Alterations in nitric oxide and cytokine production with airway inflammation in the absence of IL-10

IL-10 is an anti-inflammatory cytokine that suppresses NO synthase (NOS) and production of NO; its lack may promote NO production and alterations in cytokines modulated by NO with allergic airway inflammation (AI), such as IL-18 and IL-4. Therefore, we induced AI in IL-10 knockout ((-/-)) and IL-10-sufficient C57BL/6 (C57) mice with inhaled OVA and measured airway NO production, as exhaled NO (E(NO)) and bronchoalveolar lavage fluid nitrite levels. E(NO) and nitrite levels were elevated significantly in naive IL-10(-/-) mice as compared with C57 mice. With AI, E(NO) and nitrite levels increased in C57 mice and decreased in IL-10(-/-) mice. IL-18 production fell with both AI and addition of S-nitroso-N-acetyl-d,l-penicillamine (a NO donor) but was not significantly increased by chemical NOS inhibition by l-N(5)-(1-iminoethyl)-ornithine. IL-4 AI was increased significantly (up to 10-fold greater) in the absence of IL-10 but was reduced significantly with chemical inhibition of NOS. Airway responsiveness was lower in IL-10(-/-) mice and was associated with alteration in production of NO and IL-4. Thus, IL-4 production was increased, and likely decreased NO production, in a way not predicted by the absence of IL-10. Inhibition of IL-4 production, with inhibition of NOS in the absence of IL-10, demonstrated the importance of a NO and IL-4 feedback mechanism regulating this interaction.     

Dog bites in humans and estimating human rabies mortality in rabies endemic areas of Bhutan

Background

Dog bites in humans are a public health problem worldwide. The issues of increasing stray dog populations, rabies outbreaks, and the risk of dogs biting humans have been frequently reported by the media in Bhutan. This study aimed to estimate the bite incidence and identify the risk factors for dog bites in humans, and to estimate human deaths from rabies in rabies endemic south Bhutan.

Methods

A hospital-based questionnaire survey was conducted during 2009–2010 among dog bites victims who visited three hospitals in Bhutan for anti-rabies vaccine injection. Decision tree modeling was used to estimate human deaths from rabies following dog bite injuries in two rabies endemic areas of south Bhutan.

Results

Three hundred and twenty four dog bite victims were interviewed. The annual incidence of dog bites differed between the hospital catchment areas: 869.8 (95% CI: 722.8–1022.5), 293.8 (240–358.2) and 284.8 (251.2–323) per 100,000 people in Gelephu, Phuentsholing and Thimphu, respectively. Males (62%) were more at risk than females (P<0.001). Children aged 5–9 years were bitten more than other age groups. The majority of victims (71%) were bitten by stray dogs. No direct fatal injury was reported. In two hospital areas (Gelephu and Phuentsholing) in south Bhutan the annual incidence of death from rabies was 3.14 (95% CI: 1.57–6.29) per 100,000 population. The decision tree model predicted an equivalent annual incidence of 4.67 (95% CI: 2.53–7.53) deaths/100,000 population at risk. In the absence of post exposure prophylaxis, the model predicted 19.24 (95% CI: 13.69–25.14) deaths/year in these two areas.

Conclusions

Increased educational awareness of people about the risk of dog bites and rabies is necessary, particularly for children in rabies endemic areas of Bhutan.     

Non-invasive measurements of exhaled NO and CO associated with methacholine responses in mice

Background

Nitric oxide (NO) and carbon monoxide (CO) in exhaled breath are considered obtainable biomarkers of physiologic mechanisms. Therefore, obtaining their measures simply, non-invasively, and repeatedly, is of interest, and was the purpose of the current study.

