Podcasts about ccr7

Подкаст является продолжением серии о базовой иммунологии и посвящен активации дендритных клеток и Т-клеток. Презентация на Гугл.Диске В подкасте разбираем: Дендритные клетки как основные антиген-презентующие клетки иммунной системы; Их активация патогенами; Главный комплекс гистосовместимости (MHC) 1-го и 2-го типа; CCR7… Continue reading →

Videos zur Serie unter youtube.com/medizinmensch Lymphknoten-Schmerzen und Schwellungen sind eine Erinnerung an die wichtige Funktion dieser lymphatischen Organe bei Infektion und Entzündung. Doch wie entsteht Lymphknoten-Schwellung? Welche Rolle spielen Lymphknoten bei der Immunantwort durch Lymphozyten und andere Immunzellen wie dendritische Zellen? Bei Lymphe handelt es sich um Gewebsflüssigkeit, die nicht durch das Venen absorbiert werden kann. Das Lymphsystem dient der Drainage und vermeidet so ein Lymphödem. Das Video ist Teil der Serie "Hidden Champions" in der du über Organe lernen kannst, deren Funktion nicht allgemein bekannt ist wie Lymphknoten, Nebenniere und Milz. 0:00 Intro 0:18 Anatomie 1:00 Lymphflüssigkeit enthält Informationen, Beispiel Zahninfektion 1:50 Struktur Lymphknoten 3:10 Zusammenspiel Lymphknoten und Gewebe bei Infekt 4:24 Dendritische Zellen, Beobachter der Schlacht, CCR7 6:14 Derweil im Lymphknoten 6:49 CCL als Lockruf für CCR7 positive DC 7:20 Antigenpräsentation der DC im Lymphknoten 8:17 Auffinden der passenden Lymphzelle 10:42 Interaktion DC und T Zelle 11:30 Klonale Vermehrung aktivierte T Zelle Glossar: Antigen: Ein Ziel für eine Immunantwort, meist ein Eiweißfragment CCR7 (C-C Chemokin Rezeptor 7): Chemokinrezeptor der bei Aktivierung von dendritischen Zellen aktiv wird, und Zelle mittels CCL19 und CCL21 u.a. in den Lymphknoten transportiert Dendritische Zelle: Sternförmige Immunzelle mit ausgeprägter Fähigkeit Antigen an T Zellen zu präsentierten Granulozyt: Einfache angeborene Immunzelle und eine der ersten Verteidigungslinien gegen Infektionen. Tötet Bakterien mittels Bleiche (Wasserstoffperoxid u.ä) dem sog. oxidativem "Burst" T Zelle: Eine Art von Immunzelle, die sich bei Aktivierung mittels Klonierung vermehrt T Zell Rezeptor (TCR): Andockstelle auf T Zellen, Kann jeweils ein bestimmtes Antigen binden und ermöglicht so eine Immunreaktion Abonniere jetzt und erhalte jeden "Medizin-Mittwoch" weitere interessante Folgen zu medizinischen Themen. Folge direkt herunterladen

Wenn Krebszellen sich im Körper ausbreiten, können Tochtergeschwülste, sogenannte Metastasen, entstehen. Diese sind für etwa 90 Prozent der Todesfälle bei Krebspatienten verantwortlich. Ein wichtiger Ausbreitungsweg der Krebszellen verläuft über das Lymphgefässsystem, das, ähnlich wie das Blutgefässsystem, den ganzen Körper durchzieht und Lymphknoten miteinander verbindet. Bei der Wanderung von weißen Blutzellen durch dieses System, um beispielsweise die Abwehr von Krankheitserregern zu koordinieren, spielt ein spezielles Membranprotein, der Chemokin-Rezeptor 7 (CCR7), eine wichtige Rolle. Dieser sitzt in der Hülle der Zellen, der Zellmembran, und zwar so, dass er äussere Signale empfangen und diese in das Innere weiterleiten kann. Im Rahmen eines gemeinsamen Projekts mit dem Pharmaunternehmen F. Hoffmann-La Roche AG (Roche) haben Forschende des Paul Scherrer Instituts PSI erstmals die Struktur von CCR7 entschlüsseln und den Grundstein für die Entwicklung eines Medikaments legen können, das die Metastasierung bestimmter häufiger Krebsarten wie Darmkrebs verhindern könnte. In den Zellen aller Wirbeltiere kommen 20 verschiedene Chemokin-Rezeptoren vor, die mit mehr als 40 Signalproteinen, sogenannten Chemokinen, interagieren können. Jedes dieser Signalproteine passt nur zu ganz speziellen Rezeptoren. Bindet eines der Signalproteine an einen Rezeptor, löst das wiederum Prozesse innerhalb der Zelle aus, die zu einer spezifischen zellulären Antwort auf das Signal führt. Paul Scherrer Institut PSI/Christina Bonanati Lesen Sie den gesamten Beitrag auch auf MEDIZIN ASPEKTE Originalpublikation: Structural basis for allosteric ligand recognition in the human CC chemokine receptor 7 K. Jaeger, S. Bruenle, T. Weinert, W. Guba, J. Muehle1, T. Miyazaki, M. Weber, A. Furrer, N. Haenggi, T. Tetaz, C. Huang, D. Mattle, J.-M. Vonach, A. Gast, A. Kuglstatter, M.G. Rudolph, P. Nogly, J. Benz, R.J.P. Dawson, J. Standfuss Cell, 22. August 2019 (online) DOI: https://dx.doi.org/10.1016/j.cell.2019.07.028

