Cytokine Assay Research Articles

Nebulised mesenchymal stem cell derived extracellular vesicles ameliorate E. coli induced pneumonia in a rodent model

BackgroundMesenchymal stem cell (MSC) derived extracellular vesicles (EVs) have been proposed as an alternative to cell therapy, creating new possible delivery modalities such as nebulisation. We wished to investigate the therapeutic potential of directly nebulised MSC-EVs in the mitigation of Escherichiacoli-induced pneumonia.MethodsEV size, surface markers and miRNA content were assessed pre- and post-nebulisation. BEAS2B and A459 lung cells were exposed to lipopolysaccharide (LPS) and treated with nebulised bone marrow (BM) or umbilical cord (UC) MSC-EVs. Viability assays (MTT) and inflammatory cytokine assays were performed. THP-1 monocytes were stimulated with LPS and nebulised BM- or UC-EVs and phagocytosis activity was measured. For in vivo experiments, mice received LPS intratracheally (IT) followed by BM- or UC-EVs intravenously (IV) and injury markers assessed at 24 h. Rats were instilled with E. coli bacteria IT and BM- or UC-EVs delivered IV or by direct nebulisation. At 48 h, lung damage was assessed by physiological parameters, histology and inflammatory marker presence.ResultsMSC-EVs retained their immunomodulatory and wound healing capacity after nebulisation in vitro. EV integrity and content were also preserved. Therapy with IV or nebulised MSC-EVs reduced the severity of LPS-induced lung injury and E. coli-induced pneumonia by reducing bacterial load and oedema, increasing blood oxygenation and improving lung histological scores. MSC-EV treated animals also showed lower levels of inflammatory cytokines and inflammatory-related markers.ConclusionsMSC-EVs given IV attenuated LPS-induced lung injury, and nebulisation of MSC-EVs did not affect their capacity to attenuate lung injury caused by E. coli pneumonia, as evidenced by reduction in bacterial load and improved lung physiology.

A phase I/II trial of NOX66 in combination with nivolumab in patients (pts) with advanced cancer.

e14591 Background: NOX66 (idronoxil) targets ENOX2 and suppresses STING-mediated inflammatory pathways, reducing IFN and NFκB driven cytokine production by cancer cells. Adding NOX66 to nivolumab may help overcome resistance mechanisms to checkpoint inhibitors and improve responses in patients. Methods: IONIC-1, is an open label Phase I/II trial evaluating safety and preliminary efficacy of NOX66 (suppositories daily x 1 week, then 1 week rest) combined with nivolumab (240mg IV every 2 weeks). Three dose cohorts of NOX66 are planned (1200mg daily, 1800mg daily, 2400mg daily). Objectives include safety, tolerability, and clinical and radiological responses. Two groups are being studied: 1) Pts who have prior progression on a checkpoint inhibitor; 2) Pts naïve to checkpoint inhibitors. Results: 11 pts have been treated to date; 3 in Group 1, and 8 in Group 2. All pts have received at least 2 cycles of the combination treatment; 2 pts completed 8 cycles, and 2 pts remain on study. Tumour types include lung, prostate, gastric, pancreatic, nasopharyngeal and metastatic squamous carcinoma. Early signals of tumour response based on RECIST 1.1 criteria include stable disease in 2 pts (SD at 8 weeks and at 16 weeks); partial response (PR) in 1 patient at 16 weeks; complete response (CR) in 2 pts, both with ongoing CR at 24 weeks. Five pts had disease progression, and 1 pt was withdrawn due to suspected immune mediated myositis and hepatitis resulting from nivolumab and was not evaluable. The most common AE was local perineal irritation, readily managed with topical corticosteroid cream. Two pts developed an erythematous rash over the trunk and limbs after 2 cycles, suspected to be related to the combination treatment. Using a 32 multiplexed immune cytokine assay to evaluate the single-cell functional states of CD4+ and CD8+ T cells in patients’ peripheral blood mononuclear cells (PBMCs) at pre-treatment and at Cycle 3 Day 1, we found a significant increase in polyfunctionality in both effector CD4+ and CD8+ve T cells compared to baseline in responder patients. Conclusions: Preliminary analysis shows promising tumour responses from the combination therapy which appears to be well-tolerated, with no safety signals evident to date. Clinical trial information: ACTRN12621001537842 .

