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.

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.

Shrimp allergy: analysis of commercially available extracts for in vivo diagnosis

Background: 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 componentresolved diagnosis largely insufficient. Mites and crustaceans probably share several allergens other than tropomyosin (AU)

Introducción: Las pruebas cutáneas con extractos comerciales representan el primer paso en el diagnóstico de alergia a gamba, si bien, su eficacia clínica no está bien definida. Objetivos: El objetivo de este estudio fue analizar la utilidad clínica de todos los extractos comerciales disponibles en Italia frente a crustáceos en pruebas cutáneas. Métodos: En un estudio multicéntrico, se incluyeron 157 pacientes alérgicos a gamba a los que se realizaron pruebas cutáneas con cinco extractos comerciales de crustáceos y con ácaros del polvo doméstico. Los extractos comerciales fueron analizados mediante SDS-PAGE y comparados con un extracto de gamba preparado en fresco. Se determinó IgE frente a Pen a 1/Pen m 1; Pen m 2, y Pen m 4; y el análisis mediante inmunoblotting se realizó en un amplio número de sueros. Resultados: Los extractos de gamba comercializados dieron lugar a reacciones cutáneas muy poco homogéneas en 32 perfiles clínicos diferentes; así mismo, mostraron grandes diferencias en contenido proteico y, en algunos casos, a falta de proteína a pesos moleculares correspondientes a alérgenos mayoritarios de gamba. Únicamente los reactores más fuertes a Pen a1 /Pen m 1 reaccionaron tanto a ácaros del polvo de casa como a los cinco extractos comerciales en pruebas cutáneas. La mayoría de los pacientes, incluyendo los negativos a tropomiosina, reaccionaron a los ácaros del polvo. Los pacientes reaccionaron a un amplio y variable array de proteínas y se detectó con frecuencia reactividad de IgE en pesos moleculares altos (>50 kDa). Conclusiones: El diagnóstico in vivo de alergia a gamba todavía debe estar basado en pruebas cutáneas prick con producto fresco. Los pacientes alérgicos a gamba a menudo reaccionan a un número de alérgenos de peso molecular alto poco definido, lo que hace que las moléculas disponibles hoy en día para el diagnóstico por componentes sean muy insuficiente. Ácaros y crustáceos probablemente comparten varios alérgenos además de la tropiomiosina (AU)

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.

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 (AU)

Antecedentes: Las polcalcinas son panalérgenos de alta reactividad cruzada en pólenes capaces de sensibilizar a un 10% de los pacientes alérgicos. Todas las especies de pólenes se consideran capaces de sensibilizar pacientes mediante este panalérgeno. Objetivo: El objetivo de este trabajo fue analizar la presencia de esta polcalcina en diferentes extractos de pólenes que se utilizan en inmunoterapia. Método: El suero de pacientes reactores a polcalcina, así como frente a rPhl p 7 y a rBet v 4 fue analizado mediante ensayo de ELISA inhibición, utilizando polcalcina recombinante como sustrato y extracto de pólenes como inhibidores. Resultados: En cuanto a los resultados obtenidos, todos los extractos de pólenes indujeron una inhibición significativa de la reactividad de la IgE frente a rBet v 4, mientras que solo el extracto de polen de gramíneas inducía una marcada inhibición de la reactividad de la IgE frente a rPhl p 7. Conclusión: La polcalcina de gramíneas probablemente contiene más epítopes que las polcalcinas de otras fuentes. El polen de gramíneas podría ser responsable de la sensibilización a la polcalcina y la inmunoterapia con polen de gramíneas es probablemente una opción para los pacientes hipersensibles a polcalcina (AU)

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.

Monosensitization to a novel plane pollen allergen.

A patient monosensitized to plane pollen is described On ISAC 103 the patients showed IgE reactivity to both Pla a 1 and Pla a 2, but on immunoblot analysis the serum showed a single IgE reactivity at about 50 kDa allergen.

Clinical manifestations, co-sensitizations, and immunoblotting profiles of buckwheat-allergic patients.

