Flow Cytometry and Its Applications to Molecular Biology and Diagnosis 2.0.

Flow cytometry is a single-cell based technology aimed to quantify the scattering of light and the emission of multiple fluorescence signals by individual cells, biological vesicles, or synthetic microscopical particles when examined one by one at high speed using lasers or other suitable illumination sources [...].

Catechin versus MoS2 Nanoflakes Functionalized with Catechin: Improving the Sperm Fertilizing Ability-An In Vitro Study in a Swine Model.

Nowadays, the adoption of In Vitro Fertilization (IVF) techniques is undergoing an impressive increase. In light of this, one of the most promising strategies is the novel use of non-physiological materials and naturally derived compounds for advanced sperm preparation methods. Here, sperm cells were exposed during capacitation to MoS2/Catechin nanoflakes and catechin (CT), a flavonoid with antioxidant properties, at concentrations of 10, 1, 0.1 ppm. The results showed no significant differences in terms of sperm membrane modifications or biochemical pathways among the groups, allowing the hypothesis that MoS2/CT nanoflakes do not induce any negative effect on the parameters evaluated related to sperm capacitation. Moreover, the addition of CT alone at a specific concentration (0.1 ppm) increased the spermatozoa fertilizing ability in an IVF assay by increasing the number of fertilized oocytes with respect to the control group. Our findings open interesting new perspectives regarding the use of catechins and new materials obtained using natural or bio compounds, which could be used to implement the current strategies for sperm capacitation.

Ragweed pollen concentration predicts seasonal rhino-conjunctivitis and asthma severity in patients allergic to ragweed.

In this work, we investigate the correlation between ragweed pollen concentration and conjunctival, nasal, and asthma symptom severity in patients allergic to ragweed pollen using ambient pollen exposure in the Milan area during the 2014 ragweed season We calculate the pollen/symptom thresholds and we assess the effectiveness of ragweed allergen immunotherapy (AIT). A total of 66 participants allergic to ragweed (Amb a 1) were enrolled in the study and divided into two groups: AIT treated (24) and no AIT treated (42). Pollen counts and daily symptom/medication patient diaries were kept. Autoregressive distributed lag models were used to develop predictive models of daily symptoms and evaluate the short-term effects of temporal variations in pollen concentration on the onset of symptoms. We found significant correlations between ragweed pollen load and the intensity of symptoms for all three symptom categories, both in no AIT treated (τ = 0.341, 0.352, and 0.721; and ρ = 0.48, 0.432, and 0.881; p-value < 0.001) and in AIT treated patients ([Formula: see text]= 0.46, 0.610, and 0.66; and ρ = 0.692, 0.805, and 0.824; p-value < 0.001). In both groups, we observed a positive correlation between the number of symptoms reported and drug use. Mean symptom levels were significantly higher in no AIT treated than in AIT treated patients (p-value < 0.001) for all symptom categories. Pollen concentration thresholds for the four symptom severity levels (low, medium-low, medium-high and high) were calculated. Ragweed pollen concentration is predictive of symptom severity in patients with a ragweed (Amb a 1) allergy. Patients treated with AIT had significantly reduced mean symptom levels compared to those without AIT.

Fluorescent Silica Nanoparticles Targeting Mitochondria: Trafficking in Myeloid Cells and Application as Doxorubicin Delivery System in Breast Cancer Cells.

Fluorescent silica nanoparticles (SiNPs) appear to be a promising imaging platform, showing a specific subcellular localization. In the present study, we first investigated their preferential mitochondrial targeting in myeloid cells, by flow cytometry, confocal microscopy and TEM on both cells and isolated mitochondria, to acquire knowledge in imaging combined with therapeutic applications. Then, we conjugated SiNPs to one of the most used anticancer drugs, doxorubicin (DOX). As an anticancer agent, DOX has high efficacy but also an elevated systemic toxicity, causing multiple side effects. Nanostructures are usually employed to increase the drug circulation time and accumulation in target tissues, reducing undesired cytotoxicity. We tested these functionalized SiNPs (DOX-NPs) on breast cancer cell line MCF-7. We evaluated DOX-NP cytotoxicity, the effect on the cell cycle and on the expression of CD44 antigen, a molecule involved in adhesion and in tumor invasion, comparing DOX-NP to free DOX and stand-alone SiNPs. We found a specific ability to release a minor amount of CD44+ extracellular vesicles (EVs), from both CD81 negative and CD81 positive pools. Modulating the levels of CD44 at the cell surface in cancer cells is thus of great importance for disrupting the signaling pathways that favor tumor progression.

