Expression Of Immune-associated Genes Research Articles

Single-cell RNA sequencing highlights the role of proinflammatory fibroblasts, vascular endothelial cells, and immune cells in the keloid immune microenvironment.

Keloids, characterized by an aberrant wound-healing process and a highly complex immune microenvironment, pose significant challenges for clinical management. Fibroblasts and vascular endothelial cells (VEC) were identified as the leading cells of keloid development. However, their roles in the keloid immune landscape have yet to be thoroughly elucidated. To explore the functional state of cells in the immune landscape of keloids, we conducted a single-cell RNA sequencing analysis on the tissue from three keloid lesions and two specimens of healthy skin. We simultaneously utilized available keloid data from the public database for external validation. Specific subsets, such as proinflammatory fibroblasts (piF) and VEC, were markedly elevated in lesional skincompared to normal skin. Subsequent differential gene expression and Gene Ontology analyses indicated that these subsets may be involved in shaping the microenvironment. In keloids, there is an increased expression of immune-associated genes (P < 0.05), including TNFRSF6B, HGF, and TGFB3, alongside a decreased expression of inflammatory chemokines in the piF. Moreover, the significant upregulation of immune suppressive genes (P < 0.05), including CD39, CD73, and HIF1A, suggested the potential involvement of VEC as a conditional immune subpopulation in the keloid microenvironment. Immune cell communication analysis revealed preferential enrichment of macrophages and Tregs, highlighting intensified macrophage-centered interactions within the keloid microenvironment. Our study highlighted the role of piF and VEC in the immune microenvironment of keloids for the first time, providing potential targets for therapeutic development.

Abstract B048: Lipopolysaccharide (LPS) mediated Toll like receptor-4 (TLR4) agonism immuno-modulates pancreatic cancer niche via CD4+ T cells-Myeloid cells axis

Abstract Introduction: Pancreatic cancer is a major cause of cancer-related mortality with a dismally low overall survival. Despite advancement in the cancer immunotherapy, pancreatic cancer remains unresponsive due to its immunosuppressives. We have established that lipopolysaccharide (LPS) reduces tumor burden in pancreatic cancer mouse-models by stimulating anti-tumor immunity and infiltrating cytotoxic CD4+ T-cells in the tumor-microenvironment. Here, we are investigating how LPS immuno-modulates pancreatic cancer niche and brings anti-cancerous effect. Methods: Pancreatic cancer-cells (KPCs) were implanted in C57BL/6, TRIF KO, MyD88 KO and TLR4 flox/flox LysmCre (TLR4f/f) mice subcutaneously and were treated with LPS (1mg/kg) weekly. The animals were sacrificed at endpoints and tumors were evaluated. Subcutaneous KPC-LPS model in BL/6 mice were also coupled with clodronate treatment to deplete cells of myeloid lineage. Tumor samples were analyzed using flow-cytometry and RT- PCR for immunocyte infiltration and expression of immune-associated genes. Dendritic cells (DCs) were isolated from BL/6 and TLR4f/f mice, pulsed with KPC cancer cell antigen, treated with LPS and taken to flow cytometry. Such DCs were also co-cultured with CD4+ T-cells, which were then subjected to cytotoxicity against cancer cells Results: LPS therapy in wild-type pancreatic cancer mouse-model significantly reduced the tumor burden by stimulating anti- tumor immunity in tumor-microenvironment and caused significant upregulation of activated DCs and macrophages. The tumor regressing effect of LPS disappeared in absence of TRIF as well as MyD88 receptors in TRIF KO and MyD88 KO mice respectively. Macrophage depletion by clodronate attenuated influence of LPS on tumor burden, which again proved futile in TLR4f/f model. On antigenic stimulation and LPS treatment, DCs from BL/6 mice expressed higher frequency of co-stimulatory molecules such as CD40, CD80 and CD86 whereas TLR4f/f mice derived DCs did not show such a trend. When such BL/6 DCs were co-cultured with CD4+ T- cells, they expressed higher cytotoxicity against cancer cells. Conclusions: LPS treatment reduces tumor burden in pancreatic cancer model by stimulating anti-tumor immunity in tumor- microenvironment wherein cells of the myeloid lineage are indispensable. This anti-cancer effect executed by CD4+ T-cells in an MHC II dependent manner, seems to be mediated through dendritic cells. Designer TLR4-agonists have the potential to emerges as a novel immunotherapeutic agent and could be combined with available immune-checkpoint blockade therapies. Citation Format: Utpreksha Vaish, Tejeshwar Jain, Dhanisha S Suresh, Maria F Salcedo-Noriega, Srikanth Iyer, Troy Randall, Vikas Dudeja. Lipopolysaccharide (LPS) mediated Toll like receptor-4 (TLR4) agonism immuno-modulates pancreatic cancer niche via CD4T cells-Myeloid cells axis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B048.

