mRNA Expression Of Immune-related Genes Research Articles

Poria cocos polysaccharides alleviate cyclophosphamide-induced immunosuppression effects in silkworm and their metabolic profiling analysis

The present study investigated the immune regulatory mechanisms of Poria cocos polysaccharides (PS) in a cyclophosphamide (CTX)-induced immunosuppression silkworm model. We found that PS increased the superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities, as well as reactive oxygen species (ROS) levels in the fat body or haemocytes of CTX-treated silkworms. PS also strongly stimulated the immunity of silkworms by increasing haemocyte counts and PO activity and upregulating the mRNA expression of immune-related genes. We further investigated the metabolic profile of the fat body after PS treatment using liquid chromatography–massspectrometry/mass spectrometry (LC–MS/MS) and identified 236 differentially expressed metabolites. Among them, nine metabolites related to amino acid metabolism were present at high levels in the PS-treated group, whereas the contents of metabolites involved in sphingolipid metabolism and alpha-linolenic acid metabolism were significantly decreased after PS treatment. The amino acid metabolism pathways and lipid metabolism pathways were found to be significantly altered in the PS-treated group, which indicates that PS may regulate the immune system of silkworms by reprogramming lipid and amino acid metabolism. This study provides new insight into the immune regulatory effect of PS in animals.

The Infection Properties of Trionyx sinensis Hemorrhagic Syndrome Virus and the Antiviral Effect of Curcumin In Vivo

Trionyx sinensis hemorrhagic syndrome virus (TSHSV) is an aquatic arterivirus causing a high mortality rate for T. sinensis (Chinese soft-shelled turtle), but the detailed infection properties of TSHSV are unclear, and no effective treatment is available. In this study, cell culture and histopathology were performed to elucidate the infection properties of TSHSV. Furthermore, the anti-TSHSV and immune-enhancing effects of curcumin were evaluated using survival statistics, qPCR, and tissue immunofluorescence. The results demonstrated that TSHSV could proliferate in the spleen cell line of T. sinensis, leading to cytopathic effects. TSHSV damaged the livers, kidneys, and lungs, characterized by cell disintegration and hyperemia. Curcumin at 250 mg/kg improved the survival of T. sinensis, and significantly reduced the viral load in the spleens, kidneys, and lungs. Moreover, curcumin inhibited the mRNA expression of immune-related genes, RSAD2, IFN-γ, and TNF-α (p < 0.05). In conclusion, these results imply that TSHSV is pathogenic to the spleen cell line, liver, spleen, kidney, and lung of T. sinensis. Curcumin effectively inhibits TSHSV and modulates the immune function of T. sinensis, so it holds promise as a means to prevent TSHSV.

Change to the transcriptomic profile, oxidative stress, apoptotic and immunity in the liver of small yellow croaker (Larimichthys polyactis) under hypoxic stress

The small yellow croaker (Larimichthys polyactis) is one of the most highly cultured fish in China, contributing considerably to the country's economy. However, conditions experienced under high-density culture make the fish susceptible to hypoxia, which impacts growth and consequently product quality. To understand the effects of hypoxia stress on physiological and biochemical responses of L. polyactis, we subjected the fish to hypoxic (2.5 mg/L) stress for 96 h. We observed histological changes in the liver pre- and post-hypoxia and measured the changes in liver injury parameters. The results showed injury to the liver of L. polyactis following hypoxic stress, indicating that the liver is an important organ in the hypoxic stress response. To explore the genes and biological processes involved in hypoxic adaptation of L. polyactis at the overall transcription level, liver tissues were obtained pre- (0 h) and post- (6, 24, 48, and 96 h) hypoxia for transcriptomic analysis. GO and KEGG enrichment analysis of differentially expressed genes (DEGs) showed that DEGs were mainly enriched in redox equilibrium, metabolism, immunity, and apoptosis pathways under hypoxic stress. In addition, we examined the changes in molecular indices related to oxidative stress, cell apoptosis, and non-specific immunity in L. polyactis under hypoxic stress. The results showed that hypoxia induced reactive oxygen species overproduction, altered the activity of oxidase (MDA, PCO, 8-OhdG) and antioxidant enzymes (T-AOC, SOD, CAT, GPx, GR), and reduced the mRNA expression of key antioxidant genes (sod1, sod2, cat) in the liver, thus confirming that hypoxic stress induced oxidative stress. Additionally, we confirmed that hypoxic stress induced apoptosis in the liver by inducing the enzyme activity of Caspase-3/8/9 and the mRNA expression of key apoptotic genes (bcl2, bax, fas, and bnip3). Furthermore, hypoxic stress inhibited the activity of non-specific enzymes (AKP, ACP, and LZM) and mRNA expression of immune-related genes (tnf-α, il-1β, and il-17c); hence, hypoxic stress inhibited the non-specific immune function of L. polyactis. In conclusion, our study provides new insights into the molecular mechanisms underlying hypoxia adaptation in marine fish, which will aid in selective breeding of hypoxia-tolerant L. polyactis.

