LTA and PGN from Bacillus siamensis can alleviate soybean meal-induced enteritis and microbiota dysbiosis in Lateolabrax maculatus.

An eight-week feeding trial was designed to assess which component of commensal Bacillus siamensis LF4 can mitigate SBM-induced enteritis and microbiota dysbiosis in spotted seabass (Lateolabrax maculatus) based on TLRs-MAPKs/NF-кB signaling pathways. Fish continuously fed low SBM (containing 16 % SBM) and high SBM (containing 40 % SBM) diets were used as positive (FM group) and negative (SBM group) control, respectively. After feeding high SBM diet for 28 days, fish were supplemented with B. siamensis LF4-derived whole cell wall (CW), cell wall protein (CWP), lipoteichoic acid (LTA) or peptidoglycan (PGN) until 56 days. The results showed that a high inclusion of SBM in the diet caused enteritis, characterized with significantly (P < 0.05) decreased muscular thickness, villus height, villus width, atrophied and loosely arranged microvillus. Moreover, high SBM inclusion induced an up-regulation of pro-inflammatory cytokines and a down-regulation of occludin, E-cadherin, anti-inflammatory cytokines, apoptosis related genes and antimicrobial peptides. However, dietary supplementation with CW, LTA, and PGN of B. siamensis LF4 could effectively alleviate enteritis caused by a high level of dietary SBM. Additionally, CWP and PGN administration increased beneficial Cetobacterium and decreased pathogenic Plesiomonas and Brevinema, while dietary LTA decreased Plesiomonas and Brevinema, suggesting that CWP, LTA and PGN positively modulated intestinal microbiota in spotted seabass. Furthermore, CW, LTA, and PGN application significantly stimulated TLR2, TLR5 and MyD88 expressions, and inhibited the downstream p38 and NF-κB signaling. Taken together, these results suggest that LTA and PGN from B. siamensis LF4 could alleviate soybean meal-induced enteritis and microbiota dysbiosis in L. maculatus, and p38 MAPK/NF-κB pathways might be involved in those processes.

Effect of feeding chitosan or peptidoglycan on Nosema ceranae infection and gene expression related to stress and the innate immune response of honey bees (Apis mellifera).

Nosema ceranae is a microsporidian parasite that causes nosema disease, an infection of the honey bee (Apis mellifera) midgut. Two pathogen-associated molecular patterns (PAMPs), chitosan and peptidoglycan, and N. ceranae spores were fed to worker bees in sucrose syrup and compared to non-inoculated and N. ceranae-inoculated bees without PAMPs. Both chitosan and peptidoglycan significantly increased bee survivorship and reduced spore numbers due to N. ceranae infection. To determine if these results were related to changes in health status, expression of the immune-related genes, hymenoptaecin and defensin2, and the stress tolerance-related gene, blue cheese, was compared to that of control bees. Compared to the inoculated control, bees with the dose of chitosan that significantly reduced N. ceranae spore numbers showed lower expression of hymenoptaecin and defensin2 early after infection, higher expression mid-infection of defensin2 and lower expression of all three genes late in infection. In contrast, higher expression of defensin2 early in the infection and all three genes late in the infection was observed with peptidoglycan treatment. Changes late in the parasite multiplication stage when mature spores would be released from ruptured host cells are less likely to have contributed to reduced spore production. Based on these results, it is concluded that feeding bees chitosan or peptidoglycan can reduce N. ceranae infection, which is at least partially related to altering the health of the bee by inducing immune and stress-related gene expression.

Enhanced immunity and hemocytes proliferation by three immunostimulants in tri-spine horseshoe crab Tachypleus tridentatus.

