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In the current era of precision oncology, it is widely acknowledged that CRC is a heterogeneous disease entity. Tumor location (right- or left-sided colon cancer or rectal cancer) is a crucial factor in determining disease progression as well as prognosis and influences disease management. In the last decade, numerous works have reported that the microbiome is an important element of CRC carcinogenesis, progression and therapy response. Owing to the heterogeneous nature of microbiomes, the findings of these studies were inconsistent. The majority of the studies combined colon cancer (CC) and rectal cancer (RC) samples as CRC for analysis. Furthermore, the small intestine, as the major site for immune surveillance in the gut, is understudied compared to the colon. Thus, the CRC heterogeneity puzzle is far from being solved, and more research is necessary for prospective trials that separately investigate CC and RC. Our prospective study aimed to map the colon cancer landscape using 16S rRNA amplicon sequencing in biopsy samples from the terminal ileum, healthy colon tissue, healthy rectal tissue and tumor tissue as well as in preoperative and postoperative stool samples of 41 patients. While fecal samples provide a good approximation of the average gut microbiome composition, mucosal biopsies allow for detecting subtle variations in local microbial communities. In particular, the small bowel microbiome has remained poorly characterized, mainly because of sampling difficulties. Our analysis revealed the following: (i) right- and left-sided colon cancers harbor distinct and diverse microbiomes, (ii) the tumor microbiome leads to a more consistent cancer-defined microbiome between locations and reveals a tumor microbiome–ileal microbiome association, (iii) the stool only partly reflects the microbiome landscape in patients with CC, and (iv) mechanical bowel preparation and perioperative antibiotics together with surgery result in major changes in the stool microbiome, characterized by a significant increase in the abundance of potentially pathogenic bacteria, such as Enterococcus. Collectively, our results provide new and valuable insights into the complex microbiome landscape in patients with colon cancer.

The role of progesterone receptor A (PRA) for the survival outcome of cervical cancer patients is ambiguous. In mouse models, it has been shown that PRA plays a rather protective role in cancer development. The aim of this study was to assess its expression by immunohistochemistry in 250 cervical cancer tissue samples and to correlate the results with clinicopathological parameters including patient survival. PRA expression was positively correlated with the International Federation of Gynecology and Obstetrics (FIGO) classification scores. PRA was significantly overexpressed in adenocarcinomas compared to squamous epithelial carcinoma subtypes. Correlation analyses revealed a trend association with the HPV virus protein E6, a negative correlation with p16 and a positive correlation with EP3. PRA expression was also associated with the expression of RIP140, a transcriptional coregulator that we previously identified as a negative prognostic factor for survival in cervical cancer patients. Univariate survival analyses revealed PRA as a negative prognosticator for survival in patients with cervical adenocarcinoma. Multivariate analyses showed that simultaneous expression of RIP140 and PRA was associated with the worst survival, whereas with negative RIP140, PRA expression alone was associated with the best survival. We can therefore assume that the effect of nuclear PRA on overall survival is dependent upon nuclear RIP140 expression.

Growth Factor Independence 1 (GFI1) is a transcription factor with an important role in the regulation of development of myeloid and lymphoid cell lineages and was implicated in the development of myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). Reduced expression of GFI1 or presence of the GFI1-36N (serine replaced with asparagine) variant leads to epigenetic changes in human and murine AML blasts and accelerated the development of leukaemia in a murine model of human MDS and AML. We and other groups previously showed that the GFI1-36N allele or reduced expression of GFI1 in human AML blasts is associated with an inferior prognosis. Using GFI1-36S, -36N -KD, NUP98-HOXD13-tg mice and curcumin (a natural histone acetyltransferase inhibitor (HATi)), we now demonstrate that expansion of GFI1-36N or ���KD, NUP98-HODXD13 leukaemic cells can be delayed. Curcumin treatment significantly reduced AML progression in GFI1-36N or -KD mice and prolonged AML-free survival. Of note, curcumin treatment had no effect in GFI1-36S, NUP98-HODXD13 expressing mice. On a molecular level, curcumin treatment negatively affected open chromatin structure in the GFI1-36N or -KD haematopoietic cells but not GFI1-36S cells. Taken together, our study thus identified a therapeutic role for curcumin treatment in the treatment of AML patients (homo or heterozygous for GFI1-36N or reduced GFI1 expression) and possibly improved therapy outcome., Finanziert durch den Open-Access-Publikationsfonds der Westf��lischen Wilhelms-Universit��t M��nster (WWU M��nster).

Oral cancer often presents with aggressive behavior and a high risk of recurrence and metastasis. For oral squamous cell carcinoma (OSCC), which is the most frequent histological subtype, therapy strategies include surgery, radiation therapy, chemotherapy, immune checkpoint inhibitors, and EGFR inhibitors. Recently, a Trop-2 antibody-drug conjugate (ADC) has been approved in the United States of America for the treatment of advanced triple-negative breast cancer. However, this ADC has also been tested in other solid tumors including head & neck squamous cell carcinoma. The prognostic impact of Trop-2 has already been reported for several cancers. We studied the prognostic influence of Trop-2 protein expression on OSCC patients’ survival. The cohort comprised n = 229 OSCC patients with available archived tumor tissue and corresponding non-neoplastic oral mucosa tissue. Using immunohistochemistry, we investigated Trop-2 expression in both the central and peripheral regions of each tumor and in corresponding non-neoplastic oral mucosa. In patients suffering from OSCC with combined high central and low peripheral Trop-2 expression, five-year overall survival (OS) was 41.2%, whereas 55.6% of OSCC patients who presented lower central and/or higher peripheral tumoral Trop-2 expression were alive after five years (p = 0.075). In multivariate Cox regression, the expression pattern of high central tumoral and lower peripheral Trop-2 expression was significantly correlated with impaired OS (HR = 1.802, 95%-CI: 1.134–2.864; p = 0.013) and recurrence-free survival (RFS) (HR = 1.633, 95%-CI: 1.042–2.560; p = 0.033), respectively, when adjusting for co-variables. Hence, Trop-2 may serve as an independent prognostic biomarker in OSCC. In subsequent studies, the pathophysiological meaning of downregulated Trop-2 expression in the OSCC periphery has to be analyzed.

Myeloablative therapy with highdoses of the cytostatic drug melphalan (MEL) in preparation for hematopoietic cell transplantation is the standard of care for multiple myeloma (MM) patients. Melphalan is a bifunctional alkylating agent that covalently binds to nucleophilic sites in the DNA and effective in the treatment, but unfortunately has limited therapeutic benefit. Therefore, new approaches are urgently needed for patients who are resistant to existing standard treatment with MEL. Regulating the pharmacological activity of drug molecules by modifying their structure is one method for improving their effectiveness. The purpose of this work was to analyze the physicochemical and biological properties of newly synthesized melphalan derivatives (EE-MEL, EM-MEL, EM-MOR-MEL, EM-I-MEL, EM-T-MEL) obtained through the esterification of the carboxyl group and the replacement of the the amino group with an amidine group. Compounds were selected based on our previous studies for their improved anticancer properties in comparison with the original drug. For this, we first evaluated the physicochemical properties using the circular dichroism technique, then analyzed the zeta potential and the hydrodynamic diameters of the particles. Then, the in vitro biological properties of the analogs were tested on multiple myeloma (RPMI8226), acute monocytic leukemia (THP1), and promyelocytic leukemia (HL60) cells as model systems for hematological malignant cells. DNA damage was assessed by immunostaining γH2AX, cell cycle distribution changes by propidium iodide (PI) staining, and cell death by the activation of caspase 2. We proved that the newly synthesized derivatives, in particular EM-MOR-MEL and EM-T-MEL, affected the B-DNA conformation, thus increasing the DNA damage. As a result of the DNA changes, the cell cycle was arrested in the S and G2/M phases. The cell death occurred by activating a mitotic catastrophe. Our investigations suggest that the analogs EM-MOR-MEL and EM-T-MEL have better anti-cancer activity in multiple myeloma cells than the currently used melphalan.

