Enzymatic methods for the evaluation of the allergenic potency of house dust extracts.

House dust extracts are known to contain powerful proteolytic enzymes. The significance of these proteases is supported by the recently reported association of major dust mite allergens with serine and/or cysteine proteases. The proteolytic activity of house dust extracts, therefore, represents an allergologically relevant parameter for mite-derived allergens. However, several other major allergens in extracts of animal and insect excreta (e.g., from cockroaches) have been shown to possess protein hydrolase properties. Furthermore, acid phosphatase activities are known to correlate well with allergenic potency in several pollen extracts. The present study reports on the quantitative estimation of these two enzymes in relation to human antimite IgE-binding potency in 20 different dust samples from Spanish houses.

Estimation of IgE antibodies to the common allergens by reverse enzyme immunoassay. Comparison with the radioallergosorbent test.

A reverse enzyme immunoassay (REINA) is described, in which polystyrene microtiter wells are sensitized with murine monoclonal anti-human IgE, and then sequentially allowed to react with patient's serum, peroxidase-labeled allergens and substrate. The results obtained with the sera of patients allergic to Lolium perenne grass pollen, the tree pollens of Betula alba and Olea europea, the epithelia of cat and dog, the mite Dermatophagoides pteronyssinus, or to the foodstuffs cow's milk, chicken eggwhite or peanut were compared with the analytical data from the ratio allergosorbent test (RAST). The results show a good correlation between these two laboratory techniques.

Asthma from inhalation of Triplochiton scleroxylon (Samba) wood dust.

The inhalation of wood dust may produce allergic rhinitis, asthma, or contact dermatitis in sensitized patients. We describe a patient with occupational asthma induced by the inhalation of samba (Triplochiton scleroxylon) wood dust. A specific bronchial provocation test was conducted, and the patient showed a significant decrease in forced-expiratory volume in first second (FEV1) after the inhalation of nebulized samba wood allergens. We suggest an IgE-mediated mechanism since the patient had a positive skin test and positive specific IgE determinations using an extract of samba wood. SDS-PAGE immunoblots revealed IgE binding to proteins with molecular weights of 17 kDa, 28 kDa and less intense binding to a band with an approximate molecular weight of 60 kDa. Two asymptomatic skin test-negative subjects, also occupationally exposed to samba, did not show any signs of bronchoconstriction when challenged with the samba wood extracts. We conclude that the occupational asthma suffered by this patient is related to sensitization and occupational exposure to samba wood dust.

Double-blind, placebo-controlled clinical trial of preseasonal treatment with allergenic extracts of Olea europaea pollen administered sublingually.

In a double-blind, placebo-controlled, pilot clinical trial we evaluated the clinical efficacy and safety of immunotherapy (IT) with an extract of the pollen of the tree Olea europaea administered sublingually. The parameters tested were symptom score, dose-response bioassay of skin prick test and specific IgE and IgG, and the absolute value at a single serum dilution of each IgG subclass. Fifteen patients allergic to this pollen with symptomatology of rhinitis and/or rhinoconjunctivitis were randomly allocated to the placebo group (6 patients) or to the extract group (9 patients). Immunotherapy was administered in a short preseasonal period of time, practically no side effects being recorded. The group of patients treated with extract presented a slightly lower incidence (0.05 < p < 0.1) of nasal symptoms of sneezing and obstruction, and, more importantly, developed less dyspnea (p < 0.05) than the group treated with placebo, suggesting that IT can act as prophylaxis for the development of bronchial symptoms. No differences were observed in the immunological determinations. Differences in skin tests between the two groups displayed a slight significance (0.05 < p < 0.1) at the end of the trial; hence, a higher concentration of the allergen was needed in the group treated with extract to induce the same wheal as in the placebo group. In both groups the size of the wheal showed a time-dependent variation, which was dependent on the time of the year and independent of the type of treatment received, indicating a significant modification in the in vivo skin response to allergen challenge, demonstrated by a shift in the kinetics of allergen-ligand binding (slope) and in the magnitude of the measured response (intercept).

[Difference between preeclampsia, HELLP syndrome and eclampsia, maternal evaluation]. / Diferencias entre preeclampsia, síndrome de Hellp y eclampsia, evaluación materna.

