function in skin cancer cells. IMQ-induced caspase-8 activation contributed to the processes of lysosomal cell death. Moreover, the use of ROS scavengers significantly abolished caspase-8 activation and inhibited IMQ-induced LMP. Additionally, pharmacological inhibition of CTSD not only abrogated caspase-8 activation but also rescued IMQ-induced cell death. Finally, lysosome-alkalizing agents enhanced the cytotoxicity of IMQ in vitro and in vivo. CONCLUSIONS: IMQ-induced ROS accumulation promotes LMP, releases CTSs into the cytosol, stimulates caspase-8 activation and finally causes lysosomal cell death. Lysosomal cell death and the CTSD/caspase-8 axis may play a crucial role in IMQ-induced cell death.

Small Airway Dysfunction Measured by Impulse Oscillometry and Fractional Exhaled Nitric Oxide Is Associated With Asthma Control in Children.

Background: Impulse oscillometry (IOS) and fractional exhaled nitric oxide (FeNO) are sensitive and non-invasive methods to measure airway resistance and inflammation, although there are limited population-based studies using IOS and FeNO to predict asthma control. Objective: This study aimed to investigate the utility of IOS and FeNO for assessing childhood asthma control in terms of small airway dysfunction and airway inflammation. Methods: This prospective observational cohort study enrolled 5,018 school children (aged 6-12 years), including 560 asthmatic children and 140 normal participants. FeNO, spirometry, IOS, bronchial dilation test, total IgE, and childhood asthma control test (C-ACT) were measured. FeNO, IOS, spirometry, and C-ACT results were correlated with childhood asthma with and without control. Results: Uncontrolled asthmatic children had abnormal FeNO, IOS, and spirometric values compared with control subjects (P < 0.05). IOS parameters with R5, R5-R20, X5, Ax, △R5, and FeNO can predict lower C-ACT scales by the areas under receiver operating characteristic curves (AUCs) (0.616, 0.625, 0.609, 0.622, 0.625, and 0.714). A combination of FeNO (>20 ppb) with IOS measure significantly increased the specificity for predicting uncontrolled asthma patients compared with FeNO alone (P < 0.01). A multiple regression model showed that small airway parameter (R5-R20) was the strongest risk factor [OR (95% CI): 87.26 (7.67-993.31)] for uncontrolled asthma patients. Poor control with lower C-ACT scales correlated with high FeNO (r = -0.394), R5 (r = -0.106), and R5-R20 (r = -0.129) in asthmatic children (P < 0.05). Conclusion: A combined use of FeNO and IOS measurements strongly predicts childhood asthma with or without control.

Imiquimod Accelerated Antitumor Response by Targeting Lysosome Adaptation in Skin Cancer Cells.

Lysosomal adaptation is a cellular physiological process in which the number and function of lysosomes are regulated at the transcriptional and post-transcriptional levels in response to extracellular and/or intracellular cues or lysosomal damage. Imiquimod (IMQ), a synthetic toll-like receptor 7 ligand with hydrophobic and weak basic properties, exhibits both antitumor and antiviral activity against various skin malignancies as a clinical treatment. Interestingly, IMQ has been suggested to be highly concentrated in the lysosomes of plasmacytoid dendritic cells, indicating that IMQ could modulate lysosome function after sequestration in the lysosome. In this study, we found that IMQ not only induced lysosomal membrane permeabilization and dysfunction but also increased lysosome biogenesis to achieve lysosomal adaptation in cancer cells. IMQ-induced ROS production but not lysosomal sequestration of IMQ was the major cause of lysosomal adaptation. Moreover, IMQ-induced lysosomal adaptation occurred through lysosomal calcium ion release and activation of the calcineurin/TFEB axis to promote lysosome biogenesis. Finally, depletion of TFEB sensitized skin cancer cells to IMQ-induced apoptosis in vitro and in vivo. In summary, a disruption of lysosomal adaptation might represent a therapeutic strategy for synergistically enhancing the cytotoxicity of IMQ in skin cancer cells.

Imiquimod-induced ROS production disrupts the balance of mitochondrial dynamics and increases mitophagy in skin cancer cells.

