Endothelial Cell-Derived Extracellular Vesicles Target TLR4 via miRNA-326-3p to Regulate Skin Fibroblasts Senescence.

Backgrounds: Skin aging could be regulated by the aberrant expression of microRNAs. In this manuscript, we explain that endothelial cell-derived extracellular vesicles could act as supporters to deliver exogenous miR-326-3p to accelerate skin fibroblasts senescence. Methods: ß-galactosidase senescence staining assay, Hoechst 33258 apoptosis staining assay, and Ki67 staining assay were used to evaluate the biological function of mouse skin fibroblasts. Real-time PCR was applied to assay miRNAs and mRNAs expressions. Western blot was used to detect TLR4 protein expression. The target gene of miRNA were identified using a double luciferase reporter assay. miR-326-3p mimic/inhibitor and siRNA-TLR4 can demonstrate a nonnegligible link between miR-326-3p-TLR4 and skin aging. Results: In coculture experiment, senescence endothelial cells could promote the skin fibroblasts senescence and apoptosis via extracellular vesicles pathway. In contrast, miR-326-3p mimics accelerated senescence and apoptosis of skin fibroblasts, while miR-326-3p inhibitor could dramatically delay skin fibroblasts senescence and apoptosis. TLR4 was proved to be a miR-326-3p directly target gene via double luciferase assay. After skin fibroblasts transfected with siRNA-TLR4, cellular senescence and apoptosis were significantly increased. Furthermore, the skin tissues of aging mice were shown with overexpression of miR-326-3p and decrease of TLR4 gene and protein expression levels. Conclusions: Endothelial cell-derived extracellular vesicles delivery of miR-326-3p was found to have a function in skin fibroblasts via target TLR4. Therefore, endothelial cell-derived extracellular vesicles in antiaging therapies might be a new treatment way for delaying skin aging.

[Strategy research and analysis on 10 590 cases of invalid ambulance attendance in prehospital emergency care in Nanshan District of Shenzhen from 2014 to 2018].

OBJECTIVE: To provide a scientific basis for reducing the rate of invalid ambulance attendance and the establishment of grades dispatch system through a retrospective analysis of the invalid ambulance attendance in prehospital emergency care. METHODS: The data of the invalid ambulance attendance was collected in Huazhong University of Science and Technology Union Shenzhen Hospital (the only tertiary hospital of in Nanshan District) from 2014 to 2018, and the reasons of the invalid ambulance attendance, the time period during the invalid ambulance attendance occurred (every 3 hours was divided into one time period), and the reasons of ambulance calling were statistically analyzed. RESULTS: (1) The invalid ambulance attendance rates showed a fluctuating decrease trend year by year from 2014 to 2018, the rates were 31.22% (2 515/8 055), 26.94% (2 147/7 970), 29.80% (2 398/8 046), 25.69% (1 844/7 177) and 21.89% (1 686/7 703), respectively. The total invalid ambulance attendance rate was 27.19% (10 590/38 951) in the five years. (2) The top three reasons for the invalid ambulance attendance were cancelled calls, departure before the ambulance arrived, and going to hospital by themselves, accounting for 36.87%, 25.08%, and 17.03%, respectively. The constituent ratios of the causes of invalid ambulance attendance in each year were different with statistical significance (χ2 = 217.626, P < 0.001). (3) The top three time period of invalid ambulance attendance occurred at 21:00-23:59, 18:00-20:59 and 09:00-11:59, accounting for 16.86%, 14.95% and 13.54%, respectively. There was no statistical significance in the distribution of time period in each year (χ2 = 32.571, P = 0.252). (4) The top five reasons for ambulance calling of invalid ambulance attendance were fainting/syncope, trauma, alcoholism, traffic accident injuries, and brawls, accounting for 20.13%, 15.67%, 9.97%, 8.64%, and 6.45%, respectively, and there was statistically significant difference in the distribution of the reason for ambulance calls in each year (χ2 = 194.213, P < 0.001). CONCLUSIONS: The invalid ambulance attendance rate is high in Nanshan District of Shenzhen. Improving the professional level and triage ability of the dispatchers, improving the system construction of prehospital emergency care system and increasing social education are conducive to reduce invalid ambulance attendance rate.

TLR2 signal influences the iNOS/NO responses and worm development in C57BL/6J mice infected with Clonorchis sinensis.

