Association Between Gut Regulatory Hormones and Post-operative Weight Loss Following Gastrectomy in Patients With Gastric Cancer.

Background/Aims: Post-operative weight loss in patients with gastric cancer lead to a poor quality of life and long-term survival. This study aims to evaluate the effects of gut regulatory hormones on post-operative weight loss in patients with subtotal gastrectomy for gastric cancer. Methods: This prospective study was conducted for 12 months post-surgery in 14 controls and 13 gastrectomy patients who underwent subtotal gastrectomy for gastric cancer. Serum plasma ghrelin, glucagon-like peptide-1, gastric inhibitory peptide-1, peptide YY, insulin, and homeostatic model assessment for insulin resistance responses to a standardized test meal were recorded at multiple time points before and after gastrectomy at 4 and 12 months. Results: The mean weight difference between the pre-operative state and the 4-month period was significantly reduced to 6.6 kg (P = 0.032), but significant weight reduction was not observed from 4 months to 12 months. The plasma levels of glucagon-like peptide-1, gastric inhibitory peptide-1, and peptide YY were significantly increased 4 months postoperatively compared to the pre-operative state (all P = 0.035); however, pre-operative levels and relative changes over a period of 0-4 months of hormones were not correlated with body weight changes. Only the pre-operative ghrelin at peak had a negative correlation with changes in weight reduction in the 4 months after surgery (ρ = -0.8, P = 0.024). Conclusions: Significant weight reduction was common after subtotal gastrectomy for gastric cancer with a negative correlation pre-operative plasma ghrelin levels. Incretin hormones are modestly but significantly increased after subtotal gastrectomy; however, these changes did not affect the weight changes.

Hyperoxygenation Ameliorates Stress-induced Neuronal and Behavioral Deficits.

Hyperoxygenation therapy remediates neuronal injury and improves cognitive function in various animal models. In the present study, the optimal conditions for hyperoxygenation treatment of stress-induced maladaptive changes were investigated. Mice exposed to chronic restraint stress (CRST) produce persistent adaptive changes in genomic responses and exhibit depressive-like behaviors. Hyperoxygenation treatment with 100% O2 (HO2) at 2.0 atmospheres absolute (ATA) for 1 h daily for 14 days in CRST mice produces an antidepressive effect similar to that of the antidepressant imipramine. In contrast, HO2 treatment at 2.0 ATA for 1 h daily for shorter duration (3, 5, or 7 days), HO2 treatment at 1.5 ATA for 1 h daily for 14 days, or hyperbaric air treatment at 2.0 ATA (42% O2) for 1 h daily for 14 days is ineffective or less effective, indicating that repeated sufficient hyperoxygenation conditions are required to reverse stress-induced maladaptive changes. HO2 treatment at 2.0 ATA for 14 days restores stress-induced reductions in levels of mitochondrial copy number, stress-induced attenuation of synaptophysin-stained density of axon terminals and MAP-2-staining dendritic processes of pyramidal neurons in the hippocampus, and stress-induced reduced hippocampal neurogenesis. These results suggest that HO2 treatment at 2.0 ATA for 14 days is effective to ameliorate stress-induced neuronal and behavioral deficits.

Deficiency of glutathione peroxidase-1 and catalase attenuated diet-induced obesity and associated metabolic disorders.

