Donkey-derived anti-CDV IgG, as a passive immunotherapy agent, can effectively increase survival rates of the experimental CDV-infected dogs.

BACKGROUND: Humoral immunity plays an important role in the prevention of canine distemper. Anti-CD virus (CDV) antibody has strong antiviral activity and is widely used in the treatment of CD. However, with the increase of CD cases, the availability of therapeutic CD antibody fell short of the clinical needs. RESULTS: The high-titer antiserum with the high-titer neutralizing activity against CDV was obtained from the donkeys (Dezhou Donkey) immunized with the inactivated CDV vaccine. The donkey anti-CDV IgG was purified from the donkey serum, which was identified to significantly inhibit the CDV replication in the cultured Vero cells and effectively reduce the clinical symptoms and increase the survival rates (75%) of CDV-infected dogs (Shih-tzu Dog), similar to that treated with the dog-derived anti-CDV IgG. These results indicate that donkey-derived IgG is a potential substitute for dog-derived IgG to treat the CD in clinic. CONCLUSIONS: Administration of donkey-derived anti-CDV IgG can ameliorate clinical symptoms and inhibit virus replication, thereby increasing the survival of CDV-infected dogs. This study opens up a new source of therapeutic antibody for CD treatment.

Tanshinone IIA attenuates atherosclerosis via inhibiting NLRP3 inflammasome activation.

Tanshinone IIA (Tan IIA) possesses potent anti-atherogenic function, however, the underlying pharmacological mechanism remains incompletely understood. Previous studies suggest that oxidized LDL (oxLDL)-induced NLRP3 (NOD-like receptor (NLR) family, pyrin domain-containing protein 3) inflammasome activation in macrophages plays a vital role in atherogenesis. Whether the anti-atherogenic effect of Tan IIA relies on the inhibition of the NLRP3 inflammasome has not been investigated before. In this study, we found that Tan IIA treatment of high-fat diet fed ApoE-/- mice significantly attenuated NLRP3 inflammasome activation in vivo. Consistently, Tan IIA also potently inhibited oxLDL-induced NLRP3 inflammasome activation in mouse macrophages. Mechanically, Tan IIA inhibited NF-κB activation to downregulate pro-interleukin (IL) -1ß and NLRP3 expression, and decreased oxLDL-induced expression of lectin-like oxidized LDL receptor-1 (LOX-1) and cluster of differentiation 36 (CD36), thereby attenuating oxLDL cellular uptake and subsequent induction of mitochondrial and lysosomal damage - events that promote the NLRP3 inflammasome assembly. Through regulating both the inflammasome 'priming' and 'activation' steps, Tan IIA potently inhibited oxLDL-induced NLRP3 inflammasome activation, thereby ameliorating atherogenesis.

Porcine soluble CD83 alleviates LPS-induced abortion in mice by promoting Th2 cytokine production, Treg cell generation and trophoblast invasion.

CD83, either in its membrance-bound form (mCD83) or soluble form (sCD83), is an important immunomodulatory molecule in humans and mice. While mCD83 is immunostimulatory, sCD83 exhibits striking immunosuppressive activities, suggesting that sCD83 may be used to combat inflammatory diseases, such as rheumatoid arthritis, graft-versus-host disease and habitual abortion. Although many studies had shed lights on the role of CD83 in humans and mice, little is known about CD83 in other animals. Recently, we showed that porcine CD83 had similar biochemical characteristics and immunoregulatory functions as its human counterpart. However, whether porcine sCD83 (psCD83) is involved in maintaining the immunological tolerance at the maternal-fetal interface and thereby prevents embryo loss and abortion during pregnancy is unclear. In this study, we used LPS-induced animal model to analyze the effect of porcine sCD83 on the mouse abortion. Results showed that psCD83 could significantly alleviate LPS-induced abortion in mice, indicating that the psCD83 had the function of fetal protection. Mechanically, psCD83-mediated fetal protection was related to the promotion on Th2 cytokine production, Treg cell differentiation and trophoblast invasion. This study provides a molecular basis for the fetal protection of psCD83, as well as a potential target for the regulation of maternal-fetal interfacial immune tolerance.