Tachypleus amebocyte lysate (TAL) is crucial in medical testing, but its industry in China has been restricted due to the decline of horseshoe crab population in recent years. Exploring methods of enhancing immunity and rapid hemocytes proliferation is urgent for the industrial horseshoe crab culture. In this study, ß-glucan (G), peptidoglycan (P), and squalene (S) were injected to horseshoe crabs at two concentrations (5 and 10 mg/kg), in order to compare their effects on total hemocyte count (THC), reactive oxygen species (ROS), and non-specific immune enzyme activities. Results showed that the THC, superoxide dismutase (SOD), catalase (CAT), and total antioxidant capacity (T-AOC) were significantly increased by three immunostimulants at different points of time; ROS was significantly increased except at two squalene groups; lysozyme (LZM) and alkaline phosphatase (AKP) activity were increased except at low dose (5 mg/kg) squalene group; malondialdehyde (MDA) activity was decreased in all treatments; and hemocyanin concentration (HC) changed little during the experiment. At the 48th hour, THC, ROS, SOD, CAT, T-AOC, LZM, and AKP activities were significantly higher in the two peptidoglycan groups than those in the control group; the low dose ß-glucan and squalene groups showed significantly higher SOD and CAT, but their THC and AKP were not significantly different from those of the control group. In general, all three immunostimulants stimulated the hemolymph parameters of horseshoe crabs, notably, peptidoglycan could significantly increase the THC and enzyme activities, suggesting that peptidoglycan can be developed as an efficient immunostimulant for horseshoe crabs.

A phase I study of the safety and activity of K-001 in patients with advanced pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease that lack of effective therapeutic drugs. K-001 is an oral antitumor drug made from active ingredients of marine microorganisms. The current study aimed to evaluate safety and antitumor activity of K-001 in patients with advanced PDAC. METHODS: In this phase I, open-label trial, patients with advanced PDAC were recruited to a dose-escalation study in a standard 3 + 3 design. K-001 was administered twice daily in four-week cycles, and dose escalation from 1350 mg to 2160 mg was evaluated twice daily. Physical examination and laboratory tests were done at screening and then weekly. The safety, dose-limiting toxicity (DLT), and maximum tolerated dose (MTD) of K-001 were assessed while tumor response was estimated by Response Evaluation Criteria in Solid Tumor (RECIST). RESULTS: Eighteen patients with advanced PDAC were screened, and twelve eligible patients were analyzed in the study. No DLT was observed. Totally, 47 adverse events (AEs) presented, and 14 drug-related AEs were reported in 7 patients, including 8 grade 1 events (57.1%) and 6 grade 2 events (42.9%). There was no grade 3 or 4 drug-related AE. In these 14 drug-related AEs, the most frequent ones were dyspepsia (21.4%), followed by flatulence, constipation, and hemorrhoid bleeding (above 10% of each). Among all 12 patients, 10 patients (83.3%) maintained stable disease (SD), and 2 patients (16.7%) had progressive disease (PD). The objective response rate (ORR) was 0% and the disease control rate (DCR) was 83.3%. CONCLUSIONS: K-001 manifests satisfactory safety and tolerability, as well as meaningful antitumor activity in advanced PDAC patients. Further evaluation of K-001 in phase II/III appears warranted. TRIAL REGISTRATION: NCT02720666 . Registered 28 Match 2016 - Retrospectively registered.

Molecular characterization and functional analysis of a Rel gene in the Pacific oyster.

The nuclear factor-κB (NF-κB) signaling pathway plays a crucial role in regulating many physiological processes such as development, inflammation, apoptosis, cell proliferation, differentiation and immune responses. And the NF-κB/Rel family members were considered as the most important transcription factors in the NF-κB signaling pathway. In this study, we cloned a Rel homolog gene (named as CgRel2) from the Pacific oyster, Crassostrea gigas. The 2115-bp open reading frame (ORF) encodes 704 amino acids and CgRel2 possesses a conserved Rel Homology Domain (RHD) at the N-terminus. Phylogenetic analysis revealed that CgRel2 is most closely related to Pinctada fucata dorsal protein. CgRel2 transcripts are widely expressed in all tested tissues, with the highest expression observed in the labial palp and the gill. Moreover, the expression of CgRel2 is significantly upregulated after lipopolysaccharide (LPS), peptidoglycan (PGN), and polyinosinic-polycytidylic acid [poly(I:C)] challenge. CgRel2 transfection into human cell lines activated NF-κB, TNFα and oyster IL-17 (CgIL-17) reporter genes in a dose-dependent manner, while CgRel2 overexpression cannot induce ISRE (Interferon stimulation response element) reporter gene's transcriptional activity. Additionally, the results of co-immunoprecipitation showed that CgRel2 or CgRel1 could interact with oyster IκB1, IκB2 and IκB3 proteins strongly, which may be critical for the immune signaling transduction and the regulation of its immune functions. Together, these results suggest that CgRel2 could respond to pathogenic infection, participate in the immune signal transduction and activate NF-κB, TNFα and CgIL-17 reporter genes. Thus, CgRel2 could play an important role in the oyster immune system.

