Altersolanol A: a selective cytotoxic anthraquinone from a Phomopsis sp.

UNLABELLED: The cytotoxic compound Altersolanol A, an anthraquinone derivative was isolated from PM0409092 a fungus of Nyctanthes arbor-tristis (family Oleaceae). It was identified as a Phomopsis sp. by DNA amplification and sequencing of the ITS region. The chemical structure of Altersolanol A was elucidated from its physicochemical properties, 2D NMR spectroscopy and other spectroscopic data. The compound has in vitro cytotoxic activity against 34 human cancer cell lines with mean IC50 (IC70) values of 0.005 µg ml(-1) (0.024 µg ml(-1)) respectively. Altersolanol A, a kinase inhibitor, induces cell death by apoptosis through the cleavage by Caspase-3 and -9 and by decreased anti-apoptotic protein expression. There are several previous reports of the anticancer activity of Altersolanol A, but we report here an extensive study using 36 cell lines which gives wider spectrum of results. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms the cytotoxic potential of Altersolanol A isolated from the endophyte Phomopsis sp. (PM0409092) of the plant Nyctanthes arbor-tristis. The compound exhibits in vitro cytotoxicity against 34 human cancer cell lines with mean IC50 (IC70) value of 0.005 µg ml(-1) (0.024 µg ml(-1)). This is an in-depth report of Altersolanol A against a panel of 34 human cancer cell lines and extends observations from previous studies indicating that Altersolanol A can be used for the development of chemotherapeutics. Altersolanol A, a kinase inhibitor, induces cell death by apoptosis through the cleavage of Caspase-3 and -9 and by decreased anti-apoptotic protein expression.

Establishment of recombinant major allergens Bet v 1 and Phl p 5a as Ph. Eur. reference standards and validation of ELISA methods for their measurement. Results from feasibility studies.

The potency of allergen extracts is determined as total allergenic activity without consideration of their composition and the units differ from one manufacturer to another, making it very difficult to compare the different products. Recently, purified major allergens have been obtained by recombinant DNA technology and produced under Good Manufacturing Practice (GMP) conditions. In principle, such recombinant allergens could be established as reference standards and could help for the standardisation of the major allergen content of allergen extracts. Two recombinant major allergens, one from birch pollen, rBet v 1, and one from Timothy grass pollen, Phl p 5a, have been selected at the end of the CREATE programme as a potential starting point for the establishment as European Pharmacopoeia (Ph. Eur.) Reference Standards through a project run by the Biological Standardisation Programme (BSP) of the European Directorate for the Quality of Medicines & HealthCare (EDQM). To this end, bulk candidate recombinant materials, produced under GMP conditions, were procured from two European manufacturers and subsequently formulated and lyophilised. Four ELISA systems from three different manufacturers were included in the project, two for Bet v 1 and two for Phl p 5a with the aim of establishing reference methods for determination of the respective major antigens both in natural allergen extracts as well as in recombinant allergen products. The project was run in 3 phases: a preparatory and preliminary testing phase (feasibility phase or Phase 1), an extended feasibility phase carried out in 3 laboratories (Phase 2) to confirm the transferability of the methods and an international collaborative study with a large number of participating laboratories (Phase 3). This article describes the work done in Phase 1 and Phase 2, i.e. the physico-chemical and biological characterisation of the recombinant candidate reference standards, the assessment of their suitability for the intended purpose as well as the evaluation of the candidate ELISA systems. The results show that both candidate reference standards are suitable for the intended purpose. In addition, three out of the four ELISA systems that were included in the preliminary phase were found to be appropriate for further evaluation in the collaborative study which was organised in 2011. The results of the collaborative study will be published separately.

Telomerase activity and telomere length in human tumor cells with acquired resistance to anticancer agents.

