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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Immune activation decreases sperm viability in both sexes and influences female sperm storage.

All animals are under the constant threat of pathogenic infection. However, little is known regarding the influence of acute infection on sperm viability, particularly in female insects. This information is crucial for our understanding of mating and immune system coevolution, considering that females store sperm and serve as the site of sperm competition. Using the fruitfly, Drosophila melanogaster, we examined the influence of infection on sperm viability and storage. Twenty-four hours after haemocoel inoculation with a pathogen mimic (peptidoglycan, PGN) both sexes exhibited reduced sperm viability, indicating that systemic immune activation played a significant role in gamete survival. Surprisingly, sperm death did not appear to result from a reproductive-immune system trade-off, considering that sperm survived 24 h in vitro once removed from their somatic resources. Instead, our results are most consistent with death owing to immune effector collateral damage. We also examined the potential for sexually transmitted pathogens to influence sperm storage. Females mated with 'infected' males (created by dipping genitalia into a PGN solution) exhibited a higher proportion of empty sperm stores 48 h after mating compared to their controls. Remarkably, these data indicate that females may increase their fitness by removing 'infected' ejaculates from storage over time.

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.

A novel tissue inhibitor of metalloproteinase in blood clam Tegillarca granosa: molecular cloning, tissue distribution and expression analysis.

Tissue inhibitor of metalloproteinases (TIMPs) were originally characterized as inhibitors of matrix metalloproteinases (MMPs), but their range of activities has been found to be broader as it includes the inhibition of several of the MMPs, etc. The cDNA encoding TIMP-4-like gene from blood clam Tegillarca granosa (designated as Tg-TIMP-4-like) which is the first tissue inhibitor of metalloproteinase identified in blood clams, was cloned and characterized. It was of 1164 bp, and an open reading frame (ORF) of 666 bp encoding a putative protein of 222 amino acids. The predicted amino acid sequence comprised all recognized functional domains found in other TIMP homologues and showed the highest (30.56%) identity to the TIMP-1.3 from Crassostrea gigas. Several highly conserved motifs including several TIMP signatures, amino acid residue Cys³° responsible for coordinating the metal ions, the Cys-X-Cys motif and the putative NTR (netrin) domain were almost completely conserved in the deduced amino acid of Tg-TIMP-4 like, which indicated that Tg-TIMP-4-like should be a member of the TIMP family. The mRNA expression of Tg-TIMP-4-like in the tissues of mantle, adductor muscle, foot, gill, hemocyte and hepatopancreas was examined by quantitative real-time PCR (qT-PCR) and mRNA transcripts of Tg-TIMP-4-like were mainly detected in hemocyte, and weakly detected in the other tissues. We also observed that Tg-TIMP-4 like mRNA accumulated significantly during Vibrio parahaemolyticus, Peptidogylcan (PGN) and Lipopolysaccharide (LPS) challenge, whereas the timing and quantitative differences of mRNA expression against different challenge indicated that Tg-TIMP-4-like may play a pivotal role in mollusc defense mechanisms.

Induction of toll-like receptor 2 positive antigen-presenting cells in spleen of pristane-induced arthritis in rats.

Toll-like receptors (TLRs) have been found to contribute to the pathogenesis of rheumatoid arthritis (RA). The aim of this study is to investigate the regulation and potential role of TLR2 in spleen of pristane-induced arthritis (PIA) rat, which can be used to further understand the mechanisms of RA. Arthritis in DA rats was induced by pristane. TLR2 expression in spleen was detected by real-time quantitative PCR and western blotting, and TLR2 expression at both mRNA and protein levels was upregulated in PIA rats. Peptidoglycan (PGN) was systemically administrated to PIA rats, and arthritis severity was evaluated macroscopically and microscopically. Results showed that systemic administration of PGN to PIA rats obviously deteriorated arthritis severity. TLR2 expression on splenocytes and different types of immune cells was measured by flow cytometry. And it was found that TLR2 was mainly expressed on antigen-presenting cells (APCs) of spleen, and the proportion of TLR2(+) dendritic cells and macrophages in spleen of PIA rats was increased remarkably. Thus, we conclude that the induction of TLR2(+) APCs in spleen may participate in the maintenance of PIA.