Development of a monoclonal antibody against canine parvovirus NS1 protein and investigation of NS1 dynamics and localization in CPV-infected cells.

Canine parvovirus (CPV) non-structural protein-1 (NS1) plays crucial roles in CPV replication and transcription, as well as pathogenic effects to the host. However, the mechanism was not fully understood. Lack of NS1 antibody is one of the restricting factors for NS1 function investigation. To prepare NS1 monoclonal antibody (mAb), the NS1 epitope (AA461 ~ AA650) gene was amplified by PCR, and inserted into pGEX-4T-1vector to construct the prokaryotic expression vector of GST-tag-fused NS1 epitope gene. The NS1 fusion protein was expressed in E. coli, and purified with GSH-magnetic beads, and then used to immunize BALB/c mice. The mouse splenic lymphocytes were isolated and fused with myeloma cells (SP 2/0) to generate hybridoma cells. After several rounds of screening by ELISA, a hybridoma cell clone (1B8) stably expressing NS1 mAb was developed. A large amount of NS1 mAb was prepared from mouse ascites fluid. The isotype of NS1 mAb was identified as IgG1, which can specifically bind NS1 protein in either CPV-infected cells or NS1 vector-transfected cells, indicating the NS1 mAb is effective in detecting NS1 protein. Meanwhile, we used the NS1 mAb to investigate NS1 dynamic changes by qRT-PCR and location by confocal imaging in CPV-infected host cells and showed that NS1 began to appear in the cells at 12 h after CPV infection and reached the highest level at 42 h, NS1 protein was mainly located in nucleus of the cells. This study provided a necessary condition for further investigation on molecular mechanism of NS1 function and pathogenicity.

Detection and phylogenetic analysis of porcine circovirus 3 in part of northern China from 2016 to 2018.

Porcine circovirus 3 (PCV3) is a recently identified virus that is associated with reproductive failure, porcine dermatitis and nephropathy syndrome, and multi-systemic inflammation. To investigate the molecular epidemic characteristics and genetic evolution of PCV3 in northern China, a commercial TaqMan-based real-time quantitative PCR kit was used to detect PCV3 in 435 tissue specimens collected from pigs with various clinical signs from 105 different swine farms in northern China. The results showed that 48 out of 105 (45.7%) farms and 97 out of 435 (22.3%) samples tested positive for PCV3. Of the 97 PCV3-positive samples, 80 (82.5%) tested positive for other pathogens. PCV3 was found more frequently in pigs with reproductive failure than in those with other clinical signs. This study is the first to detect PCV3 in Tianjin. The complete genome sequences of six PCV3 isolates and the capsid (Cap) protein gene sequences of 11 isolates were determined. Based on the predicted amino acids at positions 24 and 27 of the Cap protein and their evolutionary relationships, the 17 PCV3 strains obtained from northern China and 49 reference strains downloaded from the GenBank database were divided into four major groups (3a-3d). An analysis of selection pressure and polymorphism indicated that the PCV3 Cap protein seems to be evolving under balancing selection, that the population is in dynamic equilibrium, and that no population expansion occurred during the study period. Our results provide new information about the molecular epidemiology and evolution of PCV3.

Establishment of canine macrophages stably expressing GFP-tagged canine LC3 protein for effectively detecting autophagy.

Autophagy plays a crucial role in eliminating protein aggregates, damaged organelles and invading pathogens. Genetically engineered cell line stably expressing green fluorescent protein (GFP)-tagged microtubule-associated protein light chain 3 (LC3) is extensively used to test autophagy through observing GFP puncta formation in the cells by fluorescence imaging. However, canine LC3 (cLC3) gene has not been cloned, therefore, GFP-tagged canine LC3 (GFP-cLC3) detection system has not been established. To generate GFP-cLC3 stably expressing canine-derived macrophages, the cLC3 cDNA was first amplified by RT-PCR and inserted into pEGFP-C1 plasmid to create GFP-cLC3 gene fusion. This genetic element was then transducted into canine macrophages mediated by lentivirus vector to generate the canine macrophages stably expressing fusion protein. Results showed that the sequence of cLC3 cloned in this study is highly homologous with other animals (80-95% homology). Phenotypic and functional analysis of these engineered cells revealed that GFP-cLC3 was indeed stably expressed and rapamycin or starvation can effectively induce GFP puncta formation in the cells, indicative of autophagosome formation. These GFP-cLC3-expressing cells may thus be useful to study autophagy in canine.