Telomeres and telomerase are targets for anticancer drug development and specific inhibitors are currently under clinical investigation. However, it has been reported that standard cytotoxic agents can affect telomere length and telomerase activity suggesting that they also have of a role in drug resistance. in this study, telomere lengths and telomerase activity as well as drug efflux pump expression, glutathione (GSH) levels and polyadenosine-ribose polymerase (PARP) cleavage were assessed in a panel of human tumor cell lines made resistant to vindesine, gemcitabine and cisplatin. these included two lung cancer cell lines resistant to vindesine (LXFL 529L/Vind, LXFA 526L/Vind), a renal cancer cell line (RXF944L/Gem) and an ovarian cancer cell line (AG6000) resistant to gemcitabine, and one resistant to cisplatin (ADDP). The resistant clones were compared to their parental lines and evaluated for cross resistance to other cytotoxic agents. Several drug specific resistance patterns were found, and various complex patterns of cross resistance emerged from some cell lines, but these mechanisms of resistance could not be related to drug efflux pump expression, GSH levels or pARp cleavage. However, all displayed changes in telomerase activity and/or telomere length. Our studies present evidence that telomere maintenance should be taken into consideration in efforts not only to overcome drug resistance, but also to optimize the use of telomere-based therapeutics.

Clinical effects of immunotherapy with genetically modified recombinant birch pollen Bet v 1 derivatives.

BACKGROUND: Birch pollen and pollen from related trees of the Fagales order are a major cause of allergic rhinitis, conjunctivitis, and asthma through the spring season in northern and central Europe. OBJECTIVE: To investigate the clinical effects of injection immunotherapy with genetically modified derivatives of major birch pollen allergen Bet v 1 on pollen-induced allergic symptoms. METHODS: A three-arm double-blind placebo-controlled immunotherapy study was conducted with one pre-seasonal course of treatment using two derivatives of Bet v 1, namely a recombinant Bet v 1 trimer and an equimolar mixture of two recombinant Bet v 1 fragments together representing the whole protein sequence. Analysis of local and systemic adverse events was performed for 124 patients who had received at least one dose of medication. Clinical efficacy was monitored by symptom medication scores and interval scoring in the per protocol-treated population (n=84). In addition, skin and nasal provocation responses and allergen-specific antibodies were assessed. RESULTS: There were trends towards improvement in the subjects' well-being and clinical symptoms (nasal scores), although comparisons with a placebo group did not show statistical significance in the main end-point, the combined symptom medication score. Reductions in skin and nasal sensitivity were observed for some subjects with a trend for the Bet v 1 trimer to be more effective than the fragments. Treatment induced strong IgG1 and IgG4 allergen-specific antibody responses. Local injection-site reactions were most frequent in the trimer group affecting 59.5% of patients as opposed to 37.8% and 30.6% in the fragment and placebo groups, respectively. Systemic reactions were elicited more frequently by fragments. A large proportion of adverse side-effects appeared hours following injections, and might be attributable to concurrent exposure to related pollens. CONCLUSION: Single courses of injection immunotherapy with Bet v 1 allergen derivatives showed trends towards improved well-being and reduced reactivity to specific allergen provocation, but did not yield significant improvement in the combined symptom medication score in this study.

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.

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Characterization of a hypoallergenic recombinant Bet v 1 variant as a candidate for allergen-specific immunotherapy.

BACKGROUND: Recombinant allergens and especially their hypoallergenic variants are promising candidates for a more effective and safer specific immunotherapy. METHODS: Physicochemical and immunological characteristics of a folding variant of recombinant Bet v 1 (rBet v 1-FV) were investigated in comparison to natural Bet v 1 (nBet v 1) and the correctly folded recombinant Bet v 1 (rBet v 1-WT) by SDS-PAGE, size exclusion chromatography, multi-angle light scattering, circular dichroism, immunoblotting and enzyme allergosorbent test inhibition assay for detection of IgE reactivity and ELISA with Bet v 1-specific monoclonal antibodies. The functional IgE reactivity of the different Bet v 1 proteins was investigated using basophil activation in terms of CD203c expression and histamine release. T cell reactivity was investigated using T cell lines raised from birch pollen-allergic subjects against nBet v 1. Immunogenicity was investigated in mice. RESULTS: Physicochemical characterization revealed purity, homogeneity and monomeric properties of rBet v 1-FV. Unlike nBet v 1 and rBet v 1-WT, rBet v 1-FV showed almost no IgE binding in immunoblots. The reduction of allergenicity was further proved by IgE-binding inhibition assays, basophil activation and histamine release. T cell reactivity was completely conserved, as demonstrated by proliferation of Bet v 1-specific T cell lines with multiple epitope specificities. rBet v 1-FV showed strong immunogenicity in mice. CONCLUSIONS: Due to its reduced IgE reactivity and decreased capacity to activate basophils, but retained T cell reactivity and strong immunogenicity, rBet v 1-FV proved to be a very promising candidate for specific immunotherapy in birch pollen-allergic subjects.

