Changes In Immune Gene Expression Research Articles

Complex associations between cancer progression and immune gene expression reveals early influence of transmissible cancer on Tasmanian devils.

The world's largest extant carnivorous marsupial, the Tasmanian devil, is challenged by Devil Facial Tumor Disease (DFTD), a fatal, clonally transmitted cancer. In two decades, DFTD has spread across 95% of the species distributional range. A previous study has shown that factors such as season, geographic location, and infection with DFTD can impact the expression of immune genes in Tasmanian devils. To date, no study has investigated within-individual immune gene expression changes prior to and throughout the course of DFTD infection. To explore possible changes in immune response, we investigated four locations across Tasmania that differed in DFTD exposure history, ranging between 2 and >30 years. Our study demonstrated considerable complexity in the immune responses to DFTD. The same factors (sex, age, season, location and DFTD infection) affected immune gene expression both across and within devils, although seasonal and location specific variations were diminished in DFTD affected devils. We also found that expression of both adaptive and innate immune genes starts to alter early in DFTD infection and continues to change as DFTD progresses. A novel finding was that the lower expression of immune genes MHC-II, NKG2D and CD8 may predict susceptibility to earlier DFTD infection. A case study of a single devil with regressed tumor showed opposite/contrasting immune gene expression patterns compared to the general trends observed across devils with DFTD infection. Our study highlights the complexity of DFTD's interactions with the host immune system and the need for long-term studies to fully understand how DFTD alters the evolutionary trajectory of devil immunity.

Polyquaternium polymers cause inflammatory response and alterations of the lipidome in Danio rerio larvae

Polyquaternium polymers are widely used in various applications, such as personal care products and wastewater treatment plants, and eventually end up in the aquatic environment. While polymers have been perceived of low toxicological concern due to their size, several studies have pointed towards water-soluble cationic polymers being toxic towards aquatic organisms – and that the toxicity largely is determined by the polymer charge density. The present study investigated the polyquaternium toxicological mechanism of action throughout lipidomic analysis and changes in immune-gene expression (qPCR) of zebrafish larvae exposed continuously to two water-soluble polymers; a high charge density polyquaternium-6 and a low charge density polyquaternium-10, for 5 and 12 days upon fertilization. The results showed that the investigated polyquaterniums cause both inflammatory responses and significant alterations of the zebrafish larvae lipidome. Depending on polyquaternium polymer and larvae development stage, the gene expression showed an inflammatory response (e.g. significant up-regulation of il8, il1β and tnfα) in the exposed zebrafish. Alterations of the lipidome were additionally observed, with severe depletion of lipids (e.g. lyso-glycerophosphocholines and ceramides) in the 12 days old larvae exposed to high charge density polymer. The findings furthermore support a hypothetical mechanism of action to be non-specific and lethality potentially to be narcosis-like driven.

Probiotics enhance resistance to Streptococcus iniae in Nile tilapia (Oreochromis niloticus) reared in biofloc systems.

Biofloc technology is a rearing technique that maintains desired water quality by manipulating carbon and nitrogen and their inherent mixture of organic matter and microbes. Beneficial microorganisms in biofloc systems produce bioactive metabolites that may deter the growth of pathogenic microbes. As little is known about the interaction of biofloc systems and the addition of probiotics, this study focused on this integration to manipulate the microbial community and its interactions within biofloc systems. The present study evaluated two probiotics (B. velezensis AP193 and BiOWiSH FeedBuilder Syn 3) for use in Nile tilapia (Oreochromis niloticus) culture in a biofloc system. Nine independent 3785 L circular tanks were stocked with 120 juveniles (71.4 ± 4.4 g). Tilapia were fed for 16 weeks and randomly assigned three diets: a commercial control diet or a commercial diet top-coated with either AP193 or BiOWiSH FeedBuilder Syn3. At 14 weeks, the fish were challenged with a low dose of Streptococcus iniae (ARS-98-60, 7.2 × 107 CFU mL-1 , via intraperitoneal injection) in a common garden experimental design. At 16 weeks, the fish were challenged with a high dose of S. iniae (6.6 × 108 CFU mL-1 ) in the same manner. At the end of each challenge trial, cumulative per cent mortality, lysozyme activity and expression of 4 genes (il-1β, il6, il8 and tnfα) from the spleen were measured. In both challenges, the mortalities of the probiotic-fed groups were significantly lower (p < .05) than in the control diet. Although there were some strong trends, probiotic applications did not result in significant immune gene expression changes related to diet during the pre-trial period and following exposure to S. iniae. Nonetheless, overall il6 expression was lower in fish challenged with a high dose of ARS-98-60, while tnfα expression was lower in fish subjected to a lower pathogen dose. Study findings demonstrate the applicability of probiotics as a dietary supplement for tilapia reared in biofloc systems.