Seroprevalence of porcine bocavirus in pigs in north-central China using a recombinant-NP1-protein-based indirect ELISA.

Porcine bocavirus (PBoV), which belongs the genus Bocaparvovirus, has been identified throughout the world. However, serological methods for detecting anti-PBoV antibodies are presently limited. In the present study, an indirect enzyme-linked immunosorbent assay (PBoV-rNP1 ELISA) based on a recombinant form of nucleoprotein 1 (NP1) of PBoV was established for investigating the seroprevalence of PBoV in 2025 serum specimens collected in north-central China from 2016 to 2018, and 42.3% of the samples tested positive for anti-PBoV IgG antibodies, indicating that the seroprevalence of PBoV is high in pig populations in China.

Membrane-bound and soluble porcine CD83 functions antithetically in T cell activation and dendritic cell differentiation in vitro.

Emerging evidence suggests that CD83, a dendritic cells (DCs) maturation marker in humans and mice, may prossess immunomodulatory capacities. Although porcine CD83 shares ∼75% sequence homology with its human counterpart, whether it functions as an immunoregulatory molecule remains unknown. To investigate porcine CD83 function, we deleted it in porcine DCs by RNA intereference. Results show that membrane-bound CD83 (mCD83) promotes DC-mediated T cell proliferation and cytokine production, thus confirming its immunoregulatory capacity. Intriguingly, porcine soluble CD83 (sCD83) treatment instead led to inhibition of DC-mediated T cell activation. Moreover, porcine sCD83 also inhibited differentiation of prepheral blood mononuclear cells (PBMCs) into DCs. These results collectively indicate that in addition to being a DC maturation maker, both membrane bound and souble porcine CD83 serve as immunoregulatory molecules with opposite effects on DC-mediated T cell activation and DC differentiation.

Co-Expression of Chicken IL-2 and IL-7 Enhances the Immunogenicity and Protective Efficacy of a VP2-Expressing DNA Vaccine against IBDV in Chickens.

Chicken infectious bursal disease (IBD) is still incompletely controlled worldwide. Although IBD virus (IBDV) VP2 DNA vaccine was considered a safe vaccine for IBD prevention, the immunogenicity by itself remains poor, resulting in the failure of effectively protecting chickens from infection. We and others demonstrated that chicken IL-2 (chIL-2) and chIL-7 have the capacity to enhance the immunogenicity of the VP2 DNA vaccine. However, whether chIL-2 and chIL-7 can mutually enhance the immunogenicity of VP2 DNA vaccine and thereby augment the latter's protection efficacy remains unknown. By using chIL-2/chIL-7 bicistronic gene vector to co-immunize the chickens together with the VP2 DNA vaccine, we now show that chIL-2 and chIL-7 significantly increased IBDV VP2-specific antibody titers, T cell proliferation, and IFN-γ production, resulting in the ultimate enhancement of vaccine-induced protection efficacy relative to that of chIL-2 or chIL-7 gene vectors alone. These results suggest that chIL-2 and chIL-7 can mutually enhance VP2 DNA vaccine's efficacy, thereby establishing a concrete foundation for future optimization of IBDV VP2 DNA vaccine to prevent/treat chicken IBD.

Porcine Parvovirus Infection Causes Pig Placenta Tissue Damage Involving Nonstructural Protein 1 (NS1)-Induced Intrinsic ROS/Mitochondria-Mediated Apoptosis.

