Sensitization to Gibberellin-Regulated Protein (Peamaclein) Among Italian Cypress Pollen-Sensitized Patients.

BACKGROUND AND OBJECTIVES: Peach gibberellin-regulated protein (peamaclein) has recently emerged as a relevant food allergen in cypress pollen-hypersensitive patients. Objective: We investigated monosensitization to peamaclein among Italian cypress pollen-allergic patients. MATERIAL AND METHODS: A total of 835 cypress pollen-hypersensitive patients from 28 Italian allergy centers underwent a thorough work-up to determine food-allergic reactions and performed skin prick testing with a commercial peach extract containing peamaclein. IgE to rPru p 3 was measured in peach reactors, and those with negative results were enrolled as potentially monosensitized to peamaclein. IgE reactivity to rPru p 7 was evaluated using immunoblot and an experimental ImmunoCAP with rPru p 7. RESULTS: Skin prick tests were positive to peach in 163 patients (19.5%); however, 127 (77.9%) were excluded because they reacted to Pru p 3. Twenty-four patients (14.7%) corresponding to 2.8% of the entire study population) were considered potentially monosensitized to peamaclein. No geographic preference was observed. Seventeen of the 24 patients (70.8%) had a history of food allergy, mainly to peach (n=15). Additional offending foods included other Rosaceae, citrus fruits, fig, melon, tree nuts, and kiwi. On peach immunoblot, only 3 of 18 putative peamaclein-allergic patients reacted to a band at about 7 kDa; an additional 4 patients reacted at about 50-60 kDa. Ten of 18 patients (56%) had a positive result for Pru p 7 on ImmunoCAP. CONCLUSION: Allergy and sensitization to peamaclein seem rare in Italy. Most patients react to peach, although other Rosaceae fruits and several citrus fruits may also be offending foods. Peach and cypress pollen probably also share cross-reacting allergens other than peamaclein.

Evaluation of two commercial peach extracts for skin prick testing in the diagnosis of hypersensitivity to lipid transfer protein. A multicenter study.

Summary: The clinical usefulness of two commercial peach extracts for SPT (by Lofarma SpA and ALK-Abellò, respectively) was compared in a multicenter study carried out in Italy. Peach allergic patients were tested with the two extracts in parallel and underwent the detection of IgE specific for all three peach allergens currently available (Pru p1, Pru p3, and Pru p4, respectively). The two extracts were almost identical in terms of sensitivity and specificity, being able to detect virtually all patients sensitized to stable peach allergens (lipid transfer protein (LTP) and, presumably, peamaclein) but scoring negative in patients exclusively sensitive to labile allergens (either PR-10 and/or profilin). Thus, the two extracts represent an excellent tool to carry out a preliminary component-resolved diagnosis of peach allergy at the first patient visit.

The role of allergoids in allergen immunotherapy: from injective to sublingual route.

Summary: Summary Allergen immunotherapy (AIT) is aimed at inducing tolerance to allergens, such as pollens, dust mites or moulds, by administering increasing amounts of the causative allergen through subcutaneous or sublingual route. The evidence of efficacy of AIT is high, but the issue of safety, especially for the subcutaneous route, must be taken into account. The search for safer AIT products aimed at reducing the allergenicity, and thus adverse reactions, while maintaining the immunogenicity, that is essential for effectiveness, gave rise to the introduction of allergoids, which were conceived to fulfill these requirements. In the first allergoids glutaraldehyde or formaldehyde were used as cross-linking agent to polymerize allergens, this resulting in high molecular weight molecules (200,000 to 20,000,000 daltons) which were significantly less allergenic due to a decreased capacity to bridge IgE on its specific receptor, while maintaining the immunogenicity and thus the therapeutic efficacy. In recent years further agents, acting as adjuvants, such as L-tyrosine, monophosphoryl lipid A, aluminium hydroxide, were added to polymerized extracts. Moreover, a carbamylated monomeric allergoid was developed and, once adsorbed on calcium phosphate matrix, used by subcutaneous route. At the same time, in virtue of its peculiarities, such allergoid revealed particularly suitable for sublingual administration. A lot of clinical evidences show that it is well tolerated, largely safer and effective. Importantly, the higher safety of allergoids allows faster treatment schedules that favor patient compliance and, according to pharmaco-economic studies, they might be more cost-effective than other AIT options.

