Biological characteristics of a new long-chain fatty acid transport protein 1 from Trichinella spiralis and its participation in lipid metabolism, larval moulting, and development.

Long-chain fatty acid transport protein 1 (FATP1) is a member of the fatty acid transporter family. It facilitates transmembrane transport of fatty acids and participates in lipid metabolism. Lipids are essential components of the cell and organelle membranes of Trichinella spiralis. The nematode has lost the capacity to synthesise the necessary lipids de novo and has instead evolved to obtain fatty acids and their derivatives from its host. This study aims to ascertain the primary biological characteristics and roles of T. spiralis FATP1 (TsFATP1) in lipid metabolism, larval moulting, and the development of this nematode. The results show that TsFATP1 is highly expressed at enteral T. spiralis stages, mainly localised at the cuticle, the stichosome and the intrauterine embryos of the parasite. The silencing of the TsFATP1 gene by TsFATP1-specific dsRNA significantly decreases the expression levels of TsFATP1 in the worm. It reduces the contents of ATP, triglycerides, total cholesterol, and phospholipids both in vitro and in vivo. RNAi inhibits lipid metabolism, moulting, and the growth of this nematode. The results demonstrate that TsFATP1 plays an essential role in lipid metabolism, moulting, and the development of T. spiralis. It could also be a target candidate for the anti-Trichinella vaccine and drugs.

[Role of Toll-like receptor 2/4-nuclear factor-κB signaling pathway in invasion of Mycobacterium tuberculosis to mouse dendritic cells].

OBJECTIVE: To investigate the mechanism of Mycobacterium tuberculosis invasion to mouse dendritic cells (DC). METHODS: Mycobacterium tuberculosis strain H37Rv was co-cultured with mouse DC2.4 cells.The mRNA expression of Toll-like receptor 2/4(TLR2/4) in DC2.4 cells was detected by fluorescent quantitative real-time PCR and the protein expression of nuclear factor κB(NF-κB) was assessed by Western blotting.The extracellular concentration of tumor necrosis factor α(TNF-α) was measured by ELISA methods during Mycobacterium Tuberculosis invasion.Indirect immunofluorescent staining and flow cytometry assay were used to detect the expression of CD80 and CD86 on DC2.4 cells before and after invasion. RESULTS: The invasion of Mycobacterium tuberculosis in DC2.4 cells was observed after 2 h of co-incubation.The rates of invasion were (37.9±5.6)%,(51.2±7.6)%,(57.2±8.9)% and(63.9±6.8)% at 6,8,10 and 12 h after co-incubation,respectively.The mRNA expression level of TLR2 /4 was significantly increased at 6 h but decreased at 10 h after co-incubation.The expressions of NF-κB p65 and TNF-α were higher in DC2.4 cells after being invaded by 6,8,and 10 h and then gradually decreased.CD80 and CD86 expression were increased on DC2.4 at 6 h after co-incubation. CONCLUSION: Invasion of Mycobacterium tuberculosis strain H37Rv to DC might enhance its antigen-presenting function through activation of TLR2/4-NF-kB signaling pathway.

Comparison of vonoprazan-based dual therapy with vonoprazan-based bismuth quadruple therapy for treatment-naive patients with Helicobacter pylori infection: A propensity score matching analysis.

Vonoprazan, a novel acid suppressant and the first potassium-competitive acid blocker, has the potential to enhance the eradication rate of Helicobacter pylori due to its robust acid-suppressing capacity. This study aimed to compare the efficacy of vonoprazan-based dual therapy (vonoprazan-amoxicillin, VA) with vonoprazan-based bismuth quadruple therapy (VBQT) as a first-line treatment for H pylori infection. This retrospective single-center non-inferiority study was conducted in China. Treatment-naive H pylori-positive patients aged 18 to 80 received one of the 2 treatment regimens at our center. The VA group received vonoprazan 20 mg twice daily and amoxicillin 1000 mg 3 times daily for 14 days, whereas the VBQT group received vonoprazan 20 mg, amoxicillin 1000 mg, clarithromycin 500 mg, and bismuth potassium citrate 220 mg twice daily for 14 days. The eradication rate was evaluated 4 to 6 weeks after treatment using the carbon-13/14 urea breath test. Propensity score matching was used to analyze eradication rates, adverse events (AEs), and patient compliance between the 2 groups. Initially, 501 patients were included, and after propensity score analysis, 156 patients were selected for the study. Intention-to-treat analysis showed eradication rates of 87.2% (95% CI, 79.8-94.6%) for the VA group and 79.5% (95% CI, 70.5-88.4%) for the VBQT group (P = .195). Per-protocol analysis demonstrated rates of 94.4% (95% CI, 89.2-99.7%) for the VA group and 96.8% (95% CI, 92.4-100%) for the VBQT group (P = .507). Non-inferiority was confirmed between the 2 groups, with P values < .025. The VA group showed a lower rate of AEs (10.3% vs 17.9%, P = .250) compared to the VBQT group. There were no significant differences in patient compliance between the 2 groups. In treatment-naive patients with H pylori infection, both the 14-day VA and VBQT regimens demonstrated comparable efficacy, with excellent eradication rates. Moreover, due to reduced antibiotic usage, lower rate of AEs, and lower costs, VA dual therapy should be prioritized.