Extracellular Vesicles from Campylobacter jejuni CDT-Treated Caco-2 Cells Inhibit Proliferation of Tumour Intestinal Caco-2 Cells and Myeloid U937 Cells: Detailing the Global Cell Response for Potential Application in Anti-Tumour Strategies.

Cytolethal distending toxin (CDT) is produced by a range of Gram-negative pathogenic bacteria such as Campylobacter jejuni. CDT represents an important virulence factor that is a heterotrimeric complex composed of CdtA, CdtB, and CdtC. CdtA and CdtC constitute regulatory subunits whilst CdtB acts as the catalytic subunit exhibiting phosphatase and DNase activities, resulting in cell cycle arrest and cell death. Extracellular vesicle (EV) secretion is an evolutionarily conserved process that is present throughout all kingdoms. Mammalian EVs play important roles in regular cell-to-cell communications but can also spread pathogen- and host-derived molecules during infections to alter immune responses. Here, we demonstrate that CDT targets the endo-lysosomal compartment, partially evading lysosomal degradation and exploiting unconventional secretion (EV release), which is largely involved in bacterial infections. CDT-like effects are transferred by Caco-2 cells to uninfected heterologous U937 and homologous Caco-2 cells. The journey of EVs derived from CDT-treated Caco-2 cells is associated with both intestinal and myeloid tumour cells. EV release represents the primary route of CDT dissemination, revealing an active toxin as part of the cargo. We demonstrated that bacterial toxins could represent suitable tools in cancer therapy, highlighting both the benefits and limitations. The global cell response involves a moderate induction of apoptosis and autophagic features may play a protective role against toxin-induced cell death. EVs from CDT-treated Caco-2 cells represent reliable CDT carriers, potentially suitable in colorectal cancer treatments. Our data present a potential bacterial-related biotherapeutic supporting a multidrug anticancer protocol.

Uptake and Intracellular Trafficking Studies of Multiple Dye-Doped Core-Shell Silica Nanoparticles in Lymphoid and Myeloid Cells.

INTRODUCTION: Since most biologically active macromolecules are natural nanostructures, operating in the same scale of biomolecules gives the great advantage to enhance the interaction with cellular components. Noteworthy efforts in nanotechnology, particularly in biomedical and pharmaceutical fields, have propelled a high number of studies on the biological effects of nanomaterials. Moreover, the determination of specific physicochemical properties of nanomaterials is crucial for the evaluation and design of novel safe and efficient therapeutics and diagnostic tools. In this in vitro study, we report a physicochemical characterisation of fluorescent silica nanoparticles (NPs), interacting with biological models (U937 and PBMC cells), describing the specific triggered biologic response. METHODS: Flow Cytometric and Confocal analyses are the main method platforms. However TEM, NTA, DLS, and chemical procedures to synthesize NPs were employed. RESULTS: NTB700 NPs, employed in this study, are fluorescent core-shell silica nanoparticles, synthesized through a micelle-assisted method, where the fluorescence energy transfer process, known as FRET, occurs at a high efficiency rate. Using flow cytometry and confocal microscopy, we observed that NTB700 NP uptake seemed to be a rapid, concentration-, energy- and cell type-dependent process, which did not induce significant cytotoxic effects. We did not observe a preferred route of internalization, although their size and the possible aggregated state could influence their extrusion. At this level of analysis, our investigation focuses on lysosome and mitochondria pathways, highlighting that both are involved in NP co-localization. Despite the main mitochondria localization, NPs did not induce a significant increase of intracellular ROS, known inductors of apoptosis, during the time course of analyses. Finally, both lymphoid and myeloid cells are able to release NPs, essential to their biosafety. DISCUSSION: These data allow to consider NTB700 NPs a promising platform for future development of a multifunctional system, by combining imaging and localized therapeutic applications in a unique tool.