Higher immune-related gene expression in major depression is independent of CRP levels: results from the BIODEP study

Compelling evidence demonstrates that some individuals suffering from major depressive disorder (MDD) exhibit increased levels of inflammation. Most studies focus on inflammation-related proteins, such as serum or plasma C-reactive protein (CRP). However, the immune-related modifications associated with MDD may be not entirely captured by CRP alone. Analysing mRNA gene expression levels, we aimed to identify broader molecular immune-related phenotypes of MDD. We examined 168 individuals from the non-interventional, case–control, BIODEP study, 128 with a diagnosis of MDD and 40 healthy controls. Individuals with MDD were further divided according to serum high-sensitivity (hs)CRP levels (n = 59 with CRP <1, n = 33 with CRP 1–3 and n = 36 with CRP >3 mg/L). We isolated RNA from whole blood and performed gene expression analyses using RT-qPCR. We measured the expression of 16 immune-related candidate genes: A2M, AQP4, CCL2, CXCL12, CRP, FKBP5, IL-1-beta, IL-6, ISG15, MIF, GR, P2RX7, SGK1, STAT1, TNF-alpha and USP18. Nine of the 16 candidate genes were differentially expressed in MDD cases vs. controls, with no differences between CRP-based groups. Only CRP mRNA was clearly associated with serum CRP. In contrast, plasma (proteins) IL-6, IL-7, IL-8, IL-10, IL-12/IL-23p40, IL-16, IL-17A, IFN-gamma and TNF-alpha, and neutrophils counts, were all differentially regulated between CRP-based groups (higher in CRP >3 vs. CRP <1 and/or controls), reflecting the gradient of CRP values. Secondary analyses on MDD individuals and controls with CRP values <1 mg/L (usually interpreted as 'no inflammation') confirmed MDD cases still had significantly different mRNA expression of immune-related genes compared with controls. These findings corroborate an immune-related molecular activation in MDD, which appears to be independent of serum CRP levels. Additional biological mechanisms may then be required to translate this mRNA signature into inflammation at protein and cellular levels. Understanding these mechanisms will help to uncover the true immune abnormalities in depression, opening new paths for diagnosis and treatment.

Akkaraman ve Romanov Kuzularının Alveolar Makrofajlarında Lipopolisakkarit ve Lipoteikoik Asite Yanıtta İmmün İlişkili Genlerin Ekspresyon Profilinin Araştırılması

The alveolar macrophages (AMs) are frontier of defense against foreign materials that initiate immune response in lungs. Knowledge of the expression dynamics of major immune-related genes in the alveolar macrophages in response to lipopolysaccharide (LPS) and lipoteichoic acid (LTA) challenge can help to understand disease mechanism involved in several respiratory diseases. The aim of this study was to investigate the mRNA expression of selected immune-related genes in response to lipopolysaccharide (LPS) and lipoteichoic acid (LTA) challenge in sheep alveolar macrophages in vivo. Results revealed that Romanov lambs exhibited higher mRNA expression of TLR2, TLR4, NF-ĸβ, TNFα, IL-1β, IL-6, IL-8, and IL-10 genes as compared to Akkaraman lambs along with the control of all treatments. Moreover, the expression of TLR2, TLR4, NF-ĸβ, TNFα, IL-1β, IL-6, IL-8, and IL-10 genes was higher in combine treatment of LPS and LTA as compared to separate treatments of LPS and LTA in both breeds. The results showed that the mRNA expression of immune-related genes was significantly increased in the sheep AMs in response to LPS and LTA treatment whereas a synergistic effect was observed in LPS+LTS treatment. Also, breed comparison showed that the native Akkaraman was more resistant to disease compared to exotic Romanov.

DietaryAsparagus racemosusethanolic root extract modulates immune‐biochemical response, immune gene expression and provides protection againstAeromonas hydrophilainLabeo rohitafingerlings