BACKGROUND: Buckwheat allergy is a rare food allergy in Europe and North America, whereas it is often described and studied in Asia. The aim of this study was to describe a series of patients with proven buckwheat allergy evaluated in an Italian allergy clinic. Co-sensitization to other food and inhalant allergens and immunoblotting profiles of buckwheat-allergic patients were studied. METHODS: Patients with suspected buckwheat allergy who attended the allergy clinic between January 1, 2006, and September 30, 2008, were evaluated. All patients underwent skin prick tests for a standard panel of inhalant and food allergens, prick-by-prick with buckwheat flour, buckwheat-specific IgE determinations, and double-blind placebo-controlled food challenge (DBPCFC) with buckwheat flour. Immunoblotting with buckwheat flour extract was performed on sera from buckwheat-allergic patients. RESULTS: Among 72 patients with suspected buckwheat allergy, 30 (41.7%) were sensitized to buckwheat and 24 had a positive DBPCFC. The mean buckwheat IgE level was 6.23 kUA/l (range, 0.16 to >100 kUA/l). Several IgE-binding proteins were identified and grouped into three patterns: a 16-kDa band in patients with predominantly gastrointestinal symptoms with grass and wheat flour co-sensitization, a 25-kDa band in patients with predominantly cutaneous symptoms and a low frequency of co-sensitization, and a 40-kDa band in patients with anaphylaxis and a low frequency of co-sensitization. CONCLUSIONS: Buckwheat allergy is an emerging food allergy in Italy. We identified three distinct patterns of clinical and laboratory characteristics, suggesting that specific allergens could be more frequently associated with clinical manifestations of different severity.

Detection of a novel allergen in raw tomato.

The study reports a case of "pure" tomato allergy in an adult female. The responsible allergen was partially characterized by immunoblot analysis, pepsin digestion, and heating. It had a molecular weight of about 9 kDa and was heat-labile and pepsin-resistant, thus confirming the clinical history. Unfortunately, due to the failure of both 2-dimensional electrophoresis analysis and N-terminal sequencing experiments, it was not possible to characterize the protein further. Based on a comparison with currently known tomato allergens, this seems to be a novel allergen protein.

PT with heat-processed apple peel extract to detect LTP hypersensitivity.

BACKGROUND: Allergen-resolved diagnosis of food allergy may be essential in the clinical practice, particularly in patients allergic to foods that may contain both labile and stable allergens. However, presently available diagnostic tests are not useful in this sense. OBJECTIVE: To assess the clinical usefulness of SPT with a heat-processed apple peel extract as an easily available means to detect hypersensitivity to lipid transfer protein (LTP), an extremely stable and potentially hazardous apple allergen. METHODS: Raw and heat-processed (100 degrees C x 10 min) apple peel extract (100 microg/ml) were used to carry out SPT in 23 patients with apple-allergy, 15 of which considered as probably sensitized to labile allergens (Maid 1, Mal d 4) and 8 to stable allergens (Mal d 3, LTP), respectively on the basis of the presence/absence of IgE reactivity to birch pollen. IgE reactivity to the heat-processed apple peel extract was further analyzed by immunoblot. RESULTS: Altogether SPT with raw apple extract scored positive in 20/23 (87%) patients, including 12/15 patients considered as probably sensitized to labile allergens and 8/8 patients considered as probably sensitized to LTP. In contrast, the heat-processed apple extract induced a wheal-and-flare reaction only in the 8 (100%) presumptive LTP reactors. Immunoblot analysis showed IgE reactivity to a 10 kDa protein (LTP) in heat-processed apple extract. CONCLUSION: The heat-resistance of stable apple allergens like LTP can be usefully exploited to prepare extracts for allergen-resolved diagnosis in-vivo.

Biological treatments affect the chemical composition of coffee pulp.

