Cellular evidence and spatial distribution of endosomal biosynthesis and autophagy in intestinal immune barrier cells of crucian carp (Carassiuscarassius).

Crucian carp (Carassius carassius) is an important aquatic economic animal, and the immune barrier function of its intestine has been a focus of research into oral vaccines and drugs. However, the histological structures of the intestinal barrier and its adjacent areas have not been clearly established, and little subcellular evidence is available to elucidate the spatial distribution of intracellular biological processes. In this study, the spatial distribution of autophagy and endosome formation in the intestinal epithelial cells (IECs) of crucian carp were analyzed. These two biological activities are closely related to intestinal homeostasis, immunity, and cell communication. Periodic acid-Schiff (PAS) and Masson's trichrome staining were employed to elucidate the distinctive histological framework of the Crucian carp's myoid cell network, which resides within the subepithelial layer and is characterized by gap junctions. Transmission electron microscopy (TEM), immunohistochemistry (IHC), and immunofluorescence (IF) were used to detect the structural and functional aspects of the IEC in different intestinal segments. TEM and immunohistochemical analyses captured the biogenesis and maturation of early and late endosomes as well as multivesicular bodies (MVBs), as well as the initiation and progression of autophagy, including macroautophagy and mitophagy. The endosome and MVBs-specific marker CD63 and autophagy-related protein LC3 were highly expressed in IECs and were correlated with autophagy and endosome biosynthesis in the apical and basal regions of individual cells, and differed between different intestinal segments. In summary, this study elucidated the ubiquity and morphological characteristics of autophagy and endosome formation across different intestinal segments of crucian carp. A unique myoid cell network beneath the intestinal epithelium in crucian carp was also identified, expanding the histological understanding of this animal's intestinal tract.

Chronic exposure to polystyrene nanoplastics induces intestinal mechanical and immune barrier dysfunction in mice.

Micro(nano)plastics are prevalent in the environment, and prolonged exposure to them represents a threat to human health. The goal of this study is to assess the health risk of long-term exposure to nanoplastics (NPs) at environmental concentrations on the intestinal mechanical and immune barrier in mice. In this study, mice were provided drinking water containing polystyrene NPs (PS-NPs; 0.1, 1, and 10 mg·L-1) for 32 consecutive weeks. The levels of endocytosis proteins caveolin and clathrin and of tight junctional proteins claudin-1, occludin, and ZO-1, and morphological changes, proportion of lymphocytes B in MLNs and lymphocytes T in IELs and LPLs were determined by immunohistochemistry, hematoxylin-eosin, and flow cytometry assays in the intestinal tissues of mice at 28 weeks. The activities or concentrations of ROS, SOD, MDA, and GSH-Px and inflammatory factors (IL-1ß, IL-6, and TNF-α) in the intestinal tissues of mice were measured by ELISA at 12, 16, 20, 24, and 32 weeks. Compared with the control group, oral ingested PS-NPs entered the intestinal tissues of mice and upregulated expression levels of the clathrin and caveolin. The intestinal tissue structure of mice in the PS-NPs (1 and 10 mg·L-1) exposure groups showed significant abnormalities, such as villus erosion, decreased of crypts numbers and large infiltration of inflammatory cells. Exposure to 0.1 mg·L-1 PS-NPs decreased occludin protein levels, but not claudin-1 and ZO-1 levels. The levels of these three tight junction proteins decreased significantly in the 1 and 10 mg·L-1 PS-NPs exposed groups. Exposure to PS-NPs led to a significant time- and dose-dependent increase in ROS and MDA levels, and concurrently decreased GSH-Px and SOD contents. Exposure to PS-NPs increased the proportion of B cells in MLNs, and decreased the proportion of CD8+ T cells in IELs and LPLs. The levels of pro-inflammatory cytokines IL-6, TNF-α and IL-1ß were markedly elevated after PS-NPs exposure. Long-term PS-NPs exposure impaired intestinal mechanical and immune barrier, and indicate a potentially significant threat to human health.

Identification of a tumour immune barrier in the HCC microenvironment that determines the efficacy of immunotherapy.

