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1.
Parasitol Int ; 74: 101923, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31054916

RESUMO

There is limited information regarding responses by slow cycling stem cells during T. spiralis-induced T-cell mediated intestinal inflammation and how such responses may relate to those of Paneth cells. Transgenic mice, in which doxycycline induces expression of histone 2B (H2B)-green fluorescent protein (GFP), were used. Following discontinuation of doxycycline ("chase" period), retention of H2B-GFP enabled the identification of slow cycling stem cells and long-lived Paneth cells. Inflammation in the small intestine (SI) was induced by oral administration of T. spiralis muscle larvae. Epithelial retention of H2B-GFP per crypt cell position (cp) was studied following immunohistochemistry and using the Score and Wincrypts program. Compared to non-infected controls, there was significant reduction in the number of H2B-GFP-retaining stem cells in T. spiralis-infected small intestines. H2B-GFP-retaining stem cells peaked at around cp 4 in control sections, but smaller peaks at higher cell positions (>10) were seen in sections of inflamed small intestines. In the latter, there was a significant increase in the total number of Paneth cells, with significant reduction in H2B-GFP-retaining Paneth cells, but a marked increase in unlabelled (H2B-GFP-negative) Paneth cells. In conclusion, following T. spiralis-infection, putative slow cycling stem cell numbers were reduced. A marked increase in newly generated Paneth cells at the crypt base led to higher cell positions of the remaining slow cycling stem cells.


Assuntos
Intestino Delgado/citologia , Celulas de Paneth/parasitologia , Células-Tronco/parasitologia , Triquinelose/imunologia , Animais , Ciclo Celular , Feminino , Proteínas de Fluorescência Verde/genética , Histonas/genética , Imuno-Histoquímica , Intestino Delgado/imunologia , Intestino Delgado/parasitologia , Masculino , Camundongos , Camundongos Transgênicos , Celulas de Paneth/imunologia , Células-Tronco/imunologia , Trichinella spiralis
2.
Vet Parasitol ; 273: 60-66, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31442895

RESUMO

Maternal antibody transmission via placenta and breastmilk are known to confer protection in infants. In this study, we investigated the maternal immunity transmission in pups delivered by rats infected with Trichinella spiralis and assessed the resulting resistance against subsequent parasitic infection. Our results revealed that parasite-specific IgG, IgG1 and IgG2a antibodies were present in pups prior to breastmilk ingestion (pre-milk), in which IgG and IgG1 antibodies persisted until week 8 after birth while parasite-specific IgG2a antibodies only lasted until week 4. After weaning on week 3, pups delivered by T. spiralis-infected dam and subsequently challenge-infected (immune-challenge) were found to possess higher mucosal IgG antibodies than control groups, whereas mucosal IgA levels were not significantly different across all groups. T. spiralis excretory-secretory antigen was discovered to react with pup sera until week 8, correlating with the resistance against parasitic infection which is represented by lessened worm burden. Upon T. spiralis infection at weeks 3 and 8, lower levels of eosinophil responses were detected in immune-challenge pups compared to naïve-challenge pups, indicating correlates of resistances in which ADCC may be involved. Findings from the present study demonstrate that resistances against T. spiralis infection in pups can be acquired by maternally-derived IgG, IgG1 and IgG2a antibody transmission through the placenta and breastmilk from T. spiralis-infected dam, which lasts until week 8.


Assuntos
Resistência à Doença/imunologia , Imunidade Materno-Adquirida/imunologia , Trichinella spiralis/imunologia , Triquinelose/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/metabolismo , Feminino , Proteínas de Helminto/metabolismo , Leite/imunologia , Placenta/imunologia , Gravidez , Ratos
3.
PLoS One ; 14(7): e0218198, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31291264

RESUMO

Bisphenol A (BPA) is an endocrine disruptor compound with estrogenic activity, possessing affinity for both nuclear (ERα and ERß) and membrane estrogen receptors. The main source of BPA exposure comes from the contamination of food and water by plastic storage containers or disposable bottles, among others, in which case BPA is easily ingested. Exposure to BPA during early pregnancy leads to lifelong effects; however, its effect on the immune system has not been fully studied. Since endocrine and immune systems interact in a bidirectional manner, the disruption of the former may cause permanent alterations of the latter, thus affecting a future anti-parasitic response. In this study, neonate BALB/c mice were exposed to a single dose of BPA (250 µg/kg); once sexual maturity was reached, they were orally infected with Trichinella spiralis (T. spiralis). The analyses performed after 5 days of infection revealed a decreased parasitic load in the duodenum of mice in the BPA-treated group. Flow cytometry analyses also revealed changes in the immune cell subpopulations of the infected animals when compared to the BPA-treated group. RT-PCR analyses of duodenum samples showed an increased expression of TNF-α, IFN-γ, IL-4, IL-5, and IL-9 in the BPA-treated group. These findings show a new aspect whereby early-life exposure to BPA contributes to the protection against T. spiralis by modulating the anti-parasitic immune response.


