Towards a mouse model of depression : a psychoneuroendocrine approach

Chronic stress is considered a vulnerability factor for depression. A key symptom is anhedonia; a reduced response to positive stimuli. Drugs are effective for only 20-40% of the patients and new drugs are urgently needed. The objective of the research was to develop a mouse model of depression that would express anhedonia, induced by chronic stress. Mice were repeatedly exposed to the non-physical presence of a rat. Alterations in stress system activity were measured. Anhedonia was assessed by studying the behavioral response to positive stimuli. As a potential therapeutical approach we assessed reward expectation, and studied the effect of repeated administration of mifepristone (glucocorticoid receptor antagonist), directly targeting stress system regulation. Our model induced changes in the sensitivity of the reward system that contributed to cognitive impairments underlying anhedonia. The effects could partially be restored by additional reward. Mifepristone in na_ve mice suppressed stress system activity, which could indicate a similar direction of effects in stressed mice if provided. Concluding, our chronic stress mouse model induces anhedonia. The new methodology to reduce stress by either providing additional positive stimuli or mifepristone, increases the well being of the mice and may prove a new drug target to treat depression in humans.UBL - phd migration 201

Can sleep and resting behaviours be used as indicators of welfare in shelter dogs (Canis lupusfamiliaris)?

Previous research on humans and animals suggests that the analysis of sleep patterns may reliably inform us about welfare status, but little research of this kind has been carried out for non-human animals in an applied context. This study explored the use of sleep and resting behaviour as indicators of welfare by describing the activity patterns of dogs (Canis lupus familiaris) housed in rescue shelters, and comparing their sleep patterns to other behavioural and cognitive measures of welfare. Sleep and activity patterns were observed over five non-consecutive days in a population of 15 dogs. Subsequently, the characteristics of sleep and resting behaviour were described and the impact of activity on patterns of sleep and resting behaviour analysed. Shelter dogs slept for 2.8% of the day, 14.3% less than previously reported and experienced less sleep fragmentation at night (32 sleep bouts). There were no statistically significant relationships between behaviours exhibited during the day and sleep behaviour. A higher proportion of daytime resting behaviour was significantly associated with a positive judgement bias, less repetitive behaviour and increased time spent coded as ‘relaxed’ across days by shelter staff. These results suggest that, in the context of a busy shelter environment, the ability to rest more during the day could be a sign of improved welfare. Considering the non-linear relationship between sleep and welfare in humans, the relationship between sleep and behavioural indicators of welfare, including judgement bias, in shelter dogs may be more complex than this study could detect

Post-Training Reward Partially Restores Chronic Stress Induced Effects in Mice

Reduced responsiveness to positive stimuli is a core symptom of depression, known as anhedonia. In the present study, we assessed the expression of anhedonia in our chronic stress mouse model using a subset of read-out parameters. In line with this, we investigated in how far chronic stress would affect the facilitating effect of post-training self-administration of sugar, as we previously observed in naïve mice. Male C57BL/6J mice were repeatedly and at unpredictable times exposed to rats (no physical contact) over the course of two weeks. Following novelty exploration, (non-) spatial learning and memory processes with and without post-training sugar acting as reinforcer, emotionality, reward sensitivity and corticosterone levels were determined. We found that (1) the effects of chronic stress persisted beyond the period of the actual rat exposure. (2) Post-training self-administration of sugar as reinforcer improved spatial performance in naïve mice, whereas (3) in stressed mice sugar partially “normalized” the impaired performance to the level of controls without sugar. Chronic stress (4) increased behavioral inhibition in response to novelty; (5) induced dynamic changes in the pattern of circadian corticosterone secretion during the first week after rat stress and (6) increased the intake of sucrose and water. (7) Chronic stress and sugar consumed during spatial training facilitated the memory for the location of the sucrose bottle weeks later. Concluding, our chronic stress paradigm induces the expression of anhedonia in mice, at different levels of behavior. The behavioral inhibition appears to be long lasting in stressed mice. Interestingly, sugar consumed in close context with spatial learning partially rescued the stress-induced emotional and cognitive impairments. This suggests that reward can ameliorate part of the negative consequences of chronic stress on memory

Hyperactivation of the PI3K pathway in inborn errors of immunity:current understanding and therapeutic perspectives

