I-FABP protein/mRNA and IL-6 as biomarkers of intestinal barrier dysfunction in neonates with necrotizing enterocolitis and SPF BALB/c mouse models.

OBJECTIVE: Neonatal necrotizing enterocolitis (NEC) is a serious intestinal inflammatory disease. We investigated intestinal fatty acid binding protein (I-FABP), I-FABP mRNA, and interleukin-6 (IL-6) as potential diagnostic biomarkers in NEC. METHODS: Forty mice were subjected to hypoxic-ischemic intestinal injury, and then serum I-FABP protein and mRNA levels were quantified. Ileal tissue pathological scores were determined by hematoxylin and eosin staining. I-FABP expression levels and translocation in these tissues were detected using western blotting and immunofluorescence, respectively. Samples from 30 human neonates with NEC and 30 healthy neonates had serum I-FABP protein/mRNA and IL-6 levels measured. RESULTS: The mouse ileal tissue pathological score and I-FABP levels, as well as serum I-FABP and I-FABP mRNA levels, were significantly higher in the model group than in the control group. Serum I-FABP, I-FABP mRNA, and IL-6 levels were significantly higher in human neonates with NEC than in the healthy group. Logistic regression and receiver operating curve analyses revealed that I-FABP protein/mRNA and IL-6 levels could be diagnostic biomarkers for NEC. CONCLUSIONS: I-FABP protein/mRNA and IL-6 levels are useful biomarkers of intestinal ischemic injury in neonates with NEC. The combined detection of I-FABP protein/mRNA and IL-6 is recommended rather than using a single biomarker.

Temporal metabolic profiling of erythrocytes in mice infected with Babesia microti.

BACKGROUND: Babesiosis is an emerging zoonosis worldwide that is caused by tick-borne apicomplexans, Babesia spp., which threatens the health of domesticated and wild mammals and even humans. Although it has done serious harm to animal husbandry and public health, the study of Babesia is still progressing slowly. Until now, no effective anti-Babesia vaccines have been available, and administration of combined drugs tends to produce side effects. Therefore, non-targeted metabolomics was employed in the present study to examine the temporal dynamic changes in the metabolic profile of the infected erythrocytes. The goal was to obtain new insight into pathogenesis of Babesia and to explore vaccine candidates or novel drug targets. METHODS: C57BL/6 mice were infected with B. microti and erythrocytes at different time points (0, 3, 6 , 9, 12, and 22-days post-infection) were subjected to parasitemia surveillance and then metabolomics analysis using liquid chromatography-mass spectrometry (LC-MS). Multivariate statistical analyses were performed to clearly separate and identify dysregulated metabolites in Babesia-infected mice. The analyses included principal components analysis (PCA) and orthogonal partial least squares-discrimination analysis (OPLS-DA). The time-series trends of the impacted molecules were analyzed using the R package Mfuzz and the fuzzy clustering principle. The temporal profiling of amino acids, lipids, and nucleotides in blood cells infected with B. microti were also investigated. RESULTS: B. microti infection resulted in a fast increase of parasitemia and serious alteration of the mouse metabolites. Through LC-MS metabolomics analysis, 10,289 substance peaks were detected and annotated to 3,705 components during the analysis period. There were 1,166 dysregulated metabolites, which were classified into 8 clusters according to the temporal trends. Consistent with the trend of parasitemia, the numbers of differential metabolites reached a peak of 525 at 6-days post-infection (dpi). Moreover, the central carbon metabolism in cancer demonstrated the most serious change during the infection process except for that observed at 6 dpi. Sabotage occurred in components involved in the TCA cycle, amino acids, lipids, and nucleotide metabolism. CONCLUSION: Our findings revealed a great alteration in the metabolites of Babesia-infected mice and shed new light on the pathogenesis of B. microti at the metabolic level. The results might lead to novel information about the mechanisms of pathopoiesis, babesisosis, and anti-parasite drug/vaccine development in the future.

Corrigendum: Anti-Tumoral Effect and Action Mechanism of Exosomes Derived From Toxoplasma gondii-Infected Dendritic Cells in Mice Colorectal Cancer.

