Alternaria alternata NADP-dependent mannitol dehydrogenase is an important fungal allergen.

BACKGROUND: Alternaria alternata is one of the most important allergenic fungi worldwide. Mannitol dehydrogenase (MtDH) has previously been shown to be a major allergen of Cladosporium herbarum and cross-reactivity has been demonstrated for several fungal allergens. OBJECTIVE: The present study's objective was to clone the MtDH from an A. alternata cDNA library, express and purify the recombinant non-fusion protein and test its IgE-binding properties. Methods A cDNA library prepared from A. alternata hyphae and spores was screened for mannitol dehydrogenase by DNA hybridization with the radioactively labelled C. herbarum homologue as a probe. The resulting clone was sequenced and heterologously expressed in Escherichia coli as a recombinant non-fusion protein, which was purified to homogeneity and analysed for its IgE-binding capacity. RESULTS: The coding sequence of the full-length cDNA clone comprises 798 bp encoding a protein with a molecular mass of 28.6 kDa and a predicted pI of 5.88. Protein sequence analysis revealed an identity of 75% and a homology of 86% between the MtDHs of A. alternata and C. herbarum. The functional mannitol dehydrogenase was expressed in the E. coli strain BL21(DE3) transformed with the vector pMW172 and purified to homogeneity. The enzyme catalyses the NADPH-dependent conversion of d-fructose to d-mannitol. In IgE-ELISA and immunoblots, MtDH is recognized by 41% of A. alternata-allergic patients. In vivo immunoreactivity of the recombinant MtDH was verified by skin prick testing. Finally, inhibition-ELISA experiments confirmed cross-reactivity between the MtDHs of A. alternata and C. herbarum. CONCLUSION: Mannitol dehydrogenase (Alt a 8) represents an important new allergen of the ascomycete A. alternata that might be suitable for improving diagnostic and therapeutic procedures.     

The molecular immunology of human susceptibility to fungal diseases: lessons from single gene defects of immunity

Introduction: Fungal diseases are a threat to human health. Therapies targeting the fungus continue to lead to disappointing results. Strategies targeting the host response represent unexplored opportunities for innovative treatments. To do so rationally requires the identification and neat delineation of critical mechanistic pathways that underpin human antifungal immunity. The study of humans with single-gene defects of the immune system, i.e. inborn errors of immunity (IEIs), provides a foundation for these paradigms.

Areas covered: A systematic literature search in PubMed, Scopus, and abstracts of international congresses was performed to review the history of genetic resistance/susceptibility to fungi and identify IEIs associated with fungal diseases. Immunologic mechanisms from relevant IEIs were integrated with current definitions and understandings of mycoses to establish a framework to map out critical immunobiological pathways of human antifungal immunity.

Expert opinion: Specific immune responses non-redundantly govern susceptibility to their corresponding mycoses. Defining these molecular pathways will guide the development of host-directed immunotherapies that precisely target distinct fungal diseases. These findings will pave the way for novel strategies in the treatment of these devastating infections.     

保健机构新生儿听力普遍筛查模式的建立

[目的]发挥保健机构特色建立新生儿听力普遍筛查(universal newborn hearing screening,UNHS )模式,获得本地区初生婴儿听力损失的基本资料. [方法]选择佛山市妇幼保健院出生的活产新生儿于生后48～72 h接受耳声发射(otoacoustic emissions,OAE) 测试,通过者予出院;未通过者在出生42 d复查OAE,仍未通过者行听觉脑干诱发电位反应(auditory brainstem response, ABR)检查.所有ABR检查不通过者在3个月内接受全面的听力学诊断和评估,以确定听力损失的性质和程度.耳声发射测试采用畸变产物耳声发射(distortion product otoacoustic emissions,DPOAE). [结果]3 244名新生儿中2 910名(89.7%)初筛通过OAE测试,未通过测试的313例于生后42 d复查OAE, 309例通过.OAE测试总的通过率99.2%;接受ABR检查4例,3例未通过测试.经系统的听力学诊断与评估,此3例患儿伴有不同程度的听力损伤,占受试对象的0.92‰;新生儿重症监护病房(NICU)中高危新生儿听力损伤的发生率4.8‰(2/417),显著高于爱婴区未患病新生儿的发生率0.35‰(1/2 827)(χ2=7.76,P＜0.01). [结论]充分发挥保健机构特色建立OAE两步筛查、ABR初步诊断方案,对建立新生儿听力普遍筛查模式,促进听力筛查工作的普遍开展有重要作用.     