Novel insights into the complex interplay of immune dysregulation and inflammatory biomarkers in preeclampsia and fetal growth restriction: A two-step Mendelian randomization analysis

BackgroundThe relationship between genetic immune dysregulation and the occurrence of preeclampsia (PE) or PE with fetal growth restriction (PE with FGR) has yielded inconsistent findings, and the underlying mediators of this association remain elusive. We aimed to explore the causal impact of genetic immune dysregulation on the risk of PE or PE with FGR and to elucidate the role of specific transcriptomes in mediating this relationship. MethodsA two-step Mendelian randomization (MR) analysis was performed to explore the link between immune dysregulation and PE or PE with FGR, as well as to identify potential inflammatory biomarkers that act as mediators. GWAS summary data for outcomes were obtained from the FinnGen dataset. The analyses encompassed five systemic immune-associated diseases, four chronic genital inflammatory diseases, and thirty-one inflammatory biomarkers. Summary-data-based MR (SMR) and HEIDI analysis were conducted to test whether the effect size of single nucleotide polymorphisms (SNPs) on outcomes was mediated by the expression of immune-associated genes. ResultsThe primary univariable analysis revealed a significant positive correlation between systemic lupus erythematosus (SLE), type 1 diabetes (T1D), type 2 diabetes (T2D), and rheumatoid arthritis (RA) with the risk of PE or PE with FGR. Surprisingly, a counterintuitive finding showed a significant negative association between endometriosis of pelvic peritoneum (EMoP) and the risk of PE with FGR. None of the inflammatory factors had a causal relationship with PE or PE with FGR. However, there was a significant association between lymphocyte count and the risk of PE with FGR. Within the lymphocyte subset, both the proportion of Natural Killer (NK) cells and absolute counts of naïve CD4+ T cells demonstrated significant effects on the risk of PE with FGR. Two-step MR analysis underscored the genetically predicted lymphocyte count as a significant mediator between T1D and PE with FGR. Additionally, SMR analysis indicated the potential involvement of SH2B3 in the occurrence of PE with FGR. ConclusionsOur findings provided substantial evidence of the underlying causal relationship between immune dysregulation and PE or PE with FGR and some of these diseases proved to accelerate immune cells disorders and then contribute to the risk of incident PE or PE with FGR.

Predicting therapeutic responses in head and neck squamous cell carcinoma from TP53 mutation detected by cell-free DNA.

Head and neck squamous cell carcinoma (HNSCC) is an epithelial malignant tumor originating from the oral cavity, oropharynx, nasal cavity, sinuses, nasopharynx, hypopharynx, or larynx. Mutations in TP53 are the most common of all somatic genomic changes in HNSCC, and TP53 mutations are associated with the response to immunotherapy and chemotherapy. Tumor-derived circulating cell-free DNA (cfDNA) is a minimally invasive method to determining genetic alterations in cancer. This study aimed to explore the therapeutic responses of patients with HNSCC with TP53 mutation and the accuracy of cfDNA for detecting TP53 mutation. Information on TP53 mutations, patient survival time, and clinical data in HNSCC were downloaded from The Cancer Genome Atlas database. The difference in immune infiltration between the TP53-mutant group and the wild-type group was compared. We applied the single-sample gene set enrichment analysis method on the transcriptome of HNSCC samples to assess the distribution of immune cell types between the two groups. The chemotherapy response was constructed using the R software package, "pRRophetic". Gene set enrichment analysis was performed based on the TP53 mutation. The next-generation sequencing was executed on cfDNA from nine patients with HNSCC to detect genetic alterations. Tumor biopsy (n=9) was sequenced using the same technique. TP53 was the most frequently mutated gene in HNSCC. The TP53 mutation was related to immune cells and the expression of immune-associated genes. The TP53 mutation group showed lower response to immunotherapy but high sensitivity to some chemotherapies compared with the wild-type group. TP53 was the most frequently mutated gene (6/9; 66.67%) in cfDNA. Only 27.27% of TP53 mutations in tumor tissue were detected outside of cfDNA. TP53 mutation could be used as a specific predictor of treatment response in patients with HNSCC. Using cfDNA to detect the TP53 mutations in patients with HSNCC is a feasible method. The results suggested that the therapeutic response in patients could be predicted by detecting TP53 mutations in cfDNA, and large-scale and prospective studies are needed to validate this hypothesis.

