Novel murine mAbs define specific and cross-reactive epitopes on the latex profilin panallergen Hev b 8.

The production of specific antibodies able to recognize allergens from different sources or block interactions between allergens and antibodies mediating allergic reactions is crucial for developing successful tools for diagnostics and therapeutics. Panallergens are highly conserved proteins present in widely different species, implicated in relevant cross-reactions. The panallergen latex profilin (Hev b 8) has been associated with the latex-food-pollen syndrome. We generated five monoclonal IgGs and one IgE from murine hybridomas against recombinant Hev b 8 and evaluated their interaction with this allergen using ELISA and biolayer interferometry (BLI). Affinity purified mAbs exhibited high binding affinities towards rHev b 8, with KD1 values ranging from 10-10 M to 10-11 M. Some of these antibodies also recognized the recombinant profilins from maize and tomato (Zea m 12 and Sola l 1), and the ash tree pollen (Fra e 2). Competition ELISA demonstrated that some mAb pairs could bind simultaneously to rHev b 8. Using BLI, we detected competitive, non-competitive, and partial-competition interactions between pairs of mAbs with rHev b 8, suggesting the existence of at least two non-overlapping epitopes on the surface of this allergen. Three-dimensional models of the Fv of 1B4 and 2D10 IgGs and docking simulations of these Fvs with rHev b 8 revealed these epitopes. Furthermore, these two mAbs inhibited the interaction of polyclonal IgE and IgG4 antibodies from profilin-allergic patients with rHev b 8, indicating that the mAbs and the antibodies present in sera from allergic patients bind to overlapping epitopes on the allergen. These mAbs can be useful tools for immune-localization studies, immunoassay development, or standardization of allergenic products.

Diagnosis and management of hypersensitivity reactions to vaccines.

INTRODUCTION: Many countries in Europe now recommend and enforce mandatory vaccinations to improve vaccination coverage. Thus, the number of adverse events following immunization (AEFI) may show an increase. Among these events, severe hypersensitivity reactions to vaccines are rare. However, it is important that they be identified and recognized so that they may be adequately managed. AREAS COVERED: The literature search was undertaken through PubMed and Embase to identify English-language papers focusing on hypersensitivity to vaccines. EXPERT OPINION: Hypersensitivity reactions following vaccinations are rare and are classified according to their chronology and extension: immediate when they occur within the first 4 hours following administration and non-immediate when they occur later. Local reactions are the most common adverse event following injection of vaccines and generally do not require any allergy workup. Immediate reactions, however, are potentially IgE-mediated and require an allergy workup. In general, a previously known food allergy (i.e., egg or milk) is not a contraindication to immunizations. Patients with a known allergy to gelatin, yeast, latex, antibiotics, or other specific components of vaccines require an allergy workup before administration of the vaccine.

Sublingual Immunotherapy for Other Indications: Venom Large Local, Latex, Atopic Dermatitis, and Food.

There is some evidence to support the use of sublingual immunotherapy (SLIT) in food allergy, although its role is unclear. One randomized, double-blind, placebo-controlled trial supports the safe and efficacious use of dust mite SLIT in children with mild to moderate atopic dermatitis, but these data have not been confirmed. Although there are several randomized, double-blind, placebo-controlled trials to support the use of SLIT-LATEX, this product is not available in the United States and extrapolation of these effects to latex extracts is unsubstantiated. There is also insufficient evidence to support the use of SLIT for venom hypersensitivity at this time.

Crystal structure and biochemical characterization of CJP38, a ß-1,3-glucanase and allergen of Cryptomeria japonica pollen.

A 38 kDa ß-1,3-glucanase allergen from Cryptomeria japonica pollen (CJP38) was recombinantly produced in E. coli and purified to homogeneity with the use of Ni-affinity resin. CJP38 hydrolyzed ß-1,3-glucans such as CM-curdlan and laminarioligosaccharides in an endo-splitting manner. The optimum pH and temperature for ß-1,3-glucanase activity were approximately 4.5 and 50 °C, respectively. The enzyme was stable at 30-60 °C and pH 4.0-10.5. Furthermore, CJP38 catalyzed a transglycosylation reaction to yield reaction products with a molecular weight higher than those of the starting laminarioligosaccharide substrates. The three-dimensional structure of CJP38 was determined using X-ray crystallography at 1.5 Å resolution. CJP38 exhibited the typical (ß/α)8 TIM-barrel motif, similar to allergenic ß-1,3-glucanases from banana (Mus a 5) and rubber tree latex (Hev b 2). Amino acid sequence alignment of these proteins indicated that the two-consensus IgE epitopes identified on the molecular surfaces of Mus a 5 and Hev b 2 were highly conserved in CJP38. Their conformations and surface locations were quite similar for these proteins. Sequence and structural conservation of these regions suggest that CJP38 is a candidate allergen responsible for the pollen-latex-fruit syndrome relating to Japanese cedar pollinosis.

