Podcasts about cd86

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.31.551394v1?rss=1 Authors: Fernandez-Gonzalez, A., Mukhia, A., Nadkarni, J., Willis, G., Reis, M., Zhumka, K., Vitali, S., Liu, X., Galls, A., Mitsialis, S. A., Kourembanas, S. Abstract: Objective: Macrophages play a central role in the onset and progression of vascular disease in pulmonary hypertension (PH) and cell-based immunotherapies aimed at treating vascular remodeling are lacking. This work evaluates the effect of pulmonary administration of macrophages modified to have an anti-inflammatory/pro-resolving phenotype in attenuating early pulmonary inflammation and progression of experimentally induced PH. Approach and Results: Mouse bone marrow derived macrophages (BMDMs) were polarized in vitro to a regulatory (M2reg) phenotype. M2reg profile and anti-inflammatory capacity were assessed in vitro upon lipopolysaccharide (LPS)/interferon-{gamma} (IFN{gamma}) restimulation, before their administration to 8- to 12- week-old mice. M2reg protective effect was tested at early (2 to 4 days) and late (4 weeks) time points during hypoxia (8.5% O2) exposure. Levels of inflammatory markers were quantified in alveolar macrophages and whole lung, while PH development was ascertained by right ventricular systolic pressure (RSVP) and right ventricular hypertrophy (RVH) measurements. Bronchoalveolar lavage (BAL) from M2reg-transplanted hypoxic mice was collected, and its inflammatory potential tested on naive BMDMs. M2reg macrophages demonstrated a stable anti-inflammatory phenotype upon a subsequent pro-inflammatory stimulus by maintaining the expression of specific anti-inflammatory markers (Tgf{beta}, Il10 and Cd206) and downregulating the induction of proinflammatory cytokines and surface molecules (Cd86, Il6 and Tnf). A single dose of M2regs attenuated the hypoxic monocytic recruitment and perivascular inflammation. Early hypoxic lung and alveolar macrophage inflammation leading to PH development was significantly reduced and, importantly, M2regs attenuated RVH, RVSP and vascular remodeling at 4 weeks post treatment. Conclusion: Adoptive transfer of M2regs halts the recruitment of monocytes and modifies the hypoxic lung microenvironment, potentially changing the immunoreactivity of recruited macrophages and restoring normal immune functionality of the lung. These findings provide new mechanistic insights on the diverse role of macrophage phenotype on lung vascular homeostasis that can be explored as novel therapeutic targets. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.02.518821v1?rss=1 Authors: Backstrom, F., Jimenez-Ferrer, I., Grabert, K., Belfiori, L., Luk, K., Swanberg, M. Abstract: Parkinsons disease (PD) is a heterogeneous disorder characterized by intraneuronal inclusions of alpha-synuclein (-Syn), a strong neuroinflammatory component and neurodegeneration. Human genetic association studies have shown that variants affecting quantity and quality of major histocompatibility complex II (MHCII) have implications in PD susceptibility and it was recently shown that PD patients have -Syn specific T lymphocytes in circulation. The class II transactivator (Ciita) is the major regulator of MHCII expression and reduced Ciita expression has been shown to increase -Syn induced neurodegeneration and pathology in vivo. Here we show, using flow cytometry in an -Syn overexpression model combined with -Syn pre-formed fibrils (PFF), that congenic rats with naturally occurring differences in Ciita expression have altered local and peripheral immune populations. Lower Ciita levels are associated with increased percentages of microglia and circulating myeloid cells being MHCII+ but with lower levels of MHCII on individual cells. Additionally, lower Ciita levels was associated to higher TNF levels in serum, trends of higher CD86 levels in circulating myeloid population and a lower CD4/CD8 T lymphocyte ratio. Taken together, these results indicate that Ciita regulates serum TNF levels and baseline immune populations which could mediate an increased susceptibility to PD-like pathology. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.02.518821v1?rss=1 Authors: Backstrom, F., Jimenez-Ferrer, I., Grabert, K., Belfiori, L., Luk, K., Swanberg, M. Abstract: Parkinsons disease (PD) is a heterogeneous disorder characterized by intraneuronal inclusions of alpha-synuclein (-Syn), a strong neuroinflammatory component and neurodegeneration. Human genetic association studies have shown that variants affecting quantity and quality of major histocompatibility complex II (MHCII) have implications in PD susceptibility and it was recently shown that PD patients have -Syn specific T lymphocytes in circulation. The class II transactivator (Ciita) is the major regulator of MHCII expression and reduced Ciita expression has been shown to increase -Syn induced neurodegeneration and pathology in vivo. Here we show, using flow cytometry in an -Syn overexpression model combined with -Syn pre-formed fibrils (PFF), that congenic rats with naturally occurring differences in Ciita expression have altered local and peripheral immune populations. Lower Ciita levels are associated with increased percentages of microglia and circulating myeloid cells being MHCII+ but with lower levels of MHCII on individual cells. Additionally, lower Ciita levels was associated to higher TNF levels in serum, trends of higher CD86 levels in circulating myeloid population and a lower CD4/CD8 T lymphocyte ratio. Taken together, these results indicate that Ciita regulates serum TNF levels and baseline immune populations which could mediate an increased susceptibility to PD-like pathology. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Journalist Neil Taylor in conversation - alongside Roy Carr & Adrian Thrills compiled the original NME cassette. C86 is a cassette compilation released by the British music magazine NME in 1986, featuring new bands licensed from British independent record labels of the time. As a term, C86 quickly evolved into shorthand for a guitar-based musical genre characterized by jangling guitars and melodic power pop song structures, although other musical styles were represented on the tape. In its time, it became a pejorative term for its associations with so-called "shambling" (a John Peel-coined description celebrating the self-conscious primitive approach of some of the music) and underachievement. The C86 scene is now recognized as a pivotal moment for independent music in the UK, as was recognized in the subtitle of the compilation's 2006 CD issue: CD86: 48 Tracks from the Birth of Indie Pop. 2014 saw the original compilation reissued in a 3CD expanded edition from Cherry Red Records; the 2014 box-set came with an 11,500-word book of sleevenotes by one of the tape's original curators, former NME journalist Neil Taylor. The C86 name was a play on the labelling and length of blank compact cassettes—commonly C60, C90 and C120—combined with 1986.

Crescentic glomerulonephritis is characterized by glomerular necrosis. Dying cells release intracellular proteins that act as danger-associated molecular patterns to activate the innate immune system. Previously, we have demonstrated that dying tubular cells release histones, which can kill endothelial cells and activate the toll-like receptor 2/4 (TLR2/4). This drives tubulointerstitial inflammation in septic or post-ischemic acute kidney injury (AKI). Furthermore, other groups have also reported that extracellular histones cause organ damage during acute lung injury, stroke, peritonitis and retinal dysfunction, and that blocking extracellular histones represents a beneficial approach during the disease progression. In this thesis, we investigated whether extracellular histones can elicit similar pathogenic effects during necrotizing glomerulonephritis. To do so, we used an animal model based on the necrotizing type of severe glomerulonephritis. Necrotic glomerulonephritis was induced in mice by a single intravenous injection of 100µl sheep anti-GBM antiserum. The impact of histone neutralization was studied by using an antibody isolated from the BWA-3 clone, which had the capacity to neutralize released extracellular histones in-vivo and in-vitro. After 7 days, mice were sacrificed and kidneys were collected for further data analysis. Proteinuria was assessed in spot urine samples. Anti-GBM treated mice showed increased proteinuria (albumin/creatinine ratio), plasma creatinine and BUN levels. This was associated with a reduced number of podocytes, increased crescentic glomeruli and the infiltration of neutrophils and macrophages into the kidney. Interestingly, neutralization of extracellular histones significantly reduced proteinuria leading to less podocyte damage. This was linked to an improved renal function defined by lower plasma creatinine and BUN levels, and with a decrease in neutrophil and macrophage infiltration and activation in kidney. Histone blockade also significantly reduced renal mRNA expression of TNF-α and fibrinogen in the glomerular capillaries, which was associated with less glomerulosclerosis, crescents and tubular atrophy. In-vitro studies demonstrated that extracellular histones and NETs-related histones kill glomerular endothelial cells, podocytes and parietal epithelial cells in a dose-dependent manner. Histone-neutralizing agents such as anti-histone IgG, activated protein C or heparin prevented this cytotoxic effect. Stimulation of BMDCs with histones upregulated the expression of the activation marker including MHC-II, CD48, CD80 and CD86 significantly as well as increased the production of TNF-α and IL-6. It has been previously reported by others including us that in biopsies from patients with ANCA-associated vasculitis showed an over-expression of the TLR2/4 receptor compared to the healthy glomeruli. Histone toxicity on glomeruli ex-vivo was also dependent on the TLR2/4 receptor axis given that the lack of TLR2/4 attenuated histone-induced renal thrombotic microangiopathy and glomerular necrosis in mice. Anti-GBM glomerulonephritis involved NET formation and vascular necrosis, while blocking NET formation via PAD inhibitor or pre-emptive anti-histone IgG injection significantly reduced all parameters of glomerulonephritis including vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment and activation of glomerular leukocytes, and glomerular crescent formation. Finally, to evaluate histones as a therapeutic target, mice with established glomerulonephritis were treated with three different histone-neutralizing agents such as anti-histone IgG, recombinant activated protein C and/or heparin. Interestingly, all agents were equally effective in abrogating severe glomerulonephritis, while combination therapy had no additive effect. In summary, the results of this thesis indicate that NET-related histones released during glomerulonephritis elicit cytotoxic and immunostimulatory effects and that neutralizing extracellular histones, therefore, represents a potential therapeutic approach when applied during already established glomerulonephritis.

