Differential effects of dexamethasone and roflumilast on asthma in mice with or without short cigarette smoke exposure.

Appropriate drug treatment for smoking asthmatics is uncertain because most smokers with asthma are less sensitive to treatment with glucocorticoids compared with non-smokers with asthma. We hypothesized that roflumilast (Rof), a selective phosphodiesterases-4 inhibitor regarded as an add-on therapy for chronic obstructive pulmonary disease, might be more effective than glucocorticoids for improving asthma in smokers. To investigate this hypothesis, we compared the therapeutic effects of dexamethasone (Dex) and Rof in a mouse model of ovalbumin-induced asthma with or without concurrent cigarette smoke (CS) exposure for 2 weeks. We found that recurrent asthma attacks increased lung tissue resistance. CS exposure in asthmatic mice decreased the central airway resistance, increased lung compliance, and attenuated airway hyper-responsiveness (AHR). CS exposure in asthmatic mice also increased the number of neutrophils and macrophages in the bronchoalveolar fluid. Treatment with Dex in asthmatic mice without CS exposure reduced airway resistance, AHR and airway eosinophilia. In asthmatic mice with CS exposure, however, Dex treatment unexpectedly increased lung tissue resistance and restored AHR that had been otherwise suppressed. Dex treatment in asthmatic mice with CS exposure inhibited eosinophilic inflammation but conversely exacerbated neutrophilic inflammation. On the other hand, treatment with Rof in asthmatic mice without CS exposure reduced airway resistance and airway eosinophilia, although the inhibitory effect of Rof on AHR was unremarkable. In asthmatic mice with CS exposure, Rof treatment did not exacerbate lung tissue resistance but modestly restored AHR, without any significant effects on airway inflammation. These results suggest that CS exposure mitigates sensitivity to both Dex and Rof. In asthmatic mice with CS exposure, Dex is still effective in reducing eosinophilic inflammation but increases lung tissue resistance, AHR and neutrophilic inflammation. Rof is ineffective in improving lung function and inflammation in asthmatic mice with CS exposure. This study did not support our initial hypothesis that Rof might be more effective than glucocorticoids for improving asthma in smokers. However, glucocorticoids may have a detrimental effect on smoking asthmatics.

Hypercapnia Accelerates Adipogenesis: A Novel Role of High CO2 in Exacerbating Obesity.

Obesity is a major risk factor for the development of obstructive sleep apnea (OSA) and obesity hypoventilation syndrome (OHS), which manifest as intermittent hypercapnia and sustained plus intermittent hypercapnia, respectively. In this study, we investigated whether CO2 affects adipocyte differentiation (adipogenesis) and maturation (hypertrophy). Human visceral or subcutaneous preadipocytes were grown to confluence and then induced to differentiate to adipocytes under hypocapnia, normocapnia, and hypercapnia with or without hypoxia. Adipogenesis was also induced under intermittent or sustained hypercapnia. Differentiated adipocytes were maintained to maturity under normocapnia or hypercapnia. Our main findings are as follows: (1) hypercapnia accelerated adipogenesis in visceral and subcutaneous preadipocytes, whereas hypocapnia inhibited adipogenesis; (2) hypercapnia did not affect adipocyte hypertrophy; (3) hypercapnia-accelerated adipogenesis was independent of extracellular acidosis, oxygen concentration, or either intermittent or sustained exposure to high CO2; and (4) the mechanisms underlying hypercapnia-accelerated adipogenesis involved increased production of cyclic adenosine monophosphate (cAMP) via soluble adenylyl cyclase, leading to the activation of protein kinase A and exchanger protein directly activated by cAMP, which, in turn, activated proadipogenic transcription factors, such as cAMP response element binding protein, CCAAT/enhancer binding protein ß, and peroxisome proliferator-activated receptor γ. This study reveals a novel role of high CO2 in promoting adipogenesis, which provides mechanistic clues to a pathoetiological interaction between OSA/OHS and obesity. Our data suggest a vicious cycle of disease progression via the following mechanism: OSA/OHS → hypoventilation → hypercapnia → increased adipogenesis → increased fat mass → exacerbated OSA/OHS.

