Transcription Of Immune Genes Research Articles

BRAFV600E/pTERT double mutated papillary thyroid cancers exhibit immune gene suppression

IntroductionBRAFV600E mutation (BRAFmut) is common in papillary thyroid cancer (PTC), and most patients have an excellent outcome. However, a TERT-promoter mutation (pTERTmut) in the presence of BRAFmut (BRAFmutpTERTmut) has been demonstrated to confer a more aggressive behavior to PTC. Lymphocytic infiltration is often present in PTC. In this study, we sought to decipher the relationship between the BRAF and pTERT mutations and immune gene dysregulation in tumor samples from a cohort of 147 samples of PTC.MethodsThe abundance of 770 immune gene transcripts was determined by multiprex capture/detection and digital counting of mRNA transcripts using the NanoString nCounter® PanCancer Immune Profiling Panel.ResultsWe identified 40 immune transcripts differentially expressed in BRAFmutpTERTmutvs BRAFmutpTERT wildtype (pTERTwt) (P<0.05). Transcripts induced by BRAFmut alone were significantly repressed in BRAFmutpTERTmut samples, such as genes expressed by lymphoid cells, antigen-presenting cells, and cytotoxic cells, including chemokines, cytokines, checkpoint control proteins, interferon downstream markers, TNF superfamily proteins and BMP markers. A validation analysis using 444 samples from The Cancer Genome Atlas (TCGA) PTC dataset yielded similar results. Deconvolution analysis confirmed differences in the immune cell populations such as increased presence of M2 macrophages in the BRAFmutpTERTmut Mayo cohort and a lower abundance of M1 macrophages in the BRAFmutpTERTmut TCGA cohort compared to BRAFmutpTERTwt. Most of the immune gene pathways were enriched in the BRAFmutpTERTwt tumors in both Mayo and TCGA cohorts but not in BRAFmutpTERTmut. BRAFmutpTERTwt had higher stromal lymphocytes infiltration as compared to BRAFwtpTERTwt tumors, corroborating the transcriptomic findings.DiscussionTo our knowledge this is the first report of a potential link between TERT and the immune microenvironment, offering an explanation for the aggressive nature of BRAFmutpTERTmut PTC.

Enhancing farmed fish welfare: Evaluating the effectiveness of plant-based stress mitigating agents as sedatives in sea bass (Dicentrarchus labrax) following intraperitoneal vaccination

The effectiveness of vaccines may be compromised by the stress response induced by intraperitoneal/intramuscular (IP/IM) vaccination due to an intimate interaction between the neuroendocrine and immune systems. Essential oils (EOs), known for their antibacterial, antioxidant, and sedative properties, are potential candidates to mitigate this stress response. This study investigates the short-term sedative effect of two essential oil-based products, FishEase-C (FEC) and FishEase-L (FEL) in sea bass prior to intraperitoneal vaccination (IP). Physiological stress indicators (plasma cortisol, glucose, lactate) increased 1-h post-vaccination (hpv) as expected. Cortisol remained elevated up to 24 hpv in the N.P. vaccine and FEC + vaccine groups but decreased with FEL treatment (FEL + vaccine group). However, FEC at the tested concentration appeared to induce stress. The transcription of stress (gr, hsp70, cox2), immune (il1β, il6, tnfα), and antioxidant (gpx, sod, catalase) genes confirmed the vaccination-induced stress response, with mc2r transcription indicating increased cortisol production in vaccinated groups (N.P. vaccine and FEC + vaccine). FEL reduced stress at both physiological (e.g., cortisol) and transcriptional levels (e.g., hsp70, cox2 and il6) at either 1 or 24 hpv. It is worth noting that, from an inflammatory perspective, there was a big difference between tissues in terms of magnitude and pattern (treatment and time effects). The brain was more resistant to inflammation, while the head kidney and spleen showed heightened il1β expression (860-2100-fold). These findings support the use of FEL as a sedative before IP/IM vaccination in sea bass.