Zystische Fibrose ist die häufigste vererbbare letale Erkrankung in Europa, die trotz Fortschritten in Diagnostik und Therapie weiterhin mit einer verkürzten Lebenserwartung einhergeht. Einer der Hauptgründe verfrühter Sterblichkeit betroffener Patienten sind persistierende pulmonale Infektionen mit Pseudomonas aeruginosa. Der Keim bedient sich des Quorum Sensing (QS), eines interbakterielles Kommunikationssystems, um die Ausbildung von Virulenzfaktoren zu regulieren und die Immunantwort des Patienten zu beeinflussen. In dieser Arbeit wurde die Auswirkung des P. aeruginosa QS Moleküls 3oxoC12-HSL auf die Reifung humaner Dendritischer Zellen (DZ) untersucht. DZ vermitteln als professionelle Antigen-präsentierende Zellen zwischen angeborenem und erworbenem Immunsystem. Eine Infektion wurde simuliert, indem humane DZ mit Lipopolysaccharid oder Zytokin-Cocktail aktiviert wurden. Anschliessend wurde die Expression von Maturations- und Migrationsmarkern sowie Zytokinsekretion in Anwesenheit von 3oxoC12-HSL untersucht. Bei LPS-stimulierten DZ kam es in Anwesenheit von 3oxoC12-HSL zu einer erniedrigten Expression der Reifungsmarker CD80, CD86, CD83, CD40, HLA-DR, sowie der Migrationsmarker CD184 (CXCR4) und CD197 (CCR7). Die Coinkubation mit Zytokin-Cocktail und 3oxoC12-HSL ergab eine Herabregulierung der Maturationsmarker CD80, CD86 und HLA-DR. Auf unstimulierte DZ zeigte 3oxoC12-HSL keinen Effekt, das Oberflächenmarker-Expressionsprofil dieser Zellen glich dem unreifer DZ. 3oxoC12-HSL inhibierte auch die Sekretion der pro-inflammatorischer Zytokine IL-12, IFN-gamma, MIP-1alpha, TNF-alpha durch LPS- bzw. Zytokin-Cocktail-gereifte DZ. Insgesamt zeigen unsere Ergebnisse, dass 3oxoC12-HSL die Reifung von DZ unterdrückt und somit das Zustandekommen einer effektiven Immunantwort verhindert wird.

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: Parkinson's disease (PD) is characterized at the cellular level by a destruction of neuromelanin (NM)containing dopaminergic cells and a profound reduction in striatal dopamine. It has been shown recently that antimelanin antibodies are increased in sera of Parkinson patients, suggesting that NM may act as an autoantigen. In this study we tested whether NM is being recognized by dendritic cells (DCs), the major cell type for inducing T-and B-cell responses in vivo. This recognition of NM by DCs is a prerequisite to trigger an adaptive autoimmune response directed against NM-associated structures. Results: Murine DCs were treated with NM of substantia nigra (SN) from human subjects or with synthetic dopamine melanin (DAM). DCs effectively phagocytized NM and subsequently developed a mature phenotype (CD86(hig)h/MHCII(high)). NM-activated DCs secreted the proinflammatory cytokines IL-6 and TNF-alpha. In addition, they potently triggered T cell proliferation in a mixed lymphocyte reaction, showing that DC activation was functional to induce a primary T cell response. In contrast, DAM, which lacks the protein and lipid components of NM but mimics the dopamine-melanin backbone of NM, had only very little effect on DC phenotype and function. Conclusions: NM is recognized by DCs in vitro and triggers their maturation. If operative in vivo, this would allow the DC-mediated transport and presentation of SN antigens to the adaptive immune system, leading to autoimmmunity in susceptible individuals. Our data provide a rationale for an autoimmune-based pathomechanism of PD with NM as the initial trigger.

