Safety, efficacy and repeatability of a novel house dust mite allergen challenge technique in the Fraunhofer allergen challenge chamber.

BACKGROUND: Efficacy testing of immunotherapy in field studies is often hampered by variation of airborne allergens. Standardized allergen exposure in challenge chamber settings might be an alternative. Therefore, we developed a universal technique to create an atmosphere loaded with allergen particles of adjustable size from aqueous solutions of licensed allergen extracts. OBJECTIVE: The aim of this study was to apply this technique and test the safety and efficacy of challenges with house dust mite (HDM) allergen in the Fraunhofer allergen challenge chamber. METHODS: Aerosol particles carrying HDM allergen were produced by spray-drying of an aqueous solution containing HDM allergen and lactose. In a monocenter, placebo-controlled, single-blind, dose-escalation pilot study, 18 subjects with perennial allergic rhinitis and sensitization to HDM were exposed to HDM allergen for 4 h at either 250, 500, 1000 SQE/m3 or lactose alone (0 SQE/m3 ) 7 days apart. The dose of 500 SQE/m³ was repeated to investigate reproducibility. Total nasal symptom score (TNSS) was the primary endpoint. RESULTS: Exposure to HDM increased TNSS (mean ± SD) to 3.4 ± 1.8, 3.3 ± 2.1, and 3.6 ± 2.0 at 250, 500 and 1000 SQE/m3 , respectively, while lactose alone did not change TNSS (0.7 ± 0.6). The results were reproducible at 500 SQE/m3 . Pulmonary function and adverse event frequency did not change with escalation of allergen dose. CONCLUSION: This HDM allergen particle generation is safe, specific and reproducible and can therefore be used for efficacy testing of immunotherapy and for basic clinical research.

Relevance of an academic GMP Pan-European vector infra-structure (PEVI).

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

Large scale purification of gangliosides GM3(Neu5Ac) and GM3(Neu5Gc) by trimethylaminoethyl-Fractogel high-performance liquid chromatography.

A preparative anion-exchange high-performance liquid chromatographic method for the separation of the closely allied monosialogangliosides GM3(Neu5Ac) and GM3(Neu5Gc) has been developed. Hybridoma cells, readily available material derived from industrial monoclonal antibody production, were used as ganglioside source and led to fractions with pure GM3(Neu5Ac) and GM3(Neu5Gc) in high milligram quantities. The crude ganglioside extract was loaded onto columns filled with the strong anion-exchanger trimethylaminoethyl (TMAE)-Fractogel. Gangliosides were eluted from the stationary phase with a gradient system of ammonium acetate in methanol. The scaled-up approach ranged over more than one order of magnitude from 20 to 500 mg batches of GM3 gangliosides. Thus, the high-resolution power of the strong anion-exchanger TMAE-Fractogel allowed the preparative isolation by one-step column chromatography of two GM3 specimens which only differ in one hydroxyl group at position 5 of the neuraminic acid (N-acetyl-versus N-glycolylneuraminic acid).

Enhanced thin-layer chromatographic separation of GM1b-type gangliosides by automated multiple development.

Enhancement in separation of gangliosides on silica gel precoated high-performance TLC plates has been obtained by automated multiple development chromatography. A less polar mixture of the standard solvent chloroform-methanol-20 mM aqueous CaCl2 (120:85:20, v/v) was used. Lowering the water content achieved separation of two complex monosialoganglioside fractions, isolated from murine YAC 1 T lymphoma and MDAY-D2 lymphoreticular cells. Three-fold chromatography in the solvent chloroform-methanol-20 mM aqueous CaCl2 (120:85:14, v/v) resulted in TLC separation of GM1b-type gangliosides, substituted with C24 and C16 fatty acids and with Neu5Ac and Neu5Gc as well, which could not be achieved by unidirectional standard chromatography. Compared to conventional single chromatography, the technique described allows high-resolution separation of extremely heterogenous ganglioside mixtures and offers a convenient tool for both analytical and preparative TLC.

Immobilization of microbial cells by adsorption.

Immobilized cells cover a wide area of applications and are essential components of many biotechnological processes. In general it can be distinguished between two immobilization methods: (1) entrapment into polymers and (2) natural adsorption onto porous and inert support materials. The immobilization by adsorption is discussed by the following criteria: biomass loading, strength of adhesion, enzymatic stability/specific activity of the biocatalyst, effectivity/reaction engineering and operational stability.

Rapid detection of extended ganglio-series gangliosides with terminal GalNAc beta 1-4Gal sequence on high performance thin layer chromatography plates.

The mouse monoclonal antibody 2D4, which recognizes the terminal GalNAc beta 1-4Gal-disaccharide of GgOse3Cer and GgOse5Cer, was used for the detection of ganglio-series gangliosides. The method involves separation of gangliosides on thin layer chromatography plates, followed by silica gel fixation, Arthrobacter ureafaciens neuraminidase treatment and final immunostaining of desialylated gangliosides with the monoclonal antibody 2D4. Both neuraminidase and the hybridoma 2D4 producing the specific monoclonal antibody are commercially available and therefore accessible to all researchers working in this field. Gangliosides from mouse T lymphocytes and the mouse T cell lymphoma YAC-1 have been used as examples. This technique may be used for fast screening of gangliosides with the GgOse5Cer core structure which have been described as T cell markers, antigens in human neuronal disease and receptors for certain pathogenic bacteria.

Physical methods for characterization of microbial surfaces.

There are different concepts for explaining the adsorption of microorganisms to solid surfaces: the DLVO theory and the surface free energy. Basic aspects of both theories are discussed. Established methods for determining the surface properties of microbial cells are reviewed: Electrophoretic mobility, colloid titration, electrostatic interaction chromatography, bacterial adherence to hydrocarbons, partitioning in an aqueous two-phase system, hydrophobic interaction chromatography, contact angle measurement and X-ray photoelectron spectroscopy. They are discussed and classified according to their potential for the correlation of cell surface characteristics and adsorption behavior.

Continuous production of L-phenylalanine by transamination.

L-Phenylalanine was produced continuously from L-as-partate and phenylpyruvate by transaminase from a newly screened Pseudomonas putida strain. The process was carried out with an isolated enzyme in homogeneous phase in an enzyme membrane reactor and with immobilized whole cells in a stirred tank reactor, respectively. Due to the difference in transport resistance, the productivity of the free enzyme in homogeneous phase (72 mmol/L h) was about 3 times higher than the productivity achieved using immobilized cells. However, a better stability of the biocatalyst was observed with immobilized cells.