PMC:7018936 / 13033-20583 JSONTXT

{"project":"TEST0","denotations":[{"id":"32117206-102-108-3873192","span":{"begin":124,"end":126},"obj":"[\"23383677\"]"},{"id":"32117206-106-112-3873193","span":{"begin":128,"end":130},"obj":"[\"26776889\"]"},{"id":"32117206-209-215-3873194","span":{"begin":231,"end":233},"obj":"[\"329127\", \"11386125\", \"11591543\", \"16702747\"]"},{"id":"32117206-61-66-3873195","span":{"begin":304,"end":305},"obj":"[\"26198455\"]"},{"id":"32117206-64-69-3873196","span":{"begin":307,"end":308},"obj":"[\"30430289\"]"},{"id":"32117206-88-94-3873197","span":{"begin":479,"end":481},"obj":"[\"28797493\"]"},{"id":"32117206-71-77-3873198","span":{"begin":1667,"end":1669},"obj":"[\"329127\"]"},{"id":"32117206-106-112-3873199","span":{"begin":1842,"end":1844},"obj":"[\"11386125\"]"},{"id":"32117206-138-144-3873200","span":{"begin":2030,"end":2032},"obj":"[\"11591543\"]"},{"id":"32117206-162-168-3873201","span":{"begin":2218,"end":2220},"obj":"[\"16702747\"]"},{"id":"32117206-139-145-3873202","span":{"begin":2388,"end":2390},"obj":"[\"17379495\"]"},{"id":"32117206-237-243-3873203","span":{"begin":2678,"end":2680},"obj":"[\"19490802\"]"},{"id":"32117206-128-134-3873204","span":{"begin":3130,"end":3132},"obj":"[\"26388311\"]"},{"id":"32117206-184-190-3873205","span":{"begin":3364,"end":3366},"obj":"[\"26585435\"]"},{"id":"32117206-229-235-3873206","span":{"begin":3718,"end":3720},"obj":"[\"26922860\"]"},{"id":"32117206-110-116-3873207","span":{"begin":4547,"end":4549},"obj":"[\"21338430\"]"},{"id":"32117206-114-120-3873208","span":{"begin":4551,"end":4553},"obj":"[\"25017686\", \"24697291\", \"28382604\"]"},{"id":"32117206-142-148-3873209","span":{"begin":4916,"end":4918},"obj":"[\"18005225\", \"14568857\", \"9061217\"]"},{"id":"32117206-129-135-3873210","span":{"begin":5053,"end":5055},"obj":"[\"16461148\"]"},{"id":"32117206-123-129-3873211","span":{"begin":5181,"end":5183},"obj":"[\"17379495\"]"},{"id":"32117206-127-133-3873212","span":{"begin":5185,"end":5187},"obj":"[\"19490802\"]"},{"id":"32117206-112-118-3873213","span":{"begin":5302,"end":5304},"obj":"[\"26179335\"]"},{"id":"32117206-64-70-3873214","span":{"begin":5371,"end":5373},"obj":"[\"23698813\"]"},{"id":"32117206-90-96-3873215","span":{"begin":5397,"end":5399},"obj":"[\"27838522\"]"},{"id":"32117206-92-98-3873216","span":{"begin":6294,"end":6296},"obj":"[\"23479226\"]"},{"id":"32117206-113-119-3873217","span":{"begin":6412,"end":6414},"obj":"[\"29168073\"]"},{"id":"32117206-128-134-3873218","span":{"begin":7353,"end":7355},"obj":"[\"30030925\"]"}],"text":"Discussion and Review\nFungal species demonstrated as triggers of allergic pulmonary diseases have been reviewed previously (17, 18), while cellular assays for ABPM diagnosis were reported as early as 1977, as described in Table 2 (19–22, 25). BAT can demonstrate an immediate-hypersensitivity mechanism (8, 9). Only three studies have evaluated the utility of BAT for ABPA diagnosis so far. All focused on CF patients, a background associated with the highest incidence of ABPA (26). Mirković et al. showed that BAT with Af extract identified sensitized patients and suggested that a combination of BAT and routine workup could detect ABPA. Moreover, the level of basophil activation was correlated with decreased lung function tests, suggesting that BAT could be used not only as a diagnostic assay, but also as a prognostic marker. Gernez et al. confirmed BAT as an effective and robust diagnostic assay for ABPA, although in their hands, in a contradictory manner, it could not discriminate between Af-sensitized and ABPA CF patients. Katelari et al. proposed the BAT cutoffs of 60.30 and 76.86% of CD63 and CD203c basophil activation for ABPA diagnosis with good sensitivity and specificity. In our patients, BAT with Af extract discriminated Af sensitized from ABPA patients by using a higher threshold. Thus, BAT with fungal antigens is a promising diagnostic tool for ABPM, but further studies are needed to prove the suitability in non-CF patients and by using other fungal antigens.