Involvement of extracellular vesicles in the progression, diagnosis, treatment, and prevention of whole-body ionizing radiation-induced immune dysfunction.

Acute radiation syndrome (ARS) develops after exposure to high doses of ionizing radiation and features immune suppression and organ failure. Currently, there are no diagnostics to identify the occurrence or severity of exposure and there are limited treatments and preventative strategies to mitigate ARS. Extracellular vesicles (EVs) are mediators of intercellular communication that contribute to immune dysfunction across many diseases. We investigated if EV cargo can identify whole body irradiation (WBIR) exposure and if EVs promote ARS immune dysfunction. We hypothesized that beneficial EVs derived from mesenchymal stem cells (MSC-EVs) would blunt ARS immune dysfunction and might serve as prophylactic radioprotectants. Mice received WBIR (2 or 9 Gy) with assessment of EVs at 3 and 7 days after exposure. LC-MS/MS proteomic analysis of WBIR-EVs found dose-related changes as well as candidate proteins that were increased with both doses and timepoints (34 total) such as Thromboxane-A Synthase and lymphocyte cytosolic protein 2. Suprabasin and Sarcalumenin were increased only after 9 Gy suggesting these proteins may indicate high dose/lethal exposure. Analysis of EV miRNAs identified miR-376 and miR-136, which were increased up to 200- and 60-fold respectively by both doses of WBIR and select miRNAs such as miR-1839 and miR-664 were increased only with 9 Gy. WBIR-EVs (9 Gy) were biologically active and blunted immune responses to LPS in RAW264.7 macrophages, inhibiting canonical signaling pathways associated with wound healing and phagosome formation. When given 3 days after exposure, MSC-EVs slightly modified immune gene expression changes in the spleens of mice in response to WBIR and in a combined radiation plus burn injury exposure (RCI). MSC-EVs normalized the expression of certain key immune genes such as NFκBia and Cxcr4 (WBIR), Map4k1, Ccr9 and Cxcl12 (RCI) and lowered plasma TNFα cytokine levels after RCI. When given prophylactically (24 and 3 hours before exposure), MSC-EVs prolonged survival to the 9 Gy lethal exposure. Thus, EVs are important participants in ARS. EV cargo might be used to diagnose WBIR exposure, and MSC-EVs might serve as radioprotectants to blunt the impact of toxic radiation exposure.

Abstract 700: Induction of cancer-specific T-cell responses in patients immunized with P10s-PADRE vaccine