Clinico-demographical profile of keratomycosis in Delhi,North India

This study was undertaken to evaluate the clinico-demographical profile of keratomycosis. (January 2004 to January 2012). The corneal scrapings were processed by direct microscopic methods and standard culture techniques. Of 209 cases of keratitis studied, culture yielded growth in 80 cases (38.3%). Out of these 80 cases of growth, fungi were isolated in 77.5% and bacteria in 22.5%. The spectrum of keratomycosis was Aspergillus flavus (22.5%), Fusarium solani (16.1%), A. fumigatus (11.3%), Candida albicans (6.4%), etc., Routine surveillance of fungal keratitis is necessary to know the existing and emerging pattern of pathogens and to prevent use of un-warranted anti-microbial therapy.     

Outdoor fungi and child asthma health service attendances

Asthma is a significant global public health issue. Severe asthma exacerbations can be triggered by environmental factors and require medical care from health services. Although it is known that fungal exposure may lead to allergic sensitization, little is understood about its impact on asthma exacerbations. This review aims to examine whether outdoor fungi play a significant role in child asthma exacerbations. Systematic search of seven electronic databases and hand searching for peer‐reviewed studies published in English, up to 31 August 2013. Inclusion criteria were study population aged <18 yr, diagnosis of asthma, attended a health service; outdoor fungi exposure was reported. Quality and risk of bias assessments were conducted. Due to significant heterogeneity, meta‐analysis was not conducted. Of the 1896 articles found, 15 were eligible. Findings were not consistent, possibly due to methodological variations in exposure classifications, statistical methods and inclusion of confounders. Cross‐sectional studies found no or weak associations. All but one time series studies indicated an association that varied between fungal species. Increasing evidence indicates that asthmatic children are susceptible to asthma exacerbations when exposed to outdoor fungal spores. There is limited understanding of the contributions of different fungal species. Research is needed to investigate interactions of outdoor fungi with pollen, air pollutants and respiratory viruses.     

First report of Mortierella wolfii causing fungal keratitis from a tertiary eye hospital in India

A young 33 year old male presented with non-resolving corneal infiltrate for 2 month duration in the right eye. KOH/ Calcoflour wet mount revealed sparsely septate fungal hyphae. Post therapeutic penetrating keratoplasty 3 doses of intracameral voriconazole(100μg/0.1ml) was administered suspecting recurrence. Fungal culture revealed non sporulating mould on SDA. PCR based DNA sequencing targeting the ITS region identified the fungal isolate as Mortierella wolfii (M. wolfii) belonging to zygomycetes. To the best of our knowledge, this is the first report of human fungal keratitis caused by M. wolfii.     

Neuroprotective effects of dimerumic acid and deferricoprogen from Monascus purpureus NTU 568-fermented rice against 6-hydroxydopamine-induced oxidative stress and apoptosis in differentiated pheochromocytoma PC-12 cells

Context Oxidative stress plays a key role in neurodegenerative disorders, including Parkinson’s disease (PD). Rice fermented with Monascus purpureus Went (Monascaceae) NTU 568 (red mould rice) was found to contain antioxidants, including dimerumic acid (DMA) and deferricoprogen (DFC).

Objective The effects of DMA and DFC on 6-hydroxydopamine (6-OHDA)-induced cytotoxicity and potential protective mechanisms in differentiated PC-12 pheochromocytoma cells were investigated.

Materials and methods DMA (0–60?μM) or DFC (0–10?μM) was co-treated with 6-OHDA (200?μM, 24?h exposure) in differentiated PC-12 cells. Cell viability and intercellular reactive oxygen species (ROS) were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and 2′,7′-dichlorofluorescein-diacetate (DCFH-DA) assays, respectively. Cell apoptosis was determined by DNA fragmentation analysis and propidium iodide staining by flow cytometry. Western blot analysis was used to measure the levels of cell protein expression.

Results DMA and DFC significantly increased cell viability to 72% and 81% in 6-OHDA-induced differentiated PC-12 cell cultures, respectively. Furthermore, DMA and DFC reduced 6-OHDA-induced formation of extracellular and intercellular ROS by 25% and 20%, respectively, and decreased NADPH oxidase-2 expression in differentiated PC-12 cells. DMA and DFC inhibited 6-OHDA-induced apoptosis and decreased activation of caspase-3 via regulation of Bcl-2-associated X protein (Bax) and Bcl-2 protein expression in differentiated PC-12 cells.

Conclusion DMA and DFC may protect against 6-OHDA toxicity by inhibiting ROS formation and apoptosis. These results showed that the metabolites from M. purpureus NTU 568 fermentation were potential therapeutic agents for PD induced by oxidative damage and should be encouraged for further research.