\nTable 2 Overview of reports on cellular assays as a diagnostic tool for allergic mycosis.\nReferences Population Allergen(s) Markers Main conclusions\nHollmann et al. (19) 79 C. albicans-colonized patients 30 healthy controls C. albicans LST with 3H-thymidine 57% of C. albicans-colonized patients show lymphocyte response\nBrunet et al. (20) 60 C. albicans-delayed HS patients C. albicans LST with CD25 and CD69 All the patients have CD25 T cells Only patients with syndromic reactions have CD69 T cells\nTsushima et al. (21) 7 patients L. aggregatum LST with 3H-thymidine LST is positive in peripheral blood and BAL for all patients Diagnosis of HS pneumonitis induced by L. aggregatum\nYoshikawa et al. (22) One case report P. citrinum LST with 3H-thymidine LST is positive in peripheral blood and BAL Diagnosis of HS pneumonitis induced by P. citrinum\nMatsuno et al. (23) One case report A. alternata A. fumigatus C. albicans LST with 3H-thymidine Evaluation of IL-5 production LST are positive with A. fumigatus and C. albicans Only C. albicans culture produce IL-5 in vitro Diagnosis of acute eosinophilic pneumonia caused by C. albicans\nLuong et al. (24) 10 allergic fungal rhinosinusitis 11 healthy controls A. alternata A. fumigatus C. herbarum P. notatum LST with evaluation of cytokine production after 72 h of incubation Fungal antigens stimulate T-cell activation, with a Th2 immune response (IL-4 and IL-5 production)\nOgawa et al. (25) Two case reports S. commune LST with 3H-thymidine LST is positive in peripheral blood Diagnosis between Schizophyllum-asthma and ABPM\nMirkovic et al. (12) 48 CF patients 11 healthy controls A. fumigatus BAT with CD203c BAT discriminates non-sensitized and Af-sensitized patients BAT is inversely correlated with FEV1 Antifungal therapy does not altered BAT results\nGernez et al. (11) 74 CF patients 2 asthmatic patients A. fumigatus Asp f 1 BAT with CD203c and CD63 CD203c shows better discriminated performance than CD63 BAT with Af discriminates ABPA and no-ABPA CF patients BAT with Af does not discriminate non-sensitized and Af-sensitized patients BAT with Asp f 1 discriminates ABPA and no-ABPA CF patients\nKatelari et al. (13) 56 CF patients A. alternata A. fumigatus BAT with CD203c and CD63 BAT with Af discriminates ABPA and no-ABPA CF patients with a high threshold BAT with Af discriminates Af-sensitized patients who run a higher risk of ABPA No correlation between BAT with Af and BAT with A. alternata\nA. alternata, Alternaria alternata; ABPM, Allergic bronchopulmonary mycosis; B. adusta, Bjerkandera adusta; BAL, Bronchoalveolar lavage; C. albicans, Candida albicans; C. herbarium, Cladosporium herbarum; CF, Cystic fibrosis; FEV1, Forced expiratory volume in 1 s; HS, Hypersensitivity; L. aggregatum, Lyophyllum aggregatum; P. citrinum, Penicillium citrinum; P. notatum, Penicillium notatum; S. commune, Schizophyllum commune. Evaluation of the lymphocyte responses to allergen is a useful delayed drug hypersensitivity diagnostic tool (10, 27–29). LST has long been used as a diagnostic tool for delayed hypersensitivity against several fungi (Table 2). The incorporation of tritiated thymidine was the gold standard for the detection of fungal-specific T cells. Owing to the constraints inherent to radioactive methods, new markers were developed, with CD69 and CD25 upregulation being the most popular (30–32). Currently, an alternative is the lymphocyte proliferation test with 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) (33). Few studies have evaluated T helper (Th) 2 cytokine production after fungal stimulation, notably IL-5, for ABPM diagnosis (23, 24). Stimulation with Af extract and recombinant proteins in ABPA patients has shown