Extracellular vesicles from Staphylococcus aureus promote the pathogenicity of Pseudomonas aeruginosa.

Co-infections with Staphylococcus aureus and Pseudomonas aeruginosa are common in patients with chronic wounds, but little is known about their synergistic effect mediated by extracellular vesicles (EVs). In this study, we investigated the effect of EVs derived from S. aureus (SaEVs) on the pathogenicity of P. aeruginosa. By using lipophilic dye, we could confirm the fusion between SaEV and P. aeruginosa membranes. However, SaEVs did not alter the growth and antibiotic susceptible pattern of P. aeruginosa. Differential proteomic analysis between SaEV-treated and non-treated P. aeruginosa was performed, and the results revealed that lipopolysaccharide (LPS) biosynthesis protein in P. aeruginosa significantly increased after SaEV-treatment. Regarding this result, we also found that SaEVs promoted LPS production, biofilm formation, and expression of polysaccharide polymerization-related genes in P. aeruginosa. Furthermore, invasion of epithelial cells by SaEV-pretreated P. aeruginosa was enhanced. On the other hand, uptake of P. aeruginosa by RAW 264.7 macrophages was impaired after pretreatment P. aeruginosa with SaEVs. Proteomic analysis SaEVs revealed that SaEVs contain the proteins involving in host cell colonization, inhibition of host immune response, anti-phagocytosis of the macrophages, and protein translocation and iron uptake of S. aureus. In conclusion, SaEVs serve as a mediator that promote P. aeruginosa pathogenicity by enhancing LPS biosynthesis, biofilm formation, epithelial cell invasion, and macrophage uptake impairment.

Heat Shock Protein SSA1 Enriched in Hypoxic Secretome of Candida albicans Exerts an Immunomodulatory Effect via Regulating Macrophage Function.

Candida albicans is an opportunistic pathogenic yeast that can survive in both normoxic and hypoxic environments. The involvement of C. albicans secretome on host biological processes has been demonstrated. However, the immunoregulatory function of C. albicans secretome released under hypoxic condition remains unclear. This study demonstrated the differences in cytokine responses and protein profiles between secretomes prepared under normoxic and hypoxic conditions. Furthermore, the immunoregulatory effects of heat shock protein SSA1(Ssa1), a protein candidate enriched in the hypoxic secretome, were investigated. Stimulation of mouse bone marrow-derived macrophages (BMMs) with Ssa1 resulted in the significant production of interleukin (IL)-10, IL-6, and tumor necrosis factor (TNF)-α as well as the significant expression of M2b macrophage markers (CD86, CD274 and tumor necrosis factor superfamily member 14), suggesting that C. albicans Ssa1 may promote macrophage polarization towards an M2b-like phenotype. Proteomic analysis of Ssa1-treated BMMs also revealed that Ssa1 reduced inflammation-related factors (IL-18-binding protein, IL-1 receptor antagonist protein, OX-2 membrane glycoprotein and cis-aconitate decarboxylase) and enhanced the proteins involved in anti-inflammatory response (CMRF35-like molecule 3 and macrophage colony-stimulating factor 1 receptor). Based on these results, we investigated the effect of Ssa1 on C. albicans infection and showed that Ssa1 inhibited the uptake of C. albicans by BMMs. Taken together, our results suggest that C. albicans alters its secretome, particularly by promoting the release of Ssa1, to modulate host immune response and survive under hypoxic conditions.

Salmon nasal cartilage proteoglycan stimulates hair growth.

Hair loss is a commonly encountered problem. In this study, hair growth was enhanced by daily oral ingestion of salmon nasal cartilage proteoglycan (PG) in mice. Proteoglycan stimulated vesicular endothelial growth factor production in human follicle dermal papilla cells through insulin growth factor-1 receptor signaling, suggesting the possibility of hair loss improvement by PG ingestion.

Vitamin A Promotes the Fusion of Autophagolysosomes and Prevents Excessive Inflammasome Activation in Dextran Sulfate Sodium-Induced Colitis.