Biological treatments were applied to fresh coffee pulp (CoP) to improve its nutritive value for monogastric animals by reducing its content of cellulose and antinutritional factors (ANFs) such as total phenols, tannins and caffeine. Treatments were: (1) ensiling with 0, 50 and 100 gkg(-1) molasses for 2 and 3 months, (2) aerobic decomposition for 0, 7, 14, 21, 28, 35 and 42 days, (3) aerobic bacterial inoculation (Bacillus sp.) for 0, 7, 14, 21 and 28 days. Ensiled CoP (E-CoP) showed higher fat and ash contents than oven-dried-CoP (OD-CoP; P<0.05). Similarly, true protein values tended to increase. The cellulose and total phenols levels of E-CoP were lower than OD-CoP (P<0.05). The E-CoP tannins levels tended to be lower than OD-CoP whereas caffeine levels remained unaffected. Improvement in the nutritional quality of E-CoP was associated with higher fat and protein contents and reduction of cellulose, total phenols and tannins. The aerobic decomposition treatment improved the nutritional quality of CoP by increasing true protein and fat contents. In addition, total phenols, tannins, caffeine and cellulose contents were reduced by an increase in treatment time (P<0.05). Bacterial treatment increased the protein content of CoP after 21 days (from 137 to 392 gkg(-1)) and decreased it after 28 days. Cellulose, total phenols, tannins and caffeine contents reduced with an increase in time of bacterial degradation. Bacterial treatment improved the CoP quality by increasing protein content and reducing cellulose and ANFs, especially after 21 days of treatment. Both the aerobic decomposition (after 21-28 days) and the aerobic bacterial degradation of CoP (after 21 days) appeared more suitable to improve the nutritional quality of CoP than the ensiling.

Allergenic relevance of Cupressus arizonica pollen extract and biological characterization of the allergoid.

BACKGROUND: Cupressaceae (cypress) pollens can cause pollinosis in winter. However, the lack of specific commercial extracts combined with the early pollination period of cypress trees make a precise diagnosis difficult. The need for a reliable and effective cypress extract for diagnostic and therapeutic purposes is increasingly felt. METHODS: Mixed or single Cupressus arizonica, lusitanica and sempervirens pollen extracts precipitated with ammonium sulfate (PPT) were compared by direct RAST, RAST inhibition and SDS-PAGE techniques. The major allergen of C. arizonica (Cup a 1), purified by anion exchange chromatography, was checked by immunoblotting experiments before chemical modification, in parallel with a C. arizonica extract, with potassium cyanate (KCNO) to obtain a monomeric allergoid. The allergoid extract was characterized for its biological, chemico-physical and immunological features by RAST inhibition, SDS-PAGE and ELISA assays. RESULTS: Direct RAST, RAST inhibition, and SDS-PAGE data indicated that the PPT C. arizonica pollen extract showed the most allergenic potential, and it can be considered representative of the Cupressus spp. Immunoblotting data confirmed Cup a 1 as a major allergen. RAST inhibition and ELISA showed that modified PPT C. arizonica extract had less IgE reactivity than the native, non-modified extract, while preserving the immunogenic capacity typical for an allergoid. Finally, the SDS-PAGE profile of Cup a 1 allergoid was similar to native Cup a 1 allergen, suggesting the modified C. arizonica extract shows the characteristics of a monomeric allergoid. CONCLUSIONS: The PPT C. arizonica pollen extract shows good in vitro diagnostic potential and its chemically modified form offers the features of a monomeric allergoid. It might therefore lend itself to the development of a product to be administered by the sublingual or oromucosal route for immunotherapy of individuals with cypress pollinosis.

Prognostic significance of pathological response of primary tumor and metastatic axillary lymph nodes after neoadjuvant chemotherapy for locally advanced breast carcinoma.

PURPOSE: The prognostic significance of pathological response of primary tumor and metastatic axillary lymph nodes after neoadjuvant chemotherapy was assessed in patients with noninflammatory locally advanced breast carcinoma. PATIENTS AND METHODS: Between January 1989 and April 1995, 148 consecutive patients with locally advanced breast carcinoma participated in the study. Of these, 140 fully evaluable patients (67, stage IIIA; 73, stage IIIB) were treated with three courses of 5-fluorouracil, doxorubicin, and cyclophosphamide (FAC), followed by modified radical mastectomy when technically feasible or definitive radiation therapy. The median age was 53 years (range, 26 to 75 years); 55% of patients were postmenopausal. RESULTS: Objective response was recorded in 99 of 140 patients (71%; 95% confidence interval, 63% to 79%). Complete response occurred in 11 patients (8%), and partial response occurred in 88 patients (63%). No change was recorded in 37 patients (26%), and progressive disease occurred in 4 patients (3%). One hundred and thirty-six patients underwent the planned surgery. Maximal pathological response of the primary tumor (in situ carcinoma or minimal microscopic residual tumor) was observed in 24 (18%); 112 patients (82%) presented minimal pathological response of the primary tumor (gross residual tumor). The number of metastatic axillary nodes after neoadjuvant chemotherapy was as follows: N0, 39 patients (29%); N1-N3, 35 patients (26%); > N3, 62 patients (45%). Considering the initial TNM status, 75% of the patients had decreases in tumor compartment after neoadjuvant chemotherapy. Also, 31% and 23% of patients with clinical N1 and N2, respectively, showed uninvolved axillary lymph nodes. A significant correlation was noted between pathological response of primary tumor and the number of metastatic axillary lymph nodes. Median disease-free survival was 34 months, whereas median overall survival was 66 months. Pathological responses of both primary tumor and metastatic axillary lymph nodes were strongly correlated with disease-free survival and overall survival in univariate analyses. Additionally, in a proportional hazard regression model and in an accelerated failure time model, metastatic axillary lymph nodes significantly influenced both disease-free survival and overall survival, whereas pathological response of primary tumor did so on disease-free survival only. CONCLUSION: After neoadjuvant chemotherapy, pathological responses of both primary tumor and metastatic axillary lymph nodes had a marked prognostic significance and influenced outcome for patients with locally advanced breast carcinoma. Our results suggest that maximal tumor shrinkage and sterilization of potentially involved axillary nodes may represent a major goal of neoadjuvant chemotherapy. Further studies are warranted to clarify whether these results reflect the therapeutic effect or intrinsic biologic factors of the tumor.