[This corrects the article DOI: 10.3389/fonc.2022.870528.].

Disordered Gut Microbiota in Colorectal Tumor-Bearing Mice Altered Serum Metabolome Related to Fufangchangtai.

Purpose: This study aimed to investigate the relationship between gut microbiota (GM) and serum metabolism using antineoplastic Fufangchangtai (FFCT) as the model prescription in the treatment of colorectal cancer (CRC). Methods: Tumor-bearing mice and normal mice were administered different doses of FFCT. The tumor volume of tumor-bearing mice was observed. The levels of CD4+ and CD8+ T cells in the blood, spleen, and tumor of mice were determined using a flow cytometer. The bacterial microbiota in stool samples from mice and the serum metabolomics of FFCT-treated mice and fecal microbiota transplantation mice were detected using 16s RNA sequencing and liquid chromatography-mass spectrometry (LC/MS), respectively. Results: The tumor volume of mice showed no significant decrease after FFCT intervention. The levels of CD4+ and CD8+T lymphocytes showed a significant increase under the intervention of FFCT. GM of colorectal tumor-bearing mice and healthy mice were determined, and the diversity and abundance of Firmicutes, Deferribacteres, Bacteroidetes, and Proteobacteria were significantly different between the two groups. Furthermore, we found that the levels of matrine, isogingerenone B, and armillaripin were significantly decreased in tumor-bearing mice after FFCT intervention, indicating that the tumor-induced dysbiosis of gut bacteria may affect the absorption and metabolism of FFCT. Under the intervention of FFCT, serum metabolism of mice transplanted with feces from CRC patients showed less metabolites related to FFCT than that from healthy people, indicating that GM could be a single factor affecting the metabolism of FFCT. Furthermore, we found that different doses of FFCT-treated mice had higher abundance of Roseburia, Turicibacter, and Flexispira than that in the non-intervention control group. Firmicutes and Bacteroidetes in FFCT-treated groups showed a similar trend compared to the healthy group, indicating that FFCT might correct the intestinal microenvironment by modulating gut microbiota in colorectal tumor-bearing mice. Conclusion: The dysbiosis of GM in tumor-bearing mice reduced the serum metabolites related to FFCT, and FFCT could correct the disordered GM of colorectal tumor-bearing mice to exert efficacy.

Systemic Evaluation of the Effect of Diabetes Mellitus on Breast Cancer in a Mouse Model.

Breast cancer complicated with diabetes mellitus (DM) is a common disease. To evaluate the effect of preexisting DM on breast cancer progression without drug interference, we used a streptozotocin (STZ)-induced type 2 diabetes mellitus BALB/c mouse model. We found that 4T1 breast cancer complicated with DM decreased the mouse survival time compared with 4T1-bearing mice. The diversity of gut microbiome was affected by DM. The infiltration of mucosal-associated invariant T cell (MAIT), CD8+ T cell, and CD4+ T cell in the tumor was significantly decreased in the DM-4T1 group compared with the 4T1 group. The transcriptome data of tumor tissues indicated that the expressions of inflammatory C-C chemokine- and metabolism-related genes were greatly changed. The abnormal expression of these genes may be related with the decreased T-cell infiltration in DM-4T1. In conclusion, the gut microbiome and tumor microenvironment of diabetic breast cancer patients have unique features. The effect of diabetes on breast cancer should be considered in the treatment for diabetic breast cancer patients.

Anti-Tumoral Effect and Action Mechanism of Exosomes Derived From Toxoplasma gondii-Infected Dendritic Cells in Mice Colorectal Cancer.

