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Chronic lung allograft dysfunction (CLAD) remains the major complication limiting long-term survival among lung transplant recipients (LTRs). Limited understanding of CLAD immunopathogenesis and a paucity of biomarkers remain substantial barriers for earlier detection and therapeutic interventions for CLAD. We hypothesized the airway transcriptome would reflect key immunologic changes in disease. We compared airway brush-derived transcriptomic signatures in CLAD (n = 24) versus non-CLAD (n = 21) LTRs. A targeted assessment of the proteome using concomitant bronchoalveolar lavage (BAL) fluid for 24 cytokines/chemokines and alloimmune T cell responses was performed to validate the airway transcriptome. We observed an airway transcriptomic signature of differential genes expressed (DGEs) in CLAD marked by Type-1 immunity and striking upregulation of two endogenous immune regulators: indoleamine 2, 3 dioxygenase 1 (IDO-1) and tumor necrosis factor receptor superfamily 6B (TNFRSF6B). Advanced CLAD staging was associated with a more intense airway transcriptome signature. In a validation cohort using the identified signature, we found an area under the curve (AUC) of 0.77 for CLAD LTRs. Targeted proteomic analyses revealed a predominant Type-1 profile with detection of IFN-γ, TNF-α, and IL-1β as dominant CLAD cytokines, correlating with the airway transcriptome. The airway transcriptome provides novel insights into CLAD immunopathogenesis and biomarkers that may impact diagnosis of CLAD.

In humans and animals, offspring of allergic mothers have increased responsiveness to allergens. This is blocked in mice by maternal supplementation with α-tocopherol (αT). Also, adults and children with allergic asthma have airway microbiome dysbiosis with increased Proteobacteria and may have decreased Bacteroidota. It is not known whether αT alters neonate development of lung microbiome dysbiosis or whether neonate lung dysbiosis modifies development of allergy. To address this, the bronchoalveolar lavage was analyzed by 16S rRNA gene analysis (bacterial microbiome) from pups of allergic and non-allergic mothers with a basal diet or αT-supplemented diet. Before and after allergen challenge, pups of allergic mothers had dysbiosis in lung microbial composition with increased Proteobacteria and decreased Bacteroidota and this was blocked by αT supplementation. We determined whether intratracheal transfer of pup lung dysbiotic microbial communities modifies the development of allergy in recipient pups early in life. Interestingly, transfer of dysbiotic lung microbial communities from neonates of allergic mothers to neonates of non-allergic mothers was sufficient to confer responsiveness to allergen in the recipient pups. In contrast, neonates of allergic mothers were not protected from development of allergy by transfer of donor lung microbial communities from either neonates of non-allergic mothers or neonates of αT-supplemented allergic mothers. These data suggest that the dysbiotic lung microbiota is dominant and sufficient for enhanced neonate responsiveness to allergen. Importantly, infants within the INHANCE cohort with an anti-inflammatory profile of tocopherol isoforms had an altered microbiome composition compared to infants with a pro-inflammatory profile of tocopherol isoforms. These data may inform design of future studies for approaches in the prevention or intervention in asthma and allergic disease early in life.

Epidemiological studies demonstrate that maternal obesity and maternal allergy are major risk factors for asthma in offspring. However, the impact of maternal allergy and obesity on offspring lung insulin signaling and allergen responsiveness is not known. To evaluate this, allergic and nonallergic female mice were fed a high-fat diet or low-fat diet from 7 wk before pregnancy until weaning. Neonatal pups were allergen-sensitized and allergen-challenged and then were assessed for obesity, insulin signaling, and allergic inflammation. Compared with pups of nonobese nonallergic mothers, allergen-challenged pups of obese nonallergic mothers, nonobese allergic mothers, and obese allergic mothers had bronchoalveolar lavage (BAL) eosinophilia, with the pups of obese allergic mothers having the highest BAL eosinophilia. These pups also had lower insulin-induced lung AKT phosphorylation, indicating a decrease in lung parenchymal insulin sensitivity. In cross-fostering experiments, allergen-challenged pups exposed to both pre- and postnatal obese allergic mothers had the highest level of BAL eosinophilia. Maternal obesity or allergy increased offspring serum allergen-specific IgE and interleukin-5 that was highest when the mother was both obese and allergic. Also, allergen-challenged pups exposed to both pre- and postnatal obese allergic mothers had the highest level of interleukin-5. In summary, offspring born to obese allergic mothers have decreased lung insulin sensitivity and increased lung allergic inflammation. Interestingly, our data also demonstrate that there is both a pregnancy and postpregnancy aspect of maternal allergy and obesity that enhances allergen responsiveness in offspring. [ABSTRACT FROM AUTHOR]