The impact of five years storage/biobanking at -80°C on mouse spermatozoa fertility, physiology, and function.

BACKGROUND: We previously demonstrated how mouse spermatozoa can be efficiently stored for two years in a -80°C freezer, maintaining their ability to fertilize mouse eggs. OBJECTIVES: The main objective here was to evaluate the effects of five years at -80°C compared to liquid nitrogen storage (LN2 , control condition) on mouse sperm viability, physiological parameters, and fertilization capacity. MATERIALS AND METHODS: Three different strains were used: C57BL/6N, C57BL/6J and CD1. Flow cytometry experiments were performed to analyze sperm viability (SYBR-14 + Propidium Iodide +Hoechst33342), the intracellular calcium concentration (Fluo 3-AM), the membrane lipid disorder (Merocyanine 540), and the mitochondrial activity (MitoTracker Red) in live spermatozoa. The in vitro fertilization (IVF) was used to evaluate the sperm fertilizing ability. RESULTS: Flow cytometry analysis showed that the percentage of live cells are reduced in B6N and B6J, but not in CD1 mice. However, in the live population no differences in terms of intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity were reported when comparing both biobanking methods. Spermatozoa stored at -80°C for 5 years successfully fertilized the eggs and developed mouse embryo normally both in culture and in vivo, generating live pups with no differences compared to control samples stored in LN2 . DISCUSSION: Long-term mouse sperm storage at -80°C (five years) could be considered an ideal alternative to the most common LN2 approach, giving economical and logistic advantages. Moreover, the precise information originated from the flow cytometry analysis stands up this technique as an optimal strategy to evaluate the sperm quality and ranking. CONCLUSION: It is demonstrated here the possibility to store mouse spermatozoa for up to five years in a -80°C freezer with no significant differences compared to the storage in LN2 in terms of fertilizing ability, sperm viability, intracellular calcium concentration, membrane lipid disorder, and mitochondrial activity.

Hydroxychloroquine and dexamethasone in COVID-19: who won and who lost?

BACKGROUND: On June 30, 2020, the WHO reported over 10 millions of COVID-19 cases worldwide with over half a million deaths. In severe cases the disease progresses into an Acute Respiratory Distress Syndrome (ARDS), which in turn depends on an overproduction of cytokines (IL-6, TNFα, IL-12, IL-8, CCL-2 and IL1) that causes alveolar and vascular lung damage. Clearly, it is essential to find an immunological treatment that controls the "cytokine storm". In the meantime, however, it is essential to have effective antiviral and anti-inflammatory drugs available immediately. PHARMACOLOGIC THERAPY FOR COVID-19: Hydroxychloroquine or chloroquine have been widely adopted worldwide for the treatment of SARS-CoV-2 pneumonia. However, the choice of this treatment was based on low quality of evidence, i.e. retrospective, non-randomized controlled studies. Recently, four large Randomized Controlled Trials (RCTs) have been performed in record time delivering reliable data: (1) the National Institutes of Health (NIH) RCT included 60 hospitals participating all over the world and showed the efficacy of remdesivir in reducing the recovery time in hospitalized adults with COVID-19 pneumonia; (2) three large RCTs already completed, for hydroxychloroquine, dexamethasone and Lopinavir and Ritonavir respectively. These trials were done under the umbrella of the 'Recovery' project, headed by the University of Oxford. The project includes 176 participating hospitals in the UK and was set up to verify the efficacy of some of the treatments used for COVID-19. These three 'Recovery' RCTs concluded definitely: (a) that treatment with hydroxychloroquine provides no benefits in patients hospitalized with COVID-19; (b) that treatment with dexamethasone reduced deaths by one-third in COVID-19 patients that were mechanically ventilated, and by one-fifth in patients receiving oxygen only; (c) that the combination of Lopinavir and Ritonavir is not effective in reducing mortality in COVID-19 hospitalized patients. CONCLUSIONS: The results of these four large RCTs have provided sound indications to doctors for the treatment of patients with COVID-19 and prompted the correction of many institutional provisions and guidelines on COVID-19 treatments (i.e. FDA, NIH, UK Health Service, etc.). Even though a definitive treatment for COVID-19 has not yet been found, large RCTs stand as the Gold Standards for COVID-19 therapy and offer a solid scientific base on which to base treatment decisions.