Shrimp Allergy: Analysis of Commercially Available Extracts for In Vivo Diagnosis.

BACKGROUND AND OBJECTIVE: Skin prick testing (SPT) with commercial extracts is the first step in the diagnosis of shrimp allergy, although its clinical efficiency is unknown. Objective: To analyze the clinical usefulness of all commercial crustacean extracts available for SPT in Italy. METHODS: We performed a multicenter study of 157 shrimp-allergic patients who underwent SPT with 5 commercial crustacean extracts and with house dust mite (HDM) extract. Commercial extracts were analyzed using SDS-PAGE and compared with a freshly prepared in-house shrimp extract. IgE to Pen a 1/Pen m 1, Pen m 2, and Pen m 4 was determined, and immunoblot analysis was performed on a large number of sera. RESULTS: The skin reactions caused by commercial crustacean extracts were extremely heterogeneous, resulting in 32 clinical profiles, with marked differences in protein content and missing proteins at molecular weights corresponding to those of major shrimp allergens. Only strong Pen a 1/Pen m 1 reactors reacted to both HDM and all 5 commercial extracts in SPT. Most patients, including those who were tropomyosin-negative, reacted to HDM. Patients reacted to a large and variable array of proteins, and IgE reactivity was common at high molecular weights (>50 kDa). CONCLUSIONS: The in vivo diagnosis of shrimp allergy must continue to be based on SPT with fresh material. Shrimp-allergic patients frequently react to a number of ill-defined high-molecular-weight allergens, thus leaving currently available materials for component-resolved diagnosis largely insufficient. Mites and crustaceans probably share several allergens other than tropomyosin.

IgE Reactivity to Polcalcins Varies According to Pollen Source.

BACKGROUND: Polcalcins are highly cross-reactive pollen panallergens. Less than 10% of allergic patients are sensitized to polcalcins. All pollen species are considered able to sensitize patients to this panallergen. OBJECTIVE: We aimed to assess the presence of polcalcins in various pollen extracts used in allergen immunotherapy. METHODS: ELISA inhibition experiments were performed with sera from patients sensitized to polcalcin and rPhl p 7 and rBet v 4. Recombinant polcalcin was used as the substrate and freshly prepared pollen extracts as inhibitors. RESULTS: All pollen extracts induced significant inhibition of IgE reactivity to rBet v 4, whereas only grass pollen extract induced marked inhibition of IgE reactivity to rPhl p 7. CONCLUSION: Grass polcalcin probably contains more epitopes than polcalcins from other pollen sources. Grass pollen could be responsible for sensitization to polcalcins, and grass pollen immunotherapy is likely to be an option for polcalcin-hypersensitive patients.

Unusual allergy to soy appeared in adult age.

A case of adult onset severe soy allergy is discussed. The allergen protein involved did not correspond to those presently detectable by commercial diagnostic means, but was not identified, possibly due to the insufficient level of specific IgE. Fresh foods and commercial food extracts remain an invaluable tool to support the diagnosis of food allergy, both in-vivo and in-vitro.

SERUM YKL-40 IN CHILDREN WITH ASTHMA.

Asthma is one of the most common chronic diseases in children. To date the diagnosis of asthma is mainly clinical, based on the clinical history, a careful physical examination and lung function tests. However, symptoms are often not specific and lung function tests are not very sensitive. In order to find a solution to this problem new biomarkers of airway inflammation are being developed. YKL-40 is a chitinase-like protein that has a role in the inflammation and tissue remodeling in several human diseases. The aim of this study is to evaluate serum levels of YKL40 in children with intermittent or persistent asthma. We performed a cross-sectional analysis of serum samples from a cohort of patients with asthma and healthy controls. Patients with asthma were stratified according to four levels of asthma severity (mild intermittent, mild persistent, moderate persistent, and severe persistent). The analysis of serum samples was performed with the use of a commercially available enzyme-linked immune-adsorbent assay (ELISA) kit (Quidel). The minimum detection limit of the assay for YKL-40 is 15.6 ng per milliliter (ng/ml). Our data showed that circulating YKL-40 levels are significantly higher in patients with asthma than healthy subjects (36±18.6 vs 14:41±2.88, p= 0.001). Furthermore, we found significantly higher values of YKL-40 in both groups of children with intermittent asthma (p less than 0.001) and persistent asthma (p less than 0.001) than healthy controls. However, no correlation was found with duration and severity of asthmatic disease (r = 0:18, p= 0:33, r = 0.28 P = 0:13, respectively). Our data allow us to suggest that YKL-40 represents a useful biomarker of asthma in children with intermittent or persistent asthma.

