Microarray Immunodiagnostics for Aeroallergens.

PURPOSE OF REVIEW: The impact of new technologies, especially multiplexed molecular allergy diagnostics based on allergen arrays, on the management of complex patients with respiratory allergies has been important. RECENT FINDINGS: Currently, the detailed characteristics of the IgE profile of the patient, such as sensitization to genuine or cross-reacting components or the sensitization to potentially harmful allergens, allow an allergist to tailor treatment in the context of precision medicine rules. A number of relevant articles have been published in recent years on this topic, and, in this review, the new added values of allergen array-based diagnostics are reported.

Mechanical bacterial lysate administration prevents exacerbation in allergic asthmatic children-The EOLIA study.

BACKGROUND: Despite progress in asthma management, prevention of asthma exacerbation remains challenging in school-aged children with allergic asthma. New therapeutic approaches are needed. Previously, a chemical bacterial lysate has been successfully used in preschool children to reduce wheezing attacks. We assessed the effect of Polyvalent Mechanical Bacterial Lysate (PMBL® ) Tablet on asthma clinical course and control in 6- to 16-year-old children with partly controlled or uncontrolled allergic asthma. METHODS: A randomized, double-blind, placebo-controlled, parallel-group study was performed in 152 patients exhibiting allergic asthma assigned to receive Placebo or PMBL® . Eligible patients underwent four visits during the 9-month study. Asthma control level was assessed by ACT/C-ACT score. RESULTS: The main criterion was not achieved as ACT/C-ACT changes were similar in both groups at the end of the 3-month treatment period. However, the mean number (±SD) of asthma exacerbations was significantly lower with PMBL® Tablet than with Placebo at Week 12 (0.3 ± 0.6 vs 0.8 ± 1.1, P = .009) and over the total study period (1.1 ± 1.3 vs 1.9 ± 2.0, P = .01). Consistently, the mean number of days with exacerbation per patient was significantly lower with PMBL® Tablet (13.3 ± 11.2 vs 19.8 ± 15.7 over the whole study, P = .009). Treatment with PMBL® Tablet prolonged the time to second exacerbation by 55% (Hazard Ratio [HR]=0.45; 95% Confidence Interval [CI] 0.27 to 0.77, P = .002) and to third exacerbation by 74% (HR=0.26; 95% CI 0.12 to 0.58, P < .001). No serious adverse event related to PMBL® Tablet administration was recorded. CONCLUSION: Administration of PMBL® Tablet represents a safe and effective means for significantly reducing the rate of exacerbations in school-aged allergic asthmatic children. (EudraCT 2013-000737-12 and NCT02541331).

Molecular diagnosis and precision medicine in allergy management.

Precision medicine (PM) can be defined as a structural model aimed at customizing healthcare, with medical decisions/products tailored on an individual patient at a highly detailed level. In this sense, allergy diagnostics based on molecular allergen components allows to accurately define the patient's IgE repertoire. The availability of highly specialized singleplexed and multiplexed platforms support allergists with an advanced diagnostic armamentarium. The therapeutic intervention, driven by the standard diagnostic approach, but further supported by these innovative tools may result, for instance, in a more appropriate prescription of allergen immunotherapy (AIT). Also, the phenotyping of patients, which may have relevant effects on the treatment strategy, could be take advantage by the molecular allergy diagnosis.

Sub-lingual administration of a polyvalent mechanical bacterial lysate (PMBL) in patients with moderate, severe, or very severe chronic obstructive pulmonary disease (COPD) according to the GOLD spirometric classification: A multicentre, double-blind, randomised, controlled, phase IV study (AIACE study: Advanced Immunological Approach in COPD Exacerbation).

Polyvalent mechanical bacterial lysates (PMBLs) have been shown to reduce the number of infectious episodes in patients with recurrent infections of the respiratory tract. Some previous investigations have also shown the effectiveness of PMBLs in reducing exacerbations of chronic obstructive pulmonary disease (COPD). The AIACE study, which was developed according to criteria of evidence-based medicine, evaluated whether the administration of PMBLs to COPD patients, in addition to the recommended treatment, was able to reduce the number of exacerbations by 25%. Two hundred eighty-eight patients with moderate to very severe COPD were recruited and randomly assigned to either placebo or PMBLs. The placebo or PMBLs were administered according to the standard scheme. The primary outcome of the study was not achieved. However, the number of days with fever (21 days per year versus 40.15; p < 0.001), the days of hospitalisation (65 days vs 162 days; p < 0.001), the interval between the first and second exacerbations (123.89 days vs 70.36; p = 0.03) and the number of days in poor health (109 days/year vs 171 days/year; p < 0.001) were significantly better in the PMBL group than in the placebo group. In conclusion, the results of this trials showed that Ismigen, in addition to guideline-suggested treatment, could not significantly reduce the number of exacerbations in the considered population; nevertheless, the secondary outcome results demonstrated potential benefits of this compound for relevant clinical outcomes.