[Host Defense against Bacterial Infection and Bacterial Toxin-induced Impairment of Innate Immunity].

　Whereas granulopoiesis during Gram-negative bacterial infection is accelerated through activation of toll-like receptor 4 (TLR4), it has not been elucidated whether Gram-positive bacterial infection can stimulate granulopoiesis. Using the well-known TLR2 agonist peptidoglycan (PGN), it was shown that neutrophils in bone marrow and spleen and plasma granulocyte colony-stimulating factor were increased in mice that had received intraperitoneal administration of PGN. Incorporation of bromodeoxyuridine into bone marrow neutrophils increased in mice administered PGN, demonstrating that PGN promotes granulopoiesis. These results illustrate that bacterial recognition by TLR2 facilitates granulopoiesis during Gram-positive bacterial infection. Thus, granulopoiesis is accelerated to suppress bacterial infection, but some bacteria can still cause severe infections. Clostridium perfringens is a Gram-positive, anaerobic pathogenic bacterium and causes life-threatening gas gangrene in humans. Of the many toxins produced by C. perfringens, α-toxin is known to be a major virulence factor during infection. Recently, it has been revealed that C. perfringens α-toxin impairs the innate immune system by inhibiting neutrophil differentiation, which is crucial for the pathogenesis of C. perfringens. Moreover, the toxin also attenuates erythropoiesis, which would cause severe anemia in clinical settings. The findings provide new insight to understand how hosts strengthen innate immunity to fight pathogenic bacteria and how they evade the hosts' immune systems.

Are the immunomodulatory properties of Lactobacillus rhamnosus CRL1505 peptidoglycan common for all Lactobacilli during respiratory infection in malnourished mice?

Previously, we reported that Lactobacillus rhamnosus CRL1505 peptidoglycan (PG05) improves the innate immune response in immunocompromised-malnourished mice after Streptococcus pneumoniae infection. This study extends those previous findings by demonstrating that the dietary recovery of malnourished mice with nasal administration of PG05 improves not only the innate immune response but the respiratory and systemic adaptive humoral response as well. PG05 enhanced the Th2 response, the recovery of B cells, and the concentration and opsonophagocytic activity of anti-pneumococcal antibodies. In addition, by performing comparative studies with the peptidoglycans from lactobacilli of the same species (L. rhamnosus CRL534) or with similar immunomodulatory properties (L. plantarum CRL1506), we demonstrated here that PG05 has unique immunomodulatory properties that cannot be extended to peptidoglycans from other probiotic strains. However, the knowledge of the molecular characteristics of PG05 is indispensable to understand immunomodulatory abilities of L. rhamnosus CRL1505.

SNX8 mediates IFNγ-triggered noncanonical signaling pathway and host defense against Listeria monocytogenes.

IFNγ is a cytokine that plays a key role in host defense against intracellular pathogens. In addition to the canonical JAK-STAT1 pathway, IFNγ also activates an IKKß-mediated noncanonical signaling pathway that is essential for induction of a subset of downstream effector genes. The molecular mechanisms and functional significance of this IFNγ-triggered noncanonical pathway remains enigmatic. Here, we identified sorting nexin 8 (SNX8) as an important component of the IFNγ-triggered noncanonical signaling pathway. SNX8-deficiency impaired IFNγ-triggered induction of a subset of downstream genes. Snx8-/- mice infected with Listeria monocytogenes exhibited lower serum cytokine levels and higher bacterial loads in the livers and spleens, resulting in higher lethality. Mechanistically, SNX8 interacted with JAK1 and IKKß and promoted their association. IFNγ induced JAK1-mediated phosphorylation of SNX8 at Tyr95 and Tyr126, which promoted the recruitment of IKKß to the JAK1 complex. SNX8-deficiency impaired IFNγ-induced oligomerization and autophosphorylation of IKKß at Ser177, which is critical for selective induction of downstream genes. Our findings suggest that SNX8 acts as a link for IFNγ-triggered noncanonical signaling pathway, which induces a subset of downstream genes important for host defense against L. monocytogenes infection.

Antagonistic effect of peptidoglycan of Streptococcus sanguinis on lipopolysaccharide of major periodontal pathogens.