120 cases of severe preeclampsia without HELLP Syndrome (hemolysis, elevated liver enzymes and low platelets), 120 cases of HELLP Syndrome without eclampsia and 119 eclamptic patients without HELLP were analyzed. The objective was to found differences in the maternal morbidity and mortality. Eclampsia was found in those patients 20 years or less, nulliparous and with 37 o more weeks of gestation. This difference was statistically significant. In the group with severe preeclampsia there was one death and 18 complications. In the group with eclampsia there was 5 deaths and 21 complications. In the group of patients with HELLP Syndrome there was 9 death and 63 complications. HELLP syndrome presented various causes of death. Cerebral hemorrhage was the principal one in eclamptic patients. The main complication in the three groups was acute renal insufficiency and this was presented mainly in HELLP Syndrome patients. In summary, HELLP Syndrome have different pathophysiologic characteristics than severe eclampsia and eclampsia. This fact was showed by the great difference in the maternal morbidity and mortality. We suggest a more aggressive and intensive care of HELLP Syndrome patients.

The chimerical multi-component Q protein from Leishmania in the absence of adjuvant protects dogs against an experimental Leishmania infantum infection.

The protective potential against Leishmania infection of the Leishmania chimerical Q protein administered as a single (Q) or double dose (Q+Q) without adjuvant was analyzed in a double-blind placebo controlled experiment in dogs. During vaccination the protein induced an intense early anti-Q response but no reactivity against total Leishmania infantum proteins was detected. Several end-points were taken into consideration. In the vaccinated animals the amount and intensity of clinical symptoms was lower than in the control group. Pathological signs of disease were observed in liver, kidney and spleen of all dogs from the control group in contrast to the normal appearance of the organs of the vaccinated animals. Vaccination was able to induce parasite clearance in most dogs. Only 1/7 dog was parasite DNA positive in skin in the Q group in contrast to 6/7 dogs in control and 4/7 in Q+Q. Significant anti-SLA clearance was observed in the vaccinated animals at the end of the study. Differences between control and vaccinated animals were also observed at the biochemical level, DTH and nitrite oxide production.

IgE-binding trypsin inhibitors in plant pollen extracts.

In an attempt to access the possible role of protease-antiprotease mechanisms of non-immune defence in pollinosis, only low levels of trypsin-, kallikrein- or plasmin-like proteinases could be detected in aqueous pollen extracts. In contrast, several pollen species displayed appreciable trypsin inhibitory activity, e.g. Parietaria, Olea, Ambrosia, Rumex, Chenopodium, Holcus and Poa spp. These proteins of the serpin family of anti-proteinases were found to bind specific IgE-antibodies from the serum of hay fever patients. As examples, the IgE-binding trypsin inhibitors from the pollen of Parietaria judaica and Ambrosia elatior were purified and characterized as acidic proteins with pI 4.2 and a molecular weight of 20-24 kDa.

Comparison of skin-prick test assay and reverse enzyme immunoassay competition (REINA-C) for biological activity of allergens.

An in vitro technique for measuring the relative potency of allergenic extracts has been developed and compared with the dose-response skin-prick test. The in vitro technique is a modification of an ELISA based on the method of antigen capture using polystyrene microtitre wells as solid phase (REINA-competition) and is also based on a dose-response. Both methods have been applied to allergen extracts of Dermatophagoides pteronyssinus, Phleum pratense, Secale cereale, Lolium perenne, Plantago lanceolata, Artemisia vulgaris, Salsola kali and Parietaria judaica. For each allergen, the slope of the regression log-log dose-response lines displayed by skin-prick test and REINA-C does not show a statistically significant difference, being parallel (P < 0.001), and indicating that the allergen-ligand binding kinetics are identical in both methods.

A competitive enzyme immunoassay Subclass for the determination of total IgG-subclass levels in human serum. Comparison with single radial immunodiffusion.

A quantitative immunoassay has been developed for the determination of the total IgG subclass levels in human serum. The method is based on an enzyme immunoassay in which IgG subclass proteins IgGx in an unknown serum sample compete with a known quantity of peroxidase (PO)-labelled IgGx in fluid phase for subclass-specific monoclonal antibodies coated to the solid phase of microtiter wells. Using a series of human blood samples an excellent correlation was observed with the IgG-subclass levels determined by single radial immunodiffusion.

Characterization of allergens of Anisakis simplex.

