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.
As our data pointed out the importance of ATII-LCs in the antigen uptake process, further studies on the functionality of these cells were performed. Previous studies using isolated mu- rine ATII cells showed their trans-differentiation towards ATI cells within days during in vitro cultivation [ 30 , 31 ], indicating that they are not suitable for long term experiments in vitro. We therefore used the human ATII cell line A549 for further experiments. When stimulating A549 cells alone or in a transwell co-culture with the monocyte cell line THP1 with Bv1-Pro- tein, enhanced TSLP mRNA levels, known to initiate or enhance T H 2 responses at the lung epithelial cell surface [ 32 ], were measured in A549 cells, whereas Bv1-Peptide induced the tran- scription of IL-10 mRNA ( Fig 5 ). Our data indicate a distinct polarisation pattern of A549 cells depending on the encountered antigen. Similar to our observations, it has been shown that stimulation with correctly folded allergens, such as phospholipase A 2 or OVA, induces T H 2 im- mune responses, while structurally modified allergens (e.g. heat-treated OVA) rather induce T H 1 responses [ 33 , 34 ]. The capacity of peptides to induce IL-10 as described in our study is also supported by other investigators, suggesting allergen peptides as suitable candidates for tolerance induction (33).
21 neutrophil elastase, were shown to be predisposed to an early onset of severe emphysema (67). A study on Pallid mice which are naturally deficient in AAT revealed that they suffered from an early development of emphysema compared to wild type mice with normal level of AAT (68, 69). Indeed, the imbalance may occur either by an unregulated excessive release of proteases or by a deficiency, reduced synthesis or increased breakdown of anti-proteases. The excessive proteolytic load is contributed by infiltrating phagocytic leukocytes, namely neutrophils and macrophages. These cells secrete a wide range of proteases, including serine proteases (neutrophil elastase, proteinase 3, cathepsin G), cysteine proteases (Cathepsins B, H, L, K and S) and a different types of matrix metalloproteases (MMPs) into emphysematous lungs (70-72). Among these, neutrophil elastase is a highly potent elastolytic enzyme and its intra-tracheal injection in experimental animals is capable of inducing emphysema (73). Another study demonstrated that mice lacking neutrophil elastase were 59% protected against emphysema (74). Secretory leukocyte protease inhibitor (SLPI) and elafin secreted by goblet cells, serus cells, Clara cells and alveolartypeII (ATII) cells are both potent anti-proteases that can reversibly inhibit neutrophil elastase and in addition, SLPI can inhibit cathepsin G, trypsin, chymotrypsin and chymase while elafin inhibits proteinase-3 (72).
oxidative burst [ 55 ]. As a consequence, particle-induced NO can react with the superoxide radical to form peroxynitrite [ 56 ], the homolysis of which generates the highly reactive OH-radical mediating tissue damage [ 57 ] via the initiation of lipid peroxidation. Similar to activated polymorphonuclear leukocytes [ 22 ], activated macrophages will cause the oxidative degradation of β-carotene and thus the formation of aldehydic and other breakdown products with different biological activities aggravating the impact of the oxidative burst. In fact, it has been demonstrated that β-carotene at a concentration that can be achieved in human plasma after chronic oral supplementation (5 µM) [ 58 ], and its metabolites were able to increase ~ OH formation from H 2 O 2 in the Fenton reaction and the addition of vitamin A and retinoic acid to lung epithelialcells co-cultured with activated neutrophils resulted in a significant increase of the level of oxidized purines [ 59 ], while the increase of oxidized purins was not significant after β-carotene treatment. These findings are in contrast to our findings with primary pneumocytes, as there was no indication of genotoxicity up to 10 µM. The question therefore arises as to whether DMNQ is an adequate model for oxidative stress in the lung. In this context, it has to be emphasized that DMNQ is an inducer of glutathione (GSH) [ 60 ], which is an essential element of the antioxidant defense [ 61 , 62 ]. GSH is the predominant scavenger of reactive oxygen species (ROS), particularly in the liver [ 62 ] and lung [ 63 ]. Under oxidative stress, the normal physiological ratio of ~100–1000 GSH:1 GSSG can be shifted toward the oxidized form, eventually even reaching an equimolar ratio [ 61 , 64 ]. GSSG is then exported out of the cells and metabolized [ 65 ]. Thus, relative levels of GSH and GSSG provide an efficient diagnostic option in judging the redox state of cells and hallmark oxidative stress, as demonstrated for several respiratory diseases and aging [ 62 , 66 ]. The lipid peroxidation product 4-hydroxynonenal is known to form adducts with GSH [ 67 ], and the immediate decrease of glutathione reported after smoking [ 54 , 68 ] can be attributed to this and other aldehydic lipid peroxidation products. While smoking significantly reduces cellular free glutathione (GSH) in experimental animals, especially in the lung, even after smoking periods as low as 30 days with exposures three times a day [ 69 ] DMNQ may eventually protect from oxidative damage. Therefore, the lack of an effect comparable to that found with primary hepatocytes [ 26 ] may relate to increased GSH levels in pneumocytes, and it can be assumed that co-cultivation of pneumocytes with alveolar macrophages or neutrophils, and subsequent activation will more realistically reflect the in vivo situation of smokers consuming β-carotene supplements.