The aim of this study was to investigate the dietary effects of the medicinal herb Asparagus racemosus on immune-biochemical response, immune-related gene expression and resistance of Labeo rohita fingerlings to Aeromonas hydrophila infection. The acute toxicity study showed that the dietary incorporation of the A. racemosus ethanolic root extract (AREE) at the dose up to 2000 mg kg−1 had no toxic effect on L. rohita. Three experimental diets were prepared with the inclusion of different concentrations of AREE (T1-50, T2-100 and T3-150 mg of AREE kg−1 feed). Fingerlings of L. rohita were fed with these diets and various immune-biochemical responses and immune-related gene expression were measured on the 0, 15th, and 30th day of feeding. Dietary inclusion of AREE significantly enhanced the biochemical responses (glucose, total protein, albumin and alkaline phosphatase activity), innate immune responses (superoxide anion, lysozyme and myeloperoxidase activity), and mRNA expression of immune-related genes (IL-1β, IFN-γ and C3) in the majority cases. The survivability of rohu against A. hydrophila infection was significantly higher in fish fed with the T2 diet. In conclusion, the dietary A. racemosus ethanolic root extract showed promising immunostimulatory activity, and conferred resistance against A. hydrophila challenge in L. rohita. The dietary supplementation of the extract at the rate of 100 mg kg−1 feed for 15 days is suggested for obtaining optimal immunostimulatory effect.

Vitamin C elicits the activation of immunological responses in swimming crab (Portunus trituberculatus) hemocytes against Mesanophrys sp.

Under suitable conditions, Vitamin C (vit. C) is essential for immunological responses and positively influences the function of stimulated immune cells. Therefore, this study evaluated the specific mechanisms of innate immunity in crab after supplementing the cell culture media with vit. C. We used the primary culture media of swimming crab hemocytes to detect some immune factors and immune-related signaling pathways via 24 h stimulation in vitro assays. Besides, we evaluated the anti-parasitic action of these hemocytes. Herein, the cellular and humoral factors, including intra- and extracellular phenol oxidase, nitrite oxidase synthase, lysozyme, and intracellular O2− were significantly (P < 0.05) enhanced after the culture media was supplemented with vit. C as compared to control. A two-slope broken-line analysis indicated that the optimum vit. C level for intracellular O2− was 118.91 μg mL−1 of the cell culture media (R2 = 0.73). However, for the extracellular O2−, the vit. C supplementation showed a downward quadratic relationship (R2 = 0.59). qRT-PCR demonstrated that relative mRNA expression of immune-related genes, including proPO, serine proteases, a2M, and alf-1 was significantly (P < 0.05) increased with vit. C media supplementation. Further, the results indicated that the anti-parasitic response and survival rate (%) of hemocytes after Mesanophrys sp. challenge was also significantly (P < 0.05) augmented in media supplemented with vit. C, relative to the control. Thus, the different vit. C in vitro levels were necessary for optimal immunological responses of crab hemocytes. In summary, vit. C supplementation enhanced and modulated the innate immune response of hemocytes against Mesanophrys sp., highlighting its potential application as an immunostimulant.

Innate Immune Response to Fasting and Refeeding in the Zebrafish Kidney.

Animals acquire nutrients and energy through feeding to achieve a balance between growth and organismal health. When there is a change in nutrient acquisition, the state of growth changes and may also cause changes in the intrinsic immune system. Compensatory growth (CG), a specific growth phenomenon, involves the question of whether changes in growth can be accompanied by changes in innate immunity. The zebrafish (Danio rerio), a well-known fish model organism, can serve as a suitable model. In this study, the zebrafish underwent 3 weeks of fasting and refeeding for 3 to 7 day periods. It was found that CG could be achieved in zebrafish. Zebrafish susceptibility to Streptococcus agalactiae increased after starvation. In addition, the amount of melano-macrophage centers increased after fasting and the proportion of injured tubules increased after refeeding for 3 and 5 days, respectively. Furthermore, the kidneys of zebrafish suffering from starvation were under oxidative stress, and the activity of several antioxidant enzymes increased after starvation, including catalase, glutathione peroxidases and superoxide dismutase. Innate immune parameters were influenced by starvation. Additionally, the activity of alkaline phosphatase and lysozyme increased after starvation. The mRNA expression of immune-related genes like il-1β was elevated to a different extent after fasting with or without lipopolysaccharides (LPS) challenge. This study showed that the function of the innate immune system in zebrafish could be influenced by nutrition status.

Multi-biomarker assessment in the giant freshwater prawn Macrobrachium rosenbergii after deltamethrin exposure

