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.

Natural clinical tolerance to peanut in African patients is caused by poor allergenic activity of peanut IgE.

BACKGROUND: In Africa, peanuts are frequently consumed, but severe allergic reactions are rare. We investigated immunological patterns of clinical tolerance to peanut in peanut-sensitized but asymptomatic patients from central Africa compared to peanut-allergic and peanut-sensitized but asymptomatic patients from Sweden. METHODS: Sera from allergic patients (n = 54) from Zimbabwe sensitized to peanut but without allergic symptoms to peanut, and sera from peanut-allergic (n = 25) and peanut-sensitized but asymptomatic (n = 25) patients from Sweden were analyzed toward peanut allergen components (Ara h 1-3, 6, 8-9) and other allergen molecules from important allergen sources using microarray. IgE to Ara h 2 peptide epitopes was analyzed, and allergenic activity was assessed by basophil activation assay. RESULTS: Forty-six percent of the African and all peanut-allergic Swedish patients showed IgE toward one of the highly allergenic peanut allergens (Ara h 1-3, 6, 9). However, 48% of the African patients had IgE to cross-reactive carbohydrate determinants (CCDs) with low allergenic activity and 60% of the Swedish asymptomatic patients had IgE against the PR protein Ara h 8. IgG and IgG4 specificities and levels could not discriminate between the African asymptomatic and Swedish peanut-allergic patients. Asymptomatic patients almost lacked IgE to Ara h 2 peptides, which were recognized by peanut-allergic patients. Peanut IgE from peanut asymptomatic patients showed poor allergenic activity compared with IgE from peanut-allergic patients. CONCLUSIONS: Natural clinical tolerance to peanut in the African patients can be caused by IgE to low allergenic peanut components and by poor allergenic activity of peanut-specific IgE.

Hypoallergenic derivatives of Fel d 1 obtained by rational reassembly for allergy vaccination and tolerance induction.

BACKGROUND AND OBJECTIVE: The major cat allergen Fel d 1 represents one of the most important respiratory allergens. Aim of this study was to engineer recombinant Fel d 1 derivatives with reduced IgE reactivity and preserved T cell epitopes for vaccination and tolerance induction. METHODS: Seven recombinant mosaic proteins were generated by reassembly of non-IgE-reactive peptides of Fel d 1 which contained the sequence elements for induction of allergen-specific blocking IgG antibodies and T cell epitopes. Mosaic proteins were expressed in Escherichia coli using codon-optimized synthetic genes and compared with Fel d 1 regarding structural fold by circular dichroism, IgE-binding capacity, activation of allergic patients' basophils and ability to induce allergen-specific blocking IgG antibodies upon immunization. RESULTS: Although each of the mosaic proteins had lost the alpha-helical fold typical for Fel d 1, a strong reduction in IgE reactivity as well as allergenic activity in basophil activation assays was only obtained for three constructs, two reassembled fragments (Fel d 1 MB, Fel d 1 MC) and a fusion of the latter two (Fel d 1 MF) in which the cysteines of Fel d 1 MC were replaced by serines. Immunization of rabbits with Fel d 1 MB, MC and MF induced high levels of IgG antibodies that inhibited IgE reactivity of cat-allergic patients to Fel d 1 in a comparable manner as IgG induced with the wild-type allergen. CONCLUSIONS: We report the development of hypoallergenic reassembled Fel d 1 proteins suitable for vaccination and tolerance induction in cat-allergic patients.

The high molecular weight glutenin subunit Bx7 allergen from wheat contains repetitive IgE epitopes.

BACKGROUND: Wheat is one of the most common food allergen sources for children and adults. The aim of this study was to characterize new wheat allergens using an IgE discovery approach and to investigate their IgE epitopes. METHODS: A cDNA expression library representing the wheat transcriptome was constructed in phage lambda gt11 and screened with IgE antibodies from wheat food allergic patients. IgE-reactive cDNA clones coding for portions of high molecular weight (HMW) glutenin subunits were identified by sequence analysis of positive clones. IgE epitopes were characterized using recombinant fragments from the HMW Bx7 and synthetic peptides thereof for testing of allergic patients' sera and in basophil degranulation assays. RESULTS: We found that the major IgE-reactive areas of HMW glutenins are located in the repetitive regions of the protein and could show that two independent IgE-reactive fragments from HMW Bx7 contained repetitive IgE epitopes. CONCLUSIONS: Our results demonstrate that IgE antibodies from wheat food allergic patients can recognize repetitive epitopes in one of the important wheat food allergens. Recombinant HMW Bx7 may be included into the panel of allergens for component-resolved diagnosis of wheat food allergy.

