Molecular cloning and characterization of a novel aspartyl aminopeptidase from Trichinella spiralis.

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.

Specific binding of aspartic protease and enterocytes promotes Trichinella spiralis invasion of murine intestinal epithelium cells.

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.

Molecular cloning and characterization of a novel aspartyl aminopeptidase from Trichinella spiralis

@#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.

Specific binding of aspartic protease and enterocytes promotes Trichinella spiralis invasion of murine intestinal epithelium cells

@#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.

Trichinella spiralis: RNAi-mediated silencing of serine protease results in reduction of intrusion, development and fecundity.

In previous studies, a Trichinella spiralis serine protease (TsSP) was identified in excretion/secretion (ES) products from intestinal infective L1 larvae (IIL1) using immunoproteomics. The complete cDNA sequence of TsSP gene was 1372 bp, which encoded 429 amino acids with 47.55 kDa. The TsSP was transcribed and expressed at all T. spiralis life cycle phases, as well as mainly located at the cuticle and stichosome of the parasitic nematode. Recombinant TsSP bind to intestinal epithelial cells (IEC) and promoted larva invasion, however, its exact function in invasion, development and reproduction are still unknown. The aim of this study was to confirm the biological function of TsSP during T. spiralis invasion and growth using RNA interference (RNAi) technology. The results showed that on 1 day after electroporation using 2.5 µM siRNA156, TsSP mRNA and protein expression of muscle larvae (ML) was suppressed by 48.35 and 59.98%, respectively. Meanwhile, silencing of TsSP gene by RNAi resulted in a 61.38% decrease of serine protease activity of ML ES proteins, and a significant reduction of the in vitro and in vivo invasive capacity of IIL1 to intrude into the IEC monolayer and intestinal mucosa. When mice were infected with siRNA 156-transfected larvae, adult worm and muscle larva burdens were decreased by 58.85 and 60.48%, respectively. Moreover, intestinal worm growth and female fecundity were evidently inhibited after TsSP gene was knockdown, it was demonstrated that intestinal adults became smaller and the in vitro newborn larval yield of females obviously declined compared with the control siRNA group. The results indicated that knockdown of TsSP gene by RNAi significantly reduced the TsSP expression and enzymatic activity, impaired larvae intrusion and growth, and lowered the female reproductive capacity, further verified that TsSP might participate in diverse processes of T. spiralis life cycle, it will be a new prospective candidate molecular target of anti-Trichinella vaccines.

Functional analysis of Trichinella spiralis serine protease 1.2 by siRNA mediated RNA interference.

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.

Functional analysis of Trichinella spiralis serine protease 1.2 by siRNA mediated RNA interference

@#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.

Trichinella spiralis: RNAi-mediated silencing of serine protease results in reduction of intrusion, development and fecundity

@# In previous studies, a Trichinella spiralis serine protease (TsSP) was identified in excretion/secretion (ES) products from intestinal infective L1 larvae (IIL1) using immunoproteomics. The complete cDNA sequence of TsSP gene was 1372 bp, which encoded 429 amino acids with 47.55 kDa. The TsSP was transcribed and expressed at all T. spiralis life cycle phases, as well as mainly located at the cuticle and stichosome of the parasitic nematode. Recombinant TsSP bind to intestinal epithelial cells (IEC) and promoted larva invasion, however, its exact function in invasion, development and reproduction are still unknown. The aim of this study was to confirm the biological function of TsSP during T. spiralis invasion and growth using RNA interference (RNAi) technology. The results showed that on 1 day after electroporation using 2.5 µM siRNA156, TsSP mRNA and protein expression of muscle larvae (ML) was suppressed by 48.35 and 59.98%, respectively. Meanwhile, silencing of TsSP gene by RNAi resulted in a 61.38% decrease of serine protease activity of ML ES proteins, and a significant reduction of the in vitro and in vivo invasive capacity of IIL1 to intrude into the IEC monolayer and intestinal mucosa. When mice were infected with siRNA 156-transfected larvae, adult worm and muscle larva burdens were decreased by 58.85 and 60.48%, respectively. Moreover, intestinal worm growth and female fecundity were evidently inhibited after TsSP gene was knockdown, it was demonstrated that intestinal adults became smaller and the in vitro newborn larval yield of females obviously declined compared with the control siRNA group. The results indicated that knockdown of TsSP gene by RNAi significantly reduced the TsSP expression and enzymatic activity, impaired larvae intrusion and growth, and lowered the female reproductive capacity, further verified that TsSP might participate in diverse processes of T. spiralis life cycle, it will be a new prospective candidate molecular target of anti-Trichinella vaccines.

