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The intestinal epithelium is the first natural barrier against Trichinella spiralis larval invasion, but the mechanism of larval invasion of the gut epithelium is not fully elucidated. The aim of this study was to investigate whether the excretory/secretory proteins (ESPs) of T. spiralis intestinal infective larvae (IIL) degrade tight junction (TJ) proteins, to assess the main ESP proteases hydrolysing TJ proteins using various enzyme inhibitors and to define the key invasive factors in IIL invasion of the gut epithelium. The results of immunofluorescence, Western blot and Transwell assays showed that serine proteases and cysteine proteases in the ESPs played main roles in hydrolysing occludin, claudin-1 and E-cad and upregulating claudin-2 expression. Challenge infection results showed that IIL expulsion from the gut at 12 hpi was significantly higher in mice which were infected with muscle larvae (ML) treated with a single inhibitor (PMSF, E-64, 1,10-Phe or pepstatin) or various mixtures containing PMSF and E-64 than in mice in the PBS group or the groups treated with an inhibitor mixture not containing PMSF and E-64 (P < 0.0001). At 6 days post-infection, mice which were infected with ML treated with PMSF, E-64, 1,10-Phe or pepstatin exhibited 56.30, 64.91, 26.42 and 31.85% reductions in intestinal adult worms compared to mice in the PBS group (P < 0.0001). The results indicate that serine proteases and cysteine proteases play key roles in T. spiralis IIL invasion, growth and survival in the host and that they may be main candidate target molecules for vaccines against larval invasion and development., (© 2022. The Author(s).)

Background: Fine-needle aspiration (FNA) is used to diagnose malignancies, recurrences, and metastases. The procedure is quick and well tolerated and can be facilitated by ultrasound guidance., Methods: This article describes the authors' experience in using serial FNA to harvest cellular material during 4 clinical trials of immunotherapy by in situ vaccination in patients with low-grade lymphoma., Results: Two hundred ninety-six FNA samples were collected from 44 patients over a span of approximately 6 weeks for each patient. Samples were sufficient in quantity and quality to be analyzed by flow cytometry and/or single-cell messenger RNA sequencing. FNA samples yielded an average of 12 × 10 6 cells with a mean cellular viability of 86%. Material collected from the tumor lymph nodes differed significantly in the proportions and phenotypes of cellular populations in comparison with matched peripheral blood samples. A comparison of flow cytometry results obtained by FNA directly from the patient and by FNA performed ex vivo and a dissociation of the same lymph node after surgical excision confirmed that FNA sampling of the patient accurately represented the tumor and the microenvironment. An analysis of the FNA samples from immunotherapy-treated target lymph nodes versus nodes from nontreated tumor sites provided insight into the impact of specific immunotherapy regimens., Conclusions: This is the largest study describing the use of serial FNA sampling to harvest cellular material during immunotherapy clinical trials. The success of this technique opens the door for FNA sampling to expand significantly future investigations of the dynamic effects of investigational agents, be they immunotherapies or targeted therapies., (© 2021 American Cancer Society.)

Objectives: Small-volume biopsy-fine-needle aspiration biopsy (FNAB) with or without core biopsy-is in increasing use in diagnosis and management of lymphoma patients. Our objective was to survey the current practice in small-volume biopsy diagnosis of lymphoma, focusing on the interaction among hematopathologists and cytopathologists and the integration of FNAB, core biopsy, and flow cytometry studies at sign-out., Methods: This study used a cross-sectional survey design employing the RedCap database distributed via nine pathology professional society email listservs. The survey consisted of 25 multiple-choice questions and several free text fields. In total, 128 pathologists participated., Results: Most respondents indicated that FNAB specimens in which lymphoma is a diagnostic consideration (FNAB-L) are seen daily or weekly (68/116; 58.6%). However, most institutions have separate hematopathology and cytopathology services (72/116; 62.1%) with inconsistent communication. When communication occurred, respondents were frequently inclined to reconsider their original diagnoses. Barriers identified included lack of communication, inadequate access to diagnostic studies, no formal subspecialty training, and various opinions regarding FNAB in diagnosing lymphoma., Conclusions: This survey showed that FNAB-L specimens are common, with a lack of uniformity in how complementary fine-needle aspiration and core biopsy specimens or flow immunophenotyping results are shared across hematopathology and cytopathology services., (© American Society for Clinical Pathology, 2021. All rights reserved.For permissions, please e-mail: journals.permissions@oup.com.)

Elastase belongs to the serine protease family. Previous studies showed that Trichinella spiralis elastase (TsE) was highly expressed in intestinal infective larvae (IIL). Recombinant TsE (rTsE) promoted the larval intrusion of enteral epithelium cells (IECs), whereas anti-rTsE antibodies and siRNA impeded larval intrusion. Subcutaneous vaccination of mice with rTsE showed a partial protective immunity, suggesting that TsE might be a promising vaccine target against Trichinella infection. In this study, complete TsE cDNA sequence was cloned into pcDNA3.1, and the rTsE DNA was transformed into attenuated S. typhimurium strain ΔcyaSL1344. Oral vaccination of mice with TsE DNA elicited a systemic Th1/Th2/Treg mixed immune response and gut local mucosal sIgA response. Immunized mice exhibited a significant immune protection against T. spiralis larval challenge, as demonstrated by a 52.48% reduction of enteral adult worms and a 69.43% reduction of muscle larvae. The protection might be related to the TsE-induced production of intestinal mucus, specific anti-TsE sIgA and IgG, and secretion of IFN-γ, IL-2, IL-4 and IL-10, which protected gut mucosa from larval intrusion, suppressed worm development and impeded female reproduction. The results demonstrated that attenuated Salmonella-delivered TsE DNA vaccine provided a prospective strategy for the control of Trichinella infection in food animals., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Diagnosis of histologic transformation (HT) of follicular lymphoma (FL) requires tissue biopsy. While surgical biopsy represents the gold standard, less invasive procedures such as fine-needle aspiration biopsy (FNAB) and core needle biopsy (CNB) are frequently performed. In this retrospective multi-institutional study including 269 patients with FL and suspected HT, the median time from initial clinical suspicion to final diagnostic biopsy was similar whether the workup began with FNAB, CNB, or surgical biopsy (4, 9, and 6 days, respectively; p =.27), despite more subsequent biopsies performed following initial FNAB. Periprocedural complications were uniformly minimal. Biopsy-proven HT was more common in the initial surgery group and in workups including positron emission tomography/computed tomography (PET/CT). Our findings, derived from US academic centers with specialized procedural and pathology expertise, suggest that FNAB, CNB, and surgical biopsy are all viable initial diagnostic procedures that can inform clinical decision-making in select FL patients with suspected HT.

Trichinella spiralis is a major foodborne zoonotic parasitic nematode which has a serious threat to meat food safety. Development of anti-Trichinella vaccine is requisite for control and elimination of Trichinella infection in food animals to ensure meat safety. Aminopeptidase P (TsAPP) and cathepsin X (TsCX) are two novel proteins identified in T. spiralis intestinal infectious L1 larvae (IIL1). The objective of this study was to investigate the protective immunity elicited by immunization with TsAPP and TsCX alone and TsAPP-TsCX in combination in a mouse model. The results demonstrate that subcutaneous vaccination of mice with rTsAPP, rTsCX or rTsAPP + rTsCX elicited a systemic humoral response (high levels of serum IgG, IgG1/IgG2a and IgA) and significant local gut mucosal sIgA responses. The vaccination with rTsAPP, rTsCX or rTsAPP + rTsCX also induced a systemic and local mixed Th1/Th2 response, as demonstrated by clear elevation levels of IFN-γ and IL-4 in vaccinated mice. Vaccination of mice with rTsAPP+rTsCX exhibited a 63.99 % reduction of intestinal adult worms and 68.50% reduction of muscle larva burdens, alleviated inflammation of intestinal mucosal and muscle tissues, and provided a higher immune protection than that of vaccination with rTsAPP or rTsCX alone. The results demonstrated that TsAPP and TsCX might be considered novel candidate target molecules for anti-Trichinella vaccines., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cathepsin L is one member of cysteine protease superfamily and widely distributed in parasitic organisms, it plays the important roles in worm invasion, migration, nutrient intake, molting and immune evasion. The objective of this study was to investigate the biological characteristics of a novel cathepsin L from Trichinella spiralis (TsCL) and its role in larval invasion, development and reproduction. TsCL has a functional domain of C1 peptidase, which belongs to cathepsin L family. The complete TsCL sequence was cloned and expressed in Escherichia coli BL21. The rTsCL has good immunogenicity. RT-PCR and Western blotting analysis showed that TsCL was transcribed and expressed at different T. spiralis phases (e.g., muscle larvae, intestinal infectious larvae, adult worms and newborn larvae). Immunofluorescence test revealed that TsCL was principally localized in the cuticle, stichosome, midgut and female intrauterine embryos of the nematode. rTsCL has the capacity to specially bind with intestinal epithelial cells (IECs) and the binding sites was located in the cytoplasm. rTsCL promoted larval penetration into IEC, while anti-rTsCL antibodies inhibited the invasion. The silencing of TsCL gene by specific dsRNA significantly reduced the TsCL expression and enzyme activity, and also reduced larval invasive ability, development and female reproduction. The results showed that TsCL is an obligatory protease in T. spiralis lifecycle. TsCL participates in worm invasion, development and reproduction, and may be regarded as a potential candidate vaccine/drug target against T. spiralis infection., (Copyright © 2021. Published by Elsevier B.V.)