AIMS: Oxidative stress has been considered to contribute to the development of obesity-related metabolic disorders including insulin resistance. To the contrary, deficiency of an anti-oxidizing enzyme, glutathione peroxidase (GPx)-1, was reported to enhance insulin signaling, suggesting that oxidative stress may inhibit the development of type 2 diabetes. However, the beneficial effects of the absence of GPx-1 in metabolic homeostasis, including body weight control, have not yet been clearly manifested. To clarify the relationship between oxidative stress and obesity-related metabolic disorders, we investigated another mouse deficient with both GPx-1 and catalase (Cat). METHODS: C57BL/6J wild-type and GPx-1-/- × Cat-/- mice were fed with a high-fat diet (60% fat) or a normal chow diet for 16 weeks and were investigated for metabolic and histological studies. RESULTS: Body weight gain was significantly reduced, and glucose metabolism as well as hepatic steatosis was obviously improved in the GPx-1-/- × Cat-/- mice. The serum levels of insulin and total cholesterol were also significantly lowered. For the underlying mechanism, inflammation was attenuated and expression of markers for fat browning was enhanced in the visceral white adipose tissues. CONCLUSIONS: Oxidative stress due to deficiency of GPx-1 and Cat may improve obesity-related metabolic disorders through attenuation of inflammation and fat browning.

FAK-Copy-Gain Is a Predictive Marker for Sensitivity to FAK Inhibition in Breast Cancer.

BACKGROUND: Cancers with copy-gain drug-target genes are excellent candidates for targeted therapy. In order to search for new predictive marker genes, we investigated the correlation between sensitivity to targeted drugs and the copy gain of candidate target genes in NCI-60 cells. METHODS: For eight candidate genes showing copy gains in NCI-60 cells identified in our previous study, sensitivity to corresponding target drugs was tested on cells showing copy gains of the candidate genes. RESULTS: Breast cancer cells with Focal Adhesion Kinase (FAK)-copy-gain showed a significantly higher sensitivity to the target inhibitor, FAK inhibitor 14 (F14). In addition, treatment of F14 or FAK-knockdown showed a specific apoptotic effect only in breast cancer cells showing FAK-copy-gain. Expression-profiling analyses on inducible FAK shRNA-transfected cells showed that FAK/AKT signaling might be important to the apoptotic effect by target inhibition. An animal experiment employing a mouse xenograft model also showed a significant growth-inhibitory effect of F14 on breast cancer cells showing FAK-copy-gain, but not on those without FAK-copy-gain. CONCLUSION: FAK-copy-gain may be a predictive marker for FAK inhibition therapy in breast cancer.

Hyperoxygenation revitalizes Alzheimer's disease pathology through the upregulation of neurotrophic factors.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by Aß-induced pathology and progressive cognitive decline. The incidence of AD is growing globally, yet a prompt and effective remedy is not available. Aging is the greatest risk factor for AD. Brain aging proceeds with reduced vascularization, which can cause low oxygen (O2 ) availability. Accordingly, the question may be raised whether O2 availability in the brain affects AD pathology. We found that Tg-APP/PS1 mice treated with 100% O2 at increased atmospheric pressure in a chamber exhibited markedly reduced Aß accumulation and hippocampal neuritic atrophy, increased hippocampal neurogenesis, and profoundly improved the cognitive deficits on the multiple behavioral test paradigms. Hyperoxygenation treatment increased the expression of BDNF, NT3, and NT4/5 through the upregulation of MeCP2/p-CREB activity in HT22 cells in vitro and in the hippocampus of mice. In contrast, siRNA-mediated inhibition of MeCP2 or TrkB neurotrophin receptors in the hippocampal subregion, which suppresses neurotrophin expression and neurotrophin action, respectively, blocked the therapeutic effects of hyperoxygenation on the cognitive impairments of Tg-APP/PS1 mice. Our results highlight the importance of the O2 -related mechanisms in AD pathology, which can be revitalized by hyperoxygenation treatment, and the therapeutic potential of hyperoxygenation for AD.

Reactive oxygen species-induced parthanatos of immunocytes by human cytomegalovirus-associated substance.