Characterization and pathogenicity analysis of a newly isolated strain of infectious hematopoietic necrosis virus

Rainbow trout is one of the fastest-growing aquaculture species and infectious hematopoietic necrosis virus (IHNV) is endemic throughout almost all rainbow trout farms in China nowadays. In this study, IHNV GS21 was identified as the causative pathogen, which resulted in massive mortality of rainbow trout occurring in northwest China. GS21 isolate was propagated in Chinook salmon embryonic cell line (CHSE-214) and induced apparent cytopathic effects (CPE) at 3 days post-infection (dpi). Phylogenetic analysis revealed that GS21 isolate was clustered with other reported Chinese isolates within the J genogroup. Moreover, the complete cDNA sequence of GS21 isolate was obtained and it possesses more than 98 % of ANI values and 89 % of DDH values with other Chinese IHNV isolates. The detailed sequence analysis of G gene revealed the distinct amino acid substitutions of G230, G252, G270, and I277 in GS21 isolate. Furthermore, the artificially infected rainbow trout exhibited similar clinical disease symptoms as natural infection did. The cumulative mortality infected by GS21 isolate of 104 PFU/mL reached 93 % at approximately 13.5 °C. Additionally, viral loads in tissues increased first and declined then as well as the expression of immune-associated genes. Collectively, our results characterized a novel IHNV GS21 isolate that can lead to massive mortality in juvenile rainbow trout and provided a basis to define the pathogenic characteristics and evolutionary relationship of IHNV and host immune response against IHNV infection.

Toxicity evaluation of zinc oxide nanoparticles green synthesized using papaya extract in zebrafish.

In green synthesis of zinc oxide nanoparticles (ZnO NPs), the use of papaya extract as a capping and reducing agent shows promise for potential applications of these particles in biomedicine. However, toxicity evaluation is necessary to ensure the safety of humans and the environment. The zebrafish model is used to assess toxicity with embryo developmental observation as it is a rapid, simple method for screening of toxicity. The objective of the present study was to assess the toxicological characteristics of ZnO NPs produced from papaya extract using a zebrafish model. The preparation of plant extracts from papaya using two solvents (water and methanol) and characterization of bioactive compounds in the extracts were reported. ZnO NPs were synthesized from both plant extracts and characterized with scanning electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy. Toxicity evaluation was conducted on zebrafish embryos for 96 h. ZnO NPs synthesized from aqueous and methanol extracts had mean crystallite diameters of 13 and 12 nm, respectively. Mortality, hatching rate and malformation of zebrafish embryos were assessed at different concentrations of ZnO NPs. Both NPs showed high mortality rates at high concentrations, with 100 (aqueous) and 20 mg/l (methanol extract) being lethal for all embryos. Concentrations <10 mg/l for both synthesized ZnO NPs had similar results to the negative control, indicating a safe dosage for embryos. The hatching rate and malformation were also affected, with higher concentrations of NPs causing a delayed hatching rate and malformation in pericardial and yolk sac edema. Whole embryo mRNA expression of immune-associated genes, including IL-1 and -10 and TNF-α, was upregulated following lethal concentration 50 (LC50) ZnO NP exposure. ZnO NPs synthesized from papaya extract (both in aqueous and methanol environments) had a dose- and time-dependent embryonic toxicity effect. Hence, the present study demonstrated initial toxicity screening of ZnO NPs synthesized from plant extract.

Immune responses of hemocytes in the blood clam Tegillarca granosa in response to in vivo Vibrio harveyi infection

Aquaculture of the blood clam Tegillarca granosa accounts for approximately 50% of Arcidae (ark shell) production in China. Vibrio infection severely threatens the sustainability of the clam aquaculture industry. Exposure to Vibrio induces an immune response in blood clams. However, the underlying mechanism remains poorly understood. In this study, immune responses of hemocytes in blood clams were detected after Vibrio infection; the immersion method was used in vivo to mimic the clam's natural infection process. After 24 h of exposure to Vibrio infection, the Vibrio load in hemolymph fluid in both the treatment Ⅰ (25,033.33 ± 19,563.11 CFU/mL) and treatment Ⅱ (122,163.33 ± 194,409.49 CFU/mL) groups were significantly higher, than that in the control group (13.67 ± 37.73 CFU/mL) (P < 0.05). Correspondingly, the production of intracellular reactive oxygen species was approximately 1.40 (treatment Ⅰ) and 2.12 (treatment Ⅱ) fold higher than that in the control group (P < 0.05), and the induced DNA damage showed a similar trend (P < 0.05). Vibrio infection also significantly increased lysozyme content, adenosine triphosphate content, and peroxidase isozyme activity, in both the serum and hemocyte lysates (P < 0.05). The expression of immune-associated genes (ABCA3, c-Myc, Caspase 3, and HSP70) was upregulated under infection conditions. The phagocytic activity was approximately 1.99 (treatment Ⅰ) and 2.57 (treatment Ⅱ) fold that in control clams (P < 0.05). In addition, the total hemocyte count and red granulocyte percentage both significantly decreased by approximately 75–90% after Vibrio infection. These results provided novel insights into the mechanism of hemocyte immunity in T. granosa against Vibrio infection, which may aid in the future prevention and control of Vibrio infection in vivo.

