Gut Microbiota Defines Functional Direction of Colonic Regulatory T Cells with Unique TCR Repertoires.

Intestinal microbiota and selected strains of commensal bacteria influence regulatory T (Treg) cell functionality in the colon. Nevertheless, whether and how microbiota changes the transcriptome profile and TCR specificities of colonic Tregs remain to be precisely defined. In this study, we have employed single-cell RNA sequencing and comparatively analyzed colonic Tregs from specific pathogen-free and germ-free (GF) mice. We found that microbiota shifts the activation trajectory of colonic Tregs toward a distinct phenotypic subset enriched in specific pathogen-free but not in GF mice. Moreover, microbiota induced the expansion of specific Treg clonotypes with shared transcriptional specificities. The microbiota-induced subset of colonic Tregs, identified as PD-1- CXCR3+ Tregs, displayed enhanced suppressive capabilities compared with colonic Tregs derived from GF mice, enhanced production of IL-10, and were the primary regulators of enteric inflammation in dextran sodium sulfate-induced colitis. These findings identify a hitherto unknown gut microbiota and immune cell interaction module that could contribute to the development of a therapeutic modality for intestinal inflammatory diseases.

Integrated Analysis of Microbiome and Metabolome Reveals Disease-Specific Profiles in Inflammatory Bowel Diseases and Intestinal Behçet's Disease.

The gut microbial and metabolic characteristics of intestinal Behçet's disease (BD), a condition sharing many clinical similarities with ulcerative colitis (UC) and Crohn's disease (CD), are largely unexplored. This study investigated the gut microbial and metabolic characteristics of intestinal BD as well as potential biomarkers, comparing them with those in UC, CD, and healthy controls. Colon tissue and stool samples from 100 patients (35 UC, 30 CD, and 35 intestinal BD) and 41 healthy volunteers were analyzed using 16S ribosomal RNA sequencing to assess microbial diversity, taxonomic composition, and functional profiling. Plasma metabolomic analyses were performed using gas chromatography and ultra-performance liquid chromatography-mass spectrometry. Results indicated reduced microbial diversity in CD but not in intestinal BD, with intestinal BD showing fewer changes compared to controls yet distinct taxonomic features from UC, CD, and controls. Common alterations across all diseases included a reduction in beneficial bacteria producing short-chain fatty acids. Intestinal BD-specific changes featured a decreased abundance of Bacteroides fragilis. Metabolomic profiles in intestinal BD were similar to those in CD but distinct from those in UC, displaying significant changes in energy metabolism and genetic information processing. This integrative analysis revealed both shared and unique profiles in intestinal BD compared with UC, CD, and controls, advancing our understanding of the distinctive features of these diseases.

LIN28A loss of function is associated with Parkinson's disease pathogenesis.

Parkinson's disease (PD) is neurodegenerative movement disorder characterized by degeneration of midbrain-type dopamine (mDA) neurons in the substantia nigra (SN). The RNA-binding protein Lin28 plays a role in neuronal stem cell development and neuronal differentiation. In this study, we reveal that Lin28 conditional knockout (cKO) mice show degeneration of mDA neurons in the SN, as well as PD-related behavioral deficits. We identify a loss-of-function variant of LIN28A (R192G substitution) in two early-onset PD patients. Using an isogenic human embryonic stem cell (hESC)/human induced pluripotent stem cell (hiPSC)-based disease model, we find that the Lin28 R192G variant leads to developmental defects and PD-related phenotypes in mDA neuronal cells that can be rescued by expression of wild-type Lin28A. Cell transplantation experiments in PD model rats show that correction of the LIN28A variant in the donor patient (pt)-hiPSCs leads to improved behavioral phenotypes. Our data link LIN28A to PD pathogenesis and suggest future personalized medicine targeting this variant in patients.

Bifidobacterium breve CBT BR3 is effective at relieving intestinal inflammation by augmenting goblet cell regeneration.

