Transforming Cell-Drug Interaction through Granular Hydrogel-Mediated Delivery of Polyplex Nanoparticles for Enhanced Safety and Extended Efficacy in Gene Therapy.

The utilization of hydrogels for DNA/cationic polymer polyplex nanoparticle (polyplex) delivery has significantly advanced gene therapy in tissue regeneration and cancer treatment. However, persistent challenges related to the efficacy and safety of encapsulated polyplexes, stemming from issues such as aggregation, degradation, or difficulties in controlled release during or postintegration with hydrogel scaffolds, necessitate further exploration. Here, we introduce an injectable gene therapy gel achieved by incorporating concentrated polyplexes onto densely packed hydrogel microparticles (HMPs). Polyplexes, when uniformly adhered to the gene therapy gel through reversible electrostatic interactions, can detach from the HMP surface in a controlled manner, contrasting with free polyplexes, and thereby reducing dose-dependent toxicity during transfection. Additionally, the integration of RGD cell adhesion peptides enhances the scaffolding characteristics of the gel, facilitating cell adhesion, migration, and further minimizing toxicity during gene drug administration. Notably, despite the overall transfection efficiency showing average performance, utilizing confocal microscopy to meticulously observe and analyze the cellular states infiltrating into various depths of the gene therapy gel resulted in the groundbreaking discovery of significantly enhanced local transfection efficiency, with primary cell transfection approaching 80%. This phenomenon could be potentially attributed to the granular hydrogel-mediated delivery of polyplex nanoparticles, which revolutionizes the spatial and temporal distribution and thus the "encounter" mode between polyplexes and cells. Moreover, the gene therapy gel's intrinsic injectability and self-healing properties offer ease of administration, making it a highly promising candidate as a novel gene transfection gel dressing with significant potential across various fields, including regenerative medicine and innovative living materials.

Novel insights into antimony mobilization in different high- antimony aquifers from the molecular signatures of dissolved organic matter.

The crucial role of the fluorescent components of dissolved organic matter (DOM) in controlling antimony (Sb) mobilization in groundwater has been confirmed. However, the molecular signatures contributing to Sb enrichment in DOM remain unknown. This study aims to investigate the origins and molecular compositions of DOM in different high-Sb aquifers (Sb-mining and no-Sb-mining aquifer), as well as compare different molecular signatures of DOM and mechanisms for Sb migration. The findings showed that Sb concentrations in Sb-mining aquifer exhibited a positive correlation with lignin- and tannin-like molecules characterized by high O/C and low H/C ratios, indicating an increased abundance of aromatic components with higher Humification Index and SUV-absorbance at 254 nm, compared to no-Sb-mining aquifer. Correspondingly, the complexation and competitive adsorption were considered as the predominate formation mechanisms on Sb enrichment in Sb-mining aquifer. In addition, high abundances of bioreactivity DOM may facilitated the migration of Sb via electron transfer and competitive adsorption in native no-Sb-mining aquifer. The outcomes of this investigation offer novel insights into the mechanism on Sb enrichment influenced by DOM at the molecule level.

Trichinella spiralis -induced immunomodulation signatures on gut microbiota and metabolic pathways in mice.

The hygiene hypothesis proposes that decreased exposure to infectious agents in developed countries may contribute to the development of allergic and autoimmune diseases. Trichinella spiralis, a parasitic roundworm, causes trichinellosis, also known as trichinosis, in humans. T. spiralis had many hosts, and almost any mammal could become infected. Adult worms lived in the small intestine, while the larvae lived in muscle cells of the same mammal. T. spiralis was a significant public health threat because it could cause severe illness and even death in humans who eat undercooked or raw meat containing the parasite. The complex interactions between gastrointestinal helminths, gut microbiota, and the host immune system present a challenge for researchers. Two groups of mice were infected with T. spiralis vs uninfected control, and the experiment was conducted over 60 days. The 16S rRNA gene sequences and untargeted LC/MS-based metabolomics of fecal and serum samples, respectively, from different stages of development of the Trichinella spiralis-mouse model, were examined in this study. Gut microbiota alterations and metabolic activity accompanied by parasite-induced immunomodulation were detected. The inflammation parameters of the duodenum (villus/crypt ratio, goblet cell number and size, and histological score) were involved in active inflammation and oxidative metabolite profiles. These profiles included increased biosynthesis of phenylalanine, tyrosine, and tryptophan while decreasing cholesterol metabolism and primary and secondary bile acid biosynthesis. These disrupted metabolisms adapted to infection stress during the enteral and parenteral phases and then return to homeostasis during the encapsulated phase. There was a shift from an abundance of Bacteroides in the parenteral phase to an abundance of probiotic Lactobacillus and Treg-associated-Clostridia in the encapsulated phase. Th2 immune response (IL-4/IL-5/IL-13), lamina propria Treg, and immune hyporesponsiveness metabolic pathways (decreased tropane, piperidine and pyridine alkaloid biosynthesis and biosynthesis of alkaloids derived from ornithine, lysine, and nicotinic acid) were all altered. These findings enhanced our understanding of gut microbiota and metabolic profiles of Trichinella -infected mice, which could be a driving force in parasite-shaping immune system maintenance.