BACKGROUND: Although the responses of inducible nitric oxide synthase (iNOS) and associated cytokine after Clonorchis sinensis infection have been studied recently, their mechanisms remain incompletely understood. In this study, we investigated the effects of toll-like receptor 2 (TLR2) signals on iNOS/nitric oxide (NO) responses after C. sinensis infection. We also evaluated the correlations between iNOS responses and worm development, which are possibly regulated by TLR2 signal. METHODS: TLR2 wild-type and mutant C57BL/6 J mice were infected with 60 C. sinensis metacercariae, and the samples were collected at 30, 60, 90 and 120 days post-infection (dpi). The total serum NO levels were detected using Griess reagent after nitrate was reduced to nitrite. Hepatic tissue samples from the infected mice were sliced and stained with hematoxylin and eosin (HE) to observe worm development in the intrahepatic bile ducts. The iNOS mRNA transcripts in the splenocytes were examined by real time reverse transcriptase polymerase chain reaction (qRT-PCR), and iNOS expression was detected by immunohistochemistry. RESULTS: Developing C. sinensis juvenile worms were more abundant in the intrahepatic bile ducts of TLR2 mutant mice than those of TLR2 wild-type mice. However, no eggs were found in the faeces of both mice samples. The serum levels of total NO significantly increased in TLR2 mutant mice infected with C. sinensis at 30 (t (5) = 2.595, P = 0.049), 60 (t (5) = 7.838, P = 0.001) and 90 dpi (t (5) = 3.032, P = 0.029). Meanwhile, no changes occurred in TLR2 wild-type mice compared with uninfected controls during the experiment. The iNOS expression in splenocytes showed unexpected higher background levels in TLR2 mutant mice than those in TLR2 wild-type mice. Furthermore, the iNOS mRNA transcripts in splenocytes were significantly increased in the TLR2 wild-type mice infected with C. sinensis at 30 (t (5) = 5.139, P = 0.004), 60 (t (5) = 6.138, P = 0.002) and 90 dpi (t (5) = 6.332, P = 0.001). However, the rising of iNOS transcripts dropped under the uninfected control level in the TLR2 mutant mice at 120 dpi (t (5) = -9.082, P < 0.0001). Both total NO and iNOS transcripts were significantly higher in the TLR2 mutant mice than those in the TLR2 wild-type mice at 30 (t (5) = 3.091/2.933, P = 0.027/0.033) and 60 dpi (t (5) = 2.667/6.331, P = 0.044/0.001), respectively. In addition, the remarkable increase of iNOS expressions was immunohistochemically detected in the splenic serial sections of TLR2 wild-type mice at 30 and 60 dpi. However, the expressions of iNOS were remarkably decreased in the splenocytes of both TLR2 wild-type and mutant mice at 120 dpi. CONCLUSIONS: These results demonstrate that TLR2 signal plays an important role in the regulation of iNOS expression after C. sinensis infection. TLR2 signal is also beneficial to limiting worm growth and development and contributing to the susceptibility to C. sinensis in which the iNOS/NO reactions possibly participate.

A novel genotype screening and phylogenetic analysis of Blastocystis hominis based on EF-1α.

Blastocystis hominis (B. h) is a kind of intestinal parasitic protozoa with the characteristic of worldwide distribution, morphology diversity, and diarrhea induced, etc. The traditional morphological classify was difficult to distinguish the genetic difference of B. h in different population and different geological strains. Recently, based on the small subunit ribosomal DNA sequence of B. h, the sequenced-tagged site (STS) primers was design, and successfully and widely applied to the distinguish the genotype of B. h, and however several B. h strains did not distinguish. To address it, the elongation factor-1 alpha (EF-1α) gene of B. h was screened due to its conservation here, and its specific primers were designed to distinguish the genotype of B. h. After epidemiological survey, the infection rate of B. h in boys was 14.74%, and that of girls was 15.05%, and the total infection rate of B. h was 14.93%. In total of 53 infection students, with the using of 7 pairs STS primers, 31 strains was validated by polymerase chain reaction (PCR), including 4 strains of Type 1, 17 strains of Type 3, 4 strains of Type 4, 1 strains of Type 6, and 5 strains of Type 7, and did not found the Type 2, Type 5 and mixture genotype. In the 23 unknown genotype strains of B. h, 15 strains were identified by PCR using EF-1α primers, and had a higher homology in the DNA sequence (70%), and was evolutionarily closer to the EF-1α sequence of S and H strains of B. h. This study indicated that STS primers could identify the genotype of B. h, and EF-1α primers as a novel diagnosis primers could auxiliary identify the unknown genotype strain of B. h, and exhibited a wide application on the identification of the genotype strain of B. h, and provided a significant reference on the study of B. h in clinic.