Deltamethrin (DM) is a synthetic pyrethroid used for agricultural purposes to control insects. However, its extensive use contaminates the aquatic environment and results in serious health problems in aquatic organisms. Knowledge about the toxic effect of DM in freshwater prawns is limited; therefore, this study aims to assess the toxicity of DM in Macrobrachium rosenbergii based on multiple biomarkers. Four-day acute toxicity tests showed that DM was highly toxic to M. rosenbergii with the 24 h, 48 h, 72 h and 96 h LC50 values to be 1.919, 0.603, 0.539, and 0.449 μg/L, respectively. According to 96 h LC50, prawns were exposed to DM at three concentrations (0.02, 0.08, and 0.32 μg/L) for 4 days, and then moved into fresh water for decontamination to investigate the toxic effect of DM in M. rosenbergii. At low concentration (0.02 μg/L and 0.08 μg/L), DM did not cause obvious histopathological damage to hepatopancreas and gill tissue, while at high concentration (0.32 μg/L), the histopathological harm was serious and the damage did not recover to the initial level after 7-day decontamination. 0.02 μg/L DM exposure did not induce significant changes in most of the biomarkers except the increased lactate dehydrogenase (LDH) activity, lactic acid (LD) level, and the first increased then decreased mRNA expression of immune-related genes, indicating the stimulation of DM on energy production and immunity. 0.08 μg/L and 0.32 μg/L DM exposure resulted in varying degrees of damage on prawns, but overall, their toxic effects showed similar trends based on the biomarkers. Increase in malonaldehyde (MDA) and hydrogen peroxide (H2O2) content and decrease in superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity after DM exposure demonstrated the oxidative stress caused by DM. The significantly increased acid phosphatase (ACP), alkaline phosphatase (AKP), LDH activity and LD level indicated hepatopancreatic dysfunction and respiration disruption. The first increased and then decreased expression pattern of immune-related genes indicated the immunosuppression caused by DM. After 7-day decontamination in freshwater, the activity/level of the biomarkers partly recovered. This study revealed the severe toxic effect of DM on Macrobrachium rosenbergii based on multiple biomarkers, providing fundamental knowledge for the establishment of DM toxicity assessment system with proper parameters in freshwater crustaceans.

Genomic aberration of chromatin regulatory BAF complex as predictive biomarker for immunotherapy in gastrointestinal adenocarcinoma.

4035 Background: SNF/SWI, a large ATP-dependent chromatin remodeling complex, is required for transcriptional activation of genes normally repressed by chromatin, and critical to tumor initiation and progression. Here, we analyzed the predictive utility of the mutations of the SNF/SWI members involved in BAF and PBAF complexes, and sought to explore the potential mechanisms. Methods: Clinical, genomic, transcriptional, and immunohistochemical data from immunotherapeutic cohort (MSKCC, n=185), Cancer Cell Line Encyclopedia (CCLE, n=92), The Cancer Genome Atlas (TCGA, n=925), and 3D Medicines database (3DMed, n=1812) were analyzed to explore the predictive effect of genomic aberration of BAF complex on the benefit from immunotherapy in patients with gastrointestinal adenocarcinoma. Results: In the MSKCC cohort involving 185 patients with gastrointestinal adenocarcinoma, the mutation of any member involved in BAF complex ( ARID1A, ARID1B, SMARCA4, SMARCB1, and SMARCD1) was significantly associated with prolonged OS of ICI treatment (HR 0.53, 95%CI 0.31-0.90, P=0.019), instead of the mutations of PBAF members including PBRM1 and ARID2. In addition, BAF mutation was not linked with better prognosis in TCGA database, indicating its predictive, not prognostic efficacy of immunotherapy. BAF-mutated samples exhibited higher tumour mutational burden (TMB, P&lt;0.05, Table), and increased mRNA expression of immune-related genes including chemokines and granzyme A. In the 3DMed cohort where tumour samples received both genomic sequencing and PD-L1 immunohistochemical staining, BAF mutation was associated with higher PD-L1 positive rate in tumour cells (P&lt;0.05, Table). Conclusions: Genomic aberration of members in chromatin regulatory BAF complex may serve as a predictive, not prognostic biomarker of ICI benefit in patients with gastrointestinal adenocarcinoma, partially underlying the mechanisms including higher mutational burden, transcription of immune-related genes, and protein-level PD-L1 expression. [Table: see text]