Methods

Expired NO (E_NO) and CO (E_CO) were measured non-invasively using a gas micro-analyzer on several strains of mice (C57Bl6, IL-10^-/-, A/J, MKK3^-/-, JNK1^-/-, NOS-2^-/- and NOS-3^-/-) with and without allergic airway inflammation (AI) induced by ovalbumin systemic sensitization and aerosol challenge, compared using independent-sample t-tests between groups, and repeated measures analysis of variance (ANOVA) within groups over time of inflammation induction. E_NO and E_CO were also measured in C57Bl6 and IL-10-/- mice, ages 8–58 weeks old, the relationship of which was determined by regression analysis. S-methionyl-L-thiocitrulline (SMTC), and tin protoporphyrin (SnPP) were used to inhibit neuronal/constitutive NOS-1 and heme-oxygenase, respectively, and alter NO and CO production, respectively, as assessed by paired t-tests. Methacholine-associated airway responses (AR) were measured by the enhanced pause method, with comparisons by repeated measures ANOVA and post-hoc testing.

Results

E_NO was significantly elevated in naïve IL-10^-/- (9–14 ppb) and NOS-2^-/- (16 ppb) mice as compared to others (average: 5–8 ppb), whereas E_CO was significantly higher in naïve A/J, NOS-3^-/- (3–4 ppm), and MKK3^-/- (4–5 ppm) mice, as compared to others (average: 2.5 ppm). As compared to C57Bl6 mice, AR of IL-10^-/-, JNK1^-/-, NOS-2^-/-, and NOS-3^-/- mice were decreased, whereas they were greater for A/J and MKK3^-/- mice. SMTC significantly decreased E_NO by ~30%, but did not change AR in NOS-2^-/- mice. SnPP reduced E_CO in C57Bl6 and IL-10^-/- mice, and increased AR in NOS-2^-/- mice. E_NO decreased as a function of age in IL-10^-/- mice, remaining unchanged in C57Bl6 mice.

Conclusion

These results are consistent with the ideas that: 1) E_NO is associated with mouse strain and knockout differences in NO production and AR, 2) alterations of E_NO and E_CO can be measured non-invasively with induction of allergic AI or inhibition of key gas-producing enzymes, and 3) alterations in AR may be dependent on the relative balance of NO and CO in the airway.     

西藏黄绿卷毛菇生境土壤微生物群落组成

牧草外生菌根菌黄绿卷毛菇Floccularia luteovirens具有较高的生态和经济价值,其生境微生物对其菌丝生长、菌根化和子实体形成具有促进作用。本研究利用高通量测序技术对西藏黄绿卷毛菇菌窝土壤及其周围无菇土壤微生物群落组成进行分析,挖掘促进黄绿卷毛菇生长的有益微生物资源。结果显示,西藏黄绿卷毛菇生境土壤细菌隶属于17门22纲116目161科227属,其中酸杆菌门、变形菌门、拟杆菌门和疣微菌门累计比例高达81.75%;生境土壤真菌隶属于5门17纲34目55科61属,子囊菌门和担子菌门累计比例高达80.89%。对比周围无菇土壤的微生物群落,促进黄绿卷毛菇生长的潜在细菌类群为Flavisolibacter、黄杆菌属Flavobacterium、芽单胞菌属Gemmatimonas、Haliangium、赛格特杆菌Segetibacter和鞘氨醇单胞菌属Sphingomonas;促进黄绿卷毛菇生长的潜在真菌类群为斜盖伞属Clitopilus、丝膜菌属Cortinarius、被孢霉属Mortierella和毛霉属Mucor。土壤微生物促进黄绿卷毛菇生长繁殖的机制有待进一步研究。     

Enhanced nitric oxide production associated with airway hyporesponsiveness in the absence of IL-10