Dr Carolyn Lam:                Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to the journal and its editors.                                                 I'm Dr Carolyn Lam, associate editor from the National Heart Center and Duke National University of Singapore. The ORBITA Trial of percutaneous coronary intervention and stable single vessel coronary artery disease has to be one of the most hotly discussed in the cardiology world. The featured paper of this week adds important knowledge that will help us understand the physiology stratified results of ORBITA.                                                 Coming right up after these summaries.                                                 The first original paper this week provides novel mechanistic insights that may lead to a new treatment approach for obesity and hypertriglyceridemia. Co-corresponding authors, Drs Xiang and Xia from Central South University of Xiangya in China, looked at Reticulin 3, which is an endoplasmic reticular protein that has previously shown to play a role in neurodegenerative diseases.                                                 In the current paper, the authors show that over-expression of Reticulin 3 in mice induced obesity and a greater accumulation of triglycerides. Remarkably, increased Reticulin 3 expression was also found in patients with obesity and hypertriglyceridemia. They further showed that Reticulin 3 played critical roles in regulating the biosynthesis and storage of triglycerides and in controlling lipid droplet expansion. Thus, these results suggest that inhibiting the expression of Reticulin 3 in fat tissue may be a novel therapeutic approach to treat obesity and hypertriglyceridemia in the future.                                                 The next study provides insights into the genetic determinates of residual cardiovascular risk in patients already receiving statins. First author Dr Wei, corresponding Dr Denny from Vanderbilt University Medical Center and their colleagues performed a genome-wide association study and identified that a variation at the LPA Locus was associated with coronary heart disease events during statin therapy and independent of the extent of LDL cholesterol lowering. The association of the LPA Locus with coronary heart disease events persisted in individuals with an LDL cholesterol less than 70 milligrams per deciliter. These findings, therefore, provide support for exploring strategies targeting circulating concentrations of lipoprotein(a) to reduce coronary heart disease events in patients already receiving statins.                                                 The next paper provides important mechanistic results that help us understand pathways in atherosclerotic plague regression. Co first authors, Drs Mueller and Zhu, corresponding author Dr Fazio from Oregon Health and Science University and their colleagues have previously shown that mice lacking an LDL receptor with beta protein 1 in macrophages undergo accelerated atherosclerotic plague formation. However, in the current study they sought to explore the role of macrophage LDL receptor protein 1 during plague regression. They did this by placing EPO E deficient mice on a high fat diet for 12 weeks, then reconstituting their bone marrow using wall type or macrophage LDL receptor protein 1 deficient mice as donors, and finally switching them back to a chow diet for 10 weeks. The authors found that the lack of LDL receptor protein 1 expression in macrophages unexpectedly caused more atherosclerosis regression. Mice with macrophages lacking LDL receptor protein 1 showed less M1 macrophages in the plague and increased CCR7 dependent egress of macrophages from the plague. Thus, loss of macrophage LDL receptor protein 1 has a dual and opposite effect on plague biogenesis, depending on whether the plague is growing or shrinking.                                                 The next paper highlights the intercalated disc, which is a specialized intercellular junction, coupling cardiomyocyte electrical activity in forced transmission as a mechanosensitive signaling hub for causative mutations in cardiomyopathy. First author Dr Trembley, corresponding author Dr Small from University of Rochester School of Medicine and Dentistry and their colleagues showed that myocardin related transcription factors associated with desmosome proteins of their intercalated disc in both murine and human hearts. Genetic deletion of myocardin related transcription factors in cardiomyocytes led to rapid onset of dilated cardiomyopathy in response to pressure overload hypertrophy. Furthermore, myocardin related transcription factors were required for the maintenance of sacromere and intercalated disc integrity under pathological stress. These findings, therefore, provide a unique link between the intercalated disc and mechanosensitive transcriptional regulations. Since myocardin related transcription factors redistribute from intercalated disc in human heart failure, this may represent a novel signaling complex present in cardiomyopathic characterized by desmosome dysfunction.                                                 The next paper investigated the association of blood pressure with peripheral arterial disease events, using data from the ALLHAT Trial. Co first authors Drs Itoga and Tawfik, corresponding author Dr Chang from Stanford University School of Medicine and their colleagues found that both lower systolic blood pressure of less than 120 and higher systolic blood pressure of above 160 millimeters of mercury were both associated with higher rates of peripheral arterial disease events. Diastolic blood pressure less than 70 and a pulse pressure above 65 millimeters mercury were also associated with increased rates of lower extremity peripheral arterial disease events. Given that the recent revised blood pressure guidelines advocate lower systolic blood pressure targets for overall cardiovascular risk reduction, the authors called for future, further refinement of optimal blood pressure targets, specific for peripheral artery disease.                                                 The final original paper this week provides the first integrated atherosclerotic disease risk calculator to incorporate risk factors including high sensitivity C reactive protein, family history, and coronary artery calcium data. First and corresponding author Dr Khera from UT Southwestern Medical Center and colleagues used 3 population-based cohorts to develop Cox Proportional Hazards Models for the outcome of atherosclerotic cardiovascular disease. The derived Astro-CHARM model incorporated factors like age, sex, systolic blood pressure, total and HDL cholesterol, smoking, diabetes, hypertension treatment, family history of myocardial infarction, high sensitivity c reactive protein, and coronary artery calcium scores. The model performance was validated externally in a 4th cohort, and shown to improve risk prediction compared with traditional risk factor equations, and showed good discrimination in calibration in the validation cohort. A mobile application and web based tool was developed to facilitate the clinical application of this tool, and is available at www.astrocharm.org.                                                 And that brings us to the end of this week's summaries. Now for our featured discussion.                                                 Gosh, I am learning for the first time today that it's terribly inconvenient to lose my voice when I am a podcaster. This is Carolyn Lam and our featured discussion that I am so excited about, but the cool thing is the thing we are talking about is so hot that you don't even need me to say anything. And what we are talking about is the ORBITA Trial. That was greeted with as much hype and hoopla and sensationalism since its publication in 2017. I am so proud to have the first and corresponding author Dr Rasha Al-Lamee from National Heart and Lung Institute Hammersmith Hospital in London. I also have Dr Ajay Kirtane from Columbia University Medical Center in New York Presbyterian Hospital and the Cardiovascular Foundation in New York as the editorialist for the paper. And finally, our associate editor Dr Manos Brilakis from UT Southwestern. Rasha, why don't you just take it away and just tell us, what is your paper focusing on in this week's issue? Dr Rasha Al-Lamee:         The paper that was published in this issue in circulation is basically our second analysis of the ORBITA Trial, a substudy analysis. Essentially, looking at the primary endpoint and the secondary endpoints of ORBITA, and having a look at those patients from ORBITA and seeing whether there was any association between their invasive physiological assessment using FFR and ISR at the pre-randomization stage and seeing whether the level of ischemia on ISR or FSR was associated or predicted in the way in which they performed in terms of their endpoints. To see whether there was any difference in the placebo control efficacy of angioplasty in those patients who have more or less severe ischemia on their invasive physiological assessment. Dr Manos Brilakis:            First off, that's a phenomenal paper, and I think she puts things into perspective. I know Ajay put an excellent tutorial. I think all of us were surprised about the findings. You would expect that the more ischemia, that you might see a little more response. Any thoughts as to why there wasn't such an association? Dr Rasha Al-Lamee:         I think it's so difficult because, of course, as we all know from the primary paper that was published in The Lancet, in terms of the primary endpoint, which would be change in exercise time and the difference between the two groups, the difference is actually much smaller than we expected. And when we have such a small difference in exercise time, the ability to be powered enough to be able to split that endpoint based on stratification of invasive physiology becomes very difficult, and we're perhaps underpowered to be able to do that.                                                 