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Supplementary Table 1 XLS file 22K, Tumors and reference tissues for methylation microarray analysis

Supplementary Figure 1 - PDF file 1233K, Supplemental Figure 1: Representative array comparative genomic hybridization (aCGH) results of genomic copy number near-normal GIST (left column, n=5) and genomic copy number abnormal GIST (right column, n=5) (Agilent 180K catalogue assay)

Supplementary Table 2 XLSX file 490K, Illumina GoldenGate Methylation Standard Cancer Panel I Target CpG Annotations and differential methylation significance values for group comparisons listed in Table

Supplementary Tables S1-S3 from A Pilot Study of Consolidative Immunotherapy in Patients with High-Risk Pediatric Sarcomas

Purpose: The development of new cancer drugs is slow and costly. HIV protease inhibitors are Food and Drug Administration approved for HIV patients. Because these drugs cause toxicities that can be associated with inhibition of Akt, an emerging target in cancer, we assessed the potential of HIV protease inhibitors as anticancer agents.Experimental Design: HIV protease inhibitors were screened in vitro using assays that measure cellular proliferation, apoptotic and nonapoptotic cell death, endoplasmic reticulum (ER) stress, autophagy, and activation of Akt. Nelfinavir was tested in non–small cell lung carcinoma (NSCLC) xenografts with biomarker assessment.Results: Three of six HIV protease inhibitors, nelfinavir, ritonavir, and saquinavir, inhibited proliferation of NSCLC cells, as well as every cell line in the NCI60 cell line panel. Nelfinavir was most potent with a mean 50% growth inhibition of 5.2 μmol/L, a concentration achievable in HIV patients. Nelfinavir caused two types of cell death, caspase-dependent apoptosis and caspase-independent death that was characterized by induction of ER stress and autophagy. Autophagy was protective because an inhibitor of autophagy increased nelfinavir-induced death. Akt was variably inhibited by HIV protease inhibitors, but nelfinavir caused the greatest inhibition of endogenous and growth factor–induced Akt activation. Nelfinavir decreased the viability of a panel of drug-resistant breast cancer cell lines and inhibited the growth of NSCLC xenografts that was associated with induction of ER stress, autophagy, and apoptosis.Conclusions: Nelfinavir is a lead HIV protease inhibitor with pleiotropic effects in cancer cells. Given its wide spectrum of activity, oral availability, and familiarity of administration, nelfinavir is a Food and Drug Administration–approved drug that could be repositioned as a cancer therapeutic.

Purpose: Patients with metastatic or recurrent Ewing’s sarcoma family of tumors and alveolar rhabdomyosarcoma have Experimental Design: Fifty-two patients with translocation positive, recurrent, or metastatic Ewing’s sarcoma family of tumors or alveolar rhabdomyosarcoma underwent prechemotherapy cell harvest via apheresis for potential receipt of immunotherapy. Following completion of standard multimodal therapy, 30 patients ultimately initiated immunotherapy and were sequentially assigned to three cohorts. All cohorts received autologous T cells, influenza vaccinations, and dendritic cells pulsed with peptides derived from tumor-specific translocation breakpoints and E7, a peptide known to bind HLA-A2. Cohort 1 received moderate-dose recombinant human interleukin-2 (rhIL-2), cohort 2 received low-dose rhIL-2, and cohort 3 did not receive rhIL-2.Results: All immunotherapy recipients generated influenza-specific immune responses, whereas immune responses to the translocation breakpoint peptides occurred in 39%, and only 25% of HLA-A2+ patients developed E7-specific responses. Toxicity was minimal. Intention-to-treat analysis revealed a 31% 5-year overall survival for all patients apheresed (median potential follow-up 7.3 years) with a 43% 5-year overall survival for patients initiating immunotherapy.Conclusions: Consolidative immunotherapy is a scientifically based and clinically practical approach for integrating immunotherapy into a multimodal regimen for chemoresponsive cancer. Patients receiving immunotherapy experienced minimal toxicity and favorable survival. The robust influenza immune responses observed suggest that postchemotherapy immune incompetence will not fundamentally limit this approach. Future studies will seek to increase efficacy by using more immunogenic antigens and more potent dendritic cells.

Supplementary Figures S1-S5 from Nelfinavir, A Lead HIV Protease Inhibitor, Is a Broad-Spectrum, Anticancer Agent that Induces Endoplasmic Reticulum Stress, Autophagy, and Apoptosis In vitro and In vivo

Mapping of protein signaling networks within tumors can identify new targets for therapy and provide a means to stratify patients for individualized therapy. Despite advances in combination chemotherapy, the overall survival for childhood rhabdomyosarcoma remains ∼60%. A critical goal is to identify functionally important protein signaling defects associated with treatment failure for the 40% nonresponder cohort. Here, we show, by phosphoproteomic network analysis of microdissected tumor cells, that interlinked components of the Akt/mammalian target of rapamycin (mTOR) pathway exhibited increased levels of phosphorylation for tumors of patients with short-term survival. Specimens (n = 59) were obtained from the Children's Oncology Group Intergroup Rhabdomyosarcoma Study (IRS) IV, D9502 and D9803, with 12-year follow-up. High phosphorylation levels were associated with poor overall and poor disease-free survival: Akt Ser473 (overall survival P < 0.001, recurrence-free survival P < 0.0009), 4EBP1 Thr37/46 (overall survival P < 0.0110, recurrence-free survival P < 0.0106), eIF4G Ser1108 (overall survival P < 0.0017, recurrence-free survival P < 0.0072), and p70S6 Thr389 (overall survival P < 0.0085, recurrence-free survival P < 0.0296). Moreover, the findings support an altered interrelationship between the insulin receptor substrate (IRS-1) and Akt/mTOR pathway proteins (P < 0.0027) for tumors from patients with poor survival. The functional significance of this pathway was tested using CCI-779 in a mouse xenograft model. CCI-779 suppressed phosphorylation of mTOR downstream proteins and greatly reduced the growth of two different rhabdomyosarcoma (RD embryonal P = 0.00008; Rh30 alveolar P = 0.0002) cell lines compared with controls. These results suggest that phosphoprotein mapping of the Akt/mTOR pathway should be studied further as a means to select patients to receive mTOR/IRS pathway inhibitors before administration of chemotherapy. [Cancer Res 2007;67(7):3431–40]