Generation of a low immunoglobulin E-binding mutant of the timothy grass pollen major allergen Phl p 5a.

BACKGROUND: Immunotherapy of grass pollen allergy is currently based on the administration of pollen extracts containing natural allergens. Specifically designed recombinant allergens with reduced IgE reactivity could be used in safer and more efficacious future therapy concepts. OBJECTIVES: This study aimed to generate hypoallergenic variants of the timothy grass major allergen Phl p 5a as candidates for allergen-specific immunotherapy. METHODS: Three deletion mutants were produced in Escherichia coli and subsequently purified. The overall IgE-binding capacity of the mutants was compared with the recombinant wild-type allergen by membrane blot and IgE-inhibition assays. The capacity for effector cell activation was determined in basophil activation assays. T cell proliferation assays with allergen-specific T cell lines were performed to confirm the retention of T cell reactivity. Structural properties were characterized by circular dichroism analysis and homogeneity by native isoelectric focusing. The deletion sites were mapped on homology models comprising the N- and C-terminal halves of Phl p 5a, respectively. RESULTS: The double-deletion mutant rPhl p 5a Delta(94-113, 175-198) showed strongly diminished IgE binding in membrane blot and IgE-inhibition assays. Both deletions affect predominantly alpha-helical regions located in the N- and C-terminal halves of Phl p 5a, respectively. Whereas deletion of Delta175-198 alone was sufficient to cause a large reduction of the IgE reactivity in a subgroup of allergic sera, only the combination of both deletions was highly effective for all the sera tested. rPhl p 5a Delta(94-113, 175-198) consistently showed at least an 11.5-fold reduced capacity to activate basophils compared with the recombinant wild-type molecule, and the T cell proliferation assays demonstrated retention of T cell reactivity. CONCLUSION: The mutant rPhl p 5a Delta(94-113, 175-198) fulfils the basic requirements for a hypoallergenic molecule suitable for a future immunotherapy of grass pollen allergy; it offers substantially reduced IgE binding and maintained T cell reactivity.

Preclinical pharmacology of the pyrrolobenzodiazepine (PBD) monomer DRH-417 (NSC 709119).

The pyrrolobenzodiazepine monomer DRH-417 is a member of the anthramycin group of anti-tumor antibiotics that bind covalently to the N2 of guanine within the minor groove of DNA. DRH-417 emerged from the EORTC-Drug Discovery Committee and NCI 60 cell line in vitro screening programs as a potent antiproliferative agent with differential sensitivity towards certain cancer types such as melanoma, breast and renal cell carcinoma (mean IC(50) = 3 nM). DRH-417 was therefore tested for in vivo activity. The maximum tolerated dose (MTD) was established as 0.5 mg/kg given i.p. Marked anti-tumor activity was seen in two human renal cell cancers, one breast cancer and a murine colon tumor model (p<0.01). A selective HPLC (LC/MS) analytical method was developed and plasma pharmacokinetics determined. At a dose of 0.5 mg kg(-1), the plasma AUC was 540 nM h (197.1 ng h ml(-1)) and the peak plasma concentration (171 nM [62.4 ng ml(-1)]) occurred at 30 min., reaching doses levels well above those needed for in vitro antiproliferative activity. Genomic profiling of in vivo sensitive tumors revealed that the latter have an activated insulin-like growth factor signaling pathway.

Anti-tumour activity in non-small cell lung cancer models and toxicity profiles for novel ruthenium(II) based organo-metallic compounds.