Molecular epidemiology of rotavirus gastroenteritis in Central Kenya before vaccine introduction, 2009-2014.

Between July 2009 and June 2014, a total of 1,546 fecal specimens were collected from children <5 years of age with acute gastroenteritis admitted to Kiambu County Hospital, Central Kenya. The specimens were screened for group A rotavirus (RVA) using ELISA, and RVA-positive specimens were subjected to semi-nested RT-PCR to determine the G and P genotypes. RVA was detected in 429/1,546 (27.5%) fecal specimens. RVA infections occurred in all age groups <59 months, with an early peak at 6-17 months. The infections persisted year-round with distinct seasonal peaks depending on the year. G1P[8] (28%) was the most predominant genotype, followed by G9P[8] (12%), G8P[4] (7%), G1P[4] (5%), G9P[4] (4%), and G12P[6] (3%). In the yearly change of G and P genotypes, a major shift from G9P[8] to G1P[8] was found in 2012. Phylogenetic analysis of the nucleotide sequences of the VP7 and VP4 genes of seven strains with unusual G8 or P[6] showed that the VP7 nucleotide sequences of G8 were clustered in lineage 6 in which African strains are included, and that there are at least two distinct VP4 nucleotide sequences of P[6] strains. These results represent basic data on RVA strains circulating in this region before vaccine introduction. J. Med. Virol. 89:809-817, 2017. © 2016 Wiley Periodicals, Inc.

Predominant prevalence of human rotaviruses with the G1P[8] and G8P[8] genotypes with a short RNA profile in 2013 and 2014 in Sukhothai and Phetchaboon provinces, Thailand.

Of 2,754 stool specimens collected from children with acute gastroenteritis during 2013-2014 in Sukhothai and Phetchaboon provinces, Thailand, 666 (24.2%) were positive for rotavirus A (RVA) in polyacrylamide gel electrophoresis (PAGE). The G and P types of all RVA-positive specimens were determined by semi-nested RT-PCR. G1P[8] (56.5%) was most prevalent, followed by G2P[4] (22.1%). Unusual G8P[8] human RVAs (HuRVAs) were detected at a high frequency (20.0%). Interestingly, 171 of the 376 G1P[8] HuRVAs and all of the 133 G8P[8] HuRVAs showed a short RNA pattern in PAGE. Thus, it was shown that the properties of HuRVAs have been markedly unusual in recent years in Thailand. J. Med. Virol. 89:615-620, 2017. © 2016 Wiley Periodicals, Inc.

Improvement of metabolic disorders by an EP2 receptor agonist via restoration of the subcutaneous adipose tissue in pulmonary emphysema.

Chronic obstructive pulmonary disease (COPD) is often associated with co-morbidities. Metabolic disorders like hyperlipidemia and diabetes occur also in underweight COPD patients, although the mechanism is uncertain. Subcutaneous adipose tissue (SAT) plays an important role in energy homeostasis, since restricted capacity to increase fat cell number with increase in fat cell size occurring instead, is associated with lipotoxicity and metabolic disorders. The aim of this study is to show the protective role of SAT for the metabolic disorders in pulmonary emphysema of a murine model. We found ectopic fat accumulation and impaired glucose homeostasis with wasting of SAT in a murine model of elastase-induced pulmonary emphysema (EIE mice) reared on a high-fat diet. ONO-AE1-259, a selective E-prostanoid (EP) 2 receptor agonist, improved angiogenesis and subsequently adipogenesis, and finally improved ectopic fat accumulation and glucose homeostasis with restoration of the capacity for storage of surplus energy in SAT. These results suggest that metabolic disorders like hyperlipidemia and diabetes occured in underweight COPD is partially due to the less capacity for storage of surplus energy in SAT, though the precise mechanism is uncertained. Our data pave the way for the development of therapeutic interventions for metabolic disorders in emphysema patients, e.g., use of pro-angiogenic agents targeting the capacity for storage of surplus energy in the subcutaneous adipose tissue.

Development of a simple and rapid diagnosis method for swine edema disease to specifically detect Stx2e protein by immunochromatographic test.

