Nonanaphylactic synthetic peptides derived from B cell epitopes of the major grass pollen allergen, Phl p 1, for allergy vaccination.

Worldwide more than 200 million individuals are allergic to group 1 grass pollen allergens. We have used the major timothy grass pollen allergen Phl p 1, which cross-reacts with most grass-, corn-, and monocot-derived group 1 allergens to develop a generally applicable strategy for the production of hypoallergenic allergy vaccines. On the basis of the experimentally determined B cell epitopes of Phl p 1, we have synthesized five synthetic peptides. These peptides are derived from the major Phl p 1 IgE epitopes and were between 28-32 amino acids long. We demonstrate by nuclear magnetic resonance that the peptides exhibit no secondary and tertiary structure and accordingly failed to bind IgE antibodies from grass pollen allergic patients. The five peptides, as well as an equimolar mixture thereof, lacked allergenic activity as demonstrated by basophil histamine release and skin test experiments in grass pollen allergic patients. When used as immunogens in mice and rabbits, the peptides induced protective IgG antibodies, which recognized the complete Phl p 1 wild-type allergen and group 1 allergens from other grass species. Moreover, peptide-induced antibodies inhibited the binding of grass pollen allergic patients IgE antibodies to the wild-type allergen. We thus demonstrate that synthetic hypoallergenic peptides derived from B cell epitopes of major allergens represent safe vaccine candidates for the treatment of IgE- mediated allergies.

Dissociation of allergen-specific IgE and IgA responses in sera and tears of pollen-allergic patients: a study performed with purified recombinant pollen allergens.

BACKGROUND: Trees and grass pollen allergens represent potent elicitors of allergic rhinoconjunctivitis and asthma. Little is known regarding the presence of allergen-specific IgA antibodies in sera and tears and their association with IgE responses in patients with allergic conjunctivitis. OBJECTIVE: The purpose of this study was to compare the specificities of IgE and IgA antibodies in sera and tears of pollen-allergic patients with conjunctivitis by using purified recombinant pollen allergens. METHODS: Sera and tears collected from 23 pollen-allergic and from 23 nonatopic individuals were analyzed for IgE and IgA reactivity to nitrocellulose-blotted birch and timothy grass pollen extracts. In addition, we determined the specificities of IgE, IgG(1-4), and IgA antibodies with use of a panel of purified recombinant pollen allergens (timothy grass: rPhl p 1, rPhl p 2, rPhl p 5; birch: rBet v 1, rBet v 2) in serum and tear samples by immunoblotting and ELISA. Statistical analyses of data were performed by t test and Mann Whitney U test. RESULTS: Serum and tears of many of the pollen-allergic individuals with conjunctivitis exhibited specificity for the very same pollen allergens. No allergen-specific IgE antibodies were detected in tears of nonatopic individuals. IgA antibodies in sera and tears of patients with allergic conjunctivitis were mainly directed against nonallergenic moieties and showed specificities that were significantly different from those of IgE antibodies. CONCLUSION: The dissociation of IgE and IgA responses and the lack of allergen-specific IgA antibodies in mucosal secretions (eg, tears) may contribute to allergic manifestations in target organs of atopy. Induction of allergen-specific IgA antibodies may hence be considered as a promising strategy for the treatment of mucosal forms of atopy.

B cell epitopes of the major timothy grass pollen allergen, phl p 1, revealed by gene fragmentation as candidates for immunotherapy.