Previous studies have examined various immune evasion strategies of human cytomegalovirus (HCMV) to gain understanding of its pathogenesis. Although the mechanism that underlies immunocyte destruction near HCMV-infected lesions has yet to be established, it is here shown that substances produced by HCMV-infected cells induce death in several types of immunocytes, but not in fibroblasts or astrocytomas. These substances contain HCMV proteins and were termed HCMV-associated insoluble substance (HCMVAIS). The mechanism by which HCMVAIS induces cell death was characterized to improve understanding the death of immunocytes near HCMV-infected lesions. HCMVAIS were found to trigger production of intracellular nicotinamide adenine dinucleotide phosphate oxidase-derived reactive oxygen species (ROS), resulting in cell death, this effect being reversed following treatment with ROS inhibitors. Cell death was not induced in splenocytes from NOX-2 knockout mice. It was hypothesized that DNA damage induced by oxidative stress initiates poly ADP-ribose polymerase-1 (PARP-1)-mediated cell death, or parthanatos. HCMVAIS-induced cell death is accompanied by PARP-1 activation in a caspase-independent manner, nuclear translocation of apoptosis-inducing factor (AIF), and DNA fragmentation, which are typical features of parthanatos. Treatment with an AIF inhibitor decreased the rate of HCMVAIS-induced cell death, this being confirmed by hematoxylin and eosin staining; cell death in most HCMV-positive foci in serial section samples of a large intestine with HCMV infection was TUNEL-positive, cleaved caspase 3-negative and CD45-positive. Taken together, these data suggest that HCMV inhibits local immune responses via direct killing of immunocytes near HCMV-infected cells through ROS-induced parthanatos by HCMVAIS.

Rapid Assessment of Microbiota Changes in Individuals with Autism Spectrum Disorder Using Bacteria-derived Membrane Vesicles in Urine.

Individuals with autism spectrum disorder (ASD) have altered gut microbiota, which appears to regulate ASD symptoms via gut microbiota-brain interactions. Rapid assessment of gut microbiota profiles in ASD individuals in varying physiological contexts is important to understanding the role of the microbiota in regulating ASD symptoms. Microbiomes secrete extracellular membrane vesicles (EVs) to communicate with host cells and secreted EVs are widely distributed throughout the body including the blood and urine. In the present study, we investigated whether bacteria-derived EVs in urine are useful for the metagenome analysis of microbiota in ASD individuals. To address this, bacterial DNA was isolated from bacteria-derived EVs in the urine of ASD individuals. Subsequent metagenome analysis indicated markedly altered microbiota profiles at the levels of the phylum, class, order, family, and genus in ASD individuals relative to control subjects. Microbiota identified from urine EVs included gut microbiota reported in previous studies and their up- and down-regulation in ASD individuals were partially consistent with microbiota profiles previously assessed from ASD fecal samples. However, overall microbiota profiles identified in the present study represented a distinctive microbiota landscape for ASD. Particularly, the occupancy of g_Pseudomonas, g_Sphingomonas, g_Agrobacterium, g_Achromobacter, and g_Roseateles decreased in ASD, whereas g_Streptococcus, g_Akkermansia, g_Rhodococcus, and g_Halomonas increased. These results demonstrate distinctively altered gut microbiota profiles in ASD, and validate the utilization of urine EVs for the rapid assessment of microbiota in ASD.

Exogenous Hydrogen Peroxide Induces Lipid Raft-Mediated STAT-6 Activation in T Cells.

BACKGROUND/AIMS: CD4+ T cells are a critical component of the adaptive immune response. While the mechanisms controlling the differentiation of the Th1, Th17, and regulatory T cell subsets from naïve CD4+ T cells are well described, the factors that induce Th2 differentiation are still largely unknown. METHODS: The effects of treatment with exogenous H2O2 on STAT-6 phosphorylation and activation in T cells were examined by immunoblotting, immunofluorescence and gel shift assay. Anti-CD3 antibody and methyl-ß-cyclodextrin were utilized to induce lipid raft assembly and to investigate the involvement of lipid rafts, respectively. RESULTS: Jurkat and EL-4 T cells that were exposed to H2O2 showed rapid and strong STAT-6 phosphorylation, and the extent of STAT-6 phosphorylation was enhanced by co-treatment with anti-CD3 antibody. The effect of H2O2 on STAT-6 phosphorylation and translocation was inhibited by disruption of lipid rafts. STAT-6 activation in response to H2O2 treatment regulated IL-4 gene expression, and this response was strengthened by treatment with anti-CD3. CONCLUSION: Our results indicate that reactive oxygen species such as H2O2 can act on upstream and initiating factors for activation of STAT-6 in T cells and contribute to formation of a positive feedback loop between STAT-6 and IL-4 in the Th2 differentiation process.

