Targeting Neutrophils in Severe Asthma via Siglec-9.

Severe asthma comprises only 5% of patients with asthma, but the burden it brings to the social health system accounts for more than half of all asthmatics. Clinical evidence shows that severe asthma is often linked to the recruitment and activation of neutrophils in the airways. However, the underlying molecular and immunological mechanisms of neutrophilia in severe asthma are not clear and currently available drugs exert only limited effects on neutrophilic inflammation. Great efforts are underway to address the mystery of neutrophilic inflammation in chronic respiratory disorders. Sialic acid-binding immunoglobulin-like lectins (Siglecs) are members of the immunoglobulin gene family. Of note, Siglec-9 is uniquely expressed by human neutrophils and monocytes, as well as a minor population of natural killer cells. Engaging this structure with antibodies or glycan ligands results in programmed cell death in human neutrophils. Intriguingly, the administration of Siglec-E antibody abolished the recruitment of neutrophils in mouse models of neutrophilic pulmonary inflammation in vivo. Given that neutrophils are probably a major culprit in the generation and perpetuation of inflammation, targeting Siglec-9 could be beneficial for the treatment of severe asthma, chronic obstructive pulmonary disease, and related pulmonary disorders characteristic of neutrophilia.

Development of a loop-mediated isothermal amplification assay for rapid detection of Mycoplasma hyorhinis.

BACKGROUND: To establish a method for sensitive and rapid diagnosis of Mycoplasma hyorhinis in clinical specimens, a simple, sensitive loop-mediated isothermal amplification (LAMP) assay was designed and evaluated. METHODS: Three sets of four special primers, recognizing distinct sequences of the target, were designed for sensitive, specific amplification of nucleic acid under isothermal conditions. The LAMP assay was carried out using 35 clinical specimens of bronchoalveolar lavage fluid (BALF) from pigs. For comparison, these specimens were also tested using conventional PCR, real-time PCR, and nested PCR assays. RESULTS: After optimization of the reaction condition and reaction system, the LAMP reaction successfully detected Mycoplasma hyorhinis within 40 minutes at 61 degrees C. The LAMP assay achieved a sensitivity of 10(1) copies per microL at 61 degrees C in 40 minutes, compared to real-time PCR and nested PCR, and was over 10(3) times more sensitive than conventional PCR. In the test for the specificity of the LAMP assay, only Mycoplasma hyorhinis genomic DNA was positive and no other microorganisms were positive with the primers, indicating that the LAMP assay is specific to Mycoplasma hyorhinis. Mycoplasma hyorhinis was detected in 32 samples using the LAMP and real-time PCR assays and in 27 and 11 samples using the nested PCR assay and conventional PCR assay, respectively. All the positive samples detected by real-time PCR, nested PCR and conventional PCR assays were positive in the LAMP assay. CONCLUSIONS: The LAMP assay is inexpensive, easy to perform, shows a rapid reaction and does not require complex instruments like PCR. Therefore, LAMP is a simple, accurate, fast, and economical assay suitable as an alternative in veterinary practices.

Use of serological and mucosal immune responses to Mycoplasma hyopneumoniae antigens P97R1, P46 and P36 in the diagnosis of infection.

Currently available ELISAs used to diagnose Mycoplasma hyopneumoniae infection in pigs have high specificity but low sensitivity. To develop more sensitive assays, the kinetics of specific serum IgG and respiratory mucosal sIgA responses against three M. hyopneumoniae antigens, namely, P97R1 (an adhesin protein), P46 (a membrane protein), and P36 (a cytosolic protein), were characterised over 133 days following experimental infection. Immunoglobulin G against the three proteins remained at high concentrations from 28 to 133 days post-infection (dpi), although IgG against P97R1 was detected earlier and was more reactive than the other two antigens under assessment. Mucosal sIgA appeared earlier than serum IgG but did not persist as long; sIgA concentrations against P97R1 were the highest. Seroconversion was detected 2 weeks earlier with the P97R1-based ELISA than with a commercially available ELISA. On analysis of serum samples from five pig farms that did not use a M. hyopneumoniae vaccine, the P97R1-based IgG ELISA demonstrated a 73.6% coincidence rate with the commercial kit. Moreover, this more specific P97R1-based ELISA detected more positive samples than the commercial kit (52.8% vs. 39.2%). It was concluded that the systemic immune response to M. hyopneumoniae infection in pigs was delayed in onset but persistent whereas the mucosal response developed more rapidly but was less sustained. The P97R1 antigen was identified as a suitable serological marker for diagnosing M. hyopneumoniae infection in pigs, particularly early stage infection.

[Shoot type morphology and growth characteristics of winter wheat sown at different dates].

Aiming at the delayed sowing of winter wheat induced by the drought and water logging often occurred in Huanghuai Plains of China, six sowing dates (15 October, normal sowing; 30 October, moderate delay; 15 November, delay; 30 November, seriously delay; 15 February, early spring sowing; and 1 March, spring sowing) were designed to investigate the effects of different sowing dates on the shoot type morphology and growth characteristics of winter wheat. With the delay of sowing date, the winter wheat grew and developed faster, and the growing period of the wheat sown in early spring and spring was 115-130 days shorter than that with normal sowing. As compared with those of the wheat with normal sowing, the shoot height, spike number per unit area, and productive spikelets per unit ear of the wheat sown delayed had a decrease, leaf position and canopy moved down, and leaf area reduced. When the sowing was delayed from the date 15 October (normal sowing) to 1 March (spring sowing), the harvest index increased from 0.46 to 0.53. Delaying sowing date also resulted in the significant reduction of grain yield, with the maximum decrement as high as 43. 6%. The spring-sown winter wheat not going through vernalization could still form yield.

Development and validation of an attenuated Mycoplasma hyopneumoniae aerosol vaccine.

Mycoplasma hyopneumoniae (M. hyopneumoniae) causes a chronic respiratory disease with high morbidity and low mortality in swine, and has been presented as a major cause of growth retardation in the swine industry. Aerosol vaccination presents a needle free, high throughput, and efficient platform for vaccine delivery, and has been widely applied in poultry vaccination. However, aerosol vaccines have rarely been used in swine vaccination primarily because the long and curving respiratory track of swine presents a barrier for vaccine particle delivery. To develop an effective M. hyopneumoniae aerosol vaccine, three major barriers need to be overcome: to optimize particle size for aerosol delivery, to maintain the viability of mycoplasma cells in the vaccine, and to optimize the environmental conditions for vaccine delivery. In this study, an aerosol mycoplasma vaccine was successfully developed based on a conventional live attenuated M. hyopneumoniae vaccine. Specifically, the Pari LCD nebulizer was used to produce an aerosol vaccine particle size less than 5 µm; and a buffer with 5% glycerol was developed and optimized to prevent inactivation of M. hyopneumoniae caused by aerosolization and evaporation. Before nebulization, the room temperature and relative humidity were control to 20-25 °C and 70-75%, respectively, which helped maintain the viability of aerosol vaccine. Animal experiments demonstrated that this newly developed aerosol vaccine was effectively delivered to swine low respiratory track, being confirmed by nested-PCR, in situ hybridization and scanning electron microscope. Moreover, M. hyopneumoniae specific sIgA secretion was detected in the nasal swab samples at 14 days post-immunization. To our knowledge, this is the first report on a live M. hyopneumoniae aerosol vaccine.