Vitamin A ensures intestinal homeostasis, impacting acquired immunity and epithelial barrier function; however, its role in innate immunity is mostly unknown. Here, we studied the impact of vitamin A in different dextran sulfate sodium (DSS)-induced colitis animal models. Interestingly, more severe DSS-induced colitis was observed in vitamin A-deficient (VAD) mice than in vitamin A-sufficient (VAS) mice; the same was observed in VAD severe combined immunodeficient mice lacking T/B cells. Remarkably, IL-1ß production, LC3B-II expression, and inflammasome activity in the lamina propria were significantly elevated in VAD mice. Electron microscopy revealed numerous swollen mitochondria with severely disrupted cristae. In vitro, non-canonical inflammasome signaling-induced pyroptosis, LC3B-II and p62 expression, and mitochondrial superoxide levels were increased in murine macrophages (RAW 264.7) pretreated with retinoic acid receptor antagonist (Ro41-5253). These findings suggest that vitamin A plays a crucial role in the efficient fusion of autophagosomes with lysosomes in colitis.

Extracellular vesicles of Pseudomonas aeruginosa downregulate pyruvate fermentation enzymes and inhibit the initial growth of Staphylococcus aureus.

Staphylococcus aureus and Pseudomonas aeruginosa are well-known opportunistic pathogens that frequently coexist in chronic wounds and cystic fibrosis. The exoproducts of P. aeruginosa have been shown to affect the growth and pathogenicity of S. aureus, but the detailed mechanisms are not well understood. In this study, we investigated the effect of extracellular vesicles from P. aeruginosa (PaEVs) on the growth of S. aureus. We found that PaEVs inhibited the S. aureus growth independently of iron chelation and showed no bactericidal activity. This growth inhibitory effect was also observed with methicillin-resistant S. aureus but not with Acinetobacter baumannii, Enterococcus faecalis, S. Typhimurium, E. coli, Listeria monocytogenes, or Candida albicans, suggesting that the growth inhibitory effect of PaEVs is highly specific for S. aureus. To better understand the detailed mechanism, the difference in protein production of S. aureus between PaEV-treated and non-treated groups was further analyzed. The results revealed that lactate dehydrogenase 2 and formate acetyltransferase enzymes in the pyruvate fermentation pathway were significantly reduced after PaEV treatment. Likewise, the expression of ldh2 gene for lactate dehydrogenase 2 and pflB gene for formate acetyltransferase in S. aureus was reduced by PaEV treatment. In addition, this inhibitory effect of PaEVs was abolished by supplementation with pyruvate or oxygen. These results suggest that PaEVs inhibit the growth of S. aureus by suppressing the pyruvate fermentation pathway. This study reported a mechanism of PaEVs in inhibiting S. aureus growth which may be important for better management of S. aureus and P. aeruginosa co-infections.

Differential proteomic analysis and pathogenic effects of outer membrane vesicles derived from Acinetobacter baumannii under normoxia and hypoxia.

Acinetobacter baumannii is a major causative agent of nosocomial infections and its outer membrane vesicles (AbOMVs) have been shown to be involved in pathogenicity by transporting virulence factors and transferring information for communication between pathogens and host cells. Despite the fact that the infected sites of A. baumannii such as lungs and skin soft tissues are hypoxic, most studies on AbOMV virulence have used AbOMVs prepared under aerobic conditions. The present study aims to elucidate the protein profile and pathogenic impact of AbOMVs released under hypoxic condition. AbOMVs were isolated from A. baumannii under normoxic and hypoxic conditions, and their protein profiles were compared. The different effects of both normoxic and hypoxic AbOMVs in cytokine response from mouse macrophages, cytotoxicity to the human lung epithelial cells, and bacterial invasion were then investigated. Our results showed that A. baumannii under hypoxia released larger amounts of OMVs with different protein profiles. Although the cytotoxic effect of AbOMVs from normoxia and hypoxia were comparable, AbOMVs from normoxia induced higher TNF-α production and invasion of Staphylococcus aureus and Pseudomonas aeruginosa than those from hypoxia. On the other hand, AbOMVs significantly enhanced A. baumannii invasion into lung epithelial cells in a dose-dependent manner. These results clearly demonstrate that AbOMVs released from normoxic and hypoxic have different impacts in pathogenesis. This finding provides new insight into the complex interactions between A. baumannii, coinfecting pathogens and host cells via OMVs, in particular the different pathogenic effects of AbOMVs under normoxic and hypoxic conditions.