Attenuation of collagen-induced arthritis by hyperbaric oxygen therapy through altering immune balance in favor of regulatory T cells.

Hyperbaric oxygen (HBO2) therapy is currently used for the treatment of chronic wounds, radiation-induced soft tissue necrosis, several oxygen-deficiency conditions and decompression sickness. In addition to the current indications, much empirical and experimental data suggest that HBO2 therapy may benefit autoimmune diseases by suppressing immunity, but the underlying mechanism is not well understood. Therefore, in the present study, we investigated whether HBO2 prevents the development of collagen-induced arthritis (CIA) in association with alteration of the immune balance between pro-inflammatory Th17 and anti-inflammatory regulatory T cells (Tregs). Arthritis was induced in DBA/1 mice by intradermal injection of type II collagen. Animals received either no treatment or 90 minutes of HBO2 (100% oxygen, at 2.0 ATA) daily beginning three days prior to the injection and were monitored for the development of arthritis. Six weeks later, joint tissues and spleens were analyzed for the alteration of immune balance between Th17 and Tregs by immunohistochemistry (IHC) or Western blot. Injection of collagen-induced extensive arthritis and extramedullary hematopoiesis in the spleens. Meanwhile, joint swelling and inflammatory tissue damages as well as extramedullary hematopoiesis were significantly less severe in the mice treated with HBO2. Both IHC and Western blot showed a decrease of FOXP3 and an increase of pSTAT3 expressions in the joints and spleens of the mice injected with collagen. This suggested that the systemic immune balance was biased toward Th17 cells, which was reversed by HBO2 therapy. These results suggested acute CIA associated with an immune balance favoring Th17 was attenuated by HBO2 in parallel with restoration of the immune balance to favor Tregs.

Isolation and Characterization of Vaccine Candidate Genes Including CSP and MSP1 in Plasmodium yoelii.

Malaria is an infectious disease affecting humans, which is transmitted by the bite of Anopheles mosquitoes harboring sporozoites of parasitic protozoans belonging to the genus Plasmodium. Despite past achievements to control the protozoan disease, malaria still remains a significant health threat up to now. In this study, we cloned and characterized the full-unit Plasmodium yoelii genes encoding merozoite surface protein 1 (MSP1), circumsporozoite protein (CSP), and Duffy-binding protein (DBP), each of which can be applied for investigations to obtain potent protective vaccines in the rodent malaria model, due to their specific expression patterns during the parasite life cycle. Recombinant fragments corresponding to the middle and C-terminal regions of PyMSP1 and PyCSP, respectively, displayed strong reactivity against P. yoelii-infected mice sera. Specific native antigens invoking strong humoral immune response during the primary and secondary infections of P. yoelii were also abundantly detected in experimental ICR mice. The low or negligible parasitemia observed in the secondary infected mice was likely to result from the neutralizing action of the protective antibodies. Identification of these antigenic proteins might provide the necessary information and means to characterize additional vaccine candidate antigens, selected solely on their ability to produce the protective antibodies.

Gut-Specific Delivery of T-Helper 17 Cells Reduces Obesity and Insulin Resistance in Mice.

