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Abstract: Treatment resistance of leukemia stem cells (LSCs) and suppression of the autologous immune system represent major challenges to achieve a cure in acute myeloid leukemia (AML). Although AML blasts generally retain high levels of surface CD38 (CD38pos), LSCs are frequently enriched in the CD34posCD38neg blast fraction. Here, we report that interferon gamma (IFN-γ) reduces LSCs clonogenic activity and induces CD38 upregulation in both CD38pos and CD38neg LSC-enriched blasts. IFN-γ-induced CD38 upregulation depends on interferon regulatory factor 1 transcriptional activation of the CD38 promoter. To leverage this observation, we created a novel compact, single-chain CD38-CD3 T-cell engager (BN-CD38) designed to promote an effective immunological synapse between CD38pos AML cells and both CD8pos and CD4pos T cells. We demonstrate that BN-CD38 engages autologous CD4pos and CD8pos T cells and CD38pos AML blasts, leading to T-cell activation and expansion and to the elimination of leukemia cells in an autologous setting. Importantly, BN-CD38 engagement induces the release of high levels of IFN-γ, driving the expression of CD38 on CD34posCD38neg LSC-enriched blasts and their subsequent elimination. Critically, although BN-CD38 showed significant in vivo efficacy across multiple disseminated AML cell lines and patient-derived xenograft models, it did not affect normal hematopoietic stem cell clonogenicity and the development of multilineage human immune cells in CD34pos humanized mice. Taken together, this study provides important insights to target and eliminate AML LSCs., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Background: Whether immunotherapy combined with different histone deacetylases (HDAC) inhibitors in refractory or relapsed natural killer/T-cell lymphoma (NKTCL) is superior to each agent is still lacking in head-to-head clinical trials or preclinical evidence., Methods: NKTCL cell line xenograft models (CDX) in immunocompetent, human programmed cell death protein 1 (PD1) knock-in genetically engineered mice were used to investigate the combination effects. Different types and dosages of HDAC inhibitors were investigated. We explored the underlying mechanisms by RNA-sequencing and ChIP-sequencing. Two clinical cases treated with anti-PD1/chidamide were presented., Findings: Anti-PD1/chidamide shows significant tumour rejection in two CDX models. RNA-seq and CHIP-seq revealed that chidamide is synergistic to enhance T-cell chemokine expression, augment the Ifn-γ response, and increase CD8 T-cell infiltration via histone modification. Ifn-γ neutralizing antibody can attenuate the efficacy of combination drugs. However, the anti-PD1/romidepsin failed to augment the Ifn-γ response. The expressions of Ifn-γ related gene set signatures are significantly correlated with tumour rejection in anti-PD1/chidamide. In the clinic, two NKTCL patients treated with the PD1/chidamide show promising efficacy and limited toxicity., Interpretation: Anti-PD1/chidamide enhances T-cell chemokine expression and augments the IFN-γ response in preclinical NKTCL immunocompetent models. IFN-γ signatures may be good response biomarkers for the selection of potentially benefit patients., Funding: This study was supported by the Chinese National Major Project for New Drug Innovation (2017ZX09304015) and the Chinese Society of Clinical Oncology Research Fund (Y-BMS2019-026)., Competing Interests: Declaration of interests The authors declare no potential conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

The management of pemphigus vulgaris (PV) is challenging. This study aimed to evaluate the immunomodulating effects of metformin on PV. The study was conducted in two phases: in the first phase, patients received routine first-line treatment (prednisolone plus azathioprine) for 2 months, then in the second phase, metformin was added to this regimen for another 2 months. After addition of metformin to the first-line medications, significant reductions were seen in serum IgG1 (reduced from 534.92 ± 134.83 mg/dL to 481.58 ± 130.46 mg/dL, P < 0.001), IgG4 (51.83 ± 27.26 mg/dL to 44.50 ± 26.05 mg/dL, P < 0.001) and interferon-γ (277.99 ± 108.71 pg/mL to 45.05 ± 17.080 pg/mL, P = 0.03) concentrations. The suppressant effect of metformin was greatest on IgG4 (coefficient of variation 1.28), the dominant subclass of IgG involved in PV. Metformin could have immunomodulating effects on PV with controlling effects on steroid complications., (© 2021 British Association of Dermatologists.)

Mitogen-activated protein kinase (MAPK) signalling pathways are crucial for developmental processes, oncogenesis, and inflammation, including the production of proinflammatory cytokines caused by reactive oxygen species and upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. There are no drugs that can effectively prevent excessive inflammatory responses in endothelial cells in the lungs, heart, brain, and kidneys, which are considered the main causes of severe coronavirus disease 2019 (COVID-19). In this work, we demonstrate that human MAPKs, i.e. extracellular signal-regulated kinases 1 and 2 (ERK1/2), are CO 2 sensors and CO 2 is an efficient anti-inflammatory compound that exerts its effects through inactivating ERK1/2 in cultured endothelial cells when the CO 2 concentration is elevated. CO 2 is a potent inhibitor of cellular proinflammatory responses caused by H 2 O 2 or the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. ERK1/2 activated by the combined action of RBD and cytokines crucial for the development of severe COVID-19, i.e. interferon-gamma (IFNγ) and tumour necrosis factor-α (TNFα), are more effectively inactivated by CO 2 than by dexamethasone or acetylsalicylic acid in human bronchial epithelial cells. Previously, many preclinical and clinical studies showed that the transient application of 5-8% CO 2 is safe and effective in the treatment of many diseases. Therefore, our research indicates that CO 2 may be used for the treatment of COVID-19 as well as the modification of hundreds of cellular pathways., (© 2021. The Author(s).)

Oligomannose-coated liposomes (OMLs) comprised of dipalmitoylphosphatidylcholine, cholesterol and Man3-DPPE at a molar ratio of 1:1:0.1 and particle diameters of about 1000 nm can induce liposome-encased antigen-specific strong Th1 immunity. In this study, we evaluated the effect of particle sizes of OMLs on induction of Th1 immune responses in mice. Spleen cells obtained from mice immunized with antigen-encapsulating OMLs with 1000- and 800-nm diameters secreted remarkably high levels of IFN-γ upon in vitro stimulation. In addition, sera of mice that received these OMLs had significantly higher titers of antigen-specific IgG2a than those of IgG1, which are commonly associated with Th1 responses. In contrast, treatment with antigen-encapsulating OMLs with 400- and 200-nm diameters failed to induce IFN-γ secretion from spleen cells, although these OMLs did elicit elevation of antigen-specific IgGs. In addition, the titers of serum antigen-specific IgG2a were the same as those of IgG1 in mice that received 400-nm OMLs. Resident peritoneal mononuclear phagocytes (MNPs) treated with OMLs of diameter ≥ 600 nm secreted IL-12, which is essential for induction of Th1 immune responses, while those treated with OMLs of ≤ 400 nm failed to produce this cytokine. However, 400-nm OMLs did induce enhanced expression of MHC class II and costimulatory molecules on MNPs, similarly to OMLs of ≥ 600 nm. Taken together, these results strongly indicate that OMLs of diameter ≥ 600 nm are required to induce Th1 immune responses against OML-encased antigens, although OMLs of diameter ≤ 400 nm can activate MNPs., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Endometriosis is a serious reproductive and general health consequences. Recombinant human IL-37 (rhIL-37) is an inhibitor of inflammation., Methods: ELISA assay was performed to detect the concentration of cytokines. Flow cytometry was used to analyze cell proportion. Besides, qRT-PCR and western blotting assay were used to detect the level of gene and protein, respectively. Transwell co-culture system was used for the co-culture of dendritic cells (DCs) and CD4 + T cells., Results: Our data showed that rhIL-37 inhibited the development of ectopic lesions in the mice with endometriosis, increased Th1/Th2 ratio and induced DCs maturation. The co-culture system of DCs and CD4 + T cells demonstrated that rhIL-37 increased Th1/Th2 cell ratio through promoting DCs maturation. Moreover, the expression of IL-4 in the DCs derived from healthy mice was inhibited by rhIL-37 treatment. rhIL-37 increased Th1/Th2 cell ratio through inhibiting IL-4 in DCs. Subsequently, our results proved that rhIL-37 promoted the maturation of DCs via inhibiting phosphorylation of STAT3. Activation of STAT3 could reverse rhIL-37-induced maturation of DCs., Conclusion: Overall, rhIL-37 could protect against endometriosis through increasing the ratio of Th1/Th2 cells via inducing DCs maturation and inhibiting IL-4 expression in the DCs. Furthermore, rhIL-37 induced DCs maturation by inhibiting STAT3 phosphorylation. Our data confirmed the protective effect of rhIL-37 in endometriosis. These data may provide a novel idea for the treatment of the disease., (© 2021. The Author(s).)