Bacterial exposure risk to the endoscopist's face while performing endoscopy.

Objectives: Gastrointestinal endoscopy increases the risk of bacterial exposure to endoscopists. However, before 2019, most endoscopists did not pay attention to microorganism transmission from patients. This study aimed to investigate the incidence of bacterial exposure to endoscopists' faces during gastrointestinal endoscopic procedures using the bacterial culture method. Methods: This was a single-centered, retrospective study including endoscopists who performed various gastrointestinal endoscopy procedures at the Division of Endoscopy, Hirosaki University Hospital between August 31 and October 6, 2020. Endoscopists wore surgical masks and affixed pre-sterilized films over them. Following the gastrointestinal endoscopic procedures, attached microbes were collected from the endoscopists' surface films using sterilized swabs. Collected microorganisms were cultured on tryptic soy agar and 5% sheep blood agar, and the incidence of bacterial exposure was determined by bacterial culture positivity. Cultured bacteria were identified by gram staining and 16S rRNA gene sequencing. Results: Bacterial culture positivity was 12.6%, and it was significantly higher in therapeutic than in diagnostic endoscopy. Notably, therapeutic endoscopy increased bacterial culture positivity in colonoscopy, but not in esophagogastroduodenoscopy. Staphylococci, including Staphylococcus epidermidis and Staphylococcus capitis, were the most commonly found bacteria in samples identified through 16S rRNA gene sequencing. Conclusions: The risk of bacterial exposure to the endoscopist's face was increased in colonoscopy treatment procedures. Therefore, endoscopists should be aware of the significant risk of microbial infection from scattering fluid that comes from the endoscopy's working channel.

Effect of ultraviolet C emitted from KrCl excimer lamp with or without bandpass filter to mouse epidermis.

It has been reported that 222-nm ultraviolet C (UVC) exerts a germicidal effect on bacteria and viruses as well as UV radiation emitted from a conventional germicidal lamp but is less toxic to the mammalian cells than that from a germicidal lamp. An excimer lamp filled with krypton chloride (KrCl) gas principally emits 222-nm UVC. However, the lamp also emits a wide band of wavelengths other than 222 nm, especially UVC at a longer wavelength than 222 nm and ultraviolet B, which cause DNA damage. There are some reports on the critical role of bandpass filters in reducing the harmful effect of UVC emitted from a KrCl excimer lamp in a human skin model and human subjects. However, the effectiveness of a bandpass filter has not been demonstrated in animal experiments. In the present study, mice were irradiated with UVC emitted from a KrCl excimer lamp with or without a bandpass filter. UVC emitted from an unfiltered KrCl lamp at doses of 50, 150 and 300 mJ/cm2 induced cyclobutyl pyrimidine dimer (CPD)-positive cells, whereas UVC emitted from a filtered lamp did not significantly increase CPD-positive cells in the epidermis. The present study suggested that the bandpass filter serves a critical role in reducing the harmful effect of emission outside of 222 nm to mouse keratinocytes.

Effects of salmon cartilage proteoglycan on obesity in mice fed with a high-fat diet.

