Detection of Antibody-Dependent Cell-Mediated Cytotoxicity-Supporting Antibodies by NK-92-CD16A Cell Externalization of CD107a: Recognition of Antibody Afucosylation and Assay Optimization.

Antibody-dependent cell-mediated cytotoxicity (ADCC) by natural killer (NK) lymphocytes eliminates cells infected with viruses. Anti-viral ADCC requires three components: (1) antibody; (2) effector lymphocytes with the Fc-IgG receptor CD16A; and (3) viral proteins in infected cell membranes. Fc-afucosylated antibodies bind with greater affinity to CD16A than fucosylated antibodies; individuals' variation in afucosylation contributes to differences in ADCC. Current assays for afucosylated antibodies involve expensive methods. We report an improved bioassay for antibodies that supports ADCC, which encompasses afucosylation. This assay utilizes the externalization of CD107a by NK-92-CD16A cells after antibody recognition. We used anti-CD20 monoclonal antibodies, GA101 WT or glycoengineered (GE), 10% or ~50% afucosylated, and CD20-positive Raji target cells. CD107a increased detection 7-fold compared to flow cytometry to detect Raji-bound antibodies. WT and GE antibody effective concentrations (EC50s) for CD107a externalization differed by 20-fold, with afucosylated GA101-GE more detectable. The EC50s for CD107a externalization vs. 51Cr cell death were similar for NK-92-CD16A and blood NK cells. Notably, the % CD107a-positive cells were negatively correlated with dead Raji cells and were nearly undetectable at high NK:Raji ratios required for cytotoxicity. This bioassay is very sensitive and adaptable to assess anti-viral antibodies but unsuitable as a surrogate assay to monitor cell death after ADCC.

Characterization of CD4+ and CD8+ T cells responses in the mixed lymphocyte reaction by flow cytometry and single cell RNA sequencing.

Background: The Mixed Lymphocyte Reaction (MLR) consists in the allogeneic co-culture of monocytes derived dendritic cells (MoDCs) with T cells from another donor. This in vitro assay is largely used for the assessment of immunotherapy compounds. Nevertheless, the phenotypic changes associated with lymphocyte responsiveness under MLR have never been thoroughly evaluated. Methods: Here, we used multiplex cytokine and chemokine assays, multiparametric flow cytometry and single cell RNA sequencing to deeply characterize T cells activation and function in the context of CD4+- and CD8+-specific MLR kinetics. Results: We showed that CD4+ and CD8+ T cells in MLR share common classical markers of response such as polyfunctionality, increased proliferation and CD25 expression but differ in their kinetics and amplitude of activation as well as their patterns of cytokines secretion and immune checkpoints expression. The analysis of immunoreactive Ki-67+CD25+ T cells identified PBK, LRR1 and MYO1G as new potential markers of MLR response. Using cell-cell communication network inference and pathway analysis on single cell RNA sequencing data, we also highlighted key components of the immunological synapse occurring between T cells and the stimulatory MoDCs together with downstream signaling pathways involved in CD4+ and CD8+ T cells activation. Conclusion: These results provide a deep understanding of the kinetics of the MLR assay for CD4+ or CD8+ T cells and may allow to better characterize compounds impacting MLR and eventually identify new strategies for immunotherapy in cancer.

Molecular Identification and Characterization of Five Ganoderma Species from the Lower Volta River Basin of Ghana Based on Nuclear Ribosomal DNA (nrDNA) Sequences.