Edema disease in piglets is caused by Shiga toxin 2e (Stx2e)-producing Escherichia coli. However, there is currently no available Stx2e-specific immunochromatographic test strip to differentiate Stx2e from other types of Shiga toxin 2. In the present study, to develop an Stx2e-specific immunochromatographic test strip, we isolated nine different monoclonal antibody-producing hybridoma clones from Stx2e toxoid-immunized mice and confirmed that six antibodies were A subunit-specific whereas three antibodies were B subunit-specific. Only one A subunit-specific monoclonal antibody (45B2) was cross-reactive with prototype Stx2 (Stx2a) at the same sensitivity, but the remaining eight monoclonal antibodies were not. In immunochromatographic tests using the highly sensitive antibodies, test strips using some combinations of gold colloid-conjugated monoclonal antibody with the B subunit-specific monoclonal antibody on the membrane detected Stx2e, but not other types of Shiga toxin 2. These test strips had the ability to detect Stx2e in the culture supernatant of clinically isolated Stx2e gene-positive strains, but not in those of Stx2e gene-negative strains. These results indicate that our test strip is practical for the specific detection of Stx2e to diagnose swine edema disease.

Problems of elderly patients on inhalation therapy: Difference in problem recognition between patients and medical professionals.

BACKGROUND: There is no systematic analysis to identify problems involved with instruction on inhalation therapy for elderly patients. We conducted a nationwide questionnaire survey for patients and medical professionals. METHODS: A questionnaire survey was conducted of adult patients on inhaled drugs (ages 18-92 years, 820 individuals) and medical professionals (pharmacists or nurses) who provided instruction on inhalation therapy to these patients in 23 institutions in Japan to investigate the technique and the level of understanding (knowledge) of the inhalation therapy. Changes in the recognition of performance of inhalation technique and inhalation knowledge with increasing age were analyzed. RESULTS: According to patients' subjective assessment, there was no deterioration in the performance of the inhalation technique or loss of the knowledge with increasing age. On the other hand, medical professionals' objective assessment revealed a significant loss of both inhalation technique and knowledge with increasing age. Not many elderly patients noticed their own problems themselves, revealing a great perception gap between elderly patients and medical professionals. Thus, there was concern that patients would unconsciously practice the inhalation procedure improperly. On the other hand, in comparison with non-elderly patients, elderly patients were less resistant to continuation of therapy, suggesting that they would be more likely to accept instruction on inhalation therapy. CONCLUSIONS: Elderly patients are apt to assume that they "understand well", therefore, in order to recognize and close the perception gap between elderly patients and medical professionals, it is necessary to provide them with more aggressive (frequent) instructions on inhalation therapy.

Isolation of B subunit-specific monoclonal antibody clones that strongly neutralize the toxicity of Shiga toxin 2.

Shiga toxin 2 (Stx2)-specific mAb-producing hybridoma clones were generated from mice. Because mice tend to produce small amounts of B subunit (Stx2B)-specific antibodies at the polyclonal antibody level after immunization via the parenteral route, mice were immunized intranasally with Stx2 toxoids with a mutant heat-labile enterotoxin as a mucosal adjuvant; 11 different hybridoma clones were obtained in two trials. Six of them were A subunit (Stx2A)-specific whereas five were Stx2B-specific antibody-producing clones. The in vitro neutralization activity of Stx2B-specific mAbs against Stx2 was greater than that of Stx2A-specific mAbs on HeLa229 cells. Furthermore, even at low concentrations two of the Stx2B-specific mAbs (45 and 75D9) completely inhibited receptor binding and showed in vivo neutralization activity against a fivefold median lethal dose of Stx2 in mice. In western blot analysis, these Stx2B-specific neutralization antibodies did not react to three different mutant forms of Stx2, each amino acid residue of which was associated with receptor binding. Additionally, the nucleotide sequences of the VH and VL regions of clones 45 and 75D9 were determined. Our Stx2B-specific mAbs may be new candidates for the development of mouse-human chimeric Stx2-neutralizing antibodies which have fewer adverse effects than animal antibodies for enterohemorrhagic Escherichia coli infection.