Assuntos
Compostos Benzidrílicos/imunologia , Disruptores Endócrinos/imunologia , Fenóis/imunologia , Efeitos Tardios da Exposição Pré-Natal/imunologia , Trichinella spiralis/imunologia , Triquinelose/imunologia , Animais , Feminino , Imunidade/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Fatores de Proteção , Triquinelose/prevenção & controle
4.
Parasitol Res ; 118(7): 2247-2255, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31081529

RESUMO

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.


Assuntos
Doenças Transmitidas por Alimentos/prevenção & controle , RNA Interferente Pequeno/administração & dosagem , Inibidores de Serino Proteinase/genética , Trichinella spiralis/genética , Triquinelose/prevenção & controle , Animais , Feminino , Fertilidade , Doenças Transmitidas por Alimentos/imunologia , Doenças Transmitidas por Alimentos/parasitologia , Humanos , Mucosa Intestinal/imunologia , Larva , Camundongos , Camundongos Endogâmicos BALB C , Músculos/parasitologia , Proteômica , Interferência de RNA , Inibidores de Serino Proteinase/metabolismo , Trichinella spiralis/crescimento & desenvolvimento , Trichinella spiralis/imunologia , Trichinella spiralis/patogenicidade , Triquinelose/imunologia , Triquinelose/parasitologia
5.
Acta Parasitol ; 64(3): 520-527, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31087260

RESUMO

INTRODUCTION: Trichinellosis is a severe zoonosis involving the activation of inflammatory cells, accompanied by the prominent expressions of proinflammatory cytokines in the host. Semen vaccariae, the seeds of Vaccaria segetalis (Neck.) Garcke. ex Asch. (Caryophyllaceae), is a famous traditional herb that is rich in vaccaria n-butanol extract (VNE). Vaccarin is one major active component of VNE, and it is reported in the treatment of stranguria disease. Hypaphorine is another main active component of VNE and has good anti-inflammatory effect, whereas the potential bioactivity of VNE in trichinellosis treatment is still unknown. MATERIALS AND METHODS: This study was designed to evaluate the potential anthelmintic and anti-inflammatory activity of VNE toward T. spiralis infection. ICR mice were used to assess the effect of VNE on repression larvae and adult worms in vivo. Immunohistochemistry analysis was performed to evaluate the expression levels of IL-1ß, IL-6, TNF-α, and COX-2. RESULTS: Our results showed that VNE could effectively depress the expressions of proinflammatory cytokines, including IL-1ß, IL-6, TNF-α, and COX-2. The adult worms were decreased by 79.53%, while the muscle larvae were diminished by 77.70% as compared to the control. CONCLUSION: These results demonstrated that VNE may be a promising therapeutic agent against the inflammation and diseases caused by T. spiralis infection.


Assuntos
Anti-Inflamatórios/administração & dosagem , Extratos Vegetais/administração & dosagem , Triquinelose/tratamento farmacológico , Vaccaria/química , Animais , Anti-Inflamatórios/análise , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Humanos , Interleucina-6/genética , Interleucina-6/imunologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Extratos Vegetais/análise , Trichinella/efeitos dos fármacos , Trichinella/fisiologia , Triquinelose/genética , Triquinelose/imunologia , Triquinelose/parasitologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
6.
PLoS Pathog ; 15(4): e1007657, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30998782

RESUMO

Helminths are highly prevalent metazoan parasites that infect over a billion of the world's population. Hosts have evolved numerous mechanisms to drive the expulsion of these parasites via Th2-driven immunity, but these responses must be tightly controlled to prevent equally devastating immunopathology. However, mechanisms that regulate this balance are still unclear. Here we show that the vigorous Th2 immune response driven by the small intestinal helminth Trichinella spiralis, is associated with increased TGFß signalling responses in CD4+ T-cells. Mechanistically, enhanced TGFß signalling in CD4+ T-cells is dependent on dendritic cell-mediated TGFß activation which requires expression of the integrin αvß8. Importantly, mice lacking integrin αvß8 on DCs had a delayed ability to expel a T. spiralis infection, indicating an important functional role for integrin αvß8-mediated TGFß activation in promoting parasite expulsion. In addition to maintaining regulatory T-cell responses, the CD4+ T-cell signalling of this pleiotropic cytokine induces a Th17 response which is crucial in promoting the intestinal muscle hypercontractility that drives worm expulsion. Collectively, these results provide novel insights into intestinal helminth expulsion beyond that of classical Th2 driven immunity, and highlight the importance of IL-17 in intestinal contraction which may aid therapeutics to numerous diseases of the intestine.


