In vivo imaging of the regulatory mechanisms in acute and chronic delayed type hypersensitivity reactions

Inflammatory responses are involving a complex interplay between molecular and cellular mechanisms. Here in this thesis, different players within the inflammatory tissue and draining lymph nodes such as cathepsin B, reactive oxygen species (ROS) and reactive nitrogen species (RNS) as well as the NF κB pathway were investigated in a model of acute and chronic delayed type hypersensitivity reaction (DTHR) by non invasive in vivo optical imaging. Cathepsin B represents an important intra- and extracellular protease, which is of immense importance during the establishment of an immune response e.g. in antigen processing. ROS and RNS are a byproduct of cellular metabolism but are also produced as effector and signaling molecule mainly by neutrophilic granulocytes. NF-κB is an important signaling pathway which is sensing inflammatory stimuli and leads to the expression of many genes involved in the immune response. The T-cell driven acute cutaneous DTHR was induced by sensitizing mice at the abdomen and eliciting the inflammatory response 7 days later at the right ear using the hapten 2,4,6-trinitrochlorobenzene (TNCB). Repetitive application of TNCB at the right ear for up to five times induced chronic DTHR. Beside wild-type C57BL/6 mice, we used cathepsin B-deficient (Ctsb-/- ), cathepsin Z-deficient (Ctsz-/-) and NF-κB-luciferase-reporter mice to induce the acute and chronic TNCB specific DTHR. Cathepsin B activity, ROS/RNS production and NF-κB activation were measured noninvasively by optical imaging employing protease-activatable fluorescence probes, the ROS-sensitive chemiluminescence probe L-012 and luciferin for bioluminescence imaging in NF-κB-luciferase-reporter mice. Extensive ex vivo validation was performed including histopathology, immunohistochemistry, flow cytometry, fluorescence microscopy, RT-PCR as well as active site labeling of proteases and Western blotting. Furthermore, the therapeutic effects of N-acetylcysteine (NAC) and the cathepsin-inhibitors CA-074 and Inhibitor 17 were investigated. In acute cutaneous DTHR in vivo optical imaging detected an intense cathepsin B activity as well as ROS/RNS production and NF-κB activation peaking at 24 h after the 1st TNCB ear challenge. In chronic DTHR the cathepsin B activity further increased while the peaks of ROS/RNS production and NF-κB activation were shifted to an earlier timepoint. NAC treatment decreased the ear swelling response in acute and chronic DTHR while the influence on the ROS/RNS production and NF-κB activation during acute and chronic DTHR assessed by in vivo optical imaging was divergent. The cathepsin B inhibitors CA-074 and Inhibitor 17 reduced inflammation in acute but not in chronic DTHR, while Ctsb-/- mice exhibited an even enhanced ear swelling response during acute DTHR caused by a compensatory expression of cathepsin Z. Ex vivo analysis revealed enhanced cathepsin B expression in neutrophilic granulocytes, dendritic cells, macrophages, B, T and natural killer (NK) cells within inflamed ears and draining lymph nodes. The investigated mechanisms are an essential part of the multifaceted interplay which is needed to establish and maintain inflammatory immune responses. Molecular imaging involving optical imaging is a highly capable tool to monitor these mechanisms in vivo and to asses targeted therapeutic interventions. These results could be of high importance not only to modulate inflammatory autoimmune diseases like rheumatoid arthritis but also to prevent carcinogenesis in chronic inflammation like non alcoholic steatohepatitis

A role for artificial intelligence in molecular imaging of infection and inflammation

The detection of occult infections and low-grade inflammation in clinical practice remains challenging and much depending on readers' expertise. Although molecular imaging, like [F-18]FDG PET or radiolabeled leukocyte scintigraphy, offers quantitative and reproducible whole body data on inflammatory responses its interpretation is limited to visual analysis. This often leads to delayed diagnosis and treatment, as well as untapped areas of potential application. Artificial intelligence (AI) offers innovative approaches to mine the wealth of imaging data and has led to disruptive breakthroughs in other medical domains already. Here, we discuss how AI-based tools can improve the detection sensitivity of molecular imaging in infection and inflammation but also how AI might push the data analysis beyond current application toward predicting outcome and long-term risk assessment

Children of Mentally III Parents at Risk Evaluation (COMPARE): Design and Methods of a Randomized Controlled Multicenter Study—Part I

