Seasonal Local Allergic Rhinitis in Areas With High Concentrations of Grass Pollen.

BACKGROUND: Local allergic rhinitis (LAR) is a phenotype of allergic rhinitis characterized by the presence of a localized immune response in the nasal mucosa of patients with negative skin prick test (SPT) results and undetectable serum specific IgE (sIgE). It unknown whether LAR is limited to areas with low or moderate aeroallergen exposure. OBJECTIVE: To explore the presence of LAR and the clinical and immunological characteristics of this entity in geographic areas with high grass pollen loads. METHODS: A cross-sectional observational study was carried out in 2 hospitals in central Spain (Madrid and Ciudad Real). Sixty-one patients with seasonal rhinitis and negative SPT results and undetectable serum sIgE were evaluated using a clinical questionnaire, determination of serum total IgE, and a nasal allergen provocation test (NAPT) with Phleum species. The response to NAPT was monitored using assessment of nasal symptoms, acoustic rhinometry, and determination of sIgE, tryptase, and eosinophil cationic protein in the nasal cavity. RESULTS: Seasonal LAR was detected in 37 patients (61%) using the techniques described above. Eleven percent of patients with LAR were adolescents or children, and 14% reported onset of rhinitis in childhood. Most patients reported persistent-moderate seasonal nasal symptoms, and 41% reported worsening of the disease during the last 2 years. Conjunctivitis was the most common comorbidity, affecting 95% of cases. CONCLUSIONS: LAR to grass pollen is relevant in patients with seasonal symptoms indicative of allergic rhinitis but with a negative skin test result who live in areas with high allergenic pollen loads. This entity should be included the differential diagnosis of rhinitis.

Thermomechanical transitions of egg-ceramide monolayers.

Ceramides have unique biophysical properties. Their high melting temperature and their ability to form lateral domains have converted ceramides into the paradigm of rigid lipids. Here, using shear surface rheology of egg-ceramide Langmuir monolayers, a solid to fluid transition was evidenced as a vanishing shear rigidity at lower temperatures than the lipid melting temperature. Such a mechanical transition, which depends on the lipid lateral pressure, was found in a broad range temperature (40-50 °C). The solid to fluid transition was correlated to a LC to LC+LE phase transition, as confirmed by BAM experiments. Interestingly, together with the softening transition, a supercooling process compatible with a glassy behavior was found upon freezing. A new phase scenario is then depicted that broadens the mechanical behavior of natural ceramides. The phase diversity of ceramides might have important implications in their physiological roles.

Component-resolved diagnosis in vespid venom-allergic individuals.

BACKGROUND: Hymenoptera venom-allergic patients frequently present multiple sensitisations. OBJECTIVES: To define the allergic profile by components in wasp allergic patients. To study the usefulness of specific IgE to components in cases of double sensitisation. MATERIALS AND METHODS: Wasp allergic patients who needed Polistes and/or Vespula venom immunotherapy were included. Before immunotherapy and after two years of treatment the following specific IgE (sIgE) levels were measured: Apis mellifera, Vespula spp. Polistes spp., rVes v 5, rPol d 5, nVes v 5, nPol d 5, nVes v 1, nPol d 1, nApi m 1, nApi m 2 and peroxidase. Skin tests with venoms were performed. Based on the sIgE and the skin test results, Polistes and/or Vespula immunotherapy was administered. RESULTS: Thirteen patients were included. Double sensitisation to Polistes/Vespula was detected in eight patients. Sensitisation to rVes v 5 and rPol d 5 was found in two of eight cases, to nVes v 1 and nPol d 1 in eight of 13 cases, and to nVes v 5 and nPol d 5 in 2 of 13 cases. Three patients received double immunotherapy with both wasps. One patient was treated with Vespula and nine with Polistes. sIgE levels decreased after two years of treatment. In patients who showed double sensitisation but were treated with only one venom, sIgE to both venoms decreased. CONCLUSIONS: Components analysis can be useful to study double positivity. In case of doubt, double immunotherapy should be administered. Phospholipase was found to be a major allergen in our population.