Damage of the endo-epithelial barrier is the major hallmark of acute lung injury upon bacterial infection, associated with oedema formation, alveolar flooding, impaired fluid clearance and gas exchange. Hence, to restore the normal lung function, alveolar repair processes are ultimately initiated (34). Resident alveolar macrophages have been assigned a contributing role in epithelial repair, closely associated with the transition of the pro- inflammatory into the anti-inflammatory macrophage phenotype (62, 94). In the current thesis the potential of early activated, pro-inflammatory resident alveolar macrophages to influence epithelial repair processes was investigated. Moreover, the hypothesis that pro-inflammatory resident alveolar macrophages may contribute to effective epithelial repair after LPS- and K. pneumoniae induced lung injury was tested. Hence, in vitro experiments revealed that alveolarepithelialcells co-cultured with LPS-stimulated resident alveolar macrophages express significantly higher amounts of growth factors, particularly of GM-CSF. Macrophage TNF-α released upon LPS stimulation was identified as a mediator inducing GM-CSF expression in epithelialcells, which in turn elicited autocrine proliferative signalling in typeIIalveolarepithelialcells. Genetic deletion of GM-CSF resulted in absence of macrophage-induced epithelial cell proliferation. Similarly, in vivo TNF-α neutralization after LPS-induced lung injury impaired epithelial proliferation. Furthermore, GM-CSF-deficient mice displayed reduced AEC II proliferation and sustained alveolar leakage after LPS challenge. Similarly, K. pneumoniae-induced lung injury was associated with early release of TNF-α and GM-CSF, and subsequent TNF-α-dependent AEC II proliferation during the alveolar repair phase. Altogether, these data reveal that alveolar repair processes are initiated early in the inflammatory course of pathogen-induced acute lung injury, and are mediated by macrophage TNF-α and epithelial GM-CSF (Fig. 31).
Alveolarepithelial 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.
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 .
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, typeIIalveolarepithelialcells, 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).
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 .
lacrimal glands, it was found that autoantibodies mediate apoptosis in the human salivary gland cells in a caspase-3 dependent manner . Furthermore it was shown, that autoantibodies to dsDNA and ribosomal P proteins, which are often found in SLE, are potent inhibitors of protein synthesis and are likely to mediate cellular dysfunction via this pathway . However, blockade of HisRS and Topo 1, 2α or β by antibody- binding has not been shown yet. Other mechanisms, how these autoantibodies may contribute to development of ILD in CVD, have to be taken into consideration. To this end, immune complexes might perpetuate a positive feedback loop amplifying inflammatory responses . IgG-mediated activation of complement is an important defense mechanism of the innate immune system to protect against infections. However, the same mechanisms can drive severe and harmful inflammation, when IgG antibodies react with self-antigens in solution or tissues, as described for several autoimmune diseases. More specifically, IgG immune complexes can activate all three pathways of the complement system, resulting in activation a panel of different complement receptors on innate and adaptive immune cells. Importantly, complement is often co-expressed on inflammatory immune cells such as neutrophils, monocytes, macrophages or dendritic cells and act in concert to mediate the inflammatory response in autoimmune diseases .