Understanding the Synergy of NKp46 and Co-Activating Signals in Various NK Cell Subpopulations: Paving the Way for More Successful NK-Cell-Based Immunotherapy.

The NK cell population is characterized by distinct NK cell subsets that respond differently to the various activating stimuli. For this reason, the determination of the optimal cytotoxic activation of the different NK cell subsets can be a crucial aspect to be exploited to counter cancer cells in oncologic patients. To evaluate how the triggering of different combination of activating receptors can affect the cytotoxic responses of different NK cell subsets, we developed a microbead-based degranulation assay. By using this new assay, we were able to detect CD107a+ degranulating NK cells even within the less cytotoxic subsets (i.e., resting CD56bright and unlicensed CD56dim NK cells), thus demonstrating its high sensitivity. Interestingly, signals delivered by the co-engagement of NKp46 with 2B4, but not with CD2 or DNAM-1, strongly cooperate to enhance degranulation on both licensed and unlicensed CD56dim NK cells. Of note, 2B4 is known to bind CD48 hematopoietic antigen, therefore this observation may provide the rationale why CD56dim subset expansion correlates with successful hematopoietic stem cell transplantation mediated by alloreactive NK cells against host T, DC and leukemic cells, while sparing host non-hematopoietic tissues and graft versus host disease. The assay further confirms that activation of LFA-1 on NK cells leads to their granule polarization, even if, in some cases, this also takes to an inhibition of NK cell degranulation, suggesting that LFA-1 engagement by ICAMs on target cells may differently affect NK cell response. Finally, we observed that NK cells undergo a time-dependent spontaneous (cytokine-independent) activation after blood withdrawal, an aspect that may strongly bias the evaluation of the resting NK cell response. Altogether our data may pave the way to develop new NK cell activation and expansion strategies that target the highly cytotoxic CD56dim NK cells and can be feasible and useful for cancer and viral infection treatment.

Monocyte Response to Different Campylobacter jejuni Lysates Involves Endoplasmic Reticulum Stress and the Lysosomal⁻Mitochondrial Axis: When Cell Death Is Better Than Cell Survival.

Campylobacter jejuni is a Gram-negative spiral-shaped bacterium, commonly associated with gastroenteritis in humans. It explicates its virulence also by the cytolethal distending toxin (CDT), able to cause irreversible cell cycle arrest. Infection by C. jejuni may result in the development of the Guillain⁻Barré Syndrome, an acute peripheral neuropathy. Symptoms of this disease could be caused by CDT-induced cell death and a subsequent inflammatory response. We tested C. jejuni lysates from different strains on donor monocytes: in fact, monocytes are potent producers of both pro- and anti-inflammatory cytokines, playing a major role in innate immunity and in non-specific host responses. We found, by cytometric and confocal analyses, that mitochondria and lysosomes were differently targeted: The C. jejuni strain that induced the most relevant mitochondrial alterations was the ATCC 33291, confirming an intrinsic apoptotic pathway, whereas the C. jejuni ISS 1 wild-type strain mostly induced lysosomal alterations. Lysates from all strains induced endoplasmic reticulum (ER) stress in monocytes, suggesting that ER stress was not associated with CDT but to other C. jejuni virulence factors. The ER data were consistent with an increase in cytosolic Ca2+ content induced by the lysates. On the contrary, the changes in lysosomal acidic compartments and p53 expression (occurring together from time 0, T0, to 24 h) were mainly due to CDT. The loss of p53 may prevent or impede cell death and it was not observable with the mutant strain. CDT not only was responsible for specific death effects but also seemed to promote an apoptotic stimuli-resisting pathway.

