Distinct molecular profiles of skull bone marrow in health and neurological disorders.

The bone marrow in the skull is important for shaping immune responses in the brain and meninges, but its molecular makeup among bones and relevance in human diseases remain unclear. Here, we show that the mouse skull has the most distinct transcriptomic profile compared with other bones in states of health and injury, characterized by a late-stage neutrophil phenotype. In humans, proteome analysis reveals that the skull marrow is the most distinct, with differentially expressed neutrophil-related pathways and a unique synaptic protein signature. 3D imaging demonstrates the structural and cellular details of human skull-meninges connections (SMCs) compared with veins. Last, using translocator protein positron emission tomography (TSPO-PET) imaging, we show that the skull bone marrow reflects inflammatory brain responses with a disease-specific spatial distribution in patients with various neurological disorders. The unique molecular profile and anatomical and functional connections of the skull show its potential as a site for diagnosing, monitoring, and treating brain diseases.

IRF8 Transcription-Factor-Dependent Classical Dendritic Cells Are Essential for Intestinal T Cell Homeostasis.

The role of dendritic cells (DCs) in intestinal immune homeostasis remains incompletely defined. Here we show that mice lacking IRF8 transcription-factor-dependent DCs had reduced numbers of T cells in the small intestine (SI), but not large intestine (LI), including an almost complete absence of SI CD8αß(+) and CD4(+)CD8αα(+) T cells; the latter requiring ß8 integrin expression by migratory IRF8 dependent CD103(+)CD11b(-) DCs. SI homing receptor induction was impaired during T cell priming in mesenteric lymph nodes (MLN), which correlated with a reduction in aldehyde dehydrogenase activity by SI-derived MLN DCs, and inefficient T cell localization to the SI. These mice also lacked intestinal T helper 1 (Th1) cells, and failed to support Th1 cell differentiation in MLN and mount Th1 cell responses to Trichuris muris infection. Collectively these results highlight multiple non-redundant roles for IRF8 dependent DCs in the maintenance of intestinal T cell homeostasis.

Targeted therapies in retinoblastoma: GD2-directed immunotherapy following autologous stem cell transplantation and evaluation of alternative target B7-H3.

BACKGROUND: GD2-directed immunotherapy is highly effective in the treatment of high-risk neuroblastoma (NB), and might be an interesting target also in other high-risk tumors. METHODS: The German-Austrian Retinoblastoma Registry, Essen, was searched for patients, who were treated with anti-GD2 monoclonal antibody (mAb) dinutuximab beta (Db) in order to evaluate toxicity, response and outcome in these patients. Additionally, we evaluated anti-GD2 antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) in retinoblastoma cell lines in vitro. Furthermore, in vitro cytotoxicity assays directed against B7-H3 (CD276), a new identified potential target in RB, were performed. RESULTS: We identified four patients with relapsed stage IV retinoblastoma, who were treated with Db following autologous stem cell transplantation (ASCT). Two out of two evaluable patients with detectable tumors responded to immunotherapy. One of these and another patient who received immunotherapy without residual disease relapsed 10 and 12 months after start of Db. The other patients remained in remission until last follow-up 26 and 45 months, respectively. In vitro, significant lysis of RB cell lines by ADCC and CDC with samples from patients and healthy donors and anti-GD2 and anti-CD276-mAbs were demonstrated. CONCLUSION: Anti-GD2-directed immunotherapy represents an additional therapeutic option in high-risk metastasized RB. Moreover, CD276 is another target of interest.

How Effective is a Web-Based Mental Health Intervention (Deprexis) in the Treatment of Moderate and Major Depressive Disorders when started during Routine Psychiatric Inpatient Treatment as an Adjunct Therapy? A Pragmatic Parallel-Group Randomized Controlled Trial.

