Heterogeneity of antibody responses among clinical responders during grass pollen sublingual immunotherapy.

BACKGROUND: During allergen-specific sublingual immunotherapy (SLIT), the relevance of changes in specific IgE and IgG antibody titres to treatment efficacy remains to be evaluated at an individual patient level. OBJECTIVE: To investigate whether antibody responses can be used as biomarkers for SLIT efficacy. METHODS: Comprehensive quantitative, qualitative and functional analyses of allergen-specific IgA, IgE, IgG1-4 and IgM responses were performed using purified Phl p 1 to 12 allergens in sera, saliva and nasal secretions from 82 grass pollen allergic patients. These patients were enrolled in a randomized, double-blind placebo-controlled study and assessed in an allergen challenge chamber (ClinicalTrials.gov NCT00619827). Antibody responses were monitored in parallel to clinical responses before and after daily sublingual treatment for 4 months with either a grass pollen or a placebo tablet. RESULTS: A significant mean improvement (i.e. 33-40.6%) in rhinoconjunctivitis total symptom scores was observed in SLIT recipients, irrespective of their baseline patterns of IgE sensitization (i.e. narrow, intermediate, broad) to grass pollen allergens. SLIT did not induce any de novo IgE sensitization. Clinical responders encompassed both immunoreactive patients who exhibited strong increases in titres, affinity and/or blocking activity of grass-pollen-specific IgGs (representing 17% of treated patients), as well as patients with no detectable antibody responses distinguishing them from the placebo group. No significant changes were detected in antibody titres in saliva and nasal washes, even in clinical responders. CONCLUSIONS AND CLINICAL RELEVANCE: Sublingual immunotherapy with a grass pollen tablet is efficacious irrespective of the patients' baseline sensitization to either single or multiple grass pollen allergens. Seric IgG responses may contribute to SLIT-induced clinical tolerance in a fraction (i.e. 17%) of patients, but additional immune mechanisms are involved in most patients. Consequently, antibody responses cannot be used as a marker of SLIT efficacy at an individual patient level.

Immunodominant semen proteins II: contribution of seminal proteins to female immune infertility.

Seminal fluid is a protective medium for sperm, but it also represents potential immunogenic structures for the female immune system. Anti-seminal antibodies may threaten early fertilization. The aim of our work is to detect and identify seminal proteins that are related to female isoimmunization. In this report, we quantified serum anti-seminal IgG antibodies. Seminal proteins were analysed by two-dimensional gel electrophoresis followed by immunoblotting. To identify IgG-binding proteins of interest, a proteomic approach was selected. The dominant seminal antigens were detected within the relative molecular mass ranging from 25 to 85 kDa and the isoelectric point from 5 to 7. The detected proteins were further identified as prostate-specific antigen, prostatic acid phosphatase, zinc-α-2-glycoprotein and zinc finger protein 778. Since these proteins were recognized by IgGs produced by infertile women and not by fertile women, we presume that major seminal antigens may play an important role in the pathogenesis of female immune infertility. Our study suggests the pattern of seminal proteins for further therapeutic attempts in the diagnosis of female immune infertility.

Female serum immunoglobulins G, A, E and their immunological reactions to seminal fluid antigens.

One in five couples of reproductive age has been diagnosed with infertility. Some diagnoses indicate an immunological basis for this disorder. Female immune infertility may be caused by iso-immunization by seminal components. We focused on the characterization of seminal proteins to illustrate the IgG, IgA and IgE immune responses of 31 infertile women. The biochemical characterization was performed by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis and isoelectric focusing, both of which were followed by immunoblotting analyses. IgG mainly recognized the antigens with relative molecular masses (Mr) 95 and 183 kDa and isoelectric points ranging from 6.9 to 7.0. The immunodominant antigens recognized by IgA had the Mr of 35 kDa and isoelectric points ranging from 6.2 to 7.2. The reactivity of IgE was not confirmed within our group of patients. The seminal IgG- and IgA -binding patterns were analysed immunochemically to determine the characteristics of possible seminal proteins associated with female immune infertility.

Prevalence of sensitisation to oilseed rape and maize pollens in France: a multi-center study carried out by the Allergo-Vigilance Network.