Supplementary Table 2 from Phosphoprotein Pathway Mapping: Akt/Mammalian Target of Rapamycin Activation Is Negatively Associated with Childhood Rhabdomyosarcoma Survival

Einrichtungen der medizinischen Versorgung sind bedeutende Orte fur das Erkennen und die Intervention bei Gewalt in Paarbeziehungen. Arzte, Notfallsanitater/-assistenten und Pflegekrafte am Einsatzort oder in Rettungsstellen gehoren zu den ersten und mitunter einzigen Personen, die professionelle Unterstutzung anbieten und weitere Hilfe vermitteln konnen. Grundlegende Voraussetzungen fur das Erkennen der hauslichen Gewalt stellen u. a. Kenntnisse uber die Epidemiologie, die Risikofaktoren und allem voran die Formen der hauslichen Gewalt dar. Eine fruhzeitige Erkennung kann wesentlich dazu beitragen, das Risiko weiterer Gewalt und langfristiger gesundheitlicher Schadigungen zu verhindern, von dem v. a. Frauen und Kinder betroffen sind. Fachkompetenz und Handlungssicherheit aufseiten der Fachkrafte in der Notfallversorgung sind somit haufig entscheidend dafur, ob Praventions- und Interventionschancen realisiert werden.

Einrichtungen der medizinischen Versorgung sind bedeutende Orte fur das Erkennen und die Intervention bei Gewalt in Paarbeziehungen. Arzte, Notfallsanitater/-assistenten und Pflegekrafte am Einsatzort oder in Rettungsstellen gehoren zu den ersten und mitunter einzigen Personen, die professionelle Unterstutzung anbieten und weitere Hilfe vermitteln konnen. Das Erkennen der hauslichen Gewalt sowie eine rechtzeitige und kompetente Intervention im Rahmen der Gesundheitsversorgung konnen wesentlich dazu beitragen, das Risiko weiterer Gewalt und langfristiger gesundheitlicher Schadigungen zu verhindern – von dem v. a. Frauen und Kinder betroffen sind. Fachkompetenz und Handlungssicherheit aufseiten der Fachkrafte in der Notfallversorgung sind entscheidend dafur, ob Praventions- und Interventionschancen realisiert werden.

Trauma remains a major cause of death throughout the world, especially for patients younger than 45 years. Due to rapid advances in clinical management, both in the acute and prehospital settings, trauma patients survive devastating injuries at unprecedented rates. However, these patients can often face life threatening complications that stem from the robust innate immune response induced by severe hemorrhage, leading to further tissue injury rather than repair. The complement and coagulation cascades are key mediators in this disordered reaction, which includes the development of trauma‐induced coagulopathy. There is increasing evidence that cross‐talk between these two pathways allows rapid amplification of their otherwise targeted responses and contributes to overwhelming and prolonged systemic inflammation. In this article, we summarize the initial steps of innate immune response to trauma and review the complex complement and coagulation cascades, as well as how they interact with each other. Despite progress in understanding these cascades, effective therapeutic targets have yet to be found and further research is needed both to improve survival rates as well as decrease associated morbidity.

Interstitial kidney inflammation is present in various nephritides in which serum interleukin 23 (IL-23) is elevated. Here we showed that murine and human renal tubular epithelial cells (TECs) expressing the IL-23 receptor (IL-23R) responded to IL-23 by inducing intracellular calcium flux, enhancing glycolysis, and upregulating calcium/calmodulin kinase IV (CaMK4), which resulted in suppression of the expression of the arginine-degrading enzyme arginase 1 (ARG1), thus increasing in situ levels of free L-arginine. Limited availability of arginine suppressed the ability of infiltrating T cells to proliferate and produce inflammatory cytokines. TECs from humans and mice with nephritis expressed increased levels of IL-23R and CaMK4 but reduced levels of ARG1. TEC-specific deletion of Il23r or Camk4 suppressed inflammation, whereas deletion of Arg1 exacerbated inflammation in different murine disease models. Finally, TEC-specific delivery of a CaMK4 inhibitor specifically curbed renal inflammation in lupus-prone mice without affecting systemic inflammation. Our data offer the first evidence to our knowledge of the immunosuppressive capacity of TECs through a mechanism that involves competitive uptake of arginine and signify the importance of modulation of an inflammatory cytokine in the function of nonlymphoid cells, which leads to the establishment of an inflammatory microenvironment. New approaches to treat kidney inflammation should consider restoring the immunosuppressive capacity of TECs.