Enhanced CRC Growth in Iron-Rich Environment, Facts and Speculations.

The contribution of nutritional factors to disease development has been demonstrated for several chronic conditions including obesity, type 2 diabetes, metabolic syndrome, and about 30 percent of cancers. Nutrients include macronutrients and micronutrients, which are required in large and trace quantities, respectively. Macronutrients, which include protein, carbohydrates, and lipids, are mainly involved in energy production and biomolecule synthesis; micronutrients include vitamins and minerals, which are mainly involved in immune functions, enzymatic reactions, blood clotting, and gene transcription. Among the numerous micronutrients potentially involved in disease development, the present review will focus on iron and its relation to tumor development. Recent advances in the understanding of iron-related proteins accumulating in the tumor microenvironment shed light on the pivotal role of iron availability in sustaining pathological tumor hallmarks, including cell cycle regulation, angiogenesis, and metastasis.

Translation initiation or elongation inhibition triggers contrasting effects on Caenorhabditis elegans survival during pathogen infection.

Several microbial pathogens target host protein synthesis machinery, potentially limiting the innate immune responses of the host. In response, hosts trigger a defensive response, elevating immune gene transcripts. This counterintuitive response can have either beneficial or harmful effects on host survival. In this study, we conduct a comprehensive analysis of the impact of knocking down various translation initiation and elongation factors on the survival of Caenorhabditis elegans exposed to Pseudomonas aeruginosa. Intriguingly, inhibiting initiation and elongation factors has contrasting effects on C. elegans survival. Inhibiting translation initiation activates immune responses that protect the host from bacterial infection, while inhibiting translation elongation induces aberrant immune responses that, although clear the infection, are detrimental to the host. Our study reveals divergent roles of translation initiation and elongation inhibition in C. elegans survival during P. aeruginosa infection and identifies differential transcriptional reprogramming that could underlie these differences.

Genome Restructuring around Innate Immune Genes in Monocytes in Alcohol-associated Hepatitis.

Many inflammatory genes in the immune system are clustered in the genome. The 3D genome architecture of these clustered genes likely plays a critical role in their regulation and alterations to this structure may contribute to diseases where inflammation is poorly controlled. Alcohol-associated hepatitis (AH) is a severe inflammatory disease that contributes significantly to morbidity in alcohol associated liver disease. Monocytes in AH are hyper-responsive to inflammatory stimuli and contribute significantly to inflammation. We performed high throughput chromatin conformation capture (Hi-C) technology on monocytes isolated from 4 AH patients and 4 healthy controls to better understand how genome structure is altered in AH. Most chromosomes from AH and healthy controls were significantly dissimilar from each other. Comparing AH to HC, many regions of the genome contained significant changes in contact frequency. While there were alterations throughout the genome, there were a number of hotspots containing a higher density of changes in structure. A few of these hotspots contained genes involved in innate immunity including the NK-gene receptor complex and the CXC-chemokines. Finally, we compare these results to scRNA-seq data from patients with AH challenged with LPS to predict how chromatin conformation impacts transcription of clustered immune genes. Together, these results reveal changes in the chromatin structure of monocytes from AH patients that perturb expression of highly clustered proinflammatory genes.

Pharmacoinformational, basic and clinical studies of 7-hydroxymatairesinol in the context of searching for effective and safe therapeutic approaches to the treatment of mastopathy

