These findings indicate that β-carotene cleavage products are most likely responsible for the increased lung cancer risk observed in chemoprevention trials. However, there is a lack of information on the sensitivity of the putative target cells in the lung, namely alveolartypeIIcells. AlveolartypeIIcells have a central role in the maintenance of normal lung function, reaction to injury, and response to specific toxins. They express phase I and phase II biotransformation enzymes—particularly cytochrome P450-dependent mono-oxygenases [ 30 ]—and thus are potential targets for many inhaled materials, and at the same time represent the relevant cells for the evaluation of a mutagenic and carcinogenic potential of specific agents [ 31 ]. Therefore, the goal of this investigation was the evaluation of the cyto- and genotoxic potential of β-carotene and its cleavage products in primary rat pneumocytes. 2. Materials and Methods
The complexity of the isolated lung model makes it difficult to ascribe physiological and biochemical events to specific cell types. Thus, to further confirm our hypothesis that excess albumin was taken up by the epithelium of the distal air spaces, experiments in cultured human alveolar epithelial A549 cells were performed. This cell line is a popularly-used in vitro model of typeIIalveolar epithelial cells (Foster, Oster et al. 1998). The A549 cell line exhibits many characteristics of alveolartypeIIcells, including the existence of lamellar inclusion bodies and the ability to synthesize surfactant constituents (Lieber, Smith et al. 1976). Furthermore, A549 cells were demonstrated to take up transferrin, a maker of receptor-mediated endocytosis, as well as cationised ferritin, an unspecific marker for absorption in a temperature-, time-, and concentration-dependent manner. This cell-line was also used as a tool to investigate sodium transport studies (Lazrak, Samanta et al. 2000) because of similarities in sodium channel expression when compared with primary alveolartypeIIcells. However, in contrast to typeIIcells, A549 cells neither polarize nor form fully-developed intercellular junctions, and are therefore unable to establish electrophysiologically tight monolayers (Foster, Oster et al. 1998). Consequently, transcytosis cannot be addressed in this cell-line. Additionally, A549 cells represent only one cell type of the alveolar epithelium (typeIIcells, which have been accredited with a key role in transport processes) but not type I cells. Nevertheless, A549 cells represent a powerful tool to with which investigate alveolar epithelial uptake of macromolecules such as albumin.
Interestingly, members of the polycistronic miR-17-92 cluster and its two mammalian paralogs miR-106a-363 cluster and miR-106b-25 cluster were highly represented in the ATII miRNAs. These clusters contain four seed families: miR-17, miR-18, miR-19 and miR-92 (Concepcion et al. 2012). In the present study, four miR-17-92 cluster members (miR-19a, miR-17, miR-20a, miR-18a) were detected. MiR-19a and miR-17- 5p were expressed at high levels with miR-17-5p having three targets within the canonical TGF-beta signaling pathway. Further, miR-106a from the miR-106a-363 cluster and all three members of miR-106b-25 cluster (miR-25, miR-93, miR-106b) were expressed at moderate levels. MiR-20b of the miR-106a-363 cluster was found at low level. So far, most studies have described the main role of the miR-17-92 cluster and its paralogs as oncogenes with upregulation in hematopoietic and solid cancers (Concepcion et al. 2012). However, there is growing evidence on its physiological function in normal development with loss of function of miR-17-92 cluster leading to early postnatal death (Ventura et al. 2008) and its potential role in tumor suppression. TGF-beta typeII transmembrane receptor was directly inhibited by miR-17, miR-20a and miR-20b and these miRNAs were upregulated in A549 with cisplatin sensitivity compared to cisplatin resistance (Jiang et al. 2014). Further, in oral squamous cell carcinoma miR-17 and miR-20a repressed tumor migration (Chang et al. 2013). Of special interest for the present study, in lung development miR-17, miR-20a and miR- 106b controlled E-cadherin expression and distribution, thus, provoking an epithelial phenotype. MiR-17 and miR-20a were expressed more highly during lung development than in adult lung, while miR-106b had even higher levels in adult lungs (Carraro et al. 2009). In the present study, miR-17, miR-20a and miR-106b were expressed above median levels in ATII cells in adult, healthy mice. These data suggest that not only during lung development, but also in adult mice all three miRNAs have a physiological role in maintaining epithelial homeostasis.