Where we did see a very great effect in terms of the primary assessment in The Lancet paper was in stress echo ischemia. What we saw is those patients who had angioplasty were far more likely to have an improvement, or indeed, a normalization of their ischemia on their stress echo. Where we saw a big difference the two groups we were then clearly powered to be able to stratify those patients based on their invasive physiology, and for that secondary endpoint we saw that, in fact, tied to your stenosis or the lower your ISR or FRR, the more likely you are to have an improvement in stress echo, having had placebo controlled angioplasty. Dr Manos Brilakis:            Ajay, I know you had a lot of things insight into the vision of the tutorial for the ORBITA Trial. What are your thoughts about the findings? Dr Ajay Kirtane:                 I would, first of all, congratulate Rasha and the ORBITA team, there are others, for not only doing the main trial, but for conducting these detailed analyses, which were clearly set up ahead of time, and that's been one of the critiques of the trial is why were patients with normal-ish range FFRs included. Well, part of it was to test this hypothesis, and perhaps to show that there would be a correlation between the change in the FFR, if you will, and the endpoints that were measured.                                                 So, I think that that's the first part, that this is actually a scientific experiment, and a thoughtful one in doing so. I think exactly as Rasha said though, if there is a limited signal, with respect to the overall trial, then further subsetting is less likely to show a significant signal. I think that's exactly what the investigators found. The only other comment I would make though is, I would commend Rasha and the team for producing other analyses that are novel in this manuscript including the freedom from angina analysis, as well as responding to some of the earlier critiques of the trial and not using specific methodologies to adjust the baseline differences improves. Those are also included in this analysis. Dr Manos Brilakis:            Yeah, absolutely, I think that was very enlightening to see, the freedom of angina. And I know there was some questions whether that might change the overall findings from the studies, so there is some quality of life benefit. Rasha, what is your thoughts about this? I mean, you must understand this study better than anyone else. People who have stable angina, should they undergo PCI or not? Dr Rasha Al-Lamee:         I think the freedom from angina signal was very important, and obviously not something that we had pre-specified, so it wasn't reported in the primary analysis. We're obviously much more able now, since we've published that primary analysis to do secondary analyses and look at things that perhaps we haven't pre specified. And it's interesting to see that 20% more patients are free from angina having had angioplasty vs. placebo. Having said that, to me, it's a fantastic finding, but still a little unexpected. Much less than we might expect looking at unblinded data, or our unblinded clinical experience. I would have expected much higher levels from freedom of angina. Dr Rasha Al-Lamee:         I think what we know, and what we've seen both from this paper, very importantly, and also the primary manuscript, is that the efficacy of angioplasty is very tightly linked to the improvement in ischemia. We've actually, in fact, got more papers that are coming out from our group recently. And that you can predictably tell your patients that if I sense a lesion that's causing a reduction in ISR or FFR, and potentially symptoms, then I will improve your ischemic burden.                                                 What I think is more tricky is how much I will relieve your symptoms, or make you feel better. That may be because symptom assessment itself is very tricky, and perhaps that actually just diagnosing cardiac angina is actually a very difficult thing. The easiest way to piece out improvement in symptoms is to find those patients who become free of angina because, of course, that's the binary end point. When we look at grades of symptoms, and whether their angina frequency improves, or whether the level of angina improves in terms of PCI, then I think it becomes much harder, especially in a blinded trial where, of course, when people come back, even with atypical chest pain, it will still be recorded as potentially angina because, of course, both the investigators and the patients have no idea what they've had done, which is quite different from real life where, of course, you are able to think more about whether this chest pain might indeed be from the heart or from other causes. Dr Manos Brilakis:            Perfect, thank you very much. And I would completely agree with you that, the study was perfect. And, as Ajay said, it is something that we needed, and more of them should be done. And I think you are right that this is the best way to piece out the symptom improvement.                                                 Ajay, any final comments? Dr Ajay Kirtane:                 I think that the toughest challenge with trials like this is to really enroll the patients that many of us as interventionists feel would really improve in terms of their symptom class. Even despite these efforts, if one looks at the baseline of anginal frequency in the trial, the means are relatively high, which suggest that the anginal burden, at least in terms of measurements through the anginal questionnaire is not that severe. One could argue that somebody has severe angina that is occurring all the time, that those are types of patients that are hard to randomize in a clinical trial.                                                 I think, at least my overview stepping back perspective of the context of ORBITA within clinical practice, is exactly that. The trial is an important scientific advance, but this does not encompass the answer for every single patient that comes to see us in the office that have a range of symptoms, very severe to less severe. That was something Rasha has been saying all along as well. It's not something that we could over extrapolate this to every patient that we see. So, I think that when the hype dies down, these types of scientific analyses will stand out. They emphasize the need for regular clinical research, and in that way, I think has generated a lot of attention not only to the clinical field here, but also the scientific pursuit of evidence. That's a really magical thing. Dr Rasha Al-Lamee:         I think, if I can add to that Ajay, I think it's probably also sort of the assessment of symptoms is incredibly important. I think many of us, and I'll include myself in this, when we see a very tight stenosis, are happy to essentially correlate any level of symptoms to that tight stenosis. One thing I've learned from all this, I want to see reproducible angina that very much is textbook, cardiac caused chest pain, and the atypical anginas we see, perhaps some of that pain is not from that stenosis, but from somewhere else. Therefore, by fixing that stenosis, we don't necessarily make that pain go away. Dr Manos Brilakis:            Absolutely, and I think you are absolutely, if it is something simple vessel disease, if it's something a little more straightforward, then I think you are right Ajay, that this is much harder, multiple vessel disease especially in people with reduced ejection fraction. Dr Carolyn Lam:                You've been listening to Circulation on the Run! Don't forget to tune in again next week!  