7-hydroxymatairesinol (7(OH)MR) is the main lignan of spruce extracts, characterized by oncoprotective and anti-inflammatory properties. The results of chemoreactome modeling made it possible to formulate the molecular mechanisms of the pharmacological effects of 7(OH)MR for anti-inflammatory (inhibition of 5-lipoxygenase, matrix metalloproteinase MMP2, mitogen-activated kinase p38-alpha, leukotriene b4 receptor, prostacyclin receptor), oncoprotective (antioxidant effect due to inhibition heme oxygenase-2, inhibition of cyclin-dependent kinases 3 and 4, epidermal growth factor, mTOR protein). An assessment of the transcriptomic effects of 7(OH)MR in breast cancer cells showed significant dose-dependent effects on the transcription of 3468 of 12700 genes. The 7(OH)MR molecule, reducing the expression of proliferative genes (401 genes) and chronic inflammation (148 genes), while simultaneously increasing the transcription of oncoprotective immunity genes (100 genes), inhibits the proliferation of tumor cells. Experiments on solid Ehrlich carcinoma showed a significant oncoprotective effect of 7(OH)MR at doses of 60, 120 mg/day, even while taking estrogen (p = 0.007). A clinical study showed that 7(OH)MR (60 mg/day, 1 month) helps normalize estrogen metabolism and improve the clinical symptoms of mastopathy.

Comparative Analysis of Immune Gene Transcription in Sea Bream (Sparus aurata) Challenged with RGNNV or RGNNV/SJNNV Betanodaviruses.

Gilthead sea bream and European sea bass display different resistance-susceptibility patterns during infection with different nervous necrosis virus (NNV) species, which may derive from differences in the triggered immune response. Based on this premise, we analysed the transcription of several selected immune-related genes in sea bream experimentally infected with NNV isolates obtained from sea bass (DlNNV, RGNNV) or sea bream (SaNNV, RGNNV/SJNNV). Viral replication only occurred in SaNNV-inoculated fish; therefore, the differences between the immune response elicited by both viruses may be the key to understanding the mechanism behind the inhibition of DlNNV replication. Principal component analysis clustered samples according to the viral isolate from 1 day post infection onwards and evidenced differences in the immune response against both viruses, even though no mortalities or symptoms were recorded. The response against DlNNV is characterized by higher rtp3 transcription early after the infection, longer-lasting il-10 transcription and stronger induction of casp1 and hsp70. These genes should be targets for future studies in order to elucidate their role in hampering NNV replication in sea bream, which is essential for developing effective prophylactic measures.

Genetic architecture and correlations between the gut microbiome and gut gene transcription in Chinook salmon (Oncorhynchus tshawytscha).

Population divergence through selection can drive local adaptation in natural populations which has implications for the effective restoration of declining and extirpated populations. However, adaptation to local environmental conditions is complicated when both the host and its associated microbiomes must respond via co-evolutionary change. Nevertheless, for adaptation to occur through selection, variation in both host and microbiome traits should include additive genetic effects. Here we focus on host immune function and quantify factors affecting variation in gut immune gene transcription and gut bacterial community composition in early life-stage Chinook salmon (Oncorhynchus tshawytscha). Specifically, we utilized a replicated factorial breeding design to determine the genetic architecture (sire, dam and sire-by-dam interaction) of gut immune gene transcription and microbiome composition. Furthermore, we explored correlations between host gut gene transcription and microbiota composition. Gene transcription was quantified using nanofluidic qPCR arrays (22 target genes) and microbiota composition using 16 S rRNA gene (V5-V6) amplicon sequencing. We discovered limited but significant genetic architecture in gut microbiota composition and transcriptional profiles. We also identified significant correlations between gut gene transcription and microbiota composition, highlighting potential mechanisms for functional interactions between the two. Overall, this study provides support for the co-evolution of host immune function and their gut microbiota in Chinook salmon, a species recognized as locally adapted. Thus, the inclusion of immune gene transcription profile and gut microbiome composition as factors in the development of conservation and commercial rearing practices may provide new and more effective approaches to captive rearing.