Expression Changes of MHC and Other Immune Genes in Frog Skin during Ontogeny

Simple SummaryTadpoles undergo many changes in physiology and immunology until metamorphosis into adult frogs. Major histocompatibility complex (MHC) molecules are an important part of vertebrate adaptive immunity, and our study measured the expression of two MHC genes (MHC class I and II) in skin during six tadpole stages of the Montane Brown frog (Rana ornativentris). First, using a qPCR method, we found that both MHC class I and II expression significantly increased between stage 24/25 (‘early’) and stage 28 (‘mid’) tadpole skin. Then, we conducted next-generation sequencing for ‘early’, ‘mid’ and ‘late’ stage tadpole skin mRNA of both R. ornativentris and a model species, Xenopus tropicalis, and confirmed that MHC expression increased from the ‘mid’ stage. We also performed further analyses of transcriptome data and found that several immune-related gene ontology terms were upregulated from the ‘mid’ tadpole stage. Our findings probably support that both MHC class I and II have a functional role during tadpole development.Anuran amphibians undergo major physiological and immunological changes following metamorphosis. Genes of the major histocompatibility complex (MHC) code for receptors important for vertebrate adaptive immunity. We used qPCR to measure skin MHC expression in six different ontological stages of Rana ornativentris (n = 10 per stage); normalized MHC class I and II expression at the mRNA level was significantly higher in stage 28 (mid-larval) compared to stages 24/25 (early-larval) tadpoles. Subsequent transcriptomic analyses of three tadpole (early-, mid-, and late-larval) stages of R. ornativentris and model species Xenopus tropicalis focused on mRNA expression of immune-related genes in the skin. Normalized expression of most MHC class I and II transcripts in both species were significantly higher in mid- and late-larval stages compared to early-larval stage. In addition, gene ontology (GO) analyses of differentially expressed transcripts revealed several immune-related GO terms that were significantly upregulated from the mid-larval stage. Our study provides evidence that both MHC class I and II is expressed during development in both R. ornativentris and X. tropicalis.

Effects of dietary hawthorn extracts supplementation on lipid metabolism, skin coloration and gut health of golden pompano (Trachinotus ovatus)

Hawthorn fruit has long been used as a functional food in China due to its effects of increasing appetite, promoting digestion and protecting the gastrointestinal tract. The present study was conducted to assess the impact of dietary hawthorn extract (HTE) supplementation on plasma biochemical indices, proximate body composition, skin coloration, intestinal morphology and changes in mRNA expression of immune-related genes and tight junction proteins genes in the intestine of golden pompano. Basal diets supplemented with HTE at 0, 0.50, 1.00, 2.00, 4.00 and 10.00 g kg−1 were fed to golden pompano for eight weeks. The study showed dietary HTE supplementation had hypolipidemic effects by reducing plasma cholesterol, triglyceride and low density lipoprotein content and improved skin coloration by increasing yellowness values of the ventral skin color of golden pompano. Meanwhile, dietary supplementation of 0.50 and 1.00 g kg−1 HTE could improve gut morphology by increasing villus length, villus width and muscle thickness, and enhance barrier function by increasing tight junction protein genes mRNA levels. However, dietary 10.00 g kg−1 HTE supplementation not only caused damage to intestinal morphology and barrier function, but also induced oxidative stress and inflammatory response. Our study demonstrated that dietary 0.50 and 1.00 g kg−1 HTE supplementation was beneficial for skin coloration and intestinal health of golden pompano.

Rice Husk Silica Enhances Innate Immune in Zebrafish (Danio rerio) and Improves Resistance to Aeromonas hydrophila and Streptococcus iniae Infection

Rice husk (RH) contains abundant silica such that RH silica (RHS) may be useful for possible industrial exploitation. Here, amorphous silica nanoparticles with multiple pore structures were acquired from RH by simple thermochemical processes. RHS antimicrobial activity and effects on zebrafish innate immunity against pathogen infections were evaluated. A toxicity assay showed that zebrafish exposed to an RHS dose lower than 200 μg/mL did not exhibit damage to zebrafish embryonic development or juvenile survival. RHS showed a wide spectrum of bacteriostatic activity against a variety of pathogens including antibiotic-resistant pathogens, implying its potential application as an antimicrobial agent in diverse industries. Fish exposed to 20 or 200 μg/mL RHS exhibited significantly increased mRNA expression of immune-related genes, including IL-1β, IL-6, IL-15, TNF-α, COX-2a, TLR-4a, lysozyme, and complement C3b. RHS-treated zebrafish exhibited a higher cumulative survival compared to that in control fish after infecting with Aeromonas hydrophila and Streptococcus iniae. The present results showed that a safe RHS dose enhanced innate immunity against infections without toxic effects in healthy fish, suggesting that RHS may be developed as an immunostimulant for improving health status in aquaculture.

Effects of potential probiotic Bacillus velezensis K2 on growth, immunity and resistance to Vibrio harveyi infection of hybrid grouper (Epinephelus lanceolatus♂ × E. fuscoguttatus♀)