Porcine parvovirus (PPV) is an important pathogen causing reproductive failure in pigs. PPV-induced cell apoptosis has been recently identified as being involved in PPV-induced placental tissue damages resulting in reproductive failure. However, the molecular mechanism was not fully elucidated. Here we demonstrate that PPV nonstructural protein 1 (NS1) can induce host cell apoptosis and death, thereby indicating the NS1 may play a crucial role in PPV-induced placental tissue damages and reproductive failure. We have found that NS1-induced apoptosis was significantly inhibited by caspase 9 inhibitor, but not caspase 8 inhibitor, and transfection of NS1 gene into PK-15 cells significantly inhibited mitochondria-associated antiapoptotic molecules Bcl-2 and Mcl-1 expressions and enhanced proapoptotic molecules Bax, P21, and P53 expressions, suggesting that NS1-induced apoptosis is mainly through the mitochondria-mediated intrinsic apoptosis pathway. We also found that both PPV infection and NS1 vector transfection could cause host DNA damage resulting in cell cycle arrest at the G1 and G2 phases, trigger mitochondrial ROS accumulation resulting in mitochondria damage, and therefore, induce the host cell apoptosis. This study provides a molecular basis for elucidating PPV-induced cell apoptosis and reproductive failure.

Porcine CD83 is a glycosylated dimeric protein existing naturally in membrane-bound and soluble forms.

Human and mouse CD83 have been well characteized, however, the other mammalian CD83 genes have not been cloned and characterized. In this study, the porcine CD83 (pCD83) was cloned, expressed and characterized, and showed that the pCD83 gene has 81% and 74% homologies with humans and mice, respectively, which was identified to be glycosylated when expressed in eukaryotic cells, existing naturally in two forms: membrance-bound CD83 (mCD83) and soluble CD83 (sCD83), the latter was identified to be generated mainly from mCD83 by proteolytic shedding. The pCD83 was a dimmer mediated by intermolecular disulfide bond formed by the fifth cysteine in the exrtracellular domain. Functionally, the recombinant porcine sCD83 was preliminarily tested to have the ability to inhibit DC-mediated T cell activition. This study provided necessary fundation for further investigation on pCD83 functions.

Naturally-occurring right terminal hairpin mutations in three genotypes of canine parvovirus (CPV-2a, CPV-2b and CPV-2c) have no effect on their growth characteristics.

We have isolated 4 naturally-occurring strains of CPV in mainland China and have identified them as CPV-2, 2a, 2b and 2c genotypes according to their VP2 sequences which also revealed substitutions within their right terminal regions. To determine if these substitutions affected the growth characteristics of the 4 strains, we constructed plasmids based on their genomic sequences minus their right terminal sequences, with the latter replaced by a single right terminal region. Analysis of rescued recombinants showed that the substitutions within their natural right termini had no significant effect on their growth characteristics.

Recombinant chicken interleukin-7 as a potent adjuvant increases the immunogenicity and protection of inactivated infectious bursal disease vaccine.

Our previous work showed that a plasmid-based chicken interleukin-7 (chIL-7) gene expression vector possessed potent adjuvant activity for a VP2 DNA vaccine against chicken infectious bursal disease virus (IBDV). Whether recombinant chIL-7 prepared in procaryotic expression system has the adjuvant activity for inactivated IBDV vaccine remains unknown. Here, we prepared recombinant chIL-7 using an E. coli expression system and analyzed its adjuvant activity for the inactivated IBDV vaccine. The results show that the recombinant chIL-7 was successfully prepared in E. coli using the pET20b vector, which possessed biological activity to stimulate mouse B lymphocyte proliferation. Co-administration of the chIL-7 with inactivated IBDV vaccine significantly increased specific serum antibody titers against IBDV, enhanced lymphocyte proliferation and IFN-γ and IL-4 productions, and increased protection against virulent IBDV infection.

Construction of Bacillus subtilis strain engineered for expression of porcine ß-defensin-2/cecropin P1 fusion antimicrobial peptides and its growth-promoting effect and antimicrobial activity.

OBJECTIVE: To generate recombinant Bacillus subtilis (B. subtilis) engineered for expression of porcine ß-defensin-2 (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo. METHODS: The pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli). The weight gain and diarrhea incidence of piglets were measured after challenge. RESULTS: The recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis) and gram-positive bacteria (Staphylococcus aureus). Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets. CONCLUSION: The generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial activities to a variety of bacterial species in vitro. Supplementation of the engineered B. subtilis in pig feed obviously promote piglet growth and resistance to the colibacillosis.

Chicken IL-7 as a potent adjuvant enhances IBDV VP2 DNA vaccine immunogenicity and protective efficacy.