Aim of the Study: Glioblastoma multiforme (GBM) is the most severe forms of brain cancer, eventually becoming the leading cause of brain cancer-related death worldwide. Owing to the bleak surgical interventions and resistance to the different treatment regime, GBM is a parlous disease demanding newer therapeutical perspective for its treatment. Toll-like receptors (TLRs) are well-known members of pathogen recognition receptors (PRRs) and have been extensively explored for their therapeutic and prophylactic potential in an array of disease including cancer. Recent trends in drug delivery research has shown shift towards delivering short DNA sequences (CpG DNA) to endosomal TLR9 within immune cells (macrophages, dendritic cells, etc.) for the activation of desired inflammatory response using non-agonistic β-glucan particles; a well-known ligand for Dectin-1 receptors. Our study is therefore focused to explore the role of nano-encapsulated CpG ODN as critical players in polarizing M2 scavenging to much desired pro-inflammatory type., Materials and Methods: The nanoparticles entrapping CpG ODN 1826 were prepared by using a fungal polymer Schizophyllan (SPG). The constructed nanoparticles were characterized and assessed for their efficacy on rat glioblastoma cells (C6)., Results: The constructed Schizophyllan (SPG) nanoparticles entrapping CpG ODN 1826 (95.3%) were of 25.49 nm in diameter and thus capable of crossing blood-brain barrier. The rat glioblastoma (C6) cells evaluated for intracellular oxidative burst and cytokine levels pre- and post-incubation with nanoparticles exhibited marked elevation in the expression of intracellular ROS and IFN-γ as well as IL-1β post treatment., Conclusion: The findings indicate towards potentiality of repolarizing the M2 macrophages to much desired M1 phase by inducing higgh levels of oxidative burst and inflammatory cytokines. Consequently, the apoptosis was induced in glioblastoma cells establishing the suitablity of CpG ODN carrying nanoformulations as emerging therapeutic intervention for GBM.

Aconitine, the main component in Radix Aconiti Lateralis Preparata, not only exerts the anti-tumor effect on Hepatocellular Carcinoma (HCC) but also damages on immune system. In the present study, Crude Monkshood Polysaccharide (CMP), another one natural composition component originated from the same herbal with aconitine, combined with aconitine to investigate the effects on HCC and immunity in vitro and in vivo. The combination of CMP and aconitine enhanced the ability of the immunocyte to kill the tumor cell in vitro and had an additive effect on anti-HCC in vivo. Aconitine-CMP in combination improved the spleen weights, spleen index, thymus weights, thymus index. Elevated CD4 + T and CD8 + T cells and macrophages in spleen, decreased serum IL-6 level and increased serum IFN-γ and TNF-α levels were observed in mice treated with the combination of aconitine and CMP compare with control group (P<0.05). Our results showed that the combination of aconitine and CMP exerts anti-tumor effect by directly killing tumor cells and enhancing the anti-tumor immune responses, which further implies that chemotherapy drugs combined with Chinese medicine immunopotentiator maybe a feasible and effective strategy for HCC.

Background: Toll-like receptor 4 (TLR4) is known to be involved in carcinogenesis and cancer progression. Changes in TLR4 expression are associated with changes in the expression of key cellular cytokines (transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ)), which affect cancer progression and metastasis., Aim: To study changes in the expression of TLR4, TGF-β, TNF-α, IFN-γ genes, the level of apoptosis and cell cycle distribution in human invasive urothelial carcinoma T24/83 cells under the treatment with polyphenolic adjuvant compound of fungal origin melanin, cytotoxic drug cisplatin, and combination of both., Materials and Methods: T24/83 cells were incubated with cisplatin (0.05 mM), melanin (5 µg/ml), or their combination. The expression level of TLR-4, TGF-β, INF-γ, TNF-α was evaluated by the real time polymerase chain reaction. The flow cytometry was used to study cell cycle distribution, proliferative activity and level of apoptosis. Morphological analysis of the Т24/83 cells was performed as well., Results: Melanin, cisplatin, and their combination downregulate TLR4 expression (2.67; 1.28; and 2.73-fold decrease, respectively) and TNF-α expression (6.5; 1.4; and 1.7-fold decrease, respectively). Melanin did not affect TGF-β expression while cisplatin caused 13-fold downregulation of TGF-β. The combined use of cisplatin and melanin decreased TGF-β expression by 6.5 times. The upregulation of IFN-γ by melanin, cisplatin, and their combination was demonstrated (4.3; 6.7; and 2-fold increase, respectively). All treatment modalities increased the level of apoptosis in T24/83 cells. Melanin treatment increased significantly the proportion of fibroblast-like cells in T24/83 culture with decreased cell adhesion to the substrate., Conclusions: Melanin, cisplatin, and combination of both agents affect significantly TLR4, TNF-α, TGF-β, INF-γ expression, cell cycle distribution and morphology in T24/83 cells suggesting their transition to less aggressive phenotype.

Objectives: Using QuantiFERON-TB Gold In-Tube (QFT-GIT) for monitoring tuberculosis (TB) and latent TB infection treatment effect is controversial. The present study aimed to evaluate the dynamic changes of interferon gamma (IFN-γ) levels along with latent TB infection treatment via a randomized controlled study., Methods: A total of 910 participants treated with 8 weeks of once-weekly rifapentine plus isoniazid (group A), 890 treated with 6 weeks of twice-weekly rifapentine plus isoniazid (group B) and 818 untreated controls (group C) were followed for 2 years to track active TB development. QFT-GIT tests were repeated three times for all groups: before treatment (T0), at completion of treatment (T1) and 3 months after completion of treatment (T2)., Results: Similar rates of persistent QFT-GIT reversion were observed in groups A (19.0%, 173/910), B (18.5%, 165/890) and C (20.7%, 169/818) (p 0.512). The dynamic changes of IFN-γ levels were not statistically significant among the three groups. In treated participants, individuals with higher baseline IFN-γ levels showed increased TB occurrence (1.0%, 9/896) compared to those with lower baseline levels (0.2%, 2/904) (p 0.037). A similar but statistically insignificant trend was also observed in untreated controls (1.8% (7/400) vs. 0.5% (2/418), p 0.100). When TB cases were matched with non-TB cases on baseline IFN-γ levels, no significant differences were found with respect to the dynamic changes in IFN-γ levels with time, regardless of whether they received treatment., Conclusions: QFT-GIT reversion or decreased IFN-γ levels should not be used for monitoring host response to latent TB infection treatment., (Copyright © 2020 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Increasing studies confirm that anti-angiogenesis can increase the effectiveness of immunotherapy. In this study, we found that an angiogenesis inhibitor apatinib enhanced anti-PD-1 therapy for colon cancer in mice via promoting PD-L1 expression. Apatinib treatment upregulated PD-L1 expression in various colon cancer cells both at the mRNA and protein levels. Further, apatinib-treated cancer cells hampered activation and IFN-γ secretion of T cells in the co-culture system, which was reversed by the anti-PD-1 antibody. Based on this, the combination of apatinib with anti-PD-1 on colon cancer growth in mice was examined. The combination treatment showed more significant inhibition on the growth of transplanted tumors in mice than single-drug treatment. Overall, our study here showed the enhancement of anti-PD-1 antitumor efficacy in a syngeneic mouse model (CT-26 cells in Balb/c) by the angiogenesis inhibitor apatinib via upregulating PD-L1 expression as well as angiogenesis inhibition, which may provide a rationale for the combination of apatinib and anti-PD-1 antibody for colorectal cancer treatment in the clinic., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Poultry coccidiosis is a parasitic enteric disease with a highly negative impact on chicken production. In-feed chemoprophylaxis remains the primary method of control, but the increasing ineffectiveness of anticoccidial drugs, and potential future restrictions on their use has encouraged the use of commercial live vaccines. Availability of such formulations is constrained by their production, which relies on the use of live chickens. Several experimental approaches have been taken to explore ways to reduce the complexity and cost of current anticoccidial vaccines including the use of live vectors expressing relevant Eimeria proteins. We and others have shown that vaccination with transgenic Eimeria tenella parasites expressing Eimeria maxima Apical Membrane Antigen-1 or Immune Mapped Protein-1 (EmAMA1 and EmIMP1) partially reduces parasite replication after challenge with a low dose of E. maxima oocysts. In the present study, we have reassessed the efficacy of these experimental vaccines using commercial birds reared at high stocking densities and challenged with both low and high doses of E. maxima to evaluate how well they protect chickens against the negative impacts of disease on production parameters., Methods: Populations of E. tenella parasites expressing EmAMA1 and EmIMP1 were obtained by nucleofection and propagated in chickens. Cobb500 broilers were immunised with increasing doses of transgenic oocysts and challenged two weeks later with E. maxima to quantify the effect of vaccination on parasite replication, local IFN-γ and IL-10 responses (300 oocysts), as well as impacts on intestinal lesions and body weight gain (10,000 oocysts)., Results: Vaccination of chickens with E. tenella expressing EmAMA1, or admixtures of E. tenella expressing EmAMA1 or EmIMP1, was safe and induced partial protection against challenge as measured by E. maxima replication and severity of pathology. Higher levels of protection were observed when both antigens were delivered and was associated with a partial modification of local immune responses against E. maxima, which we hypothesise resulted in more rapid immune recognition of the challenge parasites., Conclusions: This study offers prospects for future development of multivalent anticoccidial vaccines for commercial chickens. Efforts should now be focused on the discovery of additional antigens for incorporation into such vaccines.

Nanoparticle-cell-nanoparticle communication by stigmergy was demonstrated using two capped nanodevices. The first community of nanoparticles (i.e.S(RA)IFN) is loaded with 9-cis-retinoic acid and capped with interferon-γ, whereas the second community of nanoparticles (i.e.S(sulf)PIC) is loaded with sulforhodamine B and capped with poly(I:C). The uptake of S(RA)IFN by SK-BR-3 breast cancer cells enhanced the expression of TLR3 receptor facilitating the subsequent uptake of S(sulf)PIC and cell killing.