Neutrophils are an essential part of the innate immune system and the first line of defense against invading pathogens. They phagocytose, release granular contents, produce reactive oxygen species, and form neutrophil extracellular traps (NETs) to fight pathogens. With the characterization of NETs and their components, neutrophils were identified as players of the innate adaptive crosstalk. This has placed NETs at the center not only of physiological but also pathological processes. Aside from their role in pathogen uptake and clearance, NETs have been demonstrated to contribute to the resolution of inflammation by forming aggregated NETs able to degrade inflammatory mediators. On the other hand, NETs have the potential to foster severe pathological conditions. When homeostasis is disrupted, they occlude vessels and ducts, serve as sources of autoantigens and danger or damage associated molecular patterns, directly damage tissues, and exaggerate complement activity and inflammation. This review focusses on the understanding of NETs from their formation to their functions in both physiological and pathological processes.

During the onset of acute inflammation, rapid trafficking of leukocytes is essential to mount appropriate immune responses towards an inflammatory insult. Monocytes are especially indispensable for counteracting the inflammatory stimulus, neutralising the noxa and reconstituting tissue homeostasis. Thus, monocyte trafficking to the inflammatory sites needs to be precisely orchestrated. In this study, we identify a regulatory network driven by miR-125a that affects monocyte adhesion and chemotaxis by the direct targeting of two adhesion molecules, i.e., junction adhesion molecule A (JAM-A), junction adhesion molecule-like (JAM-L) and the chemotaxis-mediating chemokine receptor CCR2. By investigating monocytes isolated from patients undergoing cardiac surgery, we found that acute yet sterile inflammation reduces miR-125a levels, concomitantly enhancing the expression of JAM-A, JAM-L and CCR2. In contrast, TLR-4-specific stimulation with the pathogen-associated molecular pattern (PAMP) LPS, usually present within the perivascular inflamed area, resulted in dramatically induced levels of miR-125a with concomitant repression of JAM-A, JAM-L and CCR2 as early as 3.5 h. Our study identifies miR-125a as an important regulator of monocyte trafficking and shows that the phenotype of human monocytes is strongly influenced by this miRNA, depending on the type of inflammatory stimulus.

Aripiprazole is an atypical antipsychotic drug, which is prescribed for many psychiatric diseases such as schizophrenia and mania in bipolar disorder. It primarily acts as an agonist of dopaminergic and other G-protein coupled receptors. So far, an interaction with ligand- or voltage-gated ion channels has been classified as weak. Meanwhile, we identified aripiprazole in a preliminary test as a potent blocker of voltage-gated sodium channels. Here, we present a detailed analysis about the interaction of aripiprazole with the dominant voltage-gated sodium channel of heart muscle (hNav1.5). Electrophysiological experiments were performed by means of the patch clamp technique at human heart muscle sodium channels (hNav1.5), heterologously expressed in human TsA cells. Aripiprazole inhibits the hNav1.5 channel in a state- but not use-dependent manner. The affinity for the resting state is weak with an extrapolated Kr of about 55 µM. By contrast, the interaction with the inactivated state is strong. The affinities for the fast and slow inactivated state are in the low micromolar range (0.5–1 µM). Kinetic studies indicate that block development for the inactivated state must be described with a fast (ms) and a slow (s) time constant. Even though the time constants differ by a factor of about 50, the resulting affinity constants were nearly identical (in the range of 0.5 µM). Besides this, aripirazole also interacts with the open state of the channel. Using an inactivation deficit mutant, an affinity of about 1 µM was estimated. In summary, aripiprazole inhibits voltage-gated sodium channels at low micromolar concentrations. This property might add to its possible anticancer and neuroprotective properties.

Glycoprotein (GP) VI is the major platelet collagen receptor and a promising anti-thrombotic target. This was first demonstrated in mice using the rat monoclonal antibody JAQ1, which completely blocks the Collagen-Related Peptide (CRP)-binding site on mouse GPVI and efficiently inhibits mouse platelet adhesion, activation and aggregation on collagen. Here, we show for the first time that JAQ1 cross-reacts with human GPVI (huGPVI), but not with GPVI in other tested species, including rat, rabbit, guinea pig, swine, and dog. We further demonstrate that JAQ1 differently modulates mouse and human GPVI function. Similar to its effects on mouse GPVI (mGPVI), JAQ1 inhibits CRP-induced activation in human platelets, whereas, in stark contrast to mouse GPVI, it does not inhibit the adhesion, activation or aggregate formation of human platelets on collagen, but causes instead an increased response. This effect was also seen with platelets from newly generated human GPVI knockin mice (hGP6tg/tg). These results indicate that the binding of JAQ1 to a structurally conserved epitope in GPVI differently affects its function in human and mouse platelets.

Background: Brain-tumor-initiating cells (BTICs) of proneural and mesenchymal origin contribute to the highly malignant phenotype of glioblastoma (GB) and resistance to current therapies. Methods: BTICs of different subtypes representing tumor heterogeneity were challenged with OXPHOS (oxidative phosphorylation) inhibition to assess the differential effects of metabolic intervention on key resistance features. Results: Whereas mesenchymal BTICs were more invasive, more glycolytic and less responsive to OXPHOS-inhibition, proneural BTICs were less invasive, catabolized glucose more via the pentose phosphate pathways, and responded better to OXPHOS inhibition. Conclusion: Targeting glycolysis may be a promising approach to inhibit highly invasive tumor cells of mesenchymal origin, whereas proneural cells are more responsive to OXPHOS inhibition. Future clinical trials exploring metabolic interventions should account for metabolic heterogeneity of brain tumors to overcome resistance to current treatments.

All forms of restriction, from caloric to amino acid to glucose restriction, have been established in recent years as therapeutic options for various diseases, including cancer. However, usually there is no direct comparison between the different restriction forms. Additionally, many cell culture experiments take place under static conditions. In this work, we used a closed perfusion culture in murine L929 cells over a period of 7 days to compare methionine restriction (MetR) and glucose restriction (LowCarb) in the same system and analysed the metabolome by liquid chromatography mass spectrometry (LC-MS). In addition, we analysed the inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) over a period of 72 h. 2-DG induced very fast a low-energy situation by a reduced glycolysis metabolite flow rate resulting in pyruvate, lactate, and ATP depletion. Under perfusion culture, both MetR and LowCarb were established on the metabolic level. Interestingly, over the period of 7 days, the metabolome of MetR and LowCarb showed more similarities than differences. This leads to the conclusion that the conditioned medium, in addition to the different restriction forms, substantially reprogramm the cells on the metabolic level.

Programmed Cell Death 1 Ligand 1 (PD-L1, CD274, B7-H1) is a transmembrane protein which is strongly involved in immune modulation, serving as checkpoint regulator. Interaction with its receptor, Programmed Cell Death Protein 1 (PD-1), induces an immune-suppressive signal, which modulates the activity of T cells and other effector cells. This mediates peripheral tolerance and contributes to tumor immune escape. PD-L1 became famous due to its deployment in cancer therapy, where blockage of PD-L1 with the help of therapeutic antagonistic antibodies achieved impressive clinical responses by reactivating effector cell functions against tumor cells. Therefore, in the past, the focus has been placed on PD-L1 expression and its function in various malignant cells, whereas its role in healthy tissue and diseases apart from cancer remained largely neglected. In this review, we summarize the function of PD-L1 in non-cancerous cells, outlining its discovery and origin, as well as its involvement in different cellular and immune-related processes. We provide an overview of transcriptional and translational regulation, and expression patterns of PD-L1 in different cells and organs, and illuminate the involvement of PD-L1 in different autoimmune diseases as well as in the context of transplantation and pregnancy.