BACKGROUND & AIMS: The tumour microenvironment (TME) is a crucial mediator of cancer progression and therapeutic outcome. The TME subtype correlates with patient response to immunotherapy in multiple cancers. Most previous studies have focused on the role of different cellular components in the TME associated with immunotherapy efficacy. However, the specific structure of the TME and its role in immunotherapy efficacy remain largely unknown. METHODS: We combined spatial transcriptomics with single-cell RNA-sequencing and multiplexed immunofluorescence to identify the specific spatial structures in the TME that determine the efficacy of immunotherapy in patients with hepatocellular carcinoma (HCC) receiving anti-PD-1 treatment. RESULTS: We identified a tumour immune barrier (TIB) structure, a spatial niche composed of SPP1+ macrophages and cancer-associated fibroblasts (CAFs) located near the tumour boundary, which is associated with the efficacy of immune checkpoint blockade. Furthermore, we dissected ligand‒receptor networks among malignant cells, SPP1+ macrophages, and CAFs; that is, the hypoxic microenvironment promotes SPP1 expression, and SPP1+ macrophages interact with CAFs to stimulate extracellular matrix remodelling and promote TIB structure formation, thereby limiting immune infiltration in the tumour core. Preclinically, the blockade of SPP1 or macrophage-specific deletion of Spp1 in mice led to enhanced efficacy of anti-PD-1 treatment in mouse liver cancer, accompanied by reduced CAF infiltration and increased cytotoxic T-cell infiltration. CONCLUSIONS: We identified that the TIB structure formed by the interaction of SPP1+ macrophages and CAFs is related to immunotherapy efficacy. Therefore, disruption of the TIB structure by blocking SPP1 may be considered a relevant therapeutic approach to enhance the therapeutic effect of immune checkpoint blockade in HCC. IMPACT AND IMPLICATIONS: Only a limited number of patients with hepatocellular carcinoma (HCC) benefit from tumour immunotherapy, which significantly hinders its application. Herein, we used multiomics to identify the spatial structure of the tumour immune barrier (TIB), which is formed by the interaction of SPP1+ macrophages and cancer-associated fibroblasts in the HCC microenvironment. This structure constrains immunotherapy efficacy by limiting immune cell infiltration into malignant regions. Preclinically, we revealed that blocking SPP1 or macrophage-specific deletion of Spp1 in mice could destroy the TIB structure and sensitize HCC cells to immunotherapy. These results provide the first key steps towards finding more effective therapies for HCC and have implications for physicians, scientists, and drug developers in the field of HCC.

FoxP3 expression by retinal pigment epithelial cells: transcription factor with potential relevance for the pathology of age-related macular degeneration.

BACKGROUND: Forkhead-Box-Protein P3 (FoxP3) is a transcription factor and marker of regulatory T cells, converting naive T cells into Tregs that can downregulate the effector function of other T cells. We previously detected the expression of FoxP3 in retinal pigment epithelial (RPE) cells, forming the outer blood-retina barrier of the immune privileged eye. METHODS: We investigated the expression, subcellular localization, and phosphorylation of FoxP3 in RPE cells in vivo and in vitro after treatment with various stressors including age, retinal laser burn, autoimmune inflammation, exposure to cigarette smoke, in addition of IL-1ß and mechanical cell monolayer destruction. Eye tissue from humans, mouse models of retinal degeneration and rats, and ARPE-19, a human RPE cell line for in vitro experiments, underwent immunohistochemical, immunofluorescence staining, and PCR or immunoblot analysis to determine the intracellular localization and phosphorylation of FoxP3. Cytokine expression of stressed cultured RPE cells was investigated by multiplex bead analysis. Depletion of the FoxP3 gene was performed with CRISPR/Cas9 editing. RESULTS: RPE in vivo displayed increased nuclear FoxP3-expression with increases in age and inflammation, long-term exposure of mice to cigarette smoke, or after laser burn injury. The human RPE cell line ARPE-19 constitutively expressed nuclear FoxP3 under non-confluent culture conditions, representing a regulatory phenotype under chronic stress. Confluently grown cells expressed cytosolic FoxP3 that was translocated to the nucleus after treatment with IL-1ß to imitate activated macrophages or after mechanical destruction of the monolayer. Moreover, with depletion of FoxP3, but not of a control gene, by CRISPR/Cas9 gene editing decreased stress resistance of RPE cells. CONCLUSION: Our data suggest that FoxP3 is upregulated by age and under cellular stress and might be important for RPE function.

Lactoferrin Alleviates Lipopolysaccharide-Induced Infantile Intestinal Immune Barrier Damage by Regulating an ELAVL1-Related Signaling Pathway.