Group 1 grass pollen allergens are recognized by IgE antibodies of almost 40% of allergic individuals and therefore belong to the most important elicitors of Type I allergy worldwide. We have previously isolated the cDNA coding for the group 1 allergen from timothy grass, Phl p 1, and demonstrated that recombinant Phl p 1 contains most of the B cell as well as T cell epitopes of group 1 allergens from a variety of grass and corn species. Here we determine continuous B cell epitopes of Phl p 1 by gene fragmentation. IgE antibodies of grass pollen allergic patients identified five continuous epitope-containing areas that on an average bound 40% of Phl p 1-specific IgE antibodies and were stably recognized in the course of disease. In contrast to untreated patients, patients undergoing grass pollen immunotherapy started to mount IgG(4) antibodies to the recombinant IgE-defined fragments in the course of immunotherapy. The protective role of these IgG(4) antibodies is demonstrated by observations that 1) increases in rPhl p 1 fragment-specific IgG(4) were in parallel with decreases in Phl p 1-specific IgE, and 2) preincubation of rPhl p 1 with patients sera containing rPhl p 1 fragment-specific IgG(4) blocked histamine release from basophils of an untreated grass pollen allergic patient. We propose to use recombinant Phl p 1 fragments for active immunotherapy in order to induce protective IgG responses against IgE epitopes in grass pollen allergic patients. This concept may be applied for the development of allergy vaccines whenever the primary sequence or structure of an allergen is available.

Induction of antibody responses to new B cell epitopes indicates vaccination character of allergen immunotherapy.

Whether the modulation of antibody responses can contribute to the improvement of clinical symptoms in patients receiving allergen immunotherapy represents a controversial issue. We have used purified [seven recombinant (r) and one natural] timothy grass pollen allergens as well as recombinant B cell epitope-containing fragments of the major timothy grass pollen allergen, Phl p 1, to investigate humoral immune responses in eight allergic patients receiving grass pollen-specific immunotherapy. We found that the administration of aluminium hydroxide-adsorbed grass pollen extract induced complex changes in allergen/epitope-specific antibody responses: increases in IgG subclass (IgG1, IgG2, IgG4) responses against allergens recognized before the therapy were observed. All eight patients started to mount IgE and IgG4 responses to continuous Phl p 1 epitopes not recognized before the therapy and a de novo induction of IgE antibodies against new allergens was found in one patient. Evidence for a protective role of IgG antibodies specific for continuous Phl p 1 epitopes was provided by the demonstration that preincubation of rPhl p 1 with human serum containing therapy-induced Phl p 1-specific IgG inhibited rPhl p 1-induced histamine release from basophils of a grass pollen-allergic patient. Our finding that immunotherapy induced antibody responses against previously not recognized B cell epitopes indicates the vaccination character of this treatment. The fact that patients started to mount de novo IgE as well as protective IgG responses against epitopes may explain the unpredictability of specific immunotherapy performed with allergen extracts and emphasizes the need for novel forms of component-resolved immunotherapy.

The molecular basis for allergen cross-reactivity: crystal structure and IgE-epitope mapping of birch pollen profilin.

BACKGROUND: The profilins are a group of ubiquitous actin monomer binding proteins that are responsible for regulating the normal distribution of filamentous actin networks in eukaryotic cells. Profilins also bind polyphosphoinositides, which can disrupt the profilin-action complex, and proline-rich ligands which localize profilin to sites requiring extensive actin filament accumulation. Profilins represent cross-reactive allergens for almost 20 % of all pollen allergic patients. RESULTS: We report the X-ray crystal structure of birch pollen profilin (BPP) at 2.4 resolution. The major IgE-reactive epitopes have been mapped and were found to cluster on the N- and C-terminal alpha helices and a segment of the protein containing two strands of the beta sheet. The overall fold of this protein is similar to that of the mammalian and amoeba profilins, however, there is a significant change in the orientation of the N-terminal alpha helix in BPP. This change in orientation alters the topography of a hydrophobic patch on the surface of the molecule, which is thought to be involved in the binding of proline-rich ligands. CONCLUSIONS: Profilin has been identified as an important cross-reactive allergen for patients suffering from multivalent type I allergy. The prevalent epitopic areas are located in regions with conserved sequence and secondary structure and overlap the binding sites for natural profilin ligands, indicating that the native ligand-free profilin acts as the original cross-sensitizing agent. Structural homology indicates that the basic features of the G actin-profilin interaction are conserved in all eukaryotic organisms, but suggests that mechanistic differences in the binding of proline-rich ligands may exist. The structure of BPP provides a molecular basis for understanding allergen cross-reactivity.