#1121 Identification of kidney transcripts associated with prognosis in ANCA-associated glomerulonephritis

Abstract Background and Aims Kidney involvement in ANCA-associated vasculitis (AAV-GN) predicts poor patient and kidney survival. Deciphering the transcriptomic landscape in AAV-GN may provide insights into pathogenic mechanisms or identify biomarkers for refining diagnosis and/or prognosis, which would help stratify risk and tailor therapeutic management. We aimed to investigate the potential prognostic value of kidney transcripts associated with kidney survival. Method This study included adult patients with AAV-GN from the French Maine-Anjou Registry. Immune gene transcript analysis was performed on RNA extracted from 97 AAV-GN kidney biopsies using NanoString technology. Transcripts of interest were selected, and their prognostic performance was assessed with respect to current histological-based classifications. Following the identification of a possible role for clusterin (CLU), the relationship between serum CLU and prognosis was assessed. Results Among the 750 evaluated transcripts, we identified a 4-gene signature (XRCC6, PRKCD, TEK, and CLU) that was strongly associated with kidney survival (Fig. 1A). This signature predicted kidney survival better than histological-based classifications (global C-Index 0.87 vs. 0.65 for Berden classification or 0.81 for Renal Risk Score, with better time-dependent AUC and Brier scores, especially beyond 1 year after diagnosis) (Fig. 1B). Among these 4 transcripts, the expression level of the CLU transcript had the highest correlation with glomerular involvement, kidney function at diagnosis, and kidney survival. Serum CLU levels were associated with kidney survival, especially when assessed at 6 months from diagnosis (Fig. 1C, P = .023). Conclusion Transcriptomic analysis of kidney biopsies of AAV-GN identified potential transcripts that may improve prediction of kidney survival. This transcriptomic signature may help us gain a deeper understanding of the AAV-GN pathogenesis and provide insights for developing new therapeutic options.

#1154 Type I interferon pathway is upregulated in microscopic polyangiitis (vs granulomatosis with polyangiitis)

Abstract Background and Aims The two main types of ANCA-associated vasculitis (AAV), microscopic polyangiitis (MPA) and granulomatosis with polyangiitis (GPA), share common characteristics but also differ on many aspects (epidemiological, genetic, clinical, outcomes). These differences did not justify, to date, different patient management. To identify more specific targetable pathways, we characterized the kidney transcriptome of MPA vs. GPA patients with AAV-glomerulonephritis (AAV-GN). Method This retrospective study included adult patients with AAV-GN from the French Maine-Anjou Registry. Immune gene transcript analysis was performed on RNA extracted from 97 kidney biopsies using NanoString technology. Significant transcripts were examined to identify immune pathways of interest. We then used a publicly available dataset to explore these identified pathways (qPCR / ELISA). Results We compared kidney biopsies from GPA (n = 33) and MPA (n = 64) patients. There was no difference in eGFR between both groups at diagnosis. Of the 750 evaluated immune transcripts, 8 genes were found differentially expressed between MPA and GPA patients, all being upregulated in MPA (log2FC > 1, q-value < 0.05) (Fig. 1A). Seven (out of 8) genes belonged to the type I interferon (IFN-I) signaling pathway. A personal analysis of unpublished, publicly available, data1 identified a significantly more pronounced IFN-I signature in the peripheral blood mononuclear cells of patients with MPO-AAV when compared to those with PR3-AAV (Fig. 1B). Serum levels or CXCL10, a cytokine that mediates immune responses through the recruitment and activation of numerous immune cells under the regulation of IFN-I, were found higher in MPO-AAV patients (Fig. 1C). Conclusion We identified a type I interferon signature, both in kidneys and in blood, in patients with MPA (or MPO-AAV) in comparison to GPA (or PR3-AAV). This is in line with previous lines of evidence brough by Kessenbrock et al. and Kessler et al. suggesting a role for IFN-I in AAV and especially in MPO-AAV. This signature may help gain a deeper understanding of the AAV-GN pathogenesis and provide insights for developing new therapeutic options.

The WY Domain of an RxLr Effector Drives Interactions with a Host Target Phosphatase to Mimic Host Regulatory Proteins and Promote Phytophthora infestans Infection.