BACKGROUND: Internet-based self-help-programs like deprexis have been increasingly shown to reduce depressive symptoms if added to distinct, primarily outpatient-treatment-settings. There is limited information about the effectiveness of deprexis if started at routine psychiatric hospital inpatient treatment of moderate-to-severe major depressive disorder (MDD). SUBJECTS AND METHODS: To examine, sixty-nine adult MDD-inpatients were randomly assigned to a 12-week-period of treatment-as-usual (TAU, N=33) or TAU plus guided deprexis (TAU-PLUS, N=36). The study was planned as a pragmatic approach considering psychiatric routine conditions, particularly, offering an instant and flexible discharge management when the patients felt stabilized enough for primary/secondary care. Therefore, there was no fixed time frame for the inpatient treatment duration. Post-discharge, patients were followed by structured telephone interviews up to study-endpoint, i. e., 12 weeks after deprexis-initiation. Primary (Beck-Depression-Inventory-II, BDI-II) and secondary outcome-measures (Hamilton-Depression-Scale, Clinical-Global-Impression-Severity, WHO-Well-Being-Index, Helping-Alliance-Questionnaire) were carried out at study entry and every 2 weeks. Furthermore, the working alliance with deprexis as well as the inpatient treatment duration, the daily activity and the utilization of post-hospital care after discharge were determined. RESULTS: At week 12, modified ITT-analyses showed significant between-group differences of BDI-II scores in favor of the TAU-PLUS-patients (p=.03) corresponding to a medium effect size (d=-.73, 95% CI -1.4 to .06). TAU-PLUS-patients showed greater daily activity (p=.04, d=.70, 95% CI -.03 to 1.38) and had been discharged significantly earlier from inpatient treatment (p=.003). Post-discharge, the TAU-PLUS-group reported a lower rate of post-hospital care (p=.01) and re-admissions (p=.04). Secondary outcome-measures including the alliance with the therapists were not significantly different between the groups at study-endpoint. The patients´ working-alliance with deprexis significantly predicted MDD-improvement and wellbeing. Both groups (TAU and TAU plus deprexis) were comparable with regard to the prescribed antidepressant medication. Unfortunately, detailed data on the amount and actual duration of the psychotherapeutic and special therapeutic individual and group settings of the TAU were not collected CONCLUSION: TAU plus deprexis was superior to TAU in improving subjective depression-severity (BDI-II) and daily activity in patients having sought psychiatric inpatient MDD-treatment before. This beneficial effect appeared 12 weeks after inpatient deprexis-initiation, i. e. when the vast majority of patients were back in primary/secondary care. Adjunctive deprexis was associated with earlier discharges and a significant advantage for post-hospital stabilization. In this regard, it could be promising to include deprexis into inpatient treatment conditions, thereby also preparing its continuing outpatient use. We found no evidence that deprexis interfered negatively with the alliance between the patients and their therapists.

Orofacial conditions and oral health behavior of young athletes: A comparison of amateur and competitive sports.

PURPOSE: This retrospective cross-sectional study aimed to evaluate oral health status (dental, periodontal, and functional) and oral health behavior in young German athletes including the comparison of competitive (CA) and amateur sports (AA). METHODS: Data of CA (German national teams, perspective, and youth squads) and AA aged between 18 and 30 years with an available oral examination in 2019 were included. CLINICAL EXAMINATION: caries experience (DMF-T), non-carious wear (erosion, BEWE), partially erupted wisdom teeth, gingival inflammation (PBI), plaque index, periodontal screening (PSI), and temporomandibular dysfunction (TMD) screening. Questionnaires: oral health behavior and periodontal symptoms. RESULTS: 88 CA (w = 51%, 20.6 ± 3.5 years) of endurance sports and 57 AA (w = 51%, 22.2 ± 2.1 years) were included. DMF-T was comparable (CA: 2.7 ± 2.2, AA: 2.3 ± 2.2; p = 0.275) with more D-T in CA (0.6 ± 1.0) than AA (0.3 ± 0.7; p = 0.046; caries prevalence: CA: 34%, AA: 19%; p = 0.06). Both groups had low severity of erosion (BEWE about 3.5). CA had more positive TMD screenings (43% vs. 25%; p = 0.014). In both groups, all athletes showed signs of gingival inflammation, but on average of low severity (PBI <1). More CA needed complex periodontal treatment than AA (maximum PSI = 3 in 40% vs. 12%; p < 0.001). Oral health behavior was comparable (daily tooth brushing; regular dental check-ups in >70%). CONCLUSIONS: Young German athletes (CA and AA) generally showed signs of gingival inflammation and needed to improve their oral health behavior. CA showed slightly increased oral findings (more D-T, periodontal and TMD screening findings) than AA, but similar oral health behavior. This may imply an increased dental care need in competitive sports.