Ganoderma is a genus of biomedical fungus that is used in the development of numerous health products throughout the world. The Lower Volta River Basin of Ghana is an undulating land surface covered by extensive vegetation and water bodies and is rich in polypore mushrooms resembling various members of the Ganoderma genus. Despite the extensive biopharmaceutical benefits of Ganoderma spp., the isolates from the Lower Volta River Basin have not been properly characterized, thus limiting their use in the development of biotechnological products. In this study, Ganoderma spp. collected from the Lower Volta River Basin were genetically analyzed using the nuclear ribosomal sequences, the internal transcribed spacer 2 (ITS 2), the complete internal transcribed spacer (ITS), and the nuclear large subunit (nLSU). Blastn search and sequence analysis revealed that the sample we coded as Ganoderma LVRB-2 belongs to G. mbrekobenum, whereas Ganoderma LVRB-1, Ganoderma LVRB-14, and Ganoderma LVRB-16 belong to the species G. enigmaticum. Our analysis further demonstrates that Ganoderma LVRB-17 belongs to the species G. resinaceum. Thus, the five samples collected in the present study were positioned in three different distinct groups, namely G. mbrekobenum, G. enigmaticum, and G. resinaceum. The current data may serve as reference points for future studies.

Mucosal Immunity and the Gut-Microbiota-Brain-Axis in Neuroimmune Disease.

Recent advances in next-generation sequencing (NGS) technologies have opened the door to a wellspring of information regarding the composition of the gut microbiota. Leveraging NGS technology, early metagenomic studies revealed that several diseases, such as Alzheimer's disease, Parkinson's disease, autism, and myalgic encephalomyelitis, are characterized by alterations in the diversity of gut-associated microbes. More recently, interest has shifted toward understanding how these microbes impact their host, with a special emphasis on their interactions with the brain. Such interactions typically occur either systemically, through the production of small molecules in the gut that are released into circulation, or through signaling via the vagus nerves which directly connect the enteric nervous system to the central nervous system. Collectively, this system of communication is now commonly referred to as the gut-microbiota-brain axis. While equally important, little attention has focused on the causes of the alterations in the composition of gut microbiota. Although several factors can contribute, mucosal immunity plays a significant role in shaping the microbiota in both healthy individuals and in association with several diseases. The purpose of this review is to provide a brief overview of the components of mucosal immunity that impact the gut microbiota and then discuss how altered immunological conditions may shape the gut microbiota and consequently affect neuroimmune diseases, using a select group of common neuroimmune diseases as examples.

Optimizing NK-92 serial killers: gamma irradiation, CD95/Fas-ligation, and NK or LAK attack limit cytotoxic efficacy.

BACKGROUND: The NK cell line NK-92 and its genetically modified variants are receiving attention as immunotherapies to treat a range of malignancies. However, since NK-92 cells are themselves tumors, they require irradiation prior to transfer and are potentially susceptible to attack by patients' immune systems. Here, we investigated NK-92 cell-mediated serial killing for the effects of gamma-irradiation and ligation of the death receptor Fas (CD95), and NK-92 cell susceptibility to attack by activated primary blood NK cells. METHODS: To evaluate serial killing, we used 51Cr-release assays with low NK-92 effector cell to target Raji, Daudi or K562 tumor cell (E:T) ratios to determine killing frequencies at 2-, 4-, 6-, and 8-h. RESULTS: NK-92 cells were able to kill up to 14 Raji cells per NK-92 cell in 8 h. NK-92 cells retained high cytotoxic activity immediately after irradiation with 10 Gy but the cells surviving irradiation lost > 50% activity 1 day after irradiation. Despite high expression of CD95, NK-92 cells maintained their viability following overnight Fas/CD95-ligation but lost some cytotoxic activity. However, 1 day after irradiation, NK-92 cells were more susceptible to Fas ligation, resulting in decreased cytotoxic activity of the cells surviving irradiation. Irradiated NK-92 cells were also susceptible to killing by both unstimulated and IL-2 activated primary NK cells (LAK). In contrast, non-irradiated NK-92 cells were more resistant to attack by NK and LAK cells. CONCLUSIONS: Irradiation is deleterious to both the survival and cytotoxicity mediated by NK-92 cells and renders the NK-92 cells susceptible to Fas-initiated death and death initiated by primary blood NK cells. Therefore, replacement of irradiation as an antiproliferative pretreatment and genetic deletion of Fas and/or NK activation ligands from adoptively transferred cell lines are indicated as new approaches to increase therapeutic efficacy.

Decreasing the Likelihood of Multiple Organ Dysfunction Syndrome in Burn Injury with Early Antioxidant Treatment.