Galactomannan inhibits Trichinella spiralis invasion of intestinal epithelium cells and enhances antibody-dependent cellular cytotoxicity related killing of larvae by driving macrophage polarization.

Previous studies have shown that recombinant Trichinella spiralis galectin (rTsgal) is characterized by a carbohydrate recognition domain sequence motif binding to beta-galactoside, and that rTsgal promotes larval invasion of intestinal epithelial cells. Galactomannan is an immunostimulatory polysaccharide composed of a mannan backbone with galactose residues. The aim of this study was to investigate whether galactomannan inhibits larval intrusion of intestinal epithelial cells and enhances antibody-dependent cellular cytotoxicity (ADCC), killing newborn larvae by polarizing macrophages to the M1 phenotype. The results showed that galactomannan specially binds to rTsgal, and abrogated rTsgal facilitation of larval invasion of intestinal epithelial cells. The results of qPCR, Western blotting, and flow cytometry showed that galactomannan and rTsgal activated macrophage M1 polarization, as demonstrated by high expression of iNOS (M1 marker) and M1 related genes (IL-1ß, IL-6, and TNF-α), and increased CD86+ macrophages. Galactomannan and rTsgal also increased NO production. The killing ability of macrophage-mediated ADCC on larvae was also significantly enhanced in galactomannan- and rTsgal-treated macrophages. The results demonstrated that Tsgal may be considered a potential vaccine target molecule against T. spiralis invasion, and galactomannan may be a novel adjuvant therapeutic agent and potential vaccine adjuvant against T. spiralis infection.

Title: Le galactomannane inhibe l'invasion par Trichinella spiralis des cellules de l'épithélium intestinal et améliore la cytotoxicité cellulaire dépendante des anticorps tuant les larves en activant la polarisation des macrophages. Abstract: Des études antérieures ont montré que la galectine recombinante de Trichinella spiralis (rTsgal) est caractérisée par un motif de séquence de domaines de reconnaissance des glucides se liant au bêta-galactoside, et que la rTsgal favorise l'invasion larvaire des cellules épithéliales intestinales. Le galactomannane est un polysaccharide immunostimulateur composé d'un squelette mannane avec des résidus galactose. Le but de cette étude était de déterminer si le galactomannane inhibe l'intrusion larvaire des cellules épithéliales intestinales et améliore la cytotoxicité cellulaire dépendante des anticorps (CCDA) tuant les larves nouvelles-nées en polarisant les macrophages au phénotype M1. Les résultats ont montré que le galactomannane se liait spécialement au rTsgal et supprimait la facilitation du rTsgal sur l'invasion larvaire des cellules épithéliales intestinales. Les résultats de la qPCR, du Western blot et de la cytométrie en flux ont montré que le galactomannane et le rTsgal activaient la polarisation des macrophages M1, comme le démontre la forte expression de l'iNOS (marqueur de M1) et des gènes liés à M1 (IL-1ß, IL-6 et TNF-α), et l'augmentation des macrophages CD86+. Le galactomannane et le rTsgal ont également augmenté la production de NO. La capacité de destruction de la CCDA médiée par les macrophages sur les larves était également significativement améliorée dans les macrophages traités au galactomannane et au rTsgal. Les résultats ont démontré que Tsgal pourrait être considéré comme une molécule cible potentielle d'un vaccin contre l'invasion par T. spiralis, et que le galactomannane pourrait être un nouvel agent thérapeutique adjuvant et un adjuvant vaccinal potentiel contre l'infection à T. spiralis.