BACKGROUND AND AIM: Bifidobacterium breve was the first bacteria isolated in the feces of healthy infants and is a dominant species in the guts of breast-fed infants. Some strains of B. breve have been shown to be effective at relieving intestinal inflammation, but the modes of action have yet to be elucidated. In this study, we investigated the mechanisms of action of B. breve CBT BR3 isolated from South Korean infant feces in relieving colitis in vitro and in vivo. METHODS: Colitis was induced in mice with dextran sodium sulfate (DSS) and dinitrobenzene sulfonic acid (DNBS). Quantitative reverse-transcription polymerase chain reaction, in vitro FITC-dextran flux permeability assay, and aryl hydrocarbon receptor (AhR) luciferase assay are performed using Caco-2 cells and HT29-Lucia™ AhR cells. RESULTS: B. breve CBT BR3 was orally administered. B. breve CBT BR3 improved colitis symptoms in both DSS- and DNBS-induced colitis models. B. breve CBT BR3 increased the number of goblet cells per crypt. B. breve increased the mRNA expressions of Notch, Spdef, Muc5, and Il22. The mRNA expressions of Occludin, which encodes a membrane tight-junction protein, and Foxo3, which encodes a protein related to butyrate metabolism, were also increased in the DSS- and DNBS-induced colitis models. B. breve CBT BR3 protected inflammation-induced epithelial cell permeability and improved goblet cell function by inducing aryl hydrocarbon receptor in vitro. CONCLUSIONS: These results indicate that B. breve CBT BR3 is effective at relieving intestinal inflammation by augmenting goblet cell regeneration.

Glutathione S-transferase theta 1 protects against colitis through goblet cell differentiation via interleukin-22.

The enzyme glutathione S-transferase theta 1 (GSTT1) is involved in detoxifying chemicals, including reactive oxygen species (ROS). Here, we provide a significant insight into the role of GSTT1 in inflammatory bowel disease (IBD). We identified decreased expression of GSTT1 in inflamed colons from IBD patients compared to controls. We intrarectally or intraperitoneally delivered Gstt1 gene to mice with dextran sodium sulfate (DSS)-induced colitis and noted attenuation of colitis through gene transfer of Gstt1 via an IL-22 dependent pathway. Downregulation of GSTT1 by pathogen-associated molecular patterns (PAMPs) of microbes reduced innate defense responses and goblet cell differentiation. The GSTT1 mutation in intestinal epithelial cells (IECs) and IBD patients decreased its dimerization, which was connected to insufficient phosphorylation of signal transducer and activator of transcription-3 and p38/mitogen-activated protein kinase by their common activator, IL-22. GSTT1 ameliorated colitis and contributed as a modulator of goblet cells through sensing pathogens and host immune responses. Its mutations are linked to chronic intestinal inflammation due to its insufficient dimerization. Our results provide new insights into GSTT1 mutations that are linked to chronic intestinal inflammation due to its insufficient dimerization and their functional consequences in IBDs.

Lactobacillus plantarum CBT LP3 ameliorates colitis via modulating T cells in mice.

Lactobacillus plantarum has been identified as a probiotic bacterium owing to its role in immune regulation and maintenance of intestinal permeability. Here, we investigated the anti-colitic effects and mechanism of L. plantarum CBT LP3 (LP3). This in vivo study was performed using dextran sodium sulfate (DSS) to induce colitis in mice. Mice were randomly divided into three groups: a control supplied with normal drinking water, a DSS-treated group followed by oral administration of vehicle, and a DSS-treated group gavaged with LP3 daily for 7 days following DSS administration. An analysis of macrophages and T cell subsets harvesting from peritonium cavity cells and splenocytes was performed using a flow cytometric assay. Gene expression and cytokine profiles were measured using quantitative reverse transcriptase polymerase chain reaction. The administration of LP3 significantly attenuated disease activity and histolopathology compared to control. LP3 had anti-inflammatory effects, with increased induction of regulatory T cells and type 2 helper T cells in splenocytes and restoration of goblet cells accompanied by suppression of proinflammatory cytokine expressions. These findings suggest that L. plantarum CBT LP3 can be used as a potent immunomodulator, which has significant implications for IBD treatment.

Efficient Neural Differentiation of hPSCs by Extrinsic Signals Derived from Co-cultured Neural Stem or Precursor Cells.

