CAFs secreted exosomes promote metastasis and chemotherapy resistance by enhancing cell stemness and epithelial-mesenchymal transition in colorectal cancer.

BACKGROUND: Cancer associated fibroblasts (CAFs) are key stroma cells that play dominant roles in tumor progression. However, the CAFs-derived molecular determinants that regulate colorectal cancer (CRC) metastasis and chemoresistance have not been fully characterized. METHODS: CAFs and NFs were obtained from fresh CRC and adjacent normal tissues. Exosomes were isolated from conditioned medium and serum of CRC patients using ultracentrifugation method and ExoQuick Exosome Precipitation Solution kit, and characterized by transmission electronic microscopy, nanosight and western blot. MicroRNA microarray was employed to identify differentially expressed miRNAs in exosomes secreted by CAFs or NFs. The internalization of exosomes, transfer of miR-92a-3p was observed by immunofluorescence. Boyden chamber migration and invasion, cell counting kit-8, flow cytometry, plate colony formation, sphere formation assays, tail vein injection and primary colon cancer liver metastasis assays were employed to explore the effect of NFs, CAFs and exosomes secreted by them on epithelial-mesenchymal transition, stemness, metastasis and chemotherapy resistance of CRC. Luciferase report assay, real-time qPCR, western blot, immunofluorescence, and immunohistochemistry staining were employed to explore the regulation of CRC metastasis and chemotherapy resistance by miR-92a-3p, FBXW7 and MOAP1. RESULTS: CAFs promote the stemness, epithelial-mesenchymal transition (EMT), metastasis and chemotherapy resistance of CRC cells. Importantly, CAFs exert their roles by directly transferring exosomes to CRC cells, leading to a significant increase of miR-92a-3p level in CRC cells. Mechanically, increased expression of miR-92a-3p activates Wnt/ß-catenin pathway and inhibits mitochondrial apoptosis by directly inhibiting FBXW7 and MOAP1, contributing to cell stemness, EMT, metastasis and 5-FU/L-OHP resistance in CRC. Clinically, miR-92a-3p expression is significantly increased in CRC tissues and negatively correlated with the levels of FBXW7 and MOAP1 in CRC specimens, and high expression of exosomal miR-92a-3p in serum was highly linked with metastasis and chemotherapy resistance in CRC patients. CONCLUSIONS: CAFs secreted exosomes promote metastasis and chemotherapy resistance of CRC. Inhibiting exosomal miR-92a-3p provides an alternative modality for the prediction and treatment of metastasis and chemotherapy resistance in CRC.

[Research progress of obstructive sleep apnea syndrome caused by epiglottic collapse].

The incidence of obstructive sleep apnea syndrome (OSAS) is rising in recent years. Since OSAS is cased by collapse of the airways, while knowledge regarding the role of the epiglottic collapse in OSAS is limited. The use of DISE has led to better understanding of the relationship between epiglottis and OSAS.In order to improve the level of diagnosis and therapeutic effect,in this paper,the clinical characteristics, diagnosis, treatment and prognosis of OSAS caused by epiglottic collapse are reviewed.

Effect of procyanidine on VEGFR-2 expression and transduction pathway in rat endothelial progenitor cells under high glucose conditions.

The protective effect of procyanidine and its oligomers against high glucose-mediated oxidative stress injury in endothelial progenitor cells (EPCs), and effect of procyanidin on vascular endothelial growth factor receptor-2 (VEGFR-2) expression and downstream signal pathway were analyzed in vitro. Rat bone marrow mononuclear cells were isolated, cultured under normal and high glucose (HG) conditions, and the changes in cell morphology observed. The EPCs were identified, and the oxidative stress products produced by EPCs (under normal and HG conditions) were quantified. Subsequently, an appropriate number of EPCs were cultured with and without procyanidin (OPC), and the MDA concentration and relative expression of VEGFR-2, AKT, IκB-α, and nuclear factor (NF)-κB were detected 1, 3, 5, and 7 days post-culture. We observed minor (round, translucent, gradually adhering) and significant (fusiform morphology/pebble distribution) cell morphological changes 3 and 7 days post-culture, respectively. Apoptosis and oxidative stress product release in EPCs cultured with HG increased significantly compared to the control group (P < 0.05). The oxidative stress product generation and relative expression of VEGFR-2, AKT, IkB-α, and NF-κB were not significantly affected by OPC addition in normal glucose conditions (P > 0.05); alternately, products generated as a result of oxidative stress were significantly reduced, the relative expression of VEGFR-2, AKT, and NF-κB protein was upregulated, and that of IκB-α was downregulated (P < 0.05) in HG + OPC EPCs. Therefore, procyanidin may promote cell proliferation by alleviating oxidative damage to EPCs under HG conditions, and upregulating VEGFR-2 expression and its downstream signal pathway.