As the most important intestinal mucosal barrier of the main body, the innate immune barrier in intestinal tract plays especially pivotal roles in the overall health conditions of infants and young children; therefore, how to strengthen the innate immune barrier is pivotal. A variety of bioactivities of lactoferrin (LF) has been widely proved, including alleviating enteritis and inhibiting colon cancer; however, the effects of LF on intestinal immune barrier in infants and young children are still unclear, and the specific mechanism on how LF inhibits infantile enteritis by regulating immune signaling pathways is unrevealed. In the present study, we firstly performed pharmacokinetic analyses of LF in mice intestinal tissues, stomach tissues and blood, through different administration methods, to confirm the metabolic method of LF in mammals. Then we constructed in Vitro and in Vivo infantile intestinal immune barrier damage models utilizing lipopolysaccharide (LPS), and evaluated the effects of LF in alleviating LPS-induced intestinal immune barrier damage. Next, the related immune molecular mechanism on how LF exerted protective effects was investigated, through RNA-seq analyses of the mouse primary intestinal epithelial cells, and the specific genes were analyzed and screened out. Finally, the genes and their related immune pathway were validated in mRNA and protein levels; the portions of special immune cells (CD4+ T cells and CD8+ T cells) were also detected to further support our experimental results. Pharmacokinetic analyses demonstrated that the integrity of LF could reach mice stomach and intestine after oral gavage within 12 h, and the proper administration of LF should be the oral route. LF was proven to down-regulate the expression levels of inflammatory cytokines in both the primary intestinal epithelial cells and mice blood, especially LF without iron (Apo-LF), indicating LF alleviated infantile intestinal immune barrier damage induced by LPS. And through RNA-seq analyses of the mouse primary intestinal epithelial cells treated with LPS and LF, embryonic lethal abnormal vision Drosophila 1 (ELAVL1) was selected as one of the key genes, then the ELAVL1/PI3K/NF-κB pathway regulated by LF was verified to participate in the protection of infantile intestinal immune barrier damage in our study. Additionally, the ratio of blood CD4+/CD8+ T cells was significantly higher in the LF-treated mice than in the control mice, indicating that LF distinctly reinforced the overall immunity of infantile mice, further validating the strengthening bioactivity of LF on infantile intestinal immune barrier. In summary, LF was proven to alleviate LPS-induced intestinal immune barrier damage in young mice through regulating ELAVL1-related immune signaling pathways, which would expand current knowledge of the functions of bioactive proteins in foods within different research layers, as well as benefit preclinical and clinical researches in a long run.

Delivery of nanovaccine towards lymphoid organs: recent strategies in enhancing cancer immunotherapy.

With the in-depth exploration on cancer therapeutic nanovaccines, increasing evidence shows that the poor delivery of nanovaccines to lymphoid organs has become the culprit limiting the rapid induction of anti-tumor immune response. Unlike the conventional prophylactic vaccines that mainly form a depot at the injection site to gradually trigger durable immune response, the rapid proliferation of tumors requires an efficient delivery of nanovaccines to lymphoid organs for rapid induction of anti-tumor immunity. Optimization of the physicochemical properties of nanovaccine (e.g., size, shape, charge, colloidal stability and surface ligands) is an effective strategy to enhance their accumulation in lymphoid organs, and nanovaccines with dynamic structures are also designed for precise targeted delivery of lymphoid organs or their subregions. The recent progress of these nanovaccine delivery strategies is highlighted in this review, and the challenges and future direction are also discussed.

The liver as an immunological barrier redefined by single-cell analysis.

The liver is a front-line immune tissue that plays a major role in the detection, capture and clearance of pathogens and foreign antigens entering the bloodstream, especially from the gut. Our largest internal organ maintains this immune barrier in the face of constant exposure to external but harmless antigens through a highly specialized network of liver-adapted immune cells. Mapping the immune resident compartment in the liver has been challenging because it requires multimodal single-cell deep phenotyping approaches of often rare cell populations in difficult to access samples. We can now measure the RNA transcripts present in a single cell (scRNA-seq), which is revolutionizing the way we characterize cell types. scRNA-seq has been applied to the diverse array of immune cells present in murine and human livers in health and disease. Here, we summarize how emerging single-cell technologies have advanced or redefined our understanding of the immunological barrier provided by the liver.

Tryptophan (Trp) modulates gut homeostasis via aryl hydrocarbon receptor (AhR).

The intestinal homeostasis is an orchestrated dynamic equilibrium state composed of the coexistence and interactions among the nutrients, microbial flora, and immune system. The intestinal balance disorder can trigger a series of diseases, such as inflammatory bowel disease (IBD). Many of tryptophan (Trp) metabolites, such as kynurenine and indole, generated under a series of endogenous enzymes or microbial metabolism, have been reported enable to bind and activate the aryl hydrocarbon receptor (AhR), this series of process is termed the Trp-AhR pathway. The activated Trp-AhR pathway can induce the expression of downstream cytokines such as interleukin-22 (IL-22) and interleukin-17 (IL-17), thereby regulating the intestinal homeostasis. This review highlights the advance of Trp-AhR pathway in the regulation of intestinal homeostasis and provides some insights for the clinical strategies that expect to effectively prevent and treat gut diseases via intervening the Trp-AhR pathway.