Efficacy of IgE-targeted vs empiric six-food elimination diets for adult eosinophilic oesophagitis.

BACKGROUND: Skin testing-guided elimination diet has proved unsuccessful for adult eosinophilic oesophagitis (EoE), whereas empiric six-food elimination diet (SFED) achieves an efficacy of 70%. OBJECTIVE: To compare the efficacy of food-specific serum IgE-targeted elimination diet (sIgE-ED) and SFED. METHODS: Prospective study in adult patients with EoE. Food-specific serum IgE, skin prick test (SPT) and atopy patch test (APT) to foods included in SFED were performed. Those with ≥1 positive IgE test, defined by ≥0.1 kU/l, followed a 6-week sIgE-ED, whereas non-IgE-sensitized patients underwent a 6-week SFED. Responders to diet (<15 eos/HPF) underwent individual reintroduction of foods followed by histological assessment. RESULTS: Forty-three EoE patients were included (26 sIgE-ED and 17 SFED). Regarding sIgE-ED, the mean number of eliminated foods per patient was significantly lower than in SFED (3.81 vs 6; P < 0.001), being wheat (85%), nuts (73%) and cow's milk (61%) the most commonly foods withdrawn. No difference in histological response was observed between sIgE-ED and SFED (73% vs 53%, P = 0.17). Causative foods identified by food challenge were cow's milk (64%), wheat (28%), egg (21%) and legumes (7%), with a single food trigger in 71% of patients. sIgE exhibited the higher accuracy to predict offending foods in IgE-sensitized patients (sensitivity 87.5%, specificity 68% (κ = 0.43)), with k values of 1 for cow's milk. APT results were all negative. CONCLUSIONS: Histological remission was accomplished in 73% of patients undergoing sIgE-ED, which was nonsignificantly superior to SFED. sIgE effectively identified cow's milk as a food trigger in IgE-sensitized patients.

Alterations in the antioxidant defense system in prepubertal children with a history of extrauterine growth restriction.

PURPOSE: The role of oxidative stress is well known in the pathogenesis of acquired malnutrition. Intrauterine growth restriction has been associated with an imbalance in oxidative stress/antioxidant system. Therefore, early postnatal environment and, consequently, extrauterine growth restriction might be associated with alterations in the antioxidant defense system, even in the prepubertal stage. METHODS: This is a descriptive, analytical, and observational case-control study. The study included two groups; 38 Caucasian prepubertal children born prematurely and with a history of extrauterine growth restriction as the case group, and 123 gender- and age-matched controls. Plasma exogenous antioxidant (retinol, ß-carotene, and α-tocopherol) concentrations were measured by HPLC; antioxidant enzyme activities of catalase, glutathione reductase, glutathione peroxidase, and superoxide dismutase were determined in lysed erythrocytes by spectrophotometric techniques. RESULTS: Catalase and glutathione peroxidase concentrations were significantly lower in extrauterine growth restriction children than in controls (P < 0.001). Lower plasma retinol concentrations were found in the case group (P = 0.029), while concentrations of ß-carotene and α-tocopherol were higher (P < 0.001) in extrauterine growth restriction prepubertal children as compared with controls. After correction by gestational age, birth weight, and length, statistically significant differences were also found, except for retinol. CONCLUSIONS: Prepubertal children with a history of extrauterine growth restriction present alterations in their antioxidant defense system. Knowing these alterations may be important in establishing pharmacological and nutritional treatments as this situation might be associated with higher metabolic disorders in adulthood.

First Report of Fusarium oxysporum on Sweet Pepper Seedlings in Almería, Spain.

