Infant milk formulas differ regarding their allergenic activity and induction of T-cell and cytokine responses.

BACKGROUND: Several hydrolyzed cow's milk (CM) formulas are available for avoidance of allergic reactions in CM-allergic children and for prevention of allergy development in high-risk infants. Our aim was to compare CM formulas regarding the presence of immunoreactive CM components, IgE reactivity, allergenic activity, ability to induce T-cell proliferation, and cytokine secretion. METHODS: A blinded analysis of eight CM formulas, one nonhydrolyzed, two partially hydrolyzed (PH), four extensively hydrolyzed (EH), and one amino acid formula, using biochemical techniques and specific antibody probes was conducted. IgE reactivity and allergenic activity of the formulas were tested with sera from CM-allergic patients (n = 26) in RAST-based assays and with rat basophils transfected with the human FcεRI, respectively. The induction of T-cell proliferation and the secretion of cytokines in Peripheral blood mononuclear cell (PBMC) culture from CM allergic patients and nonallergic individuals were assessed. RESULTS: Immune-reactive α-lactalbumin and ß-lactoglobulin were found in the two PH formulas and casein components in one of the EH formulas. One PH formula and the EH formula containing casein components showed remaining IgE reactivity, whereas the other hydrolyzed formulas lacked IgE reactivity. Only two EH formulas and the amino acid formula did not induce T-cell proliferation and proinflammatory cytokine release. The remaining formulas varied regarding the induction of Th2, Th1, and proinflammatory cytokines. CONCLUSION: Our results show that certain CM formulas without allergenic and low proinflammatory properties can be identified and they may also explain different outcomes obtained in clinical studies using CM formulas.

Carrier-bound Alt a 1 peptides without allergenic activity for vaccination against Alternaria alternata allergy.

BACKGROUND: The mould Alternaria alternata is a major elicitor of allergic asthma. Diagnosis and specific immunotherapy (SIT) of Alternaria allergy are often limited by the insufficient quality of natural mould extracts. OBJECTIVE: To investigate whether recombinant Alt a 1 can be used for reliable diagnosis of Alternaria alternata allergy and to develop a safe, non-allergenic vaccine for SIT of Alternaria allergy. METHODS: The qualitative sensitization profile of 80 Alternaria-allergic patients from Austria and Italy was investigated using an allergen micro-array and the amount of Alternaria-specific IgE directed to rAlt a 1 was quantified by ImmunoCAP measurements. Peptides spanning regions of predicted high surface accessibility of Alt a 1 were synthesized and tested for IgE reactivity and allergenic activity, using sera and basophils from allergic patients. Carrier-bound peptides were studied for their ability to induce IgG antibodies in rabbits which recognize Alt a 1 and inhibit allergic patients' IgE reactivity to Alt a 1. RESULTS: rAlt a 1 allowed diagnosis of Alternaria allergy in all tested patients, bound the vast majority (i.e. >95%) of Alternaria-specific IgE and elicited basophil activation already at a concentration of 0.1 ng/mL. Four non-allergenic peptides were synthesized which, after coupling to the carrier protein keyhole limpet hemocyanin, induced Alt a 1-specific IgG and inhibited allergic patients' IgE binding to Alt a 1. CONCLUSIONS AND CLINICAL RELEVANCE: rAlt a 1 is a highly allergenic molecule allowing sensitive diagnosis of Alternaria allergy. Carrier-bound non-allergenic Alt a 1 peptides are candidates for safe SIT of Alternaria allergy.

Patients suffering from non-IgE-mediated cow's milk protein intolerance cannot be diagnosed based on IgG subclass or IgA responses to milk allergens.