First Report of Leaf Spot Disease on Walnut Caused by Colletotrichum fioriniae in China.

In May 2014, a severe leaf spot disease was observed on walnut tree (Juglans regia L.) in Hechi, Guangxi, China. Leaf spots were circular to semicircular in shape, water-soaked, later becoming grayish white in the center with a dark brown margin and bordered by a tan halo. Necrotic lesions were approximately 3 to 4 mm in diameter. Diseased leaves were collected from 10 trees in each of five commercial orchards. The diseased leaves were cut into 5 × 5 mm slices, dipped in 75% ethanol for 30 s, washed three times in sterilized water, sterilized with 0.1% (w/v) HgCl2 for 3 min, and then rinsed five times with sterile distilled water. These slices were placed on potato dextrose agar (PDA), followed by incubating at 28°C for about 3 to 4 days. Fungal isolates were obtained from these diseased tissues, transferred onto PDA plates, and incubated at 28°C. These isolates produced gray aerial mycelium and then became pinkish gray with age. Moreover, the reverse of the colony was pink. The growth rate was 8.21 to 8.41 mm per day (average = 8.29 ± 0.11, n = 3) at 28°C. The colonies produced pale orange conidial masses and were fusiform with acute ends, hyaline, sometimes guttulate, 4.02 to 5.25 × 13.71 to 15.72 µm (average = 4.56 ± 0.31 × 14.87 ± 1.14 µm, n = 25). The morphological characteristics and measurements of this fungal isolate matched the previous descriptions of Colletotrichum fioriniae (Marcelino & Gouli) R.G. Shivas & Y.P. Tan (2). Meanwhile, these characterizations were further confirmed by analysis of the partial sequence of five genes: the internal transcribed spacer (ITS) of the ribosomal DNA, beta-tubulin (ß-tub) gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, chitin synthase 3(CHS-1) gene, and actin (ACT) gene, with universal primers ITS4/ITS5, T1/ßt2b, GDF1/GDR1, CHS1-79F/CHS1-354R, and ACT-512F/ACT-783R, respectively (1). BLAST of these DNA sequences using the nucleotide database of GenBank showed a high identify (ITS, 99%; ß-tub, 99%; GAPDH, 99%; CHS-1, 99%; and ACT, 100%) with the previously deposited sequences of C. fioriniae (ITS, KF278459.1, NR111747.1; ß-tub, AB744079.1, AB690809.1; GAPDH, KF944355.1, KF944354.1; CHS-1, JQ948987.1, JQ949005.1; and ACT, JQ949625.1, JQ949626.1). Koch's postulates were fulfilled by inoculating six healthy 1-year-old walnut trees in July 2014 with maximum and minimum temperatures of 33 and 26°C. The 6-mm mycelial plug, which was cut from the margin of a 5-day-old colony of the fungus on PDA, was placed onto each pin-wounded leaf, ensuring good contact between the mycelium and the wound. Non-colonized PDA plugs were placed onto pin-wounds as negative controls. Following inoculation, both inoculated and control plants were covered with plastic bags. Leaf spots, similar to those on naturally infected plants, were observed on the leaves inoculated with C. fioriniae within 5 days. No symptoms were observed on the negative control leaves. Finally, C. fioriniae was re-isolated from symptomatic leaves; in contrast, no fungus was isolated from the control, which confirmed Koch's postulates. To our knowledge, this is the first report of leaf disease on walnut caused by C. fioriniae. References: (1) L. Cai et al. Fungal Divers. 39:183, 2009. (2) R. G. Shivas and Y. P. Tan. Fungal Divers. 39:111, 2009.

The polymorphism of catalase T/C codon 389 in exon 9 and vitiligo susceptibility: a meta-analysis.