Dr. Gwen Randolph speaks about the seminal work of Reinhold Förster and colleagues on the role of CCR7 in the primary immune response.

Interstitial lung diseases (ILD) are severe chronic lung diseases characterized by an increased deposition of extracellular matrix in the lung interstitial space, leading to a thickening of the alveolar walls and impairment of the gas exchange. One of the most common entities in this category is idiopathic pulmonary fibrosis (IPF) with a mean survival time of 2 to 3 years from diagnosis. Until now, there is no curative therapy available and the symptomatic anti- inflammatory treatment and oxygen supplementation cannot prevent the development of the end stage pulmonary fibrosis. The chemokine receptor CCR2 is important for leukocyte recruitment to inflamed tissues through interaction with CCL2 (MCP-1). The blockade of the CCR2/CCL2 pathway attenuated the development of pulmonary fibrosis in mouse models. However, CCR2+ T-lymphocytes acquired regulatory functions in experimental arthritis during the course of disease. Therefore, it is unknown whether CCR2+ T cells are involved in the pathogenesis of IPF or, on the contrary, represent an unsuccessful effort of the immune system to limit the disease. Observations in paediatric patients with different forms of ILDs suggested a role for CCR2+ T cells in pulmonary fibrosis. To characterize these T cells, flow cytometric studies were performed using the bleomycin mouse model of pulmonary fibrosis. The kinetic of CCR2+ T cells in BALF, lung tissue, and spleen following intratracheal administration of bleomycin (BLM) was assessed at time points between day 3 and day 21. To determine, if the constellation of naïve, central memory and effector memory T cells changes after BLM treatment, and to which of these subtypes CCR2+ T cells belong to, the cells were additionally stained for CD62L and CD44. For further characterization of CCR2+ T cells, chemokine receptor co-expression with CCR2 was investigated at the time point of the maximal presence of CCR2+ T cells. Total T cell numbers increased in BAL and lung tissue but not in spleen. Percentages of CD62LlowCD44hi effector memory T cells increased in lung tissue in the early phase of BLM induced fibrosis, while the CD62LhiCD44low naïve T cell population decreased. The percentage of CCR2+ T cells increased following BLM treatment with a maximum on day 12. The majority of CCR2+CD4+ T cells showed a Tem phenotype. CCR3, CCR4, CCR6, CXCR4, and CXCR5 expressing cells increased within the pulmonary CD4+ T cell population following bleomycin treatment. Among CD8+ T cells from treated mice, CCR5, CCR6, and CXCR5 positive cells were increased. CCR7 was highly co-expressed with CCR2 in saline and bleomycin treated mice, whereas co-expression of CCR3, CCR4, CCR6 and CXCR5 increased significantly in treated mice. The results indicate an activation of pulmonary T cell populations following bleomycin treatment. CCR2+CD4+ T cells probably take part on this T cell response as they exhibit an effector memory phenotype and increase following BLM treatment. In contrast, the stable percentages of the different T cell subtypes in spleens gave no hint for a systemic T cell reaction. The pattern of chemokine receptor expression argues against a Th1 polarization and towards a Th2, Th17 or TFH polarization of CCR2+ T cells.