In this study, we found that undifferentiated human pluripotent stem cells (hPSCs; up to 30% of total cells) present in the cultures of neural stem or precursor cells (NPCs) completely disappeared within several days when cultured under neural differentiation culture conditions. Intriguingly, the disappearance of undifferentiated cells was not due to cell death but was instead mediated by neural conversion of hPSCs. Based on these findings, we propose pre-conditioning of donor NPC cultures under terminal differentiation culture conditions as a simple but efficient method of eliminating undifferentiated cells to treat neurologic disorders. In addition, we could establish a new neural differentiation protocol, in which undifferentiated hPSCs co-cultured with NPCs become differentiated neurons or NPCs in an extremely efficient, fast, and reproducible manner across the hESC and human-induced pluripotent stem cell (hiPSC) lines.

Nanocomposites-based targeted oral drug delivery systems with infliximab in a murine colitis model.

BACKGROUND: Infliximab (IFX), a TNF-α blocking chimeric monoclonal antibody, induces clinical response and mucosal healing in patients with inflammatory bowel disease (IBD). However, systemic administration of this agent causes unwanted side effects. Oral delivery of antibody therapeutics might be an effective treatment strategy for IBD compared to intravenous administration. RESULTS: All three carriers had a high encapsulation efficiency, narrow size distribution, and minimal systemic exposure. There was a higher interaction between nanocomposite carriers and monocytes compared to lymphocytes in the PBMC of IBD patients. Orally administered nanocomposite carriers targeted to inflamed colitis minimized systemic exposure. All IFX delivery formulations with nanocomposite carriers had a significantly less colitis-induced body weight loss, colon shortening and histomorphological score, compared to the DSS-treated group. AC-IFX-L and EAC-IFX-L groups showed significantly higher improvement of the disease activity index, compared to the DSS-treated group. In addition, AC-IFX-L and EAC-IFX-L alleviated pro-inflammatory cytokine expressions (Tnfa, Il1b, and Il17). CONCLUSION: We present orally administered antibody delivery systems which improved efficacy in murine colitis while reducing systemic exposure. These oral delivery systems suggest a promising therapeutic approach for treating IBD.

Protective Effect of Alpha-Lipoic Acid on Salivary Dysfunction in a Mouse Model of Radioiodine Therapy-Induced Sialoadenitis.

Radioiodine (RI) therapy is known to cause salivary gland (SG) dysfunction. The effects of antioxidants on RI-induced SG damage have not been well described. This study was performed to investigate the radioprotective effects of alpha lipoic acid (ALA) administered prior to RI therapy in a mouse model of RI-induced sialadenitis. Four-week-old female C57BL/6 mice were divided into four groups (n = 10 per group): group I, normal control; group II, ALA alone (100 mg/kg); group III, RI alone (0.01 mCi/g body weight, orally); and group IV, ALA + RI (ALA at 100 mg/kg, 24 h and 30 min before RI exposure at 0.01 mCi/g body weight). The animals in these groups were divided into two subgroups and euthanized at 30 or 90 days post-RI treatment. Changes in salivary 99mTc pertechnetate uptake and excretion were tracked by single-photon emission computed tomography. Salivary histological examinations and TUNEL assays were performed. The 99mTc pertechnetate excretion level recovered in the ALA treatment group. Salivary epithelial (aquaporin 5) cells of the ALA + RI group were protected from RI damage. The ALA + RI group exhibited more mucin-containing parenchyma and less fibrotic tissues than the RI only group. Fewer apoptotic cells were observed in the ALA + RI group compared to the RI only group. Pretreatment with ALA before RI therapy is potentially beneficial in protecting against RI-induced salivary dysfunction.

ZNF133 is associated with infliximab responsiveness in patients with inflammatory bowel diseases.