Author(s): Aziguli Yigaimu [sup.1], Jiahua Chang [sup.1], Amina Hoji [sup.1], Turghun Muhammad [sup.1], Burabiye Yakup [sup.2], Wenwu Yang [sup.1], Sailemayi Aihebaier [sup.1] Author Affiliations: (1) grid.413254.5, 0000 0000 9544 7024, [...], Auxins play a very important role in plant development and growth. Herein a rapid and convenient method was developed for the quantification of total auxins including indole-3-acetic acid, indole-3-propionic acid and indole-3-butyric acid in mung bean sprouts by means of spectrofluorometry. Based on the same fluorescence properties of the compounds, a linear working curve was established between total concentrations of auxins and fluorescence intensities of their solutions. Under optimized conditions, correlation coefficient and limit of detection were found to be 0.9993 and 0.03 [mu]g/mL, respectively, in linear range of 0.10-24 [mu]g/mL. This method was successfully employed to the determination of total auxins after their extraction from mung bean sprouts, and the recovery was found to be in the range of 100-104% with RSD below 5%. This method has been proved to be simple, reproducible and accurate, also applicable for food samples without complicated apparatus and procedures.

RationaleThe mechanism by which acute allograft rejection leads to chronic rejection remains poorly understood despite its common occurrence. Exosomes, membrane vesicles released from cells within the lung allograft, contain a diverse array of biomolecules that closely reflect the biologic state of the cell and tissue from which they are released. Exosome transcriptomes may provide a better understanding of the rejection process. Furthermore, biomarkers originating from this transcriptome could provide timely and sensitive detection of acute cellular rejection (AR), reducing the incidence of severe AR and chronic lung allograft dysfunction and improving outcomes.ObjectivesTo provide an in-depth analysis of the bronchoalveolar lavage fluid exosomal shuttle RNA population after lung transplantation and evaluate for differential expression between acute AR and quiescence.MethodsSerial bronchoalveolar lavage specimens were ultracentrifuged to obtain the exosomal pellet for RNA extraction, on which RNA-Seq was performed.Measurements and main resultsAR demonstrates an intense inflammatory environment, skewed toward both innate and adaptive immune responses. Novel, potential upstream regulators identified offer potential therapeutic targets.ConclusionsOur findings validate bronchoalveolar lavage fluid exosomal shuttle RNA as a source for understanding the pathophysiology of AR and for biomarker discovery in lung transplantation.

Neonatal mice with heterozygous mutations in genes encoding the skin barrier proteins filaggrin and mattrin (flaky tail mice [FT+/−]) exhibit oral peanut-induced anaphylaxis after skin sensitization. As we have previously reported, sensitization in this model is achieved via skin co- exposure to the environmental allergen Alternaria alternata (Alt), peanut extract (PNE), and detergent. However, the function of Alt in initiation of peanut allergy in this model is little understood. The purpose of this study was to investigate candidate cytokines induced by Alt in the skin and determine the role of these cytokines in the development of food allergy, namely oncostatin M (Osm), amphiregulin (Areg), and IL-33. RT-qPCR analyses demonstrated that skin of FT+/− neonates expressed Il33 and Osm following Alt or Alt/PNE but not PNE exposure. By contrast, expression of Areg was induced by either Alt, PNE, or Alt/PNE sensitization in FT+/− neonates. In scRNAseq analyses, Osm, Areg, and Il33 were expressed by several cell types, including a keratinocyte cluster that was expanded in the skin of Alt/PNE-exposed FT+/− pups as compared to Alt/PNE-exposed WT pups. Areg and OSM were required for oral PNE-induced anaphylaxis since anaphylaxis was inhibited by administration of neutralizing anti-Areg or anti-OSM antibodies prior to each skin sensitization with Alt/PNE. It was then determined if intradermal injection of recombinant IL33 (rIL33), rAreg, or rOSM in the skin could substitute for Alt during skin sensitization to PNE. PNE skin sensitization with intradermal rIL33 was sufficient for oral PNE-induced anaphylaxis, whereas skin sensitization with intradermal rAreg or rOSM during skin exposure to PNE was not sufficient for anaphylaxis to oral PNE challenge. Based on these studies a pathway for IL33, Areg and OSM in Alt/PNE sensitized FT+/− skin was defined for IgE induction and anaphylaxis. Alt stimulated two pathways, an IL33 pathway and a pathway involving OSM and Areg. These two pathways acted in concert with PNE to induce food allergy in pups with skin barrier mutations.

