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The current paradigm in vaccine development is that nonreplicating vaccines delivered parenterally fail to induce immune responses in mucosal tissues. However, both clinical and experimental data have challenged this concept, and numerous studies have shown that induction of mucosal immune responses after parenteral vaccination is not a rare occurrence and might, in fact, significantly contribute to the protection against mucosal infections afforded by parenteral vaccines. While the mechanisms underlying this phenomenon are not well understood, the realization that parenteral vaccination can be an effective means of inducing protective mucosal responses is paradigm-shifting and has potential to transform the way vaccines are designed and delivered.

A typical lipidomics approach aims at the simultaneous analysis of a multitude of lipid species from different lipid classes with highest possible sensitivity for all target lipids. Efficient extraction of lipids from the biological matrix is a crucial step in the analytical workflow. Whereas numerous applications of classical and more recently published extraction methods have been reported for blood serum or plasma samples, very little is known about the applicability of these methods for cerebrospinal fluid (CSF). CSF though represents a highly interesting biofluid for the investigation of neurological disorders, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, or brain cancer. Since CSF comprises substantially lower endogenous lipid concentrations compared to serum or plasma, the use of highly efficient extraction methods is of utmost importance. In addition, literature on lipid extraction methods is often inconsistent in terms of methodological parameters like temperature, mixing times, or the number of repeated extraction cycles. In this study, four liquid-liquid extraction methods (Folch, Bligh & Dyer, MTBE and BUME) and one protein precipitation method (MMC method) were evaluated using a pooled CSF sample, followed by the investigation of key process parameters (temperature and mixing times) and modifications of the most promising methods, in order to achieve a broad coverage of lipid classes as well as high recoveries and repeatabilities. A modified Folch method turned out as most suitable for the efficient extraction of a broad range of lipid classes from CSF including glycerophospholipids, glycerolipids and sphingolipids. In addition, using cooled solvents and equipment was shown to significantly improve lipid extraction efficiencies. Mixing times should be thoroughly optimized for the lipid classes of interest in order to achieve high recoveries without lipid degradation due to unnecessarily long mixing. Finally, acidification led to improved extraction efficiency for acidic glycerophospholipids.

Zusammenfassung: INQUAM e.V. fördert den Excellencegedanken und unterstützt medizinische Laboratorien auf dem Weg zur ersten Stufe des European Foundation for Quality Management (EFQM)-Excellence Modells „Committed to Excellence“ mit der angepassten Selbstbewertungsmatrix für medizinische Laboratorien.

Despite the fact that the adjuvant properties of the heat-labile enterotoxins of Escherichia coli (LT) and Vibrio cholerae (CT) have been known for more than 20 years, there are no available oral vaccines containing these molecules as adjuvants, primarily because they are both very potent enterotoxins. A number of attempts with various degrees of success have been made to reduce or eliminate the enterotoxicity of LT and CT so they can safely be used as oral adjuvants or immunogens. In this report we characterize the structural, enzymatic, enterotoxic, and adjuvant properties of a novel mutant of LT, designated LT(R192G/L211A), or dmLT. dmLT was not sensitive to trypsin activation, had reduced enzymatic activity for induction of cyclic AMP in Caco-2 cells, and exhibited no enterotoxicity in the patent mouse assay. Importantly, dmLT retained the ability to function as an oral adjuvant for a coadministered antigen (tetanus toxoid) and to elicit anti-LT antibodies. In vitro and in vivo data suggest that the reduced enterotoxicity of this molecule compared to native LT or the single mutant, LT(R192G), is a consequence of increased sensitivity to proteolysis and rapid intracellular degradation in mammalian cells. In conclusion, dmLT is a safe and powerful detoxified enterotoxin with the potential to function as a mucosal adjuvant for coadministered antigens and to elicit anti-LT antibodies without undesirable side effects. Bacterially derived enterotoxins, such as the heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli (ETEC) and the closely related cholera enterotoxin (CT) produced by Vibrio cholerae, promote secretory diarrhea during microbial infection. Each enterotoxin is composed of an enzymatically active A-subunit noncovalently associated with a pentameric B-subunit. Induction of intestinal fluid secretion occurs after a series of events involving both changes to toxin structure and activation of intracellular signaling pathways (reviewed in reference 14). The B-subunit mediates binding and internalization of the toxin to cells; subsequent proteolytic cleavage and disulfide bond reduction separate the A-subunit into two domains, the enzymatically active A1 subunit and a smaller A2 peptide. Transport of A1 into the cytoplasm results in ADP-ribosylation of Gs, followed by irreversible activation of adenylate cyclase and increases in intracellular levels of cyclic AMP (cAMP). In intestinal epithelial cells, this induction of cAMP causes a disregulation of cAMP-sensitive ion transport mechanisms, inhibiting intracellular salt absorption, increasing electrolyte transport into the gut lumen, and creating an osmotic gradient favoring intestinal water secretion (12). An enzymatically active A-subunit is required for intestinal fluid secretion but also enables LT to act as an oral adjuvant, boosting the immune response to coadministered antigens and inducing both humoral and cellular immune responses in both the systemic and mucosal compartments. However, even low doses of LT (and CT) induce side effects, such as diarrhea