The effect of peptidoglycan stimulation on basophil-mediated atopic responses during pregnancy and in newborns.

BACKGROUND: Toll-like receptor (TLR) molecules play critical roles in directing the course of atopic diseases by recognizing specific microbial products that activate immune effector cell function. OBJECTIVE: We determined if basophils harvested from neonates genetically predisposed to atopic disease had different levels of TLR2 expression and determined whether putative TLR2 ligands mediated cytokine secretion. METHODS: Blood samples were collected from 10 asthmatic and 12 healthy women and their newborns. Basophil histamine was measured using the human basophil degranulation test and TLR2 expression was determined using nucleic acid hybridization in situ and flow cytometry. IL-4 levels were quantified by ELISA following allergen stimulation. RESULTS: The basophil degranulation index (DI) in granulocytes harvested from peripheral blood of asthmatic women was assessed following stimulation with either peptidoglycan (PGN) or Dermatophagoides farinae (Df) extract. The DI was significantly higher in atopic women than in healthy controls. Basophils purified from the cord blood of neonates born to atopic mothers produced more IL-4 compared with basophils purified from children born to nonatopic controls. Finally, TLR2 expression at the protein and mRNA levels was upregulated in cord blood basophils from neonates born to mothers with asthma following stimulation with PGN but not Df. CONCLUSION: These data suggested that TLR2-mediated innate immune responses play a role in augmenting allergic reactions through the modulation of basophil cytokine secretion and histamine release. Microbial components may activate basophils through TLR2 (especially for genetically predisposed infants) to release cytokines associated with an increased incidence of allergic diseases.

Percutaneous application of peptidoglycan from Staphylococcus aureus induces infiltration of CCR4+ cells into mouse skin.

BACKGROUND: The lesional skin of patients with atopic dermatitis has an increased number of type 2 helper T (TH2) cells in the dermis and is superficially colonized by Staphylococcus aureus. The purpose of this study was to determine the effects of peptidoglycan (PEG) from S aureus on TH2 cell induction in murine skin. METHODS: Mice were sensitized with house dust mite antigen (MA) by topical application to barrier-disrupted abdominal skin. Seven days after sensitization, PEG was applied to the barrier-disrupted dorsal skin. After a further 3 days, C-C chemokine receptor type 4-positive (CCR4+) cells were counted in the PEG-treated skin.The production of chemokine (C-C) motif ligand 17 (CCL17) (thymus- and activation-regulated chemokine) and CCL22 (macrophage-derived chemokine) in the skin was investigated using reverse transcriptase polymerase chain reaction and immunohistological analysis. RESULTS: Application of PEG to the dorsal skin of MA-sensitized mice led to a significant increase in the number of cells expressing CCR4 in the dermis. The skin of PEG-treated mice showed an increased level of CCL17 mRNA expression, which coincided with TH2 cytokine mRNA expression. Immunohistological analysis demonstrated that levels of CCL17 transcripts corresponded to those of protein synthesis in the epidermis. CCL17 production was induced mainly by Langerhans cells stimulated with PEG. Furthermore, intraperitoneal injection of anti-CCL17 antibody abrogated the induction of CCR4+ cells in the skin. CONCLUSION: These results suggest that PEG may induce TH2 cells in the skin through the production of CCL17 by Langerhans cells and would explain the role of colonization by S aureus in patients with atopic dermatitis.

Identification of the extracellular copper-zinc superoxide dismutase (ecCuZnSOD) gene of the mud crab Scylla serrata and its expression following beta-glucan and peptidoglycan injections.

