Peter Hammerl

Characterization of the cell death modes and the associated changes in cellular energy supply in response to AlPcS4-PDT

Photodynamic therapy (PDT) can result in both types of cell death, apoptosis or necrosis. Several steps in the induction and execution of apoptosis depend on ATP and the intracellular ATP level has been shown to be one determinant in whether apoptosis or necrosis occurs. Therefore, photochemical damage of cellular targets involved in energy supply might play a crucial role in the mode of cell death being executed. The present study is aimed at the characterization of changes in cellular energy supply and the associated cell death modes in response to PDT. Using the human epidermoid carcinoma cell line A431 and aluminium(III) phthalocyanine tetrasulfonate chloride (2.5 microM) as a photosensitizer, we studied the changes in mitochondrial function and intracellular ATP level after irradiation with different light doses. Employing assays for caspase-3 activation and nuclear fragmentation, 50% of the cells were found to undergo apoptosis after irradiation between 2.5 to 3.5 J cm(-2) while the remainder died by necrosis. At higher light doses (> 6 J cm(-2)), neither caspase-3 activation nor nuclear fragmentation was observed and this suggests that these cells died exclusively by necrosis. Necrotic cell death was also associated with a rapid decline in mitochondrial activity and intracellular ATP. By contrast, with apoptosis the loss of mitochondrial function was delayed and the ATP level was maintained at near control levels for up to eight hours which was far beyond the onset of morphological changes. These data suggest that, depending on the light dose applied, both, necrosis as well as apoptosis can be induced with AlPcS4 mediated PDT and that photodamage in energy supplying cellular targets may influence the mode of cell death. Further, it is speculated that cells undergoing apoptosis in response to PDT might maintain a high ATP level long enough to complete the apoptotic program.

Pulmonary hypertension due to chronic lung disease: Updated Recommendations of the Cologne Consensus Conference 2011

The 2009 European Guidelines on Pulmonary Hypertension did not cover only pulmonary arterial hypertension (PAH) but also some aspects of pulmonary hypertension (PH) in chronic lung disease. These guidelines point out that the drugs currently used to treat patients with PAH (prostanoids, endothelin receptor antagonists and phosphodiesterase type-5 inhibitors) have not been sufficiently investigated in other forms of PH. Therefore, the use of these drugs in patients with chronic lung disease and PH is not recommended. This recommendation, however, is not always in agreement with medical needs as physicians feel sometimes inclined to also treat other forms of pulmonary hypertension which may affect the quality of life and survival of these patients in a similar manner as in PAH. In June 2010, a consensus conference was held in Cologne, Germany, to discuss open and controversial issues surrounding the practical implementation of the European Guidelines. The conference was sponsored by the German Society of Cardiology, the German Society of Respiratory Medicine and the German Society of Pediatric Cardiology (DGK, DGP and DGPK). To this end, a number of working groups were initiated, one of which was specifically dedicated to the diagnosis and treatment of PH due to chronic lung disease. This manuscript describes in detail the results and recommendations of this working group which were last updated in October 2011.

Epidermal Langerhans Cells Are Dispensable for Humoral and Cell-Mediated Immunity Elicited by Gene Gun Immunization

Gene gun immunization, i.e., bombardment of skin with DNA-coated particles, is an efficient method for the administration of DNA vaccines. Direct transfection of APC or cross-presentation of exogenous Ag acquired from transfected nonimmune cells enables MHC-I-restricted activation of CD8+ T cells. Additionally, MHC-II-restricted presentation of exogenous Ag activates CD4+ Th cells. Being the principal APC in the epidermis, Langerhans cells (LC) seem ideal candidates to accomplish these functions. However, the dependence on LC of gene gun-induced immune reactions has not yet been demonstrated directly. This was primarily hampered by difficulties to discriminate the contributions of LC from those of other dermal dendritic cells. To address this problem, we have used Langerin-diphtheria toxin receptor knockin mice that allow for selective inducible ablation of LC. LC deficiency, even over the entire duration of experiments, did not affect any of the gene gun-induced immune functions examined, including proliferation of CD4+ and CD8+ T cells, IFN-γ secretion by spleen cells, Ab production, CTL activity, and development of protective antitumor immunity. Together, our data show that gene gun immunization is capable of inducing humoral and cell-mediated immune reactions independently of LC.