Favorable outcome of experimental islet xenotransplantation without immunosuppression in a nonhuman primate model of diabetes.

Transplantation of pancreatic islets for treating type 1 diabetes is restricted to patients with critical metabolic lability resulting from the need for immunosuppression and the shortage of donor organs. To overcome these barriers, we developed a strategy to macroencapsulate islets from different sources that allow their survival and function without immunosuppression. Here we report successful and safe transplantation of porcine islets with a bioartificial pancreas device in diabetic primates without any immune suppression. This strategy should lead to pioneering clinical trials with xenotransplantation for treatment of diabetes and, thereby, represents a previously unidentified approach to efficient cell replacement for a broad spectrum of endocrine disorders and other organ dysfunctions.

Impaired intestinal immune barrier and physical barrier function by phosphorus deficiency: Regulation of TOR, NF-κB, MLCK, JNK and Nrf2 signalling in grass carp (Ctenopharyngodon idella) after infection with Aeromonas hydrophila.

In aquaculture, the occurrence of enteritis has increased and dietary nutrition is considered as one of the major strategies to solve this problem. In the present study, we assume that dietary phosphorus might enhance intestinal immune barrier and physical barrier function to reduce the occurrence of enteritis in fish. To test this assumption, a total of 540 grass carp (Ctenopharyngodon idella) were investigated by feeding graded levels of available phosphorus (0.95-8.75 g/kg diet) and then infection with Aeromonas hydrophila. The results firstly showed that phosphorus deficiency decreased the ability to combat enteritis, which might be related to the impairment of intestinal immune barrier and physical barrier function. Compared with optimal phosphorus level, phosphorus deficiency decreased fish intestinal antimicrobial substances activities or contents and down-regulated antimicrobial peptides mRNA levels leading to the impairment of intestinal immune response. Phosphorus deficiency down-regulated fish intestinal anti-inflammatory cytokines mRNA levels and up-regulated the mRNA levels of pro-inflammatory cytokines [except IL-1ß and IL-12p35 in distal intestine (DI) and IL-12p40] causing aggravated of intestinal inflammatory responses, which might be related to the signalling molecules target of rapamycin and nuclear factor kappa B. In addition, phosphorus deficiency disturbed fish intestinal tight junction function and induced cell apoptosis as well as oxidative damage leading to impaired of fish intestinal physical barrier function, which might be partially associated with the signalling molecules myosin light chain kinase, c-Jun N-terminal protein kinase and NF-E2-related factor 2, respectively. Finally, based on the ability to combat enteritis, dietary available phosphorus requirement for grass carp (254.56-898.23 g) was estimated to be 4.68 g/kg diet.

Dietary threonine deficiency depressed the disease resistance, immune and physical barriers in the gills of juvenile grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare.

This study was conducted to investigate the effects of dietary threonine on the disease resistance, gill immune and physical barriers function of juvenile grass carp (Ctenopharyngodon idella). A total of 1080 juveniles were fed six iso-nitrogenous diets containing graded levels of threonine (3.99-21.66 g kg-1 diet) for 8 weeks, and then challenged with Flavobacterium columnare. Results showed that threonine deficiency (3.99 g kg-1 diet): (1) increased the gill rot morbidity after exposure to F. columnare; (2) attenuated the gill immune barrier function by decreasing antimicrobial substances production, up-regulating the mRNA levels of pro-inflammatory cytokines (except IL-12p40), and down-regulating the anti-inflammatory cytokines partly due to the modulation of NF-κB and TOR signaling. (3) disrupt the gill tight junction complexes by down-regulating TJs (claudin-3, -b, -c, 12, occludin, ZO-1 and ZO-2) and up-regulating TJs (claudin-7a, -7b) as well as related signaling molecule myosin light chain kinase mRNA levels (P < 0.05). (4) exacerbated the gill apoptosis by up-regulating cysteinyl aspartic acid-protease-3, 8, 9, c-Jun N-terminal kinases and mediating apoptosis related factors mRNA levels (P < 0.05); (5) exacerbated oxidative injury with increased reactive oxygen species, malondialdehyde and protein carbonyl contents (P < 0.05), decreased the antioxidant related enzymes activities and corresponding mRNA levels (except glutathione peroxidase-1b and glutathione-S-transferase-omega 2) as well as glutathione contents (P < 0.05) partly ascribe to the abridgement of NF-E2-related factor 2 signaling [Nrf2/Keap1a (not Keap1b)] in fish gill. Overall, threonine deficiency depressed the disease resistance, and impaired immune and physical barriers in fish gill. Finally, based on the gill rot morbidity and biochemical indices (immune indices LA activity and antioxidant indices MDA content), threonine requirements for juvenile grass carp (9.53-53.43 g) were estimated to be 15.32 g kg-1 diet (4.73 g 100 g-1 protein), 15.52 g kg-1 diet (4.79 g 100 g-1 protein), 15.46 g kg-1 diet (4.77 g 100 g-1 protein), respectively.