Fig 2. Uptake of Bv1-Protein and Bv1-Peptide in lungs of naive and sensitised mice in vivo. (A) Experimental design: Mice were sensitised 3 times intraperitonealy with 1 μg of Bet v 1 on days 0, 14 and 28. Intranasal challenge with 100 μg of birch pollen extract was performed one week after last sensitisation on days 35, 36 and 37. On day 40, 20 μg of Bv1-Protein-FAM and Bv1-Peptide-FAM were intranasal administered to naive and allergic BALB/c (n = 3) mice and lungs were collected after 1 (for Bv1-Peptide) or 6 hours (for Bv1-Protein). Dead cells were identified via 7-AAD staining and excluded from analysis. (B and C) FAM + cells were gated and antigen uptake capacity of macrophages (CD11b + /CD11c - ), dendritic cells (CD11b - /CD11c + ), B cells (B220 + / CD19 + ), and ATII-LCs (CD11b - /CD11c - /CD16/32 - /CD19 - /CD45 - /F4/80 - ) in lungs of naive and allergic mice was investigated by flow cytometry. Data are the pool from three independently performed experiments of identical design. Values represent means ± SEM. A value P<0.05 was considered to be significant. *P<0.05, **P<0.01 and ***P<0.001 indicate levels significantly different between naive and allergic mice. ATII-LCs = ATII-like cells; DCs = dendritic cells; M ϕ = macrophages.
Alveolar epithelial cell lines are useful tools to study biochemical aspects of healthy and diseased conditions, but concurrently exhibit dedifferentiated characteristics like impaired tight junction providing only limited application for barrier-dependent investigations. As an example, the human alveolartypeII-like cell line A549, derived from an adenocarcinoma, is widely applied for toxicity studies (Foldbjerg et al., 2011; Kreja & Seidel, 2002; Lestari et al., 2012) but lacks high TEER and is thus, not well suited for drug adsorption studies (Foster et al., 1998). The human alveolartype I-like cell line TT1 (‘transformed type-1’) was obtained through immortalization of primary ATII cells which were retrovirally transduced with hTERT and a temperature sensitive mutant of the Simian Virus 40 (SV40) large T antigen (Kemp et al., 2008; O'Hare et al., 2001). Immortalization methods and genes are addressed in the following chapter (see 1.3). In fact, TT1 cells have been used to study nanoparticle uptake (Kemp et al., 2008) as well as inflammatory responses and barrier properties (van den Bogaard et al., 2009). In the latter study, it was shown that TT1 did not develop high TEER limiting their applicability to barrier-independent experiments. This example illustrates that the genomic alterations upon cellular transduction might cause a loss of functional properties.
Acute respiratory distress syndrome (ARDS) is a devastating disease characterized by high mortality with no available pharmacological therapy. Transforming growth factor (TGF)-β mediates ARDS by promoting formation and persistence of alveolar edema. The deregulation of the Na,K-ATPase, a key Na + transporter in the alveolar epithelium, has been reported in ARDS, where impaired Na,K-ATPase function perturbs alveolar fluid clearance (AFC). In the present study, downregulation of ATP1B1, a gene encoding an essential subunit of the Na,K-ATPase, has been observed in ARDS patients, bleomycin model of ARDS and in TGF-β-treated primary mouse alveolar epithelial typeIIcells and A549 cells. A mechanism of TGF-β-regulated repression of the ATP1B1 gene relied on SMAD2, SMAD4, SNAI1 and E2F5 transcription factors. Moreover, epigenetic machinery involving DNA methylation and action of histone deacetylases (HDAC) have been found to mediate the TGF-β-controlled downregulation of the ATP1B1 gene. The class I HDAC member, HDAC2, has been identified as a critical element involved in ATP1B1 gene repression, and has been observed to bind the ATP1B1 promoter and to be activated by TGF-β signaling. The treatment with the histone deacetylase inhibitor trichostatin A (TSA) rescued expression of the Atp1b1 gene in the bleomycin model of ARDS which was accompanied by decreased lung wet-to-dry ratio. However, TSA neither decreased alveolar-capillary barrier permeability nor alleviated inflammatory responses indicating that reduction of edema was attributable to restored Na + transport and increased AFC.