Hyaluronic Acid-Based Reactive Oxygen Species-Responsive Multifunctional Injectable Hydrogel Platform Accelerating Diabetic Wound Healing.

Diabetic wounds are more likely to develop into complex and severe chronic wounds. The objective of this study is to develop and assess a reactive oxygen species (ROS)-responsive multifunctional injectable hydrogel for the purpose of diabetic wound healing. A multifunctional hydrogel (HA@Cur@Ag) is successfully synthesized with dual antioxidant, antibacterial, and anti-inflammatory properties by crosslinking thiol hyaluronic acid (SH-HA) and disulfide-bonded hyperbranched polyethylene glycol (HB-PBHE) through Michael addition; while, incorporating curcumin liposomes and silver nanoparticles (AgNPs). The HA@Cur@Ag hydrogel exhibits favorable biocompatibility, degradability, and injectivity. The outcomes of in vitro and in vivo experiments demonstrate that the hydrogel can effectively be loaded with and release curcumin liposomes, as well as silver ions, thereby facilitating diabetic wound healing through multiple mechanisms, including ROS scavenging, bactericidal activity, anti-inflammatory effects, and the promotion of angiogenesis. Transcriptome sequencing reveals that the HA@Cur@Ag hydrogel effectively suppresses the activation of the tumour necrosis factor (TNF)/nuclear factor κB (NF-κB) pathway to ameliorate oxidative stress and inflammation in diabetic wounds. These findings suggest that this ROS-responsive multifunctional injectable hydrogel, which possesses the ability to precisely coordinate and integrate intricate biological and molecular processes involved in wound healing, exhibits notable potential for expediting diabetic wound healing.

Potent gene delivery from fluorinated poly(ß-amino ester) in adhesive and suspension difficult-to-transfect cells for apoptosis and ferroptosis.

Tremendous efforts have been made to improve polymeric property in gene delivery performances, especially when obstacle of transferring gene construct into difficult-to-transfect cells occurs. Innovations in the area of fluorination and fluorinated compounds with biomedical potential in medicinal chemistry are believed to assist in the development of new therapeutics. Fluorine modified polymers have shown to navigate the gene transfection cellular barriers and promoted the transfection outcomes. Gene transfer into some liver cancer cells and human leukemia cells has always been a challenge. Here, by facile incorporation of a fluorine containing amine monomer, 1H,1H-undecafluorohexylamine, fluorinated poly(ß-amino ester) (FPAE) was synthesized to significantly improve the transfection performance, achieving high transfection efficiency of 87% and 55% in two representative difficult-to-transfect cells, HepG2 and Molt-4, which were cultured in adhesive and suspension condition, respectively. However, the potency of Lipofectamine 3000 was very limited. More importantly, functional studies revealed that FPAE can dramatically outperform Lipofectamine 3000 in delivering Bcl-xL and PKCßII to either provide the protection against apoptosis or promote the ferroptosis in HepG2 cells. This work facilitates gene therapies by overcoming biological barriers for targeting difficult-to-transfect cells and disease models when medically necessary.

Mussel-inspired self-healing hydrogel based on gelatin and oxidized tannic acid for pH-responsive controlled drug release.