BACKGROUND AND AIM: Infliximab has been widely prescribed for treating inflammatory bowel disease (IBD). However, the response rates to infliximab differ among patients. Therefore, we aimed to identify the genetic and clinical markers that predict infliximab response. METHODS: A total of 139 Korean patients with IBD who received infliximab were classified according to infliximab response as follows: (i) primary response vs nonresponse and (ii) sustained response vs loss of response. We performed an association study using whole-exome sequencing data to identify genetic variants associated with infliximab response. Candidate variants were validated in 77 German patients with IBD. Stepwise multivariate logistic regression was performed to identify predictors. RESULTS: We found five candidate variants that were associated with primary nonresponse to infliximab (P < 5 × 10-6 ). Of the five variants, rs2228273 in ZNF133 was validated in German (combined P = 6.49 × 10-7 ). We also identified the best genetic variant (rs9144, P = 4.60 × 10-6 ) associated with the loss of infliximab response. In multivariate regression analysis, rs2228273 (P = 2.10 × 10-5 ), concurrent azathioprine/6-mercaptopurine use, and bodyweight at the first infliximab use (< 50 kg) were associated with primary nonresponse. In addition, the Crohn's disease activity index at the first infliximab use and rs9144 (P = 0.001) were independently associated with the loss of response in patients with Crohn's disease. CONCLUSIONS: We identified clinical and genetic markers associated with infliximab response in IBD patients. Our findings could provide insights to maximize the efficacy of infliximab therapy in IBD patients.

Lactobacillus acidophilus suppresses intestinal inflammation by inhibiting endoplasmic reticulum stress.

BACKGROUND AND AIM: Nuclear factor kappa B (NF-κB) activation and endoplasmic reticulum (ER) stress signaling play significant roles in the pathogenesis of inflammatory bowel disease (IBD). Thus, we evaluated whether new therapeutic probiotics have anti-colitic effects, and we investigated their mechanisms related to NF-κB and ER-stress pathways. METHODS: Luciferase, nitric oxide, and cytokine assays using HT-29 or RAW264.7 cells were conducted. Mouse colitis was induced using dextran sulfate sodium and confirmed by disease activity index and histology. Macrophages and T-cell subsets in isolated peritoneal cavity cells and splenocytes were analyzed by flow cytometry. Gene and cytokine expression profiles were determined using reverse-transcription polymerase chain reaction. RESULTS: Lactobacillus acidophilus (LA1) and Pediococcus pentosaceus inhibited nitric oxide production in RAW264.7 cells, but only LA1 inhibited Tnfa and induced Il10 expression. LA1 increased the lifespan of dextran sulfate sodium-treated mice and attenuated the severity of colitis by inducing M2 macrophages in peritoneal cavity cells and Th2 and Treg cells in splenocytes. The restoration of goblet cells in the colon was accompanied by the induction of Il10 expression and the suppression of pro-inflammatory cytokines. Additionally, we found that LA1 exerts an anti-colitic effect by improving ER stress in HT-29 cells as well as in vivo. CONCLUSIONS: We showed that LA1 significantly interferes with ER stress and suppresses NF-κB activation. Our findings suggest that LA1 can be used as a potent immunomodulator in IBD treatment, and the regulation of ER stress may have significant implications in treating IBD.

Protective effects of guggulsterone against colitis are associated with the suppression of TREM-1 and modulation of macrophages.

Triggering receptor expressed on myeloid cells 1 (TREM-1)-expressing intestinal macrophages are significantly increased in the colons of patients with inflammatory bowel disease (IBD). We focused here on the effects of guggulsterone on macrophage modulation in colitis as a potential therapeutic molecule in human IBD and explore the underlying mechanisms. Gene expression in macrophages was examined and wound-healing assay using HT-29 cells was performed. Colitis in wild-type and IL-10-, Toll-like receptor 4 (TLR4)-, and myeloid differentiation primary response 88 (MyD88)-deficient mice was induced via the administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) into the colon. In both in vitro and in vivo experiments, guggulsterone suppressed intestinal inflammation amplified by TREM-1 stimulation, in which the suppression of NF-κB, activating protein-1, and proteasome pathways was involved. In the TNBS-induced colitis model, guggulsterone reduced disease activity index scores and TREM-1 expression, stimulated IL-10 production, and improved survival in wild-type mice. These effects were not observed in IL-10-, TLR4-, and MyD88-deficient mice. Guggulsterone also suppressed M1 polarization, yet induced the M2 phenotype in macrophages from IBD patients as well as from mice. These findings indicate that guggulsterone blocks the hyperactivation of macrophages via TREM-1 suppression and induces M2 polarization via IL-10 mediated by the TLR4 signaling pathway. Furthermore, this study provides a new rationale for the therapeutic potential of guggulsterone in the treatment of IBD. NEW & NOTEWORTHY We found that guggulsterone attenuates triggering receptor expressed on myeloid cells 1 (TREM-1)-mediated hyperactivation of macrophages and polarizes macrophages toward the M2 phenotype. This was mediated by IL-10 and partly Toll-like receptor 4 signaling pathways. Overall, these data support that guggulsterone as a natural plant sterol modulates macrophage phenotypes in colitis, which may be of novel therapeutic importance in inflammatory bowel disease treatment.