The alphaproteobacterium Sinorhizobium meliloti secretes two acidic exopolysaccharides (EPSs), succinoglycan (EPSI) and galactoglucan (EPSII), which differentially enable it to adapt to a changing environment. Succinoglycan is essential for invasion of plant hosts and, thus, for the formation of nitrogen-fixing root nodules. Galactoglucan is critical for population-based behaviors such as swarming and biofilm formation and can facilitate invasion in the absence of succinoglycan on some host plants. The biosynthesis of galactoglucan is not as completely understood as that of succinoglycan. We devised a pipeline to identify putative pyruvyltransferase and acetyltransferase genes, construct genomic deletions in strains engineered to produce either succinoglycan or galactoglucan, and analyze EPS from mutant bacterial strains. EPS samples were examined by 13 C cross-polarization magic-angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). CPMAS NMR is uniquely suited to defining chemical composition in complex samples and enables the detection and quantification of distinct EPS functional groups. Galactoglucan was isolated from mutant strains with deletions in five candidate acyl/acetyltransferase genes ( exoZ , exoH , SMb20810 , SMb21188 , and SMa1016 ) and a putative pyruvyltransferase ( wgaE or SMb21322 ). Most samples were similar in composition to wild-type EPSII by CPMAS NMR analysis. However, galactoglucan produced from a strain lacking wgaE exhibited a significant reduction in pyruvylation. Pyruvylation was restored through the ectopic expression of plasmid-borne wgaE . Our work has thus identified WgaE as a galactoglucan pyruvyltransferase. This exemplifies how the systematic combination of genetic analyses and solid-state NMR detection is a rapid means to identify genes responsible for modification of rhizobial exopolysaccharides. IMPORTANCE Nitrogen-fixing bacteria are crucial for geochemical cycles and global nitrogen nutrition. Symbioses between legumes and rhizobial bacteria establish root nodules, where bacteria convert dinitrogen to ammonia for plant utilization. Secreted exopolysaccharides (EPSs) produced by Sinorhizobium meliloti (succinoglycan and galactoglucan) play important roles in soil and plant environments. The biosynthesis of galactoglucan is not as well characterized as that of succinoglycan. We employed solid-state nuclear magnetic resonance (NMR) to examine intact EPS from wild-type and mutant S. meliloti strains. NMR analysis of EPS isolated from a wgaE gene mutant revealed a novel pyruvyltransferase that modifies galactoglucan. Few EPS pyruvyltransferases have been characterized. Our work provides insight into the biosynthesis of an important S. meliloti EPS and expands the knowledge of enzymes that modify polysaccharides.

Background: Trichinellosis is a serious zoonotic disease distributed around the world. It is needed to develop a safe, effective and feasible anti-Trichinella vaccine for prevention and control of trichinellosis. The aim of this study was to construct a recombinant Lactobacillus plantarum encoding Trichinella spiralis inorganic pyrophosphatase (TsPPase) and investigate its immune protective effects against T. spiralis infection., Methodology/principal Findings: The growth of recombinant L. plantarum was not affected by TsPPase/pSIP409-pgsA' plasmid, and the recombinant plasmid was inherited stably in bacteria. Western blot and immunofluorescence assay (IFA) indicated that the rTsPPase was expressed on the surface of recombinant L. plantarum. Oral vaccination with rTsPPase induced higher levels of specific serum IgG, IgG1, IgG2a and mucosal secretory IgA (sIgA) in BALB/c mice. ELISA analysis revealed that the levels of IFN-γ and IL-4 released from spleen, mesenteric lymph nodes and Peyer's patches were evidently increased at 2-4 weeks following vaccination, compared to MRS (De Man, Rogosa, Sharpe) medium control group (P < 0.05). Immunization of mice with rTsPPase exhibited a 67.18, 54.78 and 51.91% reduction of intestinal infective larvae, adult worms and muscle larvae at 24 hours post infection (hpi), 6 days post infection (dpi) and 35 dpi, respectively (P < 0.05), and the larval molting and development was significantly inhibited by 45.45% at 24 hpi, compared to the MRS group., Conclusions: TsPPase plays a crucial role in T. spiralis molting and development, oral vaccination with rTsPPase induced a significant local mucosal sIgA response and systemic Th1/Th2 immune response, and immune protection against T. spiralis infection in BALB/c mice., Competing Interests: The authors have declared that no competing interests exist.

Trichinellosis is an important zoonotic parasitic disease worldwide and is principally caused by ingesting animal meat containing Trichinella infective larvae. Aspartyl aminopeptidase is an intracytoplasmic metalloproteinase that specifically hydrolyzes the N-terminus of polypeptides free of acidic amino acids (aspartic acid and glutamate), and plays an important role in the metabolism, growth and development of organisms. In this study, a novel T. spiralis aspartyl aminopeptidase (TsAAP) was cloned and expressed, and its biological properties and roles in worm growth and development were investigated. The results revealed that TsAAP transcription and expression in diverse T. spiralis stages were detected by RT-PCR and Western blotting, and primarily localized at cuticle, stichosome and intrauterine embryos of this nematode by immunofluorescence test. rTsAAP has the enzymatic activity of native AAP to hydrolyze the substrate H-Glu-pNA. There was a specific binding between rTsAAP and murine erythrocyte, and the binding site was localized in erythrocyte membrane proteins. Silencing of TsAAP gene by specific dsRNA significantly reduced the TsAAP expression, enzymatic activity, intestinal worm burdens and female fecundity. The results demonstrated that TsAAP participates in the growth, development and fecundity of T. spiralis and it might be a potential target molecule for anti-Trichinella vaccines.

The purpose of this study was to determine the biological function of a Trichinella spiralis glutathione S-transferase (TsGST) in larval invasion and development by RNA interference (RNAi). The TsGST-specific siRNA 366 was transfected into T. spiralis muscle larvae (ML) via electroporation. At 1 day following transfection, the larval TsGST mRNA and protein expressions were reduced by 40.09 and 65.22 % (P < 0.05), respectively. The enzymatic activity of natural TsGST in siRNA-transfected ML was also suppressed by 45% compared with PBS group (P < 0.05). Silencing of the TsGST significantly inhibited the ability of larvae to invade intestinal epithelium cells (IECs) and isolated intestine. After challenge with siRNA-366-treated ML, the infected mice exhibited a 62.82% reduction of intestinal adult worms, and 65.03 % reduction of muscle larvae compared to the PBS group. Besides, the length of adults, newborn larvae and muscle larvae was significantly shorter than that of control siRNA and PBS group; the female fecundity of siRNA 366 group was lower than those of control siRNA and PBS group (P <  0.05). The results revealed that the specific RNAi significantly reduced the expression and enzymatic activity of TsGST, inhibited the larval invasive and developmental capacity, and impaired the female fecundity. The results further confirmed that TsGST plays a crucial role in the T. spiralis life cycle and it might be a potential molecular target for anti-Trichinella vaccines., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The aim of this study was to ascertain the characteristics of a Trichinella spiralis cathepsin X (TsCX) and its role on larval invasion of intestinal epithelial cells (IECs). The full-length of TsCX cDNA sequence was cloned and expressed in Escherichia coli BL21. The results of RT-PCR, IFA and Western blot revealed that TsCX was expressed at T. spiralis muscle larvae (ML), intestinal infective larvae, adult worm and newborn larvae, and it was located in whole worm section. The results of Far western and confocal microscopy demonstrated that there was a specific binding of rTsCX and IEC, and the binding site was located within the IEC cytoplasm. rTsCX promoted T. spiralis larval invasion of mouse IECs while anti-rTsCX antibody inhibited larval invasion into the IECs. Silencing TsCX by specific siRNA reduced the TsCX expression and larval invasive capacity. These results indicated that TsCX specifically binds to IECs and promotes larval invasion of intestinal epithelia, and it might be a potential target of vaccines against enteral stages of T. spiralis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The aim of this study was to investigate the biological properties of a novel gut-specific cysteine protease in Trichinella spiralis (TsGSCP) and its role in larval intrusion, development and fecundity. TsGSCP has a functional C1 peptidase domain; C1 peptidase belongs to cathepsin B family. The TsGSCP gene cloned and expressed in Escherichia coli BL21 showed intensive immunogenicity. qPCR and Western blotting revealed that TsGSCP mRNA and protein were expressed at various T. spiralis stages, but their expression levels in intestinal infectious larvae (IIL) were clearly higher than those in muscle larvae (ML), adult worms (AWs) and new-born larvae (NBL). Indirect immunofluorescence (IIF) analysis showed that TsGSCP was primarily located at the outer cuticle and the intrauterine embryos of this parasite. rTsGSCP showed the ability to specifically bind with IECs, and the binding site is within the IEC cytoplasm. rTsGSCP accelerated larval intrusion into host intestinal epithelial cells (IECs), whereas anti-rTsGSCP antibodies suppressed larval intrusion; the acceleration and suppression was induced by rTsGSCP and anti-rTsGSCP antibodies, respectively, in a dose-dependent manner. When ML were transfected with TsGSCP-specific dsRNA, TsGSCP expression and enzymatic activity were reduced by 46.82 and 37.39%, respectively, and the capacity of the larvae to intrude into IECs was also obviously impeded. Intestinal AW burden and adult female length and fecundity were significantly decreased in the group of mice infected with dsRNA-transfected ML compared to the control dsRNA and PBS groups. The results showed that TsGSCP plays a principal role in gut intrusion, worm development and fecundity in the T. spiralis lifecycle and might be a candidate target for vaccine development against Trichinella intrusion and infection., (© 2021. The Author(s).)