Comparative analysis of intestinal parasitic infections of outpatients in Guangxi medical university affiliated hospital in 2005 and 2013.

OBJECTIVE: To understand the hospital's status and trends of intestinal parasitic infections and to provide a reference for prevention. METHODS: Stool samples were treated by acid-ether centrifugation; iodine staining and direct-smearing were performed; intestinal parasites were examined under a microscope; characteristics of parasitic infections in population were analyzed using the descriptive epidemiological method. RESULTS: 10 kinds of parasites were detected; the infection rate of clonorchissinensis was the highest, followed by B. hominis, hookworm, whipworm and roundworm in order (x(2) = 131.188, 1261.928, 129.386, P < 0.01); The overall infection rates in 2013 and 2005 were 37.08% and 41.07% respectively, and the infection rate in 2013 was lower than that in 2005 (x(2) = 20.5003, P < 0.01); All the infection rates of clonorchissinensis, hookworm, whipworm and roundworm in 2013 were lower than those in 2005 (x(2) = 18.275, 45.449, 34.855, 12.435, P < 0.01); Both in 2005 and 2013, the male infection rate was higher than that in female (x(2) = 12.859, 24.924, P < 0.01); For male, the infection rate of clonorchissinensis was the highest, followed by B. hominis (x(2) = 313.621, 104.409, P < 0.01); for female, the infection rate of B. hominis was the highest, followed by clonorchissinensis (x(2) = 95.293, 43.357, P < 0.01). For male, the age group of 41~ had the highest infection rate of clonorchissinensis in 2005 (x(2) = 5.734, P < 0.05), and the age groups of 31~ and 41~ had the highest infection rate of clonorchissinensis in 2013 (x(2) = 8.908, P < 0.01); for female, both in 2005 and 2013, the age group of 21~, 31~, 41~ and 51~ had the highest infection rate of clonorchissinensis (x(2) = 6.508, 5.145, P < 0.05). There was no difference in male infection rate of B. hominis in 2005 (x(2) = 10.134, P > 0.05); in 2013, the age group of 0~ had the highest infection rate (x(2) = 3.825, P < 0.05); for women, it was the highest in the age groups of 11~, 21~ and 31~ in 2005 (x(2) = 10.459, P < 0.01), 0~ and 11~ in 2013 (x(2) = 53.669, P < 0.01). For Hookworm infection in male, the highest infection rate was found in the age group of 11~ 21~ and 61~ in 2005 (x(2) = 4.547, P < 0.05), 61~ and ≥ 71~ in 2013 (x(2) = 4.843, P < 0.05); for female, the highest infection rate was found in the age groups of 51~ and 61~ both in 2005 and 2013 (x(2) = 5.709, 5.958, P < 0.05). CONCLUSION: In Nanning city, although there was a decline in the infection rate of intestinal parasites of attenders compared with 8 years ago, the infection rate was still high and intestinal parasites were various; The infection rate of geohelminthes had been reduced to a low level; Clonorchissinensis and B. hominis were still the insect species with the highest infection rate.

[Effect of Excretory-secretory Products of Clonorchis sinensis on Nitric Oxide Production and NF-κB Activation in RAW264.7 Mouse Macrophages].

OBJECTIVE: To study the role of excretory-secretory products (ESPs) from Clonorchis sinensis in the production of nitric oxide (NO) and the activation of nuclear transcription factor kappa B (NF-κB) in the macrophages of RAW264.7 mouse. METHODS: 20 µg/ml of C. sinensis EPSs, the organic solvent extracts of EPSs (ESP-ex), and 0.1 µg/ml of lipopolysaccharide from Salmonella minnesota (LPS-SM) were used as stimulators in co-culture with RAW264.7 mouse macrophages as experimental groups. The Hank's balanced salt solution (HBSS) served as control. At the same time the RAW264.7 macrophages were stimulated with EPSs, ESP-ex, and LPS-SM, and then added 0.3 mmol/L of SMT, a specific inhibitor of iNOS as the interference groups. After co-culture for 18 days, the concentrations of NO2- in the culture supernatants were detected with Griess regents, and the activation of NF-κB was determined by transfection with a NF-κB-inducible reporter plasmid, pNiFty2-SEAP. The activities of secreted embryonic alkaline phosphatase (SEAP) in culture supernatants were quantified by using HEK-Blue™ detection medium and expressed as the value of optical density at 620 nm (A620 value). The intercellular activities of SEAP were determined by microscopic observation. RESULTS: After stimulation with both ESPs-ex and LPS-SM, the concentrations of NO2- in culture supernatants were (14.30 ± 1.62) and (14.10 ± 2.17) µmol/L, respectively, which were significantly higher than that of the control [(7.70 ± 0.95) µmol/L] (P < 0.05), and significantly decreased to (8.97 ± 0.81) and (4.96 ± 1.36) µmol/L after adding SMT, respectively (P < 0.05). However, the concentration of NO2- in ESPs stimulation group [(4.06 ± 0.62) µmol/L] was lower than that of the control (P < 0.05), and almost unchanged [(3.99 ± 0.87) µmol/L] after adding SMT (P > 0.05). SEAP activity in ESP group (0.836 ± 0.005) was significantly higher than that of the control [(0.097 ± 0.009) µmol/L] (P < 0.05). A strong blue color reaction was observed in cells of ESP group. SEAP activity of ESPs-ex and LPS groups [(0.112 ± 0.004), (0.116 ± 0.009) µmol/L] was slightly higher than that of the control (P > 0.05), and blue color reaction was observed in some cells. CONCLUSION: ESPs from C. sinensis can stimulate NF-κB activation in RAW264.7 cells. The water-soluble components of ESPs can inhibit the NO production, while ESPs-ex and LPS-SM can promote the NO production.