Abstract B038: Toll like receptor 4 (TLR4) agonism by lipopolysaccharide (LPS) in pancreatic cancer model stimulates anti-tumor immunity in CD4+T cell dependent fashion

Abstract Introduction: Pancreatic cancer, one of the most lethal malignancies, has a 5-year overall survival of approximately 10%. It is an immunologically cold tumor and is poorly responsive to immunotherapy. Globally, researchers are in quest of immunomodulatory modalities to improve the therapeutic response. lipopolysaccharide (LPS), a known toll like receptor 4 (TLR4) agonist is known to activate immune response. Here, we evaluate the effect of LPS mediated TLR4 agonism on the anti-tumor immune machinery of pancreatic cancer. Methods: We implanted KPC (Kras LSL-G12D; Trp53 LSL-R172H/+; Pdx1Cre/+) pancreatic cancer cells in C57BL/6 mice subcutaneously. The animals were treated intraperitoneally with LPS (1mg/kg) or saline (control) at day 10 and 17. Tumor samples were isolated at endpoint and were analyzed using flow cytometry and real time polymerase chain reaction (RT-PCR) for infiltration of immunocytes and expression of immune-associated genes respectively. Functional response of CD4+ T-cells to tumor antigen re-challenge was assessed by measuring interferon-γ (IFN-γ) release in-vitro. In another experiment, LPS or saline treatment was administered along with depletion of CD4+ or CD8+ T-cells employing monoclonal antibodies. CD4+ T-cells extracted from spleens of tumor bearing control or LPS treated mice were subjected to Calcein AM based cytotoxicity assay. Cytotoxicity assay was also performed by blocking major histo-compatibility II (MHCII) on KPC cells. Results: LPS administration in tumor bearing mice significantly reduced the tumor burden as compared to control group. Flow cytometry on tumor samples revealed increased infiltration of CD4+ T-cells in LPS group whereas CD8+ T-cell repertoire was unaffected. Tumor samples from the LPS group showed higher expression of genes for anti-tumor cytokines such as IFN-γ, granzymes and perforin. Depletion of CD4+ T cells, but not CD8+ T cells attenuated the effect of LPS on tumor growth. When the splenic CD4+ T-cells from both groups were pulsed with KPC cell lysate, LPS treated mice derived CD4+ T-cells demonstrated a greater ability to secrete IFN-γ. Splenic CD4+ T-cells from LPS treated mice showed significantly higher cytotoxicity against KPC cells as compared to control CD4+ T-cells. However, blocking MHCII molecules on KPC cells reduced the cytotoxicity in CD4+ T-cells derived from LPS treated mice. This suggests that CD4 cytotoxicity ensues in an MHCII dependent manner. Conclusions: LPS treatment reduces tumor burden in the subcutaneous tumor model of pancreatic cancer by upregulating the infiltration of CD4+ T-cells and translating them to a cytotoxic phenotype. It also stimulates the upregulation of anti-tumor immunity related cytokines in the tumor microenvironment. We have also found that the cytotoxic CD4+ T cells terminate the pancreatic cancer cells in an MHCII dependent manner. Therefore, our findings provide a novel insight into how “TLR4 agonism can stimulate CD4+ T-cell mediated antitumor immunity”, which can be harnessed as a potential source for pancreatic cancer immunotherapy. Citation Format: Utpreksha Vaish, Anthony R. Ferrantella, Tejeshwar Jain, Prateek Sharma, Srikanth Iyer, Vikas Dudeja. Toll like receptor 4 (TLR4) agonism by lipopolysaccharide (LPS) in pancreatic cancer model stimulates anti-tumor immunity in CD4+T cell dependent fashion [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B038.

EXTH-54. CO-CULTURE OF PATIENT-DERIVED GLIOBLASTOMA CELL LINES WITH AUTOLOGOUS PBMCS FOR THE INVESTIGATION OF ONCOLYTIC VIRUS RESPONSE

Abstract BACKGROUND The dismal prognosis of glioblastoma (GBM) patients, with a median survival of less than 15 months despite maximal therapy urgently warrants new therapeutic approaches. Clinical trials employing oncolytic viruses (OVs) have shown encouraging results, however, in each OV clinical trial only a small subset of patients responded to treatment. As inter-tumoral heterogeneity has been the key challenge in treating GBM, we hypothesized that development of an in vitro co-culture model for assessment of viral replication and subsequent immune response might ultimately predict in vivo OV efficacy for individual GBM patients. METHODS 20 patient-derived GBM cell cultures were tested with dose-ranges of two clinically relevant OVs (DNX2401 and rQnestin34.5 V1) to determine the EC50 values (half-maximal effective concentration). To discriminate between responders and non-responders six of these cultures were infected using MOI 10 for DNX2401 and 0.25 for rQnestin34.5V1, and co-cultured with autologous PBMCs. OV-induced changes in gene and protein expression of immune associated genes were assessed using targeted gene expression (NanoString Technology) and ELISA. RESULTS DNX2401 EC50 values ranged from 0.35 to &amp;gt; 600 and from 0.04 to 1.77 for rQnestin34.5V1. Induction of pro-inflammatory cytokines and chemokines differed per virus and per patient. Enhanced OV infection or oncolysis efficiency did not lead to increased levels of IFNγ production. Importantly, longer exposure to dexamethasone prior to PBMC isolation correlated with suppressed IFNγ production. CONCLUSION We established an autologous GBM cells/PBMCs co-culture model which reflects inter-tumoral heterogeneity in terms of cytokine induction and virus-specific changes in gene and protein expression upon OV infection. Immune activation is not directly related to degree of infection or oncolysis and can be hampered by prolonged prior dexamethasone use. These first results support our hypothesis that improved prediction of response rates in oncolytic virotherapy for GBM may require a personalized approach with an in vitro test system.