BACKGROUND: Anisakis simplex is an intestinal parasite of sea mammals. The larvae infect crustaceans, cephalopods and fish. Humans may consume A. simplex third stage larvae (L3) when eating infected raw or under-cooked fish. Consumed larvae cause an inflammatory reaction when they penetrate the digestive mucosa. The larvae or their secretory/excretory products can sensitize humans and induce an immunoglobulin E (IgE)-mediated allergic reaction. This parasite is now being implicated in numerous cases of allergic reactions after eating fish. The purpose of this study was to evaluate the allergenicity of proteins present in an extract of the third stage larva. METHODS: Rabbit antiserum raised to A. simplex somatic extract (L3) was reacted by crossed immunoelectrophoresis (CIE) with the same somatic extract. Crossed radioimmunoelectrophoresis (CRIE) was also performed by incubating CIE gels first in the sera of 13 individuals with positive immunoCAP to A. simplex and then in radiolabeled anti-human IgE. RESULTS: Twelve to 16 antigen-antibody precipitin peaks were visualized on Coomassie blue stained CIE gels in which somatic extract was reacted with somatic-antiserum. Autoradiography of CRIE gels showed that 18 different proteins bound IgE in patient sera. Individual patients had serum IgE directed at two to 10 different allergens. Five of these allergens were recognized by >/=50% of the patients. No allergen was recognized by every patient and no patient had serum IgE directed at all 18 allergens. CONCLUSION: Somatic extracts of A. simplex L3 larva contain a large number of allergenic molecules and there is significant variability between patients in their sensitivity and reactivity to these allergens.

Comparative study of the complement-activating and specific IgE-binding properties of ragweed pollen allergen.

Previous reports have defined the capacity of ragweed pollen extract (RWA) to activate human complement (C) in fluid phase through the classical pathway and have ascertained a strong correlation between the extent of complement activation and the severity of symptoms of allergic rhinoconjunctivitis during the ragweed blooming season. In the present study the complement-activating and specific IgE-binding capacities of various ragweed allergen preparations were compared. Elimination of physically adsorbed (flavonoid) pigments from the allergenic proteins had no significant effect on their complement-consuming capacity, although the process strongly diminished specific IgE binding. Removal of an IgE-binding trypsin inhibitor from RWA significantly enhanced RWA-induced complement activation, whereas it did not change IgE binding. These findings indicate that neither the physically adsorbed pigments nor the trypsin inhibitor are involved in complement activation by ragweed pollen allergens, and suggest that complement activation and specific IgE binding are distinct molecular properties of ragweed pollen allergen.

Allergenicity and cross-reactivity of Russian olive pollen (Eleagnus angustifolia).

BACKGROUND: The purposes of this study were: to determine the prevalence of sensitization and immunochemical characterization of Eleagnus angustifolia pollen (Russian olive) that belongs to the family Eleagnaceae. METHODS: A total of 134 patients with rhinoconjunctivitis and/or asthma were studied. Its allergenicity, cross-reactivity with olive pollen and the presence of Ole e 1 and Ole e 4-like molecules were evaluated. RESULTS: Eleagnus angustifolia pollen was detected from May to June. Seventy-three of 134 (30.5%) had positive skin test to E. angustifolia, all of them were positive to olive. There was a good correlation between specific immunoglobulin (Ig)E levels to E. angustifolia and Olea europaea (r = 0.77, P = 0.002). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) immunoblots revealed major IgE-binding bands in the E. angustifolia extract of 43 and 63.7 kDa. The E. angustifolia extract was not able to inhibit olive, whereas O. europaea inhibited E. angustifolia up to 41%. The presence of Ole e 1- and Ole e 4-like allergens in E. angustifolia extract was confirmed by enzyme-linked immunosorbent (ELISA) inhibition assays. Nasal challenge with E. angustifolia was positive in three of six patients with positive skin test to both pollens and negative in five patients with positive skin test only to O. europaea. CONCLUSIONS: This study confirms that E. angustifolia is capable of sensitizing individuals in Madrid. A minimal-to-moderate cross-reactivity with olive pollen was established, suggesting some cross-reactivity but not excluding co-sensitization.

Molecular cloning of paramyosin, a new allergen of Anisakis simplex.