Objective: We sought to investigate the effects and mechanism of lead and a high-fat diet on cognitive function and the central nervous system in mice., Methods: Eighty-four healthy male mice were randomly divided into a control group (n = 21) (fed with common diet and free drinking), a lead exposure group (n = 21) (fed with common diet and 300 mg/L lead acetate solution), a high-fat group (n = 21) (fed with high-fat diet and free drinking), and a lead + high-fat group (n = 21) (fed with high-fat diet and 300 mg/L lead acetate solution). In 10 weeks after lead exposure, the mice of all groups were tested for the cognition, learning and memory abilities, body weight, serum triglyceride (TG), low-density lipoprotein, and high-density lipoprotein, as well as for the contents of lead, interleukin 6 (IL-6), interleukin 17 (IL-17), interferon γ, advanced glycation end products (AGEs), glutathione S-transferase (GSH-ST), and hydrogen peroxide in the brain tissues., Results: Compared with the control group and the lead-exposed group, the body weights of mice in the high-fat group and the lead + high-fat group increased significantly from the sixth week of the experiment, of which the difference was statistically significant (P < 0.05). Compared with the control group and the high-fat group, the lead content in brain tissue of the lead exposure group and the lead + high-fat group increased significantly, of which the difference was statistically significant (P < 0.05). Compared with the control group, the escape latent period, triglyceride, low-density lipoprotein, IL-6, IL-17, interferon γ, and AGEs of the remaining 3 groups increased significantly, but the recognition index, passing platform times, high-density lipoprotein, and GSH-ST significantly decreased (P < 0.05); the second and third escape latent periods, IL-6, IL-17, and AGEs of lead + high-fat group, were obviously higher than the remaining 3 groups, but the passing platform times were obviously lower than the remaining 3 groups, of which the difference was statistically significant. The content of hydrogen peroxide in brain tissues had no difference among groups (P > 0.05)., Conclusions: The lead and high-fat diet resulted in lipid metabolism disorders and impaired the cognitive function and central nervous system by promoting the secretion of inflammatory factors in glial cells, inducing the inflammatory reaction of brain tissue, inhibiting GSH-ST expression, and increasing AGEs content., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The protozoan parasite Toxoplasma gondii lives inside a vacuole in the host cytosol where it is protected from host cytoplasmic innate immune responses. However, IFNγ-dependent cell-autonomous immunity can destroy the vacuole and the parasite inside. Toxoplasma strain differences in susceptibility to human IFNγ exist, but the Toxoplasma effector(s) that determine these differences are unknown. We show that in human primary fibroblasts, the polymorphic Toxoplasma-secreted effector GRA15 mediates the recruitment of ubiquitin ligases, including TRAF2 and TRAF6, to the vacuole membrane, which enhances recruitment of ubiquitin receptors (p62/NDP52) and ubiquitin-like molecules (LC3B, GABARAP). This ultimately leads to lysosomal degradation of the vacuole. In murine fibroblasts, GRA15-mediated TRAF6 recruitment mediates the recruitment of immunity-related GTPases and destruction of the vacuole. Thus, we have identified how the Toxoplasma effector GRA15 affects cell-autonomous immunity in human and murine cells., (© 2020 The Authors.)

Ovarian cancer (OC) is the fifth-leading cause of cancer-related death in women with a pathogenesis involving activation of regulatory T cells (Tregs). The T-cell immunoglobulin and ITIM domain (TIGIT) is a well-known immune checkpoint molecule that inhibits T-cell responses. However, the role of TIGIT in OC is not comprehensively understood. In this study, we revealed crucial functions of TIGIT in the development and progression of OC. ID8 cells were used to establish a murine OC model. TIGIT expression was increased in immune cells of OC mice, particularly in CD4 + Tregs. Anti-TIGIT monoclonal antibodies (mAb) were used to block the function of TIGIT in OC mice, and we found that the anti-TIGIT treatment reduced the proportion of CD4 + Tregs, but did not affect CD4 + and CD8 + T cells or natural killer cells. Splenic CD4 + Tregs from OC mice were isolated after the anti-TIGIT treatment, and their functioning was examined. Inhibition of TIGIT lowered the degree of immunosuppression induced by CD4 + Tregs. A survival curve suggested that anti-TIGIT treatment can improve the survival rate of OC in mice. We conclude that TIGIT enhanced CD4 + Tregs response and mediated immunosuppression in the OC model. Our data suggest that inhibition of TIGIT is a potential therapeutic target in OC patients., (© 2020 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Allergic asthma is a chronic inflammatory disease characterized by airflow obstruction, airway hyperresponsiveness (AHR), airway inflammation, and mucus overproduction. Cordyceps polysaccharide (CPS) is one of the main bioactive compounds of Cordyceps militarisis, a traditional Chinese medicine. In this study, we established a mouse model of asthma using ovalbumin (OVA) challenge and evaluated the potential regulatory effect of CPS (25, 50, and 100 mg/kg) on asthmatic mice. These results showed that the asthmatic mice treated with CPS suppressed the secretion of eotaxin, IL-4, IL-5, IL-13, and IFN-γ in the blood and bronchoalveolar lavage fluid (BALF), and decreased serum IgE levels compared to the vehicle-treated mice. CPS also alleviated inflammatory cell infiltration, goblet cell hyperplasia, and the increases of inflammatory cells in the mouse model of asthma. In addition, OVA-induced AHR was inhibited by CPS treatment. Further analyses of protein expression revealed that CPS inhibited the activation of transforming growth factor β1 (TGF-β1)/Smad pathway in mice with asthma. These findings indicated that CPS might serve as a potential therapeutic agent for the management of allergic asthma., Competing Interests: Declaration of Competing Interest All authors declare that they have no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Background: The purpose of the present study was to produce a pcDNA3.1(+)-ureA recombinant vector and evaluate the capacity of this vector to stimulate the immune response against H. pylori infection in infused BALB/c mice., Materials and Methods: The pcDNA3.1(+)-ureA construct was prepared and transformed into E. coli, successfully. The animals we used in the study were allotted into three groups for infusion of 1) recombinant plasmid, 2) pcDNA3.1(+)-ureA + nanoparticles, and 3) pcDNA3.1(+). Blood and tissue specimens from each group of mice were collected at days 15, 30, and 45 after the last infusion and the expression levels of cytokines such as TGF-β1, IL-4, and IFNγ genes comparing to GAPDH as well as the expression of ureA in the mice’s thigh muscle were evaluated., Results: The genes expression analysis showed that the IL4 expression significantly decreased (p<0.001) but IFNγ and TGF-β1 expression increased in the blood of infused mice (p<0.001). Also, the urea expression level in pcDNA3.1(+)-urea and pcDNA3.1(+)-ureA+ nanoparticle 15, 30, and 45 days after the last infusion was significantly different (p<0.001) and its expressions at days 15 and 30 were significantly different (p<0.001), but 45 days after the last infusion it was not significantly different (p>0.05)., Conclusion: The pcDNA3.1(+)-ureA recombinant vector with or without chitosan nanoparticles can stimulate the immune response in animal models against H. pylori infection. Also, after combining the recombinant vector with nanoparticles we observed a better immune response was observed. In future studies this recombinant construct can be used as a biomarker and therapeutic approaches in eukaryotic systems., (This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Intestinal epithelial apical membrane Cl-/HCO3- exchanger DRA (downregulated in adenoma, SLC26A3) has emerged as an important therapeutic target for diarrhea, emphasizing the potential therapeutic role of agents that upregulate DRA. All-trans retinoic acid (ATRA), a key vitamin A metabolite, was earlier shown by us to stimulate DRA expression in intestinal epithelial cells. However, its role in modulating DRA in gut inflammation has not been investigated., Aims: Our aim was to analyze the efficacy of ATRA in counteracting inflammation-induced decrease in DRA in vitro and in vivo., Methods: Interferon-γ (IFN-γ)-treated Caco-2 cells and dextran sulfate sodium (DSS)-treated C57BL/6J mice served as in vitro and in vivo models of gut inflammation, respectively. The effect of ATRA on IFN-γ-mediated inhibition of DRA function, expression, and promoter activity were elucidated. In the DSS colitis model, diarrheal phenotype, cytokine response, in vivo imaging, myeloperoxidase activity, and DRA expression were measured in the distal colon., Results: All-trans retinoic acid (10 μM, 24 h) abrogated IFN-γ (30 ng/mL, 24 h)-induced decrease in DRA function, expression, and promoter activity in Caco-2 cells. All-trans retinoic acid altered IFN-γ signaling via blocking IFN-γ-induced tyrosine phosphorylation of STAT-1. All-trans retinoic acid cotreatment (1 mg/kg BW, i.p. daily) of DSS-treated mice (3% in drinking water for 7 days) alleviated colitis-associated weight loss, diarrheal phenotype, and induction of IL-1β and CXCL1 and a decrease in DRA mRNA and protein levels in the colon., Conclusion: Our data showing upregulation of DRA under normal and inflammatory conditions by ATRA demonstrate a novel role of this micronutrient in alleviating IBD-associated diarrhea., (© 2019 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Acute exacerbations of asthma represent a major burden of disease and are often caused by respiratory infections. Viral infections are recognized as significant triggers of exacerbations; however, less is understood about the how microbial bioproducts such as the endotoxin (lipopolysaccharide (LPS)) trigger episodes. Indeed, increased levels of LPS have been linked to asthma onset, severity and steroid resistance., Objective: The goal of this study was to identify mechanisms underlying bacterial-induced exacerbations by employing LPS as a surrogate for infection., Methods: We developed a mouse model of LPS-induced exacerbation on the background of pre-existing type-2 allergic airway disease (AAD)., Results: LPS-induced exacerbation was characterized by steroid-resistant airway hyperresponsiveness (AHR) and an exaggerated inflammatory response distinguished by increased numbers of infiltrating neutrophils/macrophages and elevated production of lung inflammatory cytokines, including TNFα, IFNγ, IL-27 and MCP-1. Expression of the type-2 associated inflammatory factors such as IL-5 and IL-13 were elevated in AAD but not altered by LPS exposure. Furthermore, AHR and airway inflammation were no longer suppressed by corticosteroid (dexamethasone) treatment after LPS exposure. Depletion of pulmonary macrophages by administration of 2-chloroadenosine into the lungs suppressed AHR and reduced IL-13, TNFα and IFNγ expression. Blocking IL-13 function, through either IL-13-deficiency or administration of specific blocking antibodies, also suppressed AHR and airway inflammation., Conclusions & Clinical Relevance: We present evidence that IL-13 and innate immune pathways (in particular pulmonary macrophages) contribute to LPS-induced exacerbation of pre-existing AAD and provide insight into the complex molecular processes potentially underlying microbial-induced exacerbations., (© 2019 John Wiley & Sons Ltd.)