BACKGROUND: The exact aetiology of vitiligo has not yet been established. Oxidative stress is involved in the pathophysiology of vitiligo. It has been described that some polymorphisms in the catalase (CAT) gene may affect the risk of vitiligo. However, the results were inconsistent. OBJECTIVE: We performed a meta-analysis of the published studies to derive a more precise estimate of the association between CAT T/C at codon 389 in exon 9 polymorphisms and vitiligo risk. METHODS: The PubMed, Medline, Cochrane Library, China National Knowledge Infrastructure (CNKI) databases were searched to identify relevant published studies. RESULTS: Four case-control studies (cases, 645; controls, 689) that investigated the association between C/T polymorphisms of CAT exon 9 and the risk of vitiligo were retrieved and analysed. Our findings suggested a significant association between the CAT T/C exon 9 polymorphism and vitiligo risk (CT + TT vs. CC pooled odds ratio, 1.43; 95% confidence interval, 1.14-1.80; P = 0 .002). CONCLUSION: We found a significant correlation between the CAT T/C exon 9 polymorphism and the risk of vitiligo.

Regulatory T cells in chronic hepatitis B patients affect the immunopathogenesis of hepatocellular carcinoma by suppressing the anti-tumour immune responses.

Chronic hepatitis B (CHB) virus hepatitis B virus (HBV) infection is the key cause of hepatocellular carcinoma (HCC) in Asians. Recent studies have shown that levels of CD4(+)CD25(+) regulatory T cells (Tregs) were increased and were linked to an impaired immune response in patients with CHB. Evaluating whether Tregs are involved in the progression of CHB to HCC will provide insight into the immunopathogenesis of HCC. In the present study, we showed that circulating and liver-residing Tregs increased in CHB (n = 15) and HCC (n = 49) patients, particularly in the peripheral blood of HCC patients with HBV infection (n = 29). The increased Tregs in CHB patients suppressed the specific immune response induced by not only HBV antigen, but also by HCC tumour antigen. When peripheral blood mononuclear cells (PBMC) were co-cultured with human hepatoma cell lines that are stably transfected with HBV (HepG2.2.15), CD4(+)CD25(+) Treg populations increased and upregulated the expression of forkhead box P3 transcriptional regulator (FoxP3), cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and glucocorticoid-induced tumour necrosis factor (TNF) receptor family gene (GITR). In contrast, PBMCs co-cultured with HepG2 cells (the parental cell line of HepG2.2.15) did not. CD4(+)CD25(+) Tregs isolated from PBMCs that were co-cultured with HepG2.2.15 cells also had a greater suppressive ability with respect to the tumour antigen-specific immune response induced by NY-ESO-1 or MAGE-A3 compared with CD4(+)CD25(+) Tregs isolated from PBMCs co-cultured with HepG2 cells. The results offer evidence that the expansion of CD4(+)CD25(+) Tregs and the enhancement of the suppressor function of CD4(+)CD25(+) Tregs induced by HBV infection-related factors could suppress the anti-tumour immune response to HCC tumour antigen and inhibit tumour immuno-surveillance against HCC, which may be involved in the immunopathogenesis from CHB to HCC.

Balloon kyphoplasty versus vertebroplasty for treatment of osteoporotic vertebral compression fracture: a prospective, comparative, and randomized clinical study.