BACKGROUND: Cow's milk is one of the most common causes of food allergy. In two-thirds of patients, adverse symptoms following milk ingestion are caused by IgE-mediated allergic reactions, whereas for one-third, the mechanisms are unknown. Aim of this study was to investigate whether patients suffering from non-IgE-mediated cow's milk protein intolerance can be distinguished from persons without cow's milk protein intolerance based on serological measurement of IgG and IgA specific for purified cow's milk antigens. METHODS: We determined IgG(1-4) subclass and IgA antibody levels to purified recombinant αS1-casein, αS2-casein, ß-casein, κ-casein, α-lactalbumin, and ß-lactoglobulin in four patient groups by ELISA: Patients with IgE-mediated cow's milk allergy (CMA, n=25), patients with non-IgE-mediated cow's milk protein intolerance (CMPI, n=19), patients with gastrointestinal symptoms not associated with cow's milk ingestion (GI, n=15) and control persons without gastrointestinal problems (C, n=26). Cow's milk-specific IgE levels were determined by ImmunoCAP. RESULTS: Only CMA patients had IgE antibodies to cow's milk. Cow's milk allergic patients mounted the highest IgG(1) and IgG(4) antibody levels to αS1-casein, αS2-casein, ß-casein, κ-casein, and α-lactalbumin. No elevated levels of IgG(4) , IgA, and complement-binding IgG subclasses (IgG(1) , IgG(2) , IgG(3) ) to purified cow's milk allergens were found within the CMPI patients compared to persons without cow's milk protein intolerance (GI and C groups). CONCLUSION: Cow's milk protein intolerant patients cannot be distinguished from persons without cow's milk protein intolerance on the basis of IgG subclass or IgA reactivity to cow's milk allergens.

Microarray and allergenic activity assessment of milk allergens.

BACKGROUND: Cow's milk is one of the most common causes of food allergy affecting approximately 2.5% of infants in the first years of their life. However, only limited information regarding the allergenic activity of individual cow's milk allergens is available. OBJECTIVE: To analyse the frequency of IgE reactivity and to determine the allergenic activity of individual cow's milk allergens. METHODS: A nitrocellulose-based microarray, based on purified natural and recombinant cow's milk allergens was used to determine IgE reactivity profiles using sera from 78 cow's milk-sensitized individuals of varying ages. The allergenic activity of the individual allergens was tested using patients' sera for loading rat basophil leukaemia cells (RBL) expressing the α-chain of the human receptor FcεRI. RESULTS: Using the microarray and the RBL assay, cow's milk allergens were assessed for frequency of IgE recognition and allergenic activity. Moreover, the RBL assay allowed distinguishing individuals without or with mild clinical reactions from those with severe systemic or gastrointestinal symptoms as well as persons who grew out cow's milk allergy from those who did not. CONCLUSIONS: Component-resolved testing using milk allergen microarrays and RBL assays seems to provide useful additional diagnostic information and may represent a basis for future forms of prophylactic and therapeutic strategies for cow's milk allergy.

[+49 A/G genetic polymorphism of cytotoxic T lymphocyte associated antigen 4 (CTLA-4) in type 7 diabetes: association with autoantibodies and cytokines]. / Polimorfismo +49 A/G del gen del antígeno 4 del linfocito T citotóxico (CTLA-4) en la diabetes tipo 1: Asociación con el perfil de anticuerpos y citoquinas.

BACKGROUND: Cytotoxic T lymphocyte associated antigen 4 (CTLA-4) has been one ofthe non HLA genes more commonly studied in type 1 diabetes mellitus (TID). CTLA-4 is a co-stimulation protein that has a key role in the negative regulation ofT cells and is related with a functional cytokine imbalance, generating a T helper (Th) 1 over Th2 dominance. AIM: To analyze the association of +49 A/G polymorphism of CTLA-4 and its relationship with autoantibodies and cytokine expression in recently diagnosed TID patients. PATIENTS AND METHODS: CTLA-4 genetic variants and auto-antibody levéis were studied in 260 chiídren with TID and 255 healthy chiídren matched by age and gender +49 A/G polymorphism of CTLA-4 was studied by polymerase chain reaction and restriction fragmentpolymorphism (PCR-RFLP). Autoantibody levéis were measured by conventional ELISA. A panel of60 cytokines was studied simultaneously by serum array analysis in 15 TID and 15 healthy controls stratified according CTLA-4 genotype. RESULTS: The +49 A/G genetic frequency was similar in TID cases and healthy chiídren. A positive anti-GAD65 and anti-IA-2 level was observed in 673% of TID group. This percentage was increased among GG carriers (79.4% to GAD65 and 70.6% to IA-2). Finally, TID patients carrying this genotype showed a high expression of interleukin 2, 10, tumor necrosis factor alpha and interferon gamma. CONCLUSIONS: The +49 A/G polymorphism of CTLA-4 was similar in diabetic and control chiídren. Among patients with TID and carriers of GG genotype, a higher frequency of anti-GAD65 and a preferential Thl cytokine expression profile was observed.