Lupus nephritis (LN) is a serious complication occurring in 50% of patients with systemic lupus erythematosus (SLE) for which there is a lack of biomarkers, a lack of specific medications, and a lack of a clear understanding of its pathogenesis. The expression of calcium/calmodulin kinase IV (CaMK4) is increased in podocytes of patients with LN and lupus-prone mice, and its podocyte-targeted inhibition averts the development of nephritis in mice. Nephrin is a key podocyte molecule essential for the maintenance of the glomerular slit diaphragm. Here, we show that the presence of fucose on N-glycans of IgG induces, whereas the presence of galactose ameliorates, podocyte injury through CaMK4 expression. Mechanistically, CaMK4 phosphorylates NF-κB, upregulates the transcriptional repressor SNAIL, and limits the expression of nephrin. In addition, we demonstrate that increased expression of CaMK4 in biopsy specimens and in urine podocytes from people with LN is linked to active kidney disease. Our data shed light on the role of IgG glycosylation in the development of podocyte injury and propose the development of “liquid kidney biopsy” approaches to diagnose LN.

Protein phosphatase 2A (PP2A) composed of a scaffold subunit, a catalytic subunit, and multiple regulatory subunits is a ubiquitously expressed serine/threonine phosphatase. We have previously shown that the PP2A catalytic subunit is increased in T cells from patients with systemic lupus erythematosus and promotes IL-17 production by enhancing the activity of Rho-associated kinase (ROCK) in T cells. However, the molecular mechanism whereby PP2A regulates ROCK activity is unknown. In this study, we show that the PP2A regulatory subunit PPP2R2A is increased in T cells from people with systemic lupus erythematosus and binds to, dephosphorylates, and activates the guanine nucleotide exchange factor GEF-H1 at Ser885, which in turn increases the levels of RhoA-GTP and the activity of ROCK in T cells. Genetic PPP2R2A deficiency in murine T cells reduced Th1 and Th17, but not regulatory T cell differentiation and mice with T cell–specific PPP2R2A deficiency displayed less autoimmunity when immunized with myelin oligodendrocyte glycoprotein peptide. Our studies indicate that PPP2R2A is the regulatory subunit that dictates the PP2A-directed enhanced Th1 and Th17 differentiation, and therefore, it represents a therapeutic target for pathologies linked to Th1 and Th17 cell expansion.

BACKGROUND/OBJECTIVES: Rhabdomyosarcoma (RMS) is characterized by the expression of the myogenic regulatory protein MYOD1. Histologic types include alveolar, embryonal (ERMS), and spindle cell sclerosing RMS (SRMS). SRMS harbors MYOD1 mutations in a subset of adult cases in association with poor prognosis. DESIGN/METHODS: To study the level of MYOD1 protein expression and its clinical significance, we have analyzed variable numbers of pediatric (

Lung inflammation and damage is prominent in people infected with SARS-Cov-2 and a major determinant of morbidity and mortality. We report the deposition of complement components in the lungs of people who succumbed to COVID-19 consistent with the activation of the classical and the alternative pathways. Our study provides strong rationale for the expansion of trials involving the use of complement inhibitors to treat patients with COVID-19.

Current technologies and available scaffold materials do not support long-term cell viability, differentiation and maintenance of podocytes, the ultra-specialized kidney resident cells that are responsible for the filtration of the blood. We developed a new platform which imitates the native kidney microenvironment by decellularizing fibroblasts grown on surfaces with macromolecular crowding. Human immortalized podocytes cultured on this platform displayed superior viability and metabolic activity up to 28 days compared to podocytes cultured on tissue culture plastic surfaces. The new platform displayed a softer surface and an abundance of growth factors and associated molecules. More importantly it enabled podocytes to display molecules responsible for their structure and function and a superior development of intercellular connections/interdigitations, consistent with maturation. The new platform can be used to study podocyte biology, test drug toxicity and determine whether sera from patients with podocytopathies are involved in the expression of glomerular pathology.

The coat protein I (COPI) complex mediates retrograde trafficking from the Golgi to the endoplasmic reticulum (ER). Five siblings with persistent bacterial and viral infections and defective humoral and cellular immunity had a homozygous p.K652E mutation in the γ1 subunit of COPI (γ1-COP). The mutation disrupts COPI binding to the KDEL receptor and impairs the retrieval of KDEL-bearing chaperones from the Golgi to the ER. Homozygous Copg1(K652E) mice had increased ER stress in activated T and B cells, poor antibody responses, and normal numbers of T cells that proliferated normally, but underwent increased apoptosis upon activation. Exposure of the mutants to pet store mice caused weight loss, lymphopenia, and defective T cell proliferation that recapitulated the findings in the patients. The ER stress-relieving agent tauroursodeoxycholic acid corrected the immune defects of the mutants and reversed the phenotype they acquired following exposure to pet store mice. This study establishes the role of γ1-COP in the ER retrieval of KDEL-bearing chaperones and thereby the importance of ER homeostasis in adaptive immunity.

Protein phosphatase 2A is a ubiquitously expressed serine/threonine phosphatase that comprises a scaffold, a catalytic, and multiple regulatory subunits and has been shown to be important in the expression of autoimmunity. We considered that a distinct subunit may account for the decreased production of IL-2 in people and mice with systemic autoimmunity. We show that the regulatory subunit PPP2R2D is increased in T cells from people with systemic lupus erythematosus and regulates IL-2 production. Mice lacking PPP2R2D only in T cells produce more IL-2 because the IL-2 gene and genes coding for IL-2–enhancing transcription factors remain open, while the levels of the enhancer phosphorylated CREB are high. Mice with T cell–specific PPP2R2D deficiency display less systemic autoimmunity when exposed to a TLR7 stimulator. While genes related to Treg function do not change in the absence of PPP2R2D, Tregs exhibit high suppressive function in vitro and in vivo. Because the ubiquitous expression of protein phosphatase 2A cannot permit systemic therapeutic manipulation, the identification of regulatory subunits able to control specific T cell functions opens the way for the development of novel, function-specific drugs., PPP2R2D restrains the chromatin opening of IL-2 and its related transcription factors loci to suppress IL-2 production.