Modulation of tumor immune microenvironment by TAS-115, a multi-receptor tyrosine kinase inhibitor, promotes antitumor immunity and contributes anti-PD-1 antibody therapy

TAS-115 is an oral multi-receptor tyrosine kinase inhibitor that strongly inhibits kinases implicated in antitumor immunity, such as colony stimulating factor 1 receptor and vascular endothelial growth factor receptor. Because these kinases are associated with the modulation of immune pathways, we investigated the immunomodulatory activity of TAS-115. An in vitro cytokine assay revealed that TAS-115 upregulated interferon γ (IFNγ) and interleukin-2 secretion by T cells, suggesting that TAS-115 activated T cells. Gene expression analysis suggested that TAS-115 promoted M1 macrophage differentiation. In in vivo experiments, although TAS-115 exerted a moderate antitumor effect in the MC38 mouse colorectal cancer model under immunodeficient conditions, this effect was enhanced under immunocompetent conditions. Furthermore, combination of TAS-115 and anti-PD-1 antibody exhibited greater antitumor activity than either treatment alone. Flow cytometry analysis showed the increase in IFNγ- and granzyme B (Gzmb)-secreting tumor-infiltrating T cells by TAS-115 treatment. The combination treatment further increased the percentage of Gzmb+CD8+ T cells and decreased the percentage of macrophages compared with either treatment alone. These results highlight the potential therapeutic effect of TAS-115 in combination with PD-1 blockade, mediated via activation of antitumor immunity by TAS-115.

The association between peripheral inflammation, brain glutamate and antipsychotic response in Schizophrenia: Data from the STRATA collaboration

Glutamate and increased inflammation have been separately implicated in the pathophysiology of schizophrenia and the extent of clinical response to antipsychotic treatment. Despite the mechanistic links between pro-inflammatory and glutamatergic pathways, the relationships between peripheral inflammatory markers and brain glutamate in schizophrenia have not yet been investigated. In this study, we tested the hypothesis that peripheral levels of pro-inflammatory cytokines would be positively associated with brain glutamate levels in schizophrenia. Secondary analyses determined whether this relationship differed according to antipsychotic treatment response. The sample consisted of 79 patients with schizophrenia, of whom 40 were rated as antipsychotic responders and 39 as antipsychotic non-responders. Brain glutamate levels were assessed in the anterior cingulate cortex (ACC) and caudate using proton magnetic resonance spectroscopy (1H-MRS) and blood samples were collected for cytokine assay on the same study visit (IL-6, IL-8, IL-10, TNF- α and IFN-γ). Across the whole patient sample, there was a positive relationship between interferon-gamma (IFN-γ) and caudate glutamate levels (r = 0.31, p = 0.02). In the antipsychotic non-responsive group only, there was a positive relationship between interleukin-8 (IL-8) and caudate glutamate (r = 0.46, p = 0.01). These findings provide evidence to link specific peripheral inflammatory markers and caudate glutamate in schizophrenia and may suggest that this relationship is most marked in patients who show a poor response to antipsychotic treatment.

Parthenolide alleviates microglia‐mediated neuroinflammation via MAPK/TRIM31/NLRP3 signaling to ameliorate cognitive disorder

Background and purposeNeuroinflammation, mainly mediated by microglia, is involved in the evolution of Alzheimer's disease (AD). Parthenolide (PTL) has diverse pharmacological effects such as anti-inflammatory and antioxidative stress. However, whether PTL can modulate microglia-mediated neuroinflammation to improve cognitive impairment in amyloid precursor protein/presenilin 1 (APP/PS1) mice is unclear. MethodsLPS/IFN-γ-induced BV2 and HMC3 microglia were used for in vitro experiments; the roles of PTL on anti-inflammatory, anti-oxidative, phagocytic activity, and neuroprotection were assessed by inflammatory cytokines assays, dichlorodihydrofluorescein diacetate, phagocytosis, and cell counting kit-8 assays. Western blot and immunofluorescence（IF） were used to examine related molecular mechanisms. In vivo, IF and western blot were applied in LPS-treated wild-type (WT) mice and APP/PS1 mice models. The Morris water maze test was performed to evaluate the effects of PTL on cognitive disorders. ResultsIn vitro, PTL dramatically suppressed proinflammatory cytokines IL-6, IL-1β, and TNF-α release and increased IL-10 levels. Moreover, PTL decreased reactive oxygen species and restored microglial phagocytic activities via the AKT/MAPK/ NF-κB signaling pathway. Importantly, we discovered that PTL obviously enhanced TRIM31 expression and siTRIM31 elevated proinflammatory cytokine levels. Furthermore, we determined that the anti-inflammatory role of PTL was mostly TRIM31/NLRP3 signaling-dependent. In vivo, PTL alleviated microgliosis and astrogliosis in LPS-treated WT and APP/PS1 mice. Additionally, PTL significantly ameliorated memory and learning deficits in cognitive behaviors. ConclusionsPTL improved cognitive and behavioral dysfunction, inhibited neuroinflammation, and showed potent anti-neuroinflammatory activity and neuroprotective effects by improving the MAPK/TRIM31/NLRP3 axis. Our study emphasized the therapeutic potential of PTL for improving cognitive disorders during AD progression.