This study investigated the effects of salmon nasal cartilage proteoglycan (PG), which shows anti-inflammatory properties, on obesity induced by high-fat diet (HFD) in a mouse model. Mice were fed either a HFD or normal diet (ND), with or without PG, for 8-12 weeks. After 12 weeks, the body weight of mice fed with PG-free HFD was 54.08 ± 4.67 g, whereas that of mice fed with HFD containing PG was 41.83 ± 4.97 g. The results suggest that the increase in body weight was attenuated in mice fed with HFD containing PG. This effect was not observed in mice fed with ND. The PG administration suppressed the elevation of serum lipids (the level of serum lipids ranged between 54% and 69% compared to 100% in mice fed with PG-free HFD) and the upregulated mRNA expression of sterol regulatory element-binding protein-1c (SREBP-1c), which is a transcription factor that acts as a master regulator of lipogenic gene expression in the liver (the expression level was 77.5% compared to 100% in mice fed with PG-free HFD). High leptin levels in mice fed with PG-free HFD were observed during fasting (average at 14,376 ng/ml), and they did not increase after refeeding (average of 14,263 ng/ml), whereas serum leptin levels in mice fed with HFD containing PG were low during fasting (average of 6481 ng/ml) and increased after refeeding (average 13,382 ng/ml). These results suggest that PG feeding has an anti-obesity effect and that the regulation of SREBP-1c and leptin secretion play a role in this effect.

Extracellular vesicles from methicillin resistant Staphylococcus aureus stimulate proinflammatory cytokine production and trigger IgE-mediated hypersensitivity.

Extracellular vesicles (EVs) released from bacteria are enclosed particles carrying biological active molecules. They have been shown to play a role in bacterial communications and delivery of virulence factors to the host cells. Staphylococcus aureus is an opportunistic pathogen causing a variety of infections ranging from impetigo to septicaemia. The EVs released from S. aureus have a high potential to be used for vaccine development against S. aureus infections. However, it is important to clearly understand the impact of SaEVs on the host's immune response. Our study demonstrated that purified EVs from a clinical isolated methicillin-resistant S. aureus (SaEVs) significantly stimulated proinflammatory cytokine production in mouse immune cells and induced host cell death. An impairment of cytokine production in the Toll-like receptor (TLR)-silenced macrophages suggested that SaEVs stimulate proinflammatory response via TLRs 2, 4 and 9. In mouse infection model, the results demonstrated that SaEV immunization did not provide protective effect. In contrast, all SaEV-immunized mice died within Day 1 after methicillin-resistant S. aureus (MRSA) infection. After MRSA infection for 3 h, the production of IL-6, TNF-α and IL-17 in the spleen of SaEV-immunized mice was significantly higher than that of control mice. On Day 5 after the second immunization, total IgE in the serum was significantly enhanced, and a high titre of Th2-related cytokines was remarkably induced after ex vivo stimulation of the spleen cells with SaEVs. These results suggested that MRSA-derived EVs act as an immunostimulant that induces inflammatory response and IgE-mediated hypersensitivity after MRSA infection.

Investigation of Staphylococcus aureus positive for Staphylococcal enterotoxin S and T genes.

Staphylococcus aureus produces staphylococcal enterotoxins (SEs) and causes food poisoning. It is known that almost all SE-encoding genes are present on various types of mobile genetic elements and can mobilize among S. aureus populations. Further, plasmids comprise one of SE gene carriers. Previously, we reported novel SEs, SES and SET, harbored by the plasmid pF5 from Fukuoka5. In the present study, we analyzed the distribution of these SEs in various S. aureus isolates in Japan. We used 526 S. aureus strains and found 311 strains positive for at least one SE/SE-like toxin gene, but only two strains (Fukuoka5 and Hiroshima3) were positive for ses and set among the specimens. We analyzed two plasmids (pF5 and pH3) from these strains and found that they were different. Whereas these plasmids partially shared similar sequences involved in the ser/selj/set/ses gene cluster, other sequences were different. A comparison of these plasmids with those deposited in the NCBI database revealed that only one plasmid had the ser/selj/set/ses cluster with a stop mutation in set similar to that in pH3. In addition, the chromosomes of Fukuoka5 and Hiroshima3, positive for ses and set, were classified into different genotypes. Despite the low rate of gene positivity for these SEs, it is suggested that there is diversity in plasmids and strains carrying these two SEs. Consequently, regarding the entire feature of SE prevalence, we improved the multiplex PCR detection method for the SE superfamily to obtain further insight.

