Lycium barbarum polysaccharide promotes the immunoprotective effects of a recombinant Lactobacillus plantarum vaccine expressing the Trichinella spiralis cathepsin F-like protease 1 gene.

Trichinellosis caused by Trichinella spiralis (T. spiralis) is a zoonotic disease that poses a substantial risk to human health. At present, vaccines used to prevent trichinellosis are effective, but the production of antibody levels and immunogenicity are low. Adjuvants can increase antibody levels and vaccine immunogenicity. As a result, it is critical to develop an effective adjuvant for the T. spiralis vaccine. Recent research has shown that traditional Chinese medicine polysaccharides with low-toxicity and biodegradability can act as adjuvants in vaccines. In this study, BALB/c mice were orally inoculated with a recombinant Lactobacillus plantarum (L. plantarum) vaccine expressing the T. spiralis cathepsin F-like protease 1 gene (rTs-CPF1), which was given three times at 10-day intervals. Lycium barbarum polysaccharide (LBP) was administered orally for 37 days. At 37 days after the first immunization, mice were infected with 350 T. spiralis muscle larvae (ML). Specific IgG and sIgA antibody levels against the T. spiralis CPF1 protein were increased in mice immunized with rTs-CPF1+LBP compared to those immunized with rTs-CPF1 alone. Furthermore, LBP increased IFN-γ and IL-4 expression levels, and the number of intestinal and intramuscular worms was significantly reduced in the rTs-CPF1+LBP group compared to that in the rTs-CPF1 group. In the rTs-CPF1+LBP group, the reduction rates of adult worms and muscle larvae were 47.31 % and 68.88 %, respectively. To summarize, LBP promotes the immunoprotective effects of the T. spiralis vaccine and may be considered as a novel adjuvant in parasitic vaccines.

Treatment of pregnant mice with ABZ had no effect on the immune response of their offspring infected with Trichinella spiralis.

Trichinellosis is a food-borne parasitic disease with a worldwide distribution that not only endangers human health but also leads to economic loss. Infection of pregnant animals with Trichinella spiralis (T. spiralis) may lead to abortion and other adverse consequences, so it is necessary to treat the infection during pregnancy. Albendazole (ABZ) is an effective therapeutic drug for adult T. spiralis worms. The safety of this drug during pregnancy, especially whether it has any effect on offspring, should be fully evaluated. A change in the immune response to T. spiralis in the offspring of pregnant mice treated with ABZ may lead to a difference in susceptibility to T. spiralis compared to that of the offspring of normal mice. However, the safety of ABZ treatment in pregnant mice and the effects on the immune response and susceptibility of their offspring to T. spiralis are poorly understood. Therefore, we assessed whether maternal ABZ treatment during pregnancy affects the immune response or susceptibility to T. spiralis in infected offspring. In this study, mice were infected with T. spiralis at 10 days of pregnancy and treated with ABZ at 3 days post infection (dpi), and the specific immune response in the pregnant mice and the survival rate and worm burden of their 6-week-old offspring after T. spiralis infection were examined. The results showed that the antiparasitic immune response in pregnant mice was activated by T. spiralis infection. Treatment of pregnant mice with ABZ increased the percentage of CD4 + T cells. The percentages of Th2 and Treg cells in the PP, MLN and spleen of pregnant mice in the infection group were significantly increased compared with those of normal mice. ABZ treatment during pregnancy promoted the Th2 and Treg immune responses in pregnant mice infected with T. spiralis. The transcriptional levels of the Th2 and Treg cytokines IL-4, IL-5, IL-13, and TGF-ß in the small intestine, MLN and spleen of pregnant mice in the treatment group were significantly higher than those of pregnant mice in the T. spiralis infection only group. The results indicated that ABZ treatment did not cause abortion in pregnant mice or affect the survival rate of their offspring. Furthermore, treatment of pregnant mice with ABZ had no significant effect on the above immune responses in their T. spiralis-infected offspring compared to those of T. spiralis-infected offspring of mice in the normal group. The results also indicated that treatment of pregnant mice infected with T. spiralis with ABZ shifted the immune response to a Th2- and Treg-skewed immune response and that this drug had no effects on the offspring survival rate, immune response or worm burden after T. spiralis infection. This study further indicated that ABZ administration to treat T. spiralis infection in pregnant mice is safe for the select immune response and susceptibility of their offspring.