Molecular characterization of wheat allergens specifically recognized by patients suffering from wheat-induced respiratory allergy.

BACKGROUND: Wheat (Triticum aestivum) is an important allergen source responsible for various clinical manifestations of allergy (i.e. food allergy, pollen allergy, respiratory allergy to flour-Baker's asthma). OBJECTIVE: The objective of this study was the molecular and immunological characterization of new recombinant wheat allergens and to evaluate their usefulness for the diagnosis of allergy to wheat. METHODS: A T. aestivum cDNA library was constructed and screened with serum IgE from patients suffering from wheat allergy to identify cDNAs coding for new wheat allergens. The allergen-encoding cDNAs were expressed in Escherichia coli and purified to homogeneity. IgE reactivity of recombinant proteins was analysed in RAST-based, non-denaturing dot blot experiments and by ELISA with sera from wheat allergic patients and their allergenic activity was assessed in basophil degranulation experiments. RESULTS: We report the molecular characterization, recombinant expression and purification of five wheat allergens, a thioredoxin h isoform, glutathione transferase, 1-Cys-peroxiredoxin, profilin and dehydrin. Homologous proteins were identified by sequence comparisons in various plants. 1-Cys-peroxiredoxin appeared to be the most relevant of the newly identified wheat allergens according to prevalence of IgE recognition and results from basophil degranulation experiments. It showed IgE cross-reactivity with seed proteins from barley, rye, rice, maize, soy, oat and spelt. 1-Cys-peroxiredoxin, glutathione transferase and dehydrin were mainly recognized by patients with baker's asthma but not wheat-induced food allergy. CONCLUSION AND CLINICAL RELEVANCE: The characterized recombinant wheat allergens may be useful for the development of serological tests which allow the discrimination of different clinical manifestations of wheat allergy.

Transcutaneous immunotherapy via laser-generated micropores efficiently alleviates allergic asthma in Phl p 5-sensitized mice.

BACKGROUND: Specific immunotherapy via the subcutaneous or oral route is associated with local and, in some cases, systemic side effects and suffers from low patient compliance. Due to its unique immunological features, the skin represents a promising target tissue for effective and painless treatment of type I allergy. The current study was performed to compare the efficacy of transcutaneous immunotherapy via laser-generated micropores to subcutaneous injection. METHODS: BALB/c mice were sensitized by intraperitoneal injection of recombinant grass pollen allergen Phl p 5 together with alum. Subsequently, lung inflammation was induced by repeated intranasal challenge. During the treatment phase, adjuvant-free Phl p 5 was applied in solution to microporated skin or was subcutaneously injected. Lung function and cellular infiltration; Phl p 5-specific serum levels of IgG1, IgG2a, and IgE; and cytokine levels in bronchoalveolar lavage fluids as well as in supernatants of splenocyte cultures were assessed. RESULTS: Both therapeutic approaches reduced airway hyperresponsiveness and leukocyte infiltration into the lungs. Whereas subcutaneous immunotherapy induced a systemic increase in Th2-associated cytokine secretion, transcutaneous application revealed a general downregulation of Th1/Th2/Th17 responses. Successful therapy was associated with induction of IgG2a and an increase in FOXP3+ CD4+ T cells. CONCLUSIONS: Transcutaneous immunotherapy via laser microporation is equally efficient compared with conventional subcutaneous treatment but avoids therapy-associated boosting of systemic Th2 immunity. Immunotherapy via laser-microporated skin combines a painless application route with the high efficacy known from subcutaneous injections and therefore represents a promising alternative to established forms of immunotherapy.

Recombinant monoclonal human immunoglobulin E to investigate the allergenic activity of major grass pollen allergen Phl p 5.