Childhood obesity is a growing problem in industrial societies and associated with increased leptin levels in serum and salvia. Orthodontic treatment provokes pressure and tension zones within the periodontal ligament, where, in addition to fibroblasts, macrophages are exposed to these mechanical loadings. Given the increasing number of orthodontic patients with these conditions, insights into the effects of elevated leptin levels on the expression profile of macrophages during mechanical strain are of clinical interest. Therefore, the aim of this in vitro study was to assess the influence of leptin on the expression profile of macrophages during simulated orthodontic treatment. RAW264.7 macrophages were incubated with leptin and lipopolysaccharides (LPS) from Porphyromonas gingivalis (P. gingivalis) or with leptin and different types of mechanical strain (tensile, compressive strain). Expression of inflammatory mediators including tumor necrosis factor (TNF), Interleukin-1-B (IL1B), IL6, and prostaglandin endoperoxide synthase (PTGS2) was assessed by RT-qPCR, ELISAs, and immunoblot. Without additional mechanical loading, leptin increased Tnf, Il1b, Il6, and Ptgs2 mRNA in RAW264.7 macrophages by itself and after stimulation with LPS. However, in combination with tensile or compressive strain, leptin reduced the expression and secretion of these inflammatory factors. By itself and in combination with LPS from P. gingivalis, leptin has a pro-inflammatory effect. Both tensile and compressive strain lead to increased expression of inflammatory genes. In contrast to its effect under control conditions or after LPS treatment, leptin showed an anti-inflammatory phenotype after mechanical stress.

Biomarker development, improvement, and clinical implementation in the context of kidney disease have been a central focus of biomedical research for decades. To this point, only serum creatinine and urinary albumin excretion are well-accepted biomarkers in kidney disease. With their known blind spot in the early stages of kidney impairment and their diagnostic limitations, there is a need for better and more specific biomarkers. With the rise in large-scale analyses of the thousands of peptides in serum or urine samples using mass spectrometry techniques, hopes for biomarker development are high. Advances in proteomic research have led to the discovery of an increasing amount of potential proteomic biomarkers and the identification of candidate biomarkers for clinical implementation in the context of kidney disease management. In this review that strictly follows the PRISMA guidelines, we focus on urinary peptide and especially peptidomic biomarkers emerging from recent research and underline the role of those with the highest potential for clinical implementation. The Web of Science database (all databases) was searched on 17 October 2022, using the search terms “marker *” OR biomarker * AND “renal disease” OR “kidney disease” AND “proteome *” OR “peptid *” AND “urin *”. English, full-text, original articles on humans published within the last 5 years were included, which had been cited at least five times per year. Studies based on animal models, renal transplant studies, metabolite studies, studies on miRNA, and studies on exosomal vesicles were excluded, focusing on urinary peptide biomarkers. The described search led to the identification of 3668 articles and the application of inclusion and exclusion criteria, as well as abstract and consecutive full-text analyses of three independent authors to reach a final number of 62 studies for this manuscript. The 62 manuscripts encompassed eight established single peptide biomarkers and several proteomic classifiers, including CKD273 and IgAN237. This review provides a summary of the recent evidence on single peptide urinary biomarkers in CKD, while emphasizing the increasing role of proteomic biomarker research with new research on established and new proteomic biomarkers. Lessons learned from the last 5 years in this review might encourage future studies, hopefully resulting in the routine clinical applicability of new biomarkers.

Behçet’s syndrome (BS) is a rare systemic vasculitis characterized by different clinical manifestations. As no specific laboratory tests exist, the diagnosis relies on clinical criteria, and the differential diagnosis with other inflammatory diseases can be challenging. Indeed, in a relatively small proportion of patients, BS symptoms include only mucocutaneous, articular, gastrointestinal, and non-typical ocular manifestations, which are frequently found also in psoriatic arthritis (PsA). We investigate the ability of serum interleukin (IL)-36α—a pro-inflammatory cytokine involved in cutaneous and articular inflammatory diseases—to differentiate BS from PsA. A cross-sectional study was performed on 90 patients with BS, 80 with PsA and 80 healthy controls. Significantly lower IL-36α concentrations were found in patients with BS as compared to PsA, although in both groups IL-36α was significantly increased compared to healthy controls. An empirical cut-off of 420.6 pg/mL displayed a specificity of 0.93, with a sensitivity of 0.70 (AUC 0.82) in discriminating PsA from BS. This cut-off displayed a good diagnostic performance also in BS patients lacking highly specific BS manifestations. Our results indicate that IL-36α might be involved in the pathogenesis of both BS and PsA, and might be a candidate biomarker to support the differential diagnosis of BS.

SARS-CoV-2, the causative agent of COVID-19, has spread around the world with more than 700 million cases and 6.8 million deaths. Various variants of concern (VoC) have emerged due to mutations and recombination and concurrent selection for increased viral fitness and immune evasion. The viral protein that primarily determines the pathogenicity, infectivity, and transmissibility is the Spike protein. To analyze the specific impact of variant Spike proteins on infection dynamics, we constructed SARS-CoV-2 with a uniform B.1 backbone but with alternative Spike proteins. In addition, ORF6 was replaced by EYFP as a biological safety measure, and for use of this well-established reporter. We show that namely the delta variant Spike proteins cause a distinct phenotype from the wild type (B.1, D614G) and other variants of concern. Furthermore, we demonstrate that the omicron BA.1 Spike results in lower viral loads and a less efficient spread in vitro. Finally, we utilized viruses with the two different reporters EYFP and mCherry to establish a competitive growth assay, demonstrating that most but not all Spike variant viruses were able to outcompete wild type SARS-CoV-2 B.1.

The porcine ocular surface is used as a model of the human ocular surface; however, a detailed characterization of the porcine ocular surface has not been documented. This is due, in part, to the scarcity of antibodies produced specifically against the porcine ocular surface cell types or structures. We performed a histological and immunohistochemical investigation on frozen and formalin-fixed, paraffin-embedded ocular surface tissue from domestic pigs using a panel of 41 different antibodies related to epithelial progenitor/differentiation phenotypes, extracellular matrix and associated molecules, and various niche cell types. Our observations suggested that the Bowman’s layer is not evident in the cornea; the deep invaginations of the limbal epithelium in the limbal zone are analogous to the limbal interpalisade crypts of human limbal tissue; and the presence of goblet cells in the bulbar conjunctiva. Immunohistochemistry analysis revealed that the epithelial progenitor markers cytokeratin (CK)15, CK14, p63α, and P-cadherin were expressed in both the limbal and conjunctival basal epithelium, whereas the basal cells of the limbal and conjunctival epithelium did not stain for CK3, CK12, E-cadherin, and CK13. Antibodies detecting marker proteins related to the extracellular matrix (collagen IV, Tenascin-C), cell–matrix adhesion (β-dystroglycan, integrin α3 and α6), mesenchymal cells (vimentin, CD90, CD44), neurons (neurofilament), immune cells (HLA-ABC; HLA-DR, CD1, CD4, CD14), vasculature (von Willebrand factor), and melanocytes (SRY-homeobox-10, human melanoma black-45, Tyrosinase) on the normal human ocular surface demonstrated similar immunoreactivity on the normal porcine ocular surface. Only a few antibodies (directed against N-cadherin, fibronectin, agrin, laminin α3 and α5, melan-A) appeared unreactive on porcine tissues. Our findings characterize the main immunohistochemical properties of the porcine ocular surface and provide a morphological and immunohistochemical basis useful to research using porcine models. Furthermore, the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.

PRAME (PReferentially expressed Antigen in MElanoma) is a cancer testis antigen that is frequently expressed in melanoma compared to benign melanocytic proliferations and nevi. However, the interpretation of the intensity and distribution of PRAME immunostaining is not standardized a lot, which makes interpretation difficult. PRAME-stained histological slides of superficial spreading melanomas (SSM) and dysplastic nevi (DN) were digitized and analyzed using the digital pathology and image platform QuPath. t-tests and ROC AUCs were performed with SPSS. A p-value of 0.8 was considered a good result. A cut-off score was defined in an evaluation cohort and subsequently analyzed in an independent validation cohort. In total, 81 PRAME-stained specimens were included. The evaluation cohort included 32 (50%) SSM and 32 (50%) DN, and the mean of PRAME-positive cells/mm2 for the entire lesion was 455.3 (SD 428.2) in SSM and 60.5 (SD 130.1; p < 0.001) in DN. The ROC AUC of PRAME-positive cells of the entire lesion was 0.866, and in the epidermis it was 0.901. The defined cut-off score to distinguish between DN and SSM was 97.67 cells/mm2. In the validation cohort, 16 out of 17 cases (94.1%) were correctly classified by the cut-off score. The computer-aided assessment of PRAME immunostaining is a useful tool in dermatopathology to distinguish between DN and SSM. Lesions with a moderate expression and indifferent morphologic features will remain a challenge for dermatopathologists.

