TNF-alpha Production Research Articles

Physicochemical and Volatile Compounds Analysis of Fruit Wines Fermented with Saccharomyces cerevisiae: FTIR and Microscopy Study with Focus on Anti-Inflammatory Potential.

The growing trend in fruit wine production reflects consumers' interest in novel, diverse drinking experiences and the increasing demand for healthier beverage options. Fruit wines made from kiwi, pomegranates, and persimmons fermented using S. bayanus Lalvin strain EC1118 demonstrate the versatility of winemaking techniques. Kiwifruit, persimmon, and pomegranate wines were analyzed using HPLC and GC-TOFMS analyses to determine their concentrations of phenolic acids and volatile compounds. These results were supported by Fourier transform infrared (FTIR) spectroscopy to characterize and compare chemical shifts in the polyphenol regions of these wines. The wines' characterization included an anti-inflammatory assay based on NO, TNF-alpha, and IL-6 production in the RAW 264.7 macrophage model. FTIR spectroscopy predicted the antioxidant and phenolic contents in the wines. In terms of polyphenols, predominantly represented by chlorogenic, caffeic, and gallic acids, pomegranate and kiwifruit wines showed greater benefits. However, kiwifruit wines exhibited a highly diverse profile of volatile compounds. Further analysis is necessary, particularly regarding the use of other microorganisms in the fermentation process and non-Saccharomyces strains methods. These wines exhibit high biological antioxidant potential and health properties, providing valuable insights for future endeavors focused on designing healthy functional food products.

Immunomodulation of Dendrobium fimbriatum against blue light-induced ocular inflammation

Abstract As electronic device use increases, concerns about the harmful effects of blue light on eye health have grown. Past studies support the idea that Dendrobium species offer benefits such as reducing inflammation, providing antioxidants, and protecting against cataracts. We aim to investigate the immunomodulatory effect of Dendrobium fimbriatum Hook. extracts (DFs) in blue light-induced ocular damage. DFs decoctions were prepared from the fresh stems of 1-year, 2-year, and 3-year-old DFs. In vitro, cell viability, apoptosis, pro-inflammation cytokines and reactive oxygen species (ROS) were assessed. In vivo, ophthalmological and histological examination, systemic and local immunological changes were evaluated. Results showed that 2-year-old DF (DF-2yD) presents the maximal alleviation of blue light-induced cell death, apoptosis, interleukin (IL)-6 and 8, and ROS in retinal and corneal cells. In blue light-induced ocular damage mouse model, DF-2yD potently attenuated the fundus abnormality and retinal thickness. Furthermore, DF-2yD decreased IL-6 and TNF-alpha production in the draining lymph nodes. Collectively, the results indicate that DF hold promise as potential agents to counteract blue light-induced eye damage through the immunoregulation.

Cytokine inhibitory activity of eurycomanone in RAW 264.7 cells stimulated with viral-mimicking poly (I:C)

Poly (I:C) (Polyinosinic:polycytidylic acid) is a synthetic analog of double-stranded RNA binding toll-like receptor 3 (TLR3) and mimicking a virus infection in macrophage. Here, for the first time, we showed the inhibitory effects of eurycomanone on the production of IL-6, TNF-alpha and IL-10 of RAW 264.7 cells stimulated with poly (I:C). Eurycomanone inhibited production of pro-inflammatory cytokines including IL-6 and TNF-alpha of RAW 264.7 cells stimulated with poly (I:C) with the IC50 value being 5.14 +/- 0.60 uM and 2.32 +/- 0.40 uM, respectively. Interestingly, this compound also inhibited the production of anti-inflammatory cytokine IL-10 of RAW 264.7 cells stimulated with poly (I:C) with the IC50 value being 14.60 +/- 0.32 uM. These results suggested that eurycomanone has a potentially regulatory role on production of both pro-inflammatory and anti-inflammatory cytokines in macrophage in the mimicking context of virus infection. Whether eurycomanone inhibits the binding of poly (I:C)-TLR3 or intracellular signaling pathways needs further investigation.

Evaluation of immunomodulatory potential of probiotic conditioned medium on murine macrophages

Probiotics are a mixture of beneficial live bacteria and/or yeasts that naturally exist in our bodies. Recently, numerous studies have focused on the immunostimulatory effects of single-species or killed multi-species probiotic conditioned mediums on macrophages. This study investigates the immunostimulatory effect of commercially available active, multi-species probiotic conditioned medium (CM) on RAW264.7 murine macrophages. The probiotic CM was prepared by culturing the commercially available probiotic in a cell-culture medium overnight at 37 °C, followed by centrifugation and filter-sterilization to be tested on macrophages. The immunostimulatory effect of different dilution percentages (50%, 75%, 100%) of CM was examined using the MTT assay, proinflammatory cytokine (tumor necrosis factor TNF-alpha) production in macrophages, migration, and phagocytosis assays. For all the examined CM ratios, the percentages of cell viability were > 80%. Regarding the migration scratch, TNF-alpha and phagocytosis assays, CM demonstrated a concentration-dependent immunostimulatory effect. However, the undiluted CM (100%) showed a significant (p-value < 0.05) stimulatory effect compared to the positive and negative controls. The findings suggest that the secretions and products of probiotics, as measured in the CM, may be closely associated with their immune-boosting effects. Understanding this relationship between probiotic secretions and immune function is crucial for further exploring the potential benefits of probiotics in enhancing overall health and well-being.