Tumor heterogeneity complicates biomarker development and fosters drug resistance in solid malignancies. In lymphoma, our knowledge of site-to-site heterogeneity and its clinical implications is still limited. Here, we profiled 2 nodal, synchronously acquired tumor samples from 10 patients with follicular lymphoma (FL) using single-cell RNA, B-cell receptor (BCR) and T-cell receptor sequencing, and flow cytometry. By following the rapidly mutating tumor immunoglobulin genes, we discovered that BCR subclones were shared between the 2 tumor sites in some patients, but in many patients, the disease had evolved separately with limited tumor cell migration between the sites. Patients exhibiting divergent BCR evolution also exhibited divergent tumor gene-expression and cell-surface protein profiles. While the overall composition of the tumor microenvironment did not differ significantly between sites, we did detect a specific correlation between site-to-site tumor heterogeneity and T follicular helper (Tfh) cell abundance. We further observed enrichment of particular ligand-receptor pairs between tumor and Tfh cells, including CD40 and CD40LG, and a significant correlation between tumor CD40 expression and Tfh proliferation. Our study may explain discordant responses to systemic therapies, underscores the difficulty of capturing a patient's disease with a single biopsy, and furthers our understanding of tumor-immune networks in FL., (© 2021 by The American Society of Hematology.)

Inflammatory bowel disease (IBD) increases the risk of colorectal cancer, and it has the potential to diminish the quality of life. Recent clinical and experimental evidence demonstrate protective aspects of parasitic helminth infection against IBD. Reports have highlighted the potential use of helminths and their byproducts as potential treatment for IBD. In the current study, we studied the effect of a newborn larvae-specific serine protease from Trichinella spiralis (TsSp) on the host immune and inflammatory responses. A 49-kDa recombinant TsSp (rTsSp) was expressed in Escherichia coli BL21 (DE3) and purified. The cytotoxicity of rTsSp was analyzed. The immune protective effect of rTsSp was studied by using dextran sodium sulfate (DSS)-induced mouse colitis model. The result illustrated that rTsSp has no toxic effects on cells. We further demonstrated that administration of the rTsSp without the additional adjuvant before the induction of DSS-induced colitis reduced the severity of intestinal inflammation and the disease index; it suppressed macrophage infiltration, reduced TNF-α secretion, and induced IL-10 expression. Our findings suggest therapeutic potential of rTsSp on colitis by altering the effect of macrophages. Data also suggest immunotherapy with rTsSp holds promise for use as an additional strategy to positively modulate inflammatory processes involved in IBD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Long, Liu, Kumar, Wang and Su.)

The critical step of Trichinella spiralis infection is that the muscle larvae (ML) are activated to intestinal infective larvae (IIL) which invade the intestinal columnar epithelium to further develop. The IIL excretory/secretory (ES) proteins play an important role in host-parasite interaction. Proteolytic enzymes are able to mediate the tissue invasion, thereby increasing the susceptibility of parasites to their hosts. The aim of the current study was to screen and identify the natural active proteases in T. spiralis IIL ES proteins using Western blot and gel zymography combined with liquid chromatography tandem mass spectrometry (LC-MS/MS). The T. spiralis ML and IIL ES proteins were collected from the in vitro cultures and their enzymatic acitvities were examined by gelatin zymography and azocasein degradation. The protease activities were partially inhibited by PMSF, E-64 and EDTA. Three protein bands (45, 118 and 165 kDa) of T. spiralis IIL ES proteins were identified by shotgun LC-MS/MS because they have hydrolytic activity to gelatin compared to the ML ES proteins. Total of 30 T. spiralis proteins were identified and they are mainly serine proteinases (19), but also metalloproteinases (7) and cysteine proteinases (3). The qPCR results indicated that transcription levels of four T. spiralis protease genes (two serine proteases, a cathepsin B-like cysteine proteinase and a zinc metalloproteinase) at IIL stage were obviously higher than at the ML stage. These proteolytic enzymes are directly exposed to the host intestinal milieu and they may mediate the worm invasion of enteral epithelium and escaping from the host's immune responses. The results provide the new insights into understanding of the interaction of T. spiralis with host and the invasion mechanism., (Copyright © 2021. Published by Elsevier B.V.)

Background/purpose: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignant tumors. The aim of this study was to elucidate the effect of tumor microenvironment-related genes on the prognosis of HNSCC and to obtain tumor microenvironment-related genes that can predict poor prognosis in HNSCC patients., Materials and Methods: The ESTIMATE algorithm was applied to the HNSCC transcriptomic data downloaded from the TCGA (The cancer genome atlas), and then the samples were divided into two groups: high and low immune scoring groups, and high and low basal scoring groups to screen for differentially expressed genes (DEGs) associated with poor patient outcomes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to explore the potential functions of DEGs, and then to explore the potential prognostic value of individual DEGs. The results of survival analysis between DEGs and overall survival (OS) to explore tumor microenvironment-related genes relevant to the prognosis of HNSCC patients., Results: Fifty-nine tumor microenvironment-related genes were screened for association of OS with HNSCC (P < 0.05). The GO and KEGG enrichment analysis showed that the selected DEGs may mediate immune response, extracellular matrix, and immunoglobulin binding via neutrophil activation in HNSCC. Six of these DEGs, GIMAP6, SELL, TIFAB, KCNA3, P2RY8 and CCR4 were most significantly associated with OS (P < 0.001)., Conclusion: We identified six tumor microenvironment-related genes that were significantly associated with poor prognosis in HNSCC. These genes may inspire researchers to discover new targets and approaches for HNSCC treatment., Competing Interests: No conflicts of interest relevant to this article were disclosed., (© 2020 Association for Dental Sciences of the Republic of China. Publishing services by Elsevier B.V.)

Trichinella spiralis is an important foodborne zoonotic parasite and it is necessary to develop vaccine to prevent T. spiralis infection in food animals. T. spiralis aspartic protease-2 (TsASP2) has been demonstrated to play a crucial role in larval invasion of intestinal epithelium cells (IECs). The purpose of this study was to assess the interaction between TsASP2 and IECs and to investigate the immune protection elicited by vaccination with rTsASP2. The results showed that the enzymatic activity of native aspartic protease was detected in crude proteins of all T. spiralis development stages other than NBL stage, the highest activity was observed in the IIL stage. The results of Western blot showed that TsASP2 protein was expressed at ML, IIL and AW but not NBL, and the TsASP2 expression level at IIL stage was significantly higher than those of other three worm stages (P < 0.05). The specific binding between rTsASP2 and IECs was observed by immunofluorescence test (IFT) and confocal microscopy, and the binding site was localized at the IEC membrane and this binding ability was inhibited by aspartic protease specific inhibitor pepstain A. The results of ELISA showed that the binding ability was protein dose-dependent. Vaccination with rTsASP2 triggered a mixed Th1/Th2 humoral and mucosal immune responses, as demonstrated by the elevation levels of Th1/Th2 cytokines (IFN-γ and IL-4) secreted by the spleen and mesenteric lymph nodes (MLNs) of immunized mice. The mice vaccinated with rTsASP2 exhibited a 54.17% reduction in enteral adult worms and a 54.58% reduction in muscle larvae after T. spiralis challenge. The results demonstrated that TsASP2 might be a potential molecular target for anti-Trichinella vaccines.