Different allergenicity of pollen extracts of three Mediterranean cypress species accounted for cytological observations.

BACKGROUND: Cypresses play an important role in the urban landscape of the Mediterranean region, releasing a huge amount of allergenic airborne pollen which causes a specific pollinosis in exposed people. OBJECTIVE: The aim of this work is to evaluate, in vivo and in vitro, the potential allergenicity of Cupressus macrocarpa pollen, and to compare it with the allergenicity observed for C. arizonica and C. sempervirens. METHODS: Pollen extracts of the three species were prepared, to determine their protein profile through SDS PAGE analysis and to evaluate their allergenic potential through EAST inhibition assays and SPT. Pollen grain composition was evaluated using a cytochemical approach with optical microscopy. RESULTS: SDS PAGE, EAST inhibition and SPT indicated the higher allergenic potential of C. arizonica compared to C. sempervirens and C. macrocarpa. No significant differences in allergenic potential were found between the latter two species. Cytochemical observations reveal higher ß-glucans and protein content in the intine of C. arizonica during hydration. CONCLUSION: The higher protein content found in C. arizonica pollen grains extract may be due to higher enzyme activity leading to the movement of ß-glucans and pectins from the intine to the partially developed pollen cell wall during hydration. This could explain the higher potential allergenicity of C.arizonica in respect to C. macrocarpa and C. sempervirens.

Shrimp allergy beyond Tropomyosin in Italy: clinical relevance of Arginine Kinase, Sarcoplasmic calcium binding protein and Hemocyanin.

BACKGROUND: Little is known about the prevalence and clinical relevance of sensitization to shrimp allergens other than tropomyosin. OBJECTIVE: We detected the prevalence of arginine kinase and sarcoplasmic calcium binding protein sensitization, and identified a high molecular weight allergen that is frequently recognized by Italian shrimp-allergic patients. METHODS: Sera from 40 shrimp-allergic patients underwent the detection of IgE specific for arginine kinase (rPen m 2) and sarcoplasmic calcium-binding protein (rPen m 4) by ISAC 112 Microarray platform and immunoblot analysis. A high molecular weight shrimp allergen was identified by N-terminal amino acid sequencing. RESULTS: IgE to rPen m 2 and rPen m 4 were found in 4/40 (10%) and 6/40 (15%) sera, respectively; two sera reacted to both allergens. Clinically, 6/8 Pen m 2 and/or Pen m 4 reactors experienced severe allergies to shrimp. On immunoblot, 4/6 rPen m 4-positive sera showed IgE reactivity at about 20 kDa, whereas no rPen m 2-positive serum reacted at about 40 kDa. Nineteen (47%) sera showed IgE reactivity at molecular weights > 60 kDa. Such profile was not associated with IgE reactivity to rPen m 2 or rPen m 4. N-terminal amino acid sequencing of the high molecular weight allergen led to the identification of hemocyanin. CONCLUSION: Shrimp arginine kinase and sarcoplasmic calcium-binding protein are minor allergens sensitizing only 10%-15% of Italian shrimp-allergic patients, but are clinically relevant. Hemocyanin is a clinically relevant high molecular weight shrimp allergen possibly cross-reacting to house dust mite.

Detection of 20 kDa and 32 kDa IgE-binding proteins as the major allergens in Italian sesame seed allergic patients.

BACKGROUND: Sesame seed allergy, a potentially very severe food allergy, seems on the rise worldwide but is still uncommon in Italy. The aim of the present study was to investigate the allergenic profile of Italian sesame seed-allergic patients. METHODS: Patients with genuine sesame seed allergy diagnosed over one year in a large number of allergy centers scattered through Italy were considered for the study. Their IgE reactivity to sesame seed allergens was characterized by immunoblot analysis. RESULTS: Eleven sesame seed allergic patients were detected and studied. 10/10 patients showed IgE reactivity against a sesame allergen at about 20 kDa, and 7/10 showed an extremely strong reactivity at about 32 kDa. The same 7 sera reacted also against a 28 kDa allergen, although such reactivity was significantly weaker in 6/7 cases. Eight patients showed IgE reactivity at about 48 kDa, and 5 sera reacted against higher m.w. proteins at about 67 kDa. Two sera showed IgE reactivity at about 43 kDa as well. Only one serum appeared to react to 2S-albumin. CONCLUSIONS: Italian sesame seed-allergic patients react mostly against allergens other than those described so far as major ones. A large number of recombinant sesame allergens will be needed for a comprehensive component- resolved diagnosis of allergy to this food.