Introduction. Dendritic cells (DCs) and oxLDL play an important role in the atherosclerotic process with DCs accumulating in the plaques during plaque progression. Our aim was to investigate the role of oxLDL in the modulation of the DC homing-receptor CCR7 and endothelial-ligand CCL21. Methods and Results. The expression of the DC homing-receptor CCR7 and its endothelial-ligand CCL21 was examined on atherosclerotic carotic plaques of 47 patients via qRT-PCR and immunofluorescence. In vitro, we studied the expression of CCR7 on DCs and CCL21 on human microvascular endothelial cells (HMECs) in response to oxLDL. CCL21- and CCR7-mRNA levels were significantly downregulated in atherosclerotic plaques versus non-atherosclerotic controls 90% for CCL21 and 81% for CCR7 (P < 0.01)]. In vitro, oxLDL reduced CCR7 mRNA levels on DCs by 30% and protein levels by 46%. Furthermore, mRNA expression of CCL21 was significantly reduced by 50% (P < 0.05) and protein expression by 24% in HMECs by oxLDL (P < 0.05). Conclusions. The accumulation of DCs in atherosclerotic plaques appears to be related to a downregulation of chemokines and their ligands, which are known to regulate DC migration. oxLDL induces an in vitro downregulation of CCR7 and CCL21, which may play a role in the reduction of DC migration from the plaques.

Dendritic cells (DCs) play a key role in the initiation of adaptive immune responses and the maintenance of self-tolerance. Due to their therapeutic potential, understanding the mechanisms that guide DC differentiation and effector functions is important. DC differentiation and activation depends on transcription factor control of stage-specific gene expression. The recent identification of posttranscriptional control of gene expression by microRNAs (miRNAs) has added another layer of gene regulation that might be important in DC biology. We analyzed the miRNA expression profiles of different DC subsets and identified several miRNAs differentially expressed between plasmacytoid DCs (pDCs) and conventional DCs (cDCs). In terms of miRNA expression, pDCs were more closely related to CD4+ T cells than to cDCs. We also observed that pDCs and cDCs preferentially expressed miRNAs associated with lymphoid or myeloid lineage differentiation, respectively. By knocking down miR-221 or miR-222 during in vitro DC differentiation, we obtained a higher pDC frequency. While p27kip1 and c-kit are confirmed miR-221/222 targets, we additionally identified the pDC cell fate regulator E2-2 as a potential miR-221/222 target. Thus, our analysis points to a role for miRNAs in directing and stabilizing pDC and cDC cell fate decisions. To assess the general influence of miRNAs on DCs, we generated mice with a DC-specific conditional knockout of the key miRNA-producing enzyme Dicer. Dicer-deficient mice dis- played no alterations in short-lived spleen and lymph node DCs. However, long-lived epidermal DCs, known as Langerhans cells (LCs), showed increased turnover and apoptosis rates, leading to their progressive loss. Upon stimulation, Dicer-deficient LCs were able to properly upregulate the surface molecules MHC class I and CCR7, but not MHC class II, CD40 and CD86. In consequence, they were incapable of stimulating CD4+ T cell proliferation. The work presented in this thesis indicates a role for miRNAs in DC regulation not covered by transcription factors. Having demonstrated a role for miRNAs in DC lineage fate decisions, as well as in LC homeostasis, maturation and function, we conclude that miRNAs regulate various aspects of DC biology and thereby contribute to the control of adaptive immune responses.

