Differential expression of CSCs biomarkers in renal cell carcinoma stem cells after a combined treatment with phytochemicals and immune checkpoint inhibitors

Renal cell carcinoma (RCC) is a kind of kidney tumor that is distinguished by its tendency to stay undetected until late stages, resulting in a large rise in fatality rates. Immune checkpoint inhibitors have demonstrated potential in treating advanced RCC, either replacing or supplementing therapies with tyrosine kinase inhibitors (TKIs), with objective response rates varying from 42% to 71% for each combination regimen [1]. Not all patients respond to these therapies, only a few people with the median progression-free survival (PFS) varying from 11.6 to 15.4 months, followed by the therapy-resistance, which highlights the need for novel treatment strategies [2]. Recent studies have focused on the function of cancer stem cells (CSCs) in the development and progression of RCC, as these cells are thought to be resistant to traditional cancer therapies and contribute to tumor relapse. CSCs are a small subset of cells seen within malignancies that possess self-renewal and differentiation capabilities and are successful of initiating and sustaining tumor growth. CSCs have been recognized in more than a few cancers, together with RCC, and are related with in favorable outcomes. Therefore, figuring out CSC-specific biomarkers and growing treatments focused on these cells is necessary for enhancing affected person outcomes. One possible method for concentrated on CSCs in RCC is via the use of phytochemicals (PTCs), which are naturally happening compounds discovered in plants. PTCs have been proven to have anti-cancer properties, and some have been proven to mainly target CSCs. Therefore, there is developing amount of research in creating phytochemical-based treatment strategies for RCC that especially target CSCs. The goal of this thesis was to uncover new phytochemicals that originate from conventional herbs that target RCC CSCs, and combination with other treatments such as immune checkpoint inhibitor for enhancing the efficacy of present treatment strategies and creating new remedies for RCC. Various techniques, including viability, proliferation, migration, invasion, and apoptosis assays, have been utilized to identify promising candidate phytochemicals. Because of its exceptional ability to efficiently suppress cancer stem cells (CSCs), shikonin was chosen as the most promising and favorable partner for combination treatment with immune checkpoint inhibitors. Then, potential mechanisms of shikonin and ipilimumab involved in regulating T cell subpopulations and potential immunotherapeutic targets were investigated. Furthermore, figuring out biomarkers such as VCMA1 that predict response to immune checkpoint inhibitor remedy will permit for the improvement of personalized therapy plans for RCC patients

A gut commensal microbiome-host protein network map reveals bacterial modulation of human immune signaling

Complex diseases such as cardiovascular illnesses, cancer, respiratory diseases, and diabetes have been on the rise worldwide contributing to 70% of all deaths. While these illnesses are superficially not associated with microbial organisms accumulating research links bacteria of the gut microbiome to a diverse range of complex diseases. Especially Pseudomonadota, the third most abundant phylum in the gut, has been associated with several of those illnesses e.g., metabolic sicknesses and cancer. However, the underlying mechanisms mediating the bacterial impact on host health remain largely unknown. Pathogenic representatives of the Pseudomonadota phylum are well-known for employing a type three secretion system (T3SS) to manipulate host cells and thereby mediate infectious diseases. Prominent examples are Salmonella, Shigella, enterohemorrhagic Escherichia coli (EHEC), and enteropathogenic Escherichia coli (EPEC) as well as Yersinia pestis responsible for wiping out one-third of the European population during the plague. Yet, T3SSs have also been detected in plant mutualists e.g., rhizobia strains of the rhizobia-legume symbiosis. Furthermore, bacteria that do not exhibit a pathogenic or mutualistic lifestyle also seem to encode for T3SSs. Work outside of this thesis as part of the same project detected T3SSs also in commensal Pseudomonadota of the human gut microbiome. However, the impact of the T3SS effectors on the host cell and subsequently host health is not known. Therefore, this thesis aimed to elucidate the impact of T3SS effectors expressed by commensal gut Pseudomonadota on host functions in the context of human health and disease. To assess the impact of bacterial effectors on the human host a network map of protein-protein interactions (PPIs) between gut commensal bacterial effectors and human proteins was generated. To this end, an ORFeome collection of bacterial effectors was established by cloning 959 T3SS effectors from known, culturable strains as well as from metagenomic data of the human gut. Testing these bacterial effectors against the human ORFeome v9.1 collection consisting of 17,408 protein-coding genes with a systematic, high-throughput yeast two-hybrid (Y2H) pipeline gave rise to the human-microbiome meta-interactome map (HuMMI). The network consists of 1,263 interactions mediated by 289 effectors and 430 human proteins. HuMMI was subjected to a detailed quality control to assess the reliability of the used pipeline and the quality of the interactions. For this purpose, reference sets were assembled to benchmark the Y2H and an orthogonal assay, which was employed to re-test a subset of HuMMI. In addition, the saturation of HuMMI i.e., the percentage of discovered interactions compared to all detectable interactions, was assessed by a Y2H repeat screen. After demonstrating the reliability of the employed Y2H pipeline and the comparability of HuMMI to well-documented, literature-curated-interactions, validation experiments in vitro were conducted based on the functional analysis of the effector targets. As the bacterial effectors targeted human proteins involved in immune signaling their impact on the transcription factor nuclear factor kappa B (NF-κB) was assessed. A cell-based reporter assay was employed testing the ability of the effectors to modulate NF-κB activity. Five effectors significantly activated NF-κB, while three effectors seemed to inhibit the transcription factor significantly. Further impacts on human immune signaling by T3SS effectors were shown by collaborators reporting increased ICAM1 expression as well as up- and downregulation of pro-inflammatory cytokine secretion from a colon cell line upon effector transfection. The opposing effects of bacterial effectors on immune signaling pathways suggest different influences of gut commensal T3SS effectors on the human host. In conclusion, this study introduced a novel mechanism by which gut commensals might impact the human host. T3SS effectors potentially affect human immune signaling locally and systemically via cytokine secretion potentially affecting the risk of complex disease. Thereby, this works launches the investigation into gut commensal T3SS effectors and their impacts on host health and disease