Transplant glomerulopathy (TG) is a major cause of late allograft loss. Increased urine podocin/creatinine ratio in TG signifies accelerated podocyte loss. The mechanisms that lead to podocyte injury in TG remain unclear. We report that IgG from kidney transplant recipients with TG, but not from those without TG, cause a reduction in the expression of nephrin, significant podocyte actin cytoskeleton, and motility changes. These changes are preceded by increased expression of calcium/calmodulin kinase IV (CAMK4). Mechanistically, we found that CAMK4 phosphorylates GSK3β (glycogen synthase kinase 3 beta), activates the Wnt pathway and stabilizes the nephrin transcriptional repressor SNAIL. Silencing neonatal Fc Receptor (FcRn) or CAMK4 prevented the podocyte-damaging effects of IgG from patients with TG. Furthermore, we show that removal of N-linked glycosyl residues from these IgG did not interfere with its entry into the podocytes but eliminated its ability to upregulate CAMK4 and cause podocyte injury. The translational value of these findings is signified by the fact that CAMK4 is increased in podocytes of patients with TG but not in those without TG despite other forms of renal dysfunction. Our results offer novel considerations to limit podocyte injury in patients with kidney transplants, which may lead to eventual glomerular destabilization and transplant glomerulopathy.

Mature double negative (DN) T cells are a population of αβ T cells that lack CD4 and CD8 coreceptors and contribute to systemic lupus erythematosus (SLE). The splenic marginal zone macrophages (MZMs) are important for establishing immune tolerance, and loss of their number or function contributes to the progression of SLE. Here we show that loss of MZMs impairs the tolerogenic clearance of apoptotic cells and alters the serum cytokine profile, which in turn provokes the generation of DN T cells from self-reactive CD8+ T cells. Increased Ki67 expression, narrowed TCR V-beta repertoire usage and diluted T-cell receptor excision circles confirm that DN T cells from lupus-prone mice and patients with SLE undergo clonal proliferation and expansion in a self-antigen dependent manner, which supports the shared mechanisms for their generation. Collectively, our results provide a link between the loss of MZMs and the expansion of DN T cells, and indicate possible strategies to prevent the development of SLE., Splenic marginal zone macrophages can establish immune tolerance and limit the development of systemic lupus erythematosus (SLE). Here the authors show that these cells do this by clearing apoptotic cells, and defects in these cells result in the generation of self-reactive double negative T cells that are known to contribute to SLE pathogenesis.

Protein phosphatase 2A (PP2A), a serine/threonine phosphatase, has been shown to control T cell function. We found that in vitro–activated B cells and B cells from various lupus-prone mice and patients with systemic lupus erythematosus display increased PP2A activity. To understand the contribution of PP2A to B cell function, we generated a Cd19(Cre)Ppp2r1a(fl/fl) (flox/flox) mouse which lacks functional PP2A only in B cells. Flox/flox mice displayed reduced spontaneous germinal center formation and decreased responses to T cell-dependent and T-independent antigens, while their B cells responded poorly in vitro to stimulation with an anti-CD40 antibody or CpG in the presence of IL-4. Transcriptome and metabolome studies revealed altered nicotinamide adenine dinucleotide (NAD) and purine/pyrimidine metabolism and increased expression of purine nucleoside phosphorylase in PP2A-deficient B cells. Our results demonstrate that PP2A is required for optimal B cell function and may contribute to increased B cell activity in systemic autoimmunity.

Das Erleben sexualisierter Gewalt geht gemeinhin mit einer hohen emotionalen und korperlichen Belastung der meist weiblichen Opfer einher. Die Untersuchung entsprechend traumatisierter Patientinnen/Patienten stellt jedoch auch die behandelnden Arzte vor eine Herausforderung. Unabhangig von einer bereits erfolgten polizeilichen Anzeige erfordert die forensisch-medizinische Untersuchung nach erlebter sexualisierter Gewalt nicht nur arztliches Feingefuhl und Empathie im Rahmen einer professionellen Neutralitat des Untersuchers, sondern vor dem Hintergrund einer moglichen Strafverfolgung auch ein systematisches Vorgehen zur Sicherung gerichtsfester Beweise und Spuren. Hierfur empfiehlt sich die Verwendung eines standardisierten Spurensicherungs-Kits („Stuprum-Kit“), denn sowohl die Betroffenen als auch die Untersucher profitieren von einer Vereinfachung und Vereinheitlichung der Untersuchungsablaufe und somit von einer bestmoglichen Befunddokumentation sowie Spurensicherung.

International audience; Regulatory T (Treg) cells suppress inflammation and regulate immune system activity. In patients with systemic or organ-specific autoimmune diseases or those receiving transplanted organs, Treg cells are compromised. Approaches to strengthen Treg cell function, either by expanding them ex vivo and reinfusing them or by increasing the number or capacity of existing Treg cells, have entered clinical trials. Unlike the situation in autoimmunity, in patients with cancer, Treg cells limit the antitumour immune response and promote angiogenesis and tumour growth. Their immunosuppressive function may, in part, explain the failure of many immunotherapies in cancer. Strategies to reduce the function and/or number of Treg cells specifically in tumour sites are being investigated to promote antitumour immunity and regression. Here, we describe the current progress in modulating Treg cells in autoimmune disorders, transplantation and cancer.