Abstract Introduction: HR+/HER2− breast cancer is the most common form of breast cancer in the United States. HR+/HER2− tumors are known for a low number of tumor-infiltrating lymphocytes (TILs) and considered less immunogenic than other breast cancer subtypes. We have demonstrated that vaccination with P10s-PADRE, a carbohydrate-mimetic-based peptide, cancer vaccine in combination with standard-of-care chemotherapy in HR+/HER2− breast cancer patients led to an increase in TILs and stromal CD3+ T cells. The current study was performed to determine the vaccine specificity and anti-tumor functionality of T cells in treated patients. Methods: Peripheral blood mononuclear cells (PBMCs) collected at the baseline and after vaccination were used in T-cell assays by multi-color ELISpot, examining Th1, Th2, and cytotoxic responses. PBMCs were also interrogated for the vaccine-induced changes in immune gene expression by next-generation RNA-seq analysis. Results: We observed a significant increase in IFN-g, but not in IL-5 or IL-10, responses in post-immune PBMCs after stimulation with P10s, P10s-PADRE and polyclonal stimulation of T cells by anti-CD3/CD28 antibodies. Stimulation with cancer cell lysate resulted in a strikingly high IFN-g response in post-immune samples. Determining the trio of IFN-g, GzB, and IL-2 together with CD4/CD8 cell proliferation assays of consequent post vaccination specimens established the dynamics of effector T-cell populations. RNA-seq data clearly distinguished post-treatment samples by the increase in immune cell activation, cytokine response, and antigen presentation. Conclusions: The data indicate that immunization of HR+/HER2− breast cancer patients with P10s-PADRE in combination with chemotherapy leads to specific activation of T-cells that recognize breast cancer cells. Improving immunogenicity of such immunologically cold tumors could increase the effectiveness of standard therapeutic approaches. Citation Format: John J. Lee, Bernice C. Nounamo, Fariba Jousheghany, Issam Makhoul, Eric R. Siegel, Thomas Kieber-Emmons, Behjatolah Monzavi-Karbassi. Induction of cancer-specific T-cell responses in patients immunized with P10s-PADRE vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 700.

Abstract 2130: Lipid and immune-based biomarkers associated with clinical response to TPST-1120: A small molecule antagonist of peroxisome-proliferator activated receptor-alpha

Abstract Background: TPST-1120 is a small molecule antagonist of peroxisome-proliferator activated receptor-alpha (PPAR-α), a regulator of fatty acid oxidation and immune suppression. TPST-1120 was well tolerated and showed signs of activity in a phase I trial as monotherapy and in combination with nivolumab (NCT03829436). The objective response rate was 30% (3/10, all partial responses) in subjects treated at the two highest TPST-1120 doses in combination with nivolumab and included two subjects with renal cell carcinoma previously refractory to anti-PD-1 therapy [1]. We performed ctDNA mutational analysis at baseline and quantified lipid and gene expression changes in post-treatment whole blood to identify potential biomarkers of response. Methods: Mutational analysis of ctDNA was assessed using the PredicineCARE™ assay (Predicine Inc.), and lipid analysis was performed by tandem mass spectrometry. Gene expression changes were quantified using the nCounter® PanCancer Immune Profiling panel (NanoString Inc.) supplemented with 30 PPAR-α target genes. Putative clinical response biomarkers were identified as those differentially expressed by patients with partial response (PR) compared to those with progressive disease (p&amp;lt;0.05 by Mann-Whitney U Test). Longitudinal lipid change magnitudes were assessed by Wilcoxon paired analysis (p&amp;lt;0.05). Results: Baseline ctDNA mutational analysis revealed that patients with PR or stable disease were more likely to bear mutations in isocitrate dehydrogenase (IDH) and phosphatase and tensin homolog (PTEN) compared to patients with progressive disease. Patients with PR demonstrated significant elevations (p&amp;lt;0.05) in multiple genes including those associated with lipid transport (APOE), Th17 development (RORC) and down-regulation of CD155, a TIGIT ligand. Lipid analysis demonstrated acute changes in free fatty acids (FFA) four hours after first dose (p&amp;lt;0.05). Among patients receiving combination therapy, the highest post-dose elevations in FFA, lysophosphatidylcholine and lysophosphatidylethanolamine levels were observed in a PR patient exhibiting the longest duration of response. Conclusions: TPST-1120 treated patients with PR demonstrated fatty acid oxidation perturbations and immune gene expression changes as potential biomarkers of clinical benefit. Increased frequencies of responding patients bearing PI3K pathway or IDH mutations may reveal populations likely to benefit from treatment with TPST-1120. 1. Yarchoan M, et al., “A phase 1 study of TPST-1120 as a single agent and in combination with nivolumab in patients with advanced solid tumors.” Journ. Clin. Onc. 2022; 40 (16) suppl. Citation Format: Nathan E. Standifer, Yonchu Jenkins, Sam Whiting, Thomas W. Dubensky. Lipid and immune-based biomarkers associated with clinical response to TPST-1120: A small molecule antagonist of peroxisome-proliferator activated receptor-alpha [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2130.