Dendritic cells (DC) play a central role in connecting innate with specific adoptive immunity resulting in target specific activation T-cells. As professional antigen presenting cells (APC) DC specifically stimulate T-effector cells, especially tumor-cytotoxic T-cells. Therefore they are regarded as interesting candidates for anti-tumor or anti-leukemic vaccination strategies. The insufficient expression of costimulatory antigens, MHC molecules and tumor-associated antigens (TAA) on the surface of cancer cells and disturbed mechanisms of apoptosis are the main reason for an ineffective immune response in oncologic diseases. It was shown that acute myeloid leukemic cells can be differentiated to leukemia-derived DC (DCleu ), regaining the stimulatory capacity of professional DC while potentially presenting the whole leukemic antigen repertoire. Thus, vaccination strategies, using ex vivo or in vivo generated DC, might induce a highly specific anti-leukemic T-cell response circumventing the cumbersome identification of leukemia-associated antigens. In this thesis DC antigen (DCA) expression profiles of mononuclear cells (MNC) and dendritic cells (DC) generated from these MNC should be analyzed. The generated MNC and DC should be compared with respect to their DC antigen (DCA) expression profiles and the DCAs value to detect and quantify (leukemia-derived) DC in different AML/MDS subtypes and under different culture conditions. Therefore MNC and DC were generated from 137 patients with acute myeloid leukemia (AML) and 49 patients with myelodysplastic syndromes (MDS) under 6 different serum free culture conditions. DCA studied were: CD1a/1b/1c, CD206, CD25, CD137L, CD83, CD86, CD80 and CD40. DC-generating media were chosen according to their different mechanisms of inducing DC-differentiation: 1. ‚Basic method‘: TNF/GM-CSF/IL-4, 2. MCM-Mimic, 3. Ca Ionophore, 4. Picibanil, 5. Poly I:C and 6. Cytokines. Quality and quantity of generated DC was estimated by Flow cytometry applying a specified, ‘DC-based’ gating-strategy. Expression and coexpression profiles of 10 different DCA as well as various costimulatory molecules, maturation markers and blast antigens were evaluated. Only those DCA qualified for the quantification of leukemia-derived DC that were not expressed on uncultured MNC fractions. AML patients presented with an average of 58 % blasts, MDS patients with 13 % blasts in MNC fractions. DCA were expressed on average on less than 7% of uncultured MNC, however some of the markers could be expressed on up to 77% of uncultured cells in single AML cases. Consequently these DCA did not qualify for detection of DC in those cases. Highest expression rates were found for CD86 and CD40 in naïve AML and for CD137L and CD40 in naïve MDS samples. Other DCA (e.g. CD1a, 1b, 1c) were only rarely found on naïve blasts. DCA expression on uncultured AML and MDS MNC varied with FAB types and cytogenetic risk. After culture in different DC-differentiating media, on average 28% DC could be generated from AML MNC and 30% from MDS MNC, depending on methods used, with an average DC viability of more than 60% and an average DC maturity of 49% (AML) and 56% (MDS). On average 36% of leukemic blasts could be converted to DC. Proportions of DCleu in the total DC fraction varied from 40-58% and were on average 49% (AML) and 43% (MDS) after culture. Average results of all culture methods tested were comparable, however every method failed to create DC in some individual cases. The most important results of this thesis are: 1. It could be shown that DCA are expressed on naïve blasts in AML and MDS in individual patients. That means that the individual patients’ DCA-profiles have to be evaluated before DC-culture to find suitable DCA to detect and quantify (leukemia-derived) DC after culture. 2. Different methods of DC-generation qualify with varying individual efficiency to generate leukemic, mature, migratory and viable DC in individual cases. 3. To select the best DC-generating method the best DC-marker (no expression on naïve blasts, high expression on DC) has to be chosen to quantify DC in individual samples. 4. The use of only one method is not sufficient to create DC in every single AML and MDS sample. However, a successful, quantitative DC/DCleu -generation is possible in every case of AML and MDS by the combination of 3 different DC-generating media, but not every blast is convertible to DC leu . 5. There is a need for new, specific DC-markers that are not expressed on naïve blasts.