NLRP3 Plays a Key Role in Antihelminth Immunity in the Enteral and Parenteral Stages of Trichinella spiralis-Infected Mice.

Trichinellosis is an important foodborne zoonosis, and no effective treatments are yet available. Nod-like receptor (NLR) plays a critical role in the host response against nematodes. Therefore, we aimed to explore the role of the NLRP3 inflammasome (NLRP3) during the adult, migrating, and encysted stages of Trichinella spiralis infection. The mice were treated with the specific NLRP3 inhibitor MCC950 after inoculation with T. spiralis. Then, the role that NLRP3 plays during T. spiralis infection of mice was evaluated using enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry, histopathological evaluation, bone marrow-derived macrophage (BMDM) stimulation, and immunofluorescence. The in vivo results showed that NLRP3 enhanced the Th1 immune response in the adult and migrating stages and weakened the Th2 immune response in the encysted stage. NLRP3 promoted the release of proinflammatory factors (interferon gamma [IFN-γ]) and suppressed the release of anti-inflammatory factors (interleukin 4 [IL-4]). Pathological changes were also improved in the absence of NLRP3 in mice during T. spiralis infection. Importantly, a significant reduction in adult worm burden and muscle larvae burden at 7 and 35 days postinfection was observed in mice treated with the specific NLRP3 inhibitor MCC950. In vitro, we first demonstrated that NLRP3 in macrophages can be activated by T. spiralis proteins and promotes IL-1ß and IL-18 release. This study revealed that NLRP3 is involved in the host response to T. spiralis infection and that targeted inhibition of NLRP3 enhanced the Th2 response and accelerated T. spiralis expulsion. These findings may help in the development of protocols for controlling trichinellosis.

Pharmacological boosting of cGAS activation sensitizes chemotherapy by enhancing antitumor immunity.

Enhancing chemosensitivity is one of the largest unmet medical needs in cancer therapy. Cyclic GMP-AMP synthase (cGAS) connects genome instability caused by platinum-based chemotherapeutics to type I interferon (IFN) response. Here, by using a high-throughput small-molecule microarray-based screening of cGAS interacting compounds, we identify brivanib, known as a dual inhibitor of vascular endothelial growth factor receptor and fibroblast growth factor receptor, as a cGAS modulator. Brivanib markedly enhances cGAS-mediated type I IFN response in tumor cells treated with platinum. Mechanistically, brivanib directly targets cGAS and enhances its DNA binding affinity. Importantly, brivanib synergizes with cisplatin in tumor control by boosting CD8+ T cell response in a tumor-intrinsic cGAS-dependent manner, which is further validated by a patient-derived tumor-like cell clusters model. Taken together, our findings identify cGAS as an unprecedented target of brivanib and provide a rationale for the combination of brivanib with platinum-based chemotherapeutics in cancer treatment.

MiR-190 ameliorates glucotoxicity-induced dysfunction and apoptosis of pancreatic ß-cells by inhibiting NOX2-mediated reactive oxygen species production.

