[Regulatory framework of innovative therapies : From bench to bedside]. / Regulatorischer Rahmen für neuartige Therapien. Vom Labor zur klinischen Prüfung.

Novel therapies, e.g., cell and gene therapy or tissue engineering, are summarized in the European Union as advanced therapy medicinal products (ATMPs). In terms of composition and product properties, ATMPs are highly complex, and given their multiple potential actions they are subject to continuously developing regulatory requirements. Due to promising basic research findings, there are high expectations by the society toward the therapeutic potential of ATMPs. It is of utmost importance to develop a scientifically sound preclinical and clinical development plan before entering into the first clinical trial. Due to the complex features of ATMPs, this development plan should be discussed early with the regulatory authorities to define the specifics and challenges of each individual product. For planning as well as operational realization of the initial clinical trial involving ATMPs, specific requirements that need to be addressed are discussed in this paper.

Increased surfactant protein a and d expression in acute ovalbumin-induced allergic airway inflammation in brown norway rats.

BACKGROUND: The surfactant proteins SP-A and SP-D, components of the innate immune system, are involved in host defence. OBJECTIVE: We tested the hypothesis that ovalbumin (OVA) challenge leads to an upregulation of both proteins in alveolar epithelial type II cells (AEII) and Clara cells and to an enhanced uptake by macrophages. METHODS: After sensitization with OVA and heat-killed Bordetella pertussis challenge followed intratracheally with 0.5% OVA on day 13. One day after challenge lung tissue and bronchoalveolar lavage fluid (BALF) of sensitized NaCl- and OVA-challenged Brown Norway rats were compared with home cage controls using qRt-PCR, Western blot and immunohistochemistry. RESULTS: After OVA challenge (1) eosinophils increased significantly in the BALF, (2) the total amount of SP-A and SP-D was significantly increased in lung tissue, (3) the amount of SP-A was significantly and the amount of SP-D was remarkably elevated in BALF, and (4) the levels of SP-A and SP-D mRNA in lung tissue were significantly elevated. Using quantitative immunohistochemistry, we found (5) significantly higher surface fractions of SP-A- and SP-D-labelled AEII, (6) no differences in the surface fractions of SP-A- and SP-D-labelled bronchial Clara cells, and (7) a significantly increased cell density of unlabelled and SP-A-labelled macrophages. CONCLUSIONS: Thus, combining molecular biological and histological methods we suggest that after OVA challenge (1) AEII but not Clara cells show a significantly higher expression of SP-A and SP-D leading also to higher amounts of both SPs in BALF and (2) macrophages gather predominantly SP-A.

Pulmonary microbial infection in mice: comparison of different application methods and correlation of bacterial numbers and histopathology.

Many investigations have been performed in characterising experimental bacterial infections in the lung of mice using several pathogens. Robust experimental pulmonary infection models require a reproducible method of application with defined numbers of pathogens to the respiratory tract without contaminating extrapulmonary tissues. At the same time trauma due to the experimental procedure should be kept to a minimum. So far several routes of administration have been used but a systematic comparison of these methods is still missing. Here we provide a comprehensive evaluation of view controlled i.t. instillation, tracheotomy, intranasal application, blind instillation and aerosol infection. An infection dose of up to 5 x 10(4) bacteria (L. monocytogenes) was applied to a group of ten mice by each technique and the animals were killed after 1 h or 24h. The number of viable bacteria was estimated by plating homogenates of the lungs and spleens. In addition, pathological effects on lung tissue were examined by histology 24h after infection. The highest reproducibility was achieved after applying Listeria directly in the trachea under view or by tracheotomy. However, mice were severely affected in their vitality after tracheotomy. Thus, for topical application of bacterial suspension into the lung the view controlled i.t. instillation is most appropriate.

Recruitment of lymphocytes and dendritic cells from the blood to the bronchoalveolar space and the draining lymph nodes after a single intrabronchial application of the lipopeptide MALP-2.