BACKGROUND: Mitochondrial homeostasis is a highly dynamic process involving continuous fission and fusion cycles and mitophagy to maintain mitochondrial functionality. Imiquimod (IMQ), a Toll-like receptor (TLR) 7 ligand, is used to treat various skin malignancies. IMQ also induces apoptotic and autophagic cell death in various cancers through a TLR7-independent pathway. OBJECTIVE: To investigate whether IMQ-induced ROS production is involved in mitochondrial dysfunction, mitochondrial fragmentation and mitophagy in skin cancer cells. METHODS: BCC/KMC-1, B16F10 and A375 skin cancer cells, AGS gastric cancer cells and primary human keratinocytes were treated with 50 µg/mL IMQ. After 4 h, ROS were detected by CM-H2DCFDA, DHE, and MitoSOX Red staining. After 24 h, cell viability and the mitochondrial membrane potential were evaluated by a CCK-8 assay and JC-1 staining, respectively. Oxygen consumption was assessed with an Oroboros instrument. Mitochondrial morphology and mitophagy were evaluated by MitoTracker and LysoTracker staining. Mitochondrial dynamics markers, including MFN-1, DRP-1 and OPA1, and mitophagy markers, including LC3, S65-phosphorylated ubiquitin, PINK1 and TOM20, were detected by immunoblotting. RESULTS: IMQ not only induced severe ROS production but also resulted in increased mitochondrial membrane potential loss, mitochondrial fission and mitophagy and decreased oxygen consumption in skin cancer cells compared with normal keratinocytes. Pretreatment with the antioxidant NAC reduced IMQ-induced ROS production and attenuated IMQ-induced mitochondrial fission and mitophagy in skin cancer cells. CONCLUSIONS: IMQ-induced ROS might be associated with mitochondrial dysfunction, mitochondrial fission and mitophagy in cancer cells. Alleviating IMQ-induced ROS production would reduce mitochondrial fission-to-fusion skewing and further reduce IMQ-induced mitophagy.

Imiquimod Exerts Antitumor Effects by Inducing Immunogenic Cell Death and Is Enhanced by the Glycolytic Inhibitor 2-Deoxyglucose.

The induction of immunogenic cell death (ICD) in cancer cells triggers specific immune responses against the same cancer cells. Imiquimod (IMQ) is a synthetic ligand of toll-like receptor 7 that exerts antitumor activity by stimulating cell-mediated immunity or by directly inducing apoptosis. Whether IMQ causes tumors to undergo ICD and elicits a specific antitumor immune response is unknown. We demonstrated that IMQ-induced ICD-associated features, including the surface exposure of calreticulin and the secretion of adenosine triphosphate and HMGB1, were mediated by ROS and endoplasmic reticulum stress. In a B16F10 melanoma mouse model, vaccinating mice with IMQ-induced ICD cell lysate or directly injecting IMQ in situ reduced tumor growth that was mediated by inducing tumor-specific T-cell proliferation, promoting tumor-specific cytotoxic killing by CD8+ T cells, and increasing the infiltration of various immune cells into tumor lesions. The ICD-associated features were crucial in the induction of specific antitumor immunity in vivo. The glycolytic inhibitor 2-deoxyglucose enhanced IMQ-induced ICD-associated features and strengthened the antitumor immunity mediated by IMQ-induced ICD cell lysate in p53-mutant cancer cells, which were IMQ-resistant in vitro. We conclude that IMQ is an authentic ICD inducer and provide a concept connecting IMQ-induced cancer cell death and antitumor immune responses.

Subcutaneous injection of recombinant heat shock protein 70 ameliorates atopic dermatitis skin lesions in a mouse model.

Atopic dermatitis (AD) is a chronic inflammatory skin disease and sometimes is a tough challenge for physicians. We previously reported that in Th2 environment, the production and secretion of thymic stromal lymphopoietin (TSLP) from human keratinocytes was inhibited by recombinant heat shock protein 70 (rHSP70). The present study assessed the therapeutic effectiveness of rHSP70 in a mouse model of AD. An experimental model of AD was reproduced by systemic sensitization and local epicutaneous challenge with ovalbumin (OVA). Treatment of rHSP70 was performed by subcutaneous administration. The levels of OVA-specific IgE, as well as cytokines, were detected by ELISA. Skin samples from patch areas were also taken for histologic examination. Injection of rHSP70 improved the histologic picture by reducing the thickness of epidermis and allergic inflammation. Skin sonography revealed rHSP70 ameliorated skin remodeling. rHSP70 also significantly decreased the protein expression of TSLP of skin from patch areas. Furthermore, in ex vivo studies also showed group of rHSP70 treatment decreased IL-13, RANTES, MIP-1ß and increased IFN-γ secreted from splenocytes stimulated with OVA. The rHSP70 intervention in the mouse model of AD reduced the skin expression of TSLP and attenuated the clinical appearance of OVA-induced AD mice. The effect was achieved by suppressed Th2 immune response in injected skin tissue and enhanced systemic Th1 immune response. These results suggest that rHSP70 have potential as a promising protein for the treatment of AD.