Osteomyelitis is a difficult-to-treat disease with high chronification rates. First studies suggest increases in mitochondrial fission and mitochondrial dysfunction as possible contributors to the accumulation of intracellular reactive oxygen species and thereby to the cell death of infected bone cells. The aim of the present study is to analyze the ultrastructural impact of bacterial infection on osteocytic and osteoblastic mitochondria. Human infected bone tissue samples were visualized via light microscopy and transmission electron microscopy. Osteoblasts, osteocytes and their mitochondria were analyzed histomorphometrically and compared with the control group of noninfectious human bone tissue samples. The results depicted swollen hydropic mitochondria including depleted cristae and a decrease in matrix density in the infected samples. Furthermore, perinuclear clustering of mitochondria could also be observed regularly. Additionally, increases in relative mitochondrial area and number were found as a correlate for increased mitochondrial fission. In conclusion, mitochondrial morphology is altered during osteomyelitis in a comparable way to mitochondria from hypoxic tissues. This gives new perspectives on the treatment strategies since the manipulation of mitochondrial dynamics may improve bone cell survival as a potential new target for the therapy of osteomyelitis.

Inflammatory bowel diseases (IBDs) consist of a group of chronic inflammatory disorders with a complex etiology, which represent a clinical challenge due to their often therapy-refractory nature. In IBD, inflammation of the intestinal mucosa is characterized by strong and sustained leukocyte infiltration, resulting in the loss of epithelial barrier function and subsequent tissue destruction. This is accompanied by the activation and the massive remodeling of mucosal micro-vessels. The role of the gut vasculature in the induction and perpetuation of mucosal inflammation is receiving increasing recognition. While the vascular barrier is considered to offer protection against bacterial translocation and sepsis after the breakdown of the epithelial barrier, endothelium activation and angiogenesis are thought to promote inflammation. The present review examines the respective pathological contributions of the different phenotypical changes observed in the microvascular endothelium during IBD, and provides an overview of potential vessel-specific targeted therapy options for the treatment of IBD.

Long COVID (LC) describes the clinical phenotype of symptoms after infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnostic and therapeutic options are limited, as the pathomechanism of LC is elusive. As the number of acute SARS-CoV-2 infections was and is large, LC will be a challenge for the healthcare system. Previous studies revealed an impaired blood flow, the formation of microclots, and autoimmune mechanisms as potential factors in this complex interplay. Since functionally active autoantibodies against G-protein-coupled receptors (GPCR-AAbs) were observed in patients after SARS-CoV-2 infection, this study aimed to correlate the appearance of GPCR-AAbs with capillary microcirculation. The seropositivity of GPCR-AAbs was measured by an established cardiomyocyte bioassay in 42 patients with LC and 6 controls. Retinal microcirculation was measured by OCT–angiography and quantified as macula and peripapillary vessel density (VD) by the Erlangen-Angio Tool. A statistical analysis yielded impaired VD in patients with LC compared to the controls, which was accentuated in female persons. A significant decrease in macula and peripapillary VD for AAbs targeting adrenergic β2-receptor, MAS-receptor angiotensin-II-type-1 receptor, and adrenergic α1-receptor were observed. The present study might suggest that a seropositivity of GPCR-AAbs can be linked to an impaired retinal capillary microcirculation, potentially mirroring the systemic microcirculation with consecutive clinical symptoms.

MiRNAs are important epigenetic players with tissue- and disease-specific effects. In this study, our aim was to investigate the putative differential expression of miRNAs in adrenal tissues from different forms of Cushing’s syndrome (CS). For this, miRNA-based next-generation sequencing was performed in adrenal tissues taken from patients with ACTH-independent cortisol-producing adrenocortical adenomas (CPA), from patients with ACTH-dependent pituitary Cushing’s disease (CD) after bilateral adrenalectomy, and from control subjects. A confirmatory QPCR was also performed in adrenals from patients with other CS subtypes, such as primary bilateral macronodular hyperplasia and ectopic CS. Sequencing revealed significant differences in the miRNA profiles of CD and CPA. QPCR revealed the upregulated expression of miR-1247-5p in CPA and PBMAH (log2 fold change > 2.5, p < 0.05). MiR-379-5p was found to be upregulated in PBMAH and CD (log2 fold change > 1.8, p < 0.05). Analyses of miR-1247-5p and miR-379-5p expression in the adrenals of mice which had been exposed to short-term ACTH stimulation showed no influence on the adrenal miRNA expression profiles. For miRNA-specific target prediction, RNA-seq data from the adrenals of CPA, PBMAH, and control samples were analyzed with different bioinformatic platforms. The analyses revealed that both miR-1247-5p and miR-379-5p target specific genes in the WNT signaling pathway. In conclusion, this study identified distinct adrenal miRNAs as being associated with CS subtypes.

Human wild type (wt) cardiac \(\alpha\)-actin and its mutants p.A295S or p.R312H and p.E361G correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by using the \(\textit {baculovirus/Sf21}\) insect cell system. The c-actin variants inhibited DNase I, indicating maintenance of their native state. Electron microscopy showed the formation of normal appearing actin filaments though they showed mutant specific differences in length and straightness correlating with their polymerization rates. TRITC-phalloidin staining showed that p.A295S and p.R312H exhibited reduced and the p.E361G mutant increased lengths of their formed filaments. Decoration of c-actins with cardiac tropomyosin (cTm) and troponin (cTn) conveyed \(Ca^{2+}\)-sensitivity of the myosin-S1 ATPase stimulation, which was higher for the HCM p.A295S mutant and lower for the DCM p.R312H and p.E361G mutants than for wt c-actin. The lower \(Ca^{2+}\)-sensitivity of myosin-S1 stimulation by both DCM actin mutants was corrected by the addition of levosimendan. \(Ca^{2+}\)-dependency of the movement of pyrene-labeled cTm along polymerized c-actin variants decorated with cTn corresponded to the relations observed for the myosin-S1 ATPase stimulation though shifted to lower \(Ca^{2+}\)-concentrations. The N-terminal C0C2 domain of cardiac myosin-binding protein-C increased the \(Ca^{2+}\)-sensitivity of the pyrene-cTM movement of bovine, recombinant wt, p.A295S, and p.E361G c-actins, but not of the p.R312H mutant, suggesting decreased affinity to cTm.

Atrial fibrillation (AF), the most common cardiac arrhythmia worldwide, is driven by complex mechanisms that differ between subgroups of patients. This complexity is apparent from the different forms in which AF presents itself (post-operative, paroxysmal and persistent), each with heterogeneous patterns and variable progression. Our current understanding of the mechanisms responsible for initiation, maintenance and progression of the different forms of AF has increased significantly in recent years. Nevertheless, antiarrhythmic drugs for the management of AF have not been developed based on the underlying arrhythmia mechanisms and none of the currently used drugs were specifically developed to target AF. With the increased knowledge on the mechanisms underlying different forms of AF, new opportunities for developing more effective and safer AF therapies are emerging. In this review, we provide an overview of potential novel antiarrhythmic approaches based on the underlying mechanisms of AF, focusing both on the development of novel antiarrhythmic agents and on the possibility of repurposing already marketed drugs. In addition, we discuss the opportunity of targeting some of the key players involved in the underlying AF mechanisms, such as ryanodine receptor type-2 (RyR2) channels and atrial-selective K+-currents (IK2P and ISK) for antiarrhythmic therapy. In addition, we highlight the opportunities for targeting components of inflammatory signaling (e.g., the NLRP3-inflammasome) and upstream mechanisms targeting fibroblast function to prevent structural remodeling and progression of AF. Finally, we critically appraise emerging antiarrhythmic drug principles and future directions for antiarrhythmic drug development, as well as their potential for improving AF management.