Systemic immune checkpoint blockade and intraperitoneal chemo-immunotherapy in recurrent ovarian cancer: An interim analysis (317)

Systemic immune checkpoint blockade and intraperitoneal chemo-immunotherapy in recurrent ovarian cancer: An interim analysis (317)

The effects of dietary stachyose as prebiotic on immunity and antioxidant related genes’ expression and lipid metabolism in zebrafish (Danio rerio)

Abstract An 8-week feeding trial was conducted to examine the efficacy of stachyose as a prebiotic on immune parameters, antioxidant-/immune-related genes’ expression, and lipid metabolism of zebrafish. Three hundred zebrafish (0.45 ± 0.08 g) were fed four diets containing different stachyose levels at 0, 1, 2 and 4 g kg−1, respectively. After eight weeks of the feeding trial, immunity, antioxidant defence and lipid metabolism were tested. It was observed that the addition of stachyose to the diet induced no significant influence (P&gt;0.05) in SOD, GPX, and CAT, gene’s expression, compared to the control diet. The inclusion of stachyose resulted in no significant changes in immune gene expression (Lyz, IL-1, IL-6, and TNF) in zebrafish (P&gt;0.05) compared to the control diet. Total cholesterol, triglyceride and LDL (low-density lipoprotein) significantly (P&lt;0.05) decreased with the addition of 2 and 4 g kg−1 stachyose, while fish fed the control diet and 1 g.kg−1 recorded the highest significant value of LDL (P&lt;0.05). Fish fed diet, either control diet or diet supplemented with 0.5 g kg−1 stachyose, recorded the lowest HDL value (P&lt;0.05) compared to other treatments. In conclusion, stachyose can be potentially used as a feed additive to modulate lipid metabolism. However, this prebiotic did not benefit immune parameters and antioxidant defence.

Plasma extracellular vesicles released after severe burn injury modulate macrophage phenotype and function

Extracellular vesicles (EVs) have emerged as key regulators of immune function across multiple diseases. Severe burn injury is a devastating trauma with significant immune dysfunction that results in an ∼12% mortality rate due to sepsis-induced organ failure, pneumonia, and other infections. Severe burn causes a biphasic immune response: an early (0-72h) hyper-inflammatory state, with release of damage-associated molecular pattern molecules, such as high-mobility group protein 1 (HMGB1), and proinflammatory cytokines (e.g., IL-1β), followed by an immunosuppressive state (1-2+ wk post injury), associated with increased susceptibility to life-threatening infections. We have reported that early after severe burn injury HMGB1 and IL-1βare enriched in plasma EVs. Here we tested the impact of EVs isolated after burn injury on phenotypic and functional consequences in vivo and in vitro using adoptive transfers of EV. EVs isolated early from mice that underwent a 20% total body surface area burn injury (burn EVs) caused similar hallmark cytokine responses in naïve mice to those seen in burned mice. Burn EVs transferred to RAW264.7 macrophages caused similar functional (i.e., cytokine secretion) and immune gene expression changes seen with their associated phase of post-burn immune dysfunction. Burn EVs isolated early (24h) induced MCP-1, IL-12p70, and IFNγ, whereas EVs isolated later blunted RAW proinflammatory responses to bacterial endotoxin (LPS). We also describe significantly increased HMGB1 cargo in burn EVs purified days 1 to 7 after injury. Thus, burn EVs cause immune outcomes in naïve mice and macrophages similar to findings after severe burn injury, suggesting EVs promote post-burn immune dysfunction.

Early Changes in Interferon Gene Expression and Antibody Responses Following Influenza Vaccination in Pregnant Women.