Peripheral Blood Mononuclear Cell Immunophenotyping in Fibrodysplasia Ossificans Progressiva Patients: Evidence for Monocyte DNAM1 Up-regulation.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder caused by sporadic heterozygous mutations in ACVR1 gene which progressively leads to severe heterotopic ossification. FOP is characterized by episodic flare-ups triggered by different factors such as viral infections, tissue injuries, vaccinations, or occurring without a recognizable cause. The sporadic course of the disease, the documented presence of an important inflammatory reaction in early lesions and the partial response to corticosteroids support the idea that the immune system, and in particular the innate component, may play a role in FOP pathogenesis. However, an extensive expression profile of the peripheral blood mononuclear cells (PBMC) of FOP patients has never been done. METHODS: In this study, we carried out a wide PBMC immunophenotyping on a cohort of FOP patients and matching controls by multiparametric analysis of the expression of a panel of 37 markers associated with migration, adhesion, inhibition, activation, and cell death of circulating immune cells. RESULTS: We observed a statistically significant increase of the expression of DNAM1 receptor in patients' monocytes as compared to controls, and little but significant differences in the expression profile of CXCR1 (CD181), CD62L, CXCR4 (CD184), and HLA-DR molecules. CONCLUSIONS: DNAM1 had been previously shown to play a pivotal role in monocyte migration through the endothelial barrier and the increased expression detected in patients' monocytes might suggest a role of this surface receptor during the early phases of FOP flare-ups in which the activation of the immune response is believed to represent a crucial event. © 2017 International Clinical Cytometry Society.

The Memories of NK Cells: Innate-Adaptive Immune Intrinsic Crosstalk.

Although NK cells are considered part of the innate immune system, a series of evidences has demonstrated that they possess characteristics typical of the adaptive immune system. These NK adaptive features, in particular their memory-like functions, are discussed from an ontogenetic and evolutionary point of view.

Wheat IgE-mediated food allergy in European patients: alpha-amylase inhibitors, lipid transfer proteins and low-molecular-weight glutenins. Allergenic molecules recognized by double-blind, placebo-controlled food challenge.

BACKGROUND: Three main problems hamper the identification of wheat food allergens: (1) lack of a standardized procedure for extracting all of the wheat protein fractions; (2) absence of double-blind, placebo-controlled food challenge studies that compare the allergenic profile of Osborne's three protein fractions in subjects with real wheat allergy, and (3) lack of data on the differences in IgE-binding capacity between raw and cooked wheat. METHODS: Sera of 16 wheat-challenge-positive patients and 6 patients with wheat anaphylaxis, recruited from Italy, Denmark and Switzerland, were used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis/immunoblotting of the three Osborne's protein fractions (albumin/globulin, gliadins and glutenins) of raw and cooked wheat. Thermal sensitivity of wheat lipid transfer protein (LTP) was investigated by spectroscopic approaches. IgE cross-reactivity between wheat and grass pollen was studied by blot inhibition. RESULTS: The most important wheat allergens were the alpha-amylase/trypsin inhibitor subunits, which were present in all three protein fractions of raw and cooked wheat. Other important allergens were a 9-kDa LTP in the albumin/globulin fraction and several low-molecular-weight (LMW) glutenin subunits in the gluten fraction. All these allergens showed heat resistance and lack of cross-reactivity to grass pollen allergens. LTP was a major allergen only in Italian patients. CONCLUSIONS: The alpha-amylase inhibitor was confirmed to be the most important wheat allergen in food allergy and to play a role in wheat-dependent exercise-induced anaphylaxis, too. Other important allergens were LTP and the LMW glutenin subunits.

Clinical characteristics of soybean allergy in Europe: a double-blind, placebo-controlled food challenge study.

