MCP-1 induces migration of adult neural stem cells

As a model for brain inflammation we previously studied transcriptional profiles of tumor necrosis factor-alpha (TNF)treated U373 astroglioma cells. In previous work we were able to demonstrate that the chemokine monocyte chemoattractant protein-1 (MCP-1, SCYA2, CCL2, MCAF) expression in U373 cells was inducible by TNF-alpha treatment. Demonstrably MCP-1 mRNA and protein expression in U373 cells was sustainable over time and at the highest level of all genes analyzed (Schwamborn et al., BMC Genomics 4, 46, 2003). In the hematopoietic system MCP-1 is a CC chemokine that attracts monocytes, memory T lymphocytes, and natural killer cells. In search of further functions in brain inflammation we tested the hypothesis that MCP-1 acts as a chemokine on neural stem cells. Here we report that MCP-1 activates the migration capacity of rat-derived neural stem cells. The migration of stem cells in a Boyden chamber analysis was elevated after stimulation with MCP-1. Time-lapse video microscopy visualized the migration of single stem cells from neurospheres in MCP-1-treated cultures, whereas untreated cultures depicted no migration at all, but showed signs of sprouting. Expression of the MCP-1 receptor CCR2 in neurosphere cultures was verified by RT-PCR and immunofluorescence microscopy. Supernatants from TNF-treated U373 cells also induced migration of neural stem cells.

Granulocyte colony-stimulating factor receptor: stimulating granulopoiesis and much more

The granulocyte colony-stimulating factor receptor (G-CSFR) plays an important role in the production, survival and activation of neutrophilic granulocytes during both normal and emergency hematopoiesis. The G-CSFR also participates in the development of other myeloid lineages, the mobilization of hematopoietic stem cells and myeloid cell migration. This has lead to several important clinical applications for its ligand, G-CSF. More recently, additional important roles for G-CSFR have emerged outside the hematopoietic system, such as in the protection and repair of a diverse range of tissues, including muscle, liver and neural tissue, providing further scope for developing G-CSF as a therapeutic agent. The G-CSFR has also been implicated in the etiology of disease, with mutations/variants of G-CSFR implicated in neutropenia, myelodysplasia and leukemia. Additionally, autocrine/paracrine stimulation of G-CSFR may be important in the biology of solid tumors, including metastasis.

Analysis of the Stat5 genes in zebrafish

This dissertation identified and characterised a key genetic regulator called Stat5 using zebrafish. Up-regulation of Stat5 led to an increase in blood cells, indicative of pre-leukaemia, whilst down-regulation decreased these cells and caused other defects. This work shows that Stat5 is critical in blood cell maturation and early embryonic development.

Thymic lymphomas in Wistar rats exposed to N-methyl-N-nitrosourea (MNU)

The Brazilian Agency for the Environment (IBAMA) recently adopted an alternative medium-term multiple-organ assay system with the Wistar rat strain for detection of the carcinogenic potential of pesticides. Originally, this initiation-promotion protocol was established in Japan with the isogenic Fischer 344 male rat. Among the initiating agents used in that assay, N-methyl-N-nitrosourea (MNU) rapidly induces malignant lymphoma and leukemia and early mortality of rats from different strains. This study was developed to evaluate whether the outbred Wistar rats are also similarly susceptible to MNU. Particularly, it aimed to evaluate the dose-response relationship and to register the MNUinduced pre-neoplasia and neoplasia that may develop in the lympho-hematopoietic system (LHS) of the Wistar rat within a medium-term period. Four groups of male Wistar rats were treated during 2 weeks with vehicle or with MNU (80, 160 or 240 mg/kg body weight, i.p.). After sacrifice at the 12th and 20th weeks, the thymus, spleen, bone marrow, cervical and mesenteric lymph nodes and liver were collected for analysis. At the 20th week, LHS malignant tumors and benign vascular tumors occurred only in the high- and intermediate-dose MNU-treated animals. Four animals treated with 240 mg/kg developed diffuse thymic lymphomas; two others, treated respectively with 240 mg/kg and 160 mg/kg, developed spleen hemangiomas. The present observations indicate that the Wistar strain is as susceptible as other strains to the early development of MNU-induced LHS (pre)neoplasia. Therefore, this strain seems suitable to be used as test system in bioassay protocols that adopt MNU as an initiating agent for carcinogenesis.