Plant pathogens manipulate the cellular environment of the host to facilitate infection and colonization that often lead to plant diseases. To accomplish this, many specialized pathogens secrete virulence proteins called effectors into the host cell, which subvert processes such as immune signaling, gene transcription, and host metabolism. Phytophthora infestans, the causative agent of potato late blight, employs an expanded repertoire of RxLR effectors with WY domains to manipulate the host through direct interaction with protein targets. However, our understanding of the molecular mechanisms underlying the interactions between WY effectors and their host targets remains limited. In this study, we performed a structural and biophysical characterization of the P. infestans WY effector Pi04314 in complex with the potato Protein Phosphatase 1-c (PP1c). We elucidate how Pi04314 uses a WY domain and a specialized C-terminal loop carrying a KVxF motif that interact with conserved surfaces on PP1c, known to be used by host regulatory proteins for guiding function. Through biophysical and in planta analyses, we demonstrate that Pi04314 WY or KVxF mutants lose their ability to bind PP1c. The loss of PP1c binding correlates with changes in PP1c nucleolar localization and a decrease in lesion size in plant infection assays. This study provides insights into the manipulation of plant hosts by pathogens, revealing how effectors exploit key regulatory interfaces in host proteins to modify their function and facilitate disease. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Monkeypox virus infection of human astrocytes causes gasdermin B cleavage and pyroptosis

Monkeypox virus (MPXV) infections in humans cause neurological disorders while studies of MPXV-infected animals indicate that the virus penetrates the brain. Pyroptosis is an inflammatory type of regulated cell death, resulting from plasma membrane rupture (PMR) due to oligomerization of cleaved gasdermins to cause membrane pore formation. Herein, we investigated the human neural cell tropism of MPXV compared to another orthopoxvirus, vaccinia virus (VACV), as well as its effects on immune responses and cell death. Astrocytes were most permissive to MPXV (and VACV) infections, followed by microglia and oligodendrocytes, with minimal infection of neurons based on plaque assays. Aberrant morphological changes were evident in MPXV-infected astrocytes that were accompanied with viral protein (I3) immunolabelling and detection of over 125 MPXV-encoded proteins in cell lysates by mass spectrometry. MPXV- and VACV-infected astrocytes showed increased expression of immune gene transcripts (IL12, IRF3, IL1B, TNFA, CASP1, and GSDMB). However, MPXV infection of astrocytes specifically induced proteolytic cleavage of gasdermin B (GSDMB) (50 kDa), evident by the appearance of cleaved N-terminal-GSDMB (30 kDa) and C-terminal- GSDMB (18 kDa) fragments. GSDMB cleavage was associated with release of lactate dehydrogenase and increased cellular nucleic acid staining, indicative of PMR. Pre-treatment with dimethyl fumarate reduced cleavage of GSDMB and associated PMR in MPXV-infected astrocytes. Human astrocytes support productive MPXV infection, resulting in inflammatory gene induction with accompanying GSDMB-mediated pyroptosis. These findings clarify the recently recognized neuropathogenic effects of MPXV in humans while also offering potential therapeutic options.

Persistent immune responses in the heart determine the outcome of cardiomyopathy syndrome in Atlantic salmon (Salmo salar)