Influenza immunization during pregnancy provides protection to the mother and the infant. Studies in adults and children with inactivated influenza vaccine have identified changes in immune gene expression that were correlated with antibody responses. The current study was performed to define baseline blood transcriptional profiles and changes induced by inactivated influenza vaccine in pregnant women and to identify correlates with antibody responses. Pregnant women were immunized with inactivated influenza vaccine during the 2013-2014 and 2014-2015 seasons. Blood samples were collected on day 0 (before vaccination) and on days 1 and 7 after vaccination for transcriptional profile analyses, and on days 0 and 30, along with delivery and cord blood samples, to measure antibody titers. Transcriptional analysis demonstrated overexpression of interferon-stimulated genes (ISGs) on day 1 and of plasma cell genes on day 7. Prevaccination ISG expression and ISGs overexpressed on day 1 were significantly correlated with increased H3N2, B Yamagata, and B Victoria antibody titers. Plasma cell gene expression on day 7 was correlated with increased B Yamagata and B Victoria antibody titers. Compared with women who were vaccinated during the previous influenza season, those who were not showed more frequent significant correlations between ISGs and antibody titers. Influenza vaccination in pregnant women resulted in enhanced expression of ISGs and plasma cell genes correlated with antibody responses. Brief summary: This study identified gene expression profiles of interferon-stimulated genes and plasma cells before vaccination and early after vaccination that were correlated with antibody responses in pregnant women vaccinated for influenza.

Transcriptome analysis of the bursa of Fabricius and thymus of laying ducks reveals immune gene expression changes underlying the impacts of stocking densities

ABSTRACT 1. The thymus and bursa of Fabricius are important immune organs in poultry as they play essential roles in sustaining the normal immune function to maintain health. The following trial investigated whether the stocking density affected gene expressions in immune organs. 2. Jinding ducklings were raised in either low or high density (4 or 8 birds/m2) conditions from four to 14 weeks of age, and were then slaughtered and tissues removed. Samples were subjected to high-throughput sequencing to sequence RNA extraction. After filtering calculations with R software, a total of 508 (thymus) and 1,356 (bursa of Fabricius) differentially expressed genes (DEGs) were identified, suggesting that stocking density has an effect on gene expression in duck immune organs. 3. Out of a total of 112 immune factor genes and 112 immune pattern receptor genes in ducks, four thymus and 18 bursa of Fabricius genes were differentially expressed in ducks, which indicated that the change of stocking density could affect the expression of immune genes in poultry.

Immunotranscriptomic profiling the acute and clearance phases of a human challenge dengue virus serotype 2 infection model

About 20–25% of dengue virus (DENV) infections become symptomatic ranging from self-limiting fever to shock. Immune gene expression changes during progression to severe dengue have been documented in hospitalized patients; however, baseline or kinetic information is difficult to standardize in natural infection. Here we profile the host immunotranscriptome response in humans before, during, and after infection with a partially attenuated rDEN2Δ30 challenge virus (ClinicalTrials.gov NCT02021968). Inflammatory genes including type I interferon and viral restriction pathways are induced during DENV2 viremia and return to baseline after viral clearance, while others including myeloid, migratory, humoral, and growth factor immune regulation factors pathways are found at non-baseline levels post-viremia. Furthermore, pre-infection baseline gene expression is useful to predict rDEN2Δ30-induced immune responses and the development of rash. Our results suggest a distinct immunological profile for mild rDEN2Δ30 infection and offer new potential biomarkers for characterizing primary DENV infection.

A novel non-viral delivery method that enables efficient engineering of primary human T cells for ex vivo cell therapy applications

Background aimsNext-generation immune cell therapy products will require complex modifications using engineering technologies that can maintain high levels of cell functionality. Non-viral engineering methods have the potential to address limitations associated with viral vectors. However, while electroporation is the most widely used non-viral modality, concerns about its effects on cell functionality have led to the exploration of alternative approaches. Here the authors have examined the suitability of the Solupore non-viral delivery system for engineering primary human T cells for cell therapy applications. MethodsThe Solupore system was used to deliver messenger RNA (mRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) guide RNA ribonucleoprotein (RNP) cargos to T cells, and efficiency was measured by flow cytometry. Cell perturbation was assessed by immune gene expression profiling, including an electroporation comparator. In vitro and in vivo cytotoxicity of chimeric antigen receptor (CAR) T cells generated using the Solupore system was evaluated using a real-time cellular impedance assay and a Raji-luciferase mouse tumor model, respectively. ResultsEfficient transfection was demonstrated through delivery of mRNA and CRISPR CAS9 RNP cargos individually, simultaneously and sequentially using the Solupore system while consistently maintaining high levels of cell viability. Gene expression profiling revealed minimal alteration in immune gene expression, demonstrating the low level of perturbation experienced by the cells during this transfection process. By contrast, electroporation resulted in substantial changes in immune gene expression in T cells. CAR T cells generated using the Solupore system exhibited efficient cytotoxicity against target cancer cells in vitro and in vivo. ConclusionsThe Solupore system is a non-viral means of simply, rapidly and efficiently delivering cargos to primary human immune cells with retention of high cell viability and functionality.