BACKGROUND: Oilseed rape and maize crops represent a large part of agriculture fields in European countries. OBJECTIVE: To establish the actual prevalence of sensitization to oilseed rape and maize pollen, and to determine if this is correlated to the amount of exposure as well as to the patient's history of atopy or asymptomatic atopy. METHODS: The study was conducted by 69 allergists belonging to the Allergo-Vigilance Network, in collaboration with the French Agency for Safety of food, and compiles the results of skin prick-tests using oilseed rape and maize pollens and seeds, as well as common aeroallergens. The patients were classified into 3 groups: nonatopic, asymptomatic atopy, and actual atopic diseases. RESULTS: Among the 5372 subjects studied (2515 children, 2857 adults), 62.3% had an atopic disease, 10.2% had an asymptomatic atopy, and 27.5% were non-atopic. The level of sensitization was higher in the subjects with atopic disease, as compared to those with asymptomatic atopy: oilseed rape pollen: 11.8% vs 8%, maize pollen, 26% vs 19%, oilseed rape seeds, 7.7% vs 6.9%, corn seeds: 8.3% vs 4.8% (p < 0.001). The rate of sensitization was significantly increased in those living in high crop density regions. The association of an atopic disease with a high rate of exposure yielded a higher rate of sensitization of 13.8% and 21.3% for rapeseed pollen, and 22.9% and 30.7% for maize pollen in both children and adults, respectively. CONCLUSIONS: The incidence of sensitisation to rapeseed and maize pollen is positively correlated to the level of exposure. This prevalence is higher in patients with actual atopic disease as compared to those with asymptomatic atopy. The frequency of sensitization confirms the allergenicity of these plants destined for food supply and demonstrates the importance of monitoring for respiratory allergies to these pollens, not only in workers exposed to these types of crops, but also in atopic patients living in regions that contain a high density of rapeseed and maize fields. Cross-reactivities between pollens and seeds could potentially elicit cross-reacting food allergies.

The immune response to a hybrid protein molecule; specificity of secondary stimulation and of tolerance induction.

Upon immunization with LDH-III (subunit composition AABB) rabbits produce anti-A and anti-B antibodies in comparable amounts. These antibodies fit equally well to the hybrid enzyme and to LDH-V (AAAA) or LDH-I (BBBB) respectively, as tested by passive hemagglutination inhibition. No antibodies reacting with both LDH-I and LDH-V were detected. A minority of hybrid-specific antibodies was, however, present in the sera. Animals primed with LDH-III respond regularly to a boosting injection of LDH-V with the production of large amounts of anti-A (but not anti-B) antibodies. A similar injection of LDH-I stimulates (if it has any effect at all) the production of anti-B antibodies only. Stimulation with one of the pure types does not impair a subsequent response to the other. The majority of the animals primed with LDH-III responded not at all or weakly to a boosting injection of LDH-I, though antibodies to LDH-I were present in the sera at the time of stimulation. This effect can hardly be explained on the basis of serological sepcificity. Hyporesponsiveness to LDH-III can be induced by injection of LDH-V into the newborn. Both anti-A and anti-B titers are equally depressed. Within the dose range tested, LDH-I does not exert any tolerogenic action with respect to LDH-III. The carrier property of subunit A is evident in the induction of both immunity and tolerance to LDH-III. The early phase of the immune response to the hybrid enzyme may be carrier-specific, and receptors for the haptenic subunit B may not exist at that stage.

Evaluation of ash pollen sensitization pattern using proteomic approach with individual sera from allergic patients.

BACKGROUND: In Europe, sensitization to ash pollen induces pollinosis with cross-reactivities with other pollen sources. The aim of the study was to identify the repertoire of ash pollen allergens and evaluate the extent of the diversity of the IgE response in ash allergic patients. METHODS: The IgE reactivities of 114 ash pollen- and eight grass pollen-sensitized patients were screened by 1D immunoblot (SDS-PAGE) against ash pollen extract. The IgE reactivities of 13 ash pollen- and two grass pollen-sensitized patients were then evaluated in 2D immunoblots. Some IgE- and non-IgE-reactive proteins were identified by mass spectrometry. RESULTS: In 1D analysis, 86% of sera showed binding to Fra e 1 (18-20 kDa), 23% to Fra e 2 (14 kDa), 3% to Fra e 3 (10 kDa) and 57% to High Molecular Weight allergens (HMW, >30 kDa). Individual analysis of 2D immunoblots showed several IgE-binding protein areas among which three were more often recognized: (i) Fra e 1 comprising, at least, 15 isoforms, (ii) a series of acidic spots (45 kDa), and (iii) Fra e 2, the ash profilin. HMW allergens could be resolved in four areas; two unidentified, one homologous to beta-galactosidase and the other to sugar transport proteins. A malate deshydrogenase and calmodulin were shown to be IgE-binding proteins and 10 non-IgE reactive proteins were identified. CONCLUSIONS: No direct correlation was evidenced between IgE profile and the degree of sensitization even though 2 spectrotypes could be distinguished. Our data contribute to a better delineation of ash pollen allergens and patterns of sensitization.