Polimorfismo +49 A/G del gen del antígeno 4 del linfocito T citotóxico (CTLA-4) en la diabetes tipo 1: Asociación con el perfil de anticuerpos y citoquinas / +49 A/G genetic polymorphism of cytotoxic T lymphocyte associated antigen 4 (CTLA-4) in type 7 diabetes: Association with autoantibodies and cytokines

Background: Cytotoxic T lymphocyte associated antigen 4 (CTLA-4) has been one ofthe non HLA genes more commonly studied in type 1 diabetes mellitus (TID). CTLA-4 is a co-stimulation protein that has a key role in the negative regulation ofT cells and is related with a functional cytokine imbalance, generating a T helper (Th) 1 over Th2 dominance. Aim: To analyze the association of +49 A/G polymorphism of CTLA-4 and its relationship with autoantibodies and cytokine expression in recently diagnosed TID patients. Patients and Methods: CTLA-4 genetic variants and auto-antibody levéis were studied in 260 chiídren with TID and 255 healthy chiídren matched by age and gender +49 A/G polymorphism of CTLA-4 was studied by polymerase chain reaction and restriction fragmentpolymorphism (PCR-RFLP). Autoantibody levéis were measured by conventional ELISA. A panel of60 cytokines was studied simultaneously by serum array analysis in 15 TID and 15 healthy controls stratified according CTLA-4 genotype. Results: The +49 A/G genetic frequency was similar in TID cases and healthy chiídren. A positive anti-GAD65 and anti-IA-2 level was observed in 673 percent of TID group. This percentage was increased among GG carriers (79.4 percent to GAD65 and 70.6 percent to IA-2). Finally, TID patients carrying this genotype showed a high expression of interleukin 2, 10, tumor necrosis factor alpha and interferon gamma. Conclusions: The +49 A/G polymorphism of CTLA-4 was similar in diabetic and control chiídren. Among patients with TID and carriers of GG genotype, a higher frequency of anti-GAD65 and a preferential Thl cytokine expression profile was observed.

Characterization of Der p 21, a new important allergen derived from the gut of house dust mites.

BACKGROUND: The house dust mite (HDM) Dermatophagoides pteronyssinus is a major allergen source eliciting allergic asthma. The aim of the study was to identify new important HDM allergens associated with allergic asthma. METHODS: A cDNA coding for a new mite allergen, designated Der p 21, was isolated using immunoglobulin E (IgE) antibodies from patients with allergic asthma out of a D. pteronyssinus expression cDNA library and expressed in Escherichia coli. RESULTS: Circular dichroism analysis of the purified allergen showed that rDer p 21 (14 726 Da) is one of the few mite allergens with an alpha-helical secondary structure. The protein exhibited high thermal stability and refolding capacity, and, as determined by small angle X-ray scattering, formed a dimer consisting of two flat triangles. rDer p 21 bound high levels of patients' IgE antibodies and showed high allergenic activity in basophil activation experiments. Rabbit anti-Der p 21 IgG antibodies inhibited mite-allergic patients' IgE binding and allowed the ultrastructural localization of the allergen in the midgut (epithelium, lumen and faeces) of D. pteronyssinus by immunogold electron microscopy. Der p 21 revealed sequence homology with group 5 mite allergens, but IgE and IgG reactivity data and cross-inhibition studies identified it as a new mite allergen. CONCLUSIONS: Der p 21 is a new important mite allergen which is liberated into the environment via faecal particles and hence may be associated with allergic asthma.

Different allergenic activity of grass pollen allergens revealed by skin testing.

BACKGROUND: Grass pollen is one of the most important allergen sources. The aim of this study was to compare the in vivo allergenic activity of two recently characterized major grass pollen allergens, Phl p 4 and Phl p 13, with three established major grass pollen allergens, Phl p 1, Phl p 2 and Phl p 5 as a basis for the formulation of a grass pollen allergy vaccine based on purified allergens. MATERIAL AND METHODS: Eighty-two grass pollen allergic patients were skin prick tested with serial dilutions of approximately equimolar concentrations of the purified allergens in a double-blind study. RESULTS: Phl p 4 and Phl p 13 were identified as major grass pollen allergens according to IgE binding frequency (Phl p 4: 85%; Phl p 13: 56%), but exhibited a five to nine-fold lower allergenic skin reactivity compared to Phl p 1, Phl p 2 or Phl p 5. CONCLUSION: Our results indicate that Phl p 4 and Phl p 13 are not essential components for a therapeutic grass pollen vaccine and underpin the importance of evaluating the in vivo allergenic activity of individual allergens for the formulation of therapeutic vaccines based on purified allergens.