IL-10 is commonly regarded as an immunoregulatory cytokine, but accumulating evidence suggests that IL-10 may promote CD8 T cell expansion and proliferation. In this study, tumor infiltrating (TI) and peripheral blood (PB) CD8 T cells were collected from esophageal cancer patients. Interestingly, IL-10 concentration in the tumor microenvironment increased with advancing tumor stage, while TI CD8 T cell-mediated IL-10 production decreased with advancing tumor stage. By flow cytometry, three distinctive subsets, including IL-10 + IFNγ - , IL-10 + IFNγ + , and IL-10 - IFNγ + , could be observed in TI CD8 T cells. The former two subsets were present at much higher frequency in stage I and stage II patients than in stage III patients. IL-10 + IFNγ + TI CD8 T cells presented significantly higher IFNγ and lower PD-1 expression than the IL-10 - IFNγ + TI CD8 T cells. PB CD8 T cells, on the other hand, produced little IL-10 but potent IFNγ upon stimulation. Interestingly, intermediate level of exogenous IL-10 could significantly elevate the expression of IFNγ by PB CD8 T cells, while high level of exogenous IL-10 resulted in reduced expression of IFNγ by PB CD8 T cells. Exogenous IL-10 could not significantly reduce the frequencies of PD-1 + PB CD8 T cells, but significantly reduced the MFI of PD-1 in the PB CD8 T cells, especially in stage III patients. Together, this investigation demonstrated that IL-10 enhanced IFNγ expression and suppressed PD-1 expression in PB and TI CD8 T cells; however, the frequency of IL-10-expressing TI CD8 T cells decreased with increasing severity in esophageal cancer., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Tinea is an infectious disease by dermatophytes, of which Trichophyton species accounts for the overwhelming majority of case. Tinea often causes itching with inflammation. In terms of pruritus by fungal infection, however, tinea has not been investigated sufficiently to date., Aim: To evaluate itch caused by Trichophyton infection and the effect of antifungal agents on the infection, by measuring scratch behaviour and profiles of inflammatory cytokines and chemokines., Methods: We used a previously established mouse model of contact hypersensitivity induced by trichophytin, a crude extract from Trichophyton mentagrophytes. Scratching behaviour was recorded using a counting device that measured an electric current induced in a coil by movement of magnets that had been inserted into the hind paws of each animal. We investigated expression of various genes in lesional skin of mice and in normal human epidermal keratinocytes. We also investigated the antipruritic effects of the corticosteroid dexamethasone (DEX) and three antifungal agents: ketoconazole (KCZ), terbinafine (TBF) and liranaftate (LNF)., Results: Biphasic peaks of scratching were observed at 1 h and at 6-7 h during an observation period of 14 h after trichophytin induction. For lesional skin, RNA was extracted 24 h after trichophytin challenge, and increased expression was seen in the genes for interleukin (IL)-17A, interferon-γ, tumour necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-2 and Dectin-1, whereas there was no obvious change in the genes for IL-31 and prostaglandin (PG)E2. Furthermore, KCZ inhibited histidine decarboxylase (HDC) expression in vitro and in vivo, and inhibited scratching in the very early phase. LNF inhibited expression of thymic stromal lymphopoietin (TSLP) and IL-8 in vitro, and TSLP, TNF-α, IL-1α and MIP2 in vivo, and also scratching in the early phase. TBF did not induce any significant alterations in either gene expression or scratching. DEX suppressed expression of all the chemical mediators except HDC in vitro and in vivo, and inhibited scratching., Conclusion: Antifungals can inhibit itching induced by fungal infection through different mechanisms., (© 2018 British Association of Dermatologists.)

A pharmacodynamic assay has been previously developed to monitor ciclosporin treatment in dogs by assessing inhibition of cytokine transcription after whole blood stimulation with 12-myristate 13-1 acetate and ionomycin (PMA/I). In this study, whole blood stimulation with either PMA/I or lipopolysaccharide (LPS) was used to assess the effect of multiple drugs (azathioprine, ciclosporin, mycophenolate, leflunomide and prednisone) after a 7-day treatment course on production of cytokines measured with a multiplex assay in healthy dogs (n = 4 for each treatment). Interleukin-10 (IL-10), interferon gamma (IFNγ) and tumour necrosis factor alpha (TNFα) were significantly activated by PMA/I stimulation and IL-6, IL-10 and TNFα by LPS stimulation, in the absence of immunosuppressive drugs. After ciclosporin treatment, IL-10, IFNγ and TNFα production was significantly reduced after stimulation with PMA/I compared to pre-treatment. After prednisone treatment, TNFα production was significantly reduced after stimulation with PMA/I or LPS compared to pre-treatment. No significant change was observed after treatment with azathioprine, leflunomide or mycophenolate. This methodology may be useful to monitor dogs not only treated with ciclosporin, but also with prednisone or a combination of both. Further studies are needed to assess the use of this assay in a clinical setting., (© 2019 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-induced gene that catalyzes the oxidation of cholesterol to 25-hydroxycholesterol (25HC), which exerts broad-spectrum antiviral function. To investigate the roles of fish CH25H in Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV) infection, we cloned and characterized a CH25H homolog from orange-spotted grouper ( Epinephelus coioides ) (EcCH25H). EcCH25H encoded a 271-amino-acid polypeptide, with 86 and 59% homology with yellow croaker ( Larimichthys crocea ) and humans, respectively. EcCH25H contained a conserved fatty acid (FA) hydroxylase domain and an ERG3 domain. EcCH25H expression was induced by RGNNV or SGIV infection, lipopolysaccharide (LPS) or poly (I:C) treatment in vitro . Subcellular localization showed that EcCH25H and mutant EcCH25H-M were distributed in the cytoplasm and partly colocalized with the endoplasmic reticulum. SGIV and RGNNV replication was decreased by EcCH25H overexpression, which was reflected in the reduced severity of the cytopathic effect and a decrease in viral gene transcription, but replication of both viruses was increased by knockdown of EcCH25H. Besides, the antiviral activity was dependent on its enzymatic activity. Treatment with 25HC significantly inhibited replication of SGIV and RGNNV. EcCH25H overexpression positively regulated the IFN-related molecules and proinflammatory cytokines, and increased both IFN and ISRE promoter activities. Moreover, 25HC treatment significantly suppressed SGIV and RGNNV entry into host cells. The similar inhibitory effect on SGIV entry was observed in EcCH25H overexpression cells. Taken together, our findings demonstrated that EcCH25H inhibited SGIV and RGNNV infection by regulating IFN signaling molecules, and might also influence viral entry via an effect on cholesterol.