Amyotrophic lateral sclerosis (ALS) is an incurable and lethal neurodegenerative disease in which progressive motor neuron loss and associated inflammation represent major pathology hallmarks. Both the prevention of neuronal loss and neuro-destructive inflammation are still unmet challenges. Medical ozone, an ozonized oxygen mixture (O3/O2), has been shown to elicit profound immunomodulatory effects in peripheral organs, and beneficial effects in the aging brain. We investigated, in a preclinical drug testing approach, the therapeutic potential of a five-day O3/O2 i.p. treatment regime at the beginning of the symptomatic disease phase in the superoxide dismutase (SOD1G93A) ALS mouse model. Clinical assessment of SOD1G93A mice revealed no benefit of medical ozone treatment over sham with respect to gross body weight, motor performance, disease duration, or survival. In the brainstem of end stage SOD1G93A mice, however, neurodegeneration was found decelerated, and SOD1-related vacuolization was reduced in the motor trigeminal nucleus in the O3/O2 treatment group when compared to sham-treated mice. In addition, microglia proliferation was less pronounced in the brainstem, while the hypertrophy of astroglia remained largely unaffected. Finally, monocyte numbers were reduced in the blood, spleen, and mesenteric lymph nodes at postnatal day 60 in SOD1G93A mice. A further decrease in monocyte numbers seen in mesenteric lymph nodes from sham-treated SOD1G93A mice at an advanced disease stage, however, was prevented by medical ozone treatment. Collectively, our study revealed a select neuroprotective and possibly anti-inflammatory capacity for medical ozone when applied as a therapeutic agent in SOD1G93A ALS mice. Keywords: amyotrophic

Vulvar cancer incidence numbers have been steadily rising over the past decades. In particular, the number of young patients with vulvar cancer has recently increased. Therefore, the need to identify new prognostic factors and, in addition, therapeutic options for vulvar carcinoma is more apparent. The aim of this study was to analyze the influx of COX-2 positive tumor-infiltrating lymphocytes and monocytes and their influence on prognosis. Using subtyping by immunofluorescence, the majority of COX-2 expressing immune cells were identified as FOXP3-positive regulatory T cells. In addition, peri- and intra-tumoral macrophages in the same tumor tissue were detected simultaneously as M2-polarized macrophages. COX-2 positive immune cells were independent negative prognostic markers in long-term overall survival of patients with vulvar cancer. These results show an influence of immune cell infiltration for vulvar carcinoma patients. Immune cell infiltration and immune checkpoint expression may, therefore, become interesting targets for further research on new vulvar cancer treatment strategies.

The hypothalamic arcuate nucleus (Arc) is a central unit that controls the appetite through the integration of metabolic, hormonal, and neuronal afferent inputs. Agouti-related protein (AgRP), proopiomelanocortin (POMC), and dopaminergic neurons in the Arc differentially regulate feeding behaviors in response to hunger, satiety, and appetite, respectively. At the time of writing, the anatomical and electrophysiological characterization of these three neurons has not yet been intensively explored. Here, we interrogated the overall characterization of AgRP, POMC, and dopaminergic neurons using genetic mouse models, immunohistochemistry, and whole-cell patch recordings. We identified the distinct geographical location and intrinsic properties of each neuron in the Arc with the transgenic lines labelled with cell-specific reporter proteins. Moreover, AgRP, POMC, and dopaminergic neurons had different firing activities to ghrelin and leptin treatments. Ghrelin led to the increased firing rate of dopaminergic and AgRP neurons, and the decreased firing rate of POMC. In sharp contrast, leptin resulted in the decreased firing rate of AgRP neurons and the increased firing rate of POMC neurons, while it did not change the firing rate of dopaminergic neurons in Arc. These findings demonstrate the anatomical and physiological uniqueness of three hypothalamic Arc neurons to appetite control.

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. The standard of care in medically and physically fit patients is intensive induction therapy. The majority of these intensively treated patients achieve a complete remission. However, a high number of these patients will experience relapse. In patients older than 60 years, the results are even worse. Therefore, new therapeutic approaches are desperately needed. One promising approach in high-risk leukemia to prevent relapse is the induction of the immune system simultaneously or after reduction of the initial tumor burden. Different immunotherapeutic approaches such as allogenic stem cell transplantation or donor lymphocyte infusions are already standard therapies, but other options for AML treatment are in the pipeline. Moreover, the therapeutic landscape in AML is rapidly changing, and in the last years, a number of immunogenic targets structures eligible for specific therapy, risk assessment or evaluation of disease course were determined. For example, leukemia-associated antigens (LAA) showed to be critical as biomarkers of disease state and survival, as well as markers of minimal residual disease (MRD). Yet many mechanisms and properties are still insufficiently understood, which also represents a great potential for this form of therapy. Therefore, targeted therapy as immunotherapy could turn into an efficient tool to clear residual disease, improve the outcome of AML patients and reduce the relapse risk. In this review, established but also emerging immunotherapeutic approaches for AML patients will be discussed., publishedVersion

Transforming growth factor β (TGFβ) signaling has manifold functions such as regulation of cell growth, differentiation, migration, and apoptosis. Moreover, there is increasing evidence that it also acts in a neuroprotective manner. We recently showed that TGFβ receptor type 2 (Tgfbr2) is upregulated in retinal neurons and Müller cells during retinal degeneration. In this study we investigated if this upregulation of TGFβ signaling would have functional consequences in protecting retinal neurons. To this end, we analyzed the impact of TGFβ signaling on photoreceptor viability using mice with cell type-specific deletion of Tgfbr2 in retinal neurons and Müller cells (Tgfbr2ΔOC) in combination with a genetic model of photoreceptor degeneration (VPP). We examined retinal morphology and the degree of photoreceptor degeneration, as well as alterations of the retinal transcriptome. In summary, retinal morphology was not altered due to TGFβ signaling deficiency. In contrast, VPP-induced photoreceptor degeneration was drastically exacerbated in double mutant mice (Tgfbr2ΔOC; VPP) by induction of pro-apoptotic genes and dysregulation of the MAP kinase pathway. Therefore, TGFβ signaling in retinal neurons and Müller cells exhibits a neuroprotective effect and might pose promising therapeutic options to attenuate photoreceptor degeneration in humans.

Immune cell activity is a major factor for disease progression in inflammatory bowel diseases (IBD). Classifying the type and functional state of immune cells is therefore crucial in clinical diagnostics of IBD. Label-free optical technologies exploiting NADH and FAD autofluorescence, such as multiphoton microscopy, have been used to describe tissue morphology in healthy and inflamed colon samples. Nevertheless, a strategy for the identification of single immune cell subtypes within the tissue is yet to be developed. This work aims to initiate an understanding of autofluorescence changes depending on immune cell type and activation state. For this, NADH and FAD autofluorescence signals of different murine immune cell subtypes under native conditions, as well as upon in vitro stimulation and cell death, have been evaluated. Autofluorescence was assessed using flow cytometry and multiphoton microscopy. Our results reveal significantly increased NADH and FAD signals in innate immune cells compared to adaptive immune cells. This allowed identification of relative amounts of neutrophils and CD4+ T cells in mixed cell suspensions, by using NADH signals as a differentiation marker. Furthermore, in vitro stimulation significantly increased NADH and FAD autofluorescence in adaptive immune cells and macrophages. Cell death induced a significant drop in NADH autofluorescence, while FAD signals were hardly affected. Taken together, these results demonstrate the value of autofluorescence as a tool to characterize immune cells in different functional states, paving the way to the label-free clinical classification of IBD in the future.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation technique that has the potential to treat a variety of neurologic and psychiatric disorders. The extent of rTMS-induced neuroplasticity may be dependent on a subject’s brain state at the time of stimulation. Chronic low intensity rTMS (LI-rTMS) has previously been shown to induce beneficial structural and functional reorganisation within the abnormal visual circuits of ephrin-A2A5-/- mice in ambient lighting. Here, we administered chronic LI-rTMS in adult ephrin-A2A5-/- mice either in a dark environment or concurrently with voluntary locomotion. One day after the last stimulation session, optokinetic responses were assessed and fluorescent tracers were injected to map corticotectal and geniculocortical projections. We found that LI-rTMS in either treatment condition refined the geniculocortical map. Corticotectal projections were improved in locomotion+LI-rTMS subjects, but not in dark + LI-rTMS and sham groups. Visuomotor behaviour was not improved in any condition. Our results suggest that the beneficial reorganisation of abnormal visual circuits by rTMS can be significantly influenced by simultaneous, ambient visual input and is enhanced by concomitant physical exercise. Furthermore, the observed pathway-specific effects suggest that regional molecular changes and/or the relative proximity of terminals to the induced electric fields influence the outcomes of LI-rTMS on abnormal circuitry.