Primary assessment of a T. spiralis putative serine protease for early serological detection of experimental trichinellosis.

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.

Primary assessment of a T. spiralis putative serine protease for early serological detection of experimental trichinellosis

@#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.

[Analysis on the clinical therapeutic effects of arterial first approach pancreatoduodenectomy in the treatment of borderline resectable pancreatic adenocarcinoma].

Objective: To compare the clinical therapeutic effects of arterial first approach pancreaticoduodenectomy(AFA-PD) with standard approach pancreaticoduodenectomy(SPD) in the treatment of borderline resectable pancreatic cancer (BRPC). Methods: A retrospective analysis of the clinical data of 113 cases of pancreatic cancer patients from January 2014 to August 2015 at Department of Hepato-Biliary-Pancreatic Surgery, Changhai Hospital, the Second Military Medical University, including 43 cases in AFA-PD group and 70 cases in SPD group.Every patient had gone high-resolusion computed tomography before the surgery, when BRPC was definitely diagnosed by both experienced radiologist and pancreatic surgeon.There were 24 males and 19 females in the AFA-PD group, with average age of (61.6±10.2)years.And in the SPD group, there were 47 males and 23 females, with average age of (62.7±9.4)years. Results: The operation time was (210.7±31.5)minutes in AFA-PD group, (187.9±27.4)minutes in SPD group, and peroperative bleeding volume was (1 007.1±566.3)ml in AFA-PD group, (700.0±390.0)ml in the other group.Those two indicators of AFA-PD group, compared with SPD group, were relatively higher, the difference was statistically significant(all P<0.01). And with regard to postoperative diarrhea(9.3% vs.5.7%), postoperative 1, 3 days of white blood cells(postoperative 1 day: (13.3±1.1)×10(9)/L vs.(12.4±2.4)×10(9)/L; postoperative 3 days: (12.7±1.6)×10(9)/L vs.(11.7±2.5)×10(9)/L), postoperative 1, 3, 5 days of peritoneal drainage fluid volume(postoperative 1 day: (184±42)ml vs.(156±54)ml; postoperative 3 days: (155±48)ml vs.(133±35)ml; postoperative 5 days: (66±20)ml vs.(47±31)ml), the differences between the two groups were statistically significant (all P<0.05). One patient in the SPD group was treated with unplanned secondary surgery for postoperative intraperitoneal hemorrhage, and the patient was cured and discharged.There was no death in the two groups within 30 days after surgical operation and no patient with positive gastric margin, duodenal margin, or anterior margin.The resection rate of superiormesenteric artery(SMA) margin R0 in AFA-PD group was higher than that in SPD group (P=0.019). The two groups were followed up for 14 to 30 months.As for AFA-PD group, the average survival time, progression free survival time and median survival time was respectively (20.4±1.2)months, (21.5±1.4)months and 20 months.There were 3 cases(7.0%) with local recurrence and 8 cases(18.6%) with liver metastasis or distant metastasis.In the SPD group, the average survival time, progression free survival time and median survival time was (17.1±1.1)months, (16.4±1.3)months and 16 months, respectively.There were 13 cases(18.6%) with local recurrence and 25 cases(35.7%) with liver metastasis or distant metastasis.As a result, the AFA-PD group had longer survival time(P=0.001)and progression free survival time(P=0.002). However, the lower local recurrence and distant metastasis rate in AFA-PD group did not reach statistical standard (P>0.05). Conclusion: The arterial first approach pancreaticoduodenectomy is safe and effective in the treatment of borderline resectable pancreatic cancer, which can improve the resection rate of SMA margin R0, and prolong patient survival time.

Primary cultures of mouse small intestinal epithelial cells using the dissociating enzyme type I collagenase and hyaluronidase.

The epithelium is a highly dynamic system, which plays a crucial role in the homeostasis of the intestinal tract. However, studies on the physiological and pathophysiological functions of intestinal epithelial cells (IECs) have been hampered due to lack of normal epithelial cell models. In the present study, we established a reproducible method for primary culture of mouse IECs, which were isolated from the viable small intestinal crypts of murine fetuses (on embryonic day 19), using type I collagenase and hyaluronidase in a short span of time (≤20 min). With this method, continuously growing mouse IECs, which can be subcultured over a number of passages, were obtained. The obtained cell lines formed a tight cobblestone-like arrangement, displayed long and slender microvilli, expressed characteristic markers (cytokeratin 18 and Notch-1), and generated increasing transepithelial electrical resistance and low paracellular permeability during in vitro culture. The cells also had enzymatic activities of alkaline phosphatase and sucrase-isomaltase, and secreted various cytokines (IL-1ß, IL-6, IL-8, and monocyte chemoattractant protein-1), responding to the stimulation of Escherichia coli. These results show that the primary-cultured mouse IECs obtained by the method established here had the morphological and immunological characteristics of IECs. This culture system can be a beneficial in vitro model for studies on mucosal immunology and toxicology.

