Karin Gustafsson

Recruitment and activation of natural killer cells in vitro by a human dendritic cell vaccine.

Recruitment of circulating natural killer (NK) cells into inflamed lymph nodes is known to provide a potent, IFN-gamma-dependent boost for Th1-polarized immune responses in mouse models. Such NK cell recruitment into draining lymph nodes is induced by certain s.c. injected adjuvants, including mature vaccine dendritic cells (DC), and is mediated by a CXCR3-dependent pathway. Here, we show that monocyte-derived immature human DCs stimulated with polyinosinic acid:polycytidylic acid, IFN-alpha, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and IFN-gamma, alpha-type 1-polarized DC (alpha DC1), secrete profuse amounts of the CXCR3 ligand CXCL9/MIG and substantial amounts of CXCL10/IP-10 and CXCL11/I-TAC after withdrawal of maturation stimuli. In sharp contrast, no measurable production of these chemokines was found in DCs after maturation with the current gold standard maturation cocktail for human DC-based cancer vaccines consisting of TNF-alpha, IL-1 beta, IL-6, and prostaglandin-E(2) (PGE(2)-DC). PGE(2)-DCs preferentially produced the Th2 and regulatory T-cell-attracting chemokines CCL17/TARC and CCL22/MDC, whereas only marginal levels of these chemokines were produced by alpha DC1s. Functional studies in vitro showed that supernatants from mature alpha DC1s actively recruited CD3(-)CD56(+) NK cells and that adding anti-CXCL9/MIG antibodies to the alpha DC1 supernatant substantially reduced this recruitment. Finally, alpha DC1s were able to induce IFN-gamma production when cocultured with resting autologous NK cells, but only if concurrent CD40 ligation was provided. These novel findings indicate that injected human alpha DC1-based vaccines have the potential to recruit and activate NK cells during their arrival to draining lymph nodes and that this feature may be of relevance for efficient priming of Th1 cells and CTLs.

Tumour-loaded α-type 1-polarized Dendritic Cells from Patients with Chronic Lymphocytic Leukaemia Produce a Superior NK-, NKT- and CD8+ T Cell-attracting Chemokine Profile

Tumour‐loaded dendritic cells (DCs) from patients with chronic lymphocytic leukaemia (CLL) matured using an α‐type 1‐polarized DC cocktail (IL‐1β/TNF‐α/IFN‐α/IFN‐γ/poly‐I:C;αDC1) were recently shown to induce more functional CD8+ T cells against autologous tumour cells in vitro than DCs matured with the ‘standard’ cocktail (IL‐1β/TNF‐α/IL‐6/PGE2;PGE2DCs). However, the ability of vaccine DCs to induce a type 1‐polarized immune response in vivo probably relies on additional features, including their ability to induce a CXCR3‐dependent recruitment of NK cells into vaccine‐draining lymph nodes. Moreover, their guiding of rare tumour‐specific CD8+ T cells to sites of DC–CD4+ T cell interactions by secretion of CCL3 and CCL4 is needed. We therefore analysed the chemokine profile and the lymphocyte‐attracting ability in vitro of monocyte‐derived PGE2DCs and αDC1s from patients with CLL. αDC1s produced much higher levels of CXCR3 ligands (CXCL9/CXCL10/CXCL11) than PGE2DCs. Functional studies further demonstrated that αDC1s were superior recruiters of both NK and NKT cells. Moreover, αDC1s produced higher levels of CCL3/CCL4 upon CD40 ligation. These findings suggest that functional αDC1s, derived from patients with CLL, produce a desirable NK‐, NKT‐ and CD8+ T cell‐attracting chemokine profile which may favour a guided and Th1‐deviated priming of CD8+ T cells, supporting the idea that αDC1‐based vaccines have a higher immunotherapeutic potential than PGE2DCs.