Toxoplasma gondii is an obligate intracellular protozoan with anti-tumor activity against a variety of cancers. However, the therapeutic effect of T. gondii on colorectal cancer is unclear, and using direct Toxoplasma infection in immunotherapy involves safety concerns. This study investigated the anti-tumoral effect and mechanism of exosomes derived from dendritic cells (DCs) infected with T. gondii (Me49-DC-Exo). We used differential ultracentrifugation to isolate exosomes from uninfected DCs (DC-Exo) and T. gondii Me49-infected DCs (Me49-DC-Exo). The isolated exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Me49-DC-Exo significantly inhibited the tumor growth and reduced the proportion of M2 macrophages in the blood of tumor-bearing mice. In vitro, Me49-DC-Exo suppressed macrophage (RAW264.7) polarization to M2 phenotype. miRNA sequencing revealed that multiple miRNAs in Me49-DC-Exo were differentially expressed compared with DC-Exo, among which miR-182-5p, miR-155-5p, miR-125b-2-3p, and miR-155-3p were up-regulated, while miR-9-5p was significantly down-regulated. Transfecting mimics or inhibitors of these differential miRNAs into RAW264.7 cells showed that miR-155-5p promoted M1 macrophage polarization while inhibiting M2 macrophage polarization. Bioinformatics prediction and dual-luciferase reporter assay confirmed the suppressor of cytokine signaling 1 (SOCS1) as a direct target of miR-155-5p. Silencing SOCS1 gene expression in RAW264.7 cells increased CD86 + CD206 - M1 macrophage proportion, and inducible nitric oxide synthase and tumor necrosis factor-α mRNA levels. However, arginase-1 and transglutaminase 2 expression levels decreased. These results suggest that the exosomes inhibit macrophage polarization to M2 phenotype and regulate SOCS1 expression by delivering functional miR-155-5p. These findings provide new ideas for colorectal cancer immunotherapy.

Recombinant Costimulatory Fusion Proteins as Functional Immunomodulators Enhance Antitumor Activity in Murine B16F10 Melanoma.

Blocking inhibitory signaling and engaging stimulatory signaling have emerged as important therapeutic modalities for cancer immunotherapy. This study aimed to investigate immunomodulatory features of three recombinant costimulatory ligand proteins in a mouse model, which are extracellular domains of OX40-ligand (OX40L), 4-1BB-ligand (4-1BBL), or two domains in tandem, fused with the transmembrane domain of diphtheria toxin (DTT), named DTT-COS1, DTT-COS2, and DTT-COS12, respectively. In vitro study showed that DTT-COS1 and DTT-COS12 had immunological activity increasing the ratio of CD8/CD4 T cells. Treatments with DTT-COS1 and DTT-COS12 dramatically generated immune protection against the B16F10 tumor challenge in both prophylactic and therapeutic efficacy. Furthermore, regarding tumor microenvironment (TME) immunomodulation, DTT-COS1 treatment increased the proportion of CD4+ effector T cells (Teff) and decreased the expression of a suppressive cytokine. Meanwhile, DTT-COS12 reduced regulatory T cells (Treg) and improved the level of stimulatory cytokines. In addition, endogenous antibodies against OX40L/4-1BBL were generated, which may help with antitumor responses. Unexpectedly, DTT-COS2 lacked antitumor effects in vitro and in vivo. Importantly, serum analysis of liver-function associated factors and pro-inflammatory cytokines demonstrated that treatments were safe formulations in mice without signs of systemic toxicity. Remarkably, DTT-COS1 and DTT-COS12 are functional immunomodulators for mouse B16F10 melanoma, creating practical preclinical value in cancer immunotherapy.

Inclusion of PD-L1 into a recombinant profilin antigen enhances immunity against Babesia microti in a murine model.

Pathogens and cancer cells employ the programmed cell death-Ligand 1 (PD-L1)/ programmed cell death-1 (PD-1) signaling pathway to inhibit the immune response. Hence, blockade of PD-L1/PD-1 recognition through monoclonal antibodies enhances the immune response. Antibodies that block PD-L1 and PD-1 binding have been used for the prevention and therapy of human pathogenic diseases, but have not yet been evaluated for the treatment of infectious diseases of livestock. In the present study, a recombinant vaccine named PROF-PDL1E, was designed comprising the Babesia microti-derived vaccine candidate profilin and the host PD-L1 protein, and its effect on immunization against murine B. microti infection was evaluated. PD-L1-specific antibodies generated after vaccination blocked PD-L1 and PD-1 binding as shown by in vitro assays. PROF-PDL1E reduced the burden of B. microti in a mouse model and decreased PD-1 expression in T cells. Furthermore, no tissue damage could be observed after PROF-PDL1E vaccination as verified by hematoxylin and eosin tissue staining of essential organs. In conclusion, vaccines targeting immune checkpoints seem to be a promising strategy for anti-Babesia vaccine development.