Detection of an allergen in dog dander that cross-reacts with the major cat allergen, Fel d 1.

BACKGROUND: A considerable proportion of animal-allergic patients are sensitized to both cat and dog allergens but knowledge about cross-reactive allergens in cat and dog dander is limited. OBJECTIVE: To investigate whether dog dander contains an allergen that cross-reacts with the major cat allergen, Fel d 1. METHODS: Recombinant Fel d 1 with the same immunological properties as natural Fel d 1 was used for quantitative (CAP) IgE competition experiments performed with sera obtained from cat-allergic patients (n=36). A Fel d 1 cross-reactive dog allergen was characterized by one- and two-dimensional immunoblotting using rFel d 1 for IgE inhibition experiments and with monospecific, polyclonal rabbit anti-recombinant Fel d 1 antibodies. RESULTS: In 25% of Fel d 1-reactive cat-allergic patients, more than 50% inhibition of IgE reactivity to dog allergens was achieved with recombinant Fel d 1. An Fel d 1 cross-reactive 20 kDa allergen with a pI of approximately 3.4 was detected in dander extracts of several different dog breeds. CONCLUSION: This is the first report demonstrating the presence of an Fel d 1-like allergen in dog dander extracts, which may be responsible for double positivity to cat and dog in serology. However, the clinical relevance of this cross-sensitization needs to be confirmed. These results are important for the diagnostic and therapeutic use of dog dander allergen extracts.

A pollen-specific polygalacturonase from lily is related to major grass pollen allergens.

A pollen-specific gene from lily (Lilium longiflorum Thunb. cv. Snow Queen), designated LLP-PG, was characterized. Southern blots of lily genomic DNA indicated that LLP-PG is a member of a small gene family. A thorough sequence analysis revealed that the LLP-PG gene is interrupted by two introns and encodes a protein of 413 amino acids, with a calculated molecular mass of 44 kDa, and a pI of 8.1. Evaluation of the hydropathy profile showed that the protein has a hydrophobic segment at the N-terminus, indicating the presence of a putative signal peptide. A sequence similarity search showed a significant homology of the encoded protein to pollen polygalacturonases (PGs) from various plant species and to an important group (group 13) of grass pollen allergens. The LLP-PG transcript is pollen-specific and it accumulates only at the latest stage during pollen development, in the mature pollen. In contrast to other "late genes" LLP-PG transcript can neither be induced by abscisic acid (ABA) nor by dehydration. Immunoblot analyses of pollen protein extracts from lily, timothy grass and tobacco with IgG antibodies directed against LLP-PG and against the timothy grass pollen allergen, Phl p 13, indicated that lily LLP-PG shares surface-exposed epitopes with pollen PGs from monocotyledonous and dicotyledonous plants. Enzyme-linked immunosorbent assay (ELISA) analyses and inhibition ELISA assays with patients' IgE demonstrated a very low IgE reactivity of lily rLLP-PG and a lack of cross-reactivity between rLLP-PG and the timothy grass pollen allergen, rPhl p 13. These data demonstrated that despite the significant sequence homology and the conserved surface-exposed epitopes LLP-PG represents a low-allergenic member of pollen PGs.

Possible modes of allergen-specific sensitization and boosting in an atopic child.

Several studies document that allergen-specific IgE levels are boosted by allergen contact via the respiratory tract in allergic patients. Only few data are available on whether other routes of allergen contact have an influence on systemic IgE responses. We report the case of a boy who developed egg allergy after heavy consumption of eggs by the mother during pregnancy and breast feeding. In contrast to other children who outgrow egg allergy during the first years of life, the boy experienced further dramatic increases in hen egg-specific IgE antibodies after prolonged consumption of ostrich eggs containing cross-reactive allergens. IgE antibodies to most of the important respiratory allergens remained either low or not detectable. The dramatic increases in hen egg-specific IgE antibody levels after oral intake of allergens demonstrate that systemic IgE responses in allergic patients can be strongly boosted by allergen contact via routes other than the respiratory tract.

Unaltered expression of Fas (CD95/APO-1), caspase-3, Bcl-2 and annexins in brains of fetal Down syndrome: evidence against increased apoptosis.