Erratum to: Guidelines for the use and interpretation of diagnostic methods in adult food allergy.

[This corrects the article DOI: 10.1186/s12948-015-0033-9.].

Guidelines for the use and interpretation of diagnostic methods in adult food allergy.

Food allergy has an increasing prevalence in the general population and in Italy concerns 8 % of people with allergies. The spectrum of its clinical manifestations ranges from mild symptoms up to potentially fatal anaphylactic shock. A number of patients can be diagnosed easily by the use of first- and second-level procedures (history, skin tests and allergen specific IgE). Patients with complex presentation, such as multiple sensitizations and pollen-food syndromes, frequently require a third-level approach including molecular diagnostics, which enables the design of a component-resolved sensitization profile for each patient. The use of such techniques involves specialists' and experts' skills on the issue to appropriately meet the diagnostic and therapeutic needs of patients. Particularly, educational programs for allergists on the use and interpretation of molecular diagnostics are needed.

The administration of a polyvalent mechanical bacterial lysate in elderly patients with COPD results in serological signs of an efficient immune response associated with a reduced number of acute episodes.

The administration of a polyvalent mechanical bacterial lysate (PMBL) in elderly patients with COPD has been shown to reduce the number of exacerbation. This is largely related to the involvement of cells belonging to the innate and the adaptive immune system (including dendritic cells, granulocytes, T and B lymphocytes and NK cells) that actively cooperate inducing the production of specific opsonizing antibodies directed to the antigens of PMBL. We have evaluated the production of antibodies directed to respiratory and systemic pathogens in a group of elderly COPD patients, recruited in a clinical trial, ancillary to a larger multicenter double blind, placebo-controlled, parallel-designed clinical trial in which patients were randomized to daily receive either PMBL or placebo. The treated group not only experienced a reduced number of seroconversion, but also, better controlled the number of infectious episodes and COPD exacerbations. It was thus evident that the administration of PMBL resulted not only effective in inducing the secretion of specific antibodies, but also effective in reducing the infectious episodes trough the potentiation of the antibody-mediated arm of the immune response.

Sublingually administered bacterial lysates: rationale, mechanisms of action and clinical outcomes.

This review discusses available evidence on the mechanisms of action of bacterial lysates, and the clinical effects of their sublingual administration. Bacterial lysates act through many immunological effects, including dendritic cell activation, modification of circulating lymphocyte subsets and antibody production. The production of salivary IgA was repeatedly shown to be induced by the sublingual administration of a prototype bacterial lysate containing soluble and corpuscular antigens. Bacterial lysates are a useful tool for the prevention of recurrent respiratory tract infections. Sublingual administration should be the preferred option.

Diagnostic potential of hepcidin testing in pediatrics.

OBJECTIVES: Hepcidin, a peptide hormone released by hepatocytes into circulation is the main regulator of dietary iron absorption and cellular iron release. Although commercial tests are available, assay harmonization for hepcidin has not been yet reached, making reference intervals and consequent clinical decisions still elusive for each assay and specific population. The aim of this study is to set up hepcidin measurement in pediatric age and to investigate its potential usefulness in the diagnosis and management of iron disorders in children. METHODS: Serum hepcidin was measured by using an automated commercial immunoassay. Reference values were obtained from 86 healthy children. Hepcidin was then evaluated in 52 children with diseases where this hormone was expected to be differently regulated. RESULTS: Hepcidin values were 43.6 ng/mL median; 32-52.7 1-3 q: in males and 36.4 ng/mL median; 28.5-45.7 1-3 q: in females (P = 0.039). Hepcidin was significantly higher in postpubertal normal females than in normal males. Hepcidin resulted up-regulated in anemia of chronic disease of children affected by systemic Juvenile Idiopathic Arthritis and decreased after treatment with anakinra, an anti-interleukin-1 receptor antagonist. In iron deficiency anemia patients on oral iron supplementation and in ß-thalassemia subjects, hepcidin levels were similar to those found in healthy subjects. CONCLUSIONS: This study sets up reference values for pediatric population and shows that in normal controls serum hepcidin react differently to puberty in females vs. males. In addition, it suggests that serum hepcidin may discriminate microcytic inflammatory anemia of Juvenile Idiopathic Arthritis from iron deficiency anemia. Overall these findings may represent a helpful tool for future studies tailored to understand the role of hepcidin in management of iron disorders in children.