Characterization of peptidic and carbohydrate cross-reactive determinants in pollen polysensitization.

BACKGROUND: Cross-reactivity may be due to protein sequence or domain homologies and/or the existence of cross-reactive carbohydrate determinants (CCDs). The clinical relevance of peptidic cross-reactivities is well known, whereas that of CCDs is still a question of debate. The aim of this study is to characterize the IgE specificity of various patients suffering from pollen polysensitization to identify both peptidic and carbohydrate cross-reactive determinants. MATERIAL AND METHODS: Rapeseed, grass and Arabidopsis proteins were separated by isoelectric focusing, followed by SDS-PAGE, and transferred to a nitrocellulose sheet. The sheets were incubated either with an individual serum from a birch+grass-sensitive patient, followed by anti-human IgE, or with labelled Concanavalin A (ConA). Binding inhibition was tested by incubation of the sera with a mixture of sugar residues. RESULTS: The results showed two different patterns of cross-reacting sera: a pattern that implies few proteins, not always glycosylated and known as allergens, and a pattern that implies numerous proteins with molecular masses over 30 kDa. This second pattern was very close to the ConA -binding pattern. The IgE binding was abolished by pre-incubation with sugar residues only in the case of the second pattern. DISCUSSION: This study shows that multiple pollen sensitizations could result from multiple sensitizations to specific proteins or from a cross-sensitization to a wide range of glycoproteins. Two-D blots allow to characterize a cross-sensitization due to carbohydrate determinants, and thus to improve the diagnosis of allergy and its medical treatment.

Cloning, sequencing and immunological characterization of Dac g 3, a major allergen from Dactylis glomerata pollen.

Preliminary work showed that a 14-kDa allergen with a pI of 9 was recognized by more than 60% of sera from Dactylis glomerata (Dac g) pollen-allergic individuals. The N-terminal amino acid sequence of this Dac g allergen was determined by Edman degradation and compared with that of Lol p 3, a major allergen of Lolium perenne. A sequence identity of 65% was found, suggesting that the Dac g allergen could be the homologue of Lol p 3 and therefore named Dac g 3. We report the cloning and sequence analysis of a cDNA encoding the Dac g 3 pollen allergen. The recombinant allergen (rDac g 3) expressed in plasmid vector pGEX-2T contained IgE-reactive epitopes found in its natural counterpart, and induced histamine release from basophils of Dac g-allergic individuals, confirming that the recombinant protein has biological properties similar to the pollen extracted allergen. Computer analyses showed that, in spite of a high degree of sequence homology, even closely related allergens such as Dac g 3 and Lol p 3 have dissimilar predictive secondary structures and potential different antigenicity. Because it possesses the properties of the native counterpart, rDac g 3 could be a relevant tool for molecular studies in allergy.

Molecular basis of IgE cross-reactivity between human beta-casein and bovine beta-casein, a major allergen of milk.

Twenty patients allergic to cow's milk proteins and with high levels of specific IgE directed against bovine whole casein were selected to evaluate reactivity of their IgE antibodies with human beta-casein. Highly purified human and bovine beta-caseins were prepared by selective precipitations and FPLC separation. Their identity and purity were assessed by HPLC, analysis of amino acid composition, sequencing of the five N-terminal amino acid residues and immunochemical tests. Direct and indirect ELISAs were performed using human and bovine beta-casein coated into microtiter plates and monoclonal anti-human IgE antibody AChE labelled for revelation. Seven sera contained specific IgE directed against human beta-casein. Inhibition studies using native human and bovine beta-caseins as well as bovine beta-casein-derived peptides demonstrated that, depending on the sera, one or several common epitopes located in different parts of the molecule were shared by the two homologous proteins.

DMISA (dissociated membrane immunosorbent assay), a new ELISA technique performed with blotted samples.