Major burn trauma initiates a cascade of physiological events that cause profound stress on the body, resulting in significant complications which often lead to death. An understanding of these events may afford earlier and more precise interventions which, in turn, may reduce these complications, thus, improving patient outcomes. Burn trauma is associated with numerous inflammatory events that result in the release of free radicals, which promote oxidative stress and subsequent tissue damage. These mass-inflammatory events affect the body systemically, leading to several detrimental responses including complement activation, excessive histamine release, decrease in blood pressure, release of reactive oxygen species, and ultimately multiple organ dysfunction syndrome (MODS). However, recent studies conducted on the use of antioxidants as a part of a burn treatment protocol have shown promising results. In this review, we will discuss the current research and advancements in the treatment of burn trauma with the use of antioxidants, and how the early administration of antioxidant can possibly reduce the risk of developing MODS.

Photocytotoxicity of Thiophene- and Bithiophene-Dipicolinato Luminescent Lanthanide Complexes.

New thiophene-dipicolinato-based compounds, K2nTdpa (n = 1, 2), were isolated. Their anions are sensitizers of lanthanide ion (LnIII) luminescence and singlet oxygen generation (1O2). Emission in the visible and near-infrared regions was observed for the LnIII complexes with efficiencies (ϕLn) ϕEu = 33% and ϕYb = 0.31% for 1Tdpa2- and ϕYb = 0.07% for 2Tdpa2-. The latter does not sensitize EuIII emission. Fluorescence imaging of HeLa live cells incubated with K3[Eu(1Tdpa)3] indicates that the complex permeates the cell membrane and localizes in the mitochondria. All complexes generate 1O2 in solution with efficiencies (ϕO12) as high as 13 and 23% for the GdIII complexes of 1Tdpa2- and 2Tdpa2-, respectively. [Ln(nTdpa)3]3- (n = 1, 2) are phototoxic to HeLa cells when irradiated with UV light with IC50 values as low as 4.2 µM for [Gd(2Tdpa)3]3- and 91.8 µM for [Eu(1Tdpa)3]3-. Flow cytometric analyses indicate both apoptotic and necrotic cell death pathways.

Recruitment and Expansion of Tregs Cells in the Tumor Environment-How to Target Them?

Regulatory T cells (Tregs) are present in a large majority of solid tumors and are mainly associated with a poor prognosis, as their major function is to inhibit the antitumor immune response contributing to immunosuppression. In this review, we will investigate the mechanisms involved in the recruitment, amplification and stability of Tregs in the tumor microenvironment (TME). We will also review the strategies currently developed to inhibit Tregs' deleterious impact in the TME by either inhibiting their recruitment, blocking their expansion, favoring their plastic transformation into other CD4+ T-cell subsets, blocking their suppressive function or depleting them specifically in the TME to avoid severe deleterious effects associated with Treg neutralization/depletion in the periphery and normal tissues.

Genome-Wide Identification of Rare and Common Variants Driving Triglyceride Levels in a Nevada Population.

Clinical conditions correlated with elevated triglyceride levels are well-known: coronary heart disease, hypertension, and diabetes. Underlying genetic and phenotypic mechanisms are not fully understood, partially due to lack of coordinated genotypic-phenotypic data. Here we use a subset of the Healthy Nevada Project, a population of 9,183 sequenced participants with longitudinal electronic health records to examine consequences of altered triglyceride levels. Specifically, Healthy Nevada Project participants sequenced by the Helix Exome+ platform were cross-referenced to their electronic medical records to identify: (1) rare and common single-variant genome-wide associations; (2) gene-based associations using a Sequence Kernel Association Test; (3) phenome-wide associations with triglyceride levels; and (4) pleiotropic variants linked to triglyceride levels. The study identified 549 significant single-variant associations (p < 8.75 × 10-9), many in chromosome 11's triglyceride hotspot: ZPR1, BUD13, APOC3, APOA5. A well-known protective loss-of-function variant in APOC3 (R19X) was associated with a 51% decrease in triglyceride levels in the cohort. Sixteen gene-based triglyceride associations were identified; six of these genes surprisingly did not include a single variant with significant associations. Results at the variant and gene level were validated with the UK Biobank. The combination of a single-variant genome-wide association, a gene-based association method, and phenome wide-association studies identified rare and common variants, genes, and phenotypes associated with elevated triglyceride levels, some of which may have been overlooked with standard approaches.