Identification of early diagnostic antigens in soluble proteins of Trichinella spiralis adult worms by Western blot.

Previous studies showed that crude antigens from Trichinella spiralis adult worms (AW) can be recognized by mouse infection sera at 8 days post infection. The aim of this study was to identify the early diagnostic antigenic bands in soluble proteins from T. spiralis AW by Western blot using early infection sera. The affecting factors of adult recovery were firstly observed in this study, and the results showed that the maximum number of adults was collected from small intestine when the female BALB/c mice were orally infected with 4000 ML and sacrificed at 3 days post infection. The results of Western blot analysis showed that seven protein bands (31, 35.1, 39, 40.6, 41.9, 47 and 50.6 kDa) could be recognized by early infection sera as early as at 8-10 days post infection, and were strongly reacted with mouse infection sera at 11-12 days post infection. Our results suggested that the seven protein bands of T. spiralis AW soluble proteins might be the early expressed antigens during the intestinal stage of Trichinella infection and therefore have potential value for the early diagnosis of trichinellosis.

Identification of early diagnostic antigens in soluble proteins of Trichinella spiralis adult worms by Western blot

Previous studies showed that crude antigens from Trichinella spiralis adult worms (AW) can be recognized by mouse infection sera at 8 days post infection. The aim of this study was to identify the early diagnostic antigenic bands in soluble proteins from T. spiralis AW by Western blot using early infection sera. The affecting factors of adult recovery were firstly observed in this study, and the results showed that the maximum number of adults was collected from small intestine when the female BALB/c mice were orally infected with 4000 ML and sacrificed at 3 days post infection. The results of Western blot analysis showed that seven protein bands (31, 35.1, 39, 40.6, 41.9, 47 and 50.6 kDa) could be recognized by early infection sera as early as at 8-10 days post infection, and were strongly reacted with mouse infection sera at 11-12 days post infection. Our results suggested that the seven protein bands of T. spiralis AW soluble proteins might be the early expressed antigens during the intestinal stage of Trichinella infection and therefore have potential value for the early diagnosis of trichinellosis.

The divergence pattern of Trichinella spiralis isolates from China revealed by mitochondrial and ribosomal markers.

Trichinella spiralis is the main etiological agent of human trichinellosis. In China, trichinellosis remains a serious food-borne parasitic zoonosis and poses a serious threat to human health. However, the genetic structure of Chinese T. spiralis population is still little known. In this study, we used three molecular markers to analyze phylogeographic structure of the Chinese T. spiralis population. A total of 11 T. spiralis isolates were collected from 10 geographical locations in mainland China. The cytochrome c-oxidase gene (COI), large subunit ribosomal DNA (mt-lsrDNA) and 5S ribosomal DNA intergenic spacer region (5S ISR) genes of each isolate was amplified and sequenced. Only four haplotypes were found in these concatenated sequences. Both multimodal frequency distributions of mismatch analysis and the Bayesian skyline plot analysis rejected a possible population expansion of Chinese T. spiralis population. The phylogenetic inference based on neighbor-joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and the Bayesian estimation of divergence times under the uncorrelated log-normal relaxed molecular-clock model suggested that the Chinese T. spiralis isolates started radiating in the late Miocene.

Analysis of Structures, Functions, and Epitopes of Aminopeptidase from Trichinella spiralis.

We have previously reported that the recombinant T. spiralis aminopeptidase (rTsAP) could induce a partial protective immunity against T. spiralis infection in mice. The aim of this study was to predict the structures and functions of TsAP protein by using the full length cDNA sequence of TsAP gene. TsAP sequence was 1515 bp length with a 1515 bp biggest ORF encoding 504-amino acid protein. The molecular weight and isoelectric point of TsAP were 54.7 kDa and 6.69, respectively. TsAP structure domains contained a Peptidase_M17_N and a Peptidase_M17 domain, which has the function of catalysis of the hydrolysis of N-terminal amino acid residues. TsAP had no signal peptide site and transmembrane domain, and located in cytoplasm. The secondary structure of TsAP contained 16 α-helix, 14 ß-strand and 29 coils. The TsAP had 11 and 21 potential antigenic epitopes of T cell and B cell, respectively. Based on the phylogenetic analyses of TsAP, T. spiralis have the closest relationship with Plasmodium falciparum. TsAP was a kind of proteolytic enzyme with a variety of biological functions and its antigenic epitopes could provide important insights on the diagnostic antigens and target molecular of anti-Trichinella drugs.

