The presence of Prevotella melaninogenica within tissue and preliminary study on its role in the pathogenesis of oral lichen planus.

OBJECTIVE: Oral lichen planus (OLP) is a chronic inflammatory disease that occurs in the oral mucosa with characteristic white striations lesions, recurrent erosions, and pains. The etiology and pathogenesis of OLP are still unclear. MATERIALS AND METHODS: We analyzed the bacterial community structure of buccal mucosa in patients with OLP and normal controls by high-throughput sequencing. Fluorescence in situ hybridization (FISH) was used to detect Prevotella melaninogenica (P. melaninogenica) in 13 OLP samples and 10 controls. The amounts of P. melaninogenica in OLP buccal mucosa and the expression of inflammatory cytokines in co-culture of mouse-derived macrophages with P. melaninogenica were detected by RT-qPCR. RESULTS: The P. melaninogenica was more abundant in OLP than in healthy controls, and the differences were significant at the level of the phylum, family, genus, and species (p < .05). FISH showed that P. melaninogenica can invade the epithelium and even the lamina propria of OLP, while no invasion was found in the normal mucosa. Prevotella melaninogenica can adhere to and invade macrophages and then activate the transcription of IL-1ß, IL-6, and TNF-α in NF-κB signaling pathway. CONCLUSION: Prevotella melaninogenica may be involved in the pathogenic process of OLP, and its specific mechanism deserves further study.

[Screening and analysis of peptides specifically binding to the schistosomulum tegument of Schistosoma japonicum].

OBJECTIVE: To screen and analyze the peptides in 12 phage-display peptide library specifically binding to the schistosomulum, not cercaria, tegument of Schistosoma japonicum. METHODS: A 12 phage-display peptide library was screened with the S. japonicum schistosomula and cercariae as the target cells for biopanning by degrees, 15 positive clones were picked randomly and deduced by DNA sequencing. According the sequencing result, ELISA test, elution recovery test and immunohistochemical staining were performed to determine the specificity of the phages to the tegument. To further examine its binding properties, the positive peptide conjugated to RhB and recombinant pEGFP-C2 plasmid were similarly synthesized. RESULTS: After 3 rounds of biopanning, the phage recovery rate increased from 3.50 x 10(-5)% to 3.20 x 10(-2)%, indicating that the phage library was successfully enriched in the tegument of schistosomula. The analyzed sequences were identical with 3 peptide sequence of ZL6, ZL4 and ZL1. ELISA showed that the P/N value of MppZL4, MppZL6 and MppZL binding the schistosomulum membrane protein was 6.72, 3.65 and 2.22, while 1.58, 5.15 and 1.20 of binding the membrane protein of cercariae, respectively. Elution recovery test showed that the elution recovery rate of MppZL4 [(4.60 +/- 0.27) x 10(-2)%] was much higher than that of MppZL6 [(2.10 +/- 0.23) x 10(-3)%], MppZL1 [(1.20 +/- 0.28) x 10(-3)%] and M13KE [(1.30 +/- 0.60) x 10(-7)%] (P<0.01). Immunohistochemical staining showed that MppZL4 specifically bound to the tegument of schistosomula with a positive rate of 83.0% (83/100). Fluorescent microscopy revealed that the synthesized RhB-ZL4 bound to the tegument of schistosomula. The ZL4/pEGFP-C2 plasmid was introduced into juvenile S. japonicum and expressed in the parasite. CONCLUSION: The peptide of ZL4 specifically binds to the schistosomulum tegument but not to that of cercaria.

Inhibition of GSDMD-mediated pyroptosis triggered by Trichinella spiralis intervention contributes to the alleviation of DSS-induced ulcerative colitis in mice.