BACKGROUND: Allergen recognition by IgE antibodies is a key event in allergic inflammation. OBJECTIVE: To construct a plasmid for the expression of human monoclonal IgE antibodies of any desired specificity and to express IgE specific for the major timothy grass pollen allergen Phl p 5. METHODS: In a first step, the DNA sequence coding for the IgG(1) heavy chain was excised and replaced by the sequence coding for the human ɛ constant region gene in plasmid pLNOH2 expressing a human Phl p 5-specific IgG(1) heavy chain. Then, this construct together with a second plasmid expressing the corresponding Phl p 5-specific light chain was co-expressed in COS-7 cells. The Phl p 5-specific IgE (rhuMabEP5) was analysed for allergen-specificity and isotype by ELISA. Cross-reactivity of rhuMabEP5 was investigated by immunoblotting using pollen extracts from various grass species. The allergenic activity of Phl p 5 was studied by exposing rat basophil leukaemia (RBL) cells expressing human-FcɛRI to rhuMabEP5 and Phl p 5. RESULTS: We report the construction of vector pLNOH2-P5IgE, for the expression of human IgE and exemplify its usefulness by the production of a complete and functional human monoclonal IgE (rhuMabEP5). rhuMabEP5 is specific for the grass pollen allergen Phl p 5 and cross-reacts with group 5 allergens in natural grass pollen extracts. RBL-release assays with rhuMabEP5 demonstrated that oligomerization does not contribute to the high allergenic activity of Phl p 5. CONCLUSION AND CLINICAL RELEVANCE: Plasmid pLNOH2-P5IgE allowed the production of a fully functional human monoclonal IgE antibody specific for Phl p 5. Recombinant human IgE antibodies of defined specificity represent useful tools to investigate mechanisms underlying IgE-mediated allergies.

Molecular characterization of Der p 10: a diagnostic marker for broad sensitization in house dust mite allergy.

BACKGROUND: Tropomyosins represent clinically relevant seafood allergens but the role of mite tropomyosin, Der p 10, in house dust mite (HDM) allergy has not been studied in detail. OBJECTIVE: To express and purify a recombinant Der p 10 with equivalent IgE reactivity as natural Der p 10 and to evaluate its IgE reactivity and allergenic activity in HDM-allergic patients. METHODS: rDer p 10 was expressed in Escherichia coli, purified and characterized by mass spectrometry and circular dichroism. It was tested for IgE reactivity in 1322 HDM-allergic patients. Detailed IgE-reactivity profiles to six HDM allergens (Der p 1, 2, 5, 7, 10, 21) were established for subgroups of Der p 10-positive and -negative patients. The allergenic activity of rDer p 10 was evaluated in basophil degranulation experiments. RESULTS: rDer p 10 is an α-helical protein sharing IgE epitopes with nDer p 10. It is recognized by 15.2% of HDM-allergic patients. Der p 10-negative patients were primarily sensitized to Der p 1 and/or Der p 2, whereas Der p 10-positive patients reacted to several other HDM allergens besides the major allergens (Der p 1, Der p 2) or showed a rather selective Der p 10 reactivity. The allergenic activity of Der p 10 was generally low but patients could be identified who suffered from clinically relevant HDM allergy due to Der p 10 sensitization. CONCLUSION AND CLINICAL RELEVANCE: Der p 10 may be a diagnostic marker for HDM-allergic patients with additional sensitization to allergens other than Der p 1 and Der p 2. Such patients may require attention when allergen-specific immunotherapy is considered.

Recombinant allergen-based monitoring of antibody responses during injection grass pollen immunotherapy and after 5 years of discontinuation.

BACKGROUND: Subcutaneous injection immunotherapy (SCIT) is considered as antigen-specific and disease-modifying treatment with long-lasting effect. METHODS: We used a panel of recombinant grass pollen allergens for analyzing allergen-specific IgE, IgG(1) -IgG(4) , IgM, IgA, and light-chain (kappa, lambda) responses in grass pollen-allergic patients who had received one course of injection immunotherapy (SCIT) with an aluminum hydroxide-adsorbed grass pollen extract or only anti-inflammatory treatment. Serum samples were analyzed before and after 5 months of treatment as well as after 5 years. RESULTS: After 5 months of SCIT but not of anti-inflammatory treatment, IgG(1) > IgG(4) > IgG(2) > IgA antibody responses using both kappa and lambda light chains specific for major grass pollen allergens (Phl p 1, Phl p 5, Phl p 6, Phl p 2) increased significantly, whereas specific IgM or IgG(3) levels were unaltered. Allergen-dependent basophil degranulation was only inhibited with SCIT sera containing therapy-induced allergen-specific IgG antibodies. Likewise, decreases in Phl p 1- and Phl p 5-specific IgE levels and significant (P<0.001) reduction in symptom and medication scores were found only in the SCIT group but not in the group of patients receiving anti-inflammatory treatment. After 5 years, allergen-specific IgG antibody levels in the SCIT group had returned to baseline levels and there was no significant difference regarding symptoms between the SCIT and non-SCIT groups. CONCLUSION: The results from our observational study demonstrate that only SCIT but not anti-inflammatory treatment induces allergen-specific IgG and reduces boosts of allergen-specific IgE production but that one SCIT course was not sufficient to achieve long-term immunological and clinical effects.