Inorganic pyrophosphatase (PPase) participates in energy cycle and plays a vital role in hydrolysis of inorganic pyrophosphate (PPi) into inorganic phosphate (Pi). The aim of this study was to investigate the biological properties of a Trichinella spiralis PPase (TsPPase) and its role in larval molting and developmental process. The predicted TsPPase consisted of 367 amino acids with a molecular mass of 41.48 kDa and a pI of 5.76. Amino acid sequence alignment and phylogenetic analysis showed that the TsPPase gene encodes a functional family I soluble PPase with the same characteristics as prokaryotic, plant and animal/fungal soluble PPase. The rTsPPase was expressed and purified, it has the activity to catalyze the hydrolysis of PPi to Pi, and the activity was dependent on Mg 2+ , pH and temperature. The enzymatic activity of rTsPPase was significantly inhibited after its metal binding sites mutation. TsPPase was transcribed and expressed in all T. spiralis phases, especially in muscle larvae (ML) and intestinal infective larvae (IIL). Immunofluorescence assay (IFA) revealed that TsPPase was mainly located in cuticle and stichosome. When the ML and IIL were treated with TsPPase-specific siRNA-279, TsPPase expression and enzymatic activity were obviously reduced, the larval molting and development were also impeded. Intestinal IIL as well as AW burden, IIL molting rates from mice infected with siRNA-treated ML were obviously suppressed. The results indicated that rTsPPase possesses the enzymatic activity of native inorganic pyrophosphatase, and TsPPase plays an important role in development and molting process of intestinal T. spiralis larval stages.

The aim of this study was to characterize the biological properties of a novel aspartic protease-1 from Trichinella spiralis (TsASP1) and evaluate its potential in inducing immune response. TsASP1 gene was cloned and expressed in Escherichia coli BL21 (DE3). On Western blotting analysis with anti-rTsASP1 serum, native TsASP1 was detected in various T. spiralis phases other than newborn larvae (NBL). qPCR results showed that TsASP1 transcription was the highest in intestinal infective larvae (IIL) and the lowest in the NBL stage. Immunofluorescence test result shows that native TsASP1 was principally localized in stichosome, muscle cells of muscle larvae (ML) and IIL, and surrounded intrauterine embryos in female adult worms (AW). After silencing TsASP1 gene of the ML by siRNA, the worm development was significantly inhibited, showed by shorter AW and more wrinkles and longitudinal crack on epicuticle of AW on scanning electron microscopy; the AW and ML burdens were reduced by 41.82 and 56.36% respectively, compared with the control siRNA or PBS group (P < 0.001). Immunization of mice with rTsASP1 elicited an evident antibody response (serum IgG, IgG1/IgG2a and enteral sIgA), and systemic (spleen) and intestinal local mucosal (mesenteric lymph node) cellular immune response, demonstrated by a prominent elevation of IFN-γ and IL-4. The results suggested TsASP1 participated in T. spiralis development and survival in host, and immunization of mice with rTsASP1 induced systemic/intestinal local mucosal humoral and cellular immune response against Trichinella., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A series of novel thermoplastic elastomers based on (poly(decamethylene terephthalamide/decamethylene isophthalamide), PA10T/10I) and poly(ethylene glycol) (PEG) were synthesized via a facile one-pot, efficient and pollution-free method. The thermal analysis demonstrates that the melting points of the resultant elastomers were in the range of 217.1-233.9 °C, and their initial decomposition temperatures were in the range of 385.3-387.5 °C. That is higher than most commercial polyamide-based thermoplastic elastomers. The tensile strength of the resultant elastomers ranges from 21.9 to 41.1 MPa. According to the high-temperature bending test results, the resultant samples still maintain considerably better mechanical properties than commercial products such as Pebax ® 5533 (Arkema, Paris, France), and these novel thermoplastic elastomers could potentially be applied in high-temperature scenes. The non-isothermal crystallization kinetics of the resultant elastomers and PA10T/10I was investigated by means of Jeziorny and Mo's methods. Both of them could successfully describe the crystallization behavior of the resultant elastomers. Additionally, the activation energy of non-isothermal crystallization was calculated by the Kissinger method and the Friedman equation. The results indicate that the crystallization rates follow the order of TPAE-2000 > TPAE-1500 > PA10T/10I > TPAE-1000. From the crystallization analysis, the crystallization kinetics and activation energies are deeply affected by the molecular weight of hard segment.

Elastase-1 is one member of serine protease family, distributes in organisms widely and plays a crucial role in the invasion and development of Trichinella spiralis. In order to identify the binding of T. spiralis elastase-1 (TsEla) with host's intestinal epithelial cells (IECs) and its role in Trichinella larval intrusion, TsEla gene was cloned and expressed in our previous study. The recombinant TsEla (rTsEla) has the enzymatic activity to degrade specific peptide substrate. A specific binding between rTsEla and IECs was detected by Far Western blot and ELISA. In an in vitro invasion assay, rTsEla promoted the larval intrusion, whereas anti-rTsEla serum inhibited the larval penetration. The larval intrusion was also suppressed after the silencing of TsEla by siRNA. Silencing of TsEla gene by siRNA-291 meditated RNA interference suppressed TsEla protein expression, reduced the worm infectivity, development and reproductive capacity. These results indicated that TsEla plays an important role in the T. spiralis intrusion of host's intestinal epithelia, and it could be a prospective vaccine molecular target against T. spiralis infection., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In our previous studies, a novel T. spiralis peptidase (TsP) was identified among the excretory/secretory (ES) proteins of T. spiralis intestinal infective larvae (IIL) and T. spiralis at the adult worm (AW) stage using immunoproteomics, but the biological function of TsP in the life cycle of T. spiralis is not clear. The objective of this study was to investigate the biological properties and functions of TsP in larval intrusion and protective immunity induced by immunization with rTsP. The complete TsP cDNA sequence was cloned and expressed. The results of RT-PCR, indirect immunofluorescence assay (IIFA) and western blotting revealed that TsP is a surface and secretory protein expressed in T. spiralis at different stages (muscle larvae, IIL, AWs and newborn larvae) that is principally localized at the epicuticle of the nematode. rTsP facilitated the larval intrusion of intestinal epithelial cells (IECs) and intestinal mucosa, whereas anti-rTsP antibodies suppressed larval intrusion; these facilitative and suppressive roles were dose-dependently related to rTsP or anti-rTsP antibodies. Immunization of mice with rTsP triggered an obvious humoral immune response (high levels of IgG, IgG1/IgG2a, and sIgA) and also elicited systemic (spleen) and intestinal local mucosal (mesenteric lymph node) cellular immune responses, as demonstrated by an evident increase in the cytokines IFN-γ and IL-4. Immunization of mice with rTsP reduced the numbers of intestinal adult worms by 38.6% and muscle larvae by 41.93%. These results demonstrate that TsP plays a vital role in the intrusion, development and survival of T. spiralis in hosts and is a promising candidate target molecule for anti-Trichinella vaccines.

Trichinella spiralis is a major foodborne parasite and has a serious threat to meat safety. Development of anti- Trichinella vaccines is prospective to eliminate Trichinella infection in food animal. The aim of this study was to assess the biological properties of a novel T. spiralis trypsin (TsT) and its elicited immune protection against larval challenge. The cDNA sequence of TsT gene was cloned and expressed. Western blotting showed rTsT was identified by infection serum and anti-TsT serum. RT-PCR results revealed that TsT gene was transcribed at diverse T. spiralis lifecycle stages. The IIFT results showed that natural TsT was principally expressed at epicuticle of 5-6 day adult worms, indicating that TsT is a worm somatic antigen and adult-stage specific surface antigen. Vaccination of mice with rTsT triggered an evident humoral immune response (high levels of serum IgG, IgG1/IgG2a, and enteral sIgA), and it also induced the systemic and enteral local cellular immune response, demonstrated by an significantly elevation of cytokines IFN-γ and IL-4. The mice vaccinated with rTsT exhibited a 33.17% reduction of enteral adult worms and a 37.80% reduction of muscle larvae after larval challenge. The results showed that TsT might be considered as a candidate target antigen for anti- T. spiralis vaccines.