In March of 2013, new symptoms were observed in more than seven million nursery-grown sweet pepper (Capsicum annuum) plants in El Ejido, Almería (southern Spain). Symptoms included wilting without yellowing of leaves and stunting of plants. Plant crowns exhibited necrosis that advanced through the main root along with slight root rot. Xylem was not affected above or below the crown. Symptoms were thought to be caused by the well-known pepper pathogen Phytophthora capsici. However, sporodochia of Fusarium oxysporum were observed on plant crowns. Symptomatic seedlings (n = 200) were sampled and analyzed. Tissue from roots and epidermal crowns were plated on PDA, PARP, and Komada media, as well as stem discs on PDA and Komada. No Phytophthora sp. were observed and F. oxyporum was exclusively isolated from all 200 samples, from roots and crowns, but not from xylem. Pathogenicity of 60 of these F. oxysporum isolates was studied by inoculation onto sweet pepper plants (cv. del Piquillo) at the 2-true-leaf stage. Twelve plants per isolate, grown on autoclaved vermiculite, were inoculated by drenching with 20 ml of a conidial suspension (1 × 105 CFU/ml) of each isolate per plant. Each suspension was obtained by blending one PDA petri dish fully covered with one isolate. Non-inoculated plants served as control. Plants were maintained for 30 days in a growth chamber with a 14-h photoperiod (1.6 ×·104 lux) and temperatures at 23 to 26°C. The assay was conducted twice. Symptoms described above were reproduced on crown and roots of the inoculated plants with no symptoms in stem discs. No symptoms were observed on controls after 48 days. Host specificity was tested for 13 isolates to tomato (Solanum lycopersicum) cv. San Pedro, eggplant (S. melongena) cv. Alegria, cucumber (Cucumis sativus) cv. Marketmore, watermelon (Citrullus lanatus) cv. Sugar Baby, and Chinese cabbage (Brassica campestris subsp. condensa) cv. Kasumi (4). These plants were inoculated as previously described for pathogenicity tests (12 plants per species, repeated twice). None of the plants exhibited the characteristic symptoms after 60 days. Five isolates of F. oxysporum f. sp. radicis-cucumerinum and four isolates of F. o. f. sp radicis-lycopersici were also inoculated without any symptoms in any of the inoculated sweet pepper plants. Morphological identity of all isolates corresponded to F. oxysporum. The fungi were identified following the morphological keys and methodology provided by (1) and (2). Three isolates from the 60 tested were selected for molecular identification. Molecular identification was performed by sequencing partial TEF-1α gene (3). Subsequent database searches by BLASTn indicated that the resulting sequence of 659-bp had 100% identity with the corresponding gene sequence of F. oxysporum. The sequences were identical for the three isolates and were deposited on the EMBL Sequence Database (HG916993, HG916994, and HG916995). Results suggest that the pathogenic ability of the isolates varies from a vascular Fusarium wilt. F. oxysporum f. sp. capsici is a reported pathogen to sweet pepper (5), but the symptoms we have found are closer to those manifested by the formae speciales that causes root and crown rot of other plants. Consistent with the convention stablished for similar diseases we propose the name F. oxysporum f. sp. radicis-capsici f. sp. nov. References: (1) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell, Ames, IA, 2006. (2) P. E. Nelson et al. Fusarium species. An Ilustrated Manual for Identification. The Penn St. University Press, 1983. (3) K. O'Donnell et al. Proc. Nat. Acad. Sci. 95:2044, 1998.(4) L. M. Oelke and P. W. Bosland. Capsicum Eggplant Newsl. 20:86, 2001. (5) V. C. Rivelli. M.S. Thesis. Dep. Plant Pathol. and Crop Phys. Louisiana State Univ., Baton Rouge, 1989.

Engineered domain-based assays to identify individual antibodies in oligoclonal combinations targeting the same protein.