Streptococcus sanguinis is often found in subgingival biofilm including periodontopathogens, and is correlated with a delay in colonization by periodontopathogens. However, the effect of S. sanguinis on inflammation induced by periodontopathogens is poorly understood. Thus, this study investigated the effect of S. sanguinis peptidoglycan (PGN) on induction of TNF-α, IL-6, and IL-8 expression by lipopolysaccharide (LPS) of periodontal pathogens. LPS was extracted from Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythia, and PGN was isolated from S. sanguinis. THP-1 cells, a monocytic cell-line, were cotreated with LPS of the periodontal pathogens and S. sanguinis PGN, and then the expression of inflammatory cytokines was analyzed by real-time RT-PCR. To analyze the underlying mechanism, the binding assay of the LPS to CD14 or LPS-binding protein (LBP) was performed in the presence or absence of the PGN after coating recombinant human CD14 and LBP on EIA plate. The PGN inhibited the binding of LPS to CD14 and LBP in a dose-dependent manner. Also, THP-1 cells were co-treated with the LPS in the presence of N-acetylmuramic acid and N-acetylglucosamine, as components of PGN, and the competition binding assay to CD14 and LBP was performed. N-acetylmuramic acid inhibited the induction of inflammatory cytokine expression by LPS and the binding of LPS to CD14 or LBP whereas N-acetylglucosamine did not show such effect. Collectively, the results suggest that S. sanguinis PGN inhibited the cytokine expression induced by the LPS of periodontopathogens due to the inhibition of LPS binding to LBP and CD14. N-acetylmuramic acid of PGN may play a role in inhibition of the LPS binding of periodontopathogens to CD14 and LBP.

Radioprotector WR-2721 and mitigating peptidoglycan synergistically promote mouse survival through the amelioration of intestinal and bone marrow damage.

The identification of an agent effective for the treatment of intestinal and bone marrow injury following radiation exposure remains a major issue in radiological medicine. In this study, we evaluated the therapeutic impact of single agent or combination treatments with 2-(3-aminopropylamino) ethylsulphanyl phosphonic acid (WR-2721) and peptidoglycan (PGN, a toll-like receptor 2 (TLR-2) agonist) on radiation-induced injury of the intestine and bone marrow in lethally irradiated male C57BL/6 mice. A dose of 3 mg of WR-2721 per mouse (167 mg/kg, intraperitoneally) was given 30 min before irradiation, and 30 µg of PGN per mouse (1.7 mg/kg) was injected intraperitoneally 24 h after 10 Gy irradiation. Bone marrow cluster of differentiation (CD)45(+) and CD34(+) markers of multiple haematopoietic lineages, number of granulocyte-erythroid-macrophage-megakaryocyte (GEMM) progenitor colonies, bone marrow histopathology, leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) expression in the intestines, xylose absorption and intestinal histopathology were all assessed at various time-points after irradiation. Furthermore, nuclear factor kappa B (NF-κB) p65 protein in the ileum was stained by immunofluorescent labelling. PGN-treated irradiated mice showed an increase in CD45(+)CD34(+) cells compared with untreated mice 1.25 days after 10 Gy ionizing radiation (IR) (P < 0.05). Furthermore, combined PGN and WR-2721 treatment had an obviously synergistic radio-protective effect in nucleated cells in the bone marrow, including GEMM progenitors and CD45(+)CD34(+) cells 4 days after 10 Gy IR. Single agent PGN or WR-2721 treatment after 10 Gy IR clearly increased Lgr5-positive pit cells (P < 0.05) and xylose absorption (P < 0.05). However only PGN and WR-2721 combination treatment markedly increased villus height (P < 0.05), number of crypts (P < 0.05) and whole-body weights after 10 Gy whole-body irradiation (WBI). The NF-κB p65 subunit was translocated to the nucleus, and phosphate-IκBα (Ser32/Ser36) was detected after stimulation with either PGN or WR-2721, which indicates that these two agents act synergistically through the activation of the NF-κB pathway. Administration of PGN in combination with WR-2721 was demonstrated to have a synergistic effect on the increase in haematopoietic cells and intestinal reconstitution, as well as improved survival in lethally irradiated mice, but resulted in some degree of an immune disorder.

Function of two novel single-CRD containing C-type lectins in innate immunity from Eriocheir sinensis.

