Establishment of in vivo bioluminescence imaging models for tumor immunology

Die Überwachung des Tumorwachstums bzw. das Verfolgen von Zellen oder Pathogenen kann anhand des bildgebenden Verfahrens der in vivo Biolumineszenz (engl. in vivo bioluminscence imaging, Abk. BLI) erfolgen. Da die Methode des BLI noch in ihren Anfängen steht, konzentrierte sich diese Arbeit auf die Auswahl der optimalen Luciferase, um diese anschließend zur Etablierung eines in vivo BLI-Modells, das Studien der Tumorimmunologie dient, zu nützen. Außerdem sollte anhand von lumineszierenden Bakterien die Visualisierung von Tumoren untersucht werden. Zuerst wurde daher die meist verwendete Luciferase, die firefly luciferase (Fluc), mit der neu zur Verfügung stehenden grünen sowie roten Luciferase (CBGr99 und CBRed) des Schnellkäfers (engl. click beetle) verglichen. Zu diesem Zweck wurden equimolare Mengen der verschiedenen Proteine, CBGr99, CBRed bzw. Fluc, mit Hilfe von Zelltransfektion exprimiert. Um eine Equimolarität zu erzielen, wurde die jeweilige Luciferase mit eGFP (engl. enhanced green fluorescent protein) koexprimiert. Dies erfolgte durch Anwendung eines neuartigen bicistronischen 2A Systems, das in einer stöchiometrischen Koexpression der entsprechenden Proteine resultiert. Durch in vitro Experimente konnte die höchste absolute Photonen Ausbeute bei CBGr99 festgestellt werden. Injektion der Transfektanten an verschiedenen Stellen einer Maus zeigtendass die click beetle Luciferasen (CBLucs) besser gelisnet als Fluc fur die BLI sind. Der Vergleich zwischen den verwendeten CBLucs ergab, dass CBGr99 eine bessere oder ähnliche in vivo Sensitivität als CBRed aufwies, die jedoch vom Zeitpunkt der Analyse abhing. Die Analyse der mit CBGr99 transfizierten Zellen zeigte, dass die CBGr99 Expression mit der Zellanzahl korrelierte und somit für das Überwachen des Tumorwachstums in vivo verwendet werden konnte. Dafür wurde eine induzierbare transgene Mauslinie genereriert, die mit Hilfe des Cre/loxP Systems CBGr99 spezifisch in Leberzellen (ASC Linie) exprimiert. Diese transgene Mauslinie wurde mit der Linie AST gekreuzt, die nach Injektion eines Cre-recombinase kodierenden Adenoviruses autochthones Hepatokarzinom Wachstum entwickelt. Der Nachwuchs dieser Kreuzung, als ASCT benannt, ermöglichte das nicht-invasive Beobachten der Entwicklung des Hepatokarzinoms in lebenden Tieren durch in vivo BLI mittels der Expression von CBGr99. Das in dieser Arbeit entwickelte ASCT-Modell kann daher zur zeitlichen Überwachung des Tumorbeginns und -fortschreitens genützt und effektiv für Analysen zur Wirkug von Therapien verwendet werden. Ein weiterer Ansatz zur Visualisierung von Tumoren ist die Verwendung von Luciferaseexprimierenden Bakterien. Viele Bakterienstämme visieren nach intravenöser Injektion speziell Tumore an (engl. tumor targeting) und können dort überleben, während sie in Geweben vernichtet werden. BLI-Messungen zeigten, dass transgene Vibrio cholerae, die ein bakterielles Luciferase Gen (luxCDABE) exprimieren, Tumore in tumortragenden, immundefizienten Mäusen besiedeln („Tumor homing“). In dieser Arbeit konnte sowohl mit V. cholerae als auch mit Top10 E.coli ein effizientes „targeting“ von subkutanen Tumoren in immunkompetenten Mäusen, die eine Immunantwort gegen Bakterien erzeugen können, erzielt werden. Es konnte vorübergehend beobachtet werden, dass in verschiedenen Tumoren, wie z.B. AG104A, B16 sowie RMA, Tumorkolonisierung stattfand. Zusätzlich wurde die Fähigkeit der Bakterien, Tumore in verschiedenen spontanen Tumormodellen anzuvisieren, untersucht, z.B. in dem Hepatom-Modell (Albumin-Tag Mäuse), dem Insulinom-Modell (RIP.Tag-5 Mäuse) oder den Mammakarzinom-Modell (Her-2/neu Mäuse). Mit Hilfe des BLI konnte das bakterielle „homing“ in diese Tiermodellen untersucht werden und zeigte, dass die Effizienz des „tumor targeting“ geringer war als in subkutanen Tumoren. Außerdem konnte nachgewiesen werden, dass Bakterien als Vektoren für GM-CSF und IL-2 genützt werden können, um die Abwehr von Tumoren durch das Immunsystem zu verstärken. Damit eine Sekretion von GM-CSF und IL-2 mit Hilfe des Bakterienstammes Top10.lux im Tumorgewebe ermöglicht werden konnte, wurden die entsprechenden DNA-Sequenzen in ein Hemolysin A sekretorisches System kodierendes Plasmid inseriert. Die therapeutische Vakzinierung zeigte, dass Mäuse mit subkutanen Tumoren nach intravenöser Injektion mit Top10.lux.GM-CSF keine verstärkte Verzögerung des Tumorwachstums aufweisen, was im Gegensatz dazu bei Injektion mit Top10.lux beobachtet wird. Um die Infiltration von T-Zellen in das Tumorgewebe zu analysieren, wurde zusätzlich eine transgene Mauslinie (CAG-L2ACh) generiert, die CBGr99-positive Zellen liefert. Die CAGL2ACh Linie koexprimiert CBGr99 und ein rot fluoreszierendes Protein (mCherry) unter der Kontrolle eines ubiquitären Promoters. Anhand des BLI konnte in allen Geweben eine CBGr99 Expression festgestellt werden. Jede einzelne T-Zelle exprimierte mCherry und somit CBGr99. Daher zeichnet sich die CAG-L2ACh Linie als universeller Donor für „celltrafficking“ Studien mit Hilfe der BLI-Methode aus