Wir beschreiben und diskutieren den rechtsmedizinischen Einsatz nach dem Terroranschlag auf dem Breitscheidtplatz vor dem historischen Hintergrund des rechtsmedizinischen Bereitschaftsdienstes, die Zusammenarbeit mit den Polizeibehorden und die Verletzungsmuster der Verstorbenen. In diesem Zusammenhang ist von Bedeutung, dass nach stumpfen Trauma auserliche Verletzungen haufig nicht erkennbar sind (sog, Casper-Zeichen), was Rettungskrafte in falscher Sicherheit wiegen konnte oder – wie am Breitscheidtplatz geschehen – durch das akute Versterben scheinbar zunachst unverletzter Patient zu einer Irritation bzw. sogar Traumatisierung der Helfer fuhren kann.

Th17 cells favor glycolytic metabolism, and pyruvate dehydrogenase (PDH) is the key bifurcation enzyme, which in its active dephosphorylated form advances the oxidative phosphorylation from glycolytic pathway. The transcriptional factor, inducible cAMP early repressor/cAMP response element modulator (ICER/CREM), has been shown to be induced in Th17 cells and to be overexpressed in CD4(+) T cells from the patients with systemic lupus erythematosus (SLE). We found that glycolysis and lactate production in in vitro Th17-polarized T cells was reduced and that the expression of pyruvate dehydrogenase phosphatase catalytic subunit 2 (PDP2), an enzyme that converts the inactive PDH to its active form, and PDH enzyme activity were increased in Th17 cells from ICER/CREM-deficient animals. ICER was found to bind to the Pdp2 promoter and suppress its expression. Furthermore, forced expression of PDP2 in CD4(+) cells reduced the in vitro Th17 differentiation, whereas shRNA-based suppression of PDP2 expression increased in vitro Th17 differentiation and augmented experimental autoimmune encephalomyelitis. At the translational level, PDP2 expression was decreased in memory Th17 cells from patients with SLE and forced expression of PDP2 in CD4(+) T cells from lupus-prone MRL/lpr mice and patients with SLE suppressed Th17 differentiation. These data demonstrate the direct control of energy production during Th17 differentiation in health and disease by the transcription factor ICER/CREM at the PDH metabolism bifurcation level.

Es werden hier 2 Falle von Suiziden durch scharfe Gewalt mittels Messer beschrieben und diskutiert. Diese Variante der Selbsttotung ist an sich nicht ganz selten; die Suizidenten hatten sich jedoch beim Zufugen der Stichverletzungen durch Griff in die Messerklinge akzidentielle Verletzungen an den Handen zugezogen, die als aktive Abwehrverletzungen fehlinterpretiert wurden, sodass zunachst von einem Totungsdelikt ausgegangen werden musste. Die ausergewohnlichen Befunde werden vorgestellt und diskutiert.

Fur die Rekonstruktion eines Tathergangs ist oftmals nicht nur die Erstellung eines DNA-Profils von Bedeutung, sondern auch die Identifizierung einer Sekretart. Die heute gangigste Methode, die in den meisten forensischen Laboren angewandt wird, beruht auf der Immunchromatographie. Diesem Test liegt eine Antigen-Antikorper-Reaktion auf Proteinbasis zugrunde. Einen neuen Ansatz fur die Sekretdifferenzierung bietet eine DNA-basierte Methode. Diese unterscheidet Korperflussigkeiten anhand von sekretspezifischen Methylierungsmustern („Tissue-specific methylation pattern“, TSMP). Die DNA-Methylierungen kommen nur am Cytosin von Cytosin-phosphatidyl-Guanin (CpG) vor, die auch nur in CpG-Inseln stabil sind. Bei CpG-Inseln handelt es sich um CpG-reiche Sequenzabschnitte. Vor Genen auftretend konnen sie eine genregulatorische Funktion ubernehmen, d. h., sie konnen uber Aktivitat und Inaktivitat eines Gens entscheiden. So konnen die Untersuchung von CpG-Inseln vor sekretspezifischen Proteinen und die daraus entstehenden Methylierungsmuster zur Differenzierung einzelner Sekrete genutzt werden. Im Gegensatz zum proteingekoppelten Sekretnachweis konnen mithilfe der DNA-basierten Methode der Sekretdifferenzierung die genetische Analyse und die Sekretbestimmung aus derselben Spurenabnahme oder auch aus alteren DNA-Asservaten durchgefuhrt werden.

Acute and chronic kidney failure is common in hospitalized patients with COVID-19, yet the mechanism of injury and predisposing factors remain poorly understood. We investigated the role of complement activation by determining the levels of deposited complement components (C1q, C3, FH, C5b-9) and immunoglobulin along with the expression levels of the injury-associated molecules spleen tyrosine kinase (Syk), mucin-1 (MUC1) and calcium/calmodulin-dependent protein kinase IV (CaMK4) in the kidney tissues of people who succumbed to COVID-19. We report increased deposition of C1q, C3, C5b-9, total immunoglobulin, and high expression levels of Syk, MUC1 and CaMK4 in the kidneys of COVID-19 patients. Our study provides strong rationale for the expansion of trials involving the use of inhibitors of these molecules, in particular C1q, C3, Syk, MUC1 and CaMK4 to treat patients with COVID-19.