C-type lectin is one of the pattern-recognition proteins of the non-self-innate immune system in invertebrates. In this study, two novel C-type lectin cDNAs (EsCTL1 and EsCTL2) of Eriocheir sinensis were cloned and characterized. EsCTL1 has 169 amino acids, whereas EsCTL2 has 164 amino acids. These two lectins contain one carbohydrate-recognition domain. Phylogenetic analysis showed that EsCTL1 and EsCTL2 were not clustered with other reported lectins from crabs. EsCTL1 and EsCTL2 were expressed only in the hepatopancreas, as detected by real-time PCR. When healthy crabs were challenged with lipopolysaccharide (LPS), peptidoglycan (PGN), Staphylococcus aureus, or Aeromonas hydrophila, the expression levels of EsCTL1 and EsCTL2 were significantly regulated. The recombinant EsCTL1 and EsCTL2 can agglutinate both Gram-positive (S. aureus) and Gram-negative bacteria (Vibrio parahaemolyticus and A. hydrophila) in a Ca2+ -dependent manner. The recombinant EsCTL1 and EsCTL2 can directly bind to LPS and PGN and to all tested microorganisms (S. aureus, Bacillus thuringiensis, Bacillus subtilis, Escherichia coli, Vibrio natriegens, V. parahaemolyticus, and A. hydrophila). Furthermore, rEsCTL1 and rEsCTL2 may facilitate the clearance of V. parahaemolyticus in vivo. These results suggest that EsCTL1 and EsCTL2 may have important roles in the anti-bacterial immunity of Chinese mitten crab.

Regulation of apoptosis and innate immune stimuli in inflammation-induced preterm labor.

An innate immune response is required for successful implantation and placentation. This is regulated, in part, by the a2 isoform of V-ATPase (a2V) and the concurrent infiltration of M1 (inflammatory) and M2 (anti-inflammatory) macrophages to the uterus and placenta. The objective of the present study was to identify the role of a2V during inflammation-induced preterm labor in mice and its relationship to the regulation of apoptosis and innate immune responses. Using a mouse model of infection-induced preterm delivery, gestational tissues were collected 8 h after intrauterine inoculation on day 14.5 of pregnancy with either saline or peptidoglycan (PGN; a TLR 2 agonist) and polyinosinic-polycytidylic acid [poly(I:C); a TLR3 agonist], modeling Gram-positive bacterial and viral infections, respectively. Expression of a2V decreased significantly in the placenta, uterus, and fetal membranes during PGN+poly(I:C)-induced preterm labor. Expression of inducible NO synthase was significantly upregulated in PGN+poly(I:C)-treated placenta and uterus. PGN+poly(I:C) treatment disturbed adherens junction proteins and increased apoptotic cell death via an extrinsic pathway of apoptosis among uterine decidual cells and spongiotrophoblasts. F4/80(+) macrophages were increased and polarization was skewed in PGN+poly(I:C)-treated uterus toward double-positive CD11c(+) (M1) and CD206(+) (M2) cells, which are critical for the clearance of dying cells and rapid resolution of inflammation. Expression of Nlrp3 and activation of caspase-1 were increased in PGN+poly(I:C)-treated uterus, which could induce pyroptosis. These results suggest that the double hit of PGN+poly(I:C) induces preterm labor via reduction of a2V expression and simultaneous activation of apoptosis and inflammatory processes.

Inhibition of IκB kinase reduces the multiple organ dysfunction caused by sepsis in the mouse.

Nuclear factor κB (NF-κB) plays a pivotal role in sepsis. Activation of NF-κB is initiated by the signal-induced ubiquitylation and subsequent degradation of inhibitors of kappa B (IκBs) primarily via activation of the IκB kinase (IKK). This study was designed to investigate the effects of IKK inhibition on sepsis-associated multiple organ dysfunction and/or injury (MOD) and to elucidate underlying signaling mechanisms in two different in vivo models: male C57BL/6 mice were subjected to either bacterial cell wall components [lipopolysaccharide and peptidoglycan (LPS/PepG)] or underwent cecal ligation and puncture (CLP) to induce sepsis-associated MOD. At 1 hour after LPS/PepG or CLP, mice were treated with the IKK inhibitor IKK 16 (1 mg/kg body weight). At 24 hours, parameters of organ dysfunction and/or injury were assessed in both models. Mice developed a significant impairment in systolic contractility (echocardiography), and significant increases in serum creatinine, serum alanine aminotransferase and lung myeloperoxidase activity, thus indicating cardiac dysfunction, renal dysfunction, hepatocellular injury and lung inflammation, respectively. Treatment with IKK 16 attenuated the impairment in systolic contractility, renal dysfunction, hepatocellular injury and lung inflammation in LPS/PepG-induced MOD and in polymicrobial sepsis. Compared with mice that were injected with LPS/PepG or underwent CLP, immunoblot analyses of heart and liver tissues from mice that were injected with LPS/PepG or underwent CLP and were also treated with IKK 16 revealed: (1) significant attenuation of the increased phosphorylation of IκBα; (2) significant attenuation of the increased nuclear translocation of the NF-κB subunit p65; (3) significant attenuation of the increase in inducible nitric oxide synthase (iNOS) expression; and (4) a significant increase in the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS). Here, we report for the first time that delayed IKK inhibition reduces MOD in experimental sepsis. We suggest that this protective effect is (at least in part) attributable to inhibition of inflammation through NF-κB, the subsequent decrease in iNOS expression and the activation of the Akt-eNOS survival pathway.