Contribution of the geographic information system for groundwater to the mapping of subterranean waters in the region Djelfa (Algeria)

The Djelfa region characterized by a semi-arid climate. The quality of groundwater is among the problems that are becoming increasing concern in the region. The physico-chemical characteristics of water are severely affected by high salinity of them. This research is devoted to the use of geographic information systems GIS, for drawing maps of the main parameters that can reveal the spatial variation of physical and chemical characteristics of water to achieve a good estimate of its quality. Mapping is the first step in the creation of geographic information systems. The aim of our work is to present a tool for decision making that can establish useful maps to study the water quality of the Djelfa region

Estimation des paramètres de l'érosion hydrique par Approche SIG/USLE : cas du bassin versant de l'Oued Arab (région de Khenchela, Nord-Est de l’Algérie)

L’érosion, le transport solide et la sédimentation constituent, de par leurs importances, un problème majeur en Algérie. Le présent travail a pour objectif d’évaluer et cartographier les zones sensibles et à haut risque d’érosion ainsi que les régions d’urgence d’intervention dans le bassin versant, par une approche SIG/USLE à l’échelle du bassin versant. Le modèle USLE ( Universal Soil Loss Equation), avec lequel on peut estimer les risques d’érosion hydrique des sols, a été appliqué au bassin versant d’ oued el Arab. L'analyse, des cinq paramètres d’érosion à savoir l’érosivité de la pluie, l’érodibilité du sol, l’inclinaison et la longueur de pente, le couvert végétal et les pratiques antiérosives, ont été opérées dans un SIG. La combinaison des différentes cartes de ces paramètres a permis de déduire la carte  d’érosion à partir de laquelle, il ressort que le phénomène d’érosion touche l’ensemble du bassin versant d’oued el Arab  et varient de 0 à plus de 65 t/ha/an avec une perte moyenne annuelle relativement forte de 7.3 t/ha/an.Mots-clés : Bassin versant oued El Arab, érosion hydrique, vulnérabilité, SIG, USLE

Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications

Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient’s immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CART cell manufacturing process

Drug-inducible remote control of gene expression by probiotic Escherichia coli Nissle 1917 in intestine, tumor and gall bladder of mice.