A series of mussel-inspired pH-responsive self-healing hydrogels based on gelatin crosslinked by oxidized tannic acid (GLT-OTAs) were prepared and used as controlled drug delivery systems (CDDS). An antiphlogistic drug, indomethacin (IDMC) was used as model drug to be immobilized into the hydrogels. The obtained hydrogel samples were characterized by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM). Mechanical stability, biocompatibility and self-healing property of the hydrogels were estimated, respectively. Swelling and drug release behaviors of these hydrogels were measured in phosphate buffered saline (PBS) with pH 7.4 (simulated intestinal fluid) and HCl solution with pH 1.2 (simulated gastric fluid) at 37 °C. Effect of the OTA content on the structures and characteristics of all samples was discussed. FTIR spectra revealed the covalent cross-linking between gelatin and OTA induced by Michael addition and Schiff base reaction. Both XRD and FTIR all showed the drug (IDMC) was loaded successfully and existed steadily. GLT-OTA hydrogels had satisfactory biocompatibility, superior self-healing. Results also illustrated that the mechanical strength, internal structure, the swelling and drug release behaviors of the GLT-OTAs hydrogel were greatly affected by the OTA content. With increasing the OTA content, the mechanical stability of GLT-OTAs hydrogel became better and better, and their internal structure became more and more compact. The swelling degree (SD) and cumulative drug release of all hydrogel samples tended to decrease with increasing OTA content and both had obvious pH responsiveness. For each hydrogel sample, the cumulative drug release in PBS at pH 7.4 was greater than that in HCl solution at pH 1.2. These results indicated that the obtained GLT-OTAs hydrogel had promising potential to be used as effective pH-responsive and self-healing drug delivery materials.

Geochemical impact of dissolved organic matter on antimony mobilization in shallow groundwater of the Xikuangshan antimony mine, Hunan Province, China.

Dissolved organic matter (DOM) is widely used in aquatic systems to control the environmental fate of As. However, similar to the behavior of As, Sb mobilization driven by DOM is poorly understood. A total of 25 samples were collected from shallow groundwater in the Xikuangshan mine to compare the spectroscopic characteristics and chemical properties of DOM between high- and low-Sb groundwater and to determine the roles of DOM in Sb mobility. The concentrations of Sb and DOM varied from 0.003 to 18.402 mg/L (mean: 3.407 mg/L) and 0.38 to 9.90 mg/L (mean: 2.49 mg/L), respectively. The DOM of the D3x4 water was primarily dominated by terrestrial and microbial humic-like and fulvic acid substances, with a relatively small contribution of tryptophan-like components. Complexing agents, competitive adsorption, and photopromoted oxidation under sunlight were considered as the formation mechanisms for DOM-controlled Sb(V)-dominated Sb species in D3x4 water. The weakly alkaline and oxidizing conditions, and the presence of Fe hydroxides facilitated the promotion of Sb(V) concentration. The findings of this study further enhance our understanding of the Sb migration mechanism in oxic groundwater.

Increase in fluoride concentration in mine water in Shendong mining area, Northwest China: Insights from isotopic and geochemical signatures.

Mine water poses severe threats to the quality of the water supply and ecological environment of the Shendong mining areas owing to its excessive fluoride (F-) content. However, the geochemical behaviours and enrichment mechanisms responsible for F⁻ enrichment during mining activities are not fully understood. In total, 18 Yanan groundwater and 45 mine water samples were collected to analyse the spatial distribution, hydrogeochemical behaviours, and formation mechanisms related to elevated F- levels by analysing the stable isotopes and water-rock interactions. In this study, F- concentrations in mine water samples varied from 0.16 to 12.75 mg/L, with a mean value of 6.10 mg/L, and 77.78% of the mine water samples had a concentration that exceeded China's national standards (1.00 mg/L) for drinking water. The F- concentration was markedly high in the mine water samples, with the mean F- concentration being 1.58 times of that in the Yanan groundwater samples. The results of stable isotopes (18OH2O, D, 34SSO4, and 18OSO4) and water-rock interaction analyses suggested that cation exchange and competitive effects were the dominant factors responsible for elevated F- concentration in mine water during mining activities. Thus, the weathering of F-bearing minerals, agriculture, and domestic activities do not play a significant role in the secondary enrichment of F- concentration.

Trichinella spiralis Paramyosin Induces Colonic Regulatory T Cells to Mitigate Inflammatory Bowel Disease.