Anti-inflammatory properties of neutral lipids, glycolipids, and phospholipids isolated from Ammodytes personatus eggs in LPS-stimulated RAW264.7 cells

In the present study, total lipids were extracted from Ammodytes personatus eggs and separated into neutral lipids, glycolipids, and phospholipids. The anti-inflammatory activity of the neutral lipids, glycolipids, and phospholipids was investigated in macrophages, as well as the fatty acid profiles of the lipids. Palmitic acid, oleic acid, docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA) were the primary fatty acids in the three fractionated lipids. Among the lipids, the phospholipids contained the highest concentration of polyunsaturated fatty acids (PUFAs), particularly DHA and EPA (31.89 and 16.93% of the total fatty acids, respectively). The anti-inflammatory effects of the three lipids isolated from A. personatus eggs were analyzed in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The three lipids significantly reduced nitric oxide (NO) production and the mRNA expression of immune-associated genes in a dose-dependent manner. All lipids down-regulated the protein expression of phosphorylated NF-κB-p65 and MAPK (p38, JNK, and ERK1/2) signaling pathways, suggesting that they could inhibit cell signaling pathways by activating NF-κB and MAPK. The expression of CD40 and CD86 in LPS-stimulated RAW264.7 cells was also significantly decreased by A. personatus lipids. Consequently, the neutral lipids, glycolipids, and phospholipids from A. personatus eggs could serve as anti-inflammatory agents.

Protective immunity against spring viremia of carp virus by mannose modified chitosan loaded DNA vaccine

Spring viremia of carp virus (SVCV) usually be considered as one of the serious in viral diseases of aquaculture, and DNA vaccine with novel delivery mechanism or adjuvant has proven to be a promising and effective strategy to control aquatic animal diseases. In this study, the mannose-modified chitosan, a carrier system for vaccine delivery, were used to developed a chitosan-encapsulated DNA vaccine (CS-M-G) against SVCV, then investigated immune response induced by the vaccine. Our results showed that CS-M-G was confirmed the spherical or elliptical with even distribution and ranging from approximately 50 to 150 nm in size, the expression of the antigen gene could still be detected after 21 d post vaccination. The CS-M-G induces the highest antibody levels in the 20 μg dose group which is about 3 times than naked plasmid group at 21 d post vaccination, and still hold a higher level than control group at 28 d post vaccination. On the side, strongest protection with relative percent survival of 62.1% in the 20 μg CS-M-G group, which could produce significantly higher enzyme activities and up-regulated expression of immune-associated genes than control group. Thus, our results indicate that DNA vaccine loaded with mannose-modified chitosan induces strong immune response and provided an effective protection against SVCV infection, may be helpful and extended for developing more aquatic animal vaccines in the future.

Abstract 3560: Towards personalized oncolytic virotherapy: Differential response of four oncolytic viruses in primary glioblastoma cultures

Abstract Background: The brain tumor glioblastoma (GBM) is one of the most aggressive forms of cancer. The dismal prognosis of these patients, with a median survival of less than 15 months despite maximal therapy makes the need for new therapeutic approaches urgent. Clinical trials employing oncolytic viruses (OVs) have shown encouraging results, however, it appears that for each OV only a small group of patients responds to treatment. As inter- and intra-tumoral heterogeneity is a hallmark of GBM, we hypothesized that fresh patient-derived GBM cell cultures will reflect this inter-tumoral variability in response and allow identification of potential biomarkers of susceptibility to specific OVs. Furthermore, we established a co-culture system of primary GBM cultures with autologous peripheral blood mononuclear cells (PBMCs) to capture the degree of OV-induced oncolysis in conjunction with subsequent immune activation. Using these model systems, we attempt to develop tools which may guide future personalized trials of OV treatment for GBM. Methods: We tested the oncolytic potency of four OVs derived from different viral families (DNX2401, rQnestin34.5 V1, wild type Reovirus, lentogenic NDV-f0-GFP) on a panel of 19 molecularly characterized GBM cultures and calculated the half maximal effective concentration (EC50) for each virus on each cell culture. Quantitative PCR was performed to assess cytokine expression in tumor cells after infection with the 4 different OVs. OV-induced changes in the gene and protein expression of immune associated genes were assessed in co-cultures of GBM cells with PBMCs using Nanostring nCounter System and Elisa. Results: Screening of the 4 OVs on the panel of patient-derived GBM cell cultures revealed great inter-tumoral variability in oncolysis and cytokine response to the 4 different OVs with some degree of OV specific cytokine response profiles. Correlation analysis of transcriptome data with susceptibility to the four OVs shows that genes involved in distinct pathways are related to specific OV-sensitivity. In particular, cell cycle and immune related biological processes discriminate responders and non-responders. The co-culture of OV-infected glioma cells with PBMCs suggests that infection with different OVs leads to expression of distinct sets of genes and proteins in PBMCs; indicating that each OV mounts a specific immune response. Conclusion: Heterogeneity in OV sensitivity is demonstrated in primary GBM cultures, in terms of oncolysis, cytokine induction and in virus-specific changes in gene and protein expression in OV-infected tumor cells/PBMCs co-cultures. These results support the hypothesis that improving the response rates in oncolytic virotherapy for GBM may require a personalized approach. Citation Format: Eftychia Stavrakaki, Anne Kleijn, Wouter B. van den Bossche, Rutger K. Balvers, Lisette B. Vogelezang, Jie Ju, Andrew Stubbs, Yunlei Li, Dana Mustafa, Federica Fabro, Bernadette van den Hoogen, Rob Hoeben, William F. Goins, Hiroshi Nakashima, E. Antonio Chiocca, Clemens M. Dirven, Martine L. Lamfers. Towards personalized oncolytic virotherapy: Differential response of four oncolytic viruses in primary glioblastoma cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3560.