Apoptosis is the mechanism by which cells are programmed to die under a wide range of physiological and developmental stimuli. Accumulating evidence indicates that enhanced apoptosis (programmed cell death) in Down syndrome (DS) may play a role in mental retardation and precocious neurodegeneration of the Alzheimer-type. In this regard, alteration of several apoptosis related proteins have been reported in adult DS brain. Fetal DS neurons exhibited increased reactive oxygen species leading to early apoptosis, however, expression of apoptosis related proteins in fetal DS, has never been considered. To address this issue, we investigated the expression of proteins involved in apoptosis including Fas (CD95, APO-1), caspase-3, Bcl-2 and annexins in the cerebral cortex of control and DS fetal brain by western blot and two dimensional electrophoresis. Here, we report that no detectable changes were obtained in fetal DS brain in the expression of Fas, caspase-3, Bcl-2 and Annexins (I, II, V, and VI) compared to controls. In parallel experiment, we also examined the expression of neuron specific enolase (NSE), a neuronal marker found to be decreased in adult DS brain, to see if there is any neuronal loss and no difference was observed between the two groups. Protein expression did not correlate with age. The unchanged levels of Fas, Bcl-2 and annexins together with unaltered caspase-3 expression, a predominant caspase that executes apoptosis in the developing nervous system, suggest that enhanced apoptosis may not be apparent in fetal DS brain as demonstrated for adult DS brain.

Changes in nicotinic acetylcholine receptor subunits expression in brain of patients with Down syndrome and Alzheimer's disease.

Cholinergic deficit associated with loss of nicotinic acetylcholine receptors (nAChRs) has been described in Alzheimer's disease (AD) by receptor binding assays, positron emission tomography and immunoblotting. However, little is known about the alteration of these receptors in a related disease, Down syndrome (DS) which might be of importance for therapeutic strategies. The protein levels of neuronal nAChR alpha and beta subunits in human postmortem brain samples (frontal cortex and cerebellum) of control, adult DS, and AD were investigated by making use of western blot analysis. Two major bands at 26 and 45 kDa for alpha3, one at 50 kDa for alpha4 and beta2, and one at 45 kDa for alpha7 were detected by the respective antibodies. Specific alteration in individual subunits was also apparent in DS and AD. In frontal cortex, the 45kDa alpha3 subunit was significantly increased in DS (121%) (P < 0.05) and AD (93%) (P < 0.05), whereas the 26kDa, an isoform/truncated form of alpha3, displayed a reversed pattern. It was significantly decreased in DS (75%) (P < 0.001) and AD (52.6%) (P < 0.05). Alpha4 was comparable in all groups by contrast, alpha7 was significantly decreased in AD (64%) (P < 0.05). In DS, however, although the levels tended to be lower (17.3%) the reduction was not significant. Beta2 was unchanged in AD but showed a significant increase in DS frontal cortex (98.1%) (P < 0.01). In cerebellum, no significant alteration was observed in any of the subunits except beta2. It exhibited a significant increase (161%) (P < 0.01) in DS. Derangement in expression of nAChRs is apparent in DS, as in AD that may have some relevance to DS neuropathology. Furthermore, the increase in beta2 expression indicate that these subunits may have more than a structural role. Hence, therapeutic strategies tailored towards these end might be of some benefit for cognitive enhancement in these disorders.

Down syndrome patients start early prenatal life with normal cholinergic, monoaminergic and serotoninergic innervation.

Information on the fetal brain in Down syndrome (DS) is limited. In particular, there is no systematic study available on cholinergic, monoaminergic or serotoninergic innervation in the early second trimester. It was therefore the aim of the study to investigate whether deficits of any of these systems known to occur in adults with DS, was present at this early phase. For this purpose we determined markers for neuronal density (neuron-specific enolase, NSE), for cholinergic innervation (vesicular acetylcholine transporter, VAChT), for monoaminergic innervation (vesicular monoamine transporter 2, VMAT2; tyrosine hydroxylase, TH) and for the serotoninergic system (serotonine transporter, SERT) in brain of control and DS fetuses in the early second trimester using immunoblotting. Values for all neurotransmission systems were measurable at this time point of human development and comparable in control and DS fetuses. We conclude that during the second trimester DS patients do not differ in terms of immunoreactivity for all markers studied. This first study on that subject warrants further investigations for the determination of the time point when neurotransmitter deficits in DS brain are starting, a hallmark most important for pathogenesis and pharmacotherapy.