BACKGROUND: Soybean is a relevant allergenic food, but little is known about individual threshold doses in soy allergy. OBJECTIVE: We sought to determine the clinical characteristics of soy allergy in Europe, including a dose-response curve. METHODS: Patients with a history of soy allergy underwent a titrated, double-blind, placebo-controlled food challenge. A statistical model was used to calculate the risk of allergic consumers to experience an allergic reaction to soy. Sera were analyzed for specific IgE to soy, peanut, Bet v 1, and Gly m 4. RESULTS: All patients but one responded primarily with subjective symptoms to the challenge followed by objective symptoms in 11 subjects, ranging from rhinitis up to a decrease in blood pressure. Cumulative threshold doses for allergic reactions ranged from 10 mg to 50 g for subjective symptoms and from 454 mg to 50 g for objective symptoms. The pattern of IgE reactivity against proteins with molecular weights of between approximately 10 and 70 kd was highly individual among the patients and did not correlate with the severity of symptoms. CONCLUSIONS: When data are fitted by using a normal distribution statistical model, they predict that 1% of patients with soy allergy would react subjectively and objectively with 0.21 and 37.2 mg of soy protein, respectively. CLINICAL IMPLICATIONS: Both the clinical and immunologic basis of soy allergy in Europe are highly complex, which affects the diagnosis of soy allergy and the advice given to patients with soy allergy in regard to risk management.

Economic evaluation of sublingual immunotherapy vs symptomatic treatment in adults with pollen-induced respiratory allergy: the Sublingual Immunotherapy Pollen Allergy Italy (SPAI) study.

BACKGROUND: Few data are available on the pharmacoeconomic aspects of immunotherapy. OBJECTIVE: To evaluate, from the health care system and societal perspectives, the costs and consequences of sublingual immunotherapy (SLIT) added to pharmacotherapy compared with drugs alone for respiratory allergy. METHODS: This study compared costs, clinical outcomes, and cost-effectiveness ratios of 2 strategies in the management of allergic rhinitis and asthma, namely, SLIT associated with pharmacotherapy and pharmacotherapy alone (no SLIT). A decision tree was developed and populated with epidemiologic and resource utilization data concerning approximately 2,200 patients. Direct costs included visits, tests, pharmacotherapy, immunotherapy, and hospitalizations. Indirect costs and out-of-pocket drugs were also included. Outcome was calculated as the number of improved patients and asthma cases avoided at 6 years. Sensitivity analysis was performed by varying costs and epidemiologic data. RESULTS: SLIT improved the symptoms of 399 of 1,000 patients and prevented asthma in 229 of 1,000 patients compared with drugs alone. For SLIT added to pharmacotherapy and pharmacotherapy alone, the direct cost per patient at more than 6 years was Euro2,400 and Euro3,026, whereas the indirect cost was Euro1,913 and Euro3,400. CONCLUSION: From both perspectives and for both effectiveness end points, SLIT is less expensive and more effective than pharmacotherapy alone.

Q-FIHC: quantification of fluorescence immunohistochemistry to analyse p63 isoforms and cell cycle phases in human limbal stem cells.

Fluorescence microscopy has long been used for qualitative characterization of various parameters such as subcellular distribution of proteins, lipids, nucleic acids, and ions. However, quantification of these parameters is complicated by a variety of optical, biological, and physical factors. In the last decade, the progress achieved with powerful softwares and digital image processing systems has facilitated the development of fluorescence immunohistochemistry (FIHC) into a widely used quantitative assay (quantitative-FIHC or Q-FIHC). We describe here a rapid and sensitive Q-FIHC assay based on the use of a laser scanning confocal microscope and advanced image analysis softwares (Zeiss semi automatic LSM 510 and fully automatic Axiovision 4.4) for the detection and quantification of fluorescent intensity in human corneal tissues and cells obtained from small clinical samples. We have used this methodology to characterize and quantify the gene expression profile of p63 and its DeltaNalpha isoform, specific markers of human limbal stem cells. The validity of this method was evaluated through comparative studies with conventional approaches suggesting no significant differences and providing an alternative technique to traditional methods. Since Q-FIHC requires at least 20-fold less cells than traditional techniques, we have adopted it as the main quality control for our limbal cultures destined to clinical application.