Effect of Licorice (Glycyrrhiza glabra) Extract as an Immunostimulant on Serum and Skin Mucus Immune Parameters, Transcriptomic Responses of Immune-Related Gene, and Disease Resistance Against Yersinia ruckeri in Rainbow Trout (Oncorhynchus mykiss).

This study was designed to appraise the effect of licorice herbal supplement on the immune status of rainbow trout fingerlings. Accordingly, five diets were formulated with different levels of licorice extract (LE) including 0 (control), 0.5 g kg−1 (LE0.5), 1 g kg−1 (LE1), 2 g kg−1 (LE2), and 3 g kg−1 (LE3). The fingerlings (10.0 ± 0.1 g initial mean weight) received the diets in triplicates (30 fish in each replicate) for 56 days. The results showed that the white blood cells and their differential number (lymphocytes and monocytes) were remarkably increased by LE2 supplementation (P < 0.05). The oral administration of LE2 significantly increased the levels of serum immunoglobulin (Ig), lysozyme activity, and complement components (C3 and C4) compared with others. Meanwhile, the serum bactericidal activity against Yersinia ruckeri in LE2 and LE3 treatments was significantly higher than others except for LE1 (P < 0.05). In addition, serum alternative complement activity significantly improved in all treated groups except LE0.5 compared with the control group (P < 0.05). In terms of skin mucosal immunity, the fish fed with LE2 and LE3 diets exhibited notably higher lysozyme activity, alkaline phosphatase activity, and Ig value than other groups (P < 0.05). The highest skin mucus bactericidal activity against Y. ruckeri was obtained in LE2 treatment (P < 0.05). In addition, dietary LE2 significantly increased the relative expression of immune-associated genes including tumor necrosis factor-α, interleukin-1β, interleukin-8, and IgM and the former treatments showed higher values than the control group. The cumulative mortality of fish against Y. ruckeri infection was notably reduced from 53.6% in the control group to 29.0% in LE3 treatment. Overall, the dietary administration of LE at 2 g kg−1 had the best effects on immunocompetence in rainbow trout.

Hallmarks of Retroelement Expression in T-Cells Treated With HDAC Inhibitors

A wide spectrum of drugs have been assessed as latency reversal agents (LRA) to reactivate HIV-1 from cellular reservoirs and aid in viral eradication strategies. Histone deacetylase inhibitors (HDACi) have been studied in vitro and in vivo as potential candidates for HIV-1 latency reversion. Suberoylanilide hydroxamic acid (SAHA) and romidepsin (RMD) are two HDACi able to reverse HIV latency, however studies of potential off-target effects on retroelement expression have been limited. Retroelements constitute a large portion of the human genome, and some are considered “fossil viruses” as they constitute remnants of ancient exogenous retroviruses infections. Retroelements are reactivated during certain disease conditions like cancer or during HIV-1 infection. In this study, we analyzed differential expression of retroelements using publicly available RNA-seq datasets (GSE102187 and GSE114883) obtained from uninfected CD4+, and HIV-1 latently infected CD4+ T-cells treated with HDACi (SAHA and RMD). We found a total of 712 and 1,380 differentially expressed retroelements in HIV-1 latently infected cells following a 24-h SAHA and RMD treatment, respectively. Furthermore, we found that 531 retroelement sequences (HERVs and L1) were differentially expressed under both HDACi treatments, while 1,030 HERV/L1 were exclusively regulated by each drug. Despite differences in specific HERV loci expression, the overall pattern at the HERV family level was similar for both treatments. We detected differential expression of full-length HERV families including HERV-K, HERV-W and HERV-H. Furthermore, we analyzed the link between differentially expressed retroelements and nearby immune genes. TRAF2 (TNF receptor) and GBP5 (inflammasome activator) were upregulated in HDACi treated samples and their expression was correlated with nearby HERV (MERV101_9q34.3) and L1 (L1FLnI_1p22.2k, L1FLnI_1p22.2j, L1FLnI_1p22.2i). Our findings suggest that HDACi have an off-target effect on the expression of retroelements and on the expression of immune associated genes in treated CD4+ T-cells. Furthermore, our data highlights the importance of exploring the interaction between HIV-1 and retroelement expression in LRA treated samples to understand their role and impact on “shock and kill” strategies and their potential use as reservoir biomarkers.