Assuntos
Células Dendríticas/imunologia , Intestino Delgado/imunologia , Células Th17/imunologia , Fator de Crescimento Transformador beta/metabolismo , Trichinella spiralis/imunologia , Triquinelose/imunologia , Animais , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/parasitologia , Intestino Delgado/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Th17/parasitologia , Triquinelose/parasitologia
7.
Mucosal Immunol ; 12(1): 51-63, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30356098

RESUMO

Innate lymphoid cells (ILCs) play an important role in regulating immune responses at mucosal surfaces. The transcription factor T-bet is crucial for the function of ILC1s and NCR+ ILC3s and constitutive deletion of T-bet prevents the development of these subsets. Lack of T-bet in the absence of an adaptive immune system causes microbiota-dependent colitis to occur due to aberrant ILC3 responses. Thus, T-bet expression in the innate immune system has been considered to dampen pathogenic immune responses. Here, we show that T-bet plays an unexpected role in negatively regulating innate type 2 responses, in the context of an otherwise intact immune system. Selective loss of T-bet in ILCs leads to the expansion and increased activity of ILC2s, which has a functionally important impact on mucosal immunity, including enhanced protection from Trichinella spiralis infection and inflammatory colitis. Mechanistically, we show that T-bet controls the intestinal ILC pool through regulation of IL-7 receptor signalling. These data demonstrate that T-bet expression in ILCs acts as the key transcriptional checkpoint in regulating pathogenic vs. protective mucosal immune responses, which has significant implications for the understanding of the pathogenesis of inflammatory bowel diseases and intestinal infections.


Assuntos
Colite/imunologia , Mucosa Intestinal/imunologia , Proteínas com Domínio T/metabolismo , Trichinella spiralis/fisiologia , Triquinelose/imunologia , Animais , Células Cultivadas , Humanos , Imunidade Celular , Imunidade Inata , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-7/metabolismo , Transdução de Sinais , Proteínas com Domínio T/genética , Células Th2/imunologia
8.
J Vet Med Sci ; 81(2): 169-176, 2019 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-30541982

RESUMO

Infections with gastrointestinal nematodes provoke immune and inflammatory responses mediated by cytokines released from T-helper type-2 (Th2) cells. Infections with Trichinella species have been reported to differ by the host species. Previously, in rats, we observed acute liver inflammation in response to infection with Trichinella spiralis, and the rat hosts showed a series of biochemical changes characterized by a decrease in serum paraoxonase (PON) 1 activity associated with the down-regulation of hepatic PON1 synthesis. In the present study, we investigated the effect(s) of species differences on the immune response against T. spiralis infection by analyzing serum PON1 activity and the associated inflammatory/anti-inflammatory mediators in mice. There were inconsistent changes in the serum PON1 activity of mice infected with T. spiralis, and these changes were associated with significant increases in the serum levels of interleukin (IL)-2, IL-4, IL-10, IL-12 (p70), granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor α during the enteric phase of the infection, while the levels of IL-5 and interferon γ were significantly increased throughout the entire experimental period. Moreover, T. spiralis infection in mice was associated with little inflammatory cell infiltration in hepatic tissues. Given the zoonotic prevalence of T. spiralis, further mechanistic research in this area is warranted.


Assuntos
Fígado/parasitologia , Trichinella spiralis/imunologia , Triquinelose/veterinária , Animais , Arildialquilfosfatase/sangue , Fator Estimulador de Colônias de Granulócitos e Macrófagos/sangue , Interferon gama/sangue , Interleucina-5/sangue , Interleucinas/sangue , Fígado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Triquinelose/imunologia , Triquinelose/parasitologia , Fator de Necrose Tumoral alfa/sangue
9.
Immunobiology ; 224(1): 147-153, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30413272

RESUMO

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease (CD), are chronic autoimmune diseases with a high recurrence rate. Epidemiological data have shown that the incidence of IBD increases annually because of improved sanitary conditions and reduced parasitic infection rates. In this experiment, experimental colitis was induced in mice by administering 2,4,6-trinitrobenzene sulfonic acid (TNBS) 28 days after they were infected with Trichinella spiralis to confirm that T. spiralis infection could alleviate the severity of TNBS-induced colitis. Thirty-six male BALB/c mice aged 6-8 weeks were randomly divided into four groups: control group (with 50% ethanol, Control), T. spiralis-infected group (TS-Control), TNBS-induced colitis model group (Colitis), and T. spiralis-pre-infected and TNBS-induced colitis group (TS-Colitis). The mice were sacrificed 3, 7, and 14 days after the model was established. Changes in various colitis indicators to investigate the effect of T. spiralis infection on TNBS-induced murine CD model. Results showed that the weight, DAI score, and macroscopic and microscopic colon damage in the TS-Colitis significantly decreased compared with those in the Colitis. ELISA revealed that the IFN-γ expression decreased and the IL-4 expression increased in the TS-Colitis compared with those in the Colitis. Western Blotting results revealed that the NF-κB expression increased in the Colitis and higher than those in the TS-Colitis. And Flow cytometry results revealed that the percentage of CD4+CD25+Foxp3+ Treg cells significantly increased in the TS-Colitis. T. spiralis-infected mice induced Th2 immune responses and balanced Th1 immune responses stimulated by TNBS to ameliorate intestinal inflammation.


