Interleukin-17A up-regulates thymic stromal lymphopoietin production by nasal fibroblasts from patients with allergic rhinitis.

PURPOSE: Emerging evidence has shown that interleukin (IL)-17A is implicated in the pathogenesis of allergic rhinitis (AR). Thymic stromal lymphopoietin (TSLP) orchestrates the immune response toward a Th2 phenotype. Although increased TSLP is found in AR, the contribution of IL-17A in TSLP production by nasal fibroblasts is not well understood. We aimed to investigate the effect and mechanism of IL-17A on TSLP production by human nasal fibroblasts (HNFs) from AR patients. METHODS: HNFs from AR patients were cultured and stimulated with IL-17A in the absence or presence of a Janus kinase (JAK) 2 or JAK1/3 inhibitor. Western blotting was used to assay phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and nuclear factor-kappa B (NF-κB) p65 in HNFs. The TSLP expression in the cells and culture supernatants was evaluated by real-time polymerase chain reaction and enzyme-linked immunoassay. RESULTS: Stimulation with IL-17A induced STAT3 phosphorylation, which was inhibited by the pretreatment with JAK2 inhibitor AZD1480 or JAK1/3 inhibitor tofacitinib. IL-17A promoted the nuclear translocation of NF-κBp65 protein, leading to increased TSLP production, while the pre-incubation with AZD1480 prior to IL-17A attenuated these effects. However, the pre-incubation with tofacitinib before IL-17A stimulation had no impact on the expression of NF-κBp65 and TSLP. CONCLUSIONS: IL-17A up-regulated TSLP production by HNFs through JAK2/NF-κB pathway. Although IL-17A induced STAT3 activation through JAK1/2/3, IL-17A-mediated TSLP expression was not dependent on STAT3 signaling. These observations would provide mechanistic insight into therapeutic strategies to improve the immune and inflammation associated with Th17A in the management of AR.

Histamine H4 receptor regulates Th2-cytokine profile through thymic stromal lymphopoietin in allergic rhinitis.

PURPOSE: Epithelial thymic stromal lymphopoietin (TSLP) promotes Th2 inflammatory responses through induction of OX40 ligand (OX40L) on dendritic cells in allergic rhinitis (AR). Emerging evidence supports the important role of histamine H4 receptor (H4R) in allergic inflammation. This study aimed to investigate the effects of H4R in Th2-cytokine profile mediated by TSLP in AR. METHODS: Human nasal epithelial cells (HNECs) from AR patients were stimulated with histamine in the presence or absence of H4R agonist (4-methylhistamine, 4-MH) and antagonist (NJ7777120, JNJ) or H1R agonist (2-pyridylethylamine). TSLP protein was measured by Western blotting and ELISA. To further elucidate the role of H4R in the in vivo situation of experimental AR, rats were sensitized and treated with JNJ or 4-MH. TSLP and OX40 ligand (OX40L) in the nasal mucosa were assayed by Western blotting. Th2 cytokines including interleukin-4, 5 and 13 in nasal lavage fluids were detected by ELISA. RESULTS: Histamine alone did not induce TSLP production by HNECs. The pre-incubation with 4-MH prior to histamine promoted TSLP expression, which was inhibited by the stimulation with JNJ prior to histamine and 4-MH. The pre-incubation with 2-pyridylethylamine before histamine stimulation had no impact on TSLP production. In AR rats, the levels of TSLP and OX40L protein were increased as well as Th2 cytokines, which was further up-regulated by 4-MH treatment, while JNJ treatment attenuated these effects. CONCLUSIONS: H4R activation induced TSLP production by HNECs, which up-regulated OX40L expression in the nasal mucosa of sensitized rats. These factors promoted Th2-cytokine profile in AR.

Activation of CD3+ T cells by Helicobacter pylori DNA vaccines in potential immunotherapy of gastric carcinoma.