Italian study on buckwheat allergy: prevalence and clinical features of buckwheat-sensitized patients in Italy.

Buckwheat allergy is considered a rare food allergy outside of Asia. In Europe, buckwheat has been described mainly as a hidden allergen. Data on the prevalence of buckwheat hypersensitivity in non-Asian countries is very poor. The aim of this multicenter study was to evaluate the prevalence of buckwheat sensitization and its association with other sensitizations among patients referred to allergy clinics in different geographic areas of Italy. All patients referred to 18 Italian allergy clinics from February through April 2011 were included in the study and evaluated for sensitization to buckwheat and other allergens depending on their clinical history. A total of 1,954 patients were included in the study and 61.3 percent of them were atopic. Mean prevalence of buckwheat sensitization was 3.6 percent with significant difference between Northern (4.5 percent), Central (2.2 percent) and Southern (2.8 percent) regions. This is, to our knowledge, the largest epidemiological survey on buckwheat allergy reported outside of Asia. Buckwheat is an emerging allergen in Italy, being more frequently associated to sensitization in Northern regions.

Occupational asthma associated to the exposure to limonium tataricum flowers.

Limonium tataricum (Lt) is a plant belonging to the family of Plumbaginaceae. The role of this family and in particular, that of dried flowers (but not of the pollen) in occupational allergy has already been described. We have observed a farmer with asthma occurring in the presence of fresh flowers. Standard methacoline test demonstrated that the patient was a true asthmatic. The allergenicity of Lt pollen was thus investigated Skin prick tests (SPT) were carried out using both standard allergens and the Lt extract and the patient's mucosal reactivity was evaluated by nasal provocation test with the pollen extract. In vitro studies were also performed on the patient's serum by evaluating routine specific anti-allergen IgE on raw extracts and on Microarray Allergen Chip (ISAC). Finally, the raw extract of the fresh Lt pollen was also used in ELISA inhibition test, immunoblotting and Basophil Activation Test (BAT). The specific sensitization was demonstrated by Skin Prick test and nasal provocation test. The sensitization was also confirmed by specific IgE and by in vitro activation of basophils in the presence of the pollen. By using RAST inhibition test, the presence of cross-reactivity with other pollens was ruled out. According to our results, Lt extracts contain an allergenic activity not only as dried flowers, but also as fresh pollen. For its role in occupational asthma, this allergen should be included in any allergy screening at least in farmers or in the flower industry employers.

Effect of two doses of carbamylated allergoid extract of dust mite on nasal reactivity.

Background and Objective. Single SLIT studies with native allergen extracts support a dose-response effect for clinical and immunological outcomes. Conversely for carbamylated allergoids this dose-response effects is less evident, likely because the threshold for efficacy is more easily reached through the enhanced bioavailability of the extract consequent to the selective chemical modification. Thus this pilot study investigates the dose-response effect on nasal specific reactivity and safety of two unusual doses of carbamylated allergoid in patients mono-sensitized to house dust mites. Methods. A prospective open randomized study involved 6-65 year-old Italian patients with clinically relevant sensitization to house dust mites and positive response to nasal provocation challenge. Monomeric carbamylated allergoid was delivered once daily at the dose of 1000 AU or 2000 AU from June to September 2009, during the lowest level of mites exposure. Primary outcomes were the change of the threshold of allergen concentration for a positive nasal provocation test (NPT) before and after the treatment and the product safety. Secondary outcome was the change  in the mean percentage fall of peak nasal inspiratory flow (PNIF) following nasal challenge. Results. Thirty-four patients were enrolled. Fifteen in group 1 and 14 in group 2 concluded the study. After 12 weeks all patients treated in group 1 and all but one in group 2 showed an increase in the threshold dose provoking a positive NPT. Those with no symptoms onset with the highest dose delivered were 80% in group 1 and 78.6% in group 2 (p=0.92). From first to second challenge, the mean percentage fall of PNIF  was reduced with no statistical difference between groups (p=0.95), and with no difference between the final mean percentage falls (p=0.65). No serious adverse reactions occurred and the frequency of events, all mild, was similar in the two groups. Conclusions. Twelve weeks of carbamylated sublingual allergoid delivered at 1000AU or 2000AU once daily appear equally safe and show comparable effect in increasing  the threshold of allergen concentration for a positive nasal provocation test, confirming the apparent absence of a dose response effect for the used doses.