Assuntos
Colite/terapia , Doenças Inflamatórias Intestinais/terapia , Linfócitos T Reguladores/imunologia , Células Th2/imunologia , Terapia com Helmintos , Trichinella spiralis/fisiologia , Triquinelose/imunologia , Animais , Colite/induzido quimicamente , Colite/imunologia , Colo/patologia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Humanos , Interferon gama/sangue , Interleucina-4/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Ácido Trinitrobenzenossulfônico
10.
Parasit Vectors ; 11(1): 666, 2018 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-30587214

RESUMO

BACKGROUND: Trichinella spiralis is a tissue-dwelling parasite has developed the ability to evade the host immune attack to establish parasitism in a host. One of the strategies evolved by the nematode is to produce proteins that immunomodulate the host immune system. TsPmy is a paramyosin secreted by T. spiralis on the surface of larvae and adult worms that can interact with complement components C1q and C8/C9 to compromise their activation and functions. To better understand the mechanism of TsPmy involved in the C1q inactivation and immune evasion, the C1q-binding site on TsPmy was investigated. METHODS: The TsPmy C1q-binding site was investigated by sequential narrow-down fragment expression in bacteria and peptide binding screening. C1q binding activity was identified by Far-Western blotting and ELISA assays. RESULTS: After several runs of sequential fragment expression, the C1q binding site was narrowed down to fragments of N-terminal TsPmy226-280aa and TsPmy231-315aa, suggesting the final C1q binding site is probably located to TsPmy231-280aa. A total of nine peptides covering different amino acid sequences within TsPmy231-280aa were synthesized. The binding assay to C1q determined that only P2 peptide covering TsPmy241-280aa binds to C1q, indicating that the C1q binding domain may need both the linearized sequence and conformational structure required for binding to C1q. The binding of peptide P2 to C1q significantly inhibited both C1q-initiated complement classical activation and C1q-induced macrophage chemotaxis. CONCLUSIONS: This study identifies the C1q binding site within TsPmy which provides helpful information for developing a vaccine against trichinellosis by targeting the C1q-binding activity of TsPmy.


Assuntos
Complemento C1q/imunologia , Proteínas de Helminto/imunologia , Trichinella spiralis/imunologia , Triquinelose/imunologia , Tropomiosina/química , Tropomiosina/imunologia , Animais , Sítios de Ligação , Complemento C1q/química , Complemento C1q/genética , Proteínas de Helminto/química , Proteínas de Helminto/genética , Humanos , Evasão da Resposta Imune , Mapeamento de Peptídeos , Trichinella spiralis/química , Trichinella spiralis/genética , Triquinelose/parasitologia , Tropomiosina/genética
11.
Parasit Vectors ; 11(1): 625, 2018 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-30518426

RESUMO

BACKGROUND: Trichinella spiralis is an important foodborne zoonotic parasite and it is necessary to develop a vaccine in order to interrupt transmission from animals to humans. A 31 kDa protein from T. spiralis (Ts31) is an antigen targeted by protective antibodies, and Ts31 contains a domain of trypsin-like serine protease that might have the function of serine protease. The purpose of this study was to investigate the molecular characteristics of Ts31 and its induced immune protection. METHODS: Expression and localization of Ts31 in various T. spiralis phases were investigated using qPCR and immunofluorescent test (IFT). The specific binding between Ts31 and intestinal epithelium cells (IECs) was analyzed by Far-Western blotting, ELISA and IFT, and the cellular localization of binding sites was examined on confocal microscopy. The mice were subcutaneously vaccinated with recombinant Ts31 protein (rTs31), serum specific IgG was determined by ELISA, and immune protection induced by immunization with rTs31 was evaluated. Inhibition of anti-rTs31 IgG on IL1 invasion of IECs and ADCC-mediated killing of newborn larvae (NBL) was also determined. RESULTS: Ts31 was expressed at different life-cycle stages and located principally at the stichosome and cuticle of this parasite. rTs31 was capable to specially bond to IECs, and binding site was located in the cytoplasm of IECs. Immunization of mice with rTs31 elicited a significant humoral response and protection, as demonstrated by a 56.93% reduction of adult worms at 6 days post-infection (dpi) and a 53.50% reduction of muscle larvae at 42 dpi after larval challenge. Anti-rTs31 antibodies impeded T. spiralis penetration of enterocytes in a dose-dependent pattern, and participated in the destruction of NBL by an ADCC-mediated manner. CONCLUSIONS: Ts31 facilitated the T. spiralis penetration of intestinal epithelium, which could make it a vaccine candidate target molecule against Trichinella infection.