Gas exchange takes place in the last seven generations of branching of the respiratory tract that include respiratory bronchioles, alveolar ducts and alveolar sacs and alveoli . Airways and alveoli are lined by a continuous epithelium that provides secretive and absorptive functions. At the same time, it is a barrier for macromolecules but allows bidirectional flux of water, small solutes and gases –. In the most distal airways, the alveolar epithelium comprises two types of cells, thin squamous type I cells and cuboidal typeIIcells . Alveolartype I cells (ATI) cover the major part of the surface area of distal airways and provide a pathway for diffusion of respiratory gases. On the other hand, ATII cells produce and secrete surfactant proteins, and together with ATI cells, actively participate in trans-epithelial transport of ions and proteins generating the driving force that allow fluid clearance from the alveolar space (Reviewed in ). Other important structures for the barrier function of the alveolar epithelium are the tight junctions. These cell-to-cell contact areas are flexible and selectively control passive movement of fluid and solutes between compartments. Thus tight junctions are critical in the maintenance of gradients created by active transport across the epithelium .
IFN-I is associated with various diseases of which some examples will be discussed here (Figure 13). Several autoimmune-diseases are characterized by high levels of circulating and tissue pDCs and high levels of IFN-I subsequently. Under auto- inflammatory conditions IFN-I is detrimental for the host and driving pathology of disease by activating and enhancing antigen uptake and DC function and autoantibody production (Figure 13) . In psoriasis and other auto-inflammatory skin diseases self-DNA and self-RNA in lesions are bound by the peptide LL-37 and further transported into pDCs where an IFN-I response is induced via activation of TLR7 and TLR9 [91,136]. Systemic lupus erythematosus (SLE) patients display high levels of IFN-I in the serum and blood cells show expression of ISG. In this case immune complexes consisting of auto-antibodies and self-DNA and self-RNA stimulate pDCs via Fc-receptor (crystallizable fragment receptor) activation to secrete IFN-I . The importance of IFN-I in the pathogenesis of SLE is also demonstrated by the fact that many genes that have been associated with SLE are in fact regulating or modulating the IFN-I response or Fc receptor binding .
normoxia, induce miR-154-3p expression after hyperoxia. A more detailed examination of which cells express miR-154-3p and their dynamic change of expression level after hyperoxic lung injury has to be undertaken to fulfill this issue. Furthermore BPD has various different causative factors beside inhalation of high oxygen concentrations, such as ventilation with high pressures leading to volutrauma and barotrauma, going along with increased susceptibility for infection and inflammatory processes (Silva et al., 2015). With the BPD mouse model as we use it we can only examine the effect of high oxygen concentrations on the lung, leaving out ventilation with high pressures, and hence not considering volutrauma and barotrauma. Therefore, the present BPD mouse model only depicts the effect of a single factor (high oxygen concentration) of a complex disease, which has a multifactorial cause. Approaches examining the effect of ventilation and associated mechanical stress on the lung could add further information to the BPD research and complete the whole issue (Silva et al., 2015). It has been shown already that mechanical ventilation with room air leads to a BPD like phenotype with impaired alveolarization in C57BL/6 mice (Ratner et al., 2013). Again this shows that beside hyperoxia mechanical ventilation is another factor damaging the lung structure causing BPD. Alternatively, examining single causative factors can be regarded as advantageous, as the gathered data can be unmistakably attributed to the effect of the solely examined condition.