Quantitative analysis of potentially toxic metals in alginates for dental use

Alginate is one the materials most employed in practice to make dental impressions. Substances like zinc, cadmium and lead silicate, which are included in several alginate brands with the aim of improving their physical, chemical and mechanical properties, are a source of serious concern as regards their toxicity. The most serious chronic effect of oral exposure to cadmium is renal toxicity. Assimilation of lead has deleterious effects on the gastrointestinal tract, hematopoietic system, cardiovascular system, central and peripheral nervous systems, kidneys, immune system, and reproductive system. Chronic oral exposures to zinc have resulted in hypochromic and microcyte anemia in some individuals. The aim of the present study was to measure the cadmium, lead and zinc contents of seven brands of alginate for dental use on sale in Brazil. The samples were weighed and placed in the Teflon cups of a closed-system microwave oven. Aqua regia (4mL concentrated HCI:HNO3, 3:1 v/v) and hydrofluoric acid (2mL concentrated HF) were added to the samples, which were then subjected to heating. The samples were then cooled to room temperature and diluted to 25 mL in deionized water in a volumetric glass flask. The samples were diluted in duplicate and analyzed against a reagent blank. The analyses were performed in an atomic absorption flame spectrophotometer. Neither lead nor cadmium was detected. Zinc contents ranged from 0.001% to 1.36% by weight. The alginates exhibited low contents of the metals under study and gave no cause for concern regarding toxicity; even so, it is advisable to monitor potentially toxic materials continually and to analyze their plasmatic levels in the professionals working with them.

Mammary benign neoplasm diagnosed by fine needle aspiration biopsy in a guinea pig (Cavia porcellus)

Cytology is a well established research technique in human and veterinary medicine, but it is rarely used in small rodents. Spontaneous tumors are relatively uncommon in guinea pigs and those described in literature include a variety of organs like skin and subcutis, respiratory and reproductive tract, endocrine and hematopoietic system, and mammary gland. The objective of this article was to describe the use of the fine needle aspiration biopsy technique (FNAB) on evaluation of a mammary neoplasm in a guinea pig and describe the main cytological findings for the first time in literature.

Ação imunomodulatória da β-glucana e Saccharomyces cerevisiae em ratos (Rattus rattus) da linhagem Wistar imunossuprimidos pela administração experimental de dexametasona

Pós-graduação em Medicina Veterinária - FCAV

Elastografia acoustic radiation force impulse (ARFI), doppler e ultrassonografia contrastada do baço canino

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito do benzoato de estradiol na recuperação de oócitos, embriões e nos parâmetros hematológicos de cadelas recém acasaladas

We investigated estradiol benzoate effects on oocytes/embryos recovery rate and the influence of this drug on the hematopoietic system. Twenty four bitches were divided in two groups, Group I, 12 females that received a single shot of estradiol benzoate, 0.2 mg/kg intramuscularly, between 2 and 7 days after the date of the last mismating or insemination and, Group II (control), 12 bitches that received 0.2 ml/kg of oily diluent, in corresponding dates. The bitches were ovary-hysterectomized and the uterus/oviduct were isolated and flushed with a PBS, heparin and polyvinyl alcohol solution. Oocytes and embryos were quantified and classified according to their stage of development. Blood counts were performed on M1 (before drug administration), M2 (15 days after drug administration) and M3 (40 days after drug administration). Pearson correlation coefficient was used to analyze the variable retrieval structures, while Fisher exact test was used for the analysis of embryonic viability. ANOVA was used to analyze repeated measurements and Tukey test for hematological parameters. All tests were performed at 5% significance level. The recovery rate of total structures in group I was lower (22.88%) than group II (65.85%). A lower embryo recovery (ratio 3: 52) rate and a greater number of degenerated structures (ratio 11: 1) were observed in group I. Hematological parameters showed significant difference in erythrocytes, hematocrit and hemoglobin concentrations 15 days after drug administration and difference in leukocytes concentration 40 days after using the medication in bitches of group I, however, at the end of the experiment all bitches had blood counts considered normal.

Serial bone marrow transplantation reveals in vivo expression of the pCLPG retroviral vector

Abstract Background Gene therapy in the hematopoietic system remains promising, though certain aspects of vector design, such as transcriptional control elements, continue to be studied. Our group has developed a retroviral vector where transgene expression is controlled by p53 with the intention of harnessing the dynamic and inducible nature of this tumor suppressor and transcription factor. We present here a test of in vivo expression provided by the p53-responsive vector, pCLPG. For this, we used a model of serial transplantation of transduced bone marrow cells. Results We observed, by flow cytometry, that the eGFP transgene was expressed at higher levels when the pCLPG vector was used as compared to the parental pCL retrovirus, where expression is directed by the native MoMLV LTR. Expression from the pCLPG vector was longer lasting, but did decay along with each sequential transplant. The detection of eGFP-positive cells containing either vector was successful only in the bone marrow compartment and was not observed in peripheral blood, spleen or thymus. Conclusions These findings indicate that the p53-responsive pCLPG retrovirus did offer expression in vivo and at a level that surpassed the non-modified, parental pCL vector. Our results indicate that the pCLPG platform may provide some advantages when applied in the hematopoietic system.