Most of gastric carcinoma (GC) is attributed to infection by Helicobacter pylori (H. pylori) but there is increasing evidence that the positive H. pylori status correlates with better prognosis in GC. The H. pylori-induced cellular immune response may suppress cancer and in this work, recombinant pcDNA3 plasmids encoding various fragments of H. pylori virulence genes of cagA, vacA and babA are constructed and combined into groups to immunize BALB/c mice. The activated splenic CD3+ T cells are purified and the anticancer effects are investigated in vitro and in vivo. The H. pylori DNA vaccines induce a shift in the response from Th1 to Th2 that mimicks the immune status in patients of GC with chronic H. pylori infection. The stimulated CD3+ T cells inhibit the growth of human GC cells in vitro and adoptive transfusions of the CD3+ T cells suppress the growth of GC xenograft in vivo. The effects may be caused by the larger ratios of infiltrated CD8+/CD4+ T cells, reduced infiltration of regulatory FOXP3+ T cells, and enhanced apoptosis induced by upregulation of Caspase-9/Caspase-3 and downregulation of Survivin. Our results reveal the potential immunotherapeutic value of H. pylori vaccine-activated CD3+ T cells in those with advanced GC.

The relationship between mouse lung adenocarcinoma at different stages and the expression level of exosomes in serum.

Objective: The purpose of this study is to investigate the relationship between N-methylN'-nitro-N-nitrosoguanidine (MNNG)-induced lung adenocarcinoma mouse of different stages and the level of exosomes in serum. Methods: Fifty KM mice (4 weeks old, weighing 18-22 g, female) were selected for experiment. MNNG was applied to induce lung adenocarcinoma in mice for model establishment. HE staining method was used to observe pathological changes of lung tissues. Exosomes in serum were extracted, and observed under a Transmission Electron Microscopy (TEM). Western blot was employed to examine the protein levels of exosome markers TSG101 and CD63. Results: HE staining results suggested MNNG-induced lung adenocarcinoma model mice were successfully constructed. The morphology of exosomes in serum of mouse model was observed under an Electron Microscopy, showing clear membrane structures presenting as saucer or concave hemisphere. In addition, the protein level of exosome marker CD63 in advanced mouse model was significantly up-regulated relative to that in the control group. Meanwhile, the expression of exosome marker protein TSG101 was found to be markedly increased in mouse models by comparison with that in control cases. Conclusion: In lung adenocarcinoma model mice, the occurrence and development of lung adenocarcinoma are greatly correlated with the expression level of exosomes in serum, which provides theoretical basis for the diagnosis of pathological staging of lung adenocarcinoma by exosomes.

New Nodule Type Found in the Lungs of Pomacea canaliculata, an Intermediate Host of Angiostrongylus cantonensis.

BACKGROUND: Pomacea canaliculata (P.canaliculata) lung nodules, were commonly caused by Angiostrongylus cantonensis infection. Here, we found a new nodule type without any parasites. METHODS: Overall, 447 P. canaliculata snails were collected in Ning Bo, Zhe Jiang, China in 2018. In order to exhibit the similarities and differences between two nodules types (2018, Huzhou Zhejiang, China), both types were collected in formalin for tissue pathological sectioning. Besides, to obtain the microbial community of the new nodule, the 18S ribosomal RNA (rRNA) gene of it was amplified and analyzed using the Illumina second-generation sequencing platform. RESULTS: Although two nodules were found in the lungs of P. canaliculata, they were different in shape and pathology. Illumina sequencing indicated Poterioochromonas sp., a species of golden algae, might be the causing agent of the new nodule. CONCLUSION: We firstly found a new pathological nodule type in the lungs of P. canaliculata, and this nodule might be induced by golden algae infection, however, the direct link between the golden algae and the new nodules, as well as the nodules' impact on the snails' physiology and A. cantonensis infection require further study.

Circulating 25-hydroxyvitamin D level and prognosis of lung cancer patients: A systematic review and meta-analysis.