An evolutionary medicine approach to understanding factors that contribute to chronic obstructive pulmonary disease.

Although many studies have been published on the causes and mechanisms of chronic obstructive pulmonary disease (COPD), the reason for the existence of COPD and the reasons why COPD develops in humans have hardly been studied. Evolutionary medical approaches are required to explain not only the proximate factors, such as the causes and mechanisms of a disease, but the ultimate (evolutionary) factors as well, such as why the disease is present and why the disease develops in humans. According to the concepts of evolutionary medicine, disease susceptibility is acquired as a result of natural selection during the evolutionary process of traits linked to the genes involved in disease susceptibility. In this paper, we discuss the following six reasons why COPD develops in humans based on current evolutionary medical theories: (1) evolutionary constraints; (2) mismatch between environmental changes and evolution; (3) co-evolution with pathogenic microorganisms; (4) life history trade-off; (5) defenses and their costs, and (6) reproductive success at the expense of health. Our perspective pursues evolutionary answers to the fundamental question, 'Why are humans susceptible to this common disease, COPD, despite their long evolutionary history?' We believe that the perspectives offered by evolutionary medicine are essential for researchers to better understand the significance of their work.

Clinical factors associated with negative urinary antigen tests implemented for the diagnosis of community-acquired pneumococcal pneumonia in adult patients.

OBJECTIVE: This study investigated clinical factors associated with negative urinary antigen tests (UAT) implemented for the diagnosis of pneumococcal community-acquired pneumonia (CAP) in adult patients. SUBJECTS AND METHODS: We reviewed the medical records of 755 adult patients who completed the UAT in our hospital between 2009 and 2012. Of these, we evaluated 63 patients with bacteriologically confirmed definite pneumococcal CAP (33 were UAT-positive, and 30 were UAT-negative). RESULTS: There was no significant difference between the UAT-positive and the UAT-negative patients regarding age, dehydration, respiratory failure, orientation, blood pressure (ADROP) score (the CAP severity score proposed by the Japanese Respiratory Society), gender, white blood cell counts, liver/kidney function tests, or urinalysis. However, serum C-reactive protein (CRP) concentrations were 31% lower in the UAT-negative patients than in the UAT-positive patients (p = 0.02). Furthermore, the prothrombin time-international normalized ratio was 50% higher in the UAT-negative patients than in the UAT-positive patients, although the difference did not reach statistical significance (p = 0.06). The prevalence of comorbidities was similar in both UAT-positive and UAT-negative patients. However, warfarin had been prescribed in 8 (27%) of the UAT-negative patients compared to only 1 (3%) of the UAT-positive patients (odds ratio = 11.6; p = 0.01). CONCLUSIONS: These results suggested that low serum CRP concentrations and the use of warfarin increased the possibility with which false-negative UAT results occurred in these patients with pneumococcal CAP.

Macrophage elastase suppresses white adipose tissue expansion with cigarette smoking.

Macrophage elastase (MMP12) is a key mediator of cigarette smoke (CS)-induced emphysema, yet its role in other smoking related pathologies remains unclear. The weight suppressing effects of smoking are a major hindrance to cessation efforts, and MMP12 is known to suppress the vascularization on which adipose tissue growth depends by catalyzing the formation of antiangiogenic peptides endostatin and angiostatin. The goal of this study was to determine the role of MMP12 in adipose tissue growth and smoking-related suppression of weight gain. Whole body weights and white adipose depots from wild-type and Mmp12-deficient mice were collected during early postnatal development and after chronic CS exposure. Adipose tissue specimens were analyzed for angiogenic and adipocytic markers and for content of the antiangiogenic peptides endostatin and angiostatin. Cultured 3T3-L1 adipocytes were treated with adipose tissue homogenate to examine its effects on vascular endothelial growth factor (VEGF) expression and secretion. MMP12 content and activity were increased in the adipose tissue of wild-type mice at 2 weeks of age, leading to elevated endostatin production, inhibition of VEGF secretion, and decreased adipose tissue vascularity. By 8 weeks of age, adipose MMP12 levels subsided, and the protein was no longer detectable. However, chronic CS exposure led to macrophage accumulation and restored adipose MMP12 activity, thereby suppressing adipose tissue mass and vascularity. Our results reveal a novel systemic role for MMP12 in postnatal adipose tissue expansion and smoking-associated weight loss by suppressing vascularity within the white adipose tissue depots.