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.

Reducing allergenicity by altering allergen fold: a mosaic protein of Phl p 1 for allergy vaccination.

BACKGROUND: The major timothy grass pollen allergen, Phl p 1, resembles the allergenic epitopes of natural group I grass pollen allergens and is recognized by more than 95% of grass-pollen-allergic patients. Our objective was the construction, purification and immunologic characterization of a genetically modified derivative of the major timothy grass pollen allergen, Phl p 1 for immunotherapy of grass pollen allergy. METHODS: A mosaic protein was generated by PCR-based re-assembly and expression of four cDNAs coding for Phl p 1 fragments and compared to the Phl p 1 wild-type by circular dichroism analysis, immunoglobulin E (IgE)-binding capacity, basophil activation assays and enzyme-linked immunosorbent assay competition assays. Immune responses to the derivative were studied in BALB/c mice. RESULTS: Grass-pollen-allergic patients exhibited greater than an 85% reduction in IgE reactivity to the mosaic as compared with the Phl p 1 allergen and basophil activation experiments confirmed the reduced allergenic activity of the mosaic. It also induced less Phl p 1-specific IgE antibodies than Phl p 1 upon immunization of mice. However, immunization of mice and rabbits with the mosaic induced IgG antibodies that inhibited patients' IgE-binding to the wild-type allergen and Phl p 1-induced degranulation of basophils. CONCLUSION: We have developed a strategy based on rational molecular reassembly to convert one of the clinically most relevant allergens into a hypoallergenic derivative for allergy vaccination.

Fusion proteins consisting of Bet v 1 and Phl p 5 form IgE-reactive aggregates with reduced allergenic activity.

The cross-linking of effector cell-bound IgE antibodies by allergens induces the release of inflammatory mediators which are responsible for the symptoms of allergy. We demonstrate that a recombinant hybrid molecule consisting of the major birch (Bet v 1) and grass (Phl p 5) pollen allergen exhibited reduced allergenic activity as compared to equimolar mixes of the isolated allergens in basophil activation experiments. The reduced allergenic activity of the hybrid was not due to reduced IgE reactivity as demonstrated by IgE binding experiments using sera from allergic patients. Physicochemical characterization of the hybrid by size exclusion chromatography, dynamic light scattering, negative-stain electron microscopy and circular dichroism showed that the hybrid occurred as folded aggregate whereas the isolated allergens were folded monomeric proteins. IgG antibodies raised in rabbits against epitopes of Bet v 1 and Phl p 5 showed reduced reactivity with the hybrid compared to the monomeric allergens. Our results thus demonstrate that aggregation can induce changes in the conformation of allergens and lead to the reduction of allergenic activity. This is a new mechanism for reducing the allergenic activity of allergens which may be important for modifying allergens to exhibit reduced side effects when used for allergen-specific immunotherapy.

Immunologic analysis of monoclonal and immunoglobulin E antibody epitopes on natural and recombinant Amb a 1.