Im Mittelpunkt der Arbeit stehen die Auswirkungen des Prostanoids Prostaglandin E2 (PGE2) auf die Fähigkeit von Monozyten-abgeleiteten dendritischen Zellen (MoDC) zur Antigenpräsentation auf MHC-I- und MHC II Molekülen. Die Kreuzpräsentation von exogenem Antigen auf MHC I Molekülen ist eine entscheidende Fähigkeit der dendritischen Zellen (DC), die es ihnen ermöglicht zytotoxische T-Zellen zu aktivieren. PGE2 wird in Aktivierungsprotokollen als „Goldstandard“ in Kombination mit Zytokinen, wie TNF-α, IL-1β und IL 6, als Reifestimulus für DC in klinischen DC-basierten Tumorvakzinierungsstudien verwendet. Bisher wurde über den Effekt von PGE2 auf die Antigenpräsentation von Tumorantigen in Proteinform nicht berichtet. In dieser Arbeit wurde die Auswirkung von PGE2 und spezifischen EP Rezeptoragonisten auf verschiedene Funktionen von MoDC, die für die Antigenpräsentation auf MHC-I- und MHC-II-Molekülen relevant sind, untersucht. Zuerst wurde die Auswirkung von PGE2 und den EP Rezeptoragonisten auf die Ausreifung der MoDC untersucht. Hierbei wurde ersichtlich, dass MoDC nach Inkubation mit PGE2, vermittelt über EP2/4 Rezeptoren, die Aktivierungsmarker HLA A, HLA-B und HLA-C3, HLA-DR, CD83 und CD86 hochregulieren. Als nächstes wurde der Einfluss von PGE2 auf die Antigenaufnahmefähigkeit der MoDC untersucht. Hierbei wurde die Phagozytosefähigkeit anhand von Zelllysat und apoptotischen Tumorzellen und die Makropinozytosefähigkeit anhand von Dextran Partikeln analysiert. Es konnte gezeigt werden, dass weder PGE2 noch spezifische EP Rezeptoragonisten die Antigenaufnahme signifikant beeinflussten. Durch Präsentation von Antigen auf MHC-II-Molekülen sind DC in der Lage, tumorspezifische CD4+ T-Zellen zu aktivieren. Die MHC-II Präsentationsfähigkeit der MoDC unter dem Einfluss von PGE2 wurde mit NY-ESO-1157-170 Peptid, NY-ESO-1 Protein sowie mit NY ESO 1-transfiziertem CHO Zelllysat als Antigenquellen untersucht. PGE2 wurde zu den MoDC entweder 24 h vor (Präinkubation) oder zur gleichen Zeit wie das Antigen (Simultaninkubation) hinzugefügt. Die Versuche ergaben, dass die MHC-II-Antigenpräsentation der MoDC durch PGE2 nicht signifikant beeinflusst wird. Die Auswirkung von PGE2 auf die Kreuzpräsentationsfähigkeit der MoDC wurde mit einer Formulierung aus NY-ESO-1 Protein und dem ISCOMATRIX® adjuvant (NY ESO-1/IMX) und einem Immunkomplex bestehend aus NY-ESO-1 Protein und dem Antikörper anti NY ESO-1 mAk (Klon ES121; NY-ESO-1/IC) untersucht. Hierbei konnte gezeigt werden, dass sowohl die Kreuzpräsentation von NY-ESO-1/IMX als auch von NY ESO 1/IC durch PGE2 dosisabhängig signifikant gehemmt wurde. Dieser Effekt wird, ähnlich wie die Zell-Reifung, über die EP2/4-Rezeptoren vermittelt. Zusammenfassend erlauben die Ergebnisse den Schluss, dass PGE2 über einen EP2/4-Rezeptor-vermittelten Mechanismus die Kreuzpräsentationsfähigkeit der MoDC inhibiert, ohne jedoch die Antigenaufnahme oder die MHC-II Präsentation signifikant zu beeinflussen. Eine klinische Relevanz der Ergebnisse besteht bei der Verwendung von DC für therapeutische Tumorvakzinierungen zur Induktion einer gegen Malignome gerichteten Immunantwort. Die Benutzung von PGE2 als Reifestimulus für MoDC, die mit Protein-Antigenen gepulst werden, birgt das Risiko, die Induktion von tumorantigenspezifischen CD8+ T-Zellen negativ zu beeinflussen.