Glucotoxicity-induced pancreatic ß-cell failure contributes to the development of type 2 diabetes mellitus (T2DM). Accumulating evidence reveals that miRNAs play a critical role in regulating pancreatic ß-cell function and survival. In this study, we employed a self-assembled cell microarray (SAMcell)-based functional screening assay to identify miRNAs that are capable of regulating the dysfunction of ß-cells induced by glucotoxicity. Among 62 conserved miRNAs we tested, miR-190 was identified as a candidate regulator that could effectively restore insulin expression in NIT-1 cells under high-glucose (HG) stimulation. Further analyses demonstrated that miR-190 was significantly down-regulated in HG-treated NIT-1 cells, as well as in the pancreas of diabetic mice. Mechanistic studies showed that Cybb is the direct target gene of miR-190, which encodes the gp91phox protein, a subunit of the NOX2 complex. Furthermore, both miR-190 overexpression and Cybb knockdown inhibited apoptosis and improved glucose-stimulated insulin secretion (GSIS) in HG-stimulated NIT-1 cells by attenuating the excessive production of reactive oxygen species (ROS). More importantly, a targeted delivery of mPEG-PCL-g-PDMAEMA nanoparticles/miR-190 complexes (PECgD NPs/miR-190) to the pancreas significantly ameliorated hyperglycemia, decreased fasting serum insulin levels, and improved glucose tolerance in diabetic mice. Taken together, our findings suggest that the miR-190/Cybb axis plays an important role in glucotoxicity-induced pancreatic ß-cell failure. Restoring miR-190 expression levels may be a possible therapeutic strategy to protect ß-cells in T2DM.

Oral Vaccination of Mice With Trichinella spiralis Putative Serine Protease and Murine Interleukin-4 DNA Delivered by Invasive Lactiplantibacillus plantarum Elicits Protective Immunity.

Trichinellosis is a serious zoonotic parasitic disease caused by Trichinella spiralis (T. spiralis) that causes considerable economic losses for the global pig breeding and food industries. As such, there is an urgent need for a vaccine that can prevent T. spiralis infection. Previous studies have reported that recombinant invasive Lactococcus lactis (LL) expressing Staphylococcus aureus fibronectin binding protein A (LL-FnBPA+) can transfer DNA vaccines directly to dendritic cells (DCs) across an epithelial cell monolayer, leading to significantly higher amounts of heterologous protein expression compared to non-invasive Lactococcus lactis. In this study, the invasive bacterium Lactiplantibacillus plantarum (L. plantarum) expressing FnBPA was used as a carrier to deliver a novel oral DNA vaccine consisting of T. spiralis adult putative serine protease (Ts-ADpsp) and murine interleukin (IL)-4 DNA to mouse intestinal epithelial cells. Experimental mice were orally immunized 3 times at 10-day intervals. At 10 days after the last vaccination, mice were challenged with 350 T. spiralis infective larvae by oral inoculation. Immunization with invasive L. plantarum harboring pValac-Ts-ADpsp/pSIP409-FnBPA induced the production of anti-Ts-ADpsp-specific IgG of serum, type 1 and 2 helper T cell cytokines of mesenteric lymph node (MLN) and spleen, secreted (s) IgA of intestinal lavage, and decreased T. spiralis burden and intestinal damage compared to immunization with non-invasive L. plantarum expressing Ts-ADpsp (pValac-Ts-ADpsp/pSIP409). Thus, invasive L. plantarum expressing FnBPA and IL-4 stimulates both mucosal and cellular immune response to protect against T. spiralis infection, highlighting its therapeutic potential as an effective DNA vaccine for trichinellosis.

Oral vaccination with invasive Lactobacillus plantarum delivered nucleic acid vaccine co-expressing SS1 and murine interleukin-4 elicits protective immunity against Trichinella spiralis in BALB/c mice.

