Humanized mice efficiently engrafted with fetal hepatoblasts and syngeneic immune cells develop human monocytes and NK cells

Human liver chimeric mice are useful models of human hepatitis virus infection, including hepatitis B and C virus infections. Independently, immunodeficient mice reconstituted with CD34(+) hematopoietic stem cells (HSC) derived from fetal liver reliably develop human T and B lymphocytes. Combining these systems has long been hampered by inefficient liver reconstitution of human fetal hepatoblasts. Our study aimed to enhance hepatoblast engraftment in order to create a mouse model with syngeneic human liver and immune cells.The effects of human oncostatin-M administration on fetal hepatoblast engraftment into immunodeficient fah(-/-) mice was tested. Mice were then transplanted with syngeneic human hepatoblasts and HSC after which human leukocyte chimerism and functionality were analyzed by flow cytometry, and mice were challenged with HBV.Addition of human oncostatin-M enhanced human hepatoblast engraftment in immunodeficient fah(-/-) mice by 5-100 fold. In contrast to mice singly engrafted with HSC, which predominantly developed human T and B lymphocytes, mice co-transplanted with syngeneic hepatoblasts also contained physiological levels of human monocytes and natural killer cells. Upon infection with HBV, these mice displayed rapid and sustained viremia.Our study provides a new mouse model with improved human fetal hepatoblast engraftment and an expanded human immune cell repertoire. With further improvements, this model may become useful for studying human immunity against viral hepatitis.Important human pathogens such as hepatitis B virus, hepatitis C virus and human immunodeficiency virus only infect human cells which complicates the development of mouse models for the study of these pathogens. One way to make mice permissive for human pathogens is the transplantation of human cells into immune-compromised mice. For instance, the transplantation of human liver cells will allow the infection of these so-called liver chimeric mice with hepatitis B virus and hepatitis C virus. The co-transplantation of human immune cells into liver chimeric mice will further allow the study of human immune responses to hepatitis B virus or hepatitis C virus. However, for immunological studies it will be crucial that the transplanted human liver and immune cells are derived from the same human donor. In our study we describe the efficient engraftment of human fetal liver cells and immune cells derived from the same donor into mice. We show that liver co-engraftment resulted in an expanded human immune cell repertoire, including monocytes and natural killer cells in the liver. We further demonstrate that these mice could be infected with hepatitis B virus, which lead to an expansion of natural killer cells. In conclusion we have developed a new mouse model that could be useful to study human immune responses to human liver pathogens

Análise de mutação germinativa do Tpit em cães da raça Poodle com hiperadrenocorticismo ACTH-dependente

There is a high incidence of pituitary-dependent hyperadrenocorticism (PDH) in Poodle dogs, with family members being affected by the disease, suggesting a genetic involvement. Tpit is an obligate transcription factor for the expression of pro-opiomelanocortingene and for corticotroph terminal differentiation. The aim of the present study was to screen the Tpit gene for germline mutations in Poodles with PDH. Fifty Poodle dogs (33 female, 8.71 +/- 2.8 years) with PDH and 50 healthy Poodle dogs (32 females, 9.4241 2.8 years) were studied. Genomic DNA was isolated from peripheral blood, amplified by PCR and submitted to automatic sequence. No mutation in the coding region of Tpit was found, whereas the new single nucleotide polymorphism p.S343G, in heterozygous state, was found in the same frequency in both PDH and control groups. We concluded that Tpit gain-of-function mutations are not involved in the etiology of PDH in Poodle dogs.O hiperadrenocorticismo ACTH-dependente (HACAD) apresenta elevada incidência em cães da raça Poodle, sendo que membros da mesma família têm sido acometidos pela doença, sugerindo envolvimento genético. Tpit é um fator de transcrição obrigatório para a expressão do gene da pro-opiomelanocortina e para a diferenciação terminal dos corticotrofos. O objetivo deste trabalho foi pesquisar mutações germinativas no gene Tpit em Poodles com HACAD. Cinquenta cães da raça Poodle, 33 fêmeas, média de idade de 8,71±2,8 anos, com HACAD, e 50 cães Poodles saudáveis, 32 fêmeas, média de idade de 9,4±2,8 anos, foram estudados. Mutações na região codificadora do gene Tpit não foram identificadas. Foi observado um novo polimorfismo em heterozigose, p.S343G, com a mesma frequência no grupo de cães com HACAD e no grupo-controle. Conclui-se que a mutação ativadora no gene Tpit não está envolvida na patogênese do hiperadrenocorticismo ACTH-dependente em cães da raça Poodle