Dietary zinc deficiency reduced growth performance, intestinal immune and physical barrier functions related to NF-κB, TOR, Nrf2, JNK and MLCK signaling pathway of young grass carp (Ctenopharyngodon idella).

Our study investigated the effects of dietary zinc (Zn) deficiency on growth performance, intestinal immune and physical barrier functions of young grass carp (Ctenopharyngodon idella). A total of 630 grass carp (244.14 ± 0.40 g) were fed graded levels of zinc lactate (10.71, 30.21, 49.84, 72.31, 92.56, 110.78 mg Zn/kg diet) and one zinc sulfate group (56.9 mg Zn/kg diet) for 60 days. At the end of the feeding trial, fish were challenged with Aeromonas hydrophila for 14 days. These results indicated that compared with optimal dietary Zn level, dietary Zn deficiency (10.71 mg/kg diet) decreased the production of antibacterial compounds, up-regulated pro-inflammatory cytokines related to nuclear factor kappa B (NF-κB) and down-regulated anti-inflammatory cytokines related to target of rapamycin (TOR) in three intestinal segments of young grass carp (P < 0.05), suggesting that dietary Zn deficiency could impair intestinal immune barrier of fish; decreased the activities and mRNA levels of antioxidant enzymes related to NF-E2-related factor 2 (Nrf2), up-regulated the mRNA levels of caspase-3, -7, -8, -9 related to p38 mitogen activated protein (p38 MAPK) and c-Jun N-terminal protein kinase (JNK), down-regulated the mRNA levels of tight junction complexes (TJs) related to myosin light chain kinase (MLCK) in three intestinal segments of young grass carp (P < 0.05), demonstrating that dietary Zn deficiency could injury intestinal physical barrier of fish. Besides, the Zn requirements (zinc lactate as Zn source) based on percent weight gain (PWG), against enteritis morbidity, acid phosphatase (ACP) activity in the proximal intestine (PI) and malondialdehyde (MDA) content in the PI of young grass carp was estimated to be 61.2, 61.4, 69.2 and 69.5 mg/kg diet, respectively. Finally, based on specific growth rate (SGR), feed efficiency (FE) and against enteritis morbidity of young grass carp, the efficacy of zinc lactate relative to zinc sulfate were 132.59%, 135.27% and 154.04%, respectively.

Decreased enteritis resistance ability by dietary low or excess levels of lipids through impairing the intestinal physical and immune barriers function of young grass carp (Ctenopharyngodon idella).

The current study explores the hypothesis that low or excess levels of lipids decrease the enteritis resistance ability through impairing the intestinal physical and immune barrier function of young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp with an average initial weight of 261.41 ± 0.53 g were fed diets containing six graded levels of lipids at 5.9, 21.4, 36.0, 50.2, 66.6 and 80.1 g/kg for 8 weeks. After that, a challenge trial was conducted by injection of Aeromonas hydrophila over 2 weeks. The results show that compared with optimal lipids level, low or excess levels of lipids impair fish immune barrier function through declining humoral compounds and down-regulating the mRNA levels of interleukin 4/13A (IL-4(13)A) [not in the proximal intestine (PI)], IL-4(13)B, IL-6, IL-10, transforming growth factor ß1 (TGF-ß1), inhibitor of κBα (IκBα), TOR and ribosomal p70S6 kinase (S6K1) (P < 0.05), and up-regulating tumor necrosis factor α (TNF-α), interferon γ2 (IFN-γ2), IL-1ß, IL-8, IL-12 p40 (not p35), nuclear factor κB p65 (NF-κB p65), IκB kinase α (IKKα), IKKß, IKKγ, and eIF4E-binding protein (4EBP) in the intestine of young grass carp (P < 0.05). In addition, low or excess levels of lipids also decrease young grass carp physical barrier function through down-regulating the mRNA levels of zonula occludens (ZO-1), ZO-2b [only in the distal intestine (DI)], Claudin b, c, 3, 12, 15a, 15b (only in the DI), 7b (not 7a) and Occludin by MAPKK 6/p38 MAPK (not JNK)/MLCK signaling molecules, down-regulating B-cell lymphoma-2 (Bcl-2) and inhibitor of apoptosis protein (IAP) and up-regulating the mRNA levels of apoptotic protease activating factor-1 (Apaf-1), Caspase-3, -8 and -9 and Fas ligand (FasL) (not in the DI), and decreasing antioxidant ability via Kelch-like ECH-associating protein 1 (Keap1)/NF-E2-related factor 2 (Nrf2) signaling molecules in the intestine. Based on the quadratic regression analysis for the enteritis resistance ability, LA activities and GSH content in the MI were established to be 54.5, 49.91 and 47.83 g lipid/kg diets, respectively.