Chemical hazard for dental hygienists: a systematic review.

OBJECTIVE: Dental hygienists (DHs) are professionals responsible for oral health. They deal with professional oral hygiene, counselling, and screening patients for oral health, as well as preventing and treating oral diseases. However, DH responsibilities and duties may vary worldwide, characterising changeable occupational exposure scenarios and making it difficult to achieve a suitable evaluation of workplace risks, particularly regarding chemical exposure. Therefore, the aim of the present work was to provide a comprehensive overview on the current knowledge on DH chemical risks. MATERIALS AND METHODS: According to the PRISMA guidelines, a systematic review of PubMed, Scopus, and Isi Web of Knowledge databases was performed to retrieve all articles assessing DH occupational chemical exposures. RESULTS: Fragmented data are currently available on DH chemical risk, due to the limited number of studies on the topic and few DHs enrolled, as well as their frequent assimilation to other oral healthcare professionals. The majority of the retrieved investigations focused on possible hypersensitivity reactions caused by natural rubber latex exposure, but not on potential risks derived from other currently employed substances or innovative wide-spreading compounds. CONCLUSIONS: Future research should be focused on assessing DH chemical risks according to a more comprehensive and toxicologically standardised approach to achieve an appropriate awareness among the DH workforce concerning the possibility for hazardous exposure and adverse health effects. Overall, this may lead to the adoption/implementation of adequate preventive measures to protect the health and safety of these oral healthcare professionals.

ENEA, a peach and apricot IgE-binding protein cross-reacting with the latex major allergen Hev b 5.

Peach and apricot can cause allergic reactions with symptoms ranging from mild to very severe, including anaphylaxis. Sometimes subjects allergic to fruits of the Prunus genus have been reported to be also allergic to rubber latex products. The objective of this study is the characterization of a newly identified peach and apricot protein showing similarities with the allergens Hev b 5 from rubber latex and Man e 5 from manioc. This protein has been named ENEA on the basis of the single letter amino acid code of the first four N-terminal residues of the isolated molecule. It has been found in very variable amounts in different peach cultivars and batches. ENEA was isolated from peach pulp extracts by chromatographic separations and identified by direct protein sequencing. At that time, the full length sequence was available only for the homologous protein of the taxonomically closely related apricot, which was produced as a recombinant molecule in Escherichia coli. The following availability of the full length sequence of peach ENEA revealed a very high identity (97%) with the apricot homolog. Similarly to Hev b 5 and to Man e 5, the structural characterization indicated that ENEA is an intrinsically disordered protein. The immunological properties, investigated by dot blotting, the ABA system and the FABER test, showed that ENEA is recognized by specific IgE of allergic patients. In a selected population of 31 patients reporting allergic reactions to peach fruit and/or IgE positive to Hev b 5, 28 and 27 subjects resulted co-sensitized to rENEA and Hev b 5 in the ABA and ISAC test, respectively. In a random population of 3305 suspected allergic patients, analyzed with the FABER test, 17 of them were sensitized to rENEA and 10 of them were also positive to Hev b 5. In addition, both the natural molecule from peach and the recombinant protein of apricot partially inhibited the IgE binding to Hev b 5. In conclusion, a new peach and apricot IgE-binding protein, cross-reacting with the major latex allergen Hev b 5, has been identified. Its variable concentration in the fruit might explain some occasionally occurring allergic reactions. The apricot molecule has recently been registered by the WHO/IUIS Allergen Nomenclature Sub-Committee with the allergen name Pru ar 5. The recombinant form of apricot ENEA, now available, will contribute to allergy diagnosis.

The Clinical Relevance of Natural Rubber Latex-Specific IgE in Patients Sensitized to Timothy Grass Pollen.