Fluorine MR Imaging Monitoring of Tumor Inflammation after High-Intensity Focused Ultrasound Ablation.

Purpose To investigate whether high-intensity focused ultrasound (HIFU)-induced macrophage infiltration could be longitudinally monitored with fluorine 19 (19F) magnetic resonance (MR) imaging in a quantitative manner. Materials and Methods BALB/c mice were subcutaneously inoculated with 4T1 cells and were separated into three groups: untreated mice (control, n = 9), HIFU-treated mice (HIFU, n = 9), and HIFU- and clodronate-treated mice (HIFU+Clod, n = 9). Immediately after HIFU treatment, all mice were intravenously given perfluorocarbon (PFC) emulsion. MR imaging examinations were performed 2, 4, 7, 10, and 14 days after HIFU treatment. Two-way repeated measures analysis of variance was used to analyze the changes in 19F signal over time and differences between groups. Histologic examinations were performed to confirm in vivo data. Results Fluorine 19 signals were detected at the rims of tumors and the peripheries of ablated lesions. Mean 19F signal in tumors was significantly higher in HIFU-treated mice than in control mice up to day 4 (0.82 ± 0.26 vs 0.42 ± 0.17, P < .001). Fluorine 19 signals were higher in the HIFU+Clod group than in the control group from day 4 (0.82 ± 0.23, P < .001) to day 14 (0.55 ± 0.16 vs 0.28 ± 0.06, P < .05). Histologic examination revealed macrophage infiltration around ablated lesions. Immunofluorescence staining confirmed PFC labeling of macrophages. Conclusion Fluorine 19 MR imaging can longitudinally capture and quantify HIFU-induced macrophage infiltration in preclinical tumor models. © RSNA, 2018 Online supplemental material is available for this article.

Fecal calprotectin as a non-invasive biomarker for intestinal involvement of Behçet's disease.

BACKGROUND AND AIM: The diagnostic and prognostic values of fecal calprotectin (FC) levels in patients with inflammatory bowel diseases have been proven. However, little is known about the usefulness of FC measurement in predicting intestinal involvement of Behçet's disease (BD). METHODS: Forty-four consecutive patients with systemic BD who underwent colonoscopy for the evaluation of gastrointestinal symptoms were prospectively enrolled between November 2012 and March 2014 in a single tertiary medical center. Fecal specimens from the patients were obtained the day before bowel cleansing and 3 months after colonoscopy. RESULTS: Twenty-five patients showed intestinal ulcerations on colonoscopy (12 [48.0%] typical and 13 [52.0%] atypical ulcerations). The median FC level in the intestinal BD group was significantly higher than that in the non-diagnostic group (112.53 [6.86-1604.39] vs 31.64 [5.46-347.60] µg/g, respectively, P = 0.003). Moreover, the typical ulceration group showed a significantly higher median FC level than the atypical ulceration group in patients with intestinal BD (435.995 [75.65-1604.39] vs 71.42 [6.86-476.94] µg/g, respectively, P = 0.033). Multivariate analysis revealed higher FC as an independent predictor of intestinal BD (OR = 38.776; 95% CI = 2.306-652.021; P = 0.011). The cut-off level of FC for predicting intestinal BD was 68.89 µg/g (76% sensitivity and 79% specificity). The absolute changes between fecal calprotectin levels and the disease activity index of intestinal BD from initial diagnosis of intestinal BD to 3 months after diagnosis were significantly correlated (Pearson's correlation coefficient = 0.470, P = 0.027). CONCLUSION: The FC level might serve as a non-invasive surrogate marker of intestinal involvement of BD.