an unusual Th2 immune response (34). Patients with invasive aspergillosis displayed a Th1 phenotype (35) and a Th17 phenotype (36), corresponding to a relevant anti-infectious immune activation. Taken together, these studies show that the immune skew present in a given patient's responses to fungal antigens can be evidenced through functional tests. However, the specificity of Th2 activation induced by fungal antigens as a diagnostic marker for ABPM has not been established, restricting the relevance of this method for ABPM diagnosis. Recently, a new method was developed, based on magnetic antigen-reactive T cell enrichment (ARTE) of CD154+ (CD40L+) T cells, which allowed the identification of rare populations of antigen-specific T cells. Briefly, after PBMC and antigen coculture, antigen-specific cells were sorted via magnetic beads, separating CD154+ conventional T (Tcon) cells from CD137+ CD154− regulatory T cells. Magnetic enrichment allowed an easy and detailed flow cytometry analysis of subpopulations (37). Tcon cells from lung immunocompromised patients showed a strong Th2 activation after fungal antigen stimulation (38). In our study, LST was not performant for ABPM diagnosis. Yet, our results suggest that LST might predict the development of ABPM. Indeed, the patient with the highest results in our cohort, presenting with a strong CD4 and CD8 T cell activation to fungal extracts, developed an ABPA 3 months after the abnormal LST assay. Detection of peripheral fungal-specific T cell, which can activate downstream humoral responses, may highlight a pathological process underlying subclinical ABPA with the potential of development of full-blown ABPA. A larger study with an extended follow-up will be essential to confirm this hypothesis.\nEx vivo cellular activation against several fungi also suggests that ABPM is the result of molecular epitope spreading due to similar T cell activation against three distinct fungi. Watai et al. showed that de novo sensitization to fungal antigens is constant during life, contrary to most inhalant allergens (39). Our data showed a major correlation in LST results with all the three allergens, thus an LST cross-reactivity due to a permanent sensitization addition on the T cell repertoire in CF patients."}

{"project":"2_test","denotations":[{"id":"32117206-23383677-35386087","span":{"begin":124,"end":126},"obj":"23383677"},{"id":"32117206-26776889-35386088","span":{"begin":128,"end":130},"obj":"26776889"},{"id":"32117206-329127-35386089","span":{"begin":231,"end":233},"obj":"329127"},{"id":"32117206-11386125-35386089","span":{"begin":231,"end":233},"obj":"11386125"},{"id":"32117206-11591543-35386089","span":{"begin":231,"end":233},"obj":"11591543"},{"id":"32117206-16702747-35386089","span":{"begin":231,"end":233},"obj":"16702747"},{"id":"32117206-26198455-35386090","span":{"begin":304,"end":305},"obj":"26198455"},{"id":"32117206-30430289-35386091","span":{"begin":307,"end":308},"obj":"30430289"},{"id":"32117206-28797493-35386092","span":{"begin":479,"end":481},"obj":"28797493"},{"id":"32117206-329127-35386093","span":{"begin":1667,"end":1669},"obj":"329127"},{"id":"32117206-11386125-35386094","span":{"begin":1842,"end":1844},"obj":"11386125"},{"id":"32117206-11591543-35386095","span":{"begin":2030,"end":2032},"obj":"11591543"},{"id":"32117206-16702747-35386096","span":{"begin":2218,"end":2220},"obj":"16702747"},{"id":"32117206-17379495-35386097","span":{"begin":2388,"end":2390},"obj":"17379495"},{"id":"32117206-19490802-35386098","span":{"begin":2678,"end":2680},"obj":"19490802"},{"id":"32117206-26388311-35386099","span":{"begin":3130,"end":3132},"obj":"26388311"},{"id":"32117206-26585435-35386100","span":{"begin":3364,"end":3366},"obj":"26585435"},{"id":"32117206-26922860-35386101","span":{"begin":3718,"end":3720},"obj":"26922860"},{"id":"32117206-21338430-35386102","span":{"begin":4547,