Tissue-Engineered Hepatocyte Sheets Supplemental with Adipose-derived Stem Cells.

The ability to engineer biologically viable hepatocytes and tissue matrices with long-term functional maintenance has attracted considerable interest in the fields of hepatocyte transplantation and liver tissue engineering. Here, newly developed hepatocyte sheets supplemented with adipose-derived stem cells (ADSCs) were evaluated to assess the effects of ADSCs on hepatocyte function and engraftment into the subcutaneous space. Eight-week-old male C57BL/6J mice were used as donors and 6-week-old male C.B-17/Icr-scid/scid mice were used as recipients. Hepatocyte-ADSC composite sheets were developed using temperature-responsive culture dishes. Hepatocyte viability in the hepatocyte-ADSC composite sheets was evaluated in an in vitro assay, and the outcome of subcutaneous transplantation of the sheet was evaluated. Hepatocyte viability was sustained in the hepatocyte-ADSC composite sheets in vitro. Albumin secretion was significantly higher (p = 0.015) in the hepatocytes of the hepatocyte-ADSC composite sheets (70.5 μg/mL) than in hepatocyte-only sheets (24.0 μg/mL). Cytokine assays showed that hepatocyte growth factor and interleukin-6 were contributed by ADSCs and not hepatocytes, which were not capable of constitutively secreting them. Immunohistochemically, phosphorylated STAT3 and c-MET expression in hepatocytes in the hepatocyte-ADSC composite sheets was significantly higher than that in the hepatocyte-only sheets. Engraftment of the transplanted hepatocyte-ADSC composite sheets was significantly enhanced without pretreatment of the subcutaneous tissue to induce a vascular network. In the hepatocyte-ADSC composite sheets, the viability of the hepatocytes was significantly maintained as the co-cultured ADSCs provided cytokines, enhancing pivotal cell signaling necessary for hepatocyte activity.

CD16+ monocytes are involved in the hyper-inflammatory state of Prader-Willi Syndrome by single-cell transcriptomic analysis.

Patients with Prader-Willi syndrome (PWS) have a reduced life expectancy due to inflammation-related disease including cardiovascular disease and diabetes. Abnormal activation of peripheral immune system is postulated as a contributor. However, detailed features of the peripheral immune cells in PWS have not been fully elucidated. Serum inflammatory cytokines were measured in healthy controls (n=13) and PWS patients (n=10) using a 65- multiplex cytokine assays. Changes of the peripheral immune cells in PWS was assessed by single-cell RNA sequencing (scRNA-seq) and high-dimensional mass cytometry (CyTOF) using peripheral blood mononuclear cells (PBMCs) from PWS patients (n=6) and healthy controls (n=12). PWS patients exhibited hyper-inflammatory signatures in PBMCs and monocytes were the most pronounced. Most inflammatory serum cytokines were increased in PWS, including IL-1β, IL-2R, IL-12p70, and TNF-α. The characteristics of monocytes evaluated by scRNA-seq and CyTOF showed that CD16+ monocytes were significantly increased in PWS patients. Functional pathway analysis revealed that CD16+ monocytes upregulated pathways in PWS were closely associated with TNF/IL-1β- driven inflammation signaling. The CellChat analysis identified CD16+ monocytes transmitted chemokine and cytokine signaling to drive inflammatory process in other cell types. Finally, we explored the PWS deletion region 15q11-q13 might be responsible for elevated levels of inflammation in the peripheral immune system. The study highlights that CD16+ monocytes contributor to the hyper-inflammatory state of PWS which provides potential targets for immunotherapy in the future and expands our knowledge of peripheral immune cells in PWS at the single cell level for the first time.

Non-Redundant Roles of T Cell Costimulation Pathways in Inflammatory Arthritis Revealed by Dual Blockade of ICOS and CD28 with Acazicolcept (ALPN-101).