Complete Genome Sequence of Staphylococcus aureus Strain 834, Isolated from a Septic Patient in Japan.

Here, we report the complete genome sequence of Staphylococcus aureus strain 834, which was isolated from a septic patient in Japan and showed strong virulence and methicillin resistance. The complete genome consists of a 2,838,668-bp chromosome and a 24,653-bp plasmid. Genome annotation predicts 2,670 coding sequences, 16 rRNAs, and 61 tRNAs.

A novel staphylococcal enterotoxin SE02 involved in a staphylococcal food poisoning outbreak that occurred in Tokyo in 2004.

Staphylococcal enterotoxins (SEs) are extracellular proteins, produced mainly by Staphylococcus aureus, which cause staphylococcal food poisoning (SFP) when ingested. Here, a novel SE was identified from two strains, which were identified as the causative microbes of the SFP outbreak that occurred in Tokyo in 2004. Both strains harbored the SEA gene, but its production was lower than that of other SEA-producing SFP isolates. Whole-genome sequencing analysis demonstrated that both strains harbored a SE-like gene besides sea. Phylogenetic analysis revealed that the amino acid sequence deduced from the SE-like gene belonged to the SEB group. Therefore, this gene was presumed to be a novel SE gene and termed "SE02." The stability of SE02 against heating and proteolytic digestions was a little different from that of SEA. SE02 has both superantigenic and emetic bioactivities. Namely, SE02 activated mouse splenocytes and exhibited emetic activity in the common marmoset. SE02 mRNA was highly expressed in both isolates during the exponential phase of cultivation. In addition, SE02 protein was produced at 20 °C and 25 °C, which reflects the actual situation of SFP. SE02 appears to be a novel emetic toxin that was likely the causative toxin in combination with SEA in the SFP outbreak.

222-nm UVC inactivates a wide spectrum of microbial pathogens.

BACKGROUND: UVC has been used to inactivate several pathogens. Unlike the conventional 254-nm UVC, 222-nm UVC is harmless to mammalian cells. AIM: To investigate the disinfection efficacy of 222-nm UVC against human pathogens which are commonly found in the environment and healthcare facilities. METHODOLOGY: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica subsp. serovar Typhimurium, Campylobacter jejuni, Bacillus cereus (vegetative cells and endospores), Clostridium sporogenes (vegetative cells and endospores), Clostoridioides difficile (endospores), Candida albicans (yeast), Aspergillus niger (hyphae and spores), Trichophyton rubrum (hyphae and spores), feline calicivirus and influenza A virus were irradiated with 222-nm UVC at various doses. The remaining live bacterial and fungal cells, and the viral infectivity were evaluated. The efficiency of 222-nm UVC germicidal effect was compared to that of the conventional 254-nm UVC. RESULTS: The 222-nm UVC showed potent germicidal effect to vegetative bacterial cells, yeast and viruses as efficient as the 245-nm UVC. The 222-nm UVC exhibited more potent germicidal effect to bacterial endospores, compared with the 254-nm UVC. The fungicidal effect of 222-nm UVC against the fungal spores and hyphae was weaker than that of 254-nm UVC. CONCLUSIONS: The 222-nm UVC is able to inactivate a wide spectrum of microbial pathogens. In comparison with the conventional 254-nm UVC, the germicidal effect of 222-nm UVC to the fungal hyphae and spores is low, but the 222-nm UVC exhibits strong germicidal effect to the bacterial endospores.

Staphylococcus aureus Isolated from Skin from Atopic-Dermatitis Patients Produces Staphylococcal Enterotoxin Y, Which Predominantly Induces T-Cell Receptor Vα-Specific Expansion of T Cells.