"end":4549},"obj":"21338430"},{"id":"32117206-25017686-35386103","span":{"begin":4551,"end":4553},"obj":"25017686"},{"id":"32117206-24697291-35386103","span":{"begin":4551,"end":4553},"obj":"24697291"},{"id":"32117206-28382604-35386103","span":{"begin":4551,"end":4553},"obj":"28382604"},{"id":"32117206-18005225-35386104","span":{"begin":4916,"end":4918},"obj":"18005225"},{"id":"32117206-14568857-35386104","span":{"begin":4916,"end":4918},"obj":"14568857"},{"id":"32117206-9061217-35386104","span":{"begin":4916,"end":4918},"obj":"9061217"},{"id":"32117206-16461148-35386105","span":{"begin":5053,"end":5055},"obj":"16461148"},{"id":"32117206-17379495-35386106","span":{"begin":5181,"end":5183},"obj":"17379495"},{"id":"32117206-19490802-35386107","span":{"begin":5185,"end":5187},"obj":"19490802"},{"id":"32117206-26179335-35386108","span":{"begin":5302,"end":5304},"obj":"26179335"},{"id":"32117206-23698813-35386109","span":{"begin":5371,"end":5373},"obj":"23698813"},{"id":"32117206-27838522-35386110","span":{"begin":5397,"end":5399},"obj":"27838522"},{"id":"32117206-23479226-35386111","span":{"begin":6294,"end":6296},"obj":"23479226"},{"id":"32117206-29168073-35386112","span":{"begin":6412,"end":6414},"obj":"29168073"},{"id":"32117206-30030925-35386113","span":{"begin":7353,"end":7355},"obj":"30030925"}],"text":"Discussion and Review\nFungal species demonstrated as triggers of allergic pulmonary diseases have been reviewed previously (17, 18), while cellular assays for ABPM diagnosis were reported as early as 1977, as described in Table 2 (19–22, 25). BAT can demonstrate an immediate-hypersensitivity mechanism (8, 9). Only three studies have evaluated the utility of BAT for ABPA diagnosis so far. All focused on CF patients, a background associated with the highest incidence of ABPA (26). Mirković et al. showed that BAT with Af extract identified sensitized patients and suggested that a combination of BAT and routine workup could detect ABPA. Moreover, the level of basophil activation was correlated with decreased lung function tests, suggesting that BAT could be used not only as a diagnostic assay, but also as a prognostic marker. Gernez et al. confirmed BAT as an effective and robust diagnostic assay for ABPA, although in their hands, in a contradictory manner, it could not discriminate between Af-sensitized and ABPA CF patients. Katelari et al. proposed the BAT cutoffs of 60.30 and 76.86% of CD63 and CD203c basophil activation for ABPA diagnosis with good sensitivity and specificity. In our patients, BAT with Af extract discriminated Af sensitized from ABPA patients by using a higher threshold. Thus, BAT with fungal antigens is a promising diagnostic tool for ABPM, but further studies are needed to prove the suitability in non-CF patients and by using other fungal antigens.\nTable 2 Overview of reports on cellular assays as a diagnostic tool for allergic mycosis.\nReferences Population Allergen(s) Markers Main conclusions\nHollmann et al. (19) 79 C. albicans-colonized patients 30 healthy controls C. albicans LST with 3H-thymidine 57% of C. albicans-colonized patients show lymphocyte response\nBrunet et al. (20) 60 C. albicans-delayed HS patients C. albicans LST with CD25 and CD69 All the patients have CD25 T cells Only patients with syndromic reactions have CD69 T cells\nTsushima et al. (21) 7 patients L. aggregatum LST with 3H-thymidine LST is positive in peripheral blood and BAL for all patients Diagnosis of HS pneumonitis induced by L. aggregatum\nYoshikawa et al. (22) One case report P. citrinum LST with 3H-thymidine LST is positive in peripheral blood and BAL Diagnosis of HS pneumonitis induced by P. citrinum\nMatsuno et al. (23) One case report A. alternata A. fumigatus C. albicans LST with 3H-thymidine Evaluation of IL-5 production LST are positive with A. fumigatus and C. albicans Only C. albicans culture produce IL-5 in