Objective: This study examines polyfunctional T-cells in psoriatic arthritis (PsA) synovial tissue and their associations with clinical disease and implications for therapy., Methods: PsA synovial tissue was enzymatically/mechanically digested to generate synovial tissue single cell suspensions. Frequencies of polyfunctional CD4, CD8, T-helper 1 (Th1), Th17 and exTh17 cells, using CD161 as a marker of Th17 plasticity, were determined by flow cytometry in matched PsA synovial tissue and peripheral blood. Synovial T-cell polyfunctionality was assessed in relation to Disease Activity in PSoriatic Arthritis (DAPSA) and in synovial cell suspensions cultured with a current mode of treatment, phosphodiesterase 4 (PDE4) inhibitor., Results: PsA synovial tissue infiltrating CD4 + T-cells expressed higher levels of interleukin (IL)-17A, interferon gamma (IFN-γ), GM-CSF and CD161, with parallel enrichment of Th1, Th17 and exTh17 T-helper subsets (all p<0.05). Interestingly, a significant proportion of synovial T-cell subsets were triple-positive for GM-CSF, tumour necrosis factor (-TNF), -IL-17 or IFN-γ compared with matched blood (all p<0.05). Importantly, frequencies of polyfunctional T-cells correlated with DAPSA: Th1-GM-CSF + /TNF + /IFN-γ + (r=0.7, p<0.01), Th17-GM-CSF + /TNF + /IL-17 + (r=0.6, p<0.057) and exTh17-GM-CSF + /TNF + /IFN-γ + (r=0.7, p=0.0096), with no associations observed for single cytokine-producing T-cells. Following ex vivo culture of PsA synovial tissue cell suspensions, polyfunctional GM-CSF + TNFα + IL-17A + or/IFN-γ + -producing T-cells (p<0.05), but not single cytokine-producing T-cells, were inhibited with a PDE4 inhibitor., Conclusion: These data demonstrate enrichment of polyfunctional T-cells in PsA synovial tissue which were strongly associated with DAPSA and ex vivo therapeutic response., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The production of nitric oxide (NO) by inducible NO synthase (iNOS) and the regulation of gene expression by hypoxia-inducible factors (HIFs) are important for many aspects of human cell biology. However, little is known about whether iNOS expression is controlled by HIFs in human cells. Stimulation of A549 human lung epithelial cells with cytokines (TNF, IL-1 and IFNγ) increased the nuclear accumulation of HIF-1 in normoxic conditions. Activation of HIF-1 by hypoxia or CoCl 2 was not sufficient to induce iNOS expression. However, pharmacological inhibition of HIF-1 reduced the induction of iNOS expression in A549 cells and primary human astrocytes. Moreover, elimination of HIF-1α expression and activity by CRISPR/Cas9 gene editing significantly reduced the induction of human iNOS gene promoter, mRNA and protein expression by cytokine stimulation. Three putative hypoxia response elements (HRE) are present within the human iNOS gene promoter and elimination of an HRE at -4981 bp reduced the induction of human iNOS promoter activity in response to cytokine stimulation. These findings establish an important role for HIF-1α in the induction of human iNOS gene expression in response to cytokine stimulation., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background and Purpose- Cerebral small vessel disease (cSVD) is the major vascular cause of cognitive decline and dementia. The pathogenesis of cSVD remains largely unknown, although several studies suggest a role for systemic inflammation. In certain pathophysiological situations, monocytes can reprogram toward a long-term proinflammatory phenotype, which has been termed trained immunity. We hypothesize that trained immunity contributes to the progression of cSVD. Methods- Individuals with mild-to-severe cSVD participated in the study. Severity of cSVD was determined by the white matter hyperintensities (WMH) volume (mL) on magnetic resonance imaging in 2006, 2015, and the progression between 2006 and 2015 (ΔWMH). Cytokine production was assessed after ex vivo stimulation of peripheral blood mononuclear cells and monocytes. Additionally, monocyte subsets were identified by flow cytometry. Results- Fifty-one subjects (70±6 years, 60% men, 5.1±6.4 mL ΔWMH) were included. Circulating hsIL (high-sensitivity interleukin)-6 correlated with cSVD ( P=0.005, r s =0.40). Cytokine production capacity by monocytes was associated with cSVD progression. Basal IL-8 and IL-17 production ( P=0.08, r s =0.25; P=0.03, r s =0.30) and IL-6 production after Pam3Cys stimulation in monocytes was associated with cSVD (n=35: P=0.008, r s =0.44). Conversely, interferon (IFN)-γ production in Candida albicans stimulated peripheral blood mononuclear cells was negatively correlated with cSVD ( P=0.009, r s =-0.36). Flow cytometry revealed a correlation of the intermediate monocyte subset with cSVD ( P=0.01, r s =0.36). Conclusions- Severity and progression of cSVD are not only correlated with systemic inflammation (hsIL-6) but also with trained immunity characteristics of circulating monocytes, in terms of an altered cytokine production capacity and a shift toward the proinflammatory intermediate monocyte subset.

MHTP [2-methoxy-4-(7-methoxy-1,2,3,4-tetrahydroisoquinolin-1-yl) phenol], a synthetic isoquinolinic alkaloid, presented anti-inflammatory activity in several experimental models of acute inflammation as lipopolysaccharide (LPS)-induced acute lung injury and phlogistic agent-induced edema and presented low preclinical toxicity. The aim of this study was to determine the MHTP effect on ovalbumin (OVA)-induced pulmonary allergic inflammation. In other to realize this study, female BALFB/c mice were sensitized and challenged with OVA (OVA group) and treated with MHTP (MHTP group) by nasal instillation. Inflammatory, allergic, and immunomodulatory parameters such as migration of inflammatory cells to the lung tissue, pulmonary histological analysis, serum level of IgE-allergen specific, cytokine secretion, and lung T cell population characterization were analyzed and the data were considered statistically significant with p < 0.05. OVA-sensitized and OVA-challenged and MHTP (5.0 mg/kg)-treated mice presented reduction on total leukocyte migration into the bronchoalveolar lavage (BALF) dependent of lymphocyte and eosinophil migration (p < 0.001 and p < 0.01) as compared with the OVA group. Flow cytometric analysis showed that MHTP treatment decreased the percentage of granulocytes (p < 0.001) into the BALF and lung tissue histological analyzes demonstrated that the MHTP treatment decreased leukocyte migration and mucus production. In addition, treatment with MHTP decreased the number of CD3 + CD4 + T cells independently of CD8 + T cell reduction into the BALF. The treatment also reduced significantly (p < 0.05) the serum level of IgE-OVA specific followed by reduction of IL-4, IL-13, and IL-17 production. Surprisingly, the MHTP treatment increased significantly (p < 0.05) the IFN-γ production in the BALF of these animals. Therefore, the results presented here showed that MHTP treatment, by nasal instillation, in a mouse model of OVA-induced pulmonary allergy has anti-allergic and immunomodulatory effects dependent on a Th1-skewed cytokine production that ameliorate the pulmonary allergic inflammation.

Pro-Glu/Pro-Pro-Glu (PE/PPE) family proteins in Mycobacterium tuberculosis (Mtb) are contributors to pathogenesis and immune evasion. These proteins have a unique structure in which the sequence is conserved. We investigated the vaccine potential of ESAT-6 fused with consensus CD4 + T-cell epitopes of PE/PPE proteins against highly pathogenic Mtb strain HN878 in a murine model. We selected consensus CD4 + T-cell epitopes of PE/PPE proteins by multiple alignments, investigated their IFN-γ response during Mtb infection, and produced their fused ESAT-6 vaccine antigens. Our results showed an increased immune response in PE/PPE peptide -ESAT-6 fusion protein immunization group compared to ESAT-6 only immunization group. After challenge with Mtb strain HN878, we observed that induced CD4 + T-cells secreted double-positive cytokine IL-2 + /IFN-γ + , which is considered to be associated with protective T-cell immunity. Additionally, lower numbers of colony-forming units were observed in the spleen of fusion protein immunization groups than in those of single ESAT-6 group. Therefore, conjugation of consensus CD4 + T-cell epitopes in N terminus of PE/PPE to vaccine antigens could potentially increase the protective efficacy of subunit vaccine., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Rationale: Basiliximab and etanercept have achieved promising responses in steroid-refractory graft versus host disease (SR-GVHD). However, the in vivo immune changes following the treatment have not been elucidated., Patient Concerns: A 14-year-old boy presented with skin rash and diarrhea 20 days after haploidentical hemotopoietic stem cell transplantation., Diagnoses: We made the diagnose of grade 3 acute GVHD with skin and gastrointestinal involvement., Interventions: After the failure of the first-line treatment with methylprednisolone, combined anti-cytokine therapies with basiliximab and etanercept were prescibed., Outcomes: He achieved complete remission by basiliximab and etanercept. Furthermore, we detected that donor CD3CD56 Natural killer T(NKT)-like cells expanded gradually after the period of lymphocytopenia caused by GVHD and anti-cytokine therapy. The expansion of NKT-like cells was in association with high serum IFN-γ. NKT-like cells showed preferred proliferation in response to IFN-γ and potent cytotoxicity against leukemia cells. The expansion persisted > 2 years and the patient had a leukemia-free survival of 66 months., Lessons: Our case indicated that combined anti-cytokine treatment may reset the immune system and cause NKT-like cells to exhibit a predilection for expansion.