CD28 and inducible T cell costimulator (ICOS) appear to have nonredundant roles in T cell activation and adaptive immunity. We undertook this study to characterize in vitro and in vivo the therapeutic potential of acazicolcept (ALPN-101), an Fc fusion protein of a human variant ICOS ligand (ICOSL) domain designed to inhibit both CD28 and ICOS costimulation, in inflammatory arthritis. Acazicolcept was compared in vitro with inhibitors of either the CD28 or ICOS pathways (abatacept and belatacept [CTLA-4Ig], prezalumab [anti-ICOSL monoclonal antibody]) in receptor binding and signaling assays, and in a collagen-induced arthritis (CIA) model. Acazicolcept was also compared in cytokine and gene expression assays of peripheral blood mononuclear cells (PBMCs) from healthy donors or rheumatoid arthritis (RA) or psoriatic arthritis (PsA) patients stimulated with artificial antigen-presenting cells (APCs) expressing CD28 and ICOS ligands*. Acazicolcept bound CD28 and ICOS, prevented ligand binding, and inhibited human T cell functional interactions, matching or exceeding the activity of CD28 or ICOS costimulatory single-pathway inhibitors tested individually or in combination. Acazicolcept administration significantly reduced disease in the CIA model and more potently than abatacept. Acazicolcept also inhibited proinflammatory cytokine production from stimulated PBMCs in cocultures with artificial APCs and demonstrated unique effects on gene expression distinct from those induced by abatacept, prezalumab, or a combination of both. Both CD28 and ICOS signaling play critical roles in inflammatory arthritis. Therapeutic agents such as acazicolcept that coinhibit both ICOS and CD28 signaling may mitigate inflammation and/or disease progression in RA and PsA more effectively than inhibitors of either pathway alone.

Secreted Cytokines within the Urine of AKI Patients Modulate TP53 and SIRT1 Levels in a Human Podocyte Cell Model

Acute kidney injury (AKI) is a major kidney disease with a poor clinical outcome. It is a common complication, with an incidence of 10-15% of patients admitted to hospital. This rate even increases for patients who are admitted to the intensive care unit, with an incidence of >50%. AKI is characterized by a rapid increase in serum creatinine, decrease in urine output, or both. The associated symptoms include feeling sick or being sick, diarrhoea, dehydration, decreased urine output (although occasionally the urine output remains normal), fluid retention causing swelling in the legs or ankles, shortness of breath, fatigue and nausea. However, sometimes acute kidney injury causes no signs or symptoms and is detected by lab tests. Therefore, the identification of cytokines for the early detection and diagnosis of AKI is highly desirable, as their application might enable the prevention of the progression from AKI to chronic kidney disease (CKD). In this study, we analysed the secretome of the urine of an AKI patient cohort by employing a kidney-biomarker cytokine assay. Based on these results, we suggest ADIPOQ, EGF and SERPIN3A as potential cytokines that might be able to detect AKI as early as 24 h post-surgery. For the later stages, as common cytokines for the detection of AKI in both male and female patients, we suggest VEGF, SERPIN3A, TNFSF12, ANPEP, CXCL1, REN, CLU and PLAU. These cytokines in combination might present a robust strategy for identifying the development of AKI as early as 24 h or 72 h post-surgery. Furthermore, we evaluated the effect of patient and healthy urine on human podocyte cells. We conclude that cytokines abundant in the urine of AKI patients trigger processes that are needed to repair the damaged nephron and activate TP53 and SIRT1 to maintain the balance between proliferation, angiogenesis, and cell cycle arrest.

Predict postoperative atrial fibrillation after cardiac surgery thanks to a preoperative macrophages functional in vitro test

Postoperative atrial fibrillation (POAF) is a common complication after cardiac surgery with an incidence of 30%. Cardiac surgery induces a postoperative inflammatory response mediated by innate immunity and associated with POAF. Our aim is to associate the level of in vitro response of preoperative innate immunity with POAF. An in vitro stimulation test by LPS + ATP or R848 was achieved on macrophages derived from preoperative classic monocytes, in patients hospitalized for cardiac surgery, between February and July 2022 (POMI-AF cohort NCT03376165). Then, an assay of pro-inflammatory cytokines (IL-1β, TNFα, IL-6) was carried out by ELISA method after 24 hours of stimulation. In all, 57 patients were included, whose 18 patients who presented POAF (33%). Stimulation test induces a significant increase of IL-1, TNF and IL-6 rates with LPS + ATP or R848. In the “POAF” group, a 2 times higher production of IL-1β, TNFα and IL-6 after LPS + ATP was observed, without reaching statistical significance. After stimulation with R848, we also highlight a 4 times higher production of TNFα and 2 times higher production of IL-6 in the “POAF” group, without reaching statistical significance. Level of in vitro response of preoperative innate immunity appears to be wider after stimulation in patients with POAF after cardiac surgery, but it should be explored on a larger number of patients.