Interleukin (IL)-10 is an anti-inflammatory cytokine implicated in the regulation of airway inflammation in asthma. Among other activities, IL-10 suppresses production of nitric oxide (NO); consequently, its absence may permit increased NO production, which can affect airway smooth muscle contractility. Therefore, we investigated airway reactivity (AR) in response to methacholine (MCh) in IL-10 knockout (-/-) mice compared with wild-type C57BL/6 (C57) mice, in which airway NO production was measured as exhaled NO (E(NO)), and NO production was altered with administration of either NO synthase (NOS)-specific inhibitors or recombinant murine (rm)IL-10. AR, measured as enhanced pause in vivo, and tracheal ring tension in vitro were lower in IL-10(-/-) mice by 25-50%, which was associated with elevated E(NO) levels (13 vs. 7 ppb). Administration of NOS inhibitors N(G)-nitro-L-arginine methyl ester (8 mg/kg ip) or L-N(6)-(1-iminoethyl)-lysine (3 mg/kg ip) to IL-10(-/-) mice decreased E(NO) by an average of 50%, which was associated with increased AR, to levels similar to C57 mice. E(NO) in IL-10(-/-) mice decreased in a dose-dependent fashion in response to administered rmIL-10, to levels similar to C57 mice (7 ppb), which was associated with a 30% increment in AR. Thus increased NO production in the absence of IL-10, decreased AR, which was reversed with inhibition of NO, either by inhibition of NOS, or with reconstitution of IL-10. These findings suggest that airway NO production can modulate airway smooth muscle contractility, resulting in airway hyporesponsiveness when IL-10 is absent.     

Acoustic detection and acoustic habitat characterisation of the critically endangered white-bellied heron (Ardea insignis) in Bhutan

1. Growing developmental activities, such as hydropower construction, farm roads, and other human activities, are affecting the critically endangered white-bellied heron (WBH). Out of a known global population of 60, 28 individuals inhabit the river basin area and freshwater lakes and ponds of Bhutan. Several constraints impede continuous monitoring of endangered species, such as the isolated and cryptic nature of the species and the remoteness of its habitat; to date, there are no long-term reference data or techniques implemented for continuous monitoring of this species.
2. In this study, we designed acoustic detection and habitat characterisation methods using long-duration recordings from three habitat areas in Bhutan. Acoustic indices were extracted and used to implement a species-specific call detector and to generate habitat soundscape representations. Using WBH calls annotated in month-long recordings from a known site, a novel indices-based detector was implemented and tested. A total of 960 hr of continuous audio recordings from three habitats in Bhutan were analysed.
3. We found that a species call detector implemented using a combination of acoustic indices (that includes measures of spectral and temporal entropy and different angles of spectral ridges) has a correct detection rate of 81%. Additionally, visual inspection of the species’ acoustic habitat using long-duration false-colour spectrograms enabled qualitative assessment of acoustic habitat structure and other dominant acoustic events.
4. This study proposes a combined approach of species acoustic detection and habitat soundscape analysis for holistic acoustic monitoring of endangered species. As a direct outcome of this work, we documented acoustic reference data on the critically endangered WBH from multiple habitat areas and have analysed its temporal vocalisation patterns across sites.

     

Habitat requirements of the Himalayan red panda (Ailurus fulgens) and threat analysis in Jigme Dorji National Park,Bhutan

Understanding the influence of anthropogenic disturbances on species’ habitat use and distribution is critical to conservation managers in planning effective conservation strategies and mitigating the impact of development. Few studies have focused on the Himalayan red panda (Ailurus fulgens) in Bhutan. This study aimed to assess the habitat requirements and threats to this endangered species in the Khamaed subdistrict of the Jigme Dorji National Park, Bhutan. We employed a transect walk and plot‐sampling survey design across two seasons, that is, winter and spring. In total, we surveyed 84 × 50 m radius circular plots along 51 km of existing trails within a 25.4 km² study area. At 500 m intervals, we established plots at random distances and direction from the trail. We recorded direct sightings (n = 2) and indirect signs (n = 14), such as droppings and footprints as evidence of red panda presence within an altitudinal range of 2,414–3,618 m. We also noted 21 tree and 12 understory species within plots with red panda evidence; the dominant tree species was the Himalayan hemlock (Tsuga dumosa) and the Asian barberry (Berberis asiatica) as an understory species. Red panda presence showed a significant positive association with distance to water sources and fir forests. Plant disturbance and infrastructure, such as power transmission lines, were identified as prominent anthropogenic threats in the study area. Based on our findings, we recommend the development and implementation of local forest management plans, livestock intensification programs, and strict application of environmental impact assessment regulations to promote the conservation of the red panda in the region.