Guillain-Barré syndrome (GBS) is an immune-mediated acute disorder of the peripheral nervous system. Despite treatment, there is an associated mortality and severe disability in 9 to 17% of the cases. Decitabine (DAC) is a hypomethylating drug used in myelodisplastic syndrome, that has been shown to exert immunomodulatory effects. We have evaluated the effects of DAC in two rodent models of GBS, the Experimental Allergic Neuritis (EAN). Both prophylactic and therapeutic treatment with DAC ameliorated the clinical course of EAN, increasing the numbers of thymic regulatory T cells and reducing the production of proinflammmatory cytokines. Our data suggest the possible use of decitabine for the treatment of GBS., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Activation of the neonatal immune system may contribute to deficits in neuronal plasticity. We have reported that neonatal vaccination with a hepatitis B vaccine (HBV) transiently impairs mood status and spatial memory involving a systemic T helper (Th) 2 bias and M1 microglial activation. Here, an EE induced microglial anti-inflammatory M2 polarization, as evidenced by selectively enhanced expression of the Arginase1 gene (Arg-1) in the hippocampus. Interestingly, knock-down of the Arg-1 gene prevented the effects of EE on restoring the dendritic spine density. Moreover, levels of the Th1-derived cytokine IFN-gamma (IFN-γ) were elevated in the choroid plexus (CP), which is the interface between the brain and the periphery. IFN-γ-blocking antibodies blunted the protective effects of an EE on spine density and LTP. Furthermore, levels of complement proteins C1q and C3 were elevated, and this elevation was associated with synapse loss induced by the HBV, whereas an EE reversed the effects of the HBV. Similarly, blockade of C1q activation clearly prevented synaptic pruning by microglia, LTP inhibition and memory deficits in hepatitis B-vaccinated mice. Together, the EE-induced increase in IFN-γ levels in the CP may disrupt systemic immunosuppression related to HBV via an IFN-γ/Arg-1/complement-dependent pathway., (Copyright © 2018 Elsevier Inc. All rights reserved.)

BACKGROUND Severe sepsis is among the most common causes of death in Emergency Departments, with more than 30% mortality. Hydrocortisone is used in severe sepsis patients who were not responsive to fluid resuscitation and vasopressor therapy. However, the effect of hydrocortisone on regulating inflammation, hemodynamic stability, and preventing shock is still unclear in Chinese patients. MATERIAL AND METHODS In this prospective observational study, we included 105 severe sepsis patients. We measured the level of serum inflammatory cytokines, hemodynamic variables, and phagocytic ability of innate immune cells during the treatment. We analyzed the relationship between these variables and the hydrocortisone treatment. RESULTS We treated 43 (41.0%) patients with hydrocortisone, while the other 62 (59.0%) patients were not, based on their response to fluid resuscitation and vasopressor therapy. The hydrocortisone group had a mean simplified acute physiology score (SAPS) II score of 41.8 with standard deviation (SD) of 7.1, while the non-hydrocortisone group had a mean SAPS II score of 36.7 with SD of 7.3. The mean sequential organ failure assessment (SOFA) scores of these 2 groups were 10.6 and 9.2, respectively. We found an obvious decrease of serum pro-inflammatory cytokines, including interleukin-1β (IL-1β), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IL-6, after hydrocortisone treatment. However, these changes were not observed in the non-hydrocortisone group. What's more, amelioration of hemodynamic variables was observed after hydrocortisone treatment. No significant association between hydrocortisone treatment and innate immune cell phagocytic function was observed. CONCLUSIONS Based on these results, we believe that hydrocortisone treatment has potential anti-inflammatory, hemodynamic reversal, and stability effects on severe sepsis patients. These key benefits may help patients by preventing septic shock.

Objective: Phosphorylcholine (PC) is a structural component of a wide variety of pathogens including Streptococcus pneumoniae and Haemophilus influenzae. Here, the immune response in mice to PC immunization via the sublingual (SL) route versus the intranasal (IN) route was investigated in terms of efficacy and safety., Methods: BALB/c mice were immunized with PC-keyhole limpet hemocyanin (KLH) plus cholera toxin (CT) or CT alone via the IN or SL route. The immune response generated was studied in terms of PC-specific antibody titers, interferon (IFN)-γ and interleukin (IL)-4 production by CD4 + T cells, and cross-reactivity of PC-specific immunoglobulin (Ig)-A antibodies in nasal washes against S. pneumoniae and non-typeable H. influenzae., Results: SL and IN immunization with PC-KLH plus CT resulted in a marked increase in the levels of PC-specific, mucosal IgA and serum IgM, IgG, and IgA antibodies. Additionally, SL immunization elicited significantly higher levels of PC-specific IgG2a subclass antibodies and IFN-γ in serum. On the other hand, IN immunization with CT alone remarkably increased the total IgE level in serum compared with SL and IN immunization with PC-KLH plus CT. PC-specific IgA antibodies in nasal wash samples reacted to most strains of S. pneumoniae and non-typeable H. influenzae., Conclusion: SL immunization is as effective as IN immunization to induce PC-specific immune responses and more effective than IN immunization to reduce the production of IgE and to prevent the sensitization to allergen causing type I allergy., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The potential therapeutic value of Moringa oleifera extract (MOE), due to its anti-inflammatory and anti-oxidant effects, has been reported previously. In this study, Hymenolepis nana antigen (HNA) in combination with MOE was used in immunization against H. nana infection. Adult worm and egg counts were taken, while histological changes in the intestine were observed. Mucosal mast (MMCs) and goblet cells (GCs) were stained with specific stains, while serum and intestinal IgA were assayed using enzyme-linked immunosorbent assay (ELISA). Reduced glutathione (GSH) and lipid peroxidation (thiobarbituric acid reactive substances, TBARS) were assayed. Real-time polymerase chain reaction (PCR) was used for detection of mRNA expression in ileum tissue. The results demonstrated an improvement in the architecture of intestinal villi, decreased inducible nitric oxide synthase (iNOs) and TBARS, and increased GSH in HNA, MOE and MOE + HNA groups. In the same groups, an increase in GCs, mucin 2 (MUC2), interleukins (IL)-4, -5 and -9, and stem cell factor (SCF) versus a decrease in both interferon-gamma (IFN-γ) and transforming growth factor (TGF-β) expression appeared. HNA and MOE + HNA increased serum and intestinal IgA, respectively. MOE decreased MMCs and achieved the highest reductions in both adult worms and eggs. In conclusion, MOE could achieve protection against H. nana infections through decreased TGF-β, IFN-γ and MMC counts versus increased GC counts, T-helper cell type 2 (Th2) cytokines and IgA level.

Aims: Air particulate matter (PM) is associated with increased cardiovascular morbidity and mortality. Altered autonomic functions play a key role in PM-induced cardiovascular disease. However, previous studies have not address the impact of PM on sympathetic and parasympathetic control of heart function, independently, and using controlled conditions, i.e., increasing titration of PM of known composition, in absence of other potential confounding factors. To fill this gap, here we used symbolic analysis that is capable of detecting non-mutual changes of the two autonomic branches, thus considering them as independent, and concentrations of PM as they could be measured at peak levels in Milan during a polluted winter day., Methods and Results: In this randomized, cross-over study, we enrolled 12 healthy subjects who underwent two random sessions: inhalation of filtered air mixture or inhalation of filtered air containing particulate mixture (PM 10, PM 2.5, PM 1.0 and PM 0.5µm). ECG and respiration for autonomic analysis and blood sample for DNA Methylation were collected at baseline (T1), after air exposure (T2) and after 2h (T3). Spectral and symbolic analysis of heart rate variability (HRV) were performed for autonomic control of cardiac function, while alterations in DNA methylation of candidate genes were used to index pro-inflammatory modifications. In the PM expose group, autonomic analysis revealed a significant decrease of 2UV%, index of parasympathetic modulation (14% vs 9%, p = 0.0309), while DNA analysis showed a significant increase of interferon γ (IFN- γ) methylation, from T1 to T3. In a mixed model using T1, T2 and T3, fine and ultrafine PM fractions showed significant associations with IFN- γ methylation and parasympathetic modulation., Conclusions: Our study shows, for the first time, that in healthy subjects, acute exposure to PM affects parasympathetic control of heart function and it increases methylation of a pro-inflammatory gene (i.e. methylation of interferon γ). Thus, our study suggests that, even in absence of other co-factors and in otherwise healthy individuals, PM per se is sufficient to trigger parasympathetic dysautonomia, independently from changes in sympathetic control, and inflammation, in a dose-dependent manner., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: To explore the type of cytokine (IL-2 or IL-7) and its most optimal concentration regarding the improvement of the signal-to-noise ratio of glutamic acid decarboxylase 65 (GAD65) in enzyme-linked immunospot (ELISPOT) assay in Type 1 diabetic (T1DM) patients.
 Methods: Twenty T1DM patients (Group A) and sixteen healthy controls matched with age and sex (Group B) were enrolled in our study, and their peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll method. GAD65, internal control and Pediacel served as "five-for-one" vaccine were selected as the stimulating antigen. Different concentrations of IL-2 [0 U/mL (Group 1), 0.5 U/mL (Group 2), 2.5 U/mL (Group 3) and 12.5 U/mL (Group 4)] were added to the culture system. The CD4+ T cells of secreting interferon-gamma (IFN-γ) in the above groups were determined by ELISPOT. The spots number, net values and stimulating index (SI) were compared in GAD65 (signal) and internal control (background). Next, another 21 T1DM patients (Group C)and 12 healthy controls matched with age and sex (Group D) were enrolled, and the specific T cell response to the GAD65 antigen was detected. The net values and SI were compared between the best optimal concentration of IL-2 (2.5 U/mL, Group 5) and IL-7 (0.5 ng/mL, Group 6).
 Results: 1) After adding IL-2 into the Group A, the amount of GAD65 reactive T cells in different groups increased compared with Group A1, while the background in the internal control also increased gradually with the increased concentration of IL-2. There was no significant difference in net value (signal-noise) in the different concentration between the Group A3 and the Group A4 (P>0.05). The SI in the Group A3 (2.8), the highest one, was significantly higher than that in the Group B3 (1.3) (P<0.05). 2) Although the number of GAD65 spots in the Group C6 and the Group D6 were slightly higher than that in the Group C5 and the Group D5, respectively, the background in the Group C6 and the Group D6 also increased, without statistical significance (P>0.05). The mean net value spot and SI in the Group C5 (net value: 5.5; SI: 2.8) were both significantly higher than those in the Group C6 (net value: 4.3; SI: 1.8) (both P<0.05).
 Conclusion: The concentration of 2.5 U/mL for IL-2 is proved to be the best optimal concentration for GAD65 specific T-cell responses in ELISPOT in patients with T1DM. IL-2 is much better than IL-7 in improvement of the SI in the ELISPOT.