BACKGROUND: Anisakis simplex is a fish parasite that, when accidentally ingested by humans, may cause allergic reactions in sensitized individuals. The main objectives of our study were to: (1) construct a cDNA expression library of A. simplex; (2) identify clones producing specific IgE binding protein antigens, and (3) produce and purify the protein/s codified by the isolated clones produced in Escherichia coli. METHODS: An expression cDNA library from the third stage larvae (L3) of A. simplex was constructed. This library was first screened with a rabbit anti A. simplex hyperimmune serum. The positive clones, identified using the rabbit serum, were rescreened with a pool of human sera containing high titers of IgE antibodies against A. simplex. RESULTS: Two positive clones were isolated carrying the genes which codify for paramyosin. The paramyosin protein was produced in E. coli and purified. The partial sequence of a second paramyosin gene was also identified. The frequency of specific IgE binding to the recombinant and native forms of paramyosin using the sera of 26 A. simplex-sensitive individuals was 23 and 88%, respectively. Both paramyosins were able to inhibit 11% of the specific IgE binding to a total extract. CONCLUSIONS: We describe the primary structure of a paramyosin of A. simplex. It can be considered as an allergen based on its IgE binding capacity. We suggest that the recombinant protein does not maintain the complete allergenic properties of the native paramyosin, considering its lower IgE binding capacity of the recombinant protein. However, both proteins have the same specific IgE inhibition capacity. The recombinant protein can be produced in large quantities in E. coli. We propose the term Ani s 2 for this allergen.

Allergy to goat and sheep cheese with good tolerance to cow cheese.

BACKGROUND: We report on a patient who experienced allergic reactions after eating goat cheese and after touching goat and sheep cheese, but not after consuming cow's milk dairy products. OBJECTIVE: To assess the allergenicity and IgE-binding capacity of the caseins from the three different species. METHODS: Skin prick tests were carried out using whole milk and caseins from three different species (goat, sheep and cow), and whey fractions of cow's milk. Total serum IgE and specific IgE to cow's milk proteins were measured by CAP system and specific IgE against caseins and whole milk were determined by ELISA technique. To evaluate allergenic cross-reactivity, inhibition of the IgE ELISA activity to goat's milk and goat casein was tested for the three caseins. SDS-PAGE and immunoblotting was used to determine IgE binding bands in caseins. RESULTS: Skin tests were positive to sheep and goat's milk, sheep and goat casein, as well as to sheep and goat cheese. Total serum IgE was 66 kU/L and IgE determinations by CAP were negative. IgE ELISA against the caseins from goat and sheep was strongly positive, whereas it was negative to cow casein. ELISA inhibition assays revealed a high degree of cross-reactivity between goat casein and sheep casein. Immunoblotting showed three IgE-binding bands in goat casein at 31, 27 and 22 kDa, which may correspond to alpha-, beta- and gamma-caseins. A band at about 31 kDa was observed in sheep casein and another band at 34 kDa was recognized in cow casein. CONCLUSION: This patient developed allergy to goat and sheep cheese with good tolerance to cow's milk. We identified goat casein as the main allergen causing sensitization in this patient as demonstrated by in vivo and in vitro tests. A high degree of cross-reactivity between goat and sheep casein was observed.

Chicken serum albumin (Gal d 5*) is a partially heat-labile inhalant and food allergen implicated in the bird-egg syndrome.

BACKGROUND: Chicken serum albumin (alpha-livetin) has been implicated as the causative allergen of the bird-egg syndrome. However, the clinical relevance of sensitization to this allergen has not been confirmed by specific challenge tests and environmental sampling. We investigated whether chicken albumin can be detected in air samples collected in a home with birds, and whether sensitization to this protein may cause respiratory and food allergy symptoms. The heat resistance of chicken albumin and the possible cross-reactivity with conalbumin were also investigated. METHODS: We studied eight patients with food allergy to egg yolk who also suffered from respiratory symptoms (rhinitis and/or asthma) caused by exposure to birds. Sensitization to egg yolk and bird antigens was investigated by skin and serologic tests. Hypersensitivity to chicken albumin was confirmed by specific bronchial, conjunctival, and oral provocation tests. RESULTS: All patients had positive skin tests and serum IgE against egg yolk, chicken serum, chicken meat, bird feathers, and chicken albumin. The presence of airborne chicken albumin in the domestic environment was confirmed. Specific bronchial challenge to chicken albumin elicited early asthmatic responses in six patients with asthma. An oral challenge with chicken albumin provoked digestive and systemic allergic symptoms in the two patients challenged. IgE reactivity to chicken albumin was reduced by 88% after heating at 90 degrees C for 30 min. ELISA inhibition demonstrated only partial cross-reactivity between chicken albumin and conalbumin. CONCLUSION: Chicken albumin (Gal d 5) is a partially heat-labile allergen that may cause both respiratory and food-allergy symptoms in patients with the bird-egg syndrome.