Hyaluronic acid (HA), a component of the extracellular matrix, is the ligand for CD44 and has been implicated in the pathogenesis of kidney inflammation in patients with systemic lupus erythematosus (SLE), but its direct role and mechanism of action have not been studied. Here we show that administration of hymecromone (4-Methylumbelliferone, 4-MU), an HA synthesis inhibitor, to lupus-prone mice suppressed dramatically lupus-related pathology. Interestingly, 4-MU stopped the appearance of disease when administered prior to its onset and inhibited the progression of disease when administered after its appearance. Inhibition of HA synthesis in vivo reduced tissue damage and the number of intrarenal lymphoid cell infiltrates including double negative CD3+CD4–CD8– T cells which are known to be involved in the pathogenesis of SLE. Exposure of human peripheral blood mononuclear cells to HA in vitro increased the generation of CD3+CD4–CD8– T cells through a mechanism involving Rho-associated kinase. Our results signify the importance of the HA-rich tissue microenvironment in the activation of lymphocytes to cause tissue damage in SLE and suggest the consideration of inhibition of HA synthesis to treat patients.

Th1 and Th17 are important in the pathogenesis of autoimmune diseases and they depend on glycolysis as a source of energy. T cell antigen receptor signaling phosphorylates a serine/threonine kinase, calcium/calmodulin-dependent protein kinase IV (CaMK4), and promotes glycolysis. Based on these findings we hypothesized that CaMK4 promotes glycolysis. Camk4-deficient CD4(+) T cells and cells treated with a CaMK4 inhibitor had less glycolysis compared with their counterparts. Pull-down of CaMK4 and mass spectrometry identified pyruvate kinase muscle isozyme (PKM), the final rate-limiting enzyme in glycolysis, as a binding partner. Coimmunoprecipitation and Western blotting showed that CaMK4 interacts directly with PKM2. Camk4-deficient CD4(+) T cells displayed decreased pyruvate kinase activity. Silencing or pharmacological inhibition of PKM2 reduced glycolysis and in vitro differentiation to Th1 and Th17 cells, while PKM2 overexpression restored Th17 cell differentiation. Treatment with a PKM2 inhibitor ameliorated experimental autoimmune encephalomyelitis and CD4(+) T cells treated with PKM2 inhibitor or Pkm2-shRNA caused limited disease activity in an adoptive cell transfer model of experimental autoimmune encephalomyelitis. Our data demonstrate that CaMK4 binds to PKM2 and promotes its activity, which is requisite for Th1 and Th17 differentiation in vitro and in vivo. PKM2 represents a therapeutic target for T cell-dependent autoimmune diseases.

Tregs require IL-2 signaling for signal transducer and activator of transcription 5–mediated (STAT5-mediated) induction of Foxp3. Although phosphatase 2A (PP2A) is a negative regulator of IL-2 production in effector T cells and Tregs do not produce IL-2, it is not known whether PP2A controls IL-2 signaling in Tregs. To explore the role of PP2A in IL-2 signaling in Tregs, we studied mice engineered to lack PP2A in all Foxp3-expressing cells. We report that PP2A is required to enable Foxp3 expression and to maintain sufficient numbers of Tregs in the thymus. We show for the first time to our knowledge that PP2A prevents the selective loss of surface IL-2Rβ and preserves IL-2R signaling potency in Tregs. The loss of IL-2Rβ in thymus- and spleen-derived Tregs that lack PP2A is because of increased sheddase activity. Pan-sheddase or selective ADAM10 (a disintegrin and metalloproteinase 10) inhibition, like forced expression of IL-2Rβ in PP2A-deficient Tregs, restored IL-2Rβ expression and signaling. Thus, PP2A restrains the sheddase activity of ADAM10 in Tregs to prevent the cleavage of IL-2Rβ from the cell surface to enable competent IL-2R signaling, which is essential for Tregs’ development and homeostasis.

Intestinal ischemia followed by reperfusion leads to local and remote organ injury attributed to inflammatory response during the reperfusion phase. The extent to which ischemia contributes to ischemia/reperfusion injury has not been thoroughly studied. After careful evaluation of intestinal tissue following 30 min of ischemia, we noticed significant local mucosal injury in wild-type mice. This injury was drastically reduced in C3-deficient mice, suggesting C3 involvement. Depletion of circulating complement with cobra venom factor eliminated, as expected, injury recorded at the end of the reperfusion phase but failed to eliminate injury that occurred during the ischemic phase. Immunohistochemical studies showed that tissue damage during ischemia was associated with increased expression of C3/C3 fragments primarily in the intestinal epithelial cells, suggesting local involvement of complement. In vitro studies using Caco2 intestinal epithelial cells showed that in the presence of LPS or exposure to hypoxic conditions the cells produce higher C3 mRNA as well as C3a fragment. Caco2 cells were also noted to produce cathepsins B and L, and inhibition of cathepsins suppressed the release of C3a. Finally, we found that mice treated with a cathepsin inhibitor and cathepsin B–deficient mice suffer limited intestinal injury during the ischemic phase. To our knowledge, our findings demonstrate for the first time that significant intestinal injury occurs during ischemia prior to reperfusion and that this is due to activation of C3 within the intestinal epithelial cells in a cathepsin-dependent manner. Modulation of cathepsin activity may prevent injury of organs exposed to ischemia.

