Cytokine Analysis Research Articles

Pasteurella multocida Serotype D Infection Induces Activation of the IL-17 Signaling Pathway in Goat Lymphocytes

(1) Background: Pasteurellosis is a global zoonotic bacterial disease, which has caused significant economic impacts in animal husbandry. Nevertheless, there is limited understanding of the immune response between goat peripheral blood lymphocytes (PBLs) and goat-derived Pasteurella multocida (P. multocida). (2) Methods: To investigate the immune response of host PBLs during infection with P. multocida type D, we established an in vitro cell model utilizing isolated primary goat PBLs. Utilizing this in vitro infection model, we employed an enzyme-linked immunosorbent assay (ELISA) to assess the cytokine profile variation in goat PBLs following infection. Meanwhile, RNA sequencing and quantitative PCR (qPCR) methods were employed to analyze the gene expression profile. (3) Results: The ELISA test results indicated that the expression levels of pro-inflammatory cytokines, such as IL-6, IFN-γ, CXCL10, and IL-17A, were significantly elevated within 12 h after infection with P. multocida. In contrast, the levels of the anti-inflammatory cytokine IL-10 were found to be reduced. RNA sequencing and functional enrichment analysis identified 2114 differentially expressed genes (DEGs) that were primarily associated with cytokine-cytokine receptor interactions, viral protein-cytokine interactions, and the IL-17 signaling pathway. Furthermore, protein-protein interaction (PPI) network analysis and qPCR highlighted CD86, CCL5, CD8A, CXCL8, CTLA4, TNF, CD274, IL-10, IL-6, CXCL10, IFNG, and IL-17A that were crucial for the response of PBLs to P. multocida infection. (4) Conclusions: This study systematically revealed the characteristics of PBLs in goats following infection with goat-derived P. multocida type D through the analysis of cytokines and gene expression, providing important theoretical insights for a deeper understanding of the defense mechanisms in goats against P. multocida.

The landscape of chemokine and cytokine is associated with the distinct clinical status of leprosy patients and their respective household contacts

IntroductionLeprosy, a chronic infectious disease, is closely linked to the host immune response. According to the WHO, leprosy patients (L) and household contacts (HHC) are classified into subgroups: paucibacillary (PB) and multibacillary (MB), witch reflect the degree of infection in patients and the level of exposure of their contacts. The main goal of this study was to: i) establish a comprehensive overview of soluble mediator signatures of PBMCs upon in vitro antigen-specific stimuli and ii) identify whether the chemokine (CH) and cytokine (CY) signatures were associated with distinct clinical manifestations in (L) and immune response profiles in (HHC).MethodsLong-term PBMC cultures were carried out and supernatants collected for 12 CH and CY analisys by Cytometric Beads Array.Results and discussionThe CH and CY analysis, using continuous variable modeling, demonstrated that PBMCs from both L and HHC exhibited high levels of TNF upon M. leprae-stimuli. While lower production of IFN-γ were observed for L, low levels of CXCL8 was found for HHC. Soluble mediator signatures, analyzed using categorical variables, revealed that while high levels of TNF were observed for L, high levels of IFN-γ appeared as a hallmark of HHC. Overall, these analyses demonstrated that CXCL8, IFN-γ, and TNF were key markers differentiating L from HHC and endemic control (EC), especially considering the categorical analysis of the soluble mediator signatures. Data further demonstrated that higher levels of IFN-γ and lower levels CXCL8 was features associated with HHC(MB), whereas high levels of TNF were observed in both L subgroups. Moreover, data from integrative networks, based on correlation amongst soluble mediators, revealed that in M. leprae-stimuli, the number of correlations was lower in HHC(MB) compared to HHC(PB), but higher in L(MB) compared to L(PB). It was noted that the number of correlations decreased in the following order: EC > L > HHC. Our findings contribute to additional immunological features associated with L and HHC, witch can be useful complementary diagnostic/prognostic tools for classification of L and HHC, providing insights to enrich the research agenda about the hypothesis that HHC should be closely monitored as they may present a subclinical infection.