Double modulation of 5-fluorouracil by methotrexate and high-dose L-leucovorin in advanced colorectal cancer.

A phase II trial was performed to evaluate the efficacy and toxicity of a double modulation of 5-fluorouracil (5-FU) by methotrexate (MTX) and L-leucovorin (L-LV) in patients with advanced recurrent (inoperable) or metastatic colorectal carcinoma (ACC). Between July 1993 and October 1995, 41 patients with ACC received a regimen that consisted of MTX 150 mg/m2 i.v., infused over a 20-minute period at hour 0, followed 19 hours later by L-LV 250 mg/m2 in a 2-hour i.v. infusion. 5-FU, 900 mg/m2, was administered by i.v. push injection at hour 20. Beginning 24 hours after MTX administration, all patients received four doses of L-LV, 15 mg/m2 i.m., every 6 hours. Cycles were repeated every 15 days. Two patients were not assessable for response. Objective regression was observed in 11 of 39 (28%) patients, [95% confidence interval (CI), 14-42%]. One (2%) patient achieved complete response (CR) and 10 (26%) partial response (PR). No change was recorded in 15 (39%) patients and progressive disease was noted in 13 (33%) patients. The median time to treatment failure was 6 months and the median survival time was 10 months. Toxicity was within acceptable limits, but one therapy-related death due to severe leukopenia was observed. The dose-limiting toxicity was mucositis. Eight episodes of grade 3 or 4 stomatitis were observed, and were responsible for dosage modifications of MTX and 5-FU. In conclusion, further in experimental and clinical studies are clearly necessary in order to design the best modulatory strategy of 5-FU.

Use of azithromycin for the treatment of Campylobacter enteritis in travelers to Thailand, an area where ciprofloxacin resistance is prevalent.

We evaluated the use of azithromycin (500 mg) or ciprofloxacin (500 mg) daily for 3 days for the treatment of acute diarrhea among United States military personnel in Thailand. Stool cultures were obtained and symptoms were recorded on study days 0, 1, 2, 3, and 10. Campylobacter species were the most common pathogen isolated (44 isolates from 42 patients). All Campylobacter isolates were susceptible to azithromycin; 22 were resistant to ciprofloxacin. Among the 42 patients with campylobacter infection, there were 2 clinical and 6 bacteriologic treatment failures in the ciprofloxacin group and no treatment failures in the azithromycin group (P = .021 for bacteriologic failures). Overall, azithromycin was as effective as ciprofloxacin in decreasing the duration of illness (36.9 hours vs. 38.2 hours, respectively) and the number of stools (6.4 vs. 7.8, respectively). Among those not infected with Campylobacter species (n = 30), the duration of illness was 32.9 hours vs. 20.7 hours (P = .03) for the azithromycin and ciprofloxacin groups, respectively. Azithromycin is superior to ciprofloxacin in decreasing the excretion of Campylobacter species and as effective as ciprofloxacin in shortening the duration of illness. Azithromycin therapy may be an effective alternative to ciprofloxacin therapy in areas where ciprofloxacin-resistant Campylobacter species are prevalent.