Immunity decreases, antioxidant system damages and tight junction changes in the intestine of grass carp (Ctenopharyngodon idella) during folic acid deficiency: Regulation of NF-κB, Nrf2 and MLCK mRNA levels.

This investigation used the same growth trial as the previous study, which showed that folic acid deficiency retarded growth in young grass carp (the percent weight gain of Groups 1-6 were 102.32 ± 3.41%, 137.25 ± 10.48%, 179.78 ± 3.95%, 164.33 ± 3.21%, 143.35 ± 8.12% and 115.28 ± 2.66%) [1]. In the present study, we investigated the effects of dietary folic acid on the immune response, antioxidant status and tight junctions in the intestine of young grass carp (Ctenopharyngodon idella). A total of 540 young grass carp were fed diets containing graded levels of folic acid at 0.10, 0.47, 1.03, 1.48, 1.88 and 3.12 mg kg(-1) diet for 8 weeks. The results indicated that acid phosphatase and lysozyme activities, and the complement component 3 content in the proximal intestine (PI), mid intestine (MI) and distal intestine (DI) were decreased with folic acid deficiency (0.1 mg kg(-1)) (P < 0.05). Folic acid deficiency (0.1 mg kg(-1)) up-regulated interleukin 1ß, interleukin 8, tumor necrosis factor α, nuclear factor κB p65 (NF-κB p65), IκB kinase α (IKK-α), IKK-ß and IKK-γ gene expression, meanwhile down-regulated interleukin 10, transforming growth factor ß, IκB and target of rapamycin gene expression in the PI, MI and DI (P < 0.05). These data suggested that folic acid deficiency decreased fish intestinal innate immune function may be partly contributed to the regulation of NF-κB p65 pathway. Moreover, the activities and corresponding gene expression of glutathione content, Cu/Zn superoxide dismutase, catalase, glutathione peroxidase, glutathione s-transferases and glutathione reductase in fish intestine were depressed by deficient folic acid diet (0.1 mg kg(-1)) (P < 0.05). Furthermore, folic acid deficiency (0.1 mg kg(-1)) down-regulated NF-E2-related factor 2 (Nrf2) gene expression, up-regulated Kelch-like-ECH-associated protein 1a (Keap1a) and Keap1b gene expression in fish intestine (P < 0.05). These data indicated that deficient folic acid diet damaged fish intestinal antioxidant capacity partly by regulating Nrf2/Keap1 pathway. Additionally, folic acid deficiency (0.1 mg kg(-1)) down-regulated claudin-b, claudin-c, claudin-3, occludin and zonula occludens 1 gene expression; whereas folic acid deficiency (0.1 mg kg(-1)) up-regulated claudin-12, claudin-15, myosin light chain kinase (MLCK) and p38 mitogen activated protein kinase (p38 MAPK) gene expression in the PI, MI and DI (P < 0.05), suggesting that folic acid deficiency may damage fish intestinal tight junctions associated with the mediation of MLCK and p38 MAPK gene expression. In conclusion, folic acid deficiency (0.1 mg kg(-1)) impaired fish intestinal immunity, antioxidant capacity and tight junctions.

Dietary vitamin C deficiency depressed the gill physical barriers and immune barriers referring to Nrf2, apoptosis, MLCK, NF-κB and TOR signaling in grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare.