Objective: Trauma-hemorrhage results in depressed immune responses of antigen-presenting cells (APCs) and T-cells. Recent studies suggest a key role of depressed T-cell derived interferon (IFN)-g in this complex immune cell interaction. The aim of this study was to elucidate further the underlying mechanisms responsible for dysfunctional T-cells and their interaction with APCs following trauma-hemorrhage. Design: Adult C3H/HeN male mice were subjected to trauma-hemorrhage (3-cm midline laparotomy) followed by hemorrhage (blood pressure of 35�5mmHg for 90 min and resuscitation) or sham operation. At 24 h thereafter, spleens were harvested and T-cells (by Microbeads) and APCs (via adherence) were Isolated. Co-cultures of T-cells and APCs were established for 48 h and stimulated with concanavalin A and lipopolysaccharide. T-Cell specific cytokines known to affect APC function (i.e. interleukin(IL)-2, IL-4 and granulocyte-macrophage colony-stimulating factor (GM-CSF)) were measured in culture supernatants by Multiplex assay. The expression of MHC class II as well as co-stimulatory surface molecules on T-cells and APCs was determined by flow cytometry. Results: The release of IL-4 and GM-CSF by T-cells was suppressed following trauma-hemorrhage, irrespective of whether sham or trauma-hemorrhage APCs were present. Antigen-presenting cells from animals subjected to trauma-hemorrhage did not affect T-cell derived cytokine release by sham T-cells. In contrast, T-cells from traumahemorrhage animals depressed MHC class II expression of CD11c(þ) cells, irrespective of whether APCs underwent sham or trauma-hemorrhage procedure. Surprisingly, co-stimulatory molecules on APCs (CD80, CD86) were not affected by trauma-hemorrhage. Conclusions: These results suggest that beside IFN-g other T-cell derived cytokines contribute to immunosuppression following trauma-hemorrhage causing diminished MHC II expression on APCs. Thus, T-cells appear to play an important role in this interaction at the time-point examined. Therapeutic approaches should aim at maintenance of T-cell function and their interaction with APCs to prevent extended immunosuppression following trauma-hemorrhage.

It has been reported that interferon (IFN-) enhances humoral immunity and that dendritic cells of the myeloid lineage promote B-cell differentiation. Here we studied whether the plasmacytoid dendritic cell (PDC), a subset of dendritic cells specialized for the production of IFN-, is involved in regulating B-cell differentiation and immunoglobulin production. The recently identified class of CpG oligonucleotides (CpG-C) was used to activate both B cells and PDCs via Toll-like receptor 9 (TLR9). The presence of PDCs synergistically enhanced CD86 expression, cytokine production (interleukin 6 [IL-6], tumor necrosis factor , and IL-10) and plasma cell differentiation of isolated human peripheral blood B cells stimulated through CpG-C and B-cell antigen receptor (BCR) ligation. This stimulation protocol was sufficient to drive purified naive B cells into IgM-producing plasma cells and to trigger IgG synthesis in memory B cells. PDCs contributed to B-cell activation via IFN- secretion. Up-regulation of TLR9 on B cells was not involved. These results demonstrate that CpG-stimulated PDCs induce plasma cell differentiation in naive and memory B cells in the absence of T-cell help, providing an explanation for the excellent activity of CpG oligonucleotides as a humoral vaccine adjuvant. (Blood. 2004;103:3058-3064)

GDNF (glial cell line-derived neurotrophic factor) und NTN (Neurturin), die zwei zuerst beschriebenen Liganden der GDNF-Familie, fungieren als Überlebens- und Entwicklungsfaktoren für definierte Populationen von zentralen und peripheren Neuronen. GDNF ist darüber hinaus für die Nierenentwicklung erforderlich. Für die Vermittlung ihrer biologischen Wirkung benutzten GDNF und NTN einen Rezeptor, der aus zwei Ketten besteht: Die Signal-transduzierende Komponente RET wird sowohl von GDNF als auch von NTN benutzt. RET wird von 21 Exonen kodiert und kommt in multiplen Spleiß-Varianten vor. Für die Liganden-Spezifität ist eine zweite Rezeptorkomponente verantwortlich, ein Mitglied der GFR-Familie. GFRa-1 bindet präferentiell GDNF, während GFRa-2 NTN stärker als GDNF bindet. Ziel dieser Arbeit war es, mögliche wechselseitige Interaktionen zwischen dem Nerven- und Immunsystem durch die