Our previous work has demonstrated that the mammalian interleukin-7 (IL-7) gene can enhance the immunogenicity of DNA vaccine. Whether chicken IL-7 (chIL-7) possesses the ability to enhance the immunogenicity of VP2 DNA vaccine of infectious bursal disease virus (IBDV) remained unknown. To investigate this, we constructed a VP2 antigenic region (VP2366) gene and chIL-7 gene vectors, co-immunized chicken with these vectors and analyzed the effects of the chIL-7 gene on VP2366 gene immunogenicity. Results showed that co-administrated chIL-7 gene with VP2 DNA vaccine significantly increased specific serum antibody titers against IBDV, and enhanced lymphocyte proliferation and IFN-γ and IL-4 productions. More importantly, chIL-7 gene significantly increased VP2366 gene-induced protection against virulent IBDV infection, indicating that the chIL-7 gene possessed the capacity to enhance VP2366 DNA vaccine immunogenicity, and therefore might function as a novel adjuvant for IBDV VP2 DNA vaccine. Mechanically, chIL-7 could stimulate the common cytokine receptor γ chain (γc) expressions in vitro and in vivo, which might be involved in chIL-7 enhancement of the immunogenicity of VP2 DNA vaccine.

Molecular cloning of chicken IL-7 and characterization of its antiviral activity against IBDV in vivo.

Mammalian interleukin-7 (IL-7) is able to stimulate lymphocyte proliferation and maturation, and reverse immunosuppression. However, whether poultry IL-7 has similar functions remains unclear. Chicken infectious bursal disease virus (IBDV) causes serious immunosuppression in chicken due to virus-induced immune disorder. Whether chicken IL-7 (chIL-7) has the ability to restore the immunity during IBDV-induced immunosuppression is not clear. To test this, we amplified chIL-7 gene by RT-PCR, prepared recombinant chIL-7 using HEK293T cells and treated the chicken with the chIL-7 prior to IBDV infection. Our results indicate that chIL-7 promoted mouse B cell proliferation in vitro, and significantly reduced virus titer in bursal tissue and chicken morbidity of IBDV-infected chicken. Mechanically, chIL-7 induced chicken lymphocyte proliferation and interferon-γ production, but down-regulated TGF-ß expression, suggesting that chIL-7 has the ability to reverse IBDV-induced immunosuppression and might be a potential therapeutic agent for prevention and treatment of infectious bursal disease.

NLRP3 inflammasome activation contributes to Listeria monocytogenes-induced animal pregnancy failure.

BACKGROUND: Listeria monocytogenes (LM), a foodborne pathogen, can cause pregnancy failure in animals, especially in ruminants. Recent studies have shown that LM activates inflammasomes to induce IL-1ß release in macrophages, however, whether the inflammasome activation regulates LM-induced pregnancy failure remains largely unknown. Here we used mouse model to investigate the molecular mechanism by which LM-induced inflammsome activation contributes to LM-associated pregnancy failure RESULTS: We showed that wild-type, but not Listeriolysin O-deficient (Δhly) LM, significantly reduced mouse embryo survival, accompanied by the increase of IL-1ß release and caspase-1 activation. IL-1ß neutralization significantly reduced the LM-induced embryo losses, suggesting that LM-induced pregnancy failure was associated with LLO-induced inflammasome activation. To dissect the inflammasome sensor and components responsible for LM-induced caspase-1 activation and IL-1ß production, we used wild-type and NLRP3(-/-), AIM2(-/-), NLRC4(-/-), ASC(-/-), caspase-1(-/-) and cathepsin B(-/-) mouse macrophages to test the roles of these molecules in LM-induce IL-1ß production. We found that NLRP3 inflammasome was the main pathway in LM-induced caspase-1 activation and IL-1ß production. To explore the mechanism of LM-induced pregnancy failure, we investigated the effects of LM-infected macrophages on SM9-1 mouse trophoblasts. We found that the conditioned medium from LM-infected-macrophage or the recombinant IL-1ß significantly up-regulated TNFα, IL-6 and IL-8 productions in trophoblasts, suggesting that the LM-induced macrophage inflammasome activation increased trophoblast pro-inflammatory cytokine production, which was adverse to the animal pregnancy maintenance. CONCLUSIONS: Our data demonstrated that the LLO-induced NLRP3 inflammasome activation plays a key role in LM-induced pregnancy failure, and inflammasome-mediated macrophage dysregulation on trophoblasts might be involved in the pregnancy failure.