Nowadays, there is no suitable treatment for vibriosis in groupers. So an eco-efficient and environmentally friendly treatment is necessary for the grouper industry. Probiotic-feeding has been a promising strategy to control the bacterial pathogens in aquaculture. A new Bacillus velezensis strain named K2 was isolated from the intestinal tract of healthy grouper, and exhibited wide antimicrobial spectrum of against fish pathogens, including Vibrio harveyi, Vibrio alginolyticus, Aeromonas hydrophila, Aeromonas veronii, Aeromonas caviae, Enterococcus casseliflavus and Lactococcus garvieae. Moreover, results of the safety of B. velezensis K2 showed that intraperitoneal injection of K2 in healthy grouper did not cause any pathological abnormality or death, indicating this bacteria could be considered as a candidate probiotic in aquaculture. Groupers were fed with the diets containing 1 × 107 cfu/g of B. velezensis K2 for 4 weeks. Various immune parameters were examined at 1, 2, 3, and 4 weeks of post-feeding. Results showed that diets supplemented with K2 significantly increased serum acid phosphatase (ACP) activity (P < 0.05). Results of the mRNA expression of immune-related genes in the head kidney of hybrid grouper showed that the expression of lysozyme gene was significantly upregulated after 1 and 2 weeks of feeding (P < 0.05). A significant up-regulation of the expression of piscidin, IgM and MyD88 were detected at day 21, whereas the TLR3 and TLR5 showed lower expression compared to the controls during 21 days, and a significant decrease of TLR3 gene was found at day 28 (P < 0.05). After challenge with V. harveyi, the survival rate of fish administrated with the strain K2 for 28 days was signifiacantly higher than the controls without this strain (P < 0.05). These results collectively suggest that B. velezensis K2 is a potential probiotic species to improve health status and disease resistance and can be developed as a probiotic agent in grouper industry.

Abstract PD5-07: Comprehensive profiling of poor-risk paired primary and recurrent triple-negative breast cancers reveals immune phenotype shifts

Abstract Background: Prognosis for triple-negative breast cancer (TNBC) patients remains poor, due in part to the lack of effective targeted therapies in the advanced setting. Emerging clinical data indicates reduced efficacy of immune checkpoint inhibitors in heavily pre-treated TNBC, but the underlying mechanisms are poorly understood. To better understand the immune phenotypic evolution of paired TNBCs, we studied the genomic and transcriptomic profiles of tumors from patients undergoing treatment for TNBC. Methods: We analyzed primary and recurrent TNBCs from 55 poor-risk patients, including 44 paired primary-metastatic samples and 11 paired metastatic tumors. FoundationOne® and RNAseq was successful on 89 specimens and 97 specimens, respectively. In addition to somatic alterations, FoundationOne® provided tumor mutational burden (TMB). From RNAseq, we ascertained the TNBC molecular subtypes, and the mRNA expression of immune-related genes. Stromal tumor-infiltrating lymphocytes (stromal TILs), recurrence-free survival, and overall survival were also studied. Results: From FoundationOne® sequencing, a mutational landscape typical of TNBCs was observed across both primary and recurrent disease specimens, with TP53 mutated in 82.0% of specimens, and BRCA1 and BRCA2 mutated in 4.5% and 16.9% of specimens, respectively. Sample profiles revealed minimal shifts in copy number alterations and TMB over time, however, notable TNBC subtype shifts were observed between primary and recurrent tumors. These included an increase in the Lehmann/Pietenpol-defined basal-like 1 phenotype (BL1, 12.8% to 20.9%), an increase in the mesenchymal phenotype (M, 12.8% to 20.9%), and a significant decrease in the immunomodulatory phenotype (IM, 27.1% to 2.3%). Similarly, tumors exhibited a downward shift in gene expression delineating the Burstein-defined basal-like immune-activated phenotype (BLIA, 37.0% to 14.3%). Composite expression of immunomodulatory gene signatures representative of Th1/Th2 responses, IFNg-related inflammation, M1/M2 macrophage activation and suppression, etc., was decreased in the recurrent tumors compared to the primaries (p = 0.01), and histopathology-derived percent stromal TILs were significantly decreased in the recurrent TNBCs (p = 0.02). However, higher stromal TILs (≥30%) were not associated with improved overall survival when measured in primary specimens (p = 0.15), or with the time from relapse to death when measured in recurrent specimens (p = 0.65) in this cohort of immunotherapy-naïve patients. Conclusion: In this retrospective study of paired TNBCs, significant transcriptomic phenotype shifts were observed as patients progressed, while only minor genomic shifts were seen. Selective immune profiling showed significantly reduced TILs and immune-activating gene expression signatures in recurrent TNBCs, which may explain the lack of efficacy of immunotherapeutic agents in heavily pretreated TNBCs. Further studies are ongoing to understand the proteomic landscape shifts in TNBCs over time and to identify novel targeted agents appropriate for recurrent disease. Citation Format: Yuan Y, Yost SE, Chang C-W, Yoh KE, Johnson RM, Schmolze D, Liang J, Hutchinson KE. Comprehensive profiling of poor-risk paired primary and recurrent triple-negative breast cancers reveals immune phenotype shifts [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD5-07.