Cardiomyopathy syndrome (CMS) caused by piscine myocarditis virus (PMCV) is a severe cardiac disease in Atlantic salmon (Salmo salar) and one of the leading causes of morbidity and mortality in the Norwegian aquaculture industry. Previous research suggest a variation in individual susceptibility to develop severe disease, however the role of the immune response in determining individual outcome of CMS is poorly understood particularly in cases where fish are also challenged by stress. The present study's aim was therefore to characterize cardiac transcriptional responses to PMCV infection in Atlantic salmon responding to infection under stressful conditions with a high versus low degree of histopathological damage.The study was performed as a large-scale controlled experiment of Atlantic salmon smolts from pre-challenge to 12 weeks post infection (wpi) with PMCV, during which fish were exposed to intermittent stressors. RNA sequencing (RNAseq) was used to compare the heart transcriptome of high responders (HR) with atrium histopathology score ‘4’ and low responders (LR) with score ‘0.5’ at 12 wpi. A high-throughput quantitative PCR (qPCR) analysis was used to compare immune gene transcription between individuals sampled at 6, 9 and 12 wpi. Based on RNAseq and qPCR results, RNAscope in situ hybridization (ISH) was performed for visualization of IFN-γ - and IFNb producing immune cells in affected heart tissue.Compared to LR, the transcription of 1592 genes was increased in HR at 12 wpi. Of these genes, around.40 % were immune-related, including various chemokines, key antiviral response molecules, and genes.associated with a Th1 pro-inflammatory immune response. Further, the qPCR analysis confirmed.increased immune gene transcription in HR at both 9 and 12 wpi, despite a decrease in PMCV.transcription between these time points. Interestingly, increased IFNb transcription in HR suggests the.presence of high-quantity IFN secreting cells in the hearts of these individuals. Indeed, RNAscope.confirmed the presence of IFN-γ and IFNb-positive cells in the heart ventricle of HR but not LR.To conclude, our data indicate that in severe outcomes of PMCV infection various chemokines attract leucocytes to the salmon heart, including IFN-γ and IFNb-secreting cells, and that these cells play important roles in maintaining persistent antiviral responses and a sustained host immunopathology despite decreasing heart viral transcription.

Differential expression of antimicrobial peptide encoding genes of honey bee (Apis mellifera) after Varroa mite (Varroa destructor) infestation

Varroa destructor mite is a serious threat to the world apiculture industry, and severely hampered beekeeping practices. Innate immune systems are key defenses of animals and are particularly important in species that lack the sophisticated adaptive immune systems found in vertebrates. In honey bees, antimicrobial immune peptide genes play a significant immune response against pathogens. The present study analyzed the detrimental effects of Varroa infestation on Apis mellifera at all developmental stages in both Varroa challenged and controlled hives. Physical attributes were observed in Varroa hives at different time intervals (0, 30, and 45 days respectively). After 45 days of Varroa exposure, the samples were taken, and the AMPs immune response of bees parasitized by Varroa was detected by studying the expression levels of antibacterial peptides (defensin, apidaecin, abaecin, and hymenoptaecin). Significant alterations in the physical parameters viz. body length, body weight, wing shape, and mean numbers of brood cells were observed compared to the control. Colony density was also decreased with marked negative behavioural changes. Ct values of Real-Time PCR results showed that AMP-specific immune gene transcription level in Varroa-challenged bees was downregulated, except for abaecin and defensin (adult stage). There was a significant (p < 0.05) increase in abaecin gene transcription folds i.e., 1.064 larvae, 1.732 pupae, and 1.264 adults bees. As a whole, all these findings can be taken as critical biomarkers in the advancement of control techniques against Varroa and other pathogen infections as a part of wide-ranging programs of honey bee health and Varroa control.

SARM1 regulates NAD+-linked metabolism and select immune genes in macrophages

Sterile alpha and HEAT/armadillo motif-containing protein (SARM1) was recently described as a NAD+-consuming enzyme and has previously been shown to regulate immune responses in macrophages. Neuronal SARM1 is known to contribute to axon degeneration due to its NADase activity. However, how SARM1 affects macrophage metabolism has not been explored. Here, we show that macrophages from Sarm1-/- mice display elevated NAD+ concentrations and lower cyclic ADP-ribose, a known product of SARM1-dependent NAD+ catabolism. Further, SARM1-deficient macrophages showed an increase in the reserve capacity of oxidative phosphorylation and glycolysis compared to WT cells. Stimulation of macrophages to a proinflammatory state by lipopolysaccharide (LPS) revealed that SARM1 restricts the ability of macrophages to upregulate glycolysis and limits the expression of the proinflammatory gene interleukin (Il) 1b, but boosts expression of anti-inflammatory Il10. In contrast, we show macrophages lacking SARM1 induced to an anti-inflammatory state by IL-4 stimulation display increased oxidative phosphorylation and glycolysis, and reduced expression of the anti-inflammatory gene, Fizz1. Overall, these data show that SARM1 fine-tunes immune gene transcription in macrophages via consumption of NAD+ and altered macrophage metabolism.