Inducible nitric oxide synthase response and associated cytokine gene expression in the spleen of mice infected with Clonorchis sinensis.

Clonorchis sinensis is a food-borne parasite that induces a permanent increase of nitrosation in the body upon infection. The spleen is an important secondary lymphoid organ for the regulation of immune responses locally and in the whole body. However, the functions and mechanisms of the spleen in nitric oxide (NO) responses after C. sinensis infection remain unknown. In this study, BALB/c mice were infected with 20, 40, and 80 C. sinensis metacercariae to simulate mild, moderate, and severe infections, respectively. We examined the expression of inducible nitric oxide synthase (iNOS) in the spleen and the relevant cytokine transcription in splenocytes from the mice infected with different amounts of metacercariae. The iNOS of the mice infected with 80 metacercariae was expressed in the spleen as early as 10 days post-infection (dpi) and gradually increased until 90 dpi. The iNOS expression in the mice infected with 40 metacercariae was detected only at 45 and 90 dpi, but not in the mice infected with 20 metacercariae. The level of interferon (IFN)-γ messenger RNA (mRNA) transcription in splenocytes significantly increased at 10 and 20 dpi (P < 0.05) in response to mild/moderate infection but gradually decreased to normal levels after 45 dpi. The level of IL-12p35 mRNA transcription did not change at 10 and 20 dpi but significantly decreased after 45 dpi under moderate/severe infection (P < 0.05/0.01/0.001). The level of IL-18 mRNA transcription significantly increased at 10 dpi (P < 0.05/0.01) but significantly decreased after 20 dpi (P < 0.05/0.01/0.001). These results suggest that spleen is an important organ for iNOS/NO responses, which correspond to the severity of C. sinensis infection, but cannot be attributed to the expression of the Th1 cytokines.

Pathological Lesions and Inducible Nitric Oxide Synthase Expressions in the Liver of Mice Experimentally Infected with Clonorchis sinensis.

The nitric oxide (NO) formation and intrinsic nitrosation may be involved in the possible mechanisms of liver fluke-associated carcinogenesis. We still do not know much about the responses of inducible NO synthase (iNOS) induced by Clonorchis sinensis infection. This study was conducted to explore the pathological lesions and iNOS expressions in the liver of mice with different infection intensity levels of C. sinensis. Extensive periductal inflammatory cell infiltration, bile duct hyperplasia, and fibrosis were commonly observed during the infection. The different pathological responses in liver tissues strongly correlated with the infection intensity of C. sinensis. Massive acute spotty necrosis occurred in the liver parenchyma after a severe infection. The iNOS activity in liver tissues increased, and iNOS-expressing cells with morphological differences were observed after a moderate or severe infection. The iNOS-expressing cells in liver tissues had multiple origins.

[Identification of Clonorchis sinensis metacercariae based on PCR targeting ribosomal DNA ITS regions and COX1 gene].