BACKGROUND: Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is increasing worldwide. Although there is currently no completely curative treatment, helminthic therapy shows certain therapeutic potential for UC. Many studies have found that Trichinella spiralis (T.s) has a protective effect on UC, but the specific mechanism is still unclear. METHODS: Balb/c mice drank dextran sulfate sodium (DSS) to induce acute colitis and then were treated with T.s. In vitro experiments, the LPS combination with ATP was used to induce the pyroptosis model, followed by intervention with crude protein from T.s (T.s cp). Additionally, the pyroptosis agonist of NSC or the pyroptosis inhibitor vx-765 was added to intervene to explore the role of pyroptosis in DSS-induced acute colitis. The degree of pyroptosis was evaluated by western blot, qPCR and IHC, etc., in vivo and in vitro. RESULTS: T.s intervention significantly inhibited NLRP3 inflammasome activation and GSDMD-mediated pyroptosis by downregulating the expression of pyroptosis-related signatures in vitro (cellular inflammatory model) and in vivo (DSS-induced UC mice model). Furthermore, blockade of GSDMD-mediated pyroptosis by the caspase-1 inhibitor vx-765 has a similar therapeutic effect on DSS-induced UC mice with T.s intervention, thus indicating that T.s intervention alleviated DSS-induced UC in mice by inhibiting GSDMD-mediated pyroptosis. CONCLUSION: This study showed that T.s could alleviate the pathological severity UC via GSDMD-mediated pyroptosis, and it provides new insight into the mechanistic study and application of helminths in treating colitis.

[Preparation of agar-paraffin double-embedded longitudinal sections of Schistosoma japonicum].

Schistosoma japonicum adults are pre-embedded in a double-layer agar and made the block, then dehydrated with alcohol, isobutyl alcohol and n-butyl alcohol. Various staining procedures can be conducted after conventional sectioning and dewaxing. Complete longitudinal serial sections of the pre-embedded worms can be obtained, and the desired sections can be easily located accurately.

[Pathogen identification of 10 suspected cases of sparganosis mansoni].

OBJECTIVE: To diagnose 10 cases of clinically suspected cases of sparganosis mansoni by pathogen identification. METHODS: In the period from August 2009 to August 2011, 10 biopsy specimens were obtained from 10 patients of four hospitals to identify the pathogen. Among the 10 cases, 4 cases showed abdominal subcutaneous mass, 3 showed eyelid swelling, 1 displayed brain lesions, 1 showed pulmonary mass, and 1 showed pleural effusion. There was one parasite each from three patients with eyelid swelling, and one patient with abdominal subcutaneous mass, which were observed by naked eye and microscope morphologically and histologically. Specimens from other six cases were examined by microscope after paraffin embedding, sectioning, and HE staining. For further identification, the parasite biopsy tissue specimens were detected by immunohistochemistry with Sparganum mansoni-immunized rabbit serum as the primary antibody. RESULTS: Three intact worms, from three patients with eyelid swelling, showed typical S. mansoni morphological characteristics. One residue parasite from the abdominal subcutaneous mass showed network structures and full of calcareous corpuscles in the body under microscope same as that of S. mansoni. The histological structure in three of the six sections showed typically the body wall with folds, which was dense, thick and deeply eosine stained, part of the tegument outside was covered by micro-hairs. In the worm body there was net-like loose structure and calcareous corpuscles without cavity. The structure of the other three worm sections was atypical. The six worm sections were positive by immunohistochemical detection. CONCLUSION: The 10 clinically suspected cases are diagnosed as sparganosis mansoni.

[Screening and characterization of peptides specifically binding to the schistosomulum tegument of Schistosoma japonicum].

OBJECTIVE: To screen and analyze the peptides in 12 phage-display peptide library specifically binding to the schistosomulum tegument of Schistosoma japonicum. METHODS: A 12 phage-display peptide library was screened with the S. japonicum schistosomula as the target cells for biopanning by degrees, positive clones picked randomly were deduced by DNA sequencing. According the sequence seeing result, immunohistochemical staining was performed to determine the specificity of the phages to the tegument. To test their targeting efficacy, the interested phage clones were infused back to the mice infected with S. japonicum, mice were sacrificed 2.5 hours later, and the phage distribution in the liver and the tegument of schistosomula was appraised, respectively. RESULTS: After 3 rounds of biopanning, the phage recovery rate increased from 0.77 x 10(-8) to 0.75 x 10(-5), indicating that the phage library was successfully enriched in the tegument of schistosomula. Seventy-five percent (15/20) of the analyzed sequences were identical with a sequence of QHPRIRKOOOOO. The immunohistochemical stainings showed this sequence specifically binding to the tegument. In vivo titering displayed that this sequence selectively targeted the tegument. CONCLUSION: The peptide of QHPRIRKOOOOO specifically binds to the schistosomulum tegument.