This study describes the use of electrophoretically purified antigens blotted onto nitrocellulose, as solid phase antigens for enzyme-linked immunosorbent assays. This procedure is called DMISA, for dissociated membrane immunosorbent assay. The method is illustrated using immunoblotted antigens of Dactylis glomerata grass pollen extract. The band of interest was located on a print of nitrocellulose by light staining (India ink), then the corresponding strip of nitrocellulose was cut out. Immediately after its solubilization in ethyleneglycol monomethyl ether, the antigen coated nitrocellulose was precipitated by the addition of buffer. In this way the bulk of the antigen remained bound to the membrane. The resulting suspension was carefully washed, and used as a solid phase antigen in an enzyme-linked immunosorbent assay. Two different electrophoretic methods were used to separate the Dactylis glomerata antigens. We compared the results obtained with classical immunoblot and with DMISA, for IgG4 and IgE quantification using sera from patients allergic to D. glomerata and purified blotted antigens present at the nanogram level.

Inhibition of the passive cutaneous anaphylaxis reaction to Dactylis glomerata pollen allergens by a purified component of this pollen.

By isoelectric focusing a protein fraction of pI 4.6 was isolated from a crude water-soluble extract of Dactylis glomerata pollen (SE). This fraction was neither immunogenic nor allergenic in BALB/c mice. In one week, this protein inhibited the mouse IgE-specific antibodies to the soluble extract as measured by PCA in rats and was therefore called Dactylis inhibitory protein (DIP). Two experimental approaches which lowered IgE anti-SE titer were undertaken. Pretreatment with DIP as well as injection of DIP after the last sensitizing injection with SE resulted in an inhibition of the circulating IgE antibody level to SE. For both experiments the regulation of the immune response touched only the IgE class, whereas the titers of anti-SE IgG, IgM, IgA antibodies were not modified. DIP treatment did not alter the IgE titers, measured by PCA, in the immune response to ovalbumin.

Heterogeneity of grass pollen allergens (Dactylis glomerata) recognized by IgE antibodies in human patients sera by a new nitrocellulose immunoprint technique.

A new nitrocellulose immunoprint technique has been developed to detect specific antigens or/and allergens present among a heterogeneous solution such as a water-soluble crude extract of a grass pollen (Dactylis glomerata). The antigens are separated by isoelectric focusing (IEF) in an agarose gel and characterized by their isoelectric point (pI). These antigens are transferred and immobilized on a nitrocellulose sheet. They are recognized by the binding of specific antibodies contained in an unfractionated serum to be studied. Finally, the binding of these antibodies is visualized by species- or/and class-specific antibodies themselves labeled by an enzyme or by radioactivity. So one can detect the allergens recognized by the specific serum IgE antibodies and also the other antigens recognized by specific IgG, IgA or IgM antibodies.

Biochemical features of grain legume allergens in humans and animals.

Peanuts and soybeans are the major legumes involved in human food allergy, although some data exist on adverse reactions to temperate legumes including pea, green bean, sweet lupin, and lentil. An increasing number of legume proteins or glycoproteins have been characterized as food allergens. Limited data tend to indicate that they are usually different from legume inhalent allergens. Cross-recognition among legume allergens is immunochemically frequent but clinically less common. A common feature to most legume allergens is their natural resistance to thermal, chemical, and in some way, proteolytic denaturation. Finally, other mammals including preruminant calves, and piglets at the time of weaning, are prone to gut immune-mediated reactions to soybean and pea proteins.

Modification of bovine beta-lactoglobulin by glycation in a powdered state or in an aqueous solution: immunochemical characterization.

Bovine beta-LG was modified by glycation with lactose in a powdered state or in an aqueous solution. An immunological characterization was performed using monoclonal antibodies with defined epitopes. The results showed that the structural changes were confined to the AB loop region of the molecules when glycation was conducted in a restricted water environment and had little consequences on the association state of glycated beta-LG. The protein conformation was much more extensively modified when glycation was performed in an aqueous solution at 60 degrees C, despite a lower glycation extent. These structural changes were located at the dimer interface (AB loop, GH loop, beta-strand I, and alpha-helix). These results allowed us to establish a relationship between the conformational changes and the modification of the association state of the glycated protein (formation of disulfide bridges between the free thiol groups of two monomers), previously described.

Lactate dehydrogenase as a marker of Plasmodium infection in malaria vector Anopheles.

Lactate dehydrogenase (Ldh) electrophoresis showed the presence of Plasmodium yoelii yoelii in Anopheles stephensi and An. gambiae. The Ldh appeared as an additional band (pLdh) whose activity was more intense with 3-acetyl pyridine adenine dinucleotide as coenzyme than with beta nicotin-amide adenine dinucleotide. Several allelic forms occurred both in the vector and the host. The isoelectric point of Ldh, similar in the vector and host, differed from those of Ldh from mosquito and mouse. The presence of pLdh was detected from the 2nd to the 28th day of infection. The pLdh appeared to be proportional to the number of sporozoites present in infected salivary glands. However, pLdh was not found in salivary glands or midguts, but it was detected in the rest of the corresponding mosquito. The origin and use of pLdh as a marker of Plasmodium in its vector is discussed.