BACKGROUND: Amb a 1 is the major allergen from ragweed pollen and more than 90% of ragweed-allergic patients react with this protein. Although Amb a 1 was cloned and sequenced in 1991, little is known of the specificity of anti-Amb a 1 antibodies or of the immunologic properties of the recombinant allergen. OBJECTIVE: To compare binding of monoclonal antibodies (mAb) and IgE antibodies to purified natural Amb a 1 (nAmb a 1) and recombinant Amb a 1 (rAmb a 1). METHODS: Binding of a panel of anti-Amb a 1 mAb and IgE antibodies to nAmb a 1 or rAmb a 1 was compared by immunoblotting. Chimeric ELISA was used to measure specific IgE to these allergens using 89 ragweed-allergic sera from Austria, Italy, Canada and the United States. RESULTS: The 8 mAb bound to a 38 kDa Amb a 1 band in ragweed pollen extract and a subset of 5 mAb also bound to the 26 kDa chain of nAmb a 1. A two-site ELISA was developed using a mAb pair, which was approximately 10-fold more sensitive to rAmb a 1. There was a significant correlation between IgE antibody binding to nAmb a 1 and rAmb a 1 (n=89, r=0.79, P<0.001). A subset of approximately 40% of patients showed greater reactivity to nAmb a 1 than to rAmb a 1. CONCLUSIONS: The data suggest that there is less reactivity of human IgE to rAmb a 1 compared with nAmb a 1. The development of more sensitive, quantitative ELISA for Amb a 1 will require the production of new mAb especially directed against nAmb a 1.

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.

Antigenic competition in the immune response against protein mixtures: strain-specific non-immunogenicity of Escherichia coli antigens.

Antigenic competition is argued to impair the immune response on the level of accessory cell-dependent antigen presentation to responsive T-cells (regulated by MHC encoded Ir gene products). A possible influence of these mechanisms on in vivo immunization with antigen mixtures was investigated by using cytoplasmic extracts of four different strains of Escherichia coli as antigen sources for immunizing rabbits. The immune response against these antigen mixtures was tested by crossed immune electrophoresis (CIE) and immunoblotting (IB) in a homologous system (a given antigen extract of one strain against the corresponding antisera) and in the heterologous system (antigen extract of one strain against the antisera of different other strains). Several proteins were non-immunogenic in the extract of one strain but elicited good antibody responses in other strains. One of the strain-specific non-immunogenic proteins was purified and revealed a normal immune response upon immunization. The data suggest that different antigenic competition effects are induced by different protein compositions of antigen mixtures. This strain-specific competition seems to determine the immunogenicity of certain molecules (and not only the immunogenic properties of the molecules themselves). Furthermore this method offers a practical approach to increase the antibody production against weak immunogens in antigen mixtures.

Prophylactic mRNA Vaccination against Allergy Confers Long-Term Memory Responses and Persistent Protection in Mice.

Recently, mRNA vaccines have been introduced as a safety-optimized alternative to plasmid DNA-based vaccines for protection against allergy. However, it remained unclear whether the short persistence of this vaccine type would limit memory responses and whether the protective immune response type would be maintained during recurrent exposure to allergen. We tested the duration of protective memory responses in mice vaccinated with mRNA encoding the grass pollen allergen Phl p 5 by challenging them with recombinant allergen, 3.5, 6, and 9 months after vaccination. In a second experiment, vaccinated mice were repeatedly challenged monthly with aerosolized allergen over a period of 7 months. Antibody and cytokine responses as well as lung inflammation and airway hyperresponsiveness were assessed. mRNA vaccination induced robust TH1 memory responses for at least 9 months. Vaccination efficiently suppressed TH2 cytokines, IgE responses, and lung eosinophilia. Protection was maintained after repeated exposure to aerosolized allergen and no TH1 associated pathology was observed. Lung function remained improved compared to nonvaccinated controls. Our data clearly indicate that mRNA vaccination against Phl p 5 induces robust, long-lived memory responses, which can be recalled by allergen exposure without side effects. mRNA vaccines fulfill the requirements for safe prophylactic vaccination without the need for booster immunizations.

The lipocalin Xlcpl1 expressed in the neural plate of Xenopus laevis embryos is a secreted retinaldehyde binding protein.

The cellular and structural properties and binding capabilities of a lipocalin expressed in the early neural plate of Xenopus laevis embryos and the adult choroid plexus have been investigated. It was found that this lipocalin, termed Xlcpl1, binds retinal at a nanomolar concentration, retinoic acid in the micromolar range, but does not show binding to retinol. Furthermore, this protein also binds D/L thyroxine. The Xlcpl1 cDNA was expressed in cell culture using the vaccinia virus expression system. In AtT20 cells, Xlcpl1 was secreted via the constitutive secretory pathway. We therefore assume that cpl1 binds retinaldehyde during the transport through the compartments of the secretory pathway that are considered to be the storage compartments of retinoids. Therefore, cpl1-expressing cells will secrete the precursors of active retinoids such as retinoic acid isomers. These retinoids may enter the cytosol by diffusion or receptor-controlled mechanisms, as has been shown for exogenously applied retinoids. Based on these data, it is suggested that cpl1 is an integral member of the retinoid signaling pathway and, therefore, it plays a key role in pattern formation in early embryonic development.