Helminth infection modulates host regulatory immune responses to maintain immune homeostasis. Our previous study identified Trichinella spiralis paramyosin (TsPmy) as a major immunomodulatory protein with the ability to induce regulatory T cells (Tregs). However, whether TsPmy regulates gut Tregs and contributes to intestinal immune homeostasis remains unclear. Here we investigated the therapeutic effect of recombinant TsPmy protein (rTsPmy) on experimental colitis in mice, and elucidated the roles and mechanisms of colonic Tregs induced by rTsPmy in ameliorating colitis. Acute colitis was induced by dextran sodium sulfate (DSS) in C57BL/6J mice, and chronic colitis was induced by naïve T cells in Rag1 KO mice. Mice with colitis were pre-treated with rTsPmy intraperitoneally, and clinical manifestations and colonic inflammation were evaluated. Colonic lamina propria (cLP) Tregs phenotypes and functions in DSS-induced colitis were analyzed by flow cytometry. Adoptive transfer of cLP Tregs treated by rTsPmy into Rag1 KO chronic colitis was utilized to verify Tregs suppressive function. rTsPmy ameliorated the disease progress of DSS-induced colitis, reduced pro-inflammatory responses but enhanced regulatory cytokines production in DSS-induced colitis. Moreover, rTsPmy specifically stimulated the expansion of thymic-derived Tregs (tTregs) rather than the peripherally derived Tregs (pTregs) in the inflamed colon, enhanced the differentiation of effector Tregs (eTregs) with higher suppressive function and stability in colitis. This study describes the mechanisms of colonic Tregs induced by the Trichinella-derived protein rTsPmy in maintaining gut immune homeostasis during inflammation. These findings provide further insight into the immunological mechanisms involved in the therapeutic effect of helminth-derived proteins in inflammatory bowel diseases.

A core-shell structured alginate hydrogel beads with tunable thickness of carboxymethyl cellulose coating for pH responsive drug delivery.

pH-responsive core-shell structured composite hydrogel beads, composed of a alginate (ALG) core coated with carboxymethyl cellulose (CMC) shell (ALG@CMC), were prepared by using in-situ gel preparation technology as a drug delivery system. An anti-inflammatory drug, indomethacin was loaded into the formed hydrogels as a model drug. The resulting gel samples were characterized by Fourier transforms infrared (FTIR) spectroscopy, thermo-gravimetric (TG) analysis, and scanning electron microscopy (SEM). The mechanical stability of all samples in phosphate buffered solution (PBS, pH 7.4) was approximately measured through oscillation experiments. Swelling and controlled drug release behaviors of ALG@CMC beads compared with ALG were studied in simulating gastric fluid of pH 1.2 or intestinal fluid of pH 7.4 at 37 °C. Oscillation experiments proved that the mechanical stability of ALG@CMC beads could be significantly improved by the CMC shell layer. The swelling and drug release behaviors revealed that the swelling and drug release rate of ALG@CMC beads were obviously slower than that of simple-ALG and both have significant pH responsiveness. The cumulative drug release from ALG, ALG@CMC-1, ALG@CMC-2 and ALG@CMC-3 was about 100%, 67%, 46% and 37% in simulated intestinal fluid of pH 7.4, respectively, while the drug release reached only about 2.0% in simulating gastric fluid of pH 1.2 within 720 min. These developed materials could potentially be employed as a pH-responsive drug delivery device in vivo.[Formula: see text].

Excretory/Secretory Products From Trichinella spiralis Adult Worms Attenuated DSS-Induced Colitis in Mice by Driving PD-1-Mediated M2 Macrophage Polarization.

Helminth-modulated macrophages contribute to attenuating inflammation in inflammatory bowel diseases. The programmed death 1 (PD-1) plays an important role in macrophage polarization and is essential in the maintenance of immune system homeostasis. Here, we investigate the role of PD-1-mediated polarization of M2 macrophages and the protective effects of excretory/secretory products from Trichinella spiralis adult worms (AES) on DSS-induced colitis in mice. Colitis in mice was induced by oral administration of dextran sodium sulfate (DSS) daily. Mice with DSS-induced colitis were treated with T. spiralis AES intraperitoneally, and pathological manifestations were evaluated. Macrophages in mice were depleted with liposomal clodronate. Markers for M1-type (iNOS, TNF-α) and M2-type (CD206, Arg-1) macrophages were detected by qRT-PCR and flow cytometry. Macrophage expression of PD-1 was quantified by flow cytometry; RAW 264.7 cells and peritoneal macrophages were used for in vitro tests, and PD-1 gene knockout mice were used for in vivo investigation of the role of PD-1 in AES-induced M2 macrophage polarization. Macrophage depletion was found to reduce DSS-induced colitis in mice. Treatment with T. spiralis AES significantly increased macrophage expression of CD206 and Arg-1 and simultaneously attenuated colitis severity. We found T. spiralis AES to enhance M2 macrophage polarization; these findings were confirmed studying in vitro cultures of RAW264.7 cells and peritoneal macrophages from mice. Further experimentation revealed that AES upregulated PD-1 expression, primarily on M2 macrophages expressing CD206. The AES-induced M2 polarization was found to be decreased in PD-1 deficient macrophages, and the therapeutic effects of AES on colitis was reduced in PD-1 knockout mice. In conclusion, the protective effects of T. spiralis AES on DSS-induced colitis were found to associate with PD-1 upregulation and M2 macrophage polarization. Thus, PD-1-mediated M2 macrophage polarization is a key mechanism of helminth-induced modulation of the host immune system.