While investigating the virulence traits of Staphylococcus aureus adhering to the skin of atopic-dermatitis (AD) patients, we identified a novel open reading frame (ORF) with structural similarity to a superantigen from genome sequence data of an isolate from AD skin. Concurrently, the same ORF was identified in a bovine isolate of S. aureus and designated SElY (H. K. Ono, Y. Sato'o, K. Narita, I. Naito, et al., Appl Environ Microbiol 81:7034-7040, 2015, https://doi.org/10.1128/AEM.01873-15). Recombinant SElYbov had superantigen activity in human peripheral blood mononuclear cells. It further demonstrated emetic activity in a primate animal model, and it was proposed that SElY be renamed SEY (H. K. Ono, S. Hirose, K. Narita, M. Sugiyama, et al., PLoS Pathog 15:e1007803, 2019, https://doi.org/10.1371/journal.ppat.1007803). Here, we investigated the prevalence of the sey gene in 270 human clinical isolates of various origins in Japan. Forty-two strains were positive for the sey gene, and the positive isolates were from patients with the skin diseases atopic dermatitis and impetigo/staphylococcal scalded skin syndrome (SSSS), with a detection rate of ∼17 to 22%. There were three variants of SEY (SEY1, SEY2, and SEY3), and isolates producing SEY variants formed three distinct clusters corresponding to clonal complexes (CCs) 121, 59, and 20, respectively. Most sey+ isolates produced SEY in broth culture. Unlike SEYbov, the three recombinant SEY variants exhibited stability against heat treatment. SEY predominantly activated human T cells with a particular T-cell receptor (TCR) Vα profile, a unique observation since most staphylococcal enterotoxins exert their superantigenic activities through activating T cells with specific TCR Vß profiles. SEY may act to induce localized inflammation via skin-resident T-cell activation, facilitating the pathogenesis of S. aureus infection in disrupted epithelial barriers.

Interleukin-10 (IL-10) Produced by Mutant Toxic Shock Syndrome Toxin 1 Vaccine-Induced Memory T Cells Downregulates IL-17 Production and Abrogates the Protective Effect against Staphylococcus aureus Infection.

Development of long-term memory is crucial for vaccine-induced adaptive immunity against infectious diseases such as Staphylococcus aureus infection. Toxic shock syndrome toxin 1 (TSST-1), one of the superantigens produced by S. aureus, is a possible vaccine candidate against infectious diseases caused by this pathogen. We previously reported that vaccination with less toxic mutant TSST-1 (mTSST-1) induced T helper 17 (Th17) cells and elicited interleukin-17A (IL-17A)-mediated protection against S. aureus infection 1 week after vaccination. In the present study, we investigated the host immune response induced by mTSST-1 vaccination in the memory phase, 12 weeks after the final vaccination. The protective effect and IL-17A production after vaccination with mTSST-1 were eliminated because of IL-10 production. In the presence of IL-10-neutralizing monoclonal antibody (mAb), IL-17A production was restored in culture supernatants of CD4+ T cells and macrophages sorted from the spleens of vaccinated mice. Vaccinated mice treated with anti-IL-10 mAb were protected against systemic S. aureus infection in the memory phase. From these results, it was suggested that IL-10 produced in the memory phase suppresses the IL-17A-dependent vaccine effect through downregulation of IL-17A production.

Histamine release from intestinal mast cells induced by staphylococcal enterotoxin A (SEA) evokes vomiting reflex in common marmoset.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are known as causative agents of emetic food poisoning. We previously demonstrated that SEA binds with submucosal mast cells and evokes mast cell degranulation in a small emetic house musk shrew model. Notably, primates have been recognized as the standard model for emetic assays and analysis of SE emetic activity. However, the mechanism involved in SEA-induced vomiting in primates has not yet been elucidated. In the present study, we established common marmosets as an emetic animal model. Common marmosets were administered classical SEs, including SEA, SEB and SEC, and exhibited multiple vomiting responses. However, a non-emetic staphylococcal superantigen, toxic shock syndrome toxin-1, did not induce emesis in these monkeys. These results indicated that the common marmoset is a useful animal model for assessing the emesis-inducing activity of SEs. Furthermore, histological analysis uncovered that SEA bound with submucosal mast cells and induced mast cell degranulation. Additionally, ex vivo and in vivo pharmacological results showed that SEA-induced histamine release plays a critical role in the vomiting response in common marmosets. The present results suggested that 5-hydroxytryptamine also plays an important role in the transmission of emetic stimulation on the afferent vagus nerve or central nervous system. We conclude that SEA induces histamine release from submucosal mast cells in the gastrointestinal tract and that histamine contributes to the SEA-induced vomiting reflex via the serotonergic nerve and/or other vagus nerve.