Comparing Flow Cytometry and ELISpot for Detection of IL-10, IL-6, and TNF Alpha on Human PBMCs.

ELISpot and flow cytometry are two methods often utilized side-by-side for detecting secreted and intracellular cytokines, respectively. Each application has its own advantages and challenges. ELISpot is more sensitive compared to ELISA and appears to be more consistent in detecting IL-10 production than flow cytometry. ELISpot can be used for detecting the secretion of multiple cytokines but not from the same cells simultaneously, whereas flow cytometry allows for the concurrent detection of multiple intracellular cytokines by the same cells. Flow cytometry is a convenient technique allowing for the detection of many cytokines at the same time in a population of cells. The restimulation cocktails used for cytokine detection in flow cytometry are hard on cells and lead to decreased cell viability. Using a live dead dye allows for the exclusion of dead cells when analyzing data. We illustrated the differences between ELISpot and flow cytometry by stimulating cells with two toll-like receptor (TLR) agonists, LPS or Pam3CSK4. Both activators increase production of various cytokines, including IL-10, IL-6, and TNF-alpha. The TLR2 antagonist, MMG-11, was used to inhibit this increased cytokine production. We observed some inhibition of IL-6 and IL-10 from Pam3CSK4 stimulation in the presence of MMG-11 by flow cytometry. TNF-α remains largely unchanged as its basal expression is high, but there is some reduction in the presence of MMG-11 for both methods. However, IL-10 was difficult to detect by ELISpot given the low seeding density. Overall, both ELISpot and flow cytometry are good methods for detecting secreted and intracellular cytokines, respectively, and should be used as complimentary assays.

Electroacupuncture Stimulation Alleviates Inflammatory Pain in Male Rats by Suppressing Oxidative Stress.

In the present study, we focused on whether the analgesic effect of Electroacupuncture (EA) is related to the regulation of oxidative stress. We established a chronic inflammatory pain model in male rats by a single injection of complete Freund's adjuvant (CFA) and then treated the animals with daily EA stimulation at the site of "zusanli". The analgesic effect of EA was evaluated by measuring the paw withdrawal threshold (PWT) when rats received mechanical and thermal pain stimulation. The levels of inflammation-related molecules and oxidative stress-related markers in the spinal cord were measured by western blotting or ELISA kits. EA stimulation and antioxidants effectively increased the PWT in CFA rats. Co-treatment of CFA rats with the ROS donor t-butyl hydroperoxide (t-BOOH) further decreased the PWT and weakened the analgesic effect of EA. EA treatment inhibited inflammation and oxidative stress, as shown by decreased levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, and MDA and increased activity of SOD and catalase. Moreover, EA reduced the expression of p-p38, p-ERK, and p-p65 and simultaneously downregulated the expression of TRPV1 and TRPV4 in CFA rats. In an in vitro study, direct stimulation with t-BOOH to the C6 cells increased the production of TNF-alpha, IL-1beta, IL-6, activated p38, ERK, and p65 and up-regulated the expression of TRPV1 and TRPV4, and these effects could be prevented by the ROS scavenger PBN. Taken together, our data indicate that the inhibition of oxidative stress and the generation of ROS contribute to the analgesic effect of EA in male CFA rats.

Plasma Levels of Organochlorine Pesticides and Tumor Necrosis Factor-Alpha: A Potential Risk Factor for Developing Acquired Aplastic Anemia in the North Indian Population.

Background Pesticide exposure might have a contributory role in the development ofacquired aplastic anemia (AA). However, the precise mechanisms of pesticide-induced AA remain unknown. In this case-control study, we conducted a comparative analysis of plasma levels of organochlorine pesticides (OCP) and tumor necrosis factor-alpha (TNF-alpha) between Indian patients diagnosed with AA and an age- and sex-matched control group. Methods This is an observational case-controlstudy conducted at a tertiary care hospital in North India. In this study, 90 subjects were included, out of which 45 were diagnosed with AA according to the criteria of the International Agranulocytosis and Aplastic Anemia Study. Cases were compared with 45 controls. A trained interviewer gave all study subjects a questionnaire to collect data regarding demographic details, exposure to pesticides, and clinical history.Physical examinationand routine laboratory investigations of each subject were performed. Both cases and controls were tested for their plasma levels of organochlorines as per established protocol by gas chromatography-mass spectrometry. TNF-alpha level was measured by enzyme-linked immunosorbent assay in each subject. Results There was a significant increase in plasma levels of delta hexachlorocyclohexane(delta HCH) (p = 0.02) and heptachlor (p = 0.00) in patients with AA as compared to controls. We observed nonsignificant trends towards higher levels of beta HCH (p = 0.643), aldrin (p = 0.399), and p,p'-Dichlorodiphenyltrichloroethane (p,p'-DDT) (p = 0.453) in patients with AA when compared to the controls. There were significantly higherTNF-alpha levels (p = 0.024) in cases as compared to the controls. Conclusion Our study concludes that patients with AA exhibited higher levels ofdelta-HCH, heptachlor, andTNF-alpha in comparison to the control group. There is a significant positive correlation of TNF alpha with OCPs (alpha HCH, lindane, delta HCH, heptachlor, aldrin, p,p'- DDD, and methoxychlor pesticides). These organochlorines may have accumulated in the fatty tissue of bone marrow because of their lipophilic nature. This suggests that they might have served as a neoantigen to trigger an increase in TNF-alpha production, which may have led to disrupted bone marrow function through cell-mediated immunity, leading to AA.

