Differential Role of gp130-Dependent STAT and Ras Signalling for Haematopoiesis Following Bone-Marrow Transplantation

INTRODUCTION: Bone marrow transplantation (BMT) is a complex process regulated by different cytokines and growth factors. The pleiotropic cytokine IL-6 (Interleukin-6) and related cytokines of the same family acting on the common signal transducer gp130 are known to play a key role in bone marrow (BM) engraftment. In contrast, the exact signalling events that control IL-6/gp130-driven haematopoietic stem cell development during BMT remain unresolved. METHODS: Conditional gp130 knockout and knockin mice were used to delete gp130 expression (gp130(ΔMx)), or to selectively disrupt gp130-dependent Ras (gp130(ΔMxRas)) or STAT signalling (gp130(ΔMxSTAT)) in BM cells. BM derived from the respective strains was transplanted into irradiated wildtype hosts and repopulation of various haematopoietic lineages was monitored by flow cytometry. RESULTS: BM derived from gp130 deficient donor mice (gp130(ΔMx)) displayed a delayed engraftment, as evidenced by reduced total white blood cells (WBC), marked thrombocytopenia and anaemia in the early phase after BMT. Lineage analysis unravelled a restricted development of CD4(+) and CD8(+) T-cells, CD19(+) B-cells and CD11b(+) myeloid cells after transplantation of gp130-deficient BM grafts. To further delineate the two major gp130-induced signalling cascades, Ras-MAPK and STAT1/3-signalling respectively, we used gp130(ΔMxRas) and gp130(ΔMxSTAT) donor BM. BMT of gp130(ΔMxSTAT) cells significantly impaired engraftment of CD4(+), CD8(+), CD19(+) and CD11b(+) cells, whereas gp130(ΔMxRas) BM displayed a selective impairment in early thrombopoiesis. Importantly, gp130-STAT1/3 signalling deficiency in BM grafts severely impaired survival of transplanted mice, thus demonstrating a pivotal role for this pathway in BM graft survival and function. CONCLUSION: Our data unravel a vital function of IL-6/gp130-STAT1/3 signals for BM engraftment and haematopoiesis, as well as for host survival after transplantation. STAT1/3 and ras-dependent pathways thereby exert distinct functions on individual bone-marrow-lineages

Nanoscale Bouligand Multilayers: Giant Circular Dichroism of Helical Assemblies of Plasmonic 1D Nano-Objects

International audienceChirality is found at all length scales in nature, and chiral metasurfaces have recently attracted attention due to their exceptional optical properties and their potential applications. Most of these metasurfaces are fabricated by top-down methods or bottom-up approaches that cannot be tuned in terms of structure and composition. By combining grazing incidence spraying of plasmonic nanowires and nanorods and Layer-by-Layer assembly, we show that nonchiral 1D nano-objects can be assembled into scalable chiral Bouligand nanostructures whose mesoscale anisotropy is controlled with simple macroscopic tools. Such multilayer helical assemblies of linearly oriented nanowires and nanorods display very high circular dichroism up to 13 000 mdeg and giant dissymmetry factors up to g ≈ 0.30 over the entire visible and near-infrared range. The chiroptical properties of the chiral multilayer stack are successfully modeled using a transfer matrix formalism based on the experimentally determined properties of each individual layer. The proposed approach can be extended to much more elaborate architectures and gives access to template-free and enantiomerically pure nanocomposites whose structure can be finely tuned through simple design principles

Are Health Care Professionals’ Implicit and Explicit Attitudes Toward Conventional Disease-Modifying Antirheumatic Drugs Associated With Those of Their Patients?