GDNF-Familie zu untersuchen. Zu diesem Zweck wurde zunächst die Expression von GDNF, NTN und ihrer Rezeptoren in gereinigten Immunzell-Subtypen untersucht. Dabei zeigte sich, dass der Prototyp dieser Liganden-Familie, GDNF, von keiner der untersuchten Immunzellen exprimiert wurde. Hingegen wurde das verwandte NTN von T-Zellen, B-Zellen und Monozyten exprimiert wie mit RT-PCR, Western Blot und Immunzytochemie gesehen wurde. Transkripte für das zu NTN und GDNF verwandte Persephin (PSP) wurden in Monozyten und mononukleären Zellen des peripheren Blutes gefunden. Der Transmembran-Rezeptor RET wurde von allen untersuchten Immunzell-Subtypen exprimiert. B-Zellen und T-Zellen exprimierten unterschiedliche Isoformen von RET, sowohl im extrazellulären Liganden-bindenden als auch im intrazellulären Signal-transduzierenden Teil. Die Expression der Isoformen von RET wurde zudem in T-Zellen und B-Zellen noch stark durch Aktivierung reguliert. In CD8+ T-Zellen wurde auch eine bislang noch nicht beschriebene Spleiß-Variante am 5` Ende beobachtet. Im Gegensatz zu T-Zellen und B-Zellen exprimierten Monozyten nur die volle Länge von RET. Auch die Liganden-bindenden Ketten GFRa-1 und GFRa-2 wurden von Immunzellen exprimiert wie mit RT-PCR und FACS gesehen wurde. GFRa-2 war deutlich abundanter als GFRa-1. Von GFRa-2 wurden verschiedene Isoformen in Immunzellen gefunden. In der in T-Zellen und B-Zellen am stärksten exprimierten Isoform ist Exon 2 und 3 nicht enthalten. Dem resultierenden Protein fehlen die N-terminale Cystein-reiche Domäne und eine N-Glykosylierungsstelle, eine Region, die allerdings für die Bindung von NTN und die Interaktion mit RET entbehrlich ist. Mögliche Effekte von GDNF und NTN auf Immunzellen wurden untersucht. Dabei zeigte sich, dass GDNF und NTN an der Regulation von TNF-alpha beteiligt sind. Wenn GDNF oder NTN nach 5 oder 6 Tagen zu LPS+IFN-g stimulierten Blutzellen oder zu ConA aktivierten T-Zellen gegeben wurde, dann war nach weiteren 24 h der TNF-a-Gehalt im Überstand reduziert. Weitere Experimente wiesen daraufhin, dass diese Reduktion des TNF-a-Gehalts auf eine verstärkte Aufnahme oder Verbrauch zurückzuführen ist. Proliferation, Expression von Aktivierungsmarkern (HLA-DR, CD38, CD40, CD69, CD86) oder Produktion von IFN-g und IL-4 wurden durch GDNF und NTN nicht beeinflusst. Zusammenfassend zeigt diese Arbeit, dass Immunzellen den neurotrophen Faktor NTN produzieren und Rezeptoren für GDNF und NTN besitzen. Multiple Isoformen der Signal-transduzierenden Kette RET wurden exprimiert und durch Aktivierung reguliert. NTN und GDNF regulierten in aktivierten T-Zellen und Monozyten die Aufnahme oder den Verbrauch von TNF-a. Diese Befunde weisen daraufhin, dass Immunzellen miteinander und auch mit dem Nervensystem mit Hilfe der GDNF-Familie interagieren können.

Aufgrund der Zunahme an Organ- und Knochenmarkstransplantationen und der damit verbundenen Immunsuppression bzw. immunsuppressiven Therapie sowie der zunehmenden Zahl an AIDS-Patienten ist das Zytomegalovirus (CMV) als Pathogen in den letzten zwanzig Jahren trotz der Einführung wirksamer antiviraler Medikamente bis heute von großer klinischer Bedeutung. Während bei immunkompetenten Personen eine primäre CMV-Infektion durch das Immunsystem kontrolliert werden kann, führt eine Primärinfektion oder eine Reaktivierung einer latenten CMV-Infektion in immunsupprimierten Patienten zu lebensbedrohlichen Komplikationen. Die Pathogenese einer CMV-Infektion wird entscheidend von der Qualität der antiviralen Immunantwort des Wirtes beeinflusst und Kenntnisse über die Interaktion von CMV mit dem Immunsystem sind für die Prophylaxe und Behandlung einer CMV-Infektion von großer Bedeutung. Dendritische Zellen (DCs) sind die wichtigsten Antigen-präsentierenden Zellen des Immunsystems und spielen bei der Initiierung einer antiviralen Immunantwort eine zentrale Rolle. Die Stimulation von naiven T-Zellen durch DCs und die Auslösung einer zytotoxischen T-Lymphozyten-Antwort trägt entscheidend zur Eliminierung von viral-infizierten Zellen bei. Die Interaktion des Zytomegalovirus mit dendritischen Zellen gibt dem Virus eine Möglichkeit, seine Eliminierung durch das Immunsystem des Wirtes entscheidend zu beeinflussen. Zur Identifikation von Zielzellen für latente und lytische Infektionen durch MCMV und zur Untersuchung der Auswirkungen einer MCMV-Infektion auf den Phänotyp und die Funktion der