Background: Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) represents a diagnostic challenge on surgical excisional or incisional biopsy. Classification is further challenging on fine needle aspiration (FNA) material accompanied by needle core and/or cell block biopsy (FNA+core/CB)., Methods: The authors studied all FNA+core/CB and surgical excisional or incisional biopsies to evaluate for lymphoma in patients who had a prior history of NLPHL or subsequent diagnosis of NLPHL over a 5-year period from 2012 through 2016., Results: Patients who ultimately were diagnosed with NLPHL represented <0.5% of those who underwent FNA+core/CB for an initial suspicion of lymphoma. FNA+core/CB resulted in a definitive diagnosis in 7 of 13 cases, and surgical excisional or incisional biopsy specimens resulted in a definitive diagnosis in 13 of 13 cases (chi-square statistic, 9.6; P = .002). At initial diagnosis, FNA+core/CB was negative in 2 cases and atypical or suspicious in 3 cases; all 5 of those patients required surgical excisional or incisional biopsy for a definitive lymphoma diagnosis. By contrast, patients who underwent FNA+core/CB for recurrent lymphoma required surgical excisional or incisional biopsy in only 1 of 8 cases (chi-square statistic, 9.5; P = .002). Flow cytometry was positive for a light-chain-restricted B-cell population in only 1 of 11 biopsies that were involved by lymphoma., Conclusions: Surgical excisional or incisional biopsy remains the gold standard for NLPHL diagnosis and for distinguishing progression to a T-cell/histiocyte-rich large B-cell lymphoma pattern. At a tertiary cancer center with routine collaborative diagnosis of lymphoma on FNA+core/CB by cytopathologists and hematopathologists, FNA+core/CB performs well to assess for recurrent or transformed NLPHL, rarely requiring subsequent surgical excisional or incisional biopsy. FNA+core/CB has limited sensitivity in the initial diagnosis setting., (© 2020 American Cancer Society.)

A Trichinella spiralis aminopeptidase (TsAP) has been identified in intestinal infectious larvae (IIL) and adult worms (AW), but its biological function in the T. spiralis life cycle is unknown. The aim of this study was to characterize TsAP and ascertain its functions in the invasion, development and fecundity of T. spiralis. Recombinant TsAP (rTsAP) was expressed and purified. rTsAP has strong immunogenicity. qPCR and western blotting show that TsAP was transcribed and expressed at all T. spiralis lifecycle stages, but the expression level of TsAP mRNA and proteins at IIL and AW stages was obviously higher than those in muscle larvae (ML) and newborn larvae (NBL). The IFT results reveal that TsAP was principally located at the cuticle and the intrauterine embryos of this nematode. rTsAP had the enzymatic activity of natural aminopeptidase to hydrolyze the substrate Leu-pNA with an optimal temperature of 50 °C and optimal pH of 8.0. rTsAP promoted the larval penetration into intestinal epithelial cells, whereas anti-rTsAP antibodies suppressed the larval intrusion; the promotion and suppression was dose-dependently related to rTsAP or anti-rTsAP antibodies. TsAP protein expression level and enzymatic activity were reduced by 50.90 and 49.72% through silencing of the TsAP gene by specific siRNA 842. Intestinal AW and muscle larval burdens, worm length and female reproductive capacity were significantly declined in mice infected with siRNA-transfected ML compared to the control siRNA and PBS group. These results indicate that TsAP participates in the invasion, development and fecundity of T. spiralis and it might be a candidate target for anti-Trichinella vaccines.

Segregated conductive polymer composites have been proved to be outstanding electromagnetic interference shielding (EMI) materials at low filler loadings. However, due to the poor interfacial adhesion between the pure conductive filler layers and segregated polymer granules, the mechanical properties of the segregated composites are usually poor, which limit their application. Herein, a simple and effective approach, the partial dissolution method, has been proposed to fabricate segregated poly(arylene sulfide sulfone) (PASS)/graphene nanoplatelet (GNP) composites with superior EMI shielding effectiveness (SE) and high tensile strength. Morphology examinations revealed that the GNPs were restricted in the dissolved outer layer by the undissolved cores, and there was a strong interaction between the PASS/GNP layer and the pure PASS core. The resultant PASS/GNP composites showed excellent electrical conductivity (60.3 S m -1 ) and high EMI SE (41 dB) with only 5 wt% GNPs. More notably, the tensile strength of the PASS/GNPs prepared by partial dissolution reached 36.4 MPa, presenting 136% improvement compared to that of the conventional segregated composites prepared by mechanical mixing. The composites also exhibited high resistance to elevated temperatures and chemicals owing to the use of the special engineering polymer PASS as a matrix., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

A T. spiralis serine protease 1.2 (TsSP1.2) was identified in the muscle larvae (ML) and intestinal larvae surface/excretory-secretory (ES) proteins by immunoproteomics. The aim of this study was to determine the TsSP1.2 function in the process of T. spiralis intrusion, growth and reproduction by using RNA interference (RNAi). RNAi was used to silence the expression of TsSP1.2 mRNA and protein in the nematode. On 2 days after the ML were electroporated with 2 µM of TsSP1.2-specific siRNA 534, TsSP1.2 mRNA and protein expression declined in 56.44 and 84.48%, respectively, compared with untreated ML. Although TsSP1.2 silencing did not impair worm viability, larval intrusion of intestinal epithelium cells (IEC) was suppressed by 57.18% (P < 0.01) and the suppression was siRNA-dose dependent (r = 0.976). Infection of mice with siRNA 534 transfected ML produced a 57.16% reduction of enteral adult burden and 71.46% reduction of muscle larva burden (P < 0.05). Moreover, silencing of TsSP1.2 gene in ML resulted in worm development impediment and reduction of female fertility. The results showed that silencing of TsSP1.2 by RNAi inhibited larval intrusion and development, and reduced female fecundity. TsSP1.2 plays a crucial role for worm invasion and development in T. spiralis life cycle, and is a potential vaccine/drug target against Trichinella infection.

Cathepsin B is one member of cysteine protease family and widely distributed in organisms, it plays an important function in parasite penetrating, migrating, molting and immune escaping. The aim of this work was to investigate whether exist interaction between a Trichinella spiralis cathepsin B (TsCB) and mouse intestinal epithelium cells (IECs), and its influence in the process of larva cell invasion. The results of ELISA, indirect immunofluorescence assay (IIFA), confocal microscopy and Far western blotting showed that there was a strong specific binding of rTsCB and IEC proteins, and the binding positions were located in cytoplasm and nuclei of IECs. The results of the in vitro larva penetration test revealed that rTsCB facilitated the larva invasion of IECs, whereas anti-rTsCB antibodies impeded partially the larva intrusion of enterocytes, this promotive or inhibitory roles were dose-dependent of rTsCB or anti-rTsCB antibodies. Silencing TsCB by siRNA mediated RNA interference reduced the TsCB expression in T. spiralis larvae, and markedly inhibited the larva penetration of enterocytes. The results indicated that TsCB binding to IECs promoted larva penetration of host's enteral epithelia, and it is a promising molecular target against intestinal invasive stages of T. spiralis., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest. Statement: (i) All the authors have agreed to its submission and are responsible for its contents; (ii) All the authors have agreed that Jing Cui and Zhong Quan Wang may act on their behalf regarding any subsequent processing of the paper; (iii) The authors have declared that no competing interests exist., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: T. spiralis aspartic protease has been identified in excretion/secretion (ES) proteins, but its roles in larval invasion are unclear. The aim of this study was to characterize T. spiralis aspartic protease-2 (TsASP2) and assess its roles in T. spiralis invasion into intestinal epithelial cells (IECs) using RNAi., Methodology/principal Findings: Recombinant TsASP2 (rTsASP2) was expressed and purified. The native TsASP2 of 43 kDa was recognized by anti-rTsASP2 serum in all worm stages except newborn larvae (NBL), and qPCR indicated that TsASP2 transcription was highest at the stage of intestinal infective larvae (IIL). IFA results confirmed that TsASP2 was located in the hindgut, midgut and muscle cells of muscle larvae (ML) and IIL and intrauterine embryos of the female adult worm (AW), but not in NBL. rTsASP2 cleaved several host proteins (human hemoglobin (Hb), mouse Hb, collagen and IgM). The proteolytic activity of rTsASP2 was host-specific, as it hydrolyzed mouse Hb more efficiently than human Hb. The enzymatic activity of rTsASP2 was significantly inhibited by pepstatin A. The expression levels of TsASP2 mRNA and protein were significantly suppressed by RNAi with 5 μM TsASP2-specific siRNA. Native aspartic protease activity in ML crude proteins was reduced to 54.82% after transfection with siRNA. Larval invasion of IECs was promoted by rTsASP2 and inhibited by anti-rTsASP2 serum and siRNA. Furthermore, cell monolayer damage due to larval invasion was obviously alleviated when siRNA-treated larvae were used. The adult worm burden, length of adult worms and female fecundity were clearly reduced in mice challenged using siRNA-treated ML relative to the PBS group., Conclusions: rTsASP2 possesses the enzymatic activity of native aspartic protease and facilitates T. spiralis invasion of host IECs., Competing Interests: The authors have declared that no competing interests exist.