BACKGROUND & AIMS: Obesity and metabolic syndrome have been associated with alterations to the intestinal microbiota. However, few studies examined the effects of obesity on the intestinal immune system. We investigated changes in subsets of intestinal CD4+ T-helper (TH) cells with obesity and the effects of gut-tropic TH17 cells in mice on a high-fat diet (HFD). METHODS: We isolated immune cells from small intestine and adipose tissue of C57BL/6 mice fed a normal chow diet or a HFD for 10 weeks and analyzed the cells by flow cytometry. Mice fed a vitamin A-deficient HFD were compared with mice fed a vitamin A-sufficient HFD. Obese RAG1-deficient mice were given injections of only regulatory T cells or a combination of regulatory T cells and TH17 cells (wild type or deficient in integrin ß7 subunit or interleukin 17 [IL17]). Mice were examined for weight gain, fat mass, fatty liver, glucose tolerance, and insulin resistance. Fecal samples were collected before and after T cell transfer and analyzed for microbiota composition by metagenomic DNA sequencing and quantitative polymerase chain reaction. RESULTS: Mice placed on a HFD became obese, which affected the distribution of small intestinal CD4+ TH cells. Intestinal tissues from obese mice had significant reductions in the proportion of TH17 cells but increased proportion of TH1 cells, compared with intestinal tissues from nonobese mice. Depletion of vitamin A in obese mice further reduced the proportion of TH17 cells in small intestine; this reduction correlated with more weight gain and worsening of glucose intolerance and insulin resistance. Adoptive transfer of in vitro-differentiated gut-tropic TH17 cells to obese mice reduced these metabolic defects, which required the integrin ß7 subunit and IL17. Delivery of TH17 cells to intestines of mice led to expansion of commensal microbes associated with leanness. CONCLUSIONS: In mice, intestinal TH17 cells contribute to development of a microbiota that maintains metabolic homeostasis, via IL17. Gut-homing TH17 cells might be used to reduce metabolic disorders in obese individuals.

Inflammatory hypoxia induces syndecan-2 expression through IL-1ß-mediated FOXO3a activation in colonic epithelia.

Chronic inflammation is known to be a key causative factor in tumor progression, but we do not yet fully understand the molecular mechanism through which inflammation leads to cancer. Here, we report that the dextran sulfate sodium (DSS)-induced mouse model of chronic colitis is associated with increases in the serum level of IL-1ß and the colonic epithelial expression of the cell-surface heparan sulfate proteoglycan, syndecan-2. We further show that IL-1ß stimulated the transcription of syndecan-2 via NF-κB-dependent FOXO3a activation in CCD841CoN normal colonic epithelial cells and early-stage HT29 colon cancer cells. Inflammatory hypoxia was observed in the colonic epithelia of mice with chronic colitis, suggesting that hypoxic stress is involved in the regulation of syndecan-2 expression. Consistently, experimental inflammatory hypoxia induced hypoxia inducible factor-1α-dependent FOXO3a expression and the p38 MAPK-mediated nuclear localization of FOXO3a. FOXO3a directly mediated syndecan-2 expression in both cell lines and the colonic epithelia of mice with DSS-induced colitis. Moreover, syndecan-2 expression was detected in azoxymethane/DSS-induced colon tumors. Together, these data demonstrate that inflammatory hypoxia up-regulates syndecan-2 via the IL-1ß-NF-κB-FOXO3a pathway. These findings provide new mechanistic insights into inflammatory hypoxia-mediated syndecan-2 expression to connect chronic inflammation and the development of colon cancer.-Choi, S., Chung, H., Hong, H., Kim, S. Y., Kim, S.-E., Seoh, J.-Y., Moon, C. M., Yang, E. G., Oh, E.-S. Inflammatory hypoxia induces syndecan-2 expression through IL-1ß-mediated FOXO3a activation in colonic epithelia.

Small intestinal eosinophils regulate Th17 cells by producing IL-1 receptor antagonist.