Objectives: Mental disorders are frequent, associated with disability-adjusted life years, societal, and economic costs. Children of parents with a mental illness (COPMI) are at an increased risk to develop disorders themselves. The transgenerational transmission of mental disorders has been conceptualized in a model that takes parental and family factors, the social environment (i.e., school, work, and social support), parent-child-interaction and possible child outcomes into account. The goal of the “Children of Mentally Ill Parents At Risk Evaluation” (COMPARE) study will thus be twofold: (1) to establish the efficacy and cost-effectiveness of a high-quality randomized controlled trial (RCT) with the aim of interrupting the intergenerational transmission of mental disorders in COPMI, (2) to test the components of the trans-generational transmission model of mental disorders. Methods: To implement a randomized controlled trial (RCT: comparison of parental cognitive behavioral therapy/CBT with CBT + Positive Parenting Program) that is flanked by four add-on projects that apply behavioral, psychophysiological, and neuro-imaging methods to examine potential moderators and mediators of risk transmission (projects COMPARE-emotion/-interaction/-work/-school). COMPARE-emotion targets emotion processing and regulation and its impact on the transgenerational disorder transmission; COMPARE-interaction focuses especially on the impact of maternal comorbid diagnoses of depression and anxiety disorders and will concentrate on different pathways of the impact of maternal disorders on socio-emotional and cognitive infant development, such as parent-infant interaction and the infant's stress regulation skills. COMPARE-work analyzes the transmission of strains a person experiences in one area of life to another (i.e., from family to work; spill-over), and how stress and strain are transmitted between individuals (i.e., from parent to child; cross-over). COMPARE-school focuses on the psychosocial adjustment, school performance, and subjective well-being in COPMI compared to an adequate control group of healthy children. Results: This study protocol reports on the interdisciplinary approach of COMPARE testing the model of the transgenerational transmission of mental disorders. Conclusion: The combination of applied basic with clinical research will facilitate the examination of specific risk transmission mechanisms, promotion, dissemination and implementation of results into a highly important but largely neglected field. Clinical Trial Registration: DRKS-ID: DRKS00013516 (German Clinical Trials Register, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00013516)

Cancer immunotherapy is accompanied by distinct metabolic patterns in primary and secondary lymphoid organs observed by non-invasive in vivo18F-FDG-PET

Purpose: Cancer immunotherapy depends on a systemic immune response, but the basic underlying mechanisms are still largely unknown. Despite the very successful and widespread use of checkpoint inhibitors in the clinic, the majority of cancer patients do not benefit from this type of treatment. In this translational study, we investigated whether noninvasive in vivo positron emission tomography (PET) imaging using 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) is capable of detecting immunotherapy-associated metabolic changes in the primary and secondary lymphoid organs and whether this detection enables the prediction of a successful anti-cancer immune response. Methods: RIP1-Tag2 mice with progressed endogenous insular cell carcinomas underwent a combined cancer immunotherapy consisting of CD4+ T cells plus monoclonal antibodies (mAbs) against programmed death ligand-1 (PD-L1) and lymphocyte activation gene-3 (LAG-3) or a sham treatment after radiation-mediated immune cell depletion. A second cohort of RIP1-Tag2 mice underwent exclusive checkpoint inhibitor therapy (CIT) using anti-PD-L1/LAG-3 mAbs or sham treatment without initial immune cell depletion to mimic the clinical situation. All mice were monitored by 18F-FDG-PET combined with anatomical magnetic resonance imaging (MRI). In addition, we retrospectively analyzed PET / computed tomography (CT) scans (PET/CT) regarding 18F-FDG uptake of CIT-treated metastatic melanoma patients in the spleen (n=23) and bone marrow (BM; n=20) as well as blood parameters (n=17-21). Results: RIP1-Tag2 mice with advanced insular cell carcinomas treated with combination immunotherapy exhibited significantly increased 18F-FDG uptake in the spleen compared to sham-treated mice. Histopathology of the spleens from treated mice revealed atrophy of the white pulp with fewer germinal centers and an expanded red pulp with hyperplasia of neutrophils than those of sham-treated mice. Immunohistochemistry and flow cytometry analyses of the spleens revealed a lower number of T cells and a higher number of neutrophils compared to those in the spleens of sham-treated mice. Flow cytometry of the BM showed enhanced activation of T cells following the treatment schemes that included checkpoint inhibitors. The ratio of 18F-FDG uptake at baseline to the uptake at follow-up in the spleens of exclusively CIT-treated RIP1-Tag2 mice was significantly enhanced, but the ratio was not enhanced in the spleens of the sham-treated littermates. Flow cytometry analysis confirmed a reduced number of T cells in the spleens of exclusively CIT-treated mice compared to that of sham-treated mice. A retrospective analysis of clinical 18F-FDG-PET/CT scans revealed enhanced 18F-FDG uptake in the spleens of some successfully CIT-treated patients with metastatic melanoma, but there were no significant differences between responders and non-responders. The analysis of the BM in clinical 18F-FDG-PET/CT scans with a computational segmentation tool revealed significantly higher baseline 18F-FDG uptake in patients who responded to CIT than in non-responders, and this relationship was independent of bone metastasis, even in the baseline scan. Conclusions: Thus, we are presenting the first translational study of solid tumors focusing on the metabolic patterns of primary and secondary lymphoid organs induced by the systemic immune response after CIT. We demonstrate that the widely available 18F-FDG-PET modality is an applicable translational tool that has high potential to stratify patients at an early time point