Langerin+ Dermal Dendritic Cells Are Critical for CD8+ T Cell Activation and IgH γ-1 Class Switching in Response to Gene Gun Vaccines

The C-type lectin langerin/CD207 was originally discovered as a specific marker for epidermal Langerhans cells (LC). Recently, additional and distinct subsets of langerin+ dendritic cells (DC) have been identified in lymph nodes and peripheral tissues of mice. Although the role of LC for immune activation or modulation is now being discussed controversially, other langerin+ DC appear crucial for protective immunity in a growing set of infection and vaccination models. In knock-in mice that express the human diphtheria toxin receptor under control of the langerin promoter, injection of diphtheria toxin ablates LC for several weeks whereas other langerin+ DC subsets are replenished within just a few days. Thus, by careful timing of diphtheria toxin injections selective states of deficiency in either LC only or all langerin+ cells can be established. Taking advantage of this system, we found that, unlike selective LC deficiency, ablation of all langerin+ DC abrogated the activation of IFN-γ–producing and cytolytic CD8+ T cells after gene gun vaccination. Moreover, we identified migratory langerin+ dermal DC as the subset that directly activated CD8+ T cells in lymph nodes. Langerin+ DC were also critical for IgG1 but not IgG2a Ab induction, suggesting differential polarization of CD4+ T helper cells by langerin+ or langerin-negative DC, respectively. In contrast, protein vaccines administered with various adjuvants induced IgG1 independently of langerin+ DC. Taken together, these findings reflect a highly specialized division of labor between different DC subsets both with respect to Ag encounter as well as downstream processes of immune activation.

Design of protective and therapeutic DNA vaccines for the treatment of allergic diseases.

The DNA vaccine revolution has opened a vast scope of novel approaches for protective and therapeutic treatments of type I allergy. This review gives an overview on the current status of allergy DNA vaccines and presents advances in the design of vaccine constructs. An immense number of concurring studies have proven the stimulation of Th1 cells and the induction of a balanced Th1/Th2 cytokine milieu as the fundamental mechanisms underlying the anti-allergic effects of DNA vaccines. Basic vaccine formulations thus can be optimized by improving the cellular immunogenicity via co-administration of cytokines, co-expression or co-application of immunostimulatory DNA sequences or adapting the codon usage. The latter is a frequent and major reason for impaired vaccine expression (e.g. translation of plant allergen genes in mammal cells). Because of unwanted side effects during conventional specific immunotherapy with allergen extracts, safety is increasingly demanded for both, protein and DNA vaccines for allergy treatment. We discuss the creation of hypoallergenic DNA vaccines based on deliberate allergen gene fragmentation, the use of mutations and the routine production of hypoallergenic DNA vaccines by forced ubiquitination. Furthermore, allergen-expressing DNA replicon vaccines are introduced, which enable a drastic reduction of the vaccine dose without loss of anti-allergic efficacy. Finally, the development of DNA multi vaccines and fusion vaccines for protective and therapeutic applications against certain groups of allergens is addressed.

Quantification of histamine in blood plasma and cell culture supernatants: a validated one-step gas chromatography-mass spectrometry method.

A novel one-step ethylchloroformate (ECF) derivatization of histamine in biological liquid matrices that allows the sensitive quantification by gas chromatography and mass spectroscopic detection (GC-MS) from small volumes of blood plasma or cell culture supernatants within 15 min is described. After addition of ECF/chloroform directly to the crude sample, histamine has been found to be quantitatively derivatized within seconds. Following centrifugation, the organic phase is transferred to a fresh vial, dried by addition of anhydrous sodium sulfate, and subjected to GC-MS analysis. The reliability of the results is verified by use of two different ion pairs for detection. The method is validated according to DIN 38402. Linearity is given from 0.0054 to 13 microg/ml and the limit of detection is 2 ng/ml (10 pg absolute, at a signal to noise ratio of 3:1). The limit of quantification, as calculated at a confidence level of 95%, is 15.6 ng/ml. Practical application is exemplified by the determination of the histamine content in blood plasma of birch pollen-sensitized mice and in the culture supernatant of rat basophil leukemia cells after Ca(2+) ionophore-mediated degranulation.

Molecular and biochemical analysis of the first ARA6 homologue, a RAB5 GTPase, from green algae

RAB5 GTPases are important regulators of endosomal membrane traffic in yeast, plants, and animals. A specific subgroup of this family, the ARA6 group, has been described in land plants including bryophytes, lycophytes, and flowering plants. Here, we report on the isolation of an ARA6 homologue in a green alga. CaARA6 (CaRABF1) from Chara australis, a member of the Characeae that is a close relative of land plants, encodes a polypeptide of 237 aa with a calculated molecular mass of 25.4kDa, which is highly similar to ARA6 members from Arabidopsis thaliana and other land plants and has GTPase activity. When expressed in Nicotiana benthamiana leaf epidermal cells, fluorescently tagged CaARA6 labelled organelles with diameters between 0.2 and 1.2 µm, which co-localized with fluorescently tagged AtARA6 known to be present on multivesicular endosomes. Mutations in the membrane-anchoring and GTP-binding sites altered the localization of CaARA6 comparable to that of A. thaliana ARA6 (RABF1). In characean internodal cells, confocal immunofluorescence and immunogold electron microscopy with antibodies against AtARA6 and CaARA6 revealed ARA6 epitopes not only at multivesicular endosomes but also at the plasma membrane, including convoluted domains (charasomes), and at the trans-Golgi network. Our findings demonstrate that ARA6-like proteins have a more ancient origin than previously thought. They indicate further that ARA6-like proteins could have different functions in spite of the high similarity between characean algae and flowering plants.