Ephedrine hydrochloride inhibits PGN-induced inflammatory responses by promoting IL-10 production and decreasing proinflammatory cytokine secretion via the PI3K/Akt/GSK3ß pathway.

Approaches for controlling inflammatory responses and reducing the mortality rate of septic patients remain clinically ineffective; new drugs need to be identified that can induce anti-inflammatory responses. Ephedrine hydrochloride (EH) is a compound that is widely used in cardiovascular diseases, especially to treat hypotension caused by either anesthesia or overdose of antihypertensive drugs. In this study, we reported that EH also plays an important role in the control of the inflammatory response. EH increased IL-10 and decreased proinflammatory cytokine (IL-6, tumor-necrosis factor (TNF)-α, IL-12 and IL-1ß) expression in primary peritoneal macrophages and Raw264.7 cells treated with peptidoglycan (PGN), a Gram-positive cell wall component. The anti-inflammatory role of EH was also demonstrated in an experimental mouse model of peritonitis induced by intraperitoneal PGN injection. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway was found to be responsible for the EH-mediated increase in IL-10 production and decrease in IL-6 expression. Therefore, our results illustrated that EH can help maintain immune equilibrium and diminish host damage by balancing the production of pro- and anti-inflammatory cytokines after PGN challenge. EH may be a new potential anti-inflammatory drug that can be useful for treating severe invasive Gram-positive bacterial infection.

Intradermal delivery of TLR agonists in a human explant skin model: preferential activation of migratory dendritic cells by polyribosinic-polyribocytidylic acid and peptidoglycans.

TLR agonists are attractive candidate adjuvants for therapeutic cancer vaccines as they can induce a balanced humoral and T cell-mediated immune response. With a dense network of dendritic cells (DCs) and draining lymphatics, the skin provides an ideal portal for vaccine delivery. Beside direct DC activation, TLR agonists may also induce DC activation through triggering the release of inflammatory mediators by accessory cells in the skin microenvironment. Therefore, a human skin explant model was used to explore the in vivo potential of intradermally delivered TLR agonists to stimulate Langerhans cells and dermal DCs in their natural complex tissue environment. The skin-emigrated DCs were phenotyped and analyzed for T cell stimulatory capacity. We report that, of six tested TLR-agonists, the TLR2 and -3 agonists peptidoglycan (PGN) and polyribosinic-polyribocytidylic acid (Poly I:C) were uniquely able to enhance the T cell-priming ability of skin-emigrated DCs, which, in the case of PGN, was accompanied by Th1 polarization. The enhanced priming capacity of Poly I:C-stimulated DCs was associated with a strong upregulation of appropriate costimulatory molecules, including CD70, whereas that of PGN-stimulated DCs was associated with the release of a broad array of proinflammatory cytokines. Transcriptional profiling further supported the notion that the PGN- and Poly I:C-induced effects were mediated through binding to TLR2/nucleotide-binding oligomerization domain 2 and TLR3/MDA5, respectively. These data warrant further exploration of PGN and Poly I:C, alone or in combination, as DC-targeted adjuvants for intradermal cancer vaccines.

Trunk kidney of grass carp (Ctenopharyngodon idella) mediates immune responses against GCRV and viral/bacterial PAMPs in vivo and in vitro.