vitro Diagnosis of acute eosinophilic pneumonia caused by C. albicans\nLuong et al. (24) 10 allergic fungal rhinosinusitis 11 healthy controls A. alternata A. fumigatus C. herbarum P. notatum LST with evaluation of cytokine production after 72 h of incubation Fungal antigens stimulate T-cell activation, with a Th2 immune response (IL-4 and IL-5 production)\nOgawa et al. (25) Two case reports S. commune LST with 3H-thymidine LST is positive in peripheral blood Diagnosis between Schizophyllum-asthma and ABPM\nMirkovic et al. (12) 48 CF patients 11 healthy controls A. fumigatus BAT with CD203c BAT discriminates non-sensitized and Af-sensitized patients BAT is inversely correlated with FEV1 Antifungal therapy does not altered BAT results\nGernez et al. (11) 74 CF patients 2 asthmatic patients A. fumigatus Asp f 1 BAT with CD203c and CD63 CD203c shows better discriminated performance than CD63 BAT with Af discriminates ABPA and no-ABPA CF patients BAT with Af does not discriminate non-sensitized and Af-sensitized patients BAT with Asp f 1 discriminates ABPA and no-ABPA CF patients\nKatelari et al. (13) 56 CF patients A. alternata A. fumigatus BAT with CD203c and CD63 BAT with Af discriminates ABPA and no-ABPA CF patients with a high threshold BAT with Af discriminates Af-sensitized patients who run a higher risk of ABPA No correlation between BAT with Af and BAT with A. alternata\nA. alternata, Alternaria alternata; ABPM, Allergic bronchopulmonary mycosis; B. adusta, Bjerkandera adusta; BAL, Bronchoalveolar lavage; C. albicans, Candida albicans; C. herbarium, Cladosporium herbarum; CF, Cystic fibrosis; FEV1, Forced expiratory volume in 1 s; HS, Hypersensitivity; L. aggregatum, Lyophyllum aggregatum; P. citrinum, Penicillium citrinum; P. notatum, Penicillium notatum; S. commune, Schizophyllum commune. Evaluation of the lymphocyte responses to allergen is a useful delayed drug hypersensitivity diagnostic tool (10, 27–29). LST has long been used as a diagnostic tool for delayed hypersensitivity against several fungi (Table 2). The incorporation of tritiated thymidine was the gold standard for the detection of fungal-specific T cells. Owing to the constraints inherent to radioactive methods, new markers were developed, with CD69 and CD25 upregulation being the most popular (30–32). Currently, an alternative is the lymphocyte proliferation test with 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) (33). Few studies have evaluated T helper (Th) 2 cytokine production after fungal stimulation, notably IL-5, for ABPM diagnosis (23, 24). Stimulation with Af extract and recombinant proteins in ABPA patients has shown an unusual Th2 immune response (34). Patients with invasive aspergillosis displayed a Th1 phenotype (35) and a Th17 phenotype (36), corresponding to a relevant anti-infectious immune activation. Taken together, these studies show that the immune skew present in a given patient's responses to fungal antigens can be evidenced through functional tests. However, the specificity of Th2 activation induced by fungal antigens as a diagnostic marker for ABPM has not been established, restricting the relevance of this method for ABPM diagnosis. Recently, a new method was developed, based on magnetic antigen-reactive T cell enrichment (ARTE) of CD154+ (CD40L+) T cells, which allowed the identification of rare populations of antigen-specific T cells. Briefly, after PBMC and antigen coculture, antigen-specific cells were sorted via magnetic beads, separating CD154+ conventional T (Tcon) cells from CD137+ CD154− regulatory T cells. Magnetic enrichment allowed an easy and detailed flow cytometry analysis of subpopulations (37). Tcon cells from lung immunocompromised patients showed a strong Th2 activation after fungal antigen stimulation (38). In our study, LST was not performant for ABPM diagnosis. Yet, our results suggest that LST might predict the development of ABPM. Indeed, the patient with the highest results in our cohort, presenting with a strong CD4 and CD8 T cell activation to fungal extracts, developed an ABPA 3 months after the abnormal LST assay. Detection of peripheral fungal-specific T cell, which can activate downstream humoral responses, may highlight a pathological process underlying subclinical ABPA with the potential of development of full-blown ABPA. A larger study with an extended follow-up will be essential to confirm this hypothesis.