The pleiotropic function of 3′,5′-cyclic adenosine monophosphate (cAMP)-dependent pathways in health and disease led to the development of pharmacological phosphodiesterase inhibitors (PDE-I) to attenuate cAMP degradation. While there are many isotypes of PDE, a predominant role of PDE4 is to regulate fundamental functions, including endothelial and epithelial barrier stability, modulation of inflammatory responses and cognitive and/or mood functions. This makes the use of PDE4-I an interesting tool for various therapeutic approaches. However, due to the presence of PDE4 in many tissues, there is a significant danger for serious side effects. Based on this, the aim of this review is to provide a comprehensive overview of the approaches and effects of PDE4-I for different therapeutic applications. In summary, despite many obstacles to use of PDE4-I for different therapeutic approaches, the current data warrant future research to utilize the therapeutic potential of phosphodiesterase 4 inhibition.

During inflammatory responses, neutrophils enter the sites of attack where they execute various defense mechanisms. They (I) phagocytose microorganisms, (II) degranulate to release cytokines, (III) recruit various immune cells by cell-type specific chemokines, (IV) secrete anti-microbials including lactoferrin, lysozyme, defensins and reactive oxygen species, and (V) release DNA as neutrophil extracellular traps (NETs). The latter originates from mitochondria as well as from decondensed nuclei. This is easily detected in cultured cells by staining of DNA with specific dyes. However, in tissues sections the very high fluorescence signals emitted from the condensed nuclear DNA hamper the detection of the widespread, extranuclear DNA of the NETs. In contrast, when we employ anti-DNA-IgM antibodies, they are unable to penetrate deep into the tightly packed DNA of the nucleus, and we observe a robust signal for the extended DNA patches of the NETs. To validate anti-DNA-IgM, we additionally stained the sections for the NET-markers histone H2B, myeloperoxidase, citrullinated histone H3, and neutrophil elastase. Altogether, we have described a fast one-step procedure for the detection of NETs in tissue sections, which provides new perspectives to characterize neutrophil-associated immune reactions in disease.

BAMBI (bone morphogenetic protein and activin membrane-bound inhibitor) is a transmembrane pseudoreceptor structurally related to transforming growth factor (TGF)-β type 1 receptors (TGF-β1Rs). BAMBI lacks a kinase domain and functions as a TGF-β1R antagonist. Essential processes such as cell differentiation and proliferation are regulated by TGF-β1R signaling. TGF-β is the best-studied ligand of TGF-βRs and has an eminent role in inflammation and fibrogenesis. Liver fibrosis is the end stage of almost all chronic liver diseases, such as non-alcoholic fatty liver disease, and at the moment, there is no effective anti-fibrotic therapy available. Hepatic BAMBI is downregulated in rodent models of liver injury and in the fibrotic liver of patients, suggesting that low BAMBI has a role in liver fibrosis. Experimental evidence convincingly demonstrated that BAMBI overexpression is able to protect against liver fibrosis. Chronic liver diseases have a high risk of hepatocellular carcinoma (HCC), and BAMBI was shown to exert tumor-promoting as well as tumor-protective functions. This review article aims to summarize relevant studies on hepatic BAMBI expression and its role in chronic liver diseases and HCC.

Immunohistochemical evaluation of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2 status stratify the different subtypes of breast cancer and define the treatment course. Triple-negative breast cancer (TNBC), which does not register receptor overexpression, is often associated with worse patient prognosis. Mass spectrometry imaging transcribes the molecular content of tissue specimens without requiring additional tags or preliminary analysis of the samples, being therefore an excellent methodology for an unbiased determination of tissue constituents, in particular tumor markers. In this study, the proteomic content of 1191 human breast cancer samples was characterized by mass spectrometry imaging and the epithelial regions were employed to train and test machine-learning models to characterize the individual receptor status and to classify TNBC. The classification models presented yielded high accuracies for estrogen and progesterone receptors and over 95% accuracy for classification of TNBC. Analysis of the molecular features revealed that vimentin overexpression is associated with TNBC, supported by immunohistochemistry validation, revealing a new potential target for diagnosis and treatment.

The development of chimeric antigen receptor T cells (CAR-T cells) has marked a new era in cancer immunotherapy. Based on a multitude of durable complete remissions in patients with hematological malignancies, FDA and EMA approval was issued to several CAR products targeting lymphoid leukemias and lymphomas. Nevertheless, about 50% of patients treated with these approved CAR products experience relapse or refractory disease necessitating salvage strategies. Moreover, in the vast majority of patients suffering from solid tumors, CAR-T-cell infusions could not induce durable complete remissions so far. Crucial obstacles to CAR-T-cell therapy resulting in a priori CAR-T-cell refractory disease or relapse after initially successful CAR-T-cell therapy encompass antigen shutdown and CAR-T-cell dysfunctionality. Antigen shutdown predominately rationalizes disease relapse in hematological malignancies, and CAR-T-cell dysfunctionality is characterized by insufficient CAR-T-cell proliferation and cytotoxicity frequently observed in patients with solid tumors. Thus, strategies to surmount those obstacles are being developed with high urgency. In this review, we want to highlight different approaches to combine CAR-T cells with drugs, such as small molecules and antibodies, to pharmacologically boost CAR-T-cell therapy. In particular, we discuss how certain drugs may help to counteract antigen shutdown and CAR-T-cell dysfunctionality in both hematological malignancies and solid tumors.

There is a paucity of empirical research on the use of non-pharmacological interventions to both treat and curb the spread of Alzheimer’s disease (AD) across the globe. This paper examines the biochemical and clinical outlook and the social implications of the condition in relation to psychological aspects that may indicate a direction for further interventions. There is a scarcity of research on the effectiveness of using various psychological aspects of AD, a disease characterized by a process of transition from health and independence to a dependent state with a progressive loss of memory and functional skills. The paper investigates the biochemical and psychological aspects of AD and their significance for improving quality of life for patients with this disease. Psychological interventions based on, among other factors, biochemical studies, are conducted to improve the emotional wellbeing of AD patients and may assist in slowing down the progression of the disease. To date, however, no effective methods of AD treatment have been established.

Osteoporosis is a chronical, systemic skeletal disorder characterized by an increase in bone resorption, which leads to reduced bone density. The reduction in bone mineral density and therefore low bone mass results in an increased risk of fractures. Osteoporosis is caused by an imbalance in the normally strictly regulated bone homeostasis. This imbalance is caused by overactive bone-resorbing osteoclasts, while bone-synthesizing osteoblasts do not compensate for this. In this review, the mechanism is presented, underlined by in vitro and animal models to investigate this imbalance as well as the current status of clinical trials. Furthermore, new therapeutic strategies for osteoporosis are presented, such as anabolic treatments and catabolic treatments and treatments using biomaterials and biomolecules. Another focus is on new combination therapies with multiple drugs which are currently considered more beneficial for the treatment of osteoporosis than monotherapies. Taken together, this review starts with an overview and ends with the newest approaches for osteoporosis therapies and a future perspective not presented so far.

While type 2 immunity has traditionally been associated with the control of parasitic infections and allergic reactions, increasing evidence suggests that type 2 immunity exerts regulatory functions on inflammatory diseases such as arthritis, and also on bone homeostasis. This review summarizes the current evidence of the regulatory role of type 2 immunity in arthritis and bone. Key type 2 cytokines, like interleukin (IL)-4 and IL-13, but also others such as IL-5, IL-9, IL-25, and IL-33, exert regulatory properties on arthritis, dampening inflammation and inducing resolution of joint swelling. Furthermore, these cytokines share anti-osteoclastogenic properties and thereby reduce bone resorption and protect bone. Cellular effectors of this action are both T cells (i.e., Th2 and Th9 cells), but also non-T cells, like type 2 innate lymphoid cells (ILC2). Key regulatory actions mediated by type 2 cytokines and immune cells on both inflammation as well as bone homeostasis are discussed.