A Multiple Antigen Peptide Vaccine Containing CD4+ T Cell Epitopes Enhances Humoral Immunity against Trichinella spiralis Infection in Mice.

Multiepitope peptide vaccine has some advantages over traditional recombinant protein vaccine due to its easy and fast production and possible inclusion of multiple protective epitopes of pathogens. However, it is usually poorly immunogenic and needs to conjugate to a large carrier protein. Peptides conjugated to a central lysine core to form multiple antigen peptides (MAPs) will increase the immunogenicity of peptide vaccine. In this study, we constructed a MAP consisting of CD4+ T cell and B cell epitopes of paramyosin (Pmy) of Trichinella spiralis (Ts-Pmy), which has been proved to be a good vaccine candidate in our previous work. The immunogenicity and induced protective immunity of MAP against Trichinella spiralis (T. spiralis) infection were evaluated in mice. We demonstrated that mice immunized with MAP containing CD4+ T cell and B cell epitopes (MAP-TB) induced significantly higher protection against the challenge of T. spiralis larvae (35.5% muscle larva reduction) compared to the MAP containing B cell epitope alone (MAP-B) with a 12.4% muscle larva reduction. The better protection induced by immunization of MAP-TB was correlated with boosted antibody titers (both IgG1 and IgG2a) and mixed Th1/Th2 cytokine production secreted by the splenocytes of immunized mice. Further flow cytometry analysis of lymphocytes in spleens and draining lymph nodes demonstrated that mice immunized with MAP-TB specifically enhanced the generation of T follicular helper (Tfh) cells and germinal center (GC) B cells, while inhibiting follicular regulatory CD4+ T (Tfr) cells and regulatory T (Treg) cells. Immunofluorescence staining of spleen sections also confirmed that MAP-TB vaccination enhanced the formation of GCs. Our results suggest that CD4+ T cell epitope of Ts-Pmy is crucial in vaccine component for inducing better protection against T. spiralis infection.

A Temporally-Aware Interpolation Network for Video Frame Inpainting.

In this work, we explore video frame inpainting, a task that lies at the intersection of general video inpainting, frame interpolation, and video prediction. Although our problem can be addressed by applying methods from other video interpolation or extrapolation tasks, doing so fails to leverage the additional context information that our problem provides. To this end, we devise a method specifically designed for video frame inpainting that is composed of two modules: a bidirectional video prediction module and a temporally-aware frame interpolation module. The prediction module makes two intermediate predictions of the missing frames, each conditioned on the preceding and following frames respectively, using a shared convolutional LSTM-based encoder-decoder. The interpolation module blends the intermediate predictions by using time information and hidden activations from the video prediction module to resolve disagreements between the predictions. Our experiments demonstrate that our approach produces smoother and more accurate results than state-of-the-art methods for general video inpainting, frame interpolation, and video prediction.

Trichinella spiralis Excretory-Secretory Products Stimulate Host Regulatory T Cell Differentiation through Activating Dendritic Cells.