BACKGROUND: Previous studies suggested a possible influence of circulating 25-hydroxyvitamin D [25(OH)D] level on the prognosis of lung cancer patients, but conflicting findings were reported. A systematic review and meta-analysis was thus conducted to comprehensively assess the influence of circulating 25(OH)D level on the prognosis of lung cancer patients. METHODS: Prospective or retrospective cohort studies assessing the influence of circulating 25(OH)D level on the prognosis of lung cancer patients were considered eligible. Hazard Ratios (HR) were pooled using meta-analysis. RESULTS: Eight studies with 2166 lung cancer patients were included. Meta-analysis of unadjusted HRs from four studies showed low circulating 25(OH)D level was significantly correlated with poor overall survival in lung cancer (HR=1.30, 95%CI 1.08-1.55, P=0.004). Meta-analysis of adjusted HRs from eight studies suggested that low circulating 25(OH)D level was not significantly correlated with poor overall survival (HR=1.25; P=0.13). However, sensitivity analysis suggested an obvious change in the pooled HRs when excluding single study by turns. When the study by Liu et al. was omitted, low circulating 25(OH)D level was significantly correlated with poor overall survival (HR=1.34; P=0.04). CONCLUSION: The present systematic review and meta-analysis suggested a correlation between low circulating 25(OH)D level and poor overall survival in lung cancer. More studies are needed to further validate the finding above.

Measurement uncertainty assessment of magnesium trisilicate column for determination of Sudan colorants in food by HPLC using C8 column.

This study aimed to conduct measurement uncertainty assessment of a new method for determination of Sudan colorants (Sudan I, II, III and IV) in food by high performance liquid chromatography (HPLC). Samples were extracted with organic solvents (hexane, 20% acetone) and first purified by magnesium trisilicate (2MgO·3SiO2). The Sudan colorants (Sudan I-IV) were also initially separated on C8 by gradient elution using acetonitrile and 0.1% (v/v) formic acid aqueous solution as the mobile phases and detected with diode-array detector (DAD). The uncertainty of mathematical model of Sudan I, II, III and IV is based on EURACHEM guidelines. The sources and components of uncertainty were calculated. The experiment gave a good linear relationship over the concentration from 0.4 to 4.0 µg/mL and spiked recoveries were from 74.0% to 97.5%. The limits of determination (LOD) were 48, 61, 36, 58 µg/kg for the four analytes, respectively. The total uncertainty of Sudan colorants (Sudan I, II, III and IV) was 810±30.8, 790±28.4, 750±27.0, 730±50.0 µg/kg, respectively. The recovery uncertainty was the most significant factor contributing to the total uncertainty. The developed method is simple, rapid, and highly sensitive. It can be used for the determination of trace Sudan dyes in food samples. The sources of uncertainty have been identified and uncertainty components have been simplified and considered.

[A bite of parasite, experiences of application of documentaries into parasitology class teaching].

Medical parasitology education has been facing some difficulties, because it is a course of wide range, lacking clinical cases and concerned specimens of parasites currently. In addition, its relationship with life is not closely enough. All these reasons may impact the effect of class education negatively. Therefore, it is important to increase the vitality of parasitology education and diversify the instructional mode by using the resources from Internet. In recent years, the Discovery Channel has uploaded a documentary Monsters Inside Me online. This documentary is high professional and closely linked with parasitology. It maintains numbers of clinical cases about parasitic diseases. Each episode is about 3 minutes and shortly enough to be introduced into class teaching. However, this resource has not been fully used in domestic temporally. We found that direct introduction of the documentary into class teaching can enrich teaching forms to attract learning interest of students, and finally improve the teaching effect of class. Above that, another popular documentary A Bite of China involves many related knowledge points of parasitology. The appropriate usage of the knowledge can build up close linkage between book and life, which is extremely helpful to give students a deep impression of parasitology. In brief, it is our strong recommendation to introduce the documentary Monsters Inside Me into class.