Promotion of adipogenesis by an EP2 receptor agonist via stimulation of angiogenesis in pulmonary emphysema.

Body weight loss is a common manifestation in patients with chronic obstructive pulmonary disease (COPD), particularly those with severe emphysema. Adipose angiogenesis is a key mediator of adipogenesis and use of pro-angiogenic agents may serve as a therapeutic option for lean COPD patients. Since angiogenesis is stimulated by PGE2, we examined whether ONO-AE1-259, a selective E-prostanoid (EP) 2 receptor agonist, might promote adipose angiogenesis and adipogenesis in a murine model of elastase-induced pulmonary emphysema (EIE mice). Mice were intratracheally instilled with elastase or saline, followed after 4 weeks by intraperitoneal administration of ONO-AE1-259 for 4 weeks. The subcutaneous adipose tissue (SAT) weight decreased in the EIE mice, whereas in the EIE mice treated with ONO-AE1-259, the SAT weight was largely restored, which was associated with significant increases in SAT adipogenesis, angiogenesis, and VEGF protein production. In contrast, ONO-AE1-259 administration induced no alteration in the weight of the visceral adipose tissue. These results suggest that in EIE mice, ONO-AE1-259 stimulated adipose angiogenesis possibly via VEGF production, and thence, adipogenesis. Our data pave the way for the development of therapeutic interventions for weight loss in emphysema patients, e.g., use of pro-angiogenic agents targeting the adipose tissue vascular component.

Evaluation of Shiga toxin 2e-specific chicken egg yolk immunoglobulin: production and neutralization activity.

Chicken egg yolk immunoglobulin (IgY) against Shiga toxin 2e (Stx2e), a major cause of swine edema disease, was prepared to evaluate its possible clinical applications. The titer of Stx2e-specific IgY in egg yolk derived from three chickens that had been immunized with an Stx2e toxoid increased 2 weeks after primary immunization and remained high until 90 days after this immunization. Anti-Stx2e IgY was found to neutralize the toxicity of Stx2e by reacting with its A and B subunits, indicating that IgY is a cost-effective agent to develop for prophylactic foods or diagnosis kits for edema disease.

Systemic effects of acute cigarette smoke exposure in mice.

CONTEXT: Cigarette smoke (CS) causes both pulmonary and extrapulmonary disorders. OBJECTIVE: To determine the pulmonary and extrapulmonary effects of acute CS exposure in regard to inflammation, oxidative stress and DNA damage. MATERIALS AND METHODS: Mice were exposed to CS for 10 days and then their lungs, heart, liver, pancreas, kidneys, gastrocnemius muscle and subcutaneous (inguinal and flank) and visceral (retroperitoneum and periuterus) adipose tissues were excised. Bronchoalveolar lavage fluid samples were obtained for differential cell analysis. Inflammatory cell infiltration of the tissues was assessed by immunohistochemistry for Mac-3(+) cells, F4/80(+) cells and CD45(+) cells. Oxidative stress was determined by immunohistochemistry for thymidine glycol (a marker of DNA peroxidation) and 4-hydroxy hexenal (a marker of lipid peroxidation), by enzyme-linked immunosorbent assay for protein carbonyls (a marker of protein peroxidation) and by measurements of enzyme activities of glutathione peroxidase, superoxide dismutase and catalase. DNA double-strand breaks were assessed by immunohistochemistry for γH2AX. RESULTS: CS exposure-induced inflammatory cell infiltration, oxidative stress and DNA damage in the lung. Neither inflammatory cell infiltration nor DNA damage was observed in any extrapulmonary organs. However, oxidative stress was increased in the heart and inguinal adipose tissue. DISCUSSIONS: Induction of inflammatory cell infiltration and DNA damage by acute CS exposure was confined to the lung. However, an increased oxidative burden occurred in the heart and some adipose tissue, as well as in the lung. CONCLUSIONS: Although extrapulmonary effects of CS are relatively modest compared with the pulmonary effects, some extrapulmonary organs are vulnerable to CS-induced oxidative stress.