As the thymocytes commit to the DN4 stage, CD25 expression is downregulated. DN4 thymocytes then proliferate extensively to yield abundant progeny cells with co- expression of both CD4 and CD8 receptors and differentiate to double positive (DP) thymocytes. At the DP stage, thymocyte proliferation is again halted, which allows the initiation of somatic rearrangement at the TCRα locus. Unlike the TCRβ locus, the TCRα locus consists of numerous Vα and Jα gene segments but lacks a D segment. The rearranged TCRα chain pairs with TCRβ to form the TCRαβ complex as an intact TCR on the cell surface. During the whole expansion phase, the recombination activation genes (RAG) are turned off to prevent any premature rearrangement of the TCRα locus. During the formation of TCRαβ complexes, expression of receptors in response to cytokine signals is ceased and the thymocytes become unresponsive. Specifically for interleukin-7 receptor (IL-7R) that is dramatically downregulated to weaken the interaction with thymic stromal cells. In addition, high expression of suppressor of cytokine signaling 1 (SOCS1), an intracellular inhibitor of cytokine signaling, also occurs 28 . As the αβTCR is generated from random and flexible juxtaposition of TCR loci, positive and negative selections are conducted to test whether those TCRs are MHC-restricted and not autoreactive.
In most polarized epithelialcells, the α- and β-subunit are expressed at an equimolar ratio, assembled as heterodimers, and delivered to the basolateral membrane where they contribute to active Na + transport and maintain epithelial integrity . The abundance of Na + pump subunits and ATPase activity are tightly regulated by various stimuli. The pump is regulated by concentrations of its substrates as well as by changes in the molecular components of the surrounding environment (ions and non-ionic molecules). The Na,K- ATPase is modulated by membrane-associated components such as cytoskeletal elements and regulatory FXYD proteins, such as γ-subunit. The pump is also affected by variations in oxygen, carbon dioxide and nitrogen availability. As an important molecule in charge of various biological events, the Na,K-ATPase is regulated by a number of circulating endogenous inhibitors and hormones, such as aldosterone, thyroid hormone, glucocorticoid, catecholamines, insulin, carbachol, estrogen and androgen [34, 103-107]. All these stimuli can exert either short term or long term regulation of the Na, K-ATPase. Long term regulation of Na,K-ATPase usually involves changes in RNA and protein synthesis or degradation of the Na,K-ATPase isoforms [104, 107-111]. Short term modulation of the Na,K-ATPase function may be mediated by changes in the cellular distribution of pump units by reversible post-translational mechanisms such as phosphorylation or ubiquitination, or by changes in the intracellular Na + concentration which in turn modifies the pump kinetics [108, 111].
Microtubule-based in vitro motility and binding assays allow to study interaction of motor proteins with cytoskeletal filaments and to characterize movement of purified organelles along microtubules. In the current study, in vitro assays have been used to adjust the vesicle immunoprecipitation procedure and to confirm the presence of molecular motors on purified vesicles, which were examined thereafter biochemically. In vitro binding assays have revealed first evidence that purified post-TGN vesicles carry proteins that form a link between a vesicle itself and a microtubule. Most probably, this interaction is carried out by motor proteins. In fact, the motility assay showed that the purified vesicles do not only bind to microtubules, but also move along them (Fig. 3.1). Remarkably is, that the vesicles were moving in both directions on microtubular tracks and this fact can have several explanations. First of all, bidirectional movement can be carried out by at least two types of motor proteins (plus and minus end-directed) present on the motile organelle. For example, this could be the plus end-directed kinesin and minus end-directed dynein, as in the case of in vitro reconstitution of ATP-dependent movement of endocytic vesicles (Murray et al., 2000). On the other hand, the normal diameter of a single microtubule is 25 nm, whereas the measured diameter of microtubules in the current assay was 100-200 nm (data not shown), which means that several microtubules could have formed a bundle, probably with even orientations of plus and minus ends. Thus, the vesicles were moving always in one direction (plus or minus), but they were changing the microtubular filament within the bundle. Another possibility for bidirectional movement is so called “diffusive” movement of motor proteins. This type of motion has been shown, for example, for kinesin-13, which uses a one- dimensional diffusive search to rapidly target microtubule ends where it binds and depolymerises microtubules (Helenius et al., 2006). Recently, diffusive in vitro movement has been demonstrated also for processive kinesin-1 (Lu et al., 2009).