Mycobacterial DNA-binding protein 1 is critical for BCG survival in stressful environments and simultaneously regulates gene expression

Survival of the live attenuated Bacillus Calmette-Guérin (BCG) vaccine amidst harsh host environments is key for BCG effectiveness as it allows continuous immune response induction and protection against tuberculosis. Mycobacterial DNA binding protein 1 (MDP1), a nucleoid associated protein, is essential in BCG. However, there is limited knowledge on the extent of MDP1 gene regulation and how this influences BCG survival. Here, we demonstrate that MDP1 conditional knockdown (cKD) BCG grows slower than vector control in vitro, and dies faster upon exposure to antibiotics (bedaquiline) and oxidative stress (H2O2 and menadione). MDP1-cKD BCG also exhibited low infectivity and survival in THP-1 macrophages and mice indicating possible susceptibility to host mediated stress. Consequently, low in vivo survival resulted in reduced cytokine (IFN-gamma and TNF-alpha) production by splenocytes. Temporal transcriptome profiling showed more upregulated (81–240) than downregulated (5–175) genes in response to MDP1 suppression. Pathway analysis showed suppression of biosynthetic pathways that coincide with low in vitro growth. Notable was the deferential expression of genes involved in stress response (sigI), maintenance of DNA integrity (mutT1), REDOX balance (WhiB3), and host interactions (PE/PE_PGRS). Thus, this study shows MDP1’s importance in BCG survival and highlights MDP1-dependent gene regulation suggesting its role in growth and stress adaptation.

Mast cells are upregulated in hidradenitis suppurativa tissue, associated with epithelialized tunnels and normalized by spleen tyrosine kinase antagonism.

Mast cells have traditionally been associated with allergic inflammatory responses; however, they play important roles in cutaneous innate immunity and wound healing. The Hidradenitis Suppurativa tissue transcriptome is associated with alterations in innate immunity and wound healing-associated pathways; however, the role of mast cells in the disease is unexplored. We demonstrate that mast cell-associated gene expression (using whole tissue RNAseq) is upregulated, and in-silico cellular deconvolution identifies activated mast cells upregulated and resting mast cells downregulated in lesional tissue. Tryptase/Chymase positive mast cells (identified using IHC) localize adjacent to epithelialized tunnels, fibrotic regions of the dermis and at perivascular sites associated with Neutrophil Extracellular Trap formation and TNF-alpha production. Treatment with Spleen Tyrosine Kinase antagonist (Fostamatinib) reduces the expression of mast cell-associated gene transcripts, associated biochemical pathways and the number of tryptase/chymase positive mast cells in lesional hidradenitis suppurativa tissue. This data indicates that although mast cells are not the most abundant cell type in Hidradenitis Suppurativa tissue, the dysregulation of mast cells is paralleled with B cell/plasma cell inflammation, inflammatory epithelialized tunnels and epithelial budding. This provides an explanation as to the mixed inflammatory activation signature seen in HS, the correlation with dysregulated wound healing and potential pathways involved in the development of epithelialized tunnels.

The Immunostimulatory Effect of Probiotic Conditioned Medium on RAW264.7 Murine Macrophages

Background: Probiotics are a mixture of good live bacteria and/or yeasts that naturally survive in our bodies. Recently, loads of studies have focused on their role in the immune system and digestive tract. Accordingly, there are many commercially available probiotic products in the market. This study examines the immunostimulatory effect of commercially available-probiotic conditioned medium (PCM) on RAW264.7 murine macrophages.&#x0D; Methods: Probiotic conditioned medium has been prepared by culturing the commercially available probiotic in a cell culture medium overnight at 37°C, followed by centrifugation and filter-sterilization to be tested on RAW264.7 murine macrophages. The immunostimulatory effect of different ratios (50, 75, 100) of PCM was examined using MTT assay, pro-inflammatory cytokine (tumor necrosis factor TNF-alpha) production in macrophages., migration, and Phagocytosis assays.&#x0D; Results: At all the examined PCM ratios, the percentages of cell viability were &gt;80%. Regarding the migration scratch, TNF-alpha and phagocytosis assays, PCM demonstrated a concentration-dependent pattern in the immunostimulatory effect. However, the ratio of 100 PCM illustrated a significant (p-value&lt;0.05) stimulatory effect compared to the positive and negative control.&#x0D; Conclusion: The findings of this study confirm the stimulatory activity of probiotic conditioned medium, which may contribute directly to the immune-boosting effect of the probiotic supplements.