Photocytotoxicity of Oligothienyl-Functionalized Chelates That Sensitize LnIII Luminescence and Generate 1 O2.

Three new compounds containing a heptadentate lanthanide (LnIII ) ion chelator functionalized with oligothiophenes, nThept(COOH)4 (n=1, 2, or 3), were isolated. Their LnIII complexes not only display the characteristic metal-centered emission in the visible or near-infrared (NIR) but also generate singlet oxygen (1 O2 ). Luminescence efficiencies (ϕLn ) for [Eu1Thept(COO)4 ]- and [Eu2Thept(COO)4 ]- are ϕEu =3 % and 0.5 % in TRIS buffer and 33 % and 3 % in 95 % ethanol, respectively. 3Thept(COO)4 4- does not sensitize EuIII emission due to its low-lying triplet state. Near infra-red (NIR) luminescence is observed for all NIR-emitting LnIII and ligands with efficiencies of ϕYb =0.002 %, 0.005 % and 0.04 % for [YbnThept(COO)4 ]- (n=1, 2, or 3), and ϕNd =0.0007 %, 0.002 % and 0.02 % for [NdnThept(COO)4 ]- (n=1, 2, or 3) in TRIS buffer. In 95 % ethanol, quantum yields of NIR luminescence increase and are ϕYb =0.5 %, 0.31 % and 0.05 % for [YbnThept(COO)4 ]- (n=1, 2, or 3), and ϕNd =0.40 %, 0.45 % and 0.12 % for [NdnThept(COO)4 ]- (n=1, 2, or 3). All complexes are capable of generating 1 O2 in 95 % ethanol with ϕ1Ο2 efficiencies which range from 2 % to 29 %. These complexes are toxic to HeLa cells when irradiated with UV light (λexc =365 nm) for two minutes. IC50 values for the LnIII complexes are in the range 15.2-16.2 µm; the most potent compound is [Nd2Thept(COO)4 ]- . The cell death mechanisms are further explored using an Annexin V-propidium iodide assay which suggests that cell death occurs through both apoptosis and necrosis.

A Comprehensive Genome-Wide and Phenome-Wide Examination of BMI and Obesity in a Northern Nevadan Cohort.

The aggregation of Electronic Health Records (EHR) and personalized genetics leads to powerful discoveries relevant to population health. Here we perform genome-wide association studies (GWAS) and accompanying phenome-wide association studies (PheWAS) to validate phenotype-genotype associations of BMI, and to a greater extent, severe Class 2 obesity, using comprehensive diagnostic and clinical data from the EHR database of our cohort. Three GWASs of 500,000 variants on the Illumina platform of 6,645 Healthy Nevada participants identified several published and novel variants that affect BMI and obesity. Each GWAS was followed with two independent PheWASs to examine associations between extensive phenotypes (incidence of diagnoses, condition, or disease), significant SNPs, BMI, and incidence of extreme obesity. The first GWAS examines associations with BMI in a cohort with no type 2 diabetics, focusing exclusively on BMI. The second GWAS examines associations with BMI in a cohort that includes type 2 diabetics. In the second GWAS, type 2 diabetes is a comorbidity, and thus becomes a covariate in the statistical model. The intersection of significant variants of these two studies is surprising. The third GWAS is a case vs. control study, with cases defined as extremely obese (Class 2 or 3 obesity), and controls defined as participants with BMI between 18.5 and 25. This last GWAS identifies strong associations with extreme obesity, including established variants in the FTO and NEGR1 genes, as well as loci not yet linked to obesity. The PheWASs validate published associations between BMI and extreme obesity and incidence of specific diagnoses and conditions, yet also highlight novel links. This study emphasizes the importance of our extensive longitudinal EHR database to validate known associations and identify putative novel links with BMI and obesity.