Particulate nitrocellulose as a solid phase for protein immobilization in immuno-affinity chromatography.

The use of nitrocellulose paper as a solid phase matrix for protein immobilization and its application in immunoaffinity chromatography is described. Pieces of nitrocellulose paper were frozen in liquid nitrogen and ground to a powder (NCP) which was then fractionated according to particle size by repeated sedimentation/resuspension cycles in water. The flow properties of different NCP fractions were compared with those of Sephadex G-50. Protein binding capacity and binding dynamics were investigated in a model system with bovine serum albumin (BSA) in phosphate buffered saline. Applications of the material are illustrated by batch chromatography for the removal of anti-carrier protein antibodies from a hapten antiserum and by affinity purification of a hapten-specific antibody fraction using NaSCN elution from haptenated NCP. Furthermore, the applicability of the material in column chromatography is demonstrated by elution of a monospecific fraction of anti-fluorescein antibodies from a hapten/carrier mixed bed column by excess of soluble hapten. The results demonstrate that NCP chromatography appears to be a cheap and useful alternative to many other chromatographic media used for protein immobilization.

Structural integrity of the antigen is a determinant for the induction of T-helper type-1 immunity in mice by gene gun vaccines against E. coli beta-galactosidase.

The type of immune response is critical for successful protection and typically determined by pathogen-associated danger molecules. In contrast, protein antigens are usually regarded as passive target structures. Here, we provide evidence that the structure of the antigen can profoundly influence the type of response that is elicited under else identical conditions. In mice, gene gun vaccines induce predominantly Th2-biased immune reactions against most antigens. One exception is E. coli beta-galactosidase (βGal) that induces a balanced Th1/Th2 response. Because both, the delivered material (plasmid DNA-coated gold particles) as well as the procedure (biolistic delivery to the skin surface) is the same as for other antigens we hypothesized that Th1 induction could be a function of βGal protein expressed in transfected cells. To test this we examined gene gun vaccines encoding structural or functional variants of the antigen. Employing a series of gene gun vaccines encoding individual structural domains of βGal, we found that neither of them induced IgG2a antibodies. Even disruption of the homo-tetramer association of the native protein by deletion of a few N-terminal amino acids was sufficient to abrogate IgG2a production. However, enzymatically inactive βGal with only one point mutation in the catalytic center retained the ability to induce Th1 reactions. Thus, structural but not functional integrity of the antigen must be retained for Th1 induction. βGal is not a Th1 adjuvant in the classical sense because neither were βGal-transgenic ROSA26 mice particularly Th1-biased nor did co-administration of a βGal-encoding plasmid induce IgG2a against other antigens. Despite this, gene gun vaccines elicited Th1 reactions to antigens fused to the open reading frame of βGal. We interpret these findings as evidence that different skin-borne antigens may be differentially handled by the immune system and that the three-dimensional structure of an antigen is an important determinant for this.

TH1-promoting DNA immunization against allergens modulates the ratio of IgG1/IgG2a but does not affect the anaphylactic potential of IgG1 antibodies: No evidence for the synthesis of nonanaphylactic IgG1

BACKGROUND In mouse, IgG1 has been reported to make up 2 functionally distinct phenotypes that also differ in their induction requirements. One of these phenotypes lacks anaphylactic activity. OBJECTIVE We hypothesized that nonanaphylactic IgG1 could modulate allergic reactions and investigated whether such antibodies are induced by DNA immunization. METHODS Mice were immunized with allergen-encoding plasmid DNA or with recombinant allergens and alum. Sera were analyzed for IgG subclasses by ELISA for anaphylactic IgE by rat basophil degranulation, and after heat inactivation of IgE for anaphylactic IgG by passive cutaneous anaphylaxis assay. IFN-gamma and IL-5 from in-vitro restimulated spleen cells were quantitated by ELISA. RESULTS After protein immunization, mice produced IgG1 and IgE, whereas DNA immunization elicited IgG1 and IgG2a but no IgE. However, all sera were positive for non-IgE-mediated passive cutaneous anaphylaxis. In the presence of anaphylactic IgG1, the additional occurrence of nonanaphylactic IgG1 cannot be strictly ruled out. To circumvent this problem, we immunized IL-4 receptor-deficient mice against Bet v 1a, because anaphylactic but not nonanaphylactic IgG1 has been reported to depend on IL-4. These animals produced only low amounts of IgG1, but sera were again positive for non-IgE-mediated anaphylactic activity. CONCLUSIONS Our results revealed no evidence for the production of nonanaphylactic IgG1. Furthermore, our data indicate that the development of non-IgE-mediated anaphylaxis does not require IL-4 receptor signaling.