The phosphoinositide-3-kinase (PI3K) pathway function is crucial to the normal development, differentiation, and function of immune cells including B, T, and NK cells. Following the description of two cohorts of patients with an inboirn error of immunity (also known as primary immunodeficiency) with gain-of-function variants in the PIK3CD gene a decade ago, the disease entity activated PI3K delta syndrome (APDS) was named. Since then, many more patients with PIK3CD variants have been described, and loss-of-function variants in PIK3R1 and PTEN have also been linked to APDS. Importantly, the availability of small molecules that inhibit the PI3K pathway has enabled targeted treatment of APDS patients. In this review, we define (i) the PI3K pathway and its role in inborn errors of immunity; (ii) the clinical and immunological presentation of APDS1 (PIK3CD GOF), APDS2 (PIK3R1 LOF), and related disorders; (iii) Diagnostic approaches to identify and functionally validate the genetic causes of disease; (iv) therapeutic interventions to target PI3K hyperactivation; and finally (v) current challenges and future perspectives that require attention for the optimal treatment of patients with APDS and APDS-L diseases.</p

Side-by-side comparison of the two widely studied GRPR radiotracers, radiolabeled NeoB and RM2, in a preclinical setting

Introduction: NeoB and RM2 are the most investigated gastrin-releasing peptide receptor (GRPR)–targeting radiotracers in preclinical and clinical studies. Therefore, an extensive side-by-side comparison of the two radiotracers is valuable to demonstrate whether one has advantages over the other. Accordingly, this study aims to compare the in vitro and in vivo characteristics of radiolabeled NeoB and RM2 to guide future clinical studies. Method: The stability of the radiolabeled GRPR analogs was determined in phosphate buffered saline (PBS), and commercially available mouse and human serum. Target affinity was determined by incubating human prostate cancer PC-3 cells with [177Lu]Lu-NeoB or [177Lu]Lu-RM2, + / − increasing concentrations of unlabeled NeoB, RM2, or Tyr4-bombesin (BBN). To determine uptake and specificity cells were incubated with [177Lu]Lu-NeoB or [177Lu]Lu-RM2 + / − Tyr4-BBN. Moreover, in vivo studies were performed to determine biodistribution and pharmacokinetics. Finally, radiotracer binding to various GRPR-expressing human cancer tissues was investigated. Results: Both radiotracers demonstrated high stability in PBS and human serum, but stability in mouse serum decreased substantially over time. Moreover, both radiotracers demonstrated high GRPR affinity and specificity, but a higher uptake of [177Lu]Lu-NeoB was observed in in vitro studies. In vivo, no difference in tumor uptake was seen. The most prominent difference in uptake in physiological organs was observed in the GRPR-expressing pancreas; [177Lu]Lu-RM2 had less pancreatic uptake and a shorter pancreatic half-life than [177Lu]Lu-NeoB. Furthermore, [177Lu]Lu-RM2 presented with a lower tumor-to-kidney ratio, while the tumor-to-blood ratio was lower for [177Lu]Lu-NeoB. The autoradiography studies revealed higher binding of radiolabeled NeoB to all human tumor tissues. Conclusion: Based on these findings, we conclude that the in vivo tumor-targeting capability of radiolabeled NeoB and RM2 is similar. Additional studies are needed to determine whether the differences observed in physiological organ uptakes, i.e., the pancreas, kidneys, and blood, result in relevant differences in organ absorbed doses when the radiotracers are applied for therapeutic purposes.</p

Promoter Hypermethylation-Related Reduced Somatostatin Production Promotes Uncontrolled Cell Proliferation in Colorectal Cancer.

BACKGROUND: Somatostatin (SST) has anti-proliferative and pro-apoptotic effects. Our aims were to analyze and compare the SST expression during normal aging and colorectal carcinogenesis at mRNA and protein levels. Furthermore, we tested the methylation status of SST in biopsy samples, and the cell growth inhibitory effect of the SST analogue octreotide in human colorectal adenocarcinoma cell line. METHODS: Colonic samples were collected from healthy children (n1 = 6), healthy adults (n2 = 41) and colorectal cancer patients (CRCs) (n3 = 34) for SST mRNA expression analysis, using HGU133 Plus2.0 microarrays. Results were validated both on original (n1 = 6; n2 = 6; n3 = 6) and independent samples ((n1 = 6; n2 = 6; n3 = 6) by real-time PCR. SST expressing cells were detected by immunohistochemistry on colonic biopsy samples (n1 = 14; n2 = 20; n3 = 23). The effect of octreotide on cell growth was tested on Caco-2 cell line. SST methylation percentage in biopsy samples (n1 = 5; n2 = 5; n3 = 9) was defined using methylation-sensitive restriction enzyme digestion. RESULTS: In case of normal aging SST mRNA expression did not alter, but decreased in cancer (p<0.05). The ratio of SST immunoreactive cells was significantly higher in children (0.70%+/-0.79%) compared to CRC (0%+/-0%) (p<0.05). Octreotide significantly increased the proportion of apoptotic Caco-2 cells. SST showed significantly higher methylation level in tumor samples (30.2%+/-11.6%) compared to healthy young individuals (3.5%+/-1.9%) (p<0.05). CONCLUSIONS: In cancerous colonic mucosa the reduced SST production may contribute to the uncontrolled cell proliferation. Our observation that in colon cancer cells octreotide significantly enhanced cell death and attenuated cell proliferation suggests that SST may act as a regulator of epithelial cell kinetics. The inhibition of SST expression in CRC can be epigenetically regulated by promoter hypermethylation