Animal and human studies suggest an association between depression and aberrant immune response. Further, common inflammatory markers may change during the course of antidepressant treatment in patients. The objective of this study was to evaluate changes in inflammatory markers and clinical outcomes from subjects enrolled in the Combining Medications to Enhance Depression Outcome (CO-MED) trial. At baseline and week 12 (treatment completion), plasma samples of 102 participants were analyzed via a multiplex assay comprised of inflammatory markers using a 27-plex standard assay panel plus a 4-plex human acute phase xMAP technology based platform. We carried out analyses in two steps. First, t-tests were used to identify inflammatory marker levels that changed between baseline and week 12. For markers that were altered, logistic regression models were then conducted to look for associated changes in remission at week 12. Among the 31 inflammatory markers analyzed, several cytokines (IL-5, IFN-γ, IL-13), two chemokines (Eotaxin-1/CCL11, RANTES) and an acute-phase reactant (serum amyloid P component) showed change from baseline to week 12. However, only two indicated differential remission responses. Interestingly, increased levels of Eotaxin-1/CCL11 correlated with remission at week 12, whereas decreased levels of IFN-γ correlated with non-remission at week 12. Results suggest that these inflammatory proteins may serve as predictors of treatment response., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

S100A9 is an endogenous danger signal that promotes and exacerbates the neutrophilic inflammatory response. To investigate the role of S100A9 in neutrophilic asthma, S100A9 levels were measured in sputum from 101 steroid-naïve asthmatics using an ELISA kit and the levels were significantly correlated with percentages of neutrophils in sputum. Intranasal administration of recombinant S100A9 markedly increased neutrophil numbers at 8h and 24h later with concomitant elevation of IL-1β, IL-17, and IFN-γ levels. Treatment with an anti-S100A9 antibody restored the increased numbers of neutrophils and the increased airway resistance in OVA/CFA mice toward the levels of sham-treated mice. Concomitantly, the S100A9 and neutrophil elastase double positive cells were markedly reduced with attenuation of IL-1β, IL-17, and IFN-γ levels by the treatment with the anti-S100A9 antibody. Our data support a role of S100A9 to initiate and amplify the neutrophilic inflammation in asthma, possibly via inducing IL-1β, IL-17 and IFN-γ., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Gram-negative bacteria actively secrete outer membrane vesicles, spherical nano-meter-sized proteolipids enriched with outer membrane proteins, to the surroundings. Outer membrane vesicles have gained wide interests as non-living complex vaccines or delivery vehicles. However, no study has used outer membrane vesicles in treating cancer thus far. Here we investigate the potential of bacterial outer membrane vesicles as therapeutic agents to treat cancer via immunotherapy. Our results show remarkable capability of bacterial outer membrane vesicles to effectively induce long-term antitumor immune responses that can fully eradicate established tumors without notable adverse effects. Moreover, systematically administered bacterial outer membrane vesicles specifically target and accumulate in the tumor tissue, and subsequently induce the production of antitumor cytokines CXCL10 and interferon-γ. This antitumor effect is interferon-γ dependent, as interferon-γ-deficient mice could not induce such outer membrane vesicle-mediated immune response. Together, our results herein demonstrate the potential of bacterial outer membrane vesicles as effective immunotherapeutic agent that can treat various cancers without apparent adverse effects.Bacterial outer membrane vesicles (OMVs) contain immunogens but no study has yet examined their potential in treating cancer. Here, the authors demonstrate that OMVs can suppress established tumours and prevent tumour metastasis by an interferon-γ mediated antitumor response.

Pseudobrickellia brasiliensis (Asteraceae) is a plant commonly known as arnica-do-campo and belongs to the native flora of the Brazilian Cerrado. The alcoholic extract of the plant has been used as an anti-inflammatory agent in folk medicine, but the biological mechanism of action has not been elucidated. The present study evaluated the composition of P. brasiliensis aqueous extract and its effects on pro-inflammatory cytokine production and lymphocyte proliferation. The extracts were prepared by sequential maceration of P. brasiliensis leaves in ethanol, ethyl acetate, and water. Extract cytotoxicity was evaluated by trypan blue exclusion assay, and apoptosis and necrosis were measured by staining with annexin V-FITC and propidium iodide. The ethanolic (ETA) and acetate (ACE) extracts showed cytotoxic effects. The aqueous extract (AQU) was not cytotoxic. Peripheral blood mononuclear cells stimulated with phorbol myristate acetate and ionomycin and treated with AQU (100 μg/mL) showed reduced interferon (IFN)-γ and tumor necrosis factor (TNF)-α expression. AQU also inhibited lymphocyte proliferative response after nonspecific stimulation with phytohemagglutinin. The aqueous extract was analyzed by liquid chromatography coupled with photodiode array detection and mass spectrometry. Quinic acid and its derivatives 5-caffeoylquinic acid and 3,5-dicaffeoylquinic acid, as well as the flavonoids luteolin and luteolin dihexoside, were detected. All these compounds are known to exhibit anti-inflammatory activity. Taken together, these findings demonstrate that P. brasiliensis aqueous extract can inhibit the pro-inflammatory cytokine production and proliferative response of lymphocytes. These effects may be related to the presence of chemical substances with anti-inflammatory actions previously reported in scientific literature.

Multiple sclerosis (MS) is a disease that is characterized by immune-mediated destruction of CNS myelin. Current MS therapies aim to block peripheral immune cells from entering the CNS. Although these treatments limit new inflammatory activity in the CNS, no treatment effectively prevents long-term disease progression and disability accumulation in MS patients. One explanation for this paradox is that current therapies are ineffective at targeting immune responses already present in the CNS. To this end, we sought to understand the metabolic properties of T cells that mediate ongoing inflammation in the demyelinating CNS. Using experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice, a well-studied model of MS, we showed that the CD4 + and CD8 + T cells that invade the EAE CNS are highly glycolytic. Elevated glycolytic rates in T cells isolated from the EAE CNS correlate with upregulated expression of glycolytic machinery and is essential for inflammatory responses to myelin. Surprisingly, we found that an inhibitor of GAPDH, 3-bromopyruvic acid (3-BrPa), blocks IFN-γ, but not IL-17A, production in immune cells isolated from the EAE CNS. Indeed, in vitro studies confirmed that the production of IFN-γ by differentiated Th1 cells is more sensitive to 3-BrPa than is the production of IL-17A by Th17 cells. Finally, in transfer models of EAE, 3-BrPa robustly attenuates the encephalitogenic potential of EAE-driving immune cells. To our knowledge, these data are among the first to demonstrate the metabolic properties of T cells in the demyelinating CNS in vivo., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Influenza vaccine development is considered to be complicated and challenging. Constantly evolving influenza viruses require continuous global monitoring and reformulation of the vaccine strains. Peptides that are conserved among different strains and subtypes of influenza A virus are strongly considered to be attractive targets for development of cross protective influenza vaccines that stimulate cellular responses. In this study, three highly conserved (>90%) matrix 1 peptides that contain multiple T cell epitopes, ILGFVFTLTVPSERGLQRRRF (P M 1), LIRHENRMVLASTTAKA (P M 2) and LQAYQKRMGVQMQR (P M 3), were assessed for their immunogenic potential in vitro by subjecting peripheral blood mononuclear cells from healthy volunteers to repetitive stimulation with these chemically synthesised peptides and measuring their IFN-γ concentrations, proliferation by ELISA, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. Seven samples were screened for immunogenicity of P M 1 and P M 2, and six for that of P M 3. All six samples had positive responses (IFN-γ secretion) to P M 3 stimulation, as did five and three for P M 2 and P M 1 respectively. In contrast, seven (P M 1 and P M 2) and four (P M 3) samples showed proliferative response as compared with unstimulated cells. The encouraging immunogenic response generated by these highly conserved matrix 1 peptides indicates they are prospective candidates for development of broadly reactive influenza vaccines., (© 2017 The Societies and John Wiley & Sons Australia, Ltd.)

The poly-γ- D -glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis , provides protection of the bacterium from phagocytosis and allows its unimpeded growth in the host. We investigated crosstalk between murine natural killer (NK) cells and macrophages stimulated with the PGA capsule of Bacillus licheniformis , a surrogate of the B. anthracis capsule. PGA induced interferon-gamma production from NK cells cultured with macrophages. This effect was dependent on macrophage-derived IL-12 and cell-cell contact interaction with macrophages through NK cell receptor NKG2D and its ligand RAE-1. The results showed that PGA could enhance NK cell activation by inducing IL-12 production in macrophages and a contact-dependent crosstalk with macrophages.

