Development and evaluation of a patient education programme for children, adolescents, and young adults with differences of sex development (DSD) and their parents: study protocol of Empower-DSD

Background: Differences in sexual development (DSD) are rare diseases, which affect the chromosomal, anatomical or gonadal sex differentiation. Although patient education is recommended as essential in a holistic care approach, standardised programmes are still lacking. The present protocol describes the aims, study design and methods of the Empower-DSD project, which developed an age-adapted multidisciplinary education programme to improve the diagnosis-specific knowledge, skills and empowerment of patients and their parents. Methods: The new patient education programme was developed for children, adolescents and young adults with congenital adrenal hyperplasia, Turner syndrome, Klinefelter syndrome or XX-/or XY-DSD and their parents. The quantitative and qualitative evaluation methods include standardised questionnaires, semi-structured interviews, and participatory observation. The main outcomes (assessed three and six months after the end of the programme) are health-related quality of life, disease burden, coping, and diagnosis-specific knowledge. The qualitative evaluation examines individual expectations and perceptions of the programme. The results of the quantitative and qualitative evaluation will be triangulated. Discussion: The study Empower-DSD was designed to reduce knowledge gaps regarding the feasibility, acceptance and effects of standardised patient education programmes for children and youth with DSD and their parents. A modular structured patient education programme with four generic and three diagnosis-specific modules based on the ModuS concept previously established for other chronic diseases was developed. The topics, learning objectives and recommended teaching methods are summarised in the structured curricula, one for each diagnosis and age group. At five study centres, 56 trainers were qualified for the implementation of the training programmes. A total of 336 subjects have been already enrolled in the study. The recruitment will go on until August 2022, the last follow-up survey is scheduled for February 2023. The results will help improve multidisciplinary and integrated care for children and youth with DSD and their families. Trial registration: German Clinical Trials Register, DRKS00023096. Registered 8 October 2020 - Retrospectively registered

Chemokine Transfer by Liver Sinusoidal Endothelial Cells Contributes to the Recruitment of CD4+ T Cells into the Murine Liver

Leukocyte adhesion and transmigration are central features governing immune surveillance and inflammatory reactions in body tissues. Within the liver sinusoids, chemokines initiate the first crucial step of T-cell migration into the hepatic tissue. We studied molecular mechanisms involved in endothelial chemokine supply during hepatic immune surveillance and liver inflammation and their impact on the recruitment of CD4+ T cells into the liver. In the murine model of Concanavalin A-induced T cell-mediated hepatitis, we showed that hepatic expression of the inflammatory CXC chemokine ligands (CXCL)9 and CXCL10 strongly increased whereas homeostatic CXCL12 significantly decreased. Consistently, CD4+ T cells expressing the CXC chemokine receptor (CXCR)3 accumulated within the inflamed liver tissue. In histology, CXCL9 was associated with liver sinusoidal endothelial cells (LSEC) which represent the first contact site for T-cell immigration into the liver. LSEC actively transferred basolaterally internalized CXCL12, CXCL9 and CXCL10 via clathrin- coated vesicles to CD4+ T cells leading to enhanced transmigration of CXCR4+ total CD4+ T cells and CXCR3+ effector/memory CD4+ T cells, respectively in vitro. LSEC-expressed CXCR4 mediated CXCL12 transport and blockage of endothelial CXCR4 inhibited CXCL12-dependent CD4+ T-cell transmigration. In contrast, CXCR3 was not involved in the endothelial transport of its ligands CXCL9 and CXCL10. The clathrin-specific inhibitor chlorpromazine blocked endothelial chemokine internalization and CD4+ T-cell transmigration in vitro as well as migration of CD4+ T cells into the inflamed liver in vivo. Moreover, hepatic accumulation of CXCR3+ CD4+ T cells during T cell-mediated hepatitis was strongly reduced after administration of chlorpromazine. These data demonstrate that LSEC actively provide perivascularly expressed homeostatic and inflammatory chemokines by CXCR4- and clathrin-dependent intracellular transport mechanisms thereby contributing to the hepatic recruitment of CD4+ T-cell populations during immune surveillance and liver inflammation