Proteomic Analysis of Serum Amyloid A as a Potential Marker in Intestinal Behçet's Disease.

BACKGROUND/AIMS: Data regarding biomarkers to understand disease pathogenesis and to assess disease activity of intestinal Behçet's disease (BD) are limited. Therefore, we aimed to investigate the differentially expressed proteins in sera from patients with intestinal BD and to search for biomarkers using mass spectrometry-based proteomic analysis. METHODS: Serum samples were pooled for the screening study, and two-dimensional electrophoresis (2-DE) was performed to characterize the proteins present in intestinal BD patients. Candidate protein spots were identified using matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and bioinformatic analysis. To validate the proteomic results, serum samples from an independent cohort were assessed by enzyme-linked immunosorbent assay. RESULTS: Pooled serum samples were used for 2-DE, and approximately 400 protein spots were detected in the sera of intestinal BD patients. Of the 22 differentially expressed proteins, 3 were successfully identified using MALDI-TOF/TOF MS. The three up-regulated proteins identified in the intestinal BD group included fibrin, apolipoprotein A-IV, and serum amyloid A (SAA). Serum SAA in intestinal BD patients (2.76 ± 2.50 ng/ml) was significantly higher than that in controls (1.68 ± 0.90 ng/ml, p = 0.007), which is consistent with the proteomic results. In addition, the level of IL-1ß in patients with intestinal BD (8.96 ± 1.23 pg/ml) was higher than that in controls (5.40 ± 0.15 pg/ml, p = 0.009). SAA released by HT-29 cells was markedly increased by tumor necrosis factor-α (TNF-α) and lipopolysaccharides stimulation. CONCLUSIONS: Our proteomic analysis revealed that SAA was up-regulated in intestinal BD patients.

Comparison of the Antimicrobial Properties of Chitosan Oligosaccharides (COS) and EDTA against Fusarium fujikuroi Causing Rice Bakanae Disease.

Bakanae disease is a destructive rice disease in South Korea caused by Fusarium fujikuroi infection. Chemical fungicides have been used to manage the disease, but the emergence of fungicide-resistant strains has gradually increased. Two chelating agents, chitosan oligosaccharides (COS) and ethylenediaminetetraacetatic acid (EDTA), are well known as biosafe and biocompatible antimicrobial agents. In this study, we compared the actions of COS and EDTA to gain a better understanding of the underlying antimicrobial activities and to evaluate them as eco-friendly fungicides against F. fujikuroi. While COS exhibited a rapid fungicidal effect on hyphal growing cells within 5 min, EDTA had a fungistatic effect on reversible growth inhibition. Scanning electron microscopy revealed that COS treatment resulted in pore-formation and cellular leakage along the growing hyphae, whereas EDTA caused no significant morphological changes. COS activity was greatly suppressed by the addition of Ca(2+) to the medium, and EDTA action was largely suppressed by Mn(2+) and slightly by Ca(2+), respectively. Taken together, these results indicated that two chelating agents, COS and EDTA, have different modes of antimicrobial action on F. fujikuroi. Thus, the combination of chelating agents having different modes of action might be an effective disease management strategy to prevent or delay the development of fungicide-resistant strains.

The bifunctional autophagic flux by 2-deoxyglucose to control survival or growth of prostate cancer cells.