Effects of dietary baobab, Adansonia digitata on growth, haemato-immunological status, antioxidant biomarkers, intestinal histomorphometry, gene expression responses, and disease resistance in Nile tilapia, Oreochromis niloticus

This study explored the influences of baobab fruit extract powder (BP) on growth, oxidant-antioxidant balance, immunity, intestinal histology, expression of growth and immune genes, and disease resistance in Nile tilapia. Fish were fed on five test diets supplied with different BP levels as BP0 (control; 0 g kg−1), BP1 (1 g kg−1), BP2 (2 g kg−1), BP3 (3 g kg−1), and BP4 (4 g kg−1) for two months. Results revealed that dietary BP improved the growth in all BP groups compared with the BP0 group, with the best values apparent in the BP4 group. However, dietary BP did not affect the whole-body chemical composition and survival rates in all groups. Serum immunity biomarkers (lysozyme, myeloperoxidase, and bactericidal activities) were enhanced in all BP groups compared to the BP0 group. Hepatic antioxidant status significantly improved, as manifested by increases in the hepatic CAT, SOD, and T-AOC and decreases in MDA concentrations in BP groups compared to the controls. Of interest, dietary BP supplementation significantly increased the expression of growth-related genes (ghr1, ghr2, igf-1, and igf-2) in the liver and immunity-associated genes (inf-γ1, lyz, il-1β, il-10, and hepcidin) in the spleen of BP-treated fish in comparison with the controls. The intestinal histological sections revealed no significant variations among all groups with normal histoarchitecture and goblet cells. After experimental infection with Aeromonas hydrophila, the survival rates (%) in the BP0, BP1, BP2, BP3, and BP4 groups were 23.33, 43.33, 56.67, 63.33, and 70.00, respectively, suggesting the positive roles of BP in increasing the fish disease resistance. To conclude, dietary BP could improve growth (particularly in the BP4), serum immunity, antioxidant capacity, and transcription of growth and immune genes in O. niloticus. In addition, the highest disease resistance was also found in the BP4 group. All in all, BP at a dose rate (4 g kg−1 diet) could be regarded as a functional feed supplement for tilapia farming.

MODL-09. SCAFFOLD-BASED (MATRIGELTM) 3D CULTURE TECHNIQUE OF GLIOBLASTOMA RECOVERS A PATIENT-LIKE IMMUNOSUPPRESSIVE PHENOTYPE

Abstract 3D (organoid) models in cancer research are considered the novel gold standard for in vitro experiments. Nevertheless, a variety of different protocols exits and the models are often used uncritically in lack of further validation. In this study, we aimed to characterize a scaffold-based (MatrigelTM) 3D glioma cell culture approach. We compared the 3D technique to conventional 2D culturing in its efficacy of generating models and in respect to its exact modeling properties. Models were generated from 62 patients with CNS tumors, most of them with glioblastomas, IDH-wildtype (GBM, n = 43). Pairs of 11 GBM models (2D and 3D) together with corresponding patient material were further characterized using bulk and single-cell RNA sequencing as well as 850k methylation analyses. Functional aspects of 3D tumor organoids were analyzed using a coculture system (Incucyte® SX5 live cell imager) with tumor infiltrating lymphocytes. Compared to conventional 2D culture the MatrigelTM-based 3D protocol was not superior in terms of the successful generation of in vitro models. However, NGS-based bulk and single-cell sequencing of matched pairs of 2D and 3D models showed an altered transcription of key immune regulatory genes in the 3D models, indicating the reoccurrence of an immunosuppressive phenotype. Changes included the presentation of different HLA surface molecules as well as cellular stressors. In the coculturing approach, lymphocyte-mediated cell killing appeared less effective in clearing 3D models than their 2D counterparts. IFN-γ release as well as live cell staining and proliferation analysis pointed towards an elevated resistance of 3D models. In conclusion, we found that the Scaffold-based (MatrigelTM) 3D culture technique depicts clinically highly important aspects of tumor-immune cell interactions, with the potential to explore immunotherapeutic approaches in an easily accessible in vitro system.