Assuntos
Antígenos de Helmintos/imunologia , Proteínas de Helminto/imunologia , Trichinella spiralis/imunologia , Triquinelose/imunologia , Vacinas/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Antígenos de Helmintos/genética , Antígenos de Helmintos/metabolismo , Células Cultivadas , Feminino , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Imunização , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitologia , Larva/imunologia , Larva/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Transcrição Genética , Triquinelose/metabolismo , Triquinelose/prevenção & controle , Vacinas/genética , Vacinas/metabolismo
12.
Vet Immunol Immunopathol ; 203: 13-20, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30243368

RESUMO

The aim of the present study was to evaluate the detection of trichinellosis using ELISA, and to determine the degree of variation of IgG antibodies against excretory-secretory (E-S) antigens of T. spiralis muscle larvae. Ten young Polish Large White native breed pigs were experimentally inoculated with a low dose of 300 invasive Trichinella spiralis (T. spiralis) muscle larvae (ML). Pig sera were collected at 7 and 9 days prior to the experimental infection with T. spiralis and at 9, 14, 20, 23, 25, 27, 30, 33, 37, 41 and 46 days post-infection (d.p.i.). Western blotting was used as a follow-up test to detect anti-T. spiralis IgG and confirm the results provided by ELISA. The molecular weight of the E-S antigenic proteins of T. spiralis muscle larvae reacting with the sera from the infected pigs was determined. Finally, the intensity of T. spiralis infection (lpg) was determined as an average value of 16 muscles taken from each pig. The E-S antigen of T. spiralis muscle larvae in the in-house ELISA and immunoblotting assays did not demonstrate any cross-reaction with non-infected pig sera and sera from pigs naturally infected with Oesophagostomum spp. The ELISA assay did not recognize trichinellosis in pigs until 27 days after the T. spiralis infection. The anti-Trichinella IgG antibodies were first detected on day 30 post-infection. The immunoblotting technique confirmed the presence of anti-Trichinella IgG antibodies in all serum samples evaluated as ELISA-positive. The Western blot detected anti-Trichinella IgG antibodies on the same day they were detected for the first time by ELISA in five pigs, whereas the immunoblot assay detected seroconversion 2, 3 or 4 days earlier than ELISA in the remaining three pigs. The swine sera reacted with a broad range of T. spiralis ML E-S antigens with molecular weights ranging from 30 to 88 kDa and the most frequently recognized proteins were 45, 49 and 60 kDa.


Assuntos
Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/imunologia , Doenças dos Suínos/parasitologia , Trichinella spiralis/imunologia , Triquinelose/veterinária , Animais , Western Blotting/veterinária , Ensaio de Imunoadsorção Enzimática/veterinária , Larva , Músculo Esquelético/parasitologia , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/imunologia , Triquinelose/diagnóstico , Triquinelose/imunologia , Triquinelose/parasitologia
13.
Parasit Vectors ; 11(1): 499, 2018 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-30189888

RESUMO

BACKGROUND: Trichinella spiralis serine protease inhibitor (TsSPI) was identified in ES proteins of adult worms (AW), the TsSPI gene was highly expressed at enteral stage worms (AW and newborn larvae), distributed mainly in the cuticle and stichosome of this nematode. Vaccination of mice with rTsSPI exhibited a 62.2% reduction of intestinal AW and a 57.25% reduction of muscle larvae after larval challenge. The aim of this study was to investigate the biological characteristics of TsSPI and its roles in the process of T. spiralis invasion of host's intestinal epithelium cells (IECs). METHODS: The rTsSPI inhibition on trypsin enzymatic activity was detected by SDS-PAGE and spectrophotometry. The binding of rTsSPI with intestinal epithelium from normal mice and the primary cultured mouse intestinal epithelium cells (IECs) was examined by indirect immunofluorescent (IIF), the cellular localization of rTsSPI binding to IECs was observed by confocal microscopy. The inhibition of anti-rTsSPI serum on T. spiralis invasion of IECs was determined by an in vitro invasion assay. Anti-rTsSPI antibody cytotoxicity on the newborn larvae (NBL) was also determined. RESULTS: The rTsSPI had the inhibitory activity against porcine trypsin. The rTsSPI specifically bound to the intestinal epithelium from normal mice and primary cultured mouse IECs, and the binding sites were located in IEC membrane and cytoplasm. Anti-rTsSPI antibodies depressed the larval invasion of IECs with a dose-dependent mode. Anti-rTsSPI antibodies also participated in the destruction of T. spiralis NBL via an ADCC-mediated manner. CONCLUSIONS: TsSPI might participate in the T. spiralis larval invasion of IECs and it is likely the potential vaccine target against T. spiralis enteral stages.