This study explored the effects of vitamin C on the physical barriers and immune barriers, and relative mRNA levels of signaling molecules in the gill of grass carp (Ctenopharyngodon idella) under infection of Flavobacterium columnare. The results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg diet) (1) increased reactive oxygen species, malondialdehyde and protein carbonyl (PC) contents (P < 0.05), decreased the copper/zinc superoxide dismutase, manganese superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities and mRNA levels (P < 0.05), and glutathione and vitamin C contents (P < 0.05), down-regulated NF-E2-related factor 2 mRNA level (P < 0.05), and up-regulated Kelch-like ECH-associating protein (Keap) 1a (rather than Keap1b) mRNA level (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency induced oxidative injury in fish gill; (2) up-regulated caspase-3, -7, -8, -9, Fas ligand, B-cell lymphoma protein 2 associated X protein, apoptotic protease activating factor-1 mRNA levels (P < 0.05), and down-regulated inhibitor of apoptosis protein and B-cell lymphoma-2 (rather than myeloid cell leukemia-1) mRNA level (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency aggravated cell apoptosis in fish gill; (3) up-regulated pore-forming TJs Claudin-12, 15a, -15b, and related signaling molecules myosin light chain kinase, p38 mitogen-activated protein kinase (rather than c-Jun N-terminal kinases) mRNA levels (P < 0.05), and down-regulated barrier-forming TJs Occludin, zonula occludens (ZO) 1, ZO-2, Claudin-c, -3c, -7a, -7b mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency disrupted tight junctional complexes in fish gill; (4) decreased lysozyme and acid phosphatase (ACP) activities, and complement 3 (C3), C4 and IgM contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides liver expressed antimicrobial peptide (LEAP) 2A, LEAP-2B, Hepcidin, ß-defensin mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency decrease fish gill immune function; (5) down-regulated the mRNA levels of anti-inflammatory cytokines-related factors interleukin 10 (IL-10), IL-11, transforming growth factor (TGF) ß1, TGF-ß2, inhibitor of κBa and eIF4E-binding protein 1 (4E-BP1) (rather than 4E-BP2) (P < 0.05), and up-regulated pro-inflammatory cytokines-related factors interferon γ2, IL-1ß, IL-6, IL-8, IL-12 P35, IL-12 P40, nuclear factor κB (NF-κB) p65 (rather than NF-κB p52), IκB kinases (IKK) (only IKKα and IKKγ), target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the gill of grass carp under infection of F. columnare, suggesting that vitamin C deficiency aggravated fish gill inflammation. In conclusion, vitamin C deficiency disrupted physical barriers and immune barriers, and regulated relative mRNA levels of signaling molecules in fish gill. The vitamin C requirement for against gill rot morbidity of grass carp (264-1031 g) was estimated to be 156.0 mg/kg diet. In addition, based on the gill biochemical indices (antioxidant indices MDA, PC and vitamin C contents, and immune indices LA and ACP activity) the vitamin C requirements for grass carp (264-1031 g) were estimated to be 116.8, 156.6, 110.8, 57.8 and 134.9 mg/kg diet, respectively.

Transplantation of human islets without immunosuppression.

Transplantation of pancreatic islets is emerging as a successful treatment for type-1 diabetes. Its current stringent restriction to patients with critical metabolic lability is justified by the long-term need for immunosuppression and a persistent shortage of donor organs. We developed an oxygenated chamber system composed of immune-isolating alginate and polymembrane covers that allows for survival and function of islets without immunosuppression. A patient with type-1 diabetes received a transplanted chamber and was followed for 10 mo. Persistent graft function in this chamber system was demonstrated, with regulated insulin secretion and preservation of islet morphology and function without any immunosuppressive therapy. This approach may allow for future widespread application of cell-based therapies.

Suitable Cottonseed Protein Concentrate Supplementation in Common Carp (Cyprinus carpio) Serves as an Effective Strategy for Fish Meal Sparing Based on Improvement in Intestinal Antioxidant Capacity, Barrier and Microbiota Composition.

The application of cottonseed protein concentrate (CPC) is an effective strategy to moderate the shortage of fish meal (FM) for the aquafeed industry. However, little attention has been paid to the effects of replacing fishmeal with CPC on cyprinid fish. This study used common carp (Cyprinus carpio) as the biological model and assessed the potential of applying CPC as a substitute for fishmeal in the diet of common carp. The proportion of fish meal substituted with CPC in the six diets was 0% (CPC0), 25% (CPC25), 50% (CPC50), 75% (CPC75), and 100% (CPC100). Each diet was fed to three replicate groups of common carp (4.17 ± 0.02 g) for 56 days. Results revealed that the CPC50 group significantly increased the growth indexes via up-regulating the genes of the GH/IGF axis and the TOR pathway. The intestinal digestive ability was also elevated in the CPC50 group via markedly increasing intestinal villus height, protease and lipase activities in the whole intestine, and the amylase activity of the foregut and midgut. The CPC50 group captured significantly higher activities and gene expressions of antioxidant enzymes and lower malonaldehyde contents via evoking the Nrf2/Keap1 signal pathway. The CPC50 group enhance the intestinal mechanical barrier via up-regulating the gene expressions of tight junction proteins and heighten the intestinal biological barrier by increasing the probiotics (Lactococcus) and decreasing the harmful bacteria (Enterococcus). But excessive substitution levels (75% and 100%) would compromise growth performance, intestinal antioxidant capacity, and immune function. The optimum substitution level was estimated to be 46.47%, 47.72%, and 46.43% using broken-line regression analyses based on mass gain rate, protein efficiency ratio, and feed conversion rate. Overall, the fishmeal in common carp feed could be substituted up to 50% by CPC without negative influence on growth, feed utilization, and or intestinal health.