"Here is your mission, now own it!" The rhetoric and practice of local ownership in EU interventions

One of the core principles of EU interventions under the Common Security and Defence Policy (CSDP) has been local ownership. While the EU takes pride in fully respecting this principle, the existing research suggests that the implementation has been far from smooth. However, we still know very little how this principle is conceptualised and operationalised, let alone why its implementation has been so difficult. Drawing on document analysis and 27 in-depth interviews, the article makes 3 arguments. First, ownership is increasingly construed in the EU policy rhetoric as a middle ground between imposition and restraint. Second, in practice, ownership is operationalised as an externally driven, top-down endeavour, resulting in the low degree of local participation. Third, in addition to the obstacles normally faced by other peace-builders, the EU's efforts to implement ownership are constrained by the politics and policy-making of CSDP. The arguments are illustrated in a case study of the European Union Mission on Regional Maritime Capacity Building in the Horn of Africa (EUCAP Nestor)

A SynBio community comes of age: political, academical, industrial, and societal developments in the Netherlands

Synthetic biology (SynBio) is a rapidly growing scientific discipline. In the Netherlands, various universities and companies are tackling a variety of opportunities and challenges within this field. In this perspective article, we review the current synthetic biology landscape in the Netherlands across academia, industry, politics, and society. Especially within Dutch academia there is an active, though only partially connected, research community involved in various domains of SynBio. Mostly supported by governmental funding, academic research is focusing on top-down synthetic biology, involving the engineering of for example bacteria and yeast for bioproduction, as well as bottom-up and cell-free synthetic biology aiming to understand life and build synthetic cells. There is also a large number of talented and motivated students interested in the field, exemplified by the participation and success of Dutch teams in the international iGEM synthetic biology competition. Commercial synthetic biology activities are taking place in various large industrial companies, as well as in start-ups and spin-offs, mostly divided over several ‘SynBio hubs’ in the Netherlands. However, the investment, regulatory and public-perception landscape is not yet optimal to stimulate entrepreneurial activities in SynBio. The Dutch and global society can further benefit from the large promise of SynBio through better integration of people active in the Dutch SynBio field, frequent political and public dialogue, and more attention towards regulatory issues. The recently founded Dutch synthetic biology association SynBioNL aims to contribute to realizing a positive impact on society by stimulating advances of the field in the Netherlands and beyond.Microbial Biotechnolog

Analysis of FOXP3+ Regulatory T Cells That Display Apparent Viral Antigen Specificity during Chronic Hepatitis C Virus Infection

We reported previously that a proportion of natural CD25+ cells isolated from the PBMC of HCV patients can further upregulate CD25 expression in response to HCV peptide stimulation in vitro, and proposed that virus-specific regulatory T cells (Treg) were primed and expanded during the disease. Here we describe epigenetic analysis of the FOXP3 locus in HCV-responsive natural CD25+ cells and show that these cells are not activated conventional T cells expressing FOXP3, but hard-wired Treg with a stable FOXP3 phenotype and function. Of ∼46,000 genes analyzed in genome wide transcription profiling, about 1% were differentially expressed between HCV-responsive Treg, HCV-non-responsive natural CD25+ cells and conventional T cells. Expression profiles, including cell death, activation, proliferation and transcriptional regulation, suggest a survival advantage of HCV-responsive Treg over the other cell populations. Since no Treg-specific activation marker is known, we tested 97 NS3-derived peptides for their ability to elicit CD25 response (assuming it is a surrogate marker), accompanied by high resolution HLA typing of the patients. Some reactive peptides overlapped with previously described effector T cell epitopes. Our data offers new insights into HCV immune evasion and tolerance, and highlights the non-self specific nature of Treg during infection

Expression of CD11c Is Associated with Unconventional Activated T Cell Subsets with High Migratory Potential