Single-nucleotide polymorphisms in a cohort of significantly obese women without cardiometabolic diseases.

BACKGROUND/OBJECTIVES: Obesity is an important risk factor for the development of diseases such as diabetes mellitus, hypertension, and dyslipidemia; however, a small number of individuals with long-standing obesity do not present with these cardiometabolic diseases. Such individuals are referred to as metabolically healthy obese (MHO) and potentially represent a subgroup of the general population with a protective genetic predisposition to obesity-related diseases. We hypothesized that individuals who were metabolically healthy, but significantly obese (BMI ≥ 35 kg/m2) would represent a highly homogenous subgroup, with which to investigate potential genetic associations to obesity. We further hypothesized that such a cohort may lend itself well to investigate potential genotypes that are protective with respect to the development of cardiometabolic disease. SUBJECTS/METHODS: In the present study, we implemented this novel selection strategy by screening 892 individuals diagnosed as Class 2 or Class 3 obese and identified 38 who presented no manifestations of cardiometabolic disease. We then assessed these subjects for single-nucleotide polymorphisms (SNPs) that associated with this phenotype. RESULTS: Our analysis identified 89 SNPs that reach statistical significance (p < 1 × 10-5), some of which are associated with genes of biological pathways that influences dietary behavior; others are associated with genes previously linked to obesity and cardiometabolic disease as well as neuroimmune disease. This study, to the best of our knowledge, represents the first genetic screening of a cardiometabolically healthy, but significantly obese population.

CCR10+ ILC2s with ILC1-like properties exhibit a protective function in severe allergic asthma.

BACKGROUND: We previously showed that patients with severe allergic asthma have high numbers of circulating ILC2s expressing CCR10. METHOD: Herein, CCR10+ ILC2s were further analyzed in the blood of healthy individuals or patients with allergic and non-allergic asthma. Characteristics of human CCR10+ and CCR10- ILC2s were assessed by flow cytometry as well as single-cell multiplex RT-qPCR. The role of CCR10+ ILC2s in asthma pathophysiology was studied in allergen-treated mice. RESULTS: When compared to healthy controls, CCR10+ ILC2s are enriched in the blood of both allergic and non-allergic severe asthmatic patients, and these cells are recruited to the lungs. Plasma concentrations of the CCR10 ligand CCL27 are significantly increased in severe asthmatics when compared to non-asthmatic patients. CCR10+ ILC2s secrete little TH 2 cytokines, but exhibit ILC1-like properties, including a capacity to produce IFN-γ. Also, single-cell analysis reveals that the CCR10+ ILC2 subset is enriched in cells expressing amphiregulin. CCR10+ ILC2 depletion, as well as blocking of IFN-γ activity, exacerbates airway hyperreactivity in allergen-challenged mice, providing evidence for a protective role of these cells in allergic inflammation. CONCLUSIONS: Frequencies of circulating CCR10+ ILC2s and CCL27 plasma concentrations represent candidate markers of asthma severity. The characterization of CCR10+ ILC2s in human samples and in mouse asthma models suggests that these cells downregulate allergic inflammation through IFN-γ production.

Evaluation of four clinical laboratory parameters for the diagnosis of myalgic encephalomyelitis.

BACKGROUND: Myalgic encephalomyelitis (ME) is a complex and debilitating disease that often initially presents with flu-like symptoms, accompanied by incapacitating fatigue. Currently, there are no objective biomarkers or laboratory tests that can be used to unequivocally diagnosis ME; therefore, a diagnosis is made when a patient meets series of a costly and subjective inclusion and exclusion criteria. The purpose of the present study was to evaluate the utility of four clinical parameters in diagnosing ME. METHODS: In the present study, we utilized logistic regression and classification and regression tree analysis to conduct a retrospective investigation of four clinical laboratory in 140 ME cases and 140 healthy controls. RESULTS: Correlations between the covariates ranged between [- 0.26, 0.61]. The best model included the serum levels of the soluble form of CD14 (sCD14), serum levels of prostaglandin E2 (PGE2), and serum levels of interleukin 8, with coefficients 0.002, 0.249, and 0.005, respectively, and p-values of 3 × 10-7, 1 × 10-5, and 3 × 10-3, respectively. CONCLUSIONS: Our findings show that these parameters may help physicians in their diagnosis of ME and may additionally shed light on the pathophysiology of this disease.