According to most models of mycobacterial infection, inhibition of the pro-inflammatory macrophage immune responses contributes to the persistence of bacteria. Mycobacterium avium subsp. paratuberculosis (MAP) is a highly successful pathogen in cattle and sheep and is also implicated as the causative agent of Crohn's disease in humans. Pathogenic mycobacteria such as MAP have developed multiple strategies to evade host defence mechanisms including interfering with the macrophages' capacity to respond to IFN-γ, a feature which might be lacking in non-pathogenic mycobacteria such as M. smegmatis. We hypothesized that pre-sensitisation of macrophages with the pro-inflammatory cytokine IFN-γ would help in overcoming the inhibitory effect of MAP or its antigens on macrophage inflammatory responses. Herein we have compared a series of macrophage activation parameters in response to MAP and M. smegmatis as well as mycobacterial antigens. While IFN-γ did overcome the inhibition in immune suppressive mechanisms in response to MAP antigen as well as M. smegmatis, we could not find a clear role for IFN-γ in overcoming the inhibition of macrophage inflammatory responses to the pathogenic mycobacterium, MAP. We demonstrate that suppression of macrophage defence mechanisms by pathogenic mycobacteria is unlikely to be overcome by prior sensitization with IFN-γ alone. This indicates that IFN-γ signaling pathway-independent mechanisms may exist for overcoming inhibition of macrophage effector functions in response to pathogenic mycobacteria. These findings have important implications in understanding the survival mechanisms of pathogenic mycobacteria directed towards finding better therapeutics and vaccination strategies.

Background: Ketamine has been used as an analgesic adjuvant with morphine in the treatment of refractory cancer pain recently. But both morphine and ketamine have been reported to produce a number of immunomodulatory effects. The current study was performed to assess whether the concentration of ketamine, as adjuvant analgesics for patient with refractory cancer pain, was related to its effect on T cells interleukin-2 (IL-2)/interferon-γ (IFN-γ) expression in vitro., Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood of patients with refractory cancer pain over a Ficoll-Hypaque density gradient. T cells were isolated from by positive selection using anti-CD3 beads. T cells were then treated with vehicle (C group), morphine (200 ng/mL, M group), morphine (200 ng/mL), and different dose of ketamine (100, 200, 1000 ng/mL; MK1, MK5, MK10 group) for 24 hours before stimulation with anti-CD3 and anti-CD28. Then supernatant IL-2 and IFN-γ protein analysis, quantitative reverse transcription polymerase chain reaction (RT-PCR) for IL-2 and IFN-γ were done., Results: There were no significant difference of supernatant IL-2 and IFN-γ among C group, M group, and MK1 group, but the mRNA of M group and MK1 group were decreased compared with C group (P < .05). Compared with C group, both of the supernatant protein and the mRNA of MK5 group and MK10 group were all significantly decreased (P < .01). Compared with M group, both of the supernatant protein and the mRNA of MK5 group and MK10 group were all decreased (P < .05), while supernatant IL-2 and the mRNA of MK10 group were significantly decreased (P < .01)., Conclusion: In conclusion, we confirmed that just as morphine, ketamine dose-dependently suppressed IL-2 and IFN-γ of activated T lymphocyte of patients with refractory cancer pain in vitro, but the inhibitory action of low dose ketamine could be neglected.

The objective of this research was to examine the effects of dexamethasone (DEX) treatment on various aspects of immunity following administration of a multivalent respiratory vaccine, using a model intended to mimic acute versus chronic stress. Angus × Hereford steers ( = 32; 209 ± 8 kg) were stratified by BW and randomly assigned to 1 of 3 treatments: 1) acute stress (ACU), in which 0.5 mg/kg BW DEX was intravenously administered at 1000 h only on d 0; 2) chronic stress (CHR), in which 0.5 mg/kg BW DEX was intravenously administered at 1000 h on d -3 to 0; or 3) control (CON), in which no DEX was administered. Steers were fitted with indwelling jugular catheters and rectal temperature (RT) recording devices on d -4 relative to vaccination and placed in individual stanchions in an environmentally controlled facility. Blood samples were collected and serum was isolated at -74, -50, and -26 h; at 0.5-h intervals from -4 to 6 h; and at 12, 24, 36, 48, and 72 h relative to multivalent respiratory vaccination at 1200 h on d 0. Additional blood samples were used to analyze complete blood cell count (CBC) and functional capacities of neutrophils. There was a treatment × time interaction ( < 0.01) for RT such that DEX treatment in CHR and ACU steers decreased RT on d -3 and 0, respectively. A treatment × time interaction ( < 0.01) was observed for total white blood cells (WBC), neutrophils, lymphocytes, and monocytes. Specifically, DEX increased WBC and neutrophils in CHR and ACU steers ( < 0.001) yet decreased lymphocytes in CHR steers ( = 0.02) compared with CON steers. Neutrophil concentration increased rapidly, within 2 h of the DEX infusion, in ACU steers. Monocytes transiently increased ( < 0.001) in response to DEX treatment in CHR and ACU steers. In contrast, eosinophils were greater ( < 0.01) in CON steers than in ACU and CHR steers. A treatment × time interaction ( = 0.004) was observed for interferon-γ, with CON cattle exhibiting greater concentrations than the ACU and CHR cattle at 5 h after vaccination, through d 3. Treatment also influenced ( ≤ 0.001) the expression of L-selectin on the surface of neutrophils. The percentage of neutrophils engaging in phagocytosis and the oxidative burst were suppressed ( ≤ 0.001) among only the CHR steers, whereas the intensity of the oxidative burst was suppressed ( ≤ 0.001) for both ACU and CHR steers. These data suggest that our model induced acute and chronic immunosuppression and defined the acute response to a multivalent vaccine in CON steers.

Objective: To investigate the effect of IL-1β or TNF-α-treated human decidual cells on the balance between Th1 and Th2 or Th17 and Treg.
 Methods: Human decidual cells were isolated and treated with estrogen (E2) and progesterone (P) for 7 days and then with IL-1β or TNF-α for 24 h. The culture medium was collected to prepare conditioned medium. The T cells isolated from human decidual tissue were cultured in presence of conditioned medium for 72 h. ELISA was used to detect concentration of IFN-γ, IL-2, IL-5, IL-17 IL-10, and TGF-1β in supernatants and quantitative real-time PCR was used to detect the mRNA expression of IFN-γ, IL-17, IL-10, and TGF-1β.
 Results: The levels of IFN-γ, IL-2 and IL-17 were significantly increased when T cells were cultured in the conditioned medium compared with the control group (E2+P), while the levels of IL-5, IL-10, and TGF-1β were not significantly changed. The mRNA expressions of IFN-γ and IL-17 mRNA were significantly increased when T cells were cultured in the conditioned medium compared with the control group (E2+P), while the mRNA expression of IL-10 and TGF-1β was not significantly different.
 Conclusion: Human decidual cells treated by IL-1β or TNF-α can shift the balance of Th1 and Th2 or Th17 and Treg toward Th1 and Th17 immunity.

Background: Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite with a broad host range including most warm-blooded animals, including humans. T. gondii surface antigen 1 (SAG1) is a well-characterized T. gondii antigen. T. gondii expresses five nonmitochondrial rhomboid intramembrane proteases, TgROM1-5. TgROM4 is uniformly distributed on the surface of T. gondii and involved in regulating MIC2, MIC3, MIC6, and AMA1 during T. gondii invasion of host cells. Bioinformatics have predicted ROM4 B-cell and T-cell epitopes. Immunization strategy is also a key factor in determining the effectiveness of the immune response and has gained increasing attention in T. gondii vaccine research. In this study, we used a DNA prime-peptide boost vaccination regimen to assess the protective efficacy of various vaccination strategies using TgROM4., Methods: We identified a polypeptide (YALLGALIPYCVEYWKSIPR) using a bioinformatics approach, and immunized mice using a DNA-prime and polypeptide-boost regimen. BALB/c mice were randomly divided into six groups, including three experimental groups (peptide, pROM4 and pROM4/peptide) and three control groups (PBS, pEGFP-C1 and pSAG1). Mice were then immunized intramuscularly four times. After immunization, IgG and cytokine productions were determined using enzyme-linked immunosorbent assays. The survival time of mice was evaluated after challenge with tachyzoites of T. gondii RH strain. Additionally, the number of cysts in the brain was determined after intragastric challenge with cysts of T. gondii PRU strain., Results: Mice vaccinated with different immunization regimens (peptide, pROM4 and pROM4/peptide) elicited specific humoral and cellular responses, with high levels of IgG, IgG2a, and interferon (IFN)-γ. Moreover, IgG, IgG2a and IFN-γ levels were highest in the pROM4/peptide group. Immunized mice, especially those in the pROM4/peptide group, had prolonged survival times after challenge with tachyzoites and reduced numbers of brain cysts after infection compared with negative controls., Conclusion: A DNA prime-peptide boost regimen based on ROM4 elicited the highest level of humoral and cellular immune responses among immunization regimens, and may be a promising approach to increase the efficacy of DNA immunization.