The phenomenon of individual variability in susceptibility/resilience to stress and depression, in which the hippocampus plays a pivotal role, is attracting increasing attention. We investigated the potential role of hippocampal cyclooxygenase-2 (COX-2), which regulates plasticity, neuroimmune function, and stress responses that are all linked to this risk dichotomy. We used a four-week-long chronic mild stress (CMS) paradigm, in which mice could be stratified according to their susceptibility/resilience to anhedonia, a key feature of depression, to investigate hippocampal expression of COX-2, a marker of microglial activation Iba-1, and the proliferation marker Ki67. Rat exposure, social defeat, restraints, and tail suspension were used as stressors. We compared the effects of treatment with either the selective COX-2 inhibitor celecoxib (30 mg/kg/day) or citalopram (15 mg/kg/day). For the celecoxib and vehicle-treated mice, the Porsolt test was used. Anhedonic (susceptible) but not non-anhedonic (resilient) animals exhibited elevated COX-2 mRNA levels, increased numbers of COX-2 and Iba-1-positive cells in the dentate gyrus and the CA1 area, and decreased numbers of Ki67-positive cells in the subgranular zone of the hippocampus. Drug treatment decreased the percentage of anhedonic mice, normalized swimming activity, reduced behavioral despair, and improved conditioned fear memory. Hippocampal over-expression of COX-2 is associated with susceptibility to stress-induced anhedonia, and its pharmacological inhibition with celecoxib has antidepressant effects that are similar in size to those of citalopram.

Parkinson’s disease (PD) is neuropathologically characterized by the loss of dopaminergic neurons and the deposition of aggregated alpha synuclein (aSyn). Mounting evidence suggests that neuritic degeneration precedes neuronal loss in PD. A possible underlying mechanism could be the interference of aSyn with microtubule organization in the neuritic development, as implied by several studies using cell-free model systems. In this study, we investigate the impact of aSyn on microtubule organization in aSyn overexpressing H4 neuroglioma cells and midbrain dopaminergic neuronal cells (mDANs) generated from PD patient-derived human induced pluripotent stem cells (hiPSCs) carrying an aSyn gene duplication (SNCADupl). An unbiased mass spectrometric analysis reveals a preferential binding of aggregated aSyn conformers to a number of microtubule elements. We confirm the interaction of aSyn with beta tubulin III in H4 and hiPSC-derived mDAN cell model systems, and demonstrate a remarkable redistribution of tubulin isoforms from the soluble to insoluble fraction, accompanied by a significantly increased insoluble aSyn level. Concordantly, SNCADupl mDANs show impaired neuritic phenotypes characterized by perturbations in neurite initiation and outgrowth. In summary, our findings suggest a mechanistic pathway, through which aSyn aggregation interferes with microtubule organization and induces neurite impairments.

The clinical symptoms of shigellosis, a gastrointestinal infection caused by Shigella spp. range from watery diarrhea to fulminant dysentery. Endemic infections, particularly among children in developing countries, represent the majority of clinical cases. The situation is aggravated due to the high mortality rate of shigellosis, the rapid dissemination of multi-resistant Shigella strains and the induction of only serotype-specific immunity. Thus, infection prevention due to vaccination, encompassing as many of the circulating serotypes as possible, has become a topic of interest. However, vaccines have turned out to be ineffective so far. Outer membrane vesicles (OMVs) are promising novel targets for vaccination. OMVs are constitutively secreted by Gram-negative bacteria including Shigella during growth. They are composed of soluble luminal portions and an insoluble membrane and can contain toxins, bioactive periplasmic and cytoplasmic (lipo-) proteins, (phospho-) lipids, nucleic acids and/or lipopolysaccharides. Thus, OMVs play an important role in bacterial cell–cell communication, growth, survival and pathogenesis. Furthermore, they modulate the secretion and transport of biomolecules, the stress response, antibiotic resistance and immune responses of the host. Thus, OMVs serve as novel secretion machinery. Here, we discuss the current literature and highlight the properties of OMVs as potent vaccine candidates because of their immunomodulatory, antigenic and adjuvant properties.

The gap junction protein connexin 43 (Cx43) is associated with increased cell migration and to related changes of the actin cytoskeleton, which is mediated via its C-terminal cytoplasmic tail and is independent of its channel function. Cx43 has been shown to possess an angiogenic potential, however, the role of Cx43 in endothelial cell migration has not yet been investigated. Here, we found that the knock-down of Cx43 by siRNA in human microvascular endothelial cells (HMEC) reduces migration, as assessed by a wound assay in vitro and impaired aortic vessel sprouting ex vivo. Immunoprecipitation of Cx43 revealed an interaction with the tyrosine phosphatase SHP-2, which enhanced its phosphatase activity, as observed in Cx43 expressing HeLa cells compared to cells treated with an empty vector. Interestingly, the expression of a dominant negative substrate trapping mutant SHP-2 (CS) in HMEC, via lentiviral transduction, also impaired endothelial migration to a similar extent as Cx43 siRNA compared to SHP-2 WT. Moreover, the reduction in endothelial migration upon Cx43 siRNA could not be rescued by the introduction of a constitutively active SHP-2 construct (EA). Our data demonstrate that Cx43 and SHP-2 mediate endothelial cell migration, revealing a novel interaction between Cx43 and SHP-2, which is essential for this process.

The COVID-19 pandemic continues to spread around the world and remains a major public health threat. Vaccine inefficiency, vaccination breakthroughs and lack of supply, especially in developing countries, as well as the fact that a non-negligible part of the population either refuse vaccination or cannot be vaccinated due to age, pre-existing illness or non-response to existing vaccines intensify this issue. This might also contribute to the emergence of new variants, being more efficiently transmitted, more virulent and more capable of escaping naturally acquired and vaccine-induced immunity. Hence, the need of effective and viable prevention options to reduce viral transmission is of outmost importance. In this study, we investigated the antiviral effect of iota-, lambda- and kappa-carrageenan, sulfated polysaccharides extracted from red seaweed, on SARS-CoV-2 Wuhan type and the spreading variants of concern (VOCs) Alpha, Beta, Gamma and Delta. Carrageenans as part of broadly used nasal and mouth sprays as well as lozenges have the potential of first line defense to inhibit the infection and transmission of SARS-CoV-2. Here, we demonstrate by using a SARS-CoV-2 spike pseudotyped lentivirus particles (SSPL) system and patient-isolated SARS-CoV-2 VOCs to infect transgenic A549ACE2/TMPRSS2 and Calu-3 human lung cells that all three carrageenan types exert antiviral activity. Iota-carrageenan exhibits antiviral activity with comparable IC50 values against the SARS-CoV-2 Wuhan type and the VOCs. Altogether, these results indicate that iota-carrageenan might be effective for prophylaxis and treatment of SARS-CoV-2 infections independent of the present and potentially future variants., publishedVersion

Integrin beta 7 (β7), a subunit of the integrin receptor, is expressed on the surface of immune cells and mediates cell–cell adhesions and interactions, e.g., antitumor or autoimmune reactions. Here, we analyzed, whether the stimulation of immune cells by dendritic cells (of leukemic derivation in AML patients or of monocyte derivation in healthy donors) leads to increased/leukemia-specific β7 expression in immune cells after T-cell-enriched mixed lymphocyte culture—finally leading to improved antileukemic cytotoxicity. Healthy, as well as AML and MDS patients’ whole blood (WB) was treated with Kit-M (granulocyte–macrophage colony-stimulating factor (GM-CSF) + prostaglandin E1 (PGE1)) or Kit-I (GM-CSF + Picibanil) in order to generate DCs (DCleu or monocyte-derived DC), which were then used as stimulator cells in MLC. To quantify antigen/leukemia-specific/antileukemic functionality, a degranulation assay (DEG), an intracellular cytokine assay (INTCYT) and a cytotoxicity fluorolysis assay (CTX) were used. (Leukemia-specific) cell subtypes were quantified via flow cytometry. The Kit treatment of WB (compared to the control) resulted in the generation of DC/DCleu, which induced increased activation of innate and adaptive cells after MLC. Kit-pretreated WB (vs. the control) led to significantly increased frequencies of β7-expressing T-cells, degranulating and intracellular cytokine-producing β7-expressing immune cells and, in patients’ samples, increased blast lysis. Positive correlations were found between the Kit-M-mediated improvement of blast lysis (vs. the control) and frequencies of β7-expressing T-cells. Our findings indicate that DC-based immune therapies might be able to specifically activate the immune system against blasts going along with increased frequencies of (leukemia-specific) β7-expressing immune cells. Furthermore, β7 might qualify as a predictor for the efficiency and the success of AML and/or MDS therapies.