Isolation of an immunodominant IgE hapten from an epitope expression cDNA library. Dissection of the allergic effector reaction.

An epitope expression cDNA library was constructed from the randomly fragmented cDNA coding for Phl p I, the major grass pollen allergen. Using IgE from allergic patients, epitope clones were isolated and immunodominant fragments were selected. Among three epitope clones coding for a similar region of Phl p I, one clone expressed a 15-amino-acid epitope which was target for IgE antibodies from approximately 30% of grass pollen allergic patients. According to the prevalence of grass pollen allergy, 22% of all allergic patients are expected to display IgE reactivity with this epitope. Although the purified recombinant epitope specifically bound IgE, it did not release histamine from basophiles of most grass pollen allergic patients and thus represents an IgE hapten. Immunodominant IgE haptens may be useful as therapeutic agents to saturate mast cell-bound IgE prior to allergen exposure and may represent candidates for a safe immunotherapy of allergic diseases by reducing anaphylactic side effects.

cDNA cloning and expression of timothy grass (Phleum pratense) pollen profilin in Escherichia coli: comparison with birch pollen profilin.

Profilin, an actin-binding protein, was previously described as a ubiquitous allergen which is responsible for cross-reactivities in about 20% of pollen and food allergic patients. A complete cDNA clone coding for timothy grass (Phelum pratense) pollen profilin was isolated using allergic patients IgE. The deduced amino acid sequence of timothy grass profilin shares a sequence identity of 79% with birch profilin and other plant profilins and a lower average sequence identity of 35% with other eukaryotic profilins. The high degree of homology among different plant profilins at the DNA and protein level explains the extensive cross-reactivities observed in profilin allergic patients. Recombinant timothy grass pollen profilin was expressed in Escherichia coli as a beta-galactosidase fusion protein and shown to bind IgE from profilin allergic patients similar to recombinant birch profilin. Slight differences regarding the IgE-binding capacity of birch and timothy grass profilin indicate that not all IgE-epitopes of the two profilins are conserved. It is speculated that profilin allergic patients were initially sensitized against a certain profilin and then cross-react with the homologous proteins.

Pancreaticoenteric fistula: no longer a surgical disease?

We report our experience with eight patients with severe persistent pancreatitis associated with peripancreatic fluid collections requiring placement of drainage catheters who subsequently developed pancreatic fistula. The fistulas were diagnosed by endoscopic retrograde cholangiopancreatography, contrast tube study, or Hypaque enema at a mean of 13 weeks after diagnosis of pancreatitis and drain placement. These fistulas involved the duodenum in five patients and colon in three patients. Six patients had fistula resolution with medical therapy (after removal of percutaneous drainage catheters in three and with drain removal in conjunction with transpapillary stenting of a disrupted pancreatic duct in another three). We conclude that in patients with ongoing pancreatitis, pancreaticoenteric fistulas are probably caused by erosion of percutaneous drainage catheters. Such fistulas resolved with conservative treatment in six of eight patients.

Traumatic diaphragmatic hernia: errors in diagnosis.

Experience with 42 cases of traumatic diaphragmatic hernia is reviewed. The correct diagnosis was most readily made when: (1) the injury was recent, (2) the tear was left sided and large with readily identifiable structures herniated, (3) appropriate diagnostic procedures were carried out (chest film, upper gastrointestinal examination, barium enema study, nuclear liver scan, computed tomography), and (4) a high index of suspicion was maintained. The diagnosis was likely to be missed when: (1) the history of trauma, usually remote, was not obtained or was disregarded, (2) the hernia was right sided with herniation of the liver or other solid (water density) organs, or (3) diagnostic tests were not properly correlated (i.e., abnormal barium enema and chest film) or were not obtained. The rather characteristic appearance of herniated liver on the nuclear liver/spleen scan is noted and its use rather than pneumoperitoneum is recommended.