Objective: It is generally unknown how the attitudes and beliefs of health care professionals (HCPs) might affect the attitudes, beliefs, and medication-taking behavior of patients with rheumatoid arthritis (RA). This study aims 1) to examine the attitudes, health-related associations (both implicit and explicit), and beliefs of HCPs about conventional disease-modifying antirheumatic drugs, and 2) to assess whether these attitudes, health-related associations, and beliefs of HCPs are associated with those of their patients, with their patients’ medication-taking behavior, and disease activity. Methods: HCPs were recruited from 2 centers that specialized in rheumatology across The Netherlands, and patient recruitment followed. In this observational study, implicit outcomes were measured with single-category implicit association tests, whereas explicit outcomes were measured with a bipolar evaluative adjective scale and the Beliefs About Medicines Questionnaire–Specific. Spearman’s rank correlations were used to describe correlations between implicit and explicit measures of the attitudes of HCPs. Multilevel, mixed-effects linear models were used to examine the association of HCP-related characteristics, including the implicit and explicit outcomes of HCPs, with those of their patients, their medication-taking behaviors, and disease activity. Results: Of the 1,659 initially invited patients, 254 patients with RA (mean age 62.8 years, mean disease duration 11.8 years, and 68.1% of the patients were female) who were treated by 26 different HCPs agreed to participate in this study. The characteristics, attitudes, health-related associations, and beliefs about medicines of HCPs were not significantly associated with those of their patients, nor with their medication-taking behaviors or disease activity scores. Conclusion: This study demonstrated that the attitudes, health-related associations (as measured both implicitly and explicitly), and beliefs of HCPs were not significantly associated with the attitudes, beliefs, medication-taking behavior, and disease activity of patients with RA

Subgroup analysis of different T-cell subsets after BMT.

<p><b>A)/B</b>) Engraftment of CD4(+)/GFP(+) and CD8(+)/GFP(+) T-cells [G/l]: CD4(+)/GFP(+) (upper plots) and CD8(+)/GFP(+) T-cells (lower plots) were analysed by flow cytometry analysis 2, 4 and 6 weeks after BM transplantation. A lower percentage of CD4(+)/GFP(+) and CD8(+)/GFP(+) T-cells could be detected in gp130^loxPloxP recipients that were transplanted with GFP(+)gp130^ΔMx donor BM. Displayed are example flow cytometry plots for a recipient mouse of wildtype donor BM (A) as well as for a recipient animal of gp130 deficient BM (B) 2 weeks after BMT. <b>C)/D</b>) Engraftment of CD19(+)/GFP(+) B-cells [G/l]: CD19(+)/GFP(+) B-cells derived from GFP(+)gp130^ΔMx donor BM (Fig. 2D) engrafted decelerated compared to GFP(+)gp130^loxPloxP donor BM (Fig. 2C) 2 and 4 weeks after BM transplantation. Shown are example flow cytometry plots 2 weeks after BMT. <b>E)/F</b>) Engraftment of CD11b(+)/GFP(+) cells [G/l]: 2 weeks after BM transplantation the percentage of CD11b(+)/GFP(+) cells was lower in gp130^loxP/loxP recipients transplanted with GFP(+)gp130^ΔMx donor BM compared to controls. Displayed are example flow cytometry plots for 2 weeks transplanted mice having received wildtype (E) or gp130 deficient (F) BM respectively.</p

Survival analysis after BM transplantation.

<p>A) Survival curve after BMT of 1×10⁶ donor cells (6 mice per group): Recipient mice of all different donor genotypes survived BMT of 1×10⁶ donor cells. B) Survival curve after BMT of 2×10⁵ donor cells (8 mice per group): gp130^ΔMxSTAT transplanted mice showed a 75% survival after BMT with 2×10⁵ donor cells. All the other animals survived BMT to 100%. C) Survival curve after BM transplantation using the amount of 5×10⁴ donor cells (6 mice per group): Transplantation of gp130^loxP/loxP donor BM into gp130^loxP/loxP recipient mice led to a survival rate of 100%. 75% of gp130^loxP/loxP recipients of gp130^ΔMxRas donor BM survived the experiment. However, if gp130^loxP/loxP recipients were transplanted with gp130^ΔMx BM, they survived in 33%. Finally, transplantation of gp130^ΔMxSTAT donor BM led to 100% mortality with no (0%) surviving recipient mice. [*p<0.05].</p

Subgroup analysis of different T-cell subsets after BMT.