Clinicoepidemiological study of dengue positive patients admitted to tertiary care hospitals in the Burdwan district, using molecular seroprofiling and enzyme linked immunosorbent assays of cytokines and chemokines

Background: The World Health Organization views dengue fever as a significant worldwide public health threat in the tropics and subtropics. Dengue virus has four serotypes, DENG 1 to 4, which were responsible for outbreaks that occurred in other parts of the world from the 1980s until now. Aims and Objectives: Clinical manifestations and molecular and serological studies help determine the inflammatory response events associated with dengue severity. This study aimed to define the profiles of some cytokines and chemokines, as well as their molecular serotyping. Materials and Methods: Blood serum samples are collected, followed by serodiagnosis (NS1 and IgM) through ELISA, molecular serotyping detection of dengue by real-time PCR through SyBr green, and an immunological study of cytokines tumor necrosis factor-alpha and interferon-gamma (IFN-γ) on mother-hand chemokines CCL26 and MCP1 by ELISA. Statistical analysis was performed by SPSS (version 22). Results: The study examined 70 dengue seropositive samples, of which five were NS1 positive and 55 were IgM positive. After accounting for comorbidities and patients’ demographic classifications, the 21–30 age group has a maximum positivity of 30%. The mean concentration level of IFN-γ is elevated at 1544.438 pg/mL. Conclusion: Dengue-positive patients have a high ischemic heart disease rate, among other diseases. An elevated circulating level of osteopetrosis may cause cancer, tuberculosis, and other serious diseases. In immunological aspects, IFN-γ elevation can be used to predict the severity of dengue infection.

In VitroExpansion of Antigen-Specific CD8+ T Cells from Human PBMCs Using Artificial T-cell Stimulating Microparticles

Abstract While current T cell-based immunotherapies have shown moderate success, clinical translation of these methods has been hindered by biological variability, limited efficacy, and high expense. Here, we create a novel T cell stimulating platform for antigen-specific CD8+ T cell activation and expansion, eliminating the extensive ex vivoexpansion steps and thus making it accessible for many more cancer patients. The scaffold is formed using thiol-modified hyaluronic acid cross-linked with PEGDA, magnetic dextran nanoparticles, as well as DTT-reduced MHC-Ig dimer HLA-A*02 (A2) and anti-CD28. The gel is passed through a mesh device to form microparticles (MPs), which are then passively loaded with peptides (here, CMV and MART1) for ease of multiple expansions. They were plated with human PBMCs thawed overnight in culture media supplemented with human AB serum and a cocktail of cytokines (IL-1b, IL-2, IL-4, IL-6, and IFNγ). On days 7 and 14, cells were harvested and analyzed for proliferation of antigen-specific cells. After fourteen days of culturing PBMCs with A2 MPs, we could detect a significant expansion of antigen-specific T cells via flow cytometry. When stimulated with peptide-pulsed target cells for an intracellular cytokine assay, they expressed one or more of IFNγ, TNFα, and IL-2, demonstrating their polyfunctionality. We also observed a greater population of memory T cells, which indicates that these cells may persist longer in the body. We have developed a biomaterial-based T cell activation platform that provides critical signals, mimicking the lymph nodes. These MPs are effective in expanding antigen-specific T cells from human PBMCs, and these cells have robust killing of target splenocytes.

High fat-high sugar diet induces sex-dependent effects on the small intestine

Background: Metabolic syndrome includes a plethora of symptoms ultimately leading to obesity and type 2 diabetes. Obesity has been linked to chronic gastrointestinal dysfunction (constipation, or diarrhea) and specifically females with high BMI have been associated with increased prevalence of diarrhea. We aimed to better understand the influence of high fat-high sugar diet (Western diet) on small intestinal function and to assess sex-dependent effects. Methods: We measured transepithelial short circuit current (Isc) a measure of chloride secretory function, across freshly isolated segments of jejunum from male and female C57Bl/6J mice fed a high fat high sugar diet (HFS) for 12 weeks, compared to lean controls. At the completion of the study, segments of jejunum were frozen for western blot determination of key proteins involved in secretory and absorptive functions and senescence. Intestinal morphology was assessed. Serum cytokine assays were performed. Results: Basal Isc was significantly decreased 70% ( P<0.05) in HFS both females and males versus leans. In females, the HFS-induced decrease in Isc was attributed to a significant loss of CLC2, NKCC1 and CFTR expression whereas in males the decrease in Isc was due to a significant loss of Na/K-ATPase, KCa and NKCC1 expression (indicating interesting sex-dependent etiology). A2BR levels were also dysregulated in HFS fed male and female mice. Inflammatory state and shadowed body weight changes with feeding a western diet. Assessment of intestinal morphology and metabolic profile is being further evaluated. Conclusions: Our data suggests that the reduced basal jejunal Isc in HFS mice is attributed to sex-dependent mechanisms. Improved understanding of Western diet-induced intestinal dysfunctions may provide improved drug targets to treat gastrointestinal disturbances in obesity and diabetes. Midwestern Arizona Alzheimer’s Consortium and Diabetes Action Research and Education Foundation This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Transcription factor Bhlhe40 plays a protective role during intravaginal Chlamydia muridaruminfection in mice

