Neutrophils Affect IL-33 Processing in Response to the Respiratory Allergen Alternaria alternata.

Future precision medicine requires further clarifying the mechanisms of inflammation in the severe endotypes of chronic airway diseases such as asthma and chronic rhinosinusitis (CRS). The presence of neutrophils in the airways is often associated with severe airway inflammation, while their precise contribution to the severe inflammation is largely unknown. We aimed to study the role of neutrophils in BALB/c and C57BL/6 mice exposed to Alternaria alternata (Alt). The mice were exposed to Alt extract for twelve hours or ten days to induce allergic airway inflammation. C57BL/6 mice exposed to Alt responded with eosinophilic infiltration and the characteristic IL-5 upregulation. In contrast, the inflammatory response to Alt extract in BALB/c mice was characterized by a neutrophilic response, high levels of G-CSF, and elastase in the lungs. The lack of neutrophils affected the processing of IL-33 in BALB/c mice, as was demonstrated by depletion of neutrophils through intraperitoneal injections of anti-Ly6G antibody. Our data identifies the key role of neutrophils in airway inflammation through IL-33 cleavage in the Alt-induced airway inflammation in mice, which could potentially underline the different endotypes in human disease.

Pyruvate kinase M2 in lung APCs regulates Alternaria-induced airway inflammation.

Sensitivity to allergenic fungi (Alternaria alternata) is associated with acute, severe asthma attacks. Antigen presenting cells (APCs) in the lung sense environmental perturbations that induce cellular stress and metabolic changes and are critical for allergic airway inflammation. However, the mechanisms underlying such environmental sensing by APCs in the lung remains unclear. Here we show that acute Alternaria challenge rapidly induces neutrophil accumulation in airways, and alter expressions of Pyruvate Kinase (PKM2) and hypoxia-inducible factor -1α (Hif-1α) that correlates with proinflammatory mediator release. Blockade of IL33 signaling in vivo led to reduce oxidative stress and glycolysis in lung APCs. Lung-specific ablation of CD11c+ cells abrogates Alternaria-induced neutrophil accumulation and inflammation. Furthermore, administration of Alternaria into the airways stimulated APCs and elevate the expression of Glut-1. Mechanistically, we establish that PKM2 is a critical modulator of lung APC activation in Alternaria-induced acute inflammation. Allosteric activation of PKM2 by a small molecule ML265 or siRNA-mediated knock down correlated negatively with glycolysis and activation of APCs. These results collectively demonstrates that PKM2-mediated glycolytic reprogramming by fungal allergen Alternaria influences lung APC activation, thereby promotes acute airway inflammation. Our data support a model in which Alternaria sensitization in airways induce a circuitry of glycolysis and PKM2 regulation that confers an acute activation of APCs in the lung, whose targeting might represent a strategy for asthma treatment.

[Protection by some plant methanol extracts of cherry tomatoes (Solanum lycopersicum var. Cerasiforme) from fungic infection by Alternaria alternata]. / Utilisation d'extraits méthanoliques de plantes pour la protection des cultures de tomates-cerises (Solanum lycopersicum var. cerasiforme) contre l'infection fongique par Alternaria alternata.

Cherry tomato is very susceptible to fungal infections that can cause considerable damage in crops and during storage. Alternaria infection is one of the most common and dangerous alterations for this fruit. They are caused by Alternaria alternata or some other species belonging to the same genus. In this work, we tested the antifungal activity of methanol extracts from five plants harvested in the region of Jijel (Algeria) on A. alternata. The activity was first tested in vitro and then on greenhouse cherry tomato plants: extracts were applied to healthy plants before infection in order to test their preventive action, and after infection to determine whether they are able to knock out Alternaria. Results showed that Rosmarinus officinalis and Lavandula angustifolia extracts were the most active in vitro on A. alternata. Microscopic observations of the mold indicated that these extracts inhibited the dictyospores production. The antifungal activity tested on the plants grown in greenhouse revealed that R. officinalis extract still was the most active. Extracts of L. angustifolia and Punica granatum did not protect the plants from Alternaria infection, but provided a total cure at the end of the treatment. Extracts from Quercus suber and Eucalyptus globulus were the least active. They did not bestow any protection nor complete healing of the plants. Dictyospores counting on fruits at the end of the treatment confirmed the results obtained for the greenhouse crops.