Application of a recombinant novel trypsin from Trichinella spiralis for serodiagnosis of trichinellosis.

BACKGROUND: The excretory/secretory (ES) antigen of Trichinella spiralis muscle larvae (ML) is currently the most widely used diagnostic antigen to detect T. spiralis infection. However, this antigen has certain drawbacks, such as a complicated ES antigen preparation process and lower sensitivity during the early phase of infection. The aim of this study was to investigate the features of a novel T. spiralis trypsin (TsTryp) and evaluate its potential diagnostic value for trichinellosis. METHODS: The TsTryp gene was cloned and recombinant TsTryp (rTsTryp) expressed. Western blotting and an enzyme-linked immunosorbent assay (ELISA) were performed to confirm the antigenicity of rTsTryp. The expression pattern and distribution signature of TsTryp at various life-cycle stages of T. spiralis were analyzed by quantitative PCR, western blotting and the immunofluorescence test. An ELISA with rTsTryp and ML ES antigens was used to detect immunoglobulins G and M (IgG, IgM) in serum samples of infected mice, swine and humans. The seropositive results were further confirmed by western blot with rTsTryp and ML ES antigens. RESULTS: TsTryp expression was observed in diverse T. spiralis life-cycle phases, with particularly high expression in the early developmental phase (intestinal infectious larvae and adults), with distribution observed mainly at the nematode outer cuticle and stichosome. rTsTryp was identified by T. spiralis-infected mouse sera and anti-rTsTryp sera. Natural TsTryp protease was detected in somatic soluble and ES antigens of the nematode. In mice infected with 200 T. spiralis ML, serum-specific IgG was first detected by rTsTryp-ELISA at 8 days post-infection (dpi), reaching 100% positivity at 12 dpi, and first detected by ES-ELISA at 10 dpi, reaching 100% positivity at 14 dpi. Specific IgG was detected by rTsTryp 2 days earlier than by ES antigens. When specific IgG was determined in serum samples from trichinellosis patients, the sensitivity of rTsTryp-ELISA and ES antigens-ELISA was 98.1% (51/52 samples) and 94.2% (49/52 samples), respectively (P = 0.308), but the specificity of rTsTryp was significantly higher than that of ES antigens (98.7% vs. 95.4%; P = 0.030). Additionally, rTsTryp conferred a lower cross-reaction, with only three serum samples in total testing positive from 11 clonorchiasis, 20 cysticercosis and 24 echinococcosis patients (1 sample from each patient group). CONCLUSIONS: TsTryp was shown to be an early and highly expressed antigen at intestinal T. spiralis stages, indicating that rTsTryp represents a valuable diagnostic antigen for the serodiagnosis of early Trichinella infection.

A novel trypsin of Trichinella spiralis mediates larval invasion of gut epithelium via binding to PAR2 and activating ERK1/2 pathway.

BACKGROUND: Proteases secreted by Trichinella spiralis intestinal infective larvae (IIL) play an important role in larval invasion and pathogenesis. However, the mechanism through which proteases mediate larval invasion of intestinal epithelial cells (IECs) remains unclear. A novel T. spiralis trypsin (TsTryp) was identified in IIL excretory/secretory (ES) proteins. It was an early and highly expressed protease at IIL stage, and had the potential as an early diagnostic antigen. The aim of this study was to investigate the biological characteristics of this novel TsTryp, its role in larval invasion of gut epithelium, and the mechanisms involved. METHODOLOGY/PRINCIPAL FINDING: TsTryp with C-terminal domain was cloned and expressed in Escherichia coli BL21 (DE3), and the rTsTryp had the enzymatic activity of natural trypsin, but it could not directly degrade gut tight junctions (TJs) proteins. qPCR and western blotting showed that TsTryp was highly expressed at the invasive IIL stage. Immunofluorescence assay (IFA), ELISA and Far Western blotting revealed that rTsTryp specifically bound to IECs, and confocal microscopy showed that the binding of rTsTryp with IECs was mainly localized in the cytomembrane. Co-immunoprecipitation (Co-IP) confirmed that rTsTryp bound to protease activated receptors 2 (PAR2) in Caco-2 cells. rTsTryp binding to PAR2 resulted in decreased expression levels of ZO-1 and occludin and increased paracellular permeability in Caco-2 monolayers by activating the extracellular regulated protein kinases 1/2 (ERK1/2) pathway. rTsTryp decreased TJs expression and increased epithelial permeability, which could be abrogated by the PAR2 antagonist AZ3451 and ERK1/2 inhibitor PD98059. rTsTryp facilitated larval invasion of IECs, and anti-rTsTryp antibodies inhibited invasion. Both inhibitors impeded larval invasion and alleviated intestinal inflammation in vitro and in vivo. CONCLUSIONS: TsTryp binding to PAR2 activated the ERK1/2 pathway, decreased the expression of gut TJs proteins, disrupted epithelial integrity and barrier function, and consequently mediated larval invasion of the gut mucosa. Therefore, rTsTryp could be regarded as a potential vaccine target for blocking T. spiralis invasion and infection.