\nEx vivo cellular activation against several fungi also suggests that ABPM is the result of molecular epitope spreading due to similar T cell activation against three distinct fungi. Watai et al. showed that de novo sensitization to fungal antigens is constant during life, contrary to most inhalant allergens (39). Our data showed a major correlation in LST results with all the three allergens, thus an LST cross-reactivity due to a permanent sensitization addition on the T cell repertoire in CF patients."}

{"project":"MyTest","denotations":[{"id":"32117206-23383677-35386087","span":{"begin":124,"end":126},"obj":"23383677"},{"id":"32117206-26776889-35386088","span":{"begin":128,"end":130},"obj":"26776889"},{"id":"32117206-329127-35386089","span":{"begin":231,"end":233},"obj":"329127"},{"id":"32117206-11386125-35386089","span":{"begin":231,"end":233},"obj":"11386125"},{"id":"32117206-11591543-35386089","span":{"begin":231,"end":233},"obj":"11591543"},{"id":"32117206-16702747-35386089","span":{"begin":231,"end":233},"obj":"16702747"},{"id":"32117206-26198455-35386090","span":{"begin":304,"end":305},"obj":"26198455"},{"id":"32117206-30430289-35386091","span":{"begin":307,"end":308},"obj":"30430289"},{"id":"32117206-28797493-35386092","span":{"begin":479,"end":481},"obj":"28797493"},{"id":"32117206-329127-35386093","span":{"begin":1667,"end":1669},"obj":"329127"},{"id":"32117206-11386125-35386094","span":{"begin":1842,"end":1844},"obj":"11386125"},{"id":"32117206-11591543-35386095","span":{"begin":2030,"end":2032},"obj":"11591543"},{"id":"32117206-16702747-35386096","span":{"begin":2218,"end":2220},"obj":"16702747"},{"id":"32117206-17379495-35386097","span":{"begin":2388,"end":2390},"obj":"17379495"},{"id":"32117206-19490802-35386098","span":{"begin":2678,"end":2680},"obj":"19490802"},{"id":"32117206-26388311-35386099","span":{"begin":3130,"end":3132},"obj":"26388311"},{"id":"32117206-26585435-35386100","span":{"begin":3364,"end":3366},"obj":"26585435"},{"id":"32117206-26922860-35386101","span":{"begin":3718,"end":3720},"obj":"26922860"},{"id":"32117206-21338430-35386102","span":{"begin":4547,"end":4549},"obj":"21338430"},{"id":"32117206-25017686-35386103","span":{"begin":4551,"end":4553},"obj":"25017686"},{"id":"32117206-24697291-35386103","span":{"begin":4551,"end":4553},"obj":"24697291"},{"id":"32117206-28382604-35386103","span":{"begin":4551,"end":4553},"obj":"28382604"},{"id":"32117206-18005225-35386104","span":{"begin":4916,"end":4918},"obj":"18005225"},{"id":"32117206-14568857-35386104","span":{"begin":4916,"end":4918},"obj":"14568857"},{"id":"32117206-9061217-35386104","span":{"begin":4916,"end":4918},"obj":"9061217"},{"id":"32117206-16461148-35386105","span":{"begin":5053,"end":5055},"obj":"16461148"},{"id":"32117206-17379495-35386106","span":{"begin":5181,"end":5183},"obj":"17379495"},{"id":"32117206-19490802-35386107","span":{"begin":5185,"end":5187},"obj":"19490802"},{"id":"32117206-26179335-35386108","span":{"begin":5302,"end":5304},"obj":"26179335"},{"id":"32117206-23698813-35386109","span":{"begin":5371,"end":5373},"obj":"23698813"},{"id":"32117206-27838522-35386110","span":{"begin":5397,"end":5399},"obj":"27838522"},{"id":"32117206-23479226-35386111","span":{"begin":6294,"end":6296},"obj":"23479226"},{"id":"32117206-29168073-35386112","span":{"begin":6412,"end":6414},"obj":"29168073"},{"id":"32117206-30030925-35386113","span":{"begin":7353,"end":7355},"obj":"30030925"}],"namespaces":[{"prefix":"_base","uri":"https://www.uniprot.org/uniprot/testbase"},{"prefix":"UniProtKB","uri":"https://www.uniprot.org/uniprot/"},{"prefix":"uniprot","uri":"https://www.uniprot.org/uniprotkb/"}],"text":"Discussion