Food allergies and food intolerances.

Adverse reactions to foods, aside from those considered toxic, are caused by a particular individual intolerance towards commonly tolerated foods. Intolerance derived from an immunological mechanism is referred to as Food Allergy, the non-immunological form is called Food Intolerance. IgE-mediated food allergy is the most common and dangerous type of adverse food reaction. It is initiated by an impairment of normal Oral Tolerance to food in predisposed individuals (atopic). Food allergy produces respiratory, gastrointestinal, cutaneous and cardiovascular symptoms but often generalized, life-threatening symptoms manifest at a rapid rate-anaphylactic shock. Diagnosis is made using medical history and cutaneous and serological tests but to obtain final confirmation a Double Blind Controlled Food Challenge must be performed. Food intolerances are principally caused by enzymatic defects in the digestive system, as is the case with lactose intolerance, but may also result from pharmacological effects of vasoactive amines present in foods (e.g. Histamine). Prevention and treatment are based on the avoidance of the culprit food.

Wheat allergy: a double-blind, placebo-controlled study in adults.

BACKGROUND: Wheat is believed to be an uncommon cause of food allergy in adults; the number of studies that address IgE mediated wheat allergy in adults is all too few. OBJECTIVE: Determine how many subjects with a history of wheat allergy have real allergy by double-blind, placebo-controlled food challenge; identify the symptoms manifested during the challenge; determine the lowest provocation dose; determine the performance characteristics of wheat skin prick test and specific IgE; identify subjects with real wheat allergy for potential immunoblotting studies. METHODS: Patients underwent skin test with commercial wheat extract; specific wheat IgE was determined. Subjects were challenged with 25 g wheat. Subjects who were positive to raw wheat challenge underwent cooked wheat challenge. RESULTS: Thirty-seven double-blind placebo-controlled wheat challenges were performed on 27 patients. A total of 13 of 27 (48%) patients had a positive result. Eleven subjects with positive raw wheat challenge underwent cooked wheat challenge: 10 were positive. The provocation dose range was 0.1 to 25 g. Twenty-seven percent of the subjects allergic to wheat had a provocation dose that was < or =1.6 g. CONCLUSION: Wheat causes real food allergy in adults. More than a quarter of the patients allergic to wheat reacted to less than 1.6 g wheat. Specific IgE was more sensitive than skin test for wheat; however, specificity and predictive values were low for both tests. Thus, these tests should not be used to validate diagnosis of wheat allergy.

Lipid transfer protein and vicilin are important walnut allergens in patients not allergic to pollen.

BACKGROUND: Walnut is the most common cause of allergic reactions to tree nuts, as reported by large population studies. Two major allergens of walnut have been identified up until now: a 2S albumin and a vicilin-like protein. OBJECTIVE: This study was designed to identify the walnut major allergens in the Italian population and to compare the walnut IgE-binding profile in patients with or without pollen allergy. METHODS: We selected 46 patients either with oral allergy syndrome confirmed by open oral challenge or with systemic symptoms after ingestion of walnut. These patients' sera were used for the immunoblotting of walnut extract; the identified allergens were purified by HPLC and sequenced. A peach-walnut cross-inhibition study was then performed. RESULTS: The only major allergen recognized by our study population was a 9-kd lipid transfer protein (LTP), recognized by 37 patients. Two other minor allergens of approximately 9-kd molecular weight, both belonging to the vicilin family, were recognized by 10 patients. IgE binding to walnut LTP was completely inhibited by peach LTP. CONCLUSION: In Italian patients with walnut allergy confirmed by documented history of severe systemic reactions or by open oral food challenge, the major allergen is an LTP. The sensitization to this protein seems to be secondary to the sensitization to peach LTP, which acts as the primary sensitizer. LTP and vicilins were able to sensitize patients not allergic to pollen.