Assuntos
Interações Hospedeiro-Parasita , Mucosa Intestinal/parasitologia , Larva/fisiologia , Inibidores de Serino Proteinase/genética , Trichinella spiralis/química , Triquinelose/prevenção & controle , Animais , Anticorpos Anti-Helmínticos/sangue , Contagem de Células , Linhagem Celular , Feminino , Proteínas de Helminto/administração & dosagem , Proteínas de Helminto/imunologia , Intestino Delgado/citologia , Intestino Delgado/parasitologia , Larva/genética , Camundongos , Camundongos Endogâmicos BALB C , Carga Parasitária , Inibidores de Serino Proteinase/administração & dosagem , Inibidores de Serino Proteinase/imunologia , Inibidores de Serino Proteinase/metabolismo , Trichinella spiralis/imunologia , Trichinella spiralis/metabolismo , Triquinelose/imunologia , Vacinas/administração & dosagem , Vacinas/imunologia
14.
Vet Res ; 49(1): 59, 2018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-30001738

RESUMO

In our previous work, a Trichinella spiralis putative serine protease (TsSP) was identified from ES products of T. spiralis intestinal infective larvae (IIL) and adult worms (AW) by immunoproteomics: it was highly expressed in IIL compared with muscle larvae (ML). In this study, the TsSP biological characteristics in larval invasion and growth were identified and its potential as a vaccine target against Trichinella infection were investigated. Expression of TsSP at various developmental phases (newborn larvae, ML, IIL, and AW) was detected by qPCR, immunofluorescent test and Western blotting. The rTsSP could specifically bind to the intestinal epithelial cell (IEC) membrane and enter into the cytoplasm. Anti-rTsSP serum suppressed the larval invasion of enterocytes in a dose-dependent mode, and killed newborn and ML of T. spiralis, decreased larval infectivity and development in the host by an ADCC-mediated mechanism. Immunization of mice with rTsSP produced a Th2 predominant immune response, and resulted in a 52.70% reduction of adult worms at 5 days post-infection (dpi) and a 52.10% reduction of muscle larvae at 42 dpi. The results revealed there was an interaction between TsSP and the host's IEC; TsSP might be a pivotal protein for the invading, growing and parasiting of this nematode in the host. Vaccination of mice with rTsSP elicited immune protection, and TsSP is a potential target molecule for vaccines against enteral Trichinella infection.


Assuntos
Proteínas de Helminto/imunologia , Imunização/veterinária , Serina Proteases/imunologia , Trichinella spiralis/fisiologia , Triquinelose/veterinária , Animais , Feminino , Proteínas de Helminto/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Serina Proteases/genética , Trichinella spiralis/crescimento & desenvolvimento , Triquinelose/imunologia
15.
Transbound Emerg Dis ; 65(6): 1447-1458, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29873198

RESUMO

Trichinella spiralis, the causative agent of trichinellosis, is able to infect a wide range of carnivores and omnivores including human beings. In the past 30 years, a mass of vaccination efforts has been performed to control T. spiralis infection with the purpose of reduction in worm fecundity or decrease in muscle larval and adult burdens. Here, we summarize the development of veterinary vaccines against T. spiralis infection. During recent years, increasing numbers of new vaccine candidates have been developed on the protective immunity against T. spiralis infection in murine model. The vaccine candidates were not only selected from excretory-secretory (ES) antigens, but also from the recombinant functional proteins, such as proteases and some other antigens participated in T. spiralis intracellular processes. However, immunization with a single antigen generally revealed lower protective effects against T. spiralis infection in mice compared to that with the inactivated whole worms or crude extraction and ES productions. Future study of T. spiralis vaccines should focus on evaluation of the protective efficacy of antigens and/or ligands delivered by nanoparticles that could elicit Th2-type immune response on experimental pigs.


Assuntos
Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/imunologia , Trichinella spiralis/imunologia , Triquinelose/veterinária , Vacinas de DNA/imunologia , Animais , Feminino , Humanos , Imunização/veterinária , Larva , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologia , Suínos , Triquinelose/imunologia , Triquinelose/prevenção & controle , Vacinação/veterinária
16.
Immunol Invest ; 47(6): 605-614, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29723085

RESUMO

Trichinella spiralis infection induces Trichinella-specific IgG antibody and high level of blood eosinophil. However, the kinetics induced by different parasite burdens during infectious periods remains unclear. In this study, rats were infected with 100, 1000, or 3000 larvae of T. spiralis (100 TS, 1000 TS, or 3000 TS). Correlates of eosinophils, antibody responses, and Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) with worm burdens were evaluated at 1 week, 2 weeks, 1 month, and 2 months postinfection. Heavy infections (1000 TS and 3000 TS) showed significantly higher levels of eosinophil, IgG, and IgG1 antibody responses at 2 weeks postinfection compared to light infection (100 TS). The highest RANTES mRNA expression was also found in the heavy infection group (3000 TS). The results indicate, at early stage of infection (week 2), heavy infection induced higher levels of IgG, IgG1, eosinophil, and RANTES responses. However, at late stage of infection (month 2), there were no correlates of immunity with parasite burdens. Higher levels of IgG and IgG1 antibody responses are critical in heavy T. spiralis infection. These results provide important information in evaluating immune responses by T. spiralis infective stage during the T. spiralis infection.