PurposeWHO grade 2 low-grade gliomas (LGG) with high risk factors for recurrence are mostly lethal despite current treatments. We conducted a phase I study to evaluate the safety and immunogenicity of subcutaneous vaccinations with synthetic peptides for glioma-associated antigen (GAA) epitopes in HLA-A2(+) adults with high-risk LGGs in the following three cohorts: (i) patients without prior progression, chemotherapy, or radiotherapy (RT); (ii) patients without prior progression or chemotherapy but with prior RT; and (iii) recurrent patients.Experimental designGAAs were IL13Rα2, EphA2, WT1, and Survivin. Synthetic peptides were emulsified in Montanide-ISA-51 and given every 3 weeks for eight courses with intramuscular injections of poly-ICLC, followed by q12 week booster vaccines.ResultsCohorts 1, 2, and 3 enrolled 12, 1, and 10 patients, respectively. No regimen-limiting toxicity was encountered except for one case with grade 3 fever, fatigue, and mood disturbance (cohort 1). ELISPOT assays demonstrated robust IFNγ responses against at least three of the four GAA epitopes in 10 and 4 cases of cohorts 1 and 3, respectively. Cohort 1 patients demonstrated significantly higher IFNγ responses than cohort 3 patients. Median progression-free survival (PFS) periods since the first vaccine are 17 months in cohort 1 (range, 10-47+) and 12 months in cohort 3 (range, 3-41+). The only patient with large astrocytoma in cohort 2 has been progression-free for more than 67 months since diagnosis.ConclusionThe current regimen is well tolerated and induces robust GAA-specific responses in WHO grade 2 glioma patients. These results warrant further evaluations of this approach. Clin Cancer Res; 21(2); 286-94. ©2014 AACR.

Bronchiolitis obliterans syndrome (BOS) is the major obstacle to long-term survival after lung transplantation, yet markers for early detection and intervention are currently lacking. Given the role of regulatory T cells (Treg) in modulation of immunity, we hypothesized that frequencies of Treg in bronchoalveolar lavage fluid (BALF) after lung transplantation would predict subsequent development of BOS. Seventy BALF specimens obtained from 47 lung transplant recipients were analyzed for Treg lymphocyte subsets by flow cytometry, in parallel with ELISA measurements of chemokines. Allograft biopsy tissue was stained for chemokines of interest. Treg were essentially all CD45RA(-), and total Treg frequency did not correlate to BOS outcome. The majority of Treg were CCR4(+) and CD103(-) and neither of these subsets correlated to risk for BOS. In contrast, higher percentages of CCR7(+) Treg correlated to reduced risk of BOS. Additionally, the CCR7 ligand CCL21 correlated with CCR7(+) Treg frequency and inversely with BOS. Higher frequencies of CCR7(+) CD3(+)CD4(+)CD25(hi)Foxp3(+)CD45RA(-) lymphocytes in lung allografts is associated with protection against subsequent development of BOS, suggesting that this subset of putative Treg may down-modulate alloimmunity. CCL21 may be pivotal for the recruitment of this distinct subset to the lung allograft and thereby decrease the risk for chronic rejection.

Bronchiolitis obliterans syndrome (BOS) is the major obstacle to long-term survival after lung transplantation, yet markers for early detection and intervention are currently lacking. Given the role of regulatory T cells (Treg) in modulation of immunity, we hypothesized that frequencies of Treg in bronchoalveolar lavage fluid (BALF) after lung transplantation would predict subsequent development of BOS. Seventy BALF specimens obtained from 47 lung transplant recipients were analyzed for Treg lymphocyte subsets by flow cytometry, in parallel with ELISA measurements of chemokines. Allograft biopsy tissue was stained for chemokines of interest. Treg were essentially all CD45RA(-), and total Treg frequency did not correlate to BOS outcome. The majority of Treg were CCR4(+) and CD103(-) and neither of these subsets correlated to risk for BOS. In contrast, higher percentages of CCR7(+) Treg correlated to reduced risk of BOS. Additionally, the CCR7 ligand CCL21 correlated with CCR7(+) Treg frequency and inversely with BOS. Higher frequencies of CCR7(+) CD3(+)CD4(+)CD25(hi)Foxp3(+)CD45RA(-) lymphocytes in lung allografts is associated with protection against subsequent development of BOS, suggesting that this subset of putative Treg may down-modulate alloimmunity. CCL21 may be pivotal for the recruitment of this distinct subset to the lung allograft and thereby decrease the risk for chronic rejection.