Eosinophils play proinflammatory roles in helminth infections and allergic diseases. Under steady-state conditions, eosinophils are abundantly found in the small intestinal lamina propria, but their physiological function is largely unexplored. In this study, we found that small intestinal eosinophils down-regulate Th17 cells. Th17 cells in the small intestine were markedly increased in the ΔdblGATA-1 mice lacking eosinophils, and an inverse correlation was observed between the number of eosinophils and that of Th17 cells in the small intestine of wild-type mice. In addition, small intestinal eosinophils suppressed the in vitro differentiation of Th17 cells, as well as IL-17 production by small intestinal CD4(+)T cells. Unlike other small intestinal immune cells or circulating eosinophils, we found that small intestinal eosinophils have a unique ability to constitutively secrete high levels of IL-1 receptor antagonist (IL-1Ra), a natural inhibitor of IL-1ß. Moreover, small intestinal eosinophils isolated from IL-1Ra-deficient mice failed to suppress Th17 cells. Collectively, our results demonstrate that small intestinal eosinophils play a pivotal role in the maintenance of intestinal homeostasis by regulating Th17 cells via production of IL-1Ra.

Extracellular vesicle-derived protein from Bifidobacterium longum alleviates food allergy through mast cell suppression.

BACKGROUND: The incidence of food allergies has increased dramatically during the last decade. Recently, probiotics have been studied for the prevention and treatment of allergic disease. OBJECTIVE: We examined whether Bifidobacterium longum KACC 91563 and Enterococcus faecalis KACC 91532 have the capacity to suppress food allergies. METHODS: B longum KACC 91563 and E faecalis KACC 91532 were administered to BALB/c wild-type mice, in which food allergy was induced by using ovalbumin and alum. Food allergy symptoms and various immune responses were assessed. RESULTS: B longum KACC 91563, but not E faecalis KACC 91532, alleviated food allergy symptoms. Extracellular vesicles of B longum KACC 91563 bound specifically to mast cells and induced apoptosis without affecting T-cell immune responses. Furthermore, injection of family 5 extracellular solute-binding protein, a main component of extracellular vesicles, into mice markedly reduced the occurrence of diarrhea in a mouse food allergy model. CONCLUSION: B longum KACC 91563 induces apoptosis of mast cells specifically and alleviates food allergy symptoms. Accordingly, B longum KACC 91563 and family 5 extracellular solute-binding protein exhibit potential as therapeutic approaches for food allergies.

The value of cytoplasmic Y-box-binding protein 1 as a prognostic marker for breast cancer in Korean.

BACKGROUND: The human Y-box-binding protein 1 (YB-1) is a member of the DNA/RNA-binding family of proteins that regulates transcription and translation of genes. Previous studies suggest that YB-1 may have an oncogenic role in various cancers. In this study, we evaluate the prognostic value of cytoplasmic YB-1 with respect to breast cancer. METHODS: Immunohistochemical staining study was performed with YB-1 using tissue block from 233 patients with invasive ductal carcinoma. Patients were divided into two groups according to expression of cytoplasmic YB-1 in tumor cell (high versus low). The relationship between the expression of YB-1, clinicopathological characteristics and breast cancer prognosis was analyzed. RESULTS: Hormone receptor negativity, worse histologic and nuclear grade, high tumor stage, lymphovascular invasion and high Ki67 (≥14 %) were related with the increased expression of cytoplasmic YB-1 in tumor cell (p < 0.05). Although there was no significant difference in relapse-free survival (RFS) between the two groups (p = 0.412), difference in overall survival (OS) was statistically significant (p = 0.035). In multivariate analysis for OS, YB-1 was an independent prognostic factor (p = 0.043). CONCLUSION: This suggests that the increased expression of cytoplasmic YB-1 in tumor cells can be regarded as an independent prognostic factor for breast cancer, related to poor prognostics. Expression of cytoplasmic YB-1 in cancer cell could be used as an independent prognostic marker for predicting OS in breast cancer.

Functional Modulation of Regulatory T Cells by IL-2.