Transmigration of CD4+ T-Cells through liver sinusoidal endothelium

Bei Immunreaktionen in der Leber ist die Entstehung und Verteilung von T-Zell- Infiltraten entscheidend. Ein Großteil der T-Zellen gelangt chemokinabhängig über das Endothel der Lebersinusoide in das Leberparenchym. Über die Mechanismen, die die Entstehung und Verfügbarkeit von Chemokingradienten über das Lebersinusendothel kontrollieren, ist wenig bekannt. In der vorliegenden Arbeit wurde untersucht, wie Lebersinusendothelzellen (LSEC) durch die Interaktion mit Chemokinen die T-Zell-Transmigration modulieren. Im Transwell- Assay in vitro wurde die Transmigration von CD4+ T-Zellen über LSEC-Monolayer nach basaler Inkubation des Endothels mit den Chemokinen CXCL9, CXCL10 und CXCL12 gemessen. Es zeigte sich, dass LSEC die Chemokine aufnehmen und anschließend für CD4+ T-Zellen gezielt bereitstellen können. Die chemotaktische Wirkung von CXCL12 und CXCL9 wurde durch diese Interaktion verstärkt. Hierbei erfolgte eine Internalisierung von CXCL12 mittels des CXCR4-Rezeptors, der Transport in Clathrin-Vesikeln und die anschließende Immobilisation auf der Endothelzelloberfläche mittels der Glykosaminoglykane Heparansulfat und Chrondroitinsulfat. Bei CXCL9 war der Clathrin-Vesikel abhängige Transport und die apikale Präsentation an Glykosaminoglykanen Teil der Chemokintranszytose. Die Ergebnisse dieser Arbeit tragen zum Verständnis der Endothel-Chemokin Interaktion in den Lebersinusoiden bei. Dies könnte zur Entwicklung von therapeutischen Strategien in entzündlichen Lebererkrankungen beitragen.The recruitment of T-lymphocytes is central in the immune surveillance and immune defense of the liver. The extravasation of T-cells mainly occurs in the liver sinusoids and is regulated by chemokines and their receptors on the lymphocyte and endothelial cell surface. Little is known about the mechanisms that influence the chemokine availability on the sinusoidal endothelium. This study investigated how the interaction of liver sinusoidal endothelial cells (LSEC) with chemokines differentially influences the transmigration of T cells. The CXCL9, CXCL10 and CXCL12 driven transmigration of CD4+ T cells across the LSEC monolayer was analyzed in transwell assays in vitro. The results showed that LSEC can take up chemokines and make them available for CD4+ T cells on the endothelial cell surface, thereby enhancing the chemotactic activity of CXCL9 and CXCL12. CXCL12 was internalized in clathrin vesicles, transported via the CXCR4 receptor and presented by the glycosaminoglycans heparane sulfate and chondroitine sulfate. The internalization of CXCL9 was mediated by clathrin vesicles and chemokine presentation occurred via glycosaminoglycans. The results of this study enhance the understanding of chemokine-endothelial interactions in the liver sinusoids which might contribute to the development of new therapeutic strategies in hepatic inflammation

Enhanced T cell transmigration across the murine liver sinusoidal endothelium is mediated by transcytosis and surface presentation of chemokines

Transmigration through the liver endothelium is a prerequisite for the homeostatic balance of intrahepatic T cells and a key regulator of inflammatory processes within the liver. Extravasation into the liver parenchyma is regulated by the distinct expression patterns of adhesion molecules and chemokines and their receptors on the lymphocyte and endothelial cell surface. In the present study, we investigated whether liver sinusoidal endothelial cells (LSEC) inhibit or support the chemokine-driven transmigration and differentially influence the transmigration of pro-inflammatory or anti-inflammatory CD4(+) T cells, indicating a mechanism of hepatic immunoregulation. Finally, the results shed light on the molecular mechanisms by which LSEC modulate chemokine-dependent transmigration. LSEC significantly enhanced the chemotactic effect of CXC-motif chemokine ligand 12 (CXCL12) and CXCL9, but not of CXCL16 or CCL20, on naive and memory CD4(+) T cells of a T helper 1, T helper 2, or interleukin-10-producing phenotype. In contrast, brain and lymphatic endothelioma cells and ex vivo isolated lung endothelia inhibited chemokine-driven transmigration. As for the molecular mechanisms, chemokine-induced activation of LSEC was excluded by blockage of G(i)-protein-coupled signaling and the use of knockout mice. After preincubation of CXCL12 to the basal side, LSEC took up CXCL12 and enhanced transmigration as efficiently as in the presence of the soluble chemokine. Blockage of transcytosis in LSEC significantly inhibited this effect, and this suggested that chemokines taken up from the basolateral side and presented on the luminal side of endothelial cells trigger T cell transmigration. CONCLUSION: Our findings demonstrate a unique capacity of LSEC to present chemokines to circulating lymphocytes and highlight the importance of endothelial cells for the in vivo effects of chemokines. Chemokine presentation by LSEC could provide a future therapeutic target for inhibiting lymphocyte immigration and suppressing hepatic inflammation

Co-localization of CXCL12 and CXCL10 with components of the endocytic pathway.

<p>LSEC were incubated with AlexaFluor 647-labeled CXCL12 or CXCL10 present in the lower chamber of the transwell. LSEC were stained with (A) anti-EEA1, (B) anti-TfR or (C) anti-LAMP-1 antibody. (D) LSEC were incubated with AlexaFluor 488-labeled AcLDL present in the lower chamber of the transwell and stained with anti-LAMP-1 antibody. (E) LSEC were incubated with AlexaFluor 647-labeled chemokine and AcLDL-AlexaFluor 488 present in the lower chamber of the transwell. Representative images of two independent experiments are shown. Bars represent 5 μm.</p

Receptor-mediated chemokine internalization by LSEC.