Update on inborn errors of immunity

Ever since the first description of an inherited immunodeficiency in 1952 in a boy with gammaglobulin deficiency, new insights have progressed rapidly in disorders that are now referred to as inborn errors of immunity. In a field where fundamental molecular biology, genetics, immune signaling, and clinical care are tightly intertwined, 2022-24 saw a multitude of advances. Here we report a selection of research updates with a main focus on (1) diagnosis and screening, (2) new genetic defects, (3) susceptibility to severe coronavirus disease 2019 infection and impact of vaccination, and (4) treatment. Importantly, new pathogenic insights more rapidly affect treatment outcomes, either through an earlier and more precise diagnosis or through implementation of novel, personalized treatment. The field is growing rapidly, so awareness, communication, and collaboration are key to improving treatment outcomes.</p

Simultaneous Protein Quantitation and Glycosylation Profiling of Antigen-Specific Immunoglobulin G1 in Large Clinical Studies

Antibodies have a key role in the immune system, making their characterization essential to biomedical, biopharmaceutical, and clinical research questions. Antibody effector functions are mainly controlled by quantity, subclass, and Fc glycosylation. We describe an integrated method to measure these three critical dimensions simultaneously. The subclass-specific immunoglobulin G (IgG) Fc glycosylation analysis combines immunosorbance with glycopeptide-centered LC-MS detection. For integrated IgG1-specific quantitation, a commercial, stable isotope labeled IgG1 protein standard was spiked into the immunosorbent eluates. Robust quantitation was achieved, relying on a combination of a proteotypic peptide and the most abundant glycopeptides, generated through proteolytic cleavage from a mixture of natural IgG1 and the recombinant IgG1 standard. Method performance was demonstrated in a large coronavirus vaccination cohort at a throughput of 100 samples/day. LC-MS-derived, anti-SARS-CoV-2 spike protein IgG1 concentrations ranged from 100 to 10000 ng/mL and correlated well with a clinically relevant immunoassay. Technical variation was 200 times lower than biological variation; intermediate precision was 44%. In conclusion, we present a method capable of robustly and simultaneously assessing quantity, subclass, and Fc glycosylation of antigen-specific IgG in large clinical studies. This method will facilitate a broader understanding of immune responses, especially the important interplay among the three dimensions

Bioinformatic meta-analysis reveals novel differentially expressed genes and pathways in sarcoidosis

Introduction: Sarcoidosis is a multi-system inflammatory disease of unknown origin with heterogeneous clinical manifestations varying from a single organ non-caseating granuloma site to chronic systemic inflammation and fibrosis. Gene expression studies have suggested several genes and pathways implicated in the pathogenesis of sarcoidosis, however, due to differences in study design and variable statistical approaches, results were frequently not reproducible or concordant. Therefore, meta-analysis of sarcoidosis gene-expression datasets is of great importance to robustly establish differentially expressed genes and signalling pathways. Methods: We performed meta-analysis on 22 published gene-expression studies on sarcoidosis. Datasets were analysed systematically using same statistical cut-offs. Differentially expressed genes were identified by pooling of p-values using Edgington’s method and analysed for pathways using Ingenuity Pathway Analysis software. Results: A consistent and significant signature of novel and well-known genes was identified, those collectively implicated both type I and type II interferon mediated signalling pathways in sarcoidosis. In silico functional analysis showed consistent downregulation of eukaryotic initiation factor 2 signalling, whereas cytokines like interferons and transcription factor STAT1 were upregulated. Furthermore, we analysed affected tissues to detect differentially expressed genes likely to be involved in granuloma biology. This revealed that matrix metallopeptidase 12 was exclusively upregulated in affected tissues, suggesting a crucial role in disease pathogenesis. Discussion: Our analysis provides a concise gene signature in sarcoidosis and expands our knowledge about the pathogenesis. Our results are of importance to improve current diagnostic approaches and monitoring strategies as well as in the development of targeted therapeutics.</p