Micromethod for quantification of cinacalcet in human plasma by liquid chromatography-tandem mass spectrometry using a stable isotope-labeled internal standard.

BACKGROUND: Cinacalcet hydrochloride is a calcimimetic agent indicated for the treatment of secondary hyperparathyroidism in dialysis-dependent patients with chronic kidney disease. In the context of a pharmacokinetic (PK)/pharmacodynamic study of cinacalcet in dialysis-dependent chronic kidney disease children with secondary hyperparathyroidism, we describe the development and validation of a new, rapid, simple, and economical liquid chromatography-tandem mass spectrometry (LC-MS/MS) micromethod for quantifying cinacalcet plasma concentrations. METHODS: Cinacalcet was analyzed in 50-µL plasma samples over a wide range of concentrations (0.1-100 ng/mL) by LC-MS/MS after protein precipitation and addition of deuterated cinacalcet as the internal standard. Cinacalcet was quantified using selective reaction monitoring of the specific transition m/z 358.1 > 155.1, with the 361.1 > 158.1 transition used for the internal standard. The suitability of the assay for clinical PK studies was evaluated using data from a pilot PK study in a pediatric patient. RESULTS: The overall turnaround time for the assay was 20 minutes. The lower limit of quantification of the method was 0.1 ng/mL. Intraassay imprecision and inaccuracy for quality control samples ranged from 2.8% to 9% and 100% to 102%, respectively. Interassay imprecision and inaccuracy ranged from 6.9% to 8.5% and 99% to 103%, respectively. The overall recovery ranged from 90% to 106%. No ion suppression due to matrix effects was found with different preanalytical conditions, such as hemolysis, lipemia, and hyperuricemia. CONCLUSIONS: This LC-MS/MS micromethod provides high specificity, precision, and accuracy for rapid quantification of cinacalcet plasma concentrations, and it is suitable for application in pediatric PK studies; it also has potential for use in the establishment of target ranges and ultimately routine therapeutic drug monitoring to optimize cinacalcet dosing.

Interchangeability between 24-hour collection and single spot urines for vanillylmandelic and homovanillic acid levels in the diagnosis of neuroblastoma.

The determination of the two urinary catecholamine metabolites homovanillic acid (HVA) and vanillylmandelic acid (VMA) is of crucial importance for the diagnosis and follow-up of neuroblastoma (NB). The standard practice for their measurement requires the use of 24-hour collections that are time consuming and difficult to obtain. In this article, we directly demonstrate that 24-hour collections and single spot urines are interchangeable for the determination of HVA and VMA expressed as ratio on creatinine concentration. This study can be useful for a faster management of NB at onset.

Evaluation of a mobile clinical pathology laboratory developed for the home care of pediatric patients following transplantation of peripheral blood precursor cells.

BACKGROUND: A mobile clinical pathology laboratory (MoLab) was designed, outfitted and evaluated to improve the turnaround time (TAT) of laboratory tests performed in patients who receive follow-up care at home. METHODS: Full blood counts (FBCs), basic clinical biochemistry tests on blood and urine, and basic coagulation and blood gases were measured using bench-top laboratory analyzers to perform point-of-care tests in a mobile setting. The quality of the results was evaluated on the instruments both while the vehicle was at rest and following movement during the course of routine activity. RESULTS: The equivalence of the values produced by the point-of-care testing (POCT) and central laboratory (CL) measurement procedures was demonstrated and remained stable after movement in city traffic. The TATs ranged from a few minutes for FBCs to <20 min for extended clinical biochemistry tests. CONCLUSIONS: During the first 6 months of activity, the MoLab assured the provision of laboratory results for home care patients in a matter of minutes. This approach not only allowed for real-time modifications in therapy but also reduced the number of second visits by the home care medical team. Point-of-care tests performed on the MoLab were significantly more expensive than the same tests performed in the CL. However, the savings in patient hospitalization expenses and the reduced costs resulting from fewer second daily visits completely offset the costs of using the MoLab during the first 6-month pilot phase.