and Review\nFungal species demonstrated as triggers of allergic pulmonary diseases have been reviewed previously (17, 18), while cellular assays for ABPM diagnosis were reported as early as 1977, as described in Table 2 (19–22, 25). BAT can demonstrate an immediate-hypersensitivity mechanism (8, 9). Only three studies have evaluated the utility of BAT for ABPA diagnosis so far. All focused on CF patients, a background associated with the highest incidence of ABPA (26). Mirković et al. showed that BAT with Af extract identified sensitized patients and suggested that a combination of BAT and routine workup could detect ABPA. Moreover, the level of basophil activation was correlated with decreased lung function tests, suggesting that BAT could be used not only as a diagnostic assay, but also as a prognostic marker. Gernez et al. confirmed BAT as an effective and robust diagnostic assay for ABPA, although in their hands, in a contradictory manner, it could not discriminate between Af-sensitized and ABPA CF patients. Katelari et al. proposed the BAT cutoffs of 60.30 and 76.86% of CD63 and CD203c basophil activation for ABPA diagnosis with good sensitivity and specificity. In our patients, BAT with Af extract discriminated Af sensitized from ABPA patients by using a higher threshold. Thus, BAT with fungal antigens is a promising diagnostic tool for ABPM, but further studies are needed to prove the suitability in non-CF patients and by using other fungal antigens.\nTable 2 Overview of reports on cellular assays as a diagnostic tool for allergic mycosis.\nReferences Population Allergen(s) Markers Main conclusions\nHollmann et al. (19) 79 C. albicans-colonized patients 30 healthy controls C. albicans LST with 3H-thymidine 57% of C. albicans-colonized patients show lymphocyte response\nBrunet et al. (20) 60 C. albicans-delayed HS patients C. albicans LST with CD25 and CD69 All the patients have CD25 T cells Only patients with syndromic reactions have CD69 T cells\nTsushima et al. (21) 7 patients L. aggregatum LST with 3H-thymidine LST is positive in peripheral blood and BAL for all patients Diagnosis of HS pneumonitis induced by L. aggregatum\nYoshikawa et al. (22) One case report P. citrinum LST with 3H-thymidine LST is positive in peripheral blood and BAL Diagnosis of HS pneumonitis induced by P. citrinum\nMatsuno et al. (23) One case report A. alternata A. fumigatus C. albicans LST with 3H-thymidine Evaluation of IL-5 production LST are positive with A. fumigatus and C. albicans Only C. albicans culture produce IL-5 in vitro Diagnosis of acute eosinophilic pneumonia caused by C. albicans\nLuong et al. (24) 10 allergic fungal rhinosinusitis 11 healthy controls A. alternata A. fumigatus C. herbarum P. notatum LST with evaluation of cytokine production after 72 h of incubation Fungal antigens stimulate T-cell activation, with a Th2 immune response (IL-4 and IL-5 production)\nOgawa et al. (25) Two case reports S. commune LST with 3H-thymidine LST is positive in peripheral blood Diagnosis between Schizophyllum-asthma and ABPM\nMirkovic et al. (12) 48 CF patients 11 healthy controls A. fumigatus BAT with CD203c BAT discriminates non-sensitized and Af-sensitized patients BAT is inversely correlated with FEV1 Antifungal therapy does not altered BAT results\nGernez et al. (11) 74 CF patients 2 asthmatic patients A. fumigatus Asp f 1 BAT with CD203c and CD63 CD203c shows better discriminated performance than CD63 BAT with Af discriminates ABPA and no-ABPA CF patients BAT with Af does not discriminate non-sensitized and Af-sensitized patients BAT