A TP53 mutation model for the prediction of prognosis and therapeutic responses in head and neck squamous cell carcinoma

BackgroundTumor protein p53 (TP53) is the most frequently mutated gene in head and neck squamous cell carcinoma (HNSC), and TP53 mutations are associated with inhibited immune signatures and poor prognosis. We established a TP53 mutation associated risk score model to evaluate the prognosis and therapeutic responses of patients with HNSC.MethodsDifferentially expressed genes between patients with and without TP53 mutations were determined by using data from the HNSC cohort in The Cancer Genome Atlas database. Patients with HNSC were divided into high- and low-risk groups based on a prognostic risk score that was generated from ten TP53 mutation associated genes via the multivariate Cox regression model.ResultsTP53 was the most common mutant gene in HNSC, and TP53 mutations were associated with immunogenic signatures, including the infiltration of immune cells and expression of immune-associated genes. Patients in the high-risk group had significantly poorer overall survival than those in the low-risk group. The high-risk group showed less response to anti-programmed cell death protein 1 (PD-1) therapy but high sensitivity to some chemotherapies.ConclusionThe risk score based on our TP53 mutation model was associated with poorer survival and could act as a specific predictor for assessing prognosis and therapeutic response in patients with HNSC.

Molecular characterization and functional analysis of the lysosomal cathepsin D-like gene in red swamp crayfish, Procambarus clarkii.

Aspartic proteinases are one of the four families of proteinase enzymes that are widely present in living organisms. They are involved in various physiological events, such as protein degradation, development, and host defense. However, the characterization and functional roles of aspartic proteinases remain to be elucidated in crustaceans. Here, we characterized a fragment of cathepsin D-like cDNA from red swamp crayfish, Procambarus clarkii (Pc-cathepsin D-like). The open reading frame of the Pc-cathepsin D-like gene contained 1152 bp, encoding a protein of 383 amino acid residues. We also evaluated the immunological role of the Pc-cathepsin D-like gene in vivo. Spatial distribution analysis revealed that the Pc-cathepsin D-like mRNA was high in the hepatopancreas, followed by the gut, gills, and hemocytes of P. clarkii. The expression levels of the Pc-cathepsin D-like gene increased following challenge with viral (polyinosinic: polycytidylic acid) and bacterial (lipopolysaccharides, peptidoglycan) PAMPs compared with PBS injection. The suppression of the Pc-cathepsin D-like gene by RNA interference significantly increased the expression of immune-associated genes. These results showed that the Pc-cathepsin D-like gene has an essential biological role in innate immune responses because it regulates the expression of immune-associated genes.

Immune Microenvironment Signatures as Biomarkers to Predict Early Recurrence of Stage Ia-b Lung Cancer.

IntroductionApproximately 30% of patients diagnosed with stage Ia-b NSCLC die of recurrent disease after surgery. This study aimed to identify immune-related biomarkers that might predict tumor recurrence in stage Ia-b NSCLC within 40 months after curative resection.MethodsGene expression data of stage Ia-b NSCLC samples was retrieved from the TCGA database, the GEO databases, and the Second Xiangya hospital (XXEYY) database. 22 types of tumors infiltrating immune cells and the expression of immune-associated genes were investigated using CIBERSORT, immunohistochemical staining, and GSEA analyses in a total of 450 patients (80 in the training cohort and 370 in the validation cohorts). Recurrence-related immune features were selected based on the LASSO Cox regression model.ResultsHigh density of Tregs, Macrophages M0 and M1 cell could be observed in recurrence group while the memory B cell was more frequently enriched in controls, yet Tregs alone was significantly associated with tumor early recurrence in TCGA cohort, XYEYY cohort and GSE37745 dataset. A handful of immune-related genes were identified in the recurrence group. Based on Lasso regression analysis, the expressions of five immune-related genes, RLTPR, SLFN13, MIR4500HG, HYDIN and TPRG1 were closely correlated with tumor early recurrence. In the training cohort (TCGA), the combination of these five genes has sensitivity and specificity of 85% and 85%, with AUC of 0.91 (95% CI 0.84-0.98) for lung cancer early recurrence prediction, whereas in validation cohorts, the sensitivity and specificity using this panel was 61-89% and 54-82%, with AUC of 0.62-0.84.ConclusionOur study demonstrated that the immune microenvironment signatures were closely related to tumor early recurrence. Compared to tumor-infiltrating lymphocytes, the expression of five immune-related genes could be robust biomarkers to predict early recurrence of stage Ia-b NSCLC after curative resection.