The probiotic bacterium Escherichia coli Nissle 1917 (EcN) constitutes a prospective vector for delivering heterologous therapeutic molecules to treat several human disorders. To add versatility to this carrier system, bacteria should be equipped with expression modules that can be regulated deliberately in a temporal and quantitative manner. This approach is called in vivo remote control (IVRC) of bacterial vectors. Here, we have evaluated promoters P(araBAD), P(rhaBAD) and P(tet), which can be induced with L-arabinose, L-rhamnose or anhydrotetracycline, respectively. EcN harboring promoter constructs with luciferase as reporter gene were administered either orally to healthy mice or intravenously to tumor bearing animals. Subsequent to bacterial colonization of tissues, inducer substances were administered via the oral or systemic route. By use of in vivo bioluminescence imaging, the time course of reporter gene expression was analyzed. Each promoter displayed a specific in vivo induction profile depending on the niche of bacterial residence and the route of inducer administration. Importantly, we also observed colonization of gall bladders of mice when EcN was administered systemically at high doses. Bacteria in this anatomical compartment remained accessible to remote control of bacterial gene expression

Remote control of tumour-targeted Salmonella enterica serovar Typhimurium by the use of L-arabinose as inducer of bacterial gene expression in vivo.

We have used Salmonella enterica serovar Typhimurium (S. typhimurium) which are able to colonize tumours besides spleen and liver. Bacteria were equipped with constructs encoding green fluorescent protein or luciferase as reporters under control of the promoter PBAD that is inducible with L-arabinose. Reporter genes could be induced in culture but also when the bacteria resided within the mouse macrophages J774A.1. More important, strong expression of reporters by the bacteria could be detected in mice after administration of L-arabinose. This was especially pronounced in bacteria colonizing tumours. Histology demonstrated that the bacteria had accumulated in and close to necrotic areas of tumours. Bacterial gene induction was observed in both regions. PBAD is tightly controlled also in vivo because gene E of bacteriophage PhiX174 could be introduced as inducible suicide gene. The possibility to deliberately induce genes in bacterial carriers within the host should render them extremely powerful tools for tumour therapy

Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications

Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient’s immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CART cell manufacturing process

Advanced Flow Cytometry Assays for Immune Monitoring of CAR-T Cell Applications

Adoptive immunotherapy using chimeric antigen receptor (CAR)-T cells has achieved successful remissions in refractory B-cell leukemia and B-cell lymphomas. In order to estimate both success and severe side effects of CAR-T cell therapies, longitudinal monitoring of the patient’s immune system including CAR-T cells is desirable to accompany clinical staging. To conduct research on the fate and immunological impact of infused CAR-T cells, we established standardized 13-colour/15-parameter flow cytometry assays that are suitable to characterize immune cell subpopulations in the peripheral blood during CAR-T cell treatment. The respective staining technology is based on pre-formulated dry antibody panels in a uniform format. Additionally, further antibodies of choice can be added to address specific clinical or research questions. We designed panels for the anti-CD19 CAR-T therapy and, as a proof of concept, we assessed a healthy individual and three B-cell lymphoma patients treated with anti-CD19 CAR-T cells. We analyzed the presence of anti-CD19 CAR-T cells as well as residual CD19+ B cells, the activation status of the T-cell compartment, the expression of co-stimulatory signaling molecules and cytotoxic agents such as perforin and granzyme B. In summary, this work introduces standardized and modular flow cytometry assays for CAR-T cell clinical research, which could also be adapted in the future as quality controls during the CART cell manufacturing process

A Granulocytic Signature Identifies COVID-19 and Its Severity

International audienceBackground An unbiased approach to SARS-CoV-2–induced immune dysregulation has not been undertaken so far. We aimed to identify previously unreported immune markers able to discriminate COVID-19 patients from healthy controls and to predict mild and severe disease.Methods An observational, prospective, multicentric study was conducted in patients with confirmed mild/moderate (n = 7) and severe (n = 19) COVID-19. Immunophenotyping of whole-blood leukocytes was performed in patients upon hospital ward or intensive care unit admission and in healthy controls (n = 25). Clinically relevant associations were identified through unsupervised analysis.Results Granulocytic (neutrophil, eosinophil, and basophil) markers were enriched during COVID-19 and discriminated between patients with mild and severe disease. Increased counts of CD15+CD16+ neutrophils, decreased granulocytic expression of integrin CD11b, and Th2-related CRTH2 downregulation in eosinophils and basophils established a COVID-19 signature. Severity was associated with emergence of PD-L1 checkpoint expression in basophils and eosinophils. This granulocytic signature was accompanied by monocyte and lymphocyte immunoparalysis. Correlation with validated clinical scores supported pathophysiological relevance.Conclusions Phenotypic markers of circulating granulocytes are strong discriminators between infected and uninfected individuals as well as between severity stages. COVID-19 alters the frequency and functional phenotypes of granulocyte subsets with emergence of CRTH2 as a disease biomarker