Novel ruthenium(II) organo-metallic compounds are active in ovarian cancer models [Aird RE, Cummings J, Ritchie AA, Muir M, Morris RE, Chen H, et al. In vitro and in vivo activity and cross resistance profiles of novel ruthenium(II) organometallic arene complexes in human ovarian cancer. Br J Cancer 2002;86(10):1652-7]. [(eta6-C6H5C6H5)Ru(en)Cl]+ (as a PF6 salt, where en=ethylenediamine (RM175)) has been evaluated in a 13-cell line panel. Particular sensitivity (approximately 10-fold lower than mean IC50) was noted in breast cancer and non-small cell lung cancer cell lines. In addition, IC50 in the A549 was 2 microM and RM175 (25 mg kg-1, days 1 and 5, i.p.) caused a significant (p=0.004) growth delay in a xenograft model. HC11 [(eta6-tetrahydroanthracene)Ru(en)Cl]PF6 was more potent in the A549 cell line (IC50 0.5 microM). HC11 (25 mg kg-1, days 1, 8 and 15, i.p.) was also active in vivo. Following RM175 25 mg kg-1, days 1 and 5, and 15 mg kg-1, days 1-5, HC11 25 and 40 mg kg-1, day 1, elevated alanine transaminase levels were detected, suggesting hepatotoxicity. No changes were observed in kidney or haematological parameters. In liver sections, multi-focal hepatic necrosis was seen, becoming confluent at high doses of HC11. In vitro studies confirmed that HC11 was more toxic than RM175 to fresh human hepatocytes and equitoxic to mithramycin. Liver toxicity may be related to the arene ligand and modification may reduce the potential for hepatic toxicity, while maintaining the anti-tumour activity seen.

Anticancer activity of a lectin-rich mistletoe extract injected intratumorally into human pancreatic cancer xenografts.

BACKGROUND: In single case observations, tumour remissions after intratumoral injections of mistletoe extracts have been described. MATERIALS AND METHODS: We investigated the antitumour activity of intratumorally (i.t.)-injected lectin-rich mistletoe extract at different dosages and i.t.-injected mistletoe lectin I in comparison to intravenous (i.v.) Gemcitabine and i.t. treatment with placebo in a human pancreatic cancer xenograft. RESULTS: In a preliminary dose-response experiment, the most marked tumour inhibition was induced when mistletoe extract was given at 8 mg/kg body weight (BW) and mistletoe lectin I at 5.3 microg/kg BW. In a second experiment, bi-weekly i.t. injections of mistletoe extract over 8 weeks resulted in a very high antitumour activity with an optimal T/C value (=median relative tumour volume of the test group vs. the control) of 0.4% combined with 3/8 partial and 3/8 complete remissions. Gemcitabine was less active with 2/8 partial and 1/8 complete remissions and an optimal TIC of 4.6%. CONCLUSION: I.t.-injected lectin-rich mistletoe extract should be further evaluated in patients with inoperable locally advanced pancreatic cancer.

Vaccination with genetically engineered allergens prevents progression of allergic disease.

IgE-mediated allergy affects >25% of the population in industrialized countries. Repeated contact with the disease-eliciting allergens induces rises of allergen-specific IgE Abs and progression of the disease to more severe manifestations. Our study uses a type of vaccine that is based on genetically modified allergen derivatives to treat allergic patients. We developed hypoallergenic derivatives of the major birch pollen allergen, Bet v 1, by genetic engineering and vaccinated birch pollen-allergic patients (n = 124) in a double-blind, placebo-controlled study. Active treatment induced protective IgG Abs that inhibited allergen-induced release of inflammatory mediators. We also observed a reduction of cutaneous sensitivity as well as an improvement of symptoms in actively treated patients. Most important, rises of allergen-specific IgE induced by seasonal birch pollen exposure were significantly reduced in vaccinated patients. Vaccination with genetically engineered allergen derivatives is a therapy for allergy that not only ameliorates allergic reactions but also reduces the IgE production underlying the disease.

Antitumor activity of imidazothioxanthones in murine and human tumor models in vitro and in vivo.