Coronary artery disease (CAD) causes mortality and morbidity worldwide. We used near-infrared erythrocyte-derived transducers (NETs), a contrast agent, in combination with a photoacoustic imaging system to identify the locations of atherosclerotic lesions and occlusion due to myocardial-infarction (MI). NETs (≈90 nm diameter) were fabricated from hemoglobin-depleted mice erythrocyte-ghosts and doped with Indocyanine Green (ICG). Ten weeks old male C57BL/6 mice (n = 9) underwent left anterior descending (LAD) coronary artery ligation to mimic vulnerable atherosclerotic plaques and their rupture leading to MI. 150 µL of NETs (20 µM ICG,) was IV injected via tail vein 1-hour prior to photoacoustic (PA) and fluorescence in vivo imaging by exciting NETs at 800 nm and 650 nm, respectively. These results were verified with histochemical analysis. We observed ≈256-fold higher PA signal from the accumulated NETs in the coronary artery above the ligation. Fluorescence signals were detected in LAD coronary, thymus, and liver. Similar signals were observed when the chest was cut open. Atherosclerotic lesions exhibited inflammatory cells. Liver demonstrated normal portal tract, with no parenchymal necrosis, inflammation, fibrosis, or other pathologic changes, suggesting biocompatibility of NETs. Non-invasively detecting atherosclerotic plaques and stenosis using NETs may lay a groundwork for future clinical detection and improving CAD risk assessment.

The aim of this work was to identify the molecular characteristics of a chymotrypsin-like enzyme from Trichinella spiralis (Tschy) and its facilitation of larval penetration into enteral epithelial cells (EECs). The complete Tschy cDNA sequence was cloned and expressed in Escherichia coli BL21. RT-PCR, IIFA and western blotting showed that Tschy was expressed at the T. spiralis muscle larvae (ML), intestinal infective L1 larvae (IL1), adult worms (AW) and embryo stages and was primarily located in the stichosome of this parasite. The results of ELISA, IIFA and Far-western assays showed that there was a specific binding between rTschy and EECs, and the binding was dependent on the dose of both rTschy and EEC proteins. Confocal microscopy demonstrated that the binding was located in the EEC cytoplasm. rTschy facilitated T. spiralis larval penetration of EECs, and anti-rTschy antibodies impeded the larval intrusion of EECs. These results demonstrate that Tschy facilitated the larval intrusion of the host's enteral epithelium and could be a candidate molecular target for vaccine against the enteral invasive phase of T. spiralis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Liquid biopsy using cell-free DNA (cfDNA) presents new opportunities for solid tumor genotyping. While studies have demonstrated the utility of cfDNA from plasma, cfDNA from other body fluids remains underexplored., Methods: We evaluated the molecular features and clinicopathologic correlates of cfDNA from serous body cavity fluids by performing hybrid capture-based next-generation sequencing (NGS) on cfDNA isolated from residual effusion supernatants. Twenty-one serous effusions from pleural (n = 15), peritoneal (n = 5), and pericardial (n = 1) cavity were analyzed., Results: The supernatants provided a median cfDNA concentration of 10.3 ng/µL. Notably, all effusions were sequenced successfully to a median depth >1000×, revealing a broad range of genetic alterations including single nucleotide variants, small insertions and deletions, amplifications, and fusions. Specifically, pathogenic alterations were identified in all malignant fluids (13/13), all fluids suspicious for malignancy (2/2), and 1 benign fluid (1/6) from a patient with metastatic cancer. To validate our findings, we examined matching results from 11 patients who underwent additional testing using formalin-fixed, paraffin-embedded (FFPE) specimens. In 8 patients, the paired results between FFPE and supernatant testing were concordant, whereas in the remaining 3 patients, supernatant analysis identified additional variants likely associated with resistance to targeted therapies. Additional comparison between FFPE and supernatant testing showed no difference in DNA concentration (P = .5), depth of coverage (P = .6), or allele frequency of pathogenic mutations (P = .7)., Conclusion: cfDNA isolated from serous body cavity fluids represents a promising source of genomic input for targeted NGS., (© 2019 American Cancer Society.)

Background & Aims: Epidemiological and animal studies have indicated an inverse correlation between the rising prevalence of obesity and metabolic syndrome and exposure to helminths. Whether helminth-induced immune response contributes to microbiota remodeling in obesity remains unknown. The aim of this study is to explore the immune-regulatory role of helminth in the prevention of HFD-induced obesity through remodeling gut microbiome., Methods: C57BL/6J WT and STAT6 -/- mice were infected with Heligmosomoides polygyrus and followed by high fat diet (HFD) feeding for 6 weeks. The host immune response, body weight, and fecal microbiota composition were analyzed. We used adoptive transfer of M2 macrophages and microbiota transplantation approaches to determine the impact of these factors on HFD-obesity. We also examined stool microbiota composition and short chain fatty acids (SCFAs) concentration and determined the expression of SCFA-relevant receptors in the recipient mice., Results: Helminth infection of STAT6 -/- (Th2-deficient) mice and adoptive transfer of helminth-induced alternatively activated (M2) macrophages demonstrated that the helminth-associated Th2 immune response plays an important role in the protection against obesity and induces changes in microbiota composition. Microbiota transplantation showed that helminth-induced, Th2-dependent alterations of the gut microbiota are sufficient to confer protection against obesity. Collectively, these results indicate that helminth infection protects against HFD-induced obesity by Th2-dependent, M2 macrophage-mediated alterations of the intestinal microbiota., Conclusion: Our findings provide new mechanistic insights into the complex interplay between helminth infection, the immune system and the gut microbiota in a HFD-induced obesity model and holds promise for gut microbiome-targeted immunotherapy in obesity prevention., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Intestinal helminth infections elicit Th2-type immunity, which influences host immune responses to additional threats, such as allergens, metabolic disease, and other pathogens. Th2 immunity involves a shift of the CD4 + T-cell population from type-0 to type-2 (Th2) with increased abundance of interleukin (IL)-4 and IL-13. This study sought to investigate if existing gut-restricted intestinal helminth infections impact bacterial-induced acute airway neutrophil recruitment. C57BL/6 mice were divided into four groups: uninfected; helminth-Heligmosomoides polygyrus infected; Pseudomonas aeruginosa infected; and coinfected. Mice infected with H. polygyrus were incubated for 2 weeks, followed by P. aeruginosa intranasal inoculation. Bronchial alveolar lavage, blood, and lung samples were analyzed. Interestingly, infection with gut-restricted helminths resulted in immunological and structural changes in the lung. These changes include increased lung CD4 + T cells, increased Th2 cytokine expression, and airway goblet cell hyperplasia. Furthermore, coinfected mice exhibited significantly more airspace neutrophil infiltration at 6 hours following P. aeruginosa infection and exhibited an improved rate of survival compared with bacterial infected alone. These results suggest that chronic helminth infection of the intestines can influence and enhance acute airway neutrophil responses to P. aeruginosa infection.

Pterostilbene (PTE) is a natural dimethylated analog of resveratrol, which exerts antioxidative, hypolipidemic and hypoglycemic effects; however, the underlying mechanism is not yet clear. In this study, we evaluated the effects of PTE on diabetic rats and clarified the underlying mechanism. Diabetes was induced in rats by streptozotocin (STZ) and a high-sugar and high-fat diet. Rats were then treated with PTE (20, 40 and 80 mg/kg/d) for 8 weeks. Oral glucose tolerance test (OGTT) was performed to measure the glycometabolism of the diabetic rats at the end of the treatment. Fasting blood glucose (FBG), fasting insulin (FINS) and lipid profile were determined using an automatic biochemistry analyzer and serum inflammatory factors were analyzed by enzyme-linked immunosorbent assay. Serum superoxide dismutase (SOD) and malondialdehyde (MDA) were also analyzed by spectrophotometry to evaluate the anti-oxidant effects. The expression of proteins of PPARγ and PI3K/Akt signaling pathway related proteins in adipose tissue of the diabetic rats was analyzed by Western blotting. PTE treatment significantly reduced weight loss, FBG, insulin resistance, serum lipid levels and inflammatory factors. PTE treatment also inhibited oxidative stress by decreasing MDA expression and increasing SOD expression. Simultaneously, PTE treatment significantly ameliorated morphological impairment of the pancreas in diabetic rats. Furthermore, PTE treatment significantly increased the protein expression of PPARγ, PI3K, p-Akt, GLUT4 and IRS-1 in adipose tissues of diabetic rats. This study suggests that PTE can exert antidiabetic effects via the PI3K/Akt signaling pathway., (Copyright © 2019. Published by Elsevier B.V.)