OBJECTIVE: It has been shown previously that the synthetic macrophage-activating lipopeptide, MALP-2, is a potent stimulator of the respiratory immune system and an effective adjuvant in the induction of mucosal immune responses. In this study, the migration route of leukocytes from the blood to the bronchoalveolar space and then to the draining lymph nodes was investigated. METHODS: MALP-2 was intratracheally instilled into lungs of Lewis rats. Bronchoalveolar lavage cells as well as cell preparations of other lung compartments such as the marginal vascular pool, the interstitial pool and also the draining lymph nodes were examined 3 days later. RESULTS: The application of MALP-2 induced a pronounced leukocyte accumulation in the bronchoalveolar space and the lung interstitium but not in the marginal vascular pool. A tendency to increased lymphocyte and dendritic cell numbers was observed in the draining lymph nodes. CONCLUSION: Our data indicate the migration of blood cells into the lung interstitium and the bronchoalveolar space in response to MALP-2. Thus, the immune reaction induced by MALP-2 might be of relevance as an adjuvant treatment in inhalant vaccination strategies in the lung.

Stimulation of bronchus-associated lymphoid tissue in rats by repeated inhalation of aerosolized lipopeptide MALP-2.

OBJECTIVE: Bronchus-associated lymphoid tissue (BALT) is a part of the integrated mucosal immune system. It may play an important functional role for antigen uptake and induction of specific immune reactions. The aim of this study was to investigate whether it is possible to induce or modulate BALT by the repetitive inhalation of the synthetic lipopeptide MALP-2. METHODS: Female Lewis rats (245 +/- 19 g) inhaled 25 microg of MALP-2 six times at intervals of 1 week. One week after the last inhalation, they were sacrificed. Cells of the bronchoalveolar lavage and the left lung were investigated by flow cytometry. The middle lobe of the right lung was embedded in paraffin. BALT was semiquantitatively measured in 15 serial cross sections per animal. RESULTS: After repetitive inhalation of the diluent as well as MALP-2, BALT was found. The total area was increased after repetitive treatment with MALP-2. In addition, the preferential incidence of BALT was higher after MALP-2 application, in association with a bronchial diameter of 0.6-1 mm. The cellular analysis revealed no differences in the number of leukocyte subsets between the control and MALP-2 group. CONCLUSION: MALP-2 is a potent local stimulator and can be used to modulate BALT by repetitive inhalant treatment. The functional significance of enlarged or activated BALT has to be elucidated in future studies.

Afipia felis induces uptake by macrophages directly into a nonendocytic compartment.

Afipia felis is a Gram-negative bacterium that causes some cases of human Cat Scratch Disease. A. felis can survive and multiply in several mammalian cell types, including macrophages, but the precise intracellular compartmentalization of A. felis-containing phagosomes is unknown. Here, we demonstrate that, in murine macrophages, most A. felis-containing phagosomes exclude lysosomal tracer loaded into macrophage lysosomes before, as well as endocytic tracer loaded after, establishment of an infection. Established Afipia-containing phagosomes possess neither early endosomal marker proteins [early endosome antigen 1 (EEA1), Rab5, transferrin receptor, trytophane aspartate containing coat protein (TACO)] nor late endosomal or lysosomal proteins [cathepsin D, beta-glucuronidase, vacuolar proton-pumping ATPase, rab7, mannose-6-phosphate receptor, vesicle-associated membrane protein 8, lysosome-associated membrane proteins LAMP-1 and LAMP-2]. Those bacteria that will be found in a nonendosomal compartment enter the macrophage via an EEA1-negative compartment, which remains negative for LAMP-1. The smaller subpopulation of afipiae whose phagosomes will be part of the endocytic system enters into an EEA1-positive compartment, which also subsequently acquires LAMP-1. Killing of Afipia or opsonization with immune antibodies leads to a strong increase in the percentage of A. felis-containing phagosomes that interact with the endocytic system. We conclude that most phagosomes containing A. felis are disconnected from the endosome-lysosome continuum, that their unusual compartmentalization is decided at uptake, and that this compartmentalization requires bacterial viability.