The effect of passive insulin antibody transfer into recipient mice was reevaluated. Mice of inbred strains BALB/c, C57Bl/6 and DBA/2 received 10 injections of hyperimmune guinea pig insulin antiserum within 2 weeks. Ten days later the animals were sacrificed. It was found that up to 40% of animals had developed chronic lymphocytic infiltrations of pancreatic islets (insulitis) without showing diabetic symptoms. It is concluded that repeated damaging of islet tissue by passive insulin antibody transfer may result in chronic, possibly autoimmune insulitis. Our observation supports the concept that partial damage of pancreatic islets by environmental factors may trigger an organ-specific progressive islet cell destruction process mediated by immune cells.

Having the ability to alter the genetic makeup of a cancer cell by gene transfer is a potentially powerful strategy for treating human cancer. However, a low efficiency of gene delivery in vivo and poor tumour specificity has prevented the widespread implementation of this technology in the clinic. Despite these formidable obstacles, the first successful application of gene therapy in the treatment of cancer may occur when it is combined with local modalities such as radiation therapy. A small number of gene therapy strategies have been evaluated in clinical trials in combination with external beam radiation therapy. The combined therapy has been well-tolerated and has not exacerbated the side effects of radiation therapy. Gene transfer and tumour cell destruction has been demonstrated in vivo. Although the results await confirmation in larger, prospective Phase III trials, there is suggestive evidence that the combined therapies may be demonstrating better than expected antitumour activity. Our vast knowledge of the molecular defects that drive the cancer process, coupled with our expanding understanding of the genes responsible for tumour cell radioresistance, have spawned the development of rational, targeted gene therapies designed to increase tumour cell radiosensitivity. Here, the results of the clinical trials conducted so far will be reviewed, followed by a description of new approaches under development at present.

We describe a novel liposome-based immunoassay in which covalently linked hapten-cytolysin conjugates are used instead of complement and surface-immobilized immunoreagents. Stable, unilamellar liposomes containing entrapped alkaline phosphatase as a marker enzyme were prepared by dialysis of octyl glucoside from suspensions of cholesterol and egg yolk lecithin. The resulting vesicles could be immediately lysed by addition of either bee venom melittin or hapten-melittin conjugates. Using ouabain, an analog of digoxin, we synthesized conjugates that were more lytic than mellitin alone but that were inhibited in the presence of antibody. This inhibition was affected by adding competing free digoxin at various concentrations to obtain standard curves. The same liposome preparations could be lysed with a biotin-melittin conjugate, which was inhibited by avidin. The latter system was affected by free biotin and might be used to couple this approach to various heterogeneous immunoassays.

We describe a highly sensitive heterogeneous enzyme-linked immunoassay in which digoxin is used as the model analyte. An excess of enzyme-labeled monovalent antibody is incubated with sample containing the analyte such that all analyte is rapidly and quantitatively bound. Excess antibody that does not acquire a antigen in its binding site is rapidly removed from the mixture by passage through a porous affinity column containing immobilized analyte (or analog), present in vast excess. Only the labeled monovalent antibody that possesses an antigen in its binding site elutes from the column in the unbound fraction. The label present in this fraction is then quantified. Such an assay is extremely sensitive and obviates the limitations imposed by antibody affinity constants on homogeneous and competitive heterogeneous immunoassays. This assay can be performed rapidly and is readily amenable to automation.

Die Moglichkeit, durch konduktometrische Masanalyse die Untersuchung von Losungen, die neben freien Basen (Ammoniak, Natronoder Kalilauge) deren Thioglykolate oder Sulfite enthalten, sicherer und in mancher Hinsicht auch einfacher zu gestalten, wurde experimentell modellmasig gepruft, die grundsatzlichen Ergebnisse wurden ausfuhrlich besprochen. Es wurde gefunden, das zur Titration von Thioglykolat allein in isopropanolischer Losung bei Raumtemperatur oder ebenso in Gegenwart von Sulfit, aber bei 48–53° C, die Verdrangungstitration nur mit Schwefelsaure zur Bestimmung geeignet ist. Sulfit wird in wasriger Losung nach Oxydation mit Wasserstoffperoxyd zu Sulfat in ublicher Weise mit Bariumacetat in Gegenwart von Salpetersaure titriert. Eine erste Arbeitsvorschrift wird mitgeteilt.