blebbing was observed. A time course of one blebbing cell with frame intervals of 3 sec was recorded. In Figure 45, the beginning of the image sequence is shown and blebbing was visible by the strong green membrane signal. Within the first 21 sec of image acquisition, ruffling of the cell membrane occurred and a vesicle was formed inside this region. The diameter of the vesicle was approx. 1.5 µm. Membrane ruffling is typical for an inefficient lamellipodia adhesion (Borm et al., 2005). After 2 min, the vesicle fused with a second vesicle (Figure 46). Another 2 min later, the newly formed compartment moved into a region aside from the ruffles (Figure 47).The morphology of the compartment changed from elongated to circular. Additionally, the vesicle increased in diameter, up to 3 µm, and did not move further after uptake. It only moved slightly back and forth, but stayed close to the perinuclear region. Frame 191 in Figure 47 was the last image of the recorded time series. Another two vesicles were generated from the membrane as indicated by the two smaller arrows in that image. Their diameter was 1 µm and slightly below the diameter of the first synthesized vesicle. Frame 191 in Figure 47 shows further that once vesicles have been internalized membrane blebbing disappeared and the cell showed again a normal morphology without any membrane ruffles or blebs. Some studies provide evidence that besides actin polymerization also polarized endo- and exocytic cycles can cause cell migration (Bretscher 1996a, 1996b) or even macropinocytosis (Gu et al., 2011), which could explain the vesicle formation at the leading edge of the cell. Hence, the live cell data indicated that the observed blebbing potentially could be attributed to lamellipodia formation. The type of the internalized vesicles could not be clarified within this experimental setup. However, the observed vesicle sizes of > 1 µm were similar to sizes known for macropinosomes (Mercer and Helenius 2009). These structures were usually formed at sites of membrane ruffling, e.g. margins of spread cells (Swanson and Watts 1995). Furthermore, typeIIalveolar epithelial cells, like A549 cells, synthesize LB that also have sizes of 0.1 - 2.4 µm, a circular morphology, and are able to fuse with the cell membrane (Schmitz and Müller 1991).
The results obtained with the microspatula, where the indirect responders could easily be eliminated by excluding the cells with a delayed onset of the Ca 2+ signal, confirmed the findings obtained with the SC-device. The only difference compared to the SC-device was observed in cells treated with SKF96365, which had a significant effect on cells stimulated with the SC-device but no effect in cells stimulated with the microspatula. However, in the first case, the number of experiments was quite low and together with the high variability between the experiments, this might explain this difference. In addition these experiments included indirect responders that we could not identify and exclude from the analysis (please refer to 3.4.1). In cells treated with Ruthenium red, we were able to reduce the stretch response using both devices. This point at TRPV channels that (Clapham, 2007) have been shown to be blocked by Ruthenium red in literature. We neglected possible effects of Ruthenium red on channel present in the intracellular stores, like the RyR, as Ruthenium red has multiple positive charges that make it make highly membrane impermeable. Membrane permeabilization or other methods are necessary to allow the compound to pass through the plasma membrane.