Identifizierung, Sequenzierung und Charakterisierung des Dmxl1-Gen in Mus musculus sowie die funktionelle Analyse durch Knock-Out

Identifizierung, Sequenzierung und Charakterisierung des Dmxl1-Gen in Mus musculus sowie die funktionelle Analyse durch Knock-OutrnrnBei Dmxl1 handelt es sich um ein neuartiges Gen aus Mus musculus. Das ebenfalls in der vorliegenden Arbeit bioinformatisch untersuchte Gen DMXL1 ist das zu Dmxl1 homologe Gen des Menschen. Beide Gene bestehen aus 43 Exons, das murine Dmxl1 codiert für eine mRNA von 10992 bp bzw. 12210 bp, das humane DMXL1 kodiert für eine cDNA von 11082 bp, der offene Leserahmen umfasst bei der Maus 9042 bp. In der Maus konnte ein mögliches alternatives Polyadenylierungssignal identifiziert werden. Zwischen beiden Spezies sind die Exonpositionen und ihre Längen hoch konserviert. Dmxl1 liegt auf dem Crick-Strang von Chromosom 18 Bande C, der translatierte Bereich erstreckt sich auf genomischer Ebene über 129558 bp und die Orientierung verläuft in Richtung Centromer. Dmxl1 und DMXL1 gehören damit zu den größten bekannten Genen in Maus und Mensch. Bei beiden Spezies liegen die DmX-Homologen genomisch innerhalb eines Bereichs der Isochoren-Klasse L1 in einer Gen-armen Region. Die Anzahl der repetitiven Elemente innerhalb der Genregion von Dmxl1 liegt 6% unter dem erwarteten Wert eines L1 Isochors, die Anzahl beim Menschen liegt 4% über dem erwarteten Wert. Um die mögliche Promotorstruktur von Dmxl1 darzustellen, wurden umfangreiche in silico-Analysen der Region um den putativen Transkriptionsstart vorgenommen. Mit Hilfe der gewonnenen Daten konnte ein Transkriptionstartpunkt identifiziert werden. Zudem wurde eine Promotorstruktur erarbeitet, bei der angenommen werden kann, dass sie eine gute Näherung an die tatsächlich vorhandenen Bindungsstellen von Transkriptionsfaktoren darstellt. Die mit bioinformatischen Werkzeugen erzeugte virtuelle Promotor- und Enhancerstruktur zeigt das Potenzial, Dmxl1 basal und ubiquitär zu exprimieren. Gleichzeitig zeigen diese Daten, dass Dmxl1 vermutlich in einigen Geweben der Keimbahn, im Fettgewebe, dem blutbildenen System und während der Embryogenese hochkomplex reguliert werden kann. Eine regulierte Expression zur Steuerung des Energiestoffwechsels ist ebenfalls wahrscheinlich. Diese Ergebnisse passen sehr gut zu den experimentell ermittelten Daten und den beobachteten Phänotypen Dmxl1-chimärer Mäuse.rnDie abgeleitete Aminosäuresequenz umfasst in der Maus 3013 AS, im Menschen 3027 AS, der Vergleich der abgeleiteten Aminosäuresequenzen zeigt eine Identität von 89,3 % und eine Similarität von 94,7 % zwischen beiden Spezies. Im Dmxl1/DMXL1-Protein von Maus und Mensch konnten mindestens 24 und maximal 36 WD-Wiederholungseinheiten identifiziert werden, zudem wurden eine Reihe weiterer konservierter Proteinmotive gefunden. Die in silico-Strukturanalysen beider abgeleiteter Aminosäuresequenzen lässt vermuten, dass sich C- und N-terminal WD-Propellerstrukturen befinden. In dieser Arbeit gelang eine C-terminale Rekonstruktion einer 10-blättrigen Propellerstruktur, denkbar ist jedoch auch eine Struktur mit mindestens drei WD-Propellern, wenn eine prädominante Struktur mit Propellern aus jeweils sieben Propellerblättern angenommen wird.rnDas primäre Ziel dieser Arbeit, die Etablierung einer stabilen Mauslinie mit diruptiertem Dmxl1-Gen konnte aufgrund einer beobachteten Haploinsuffizienz nicht erreicht werden. Trotz zahlreicher Transformationen von Maus-Stammzelllinien konnte letztlich nur eine stabil transformierte Linie mit einem Dmxl1-Null-Allel identifiziert werden, was auch zu den theoretischen Daten und den angenommenen Aufgaben von Dmxl1 als komplex und diffizil reguliertes Multifunktions-Protein passt. Aus der transformierten Mauszelllinie konnten chimäre Mäuse entwickelt werden, die in Abhängigkeit von dem Ausmaß des Chimärismus phänotypisch massive Schädigungen aufwiesen. Neben einer Teilsterilität wurden massive Fettleibigkeit und ein ausgeprägter Hypogonadismus beobachtet. Keines der Tiere war in der Lage das Dmxl1-Null-Allel zu transduzieren. Die Tiere waren nur sehr eingeschränkt fertil, die wenigen Nachkommen entsprachen genotypisch und phänotypisch ausschließlich den verwendeten Blastocysten.rn