Design-based stereologische post mortem Studien zur Bestimmung von kortikalem Volumen, Neuronenzahl und Neuronendichte im Gehirn von Patienten mit Schizophrenie und gesunden Kontrollpersonen

Natur und Bedeutung der neuropathologischen Veränderungen bei Schizophrenie sind wenig verstanden. Bildgebungsstudien legen ein bei Erkrankten im Vergleich zu Gesunden niedrigeres mittleres kortikales Volumen nahe, während post mortem Untersuchungen Hinweise auf eine unveränderte mittlere kortikale Neuronenzahl bei zugleich höherer mittlerer kortikaler Neuronendichte fanden. Zusammen führten diese Ergebnisse zur Reduced-Neuropil-Hypothese, nach der es bei der Schizophrenie nicht zu einer Verringerung der Neuronenzahl, sondern lediglich zu einem Verlust des interneuronalen Neuropils mit einer folglich dichteren Packung kortikaler Neurone kommt. Diese Daten stammen allerdings aus Untersuchungen an abgegrenzten kortikalen Bereichen und sind nicht durch Untersuchungen des gesamten Kortex mit Berücksichtigung neuropathologischer Störvariablen wie dem post mortem Intervall und der Fixationszeit gestützt. In der vorliegenden Arbeit wird ein Hinweis darauf präsentiert, dass die Schizophrenie entgegen bisheriger Annahmen mit einer niedrigeren mittleren kortikalen Neuronenzahl und -dichte einhergeht. Mittels einer design-based stereologischen Untersuchung der gesamten kortikalen grauen Substanz (KGS) von post mortem Gehirnen von 11 Patienten mit Schizophrenie und 10 gesunden Kontrollpersonen wurde in der Gruppe der Patienten eine statistisch signifikant niedrigere mittlere Neuronenzahl (−14,9%; adjustierte Effektstärke d_adj = −0,94; P = 0,007) sowie eine statistisch signifikant niedrigere mittlere Neuronendichte (−9,6%; d_adj = −0,57; P = 0,041) in der KGS festgestellt. Zudem wurde ein statistisch hochsignifikanter Zusammenhang zwischen der Neuronenzahl in der KGS und dem post mortem Intervall (P < 0,001) festgestellt, was auf einen Störeinfluss durch diesen Parameter mit Notwendigkeit einer statistischen Berücksichtigung hinwies. Zusammenfassend fordern die Ergebnisse der vorliegenden Arbeit die Reduced-Neuropil-Hypothese heraus und stützen das Modell von Schizophrenie als komplexer Störung der Gehirnentwicklung, indem sie auf einen Verlust kortikaler Neurone möglicherweise im Rahmen eines überaktiven Pruning hindeuten.Nature and significance of neuropathological changes in schizophrenia are poorly understood. Imaging studies have suggested a lower mean cortical volume in patients with schizophrenia compared to healthy controls, while post mortem studies found evidence of unchanged mean cortical neuron number but increased mean cortical neuron density. Together, these results led to the reduced neuropil hypothesis of schizophrenia, according to which there is no reduction in the number of neurons in schizophrenia, but only a loss of interneuronal neuropil with a consequently denser packing of cortical neurons. However, these data come from studies of individual cortical areas and are not supported by studies of the whole cortex with adjustment by neuropathological confounders such as the post mortem interval and the fixation time. In this study, evidence is presented that, contrary to previous belief, schizophrenia is associated with lower mean cortical neuron number and density. Using a design-based stereologic examination of the total cortical grey matter (CGM) of post mortem brains of 11 patients with schizophrenia and 10 healthy controls, a statistically significantly lower mean neuron number (−14.9%; adjusted Cohen’s d d_adj = −0.94; P = 0.007) as well as a statistically significantly lower mean neuron density (−9.6%; d_adj = −0.57; P = 0.041) was found in the CGM of the patients with schizophrenia. In addition, a statistically significant correlation between the neuron number in the CGM and the post mortem interval (P < 0.001) was found, indicating a confounding relationship with a need for statistical adjustment. Collectively, the results of this study challenge the reduced neuropil hypothesis of schizophrenia and support the model of schizophrenia as a complex neurodevelopmental disorder by suggesting a loss of cortical neurons possibly in the context of overactive pruning