The Role of High Mobility Group Box B-1 in the Prognosis of Colorectal Cancer Based on the Changes in the Intestinal Mucosal Barrier.

Background: To investigate the expression of high mobility group box B-1 (HMGB-1) in patients with colorectal cancer (CRC) and its association with clinicopathological features and prognosis in colorectal carcinoma by combining bioinformatics and clinical data analysis, and to clarify the role of HMGB-1. To examine whether HMGB-1 expression is related to the damage of the intestinal mucosal barrier, and then explore the potential HMGB-1-dependent mechanisms affecting the progression of CRC. Methods: CRC datasets of GSE12945, GSE17536, and GSE17537 from the public gene chip database were screened and downloaded. Clinical information and CRC tissue samples from patients with stage I-III CRC from the hospital were collected. Serum samples of patients were applied by enzyme-linked immunosorbent assay on HMGB-1, and were divided into high and low HMGB-1 expression, which was examined by 16S rDNA sequencing. Immunohistochemistry was performed to examine the relationship between the expression of HMGB-1 and tight junction protein, occludin, tumor necrosis factor-α, and interferon-γ. Results: Based on the Cutoff value of 10.24 ng/mL, the CRC patients were divided into high and low expression groups. In the HMGB-1H patient group, the TNM staging, overall survival, disease-free survival, recurrence, and metastasis were inferior to the HMGB-1L group. The results of 16S rDNA sequencing demonstrated that the Providencia genus was found to be enriched in the HMGB-1L group. Immunohistochemical results showed that HMGB-1 expression was negatively correlated with the expression of ZO-1 and occludin (R = 0.035, R = 0.003, P < .05), but was positively correlated with the expression of TNF-α and IFN-γ (R = 0.016, R = 0.001, P < .05). Conclusion: The survival of CRC patients with positive HMGB-1 expression was significantly shortened, which may be related to the decrease of Rovitensis content, the decreased expression of ZO-1 and occludin, and the increased levels of TNF-α and IFN-γ, which in turn damage the intestinal mucosal barrier, leading to the development of CRC.

Mucosal-associated invariant T cells in infectious diseases of respiratory system: recent advancements and applications.

Mucosal-associated invariant T (MAIT) cells are an atypical subset of T lymphocytes, which have a highly conserved semi-constant αß chain of T-cell receptor (TCR) and recognize microbe-derived vitamin B metabolites via major histocompatibility complex class I related-1 molecule (MR1). MAIT cells get activated mainly through unique TCR-dependent and TCR-independent pathways, and express multiple functional and phenotypic traits, including innate-like functionality, T helper (Th) 1 cell immunity, Th 17 cell immunity, and tissue homing. Given the functions, MAIT cells are extensively reported to play a key role in mucosal homeostasis and infectious diseases. In the current work, we review the basic characteristics of MAIT cells and their roles in mucosal homeostasis and development of respiratory infectious diseases as well as their potential therapeutic targets.

Intestinal Mucosal Immune Barrier: A Powerful Firewall Against Severe Acute Pancreatitis-Associated Acute Lung Injury via the Gut-Lung Axis.

The pathogenesis of severe acute pancreatitis-associated acute lung injury (SAP-ALI), which is the leading cause of mortality among hospitalized patients in the intensive care unit, remains incompletely elucidated. The intestinal mucosal immune barrier is a crucial component of the intestinal epithelial barrier, and its aberrant activation contributes to the induction of sustained pro-inflammatory immune responses, paradoxical intercellular communication, and bacterial translocation. In this review, we firstly provide a comprehensive overview of the composition of the intestinal mucosal immune barrier and its pivotal roles in the pathogenesis of SAP-ALI. Secondly, the mechanisms of its crosstalk with gut microbiota, which is called gut-lung axis, and its effect on SAP-ALI were summarized. Finally, a number of drugs that could enhance the intestinal mucosal immune barrier and exhibit potential anti-SAP-ALI activities were presented, including probiotics, glutamine, enteral nutrition, and traditional Chinese medicine (TCM). The aim is to offer a theoretical framework based on the perspective of the intestinal mucosal immune barrier to protect against SAP-ALI.