Trichinella spiralis maintains chronic infections within its host, involving a variety of immunomodulatory properties, the mechanisms of which have not been completely elucidated. In this study, we found that T. spiralis infection induced strong regulatory T cell responses through parasite excretory-secretory (ES) products, characterized by increase of CD4+CD25+Foxp3+ and CD4+CD25-Foxp3+ Treg cells accompanied by high levels of IL-10 and TGF-ß. T. spiralis adult worm excretory-secretory products (AES) and muscle larvae excretory-secretory products (MES) were both able to activate BMDCs in vitro to facilitate their maturation and to create regulatory cytokines IL-10 and TGF-ß. The T. spiralis AES- and MES-pulsed dendritic cells (DCs) possessed abilities not only to present antigens to sensitized CD4+ T cell to stimulate their proliferation but also to induce naive CD4+ T cells to differentiate to Treg cells secreting IL-10 and TGF-ß. The passive transfer of T. spiralis AES- and MES-pulsed bone marrow-derived dendritic cells (BMDCs) conferred the naive mice to acquire the differentiation of Treg cells. T. spiralis AES possesses a better ability to induce Treg cells than did MES, although the latter has the ability to induce CD4+CD25-Foxp3+ Treg cells. The results obtained in this study suggested that T. spiralis ES products stimulate the differentiation of host Treg cells possibly through activating dendritic cells to create a regulatory environment that benefits the survival of the parasite in the host.

Complement Evasion: An Effective Strategy That Parasites Utilize to Survive in the Host.

Parasitic infections induce host immune responses that eliminate the invading parasites. However, parasites have evolved to develop many strategies to evade host immune attacks and survive in a hostile environment. The complement system acts as the first line of immune defense to eliminate the invading parasites by forming the membrane attack complex (MAC) and promoting an inflammatory reaction on the surface of invading parasites. To date, the complement activation pathway has been precisely delineated; however, the manner in which parasites escape complement attack, as a survival strategy in the host, is not well understood. Increasing evidence has shown that parasites develop sophisticated strategies to escape complement-mediated killing, including (i) recruitment of host complement regulatory proteins on the surface of the parasites to inhibit complement activation; (ii) expression of orthologs of host RCA to inhibit complement activation; and (iii) expression of parasite-encoded proteins, specifically targeting different complement components, to inhibit complement function and formation of the MAC. In this review, we compiled information regarding parasitic abilities to escape host complement attack as a survival strategy in the hostile environment of the host and the mechanisms underlying complement evasion. Effective escape of host complement attack is a crucial step for the survival of parasites within the host. Therefore, those proteins expressed by parasites and involved in the regulation of the complement system have become important targets for the development of drugs and vaccines against parasitic infections.

Trichinella spiralis Infection Mitigates Collagen-Induced Arthritis via Programmed Death 1-Mediated Immunomodulation.

Helminth infection induces Th2-biased immune responses and inhibitory/regulatory pathways that minimize excessive inflammation to facilitate the chronic infection of helminth in the host and in the meantime, prevent host hypersensitivity from autoimmune or atopic diseases. However, the detailed molecular mechanisms behind modulation on inflammatory diseases are yet to be clarified. Programmed death 1 (PD-1) is one of the important inhibitory receptors involved in the balance of host immune responses during chronic infection. Here, we used the murine model to examine the role of PD-1 in CD4+ T cells in the effects of Trichinella spiralis infection on collagen-induced arthritis (CIA). Mice infected with T. spiralis demonstrated higher expression of PD-1 in the spleen CD4+ T cells than those without infection. Mice infected with T. spiralis 2 weeks prior to being immunized with type II collagen displayed lower arthritis incidence and significantly attenuated pathology of CIA compared with those of uninfected mice. The therapeutic effect of T. spiralis infection on CIA was reversed by blocking PD-1 with anti-PD-1 antibody, associated with enhanced Th1/Th17 pro-inflammatory responses and reduced Th2 responses. The role of PD-1 in regulating CD4+ T cell differentiation and proliferation during T. spiralis infection was further examined in PD-1 knockout (PD-1-/-) C57BL/6 J mice. Interestingly, T. spiralis-induced alteration of attenuated Th1 and enhanced Th2/regulatory T cell differentiation in wild-type (WT) mice was effectively diminished in PD-1-/- mice characterized by recovered Th1 cytokine levels, reduced levels of Th2 and regulatory cytokines and CD4+CD25+Foxp3+ cells. Moreover, T. spiralis-induced CD4+ T cell proliferation suppression in WT mice was partially restored in PD-1-/- mice. This study introduces the first evidence that PD-1 plays a critical role in helminth infection-attenuated CIA in a mouse model by regulating the CD4+ T cell function, which may provide the new insights into the mechanisms of helminth-induced immunomodulation of host autoimmunity.