In vitro characterisation and expansion of human regulatory T cells for their in vivo application in the induction of tolerance in haematopoietic stem cell and solid organ transplantation

Solid organ transplantation (SOT) is considered the treatment of choice for many end-stage organ diseases. Thus far, short term results are excellent, with patient survival rates greater than 90% one year post-surgery, but there are several problems with the long term acceptance and use of immunosuppressive drugs. Hematopoietic Stem Cells Transplantation (HSCT) concerns the infusion of haematopoietic stem cells to re-establish acquired and congenital disorders of the hematopoietic system. The main side effect is the Graft versus Host Disease (GvHD) where donor T cells can cause pathology involving the damage of host tissues. Patients undergoing acute or chronic GvHD receive immunosuppressive regimen that is responsible for several side effects. The use of immunosuppressive drugs in the setting of SOT and GvHD has markedly reduced the incidence of acute rejection and the tissue damage in GvHD however, the numerous adverse side effects observed boost the development of alternative strategies to improve the long-term outcome. To this effect, the use of CD4+CD25+FOXP3+ regulatory T cells (Treg) as a cellular therapy is an attractive approach for autoimmunity disease, GvHD and limiting immune responses to allograft after transplantation. Treg have a pivotal role in maintaining peripheral immunological tolerance, by preventing autoimmunity and chronic inflammation. Results of my thesis provide the characterization and cell processing of Tregs from healthy controls and patients in waiting list for liver transplantation, followed by the development of an efficient expansion-protocol and the investigation of the impact of the main immunosuppressive drugs on viability, proliferative capacity and function of expanded cells after expansion. The conclusion is that ex vivo expansion is necessary to infuse a high Treg dose and although many other factors in vivo can contribute to the success of Treg therapy, the infusion of Tregs during the administration of the highest dose of immunosuppressants should be carefully considered.

Die Regulation des Enzyms Paraoxonase-2 sowie dessen Einfluss auf die Hämatopoese

In der vorliegenden Arbeit wurden durch den Einsatz von drei unabhängigen Methoden Proteine und Faktoren identifiziert, die die PON2-mRNA-Expression beeinflussen. Anhand der erhaltenen Faktoren wurden verstärkt solche ausgewählt, die eine Rolle in der Tumorbiologie spielen. Unter Verwendung verschiedener Zellmodelle wurde schließlich der Effekt dieser Faktoren auf die PON2-Expression analysiert. Die Ergebnisse dieser Arbeit zeigen, dass die PON2-Expression in K562-Zellen durch den PI3K / Akt-Signalweg, der in vielen Tumoren übermäßig aktiviert vorliegt, gesteigert wird. Auch eine Beteiligung des Wnt / β-Catenin-Signalweges kann nicht ausgeschlossen werden. Pharmakologische Inhibitoren von GSK-3β, einer Kinase die in beiden Signalwegen involviert ist, führt zu einer Steigerung der PON2-Expression durch den Transkriptionsfaktor LEF-1. Des Weiteren wurde gezeigt, dass die Familie der FoxO-Transkriptionsfaktoren an der Regulation der PON2-Expression in K562-Zellen beteiligt sind, wenn gleich es für die jeweiligen FoxO-Isoformen Unterschiede gibt.rnIm Hinblick auf die Assoziation von PON2 mit Leukämien wurde anhand eines PON2-/--Mausmodells, der Einfluss von PON2 auf die Hämatopoese untersucht. Dabei wurden signifikante Unterschiede in einigen Stammzellkompartimenten festgestellt. Ferner scheint PON2 die Entwicklung von Erythrozyten und Thrombozyten zu beeinflussen. Dies äußert sich in einer offensichtlichen Splenomegalie, zumindest bei alten weiblichen PON2-/--Mäusen.rnAbschließend wurde zur Generierung eines konditionalen PON2-Überexpressionsmausmodells erfolgreich ein Gene-Targeting-Vektor entwickelt. Durch eine gewebe-, zeit- und zellspezifische Steigerung der PON2-Expression ist es möglich, den Effekt einer PON2-Überexpression im Hinblick auf verschiedene Erkrankungen zu untersuchen.rnBisher war wenig über die Regulation des humanen PON2 bekannt. Die Ergebnisse der vorliegenden Arbeit zeigen erstmals, durch welche Signalwege und Transkriptionsfaktoren PON2 in Leukämiezellen reguliert wird. Im Hinblick auf die Rolle von PON2 in der Tumorbiologie ist es erstmals möglich, die PON2-Expression gezielt durch die Inhibition bzw. Aktivierung der PON2-regulierenden Faktoren zu beeinflussen, und damit neue Wege in der Krebstherapie zu beschreiten. rn