Trunk kidney is a vital organ for excretion in teleosts. There have been sporadic reports of processing pathogens for the immune function in trunk kidney. However, molecular processes of pathogen recognition receptors (PRRs) responding to virus and viral/bacterial pathogen-associated molecular patterns (PAMPs) are poorly elucidated in trunk kidney. In the present study, we investigated transcriptional profiles of twelve representative immune-related genes (TLRs (TLR3, TLR7 and TLR22); RLRs (RIG-I, MDA5 and LGP2); NLRs (NOD1 and NOD2); adapter molecules (MyD88 and IPS-1); effector molecule type I interferon (IFN-I) and immunoglobulin M (IgM)) in trunk kidney tissue of grass carp (Ctenopharyngodon idella) (designated as Ci) injection of grass carp reovirus (GCRV) utilizing quantitative real-time RT-PCR (qRT-PCR). Furthermore, mRNA expression patterns of these genes (IgM excepted) were examined post GCRV infection and polyinosine-polycytidylic acid (poly(I:C)), lipopolysaccharide (LPS) or peptidoglycan (PGN) stimulation in primary trunk kidney cells of grass carp. The relative values of CiTLR3, CiTLR22 and CiMyD88 were increased post GCRV challenge and viral/bacterial PAMPs stimulation. The mRNA transcriptions of CiTLR7 were obviously activated with GCRV challenge. Remarkably, the mRNA expressions of CiRIG-I, CiMDA5, CiLGP2 and CiIPS-1 were largely up-regulated with GCRV challenge and viral/bacterial PAMPs stimulation. Interestingly, the expression tendencies of CiNOD1 and CiNOD2 were differential not only in GCRV challenge and poly(I:C) stimulation, but also in LPS and PGN stimulation. It was demonstrated that CiIFN-I induced powerful anti-viral and anti-bacterial effects in trunk kidney. In addition, the expression of CiIgM was induced at 72 h post GCRV injection in vivo. Collectively, these results suggest that trunk kidney of grass carp serves as an important immune organ, and plays crucial roles in triggering anti-viral and anti-bacterial immune responses both in vivo and in vitro.

Water soluble polymer films for intravascular drug delivery of antithrombotic biomolecules.

Over the past 10 years, the number of percutaneous coronary intervention (PCI) procedures performed in the United States has increased by 33%; however, restenosis, which inhibits complete functional recovery of the vessel wall, remains a complication of this procedure. To traverse the complications associated with PCI, the investigation of therapeutic delivery has become an integral topic in modern research. One such therapeutic, a mimic of the proteoglycan decorin, termed DS-SILY, can mask exposed collagen and thereby effectively decrease platelet activation, has recently been developed by our lab. Drawing inspiration from coating technologies developed by the pharmaceutical industry, a fast-dissolving polymer film has been developed to deliver active therapeutic agents from a balloon catheter during PCI. This research investigates the release of DS-SILY from fast-dissolving polymer films composed of poly(vinyl alcohol) (PVA) and poly(ethylene glycol) (PEG). Thin, uniform polymer films were produced via spin coating technique. The dissolution speed of the polymer films was found to be dependent on the concentration of polymer solution, where at least 65% of the films were shown to dissolve into nanometer sized polymer fragments within 2 min. DS-SILY, up to 6.26 µg/cm(2), was loaded into the films and functional release of the mimic was demonstrated by its successful binding to collagen upon release. Furthermore, DS-SILY released from films resulted in increased platelet inhibition. These results indicate that use of fast-dissolving polymer films allow for the successful release of biomolecules and further investigation of their use for localized drug delivery during PCI procedures is warranted.

High mobility group box 1 protein synergizes with lipopolysaccharide and peptidoglycan for nitric oxide production in mouse peritoneal macrophages in vitro.