Background: The fetal immune system is characterized by a Th2 bias but it is unclear how the Th2 predominance is established. Natural killer T (NKT) cells are a rare subset of T cells with immune regulatory functions and are already activated in utero. To test the hypothesis that NKT cells are part of the regulatory network that sets the fetal Th2 predominance, percentages of Vα24(+)Vβ11(+) NKT cells expressing Th1/Th2-related chemokine receptors (CKR) were assessed in cord blood. Furthermore, IL-4 and IFN-γ secreting NKT cells were quantified within the single CKR(+) subsets. Results: Cord blood NKT cells expressed the Th2-related CCR4 and CCR8 at significantly higher frequencies compared to peripheral blood NKT cells from adults, while CXCR3+ and CCR5+ cord blood NKT cells (Th1-related) were present at lower percentages. Within CD4negCD8neg (DN) NKT cells, the frequency of IL-4 producing NKT cells was significantly higher in cord blood, while frequencies of IFN-γ secreting DN NKT cells tended to be lower. A further subanalysis showed that the higher percentage of IL-4 secreting DN NKT cells was restricted to CCR3+, CCR4+, CCR5+, CCR6+, CCR7+, CCR8+ and CXCR4+ DN subsets in cord blood. This resulted in significantly decreased IFN-γ /IL-4 ratios of CCR3+, CCR6+ and CCR8+ cord blood DN NKT cells. Sequencing of VA24AJ18 T cell receptor (TCR) transcripts in sorted cord blood Vα24Vβ11 cells confirmed the invariant TCR alpha-chain ruling out the possibility that these cells represent an unusual subset of conventional T cells. Conclusions: Despite the heterogeneity of cord blood NKT cells, we observed a clear Th2-bias at the phenotypic and functional level which was mainly found in the DN subset. Therefore, we speculate that NKT cells are important for the initiation and control of the fetal Th2 environment which is needed to maintain tolerance towards self-antigens as well as non-inherited maternal antigens.

Background: Active dendritic cell (DC) immunization protocols are rapidly gaining interest as therapeutic options in patients with acute myeloid leukemia (AML). Here we present for the first time a GMP-compliant 3-day protocol for generation of monocyte-derived DCs using different synthetic Toll-like receptor (TLR) agonists in intensively pretreated patients with AML. Methods: Four different maturation cocktails were compared for their impact on cell recovery, phenotype, cytokine secretion, migration, and lymphocyte activation in 20 AML patients and 25 healthy controls. Results: Maturation cocktails containing the TLR7/8 agonists R848 or CL075, with and without the addition of the TLR3 agonist poly(I:C), induced DCs that had a positive costimulatory profile, secreted high levels of IL-12(p70), showed chemotaxis to CCR7 ligands, had the ability to activate NK cells, and efficiently stimulated antigen-specific CD8(+) T cells. Conclusions: Our results demonstrate that this approach translates into biologically improved DCs, not only in healthy controls but also in AML patients. This data supports the clinical application of TLR-matured DCs in patients with AML for activation of innate and adaptive immune responses.