Nutritional modulation of the intestinal microbiota; future opportunities for the prevention and treatment of neuroimmune and neuroinflammatory disease.

The gut-brain axis refers to the bidirectional communication between the enteric nervous system and the central nervous system. Mounting evidence supports the premise that the intestinal microbiota plays a pivotal role in its function and has led to the more common and perhaps more accurate term gut-microbiota-brain axis. Numerous studies have identified associations between an altered microbiome and neuroimmune and neuroinflammatory diseases. In most cases, it is unknown if these associations are cause or effect; notwithstanding, maintaining or restoring homeostasis of the microbiota may represent future opportunities when treating or preventing these diseases. In recent years, several studies have identified the diet as a primary contributing factor in shaping the composition of the gut microbiota and, in turn, the mucosal and systemic immune systems. In this review, we will discuss the potential opportunities and challenges with respect to modifying and shaping the microbiota through diet and nutrition in order to treat or prevent neuroimmune and neuroinflammatory disease.

Humoral Immunity Profiling of Subjects with Myalgic Encephalomyelitis Using a Random Peptide Microarray Differentiates Cases from Controls with High Specificity and Sensitivity.

Myalgic encephalomyelitis (ME) is a complex, heterogeneous illness of unknown etiology. The search for biomarkers that can delineate cases from controls is one of the most active areas of ME research; however, little progress has been made in achieving this goal. In contrast to identifying biomarkers that are directly involved in the pathological process, an immunosignature identifies antibodies raised to proteins expressed during, and potentially involved in, the pathological process. Although these proteins might be unknown, it is possible to detect antibodies that react to these proteins using random peptide arrays. In the present study, we probe a custom 125,000 random 12-mer peptide microarray with sera from 21 ME cases and 21 controls from the USA and Europe and used these data to develop a diagnostic signature. We further used these peptide sequences to potentially uncover the naturally occurring candidate antigens to which these antibodies may specifically react with in vivo. Our analysis revealed a subset of 25 peptides that distinguished cases and controls with high specificity and sensitivity. Additionally, Basic Local Alignment Search Tool (BLAST) searches suggest that these peptides primarily represent human self-antigens and endogenous retroviral sequences and, to a minor extent, viral and bacterial pathogens.

Serum Cytokine Profiles Differentiating Hemorrhagic Fever with Renal Syndrome and Hantavirus Pulmonary Syndrome.

Hantavirus infection is an acute zoonosis that clinically manifests in two primary forms, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). HFRS is endemic in Europe and Russia, where the mild form of the disease is prevalent in the Tatarstan region. HPS is endemic in Argentina, as well as other countries of North and South American. HFRS and HPS are usually acquired via the upper respiratory tract by inhalation of virus-contaminated aerosol. Although the pathogenesis of HFRS and HPS remains largely unknown, postmortem tissue studies have identified endothelial cells as the primary target of infection. Importantly, cell damage due to virus replication, or subsequent tissue repair, has not been documented. Since no single factor has been identified that explains the complexity of HFRS or HPS pathogenesis, it has been suggested that a cytokine storm may play a crucial role in the manifestation of both diseases. In order to identify potential serological markers that distinguish HFRS and HPS, serum samples collected during early and late phases of the disease were analyzed for 48 analytes using multiplex magnetic bead-based assays. Overall, serum cytokine profiles associated with HPS revealed a more pro-inflammatory milieu as compared to HFRS. Furthermore, HPS was strictly characterized by the upregulation of cytokine levels, in contrast to HFRS where cases were distinguished by a dichotomy in serum cytokine levels. The severe form of hantavirus zoonosis, HPS, was characterized by the upregulation of a higher number of cytokines than HFRS (40 vs 21). In general, our analysis indicates that, although HPS and HFRS share many characteristic features, there are distinct cytokine profiles for these diseases. These profiles suggest a strong activation of an innate immune and inflammatory responses are associated with HPS, relative to HFRS, as well as a robust activation of Th1-type immune responses. Finally, the results of our analysis suggest that serum cytokines profiles of HPS and HFRS cases are consistent with the presence of extracellular matrix degradation, increased mononuclear leukocyte proliferation, and transendothelial migration.