The danger signal plus DNA damage two-hit hypothesis for chronic inflammation in COPD.

Inflammation in chronic obstructive pulmonary disease (COPD) is thought to originate from the activation of innate immunity by a danger signal (first hit), although this mechanism does not readily explain why the inflammation becomes chronic. Here, we propose a two-hit hypothesis explaining why inflammation becomes chronic in patients with COPD. A more severe degree of inflammation exists in the lungs of patients who develop COPD than in the lungs of healthy smokers, and the large amounts of reactive oxygen species and reactive nitrogen species released from inflammatory cells are likely to induce DNA double-strand breaks (second hit) in the airways and pulmonary alveolar cells, causing apoptosis and cell senescence. The DNA damage response and senescence-associated secretory phenotype (SASP) are also likely to be activated, resulting in the production of pro-inflammatory cytokines. These pro-inflammatory cytokines further stimulate inflammatory cell infiltration, intensifying cell senescence and SASP through a positive-feedback mechanism. This vicious cycle, characterised by mutually reinforcing inflammation and DNA damage, may cause the inflammation in COPD patients to become chronic. Our hypothesis helps explain why COPD tends to occur in the elderly, why the inflammation worsens progressively, why inflammation continues even after smoking cessation, and why COPD is associated with lung cancer.

Large-scale preparation of Shiga toxin 2 in Escherichia coli for toxoid vaccine antigen production.

Enterohemorrhagic Escherichia coli (EHEC) causes hemorrhagic colitis, and in more severe cases, a serious clinical complication called hemolytic uremic syndrome (HUS). Shiga toxin (Stx)is one of the factors that cause HUS. Serotypes of Stx produced by EHEC include Stx1 and Stx2. Although some genetically mutated toxoids of Stx have been developed, large-scale preparation of Stx that is practical for vaccine development has not been reported. Therefore, overexpression methods for Stx2 and mutant Stx2 (mStx2) in E. coli were developed. The expression plasmid pBSK-Stx2(His) was constructed by inserting the full-length Stx2 gene, in which a six-histidine tag gene was fused at the end of the B subunit into the lacZα fragment gene of the pBluescript II SK(+) vector. An E. coli MV1184 strain transformed with pBSK-Stx2(His) overexpressed histidine-tagged Stx2 (Stx2-His) in cells cultured in CAYE broth in the presence of lincomycin. Stx2-His was purified using TALON metal affinity resin followed by hydroxyapatite chromatography. From 1 L of culture, 68.8 mg of Stx2-His and 61.1 mg of mStx2-His, which was generated by site-directed mutagenesis, were obtained. Stx2-His had a cytotoxic effect on HeLa cells and was lethal to mice. However, the toxicity of mStx2-His was approximately 1000-fold lower than that of Stx2-His. Mice immunized with mStx2-His produced specific antibodies that neutralized the toxicity of Stx2 in HeLa cells. Moreover, these mice survived challenge with high doses of Stx2-His. Therefore, the lincomycin-inducible overexpression method is suitable for large-scale preparation of Stx2 vaccine antigens.

DNA damage as a molecular link in the pathogenesis of COPD in smokers.

In this study, we investigated whether DNA double-strand breaks (DSBs) contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD). We immunofluorescence-stained lung tissue samples obtained from COPD patients, asymptomatic smokers and nonsmokers for markers of DSBs. The numbers of DSB foci (phosphorylated histone 2AX (γH2AX), phosphorylated ATM (ataxia telangiectasia mutated) substrate and phosphorylated p53-binding protein-1 foci) per cell in alveolar type I and II cells and endothelial cells were higher in the COPD patients than in the asymptomatic smokers and nonsmokers. The lung tissue in which type II cells contained higher numbers of γH2AX foci per cell had higher percentages of type II cells that expressed p16(INK4a) (p16), phosphorylated nuclear factor (NF)-κB and interleukin (IL)-6, and of alveolar wall cells that expressed active caspase-3. The type II cells that contained higher numbers of γH2AX foci per cell had higher rates of expression of p16, phosphorylated NF-κB, and IL-6. Half of the alveolar wall cells that expressed active-caspase-3 contained γH2AX foci. Type II cells that stained positive for 8-hydroxy-2-deoxyguanosine contained a higher number of γH2AX foci per cell than the type II cells that stained negative. In conclusion, DSBs, at least in part caused by oxidative stress, appear to contribute to the pathogenesis of COPD by inducing apoptosis, cell senescence and pro-inflammatory responses.