Staphylococcal Enterotoxin C Is an Important Virulence Factor for Mastitis.

Staphylococcus aureus is an important bacterial pathogen causing bovine mastitis, but little is known about the virulence factor and the inflammatory responses in the mammary infection. Staphylococcal enterotoxin C (SEC) is the most frequent toxin produced by S. aureus, isolated from bovine mastitis. To investigate the pathogenic activity of SEC in the inflammation of the mammary gland and the immune responses in an animal model, mouse mammary glands were injected with SEC, and the clinical signs, inflammatory cell infiltration, and proinflammatory cytokine production in the mammary glands were assessed. SEC induced significant inflammatory reactions in the mammary gland, in a dose-dependent manner. SEC-injected mammary glands showed a severe inflammation with inflammatory cell infiltration and tissue damage. In addition, interleukin (IL)-1ß and IL-6 production in the SEC-injected mammary glands were significantly higher than those in the PBS control glands. Furthermore, the SEC-induced inflammation and tissue damage in the mammary gland were specifically inhibited by anti-SEC antibody. These results indicated, for the first time, that SEC can directly cause inflammation, proinflammatory cytokine production, and tissue damage in mammary glands, suggesting that SEC might play an important role in the development of mastitis associated with S. aureus infection. This finding offers an opportunity to develop novel treatment strategies for reduction of mammary tissue damage in mastitis.

Development of an Immunoassay for Detection of Staphylococcal Enterotoxin-Like J, A Non-Characterized Toxin.

Staphylococcal enterotoxins (SEs) are the cause of staphylococcal food poisoning (SFP) outbreaks. Recently, many new types of SEs and SE-like toxins have been reported, but it has not been proved whether these new toxins cause food poisoning. To develop an immunoassay for detection of SE-like J (SElJ), a non-characterized toxin in SFP, a mutant SElJ with C-terminus deletion (SElJ∆C) was expressed and purified in an E. coli expression system. Anti-SElJ antibody was produced in rabbits immunized with the SElJ∆C. Western blotting and sandwich enzyme-linked immunosorbent assay (ELISA) detection systems were established and showed that the antibody specifically recognizes SElJ without cross reaction to other SEs tested. The limit of detection for the sandwich ELISA was 0.078 ng/mL, showing high sensitivity. SElJ production in S. aureus was detected by using the sandwich ELISA and showed that selj-horboring isolates produced a large amount of SElJ in the culture supernatants, especially in that of the strain isolated from a food poisoning outbreak in Japan. These results demonstrate that the immunoassay for detection of SElJ is specific and sensitive and is useful for determining the native SElJ production in S. aureus isolated from food poisoning cases.

Salmon cartilage proteoglycan attenuates allergic responses in mouse model of papain­induced respiratory inflammation.

Proteoglycan (PG) is a complex glycohydrate, which is widely distributed in the extracellular matrix. It has been reported that daily oral administration of PG (extracted from salmon nasal cartilage) modulates the severity of proinflammatory cytokine responses in mouse experimental colitis, autoimmune encephalomyelitis, collagen­induced arthritis and obesity­induced inflammation. The present study investigated the effect of salmon nasal cartilage PG on allergic responses using a mouse model of papain­induced respiratory inflammation. Low titers of immunoglobulin E were identified in the sera of the PG­administered mice. Oral administration of PG attenuated eosinophil infiltration in the lung. In the acute model of papain­induced allergic inflammation, PG­administered mice exhibited low titers of epithelium­derived and T helper 2­associated cytokines. The results of the present study demonstrated that salmon cartilage PG has an immunomodulatory effect on intranasally delivered papain. These results suggest a potential role for PG as a prophylactic agent which may attenuate allergic respiratory inflammation.