Trichinellosis is a foodborne zoonosis caused by Trichinella spiralis (T. spiralis) that not only causes considerable economic losses for the global pig breeding and food industries, but also seriously threats the health of human. Therefore, it is very necessary to develop an effective vaccine to prevent trichinellosis. In this study, the invasive Lactobacillus plantarum (L. plantarum) expressing fibronectin-binding protein A (FnBPA) was served as a live bacterial vector to deliver DNA to the host to produce a novel oral DNA vaccine. Co-expressing T. spiralis SS1 and murine interleukin-4 (mIL-4) of DNA vaccine were constructed and subsequently delivered to intestinal epithelial cells via invasive L. plantarum. At 10 days after the third immunization, the experimental mice were challenged with 350 T. spiralis infective larvae. The results found that the mice orally vaccinated with invasive L. plantarum harboring pValac-SS1/pSIP409-FnBPA not only stimulated the production of anti-SS1-specific IgG, Th1/Th2 cell cytokines, and secreted(s) IgA but also decreased worm burden and intestinal damage. However, the mice inoculated with invasive L. plantarum co-expressing SS1 and mIL-4 (pValac-SS1-IL-4/pSIP409-FnBPA) induced the highest protective immune response against T. spiralis infection. The DNA vaccine delivered by invasive L. plantarum provides a novel idea for the prevention of T. spiralis infection.

Protocadherin clusters and cell adhesion kinase regulate dendrite complexity through Rho GTPase.

Dendritic patterning and spine morphogenesis are crucial for the assembly of neuronal circuitry to ensure normal brain development and synaptic connectivity as well as for understanding underlying mechanisms of neuropsychiatric diseases and cognitive impairments. The Rho GTPase family is essential for neuronal morphogenesis and synaptic plasticity by modulating and reorganizing the cytoskeleton. Here, we report that protocadherin (Pcdh) clusters and cell adhesion kinases (CAKs) play important roles in dendritic development and spine elaboration. The knockout of the entire Pcdhα cluster results in the dendritic simplification and spine loss in CA1 pyramidal neurons in vivo and in cultured primary hippocampal neurons in vitro. The knockdown of the whole Pcdhγ cluster or in combination with the Pcdhα knockout results in similar dendritic and spine defects in vitro. The overexpression of proline-rich tyrosine kinase 2 (Pyk2, also known as CAKß, RAFTK, FAK2, and CADTK) recapitulates these defects and its knockdown rescues the phenotype. Moreover, the genetic deletion of the Pcdhα cluster results in phosphorylation and activation of Pyk2 and focal adhesion kinase (Fak) and the inhibition of Rho GTPases in vivo. Finally, the overexpression of Pyk2 leads to inactivation of Rac1 and, conversely, the constitutive active Rac1 rescues the dendritic and spine morphogenesis defects caused by the knockout of the Pcdhα cluster and the knockdown of the Pcdhγ cluster. Thus, the involvement of the Pcdh-CAK-Rho GTPase pathway in the dendritic development and spine morphogenesis has interesting implications for proper assembly of neuronal connections in the brain.

[Effect of casamino acid on intergeneric conjugation in rapamycin-producing Streptomyces hygroscopicus ATCC29253].

OBJECTIVE: Streptomyces hygroscopicus ATCC29253 has attracted much interests due to its capacity of producing various secondary metabolites with strong bioactivities, including immunosuppressant rapamycin, nigericin, hexaenes, elaiophylin and hygrocins. OBJECTIVE: To investigate biosynthetic pathway of these metabolites and construct high-yield strains by genetic engineering, establishment of a highly efficient genetic manipulation system is critically required in this strain. METHODS: We tested the effects of conjugation media and donor strains on conjugal transfer from Escherichia coli to S. hygroscopicus ATCC29253 and other Streptomycetes. RESULTS: We found that both casamino acid and MgCl2 supplemented in conjugation media improved conjugation frequency in S. hygroscopicus ATCC29253. A random experiment led to the disclosure of an optimal combination of casamino acid and MgCl2 by which the conjugation frequency in S. hygroscopicus reached 1.5 x 10(-4). Meanwhile, we also found significant changes in conjugation frequencies of S. lividans, S. albus and S. avermitilis when casamino acid was supplemented in conjugation media. CONCLUSION: Casamino acid has significant influence on conjugation frequency in not only S. hygroscopicus ATCC29253 but also other Streptomyces such as S. lividans, S. albus and S. avermitilis.