Combinatorial inhibition of Tec kinases BTK and ITK is beneficial in ameliorating murine sclerodermatous chronic graft versus host disease.

Graft-versus-host disease (GVHD) is the major factor limiting the widespread use of potentially curative allogeneic hematopoietic stem cell transplant (allo-HSCT). Chronic GVHD is characterized by the activation of alloreactive donor immune cells, especially B- and T-cells, leading to tissue damage and pathogenic fibrosis. In this study, we used highly specific next-generation inhibitors of ITK (PCYC-274), BTK (PCYC-804), and ibrutinib-like BTK/ITK inhibitors (PCYC-914 and PCYC-401) in the B10.D2 → BALB/C model of murine sclerodermatous cGVHD. From the third week onward, allogeneic recipients in each group of respective Tec kinase inhibitors were treated three times weekly with inhibitors at doses of 10 and 30 mg/kg or with saline control via oral gavage. Overall, we found that selective BTK inhibition was less effective than combined ITK/BTK or ITK inhibition in lengthening survival and reducing symptoms of cGVHD. ITK inhibition was most efficacious, with PCYC-274 and PCYC-401 demonstrating a nearly 50 percent reduction in GVHD scoring even at the 10 mg/kg dose, while 30 mg/kg of these compounds almost completely ameliorated GVHD symptomology. BTK/ITK and ITK-treated mice showed significant reductions in overall pathology. Significant reductions in dermal thickness and fibrosis were shown for all treatment groups. There was evidence of mixed Th1 and Th2 cytokine profiles in the skin of mice with dermal cGVHD, as both IFN-gamma and IL-4 were upregulated in the allogeneic control group, while kinase inhibition significantly reduced levels of these cytokines. Using an in vitro model of T-cell polarization, Th1 cell production of TNF-alpha and IFN-gamma were partially blocked by ITK. Th2 cell production of IL-4 was almost completely blocked synergistically by ITK and BTK inhibition. BTK-specific inhibition was unable to block either Th1 or Th2 cytokine production. Taken together, these results confirm previous reports that ITK-focused inhibition inhibits Th1 and Th2 cells. Additionally, the compound's effects on T-cell proliferation were tested by CFSE assay. Pure ITK inhibition was most effective at blocking T-cell proliferation, with no proliferation in PCYC-274-treated cells even at 0.1uM. PCYC-401 and PCYC-914 showed some inhibition at lower doses, with complete inhibition evident at 10uM. PCYC-804 was only partially able to block proliferation even at 10uM. In conclusion, we observed substantial benefit for differential inhibition of Tec kinases in GVHD, with ITK being most efficacious and Th1 cells being more resistant to inhibition, matching the previously reported findings of a Th2 to Th1 selective pressure in cells treated with ibrutinib. Our data warrants the further development of ITK and ITK/BTK inhibitors with specific inhibitory ratios to improve the treatment of GVHD and other T-cell mediated diseases.

PPP1R13L-Related Cardiomyopathy Stability Following IL1 Inhibition

IntroductionVariants in PPP1R13L are associated with severe childhood-onset cardiomyopathy resulting in rapid progression to death or cardiac transplantation. PPP1R13L is proposed to encode a protein that limits the transcriptional activity of the NFkappaB pathway leading to elevated IL-1, IL-6, and TNF-alpha production in murine models. Optimal medical management for PPP1R13L-related cardiomyopathy is unknown. Here we report usage of a targeted anti-IL-1 immuno-modulatory therapy resulting in cardiac stabilization in a pediatric patient with congenital cardiomyopathy secondary to PPP1R13L variants. Case ReportA 4-year-old boy presented acutely with fever in the setting of persistent abdominal pain, vomiting, fatigue, and decreased appetite for two months following a mild COVID-19 related illness. Echocardiogram revealed severely depressed biventricular systolic function with an ejection fraction of 30%. Due to acute decompensated heart failure symptoms with hemodynamic instability, he was intubated and placed on continuous inotropic infusions with aggressive diuresis. Cardiac MRI demonstrated extensive subepicardial to near transmural fibrosis by late gadolinium enhancement in right and left ventricles. An implantable cardioverter-defibrillator (ICD) was placed due to frequent runs of polymorphic non-sustained ventricular tachycardia. Testing for viral pathogens was positive for rhino/enterovirus. Initial genetic testing was non-diagnostic (82-gene cardiomyopathy panel) but given the patient’s significant presentation whole genome sequencing was pursued that showed two separate PPP1R13L variants in trans (c.2167A>C,p.T723P and c.2179_2183del,p. G727Hfs*25, NM_006663.4). Patient serum cytokine testing revealed elevations in IL-10 (4.7 pg/mL) and IL-1beta (20.9 pg/mL). Given the patient’s tenuous circumstances and concern for continued progression of his cardiac disease, a trial of IL-1 inhibition via anakinra dosed at 3 mg/kg or 45 mg daily was initiated following hospital discharge. With approximately 6 months of therapy, the patient’s cardiac function is stable with normalization of IL-10 and IL-1beta serum levels. Notably, the ventricular arrhythmia decreased after initiation of anakinra with no ICD shocks given. Therapy overall has been well tolerated without infectious concerns. ConclusionIn patients with PPP1R13L-related cardiomyopathy, immuno-modulatory therapies should be considered in an attempt to slow cardiac disease progression.