An extracellular copper-zinc superoxide dismutase (ecCuZnSOD, or EC-SOD) gene was cloned from haemocytes of mud crab Scylla serrata. The ecCuZnSOD cDNA consists of 939 bp with an open reading frame (ORF) of 585 bp that encodes 195 amino acids (aa) with 31 residues of a signal peptide sequence. The predicted molecular mass of the mature protein (164aa) is 17.1 kDa with an estimated pI of 6.89. Amino acids responsible for binding Cu (His84, 86, 101, and 163) and Zn (His101, 109, and 121, and Asp 124), two cysteines (95 and 189) that form a disulfide bond, two CuZnSOD signatures (GFHVHAEGDLS) from 82 to 92 and (GNAGGRAGCGLI) from 181 to 192, as well as a N-linked glycosylation site (NVSG) were conserved. The deduced amino acid sequence of S. serrata ecCuZnSOD showed similarity of 84% and 54% with the ecCuZnSOD of blue crab Callinectes sapidus, and crayfish Pacifastacus leniusculus, respectively. The phylogenetic analysis revealed that the ecCuZnSOD of S. serrata grouped together with ecCuZnSODs of crustaceans and insects, but was far way from the intracellular (ic)CuZnSODs of invertebrates and vertebrates. The ecCuZnSOD transcript in haemocytes significantly increased in 6-48 h, and had returned to the original value by 72 h after the beta-glucan (betaG) injection, whereas the ecCuZnSOD transcript significantly increased 6-72 h after the peptidoglycan (PG) injection indicating induction of the immune system within a short time.

Pretreatment with the Gram-positive bacterial cell wall molecule peptidoglycan improves bacterial clearance and decreases inflammation and mortality in mice challenged with Pseudomonas aeruginosa.

The objective of this study was to determine if inflammatory tolerance and enhancement of innate immune function could be induced by the Gram-positive cell wall component peptidoglycan (PGN). Male mice (C57BL6/J or C3H/HeJ, 8-12 weeks of age) were given intraperitoneal injections of 1mg PGN on 2 consecutive days. The mice were then challenged with lipopolysaccharide (LPS) or live Pseudomonas aeruginosa (1 x 10(8) colony-forming units) 2 days after the second pretreatment. Mice pretreated with PGN had diminished plasma concentrations of TNFalpha and IFNgamma and elevated concentrations of IL-10 in response to a subsequent LPS or Pseudomonas challenge when compared to untreated controls. Bacterial clearance was improved in mice pretreated with PGN, and mortality in response to a subsequent Pseudomonas challenge was significantly attenuated. PGN pretreatment of LPS-unresponsive mice (C3H/HeJ) verified that the effect of PGN pretreatment was not due to any LPS contamination. We have previously demonstrated that PGN pretreatment induced resistance to a Gram-positive bacterial challenge. The present study extends those results by showing that exposure to the Gram-positive bacterial cell wall component peptidoglycan also induces cross-tolerance to LPS and non-specifically enhances innate immune function in that PGN-pretreated mice had increased resistance to Gram-negative bacterial challenge.

Peptidoglycan from Staphylococcus aureus induces IL-4 production from murine spleen cells via an IL-18-dependent mechanism.

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease with immunopathologic features that vary depending on the duration of the lesion. Skin lesions of AD patients show an increased number of Th2 cells in the dermis and superficial Staphylococcus aureus colonization. The purpose of this study was to predict the effects of peptido- glycan (PEG) from S. aureus on the induction of interleukin (IL)-4 production in AD patients. METHODS: PEG was applied to barrier-disrupted abdominal mouse skin every 5 days. Twenty days later, IL-4 production in the spleen was investigated by reverse transcription-polymerase chain reaction (RT-PCR). Spleen cells from normal mice were also treated in vitro with PEG and processed for IL-4 production by RT-PCR and enzyme-linked immunosorbent assay. RESULTS: IL-4 production was significantly increased in the spleen of PEG-treated mice compared with that of PBS-applied mice. In addition, in vitro experiments demonstrated that PEG was able to induce IL-4 production in murine spleen cells. Furthermore, IL-4 production was associated with IL-18 production, but not with IL-2 production from PEG-stimulated spleen cells, and IL-4 mRNA was expressed in CD4+ lymphocytes in the spleen cells. In in vivo experiments, percutaneous treatment with PEG induced mRNA expression not only for IL-4 but also for IL-18 in the spleen. CONCLUSION: These results suggest that PEG may influence the induction of a Th2-dominant cytokine response in AD patients through IL-4 production from CD4+ T cells stimulated with PEG-induced IL-18.