Mugwort-fennel-allergy-syndrome associated with sensitization to an allergen homologous to Api g 5.

BACKGROUND: The cross-reactive allergen responsible for the so called "mugwort-celery-spice-syndrome", a pollen-food allergy that occurs in a minority of mugwort pollen-allergic patients, is still undefined. OBJECTIVE: To identify the allergen responsible for the cross-reactivity between mugwort pollen and plant-derived foods. METHODS: The serum from one index patient with both fennel and mugwort pollen allergy was used to identify IgE-reactive allergens by direct ELISA and Immunoblot analysis. Cross-reactivity between mugwort pollen and fennel was checked by cross-inhibition experiments. Fennel and mugwort allergens selected on the basis of IgE reactivity and inhibition tests were excised from SDS-PAGE gels and microsequenced. The amino acid sequences obtained were used to screen the NCBI database using the protein BLAST software. RESULTS: On ELISA inhibition experiments, serum absorption with fennel extract completely inhibited the IgE response to mugwort. On immmunoblot analysis periodate treatment caused the disappearance of all bands of IgE reactivity except one at about 60 kDa. The 60 kDa bands from both mugwort and fennel PAGE-SDS gels revealed the presence of distinct proteins. The N-terminal amino acid sequencing gave the same major amino acid sequence corresponding to an Api g 5-like allergen. The MS/MS spectra were analyzed and a provided evidence of a fennel-specific protein with sequence similarity to phosphoglyceromutase from Apium graveolens. CONCLUSION: A 60 kDa allergen, highly homologous to Api g 5, was recognized in fennel by patient's IgE. Inhibition experiments showed a high degree of cross-reactivity between this fennel allergen and the homologous mugwort pollen allergen. This allergen might be responsible for the mugwort-celery-spice syndrome.

Heterogenity of IgE response to walnut and hazelnut in Italian allergic patients.

BACKGROUND: The prevalence of IgE reactivity against genuine walnut and hazelnut allergens is poorly defined. OBJECTIVE: The IgE response to walnut and hazelnut was investigated in Italian patients with primary allergy to these nuts. METHODS: Sera from 36 patients allergic to hazelnut and/or walnut, not reactive to PR-10, profilin, and LTP, underwent immunoblot analysis with extracts of both nuts. RESULTS: Most patients had a history of systemic symptoms following the ingestion of the offending food(s). Twelve patients were sensitized to both walnut and hazelnut, and 13 were sensitized to other nuts and seeds (cashew, peanut, sesame, pine nut, almond, Brazil nut, and pistachio). On walnut immunoblot, the 7 sera which scored positive showed much variability in their IgE profile. Two reacted uniquely at 10 kDa, and the others at 35 , 40, 45, 50, 67, and > 67 kDa. The profiles obtained under reducing and non-reducing conditions showed several differences. The 7 sera positive on hazelnut immunoblot under reducing conditions recognized sera at 10 kDa and at <10 kDa (n=1), 20 kDa (n=4), at about 22, 24, 30, 40, 43, 58, 60, and 90 kDa, and higher m.w. in other cases. Under non-reducing conditions IgE reactivity at 20, 28, 35, 40, 45, 60, 90, and 100 kDa, was detected. Only two sera scored positive under both conditions and showed an IgE profile that partly changed from one assay to another. CONCLUSION: The current list of walnut and hazelnut allergens is far from being complete. Both reducing and non-reducing conditions are needed to detect IgE reactivity in individual patients.

Allergy to beer in LTP-sensitized patients: beers are not all the same.