Reference values for urinary neutrophil gelatinase-associated lipocalin (NGAL) in pediatric age measured with a fully automated chemiluminescent platform.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) has been suggested as the most promising biomarker of acute kidney injury. However, there are no reliable data on analytical sensitivity and reference limits of urinary NGAL (uNGAL) assay in pediatric age. The aim of the present study is to evaluate the analytical sensitivity and the reference range of uNGAL measured in urine specimens of pediatric age with the fully automated platform ARCHITECT i1000. METHODS: A total of 333 urine samples were collected from 25 healthy newborns (16 males and 9 females; age 1-4 days) and 308 children (150 males and 147 females; mean age 80.7 months, range 0.63-248 months) and assayed for uNGAL by two different Italian centers (Department of Laboratory Medicine of the Fondazione Toscana G. Monasterio of Pisa and Massa and the Clinical Pathology Laboratory Unit of Istituto Giannina Gaslini of Genova). RESULTS: The calculated limits of blank (LOB) and detection (LOD) values were 0.5 ng/mL and 0.95 ng/mL, respectively. The distribution of uNGAL values approximated a log-normal distribution (median 5.2 ng/mL, interquartile range 2.5-12.8 ng/mL, 99th percentile 117.6 ng/mL). uNGAL values of the 25 neonates were significantly higher than those of 308 children (neonates: mean 44.2 ng/mL, median 30.3 ng/mL, range 5.2-137.4 ng/mL; children: mean 10.2 ng/mL, median 4.6 ng/mL, range 0.2-146.7 ng/mL; p<0.0001). CONCLUSIONS: uNGAL assay shows a good analytical sensitivity and imprecision, which allows the measurement of uNGAL values around the cut-off value (i.e., 117.6 ng/mL) with an imprecision <5 CV%. The distribution of uNGAL values in pediatric age approximates a log-normal distribution, with values which are higher in neonates compared to children.

Development and validation of a multiplex quantitative polymerase chain reaction assay for the detection of Mollicutes impurities in human cells, cultured under good manufacturing practice conditions, and following European Pharmacopoeia requirements and the International Conference on Harmonization guidelines.

BACKGROUND AIMS: The clinical applications of in vitro manipulated cultured cells and their precursors are often made use of in therapeutic trials. However, tissue cultures can be easily contaminated by the ubiquitous Mollicutes micro-organisms, which can cause various and severe alterations in cellular function. Thus methods able to detect and trace Mollicutes impurities contaminating cell cultures are required before starting any attempt to grow cells under good manufacturing practice (GMP) conditions. METHODS: We developed a multiplex quantitative polymerase chain reaction (qPCR) assay specific for the 16S-23S rRNA intergenic spacer regions, for the Tuf and P1 cytoadhesin genes, able to detect contaminant Mollicutes species in a single tube reaction. The system was validated by analyzing different cell lines and the positive samples were confirmed by 16S and P1 cytoadhesin gene dideoxy sequencing. RESULTS: Our multiplex qPCR detection system was able to reach a sensitivity, specificity and robustness comparable with the culture and the indicator cell culture method, as required by the European Pharmacopoeia guidelines. CONCLUSIONS: We have developed a multiplex qPCR method, validated following International Conference on Harmonization (ICH) guidelines, as a qualitative limit test for impurities, assessing the validation characteristics of limit of detection and specificity. It also follows the European Pharmacopoeia guidelines and Food and Drug Administration (FDA) requirements.

The IgE repertoire in children and adolescents resolved at component level: a cross-sectional study.

BACKGROUND: It is well known that allergy evolves at clinical level from the birth to adulthood, and this has been clearly demonstrated also at a level of sensitization. However, little information is available on the evolution of the IgE repertoire directed to single allergenic components. In this cross-sectional, observational study, the evolution of the IgE repertoire was analysed at component level. METHODS: Serum samples from 901 allergic patients, stratified in 6 groups according to age, were analysed by ImmunoCAP ISAC, a microarray chip that allows to identify the presence of specific IgE towards 103 different allergen components. Total IgE were also evaluated. RESULTS: The behaviour of total IgE according to age strictly paralleled that of the sum of specific IgE directed to molecular components. As expected, food-related components (in particular those of milk and egg) were the most frequently recognized in the earliest ages, whereas specific IgE to plant allergens appeared invariably later. Nonetheless, IgE specific to mite components was the most represented in all age classes. Of note, specific IgE against cross-reacting allergens was virtually absent in the first years and tended to appear only after the age of 6. CONCLUSION: Despite this was not a study performed on a cohort of patients followed up from birth to adolescence, the molecular patterns of allergen recognition resulted modified according to age. These findings may support, at molecular level, the clinical features of the allergic march.

Erythrocyte Galactose-1-phosphate measurement by GC-MS in the monitoring of classical galactosemia.