Effector and regulatory dendritic cells display distinct patterns of miRNA expression.

INTRODUCTION: MicroRNAs (miRNAs) contribute to the regulation of dendritic cell (DC) polarization, thereby influencing the balance of adaptive immune responses. Herein, we studied the expression of miRNAs in polarized DCs and analyzed whether expression of these miRNAs could be associated with allergic rhinitis and allergen immunotherapy (AIT) outcome. METHOD: Using specific culture conditions, we differentiated immature human monocyte-derived DCs into DC1, DC2, and DCreg subsets (supporting the differentiation of TH 1, TH 2 or regulatory T cells, respectively). Profiling of miRNA expression was performed in these DC subpopulations using microarrays. Levels of miRNAs specific for polarized DCs were then evaluated in a cohort of 58 patients with allergic rhinitis and 25 non-allergic controls, as well as in samples from 30 subjects treated with sublingual grass pollen tablets or placebo for four months. RESULTS: We successfully identified 16 miRNAs differentially regulated between immature DCs, DC1, DC2, and DCreg cells. In allergic rhinoconjunctivitis patients, the expression of two of those miRNAs (miR-132 and miR-155), was down-regulated compared to non-allergic individuals. However, the levels of these miRNAs were not significantly modified following four months of grass pollen immunotherapy. CONCLUSIONS: Studying polarized DCs and clinical samples from subjects with or without allergic rhinoconjunctivitis, we demonstrated that the expression of two miRNAs linked to effector DCs (i.e., DC1 and/or DC2 cells), was reduced in the blood of patients with allergic rhinoconjunctivitis. Nevertheless, these miRNAs did not represent relevant biomarkers to predict or follow-up AIT efficacy.

Sialylated Fetuin-A as a candidate predictive biomarker for successful grass pollen allergen immunotherapy.

BACKGROUND: Eligibility to immunotherapy is based on the determination of IgE reactivity to a specific allergen by means of skin prick or in vitro testing. Biomarkers predicting the likelihood of clinical improvement during immunotherapy would significantly improve patient selection. METHODS: Proteins were differentially assessed by using 2-dimensional differential gel electrophoresis and label-free mass spectrometry in pretreatment sera obtained from clinical responders and nonresponders within a cohort of 82 patients with grass pollen allergy receiving sublingual immunotherapy or placebo. Functional studies of Fetuin-A (FetA) were conducted by using gene silencing in a mouse asthma model, human dendritic cell in vitro stimulation assays, and surface plasmon resonance. RESULTS: Analysis by using quantitative proteomics of pretreatment sera from patients with grass pollen allergy reveals that high levels of O-glycosylated sialylated FetA isoforms are found in patients exhibiting a strong decrease in rhinoconjunctivitis symptoms after sublingual immunotherapy. Although FetA is involved in numerous inflammatory conditions, its potential role in allergy is unknown. In vivo silencing of the FETUA gene in BALB/c mice results in a dramatic upregulation of airway hyperresponsiveness, lung resistance, and TH2 responses after allergic sensitization to ovalbumin. Both sialylated and nonsialytated FetA bind to LPS, but only the former synergizes with LPS and grass pollen or mite allergens to enhance the Toll-like receptor 4-mediated proallergic properties of human dendritic cells. CONCLUSIONS: As a reflection of the patient's inflammatory status, pretreatment levels of sialylated FetA in the blood are indicative of the likelihood of clinical responses during grass pollen immunotherapy.