Pathogenesis and expression profile of selected immune genes to experimental Edwardsiella tarda infection in iridescent shark Pangasianodon hypophthalmus

Pathogenesis and immune gene expression of the iridescent shark catfish Pangasianodon hypophthalmus to experimental Edwardsiella tarda infection was studied. Healthy Pangasianodon were inoculated intramuscularly with a sub-lethal dose of 1.77 × 107 cells/fish of E. tarda to induce edwardsiellosis. Petechial haemorrhages, inflammation at the site of injection, vertical hanging from the water surface, swollen abdomen and anorexia were the common signs observed in challenged catfish fingerlings. The morbid catfish also exhibited typical signs of septicaemia with haemorrhages on ventral parts of the body and fin bases and congested vent prior to death. The mortality was observed 48 h post injection. The average percent morality was 42 % in fish exposed to 1.77 × 107 cells of E. tarda, whereas, only6.6 % mortality was observed in placebo control (catfish received 0.1 mL of 0.85 % sterile physiological saline) and no mortality in negative control group (catfish received no injection) over an observational period of 20 days. The kidney and liver of moribund fish demonstrated different necrobiotic changes. In kidney, cellular and cloudy swelling, degeneration of renal tubules, necrosis of glomerulus, dilation of Bowman’s space, hypertrophy, fibrosis of renal tubules and increased melano-macrophage reaction were frequently observed. In liver, disorganized hepatocytes, blood congestion and nuclear pyknosis were evident. To ascertain the host response to E. tarda infection, mRNA levels of three different classes of innate immune components viz. cytokine interleukin-1β (IL-1β), acute phase protein transferrrin and complement C3 were assessed by quantitative real-time PCR. Significant up-regulation (P < 0.05) of IL-1β and C3 gene expression was observed in infected fish upto 15 days post infection (dpi) in liver, kidney, spleen and blood though the level of increase differed among organs and dpi. Significant up-regulation (P < 0.05) of transferrin gene expression was seen at 1 dpi in kidney, spleen and blood while no significant variation could be observed in liver. The tissue specific pathology and changes in immune gene expression may help to improve the knowledge of E. tarda pathogenesis and role of the innate immune genes in this tropical catfish. The innate immune response of host to E. tarda which is evident from increased expression of innate immune genes may be further enhanced through immunostimulants and nutraceuticals to prevent disease onset and use of antibiotics.

Immune response of human cultured cells towards macrocyclic Fe2PO and Fe2PC bioactive cyclophane complexes.

Synthetic molecules that mimic the function of natural enzymes or molecules have untapped potential for use in the next generation of drugs. Cyclic compounds that contain aromatic rings are macrocyclic cyclophanes, and when they coordinate iron ions are of particular interest due to their antioxidant and biomimetic properties. However, little is known about the molecular responses at the cellular level. This study aims to evaluate the changes in immune gene expression in human cells exposed to the cyclophanes Fe2PO and Fe2PC. Confluent human embryonic kidney cells were exposed to either the cyclophane Fe2PO or Fe2PC before extraction of RNA. The expression of a panel of innate and adaptive immune genes was analyzed by quantitative real-time PCR. Evidence was found for an inflammatory response elicited by the cyclophane exposures. After 8 h of exposure, the cells increased the relative expression of inflammatory mediators such as interleukin 1; IRAK, which transduces signals between interleukin 1 receptors and the NFκB pathway; and the LPS pattern recognition receptor CD14. After 24 h of exposure, regulatory genes begin to counter the inflammation, as some genes involved in oxidative stress, apoptosis and non-inflammatory immune responses come into play. Both Fe2PO and Fe2PC induced similar immunogenetic changes in transcription profiles, but equal molar doses of Fe2PC resulted in more robust responses. These data suggest that further work in whole animal models may provide more insights into the extent of systemic physiological changes induced by these cyclophanes.