UNLABELLED: Bone pain and spinal axial deformity are major concerns in aged patients suffering from osteoporotic vertebral compression fracture (VCF). Pain can be relieved by vertebroplasty or kyphoplasty procedures, in which the compressed vertebral body is filled with substitutes. We randomly assigned 100 patients with osteoporotic compression fracture at the thoraco-lumbar (T-L) junction into two groups: vertebroplasty and kyphoplasty; we used polymethylmethacrylate (PMMA) as the bone filler. Pain before and after treatment was assessed with visual analog scale (VAS) scores and vertebral body height and kyphotic wedge angle were measured from reconstructed computed tomography images. More PMMA was used in the kyphoplasty group than in the vertebroplasty group (5.56 +/- 0.62 vs. 4.91 +/- 0.65 mL, p < 0.001). Vertebral body height and kyphotic wedge angle of the T-L spine were also improved (p < 0.001). VAS pain scores did not differ significantly between the treatment groups. The duration of follow-up was 6 months. Two patients in the kyphoplasty group had an adjacent segment fracture. In terms of clinical outcome there was little difference between the treatment groups. Thus, owing to the higher cost of the kyphotic balloon procedure, we recommend vertebroplasty over kyphoplasty for the treatment of osteoporotic VCFs. INTRODUCTION: Spinal axial deformities are major concerns in aged patients suffering from osteoporotic vertebral compression fracture. Pain may be relieved by vertebroplasty or kyphoplasty. We investigated the radiological and clinical outcomes of these procedures. METHODS: One hundred cases of VCF at the thoraco-lumbar junction were randomly assigned into two groups: vertebroplasty or kyphoplasty (50 cases each). We used polymethylmethacrylate as the bone filler. Pain before and after treatment was assessed with visual analog scale scores and vertebral body height and kyphotic wedge angle were measured from reconstructed computed tomography images. RESULTS: More PMMA was used in the kyphoplasty group than in the vertebroplasty group (5.56 +/- 0.62 vs. 4.91 +/- 0.65 mL, p < 0.001). Vertebral body height and kyphotic wedge angle of the T-L spine were also improved (p < 0.001). VAS pain scores did not differ significantly between the treatment groups. The duration of follow-up was 6 months. Two patients in the kyphoplasty group had an adjacent segment fracture. CONCLUSIONS: In terms of clinical outcome there was little difference between the treatment groups. Thus, with the higher cost of the kyphotic balloon procedure, we recommend vertebroplasty over kyphoplasty for the treatment of osteoporotic VCFs.

Effects of electrical cervical spinal cord stimulation on cerebral blood perfusion, cerebrospinal fluid catecholamine levels, and oxidative stress in comatose patients.

OBJECTIVES: Electrical spinal cord stimulation (SCS) is used to treat of chronic pain, obstructive arterial-related ischemia, and anginal pain. This study investigated cerebral blood perfusion, cerebrospinal fluid (CSF) catecholamine levels, and oxidative stress before and after cervical SCS in comatose patients. METHODS: We evaluated cerebral blood perfusion, catecholamine (dopamine, norepinephrine, and epinephrine) levels, and oxidative stress in 20 comatose patients before and after SCS. After SCS for six months, cerebral blood perfusion (SPECT index, 2.293 +/- 0.255 vs. 2.779 +/- 0.209, p < 0.001), dopamine (49.0 +/- 12.1 vs. 198.9 +/- 62.6, p = 0.025), and norepinephrine (197.6 +/- 62.9 vs. 379.6 +/- 52.6, p = 0.021) but not epinephrine were significantly increased. Moreover, superoxide free radicals in whole blood were significantly decreased (210,079 +/- 47,763 vs. 109,212 +/- 20,086, p = 0.011) after SCS. Nine patients recovered from the consciousness within 71-287 days. CONCLUSIONS: Increase of cerebral blood perfusion and catecholamines (dopamine and norepinephrine) in CSF after SCS was observed, whereas epinephrine level was unchanged. The superoxide free radicals were decreased after SCS. The results suggest that SCS increases cerebral blood perfusion, attenuates oxidative stress and increases biogenic amines in comatose patients.

Determination of clindamycin in human plasma by liquid chromatography-electrospray tandem mass spectrometry: application to the bioequivalence study of clindamycin.

A simple, sensitive and specific liquid chromatography-electrospray tandem mass spectrometry (LC-MS-MS) method for the determination of clindamycin (I) was developed. Both I and verapamil (II, internal standard) were analyzed using a C18 column with a mobile phase of 80% acetonitrile-0.01% trifluoroacetic acid. Column eluents were monitored by electrospray tandem mass spectrometry. Multiple reaction monitoring (MRM) using the parent to daughter combinations of m/z 425-->126 and 455-->165 was used to quantitate I. A limit of quantitation of 0.0500 microgram/ml was found. The assay exhibited a linear dynamic range of 0.0500-20.0 micrograms/ml and gave a correlation coefficient (r2) of 0.998 or better. The chromatographic run time was approximately 2 min. The intra-batch precision and accuracy of the quality controls (QCs, 0.0500, 0.150, 1.50, 15.0 and 20.0 micrograms/ml) were characterized by coefficients of variation (CVs) of 5.13 to 13.7% and relative errors (REs) of -4.34 to 4.58%, respectively. The inter-batch precision and accuracy of the QCs were characterized by CVs of 4.35 to 8.32% and REs of -10.8 to -4.17%, respectively. The method has successfully been applied to the analysis of samples taken up to 12 h after oral administration of 300 mg of I in healthy volunteers.