BACKGROUND: A new series of imidazothioxanthones has recently been synthesized as potential anticancer agents with the aim of overcoming drug resistance. The route of synthesis and DNA-binding properties of the compounds were reported previously. This paper describes the general structure-activity relationships for the class of imidazothioxanthones in panels of human and murine tumor cell lines in vitro, and the in vivo activity against human and murine solid tumors of the most potent compound, N-[3-(Dimethylamino)propylo]-11-oxo-11H-benzothiopyrano [3',2':2,3]pyrido[1,2-a]imidazo-2-carboxamide (10a). In addition, the interaction between compound 10a and DNA is also considered in terms of molecular mechanics methods and flexible docking techniques. MATERIALS AND METHODS: The cytotoxicity of compounds 10a, 11-oxo-N-[2-(pyrrolidino)ethylo]-11H-benzothiopyrano [3',2':2,3]pyrido[1,2-a]imidazo-2-carboxamide, 11-oxo-N-[2-(piperidino)ethylo]-11H-benzothiopyrano [3',2':2,3]pyrido[1,2-a]imidazo-2-carboxamide and N-[2-(morpholino)ethylo]-11-oxo-11H-benzothiopyrano [3',2':2,3]pyrido[1,2-a]imidazo-2-carboxamide (10c-10e) was assessed in human tumor cell lines and xenografts using the sulforhodamine B assay, MTT assay and the clonogenic assay. The human ovarian xenograft, PXN/109TC, two human breast carcinomas, MT-1 and MCF-7, and the murine colon adenocarcinoma, MAC15A were used for the in vivo testing of compound 10a. In addition, the interaction between compound 10a and DNA is also considered in terms of molecular mechanics methods and flexible docking techniques. RESULTS: Two compounds, 10a and 10c, showed cytotoxic activity below 10 mM in the NCI in vitro screen of 60 human tumor cell lines. The IC50 value of compound 10a was 6.8 mM and that of 10c, 8.3 mM. In addition, both compounds possessed differential activity against leukemia, colon and mammary cancer. The activity pattern was confirmed in two further screens using monolayer and clonogenic, assays. In vivo antitumor studies showed that 10a was active against the human mammary carcinoma MT-1 and murine colon cancer MAC15A. Marginal activity was observed in human ovarian cancer model PXN/109T/C and the compound was inactive in human mammary cancer MCF-7. CONCLUSION: The results warrant further in vivo testing of 10a in additional human solid tumor models. The molecular modeling showed that the planarity of the chromophore and the side-chain conformation could assist the insertion of compound 10a between the base pairs of the double helix. On the other hand, docking to the nucleotide sequence GGAATTGCCTCA suggested that the molecule could also act as a minor groove binder.

Clonogenic assay with established human tumour xenografts: correlation of in vitro to in vivo activity as a basis for anticancer drug discovery.

Pluripotent cells can be grown in clonogenic assays. The tumour stem-cell fraction, which accounts for <0.4% of the total cells, and which is considered the most relevant cell type in the development of metastases and recurrences, is able to divide and to form colonies in a semisolid matrix (agar or methylcellulose). Major applications of the tumour clonogenic assay (TCA) are chemosensitivity testing of tumours and xenografts, and for assessments within drug discovery programmes. Of critical relevance for the usefulness of the TCA is whether it can predict sensitivity or resistance towards clinically used agents. When we compared the response of human tumours established as xenografts in nude mice in the TCA in vitro to that of the clinical response, 62% of the comparisons for drug sensitivity, and 92% of the comparisons for drug resistance were correct. The same percentage of true/false observations was found when tumours were tested after serial passage in nude mice in the TCA in vitro and their response compared to in vivo activity in corresponding xenografts (60% and 90%, respectively). The highest correct predictive values were, however, found when the clinical response of tumours was compared to their explants established in the nude mouse and treated in vivo. Of 80 comparisons performed, we observed a correct prediction for tumour resistance in 97% and for tumour sensitivity in 90%. In our opinion, the TCA with established human tumour xenografts has an important role in current drug discovery strategies. We therefore included the TCA as secondary assay in our approach to anticancer drug discovery and found that a number of novel agents were active; these are now in advanced preclinical development or clinical trials. Thus, the tumour clonogenic assay has proven predictive value in the chemosensitivity testing of standard and experimental anticancer drugs.