Genetically engineered T cells expressing CD19-specific chimeric antigen receptors (CAR) have shown impressive activity against B-cell malignancies, and preliminary results suggest that T cells expressing a first-generation disialoganglioside (GD2)-specific CAR can also provide clinical benefit in patients with neuroblastoma. We sought to assess the potential of GD2-CAR therapies to treat pediatric sarcomas. We observed that 18 of 18 (100%) of osteosarcomas, 2 of 15 (13%) of rhabdomyosarcomas, and 7 of 35 (20%) of Ewing sarcomas expressed GD2. T cells engineered to express a third-generation GD2-CAR incorporating the 14g2a-scFv with the CD28, OX40, and CD3ζ signaling domains (14g2a.CD28.OX40.ζ) mediated efficient and comparable lysis of both GD2+ sarcoma and neuroblastoma cell lines in vitro. However, in xenograft models, GD2-CAR T cells had no antitumor effect against GD2+ sarcoma, despite effectively controlling GD2+ neuroblastoma. We observed that pediatric sarcoma xenografts, but not neuroblastoma xenografts, induced large populations of monocytic and granulocytic murine myeloid-derived suppressor cells (MDSC) that inhibited human CAR T-cell responses in vitro. Treatment of sarcoma-bearing mice with all-trans retinoic acid (ATRA) largely eradicated monocytic MDSCs and diminished the suppressive capacity of granulocytic MDSCs. Combined therapy using GD2-CAR T cells plus ATRA significantly improved antitumor efficacy against sarcoma xenografts. We conclude that retinoids provide a clinically accessible class of agents capable of diminishing the suppressive effects of MDSCs, and that co-administration of retinoids may enhance the efficacy of CAR therapies targeting solid tumors. Cancer Immunol Res; 4(10); 869–80. ©2016 AACR.

Zwischen Rechts- und Notfallmedizin bestehen erhebliche Gemeinsamkeiten und Schnittmengen. Insbesondere gilt dies fur frustran reanimierte Patienten oder andere letale klinische Verlaufe, die einer rechtsmedizinischen Obduktion zugefuhrt werden. Eine Kooperation zwischen Tragern von notfall- und rechtsmedizinischen Einrichtungen weist dabei nicht nur notfallmedizinisches Fortbildungspotenzial auf, sondern auch die Moglichkeit der retrospektiven Evaluation medizinischer Notfallmasnahmen – sowohl im Einzelfall als auch unter epidemiologischen Gesichtspunkten. Insbesondere die flachendeckende Erfassung rechtsmedizinisch untersuchter Traumatodesfalle, die trotz Reanimationsversuchen praklinisch verstarben, ist sinnvoll, da die praklinische Situation einen Brennpunkt, gleichzeitig aber auch einen „blinden Fleck“ in der Gesamttraumaletalitat darstellt. Der Nutzen klinischer Register ist bereits heute unstrittig, obgleich in diesen praklinische Todesfalle nicht abgebildet werden.

In Berlin waren in den Jahren 2009–2012 (Untersuchungszeitraum) 127.545 und im Land Brandenburg 111.457 Todesfalle zu verzeichnen (insgesamt 239.002 Verstorbene). Dieses bedeutet zugleich, dass fur diese Verstorbenen auch eine den Bestattungsgesetzen der beiden Bundeslander entsprechende Todesbescheinigung von einem Arzt ausgestellt werden musste. Aufgrund der Gesetzgebungskompetenz der Lander ist die Regelung des Bestattungs- und damit des Leichenschaurechts Landersache mit der Folge, dass in Berlin und Brandenburg fur den hier untersuchten Teil erhebliche gesetzliche Unterschiede bestehen. Eine – bedauerlicherweise – unbekannte Anzahl dieser Todesbescheinigungen wurde durch Notarzte im Rettungsdienst ausgestellt. Dabei gelangt der Notarzt im Rettungsdienst regelmasig in ein Spannungsverhaltnis zwischen seiner Garantenstellung zur Erhaltung von Leben und Gesundheit der Notfallpatienten, den moglichen Ermittlungsinteressen der (Kriminal-)Polizei und ggfs. der unteren Gesundheitsbehorde. Dieses Spannungsverhaltnis endet moglicherweise in einem Busgeldverfahren bei der unteren Gesundheitsbehorde. Um die diesbezugliche Busgeldrelevanz zu ermitteln, wurden alle 12 Berliner Gesundheitsamter und die 18 Gesundheitsamter der Brandenburger Landkreise und kreisfreien Stadte mittels eines strukturierten Fragebogens um Auskunft uber Busgeldverfahren im Zusammenhang mit der arztlichen Leichenschau und/oder der Ausstellung von Leichenschauscheinen in den Jahren 2009–2012 gebeten. Es wurden im Untersuchungszeitraum insgesamt (nur) 15 Busgeldverfahren gefuhrt, davon 9 in Berlin und 6 in Brandenburg; dies entspricht einem Anteil von 0,0062 % bezogen auf alle Sterbefalle in beiden Bundeslandern in den Jahren 2009–2012 (n = 239.002). In beiden Bundeslandern war nur je 1 Notarzt betroffen. Gravierende Mangel bei der (not- )arztlichen Leichenschau wurden im Rahmen der Untersuchung nicht gefunden. Die Ergebnisse zeigen, dass Befurchtungen der Notarzte, im Zusammenhang mit einer fehlerhaften (not- )arztlichen Leichenschau juristischen Weiterungen ausgesetzt zu sein, aktuell gering sind. Allerdings offenbarte die Studie zum Teil erhebliche strukturelle Probleme bei der Durchfuhrung der (not- )arztlichen Leichenschau. Sie zeigt aber auch, dass Busgeldverfahren als Instrument der Qualitatssicherung nicht geeignet sind, da eine Uberprufung der Leichenschauscheine durch die Gesundheitsamter, bis auf wenige Ausnahmen, nur formal erfolgt bzw. erfolgen kann.