The suppressive function of regulatory T cells (Tregs) is critical to the maintenance of immune homeostasis in vivo and yet, the specific identification of Tregs by phenotypic markers is not perfect. Tregs were originally identified in the CD4+CD25+ fraction of T cells, but FoxP3 expression was later included as an additional marker of Tregs as FoxP3 expression was identified as being critical to the development and function of these cells. Intracellular expression of FoxP3 makes it difficult in using to isolate live and not permeabilized cells for functional assays. As such CD4+CD25+ fraction is still frequently used for functional assays of Tregs. Although, the CD4+CD25+ fraction substantially overlaps with the FoxP3+ fraction, the minor mismatch between CD4+CD25+ and FoxP3+ fractions may confound the functional characteristics of Tregs. In this study, we isolated CD4+FoxP3+ as well as CD4+CD25+ fractions from Foxp3 knock-in mice, and compared their proliferative and suppressive activity in the presence or absence of various concentrations of IL-2. Our results showed comparable patterns of proliferative and suppressive responses for both fractions, except that contrary to the CD4+CD25+ fraction the FoxP3+ fraction did not proliferate in an autocrine fashion even in response to a strong stimulation. In presence of exogenous IL-2, both CD4+CD25+ and CD4+FoxP3+ fractions were more sensitive than the CD4+CD25- responder cells in proliferative responsiveness. In addition, a low dose IL-2 enhanced whereas a high dose abrogated the suppressive activities of the CD4+CD25+ and CD4+FoxP3+ fractions. These results may provide an additional understanding of the characteristics of the various fractions of isolated Tregs based on phenotype and function and the role of varying levels of exogenous IL-2 on the suppressive activity of these cells.

Phenotypic and functional analysis of HL-60 cells used in opsonophagocytic-killing assay for Streptococcus pneumoniae.

Differentiated HL-60 is an effector cell widely used for the opsonophagocytic-killing assay (OPKA) to measure efficacy of pneumococcal vaccines. We investigated the correlation between phenotypic expression of immunoreceptors and phagocytic ability of HL-60 cells differentiated with N,N-dimethylformamide (DMF), all-trans retinoic acid (ATRA), or 1α, 25-dihydroxyvitamin D3 (VitD3) for 5 days. Phenotypic change was examined by flow cytometry with specific antibodies to CD11c, CD14, CD18, CD32, and CD64. Apoptosis was determined by flow cytometry using 7-aminoactinomycin D. Function was evaluated by a standard OPKA against serotype 19F and chemiluminescence-based respiratory burst assay. The expression of CD11c and CD14 gradually increased upon exposure to all three agents, while CD14 expression increased abruptly after VitD3. The expression of CD18, CD32, and CD64 increased during differentiation with all three agents. Apoptosis remained less than 10% until day 3 but increased after differentiation by DMF or ATRA. Differentiation with ATRA or VitD3 increased the respiratory burst after day 4. DMF differentiation showed a high OPKA titer at day 1 which sustained thereafter while ATRA or VitD3-differentiated cells gradually increased. Pearson analysis between the phenotypic changes and OPKA titers suggests that CD11c might be a useful differentiation marker for HL-60 cells for use in pneumococcal OPKA.

Hyperoxygenation attenuated a murine model of atopic dermatitis through raising skin level of ROS.

Atopic dermatitis (AD) is a chronic inflammatory skin disease resulting from excessive stimulation of immune cells. Traditionally, reactive oxygen species (ROS) have been implicated in the progression of inflammatory diseases, but several opposing observations suggest the protective role of ROS in inflammatory disease. Recently, we demonstrated ROS prevented imiquimod-induced psoriatic dermatitis through enhancing regulatory T cell function. Thus, we hypothesized AD might also be attenuated in elevated levels of ROS through tissue hyperoxygenation, such as by hyperbaric oxygen therapy (HBOT) or applying an oxygen-carrying chemical, perfluorodecalin (PFD). Elevated levels of ROS in the skin have been demonstrated directly by staining with dihydroethidum as well as indirectly by immunohistochemistry (IHC) for indoleamine 2,3-dioxygenase (IDO). A murine model of AD was developed by repeated application of a chemical irritant (1% 2,4-dinitrochlorobenzene) and house dust mite (Dermatophagoide farinae) extract on one ear of BALB/c mice. The results showed treatment with HBOT or PFD significantly attenuated AD, comparably with 0.1% prednicarbate without any signs of side effects, such as telangiectasia. The expressions of interleukin-17A and interferon-γ were also decreased in the AD lesions by treatment with HBOT or PFD. Enhanced expression of IDO and reduced level of hypoxia-inducible factor-1α, in association with increased frequency of FoxP3+ regulatory T cells in the AD lesions, might be involved in the underlying mechanism of oxygen therapy. Taken together, it was suggested that tissue hyperoxygenation, by HBOT or treatment with PFD, might attenuate AD through enhancing skin ROS level.