Quantitation of individual monoclonal antibodies (mAbs) within a combined antibody drug product is required for preclinical and clinical drug development. We have developed two antitoxins, XOMA 3B and XOMA 3E, each consisting of three mAbs that neutralize type B and type E botulinum neurotoxin (BoNT/B and BoNT/E) to treat serotype B and E botulism. To develop mAb-specific binding assays for each antitoxin, we mapped the epitopes of the six mAbs. Each mAb bound an epitope on either the BoNT light chain (LC) or translocation domain (H(N)). Epitope mapping data were used to design LC-H(N) domains with orthogonal mutations to make them specific for only one mAb in either XOMA 3B or XOMA 3E. Mutant LC-H(N) domains were cloned, expressed, and purified from Escherichia coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. Further engineering of domains allowed construction of enzyme-linked immunosorbent assays (ELISAs) that could characterize the integrity, binding affinity, and identity of each of the six mAbs in XOMA 3B and 3E without interference from the three BoNT/A mAbs in XOMA 3AB. Such antigen engineering is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that bind the same protein.

Nuclear factor of activated T cells (NFAT)-dependent transactivation regulated by the coactivators p300/CREB-binding protein (CBP).

p300 and cAMP response element-binding protein (CREB)-binding protein (CBP) are members of a family of coactivators involved in the regulation of transcription and chromatin. We show that transcription factors of the nuclear factor of activated T cells (NFAT) family bind p300/CBP and recruit histone acetyltransferase activity from T cell nuclear extracts. The NH2-terminal transactivation domain of NFAT1 and the phospho-CREB- and E1A-binding sites of p300/CBP are involved in the interaction. The viral oncoprotein E1A inhibits NFAT-dependent transactivation in a p300-dependent manner. Recruitment of the coactivators p300/CBP by the transactivation domains of NFAT proteins is likely to play a critical role in NFAT-dependent gene expression during the immune response.

Distal biceps tendon rupture: diagnostic strength of ultrasonography and magnetic resonance. / Rotura del tendón distal del bíceps: potencia diagnóstica de la ecografía y la resonancia magnética.

OBJECTIVE: The main purpose of this study is to determine the agreement between two radiological tests, ultrasonography and magnetic resonance, in the diagnosis of distal brachial biceps tendon injury. MATERIAL AND METHOD: A retrospective study was made of patients over 3 years who underwent distal brachial biceps tendon reattachment open surgery, and whose preoperative diagnosis was complemented either by ultrasonography, by magnetic resonance or both. The agreement was calculated by Cohen's kappa coefficient (k). RESULTS: Information of 79 patients was analyzed. The concordance was excellent between resonance and surgical findings (k=.950), and a good Cohen's kappa coefficient was also achieved between both ultrasonography and surgery (k=.706) and between information of sonography and magnetic resonance (k=.667). CONCLUSIONS: Resonance and ultrasonography could be considered reliable diagnostic tests to determine the acute rupture of the distal brachial biceps tendon in patients with consistent aetiology and physical examination.

Varicose veins show enhanced chemokine expression.

OBJECTIVES: Leucocyte infiltration in the wall of varicose veins has been reported previously. This study was designed to investigate the expression of pro-inflammatory cytokines and chemokines in control and in patients with varicose veins and to test the effect of treating varicose vein patients with acetylsalicylic acid (ASA) on cytokine expression prior to removal of varices. MATERIAL AND METHODS: Sections of vein were removed during operation from both patient groups, and ribonuclease protection assays (RPAs) were performed to assess the expression of chemokines. Group I included non-varicose saphenous veins from healthy patients undergoing amputation for trauma. Varicose veins were obtained from patients with primary varicose undergoing surgical treatment who received no drug (group II) or treatment with 300 mg day(-1) of ASA for 15 days before surgery (group III). RESULTS: Non-varicose veins constitutively expressed low levels of monocyte-chemoattractant protein (MCP-1) and interleukin (IL)-8 mRNA. Varicose veins had a distinct chemokine expression pattern, since significant up-regulation of MCP-1 and IL-8 and a marked expression of IP-10, RANTES, MIP-1alpha and MIP-1beta mRNA were detected. Removal of the endothelium did not alter this pattern. Varicose veins obtained from patients treated with ASA showed a consistent decrease in chemokine expression, although it did not reach statistical significance. CONCLUSIONS: Varicose veins showed increased expression of several chemokines compared to control veins. A non-significant reduction of activation was observed following treatment with ASA for 15 days.