BACKGROUND: Recent reports using metabolism regulating drugs showed that nutrient deprivation was an efficient tool to suppress cancer progression. In addition, autophagy control is emerging to prevent cancer cell survival. Autophagy breaks down the unnecessary cytoplasmic components into anabolic units and energy sources, which are the most important sources for making the ATP that maintains homeostasis in cancer cell growth and survival. Therefore, the glucose analog 2-deoxyglucose (2DG) has been used as an anticancer reagent due to its inhibition of glycolysis. METHODS: Prostate cancer cells (PC3) were treated with 2DG for 6 h or 48 h to analyze the changing of cell cycle and autophagic flux. Rapamycin and LC3B overexpressing vectors were administered to PC3 cells for autophagy induction and chloroquine and shBeclin1 plasmid were used to inhibit autophagy in PC3 cells to analyze PC3 cells growth and survival. The samples for western blotting were prepared in each culture condition to confirm the expression level of autophagy related and regulating proteins. RESULTS: We demonstrated that 2DG inhibits PC3 cells growth and had discriminating effects on autophagy regulation based on the different time period of 2DG treatment to control cell survival. Short-term treatment of 2DG induced autophagic flux, which increased microtubule associated protein 1 light chain 3B (LC3B) conversion rates and reduced p62 levels. However, 2DG induced autophagic flux is remarkably reduced over an extended time period of 2DG treatment for 48 h despite autophagy inducing internal signaling being maintained. The relationship between cell growth and autophagy was proved. Increased autophagic flux by rapamycin or LC3B overexpression powerfully reduced cell growth, while autophagy inhibition with shBeclin1 plasmid or chloroquine had no significant effect on regulating cell growth. CONCLUSION: Given these results, maintaining increased autophagic flux was more effective at inhibiting cancer cell progression than inhibition of autophagic flux, which is necessary for the survival of PC3 cells. Autophagic flux should be tightly regulated to maintain metabolic homeostasis for cancer cell growth and survival in PC3 cells and is a suitable target for cancer therapy.

The novel histone deacetylase inhibitor, N-hydroxy-7-(2-naphthylthio) hepatonomide, exhibits potent antitumor activity due to cytochrome-c-release-mediated apoptosis in renal cell carcinoma cells.

BACKGROUND: Epigenetic modifications play a critical role in the regulation of all DNA-based processes, such as transcription, repair, and replication. Inappropriate histone modifications can result in dysregulation of cell growth, leading to neoplastic transformation and cell death. Renal tumors have been shown to have a higher global methylation percentage and reduced histone acetylation. Preclinical models have revealed that histone gene modifiers and epigenetic alterations play important roles in renal cell carcinoma (RCC) tumorigenesis. Recently, a novel HDAC inhibitor, N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA), has been introduced as an example of a new class of anti-cancer agents. The anti-cancer activity of HNHA and the underlying mechanisms of action remain to be clarified. METHODS: The MTS assay using a panel of RCC cells was used to evaluate the anti-proliferative effects of HNHA. The established HDAC inhibitors, SAHA and TSA, were used for comparison. Western blotting analysis was performed to investigate the acetylation of histone H3 and the expression of apoptotic markers in vitro and in vivo. Subcellular fractionation was performed to evaluate expression of Bax and cytochrome c in the cytosol and mitochondria, and also translocation of cytochrome c from the cytoplasm to the nucleus. A confocal microscopic evaluation was performed to confirm inhibition of cell proliferation, induction of apoptosis, and the nuclear translocation of cytochrome c in RCC cells. RESULTS: In this study, we investigated the apoptosis-inducing activity of HNHA in cultured kidney cancer cells. Apoptosis in the HNHA-treated group was induced significantly, with marked caspase activation and Bcl-2 suppression in RCC cells in vitro and in vivo. HNHA treatment caused cytochrome c release from mitochondria, which was mediated by increased Bax expression and caspase activation. HNHA also induced nuclear translocation of cytochrome c, suggesting that HNHA can induce caspase-independent nuclear apoptosis in RCC cells. An in vivo study showed that HNHA had greater anti-tumor and pro-apoptotic effects on RCC xenografts than the established HDAC inhibitors. CONCLUSIONS: HNHA has more potent anti-tumor activity than established HDAC inhibitors. Its activities are mediated by caspase-dependent and cytochrome-c-mediated apoptosis in RCC cells. These results suggest that HNHA may offer a new therapeutic approach to RCC.

Evaluation of pump pulsation in respirable size-selective sampling: part II. Changes in sampling efficiency.