CD137-inducing factors from T cells and macrophages accelerate the destabilization of atherosclerotic plaques in hyperlipidemic mice.

CD137 (4-1BB), a member of the tumor necrosis factor receptor superfamily, has been reported to be expressed in atherosclerotic plaques, and to promote lesion formation. However, the role of CD137 in mediating atherosclerotic plaque stability and the possible underlying molecular and cellular mechanisms are poorly understood. Here, apolipoprotein E-deficient (ApoE(-/-)) and CD137-deficient ApoE(-/-) (ApoE(-/-)CD137(-/-)) mice fed a chow diet for 66 wk were used. CD137 induces plaque instability, which is characterized by increased plaque necrosis, decreased collagen content, decreased vascular smooth muscle cell (VSMC) content, and increased macrophage infiltration. CD137 also increases the infiltration of effector T (Teff) cells into plaque lesion sites, resulting in increased interferon-γ (IFN-γ) expression. Interestingly, Teff-cell-derived IFN-γ inhibits collagen synthesis in atherosclerotic plaques. Furthermore, CD137 activation increases the apoptosis of VSMCs, possibly by decreasing the antiapoptotic regulator, Bcl-2, and subsequently up-regulating cleaved caspase-3. In macrophages, activation of CD137 signaling boosted the oxidized low density lipoprotein-induced expression of matrix metalloproteinase 9 via the p38 mitogen-activated protein kinase and extracellular signal-regulated kinase1/2 signaling pathways. In summary, activation of CD137 signaling decreases the stability of advanced atherosclerotic plaques via its combined effects on Teff cells, VSMCs, and macrophages.

Targeted next-generation sequencing at copy-number breakpoints for personalized analysis of rearranged ends in solid tumors.

BACKGROUND: The concept of the utilization of rearranged ends for development of personalized biomarkers has attracted much attention owing to its clinical applicability. Although targeted next-generation sequencing (NGS) for recurrent rearrangements has been successful in hematologic malignancies, its application to solid tumors is problematic due to the paucity of recurrent translocations. However, copy-number breakpoints (CNBs), which are abundant in solid tumors, can be utilized for identification of rearranged ends. METHOD: As a proof of concept, we performed targeted next-generation sequencing at copy-number breakpoints (TNGS-CNB) in nine colon cancer cases including seven primary cancers and two cell lines, COLO205 and SW620. For deduction of CNBs, we developed a novel competitive single-nucleotide polymorphism (cSNP) microarray method entailing CNB-region refinement by competitor DNA. RESULT: Using TNGS-CNB, 19 specific rearrangements out of 91 CNBs (20.9%) were identified, and two polymerase chain reaction (PCR)-amplifiable rearrangements were obtained in six cases (66.7%). And significantly, TNGS-CNB, with its high positive identification rate (82.6%) of PCR-amplifiable rearrangements at candidate sites (19/23), just from filtering of aligned sequences, requires little effort for validation. CONCLUSION: Our results indicate that TNGS-CNB, with its utility for identification of rearrangements in solid tumors, can be successfully applied in the clinical laboratory for cancer-relapse and therapy-response monitoring.