Citrus greening disease, also known as huanglongbing (HLB), is the most devastating disease of Citrus worldwide. This incurable disease is caused primarily by the bacterium Candidatus Liberibacter asiaticus and spread by feeding of the Asian Citrus Psyllid, Diaphorina citri Ca L. asiaticus cannot be cultured; its growth is restricted to citrus phloem and the psyllid insect. Management of infected trees includes use of broad-spectrum antibiotics, which have disadvantages. Recent work has sought to identify small molecules that inhibit Ca L. asiaticus transcription regulators, based on a premise that at least some regulators control expression of genes necessary for virulence. We describe a synthetic, high-throughput screening system to identify compounds that inhibit activity of Ca L. asiaticus transcription activators LdtR, RpoH, and VisNR. Our system uses the closely related model bacterium, Sinorhizobium meliloti , as a heterologous host for expression of a Ca L. asiaticus transcription activator, the activity of which is detected through expression of an enhanced green fluorescent protein (EGFP) gene fused to a target promoter. We used this system to screen more than 120,000 compounds for compounds that inhibited regulator activity, but not growth. Our screen identified several dozen compounds that inhibit regulator activity in our assay. This work shows that, in addition to providing a means of characterizing Ca L. asiaticus regulators, an S. meliloti host can be used for preliminary identification of candidate inhibitory molecules., Competing Interests: The authors declare no conflict of interest.

A putative serine protease of T. spiralis (TsSP) was expressed in Escherichia coli and its potential as a diagnostic antigen was primarily assessed in this study. Anti-Trichinella IgG in serum samples from T. spiralis different animal hosts (mice, rats, pigs and rabbits) were detected on Western blot analysis with rTsSP. Anti-Trichinella antibodies were detected in 100% (30/30) of experimentally infected mice by rTsSP-ELISA. Cross-reactions of rTsSPELISA were not found with sera from mice infected with other parasites (S. erinaceieuropaei, S. japonicum, C. sinensis, A. cantonensis and T. gondii) and sera from normal mice. There was no statistical difference in antibody detection rate among mice infected with the encapsulated Trichinella species (T. spiralis, T. nativa, T. britovi, and T. nelsoni) (P>0.05). The results of rTsSP-ELISA showed that serum specific antibody IgG in mice infected with 100 or 500 T. spiralis muscle larvae (ML) were detectable early at 7-8 dpi, but not detected by ML ES antigen-ELISA prior to 10-12 dpi. Specific anti-Trichinella IgG was detected in 100% (18/18) of infected pigs by rTsSP-ELISA and ES-ELISA, but no specific antibodies was not detected in 20 conventionally raised normal pigs by two antigens. The results showed the rTsSP had the potential for early serodiagnosis of animal Trichinella infection, however it requires to be assayed with early infection sera of swine infected with Trichinella and other parasites.

Thin-cap fibroatheroma (TCFA) are the unstable lesions in coronary artery disease that are prone to rupture, resulting in substantial morbidity and mortality worldwide. However, their small size and complex morphologic and biologic features make early detection and risk assessment difficult. We tested our newly developed catheter-based C ircumferential- I ntravascular- R adioluminescence- P hotoacoustic- I maging (CIRPI) system in vivo to enable detection and characterization of vulnerable plaque structure and biology in rabbit abdominal aorta. Methods: The CIRPI system includes a novel optical probe combining circumferential radioluminescence imaging and photoacoustic tomography (PAT). The probe's CaF 2 :Eu-based scintillating imaging window captures radioluminescence images (360° view) of plaques by detecting β-particles during 18 F-FDG decay. A tunable laser-based PAT characterizes tissue constituents of plaque at 7 different wavelengths-540 and 560 nm (calcification), 920 nm (cholesteryl ester), 1040 nm (phospholipids), 1180 nm (elastin/collagen), 1210 nm (cholesterol), and 1235 nm (triglyceride). A single B-scan is concatenated from 330 A-lines captured during a 360° rotation. The abdominal aorta was imaged in vivo in both atherosclerotic rabbits (Watanabe Heritable Hyper Lipidemic [WHHL], 13-mo-old male, n = 5) and controls (New Zealand White, n = 2). Rabbits were fasted for 6 h before 5.55 × 10 7 Bq (1.5 mCi) of 18 F-FDG were injected 1 h before the imaging procedure. Rabbits were anesthetized, and the right or left common carotid artery was surgically exposed. An 8 French catheter sheath was inserted into the common carotid artery, and a 0.035-cm (0.014-in) guidewire was advanced to the iliac artery, guided by x-ray fluoroscopy. A bare metal stent was implanted in the dorsal abdominal aorta as a landmark, followed by the 7 French imaging catheters that were advanced up to the proximal stent edge. Our CIRPI and clinical optical coherence tomography (OCT) were performed using pullback and nonocclusive flushing techniques. After imaging with the CIRPI system, the descending aorta was flushed with contrast agent, and OCT images were obtained with a pullback speed of 20 mm/s, providing images at 100 frames/s. Results were verified with histochemical analysis. Results: Our CIRPI system successfully detected the locations and characterized both stable and vulnerable aortic plaques in vivo among all WHHL rabbits. Calcification was detected from the stable plaque (540 and 560 nm), whereas TCFA exhibited phospholipids/cholesterol (1040 nm, 1210 nm). These findings were further verified with the clinical OCT system showing an area of low attenuation filled with lipids within TCFA. PAT images illustrated broken elastic fiber/collagen that could be verified with the histochemical analysis. All WHHL rabbits exhibited sparse to severe macrophages. Only 4 rabbits showed both moderate-to-severe level of calcifications and cholesterol clefts. However, all rabbits exhibited broken elastic fibers and collagen deposition. Control rabbits showed normal wall thickness with no presence of plaque tissue compositions. These findings were verified with OCT and histochemical analysis. Conclusion: Our novel multimodality hybrid system has been successfully translated to in vivo evaluation of atherosclerotic plaque structure and biology in a preclinical rabbit model. This system proposed a paradigm shift that unites molecular and pathologic imaging technologies. Therefore, the system may enhance the clinical evaluation of TCFA, as well as expand our understanding of coronary artery disease., (© 2019 by the Society of Nuclear Medicine and Molecular Imaging.)

In a previous study, immunoproteomics was used to identify a serine protease inhibitor (TsSPI) of T. spiralis excretory/secretory (ES) proteins that exhibited an inhibitory effect on trypsin enzymatic activity, but the precise role of TsSPI on worm infection and development in its host is not well understood. The objective of the present study was to use RNA interference to ascertain the function of TsSPI in larval invasion and growth. TsSPI-specific small interference RNAs (siRNAs) were delivered to muscle larvae (ML) to silence TsSPI expression by electroporation. Four days after electroporation, the ML transfected with 2 μM siRNA-653 exhibited a 75.75% decrease in TsSPI transcription and a 69.23% decrease in TsSPI expression compared with control ML. Although the silencing of TsSPI expression did not decrease worm viability, it significantly suppressed the larval invasion of intestinal epithelium cells (IEC) (P < 0.01), and the suppression was siRNA dose-dependent (r = 0.981). The infection of mice with siRNA-653-treated ML produced a 63.71% reduction of adult worms and a 72.38% reduction of muscle larvae. In addition, the length of the adults, newborn larvae, and ML and the fecundity of female T. spiralis from mice infected with siRNA-treated ML were obviously reduced relative to those in the control siRNA or PBS groups. These results indicated that the silencing of TsSPI by RNAi suppressed larval invasion and development and decreased female fecundity, further confirming that TsSPI plays a crucial role during the T. spiralis lifecycle and is a promising molecular target for anti-Trichinella vaccines.