<p>(A) Expression of CXCR3 and CXCR4 mRNA in resting and <i>in vitro</i> activated LSEC was quantified in relation to GAPDH. (B) LSEC were treated with AMD3100 prior to incubation with fluorochrome-labeled CXCL12 for 60 min and analyzed by flow cytometry. (C) LSEC were treated with AMD3100 present in the lower chamber of the transwell prior to incubation with AlexaFluor 647-labeled CXCL12 added to the lower chamber for 60 min. Nuclei were stained with DAPI. Representative images of three independent experiments are shown. Bars represent 20 μm. (D) LSEC were treated with AMD3100 present in the lower chamber of the transwell prior pre-incubation with CXCL12 added to the lower chamber for 120 min. After removal of unbound chemokine, transmigration assays with total CD4⁺ T cells were performed. (E) LSEC from CXCR3^-/- mice were incubated with fluorochrome-labeled CXCL10 for 120 min. (F) CXCR3-deficient LSEC were pre-incubated with CXCL10 present in the lower chamber of the transwell for 120 min. Transmigration assays with effector/memory CD4⁺ T cells were performed. Mean values ± SD of 2–4 independent experiments are shown. * p< 0.05; ** p< 0.01; ns, not significant.</p

Hepatic chemokine expression during T cell-mediated hepatitis.

<p>Con A was intravenously injected into C57BL/6 mice. (A) Quantitative RT-PCR analysis of CXCL9, CXCL10 and CXCL12 mRNA expression in liver tissue of healthy or Con A-treated mice was performed. The chemokine expression was quantified in relation to GAPDH as a housekeeping gene. Mean values ± SD of 4–5 mice per group are shown. (B) Plasma ALT levels were determined at indicated time points. Medians of three independent experiments with 4–5 mice per group are shown. (C) Liver samples were stained with anti-CD146 and anti-CXCL9 antibody 12 h after hepatitis induction. Nuclei were stained with DAPI. Arrows indicate co-localization of CXCL9 and CD146. Bars represent 50 μm. Images are representative of three independent experiments. * p< 0.05; *** p< 0.001; ns, not significant.</p

Migration of CD4⁺ T cells into the inflamed and healthy liver after administration of CPZ.

<p>C57BL/6 mice were treated with Con A and received CPZ 7 h after hepatitis induction. Healthy mice also received CPZ. Radioactively labeled total CD4⁺ T cells were intravenously transferred into mice 120 min after administration of CPZ. Liver-specific radioactivity in relation to total radioactivity of the body was determined after 60 min migration time. Mean values ± SD of 4 independent experiments with three mice per group are shown. ** p< 0.01; *** p< 0.001; ns, not significant.</p

Chemokine expression and internalization by LSEC.

<p>(A) Chemokine mRNA expression was quantified in resting and TNF-α/IFN-γ activated LSEC in relation to GAPDH. (B) LSEC layer were incubated with fluorochrome-labeled CXCL10 or CXCL12 at 37°C or 4°C. Histograms show chemokine uptake determined by flow cytometry. Filled graph, no chemokine at 37°C; thin line overlapping with filled graph, chemokine incubation at 4°C; bold line, chemokine incubation at 37°C. (C) Diagram shows fold increase of geometric mean fluorescence intensity (GMFI) of LSEC incubated with chemokine in relation to GMFI without chemokine incubation at 37°C. Mean values ± SD of 4 independent experiments are shown. (D) LSEC were incubated with fluorochrome-labeled CXCL10 or CXCL12 added to the lower chamber of the transwell for 30 min. Nuclei were stained with DAPI. Representative images of three independent experiments are shown. Bars represent 10 μm. Mean values ± SD of 2–4 independent experiments are shown. ** p< 0.01.</p

Hepatic accumulation of CXCR3⁺ CD4⁺ T cells during T cell-mediated hepatitis after administration of CPZ.

<p>Mice were treated with Con A and received CPZ 60 min after hepatitis induction. (A) Liver samples were stained with anti-CD3 antibody 24 h after hepatitis induction. Portal CD3⁺ T-cell numbers were counted per hpf. Arrows indicate T cells. Representative images and medians of two independent experiments with 5 mice per group. Bars represent 50 μm. (B) NPC were isolated 24 h after hepatitis induction, stained with anti-CD4 and anti-CXCR3 antibody and analyzed by flow cytometry. Representative dot plots and medians of two independent experiments with 3–4 mice per group. * p< 0.05; ** p< 0.01; *** p< 0.001.</p