with Asp f 1 discriminates ABPA and no-ABPA CF patients\nKatelari et al. (13) 56 CF patients A. alternata A. fumigatus BAT with CD203c and CD63 BAT with Af discriminates ABPA and no-ABPA CF patients with a high threshold BAT with Af discriminates Af-sensitized patients who run a higher risk of ABPA No correlation between BAT with Af and BAT with A. alternata\nA. alternata, Alternaria alternata; ABPM, Allergic bronchopulmonary mycosis; B. adusta, Bjerkandera adusta; BAL, Bronchoalveolar lavage; C. albicans, Candida albicans; C. herbarium, Cladosporium herbarum; CF, Cystic fibrosis; FEV1, Forced expiratory volume in 1 s; HS, Hypersensitivity; L. aggregatum, Lyophyllum aggregatum; P. citrinum, Penicillium citrinum; P. notatum, Penicillium notatum; S. commune, Schizophyllum commune. Evaluation of the lymphocyte responses to allergen is a useful delayed drug hypersensitivity diagnostic tool (10, 27–29). LST has long been used as a diagnostic tool for delayed hypersensitivity against several fungi (Table 2). The incorporation of tritiated thymidine was the gold standard for the detection of fungal-specific T cells. Owing to the constraints inherent to radioactive methods, new markers were developed, with CD69 and CD25 upregulation being the most popular (30–32). Currently, an alternative is the lymphocyte proliferation test with 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) (33). Few studies have evaluated T helper (Th) 2 cytokine production after fungal stimulation, notably IL-5, for ABPM diagnosis (23, 24). Stimulation with Af extract and recombinant proteins in ABPA patients has shown an unusual Th2 immune response (34). Patients with invasive aspergillosis displayed a Th1 phenotype (35) and a Th17 phenotype (36), corresponding to a relevant anti-infectious immune activation. Taken together, these studies show that the immune skew present in a given patient's responses to fungal antigens can be evidenced through functional tests. However, the specificity of Th2 activation induced by fungal antigens as a diagnostic marker for ABPM has not been established, restricting the relevance of this method for ABPM diagnosis. Recently, a new method was developed, based on magnetic antigen-reactive T cell enrichment (ARTE) of CD154+ (CD40L+) T cells, which allowed the identification of rare populations of antigen-specific T cells. Briefly, after PBMC and antigen coculture, antigen-specific cells were sorted via magnetic beads, separating CD154+ conventional T (Tcon) cells from CD137+ CD154− regulatory T cells. Magnetic enrichment allowed an easy and detailed flow cytometry analysis of subpopulations (37). Tcon cells from lung immunocompromised patients showed a strong Th2 activation after fungal antigen stimulation (38). In our study, LST was not performant for ABPM diagnosis. Yet, our results suggest that LST might predict the development of ABPM. Indeed, the patient with the highest results in our cohort, presenting with a strong CD4 and CD8 T cell activation to fungal extracts, developed an ABPA 3 months after the abnormal LST assay. Detection of peripheral fungal-specific T cell, which can activate downstream humoral responses, may highlight a pathological process underlying subclinical ABPA with the potential of development of full-blown ABPA. A larger study with an extended follow-up will be essential to confirm this hypothesis.\nEx vivo cellular activation against several fungi also suggests that ABPM is the result of molecular epitope spreading due to similar T cell activation against three distinct fungi. Watai et al. showed that de novo sensitization to fungal antigens is constant during life, contrary to most inhalant allergens (39). Our data showed a major correlation in LST results with all the three allergens, thus an LST cross-reactivity due to a permanent sensitization addition on the T cell repertoire in CF patients."}