Rainbow trout (Oncorhynchus mykiss) adipose tissue undergoes major changes in immune gene expression following bacterial infection or stimulation with pro-inflammatory molecules

In mammals, visceral adipose is increasingly seen as playing an important role in immune function with numerous pro-inflammatory, anti-inflammatory and immune-modulating proteins and peptides being identified in adipocytes. Adipose is also now known as a tissue that has an important role in the regulation of peritoneal immune responses. Despite this, only lately has consideration been given to visceral adipose as an important immune tissue in fish, especially in the context of intraperitoneal vaccination. The present study demonstrates that fish visceral adipose is capable of expressing a large range of immune molecules in response to stimulation with a live bacterium (A. salmonicida), a bacterial PAMP (Y. ruckeri flagellin), and the pro-inflammatory cytokines IL-1β, TNF-α3 and IFN-γ. Following infection and stimulation with flagellin and IL-1β a large upregulation of pro-inflammatory and antimicrobial molecules was seen, with a high degree of overlap. TNF-α treatment affected relatively few genes and the effects were more modest. IFN-γ had the smallest impact on adipose but IFN-γ inducible genes showed some of the largest effects. Overall, it is clear that adipose tissue should be considered an active immune site in fish, capable of participating in and influencing immune responses.

Analysis of adipose tissue immune gene expression after vaccination of rainbow trout with adjuvanted bacterins reveals an association with side effects

Most existing fish vaccines are presented in the form of oil-based emulsions delivered by intraperitoneal injection. Whilst very effective they are frequently associated with inflammatory responses that can result in clinically significant side-effects often involving the adipose tissue that is in direct contact with the vaccine. To explore the potential of immune gene expression changes in the adipose tissue of fish to be markers of vaccination efficacy or development of side-effects we have studied the response to a bacterial (Aeromonas salmonicida) vaccine administered with two different adjuvants. The first adjuvant was Montanide™ ISA 763A VG, thought to induce a mostly humoral response, and the second was Montanide™ ISA 761 VG that gives a more balanced humoral and cell mediated response. Following vaccination tissue samples were collected at days 3, 14 and 28 for RTqPCR analysis. Fifty immune genes were studied with a focus on a) pro-inflammatory associated molecules and b) adaptive immune response related molecules linked with possible Th1, Th2, Th17 and T-regulatory pathways, with the expression data analysed for associations with Speilberg post-vaccination side effect scores. The results showed that the adipose tissue is a particularly sensitive and discriminatory tissue for studying adjuvant effects. A clear upregulation of many immune genes occurred in response to both vaccine groups, which persisted over time and overlapped with the appearance of visible adhesions. Our analysis revealed a relationship between adipose tissue immune function and the development of vaccine-induced adhesions giving the potential to use immune gene expression profiling in this tissue to predict the side-effects seen.

An Extract of Sericea Lespedeza Modulates Production of Inflammatory Markers in Pathogen Associated Molecular Pattern (PAMP) Activated Ruminant Blood

&lt;p&gt;Programs based on antibiotics are failing to control diseases due to increase in resistance of pathogens to antibiotics. Food safety, animal welfare and public health concerns have fueled interest in the use of plant-based alternatives. This study was conducted to evaluate the effect of a plant (Sericea Lespedeza, SL), and pathogen associated molecular patterns (PAMPs) (Lipopolysaccharide (LPS) and peptidoglycan (PGN)) on gene activation in ruminant blood. A water extract of SL, was used as a source of plant-derived tannins. Blood was collected from Holstein-Friesian cows (N = 4), Spanish × Boer goats (N = 4), St Croix sheep (N = 4) and incubated with 100 ng/mL of SL in the presence or absence of LPS or PGN. Samples maintained in Phosphate-buffered saline (PBS) served as negative control. The total protein concentration, WNT5a, and prostaglandin E2 in plasma were determined. Total RNA was isolated, reverse transcribed and Real time-PCR was performed using gene specific primers for TLR2, TLR4, WNT5a, and FZD. TLR2 and FZD were up-regulated in response to PAMPs. WNT5a and TLR4 genes were undetected in PAMP treated blood. SL regulated protein and prostaglandin concentration in all species. SL reduced PGE2 in sheep and cow blood. WNT5a was only secreted in LPS treated cow blood. Transcription and translation of genes involved in innate and adaptive immunity and the WNT signaling pathway in ruminant blood were responsive to diverse PAMPS, and can be modulated by SL. This suggests that dietary tannins may promote the health of ruminants. Further studies are needed to determine the significance of these changes in immune gene expression on ruminant health.&lt;/p&gt;