Gut microbiota contributes to the distinction between two traditional Chinese medicine syndromes of ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is considered to be closely associated with alteration of intestinal microorganisms. According to the traditional Chinese medicine (TCM) theory, UC can be divided into two disease syndromes called Pi-Xu-Shi-Yun (PXSY) and Da-Chang-Shi-Re (DCSR). The relationships among gut microbiota, TCM syndromes, and UC pathogenesis have not been well investigated. AIM: To investigate the role of gut microbiota in UC and the distinction of microbiota dysbiosis between PXSY and DCSR syndromes. METHODS: From May 2015 to February 2016, UC patients presenting to LongHua Hospital who met the established inclusion and exclusion criteria were enrolled in this retrospective study. Fresh stool specimens of UC patients with PXSY or DCSR were collected. The feces of the control group came from the health examination population of Longhua Hospital. The composition of gut bacterial communities in stool samples was determined by the pyrosequencing of 16S ribosomal RNA. The high-throughput sequencing reads were processed with QIIME, and biological functions were predicted using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States. RESULTS: The composition of gut bacterial communities in 93 stool samples (30 healthy controls, 32 patients with PXSY syndrome, and 31 patients with DCSR syndrome) was determined by the pyrosequencing of 16S ribosomal RNA. Beta diversity showed that the composition of the microbiota was different among the three groups. At the family level, Porphyromonadaceae, Rikeneliaceae, and Lachnospiraceae significantly decreased while Enterococcus, Streptococcus, and other potential pathogens significantly increased in UC patients compared to healthy subjects. At the genus level, Parabacteroides, Dorea, and Ruminococcus decreased while Faeca-libacterium showed increased abundance in UC compared to healthy controls. Five differential taxa were identified between PXSY and DCSR syndromes. At the genus level, a significantly increased abundance of Streptococcus was observed in DCSR patients, while Lachnoclostridium increased in PXSY patients. The differential functional pathways of the gut microbiome between the PXSY and DCSR groups mainly included lipid metabolism, immunity, and the metabolism of polypeptides. CONCLUSION: Our study suggests that the gut microbiota contributes to the distinction between the two TCM syndromes of UC.

[Biological identification on sub-cultivation cells of Schistosoma japonicum adult worms in vitro].

Cultivation of cells from 30-day old Schistosoma japonicum (S.j) adult worms showed that the growth features of the cells were semi-floating and accumulative. The survival rate of the primary cells, passage cells prior to the 5th generation and recovered cells was all up to 90%. Phases of cell division were observed during cultivation. Chromosome karyotype of the 5th generation cells possessed diploid feature of the blood-flukes (2n=8 in number). Ultrastructure of the 5th generation cells showed that four types of cells in normal morphology and three types of cells in abnormal morphology were both viewed. It is suggested that some of the cells from S.j adult worms were subcultured successfuly in the 1640-40 defined medium.

[Immunological screening of Toxoplasma tachyzoite cDNA expression libraries with serum from infected rats].