Abstract The protein basic helix-loop-helix family member e40 (BHLHE40) is a transcription factor involved in cell cycle regulation and differentiation. It has recently emerged as a key player in immunity regulating T cell responses and cytokine production during infections. To address the role of Bhlhe40in Chlamydiainfections, we infected WT and Bhlhe40−/−mice intravaginally with Chlamydia muridarumand monitored infection kinetics. Bhlhe40−/−mice exhibited a significant defect in their ability to clear infection compared to their WT counterparts. Single-cell RNA sequencing revealed distinct heterogeneity in the CD4 +T cell population in the female reproductive tract in WT and Bhlhe40−/−mice following C. muridaruminfection. Accordingly, CD4 +T cell population in Bhlhe40−/−mice showed a marked increase in IL-10-producing T regulatory type 1 (Tr1) cells and decreased IFN-γ and GM-CSF-producing cells as assessed by flow cytometry. The overall CD4 +T cell cytokine response was diminished in the absence of Bhlhe40, as indicated by decreased levels of IFN-γ, GM-CSF and IL-17A measured by a multiplex cytokine assay. In contrast, Bhlhe40did not appear to play an essential role in antibody responses, development of pathology, or metabolic fitness of CD4 T cells during C. muridaruminfection. Lastly, IL-10 deficiency restored optimal bacterial clearance in Bhlhe40−/−mice. Altogether, our findings identified Bhlhe40as a novel player in protective immune response against C. muridaruminfection in mice. NIH/NIAID R01 (AI139124) NIH/NIGMS COBRE (GM103625)The American Association of Immunologists through a Careers in Immunology Fellowship

Deciphering molecular and functional codes of human memory lymphocytes

Abstract Long-term immunity against infections and effective response to vaccination are dependent on unique feature of lymphocytes referred as “memory generation”. Majority of our understanding is established on murine data and our understanding of human naive to memory differentiation is limited. In our ongoing study we explored epigenetics (DNA methylation and accessible chromatin), transcriptomics (basal, activation and proliferation induced gene expression), intracellular cytokine assay (high-parameter flow cytometry) of naive and memory B and T lymphocytes subsets from the healthy donors. We explored the epigenetic changes associated with memory differentiation. However, relationship between epigenetics and transcriptomic outcomes didn’t explain entirely the properties of being memory or naïve lymphocyte. To understand mechanistic differences, naive and memory cells of B and CD4 T lymphocytes were activated in vitro using either antigen receptor (BCR or TCR crosslinking with antibodies) or PMA+Ionomycin (pan-activation) and assayed for early or late gene expression and for intracellular cytokine profiles. Surprisingly, majority of transcriptomic patterns were similar between naive and memory lymphocytes while their cytokine expression profiles were significantly different. These findings indicate the significant role of translational machinery influencing the memory and naïve lymphocyte functions. Our results are in direction of segregating transcription and translation mediated dependencies of adaptive memory. These findings will provide the foundation for future studies related to dysregulation of memory cell differentiation and function in aging and infections. This project was entirely supported by intramural funding of NIA-NIH.

Effect of Lactobacillus Rhamnosus GG for Regulation of Inflammatory Response in Radiation-Induced Enteritis.

The purpose of this study was to investigate the role of Lactobacillus rhamnosus GG (LGG) probiotics in radiation enteritis using in vivo mice. A total of 40 mice were randomly assigned to four groups: control, probiotics, radiotherapy (RT), and RT + probiotics. For the group of probiotics, 0.2mL of solution that contained 1.0 × 108 colony-forming units (CFU) of LGG was used and orally administered daily until sacrifice. For RT, a single dose of 14Gy was administered using a 6 mega-voltage photon beam to the abdominopelvic area. Mice were sacrifice at day 4 (S1) and day 7 (S2) after RT. Their jejunum, colon, and stool were collected. A multiplex cytokine assay and 16s ribosomal RNA amplicon sequencing were then performed. Regarding cytokine concentrations in tissues, pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin-6 and monocyte chemotactic protein-1, showed significantly decreased protein levels in colon tissues of the RT + probiotics group than in the RT alone group (all p < 0.05). As for comparing microbial abundance through alpha-diversity and beta-diversity, no significant differences were observed between the RT + probiotics and RT alone groups, except for an increase in alpha-diversity in the stool of the RT + probiotics group. Upon analysis of differential microbes based on treatment, the dominance of anti-inflammatory-related microbes, such as Porphyromonadaceae, Bacteroides acidifaciens, and Ruminococcus, was observed in the jejunum, colon, and stool of the RT + probiotics group. With regard to predicted metabolic pathway abundances, the pathways associated with anti-inflammatory processes, such as biosynthesis of pyrimidine nucleotides, peptidoglycans, tryptophan, adenosylcobalamin, and propionate, were differentially identified in the RT + probiotics group compared to the RT alone group. Protective effects of probiotics on radiation enteritis were potentially derived from dominant anti-inflammation-related microbes and metabolites.