development of a cellular in vitro model addressing the alveolar space. The human alveolar autologous coculture consists of primary alveolartype I-like pneumocytes cocultured with primary alveolar macrophages from the same human donor. The model demonstrated its use to investigate cell-particle inreractions at the air-liquid interface. Only macrophages engulfed foreign particles in the in vitro model visualized by CLEM. The ability of the autologous co- culture to mimic inflammatory processes in the lung is the focus of the third chapter. The in
of the ion via the IP3R receptors of the ER. In line with this notion, the treatment of the alveolar epithelium with an IP3R inhibitor (2-APB) prevented the hypercapnia-induced ERAD and phosphorylation of IRE1α. These results are consistent with previously published reports, suggesting that the hypercapnia-induced Ca 2+ elevation is due to the activation of IP3R receptors (100,196). Furthermore, it has been previously reported that the Na,K-ATPase through the scaffold protein, ankyrin may establish an interaction between the Na,K-ATPase α- subunit and the IP3R receptors in ER, suggesting direct Ca 2+ sensing by the Na + pump (203,204). Of note, the dysfunction of SERCA or store-operated channels can also promote an imbalance in ER homeostasis. While we have now established that the IP3R receptors are involved in the hypercapnia-induced signaling cascade, the exact mechanisms of their activation by CO 2 remain unknown. Based on our findings, we postulate that the GPCR receptor coupling pathway may be involved in the detection of hypercapnia. Future studies will be conducted to address this hypothesis. Interestingly, recent reports suggested that a metabolic sensor, AMPK that we have previously implicated in the hypercapnia-induced downregulation of the Na,K-ATPase and ENaC (51,98), might be activated by IRE1α (205). However, we have not dissected any AMPK-mediated ERAD mechanisms, and thus, future studies will be essential to tease out whether AMPK affects the ERAD of the NKA β-subunit and whether there is also a link between AMPK and IRE1α activation upon hypercapnia.
ILC2s have emerging roles in infection by cytokine production, but they are also able to interact with other immune cells directly. They are potent sources of type 2 cytokines upon activation. They produce predominantly IL-5 and IL-13 but further IL-4, IL-9 and the epithelial growth factor amphiregulin (Moro et al., 2010, Neill et al., 2010, Price et al., 2010, Turner et al., 2013). The expression of MHC II, CD80 and CD86, OX-40- ligand (OX-40L), KLRG1, inducible T-cell costimulator (ICOS) enable activation and modulation of other immune cells (see also chapter 1.1.4. and 1.1.5.) and interaction with epithelial cells (Oliphant et al., 2014, Mirchandani et al., 2014, Drake et al., 2014). In early stages of infection, innate immune cells such as macrophages, natural killer T cells and mast cells produce IL-33 (Hsu et al., 2010, Gorski et al., 2013). IL-33 is also released as a result of danger associated molecular pattern (DAMPs)-signaling (Patel et al., 2014), during necrosis. Further DAMP-signaling activates ILC2s as well as mast cells directly (Cayrol and Girard, 2014, Lefrançais et al., 2014). Activation of mast cells leads to production of prostaglandin D2, another potent activator of ILC2s, and release of non-caspase protease chymase as well as tryptase, which fortify the effect of IL-33 on ILC2s (Xue et al., 2014). Further, basophil-derived IL-4 enhances ILC2 proliferation (Kim et al., 2014). In this way, ILC2 activation is facilitated in early stages of inflammation and ILC2s rapidly initiate and orchestrate immune responses in a direct and indirect manner. ILC2s directly promote the Th2 response in early stages of infection, by production of type 2 cytokines. Moreover, ILC2-derived IL-5 can act on eosinophils to promote their activation and recruitment (Nussbaum et al., 2013). ILC2s are also able to interact with CD4 + T cells to mediate the Th2 immune responses (see
The wakefield effects in accelerator sections for future linear colliders will be reduced either by damping by detuning or by a combination of both. For the DESY/THD linac  it is forseen to employ heavily HOM-damped cells to provide a strong coupling to the TE/TM 11 -dipole passband
Our knowledge is limited regarding the factors that alter open probability of ENaC. However, mutations of specific residues within the intracellular N-terminal tail of α-ENaC result in low Po, suggesting that this segment of the channel structure is important for gating control (Grunder et al. 1999). For example, regulation of ENaC activity by phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown in studies in renal A6 collecting duct cells in which PIP2 binding to the N-terminal domain of β and γ-ENaC increased single channel open probability (Yue et al. 2002). Furthermore, an increase in extracellular Na + concentration decreases ENaC activity by altering channel gating. This process is called Na + self-inhibition, occurs over seconds and can be blunted by treatment with external trypsin (Chraïbi and Horisberger 2002). In contrast, an increase in intracellular Na + levels (feedback inhibition) results in slower changes in ENaC activity. Interestingly, Kellenberger et al. demonstrated that channels with Liddle's syndrome mutation expressed in oocytes did not respond to Na + -dependent inhibition, suggesting that the enhancement of ENaC activity in patients with Liddle's syndrome may be driven by a mechanism that decreases feedback inhibition (Kellenberger et al. 1998).