Is Indoleamine 2,3-Dioxygenase Important for Graft Acceptance in Highly Sensitized Patients After Combined Auxiliary Liver-Kidney Transplantation?

Background. In the clinical setting, transplanted liver seems to protect other grafts from the same donor from rejection. Our previous findings suggest that an auxiliary liver transplantation a few hours before a renal transplantation not only inhibits hyperacute antibody-mediated rejection but also improves long-term kidney graft survival in sensitized recipients. Here, we investigated indoleamine 2,3-dioxygenase (IDO) activity, as one potential mechanism for liver-induced long-term acceptance of kidney grafts. Methods. Tryptophan degradation was measured to estimate IDO activity in patient sera and cell culture supernatants with high performance liquid chromatography. Gene expression in the grafted organs and cell lysates was studied using real time polymerase chain reaction analysis. Results. Tryptophan degradation increased in peripheral blood from patients undergoing combined auxiliary liver-kidney transplantation, whereas it decreased in patients after regular renal transplantation. A 100-fold increase in IDO mRNA, preceded by upregulation of the IDO-inducing cytokines tumor necrosis factor-α, interleukin-1β, and interferon-&ggr;, was observed in the transplanted organs after graft reperfusion in patients undergoing combined graft transplantation. Subsequent studies in vitro revealed that immature dendritic cells, but not hepatocytes, strongly activated IDO on maturation with tumor necrosis factor-α, interleukin-1β, and interferon-&ggr;. Finally, serum from liver-transplanted patients elicited an even stronger IDO-activity in such cytokine-stimulated dendritic cells. Conclusions. Taken together these findings suggest that the liver-induced long-term acceptance seen in human combined auxiliary liver and kidney transplantation is at least partly mediated by IDO activity.

The bone-sparing effects of estrogen and WNT16 are independent of each other

Significance Previous studies demonstrate that endogenous wingless-type MMTV integration site family (WNT)16 is a crucial regulator of cortical bone mass. Surprisingly, we demonstrate that overexpression of WNT16 increases mainly trabecular bone mass. Both estrogen and WNT16 are crucial regulators of bone mass, but the possible interaction between WNT16-signaling and estrogen-signaling is unknown. To determine the possible interaction between WNT16 and estrogen signaling in bone, we developed and used two genetically modified mouse models with either constantly high osteoblastic Wnt16 expression or no Wnt16 expression. We demonstrated that the bone-sparing effects of estrogen and WNT16 are independent of each other. As WNT16 signaling in bone does not require normal estrogen action, we propose that WNT16-targeted therapies might be useful for treatment of postmenopausal trabecular bone loss. Wingless-type MMTV integration site family (WNT)16 is a key regulator of bone mass with high expression in cortical bone, and Wnt16−/− mice have reduced cortical bone mass. As Wnt16 expression is enhanced by estradiol treatment, we hypothesized that the bone-sparing effect of estrogen in females is WNT16-dependent. This hypothesis was tested in mechanistic studies using two genetically modified mouse models with either constantly high osteoblastic Wnt16 expression or no Wnt16 expression. We developed a mouse model with osteoblast-specific Wnt16 overexpression (Obl-Wnt16). These mice had several-fold elevated Wnt16 expression in both trabecular and cortical bone compared with wild type (WT) mice. Obl-Wnt16 mice displayed increased total body bone mineral density (BMD), surprisingly caused mainly by a substantial increase in trabecular bone mass, resulting in improved bone strength of vertebrae L3. Ovariectomy (ovx) reduced the total body BMD and the trabecular bone mass to the same degree in Obl-Wnt16 mice and WT mice, suggesting that the bone-sparing effect of estrogen is WNT16-independent. However, these bone parameters were similar in ovx Obl-Wnt16 mice and sham operated WT mice. The role of WNT16 for the bone-sparing effect of estrogen was also evaluated in Wnt16−/− mice. Treatment with estradiol increased the trabecular and cortical bone mass to a similar extent in both Wnt16−/− and WT mice. In conclusion, the bone-sparing effects of estrogen and WNT16 are independent of each other. Furthermore, loss of endogenous WNT16 results specifically in cortical bone loss, whereas overexpression of WNT16 surprisingly increases mainly trabecular bone mass. WNT16-targeted therapies might be useful for treatment of postmenopausal trabecular bone loss.