Zellen wurde die murine hämatopoetische Stammzelllinie FDCP-Mix als Modellsystem verwendet. Definierte Differenzierungsstadien der Zellen entlang der dendritischen Reihe wurden hierzu mit einer GFP-exprimierenden MCMV-Mutante infiziert. Während undifferenzierte FDCP-Mix-Zellen und von FDCP-Mix-Zellen abgeleitete reife DCs nicht produktiv infizierbar waren, setzten unreife DCs infektiöse Virusnachkommen frei. In reifen DCs wurden nur virale Proteine der sehr frühen und frühen Phase der viralen Genexpression synthetisiert, während späte Genprodukte nicht nachgewiesen werden konnten. Die Infektion unreifer und reifer DCs resultierte anfänglich in deren Aktivierung, erkennbar an der vorübergehend verstärkten Expression der Oberflächenmoleküle CD80, CD86, CD40, MHC-Klasse-I und Klasse-II. Die verstärkte Expression der MHC- und ko-stimulatorischen Moleküle auf reifen DCs einige Stunden nach Infektion spiegelte sich in einer gesteigerten Stimulation naiver autologer T-Zellen durch infizierte DCs wider. In der späten Phase der Infektion war die Aktivierung von autologen T-Zellen beeinträchtigt. Dies korrelierte mit der reduzierten Oberflächenexpression der MHC- und ko-stimulatorischen Moleküle auf infizierten reifen DCs. Allogene T-Zellen konnten durch MCMV-infizierte DCs weder in der frühen noch in der späten Phase der Infektion stimuliert werden. Diese Ergebnisse sprechen dafür, dass DCs im Laufe einer MCMV-Infektion mehrere Rollen spielen: (1) unreife DCs produzieren MCMV-Nachkommen und können so zur Verbreitung des Virus im Wirt beitragen; (2) in einem frühen Stadium der Infektion aktivieren DCs naive T-Zellen und initiieren damit eine antivirale Immunantwort, die einer Ausbreitung der viralen Infektion entgegenwirkt. (3) Zu einem späteren Zeitpunkt der Infektion ist die Stimulation der T-Zell-Proliferation durch MCMV-infizierte DCs beeinträchtigt. Dies ist einer der Mechanismen, welche die Persistenz des Virus in seinem Wirt ermöglichen. Unabhängig vom Zeitpunkt der Infektion ist bei der allogenen Transplantation die Induktion der T-Zell-Antwort immer beeinträchtigt. Die Unfähigkeit der CMV-infizierten DCs, naive allogene T-Zellen zu stimulieren, trägt so zu einer reduzierten antiviralen Kontrolle bei, was CMV-verbundene Krankheiten nach allogenen Knochenmarkstransplantationen begünstigt und gravierende gesundheitliche Probleme zur Folge hat.

Dendritic cells (DC) constitute a heterogeneous leukocyte population having in common a unique capacity to induce primary T cell responses and are therefore most attractive candidates for immunomodulatory strategies. Two populations of blood DC (CD11c+ CD123(dim) and CD11c- CD123(high)) have been defined so far. However, their direct isolation for experimental purposes is hampered by their low frequency and by the lack of selective markers allowing large scale purification from blood. Here we describe the monoclonal antibody (mAb) M-DC8, which was generated by immunizing mice with highly enriched blood DC. This mAb specifically reacts with 0.2-1% of blood leukocytes and enables their direct isolation by a one-step immunomagnetic procedure from fresh mononuclear cells. These cells can be differentiated from T cells, B cells, NK cells and monocytes using lineage-specific antibodies. M-DC8+ cells express HLA class It molecules, CD33 and low levers of the costimulatory molecules CD86 and CD40. Upon in vitro culture M-DC8+ cells spontaneously mature into cells with the phenotype of highly stimulatory cells as documented by the upregulation of HLA-DR, CD86 and CD40; in parallel CD80 expression is induced. M-DC8+ cells display an outstanding capacity to present antigen. In particular, they proved to be excellent stimulators of autologous mixed leukocyte reaction and to activate T cells against primary antigens such as keyhole limpet hemocyanin. Furthermore, they induce differentiation of purified allogeneic cytotoxic T cells into alloantigen-specific cytotoxic effector cells. While the phenotypical analysis reveals similarities with the two known blood DC populations, the characteristic expression of Fc gamma RIII (CD16) and the M-DC8 antigen clearly defines them as a novel population of blood DC. The mAb M-DC8 might thus be a valuable tool to determine circulating DC for diagnostic purposes and to isolate these cells for studies of antigen-specific T cell priming. Copyright (C) 2000 S. Karger AG, Basel.