Renaissance of the biologically active vitamin A derivatives: established and novel directed therapies for cancer and chemoprevention.

Vitamin A and its biologically active derivatives are involved in a complex arrangements of physiological and developmental responses in many tissue of higher vertebrates. Retinoids are natural and synthetic compounds related to retinoic acid that act through interaction with two basic types of nuclear receptors: retinoic acid receptors (RAR alpha, RARbeta and RARgamma) and retinoid X receptors (RXRalpha, RXRbeta and RXRgamma) as retinoid-inducible transcription factors. Thus, the retinoid receptors are considered to be ligand-activated, DNA-binding, trans-acting, transcription-modulating proteins involved in a general molecular mechanism responsible for transcriptional responses in target genes. They exert both beneficial and detrimental activity; they have tumor-suppressive activity but on the other hand they are teratogenic. Retinoids inhibit carcinogenesis, suppress premalignant epithelial lesions and tumor growth and invasion in a variety of tissues. Natural and synthetic retinoids have therapeutical effects due to their antiproliferative and apoptosis-inducing effects. They are known to cause redifferentiation or to prevent further dedifferentiation of various neoplastic tissues. A number of novel chemical compounds, receptor selective retinoids and rexinoids, have been synthesized up to now and tested both in vitro and in vivo, using animal models against different cancer cells. In spite of that progress, there is still an urgent call for novel synthetic retinoids and rexinoids with greater retinoid receptor selectivity, reasonable chemotherapeutic or chemopreventive effects and reduced toxicity and side effects. This article summarizes selected effects of biologically active natural or synthetic retinoids and rexinoids, acting through their cognate nuclear receptors, and their use in chemotherapy and chemoprevention of various types of cancer.

Nucleic acid for the treatment of cancer: genetic vaccines and DNA adjuvants.

Despite some interesting pilot experiments more than a century ago, nucleic acid has only recently been added to the list of agents used for the prevention and therapy of cancer. Two distinct features of nucleic acids are used for this purpose: in DNA and RNA vaccines, genetic information for pathogen- or tumor-derived antigens is delivered to the host who then produces the encoded antigen and initiates an immune response. In DNA adjuvants, immunostimulatory sequences (CpG motifs) present in DNA of bacterial origin are used. Such sequences are delivered in the form of oligonucleotides or within the sequence of DNA vaccine. In addition, CpG oligonucleotides by themselves have successfully been used to stimulate the immune system in an antigen-independent manner for the treatment of experimental tumors. DNA and RNA vaccines for the treatment and prevention of cancer and other diseases suffer from two some shortcomings: insufficient immunogenicity and--in the case of RNA--low stability. A variety of strategies are being explored to improve the efficacy of nucleic acid vaccines (genetic vaccines) especially for self-antigens in the case of cancer. Among the most recent improvements are self-replicating RNA vaccines and replicase-based DNA-vaccines in which antigen expression is under the control of an alphaviral replicase. Despite highly promising results in many animal tumor models the efficacy of nucleic acid vaccines and adjuvants in the clinic remains to be seen.

Cascade immunization: a method of obtaining polyspecific antisera against crude fractions of antigens.

Between 20 and 30 precipitation lines are usually obtained by crossed immune electrophoresis of an Escherichia coli cytoplasmic extract against antisera produced against that extract in individual rabbits. With a combination of several such antisera, the number of precipitation lines increases to 30-40. Nevertheless, extracts as used in this work contain many antigens in addition to those thus detected. Intermolecular immune competition may be avoided by removing strong immunogens from the extracts. The remaining antigens which give no immune response in the primary immunization are used for further immunization. New antibody production against 8-14 additional antigens occurs after one such 'cascade immunization' step. Separation of strong from weak immunogens is performed by preparative CIE and the use of immuno-affinity columns. The procedure is called cascade immunization because it involves repeated removal of antigens and production of further antisera directed against antigens in the remainder.