The role of membrane ERα signaling in bone and other major estrogen responsive tissues

Estrogen receptor α (ERα) signaling leads to cellular responses in several tissues and in addition to nuclear ERα-mediated effects, membrane ERα (mERα) signaling may be of importance. To elucidate the significance, in vivo, of mERα signaling in multiple estrogen-responsive tissues, we have used female mice lacking the ability to localize ERα to the membrane due to a point mutation in the palmitoylation site (C451A), so called Nuclear-Only-ER (NOER) mice. Interestingly, the role of mERα signaling for the estrogen response was highly tissue-dependent, with trabecular bone in the axial skeleton being strongly dependent (>80% reduction in estrogen response in NOER mice), cortical and trabecular bone in long bones, as well as uterus and thymus being partly dependent (40–70% reduction in estrogen response in NOER mice) and effects on liver weight and total body fat mass being essentially independent of mERα (<35% reduction in estrogen response in NOER mice). In conclusion, mERα signaling is important for the estrogenic response in female mice in a tissue-dependent manner. Increased knowledge regarding membrane initiated ERα actions may provide means to develop new selective estrogen receptor modulators with improved profiles.

Rapid increase of interleukin-10 plasma levels after combined auxiliary liver-kidney transplantation in presensitized patients.

Background After transplantation, donor dendritic cells (DCs) in the grafted organ are activated by an ischemia/reperfusion–induced inflammatory process that induces their migration to the recipient’s secondary lymphoid tissues. The subsequent interaction between migrated and mature donor DCs, recipient T cells, and natural killer (NK) cells is proposed to be crucial in directing host immune reactions toward allograft rejection. A liver transplant is less prone to induce rejection compared with most other solid organ transplants, and simultaneous transplantation of liver and kidney is known to improve the clinical outcome of kidney transplantation. Methods and Results Here we show that liver as well as combined auxiliary liver-kidney transplantation in patients induces a rapid increase in plasma interleukin-10 (IL-10) to levels that are significantly higher than those seen after standard kidney transplantation. Addition of IL-10 during in vitro maturation of human monocyte–derived DCs with ischemia/reperfusion-associated factors was found to affect phenotypic DC maturation significantly. Addition of IL-10 inhibited DC production of the NK cell- and T cell-recruiting chemokines CXCL9, CXCL10 and CXCL11. Conclusion Our findings indicate that liver transplantation induces a substantial systemic release of IL-10, which may inhibit T cell- and NK cell-mediated rejection processes toward the transplanted liver and concurrently transplanted kidney.

Using Text in Pre-school: A learning environment

The purpose of this study is to observe and describe the use of texts in the pre-school, to document the interaction between the child, the teacher and the environment and the emergent literacy in the daily practices. The presentation of the results is a description of two different textual environments: the narrative and the passive. The frame of reference in this study is the Vygotskij theory (1982) about thought and language in a cultural and historic perspective. The frame of reference is also the phenomenographic research in learning in general and learning the written language in a perspective of meta-cognition (Marton and Booth, 1997; Dahlgren and Olsson, 1985; Pramling, 1983; 1994). The immediate background to this study is an action research inspired developmental project to implement the curriculum (Ministry of Education, 1998) in the pre-school.