Cutaneous infection by Scedosporium apiospermum and its successful treatment with itraconazole.

We report the case of a 42-year-old man with a 13-year history of bilateral faciocervical infiltrative erythema, which had been misdiagnosed as tuberculoderma and which had failed to respond to treatment with adrenal corticosteroids and antituberculotics. On admission to the department, Scedosporium apiospermum was identified on lesion biopsies and fungus cultures as the causative agent and a diagnosis of cutaneous infection by S. apiospermum was made. This is the first report of chronic skin granuloma caused by S. apiospermum in China. Treatment with oral itraconazole (100-400 mg/day) led to clinical cure within 4 months.

Supplementation with vitamins C and E enhances cytokine production by peripheral blood mononuclear cells in healthy adults.

The effect of supplementation with vitamins C and E on cytokine production of healthy adult volunteers was studied in a single-blind trial. Ten subjects in each group received daily vitamin C (1 g ascorbic acid), vitamin E (400 mg dl-alpha-tocopheryl acetate), or vitamins C and E for 28 d. Plasma concentrations of alpha-tocopherol, ascorbate, and lipid peroxides as well as the production of cytokines by peripheral blood mononuclear cells (PBMCs) were measured before, during, and at the end of the supplementation and 1 wk later. PBMCs were cultured in the presence of absence of lipopolysaccharide for 24 h. The interleukin 1 (IL-1), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) in the culture supernates were assayed by enzyme-linked immunosorbent assay methods. Production of IL-1 beta and TNF-alpha in the group supplemented with vitamins C and E was significantly higher (P < 0.05) than that of the groups given vitamin E or vitamin C alone. The enhancing effect of supplementation with a combination of vitamins E and C coincided with peak plasma alpha-tocopherol and ascorbate concentrations and the lowest plasma lipid peroxide concentrations (P < 0.05) on day 14. In addition, an in vitro experiment with PBMCs showed that vitamins E and C reduced lipopolysaccharide-induced prostaglandin E2 production and enhanced TNF-alpha production. These results indicate that combined supplementation with vitamins C and E is more immunopotentiating than supplementation with either vitamin alone in healthy adults.

[Clinical study on treating asthma of cold type with wenyang tongluo mixture].

Sixty-eight asthma patients of Cold type were randomly divided into two groups, 34 for each group. The treated group was treated with Chinese herbal medicine Wenyang Tongluo Mixture (WYTLM), the control group was treated with Salbutamol orally and beclomethasone dipropionate aerosol. After 8 weeks of treatment, the results showed that there was no significant difference between the short-term total effective rate of the two groups (P > 0.05). Results of followup 1 year after withdrawal of treatment, showed that 9 patients (26.47%) in the treated group and 2 (5.88%) in the control group were cured clinically, it indicated that the long-term curative rate of the former group was higher than that of the latter group significantly (P < 0.05). And the effect of treated group on eliminating Asthenia-Cold symptoms, improving pulmonary ventilation function, regulating adrenergic beta-receptors of peripheral blood lymphocyte and decreasing the serum level of 5-hydroxytryptamine was more superior to that of control group (P < 0.05-0.01). This study provided some objective basis for using WYTLM in preventing and treating asthma of Cold type.

[Comparison of pharmacological effects of L- and DL-n-butyl-scopolamine in rat uterus].

The affinities of L-n-butyl-scopolamine, DL-n-butyl-scopolamine and atropine for the M-cholinergic receptors of rat uterus were compared using radioligand binding assay. Results showed that the affinity of L-n-butyl-scopolamine for the M-receptors was similar to that of DL-n-butyl-scopolamine, but lower than that of atropine. In isolated rat uterus, L- and DL-n-butyl-scopolamine were found not to influence the automatic contraction but antagonize the contraction induced by acetylcholine with nearly the same PA2, although the PA2 of both compounds were still lower than that of atropine. Therefore, no difference between L- and DL-n-butyl-scopolamine in either biological activity or receptor affinity was observed.