BACKGROUND: In pollen-allergic patients, cross-reacting allergens including cross-reactive carbohydrate determinants (CCDs) and profilins may result in positive natural rubber latex (NRL)-specific IgE (sIgE) antibody tests but the relationship between this sensitization and clinical NRL type 1 allergy is poorly described. OBJECTIVE: The aims of this study were to determine the frequency and clinical relevance of NRL sIgE in grass pollen-sensitized individuals and to investigate which NRL allergen components these individuals were sensitized to. METHODS: A total of 383 grass-sensitized patients answered questions about NRL allergy symptoms and their stored sera from previous investigations were analyzed for NRL sIgE. Patients with NRL sIgE (n = 32) underwent further investigations comprising medical history, skin prick test with NRL and inhalational allergens, and an additional blood sample. The additional blood samples were analyzed for total IgE and sIgE against NRL, timothy grass, birch, rHev b 1, 3, 5, 6.01, 6.02, 8, 9, 11, rPhl p 12, and MUXF3, which was used as a marker of CCD sensitization. RESULTS: Overall, 9.4% of all grass pollen-sensitized individuals showed IgE sensitization to NRL but only 1.6% had a confirmed type I NRL allergy. CCD and Hev b 8 explained the clinically irrelevant NRL IgE sensitization in 65% of the cases. We found a highly significant correlation between NRL profilin (Hev b 8) sensitization and grass profilin (Phl p 12) sensitization (p < 0.0001). CONCLUSIONS: Data from this study support the hypothesis that in patients with grass pollen sensitization, Hev b 8 mono-sensitization has little or no clinical relevance and is caused by cross sensitization from grass profilin (Phl p 12).

Skin reactions to latex in dental professionals - first Croatian data.

Purpose. To determine the prevalence of undesirable skin reactions to latex in dental professionals and students of the School of Dental Medicine in Zagreb, Croatia. Methods. Our research included 444 participants, of which 200 agreed to undergo a skin prick test (SPT). All participants answered a questionnaire in which we asked about incidence of skin lesions, duration of occupational exposure to latex, localization of skin lesions and symptoms. Statistical analysis of the questionnaire and test results was then carried out. Results. Of the total 444 participants surveyed, 249 (56.1%) reported lesions on their skin (professionals 64.8%, students 6.1-58.5%). From the questionnaire, 239 (96.0%) respondents reported lesions on the hands and fingers, mostly in the form of erythema (37.0%) and occasional dryness of skin (29.0%). Positive SPT results were found in 14 (7.0%) out of the 200 respondents who underwent the test. Conclusions. While a large number of subjects (56.1%) reported skin lesions when using latex products at their workplace, the SPT test was positive only in 7.0%. The results show that the prevalence of self-reported skin lesions was significantly related to the length of occupational exposure, with a substantial effect size (p < 0.001; V = 0.334).

Changes in Functional Activity of Neutrophils and Monocytes Isolated from the Peripheral Blood of Women at Different Phases of the Menstrual Cycle.

We studied functional activity of neutrophilic granulocytes and monocytes isolated from the peripheral blood of women during the follicular and luteal phases of the menstrual cycle. It was shown that phagocytic activity of neutrophilic granulocytes increases, their intracellular oxygen-dependent bactericidal activity decreases, and the number of monocyte extracellular traps increases in women in the luteal phase of the menstrual cycle in comparison with the follicular phase.

An Analysis of Skin Prick Tests to Latex and Patch Tests to Rubber Additives and other Causative Factors among Dental Professionals and Students with Contact Dermatoses.

BACKGROUND: Dental workers often experience unwanted allergic and nonallergic skin reactions resulting in different contact dermatoses (e.g., contact urticaria, irritant and allergic contact dermatitis) that are often attributed to rubber gloves. OBJECTIVE: To examine allergic and nonallergic contact dermatoses by different methods amongst dental professionals and dental students, more specifically, reactions to natural rubber latex (NRL), rubber additives, and other causative factors. METHODS: In this cross-sectional study we surveyed a total of 444 subjects (dentists, assistants, technicians, and students); 200 agreed to be tested to latex by the standard skin prick test (SPT) and prick-by-prick test, of whom 107 were patch tested to rubber additives (mercapto mix, thiuram mix, carba mix, and N-isopropyl-N-phenyl-4-phenylenediamine [IPPD]). RESULTS: Skin lesions appeared significantly more frequently with longer work experience (p = 0.002; V = 0.181), frequent glove changes (p < 0.001; V = 0.310), and hand washing (p < 0.001; V = 0.263), and in subjects with a history of allergies (atopic dermatitis, allergic rhinitis, allergic conjunctivitis, and others) (p < 0.001; V = 0.183). Positive SPTs to latex occurred in 14/200 subjects (7%), of whom 5/14 subjects (35.7%) were also positive in prick-by-prick tests. Patch tests were positive in 5/104 subjects (4.8%) (mercapto mix 1%, thiuram mix 1.9%, and carba mix 1.9%). CONCLUSION: Only a small number of our subjects were allergic to latex (7%) or rubber additives (4.8%). Thus, self-reported contact dermatoses (during NRL product use) in dental professionals and students are not commonly caused by allergies to latex and rubber additives, as is often assumed, but by other factors.