Excess dietary salt reduces resting cerebral blood flow (CBF) and vascular reactivity, which can limit the fueling of neuronal metabolism. It is hitherto unknown whether metabolic derangements induced by high-salt-diet (HSD) exposure during adulthood are reversed by reducing salt intake. In this study, male and female mice were fed an HSD from 9 to 16 months of age, followed by a normal-salt diet (ND) thereafter until 23 months of age. Controls were continuously fed either ND or HSD. CBF and metabolite profiles were determined longitudinally by arterial spin labeling magnetic resonance imaging and magnetic resonance spectroscopy, respectively. HSD reduced cortical and hippocampal CBF, which recovered after dietary salt normalization, and affected hippocampal but not cortical metabolite profiles. Compared to ND, HSD increased hippocampal glutamine and phosphocreatine levels and decreased creatine and choline levels. Dietary reversal only allowed recovery of glutamine levels. Histology analyses revealed that HSD reduced the dendritic arborization and spine density of cortical and hippocampal neurons, which were not recovered after dietary salt normalization. We conclude that sustained HSD exposure throughout adulthood causes permanent structural and metabolic alterations to the mouse brain that are not fully normalized by lowering dietary salt during aging.

BRAF and MEK inhibitor (BRAFi/MEKi) combinations are currently the standard treatment for patients with BRAFV600 mutant metastatic melanoma. Since the RAS/RAF/MEK/ERK-pathway is crucial for the function of different immune cells, we postulated an effect on their function and thus interference with anti-tumor immunity. Therefore, we examined the influence of BRAFi/MEKi, either as single agent or in combination, on the maturation of monocyte-derived dendritic cells (moDCs) and their interaction with T cells. DCs matured in the presence of vemurafenib or vemurafenib/cobimetinib altered their cytokine secretion and surface marker expression profile. Upon the antigen-specific stimulation of CD8+ and CD4+ T cells with these DCs or with T2.A1 cells in the presence of BRAFi/MEKi, we detected a lower expression of activation markers on and a lower cytokine secretion by these T cells. However, treatment with any of the inhibitors alone or in combination did not change the avidity of CD8+ T cells in peptide titration assays with T2.A1 cells. T-helper cell/DC interaction is a bi-directional process that normally results in DC activation. Vemurafenib and vemurafenib/cobimetinib completely abolished the helper T-cell-mediated upregulation of CD70, CD80, and CD86 but not CD25 on the DCs. The combination of dabrafenib/trametinib affected DC maturation and activation as well as T-cell activation less than combined vemurafenib/cobimetinib did. Hence, for a potential combination with immunotherapy, our data indicate the superiority of dabrafenib/trametinib treatment.

The aim of this study was to analyze the expression of peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RxRα), a binding heterodimer playing a pivotal role in the successful trophoblast invasion, in the placental tissue of preeclamptic patients. Furthermore, we aimed to characterize a possible interaction between PPARγ and H3K4me3 (trimethylated lysine 4 of the histone H3), respectively H3K9ac (acetylated lysine 9 of the histone H3), to illuminate the role of histone modifications in a defective trophoblast invasion in preeclampsia (PE). Therefore, the expression of PPARγ and RxRα was analyzed in 26 PE and 25 control placentas by immunohistochemical peroxidase staining, as well as the co-expression with H3K4me3 and H3K9ac by double immunofluorescence staining. Further, the effect of a specific PPARγ-agonist (Ciglitazone) and PPARγ-antagonist (T0070907) on the histone modifications H3K9ac and H3K4me3 was analyzed in vitro. In PE placentas, we found a reduced expression of PPARγ and RxRα and a reduced co-expression with H3K4me3 and H3K9ac in the extravillous trophoblast (EVT). Furthermore, with the PPARγ-antagonist treated human villous trophoblast (HVT) cells and primary isolated EVT cells showed higher levels of the histone modification proteins whereas treatment with the PPARγ-agonist reduced respective histone modifications. Our results show that the stimulation of PPARγ-activity leads to a reduction of H3K4me3 and H3K9ac in trophoblast cells, but paradoxically decreases the nuclear PPARγ expression. As the importance of PPARγ, being involved in a successful trophoblast invasion has already been investigated, our results reveal a pathophysiologic connection between PPARγ and the epigenetic modulation via H3K4me3 and H3K9ac in PE.

There is a lack of knowledge regarding the connection between the ocular and nasal epithelia. This narrative review focuses on conjunctival, corneal, ultrastructural corneal stroma, and nasal epithelia as well as an introduction into their interconnections. We describe in detail the morphology and physiology of the ocular surface, the nasolacrimal ducts, and the nasal cavity. This knowledge provides a basis for functional studies and the development of relevant cell culture models that can be used to investigate the pathogenesis of diseases related to these complex structures. Moreover, we also provide a state-of-the-art overview regarding the development of 3D culture models, which allow for addressing research questions in models resembling the in vivo situation. In particular, we give an overview of the current developments of corneal 3D and organoid models, as well as 3D cell culture models of epithelia with goblet cells (conjunctiva and nasal cavity). The benefits and shortcomings of these cell culture models are discussed. As examples for pathogens related to ocular and nasal epithelia, we discuss infections caused by adenovirus and measles virus. In addition to pathogens, also external triggers such as allergens can cause rhinoconjunctivitis. These diseases exemplify the interconnections between the ocular surface and nasal epithelia in a molecular and clinical context. With a final translational section on optical coherence tomography (OCT), we provide an overview about the applicability of this technique in basic research and clinical ophthalmology. The techniques presented herein will be instrumental in further elucidating the functional interrelations and crosstalk between ocular and nasal epithelia.

Human cytomegalovirus (HCMV) is a major pathogenic herpesvirus that is prevalent worldwide and it is associated with a variety of clinical symptoms. Current antiviral therapy options do not fully satisfy the medical needs; thus, improved drug classes and drug-targeting strategies are required. In particular, host-directed antivirals, including pharmaceutical kinase inhibitors, might help improve the drug qualities. Here, we focused on utilizing PROteolysis TArgeting Chimeras (PROTACs), i.e., hetero-bifunctional molecules containing two elements, namely a target-binding molecule and a proteolysis-inducing element. Specifically, a PROTAC that was based on a cyclin-dependent kinase (CDK) inhibitor, i.e., CDK9-directed PROTAC THAL-SNS032, was analyzed and proved to possess strong anti-HCMV AD169-GFP activity, with values of EC50 of 0.030 µM and CC50 of 0.175 µM (SI of 5.8). Comparing the effect of THAL-SNS032 with its non-PROTAC counterpart SNS032, data indicated a 3.7-fold stronger anti-HCMV efficacy. This antiviral activity, as illustrated for further clinically relevant strains of human and murine CMVs, coincided with the mid-nanomolar concentration range necessary for a drug-induced degradation of the primary (CDK9) and secondary targets (CDK1, CDK2, CDK7). In addition, further antiviral activities were demonstrated, such as the inhibition of SARS-CoV-2 replication, whereas other investigated human viruses (i.e., varicella zoster virus, adenovirus type 2, and Zika virus) were found insensitive. Combined, the antiviral quality of this approach is seen in its (i) mechanistic uniqueness; (ii) future options of combinatorial drug treatment; (iii) potential broad-spectrum activity; and (iv) applicability in clinically relevant antiviral models. These novel data are discussed in light of the current achievements of anti-HCMV drug development.