Sensitization to Olea europaea: geographical differences and discrepancies.

Thirty eight patients from two geographical areas of Spain, with great differences in Olea europaea pollen counts were studied to investigate their in vivo and in vitro immune response to this pollen as a consequence of their different environmental allergen exposure. They were distributed in two groups (13 from Madrid, and 25 from Jaén). Skin sensitivity was assessed by a prick-test dose-response bioassay using serial dilutions of a biologically standardized allergen extract of O. europaea. Serological immune response was evaluated measuring specific antibody levels (IgE, IgG, IgG1 and IgG4). The patients from Jaén, who have a higher exposure to olive pollen, had higher levels of specific antibodies but significantly smaller wheal sizes than a similar patient population form the Madrid area, where olive pollen is not so copious. There is a great discrepancy between the results of skin prick tests (low cutaneous reactivity associated with high allergenic environmental load) and the levels of specific IgE to the olive pollen. While the level of specific antibodies increases with the allergenic load, the capacity to release mediators seems to be decreased, at least in the skin. Further studies are needed to evaluate if these findings also occur in other target organs with appropriate challenge tests (conjunctival, nasal and bronchial). This pattern should be studied with other allergens in large patient populations.

A double-blind, placebo-controlled oral challenge study with lyophilized larvae and antigen of the fish parasite, Anisakis simplex.

BACKGROUND: The third-stage larvae of Anisakis simplex may be a hidden source of allergens in fish. The objective was to determine whether the ingestion of lyophilized A. simplex larvae, or antigen, induces clinical symptoms in a group of A. simplex-sensitized patients. METHODS: Double-blind, placebo-controlled oral challenges were conducted in 11 individuals who had experienced allergic reactions after eating fish. Another patient had chronic urticaria unrelated to the ingestion of fish. All patients had positive skin tests and specific IgE determinations for A. simplex and negative skin tests to a battery of fish species. Conjunctival tests with A. simplex extracts were conducted in all patients and in five controls. The 12 patients received capsules containing either lactose or one, five, or 25 lyophilized larvae of A. simplex at 2-h intervals in a double-blind fashion. The highest single dose was 100 larvae. ECP and tryptase levels in serum were measured before and after the last oral challenge. Lyophilized antigen was also given to five patients. RESULTS: None of the 12 patients experienced a positive reaction after the ingestion of the placebo, the lyophilized larvae, or the antigen. Tryptase and ECP levels before and after challenges did not change significantly. Conjunctival provocation tests were positive in 11 out of the 12 patients and in none of the controls. CONCLUSIONS: The ingestion of 100 lyophilized A. simplex larvae, or its equivalent in antigen, does not induce clinical symptoms in individuals with a clinical history and laboratory findings of hypersensitivity to A. simplex. The data suggest that only the ingestion of live larvae may be capable of inducing allergic manifestations.

The diagnostic value of crude or boiled extracts to identify tolerant versus nontolerant lentil-sensitive children.

OBJECTIVE: The aim of this study was to compare two types of lentil extracts for use in skin prick tests for the diagnosis of lentil clinical allergy. METHODS: Thirty-six patients with a history of allergic reactions after the ingestion of lentils were skin tested with two types of lentil extracts at 0.05, 0.5, 5, and 10 mg/mL. Both extracts were extracted at 40 degrees C and afterward, one of them was boiled for 15 minutes. Thirty-three of these patients underwent oral challenges with lentils and three had a convincing recent history of lentil anaphylaxis. RESULTS: Twenty patients had a positive oral challenge; 13 were negative. Skin prick tests performed with the boiled extract at 0.5 and 5 mg/mL were positive in 96% and 100% of patients with positive food challenge, and in 31% and 85% of those with negative food challenge, respectively; positive skin test results were similar in both groups using the crude extract. Mean wheal sizes using the boiled extract at 0.5, 5, and 10 mg/mL were significantly greater in patients with a positive oral challenge than in those with a negative one (4.9, 6.8, and 7.4 mm versus 1.9, 3.5, and 5.1 mm, respectively; P < 0.05) These mean values were not statistically different using the crude extract. CONCLUSIONS: These data suggest that lentil extracts for the diagnosis of lentil hypersensitivity should be heated, since boiled extracts, used at a concentration of 0.5 or 5 mg/mL, best identify clinically sensitive individuals.