Mutual enhancement of IL-2 and IL-7 on DNA vaccine immunogenicity mainly involves regulations on their receptor expression and receptor-expressing lymphocyte generation.

Our previous study showed that IL-2 and IL-7 could mutually enhance the immunogenicity of canine parvovirus VP2 DNA vaccine, although the underlying mechanism remained unknown. Here, we used the OVA gene as a DNA vaccine in a mouse model to test their enhancement on DNA vaccine immunogenicity and to explore the molecular mechanism. Results showed that both IL-2 and IL-7 genes significantly increased the immunogenicity of OVA DNA vaccine in mice. Co-administration of IL-2 and IL-7 genes with OVA DNA significantly increased OVA-specific antibody titers, T cell proliferation and IFN-γ production compared with IL-2 or IL-7 alone, confirming that IL-2 and IL-7 mutually enhanced DNA vaccine immunogenicity. Mechanistically, we have shown that IL-2 significantly stimulated generation of IL-7 receptor-expressing lymphocytes, and that IL-7 significantly induced IL-2 receptor expression. These results contribute to an explanation of the mechanism of the mutual effects of IL-2 and IL-7 on enhancing DNA vaccine immunogenicity and provided a basis for further investigation on their mutual effects on adjuvant activity and immune regulation.

Preparation of North American type II PRRSV infectious clone expressing green fluorescent protein.

Porcine reproductive and respiratory syndrome virus (PRRSV) is still one of the most important infectious diseases threatening the swine industry. To construct North American type II PRRSV infectious clone containing green fluorescent protein (GFP) gene, we amplify gfp gene, flanked by PRRSV Nsp2 gene fragments upstream and downstream, using overlap PCR method from pcDNA-EF1-GFP plasmid and FL12 plasmid containing PRRSV infectious genome as the templates. The Nsp2 fragment-flanked gfp gene was inserted into Nsp2 gene of the FL12 plasmid by Spe I and Xho I sites to generate PRRSV infectious recombinant plasmid (FL12-GFP) containing gfp gene. The recombinant PRRSV expressing GFP (PRRSV-GFP) was rescued in baby hamster kidney-21 (BHK-21) cells by transfecting PRRSV mRNA synthesized in vitro and amplified in Marc-145 cells. The PRRSV-GFP infectivity and replication capacity were identified. Results showed that, by adopting overlap PCR strategy, the gfp gene was successfully inserted into and fused with PRRSV Nsp2 gene in the PRRSV infectious clone plasmid FL-12 to generate FL12-GFP plasmid. The recombinant PRRSV-GFP was generated through transfecting PRRSV mRNA in BHK-2 cells. Like its parental virus, the recombinant PRRSV-GFP maintains its infectivity to Marc-145 cells and porcine alveolar macrophages (PAMs). This study provides essential conditions for further investigation on PRRSV.

Effects of toosendanin on pregnancy and uterine immunity alterations in mice.

This study was conducted to explore the abortifacient effect and the mechanisms of the Chinese herbal medicine component toosendanin, and to elucidate the significance of the Th1 cytokines IFN-gamma and TNF-alpha, CD4+ and CD8+ T lymphocytes in the occurrence of abortion. Graded doses of toosendanin were given by intraperitoneal injection (i.p.) to mice at day 5, 6, 7 of gestation. The levels of Th1 cytokines (IFN-gamma, TNF-alpha) in serum and uterine tissues from mice sacrificed at day 8 were analyzed by enzyme linked immunosorbent assay (ELISA). Presence of T lymphocytes in endometrium was detected by immunohistochemistry. The results revealed that injection of toosendanin could produce a dose-dependent toxicity. The IFN-gamma, TNF-alpha content in serum and uterine tissues were increased significantly. The CD4+ and CD8+ T lymphocytes were also increased in the endometrium of toosendanin treated groups. In conclusion, toosendanin is pregnancy-toxic to animals and it is relevant to the increased contents of IFN-gamma, TNF-alpha and CD4+, CD8+ T lymphocytes.