BACKGROUND: Most cases of beer allergy reported so far have been associated with hypersensitivity to the non-specific lipid transfer protein (LTP). In view of the marked differences in brewing processes we assessed IgE reactivity as well as tolerance to many different beers in an allergic patient. METHODS: A 45 year-old man hypersensitive to grass pollen, cat dander and Alternaria tenuis with a history of urticaria and dyspnoea after drinking beer and a weak skin reactivity to commercial corn extract was studied. The patient underwent SPT with 36 different brands of beer and an open challenge with those scoring negative was performed. An immunoblot analysis was carried out using 2 SPT-positive beers, 2 SPT-negative beers, and barley, wheat, and maize extracts using both patient's serum and a maize LTP-specific in-house developed polyclonal antibody from rabbit. Further, the immune reactive LTP of one beer was separated by HPLC and the chromatogram was compared to that of purified maize LTP. RESULTS: Beer SPT scored positive in 30/36 cases. The immunoblot analysis showed IgE reactivity at about 10 kDa against the two SPT-positive beers and against maize with both patient's serum and the polyclonal anti-LTP rabbit serum, whereas the two SPT-negative beers, and barley extract scored negative. The immunodetected protein co-migrated with maize LTP. CONCLUSION: In beer-allergic patients the diagnostic workup may point to the detection of some tolerated products that can be consumed risk-free.

Shrimp allergy in Italian adults: a multicenter study showing a high prevalence of sensitivity to novel high molecular weight allergens.

BACKGROUND: Shrimp is a frequent cause of food allergy worldwide. Besides tropomyosin, several allergens have been described recently. OBJECTIVE: We investigated which allergens are involved in Italian shrimp-allergic adults. METHODS: Sera from 116 shrimp-allergic patients selected in 14 Italian allergy centers were studied. Skin prick tests with house dust mite (HDM) as well as measurements of IgE to Pen a 1 (shrimp tropomyosin) and whole shrimp extract were performed. All sera underwent shrimp immunoblot analysis, and inhibition experiments using HDM extract as inhibitor were carried out on some Pen a 1-negative sera. RESULTS: Immunoblots showed much variability. IgE reactivity at about 30 kDa (tropomyosin) was found in <50% of cases, and reactivity at about 67 kDa and >90 kDa was frequent. Further reactivities at 14-18, 25, 43-50, about 60 and about 80 kDa were detected. Most subjects had a history of shrimp-induced systemic symptoms irrespective of the relevant allergen protein. IgE to Pen a 1 were detected in sera from 46 (41%) patients. Skin reactivity to HDM was found in 43/61 (70%) Pen 1-negative subjects and inhibition studies showed that pre-adsorption of sera with HDM extract induced a marked weakening of the signal at >67 kDa. CONCLUSIONS: Several allergens other than tropomyosin are involved in shrimp allergy in adult Italian patients. Some hitherto not described high molecular weight allergens seem particularly relevant in this population and their cross-reactivity with HDM allergens makes them novel potential panallergens of invertebrates.

Dose-dependent clinical and immunological efficacy of sublingual immunotherapy with mite monomeric allergoid.

Sublingual immunotherapy with monomeric carbamylated allergoid (LAIS) is an effective and well tolerated treatment of respiratory allergy. The aim of the present study was to correlate the efficacy of two maintenance doses (1000 AU vs 3000 AU) of LAIS with the immunological modulation of allergen-driven Th1, Th2 and T regulatory cytokines produced in vitro by PBMCs, in patients suffering from mite allergic rhinitis. Forty-eight consecutive patients with mite allergic rhinitis were recruited. Patients were randomly assigned to group A (n=24) or group B (n=24), respectively receiving 1000 AU or 3000 AU weekly during one-year maintenance phase. Each patient was evaluated for rhinitis severity (ARIA protocol), and for drug consumption at the time of the inclusion and after 6 and 12 months of treatment. Patients were also asked to report the perceived severity of the disease and the tolerability of the treatment in a visual analogical scale (VAS). Before and at the end of the treatment allergen-driven release of cytokines by PBMCs in vitro was measured. After 1-year treatment, a statistically significant reduction of all clinical parameters was observed in all patients, associated with reduction of IL-4 and increase of INF-γ secreted in vitro by mite-challenged PBMCs. Notably, the group treated with the higher dose showed significantly better clinical and immunological results. The efficacy of LAIS is correlated to the immune modulation in a clear dose-dependent effect.