autophagosomes with the lysosomes and the subsequent degradation of the substrates. So it is feasible that a bacterial factor could interact with HDAC6 to block autophagosme maturation. Y. enterocolitica biogroup 1B posses several chromosomal virulence factors that should be further studied in the context of host-bacterium interaction and autophagy. For example, it is known that Y. enterocolitica biogroup 1B has a chromosome-encoded type three secretion system, called Ysa, for the delivery of protein effectors into host cells (Haller et al. 2000; Matsumoto & Young 2009). Studies in mice demonstrated that the Ysa TTSS plays a role in the colonization of the gastrointestinal tissues by Yersinia in the earliest stages of infection (Venecia & Young 2005). Furthermore, pathogenic and non-pathogenic Yersinia species possess one or two type two secretion systems, called Yts1 and Yts2 (von Tils et al. 2012). In Y. enterocolitica, Yts1 is speculated to be related with the interaction of free-living bacteria with their environment (Shutinoski et al. 2010). Additionally, it was described that Yts1 is involved in the dissemination and colonization of liver and spleen in orally infected mice, although the secreted substrates responsible for these effects are not determined (Iwobi et al. 2003). On the other side, it has recently shown that Yts2 is important for intracellular survival of Y. enterocolitica within macrophages (Bent et al. 2015). Therefore, it is possible that effector/s proteins delivered by the Ysa system or the type two secretion systems may interfere with the degradation of yersiniae within epithelialcells to favour the subsequent steps of the infection process. Finally, studies using in vivo expression technology (IVET) and signature-tagged mutagenesis (STM) have identified bacterial genes required for growth within the host (Darwin 2005) that could be also be important for Yersinia-induced autophagy and/or the blockage of the autophagic flux imposed by yersiniae. These studies have shown that chromosomal factors like the protease HreP, and the phospholipases PldA and YplA, among others, are specifically induced after infection of mice (Darwin 2005).
Recently, a role of miRNAs in the response against bacterial pathogens has been proposed. miRNAs were shown to be effective against Pseudomonas syringae infection in plants . Similar to viruses, P. syringae was found to secrete proteins that bind host miRNA and subsequently modulate immune response . Furthermore, Rao and colleagues described the presence miRNAs expressed by pathogenic Pseudomonas aeruginosa strains which were isolated from adult patients with cystic fibrosis . Xiao et al. uncovered a Helicobacter pylori-dependent induction of miR-146b and miR-155 in gastric epithelialcells with subsequent inhibition of IL-8, a central cytokine in the chemotaxis of leukocytes . Further investigation revealed that miRNAs control major inflammatory pathways, such as the TLR-mediated activation of the NF-kB pathway . While P. syringae and H. pylori remain extracellular during infection, a recent study showed altered immune response of mice deficient in miR-155 to the facultative intracellular pathogen Salmonella . Schulte et al. uncovered the regulation of IL-6 and IL-10 by miRNAs of the let-7 family and miR-155 induction by secreted effector proteins of Salmonella rather than the invading pathogen .
Mucus is secreted by goblet cells and consists of two layers: an inner layer (approximately 50 µm thick) firmly attached to the epithelium and a more loosely attached outer layer (100-500 µm thick) facing the intestinal lumen (Corazziari, 2009; Leser and Mølbak, 2009). Mucus is secreted at top of the crypts and then is pushed upwards along the crypt-villus axis by the newly secreted mucus underneath (Johansson et al., 2011). The tips of the villi are not always covered with mucus (Johansson et al., 2011). While some bacteria are able to colonize the outer layer, the inner firmly attached layer is devoid of bacteria (Atuma et al., 2001; Johansson et al., 2008). The main components of intestinal mucus are mucins, which are polymeric glycoproteins that are responsible for the gel-like structure (Hollingsworth and Swanson, 2004). Different mucins are expressed in an organ- specific manner with MUC2 being the predominant intestinal mucin. Some mucins, in particular MUC1, do not form polymers and stay covalently attached to the epithelium where they form the glycocalyx (Wilson, 2008). Other components of mucus are lipid vesicles, antibodies, ions, dietary products, entrapped microbes and water (Ofek et al., 2003).