TITLE: Utilisation d'extraits méthanoliques de plantes pour la protection des cultures de tomates-cerises (Solanum lycopersicum var. cerasiforme) contre l'infection fongique par Alternaria alternata. ABSTRACT: La tomate-cerise est un fruit très sujet aux infections fongiques qui peuvent causer des dégâts considérables dans les cultures et lors de la conservation. Les alternarioses comptent parmi les altérations les plus répandues et dangereuses pour ce fruit. Elles sont causées par Alternaria alternata ou d'autres espèces appartenant au même genre. Dans ce travail, nous avons testé l'activité antifongique d'extraits méthanoliques de cinq plantes récoltées dans la région de Jijel (Algérie) sur A. alternata. L'activité a d'abord été testée in vitro, puis sur des plants de tomates-cerises cultivés sous serre : les extraits ont été appliqués sur des plants sains, avant l'infection, afin de tester leur action préventive, et après l'infection pour déterminer s'ils sont capables de traiter l'alternariose. Les résultats ont montré que les extraits de Rosmarinus officinalis et Lavandula angustifolia étaient les plus actifs in vitro sur A. alternata. L'observation microscopique de la moisissure a indiqué que ces extraits agissaient en inhibant sa production de dictyospores. L'activité antifongique testée sur les plants cultivés sous serre a révélé que l'extrait de R. officinalis était toujours le plus actif. Venaient ensuite les extraits de L. angustifolia et Punica granatum qui n'ont pas permis la protection des plants contre l'alternariose, mais qui ont néanmoins donné une guérison totale à la fin du traitement. Les extraits de Quercus suber et Eucalyptus globulus étaient les moins actifs. Ils n'ont permis ni la prévention, ni la guérison complète des plants. Le comptage des dictyospores réalisé sur les fruits à la fin du traitement a confirmé les résultats obtenus pour les cultures sous serre.

COX Inhibition Increases Alternaria-Induced Pulmonary Group 2 Innate Lymphoid Cell Responses and IL-33 Release in Mice.

The cyclooxygenase (COX) metabolic pathway regulates immune responses and inflammation. The effect of the COX pathway on innate pulmonary inflammation induced by protease-containing fungal allergens, such as Alternaria alternata, is not fully defined. In this study, we tested the hypothesis that COX inhibition augments Alternaria-induced pulmonary group 2 innate lymphoid cell (ILC2) responses and IL-33 release. Mice were treated with the COX inhibitors indomethacin, flurbiprofen, or vehicle and challenged intranasally with Alternaria extract for four consecutive days to induce innate lung inflammation. We found that indomethacin and flurbiprofen significantly increased the numbers of ILC2 and IL-5 and IL-13 expression by ILC2 in the lung. Indomethacin also increased ILC2 proliferation, the percentages of eosinophils, and mucus production in the lung. Both indomethacin and flurbiprofen augmented the release of IL-33 in bronchoalveolar lavage fluid after Alternaria challenge, suggesting that more IL-33 was available for ILC2 activation and that a COX product(s) inhibited IL-33 release. This is supported by the in vitro finding that the COX product PGE2 and the PGI2 analogs cicaprost decreased Alternaria extract-induced IL-33 release by human bronchial epithelial cells. Although contrasting effects of PGD2, PGE2, and PGI2 on ILC2 responses have been previously reported, the overall effect of the COX pathway on ILC2 function is inhibitory in Alternaria-induced innate airway inflammation.

