Harald Renz

Development and clinical application of a LC-MS/MS method for simultaneous determination of various tyrosine kinase inhibitors in human plasma.

BACKGROUND Increasing numbers of tyrosine kinase inhibitors (TKIs) were studied and approved for therapy of malignancies and other diseases. The aim of this study was to develop and validate a specific, simple and rapid quantification method for various TKIs in human plasma. METHODS A simultaneous test for six TKIs (erlotinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib) was developed using liquid chromatography tandem mass spectrometry in a multiple reaction monitoring mode. After protein precipitation the specimens were applied to the HPLC system and separated using a gradient of acetonitrile containing 1% formic acid with 10 mM ammoniumformiate on an analytic RP-C18 column. RESULTS The calibration range was 10-1000 ng/mL for sunitinib and 50-5000 ng/mL for the other TKIs with coefficients of determination ≥0.99 for all analytes. The intra- and inter day coefficients of variation were ≤15% and the chromatographic run time was 12 min. Plasma specimens were stable for measurement for at least 1 week at 4°C. Clinical applications of the assay are exemplarily discussed. CONCLUSIONS This novel high-throughput method is suitable for specific simultaneous determination of different TKIs in routine clinical practice.

Enhancing in vivo circulation and siRNA delivery with biodegradable polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol) copolymers

The purpose of this study was to enhance the in vivo blood circulation time and siRNA delivery efficiency of biodegradable copolymers polyethylenimine-graft-polycaprolactone-block-poly(ethylene glycol) (hy-PEI-g-PCL-b-PEG) by introducing high graft densities of PCL-PEG chains. SYBR(®) Gold and heparin assays indicated improved stability of siRNA/copolymer-complexes with a graft density of 5. At N/P 1, only 40% siRNA condensation was achieved with non-grafted polymer, but 95% siRNA was condensed with copolymer PEI25k-(PCL570-PEG5k)(5). Intracellular uptake studies with confocal laser scanning microscopy and flow cytometry showed that the cellular uptake was increased with graft density, and copolymer PEI25k-(PCL570-PEG5k)(5) was able to deliver siRNA much more efficiently into the cytosol than into the nucleus. The in vitro knockdown effect of siRNA/hyPEI-g-PCL-b-PEG was also significantly improved with increasing graft density, and the most potent copolymer PEI25k-(PCL570-PEG5k)(5) knocked down 84.43% of the GAPDH expression. Complexes of both the copolymers with graft density 3 and 5 circulated much longer than unmodified PEI25 kDa and free siRNA, leading to a longer elimination half-life, a slower clearance and a three- or fourfold increase of the AUC compared to free siRNA, respectively. We demonstrated that the graft density of the amphiphilic chains can enhance the siRNA delivery efficiency and blood circulation, which highlights the development of safe and efficient non-viral polymeric siRNA nanocarriers that are especially stable and provide longer circulation in vivo.

Davos declaration: allergy as a global problem.