Ts-Hsp70 induces protective immunity against Trichinella spiralis infection in mouse by activating dendritic cells through TLR2 and TLR4.

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.

Heterologous Prime-Boost Vaccination Enhances TsPmy's Protective Immunity against Trichinella spiralis Infection in a Murine Model.

TsPmy is a paramyosin expressed by parasitic Trichinella spiralis and confers a protective immunity when its recombinant protein or DNA was used as an immunogen. To improve its immunogenicity and vaccine efficacy, we conducted a heterologous prime-boost strategy by orally delivering one dose of TsPmy DNA carried by attenuated Salmonella typhimurium (SL7207), followed by two doses of recombinant TsPmy intramuscularly. This strategy effectively induced intestinal mucosal sIgA response and an enhanced and balanced Th1/Th2 immune responses that improve protection against T. spiralis larval challenge, with 55.4% muscle larvae reduction and 41.8% adult worm reduction compared to PBS control. The muscle larvae reduction induced by heterologous prime-boost regimen was significant higher than that induced by the homologous DNA or protein prime-boost regimens, which could act as a practical prophylactic approach to prevent T. spiralis infection.

Vaccination with a Paramyosin-Based Multi-Epitope Vaccine Elicits Significant Protective Immunity against Trichinella spiralis Infection in Mice.

Trichinellosis is a worldwide zoonosis and remains a serious public health problem. Interrupting parasite transmission via vaccination of livestocks with a potent vaccine is a practical approach to prevent human Trichinellosis. Our previous studies have identified that paramyosin of Trichinella spiralis (Ts-Pmy) is a good vaccine candidate against Trichinellosis. In this study, a novel multi-epitope vaccine (MEP) was constructed by using four CD4+ T cell epitopes (P2, P3, P4, and P5) and one B cell epitope (YX1) from Ts-Pmy and expressed as a soluble recombinant protein (rMEP) in Escherichia coli. Mice immunized with rMEP vaccine produced significant higher muscle larval reduction (55.4%) than that induced by immunization of parental rTs-Pmy (34.4%) against T. spiralis infection. The better protection is associated with rMEP induced high levels of anti-rMEP specific IgG and subclass IgG1/IgG2a, elevated T cell proliferation of splenocytes and secretion of IFN-γ, IL-4 and IL-5. The cellular response to individual T cell epitope also showed that splenocytes from mice immunized with rMEP strongly response to the stimulation of synthetic epitope peptide P2, P3, and P4, but not to P5, suggesting that most of T cell epitopes are exposed and processed well during immunization that may contribute to the high protection induced by the immunization of rMEP. This study implies that epitope vaccine is a promising approach for the development of vaccines against Trichinellosis.

Trichinella spiralis Calreticulin Binds Human Complement C1q As an Immune Evasion Strategy.

As a multicellular parasitic nematode, Trichinella spiralis regulates host immune responses by producing a variety of immunomodulatory molecules to escape from host immune attack, but the mechanisms underlying the immune evasion are not well understood. Here, we identified that T. spiralis calreticulin (Ts-CRT), a Ca2+-binding protein, facilitated T. spiralis immune evasion by interacting with the first component of human classical complement pathway, C1q. In the present study, Ts-CRT was found to be expressed on the surface of different developmental stages of T. spiralis as well as in the secreted products of adult and muscle larval worms. Functional analysis identified that Ts-CRT was able to bind to human C1q, resulting in the inhibition of C1q-initiated complement classical activation pathway reflected by reduced C4/C3 generation and C1q-dependent lysis of antibody-sensitized sheep erythrocytes. Moreover, recombinant Ts-CRT (rTs-CRT) binding to C1q suppressed C1q-induced THP-1-derived macrophages chemotaxis and reduced monocyte-macrophages release of reactive oxygen intermediates (ROIs). Blocking Ts-CRT on the surface of newborn larvae (NBL) of T. spiralis with anti-Ts-CRT antibody increased the C1q-mediated adherence of monocyte-macrophages to larvae and impaired larval infectivity. All of these results suggest that T. spiralis-expressed Ts-CRT plays crucial roles in T. spiralis immune evasion and survival in host mostly by directly binding to host complement C1q, which not only reduces C1q-mediated activation of classical complement pathway but also inhibits the C1q-induced non-complement activation of macrophages.