Trichinellaspiralis C-type lectin mediates larva invasion of gut mucosa via binding to syndecan-1 and damaging epithelial integrity in mice.

C-type lectin (CTL) plays a vital role in parasite adhesion, invading host's cells and immune escape. The objective of this research was to explore whether recombinant T. spiralis CTL (rTsCTL) binding with syndecan-1 damages intestine epithelial integrity and mediates T. spiralis intrusion in mice. The results showed that rTsCTL interacted with syndecan-1 and activated STAT3 pathway in gut epithelium, decreased tight junctions (TJs) expressions and damaged gut epithelium integrity, promoted T. spiralis intrusion, and increased expression level of inflammatory cytokine and mucin. The syndecan-1 inhibitor (ß-xyloside) and STAT3 phosphorylation inhibitor (Stattic) significantly suppressed syndecan-1 expression and STAT3 pathway activation, reduced the expression levels of TJs, pro-inflammatory cytokines (TNF-α and IL-1ß), Muc2 and Muc5ac, and declined intestinal permeability in T. spiralis-infected mice. These results revealed that the inhibitors suppressed T. spiralis invasion and development in gut mucosa, decreased intestinal adult burdens and relieved gut inflammation. These findings further testified that the in vivo binding of TsCTL with syndecan-1 destroyed enteral mucosal epithelial integrity and promoted T. spiralis intrusion of gut mucosa via activating STAT3 pathway and decreasing TJs expression. TsCTL could be deemed as a promising vaccine target to interrupt T. spiralis infection.

Pediatric case of colonic perivascular epithelioid cell tumor complicated with intussusception and anal incarceration: A case report.

BACKGROUND: Perivascular epithelioid cell tumor (PEComa) represents a group of rare mesenchymal tumors. PEComa can occur in many organs but is rare in the colorectum, especially in children. Furthermore, PEComa is a rare cause of intussusception, the telescoping of a segment of the gastrointestinal tract into an adjacent one. We describe a rare case of pediatric PEComa complicated with intussusception and anal incarceration, and conduct a review of the current literature. CASE SUMMARY: A 12-year-old girl presented with abdominal pain and abdominal ultrasound suggested intussusception. Endoscopic direct-vision intussusception treatment and colonoscopy was performed. A spherical tumor was discovered in the transverse colon and removed by surgery. Postoperative pathologic analyses revealed that the tumor volume was 5.0 cm × 4.5 cm × 3.0 cm and the tumor tissue was located in the submucosa of the colon, arranged in an alveolar pattern. The cell morphology was regular, no neoplastic necrosis was observed, and nuclear fission was rare. The immunohistochemical staining results were as follows: Human melanoma black 45 (HMB 45) (+), cluster of differentiation 31 (CD31) (+), cytokeratin (-), melanoma-associated antigen recognized by T cells (-), smooth muscle actin (-), molleya (-), desmin (-), S-100 (-), CD117 (-), and Ki67 (positive rate in hot spot < 5%). Combined with the results of pathology and immunohistochemistry, we diagnosed the tumor as PEComa. Postoperative recovery was good at the 4 mo follow-up. CONCLUSION: The diagnosis of PEComa mainly depends on pathology and immunohistochemistry. Radical resection is the preferred treatment method.

Phlorizin Exerts Direct Protective Effects on Palmitic Acid (PA)-Induced Endothelial Dysfunction by Activating the PI3K/AKT/eNOS Signaling Pathway and Increasing the Levels of Nitric Oxide (NO).