Effects of vitamin B-6 supplementation on oxidative stress and inflammatory response in neonatal rats receiving hyperoxia therapy.

Hyperoxia is often used in the treatment of neonates. However, protracted use of hyperoxia leads to significant morbidity. The purpose of this study was to evaluate the effects of vitamin B-6 supplementation on oxidative stress and inflammatory responses in neonatal rats undergoing hyperoxia therapy. The study consisted of 2 parts: a survival study and a vitamin B-6 efficacy study for 16 days. Neonatal rats were randomly divided into either the control group, B-6 group (subcutaneously injected with 90 mg/kg/d of pyridoxal 5'-phosphate [PLP]), O2 group (treated with 85% oxygen), or O2 + B-6 group (simultaneously treated with 85% oxygen and 90 mg/kg/d PLP). After the survival study was done, the vitamin B-6 efficacy study was performed with duplicate neonatal rats sacrificed on the 3rd, 6th, 9th, and 16th day. Serum inflammatory cytokines, tissue pathology, and malondialdehyde (MDA) levels were measured. In the survival study, the survival rate of neonatal rats in the control, B-6, O2, and O2 + B-6 group on the 16th day were 100%, 100%, 25%, and 62.50%, respectively. The efficacy study showed lung polymorphonuclear granulocyte (PMN) and macrophage infiltration, increased liver hemopoiesis, and higher MDA levels in liver homogenates at days 3 through 16 in the O2 group. Vitamin B-6 supplementation considerably increased serum inflammatory cytokines in either the 6th or 9th day and decreased liver MDA level before the 6th day. These results indicate that neonatal rats receiving hyperoxia treatment suffered divergent serum inflammatory responses and were in increased liver oxidative stress. Vitamin B-6 supplementation seemed to improve survival rates, change systemic inflammatory response, and decrease liver oxidative stress while neonatal rats were under hyperoxia treatment.

Role of new population of peripheral CD11c(+)CD8(+) T cells and CD4(+)CD25(+) regulatory T cells during acute and remission stages in rheumatoid arthritis patients.

BACKGROUND AND PURPOSE: Rheumatoid arthritis (RA) is a CD4(+)-dependent chronic systemic inflammatory disease with autoimmune features. Autoreactive CD4(+) T-cell activation can result in autoimmune diseases. One of the key regulators is the CD4(+)CD25(high) regulatory T (Treg) cell. In an animal arthritis model, CD11c(+)CD8(+) T cells were found to be elevated, and could suppress pathogenic CD4(+) T cells after cross-linking with CD137. The purpose of this study was to compare the expression of CD137, CD4(+)CD25(high) Treg cells, and CD11c(+)CD8(+) in the peripheral blood T lymphocytes of RA patients during active and remissive states, and evaluate the correlation with disease activity. METHODS: Thirty nine RA patients treated at the rheumatology outpatient clinic at the Changhua Christian Hospital were assessed clinically for disease activity and classified as either highly active or remissive by the Disease Activity Score 28. Peripheral blood mononuclear cells were isolated from these patients and compared against normal controls. RESULTS: The presence of CD11c(+)CD8(+) T cells or the expression of CD137 molecules in peripheral blood cells was not related to disease activity. In contrast, CD4(+)CD25(high) Treg cell levels were increased significantly in patients with active RA compared with patients with remissive RA or controls (p<0.05). These lymphocytes were intact, without evidence of apoptosis. CONCLUSIONS: Our results indicate that CD4(+)CD25(high) Treg cells play an important role in modulating RA disease activity and can serve as a parameter of disease activity.