Staphylococcal enterotoxin A has potent superantigenic and emetic activities but not diarrheagenic activity.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus are pyrogenic superantigenic toxins that are involved in human diseases including food poisoning and toxic shock syndrome. Although the superantigenic activity of SEs has been well characterized, its role and mechanism in clinical symptoms of food poisoning remain poorly understood. In this study, house musk shrews (Suncus murinus), a small emetic animal model, were used to study the role of SEs in clinical manifestations of food poisoning. Administration of SEA induced a potent emetic response in vivo and showed significant superantigenic activity in vitro in house musk shrews. However, SEA revealed no diarrheagenic activity. SEA directly injected into the intestinal loops of house musk shrews failed to induce fluid exudation and consequent dilation of the intestinal segments. Rabbit intestinal loop experiments were further carried out to confirm the results and also showed that SEA induced no fluid exudation and consequent dilation. Furthermore, the SEA-producing S. aureus also failed to induce fluid exudation in the administered loops of these animal models. These results indicate that SEA has potent superantigenic and emetic activities, but does not have a diarrheagenic activity.

P19 embryonal carcinoma cells exhibit high sensitivity to botulinum type C and D/C mosaic neurotoxins.

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release at peripheral nerve terminals. They are serologically classified from A to G, C/D and D/C mosaic neurotoxins forming further subtypes of serotypes C and D. Cultured primary neurons, as well as neuronal cell lines such as PC12 and Neuro-2a, are often utilized in cell-based experiments on the toxic action of botulinum toxins. However, there are very few reports of the use of neural cell lines for studying BoNTs/C and D. In addition, the differentiated P19 neuronal cell line, which possesses cholinergic properties, has yet to be tested for its susceptibility to BoNTs. Here, the responsiveness of differentiated P19 cells to BoNT/C and BoNT/DC is reported. Both BoNT/C and BoNT/DC were shown to effectively bind to, and be internalized by, neurons derived from P19 cells. Subsequently, the intracellular substrates for BoNT/C and BoNT/DC were cleaved by treatment of the cells with the toxins in a ganglioside-dependent manner. Moreover, P19 neurons exhibited high sensitivity to BoNT/C and BoNT/DC, to the same extent as cultured primary neurons. These findings suggest that differentiated P19 cells possess full sensitivity to BoNT/C and BoNT/DC, thus making them a novel susceptible cell line for research into BoNTs.

Recombinant Shiga toxin B subunit can induce neutralizing immunoglobulin Y antibody.

Previously, we have shown that chickens immunized with Shiga toxin (Stx) produce Stx-neutralizing egg yolk immunoglobulin Y (IgY) antibody. The anti-Stx-1 IgY and anti-Stx-2 IgY exert their neutralizing activity through their antibody activity against the B subunit of the toxin but not the A subunit. In the present study, chickens were immunized with recombinant Stx-1 B subunit (rStx-1B) and recombinant Stx-2 B subunit (rStx-2B). Induced anti-rStx-1B and anti-rStx-2B IgY neutralized the toxicity of Stx-1 and Stx-2 against HeLa 229 cells. The neutralizing activity of anti-rStx-1B IgY on Stx-1 was almost 10 times stronger than that of anti-Stx-1 IgY, and that of anti-rStx-2B IgY was 2.6 times stronger than that of anti-Stx-2 IgY. Anti-rStx-1B and anti-rStx-2B IgY reacted with multimeric and monomeric forms of the B subunits in contrast to anti-Stx-1 and anti-Stx-2 IgY that reacted with only the multimeric form. These results indicated that recombinant B subunits were promising antigens for induction of neutralizing antibodies in chickens.