Cobra Venom Factor Boosts Arteriogenesis in Mice.

Arteriogenesis, the growth of natural bypass blood vessels, can compensate for the loss of arteries caused by vascular occlusive diseases. Accordingly, it is a major goal to identify the drugs promoting this innate immune system-driven process in patients aiming to save their tissues and life. Here, we studied the impact of the Cobra venom factor (CVF), which is a C3-like complement-activating protein that induces depletion of the complement in the circulation in a murine hind limb model of arteriogenesis. Arteriogenesis was induced in C57BL/6J mice by femoral artery ligation (FAL). The administration of a single dose of CVF (12.5 µg) 24 h prior to FAL significantly enhanced the perfusion recovery 7 days after FAL, as shown by Laser Doppler imaging. Immunofluorescence analyses demonstrated an elevated number of proliferating (BrdU+) vascular cells, along with an increased luminal diameter of the grown collateral vessels. Flow cytometric analyses of the blood samples isolated 3 h after FAL revealed an elevated number of neutrophils and platelet-neutrophil aggregates. Giemsa stains displayed augmented mast cell recruitment and activation in the perivascular space of the growing collaterals 8 h after FAL. Seven days after FAL, we found more CD68+/MRC-1+ M2-like polarized pro-arteriogenic macrophages around growing collaterals. These data indicate that a single dose of CVF boosts arteriogenesis by catalyzing the innate immune reactions, relevant for collateral vessel growth.

Plasma Cells Are the Most Abundant Gluten Peptide MHC-expressing Cells in Inflamed Intestinal Tissues From Patients With Celiac Disease.

BACKGROUND & AIMS: Development of celiac disease is believed to involve the transglutaminase-dependent response of CD4+ T cells toward deamidated gluten peptides in the intestinal mucosa of individuals with specific HLA-DQ haplotypes. We investigated the antigen presentation process during this mucosal immune response. METHODS: We generated monoclonal antibodies (mAbs) specific for the peptide-MHC (pMHC) complex of HLA-DQ2.5 and the immunodominant gluten epitope DQ2.5-glia-α1a using phage display. We used these mAbs to assess gluten peptide presentation and phenotypes of presenting cells by flow cytometry and enzyme-linked immune absorbent spot (ELISPOT) in freshly prepared single-cell suspensions from intestinal biopsies from 40 patients with celiac disease (35 untreated and 5 on a gluten-free diet) as well as 18 subjects with confirmed noninflamed gut mucosa (controls, 12 presumed healthy, 5 undergoing pancreatoduodenectomy, and 1 with potential celiac disease). RESULTS: Using the mAbs, we detected MHC complexes on cells from intestinal biopsies from patients with celiac disease who consume gluten, but not from patients on gluten-free diets. We found B cells and plasma cells to be the most abundant cells that present DQ2.5-glia-α1a in the inflamed mucosa. We identified a subset of plasma cells that expresses B-cell receptors (BCR) specific for gluten peptides or the autoantigen transglutaminase 2 (TG2). Expression of MHC class II (MHCII) was not restricted to these specific plasma cells in patients with celiac disease but was observed in an average 30% of gut plasma cells from patients and controls. CONCLUSIONS: A population of plasma cells from intestinal biopsies of patients with celiac disease express MHCII; this is the most abundant cell type presenting the immunodominant gluten peptide DQ2.5-glia-α1a in the tissues from these patients. These results indicate that plasma cells in the gut can function as antigen-presenting cells and might promote and maintain intestinal inflammation in patients with celiac disease or other inflammatory disorders.