The Immunogenicity and Anti-tumor Efficacy of a Rationally Designed Neoantigen Vaccine for B16F10 Mouse Melanoma.

Tumor neoantigens are ideal targets for cancer immunotherapy as they are recognized by host immune system as foreigners and can elicit tumor-specific immune responses. However, existing strategies utilizing RNA or long peptides for the neoantigen vaccines render limited immune responses since only 20-30% of neoantigens predicted in silico to bind MHC I molecules are capable of eliciting immune responses with the majority of responding T cells are CD4+. Therefore, it warrants further exploration to enhance neoantigen-specific CD8+ T cells responses. Since neoantigens are naturally weak antigens, we asked whether foreign T help epitopes could enhance their immunogenicity. In present study, we chose 4 weak B16F10 neoantigens as vaccine targets, and fused them to the transmembrane domain of diphtheria toxin, namely DTT-neoAg. Strikingly, the vaccine elicited anti-tumor CD8+ T cells responses and enhanced tumor infiltration of both T cells and NK cells. Impressively, DTT-neoAg vaccine significantly deterred tumor growth with the inhibition rate reached 88% in the preventive model and 100% in the therapeutic model at low dose of tumor challenge. Furthermore, after second challenge with higher dose of tumor cells, 33.3% of the immunized mice remained tumor-free for 6 months in the therapeutic model. Because DTT is a non-toxic domain of diphtheria toxin, it may be not of great concern in terms of safety as a Th epitope provider. Thus, the fusion strategy employed by this study may become a feasible and powerful approach for development of personalized cancer vaccines.

A PD-L1-Based Cancer Vaccine Elicits Antitumor Immunity in a Mouse Melanoma Model.

Engagement of programmed death 1 receptor (PD-1) and its ligand PD-L1/2 induces a signal transduction pathway that inhibits the activity of tumor-infiltrating cytotoxic T lymphocytes and promotes tumor growth and metastasis. Antibodies blocking PD-1 or PD-L1 can restore antitumor T cell responses and cause long-term remission in a subset of cancer patients with advanced or refractory tumors. In this study, we asked whether PD-L1 vaccination could confer tumor control in mouse tumor models. To address the central tolerance toward self-molecules, we fused the extracellular domain of PD-L1 (PD-L1E) to the C-terminal of the translocation domain of diphtheria toxin (DTT). DTT is able to elicit CD4+ T cell responses required for inducing robust immune responses against self-molecules. The fusion molecule is called DPDL1E. When formulated with incomplete Freund's adjuvant (IFA), DPDL1E elicited robust immune responses biased toward the Th1 type and inhibited tumor growth in both preventive and therapeutic mouse tumor models. We further showed that the anti-DPDL1E sera blocked PD-L1 binding to PD-1 in vitro. The DPDL1E vaccination increased the levels of tumor-infiltrating T lymphocytes (TILs) and reduced the levels of myeloid-derived suppressor cells (MDSCs) as well as exhausted LAG3+PD-1+ CD8+ T cells. All of these data suggest that DPDL1E vaccination reverses the suppressive phenotype of the tumor microenvironment and that it is a promising strategy for cancer therapy.

Immunomodulatory effects of Rhipicephalus haemaphysaloides serpin RHS2 on host immune responses.