Comparison of electrochemiluminescence immunoassay and latex agglutination turbidimetric immunoassay for evaluation of everolimus blood concentrations in renal transplant patients.

WHAT IS KNOWN AND OBJECTIVE: For analysis of blood concentrations of everolimus, many hospital laboratories use either latex agglutination turbidimetric immunoassay (LTIA) or electrochemiluminescence immunoassay (ECLIA). However, no studies have compared both immunoassay methods under the same conditions. Accordingly, in this study, we compared everolimus blood concentrations obtained by LTIA and ECLIA in renal transplant patients. METHODS: Blood samples (n = 230) from 60 renal transplant patients (19 female and 41 male) were evaluated using both immunoassays. Subsequently, we switched the assay for detection of everolimus blood concentrations from LTIA to ECLIA as a clinical application. Three quality control (QC) samples for LTIA were analysed using ECLIA, and 3 QC samples for ECLIA were analysed using LTIA. RESULTS: The Deming regression of ECLIA versus LTIA generated the following parameters: slope, 1.0067 and intercept, 1.7489 ng/mL, in the analysis of 230 samples. Bland-Altman analysis showed an average positive bias of 1.73 ng/mL between ECLIA and LTIA. When the clinical apparatus was switched from LTIA to ECLIA, the average everolimus blood concentration assayed by LTIA before switching was 3.57 ng/mL, whereas that by ECLIA after switching in the same patients taking the same daily dose (mean: 1.43 mg/day) was 5.85 ng/mL. The QCs assayed using LTIA were lower by an average of 67.3% (range: 55.8%-79.5%) for ECLIA, and in the same 230 samples from patients, the everolimus blood concentrations assayed by LTIA were lower by an average of 67.4% (range: 37.1%-114.5%) of ECLIA. WHAT IS NEW AND CONCLUSION: Analysis of everolimus concentrations by immunoassays with high precision and accuracy is required to ensure long-term survival of transplant recipients. Although the concentrations of QCs and calibrators of everolimus in LTIA were previously corrected to 70% concentration because of cross-reactivity with everolimus metabolites, these adjustments may need to be reviewed.

Latex sensitization in patients with myelomeningocele: contribution of microarray technique.

Summary: Background. Microarray technique employing molecular allergens is pointed out as a new method to evaluate allergic patients. Objective. To evaluate if microarray technique (ImmunoCAP-ISAC®; I-ISAC®) is similar to fluorescence enzyme immunoassay (FEIA; ImmunoCAP®) in the diagnosis of latex allergy (specific IgE to latex plus symptoms) and latex sensitization (only antibody) in pediatric (9-mo to 14-yrs) patients with myelomeningocele undergone to surgical repair. Methods. Patients underwent skin prick testing (SPT) to latex and food (prick to prick), and dosage of serum specific IgE to latex and recombinant latex allergens (rHev b 1, rHev b 3, rHev b 5, rHev b 6.01, rHev b 6.02, rHev b 8, rHev b 9, and rHev b 11) by ImmunoCAP® and I-ISAC®. Results. The comparison between the two methods showed high level of concordance considering positive and negative results. A statistically significant correlation for rHev b 3 and rHev b 6.01 for the allergic patients, and for rHev b5 and rHev b6.01 for those sensitized to latex, was observed. I-ISAC® is limited to 5 recombinant latex allergens (rHev b 1, 3, 5, 6.01 and 8). Despite the presence of antibodies against pollens, LTP and profilins (I-ISAC®) in some patients, none of them reported symptoms related with intake of fruits and/or vegetables. Conclusion. Both methods are effective in assisting the diagnosis of latex allergy, but differ in the assessment of sensitized pediatric patients with myelomeningocele. The assessment of latex sensitized patients is more complete using the 8 recombinant latex fractions available for ImmunoCAP®, instead of I-ISAC®.

Profilin, a Change in the Paradigm.