good spray was already produced with a pressure of 0.4 bar. In general, next to the nozzle dimension, the pressure is a process parameter that is related to the shear the cells may experience during spray formation. Veazey et al. tested air pressures between 0.41 and 1.24 bar and showed a direct dependence of the survival of bovine dermal ﬁbroblasts. 4 Similarly, Nahmias et al. re- port survival rates of NIH 3T3 cells of 64% at a pressure of 0.97 bar till 90% for a pressure less than 0.34 bar. 3 Tritz et al. have shown chondrocyte survival rates of 88% and 80% in alginate gels 3 days after spraying for pressures of 0.9 and 1.2 bar. 2 In another study, the same group reports a survival rate of 52% for human mesenchymal stem cells in the same setup with a pressure of 0.9 bar. 24 Roberts et al. report a chon- drocyte survival of 70–84% depending on air ﬂow rates between 4 and 8 L/min (which corresponds to different air pressures as well). 6 Thus, higher pressures evoke higher shear and elongation stresses on the cells and re- duce their survival. Hence, the unchanged survival of vSMCs and 88.5% survival rate of our RECs at a pressure of 0.4 bar are very good results.
non-lymphoid tissues. 21 Although we cannot rule out the possibility that some CD103 þ CD11b CD8a þ LDCs originate in the lymphoid compartments of the intestine, it is likely that they largely represent LP-derived migratory DCs. Crucially, CD103 þ CD11b CD8a þ DCs are also able to cross-present IEC antigen to CD8 þ T cells in vivo. By adapting a technique described by the Pabst laboratory, 29 we showed that subcapsular injection of CD103 þ CD11b CD8a þ LDCs, but not other DC subsets, from the lymph of steady-state 232-4 mice into the MLNs led to clonal expansion of OT-I T cells. The capacity of intestinal DCs to induce gut tropism in a retinoic acid-dependent manner has been well character- ized. 24,30 However, it is not clear which intestinal DCs are primarily responsible for inducing this phenotype in CD8 þ T cells. In contrast with an earlier report, 11 we have recently shown that CD103 þ CD11b CD8a þ DCs have high aldehyde dehydrogenase activity and can induce CCR9, a marker of gut tropism on T cells, with a similar efficiency to the other intestinal DC subsets. 10 Here we show that, in vivo, cross- presentation by CD103 þ CD11b CD8a þ DCs also induces CCR9 expression on responding OT-I T cells, and that, in this experimental system, fully differentiated CCR9 þ OT-I cells are only detectable in the intestinal LP of mice after subcapsular injection of CD103 þ CD11b CD8a þ LDCs, but not other LDC subsets.
The concentration of ECM proteins in the ADSC secretome was nearly equal in all 5 patients, e.g. FN/ fibronectin and THBS1/thrombospondin-1. ADSC secretion of several types of ECM proteins 60 , supports adhe- sion and proliferation of MEC. Although, the ADSC secretome did not influence ECM COL4A1 and FN1 gene expression of NORMA MEC, the integrin receptors ITGA5/6 were induced. This supports ADSC mediated MEC ECM interactions are essential for development. Normal mammary breast tissue contains low levels of FN, com- pared to higher levels in breast tumors 61 . Recently, it could be shown that high FN concentration in breast tumors induce EMT and might be both, a cause and result of tumor initiation and/or progression 61 . In contrast, THBS1 is an ECM glycoprotein that inhibits tumor cell growth and metastasis partly due to its anti-angiogenic effect 62 . However, prolonged delivery of THBS1 can result in emerging resistant cells that lead to tumor insensitivity 62 .