Durable intracranial and extracranial response to nivolumab with appearance of secondary resistance in a heavily pretreated patient with head and neck cancer

Recently, nivolumab was approved in the second-line setting of squamous cell cancer of the head and neck (SCCHN). The benefits of PD-(L)1 inhibitors in PD-L1(-) tumors are unclear, and no reports exist on the activity of these agents in brain metastases from SCCHN. Little is known regarding the mechanisms underlying acquired resistance to PD-(L)1 inhibition. A patient with PD-L1(-) metastatic SCCHN progressing to cetuximab-based chemotherapy received third-line nivolumab. T cell infiltration and mRNA expression of immune-related genes were compared in prenivolumab and postnivolumab biopsies from a progressing tumor lesion. An exceptional local and systemic response was achieved, including complete devitalization of brain metastases that lasted for more than a year. Increased T cell infiltration and upregulation of genes related to T cell exhaustion and resistance to PD-1 inhibition were found. Durable responses to PD-(L)1 inhibitors may be observed in biomarker-negative SCCHN. Mechanisms of resistance should be studied.

Probiotic potential of Bacillus velezensis JW: Antimicrobial activity against fish pathogenic bacteria and immune enhancement effects on Carassius auratus

This study evaluated the probiotic potential of B. velezensis JW through experimental and genomic analysis approaches. Strain JW showed antimicrobial activity against a broad range of fish pathogenic bacteria including Aeromonas hydrophila, Aeromonas salmonicida, Lactococcus garvieae, Streptococcus agalactiae, and Vibrio Parahemolyticus. Fish (Carassius auratus) were fed with the diets containing 0 (control), 107, and 109 cfu/g of B. velezensis JW for 4 weeks. Various immune parameters were examined at 1, 2, 3, and 4 weeks of post-feeding. Results showed that JW supplemented diets significantly increased acid phosphatase (ACP), alkaline phosphatase (AKP), and glutathione peroxidase (GSH-PX) activity. The mRNA expression of immune-related genes in the head kidney of C. auratus was measured. Among them, the interferon gamma gene (IFN- γ) and tumor necrosis factor-α (TNF-α) showed higher expression after 3 and 4 weeks of feeding (P < 0.05). The expression of interleukin-1 (IL-1) only being significantly upregulated by 109 cfu/g of JW after 1 week of feeding (P < 0.05). The upregulation of interleukin-4 (IL-4) increased over time from 1st to 4th week. The expression of interleukin-10 (IL-10) and interleukin-12 (IL-12) showed an opposite expression pattern with IL-10 significantly upregulated and IL-12 significantly downregulated by JW containing diets at 2, 3, and 4 weeks of post-feeding (P < 0.05). Moreover, fish fed with JW supplemented diets showed significantly improved survival rate after A. hydrophila infection. The analysis of the genome of JW revealed several features aiding host health and being relevant to the GIT adaptation. Four bacteriocins, three Polyketide Synthetase (PKS), and five Nonribosomal Peptide-Synthetase (NRPS) gene clusters were identified in the genome. In summary, the above results clearly proved that B. velezensis JW has the potential to be developed as a probiotic agent in aquaculture.

Reticulo-rumen mass, epithelium gene expression, and systemic biomarkers of metabolism and inflammation in Holstein dairy cows fed a high-energy diet

Feeding a higher-energy diet by increasing cereal grains at the expense of forage during the last 3 to 4 wk prepartum is a traditional approach to help the rumen "adapt" to the traditional diets fed at the onset of lactation. Increasing grain/concentrate in the diet changes ruminal fermentation and in sheep and goats elicits marked changes in mRNA expression of immune-related genes in ruminal epithelium. Whether such changes at the epithelial and systemic levels occur in dairy cows when the dietary energy content increases at a fixed level of concentrate is unknown. Fourteen nonpregnant, nonlactating Holstein cows were fed a control lower-energy (CON, 1.30 Mcal/kg of dry matter) diet to meet 100% of estimated nutrient requirements for 3 wk, after which half of the cows were assigned to a higher-energy diet (OVE, 1.60 Mcal/kg of dry matter) and half of the cows continued on CON for 6 wk. Levels of forage and concentrate for CON and OVE were 80 and 79% and 20 and 21%, respectively. Plasma samples were collected 1 d before slaughter to examine biomarkers of metabolism, liver function, inflammation, and oxidative stress. The reticulo-rumen mass was recorded at slaughter, and samples of epithelium were harvested from all cows. The expression of 29 genes associated with tight junctions, immune function, and nutrient transport (volatile fatty acids, urea, and trace minerals) was examined. Overfeeding energy led to consistently greater dry matter intake over time, and lowered plasma concentrations of haptoglobin, paraoxonase, bilirubin, fatty acids, and myeloperoxidase (secreted by neutrophils). In contrast, OVE resulted in greater hydroxybutyrate and cholesterol concentrations. A greater reticulo-rumen mass in cows fed OVE did not alter genes associated with tight junctions (CDLN1, CDNL4, OCLN, TJP1), immune function (IL1B, IL10, NFKB1, TLR2, TLR4, TNF), oxidative stress (SOD1, SOD2), or most nutrient transporters. However, feeding OVE upregulated the acute-phase protein SAA3 by 3.5-fold and downregulated a volatile fatty acid transporter (SLC16A1) and a Fe and Cu transporter (SLC11A2). The lack of effect on mRNA expression along with lower plasma concentrations of inflammation biomarkers indicates that long-term intake of a higher-energy diet ad libitum was not detrimental to ruminal epithelium integrity. In that context, a protective function of SAA3 could be envisioned with a role in opsonizing gram-negative bacteria that produce endotoxins. The long-term control of volatile fatty acid absorption and trace minerals from the rumen in cows overfed energy does not seem to be controlled at the gene transcription level. The relevance of these findings to the nutritional management of pregnant dry cows merits further research.