This second, and concluding, part of this study evaluated changes in sampling efficiency of respirable size-selective samplers due to air pulsations generated by the selected personal sampling pumps characterized in Part I (Lee E, Lee L, Möhlmann C et al. Evaluation of pump pulsation in respirable size-selective sampling: Part I. Pulsation measurements. Ann Occup Hyg 2013). Nine particle sizes of monodisperse ammonium fluorescein (from 1 to 9 µm mass median aerodynamic diameter) were generated individually by a vibrating orifice aerosol generator from dilute solutions of fluorescein in aqueous ammonia and then injected into an environmental chamber. To collect these particles, 10-mm nylon cyclones, also known as Dorr-Oliver (DO) cyclones, were used with five medium volumetric flow rate pumps. Those were the Apex IS, HFS513, GilAir5, Elite5, and Basic5 pumps, which were found in Part I to generate pulsations of 5% (the lowest), 25%, 30%, 56%, and 70% (the highest), respectively. GK2.69 cyclones were used with the Legacy [pump pulsation (PP) = 15%] and Elite12 (PP = 41%) pumps for collection at high flows. The DO cyclone was also used to evaluate changes in sampling efficiency due to pulse shape. The HFS513 pump, which generates a more complex pulse shape, was compared to a single sine wave fluctuation generated by a piston. The luminescent intensity of the fluorescein extracted from each sample was measured with a luminescence spectrometer. Sampling efficiencies were obtained by dividing the intensity of the fluorescein extracted from the filter placed in a cyclone with the intensity obtained from the filter used with a sharp-edged reference sampler. Then, sampling efficiency curves were generated using a sigmoid function with three parameters and each sampling efficiency curve was compared to that of the reference cyclone by constructing bias maps. In general, no change in sampling efficiency (bias under ±10%) was observed until pulsations exceeded 25% for the DO cyclone. However, for three models of pumps producing 30%, 56%, and 70% pulsations, substantial changes were confirmed. The GK2.69 cyclone showed a similar pattern to that of the DO cyclone, i.e. no change in sampling efficiency for the Legacy producing 15% pulsation and a substantial change for the Elite12 producing 41% pulsation. Pulse shape did not cause any change in sampling efficiency when compared to the single sine wave. The findings suggest that 25% pulsation at the inlet of the cyclone as measured by this test can be acceptable for the respirable particle collection. If this test is used in place of that currently in European standards (EN 1232-1997 and EN 12919-1999) or is used in any International Organization for Standardization standard, then a 25% pulsation criterion could be adopted. This work suggests that a 10% criterion as currently specified in the European standards for testing may be overly restrictive and not able to be met by many pumps on the market. Further work is recommended to determine which criterion would be applicable to this test if it is to be retained in its current form.

Exposure to chlorpyrifos in gaseous and particulate form in greenhouses: a pilot study.

Phase distribution of airborne chemicals is important because intake and uptake mechanisms of each phase are different. The phase distribution and concentrations are needed to determine strategies of exposure assessment, hazard control, and worker protection. However, procedures for establishing phase distribution and concentration have not been standardized. The objective of this study was to compare measurements of an airborne semivolatile pesticide (chlorpyrifos) by phase using two different procedures. Six pesticide applications in two facilities were studied and at each site, samples were collected for three time slots: T1, the first 1 or 2 hr after the commencement of application; T2, a 6-hr period immediately following T1; and T3, a 6-hr period after the required re-entry interval (24 hr for chlorpyrifos).Two phase-separating devices were co-located at the center of each greenhouse: semivolatile aerosol dichotomous sampler (SADS) using flow rates of 1.8 l x min(-1) and 0.2 l x min(-1), corresponding to a total inlet flow rate of 2.0 l x min(-1) with a vapor phase flow fraction of 0.1; and an electrostatic precipitator (ESP), along with a standard OVS XAD-2 tube. Chlorpyrifos in vapor and particulate form in a SADS sampling train and that in vapor form in an ESP sampling train were collected in OVS tubes. Chlorpyrifos in particulate form in the ESP setting would have been collected on aluminum substrate. However, no chlorpyrifos in particulate form was recovered from the ESP. Overall (vapor plus particle) concentrations measured by OVS ranged 11.7-186.6 µg/m(3) at T1 and decreased on average 77.1% and 98.9% at T2 and T3, respectively. Overall concentrations measured by SADS were 66.6%, 72.7%, and 102% of those measured by OVS on average at T1, T2, and T3, respectively. Particle fractions from the overall concentrations measured by SADS were 60.0%, 49.2%, and 13.8%, respectively, for T1, T2, and T3. SADS gives better guidance on the distribution of chlorpyrifos than does the ESP, although the accuracy of the concentration distribution cannot be verified in the absence of a standardized procedure for determining phase division.