Vaccines for birch pollen allergy based on genetically engineered hypoallergenic derivatives of the major birch pollen allergen, Bet v 1.

BACKGROUND: We have recently engineered recombinant derivatives of the major birch pollen allergen Bet v 1 (rBet v 1 fragments and trimer) with strongly reduced allergenic activity. OBJECTIVE: The aim of this study was the in vivo characterization of potential allergy vaccines based on Al(OH)3-adsorbed genetically modified rBet v 1 derivatives in mice. METHODS: BALB/c mice were immunized either with courses of nine injections of increasing doses of Al(OH)3-adsorbed rBet v 1 wild-type, rBet v 1 fragments, rBet v 1 trimer or Al(OH)3 alone in weekly intervals or with three high-dose injections applied in intervals of 3 weeks. Humoral immune responses to rBet v 1 wild-type and homologous plant allergens were measured by ELISA and Western blotting, and the ability of mouse antibodies to inhibit the binding of allergic patients IgE to Bet v 1 was studied by ELISA competition experiments. RESULTS: In both schemes, hypoallergenic rBet v 1 derivatives induced low IgE but high IgG1 responses against rBet v 1 wild-type. The IgG1 antibodies induced by genetically modified rBet v 1 derivatives cross-reacted with natural Bet v 1 and its homologues from alder (Aln g 1) as well as hazel (Cor a 1) and strongly inhibited the binding of birch pollen allergic patients' IgE to Bet v 1 wild-type. CONCLUSION: Genetically modified hypoallergenic rBet v 1 derivatives induce blocking antibodies in vivo. Their safety and efficacy for the treatment of birch pollen and associated plant allergies can now be evaluated in clinical immunotherapy studies.

No evidence of stimulation of human tumor cell proliferation by a standardized aqueous mistletoe extract in vitro.

The in vitro antiproliferative or stimulatory activity of an aqueous mistletoe extract (AME) with a defined content of bioactive mistletoe lectin (ML) was investigated in 6 human tumor cell lines, including two melanomas and leiomyosarcomas, each of which had previously been reported to show evidence of growth stimulation if treated with low concentrations of isolated ML. The effects of AME were compared to that of the standard cytotoxic agent adriamycin (ADR) using the well established propidium iodide and sulforhodamin B proliferation assays. The AME concentrations used ranged from 0.5 pg to 5 ng (0.82 fMol-85 pM) bioactive ML/ml in melanoma (HT-144, SK-MEL-28) and leiomyosarcoma (SK-MLS-1, S-UT-1B) cell lines and from 0.1-100 ng ML/ml (1.7 pM-1.7 nM) in MCF-7 breast cancer and SW620 colon carcinoma cell lines, respectively. The influence of AME on cell growth was determined at various time-points from 24 hours to 6 days of exposure. We found a time- and cell line-dependent inhibition of tumor cell growth, but no reproducible stimulation of tumor cell proliferation. Inhibitory concentrations 50% (IC50) for e.g. the SK-MEL-28 melanoma cell line, decreased from 4.1 ng ML/ml at 24 hours to 0.16 ng ML/ml at 72 hours and 0.18 ng ML/ml at 5 days. Our data clearly demonstrate that, by applying scientifically valid methods and procedures, the standardized AME did not stimulate tumor cell proliferation but showed time- and concentration-dependent antiproliferative effects.

Measurement of basophil-activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivatives by flow cytometry using anti-CD203c.