Analysis of Structures, Functions, and Epitopes of Aminopeptidase from Trichinella spiralis

We have previously reported that the recombinant T. spiralis aminopeptidase (rTsAP) could induce a partial protective immunity against T. spiralis infection in mice. The aim of this study was to predict the structures and functions of TsAP protein by using the full length cDNA sequence of TsAP gene. TsAP sequence was 1515 bp length with a 1515 bp biggest ORF encoding 504-amino acid protein. The molecular weight and isoelectric point of TsAP were 54.7 kDa and 6.69, respectively. TsAP structure domains contained a Peptidase_M17_N and a Peptidase_M17 domain, which has the function of catalysis of the hydrolysis of N-terminal amino acid residues. TsAP had no signal peptide site and transmembrane domain, and located in cytoplasm. The secondary structure of TsAP contained 16 α-helix, 14 β-strand and 29 coils. The TsAP had 11 and 21 potential antigenic epitopes of T cell and B cell, respectively. Based on the phylogenetic analyses of TsAP, T. spiralis have the closest relationship with Plasmodium falciparum. TsAP was a kind of proteolytic enzyme with a variety of biological functions and its antigenic epitopes could provide important insights on the diagnostic antigens and target molecular of anti-Trichinella drugs

Identification of surface proteins of Trichinella spiralis muscle larvae using immunoproteomics.

Trichinella spiralis surface proteins are directly exposed to the host's immune system, making them the main target antigens which induce the immune responses and may play an important role in the larval invasion and development process. The analysis and characterization of T. spiralis surface proteins could provide useful information to elucidate the host-parasite interaction, identify the early diagnostic antigens and the targets for vaccine. The purpose of this study was to identify the surface proteins of T. spiralis muscle larvae by two-dimensional gel electrophoresis (2-DE) Western-blot analysis and mass spectrometry. The 2-DE results showed that a total of approximately 33 proteins spots were detected with molecular weights varying from 10 to 66 kDa and isoelectric point (pI) from 4 to 7. Fourteen protein spots were recognized by sera of mice infected with T. spiralis at 42 dpi or at 18 dpi, and 12 spots were successfully identified by MALDI-TOF/TOF-MS, which represented 8 different proteins of T. spiralis. Out of the 8 T. spiralis proteins, 5 proteins (partial P49 antigen, deoxyribonuclease II family protein, two serine proteases, and serine proteinase) had catalytic and hydrolase activity, which might be the invasion-related proteins and the targets for vaccine. The 4 proteins (deoxyribonuclease II family protein, serine protease, 53 kDa ES antigen and hypothetical protein Tsp_08444) recognized by infection sera at 18 dpi might be the early diagnostic antigens for trichinellosis.

[Diagnostic value of ultrasonically guided lung aspiration in pneumonia].

To determine the diagnostic value of ultrasonic lung aspiration for patients with pneumonia, 60 patients with a tentative diagnosis of pneumonia were included in this study. After recording ultrasonographic findings, lung aspiration was done with a spinal needle and aspirated specimens were sent for Papanicolaou, May-Giemsa, acid fast, and Gram stains. The remaining specimens were sent for bacterial, mycobacterial and fungal culture. Twelve patients were excluded from the study because of the final diagnosis of non-infectious pulmonary diseases. In 28 cases of bacterial pneumonia, the diagnostic sensitivity of smear was 50% and culture 61%. The overall sensitivity of needle aspiration and culture was 71%. In 11 cases of bacterial pneumonia with a negative bacterial culture result, 7 cases were afebrile at the time of examination. To increase the diagnostic yield, needle aspiration should be performed at the acute stage of bacterial pneumonia. In 15 cases of pulmonary tuberculosis, the diagnostic rate of acid-fast smear was 47% and mycobacterial culture was 46%. The overall sensitivity of smear and culture was 60%. The diagnostic rate of needle biopsy was 75% and cytologic examination was 77%. Needle biopsy and cytologic examination enhanced the diagnostic rate of sputum-negative pulmonary tuberculosis. Cryptococcosis was documented by smear and needle biopsy in all of the five cases of cryptococcosis. Cryptococcosis is not easily detected by routine cytologic examination, and clinical information is still necessary to enhance the diagnostic rate. Our results show that ultrasonically guided lung aspiration is a technique with a high diagnostic yield and a low complication rate for various types of pneumonia. It is especially useful for patients without satisfactory clinical responses or without accurate microbiologic diagnosis.