Extracellular high mobility group box 1 (HMGB1) protein and nitric oxide (NO) has been credited with multiple inflammatory functions using in vivo and in vitro systems. Therefore, delineating their regulation may be an important therapeutic strategy for the treatment of sepsis. In the present study, it is demonstrated that recombinant HMGB1 (rHMGB1) synergizes with sub threshold concentration of TLR2 agonist (PGN; 1 µg/ml) as well as with TLR4 agonist (LPS; 1 ng/ml) to induce NO release in mouse peritoneal macrophages. The enhanced iNOS expression was also observed at the transcription and translational level. Co-incubation of macrophages with rHMGB1 with either PGN or LPS showed enhanced expression of TLR2, TLR4 and RAGE. TLR2, TLR4 or RAGE knockdown macrophages effectively inhibited the rHMGB1+PGN or LPS induced NO synergy. It was further observed that the JNK MAPK inhibitor SP600125 attenuated the PGN+rHMGB1 induced iNOS/NO synergy whereas p38 MAPK inhibitor SB908912 inhibited iNOS/NO synergy induced by LPS+rHMGB1. It was also observed that the activation of NF-κB is essential for the synergy as the pharmacological inhibition or siRNA knockdown of NF-κB (cRel) significantly reduced the rHMGB1+PGN or rHMGB1+LPS induced enhanced iNOS/NO expression. Altogether, the data suggests that the co-incubation of macrophages with rHMGB1 with either LPS or PGN induces the synergistic effect on iNOS expression and NO release by the upregulation of surface receptors (TLR2, TLR4 and RAGE) which in turn amplifies the MAPKs (p38 and JNK) and NF-κB activation and results in enhanced iNOS expression and NO production.

The effect of peptidoglycan enriched diets on antimicrobial peptide gene expression in rainbow trout (Oncorhynchus mykiss).

The aim of this study was to investigate the effect of feeding rainbow trout (Oncorhynchus mykiss) peptidoglycan (PG) enriched diets on antimicrobial peptide (AMP) gene expression. Fish were divided into 5 groups and fed diets containing 0, 5, 10, 50 and 100 mg PG/Kg, and sampled 1, 7 and 14 days later. The expression of eight AMP genes (four defensins, two cathelicidins and two liver expressed AMPs) was determined in skin, gill, gut and liver, tissues important for first lines of defence or production of acute phase proteins. Up-regulation of many AMPs was found after feeding the PG enriched diets, with sequential expression seen over the time course studied, where defensins were typically expressed early and cathelicidins and LEAPs later on. A number of clear differences in AMP responsiveness between the tissues examined were also apparent. Of the four PG concentrations used, 5 mg PG/Kg did not always elicit AMP gene induction or to the same degree as seen with the other diets. The three higher dose groups generally showed similar trends although differences in fold change were more pronounced in the 50 and 100 mg PG/Kg groups. Curiously several AMPs were down-regulated after 14 days of feeding in gills, gut and liver. Nevertheless, overall the PG enriched diets had a positive effect on AMP expression. Further investigations now need to be undertaken to confirm whether this higher AMP gene expression correlates with protection against common bacterial diseases and if PG enriched diets have value as a means to temporarily boost the piscine immune system.

Adjuvant selection for vaccination against murine schistosomiasis.

Schistosoma mansoni cercariae penetrate mouse epidermis, detach the glycocalyx and transform into schistosomula, triggering innate immune responses by host keratinocytes and Langerhans cells. Schistosomula leave the dermis and enter blood capillaries, releasing excretory/secretory products (ESP), which induce readily detectable primary adaptive immunity responses, dominated by T helper (Th) 1 and 17 cytokines. Partial protection against murine schistosomiasis may be achieved using subunit antigens and Th1 cytokine-inducing adjuvants. Conversely, resistance to primary and/or secondary schistosomiasis in rats, mice and humans is associated with production of Th2 cytokines. Accordingly, we reasoned that effective vaccination against murine primary schistosomiasis might be achieved provided selection of an adjuvant capable of skewing the S. mansoni larval ESP-mediated Th1/Th17 immune responses towards a Th2 profile. In an aim to select such an adjuvant, we administered the prototypical Th1 and Th2, respectively, C57BL/6 and BALB/c mice with polyinosinic-polycytidylic acid (Poly I/C), peptidoglycan (PGN), or thymic stromal lymphopoietin (TSLP) before exposure to S. mansoni cercariae. Serum antibody reactivity and ex vivo spleen cells (SC) immune responses to larval ESP, in a recombinant or multiple antigen peptide form, were assessed 1 week after infection. Injection with Poly I/C failed to increase interleukin (IL)-4 and led to elevated gamma interferon (IFN-γ) levels released by unstimulated or ESP-stimulated SC. Treatment with PGN triggered hightened amounts of IL-4, IL-17 and IFN-γ released by unstimulated or ESP-stimulated C57BL/6 SC. In contrast, TSLP succeeded in directing the ESP-mediated immune responses towards a Th2-biased profile in prototypical Th1 and Th2 mice.