Tissue Phthalate Levels Correlate With Changes in Immune Gene Expression in a Population of Juvenile Wild Salmon.

Phthalates have detrimental effects on health and have been shown to dysregulate the immune system of mammals, birds, and fish. We recently reported that di(2-ethylhexyl) phthalate exposure reduces the abundance and inhibits the proliferation of rainbow trout (Oncorhynchus mykiss) IgM(+) B lymphocytes and expression of secreted immunoglobulin heavy-chain mu transcripts in an in vitro culture system. We proposed that phthalates act as immunomodulators by modifying the normal B cell-activation pathways by accelerating B cell differentiation while suppressing plasmablast expansion, thus resulting in fewer IgM-secreting plasma cells. This hypothesis was tested here in an in vivo field study of juvenile Dolly Varden (Salvelinus malma) from a plastic-polluted lake in the Gulf of Alaska. Fish tissues were analyzed for both phthalate levels using liquid chromatography-coupled tandem mass spectrometry and for changes in immune gene expression using reverse transcriptase-real time polymerase chain reaction. Results showed that fish with higher tissue levels of di(2-ethylhexyl) phthalate, di(n-butyl) phthalate, and/or dimethyl phthalate expressed significantly fewer secreted and membrane-bound immunoglobulin heavy-chain mu and Blimp1 transcripts in their hematopoietic tissue. This suggests that in vivo uptake of phthalates in fish changes the expression of B cell-specific genes. Chronic exposure to phthalates likely dysregulates normal B-lymphoid development and antibody responses in salmonids and may increase susceptibility to infection. Given the conserved nature of B-lineage cells in vertebrate animals, other marine species may be similarly affected by chronic phthalate exposure.

Biomarker Development for Cancer Immuno-oncology/Immunotherapy: Simultaneous Digital Counting of Nucleic Acids and Proteins at 800-plex

Abstract The ability to measure changes to the DNA, RNA, and protein is crucial to understanding both tumor and immune responses to therapy. NanoString® has pioneered 3D Biology™ – the ability to measure any combination of DNA, RNA, and protein simultaneously using a single detector from a small clinical sample – in order to characterize these complex interactions. Analytes of interest are labeled with fluorescent nucleic acid barcode (via direct hybridization for nucleic acids or antibody conjugated barcodes for protein) and detected via digital counting. This enables quantification of up to 800 analytes from a small sample (50,000 cells for DNA/RNA/protein, or 1–2 5μm FFPE slides for RNA alone). The cancer immunology profiling panel measures 770 mRNAs (unique signatures for 24 infiltrating immune cell types plus extensive immune-signaling pathways) plus 30 key IO proteins. We present two examples of 3D Biology in action. First, a targeted BRAFV600E inhibitor induces expression changes of immune genes which can be detected at both the RNA and protein levels. Second, in work from ongoing collaborations with the Cancer Immunotherapy Trials Network, clinical samples from patients treated with either mono- or combination immunotherapy can be stratified by gene expression. We also show that gene signatures predict presence of immune cell populations with a high degree of concordance to flow cytometry and immunohistochemistry measurements. Multiplexed RNA biomarkers have achieved success in predicting patient response to therapy, e.g. NanoString signatures predict response to pembrolizumab (anti-PD-1). Measuring DNA, RNA, and proteins in unison greatly expands the power of biomarker signatures to characterize responses to immunotherapy.