In vitro characterization of human AML-reactive CTL clones generated from the naive subset of healthy donors and adoptive transfer into leukemia-engrafted NSG mice

Acute myeloid leukemia (AML) is a very aggressive cancer of the hematopoietic system. Chemotherapy and immunotherapeutical approaches including hematopoietic stem cell transplantation (HSCT) and donor lymphocyte infusion (DLI) are the only curative options available. The beneficial graft-versus-leukemia (GVL) effect of cellular immunotherapy is mostly mediated by donor-derived CD8+ T lymphocytes that recognize minor histocompatibility antigens (mHags) and leukemia-associated antigens (LAAs) presented on the surface of AML blasts (Falkenburg et al. 2008; Kolb 2008). A main complication is graft-versus-host disease (GVHD) that can be induced when cytotoxic T lymphocytes (CTLs) recognize broadly expressed antigens. To reduce the risk of GVHD, specific allogeneic T-cell therapy inducing selective GVL responses could be an option (Barrett & Le Blanc 2010; Parmar et al. 2011; Smits et al. 2011). This requires efficient in vitro strategies to generate AML-reactive T cells with an early differentiation phenotype as well as vigorous effector functions and humanized mouse models to analyze the anti-leukemic potential of adoptively transferred T cells in vivo. In this study, AML-reactive CTL clones and oligoclonal T-cell lines could be reliably generated from the naive subset of healthy HLA-class I-identical donors by stimulation with primary AML blasts in mini-mixed-lymphocyte / leukemia cultures (MLLCs) in eight different patient / donor pairs. These CTLs were promising candidates for cellular immunotherapy because of their relatively early differentiation phenotype and strong proliferative and lytic capabilities. The addition of the common γ-chain cytokine IL-21 to the stimulation protocol enabled more precursors to develop into potent leukemia-reactive CTLs, presumably by its beneficial effects on cell survival and antigen-specific proliferation during the first weeks of cultures. It also strengthened the early-stage phenotype. Three long-term cultured CTLs exemplarily transferred into leukemia-engrafted immunodeficient NSG mice mediated a significant reduction of the leukemic burden after a single transfusion. These results demonstrate that CTL clones with reactivity to patient-derived AML blasts can be isolated from the naive compartment of healthy donors and show potent anti-leukemic effects in vivo. The herein described allo-MLLC approach with in vitro “programmed” naive CTL precursors independent of a HSCT setting is a valuable alternative to the conventional method of isolating in vivo primed donor CTLs out of patients after transplantation (Kloosterboer et al. 2004; Warren et al. 2010). This would make leukemia-reactive CTLs already available at the time point of HSCT, when residual leukemia disease is minimal and the chances for complete leukemia eradication are high. Furthermore, leukemia-reactive CTLs effectively expanded by this in vitro protocol can be used as screening populations to identify novel candidate LAAs and mHags for antigen-specific immunotherapy.

Agrin is required for survival and function of monocytic cells

Agrin, an extracellular matrix protein belonging to the heterogeneous family of heparan sulfate proteoglycans (HSPGs), is expressed by cells of the hematopoietic system but its role in leukocyte biology is not yet clear. Here we demonstrate that agrin has a crucial, nonredundant role in myeloid cell development and functions. We have identified lineage-specific alterations that affect maturation, survival and properties of agrin-deficient monocytic cells, and occur at stages later than stem cell precursors. Our data indicate that the cell-autonomous signals delivered by agrin are sensed by macrophages through the α-DC (DG) receptor and lead to the activation of signaling pathways resulting in rearrangements of the actin cytoskeleton during the phagocytic synapse formation and phosphorylation of extracellular signal-regulated kinases (Erk 1/2). Altogether, these data identify agrin as a novel player of innate immunity.