Ajudes rebudes: Marie Curie Career Integration Grant; Dexeus Foundation for Women's Health Research; i Contratos Ramón y CajalCD11c is an α integrin classically employed to define myeloid dendritic cells. Although there is little information about CD11c expression on human T cells, mouse models have shown an association of CD11c expression with functionally relevant T cell subsets. In the context of genital tract infection, we have previously observed increased expression of CD11c in circulating T cells from mice and women. Microarray analyses of activated effector T cells expressing CD11c derived from naïve mice demonstrated enrichment for natural killer (NK) associated genes. Here we find that murine CD11c+ T cells analyzed by flow cytometry display markers associated with non-conventional T cell subsets, including γδ T cells and invariant natural killer T (iNKT) cells. However, in women, only γδ T cells and CD8+ T cells were enriched within the CD11c fraction of blood and cervical tissue. These CD11c+ cells were highly activated and had greater interferon (IFN)-γ secretory capacity than CD11c- T cells. Furthermore, circulating CD11c+ T cells were associated with the expression of multiple adhesion molecules in women, suggesting that these cells have high tissue homing potential. These data suggest that CD11c expression distinguishes a population of circulating T cells during bacterial infection with innate capacity and mucosal homing potential

Chemokine gene expression in lung CD8 T cells correlates with protective immunity in mice immunized intra-nasally with Adenovirus-85A

<p>Abstract</p> <p>Background</p> <p>Immunization of BALB/c mice with a recombinant adenovirus expressing <it>Mycobacterium tuberculosis </it>(<it>M. tuberculosis</it>) antigen 85A (Ad85A) protects against aerosol challenge with <it>M. tuberculosis </it>only when it is administered intra-nasally (i.n.). Immunization with Ad85A induces a lung-resident population of activated CD8 T cells that is antigen dependent, highly activated and mediates protection by early inhibition of <it>M. tuberculosis </it>growth. In order to determine why the i.n. route is so effective compared to parenteral immunization, we used microarray analysis to compare gene expression profiles of pulmonary and splenic CD8 T cells after i.n. or intra-dermal (i.d.) immunization.</p> <p>Method</p> <p>Total RNA from CD8 T cells was isolated from lungs or spleens of mice immunized with Ad85A by the i.n. or i.d. route. The gene profiles generated from each condition were compared. Statistically significant (p ≤ 0.05) differentially expressed genes were analyzed to determine if they mapped to particular molecular functions, biological processes or pathways using Gene Ontology and Panther DB mapping tools.</p> <p>Results</p> <p>CD8 T cells from lungs of i.n. immunized mice expressed a large number of chemokines chemotactic for resting and activated T cells as well as activation and survival genes. Lung lymphocytes from i.n. immunized mice also express the chemokine receptor gene <it>Cxcr6</it>, which is thought to aid long-term retention of antigen-responding T cells in the lungs. Expression of CXCR6 on CD8 T cells was confirmed by flow cytometry.</p> <p>Conclusions</p> <p>Our microarray analysis represents the first <it>ex vivo </it>study comparing gene expression profiles of CD8 T cells isolated from distinct sites after immunization with an adenoviral vector by different routes. It confirms earlier phenotypic data indicating that lung i.n. cells are more activated than lung i.d. CD8 T cells. The sustained expression of chemokines and activation genes enables CD8 T cells to remain in the lungs for extended periods after i.n. immunization. This may account for the early inhibition of <it>M. tuberculosis </it>growth observed in Ad85A i.n. immunized mice and explain the effectiveness of i.n. compared to parenteral immunization with this viral vector.</p

A microRNA profile of human CD8(+) regulatory T cells and characterization of the effects of microRNAs on Treg cell-associated genes.

Recently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8(+)CD25(+) Treg cells and the impact of microRNAs on molecules associated with immune regulation. We purified human natural CD8(+) Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA 'signature' for CD8(+)CD25(+)FOXP3(+)CTLA-4(+) natural Treg cells. We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3'-UTR of important Treg cell-associated genes. The human CD8(+)CD25(+) natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo. We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer

Altered Development of NKT Cells, γδ T Cells, CD8 T Cells and NK Cells in a PLZF Deficient Patient

In mice, the transcription factor, PLZF, controls the development of effector functions in invariant NKT cells and a subset of NKT cell-like, γδ T cells. Here, we show that in human lymphocytes, in addition to invariant NKT cells, PLZF was also expressed in a large percentage of CD8+ and CD4+ T cells. Furthermore, PLZF was also found to be expressed in all γδ T cells and in all NK cells. Importantly, we show that in a donor lacking functional PLZF, all of these various lymphocyte populations were altered. Therefore, in contrast to mice, PLZF appears to control the development and/or function of a wide variety of human lymphocytes that represent more than 10% of the total PBMCs. Interestingly, the PLZF-expressing CD8+ T cell population was found to be expanded in the peripheral blood of patients with metastatic melanoma but was greatly diminished in patients with autoimmune disease