Die Rekrutierung von Zellen ist ein komplexer, in mehreren Schritten ablaufender Mechanismus, der eine zentrale Bedeutung für zahlreiche biologische Prozesse, wie z.B. Entzündung, Transplantatabstoßung, Tumormetastasierung und Stammzell¬migration hat. Die Migration von Zellen aus dem Blutstrom oder einem Reservoir in ein Zielgewebe bzw. Zielorgan und umgekehrt wird durch zahlreiche spezifische und unspezifische Reize ausgelöst und orchestriert. Dies erfolgt zu einem großen Teil durch von Chemokinen regulierte Mechanismen. Chemokine sind chemotaktische Zytokine, welche an spezifische auf der Zelloberfläche exprimierte Chemokinrezeptoren (CCR) binden. Zellen mit entsprechenden Chemokinrezeptoren wandern entlang eines Chemokingradienten zum jeweiligen Ziel, z.B. einem Entzündungsherd oder einem Zielorgan. Erstes Ziel dieser Arbeit war die Analyse der Chemokinrezeptorexpression im kutanen T-Zell Lymphom (CTCL), einem Non-Hogkin-Lymphom mit primärer kutaner Manifestation. Der Nachweis von Chemokinrezeptoren erfolgte in vitro mit der Polymerasekettenreaktion (PCR), der Durchflusszytometrie und mit Migrations-versuchen. Der Chemokinrezeptornachweis auf Hautschnitten von CTCL-Patienten erfolgte mit der Immunhistochemie. Erstmals konnte der hautassoziierte Chemokinrezeptor CCR10 im Rahmen des CTCL nachgewiesen werden. Außerdem gelang der Nachweis der Chemokinrezeptoren CCR4, CCR7 und CXCR3 in Hautschnitten und Lymphknotenbiopsien. CXCR3 wurde erstmals im Sezary Syndrom, einer fortgeschrittenen und aggressiven CTCL-Unterform, beschrieben. In der Immunhistochemie wurde die stärkste CCR10-Expression in Sezary Syndrom-Hautschnitten festgestellt. In Biopsien von befallenen Lymphknoten zeigte sich ein auffälliges CCR10-Verteilungsmuster: CCR10-positive Zellen wurden im Lymphsinus nachgewiesen, drangen aber nur vereinzelt in den Lymphknoten ein. In peripheren, nicht-kutanen Lymphomen wurde CCR10 nicht nachgewiesen und ist somit vermutlich exklusiv auf dem primär kutanen CTCL exprimiert. Es ist davon auszugehen, dass CCR10 den Epidermotropismus vor allem in aggressiveren Stadien reguliert. Die Bedeutung von CCR10 für die lymphatische Metastasierung des CTCL ist noch nicht geklärt. CCR10 könnte in der Zukunft als Faktor für die klinische Einstufung des CTCL oder als Ziel für eine gezielte Tumortherapie dienen. Die gezielte Tumortherapie ist u.a. mit Chemokinantagonisten möglich. Sie erlauben die gezielte Beeinflussung der chemokingesteuerten Rekrutierung von Leukozyten, Stammzellen oder Tumorzellen. Deshalb wurde ein membranbindender Antagonist des Chemokins CCL5, als potentielles Agens für die lokale Therapie von Tumoren oder von Transplantatabstoßungen, generiert. Das Chemokin CCL5 und seine Rezeptoren spielen in der akuten Transplantatabstoßung und in der Tumorprogression, z.B. im Mammakarzinom, eine zentrale Rolle. Der CCL5-Antagonist Met-RANTES inhibiert in Transplantatabstoßungsmodellen die Rekrutierung von Leukozyten. Der akute Entzündungsprozess und der daraus resultierende chronische Gefäßschäden werden so vermindert. Auch in einem Tumormodell ist ein Effekt auf die lokale Tumorprogression wahrscheinlich. Der in dieser Arbeit hergestellte CCL5-Antagonist Met-RANTES(Dimer)-GPI soll eine lokale Therapie ohne systemische Nebenwirkungen ermöglichen. Durch die erstmals beschriebene Bindung eines Chemokins oder Chemokinderivats an einen Glykosylinositolphosphatidyl (GPI)-Anker soll der Antagonist effektiv in die Zellmembranen von Endothelzellen inkorporiert werden, länger auf dem Endothel verbleiben und die benötigte Proteinmenge vermindern. Zunächst wurde durch die Erweiterung des signalgebenden N-Terminus von CCL5 der CCL5-Antagonist Met-RANTES generiert. Ein Aminosäureaustausch erzeugte ein dimerisierendes Molekül, welches einfacher als die zur Polymerisierung neigende Wildform zu isolieren war. Das Protein wurde mit der PCR mit einem GPI-Anker fusioniert und in Chinese Hamster Ovary (CHO)-Zellen subkloniert. Met-RANTES(Dimer)-GPI wurde erfolgreich aus den CHO-Zellen isoliert und mit der Säulenchromatographie gereinigt. In in vitro-Versuchen wurde Met-RANTES(Dimer)-GPI effektiv in die Oberfläche von humanen Endothelzellen reinkorporiert und hemmte die transendotheliale Migration von Monozyten, welche bei der Transplantat¬abstoßung und bei der Tumorprogression eine wichtige Rolle spielen. Mit Met-RANTES(Dimer)-GPI präperfundierte Transplantate zeigen möglicherweise einen geringeren vaskulären Schaden bei der akuten Transplantatabstoßungsreaktion. Im Tumormodell soll eine Hemmung der Tumorinfiltration durch Monozyten, welche eine beschleunigte Tumorprogression verursachen, erreicht werden. Im Vergleich zu nicht GPI-gebundenen CCL5-Antagonisten würde eine lokale fokussierte Therapie ermöglicht und eine eventuell geringere zu applizierende Proteinmenge bei längerer Verweildauer erzielt. Die Ergebnisse dieser Arbeit erlauben zunächst einen genaueren Einblick in die Pathogenese des CTCL. Der Chemokinrezeptor CCR7 wird vor allem von fortgeschrittenen Formen mit lymphatischer Infiltration exprimiert. CCR10 wird erstmals im Zusammenhang mit dem CTCL beschrieben und vor allem von fortgeschrittenen Unterformen exprimiert. Desweiteren wurde ein membranbindender Chemokinantagonist hergestellt. Erstmals wird die Kombination eines Chemokins oder Chemokinderivats mit einem GPI-Anker beschrieben. Der Antagonist erlaubt eine hohe lokale Applikation ohne systemische Zirkulation des Agens. Mögliche Einsatzgebiete sind die gezielte Tumortherapie oder die Behandlung der Transplantatabstoßung.