Immune response induced by coinfection of the sea louse Caligus rogercresseyi and the intracellular bacteria Piscirickettsia salmonis in vaccinated Atlantic salmon.

Recently, we showed that Atlantic salmon vaccinated against Piscirickettsia salmonis lose their protection upon coinfection with Caligus rogercresseyi (sea lice). However, the causes of the overriding effect of C. rogercresseyi infection have not been elucidated, and the molecular basis of the cellular and humoral immune responses upon C. rogercresseyi infection has not been described for vaccinated salmon. Therefore, we studied changes in the transcription of immune genes in vaccinated Atlantic salmon that were experimentally challenged by co-infecting them with C. rogercresseyi and P. salmonis. In general, coinfection treatments showed immune gene expression similar to treatments with a single P. salmonis infection, showing a decreased cellular response. However, a high variance was found between individual fish in the case of crucial cellular immune genes, with a few fish reacting overwhelmingly highly compared to the majority. This supports our previous findings on vaccination response variation and reinforces the idea that vaccination failures in the field might be caused by an overwhelming amount of vaccinated fish that display a deficient immune response to the infection.

Scaffold-Based (Matrigel™) 3D Culture Technique of Glioblastoma Recovers a Patient-like Immunosuppressive Phenotype.

Conventional 2D cultures are commonly used in cancer research though they come with limitations such as the lack of microenvironment or reduced cell heterogeneity. In this study, we investigated in what respect a scaffold-based (Matrigel™) 3D culture technique can ameliorate the limitations of 2D cultures. NGS-based bulk and single-cell sequencing of matched pairs of 2D and 3D models showed an altered transcription of key immune regulatory genes in around 36% of 3D models, indicating the reoccurrence of an immune suppressive phenotype. Changes included the presentation of different HLA surface molecules as well as cellular stressors. We also investigated the 3D tumor organoids in a co-culture setting with tumor-infiltrating lymphocytes (TILs). Of note, lymphocyte-mediated cell killing appeared less effective in clearing 3D models than their 2D counterparts. IFN-γ release, as well as live cell staining and proliferation analysis, pointed toward an elevated resistance of 3D models. In conclusion, we found that the scaffold-based (Matrigel™) 3D culture technique affects the transcriptional profile in a subset of GBM models. Thus, these models allow for depicting clinically relevant aspects of tumor-immune interaction, with the potential to explore immunotherapeutic approaches in an easily accessible in vitro system.

Structural mechanism of dsDNA recognition by the hMNDA HIN domain: New insights into the DNA-binding model of a PYHIN protein

The hematopoietic interferon-inducible nuclear (HIN) domain of the PYHIN family of proteins recognizes double-stranded DNA (dsDNA) through different dsDNA-binding modes. These modes apparently confer different roles upon these proteins in the regulation of innate immune responses, gene transcription, and apoptosis. Myeloid cell nuclear differentiation antigen (MNDA), a member of the human PYHIN family, binds DNA and regulates gene transcription in monocytes. However, the mechanism of DNA recognition and DNA-binding modes of human MNDA (hMNDA) remain unclear. Here, we determined the crystal structure of the hMNDA–HIN domain in complex with dsDNA at 2.4 Å resolution, and reveal that hMNDA–HIN binds to dsDNA in a sequence-independent manner. Structure and mutation studies indicated that hMNDA–HIN binds to dsDNA through a unique mode, involving two dsDNA-binding interfaces. Interface I exhibits an AIM2-like dsDNA-binding mode, and interface II has a previously unreported mode of dsDNA-binding. These results provide new insights into the DNA-binding modes of this PYHIN protein.