Allergy and allergic diseases represent a major health problem not only in industrialized ‘modern’ societies, but worldwide. There has been an epidemic increase in prevalence of allergic diseases in the last few decades with 10–30% of the population affected. Apart from individual suffering, because of their life-threatening or chronic course, these diseases present a high socioeconomic burden. In many countries, patient care of affected individuals is insufficient and/or inadequate. In spite of great progress in research into the causes and treatment of allergy in the last decades, there are still many problems to be solved in moving to reach our goals of more effective therapies and eventual prevention (1–3). Therefore, a group of 40 scientists and clinicians from four continents and all fields of allergy and related disciplines gathered under the sponsorship of the Christine-Kühne Center of Allergy Research and Education (CK-CARE) in Davos, Switzerland from 17 to 20 July 2011 for the first ‘Global Allergy Forum’. Under the topic ‘Allergy and Allergic Diseases: Barriers to Cure’, the participants formed five working groups to discuss and define the most urgent problems in the field and seek solutions and recommend an action plan. There are numerous unmet clinical needs and millions of patients are undertreated or not treated with the most appropriate methods. Accessibility to and affordability of effective therapeutic regimens are not provided in many countries. The development of innovative therapies is slow compared to most other fields of medicine. Allergic diseases encompass broad fields of medicine and show a wide heterogeneity involving different organs such as eyes, respiratory tract, gut, and skin. Diseases include rhinoconjunctivitis, asthma, anaphylaxis, eczema, urticaria, and angioedema as well as drug and food allergies. Allergic diseases show variability in severity and clinical course which at the moment are only poorly defined. Much better definition of the subtypes of allergic patients (phenotyping) appears crucial and very much needed to address the right therapy to the right patient. A new integrative approach is needed to understand how a complex network of immunological, genetic, and environmental factors leads to a complex allergic phenotype (1). There is a tremendous lack of knowledge regarding many unsolved issues such as: • The causes of the epidemic increase in allergic diseases are unknown. Environmental exposures that appear to be critical factors include factors as diverse as air quality, diet and nutrition, climate, UV radiation, and direct skin contact as well as psycho-social interactions. Moreover, when genetic predisposition is taken into account, environment can provide either risk or protection. • The effects of changes in climate, urbanization, etc. have to be anticipated. Better ways to assess spatial and temporal environmental exposure at population and individual levels are much needed and should be related to the assessment of individual genetic susceptibility. • The interactions between microbes, pollutants, and the immune system are marginally understood. • There is inadequate understanding of the natural mechanisms that limit acute vs. chronic disease or spontaneous resolution. • There is a need for better subclassification of allergic disorders based on pathobiology. • There is a need for new agents acting on specific pathways in pathogenesis with regard to new therapeutic approaches. • There is a need for better preclinical models for translational research. • There is a need to develop better tools for complex data analysis. • There is a need for efficient strategies for primary and secondary allergy prevention. • There is a need for better approaches in diagnosis and prediction of treatment responses and the monitoring of therapeutic effectiveness. Apart from true lack of information, there is a tremendous gap between actual existing knowledge and its effective application for the millions of people in need. • There is a shortage of well-trained allergy specialists in most countries. • Education and training efforts should also be directed toward medical students at the curricular level and extended to primary care physicians who have to be involved in a strategy for diagnosing and managing allergic diseases with such high prevalence rates of 20% of the population. • Awareness campaigns for targeted public groups should be performed. Allied health professionals, such as nurses, school teachers, etc., should be included. Better and more effective tools to spread the available information should be developed. • Close cooperation with patient organizations is highly recommended. • Decision makers involved in developing and approving health policies and administration must be made more aware of the problem of allergic diseases. Action should be taken at various levels and through existing doctors, scientists, and lay organizations to solve these problems. The global expertise from clinical allergists, immunologists, microbiologists, biologists, nutritionists,

Exposure to microbial agents in house dust and wheezing, atopic dermatitis and atopic sensitization in early childhood: a birth cohort study in rural areas

Early‐life exposure to environmental microbial agents may be associated with development of wheezing and allergic diseases.

Obesity lowers the threshold of allergic sensitization and augments airway eosinophilia in a mouse model of asthma

Clinical and epidemiological studies show a close association between obesity and the risk of asthma development. The underlying cause–effect relationship between metabolism, innate and adaptive immunity, and inflammation remains to be elucidated.

High indoor microbial levels are associated with reduced Th1 cytokine secretion capacity in infancy.

Background: Exposure to microbes and their components may affect the maturation of the immune system. We examined the association of house dust microbial content with cytokine-producing capacity at birth and at the age of 1 year. Methods: Production of TNF-α, IFN-γ, IL-5, IL-8 and IL-10 at birth (n = 228) and at the age of 1 year (n = 200) following 24- and 48-hour whole-blood stimulation with staphylococcal enterotoxin B (SEB), lipopolysaccharide and the combination of phorbol ester and ionomycin was measured. Concentrations of ergosterol (marker for fungal biomass), muramic acid (marker for Gram-positive bacteria) and 3-hydroxy fatty acids with a carbon chain length from 10 to 14 (marker for Gram-negative bacteria) in living room floor dust were analyzed using gas chromatography-tandem mass spectrometry. Five single microbial species or groups were determined using a quantitative polymerase chain reaction method. Results: A high total level of the studied Gram-positive bacteria in general or Mycobacterium spp. in house dust was associated with decreased SEB-stimulated IFN-γ production, especially at the age of 1 year. The total level of indoor fungi analyzed (Penicillium spp., Aspergillus spp. and Paecilomyces variotii group, Trichoderma viride/atroviride/koningii,Wallemia sebi) was also inversely associated with IFN-γ production at the age of 1 year, but this association did not remain significant after adjustment for potential confounders. A few associations were found between microbial exposures and other measured cytokines. Conclusions: High indoor microbial exposures may affect immune development in early life by reducing T helper type 1 cytokine secretion capacity. The observed hyporesponsiveness may reflect the adaptation of the immune system to environmental antigens. In future, more attention should be paid especially to the immunomodulatory role of exposures to Gram-positive bacteria.