Hyaluronan (HA), an extracellular matrix glycosaminoglycan, is implicated in the pathogenesis of both type 1 diabetes (T1D) as well as type 2 diabetes (T2D) and has been postulated to be increased in these diseases due to hyperglycemia. We have examined the serum and tissue distribution of HA in human subjects with T1D and T2D and in mouse models of these diseases and evaluated the relationship between HA levels and glycemic control. We found that serum HA levels are increased in T2D but not T1D independently of hemoglobin-A1c, C-peptide, body mass index, or time since diabetes diagnosis. HA is likewise increased in skeletal muscle in T2D subjects relative to non-diabetic controls. Analogous increases in serum and muscle HA are seen in diabetic db/db mice (T2D), but not in diabetic DORmO mice (T1D). Diabetes induced by the β-cell toxin streptozotozin (STZ) lead to an increase in blood glucose but not to an increase in serum HA. These data indicate that HA levels are increased in multiple tissue compartments in T2D but not T1D independently of glycemic control. Given that T2D but not T1D is associated with systemic inflammation, these patterns are consistent with inflammatory factors and not hyperglycemia driving increased HA. Serum HA may have value as a biomarker of systemic inflammation in T2D., (Copyright © 2018 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

Host specificity in the root-nodule symbiosis between legumes and rhizobia is crucial for the establishment of a successful interaction and ammonia provision to the plant. The specificity is mediated by plant-bacterial signal exchange during early stages of interaction. We observed that a Sinorhizobium meliloti mutant ∆ relA , which is deficient in initiating the bacterial stringent response, fails to nodulate Medicago sativa (alfalfa) but successfully infects Medicago truncatula . We used biochemical, histological, transcriptomic, and imaging approaches to compare the behavior of the S. meliloti ∆relA mutant and wild type (WT) on the two plant hosts. ∆ relA performed almost WT-like on M. truncatula , except for reduced nitrogen-fixation capacity and a disorganized positioning of bacteroids within nodule cells. In contrast, ∆ relA showed impaired root colonization on alfalfa and failed to infect nodule primordia. Global transcriptome analyses of ∆ relA cells treated with the alfalfa flavonoid luteolin and of mature nodules induced by the mutant on M. truncatula revealed normal nod gene expression but overexpression of exopolysaccharide biosynthesis genes and a slight suppression of plant defense-like reactions. Many RelA-dependent transcripts overlap with the hypo-osmolarity-related FeuP regulon or are characteristic of stress responses. Based on our findings, we suggest that RelA is not essential until the late stages of symbiosis with M. truncatula , in which it may be involved in processes that optimize nitrogen fixation.

The aim of this study was to observe the intestinal mucosal/systemic responses triggered by intranasal vaccination using recombinant Trichinella spiralis serine protease (rTsSP) and its capacity to elicit immune protection against larva challenge in a murine model. rTsSP coupled with cholera toxin B subunit (CTB) was used to vaccinate mice via intranasal route. The results revealed that intranasal vaccination with rTsSP plus CTB elicited significantly intestinal local sIgA response and a TsSP-specific systemic antibody response in vaccinated mice. Furthermore, more goblet cells/acidic mucins and IgA-secreting cells were observed in jejunum from vaccinated mice. Anti-rTsSP immune serum strongly recognized the cuticle of various worm stages (muscle larva, intestinal infective larva and adult worm). The level of IFN-γ, IL-4 and IL-10 of rTsSP-vaccinated mice was significantly elevated relative to CTB and PBS control groups. The vaccinated mice exhibited a 71.10% adult reduction at 9 days pi and a 62.10% muscle larva reduction at 42 days pi following larva challenge. Additionally, vaccination with rTsSP also dampened intestinal T. spiralis development and decreased the female fecundity. Our results showed that intranasal vaccination using rTsSP adjuvanted with CTB triggered significantly local sIgA response and systemic concurrent Th1/Th2 response that induced an obvious protection against Trichinella infection., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Introduction: Fine needle aspiration biopsy (FNAB) is a minimally invasive modality to evaluate salivary gland neoplasms and help guide clinical management. However, significant overlap in the cytomorphology findings among salivary gland neoplasms often renders the definitive diagnosis challenging. Recently, a number of benign and malignant salivary gland tumors have been characterized by specific chromosomal aberrations detectable using fluorescence in situ hybridization (FISH) testing. In the present study, we evaluated the role of FISH testing performed on cytology cell blocks in the diagnosis of salivary gland neoplasms by FNAB., Materials and Methods: The data from 57 cases of primary salivary gland tumors diagnosed using FNAB at our institution and sent for ancillary FISH testing between 2012 and 2017 were retrospectively reviewed. The FISH studies were performed on cytology cell blocks, and break-apart probes were used to detect characteristic gene rearrangements for PLAG1, MYB, MAML2, and ETV6 for pleomorphic adenoma, adenoid cystic carcinoma, mucoepidermoid carcinoma, and secretory carcinoma (mammary analogue secretory carcinoma), respectively. Of the 57 cases sent for FISH testing, 6 were excluded because of FISH analysis failure (insufficient cell block cellularity)., Results: Of the 51 cases included in the analysis, 15 samples were successfully subclassified after FISH testing, and 10 of these 15 FISH-positive cases were diagnostically confirmed by the surgical pathology review of excision material. Forty cases overall had undergone subsequent excision with the histopathologic follow-up diagnosis available, and all subclassified cases had concordant FNAB, FISH, and excision diagnoses., Conclusions: FISH testing performed on cytology cell blocks is a useful adjunct in establishing the diagnosis of salivary gland neoplasms by FNAB., (Copyright © 2019 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

Purpose: To design and evaluate a small engineered protein binder targeting human programmed death-1 ligand (hPD-L1) in vivo for PET imaging in four mouse tumor models, and in situ in human cancer specimens. Experimental Design: The hPD-L1 protein binder, FN3 hPD-L1 , was engineered using a 12-kDa human fibronectin type-3 domain (FN3) scaffold. The binder's affinity was assayed in CT26 mouse colon carcinoma cells stably expressing hPD-L1 (CT26/hPD-L1). 64 Cu-FN3 hPD-L1 was assayed for purity, specific activity, and immunoreactivity. Four groups of NSG mice ( n = 3-5/group) were imaged with 64 Cu-FN3 hPD-L1 PET imaging (1-24 hours postinjection of 3.7 MBq/7 μg of Do-FN3 in 200 μL PBS): Nod SCID Gamma (NSG) mice bearing (i) syngeneic CT26/hPD-L1tumors, (ii) CT26/hPD-L1 tumors blocked (blk) by preinjected nonradioactive FN3 hPD-L1 binder, (iii) hPD-L1-negative Raji xenografts, and (iv) MDA-MB-231 xenografts. The FN3 hPD-L1 binder staining was evaluated against validated hPD-L1 antibodies by immunostaining in human cancer specimens., Results: FN3 hPD-L1 bound hPD-L1 with 1.4 ± 0.3 nmol/L affinity in CT26/hPD-L1 cells. 64 Cu-FN3 hPD-L1 radiotracer showed >70% yield and >95% purity. 64 Cu-FN3 hPD-L1 PET imaging of mice bearing CT26/hPD-L1 tumors showed tumor-to-muscle ratios of 5.6 ± 0.9 and 13.1 ± 2.3 at 1 and 4 hours postinjection, respectively. The FN3 hPD-L1 binder detected hPD-L1 expression in human tissues with known hPD-L1 expression status based on two validated antibodies., Conclusions: The 64 Cu-FN3 hPD-L1 radiotracer represents a novel, small, and high-affinity binder for imaging hPD-L1 in tumors. Our data support further exploration and clinical translation of this binder for noninvasive identification of cancer patients who may respond to immune checkpoint blockade therapies., (©2018 American Association for Cancer Research.)

Background: While complications of deep inferior epigastric artery perforator flaps are known and well documented, a thorough literature review revealed no other reports of a patient developing a chyle leak following the use of the internal mammary vessels for recipient vessels in autologous breast reconstruction., Case: A 55-year-old woman underwent free autologous breast reconstruction. She developed a chyle leak during the postoperative period. This was verified through a computed tomography scan and fluid analysis demonstrating a high triglyceride count and the presence of chylomicrons. The leak resolved with conservative measures including compression and a low-fat, high-protein diet., Discussion: The presence of chyle leak following dissection of the internal mammary vessels is a unique complication of autologous breast reconstruction. There have been reports of lymph leaks following mastectomy, but these are mostly reported in the axilla. A history of radiation to the contralateral breast and aberrant anatomy may have contributed to the complication. Treatment of chyle leaks ranges from conservative management to the use of total parenteral nutrition and somatostatin analogs to surgical intervention., Conclusion: While altering practice patterns based on a single case is not usually suggested, this complication does intimate that dealing with lymphatic vessels and lymph nodes in the chest should be done deliberately to prevent lymphatic leaks.