BACKGROUND: Rhipicephalus haemaphysaloides is a widespread tick species in China and other South East Asian countries, where it is the vector of many pathogens. The objective of this study was to study the role of serpin (serine protease inhibitor) during the tick-host interaction. METHODS: The differentiation of bone marrow-derived dendritic cells (BMDC) was induced in vitro, and the effect of RHS2 on the maturation of DCs was evaluated. The effects of RHS2 on T cell activation and cytotoxic T lymphocytes' (CTLs) activity were analyzed by flow cytometry. Antibody subtypes after immunization of mice with RHS2 and OVA were determined. RESULTS: RHS2 can inhibit the differentiation of bone marrow-derived cells into DCs and promote their differentiation into macrophages. RHS2 can inhibit the maturation of DCs and the expression of CD80, CD86 and MHCII. The number of CD3+CD4+ and CD3+CD8+ T cells secreting IFN-γ, IL-2 and TNF-α was decreased, and the number of CD3+CD4+ T cells secreting IL-4 was increased, indicating that RHS2 can inhibit the activation of CD4 T cells and CD8 T cells, leading to inhibition of Th1 immune response. RHS2 inhibits the elimination of target cells by cytotoxic T lymphocytes. After immunization of mice with RHS2 and OVA, serum IgG2b was significantly reduced and IgM was increased. CONCLUSIONS: The results show that RHS2 has an inhibitory effect on the host immune response. Ticks have evolved various ways to circumvent adaptive immunity. Their serpin inhibits BMDC differentiation to reduce immune responses.

Immunosuppressive effects of tick protein RHcyst-1 on murine bone marrow-derived dendritic cells.

BACKGROUND: Ticks, as blood-feeding arthropod vectors, have evolved their own unique mechanism to suppress host immune responses and evade immune defenses in order to complete blood-feeding. The immunoregulatory effect of tick bioactive molecules on hosts has been widely reported, and the cystatin family has been identified as one of the major immunomodulators. In previous studies, we obtained a novel tick salivary bioactive protein named RHcyst-1, which belongs to the type 1 cystatin family. Here, we demonstrated the effects of RHcyst-1 on the host immune response mainly on dendritic cell (DC) function. Understanding the function of tick-derived bioactive molecule may help to clarify the mechanisms of how ticks escape the host immune response and help to control ticks and tick-borne disease transmission. METHODS: Bone marrow-derived DCs (BMDCs) were generated and induced by GM-CSF and IL-4 with or without RHcyst-1 addition. Flow cytometry was used to analyze the differentiation and maturation of BMDCs and T cell cytokine production. Quantitative real-time PCR (qRT-PCR) and western blot were used to measure changes in expression within STAT and p38 MAPK signaling pathways. RESULTS: Flow cytometry analysis revealed that RHcyst-1 inhibited the differentiation of BMDCs, but had no effect on the maturation of BMDCs. T cells co-cultured with DCs treated with RHcyst-1 produced significantly less TNF-α, IFN-γ and IL-2 than the control group. Further analysis showed that the mRNA level and phosphorylation of p38, ERK and STAT were significantly changed after RHcyst-1 added to bone marrow monocytes during the differentiation stage. CONCLUSIONS: Our results suggest that RHcyst-1 is one of the major immunosuppressive proteins of BMDC function from blood-feeding ticks.

A Tick Cysteine Protease Inhibitor RHcyst-1 Exhibits Antitumor Potential.

BACKGROUND/AIMS: We previously identified a potent and tight-binding inhibitor of cysteine proteases from Rhipicephalus haemaphysaloides, RHcyst-1, which belongs to the cystatin type 1 family. Cathepsins, which are members of the cysteine protease family, participate in various pathological processes, including the initiation and development of cancers. The present study aimed to investigate the antitumor effects of RHcyst-1 and to explore the underlying mechanism of these effects. METHODS: Different tumor cells were treated with RHcyst-1 in vitro. Proliferation activity was evaluated using Cell Counting Kit-8, and migration and invasion were determined by wound healing and Transwell® invasion assays. In addition, a mouse tumor therapy model was established by inoculating the left forelimb of mice with B16-F10 cells, and tumor progression was evaluated by assessing tumor volume and survival. Flow cytometry was conducted to evaluate myeloid-derived suppressor cells (MDSCs), CD4+, and CD8+ T cell levels in PBMCs and spleens. Immunohistochemistry was performed to analyze immune cell infiltration and angiogenesis in the tumors. RESULTS: RHcyst-1 significantly inhibited the proliferation, migration, and invasion of all four different tumor cells in vitro. Additionally, it inhibited tumor growth and improved survival in vivo. A decrease and an increase in MDSCs levels were observed in PBMCs and in the spleen, respectively, after RHcyst-1 application. CONCLUSIONS: Tick RHcyst-1 has potential antitumor efficacy, and the observed antitumor activities may be partly attributable to changes in cathepsin expression and MDSCs levels in the PBMCs and spleens. The findings of the present study suggest that RHcyst-1 may have the potential to be utilized in cancer treatment.