Analysis of immune response of stressed male mice during Chlamydia muridarum lung infection

Chlamydia lung infection caused by Chlamydia trachomatis is a serious lung infection particularly in infants but there are a few studies about lung infection. Chlamydial genital and lung infections in mice is frequently done with Chlamydia muridarum. The purpose of our study was to explore the effect of stress in lung chlamydia infection in male mice. We hypothesize that stress changes the level of cytokine production in monocytes during lung infection. As a The result from these studies include counting of live cells, differentiation of proliferation of bone marrow derived monocytes where the result showed that non-stressed dendritic cells (DCs) WT mice and stressed macrophages (MO) WT mice had sufficient live cells to study. No difference in IL-b production of macrophages of stressed and non-stress mice was observed. Beta2-adrenergic receptor (b2-AR agonist (fenoterol) and antagonist (ICI 118 551) treatment resulted in difference in cytokine production the effect of agonists and antagonists. TNF-alpha production was high in stressed mice compared to non-stress mice for MO. LPS stimulated TNF-alpha production in DC but showed no difference in stressed and non-stressed mice. However, TNF-alpha production in macrophages of non-stressed mice was decreased. The production of IFN-g, in Con A- and LPS-treated splenic T cells. In contrast IL-5, IL-10, and IL-23 production was high in T cells of stressed mice. T cells treated with norepinephrine and bet2-adrenergic receptor agonist, Fenoterol. Overall, the production of cytokines in stressed and non-stressed mice shows variation that may have roles in enhancing protection or increased lung infection.

Abstract 4429: ‘Free’ ISG15 acts as an immunoadjuvant to enhance CD8+ T cell-mediated anti-tumor immunity

Abstract Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like molecule that is primarily involved in antiviral responses. ISG15 can either exist in the conjugated form, where it is covalently bound to proteins in a process termed ISGylation or can be released extracellularly in the cytokine-like “free” form. Although conjugated ISG15 often indicates poor prognosis in cancers, ‘free’ ISG15 is suggested to have immunostimulatory properties, owing to its ability to stimulate the production of IFN-gamma from lymphocytes and natural killer (NK) cells. While the immunomodulatory properties of free ISG15 are known, its ability to improve the antigen-specific T-cell response mediated by tumor immunotherapies has not been fully elucidated. The need for CD8+ T cell-based immunoadjuvant in cancer immunotherapy is underscored by the current failure in the generation of optimum CD8+ T cell response by the multitude of cancer vaccines in clinical trials. In this study, we demonstrated that ISG15 is vital for the generation of antigen-specific adaptive immunity; we showed that ISG15 deficient (ISG15-/-) mice do not develop effective systemic CD8+ T cell response after ovalbumin protein vaccination in non-tumor bearing mice. Upon tumor challenge of ISG15-/- mice and wild type C57/BL6 mice with B16-OVA melanoma tumors, effective OVA-specific CD8+ T cell-mediated anti-tumor immunity was not elicited, and the tumors progressed at a faster rate in the ISG15-/- mice. We then utilized a clinically relevant model to evaluate the therapeutic benefits of ‘free’ ISG15. We used a synergistic therapeutic approach that involved the concomitant administration of a DNA-construct encoding ‘free’ ISG15(mutISG15) and a Listeria-monocytogenes(Lm)-based cancer vaccine directed against the tumor vasculature (Lm-LLO-CD105A) to intervene in murine models of renal cell carcinoma (Renca) and breast cancer (4T1-Luc). The combination of Lm-LLO-CD105A and mutISG15 led to a significant reduction in tumor burden in both syngeneic models. This tumor control was mediated by the expansion of a population of highly polyfunctional CD8+ T cells in the combinatorial therapy group. These CD8+ T cells were characterized by the robust production of multiple cytokines, including IFN-gamma, IL-2, and TNF-alpha.Moreover, we confirmed that mutISG15 significantly increased the antigen (CD105)-specific anti-tumor response generated by Lm-LLO-CD105A. Not surprisingly, in-vitro studies demonstrated that mutISG15 probably potentiates the induction of maturation, activation, and antigen-presentation of dendritic cells to facilitate the activation and effective functionality of tumor-specific T cells. Conclusively, our results positions ‘free’ ISG15 as a powerful T-cell stimulant that may provide a suitable therapeutic option for improving the effectiveness of therapeutic cancer vaccines. Citation Format: Mariam Oladejo, Laurence Wood. ‘Free’ ISG15 acts as an immunoadjuvant to enhance CD8+ T cell-mediated anti-tumor immunity. [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 4429.