[Ball and socket ankle associated with congenital synostosis of the tarsus. Report of a case (author's transl)]. / Déformation en "cupule et dôme" de l'articulation tibio-tarsienne révélatrice d'une synostose congénitale du tarse.

The author describes a case of ball and socket ankle joint in a man aged 30 years. The deformity was associated with a congenital synostosis between the talus and calcaneus, absence of the fifth toe and 2 centimetres of shortening of the limb. The literature is reviewed.

[Study of loosening and wear of total hip prosthesis. Apropos of a series followed-up over 10 years]. / Etude des descellements et de l'usure des prothèses totales de hanche. A propos d'une série de plus de dix ans de recul.

After reviewing 30 cases of patients having had a total hip arthroplasty by the Charnley's technique between 1970 and 1978, the authors study the radiographic parameters of the cup and of the femoral stem, using geometric buildings described by Sutherland. On the acetabulum, the inclinaition angle, the medialisation and the wear of the cup were calculated. The varisation of the stem and its migration were looked for on the femur. The presence of a radiolucent line, prosthesis--bone or cement--bone and the importance of peri-articular calcifications, was also noted. The verticalisation of the cup and its anteversion were a pejorative factor. The medialisation was disquieting only if it was associated with a verticalisation. The wear more than 2 mm was noted only in 5 cases; the radiolucent line was pejorative only if it was type cement--bone; the type cement--cup, rare, was of less bad prognostic. On the femur, the most failing was the varus of the stem which determined a loosening most often when it was present. The disquieting radiolucent line was always type cement--bone and was always associated with a bicortical reaction showing a mobility of the stem. In a last part, the authors studied the wear of expanted cups in polyethylene which was different of the fluage. A distinction between the theoretical wear and the clinical wear was made. Photographs in scanning electron microscopy which permit to describe the different types of wear are presented.

[Treatment of primary vesicorenal reflux in adults. Apropos of 71 case reports]. / Traitement du reflux vésico-rénal primitif de l'adulte. A propos de soixante et onze observations.

The authors report a series of seventy one cases of vesico-renal reflux in adults. Renal scarring was present in 76% of the cases, and in 11.5%, the kidneys were destroyed. The reflux was eliminated in 95.5% of the cases treated by the Leadbetter-Politano, Cohen and Glenn Anderson procedures, but a failure rate of 26% was registered with the Lich Grégoir technique. 26.5% of the patients presented with persistent urinary tract infection six months after the successful surgical correction of the reflux.

[Inter-relationship between allergenic pollens and air pollution]. / Interrelation entre les pollens allergisants et la pollution de l'air.

Morbidity to pollens is increasing in the French population. Pollen is the vector of the male genome of the plant. It is the wind-borne pollens that are the most allergenic by release of allergen molecules that make contact with the mucosae. So every individual who is genetically allergic (atopic) may develop rhinitis and/or conjunctivitis and/or asthma.... More than 200 allergens have been identified, of which the three-dimensional structure is well-known. The urban pollution that is specific to a quarter under consideration (residential zone, pedestrianised, mid-town roadway and industrial zone) plays an important role in morbidity to pollen allergy. A significant coating of pollens is to be found on urban roads. Pollen sensitisation is increased by exposure to pollutants from 24 to 48 hours. The pollutants seem to make the surface of the exine more fragile, so triggering a mucosal reaction, making them more exposed to pollen allergens. The pollutant also plays the role of an adjuvant to the pollen allergen, and so is the origin of a greater production of IgE.

[Desensitization for 4 successive years using calcium phosphate-adsorbed pollen extracts: study of sera with RAST and PRIST immunoimprints]. / Désensibilisation par des extraits de pollen adsorbés sur phosphate de calcium pendant 4 années consécutives: étude des sérums par immunoempreintes, RAST et PRIST.

The clinical study for four years running of desensitization treatment to adsorbed pollen calcium phosphate, prepared "at request" by Institut Pasteur, shows that these preparations produced particularly good results. The population of allergic subjects is divided into two categories as from first year of treatment: good responders and non responders. A study on the serums of these patients, in view of a different "immuno-imprint" in these two groups, has not yet been conclusive, whereas we were expecting much from this prognosis test, which would have given us better treatment information.