Efficacy of PLGA microparticle-encapsulated formalin-killed Aeromonas hydrophila cells as a single-shot vaccine against A. hydrophila infection

Control and prevention of disease is a high priority in aquaculture, and vaccination is important to prevent outbreaks. Here, poly(d,l-lactide-co-glycolic acid) (PLGA) microparticles (MPs) approximately 36μm in diameter were used to encapsulate and deliver Aeromonas hydrophila formalin-killed cells (FKC) as an antigen, and the innate and adaptive immune responses of cyprinid loaches and common carp were assessed following vaccination. The antigen was confirmed to be well encapsulated by scanning electron microscopy analysis of PLGA MP sections. Blood and head kidney specimens were collected and analyzed for bacterial agglutination activity and relative mRNA expression of immune-related genes (IL-1β, IL-10, TNF-α, lysozyme C, TGF-β, and IgM) at 2, 4, 6, and 8weeks post vaccination (wpv). For both fish species, the curve of antibody titer over time was shallower in the PLGA group than the FKC group. These titers in loaches and carp were very similar in the two vaccination groups until 8 and 6 wpv, respectively, but differences were subsequently noted in both species until the end of experiment. Loaches and carp were then challenged with A. hydrophila at 12 and 20 wpv, and 10 and 14 wpv, respectively, and relative survival rates were calculated. For both species, the PLGA groups demonstrated higher survival rates at all time points. Relative expression of IL-1β and TNF-α mRNA was significantly upregulated in the PLGA group at 2 and 4 wpv. Moreover, PLGA-MP vaccination increased relative mRNA levels of lysozyme C and IgM, which were significantly higher than those observed with FKC treatment at 2 wpv and 4, 6, and 8 wpv, respectively. In conclusion, PLGA-MP vaccines have the potential to induce longer and more potent immune responses than FKCs alone, and protect both cyprinid loaches and common carp with greater efficiency.

Efficacy of Spirulina platensis diet supplements on disease resistance and immune-related gene expression in Cyprinus carpio L. exposed to herbicide atrazine

The present study evaluated the immunotoxicological effects of the herbicide atrazine (ATZ) at sub-lethal concentrations and the potential ameliorative influence of Spirulina platensis (SP) over a sub-chronic exposure period on Cyprinus carpio L., also known as common carp. Common carp was sampled after a 40-days exposure to ATZ (428 μg/L) and SP (1%), individually or in combination to assess the non-specific immune response, changes in mRNA expression of immune-related genes [lysozyme (LYZ), immunoglobulin M (IgM), and complement component 3 (C3)] in the spleen, and inflammatory cytokines (interleukins IL–1ß and IL–10) in the head kidney using real-time PCR. Additionally, disease resistance to Aeromonas sobria was evaluated. The results revealed that ATZ exposure caused a significant decline in most of the hematological variables, lymphocyte viability, and lysozyme and bactericidal activity. Moreover, ATZ increased the susceptibility to disease, reflected by a significantly lower post-challenge survival rate of the carp. ATZ may induce dysregulated expression of immune-related genes leading to downregulation of mRNA levels of IgM and LYZ in the spleen. However, expression of C3 remained unaffected. Of the cytokine-related genes examined, IL–1B was up-regulated in the head kidney. In contrast, the expression of IL–10 gene was down-regulated in the ATZ-exposed group. The SP supplementation resulted in a significant improvement in most indices; however, these values did not match with that of the controls. These results may conclude that ATZ affects both innate and adaptive immune responses through the negative transcriptional effect on genes involved in immunity and also due to the inflammation of the immune organs. In addition, dietary supplements with SP could be useful for modulation of the immunity in response to ATZ exposure, thereby presenting a promising feed additive for carps in aquaculture.