BACKGROUND Sodium glucose transporter-2 inhibitors are the newest antidiabetic drugs that seem to be cardioprotective and can prevent type 2 diabetes in patients with high cardiovascular risks. Previous clinical trials have shown that these inhibitors can alleviate endothelial dysfunction, but the mechanism of action remains unknown. How SGLT inhibitor influences the release of NO in PA-induced HUVECs has never been reported. MATERIAL AND METHODS To explore the potential effects of the endothelial-protective mechanism of phlorizin and its impact on nitric oxide (NO), human umbilical vein endothelial cells (HUVECs) were incubated with palmitic acid (PA) and then treated with phlorizin. Western blotting was performed to assess the phosphorylation of AKT, eNOS, and IRS-1. To further explore potential targets, siRNA transfection was used to demonstrate the role of SGLT1 and SGLT2. RESULTS Phlorizin suppressed the expression of SGLT1 and SGLT2, activated the PI3K/AKT/eNOS signaling pathway, increased the output of NO, and promoted the consumption of glucose in PA-induced HUVECs. Through demonstrating siRNA suppression of the expression of SGLT1 and SGLT2 in PA-induced HUVECs, this study provides a new understanding of the mechanism behind SGLT1 and SGLT2. CONCLUSIONS Our data demonstrate that phlorizin ameliorates the endothelial dysfunction link with the activation of the PI3K/AKT/eNOS signaling pathway and augmentation of the release of NO, partially through suppressing the expression of SGLT1 and SGLT2 in PA-induced HUVECS.

Walking the straight and narrow: the moderating effect of evaluation apprehension on the relationship between collectivism and corruption.

Previous studies obtained mixed results regarding the association between collectivism and corruption. To make sense of the mixed results, the current research examined the moderating role of evaluation apprehension on the relationship between collectivism and corruption. Study 1, using a bribery scenario, indicated that collectivism facilitated corruption only when evaluation apprehension was low. Study 2, using a real money bribery game, confirmed the moderated model found in Study 1. Study 3 further demonstrated the different effects of vertical/horizontal collectivism on corruption. Our results suggest that a society may effectively combat corruption by increasing its social costs while, at the same time, retaining its collectivistic values.

Correlation between microRNA­21 and sprouty homolog 2 gene expression in multiple myeloma.

The aim of the present study was to investigate the expression level of microRNA 21 (miR­21) in the peripheral blood of patients with multiple myeloma (MM) and to investigate the correlation between miR­21 and sprouty homolog 2 (SPRY2) gene expression levels in MM. A total of 30 patients with MM, 15 with monoclonal gammopathy of undetermined significance (MGUS) and 20 normal control (NC) outpatients were selected for the detection of miR­21 and SPRY2 expression using reverse transcription-quantitative polymerase chain reaction. In addition, western blot analysis was performed to detect the expression of miR­21 and SPRY2 in MM cell lines. The expression of miR­21 in U­266 cells following lipofectamine transfection of fluorescence­labeled miR­21 mimic/inhibitor was observed using a fluorescence microscope and the expression level of SPRY2 in the miR­21 mimic/inhibitor­transfected U­266 cells was detected using western blot analysis. The miR­21 expression level in the circulating serum of the MM patient group was significantly higher (P<0.01) than that of the MGUS and NC groups. The MM cell lines with high endogenous miR­21 expression exhibited an expression level of SPRY2 that was significantly lower than that in the MM cells with low endogenous miR­21 expression. The transfection efficiency of fluorescence­labeled miR­21 mimic/inhibitor was >90%. Compared with the miR­21 expression level in untreated U­266 cells (0.82±0.13), the expression level of miR­21 was increased by 120.2­fold in miR­21 mimic­transfected cells (98.6±14.2; P<0.001) and was decreased by 61.9% in the miR­21 inhibitor­transfected cells (0.37±0.06; P<0.05). The grayscale value of protein bands demonstrated that SPRY2 protein expression significantly decreased in miR­21 mimic­transfected U­266 cells compared with that in the inhibitor­transfected, siRNA­transfected and untreated cells (P<0.01). miR­21 may represent a negative regulator involved in the downregulation of SPRY2 in MM. miR­21 is closely associated with the pathogenesis, progression and prognosis of MM and may thus be used as an indicator of poor MM prognosis.