To study the unknown mechanisms in- volved in typeII resistance on transcrip- tion and replication levels, a series of infection experiments were conducted on CpR5M larvae with the baculovirus isolates CpGV-S, -M, -E2 (resistance breaking, positive control), as well as
Research on alveolar bone healing after tooth extrac- tion is mainly based on large preclinical models that have provided insight into the conserved post-extraction heal- ing sequence. 1 This sequence triggered by the tooth extrac- tion induces the formation of a blood clot. Subsequently, this blood clot is organized into a connective tissue matrix that is later reinforced by woven bone and finally replaced by organized lamellar bone. 1 This sequence of events also occurs when dental implants are placed into the alveo- lar bone as documented in canine models. The healing of tooth extraction has also been studied in rodent models, particularly in molar teeth of rats. 7 It should be mentioned however, that rat models are reliable in simulating osteo- porosis and diabetes but are not suitable when studying the impact of certain genes on the healing of the alveolar socket. Therefore, mice models were introduced by Vieira et al. 8 to test the effect of certain genes on socket healing. 9 Based on this concept, it was demonstrated that knockout of CD24 impairs bone healing following tooth extraction, 10
Fig. 1 Microphotographs of brain sections of hepatic encephalopathy (A-L) and mito- chondrial encephalopathy/Leigh syndrome (M-O). Laminar microvacuolation of the neuropil in cortical layers of the cerebrum is observed in a case of severe hepatic encephalopathy at a low magni ﬁcation (arrows in A, B) and at a higher magni ﬁcation (D, E). Abundant p62-positive nuclei are identi ﬁed in deep layers at a low magni ﬁcation (arrows in C) and at a higher magni ﬁcation (F). AA II have the characteris- tic enlarged cell nuclei and clear chromatin (G). AA II forming a triplet of nuclei (H). Small punc- tate condensations along the nuclear membrane in an AA II (I). p62 immunohistochemistry strongly labels the nuclei of AA II, including the peripheral nuclear membrane condensa- tions (arrows in L). (M-O) In a case of Leigh syndrome, glial nuclei in the basal ganglia are also enlarged but show more prominent cytoplasm on HE sta- ined sections than typical AA II (M). p62 shows intense and dif- fuse labeling of enlarged glial nuclei (N, O). HE (A, D, E, G, H, M), LFB-HE (B), p62 immu- nohistochemistry (C, F, J-L, N, O). Scale bars: 200 μm (A–C), 50 μm (N), 20 μm (D–H, J, K–M), 10 μm (I).
The three conjugated enam ine com pounds ex am ined in this study exhibited high potency in in hibiting PS II electron transport. D espite the similar ity in their chemical structure (conjugated enamine system ), their m ode of action was not the same. Two of them , cyanoacrylate and A C m l2 induced a ther moluminescence glow peak identical with the DCM U -induced Q -band in all-or-none m ode (Fig. 2 and 3), indicating that they bind to the Q B-site in D 1 protein to block Q A to Q B electron transport. In sharp contrast to these, A Pp 12 induced an unusual therm olum inescence glow peak located betw een the Q -band and B-band (Fig. 2). From the titration (Fig. 4) and oscillation (Fig. 5) experim ents, it was infer red that APp 12 induces a modified charge pair (S2Q i and S3Q i after the 1st and 2nd flashes, respectively), and thereby interrupts the S-state turnover by block