Wir berichten uber 3 todlich verlaufende Falle von akuten Aortendissektionen des Typs A nach Stanford, die samtlich im Rahmen der notfallmedizinischen Diagnostik nicht bzw. zu spat erkannt wurden. Eine Aortendissektion sollte beim akuten Brustschmerz bereits praklinisch verstarkt in differenzialdiagnostische Uberlegungen miteinbezogen werden. Vor allem sollte der Versuch einer Abgrenzung zum akuten Koronarsyndrom erfolgen, um praklinisch von einer – bei der akuten Aortendissektion prognostisch ungunstigen – medikamentosen Alteration der Blutgerinnung Abstand zu nehmen.

Simon-Blutungen gelten als klassisches Vitalitatszeichen des Erhangens. Die charakteristischen Einblutungen in die ventralen Bandscheibenanteile der Lendenwirbelsaule finden sich in geringerer Zahl jedoch auch bei anderen Todesursachen (z. B. traumatische Entstehungen im Rahmen eines Verkehrsunfalls oder eines Sturzes aus der Hohe) oder im Zusammenhang mit Faulnisveranderungen. Selten konnen entsprechende Einblutungen auch bei naturlichen Todesursachen wie z. B. einem plotzlichen Herztod festgestellt werden. Einen solchen Fall mochten wir in dieser Kasuistik vorstellen.

Cardiovascular manifestations in patients with systemic lupus erythematosus (SLE) are common, but aortic aneurysm formation is rare. We present a 63-year-old male SLE patient with a two-year history of skin lesions, leucopenia, pericarditis, mitral valve vegetations consistent with Liebman-Sacks endocarditis, and an aneurysm of the ascending aorta, which was successfully repaired surgically. Histologic examination of the aneurysm showed medial cystic degeneration, smooth muscle necrosis, and mild adventitial perivascular lymphocytic aggregates. This histology is typical of thoracic aneurysms that carry a high risk for aortic dissection and patient death. The case highlights the importance of early detection and treatment of thoracic aortic aneurysms in patients with SLE.

This study examined the sexual dimorphism in variables of the foramen magnum in a modern German population. This includes measurements of the maximum width, the maximum length, the circumference and the area of the foramen magnum, which were taken using the imaging software OsiriX (v.4.1.1. 64 bit). The data set includes 200 cadavers (100 males, 100 females) that were autopsied during the years 2011–2013 at the Institute of Legal Medicine and Forensic Sciences, Charite–Berlin. Each variable examined showed clear sexual dimorphism with p

Calcium calmodulin kinase IV (CaMK4) regulates multiple processes that significantly contribute to the lupus-related pathology by controlling the production of IL-2 and IL-17 by T cells, the proliferation of mesangial cells, and the function and structure of podocytes. CaMK4 is also upregulated in podocytes from patients with focal segmental glomerulosclerosis (FSGS). In both immune and non-immune podocytopathies, CaMK4 disrupts the structure and function of podocytes. In lupus-prone mice, targeted delivery of a CaMK4 inhibitor to CD4+ T cells suppresses both autoimmunity and the development of nephritis. Targeted delivery though to podocytes averts the deposition of immune complexes without affecting autoimmunity in lupus-prone mice and averts pathology induced by adriamycin in normal mice. Therefore, targeted delivery of a CaMK4 inhibitor to podocytes holds high therapeutic promise for both immune (lupus nephritis) and non-immune (FSGS) podocytopathies.

Defective DNA methylation in T cells leads to a series of T cell abnormalities in lupus; however, the full effect of T cell lineage–specific DNA methylation on disease expression has not been explored. Here, we show that 5-azacytidine, a DNA methyltransferase inhibitor, targeted to either CD4 or CD8 T cells in mice with established disease using a nanolipogel delivery system dramatically ameliorates lupus-related pathology through distinct mechanisms. In vivo targeted delivery of 5-azacytidine into CD4 T cells favors the expansion and function of Foxp3+ Tregs, whereas targeted delivery to CD8 T cells enhances the cytotoxicity and restrains the expansion of pathogenic TCR-αβ+CD4–CD8– double-negative T cells. Our results signify the importance of cell-specific inhibition of DNA methylation in the treatment of established lupus.

Rhabdomyosarcoma (RMS) is a common malignancy of soft tissue, subclassified as alveolar (ARMS), pleomorphic (PRMS), spindle cell/sclerosing (SRMS), and embryonal (ERMS) types. The Yes-associated protein (YAP) is a member of the Hippo pathway and a transcriptional regulator that controls cell proliferation. We have studied the immunohistochemical expression of YAP in different RMSs, arranged in tissue microarray (TMA) and whole slide formats. Pertinent clinical data including patient age, gender, tumor location, and clinical stage were collected. Out of 96 TMA cases, 30 cases (31%) were pleomorphic, 27 (28%) were embryonal, 24 (25%) alveolar, and 15 (16%) spindle cell. Positive nuclear YAP staining was seen in the PRMS (17/30, 56.7%), SRMS (7/15, 46.7%), ERMS (19/27 or 70%), and less in ARMS (37.5%). YAP nuclear staining was significantly more prevalent in ERMS than ARMS ( p=0.02). Of the 41 whole slide cases, nuclear staining was detected in all ARMS but was restricted in distribution to 30% staining. These results highlight the role of YAP in RMS tumorigenesis, a fact that can be useful in engineering targeted therapy. Restricted nuclear YAP staining (

Recovery injuries can be misinterpreted as vital injuries during the external examination or the autopsy of a body. While usually – if the finding situation and the circumstances of recovery are known – there should be no problem for the experienced forensic pathologist to differentiate between vital injuries and recovery injuries, in some cases with missing information there might be pitfalls in the interpretation of the findings. We report a case of an unusual recovery injury which could be solved through careful reconstruction of the circumstances.