Assuntos
Anticorpos Antiprotozoários/sangue , Quimiocina CCL5/sangue , Eosinófilos/imunologia , Imunoglobulina G/sangue , Trichinella spiralis/imunologia , Triquinelose/imunologia , Animais , Quimiocina CCL5/genética , Feminino , Contagem de Leucócitos , Carga Parasitária , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Triquinelose/parasitologia
17.
PLoS Negl Trop Dis ; 12(5): e0006502, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29775453

RESUMO

BACKGROUND: Trichinellosis is a serious food-borne parasitic zoonosis worldwide. In the effort to develop vaccine against Trichinella infection, we have identified Trichinella spiralis Heat shock protein 70 (Ts-Hsp70) elicits partial protective immunity against T. spiralis infection via activating dendritic cells (DCs) in our previous study. This study aims to investigate whether DCs were activated by Ts-Hsp70 through TLR2 and/or TLR4 pathways. METHODS AND FINDINGS: After blocking with anti-TLR2 and TLR4 antibodies, the binding of Ts-Hsp70 to DCs was significantly reduced. The reduced binding effects were also found in TLR2 and TLR4 knockout (TLR2-/- and TLR4-/-) DCs. The expression of TLR2 and TLR4 on DCs was upregulated after treatment with Ts-Hsp70 in vitro. These results suggest that Ts-Hsp70 is able to directly bind to TLR2 and TLR4 on the surface of mouse bone morrow-derived DCs. In addition, the expression of the co-stimulatory molecules (CD80, CD83) on Ts-Hsp70-induced DCs was reduced in TLR2-/- and TLR4-/- mice. More evidence showed that Ts-Hsp70 reduced its activation on TLR2/4 knockout DCs to subsequently activate the naïve T-cells. Furthermore, Ts-Hsp70 elicited protective immunity against T. spiralis infection was reduced in TLR2-/- and TLR4-/- mice correlating with the reduced humoral and cellular immune responses. CONCLUSION: This study demonstrates that Ts-Hsp70 activates DCs through TLR2 and TLR4, and TLR2 and TLR4 play important roles in Ts-Hsp70-induced DCs activation and immune responses.


Assuntos
Células Dendríticas/imunologia , Proteínas de Choque Térmico HSP70/imunologia , Proteínas de Helminto/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Trichinella spiralis/imunologia , Triquinelose/imunologia , Animais , Células Dendríticas/parasitologia , Feminino , Proteínas de Choque Térmico HSP70/genética , Proteínas de Helminto/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linfócitos T/imunologia , Linfócitos T/parasitologia , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Trichinella spiralis/genética , Triquinelose/genética , Triquinelose/parasitologia
18.
PLoS Negl Trop Dis ; 12(5): e0006485, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29758030

RESUMO

BACKGROUND: Trichinellosis is a serious zoonositc parasitosis worldwide. Because its clinical manifestations aren't specific, the diagnosis of trichinellosis is not easy to be made. Trichinella spiralis muscle larva (ML) excretory-secretory (ES) antigens are the most widely applied diagnostic antigens for human trichinellosis, but the major drawback of the ES antigens for assaying anti-Trichinella antibodies is the false negative in the early Trichinella infection period. The aim of this study was to characterize the T. spiralis putative serine protease (TsSP) and to investigate its potential use for diagnosis of trichinellosis. METHODOLOGY/PRINCIPAL FINDINGS: The full-length TsSP sequence was cloned and expressed, and recombinant TsSP (rTsSP) was purified by Ni-NTA-Sefinose Column. On Western blotting analysis the rTsSP was recognized by T. spiralis-infected mouse serum, and the natural TsSP was identified in T. spiralis ML crude and ES antigens by using anti-rTsSP serum. Expression of TsSP was detected at various T. spiralis developmental stages (newborn larvae, muscle larvae, intestinal infective larvae and adult worms). Immunolocalization identified the TsSP principally in cuticles and stichosomes of the nematode. The sensitivity of rTsSP-ELISA and ES-ELISA was 98.11% (52/53) and 88.68% (47/53) respectively (P > 0.05) when the sera from trichinellosis patients were examined. However, while twenty-one serum samples of trichinellosis patients' sera at 19 days post-infection (dpi) were tested, the sensitivity (95.24%) of rTsSP-ELISA was distinctly higher than 71.43% of ES-ELISA (P < 0.05). The specificity (99.53%) of rTsSP-ELISA was remarkably higher than 91.98% of ES-ELISA (P < 0.01). Only one out of 20 serum samples of cysticercosis patients cross-reacted with the rTsSP. Specific anti-Trichinella IgG in infected mice was first detected by rTsSP-ELISA as soon as 7 dpi and antibody positive rate reached 100% on 10 dpi, whereas the ES-ELISA did not permit detection of 100% of infected mice before 16 dpi. CONCLUSIONS: The rTsSP is a potential early diagnostic antigen for human trichinellosis.