BACKGROUND: Classical galactosemia is a rare but very severe disease characterized by a deficiency of the galactose-1-phosphate uridyltransferase enzyme. The confirmed galactosemic patients are treated with a galactose-restricted diet. Nevertheless, metabolites such as galactose-1-phosphate can accumulate in red blood cells of treated patients and its measurement is a standard practice for their monitoring. At present, no commercial methods for measuring galactose-1-phosphate in erythrocytes are available. METHODS: In this study, we will describe the optimization and laboratory validation of a previously published quantitative gas chromatographic-mass spectrometric method and its clinical validation on normal donors and galactosemic patients both at the diagnosis and during the follow-up. RESULTS: The method was technically optimized and validated for its clinical use on normal donors and galactosemic newborns, children and adults. The method was suitable for the monitoring of dietary compliance. Galactose-1-phosphate levels were found to be well correlated with the clinical signs in the galactosemic patients at the follow-up. CONCLUSIONS: This paper provides information on the measurement of Galactose-1-phosphate levels that can be very useful for the management of classical galactosemia.

Erratum to "IgE allergy diagnostics and other relevant tests in allergy, a World Allergy Organization position paper" [World Allergy Organ J 13/2 (2020) 100080].

[This corrects the article DOI: 10.1016/j.waojou.2019.100080.].

Human dendritic cells activate resting natural killer (NK) cells and are recognized via the NKp30 receptor by activated NK cells.

During the innate response to many inflammatory and infectious stimuli, dendritic cells (DCs) undergo a differentiation process termed maturation. Mature DCs activate antigen-specific naive T cells. Here we show that both immature and mature DCs activate resting human natural killer (NK) cells. Within 1 wk the NK cells increase two-- to fourfold in numbers, start secreting interferon (IFN)-gamma, and acquire cytolytic activity against the classical NK target LCL721.221. The DC-activated NK cells then kill immature DCs efficiently, even though the latter express substantial levels of major histocompatibility complex (MHC) class I. Similar results are seen with interleukin (IL)-2--activated NK cell lines and clones, i.e., these NK cells kill and secrete IFN-gamma in response to immature DCs. Mature DCs are protected from activated NK lysis, but lysis takes place if the NK inhibitory signal is blocked by a human histocompatibility leukocyte antigen (HLA)-A,B,C--specific antibody. The NK activating signal mainly involves the NKp30 natural cytotoxicity receptor, and not the NKp46 or NKp44 receptor. However, both immature and mature DCs seem to use a NKp30 independent mechanism to act as potent stimulators for resting NK cells. We suggest that DCs are able to control directly the expansion of NK cells and that the lysis of immature DCs can regulate the afferent limb of innate and adaptive immunity.

Evaluation of bacterial proliferation with a microfluidic-based device: Antibiochip.

The measurement of the proliferation (and the relevant inhibition of proliferation) of microbes is used in different settings, from industry to laboratory medicine. Thus, in this study, the capacity of the Antibiochip (ELTEK spa), a microfluidic-based device, to measure the amount of E. coli in certain culture conditions, was evaluated. An Antibiochip is composed of V-shaped microchannels, and the amount of microparticles (such as microbes) is measured by the surface of the pellet after centrifugation. In the present study, different geometries, volumes and times were analyzed. When the best conditions were identified, serial dilutions of microbial cultures were tested to validate the linearity of the results. Then, with the use of wild E. coli strains isolated from medical samples, the relationship between bacterial susceptibility to antibiotics measured by standard methods and that measured by the Antibiochip was evaluated. In this report, the good quality performances of the methods, their linearity and the capacity to identify susceptible microbial strains after 60 minutes of incubation are shown. These results represent a novel approach for ultrarapid antibiograms in clinics.

IgE allergy diagnostics and other relevant tests in allergy, a World Allergy Organization position paper.

Currently, testing for immunoglobulin E (IgE) sensitization is the cornerstone of diagnostic evaluation in suspected allergic conditions. This review provides a thorough and updated critical appraisal of the most frequently used diagnostic tests, both in vivo and in vitro. It discusses skin tests, challenges, and serological and cellular in vitro tests, and provides an overview of indications, advantages and disadvantages of each in conditions such as respiratory, food, venom, drug, and occupational allergy. Skin prick testing remains the first line approach in most instances; the added value of serum specific IgE to whole allergen extracts or components, as well as the role of basophil activation tests, is evaluated. Unproven, non-validated, diagnostic tests are also discussed. Throughout the review, the reader must bear in mind the relevance of differentiating between sensitization and allergy; the latter entails not only allergic sensitization, but also clinically relevant symptoms triggered by the culprit allergen.