Molecular evolution of antibody affinity for sensitive detection of botulinum neurotoxin type A.

Botulism is caused by botulinum neurotoxin (BoNT), the most poisonous substance known. Potential use of BoNT as a biothreat agent has made development of sensitive assays for toxin detection and potent antitoxin for treatment of intoxication a high priority. To improve detection and treatment of botulism, molecular evolution and yeast display were used to increase the affinity of two neutralizing single chain Fv (scFv) antibodies binding BoNT serotype A (BoNT/A). Selection of yeast displayed scFv libraries was performed using methods to select for both increased association rate constant (k(on)) and decreased dissociation rate constants (k(off)). A single cycle of error prone mutagenesis increased the affinity of the 3D12 scFv 45-fold from a K(D) of 9.43x10(-10)M to a K(D) of 2.1x10(-11)M. Affinity of the HuC25 scFv was increased 37-fold from 8.44x10(-10)M to 2.26x10(-11)M using libraries constructed by both random and site directed mutagenesis. scFv variable region genes were used to construct IgG for use in detection assays and in vivo neutralization studies. While IgG had the same relative increases in affinity as scFv, (35-fold and 81-fold, respectively, for 3D12 and HuC25) higher solution equilibrium binding constants were observed for the IgG, with the 3D12 K(D) increasing from 6.07x10(-11)M to 1.71x10(-12)M and the HuC25 K(D) increasing from 4.51x10(-11)M to 5.54x10(-13)M. Affinity increased due to both an increase in k(on), as well as slowing of k(off). Higher affinity antibodies had increased sensitivity, allowing detection of BoNT/A at concentrations as low as 1x10(-13)M. The antibodies will also allow testing of the role of affinity in in vivo toxin neutralization and could lead to the generation of more potent antitoxin.

The Calcium-Sensing Receptor in Health and Disease.

The extracellular calcium-sensing receptor (CaSR) is a unique G protein-coupled receptor (GPCR) activated by extracellular Ca2+ and by other physiological cations including Mg2+, amino acids, and polyamines. CaSR is the most important master controller of the extracellular Ca2+ homeostatic system being expressed at high levels in the parathyroid gland, kidney, gut and bone, where it regulates parathyroid hormone (PTH) secretion, vitamin D synthesis, and Ca2+ absorption and resorption, respectively. Gain and loss of function mutations in the CaSR are responsible for severe disturbances in extracellular Ca2+ metabolism. CaSR agonists (calcimimetics) and antagonists (calcilytics) are in use or under intense research for treatment of hyperparathyroidism secondary to kidney failure and hypocalcemia with hypercalciuria, respectively. Expression of the CaSR extends to other tissues and systems beyond the extracellular Ca2+ homeostatic system including the cardiovascular system, the airways, and the nervous system where it may play physiological functions yet to be fully understood. As a consequence, CaSR has been recently involved in different pathologies including uncontrolled blood pressure, vascular calcification, asthma, and Alzheimer's disease. Finally, the CaSR has been shown to play a critical role in cancer either contributing to bone metastasis and/or acting as a tumor suppressor in some forms of cancer (parathyroid cancer, colon cancer, and neuroblastoma) and as oncogene in others (breast and prostate cancers). Here we review the role of CaSR in health and disease in calciotropic tissues and others beyond the extracellular calcium homeostatic system.

Carotid artery puncture, airway obstruction and the laryngeal mask airway in a preeclamptic patient.

We report a case of a preeclamptic patient with a difficult airway in whom attempted central venous cannulation led to inadvertent carotid artery puncture and dilatation causing immediate life threatening upper airway obstruction. The use of the laryngeal mask airway was life-saving on two occasions when other techniques of airway management had failed. We discuss the series of events that led to this critical incident and suggest areas in which management might have been improved.