Absence of unspecific innate immune cell activation by GATA-3-specific DNAzymes.

DNAzymes of the 10-23 family represent an important class of antisense molecules with implications for therapeutic treatment of diseases. These molecules are single-stranded oligodeoxynucleotides combining the high specificity of oligonucleotide base pairing with an inherent RNA-cleaving enzymatic activity. However, like other oligonucleotide-based molecules these substances might exert so-called off-target effects, which have not been investigated so far for this molecule class. Therefore, the present study investigates putative off-target effects of DNAzymes on innate immune mechanisms using GATA-3-specific DNAzymes that have recently been developed as novel therapeutic approach for the treatment of allergic diseases including allergic asthma. The conserved catalytic domain of 10-23 DNAzymes contains a CpG motif that may stimulate innate immune cells via Toll-like receptor 9 (TLR-9). Therefore, potential TLR-9-mediated as well as TLR-9 independent cell activation was investigated using TLR-9-transfected HEK293 cells, macrophage cell lines and primary innate immune cells. Furthermore, putative effects of GATA-3-specific DNAzymes on the activation of neutrophil granulocytes and degranulation of mast cells/basophils were analyzed. In summary, no innate immune cell-stimulating activities of the tested DNAzymes were observed in any of the systems. Consequently, use of GATA-3-specific DNAzymes may represent a novel and highly specific approach for the treatment of allergic diseases.

Clara cells drive eosinophil accumulation in allergic asthma

Development of allergic asthma is a complex process involving immune, neuronal and tissue cells. In the lung, Clara cells represent a major part of the “immunomodulatory barrier” of the airway epithelium. To understand the contribution of these cells to the inflammatory outcome of asthma, disease development was assessed using an adjuvant-free ovalbumin model. Mice were sensitised with subcutaneous injections of 10 &mgr;g endotoxin-free ovalbumin in conjunction with naphthalene-induced Clara cell depletion. Clara epithelial cell depletion in the lung strongly reduced eosinophil influx, which correlated with decreased eotaxin levels and, moreover, diminished the T-helper cell type 2 inflammatory response, including interleukin (IL)-4, IL-5 and IL-13. In contrast, airway hyperresponsiveness was increased. Further investigation revealed Clara cells as the principal source of eotaxin in the lung. These findings are the first to show that Clara airway epithelial cells substantially contribute to the infiltration of eotaxin-responsive CCR3+ immune cells and augment the allergic immune response in the lung. The present study identifies Clara cells as a potential therapeutic target in inflammatory lung diseases such as allergic asthma.

Neurotrophins in Chronic Allergic Airway Inflammation and Remodeling

Allergic asthma is a chronic inflammatory disease characterized by the production of allergen-specific IgE antibodies, TH2 inflammation, airway hyperresponsiveness and airway remodeling. Airway remodeling represents the disease-limiting stage during disease progression, and the underlying cellular molecular network resulting in airway remodeling are still poorly defined. In addition to the well-established TH2-dependent inflammatory response, several lines of investigation reveal that this regulation in the peripheral central nervous system contributes to disease development, exacerbation and progression. Several members of the neurotrophin family (e.g. nerve growth factor, brain-derived neurotrophic factor) are important transmitters of signals between the immune and the nervous system. Recent data indicate that NGF contributes to the development of airway remodeling in an inflammation and TGF-independent manner. These and other data open the opportunity to therapeutically interfere also on this level of regulation as a novel approach.

Few associations between high-sensitivity C-reactive protein and environmental factors in 4.5-year-old children

To cite this article: Mustonen K, Keski‐Nisula L, Vaarala O, Pfefferle PI, Renz H, Riedler J, Dalphin J‐C, Buechele G, Lauener R, Braun‐Fahrländer C, von Mutius E, Pekkanen J and the PASTURE Study Group. Few associations between high‐sensitivity C‐reactive protein and environmental factors in 4.5‐year‐old children. Pediatr Allergy Immunol 2012: 23: 522–528.