Polyphasic identification of three new species in Alternaria section Infectoriae causing human cutaneous infection.

BACKGROUND: Cutaneous phaeohyphomycosis is an emerging disease in immunocompromised patients, being Alternaria one of the most common genera reported as a causative agent. Species identification is not carried out mainly due to the complexity of the genus. Analysis of the ITS barcode has become standard for fungal identification, but in Alternaria it is only able to discriminate among species-groups or sections. METHODS: We present three cases of cutaneous infection caused by Alternaria isolates morphologically identified as belonging to section Infectoriae. They have been morphologically characterised and phylogenetically delineated with five molecular markers (ITS, ATPase, gapdh, rpb2 and tef1). RESULTS: Mycotic infections have been diagnosed by repeated cultures and histopathological examination in two of the cases. The polyphasic approach has allowed to delineate three new species of Alternaria section Infectoriae, that is A anthropophila, A atrobrunnea and A guarroi. ATPase has been the only locus able to discriminate most of the species (29 out of 31) currently sequenced in this section, including A infectoria the commonest reported species causing alternariosis. Susceptibility test showed different antifungal patterns for the three species, although terbinafine was the most active in vitro drug against these fungi. CONCLUSIONS: The ATPase gene is recommended as an alternative barcode locus to identify Alternaria clinical isolates in section Infectoriae. Our results reinforce the relevance of identification of Alternaria isolates at the species level and the necessity to carry out antifungal susceptibility testing to determine the most adequate drug for treatment.

Onset of disseminated cutaneous nodules following toe amputation in heart transplant patient.

Alternaria spp. infections are rare, but organ transplant recipients and immunosuppressed patients are particularly at risk of developing cutaneous alternariosis. Although cutaneous alternariosis is well-defined, instances of disseminated infection are exceedingly rare. We report a case of disseminated Alternaria infection in an immunocompromised patient from a primary focus of ungual phaeohyphomycosis.

CARMA3 Mediates Allergic Lung Inflammation in Response to Alternaria alternata.

The airway epithelial cell (AEC) response to allergens helps initiate and propagate allergic inflammation in asthma. CARMA3 is a scaffold protein that mediates G protein-coupled receptor-induced NF-κB activation in airway epithelium. In this study, we demonstrate that mice with CARMA3-deficient AECs have reduced airway inflammation, as well as reduced type 2 cytokine levels in response to Alternaria alternata. These mice also have reduced production of IL-33 and IL-25, and reduced numbers of innate lymphoid cells in the lung. We also show that CARMA3-deficient human AECs have decreased production of proasthmatic mediators in response to A. alternata. Finally, we show that CARMA3 interacts with inositol 1,4,5-trisphosphate receptors in AECs, and that inhibition of CARMA3 signaling reduces A. alternata-induced intracellular calcium release. In conclusion, we show that CARMA3 signaling in AECs helps mediate A. alternata-induced allergic airway inflammation, and that CARMA3 is an important signaling molecule for type 2 immune responses in the lung.

Identification and expression of CAMTA genes in Populus trichocarpa under biotic and abiotic stress.

The calmodulin-binding transcription activators (CAMTAs) transcription factor family plays an important role in normal plant growth and development, as well as in biotic and abiotic stress resistance. In this study, we identified seven CAMTA genes across the whole genome of Populus trichocarpa and analyzed the expression patterns of PtCAMTAs in the root and leaf tissues. Promoter cis-element analysis indicated that most CAMTA genes contained stress- or phytohormone-related cis-elements. Quantitative real-time reverse transcription-PCR (qRT-PCR) indicated indicated that PtCAMTAs were induced by mannitol, NaCl, cold stress, pathogenic infection with A. alternata, and phytohormone treatments with abscisic acid, salicylic acid, and methyl jasmonate. We analyzed the expression of homologous genes between P. trichocarpa and P. ussuriensis and alternative splicing forms of PtCAMTA genes under cold stress. We also performed a network interaction analysis for PtCAMTA proteins to predict their interactions and associations. The results of the present study serve as a basis for future functional studies on the Populus CAMTA family.