Leave Me Alone With Your Symptoms! Social Exclusion at the Workplace Mediates the Relationship of Employee's Mental Illness and Sick Leave

Although a substantial part of employees suffers from a mental illness, the work situation of this population still is understudied. Previous research suggests that people with a mental illness experience discrimination in the workplace, which is known to have detrimental effects on health. Building on the stereotype content model and allostatic load theory, the present study investigated whether employees with a mental illness become socially excluded at the workplace and therefore show more days of sick leave. Overall, 86 employees diagnosed with a mental disorder were interviewed and completed online-surveys. Path analyses supported the hypotheses, yielding a serial mediation: The interview-rated severity of the mental disorder had an indirect effect on the days of sick leave, mediated by the symptomatic burden and the social exclusion at the workplace. In the light of the costs associated with absenteeism the present paper highlights the harmfulness of discrimination. Organizations and especially supervisors need to be attentive for signs of exclusion within their teams and try to counteract as early as possible

Preclinical Identification Of Tumor-Draining Lymph Nodes Using a Multimodal Non-invasive In vivo Imaging Approach

Purpose: Resection of the tumor-draining lymph -node (TDLN) represents a standard method to identify metastasis for several malignancies. Interestingly, recent preclinical studies indicate that TDLN resection diminishes the efficacy of immune checkpoint inhibitor-based cancer immunotherapies. Thus, accurate preclinical identification of TDLNs is pivotal to uncovering the underlying immunological mechanisms. Therefore, we validated preclinically, and clinically available non-invasive in vivo imaging approaches for precise TDLN identification. Procedures: For visualization of the lymphatic drainage into the TDLNs by non-invasive in vivo optical imaging, we injected the optical imaging contrast agents Patent Blue V (582.7 g mol−1) and IRDye® 800CW polyethylene glycol (PEG; 25,000–60,000 g mol−1), subcutaneously (s.c.) in close proximity to MC38 adenocarcinomas at the right flank of experimental mice. For determination of the lymphatic drainage and the glucose metabolism in TDLNs by non-invasive in vivo PET/magnetic resonance imaging (PET/MRI), we injected the positron emission tomography (PET) tracer (2-deoxy-2[18F]fluoro-D-glucose (18F-FDG) [181.1 g mol−1]) in a similar manner. For ex vivo cross-correlation, we isolated TDLNs and contralateral nontumor-draining lymph nodes (NTDLNs) and performed optical imaging, biodistribution, and autoradiography analysis. Results: The clinically well-established Patent Blue V was superior for intraoperative macroscopic identification of the TDLNs compared with IRDye® 800CW PEG but was not sensitive enough for non-invasive in vivo detection by optical imaging. Ex vivo Patent Blue V biodistribution analysis clearly identified the right accessory axillary and the proper axillary lymph node (LN) as TDLNs, whereas ex vivo IRDye® 800CW PEG completely failed. In contrast, functional non-invasive in vivo18F-FDG PET/MRI identified a significantly elevated uptake exclusively within the ipsilateral accessory axillary TDLN of experimental mice and was able to differentiate between the accessory axillary and the proper LN. Ex vivo biodistribution and autoradiography confirmed our in vivo18F-FDG PET/MRI results. Conclusions: When taken together, our results demonstrate the feasibility of 18F-FDG-PET/MRI as a valid method for non-invasive in vivo, intraoperative, and ex vivo identification of the lymphatic drainage and glucose metabolism within the TDLNs. In addition, using Patent Blue V provides additive value for the macroscopic localization of the lymphatic drainage both visually and by ex vivo optical imaging analysis. Thus, both methods are valuable, easy to implement, and cost-effective for preclinical identification of the TDLN