Abstract 763: Minimally invasive colorectal resection is associated with significantly elevated plasma levels of extracellular cold inducible RNA binding protein for a month after colorectal resection &amp; may promote the growth of residual metastases

Abstract Introduction: Cold-Inducible RNA-Binding Protein (CIRP) is similar to a class of stress-induced RNA binding proteins and is mostly found in the nucleus where it regulates transcription. In response to stress, CIRP moves to the cytoplasm where it regulates translation; a fraction of the cytoplasmic CIRP is released extracellularly (eCIRP). eCIRP is a damage-associated molecular pattern (DAMP) molecule that promotes inflammatory responses including TNF-alpha, IL-6, and HMGB production. Lymphocytes, macrophages, and PMN’s are activated after surgery and can generate eCIRP. Elevated eCIRP blood levels may impact macrophage function and induce immune tolerance that may promote tumor growth and progression. This study’s purpose was to measure plasma eCIRP levels before and for a month after MICR for CRC. Methods: CRC patients (pts) in an IRB approved data/tissue bank who underwent MICR for whom plasma was available were selected. Demographic, clinical and pathology data were reviewed. Plasma samples were collected preoperatively (preOp) and at 6 post-operative (Postop) time points (POD 1, 3, 7-13, 14-20, 21-27, 28-41); samples were processed and frozen until used. Late samples were bundled into 7-day blocks and considered as single time points. eCIRP levels were analyzed in duplicate using ELISA. The Wilcoxon paired t-test was used for analysis. Results: 83 CRC pts who underwent MICR were studied (colon 66%, rectal 34%; mean age 65); 26% had Hand assisted (HA) MIS (mean incision length[IL] 10.0±2.9cm) and 69% laparoscopic (LAP, mean IL 6.9±4.1cm) MICR. The cancer stage breakdown was; I, 30%; II, 32%; III, 35% and IV, 3%. The mean PreOp eCIRP level was 896.8± 757.0pg/ml; vs. preop levels increased mean plasma levels (p=&amp;lt;0.001) were noted on POD1 (2549± 2632pg/ml, n= 83), POD3 (1871± 1362pg/ml, n= 77), POD7-13 (1788± 1403pg/ml, n=57), POD14-20 (1473± 738.8pg/ml, n=30), and POD 21-27 (1681± 1375pg/ml, n= 21). With regard to MIS method, greater eCIRP increases were noted in the HA vs the Lap group from POD1-POD20 (p=0.01-0.04). Conclusions: Plasma eCIRP levels are elevated for a month after MICR for CRC (% change range 100-184) and peaked on POD1. The cause of the early increase may be the acute inflammatory response whereas the late elevations may be related to wound healing-related tissue remodeling. Higher mean levels in the HA vs Lap group suggests that the extent of surgical trauma impacts eCIRP levels postop. Previous studies noted increased post-MICR plasma levels of MCP-1, CHI3L1 and IL8 that stimulate macrophage activity. eCIRP, together with those proteins may promote macrophage recruitment and function and might promote the growth of residual tumor deposits postop. Further studies are warranted. Citation Format: Chandana S. K. Herath Mudiyanselage, Xiaohong Yan, Neil Mitra, Hansani N. Angammana, Doaa F. Morrar, Vincent Chia, Yanni Hedjar, Vesna Cekic, Jennifer L. Agnew, Richard L. Whelan. Minimally invasive colorectal resection is associated with significantly elevated plasma levels of extracellular cold inducible RNA binding protein for a month after colorectal resection &amp; may promote the growth of residual metastases [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 763.