<p><b>A</b>) Engraftment of CD4(+)/GFP(+) T-cells [G/l]: CD4(+)/GFP(+) T-cells were analysed by flow cytometry analysis 2, 4 and 6 weeks after BM transplantation. A significant lower number of CD4(+)/GFP(+) T-cells could be detected in gp130^loxPloxP recipients that were transplanted with GFP(+)gp130^ΔMxSTAT donor BM. Transplantation of GFP(+)gp130^ΔMxRas donor BM resulted in the same number of CD4(+)/GFP(+) T-cells as transplantation of gp130^loxP/loxP BM into gp130^loxP/loxP littermates. <b>B</b>) Engraftment of CD8(+)/GFP(+) T-cells [G/l]: The absolute number of CD8(+)/GFP(+) T-cells was determined by flow cytometry analysis 2, 4 and 6 weeks after BM transplantation. The number of CD8(+)/GFP(+) T-cells was significantly lower in wildtype mice that were transplanted with GFP(+)gp130^ΔMxSTAT 2 and 4 weeks after BMT. <b>C</b>) Engraftment of CD19(+)/GFP(+) B-cells [G/l]: CD19(+)/GFP(+) B-cells derived from GFP(+)gp130^ΔMxSTAT donor BM engrafted into recipient mice with significant differences at the 2 and 4 week time point. Gp130^loxP/loxP recipients of GFP(+)gp130^ΔMxRas donor BM also displayed a somewhat delayed engraftment 2 weeks after BMT but recovered faster as shown 4 and 6 weeks after BMT. <b>D</b>) Engraftment of CD11b(+)/GFP(+) cells [G/l]: Transplantation of GFP(+)gp130^ΔMxRas donor BM into gp130^loxP/loxP resulted in a significant increase of CD11b(+)/GFP(+) cells at all indicated time points (2, 4, 6 weeks) after BMT. GFP(+)gp130^ΔMxSTAT donor BM led to the same number of CD11b(+)/GFP(+) cells as the transplantation of GFP(+)gp130^ΔMx donor BM. [*p<0.05, **p<0.01].</p

Dissection of intracellular gp130 signalling pathways.

<p><b>A</b>) Number of total white blood cells (WBC) [G/l] after BMT: Displayed are the total white blood cell counts after BM transplantation at the indicated time points (2, 4 and 6 weeks). Less total white blood cells could be detected in gp130^loxP/loxP mice that were transplanted with GFP(+)gp130^ΔMxSTAT BM. Transplantation of GFP(+)gp130^ΔMxRas donor BM did not result in any significant difference concerning the total WBC count. <b>B</b>) Delayed engraftment of CD45(+)/GFP(+) white blood cells (WBC) [G/l] after BMT is STAT-dependent: Displayed are the CD45(+)/GFP(+) (donor derived) cells after BM transplantation at the indicated time points (2, 4 and 6 weeks). Whereas transplantation of GFP(+)gp130^ΔMxRas donor BM into gp130^loxP/loxP animals did not lead to a delayed engraftment of CD45(+)/GFP(+) cells, transplantation of gp130^ΔMxSTAT into gp130^loxP/loxP resulted in a significant decrease of CD45(+)/GFP(+) cells. <b>C</b>) Defective Ras and STAT signalling in donor mice leads to thrombocytopenia: Depicted are the platelet counts 2, 4 and 6 weeks after BMT. Transplantation of GFP(+)gp130^ΔMxRas as well as GFP(+)gp130^ΔMxSTAT donor BM led to a significant thrombocytopenia 2 weeks after BM transplantation. <b>D</b>) STAT-deficiency in donor mice leads to anaemia after BM transplantation: Depicted are the haemoglobin values 2, 4 and 6 weeks after BM transplantation with a significant anaemia in gp130^loxP/loxP recipients transplanted with GFP(+)gp130^ΔMxSTAT donor BM 4 weeks after BMT. [**p<0.01, ***p<0.001].</p