The Effects of Macrophage Phenotype on Osteogenic Differentiation of MSCs in the Presence of Polyethylene Particles.

Wear debris generated from the bearing surfaces of joint arthroplasties leads to acute and chronic inflammation, which is strongly associated with implant failure. Macrophages derived from monocytes recruited to the local tissues have a significant impact on bone healing and regeneration. Macrophages can adopt various functional phenotypes. While M1 macrophages are pro-inflammatory, M2 macrophages express factors important for tissue repair. Here, we established a 3D co-culture system to investigate how the immune system influences the osteogenic differentiation of mesenchymal stem cells (MSCs) in the presence of micron-sized particles. This system allowed for the simulation of an inflammatory reaction via the addition of Lipopolysaccharide-contaminated polyethylene particles (cPE) and the characterization of bone formation using micro-CT and gene and protein expression. Co-cultures of MSCs with M2 macrophages in the presence of cPE in a 3D environment resulted in the increased expression of osteogenic markers, suggesting facilitation of bone formation. In this model, the upregulation of M2 macrophage expression of immune-associated genes and cytokines contributes to enhanced bone formation by MSCs. This study elucidates how the immune system modulates bone healing in response to an inflammatory stimulus using a unique 3D culture system.

Vector Competence for DENV-2 Among Aedes albopictus (Diptera: Culicidae) Populations in China

Aedes albopictus is a vector of over 20 arboviruses that has spread throughout the world, mainly in the second half of the twentieth century. Approximately 50–100 million people are infected with dengue virus (DENV) transmitted by Aedes mosquitoes each year, leading to heavy economic burdens for both governments and individuals, among countless other negative consequences. Understanding the vector competence of vector species is critical for effectively preventing and controlling vector-borne diseases. Accordingly, in this study, vector competence was evaluated by quantitative analysis of DENV-2 loads in mosquito tissues (midguts, heads, and salivary glands) and whole mosquitoes through real-time quantitative polymerase chain reaction (RT-qPCR) analysis. Wolbachia and the expression of immune-associated genes (Rel1, Rel2, Dicer2, and STAT) in mosquitoes were also detected by RT-qPCR to explore their impact on vector competence. The amount of DENV-2 in the mosquito midguts, heads, and salivary glands from southern-western China were found to be lower than those from eastern-central-northern China. The DENV-2 loads in whole mosquitoes showed a negative correlation with Rel1 gene (r = -0.285, P = 0.011) and STAT gene expression levels (r = -0.289, P = 0.009). In terms of Wolbachia strains, the density of the wAlbB strain was found to be significantly higher than that of the wAlbA strain in the eight Ae. albopictus populations, and the relative density of the wAlbB strain in mosquitoes from southern-western China was higher than those from eastern-central-northern China. The relative density of the wAlbB strain showed a negative correlation with the mean loads of DENV-2 in the heads (r = -0.729, P = 0.040), salivary glands (r = -0.785, P = 0.021), and whole mosquitoes (r = -0.909, P = 0.002). Thus, there are lower DENV-2 loads in the mosquitoes from southern-western China, which may be related to the innate immunity of mosquitoes as affected by Rel1 in the Toll pathway, STAT in the JAK-STAT pathway, and the relative density of the wAlbB strain.

An independent poor-prognosis subtype of hepatocellular carcinoma based on the tumor microenvironment.

BackgroundHepatocellular carcinoma (HCC) is a highly malignant tumor with a particularly poor prognosis. The tumor microenvironment (TME) is closely associated with tumorigenesis, progression, and treatment. However, the relationship between TME genes and HCC patient prognosis is poorly understood.MethodsIn this study, we identified two prognostic subtypes based on the TME using data from The Cancer Genome Atlas and Gene Expression Omnibus. The Microenvironment Cell Populations-counter method was used to evaluate immune cell infiltration in HCC. Differentially expressed genes between molecular subtypes were calculated with the Limma package, and clusterProfiler was used for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional enrichment analyses to identify genes related to the independent subtypes. We also integrated mRNA expression data into our bioinformatics analysis.ResultsWe identified 4227 TME-associated genes and 640 genes related to the prognosis of HCC. We defined two major subtypes (Clusters 1 and 2) based on the analysis of TME-associated gene expression. Cluster 1 was characterized by increased expression of immune-associated genes and a worse prognosis than Cluster 2.ConclusionsThe identification of these HCC subtypes based on the TME provides further insight into the molecular mechanisms and prediction of HCC prognosis.