LBA02-05 DEFINING INNATE IMMUNE MEMORY IN THE MECHANISM OF BCG IMMUNOTHERAPY FOR BLADDER CANCER

You have accessJournal of UrologyCME1 Apr 2023LBA02-05 DEFINING INNATE IMMUNE MEMORY IN THE MECHANISM OF BCG IMMUNOTHERAPY FOR BLADDER CANCER Song Jiang, Gil Redelman-Sidi, Eugene J. Pietzak, Bernard H. Bochner, and Michael Glickman Song JiangSong Jiang More articles by this author , Gil Redelman-SidiGil Redelman-Sidi More articles by this author , Eugene J. PietzakEugene J. Pietzak More articles by this author , Bernard H. BochnerBernard H. Bochner More articles by this author , and Michael GlickmanMichael Glickman More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003361.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Intravesical instillation of the live attenuated mycobacterium Bacillus Calmette-Guerin (BCG) remains the most effective therapy for the treatment of non-muscle invasive bladder cancer. An increasing body of evidence suggests that the innate immune system has characteristics that involve a heterologous memory of past insults through a process thought to involve epigenetic reprogramming, termed trained immunity. We seek to investigate the potential for innate immune training as a predictor of the anti-tumor effects of BCG. METHODS: A prospective biospecimen collection was performed on venous blood from non-muscle invasive bladder cancer patients prior to receiving intravesical BCG therapy. An ex vivo training protocol was performed on peripheral blood monocytes as previously described. Subsequently, cells were stimulated with 25 ng/ml lipopolysaccharide (a TLR4 agonist) for 24 hours. Cell free supernatant was measured for TNF-alpha production by enzyme-linked immunosorbent assay. RESULTS: In vitro training of primary monocytes induced an increase in proinflammatory cytokine production upon re-stimulation. In our in vitro training experiments, there was demonstrable interpatient variability seen pre-BCG exposure in a cohort of 30 NMIBC patients (Figure 1). Kaplan Meier analysis of this NMIBC patient cohort demonstrated improved recurrence free survival in individuals with a higher innate immune memory response. CONCLUSIONS: We present data on a trained innate immune phenotype on primary monocytes collected from patient samples. We found that innate immune training correlated improved recurrence free survival in NMIBC patients treated with BCG. Such insight will have potential clinical impact on the development of biomarkers and clinical assays that could predict immune responsiveness to BCG a priori, as well as identify possible immunological adjuvants that could enhance the effect of intravesical BCG. Source of Funding: ASCO YIANCI SPORE in Bladder Cancer © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1188 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Song Jiang More articles by this author Gil Redelman-Sidi More articles by this author Eugene J. Pietzak More articles by this author Bernard H. Bochner More articles by this author Michael Glickman More articles by this author Expand All Advertisement PDF downloadLoading ...

Effect of Combination of Soybean and Phaleria macrocarpa Ethanol Extract on IL6, TNFα, VEGF and Fibroblasts in Mice Exposed to UVB

UV exposure causes inflammation and the generation of reactive oxygen species, both of which contribute to skin aging. The purpose of this research was to determine how a combination of Phaleria macrocarpa extract and soybean extract affected the number of fibroblasts, VEGF, IL-6, and TNF alpha expression, and blood levels of IL-6 and TNF alpha in UV-B-exposed mice. In this study, mice were placed into four groups: one control group, three treatment groups, and a combination of Phaleria macrocarpa:soybeans at a 1:1 ratio (com group). The mice were euthanized on days 5 and 21 for histological preparations and then examined under a light microscope. Using an Olympus C-21 microscope with an Optilab Advances camera at 1000x magnification, the fibroblast was studied by counting the number of fibroblast cells per field of view. The immunohistochemical approach was performed to analyze the expression of VEGF, IL-6, and TNF- in skin tissue. The ELISA technique was used to quantify the levels of IL-6 and TNF-alpha. SPSS ver 21 was used to analyze the data. On days 5 and 21, the number of fibroblasts and expression of VEGF, IL-6, and TNF alpha were significantly higher in the combination group than in the control, Phaleria macrocarpa, and soybean treatment groups. However, there was no significant change in IL-6 and TNF alpha levels across groups on days 5 and 21 (p > 0.05). Finally, a 1:1 mixture of Phaleria macrocarpa and soybeans reduced the number of fibroblasts and the production of VEGF, IL-6, and TNF alpha on days 5 and 21, but not in serum levels.

Neuroimmune interactions with binge alcohol drinking in the cerebellum of IL-6 transgenic mice

The neuroimmune system of the brain, which is comprised primarily of astrocytes and microglia, regulates a variety of homeostatic mechanisms that underlie normal brain function. Numerous conditions, including alcohol consumption, can disrupt this regulatory process by altering brain levels of neuroimmune factors. Alcohol and neuroimmune factors, such as proinflammatory cytokines IL-6 and TNF-alpha, act at similar targets in the brain, including excitatory and inhibitory synaptic transmission. Thus, alcohol-induced production of IL-6 and/or TNF-alpha could be important contributing factors to the effects of alcohol on the brain. Recent studies indicate that IL-6 plays a role in alcohol drinking and the effects of alcohol on the brain activity following the cessation of alcohol consumption (post-alcohol period), however information on these topics is limited. Here we used homozygous and heterozygous female and male transgenic mice with increased astrocyte expression of IL-6 to examined further the interactions between alcohol and IL-6 with respect to voluntary alcohol drinking, brain activity during the post-alcohol period, IL-6 signal transduction, and expression of synaptic proteins. Wildtype littermates (WT) served as controls. The transgenic mice model brain neuroimmune status with respect to IL-6 in subjects with a history of persistent alcohol use. Results showed a genotype dependent reduction in voluntary alcohol consumption in the Drinking in the Dark protocol and in frequency-dependent relationships between brain activity in EEG recordings during the post-alcohol period and alcohol consumption. IL-6, TNF-alpha, IL-6 signal transduction partners pSTAT3 and c/EBP beta, and synaptic proteins were shown to play a role in these genotypic effects.