Assuntos
Proteínas de Helminto/imunologia , Serina Proteases/imunologia , Trichinella spiralis/enzimologia , Triquinelose/parasitologia , Animais , Anticorpos Anti-Helmínticos/análise , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/análise , Antígenos de Helmintos/genética , Antígenos de Helmintos/imunologia , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas de Helminto/análise , Proteínas de Helminto/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Serina Proteases/análise , Serina Proteases/genética , Trichinella spiralis/genética , Trichinella spiralis/isolamento & purificação , Triquinelose/diagnóstico , Triquinelose/imunologia
19.
Parasitol Res ; 117(1): 201-212, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29189952

RESUMO

The present study explored potentially immunogenic proteins of the encapsulated (Trichinella spiralis) and non-encapsulated (T. pseudospiralis, T. papuae) species within the genus Trichinella. The somatic muscle larval extracts of each species were subjected to immunoblotting analysis using human T. spiralis-infected serum samples. Fifteen reactive bands of all three species were selected for further protein identification by liquid chromatography-tandem mass spectrometry, and their possible functions were ascertained using the gene ontology. Our findings showed immunogenic protein patterns with molecular mass in the range of 33-67 kDa. Proteomic and bioinformatic analysis revealed a wide variety of functions of 17 identified proteins, which are associated with catalytic, binding, and structural activities. Most proteins were involved in cellular and metabolic processes that contribute in the invasion of host tissues and the larval molting processes. The parasite proteins were identified as actin-5C, serine protease, deoxyribonuclease-2, and intermediate filament protein ifa-1. This information may lead to alternative tools for selection of potential diagnostic protein markers or aid in the design of vaccine candidates for prevention and control of Trichinella infection.


Assuntos
Trichinella spiralis/imunologia , Triquinelose/imunologia , Animais , Antígenos de Helmintos/imunologia , Cromatografia Líquida , Gerbillinae , Proteínas de Helminto/imunologia , Humanos , Immunoblotting , Larva/metabolismo , Músculos/parasitologia , Oxirredução , Proteômica , Trichinella/imunologia , Trichinella spiralis/metabolismo , Triquinelose/parasitologia
20.
Vaccine ; 36(2): 248-255, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29199042

RESUMO

Trichinellosis is a foodborne disease that remains a public health hazard and an economic problem in food safety. Vaccines against the parasite can be an effective way to control this disease; however, commercial vaccines against Trichinella infection are not yet available. Trichinella cathepsin B proteins appear to be promising targets for vaccine development. Here, we reported for the first time the characterization of a novel cDNA that encodes Trichinella spiralis (T. spiralis) cathepsin B-like protease 2 gene (TsCPB2). The recombinant mature TsCPB2 protein was successfully expressed in E. coli system and purified with Ni-affinity chromatography. TsCPB2 expression was detected at all the developmental stages of T. spiralis and it was expressed as an excretory-secretory protein of T. spiralis muscle larvae. Immunization with TsCPB2 antigen induced a combination of humoral and cellular immune responses, which manifested as a mixed Th1/Th2 response, as well as remarkably elevated IgE level. Moreover, vaccination of mice with TsCPB2 that were subsequently challenged with T. spiralis larvae resulted in a 52.3% (P < .001) reduction in worm burden and a 51.2% (P < .001) reduction in muscle larval burden. Our results suggest that TsCPB2 induces protective immunity in Trichinella-infected mice and might be a novel vaccine candidate against trichinellosis.


Assuntos
Antígenos de Helmintos/imunologia , Catepsina B/imunologia , Proteínas Recombinantes/imunologia , Trichinella spiralis/imunologia , Triquinelose/prevenção & controle , Animais , Antígenos de Helmintos/administração & dosagem , Catepsina B/administração & dosagem , Modelos Animais de Doenças , Feminino , Imunidade Celular , Imunidade Humoral , Camundongos Endogâmicos BALB C , Músculos/parasitologia , Carga Parasitária , Proteínas Recombinantes/administração & dosagem , Triquinelose/imunologia
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