OBJECTIVE: To screen and identify the potential candidates for the development of toxoplasmosis vaccine. METHODS: Rats were infected with Toxoplasma gondii (T. gondii) RH strain and their sera were used as a probe to screen T. gondii tachyzoite cDNA expression libraries. The positive clones were analyzed by PCR amplification and DNA sequencing. RESULTS: Thirteen positive clones were obtained from about 4 x 10(5) phage plagues after three rounds of screening. The size of the inserts ranged from 0.45 kb to 2.4 kb. A BLAST search of all available sequence databases using the partial sequences from four positive clones (L1, L2, L4, L5) showed that the sequence of L2 clone was identical with T. gondii P24 major antigen gene (TgP24). Clone L4 had a high homology with Saccharum officinarum pyruvate orthophosphate dikinase. There is no significant hit of any sequences to clone L1, suggesting that L1 could be a novel gene(GenBank accession number AY180109), named T.g-R1, which encodes a non-transmembrane protein with 134 amino acid open reading frame. PROSCAN analysis of the T.g-R1 amino acid sequence showed that this gene product contains two protein kinase C phosphorylation site, two casein kinase II phosphorylation site, one N-myristoylation site and one microbodies C-terminal targeting signal. Clone L5 was a small partial fragment. CONCLUSION: The identification of positive clones provides a possible way for the development of toxoplasmosis vaccine.

Multiplex real-time PCR for RRM1, XRCC1, TUBB3 and TS mRNA for prediction of response of non-small cell lung cancer to chemoradiotherapy.

BACKGROUND: This study was aimed to establish a novel method to simultaneously detect expression of four genes, ribonucleotide reductase subunit M1(RRM1), X-ray repair cross-complementing gene 1 (XRCC1), thymidylate synthase (TS) and class III ß-tubulin (TUBB3), and to assess their application in the clinic for prediction of response of non-small cell lung cancer (NSCLC) to chemoradiotherapy. MATERIALS AND METHODS: We have designed four gene molecular beacon (MB) probes for multiplex quantitative real-time polymerase chain reactions to examine RRM1, XRCC1, TUBB3 and TS mRNA expression in paraffin-embedded specimens from 50 patients with advanced or metastatic carcinomas. Twenty one NSCLC patients receiving cisplatin- based first-line treatment were analyzed. RESULTS: These molecular beacon probes could specially bind to their target genes in homogeneous solutions. Patients with low RRM1 and XRCC1 mRNA levels were found to have apparently higher response rates to chemoradiotherapy compared with those with high levels of RRM1 and XRCC1 expression (p<0.05). The TS gene expression level was not significantly associated with chemotherapy response (p>0.05). CONCLUSIONS: A method of simultaneously detecting four molecular markers was successfully established and applied for evaluation of chemoradiotherapy response. It may be a useful tool in personalized cancer therapy.

[Improvement in the dot immunogold filtration assay].

OBJECTIVE: To improve the filtrating unit of the dot immunogold filtration assay (DIGFA) to reduce its cost and make it suitable for fieldwork. METHODS: A thick round filtrating paper disc was made to replace the plastic filtrating box, and their results were compared. RESULTS: The size of the self-made filtrating paper disc was smaller and the cost was lower. The efficacy of antibody examination by our paper disc was the same as that of the usual plastic filtrating box. CONCLUSION: In some aspects, the round filtrating disc is better than the plastic filtrating box.

[Construction and sequencing of recombinant plasmid pcDNA3/GRA1 from Toxoplasma gondii].

OBJECTIVE: To construct a mammalian expression plasmid pcDNA3/GRA1 to express dense granules antigen-1 (GRA1) of Toxoplasma gondii, and to lay a foundation for further studying the protective immunity of pcDNA3/GRA1 as a DNA vaccine. METHODS: The GRA1 opening reading frame (ORF) was amplified with two specific primers. The ORF and plasmid pcDNA3 were digested with EcoR I and Xho I respectively and the ORF was ligated into the pcDNA3 at polylinker. The recombinant vector pcDNA3/GRA1 was characterized by PCR, restriction enzyme digestion, and sequencing analysis. RESULTS: The expected ORF, 573 bp long, was amplified by PCR, and inserted into plasmid pcDNA3. PCR, restriction enzyme digestion and sequencing analysis showed that pcDNA3/GRA1 contained GRA1 ORF with the right orientation. CONCLUSION: The mammalian expression vector pcDNA3/GRA1 is successfully constructed.