Alternaria alternata peritonitis in a patient undergoing continuous ambulatory peritoneal dialysis.

Fungal peritonitis is a serious complication of peritoneal dialysis (PD) leading to loss of ultrafiltration and discontinuation of PD treatment. The most frequently isolated fungi are Candida albicans and, filamentous fungi such Alternaria alternata species are found only rarely. We report the case of a 75-year-old woman who developed peritonitis due to this black fungus.

Cutaneous phaeohyphomycosis in a hematopoietic stem cell transplant patient caused by Alternaria rosae: First case report.

Alternaria species have been reported as a rare cause of fungal infection in organ and stem cell transplant recipients, but to date, no reports have been published of infection in humans caused by Alternaria rosae. Here, we report cutaneous A. rosae infection in a 66-year-old farmer with a history of primary myelofibrosis who had undergone allogeneic unrelated donor hematopoietic stem cell transplantation. Forty-nine days post transplant, he presented with a nodule on the thumb with no findings suggestive of disseminated infection. Pathology, culture, and molecular speciation showed the nodule was caused by cutaneous A. rosae. He had been on voriconazole as antifungal prophylaxis, but was found to have a subtherapeutic voriconazole level. He was switched to posaconazole based on published in vitro data showing its superior efficacy in Alternaria treatment. Susceptibility testing showed that the A. rosae isolate was indeed susceptible to posaconazole. His cutaneous lesion remained stable, but he died from respiratory failure secondary to lobar pneumonia. At lung autopsy, A. rosae was not identified in the lungs. We believe this to be the first published report, to our knowledge, of A. rosae infection in humans.

[Sinonasal fungal infections are not exclusively due to mucorales and Aspergillus!]. / Les infections fongiques nasosinusiennes ne sont pas uniquement liées aux mucorales et aux Aspergillus !

Rhino-sinusal infections are serious diseases and possibly lethal. When they are invasive, we easily discuss apergilloses and mucormycoses. The confirmation of the diagnosis of mucormycosis need an extensive surgery for precise histopathological and mycological evaluation. The pathologist may be faced to other rare mycoses such as phaeohyphomycoses, which present different morphological features than mucormycoses and Aspergillus. Once the diagnosis is established, an appropriate antifungal treatment is quickly started. The aim of our work is to report two observations of phaeohyphomycoses, to describe their histopathological features, to discuss complementary diagnostic methods and to present the main differential diagnoses.

Preclinical Experimental and Mathematical Approaches for Assessing Effective Doses of Inhaled Drugs, Using Mometasone to Support Human Dose Predictions.

BACKGROUND: Understanding the relationship between dose, lung exposure, and drug efficacy continues to be a challenging aspect of inhaled drug development. An experimental inhalation platform was developed using mometasone furoate to link rodent lung exposure to its in vivo pharmacodynamic (PD) effects. METHODS: We assessed the effect of mometasone delivered directly to the lung in two different rodent PD models of lung inflammation. The data obtained were used to develop and evaluate a mathematical model to estimate drug dissolution, transport, distribution, and efficacy, following inhaled delivery in rodents and humans. RESULTS: Mometasone directly delivered to the lung, in both LPS and Alternaria alternata rat models, resulted in dose dependent inhibition of BALf cellular inflammation. The parameters for our mathematical model were calibrated to describe the observed lung and systemic exposure profiles of mometasone in humans and in animal models. We found that physicochemical properties, such as lung fluid solubility and lipophilicity, strongly influenced compound distribution and lung retention. CONCLUSIONS: Presently, we report on a novel and sophisticated mathematical model leading to improvements in a current inhaled drug development practices by providing a quantitative understanding of the relationship between PD effects and drug concentration in lungs.