P048 Deep phenotyping of the peripheral immune cell compartment in Crohn‘s Disease

Abstract Background Crohn’s disease (CD) is characterized by chronic inflammation that can discontinuously affect any segment of the gastrointestinal tract. New findings concerning clinical behaviour, epidemiology, genetics, and gut microbiota suggest that CD in the ileum (iCD) and the colon (cCD) should be considered as two distinct subtypes of IBD. As systemic immune cell signatures differ between CD and UC, we propose a similar strategy for the differentiation between iCD and cCD. The aim of this study is to analyse and compare peripheral blood mononuclear cell (PBMC) subsets of iCD and cCD patients. For a robust analysis of the peripheral immune compartment in inflammatory bowel diseases (IBD), PBMC subsets of ileocolonic CD (icCD), ulcerative colitis (UC) and healthy controls (HC) were included as well. Methods In this study a total of 50 adults, 10 subjects per subgroup – iCD, cCD, icCD, UC and HC – were included. From each individual, both naïve and PMA/Ionomycin stimulated samples were stained for phenotypic and functional characterization using mass cytometry. For data analysis pre-gating was performed in Cytobank followed by debarcoding, compensation, normalization, clustering, and analysis on differential abundances edge R (DA_edgeR) and differential states limma (DS_limma) with CATALYST in R. Results FlowSOM allowed us to cluster CD45+ PBMCs of all samples into 16 major cell subtypes as visualized via UMAP (Fig. 1). DA testing showed significant differences between iCD and cCD, iCD and icCD but not cCD and icCD (Fig 2). Significantly different abundances among CD subgroups and HC included cluster 1 CD33+ myeloid cells, 7 CD45RO+ CD16+ cells, 8 CD11b+ cells, 9 CD4+ IL7R+ T cells and 12 CD27+ CD38+ CD45RA+ B cells (Fig. 3). When focussing on differences between iCD, cCD and icCD, DS testing revealed significantly higher IFNgamma and TNFalpha production in cluster 9 (CD4+ IL7R+ T cells), 15 (CD8+ T cells) and 16 (CD8+ CD45RO+ T cells) in iCD compared to icCD. Moreover, cluster 9 showed significantly higher levels of IFNgamma in iCD compared to cCD, and for TNFalpha cluster 16 revealed significantly higher levels in cCD compared to icCD (Fig. 4). Fig. 1 UMAP clustering of stimulated cells. Fig. 2 Differential analysis of stimulated samples with DA_edgeR and DS_limma. Fig. 3 Relative population abundances with DA_edge R testing. Fig. 4 Relative state marker abundance within clusters with DS_limma testing. Conclusion Deep phenotyping of the peripheral immune cell compartment in CD might serve as a confirmatory marker for disease location and therefore as predictor of clinical behaviour.

Helminth species dependent effects on Th1 and Th17 cytokines in active tuberculosis patients and healthy community controls.

Despite that the impact of different helminth species is not well explored, the current dogma states that helminths affect the Th1/Th2 balance which in turn affects the risk of tuberculosis (TB) reactivation and severity of disease. We investigated the influence of helminth species on cytokine profiles including IL-17A in TB patients and healthy community controls (CCs). In total, 104 newly diagnosed pulmonary TB patients and 70 HIV negative and QuantiFERON negative CCs in Gondar, Ethiopia were included following helminth screening by stool microscopy. Plasma samples and ex vivo stimulation of peripheral blood mononuclear cells (PBMCs) with purified protein derivative (PPD) and Staphylococcus enterotoxin B (SEB) was used to determine cytokine profiles by cytometric bead array. In CCs, Ascaris lumbricoides or Schistosoma mansoni infections were associated with an impaired Th1-type response (IFN-gamma, IL-6 and TNF-alpha) in PBMCs mainly with SEB stimulations, whereas in TB patients only hookworm infection showed a similar pattern. Among CCs, the IL-17A response in PBMCs stimulated with SEB was higher only for S. mansoni, whereas in TB patients, the elevated systemic IL-17A plasma level was significantly suppressed in hookworm infected TB patients compared to patients without helminth coinfection. Following treatment of TB and helminth infection there was a general decrease in ex vivio IL-10 and TNF-alpha production in unstimulated, PPD or SEB stimulated PBMCs that was the most pronounced and significant in TB patients infected with S. mansoni, whereas the follow-up levels of IFN-gamma and IL-17A was significantly increased only in TB patients without helminth coinfection from PBMCs stimulated mainly with SEB. In summary, in addition to confirming helminth specific effects on the Th1/Th2 response before and after TB treatment, our novel finding is that IL-17A was impaired in helminth infected TB patients especially for hookworm, indicating a helminth species-specific immunoregulatory effect on IL-17A which needs to be further investigated.