PP19128R, a Multiepitope Vaccine Designed to Prevent Latent Tuberculosis Infection, Induced Immune Responses In Silico and In Vitro Assays.

Background: Latent tuberculosis infection (LTBI) is the primary source of active tuberculosis (ATB), but a preventive vaccine against LTBI is lacking. Methods: In this study, dominant helper T lymphocyte (HTL), cytotoxic T lymphocyte (CTL), and B-cell epitopes were identified from nine antigens related to LTBI and regions of difference (RDs). These epitopes were used to construct a novel multiepitope vaccine (MEV) based on their antigenicity, immunogenicity, sensitization, and toxicity. The immunological characteristics of the MEV were analyzed with immunoinformatics technology and verified by enzyme-linked immunospot assay and Th1/Th2/Th17 cytokine assay in vitro. Results: A novel MEV, designated PP19128R, containing 19 HTL epitopes, 12 CTL epitopes, 8 B-cell epitopes, toll-like receptor (TLR) agonists, and helper peptides, was successfully constructed. Bioinformatics analysis showed that the antigenicity, immunogenicity, and solubility of PP19128R were 0.8067, 9.29811, and 0.900675, respectively. The global population coverage of PP19128R in HLA class I and II alleles reached 82.24% and 93.71%, respectively. The binding energies of the PP19128R-TLR2 and PP19128R-TLR4 complexes were -1324.77 kcal/mol and -1278 kcal/mol, respectively. In vitro experiments showed that the PP19128R vaccine significantly increased the number of interferon gamma-positive (IFN-γ+) T lymphocytes and the levels of cytokines, such as IFN-γ, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10. Furthermore, positive correlations were observed between PP19128R-specific cytokines in ATB patients and individuals with LTBI. Conclusions: The PP19128R vaccine is a promising MEV with excellent antigenicity and immunogenicity and no toxicity or sensitization that can induce robust immune responses in silico and in vitro. This study provides a vaccine candidate for the prevention of LTBI in the future.

Abstract CT275: Phase 1 clinical update of allogeneic invariant natural killer T cells (iNKTs), agenT-797, alone or in combination with pembrolizumab or nivolumab in patients with advanced solid tumors

Abstract Introduction: AgenT-797 is an allogeneic iNKT cell therapy and represents a novel, scalable, off-the-shelf approach against solid tumors. iNKTs are a unique subset of T cells, that mediate antitumor responses by direct killing, targeting CD1d and other ligands in the tumor microenvironment, and by activating host immune cells. We conducted a clinical trial to investigate agenT-797 activity as single agent and in combination (combo) with PD-1 blockade after prior progression on PD-1 therapy. Methods: Patients (pts) with relapsed or refractory solid tumors were treated with single IV infusion of agenT-797 (no lymphodepletion) at 4.3 × 10^6 or 1.4 × 10^7 cells/kg, as monotherapy or in combo with pembrolizumab (pembro) or nivolumab (nivo). Dose escalation followed 3+3 scheme. Endpoints included safety, persistence of agenT-797, objective responses, duration of response, progression-free survival, and time to response. Adverse events (AEs) were reported per CTCAE v5.0. Dose limiting toxicities (DLTs) were evaluated. AgenT-797 persistence was assessed by assays utilizing SNPs and cfDNA analysis. Serum biomarkers were measured with MSD V-PLEX cytokine assays. On-treatment tumor biopsy was obtained for multiplex immunofluorescence staining and next generation sequencing. Results: As of February 5th, 32 pts (median age 62y, range 30-83) were treated with agenT-797 monotherapy (n=26) or combo (n=6) with pembro or nivo. Pts had median 4 lines of prior therapy (range 1-13). Tumor types included pancreatic (6), NSCLC (4), rectal (4), cholangiocarcinoma/biliary duct (4), and other (14)*. Tolerability was favorable, with no cytokine release syndrome, neurotoxicity, and no DLTs. Treatment-related AEs included 16.7% (5) grade 1, 3.3% (1) grade 2, and 3.3% (1) grade 3 (anemia). At data cutoff, agenT-797 monotherapy and combo revealed early clinical activity. Among 29 evaluable pts, a confirmed partial response in MSI-H gastric cancer refractory to PD-1 treated with agenT-797 + nivo (remains ongoing &amp;gt;6 months) and 8 pts had stable disease (SD). Prolonged SD (&amp;gt;3 months) for 3 monotherapy pts. Overall response rate 20% (1/5) for pts treated with agenT-797 + pembro or nivo. AgenT-797 was detected in peripheral blood up to day 8 post infusion. Preliminary data identified transient increase in serum IFNgamma levels day 2 post infusion. Conclusion: AgenT-797 was well tolerated as monotherapy and in combo with PD-1 (pembro or nivo). Anti-tumor activity in combo with nivo was observed in gastric cancer. Results support the potential of a novel therapeutic strategy employed by agenT-797 to enhance antitumor immunity in PD-1 refractory tumors. Enrollment ongoing and correlative studies will be presented. *PD-1 refractory NSCLC, pancreatic, rectal, cholangiocarcinoma, cervical, gastric, ocular melanoma, renal, and upper tract urothelial Citation Format: Benedito Carneiro, Benjamin Garmezy, John T. Hamm, Rachel E. Sanborn, Trisha Wise-Draper, Anthony El- Khoueiry, Breelyn Wilky, Dave S.B Hoon, Alexa Buffa, Xavier Michelet, Marco Purbhoo, Mark A. Exley, David Einstein. Phase 1 clinical update of allogeneic invariant natural killer T cells (iNKTs), agenT-797, alone or in combination with pembrolizumab or nivolumab in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT275.