OBJECTIVE: To identify Clonorchis sinensis metacercariae using PCR targeting ribosomal DNA ITS region and COX1 gene. METHODS: Pseudorasbora parva were collected from Hengxian County of Guangxi at the end of May 2013. Single metacercaria of C. sinensis and other trematodes were separated from muscle tissue of P. parva by digestion method. Primers targeting ribosomal DNA ITS region and COX1 gene of C. sinensis were designed for PCR and the universal primers were used as control. The sensitivity and specificity of the PCR detection were analyzed. RESULTS: C. sinensis metacercariae at different stages were identified by PCR. DNA from single C. sinensis metacercaria was detected by PCR targeting ribosomal DNA ITS region and COX1 gene. The specific amplicans have sizes of 437/549, 156/249 and 195/166 bp, respectively. The ratio of the two positive numbers in PCR with universal primers and specific primers targeting C. sinensis ribosomal DNA ITS1 and ITS2 regions was 0.905 and 0.952, respectively. The target gene fragments were amplified by PCR using COX1 gene-specific primers. The PCR with specific primers did not show any non-specific amplification. However, the PCR with universal primers targeting ribosomal DNA ITS regions performed serious non-specific amplification. CONCLUSION: C. sinensis metacercariae at different stages are identified by morphological observation and PCR method. Species-specific primers targeting ribosomal DNA ITS region show higher sensitivity and specificity than the universal primers. PCR targeting COX1 gene shows similar sensitivity and specificity to PCR with specific primers targeting ribosomal DNA ITS regions.

Systemic and local mucosal immune responses induced by orally delivered Bacillus subtilis spore expressing leucine aminopeptidase 2 of Clonorchis sinensis.

Human clonorchiasis caused by Clonorchis sinensis (C. sinensis) has been increasingly prevalent in recent years so that an effective measure is essential and urgent to control the infectious disease. Oral delivery of antigens from C. sinensis may be an important approach to effectively induce both systemic and local immune responses to anti-infection of the parasite. In the current study, we used Bacillus subtilis (B. subtilis) spores as a delivery vehicle to introduce leucine aminopeptidase 2 of C. sinensis (CsLAP2), an excretory/secretory antigen with high immunogenicity, expressing on their surface. SDS-PAGE, western blotting, and flow cytometry indicated that CsLAP2 was successfully expressed on the surface of B. subtilis spores (CotC-CsLAP2 spores). BALB/c mice were treated with spores intragastrically. On day 31 after the treatment, we found that mice intragastrically treated with CotC-CsLAP2 spores exhibited higher IgG, IgG1, IgG2a, and IgA level in sera as well as higher sIgA level in bile and intestinal lavage fluid compared to mice orally administrated with spores not expressing CsLAP2 (CotC spores) and naïve mice. The peak titer of IgG/IgA presented on day 31/49 after oral administration. IgG1 level was lower than IgG2a in group administrated with CotC-CsLAP2 spores. sIgA-secreting cells were obviously observed in intestinal epithelium of mice orally treated with CotC-CsLAP2 spores. After incubated with CotC-CsLAP2, the levels of IFN-γ, IL-6, IL-10, IL-17A, and TNF significantly increased in the supernatant of splenocytes isolated from mice orally treated with CotC-CsLAP2 spores, while there was no statistically significant difference of IL-4 level representing Th2 response among the groups. Our study demonstrated that oral administration of CsLAP2 delivered by B. subtilis spore elicited obvious systemic and local mucosal immunity. Secretory IgA and Th1-Th17 cellular immunity might involved in mechanisms of the immune response.

[Relationship between morphology and pathogenicity of Blastocystis hominis trophozoites].

Six hundred and eighty-six fresh fecal specimens were collected from outpatients (663 well-formed feces and 23 watery feces) during March 2011 to March 2012. All specimens were examined microscopically by direct smear and iodine stained method. B. hominis obtained from the human positive fecal specimens were cultured in LES medium, and inoculated into the abdominal cavity of 10 female mice of 6-8-week old. The abdominal fluid was examined with same methods. 103 of 686 patients were positive (80 well-formed feces and 23 watery feces). Micro-scopically, the granular form and vacuolated form of B. hominis trophozoites could be easily identified by direct smear and iodine staining in well-formed fecal specimens, showing ovoid in shape and about (13.2 +/- 0.2) microm in size. The trophozoites cultured in LES medium showed similar feature. But in the watery fecal specimens and mice ascites specimen, they were amorphous containing more granules. And their average size was (28.0 +/- 0.3) microm which was larger than the former. Moreover, the ameba form of B. hominis trophozoites was also detected in the 23 watery fecal specimen and mice ascites specimen. The trophozoites of B. hominis were varying in shape and size depending on their living environment.

[Pathogen associated molecular patterns of parasite].

With the research progress of classical pathogen associated molecular patterns (PAMPs) from pathogenic microbes, the presence of the parasite PAMPs has been confirmed. The parasite PAMPs show some differences from classical PAMPs in molecular structures, receptor binding ways, intracellular signal transduction pathways, and induced effects. This review focuses on the issues of parasite PAMPs.