Immunogenicity and efficacy of a rationally designed vaccine against vascular endothelial growth factor in mouse solid tumor models.

Vascular endothelial growth factor (VEGF) plays an important role in the progression of various cancers. The VEGF-specific antibody bevacizumab combined with chemotherapy was shown to significantly improve progression-free survival in certain cancers. However, repeated administration is necessary for effective suppression of VEGF, thereby making the therapy expensive and cumbersome. Thus, it is urgent to develop alternative reagents such as VEGF vaccines. Here we report that DTT-VEGF, a VEGF-based antigen consisting of the receptor-binding domain of VEGF and diphtheria toxin T domain (DTT), not only stimulated neutralizing antibody response, but also induced type 1 immune response as well as anti-tumor cytotoxic T lymphocytes in mice when administered with aluminum hydroxide adjuvant. The antibodies triggered by DTT-VEGF immunization inhibited the binding of VEGF to VEGF receptor and downregulated the serum VEGF levels in tumor-bearing mice. VEGF-specific IgG2a and IgG2b antibodies as well as type 1 cytokines were stimulated by DTT-VEGF vaccination. The splenocytes from DTT-VEGF-immunized mice showed cytotoxic activity against B16-F10 cells expressing VEGF. Extensive necrosis with severe hemorrhage and enhanced CD8+ T cell infiltration were observed in tumors from DTT-VEGF-immunized mice. The percentages of CD31+ vascular areas in the tumor sections from DTT-VEGF-immunized mice were significantly lower than those of control mice. DTT-VEGF significantly inhibited tumor growth in preventive and therapeutic vaccination settings in mouse models. Our data suggest that DTT is an effective antigen carrier to break immune self-tolerance and our vaccine design has potential to be used for human cancer therapy.

Cytotoxic T cell responses are enhanced by antigen design involving the presentation of MUC1 peptide on cholera toxin B subunit.

Induction of cytotoxic T lymphocytes (CTL) is critical to cancer vaccine based immunotherapy. Efforts to elicit CTLs against tumor MUC1 with peptide based vaccine have not been successful in clinical application. We have design a MUC1 vaccine by replacing B cell epitope of CTB with MUC1 VNTR peptide. Immunization with hybrid CTB-MUC1 plus aluminum hydroxide and CpG adujuvant (CTB-MUC1-Alum-CpG) induce MUC1-specific CTLs in mice. Moreover, this vaccination can prevent tumor growth and reduce tumor burden in MUC1+B16 mice model. Meanwhile, CTB-MUC1-Alum-CpG vaccination can promote Th1 cells and CD8+ T cells inflate to tumor tissue. Our approach might be applicable to other cancer vaccine design.

[Expression of T-bet in nasopharyngeal associated lymphatic tissue of patients with allergic rhinitis].

OBJECTIVE: To investigate the pattern and value of the expression of T-bet in nasopharyngeal-associated lymphatic tissue (NALT) of patients with or without allergic rhinitis (AR). METHOD: T-bet and CD4/T-bet expression in adenoids, tonsils and nasal mucosa was respectively detected with single stain and double stain of immunohistochemistry. RESULT: The positive ratio of T-bet expression in tonsils, adenoids and nasal mucosa between AR group and the control group had statistically different (P < 0.05). There was statistical difference of T-bet expression in tonsils from AR group in three different age groups (P < 0.05). The difference was that T-bet expression in adults group was significantly lower than that in children group and adolescent group. There was no difference of T-bet expression among different age groups in non-AR group. There were some positive expression of CD4 and T-bet in tonsils, adenoids and nasal mucosa from two groups and most positive expression of T-bet on CD4 positive cell. CONCLUSION: T-bet expression was down-regulated in NALT of patients with AR. T-bet expression of NALT is associated with allergic mucosal inflammation and functional status of NALT, as well as the weak Th1 response at the level of transfer factor in local mucosa of respiratory tract in AR patients.