Subconjunctival Injection of Fluconazole in the Treatment of Fungal Alternaria Keratitis.

PURPOSE: We report two cases of Alternaria keratitis refractory to the conventional antifungal medical treatment successfully treated with subconjunctival fluconazole injection. METHODS: Report of two cases. RESULTS: After subconjunctival injection of fluconazole (2 mg/mL) 0.5 mL twice a day for 5 days then once a day till 14 days, two cases of Alternaria keratitis refractory to the conventional antifungal medical treatment were successfully treated. No severe local and systemic side effects were found in these two patients. CONCLUSIONS: Alternaria keratitis has a varied clinical presentation and suspicion must be maintained for unusual causes of infectious keratitis. Alternaria keratitis can be difficult to eradicate even with traditional antifungals such as amphotericin B and natamycin. Subconjunctival injection of fluconazole could be effective for Alternaria keratitis unresponsive to conventional antifungal medical treatment.

Unusual fungal sepsis of Alternaria alternata in acute lymphoblastic leukaemia in an adult patient.

We report a case of unusual fungal sepsis of Alternaria alternata in a patient of acute lymphoblastic leukaemia in 62-year-old male who presented with complaints of 'off and on' fever with decreased oral intake. On evaluation, haemogram showed low platelet count and 68% blast cells in peripheral blood. On flow cytometry of peripheral blood, the gated blasts (approximately 55%) highly express CD45, CD10, CD19, CD22 and condition was diagnosed as acute lymphoblastic leukaemia. He was started on standard induction treatment along with supportive therapies. During the course of treatment, two sets of paired blood cultures were sent 48 h apart. All of blood cultures were done on Bac-T alert 3D system. All of them yielded fungus. The fungus was then grown on Sabouraud's Dextrose agar media. It was identified as A. alternata. The patient condition worsened and later had cardiac arrest in ICU and could not be revived.

Candidate Resistant Genes of Sand Pear (Pyrus pyrifolia Nakai) to Alternaria alternata Revealed by Transcriptome Sequencing.

Pear black spot (PBS) disease, which is caused by Alternaria alternata (Aa), is one of the most serious diseases affecting sand pear (Pyrus pyrifolia Nakai) cultivation worldwide. To investigate the defense mechanisms of sand pear in response to Aa, the transcriptome of a sand pear germplasm with differential resistance to Aa was analyzed using Illumina paired-end sequencing. Four libraries derived from PBS-resistant and PBS-susceptible sand pear leaves were characterized through inoculation or mock-inoculation. In total, 20.5 Gbp of sequence data and 101,632,565 reads were generated, representing 44717 genes. Approximately 66% of the genes or sequenced reads could be aligned to the pear reference genome. A large number (5213) of differentially expressed genes related to PBS resistance were obtained; 34 microsatellites were detected in these genes, and 28 genes were found to be closely related to PBS resistance. Using a transcriptome analysis in response to PBS inoculation and comparison analysis to the PHI database, 4 genes (Pbr039001, Pbr001627, Pbr025080 and Pbr023112) were considered to be promising candidates for sand pear resistance to PBS. This study provides insight into changes in the transcriptome of sand pear in response to PBS infection, and the findings have improved our understanding of the resistance mechanism of sand pear to PBS and will facilitate future gene discovery and functional genome studies of sand pear.

Alternaria arborescens infection in a healthy individual and literature review of cutaneous alternariosis.

A 28-year-old man presented at our clinic with 1-month history of an ulcer covered with crust on his left anterior tibia. Based on the morphological features and molecular identification, the patient was diagnosed as cutaneous alternariosis caused by Alternaria arborescens. He was successfully cured by oral itraconazole and topical use of 0.25% liposomal amphotericin B. A review of published studies revealed 29 cases of cutaneous alternariosis. Most cases (90%) occurred in immunosuppressed patients; itraconazole (59%) and voriconazole (24%) are the most effective treatments of choices.