Profilin is a protein that is present in all eukaryotic cells and is responsible for cross-reactivity between pollen, latex, and plant foods. It has been classically acknowledged as a minor or nearly irrelevant allergen, although recent data are changing this conception. The objective of this manuscript is to provide a comprehensive review of published data on the role of this ubiquitous allergen in pollen, latex, and plant food allergy. The patterns of recognition of this minor allergen follow a north-south gradient. Although present in all pollens and vegetables, profilin is significantly associated with allergy to grass pollen and to Cucurbitaceae fruits. Heb v 8, the latex profilin, is usually a marker of profilin allergy in plant food-allergic patients, although it has no clinical relevance in latex allergy. Sensitization to profilin jeopardizes the diagnosis of pollen allergy and selection of immunotherapy, and although component-resolved diagnosis can identify its impact, there are no tailored treatments available. In recent years, several new publications have shown how profilin should be taken into account and, under certain circumstances, considered a marker of severity, an allergen capable of inducing respiratory symptoms, and, in its natural purified form, a potential candidate for etiological treatment of food allergy. Current data on profilin strongly support the need for a shift in the previously accepted paradigm for this allergen. More research should be done to assess the real clinical impact of sensitization in specific populations and to develop therapeutic strategies.

Latex proteins from Calotropis procera: Toxicity and immunological tolerance revisited.

Many thousands of plants are disseminated worldwide in traditional and folk medicines based on the belief that their leaves, roots, seeds, bark or secretions, when adequately handled, can treat, alleviate or ameliorate numerous disease symptoms. Calotropis procera (Apocynaceae) is a popular medicinal plant and the claims of this shrub's phytomedicinal properties have been scientifically validated. In this study, further prospects towards the in vivo toxicity and oral immunological tolerance of phytomodulatory proteins isolated from the latex of C. procera are reported. Acute toxicity was determined in mice by oral and intraperitoneal administration of latex proteins (LP) and was followed behavioral, hematological and histological analyses. Oral immunological tolerance to LP was assessed by intraperitoneal immunization in mice that had received LP orally before. Animals given 5000 mg/kg orally exhibited only discrete behavioral alterations and augmentation of monocytes. Death was not notified 14 days after exposure. However, all animals receiving LP 150 mg/kg by i.p. died in 1 h. Death (20%) was documented when LP (75 mg/kg) was given in the peritoneum and signs of harmful effects were observed in the survivors (80%). Oral immunological tolerance was observed in animals previously given LP orally, when they were further immunized/challenged with peritoneal exposure to different doses of LP. This was confirmed by the lowering of IgE and IgG in the serum, IL-4 and IFN-γ in spleen homogenates and the absence of anaphylaxis signs. It is therefore concluded that LP exhibited quite discrete adverse effects when orally administrated at higher concentrations and this route of administration did not stimulate adverse immunological reactions. Instead it was observed immunological tolerance. The present study contributes very important information concerning the safe use of C. procera as a phytotherapeutic agent.

Performance characteristics of an automated latex immunoturbidimetric assay [HemosIL® HIT-Ab(PF4-H)] for the diagnosis of immune heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by platelet-activating anti-PF4/heparin antibodies. Given time-sensitive treatment considerations, a rapid and accurate laboratory test for HIT antibodies is needed. AIMS: To determine operating characteristics for the HemosIL® HIT-Ab(PF4/H), a rapid, on-demand, fully-automated, latex immunoturbidimetric assay (LIA), for diagnosis of HIT. METHODS: We evaluated LIA sensitivity, specificity, negative (NPV) and positive predictive value (PPV), negative (LR-) and positive likelihood ratio (LR+), using citrated-plasma from 429 patients (prospective cohort study of 4Ts scoring; HIT, n=31), and from consecutive HIT patients (n=125), using reference standard serotonin-release assay (SRA). Comparators included two PF4-dependent enzyme-immunoassays (EIAs). We used stratum-specific likelihood ratios (SSLRs) to determine how differing magnitudes of LIA-positivity influenced post-test probability of HIT. RESULTS: LIA operating characteristics were: sensitivity=97.4% (152/156); specificity=94.0% (374/398); PPV=55.6% (30/54); and NPV=99.7% (374/375). At manufacturers' cutoffs, LIA specificity and PPV were superior to the EIAs. Although a negative LIA pointed strongly against HIT (LR-, 0.034), the post-test probability was ~2% with high 4Ts score. The LIA's LR+ was high (16.0), with SSLRs rising substantially with greater LIA-positivity: 5.7 (1.0-4.9U/mL), 31 (5.0-15.9U/mL), and 128 (≥16U/mL). A LIA-positive result (at 1.0 cutoff) indicated at least 24% HIT probability (low 4Ts score), rising to 90% with high 4Ts score. CONCLUSIONS: Although approximately 1 in 40 SRA-positive patients tested LIA-negative, the LIA's high NPV and PPV indicate that this rapid assay is useful for the diagnostic evaluation of HIT, including in low pre-test situations.