Nesta edição discuto o artigo publicado por Bayry e colaboradores no jornal PNAS, vol 105 de 2008. Neste artigo, antagonistas do receptor de quimiocinas CCR4 modelizados in silico foram testados in vitro e in vivo com o objectivo de inibirem a actividade imunosupressiva das células T reguladoras.

Nesta edição presento-vos um artigo submetido por Jandus et al ao jornal Cancer Immunol. Immunotherapy, 2008, vol 58, pag. 1795, e onde os autores avaliam por citometria de fluxo a presença de células T reguladoras CD4+ FOXP3+ em lesões primárias de melanoma (TIL), Nódulos linfáticos metastisados (TILN) e sangue periférico. Os autores descrevem que a frequência destas células T reguladoras se encontra sobre-representada em doentes em melanoma em relação a indivíduos sem doença aparente; que TIL e TILN apresentam frequências superioes de células T reg em número superior ao sangue periférico e que finalmente, o as células T reguladoras tem um fenótipo memória expressando preferencialmente CCR7 e CD45RA Um artigo de referência pela metodologia aplicada!

Background: Osteosarcoma is the most frequent bone tumor in childhood and adolescence. Patients with primary metastatic disease have a poor prognosis. It is therefore important to better characterize the biology of this tumor to define new prognostic markers or therapeutic targets for tailored therapy. Chemokines and their receptors have been shown to be involved in the development and progression of malignant tumors. They are thought to be active participants in the biology of osteosarcoma. The function of specific chemokines and their receptors is strongly associated with the biological context and microenvironment of their expression. In this report we characterized the expression of a series of chemokine receptors in the complex environment that defines osteosarcoma. Methods: The overall level of chemokine receptor mRNA expression was determined using TaqMan RT-PCR of microdissected archival patient biopsy samples. Expression was then verified at the protein level by immunohistochemistry using a series of receptor specific antibody reagents to elucidate the cellular association of expression. Results: Expression at the RNA level was found for most of the tested receptors. CCR1 expression was found on infiltrating mononuclear and polynuclear giant cells in the tumor. Cells associated with the lining of intratumoral vessels were shown to express CCR4. Infiltrating mononuclear cells and tumor cells both showed expression of the receptor CCR5, while CCR7 was predominantly expressed by the mononuclear infiltrate. CCR10 was only very rarely detected in few scattered infiltrating cells. Conclusion: Our data elucidate for the first time the cellular context of chemokine receptor expression in osteosarcoma. This is an important issue for better understanding potential chemokine/chemokine receptor function in the complex biologic processes that underlie the development and progression of osteosarcoma. Our data support the suggested involvement of chemokines and their receptors in diverse aspects of the biology of osteosarcoma, but also contradict aspects of previous reports describing the expression of these receptors in this tumor.

Background: For optimal T cell activation it is desirable that dendritic cells (DCs) display peptides within MHC molecules as signal 1, costimulatory molecules as signal 2 and, in addition, produce IL-12p70 as signal 3. IL-12p70 polarizes T cell responses towards CD4(+) T helper 1 cells, which then support the development of CD8(+) cytotoxic T lymphocytes. We therefore developed new maturation cocktails allowing DCs to produce biologically active IL-12p70 for large-scale cancer vaccine development. Methods: After elutriation of leukapheresis products in a closed bag system, enriched monocytes were cultured with GM-CSF and IL-4 for six days to generate immature DCs that were then matured with cocktails, containing cytokines, interferon-gamma, prostaglandin E2, and a ligand for Toll-like receptor 8, with or without poly (I: C). Results: Mature DCs expressed appropriate maturation markers and the lymph node homing chemokine receptor, CCR7. They retained full maturity after culture for two days without maturation cocktails and following cryopreservation. TLR ligand stimulation induced DCs capable of secreting IL-12p70 in primary cultures and after one day of coculture with CD40L-expressing fibroblasts, mimicking an encounter with T cells. DCs matured with our new cocktails containing TLR8 ligand, with or without poly (I: C), induced alloresponses and stimulated virus-specific T cells after peptide-pulsing. DCs matured in cocktails containing TLR8 ligand without poly (I: C) could also be loaded with RNA as a source of antigen, whereas DCs matured in cocktails containing poly (I: C) were unable to express proteins following RNA transfer by electroporation. Conclusion: Our new maturation cocktails allowed easy DC harvesting, stable maturation and substantial recoveries of mature DCs after cryopreservation. Our procedure for generating DCs is easily adaptable for GMP-compliance and yields IL-12p70-secreting DCs suitable for development of cancer vaccines using peptides or RNA as sources of immunizing antigens.