BACKGROUND: The assessment of the basophil-activating potential is an important aspect in the development of improved preparations for specific immunotherapy. The aim of the study was to evaluate the suitability of CD203c expression as a measure of basophil activation to compare allergoids with original allergen extracts, and recombinant hypoallergenic allergen derivatives with recombinant wild-type and natural allergens. METHODS: Heparinized whole blood samples from grass pollen allergic subjects were stimulated with grass pollen allergens and allergen derivatives followed by labelling of the basophils with PE-conjugated anti-CD203c. After lysis of the erythrocytes and fixation, the basophils were detected by flow cytometry. In some experiments, histamine release was determined simultaneously. RESULTS: Grass pollen allergoids revealed a 10-10 000-fold reduction of basophil-activating capacity measured by CD203c expression. The deletion mutant DM4 of rPhl p 5b showed stronger hypoallergenic characteristics in a range of 50-10 000-fold reduction, whereas a combination mutant of rPhl p 5b and Phl p 6 revealed less hypoallergenic features. Histamine release experiments led to a similar outcome as CD203c measurement. CONCLUSIONS: The measurement of CD203c expression on basophils by flow cytometry provides a rapid and sensitive method for the estimation of the allergic or hypoallergenic features of allergen preparations. The results demonstrated the hypoallergenicity of grass pollen allergoids and of the rPhl p 5b variant DM4, which may be a candidate in future preparations for specific immunotherapy.

5-Fluorouracil in vesicular phospholipid gels for anticancer treatment: entrapment and release properties.

Vesicular phospholipid gels (VPG), i.e. highly concentrated liposomal dispersions, are suitable for entrapping substances such as anticancer drugs with particular high encapsulation efficiencies (EE). We prepared different formulations of VPG with 30% (w/w) lipid containing 5-fluorouracil (5-FU) by high pressure homogenization and analysed their EE and drug release. Using mixtures of hydrogenated soy phosphatidylcholine and cholesterol with molar ratios ranging from 55/45 to 75/25, a decreasing amount of cholesterol correlated with an increasing EE, which is probably due to a reduced amount of smaller vesicles and number of lamellae. Using a 5-FU solution of pH 8.6 for VPG preparation, an EE of approximately 40% was found after redispersion of the gel to a liposomal dispersion and separation of free drug from liposomal drug by size exclusion chromatography. The reduced EE for preparations with lower pH values was attributed to a fast initial drug release due to the increased drug lipophilicity below the pK(a) value of 8. After redispersion of a VPG of pH 8.0, an initially faster release of about a third of the entrapped drug was found during the first 20 min, followed by stable entrapment over many hours. The rapid initial release may be due to the portion of liposomes smaller than 40 nm in diameter, determined by photon correlation spectroscopy. Cryo electron microscopic pictures show a lentil-like shape of these small liposomes. The membrane defects on the edges are probably the reason for the very high initial drug release rate. The half-life time of the release of 5-FU from intact FU-VPG at both pH 7.4 and 8.0 was found to be in the order of 4-5 h and the kinetics are typical for matrix-controlled drug diffusion. The in vitro data of 5-FU loaded VPG suggest their applicability as implants with controlled release properties or, after redispersion, as intravenously injected liposomal formulations.

Rapid method for arrayed investigation of IgE-reactivity profiles using natural and recombinant allergens.

BACKGROUND: The availability of increasing numbers of purified natural and recombinant allergens offer the possibility for component-resolved characterization of IgE binding. To make use of this potential, fast and simple methods with high capacity have to be developed. METHODS: A laboratory multiscreen device was used in an innovative two-dimensional approach. In the first step, natural and recombinant allergens were immobilized onto the membrane using the sample chambers as application mask and, after blocking and rotating the membrane through 90 degrees, the same device was used to apply and incubate sera of allergic patients. Enzyme-linked immunoassay (ELISA) quantification of specific IgE was performed for purposes of comparison. RESULTS: Proteins were most efficiently bound onto nitrocellulose in 20 mM sodium hydroxide (NaOH). Up to 45 proteins or extracts could be investigated with a maximum of 45 sera in a single application, resulting in a resolution of 2,025 spots on one membrane with a size comparable to a standard Western blot. A high correlation for IgE-binding between natural and recombinant allergens was observed. Development of the membrane resulted in very evenly distributed square patterns. The results corresponded with the conventional ELISA measurements of specific IgE. CONCLUSIONS: The innovative usage of a standard incubation device for both application of proteins as well as screening of sera provides a simple high throughput method for the characterization of IgE binding to allergens. The results are important for component resolved diagnosis of allergy by means of fast monitoring of IgE- and IgG-reactivity spectra. Recombinant allergens may be used as targets for these purposes.