Spectrophotometric determination of silicon tetrahydride in the air of workplace.

A new, simple and sensitive method was developed for the determination of silicon tetrahydride in the air of workplace in this study. The alkaline resin-based spherical activated carbon was used to collect sample of silicon tetrahydride at workplace. Silicon tetrahydride was then desorbed from active carbon in 100°C hot water. After reacting with ammonium molybdate, oxalic acid and 1,2,4-trichlorobenzene alpha-naphthol amino sulfonic acid under acid condition, silicon tetrahydride was transformed into silicon molybdenum blue. The absorbance of silicon molybdenum blue was quantitatively measured at the wavelength of 680 nm. The results showed that the average sampling efficiency and desorption efficiency were 97.53% and 94.94%, respectively by this method. Detection limits were 0.054 µg/mL for the spectrophotometric method and 0.14 mg/m(3) for the determination of silicon tetrahydride in the air of workplace (sampling volume was 7.5 L). The conversion rate of silicon tetrahydride gradually decreased when storage time of samples was extended. The descent rate of sample was less than 10% when the sample was sealed for 7 days in the room temperature. It was concluded that this spectrophotometric method can be successfully used to determine silicon tetrahydride in the worksites.

[Evaluation of transfection effectiveness using fluorescein-labelled oligonucleotides and entraster-R siRNA transfection into Plasmodium falciparum].

The cultured Plasmodium falciparum parasites were synchronized twice by 5% sorbitol treatment twice (8-hour window), and then incubated at 37 degrees C for 16 h. Parasites were transfected with fluorescein-labelled oligonucleotides (group A) or fluorescein-labelled oligonucleotides+Entranster-R siRNA transfection reagent (group B). After 5 h a part of parasites was evaluated by fluorescence microscopy and flow cytometry. The rest of parasites were washed with RPMI 1640 medium, and then incubated with 500 microl new medium containing 2% fresh erythrocytes for another 12 h, and detected by flow cytometry. The fluorescein-labelled oligonucleotides were localized in erythrocytes in group B, but nearly no fluorescence was observed for group A. Flow cytometry analysis indicated that the transfection efficiency of group B [(47.40 +/- 3.39)%] was higher than that of group A [(0.60 +/- 0.27)%]. In the second cell cycle, the transfection efficiency in group B was (26.85 +/- 2.90)%, while that of group A was nearly zero. The results indicated that Entranster-R siRNA transfection reagent may increase the oligonucleotides transfection efficiency.

[Application of mind map in teaching of medical parasitology].

To improve the teaching quality of medical parasitology, mind map, a simple and effective learning method, was introduced. The mind map of each chapter was drawn by teacher and distributed to students before the class. It was helpful for teacher to straighten out the teaching idea, and for students to grasp the important learning points, perfect the class notes and improve learning efficiency. The divergent characteristics of mind map can also help to develop the students' innovation ability.

Hepatitis C virus F protein inhibits cell apoptosis by activation of intracellular NF-kappaB pathway.