Extra-nuclear effects of estrogen on cortical bone in males require ERαAF-1

Estradiol (E2) signaling via estrogen receptor alpha (ERα) is important for the male skeleton as demonstrated by ERα inactivation in both mice and man. ERα mediates estrogenic effects not only by translocating to the nucleus and affecting gene transcription but also by extra-nuclear actions e.g., triggering cytoplasmic signaling cascades. ERα contains various domains, and the role of activation function 1 (ERαAF-1) is known to be tissue specific. The aim of this study was to determine the importance of extra-nuclear estrogen effects for the skeleton in males and to determine the role of ERαAF-1 for mediating these effects. Five-month-old male wild-type (WT) and ERαAF-1-inactivated (ERαAF-10) mice were orchidectomized and treated with equimolar doses of 17β-estradiol (E2) or an estrogen dendrimer conjugate (EDC), which is incapable of entering the nucleus and thereby only initiates extra-nuclear ER actions or their corresponding vehicles for 3.5 weeks. As expected, E2 treatment increased cortical thickness and trabecular bone volume per total volume (BV/TV) in WT males. EDC treatment increased cortical thickness in WT males, whereas no effect was detected in trabecular bone. In ERαAF-10 males, E2 treatment increased cortical thickness, but did not affect trabecular bone. Interestingly, the effect of EDC on cortical bone was abolished in ERαAF-10 mice. In conclusion, extra-nuclear estrogen signaling affects cortical bone mass in males, and this effect is dependent on a functional ERαAF-1. Increased knowledge regarding estrogen signaling mechanisms in the regulation of the male skeleton may aid the development of new treatment options for male osteoporosis.

Early Childhood Literacy and Children’s Multimodal Expressions in Preschool

In this chapter we will discuss how to provide a developmental practice (a didactics) in early childhood literacy. Our reasoning is based on video observationsSabs made in a study in which a preschool teacher reads a picture book for one child at a time (children aged 1–3 years) and then lets the child play with figures and objects that are related to the story. We will discuss some important aspects of the story reading and multimodal forms of expression in preschool and how to facilitate children’s literacy-learning. The aim of this chapter is to illustrate how teachers can observe and describe children’s literacy events and the multimodal forms of expression they use in preschool. The observations provide an opportunity to develop teaching and challenge children’s literacy-learning in a perspective of participation and democratic values.

WNT16 overexpression partly protects against glucocorticoid-induced bone loss

Therapeutic use of glucocorticoids (GCs) is a major cause of secondary osteoporosis, but the molecular mechanisms responsible for the deleterious effects of GCs in bone are only partially understood. WNT16 is a crucial physiological regulator of bone mass and fracture susceptibility, and we hypothesize that disturbed WNT16 activity might be involved in the deleterious effects of GC in bone. Twelve-week-old female Obl-Wnt16 mice (WNT16 expression driven by the rat procollagen type I α1 promoter) and wild-type (WT) littermates were treated with prednisolone (7.6 mg·kg-1·day-1) or vehicle for 4 wk. We first observed that GC treatment decreased the Wnt16 mRNA levels in bone of female mice (-56.4 ± 6.1% compared with vehicle, P < 0.001). We next evaluated if WNT16 overexpression protects against GC-induced bone loss. Dual-energy X-ray absorptiometry analyses revealed that GC treatment decreased total body bone mineral density in WT mice (-3.9 ± 1.2%, P = 0.028) but not in Obl-Wnt16 mice (+1.3 ± 1.4%, nonsignificant). Microcomputed tomography analyses showed that GC treatment decreased trabecular bone volume fraction (BV/TV) of the femur in WT mice ( P = 0.019) but not in Obl-Wnt16 mice. Serum levels of the bone formation marker procollagen type I N-terminal propeptide were substantially reduced by GC treatment in WT mice (-50.3 ± 7.0%, P = 0.008) but not in Obl-Wnt16 mice (-3.8 ± 21.2%, nonsignificant). However, the cortical bone thickness in femur was reduced by GC treatment in both WT mice and Obl-Wnt16 mice. In conclusion, GC treatment decreases Wnt16 mRNA levels in bone and WNT16 overexpression partly protects against GC-induced bone loss.