Cytokine expression in gingival crevicular fluid around teeth opposing dental implants and 3-unit fixed partial dentures in a cross-sectional study

ObjectiveThis study aimed to study the cytokines in gingival crevicular fluid (GCF) of the teeth opposing to dental implants and 3-unit fixed partial dentures (FPDs).Materials and methodsA total of 74 participants were recruited for this cross-sectional study. Based on the status of lower first molars, the participants were divided into dental implants group and 3-unit FPDs group. Social index and oral hygiene were recorded. Occlusal loading was evaluated with a T-scan. GCF was sampled from the upper first molar and assessed with a commercial cytokine assay kit.ResultsForty three dental implants patients and 31 3-unit FPDs patients received all of the clinical and laboratory evaluation. The dental implants group had a higher occlusion force distribution on first molars region. IL-10, IL-17, RANK had a higher mean in dental implants group and was associated with occlusion force of first molar. There was a weakly association between IL-10 and dental implants in the binary logistic regression analyses.ConclusionsIn this study, the teeth opposing implants have a higher level of cytokines in the GCF than teeth opposing to 3-unit FPDs in periodontal healthy participants because of the poor osseoperception of dental implants. IL-10 might reflect a higher occlusion force in dental implants region.Clinical relevanceThis study provided that different tooth restoration methods could influence the periodontal status of the contact teeth.

The protective effect of 999 XiaoErGanMao granules on the lungs and intestines of influenza A virus-infected mice

Context Gastrointestinal symptoms are a common complication of influenza virus infection in children, which the gut-lung axis become involved in its biological progress. The protective effect of 999 XiaoErGanMao granules (XEGMG) on multi-organ injury in viral pneumonia remains unclear. Objective To investigate the therapeutic effect of XEGMG on lungs and intestines injury in A/FM/1/47 (H1N1) influenza virus-infected mice. Materials and methods Male BALB/c mice were infected with the 2LD50 H1N1 influenza virus and then treated with XEGMG (6 or 12 g/kg) intragastrically once a day for 4 days. The lung and colon samples were then collected for pathological observation, and assays for inflammatory cytokines and intestinal barrier. Mouse feces were collected to evaluate the intestinal microbiota. Results Treating with XEGMG (12 g/kg) can mitigate body weight loss caused by 2LD50 H1N1 infection. It can also reduce lung index and pathological damage with the decreased inflammatory cytokines such as IL-6 and IL-1β. Furthermore, XEGMG (12 g/kg) can maintain the goblet cell number in the colons to protect the intestinal barrier and regulate the major flora such as Firmicutes, Bacteroidetes, and Muribaculaceae back to normal. Meanwhile, the expression of IL-17A in the colon tissues was significantly lower in the group of XEGMG (6, 12 g/kg) compared to H1N1 group. Discussion and conclusions XEGMG can protect against H1N1 invasion involved in gut-lung axis regulation. The results provide new evidence for the protective effect of XEGMG, which is beneficial to vulnerable children.