AIM: To observe the influence of HCV F protein on apoptosis of HepG2 cells, and explore the association between F protein and NF-kappaB signal pathway. METHODS: HCV 1b F gene containing HepG2-F cells and HCV 1b C gene containing HepG2-C cells were treated with 100 IU/mL TNF-alpha, and analyzed by flow cytometry, Western blotting, and dual luciferase reporter assay. Empty plasmid pcDNA3.1(+) containing HepG2-3.1 cells were used as control. RESULTS: (i) With the treatment of TNF-alpha for 18 h, the apoptosis rates (AR) of HepG2-F and HepG2-3.1 cells were 0.41% (+/- 0.11%) and 37.43% (+/- 2.03%) respectively, while that of HepG2-C was 4.07% (+/- 0.18%). At 36 h after TNF-alpha treatment, the AR of HepG2-F and HepG2-3.1 cells were 10.03% (+/- 0.41%) and 44.63% (+/- 3.37%), and that of HepG2-C was 14.95% (+/- 0.85%). (ii) After the treatment of TNF-alpha for 0.5-18 h, the p65 contents in the whole cells of HepG2-F and HepG2-3.1 showed no significant difference (P = 0.34, t = 1.08), while the p65 contents in the nucleus of HepG2-F and HepG2-3.1 cells were 3.8-1.9 times and 1.8-1.0 times higher than that in the non-treated cells (P = 0.013, t = 4.25). (iii) The relative luciferase unit (RLU) of the HepG2 cells, co-transfected with pcDNA3.1-F and pNF-kappaB-luc, and then treated with TNF-alpha (100 IU/mL) for 18 h, showed a pcDNA3.1-F dose-dependent increase. CONCLUSION: HCV F protein can over-activate NF-kappaB signal pathway, which makes HepG2-F cells able to resist TNF-alpha induced apoptosis.

Hepatitis C virus F protein up-regulates c-myc and down-regulates p53 in human hepatoma HepG2 cells.

OBJECTIVES: Hepatitis C virus (HCV) F protein is a newly identified protein encoded by an alternative open reading frame that +1 overlaps core-encoding gene. It has been found that regulation of c-myc and p53 genes by HCV core protein is involved in liver cancer genesis. We wondered whether HCV F protein exerts similar or adverse regulatory effects on the transcription of c-myc and p53 genes. METHODS: HCV F gene-containing, plasmid pcDNA3.1-F and HCV core gene-containing pcDNA3.1-C were constructed and transiently transfected into HepG(2) cells. Real-time quantitative PCR or Western blotting was used to determine the changes at transcription or translation levels of c-myc and p53 genes. RESULTS: The transcription level of c-myc was much higher in pcDNA3.1-F transfected cells than those without plasmid transfected. Whereas the level of p53 transcription in pcDNA3.1-F transfected cells was lower than those in the parental cells. Moreover, levels of c-myc expression were up-regulated and those of p53 expression were down-regulated by HCV F protein. CONCLUSIONS: HCV F protein is of regulatory properties in cellular oncogene c-myc and anti-oncogene p53, which may be implicated in the formation of hepatocellular carcinoma.

[Antigenicity of hepatitis C virus F protein and serum prevalence of anti-F in HCV-infected patients].

OBJECTIVE: To examine the antigenicity of hepatitis C virus (HCV) F protein and investigate serum prevalence of anti-F in HCV-infected patients. METHODS: Eleven pairs of overlapping primers were used to synthesize the full-length HCV f gene, from which the truncated HCV f65 gene fragment was amplified by PCR. HCV f65 gene was then cloned into pET32a(+), and transformed into E. coli strain Plyss (DE3). This recombinant E.coli was induced by IPTG for the production of HCV F65 protein. The expressed HCV F65 protein, purified by Ni-NTA agarose, was further used in ELISA to detect serum anti-F, and to immunize rabbits for making polyclonal anti-F. The rabbit polyclonal anti-F was purified by Staphylococcus aureus protein A agarose. RESULTS: After recombinant pET32a(+)-f65 was constructed successfully, HCV F65 protein was expressed and purified. The purified HCV F65 protein was used as a capture antigen in ELISA to detect serum anti-F in HCV infected patients (n = 30). The result showed that the mean A450 value and the positive rate of serum anti-F were 0.125+/-0.061 and 63.3%, respectively. The rabbit-derived polyclonal anti-F reacted specifically with HCV F65 protein, of which the titer was 1:30,000. CONCLUSION: Our expressed HCV F65 protein is of antigenicity, and can be used to determine serum anti-F. Anti-F IgG does exist in the sera of the HCV-infected patients. Moreover, the rabbit-derived polyclonal anti-F can be used to detect HCV F protein.