A Simple and Effective Flow Cytometry-Based Method for Identification and Quantification of Tissue Infiltrated Leukocyte Subpopulations in a Mouse Model of Peripheral Arterial Disease.

Arteriogenesis, the growth of a natural bypass from pre-existing arteriolar collaterals, is an endogenous mechanism to compensate for the loss of an artery. Mechanistically, this process relies on a locally and temporally restricted perivascular infiltration of leukocyte subpopulations, which mediate arteriogenesis by supplying growth factors and cytokines. Currently, the state-of-the-art method to identify and quantify these leukocyte subpopulations in mouse models is immunohistology. However, this is a time consuming procedure. Here, we aimed to develop an optimized protocol to identify and quantify leukocyte subpopulations by means of flow cytometry in adductor muscles containing growing collateral arteries. For that purpose, adductor muscles of murine hindlimbs were isolated at day one and three after induction of arteriogenesis, enzymatically digested, and infiltrated leukocyte subpopulations were identified and quantified by flow cytometry, as exemplary shown for neutrophils and macrophages (defined as CD45+/CD11b+/Ly6G+ and CD45+/CD11b+/F4/80+ cells, respectively). In summary, we show that flow cytometry is a suitable method to identify and quantify leukocyte subpopulations in muscle tissue, and provide a detailed protocol. Flow cytometry constitutes a timesaving tool compared to histology, which might be used in addition for precise localization of leukocytes in tissue samples.

A mouse model-based screening platform for the identification of immune activating compounds such as natural products for novel cancer immunotherapies.

The therapy of cancer continues to be a challenge aggravated by the evolution of resistance against current medications. As an alternative for the traditional tripartite treatment options of surgery, radiation and chemotherapy, immunotherapy is gaining increasing attention due to the opportunity of more targeted approaches. Promising targets are antigen-presenting cells which drive innate and adaptive immune responses. The discovery and emergence of new drugs and lead structures can be inspired by natural products which comprise many highly bioactive molecules. The development of new drugs based on natural products is hampered by the current lack of guidelines for screening these structures for immune activating compounds. In this work, we describe a phenotypic preclinical screening pipeline for first-line identification of promising natural products using the mouse as a model system. Favorable compounds are defined to be non-toxic to immune target cells, to show direct anti-tumor effects and to be immunostimulatory at the same time. The presented screening pipeline constitutes a useful tool and aims to integrate immune activation in experimental approaches early on in drug discovery. It supports the selection of natural products for later chemical optimization, direct application in in vivo mouse models and clinical trials and promotes the emergence of new innovative drugs for cancer treatment.

Targeting Influenza Virus Hemagglutinin to Xcr1+ Dendritic Cells in the Absence of Receptor-Mediated Endocytosis Enhances Protective Antibody Responses.

Targeting Ags to conventional dendritic cells can enhance Ag-specific immune responses. Although most studies have focused on the induction of T cell responses, the mechanisms by which targeting improves Ab responses are poorly understood. In this study we present data on the use of human XCL1 (hXCL1) and hXCL2 fusion vaccines in a murine model. We show that the human chemokines bound type 1 conventional dendritic cells (cDC1), and that immunization with influenza virus hemagglutinin fused to hXCL1 or hXCL2 induced full protection against influenza challenge. Surprisingly, the hXCL1- and hXCL2-fusion vaccines induced better long-term protection associated with stronger induction of neutralizing Abs, and more Ab-secreting cells in bone marrow. In contrast, murine Xcl1 fusion vaccines induced stronger CD8+ T cell responses compared with hXCL1. Further analysis revealed that although murine Xcl1 fusion vaccines induced chemotaxis and were rapidly endocytosed by cDC1, hXCL1 and hXCL2 fusion vaccines did not induce chemotaxis, were less efficiently endocytosed, and consequently, remained on the surface. This difference may explain the enhanced induction of Abs when targeting Ag to cDC1 using hXCL1 and hXCL2, and suggests that immune responses can be manipulated in directing Abs or T cells based on how efficiently the targeted Ag is endocytosed by the DC.