[Recruitment of immunocompetent cells in a transplanted lung is not impaired despite incompletel reinnervation]. / Das Rekruitment immunkompetenter Zellen in eine transplantierte Lunge ist trotz inkompletter Reinnervierung nicht behindert.

BACKGROUND: It is not clear whether surgical intervention during lung transplantation which includes cutting vegetative nerves, lymphatic vessels and bronchial arteries, leads to alterations in immune responses. Thus, it was studied in an animal model whether an induced pulmonary immune reaction after syngenic lung transplantation was impaired without the influence of immunosuppression and rejection. The recruitment of leukocytes and the status of reinnervation was examined. METHODS: Syngenic transplantation of the left lung was performed in Lewis rats without rejection and therefore without immunosuppressive therapy. In a subgroup of animals host and donor leukocytes were distinguished. An ovalbumin (OVA)-specific pulmonary immune response was induced four months after transplantation. Bronchoalveolar lavage (BAL) and interstitial leukocytes were examined using flow cytometry and immunocytology, comparing the right lung and the grafted left lung. Immunohistology was performed to detect nerve fibers on cryostat sections. RESULTS: An induced cellular inflammation was observed in the right host lung as well as in the grafted left lung. However, the CD4 T cell numbers in the BAL were increased in the left lung. Single donor-type leukocytes could still be observed four months after transplantation. A partial reinnervation was found. CONCLUSIONS: The recruitment of immune cells into the lung interstitium and bronchoalveolar space of grafted lungs is not impaired. The incomplete reinnervation has no influence on leukocyte recruitment.

A method to purify bacteria-containing phagosomes from infected macrophages.

When small particles, such as microorganisms, are taken up by macrophages, they are wrapped with a portion of the host cell plasma membrane and ingested, creating a new organelle, the phagosome. This phagosome matures stepwise as newly formed endosomes do, finally forming a phagolysosome, a process that contributes to killing of ingested microbes and to the presentation of microbial antigens on the surface of the phagocyte. Some pathogenic bacteria, however, reprogram the phagocytic cell in such a way that the phagosome will either be arrested in an early stage of maturation or will be diverted and create an unusual, novel phagosomal compartment. To study the molecular processes that underly biogenesis of bacteria-containing phagosomes, we have established a method to isolate and to biochemically analyse bacteria- containing phagosomes. This method consists of mechanical lysis of infected macrophages, production of a postnuclear supernatant followed by fractionation in a discontinuous sucrose density gradient, separation through a Ficoll cushion, and by a final concentration step. These phagosome preparations contain very little endosomal or lysosomal contamination (the organelles of most concern when studying phagosome biogenesis) and very little Golgi- and plasma membrane-derived contamination, but do contain some mitochondrial and ER contamination. This method could also be used to study bacterial factors (proteins, RNA) produced while in phagosomes.

Immunomodulating properties of the antibiotic novobiocin in human monocytes.

We show that the coumeromycin antibiotic novobiocin, a potent inhibitor of ADP ribosylation, prevents lipopolysaccharide (LPS)-induced tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-6, and IL-10 secretion in human peripheral blood mononuclear cells. It shares these cytokine-suppressing properties with other inhibitors of ADP ribosylation. We found that novobiocin prevents TNF-alpha production by inhibiting translation of the TNF-alpha mRNA. Elevated TNF-alpha levels in mice treated with D-galactosamine (GalN)-LPS or GalN-TNF were not reduced by novobiocin; however, the drug exhibited hepatoprotective properties. Novobiocin causes downregulation of the surface molecules on monocytes, among which CD14 was the most affected. The diminished expression of surface molecules was not observed on T and B lymphocytes. Similar to other inhibitors of ADP ribosylation, novobiocin prevents LPS-induced phosphate labelling of gamma-actins.