Immunological mechanisms of tuberculosis susceptibility in TB-infected individuals with type 2 diabetes mellitus: insights from mycobacterial growth inhibition assay and cytokine analysis.

This study is important because it sheds light on the impaired immune response in individuals with type 2 diabetes mellitus (T2DM) who are infected with Mycobacterium tuberculosis (M.tb), offering critical insights into why they are at higher risk of developing active tuberculosis (TB). By demonstrating that T2DM individuals exhibit a weakened ability to control M.tb growth and a compromised cytokine profile, the research underscores the need for better diagnostic tools, such as the mycobacterial growth inhibition assay (MGIA), to identify those at greater risk of progression to active TB. The findings also highlight the importance of integrated care strategies for managing both T2DM and TB, particularly in TB-endemic regions, and point to the need for further research to develop more effective interventions tailored to this vulnerable population.

Mapping the HPV Landscape in South African Women: A Systematic Review and Meta-Analysis of Viral Genotypes, Microbiota, and Immune Signals

This systematic review and meta-analysis evaluate human papillomavirus (HPV) prevalence, genotype distribution, and associations with cervicovaginal microbiota and cytokine profiles among South African women, where cervical cancer ranks as the second most common cancer. PubMed, SCOPUS, and Web of Science were searched for studies on HPV infection up to 21 September 2024. The pooled prevalence was estimated using a random-effects model, with subgroup analyses by province, sample type, and HIV status. Publication bias was evaluated using funnel plots and Egger’s test. Of the 19,765 studies screened, 120 met the inclusion criteria, comprising 83,266 participants. Results indicate a high HPV burden, with a pooled prevalence of 58% (95% CI: 52–64%), varying regionally from 53% (95% CI: 41–65%) to 64% (95% CI: 55–73%), with some regions under-researched. Cervical samples had the highest HPV prevalence (60% (95% CI: 54–66%)), while non-genital samples were less studied. High-risk (HR) HPV types, notably HPV 16 (7.5%), HPV 35 (4.1%), and HPV 18 (3.9%), were prominent, with HPV 35 emphasizing the need for expanded vaccine coverage. HIV-positive women had a higher pooled HPV prevalence (63% (95% CI: 55–71%)). Funnel plot analysis and Egger’s test suggested a potential publication bias (p = 0.047). HPV-positive women exhibited lower Lactobacillus levels and an increase in Bacterial Vaginosis (BV)-associated species like Gardnerella, potentially supporting HPV persistence. Cytokine analysis showed elevated MIP-1α and MIP-1β in HPV infections, though cytokine profiles may depend on HPV genotypes. These findings underscore the need for research on HPV–microbiome-immune interactions and call for comprehensive HPV-prevention strategies, including vaccines targeting regional HPV types and tailored interventions for HIV-positive populations.

Clinical, Immunologic, and Genetic Characteristics in Patients with Syndrome of Undifferentiated Recurrent Fevers (SURF).

Syndrome of Undifferentiated Recurrent Fevers (SURF) is characterized by recurrent fevers and autoinflammation without a confirmed molecular diagnosis of a Hereditary Recurrent Fever syndrome (HRF), and not fulfilling criteria for Periodic Fever, Adenitis, Pharyngitis, Aphthous stomatitis syndrome (PFAPA). The goal of this study was to characterize clinical features of SURF patients compared to PFAPA, and to analyze their cytokine signature, genetic variations, and responses to treatment. We enrolled 46 patients followed at Cincinnati Children's Hospital Medical Center. Baseline data and inflammatory cytokines were collected at enrollment, and their clinical course was followed. Cytokine analysis was performed using a cytokine multiplex assay. Many patients had specific or whole exome genetic testing. The prevalence of rash and arthralgias were higher in SURF compared to PFAPA. Pharyngitis and adenopathy were less frequent. A subset of SURF patients clustered together with elevated pro-inflammatory cytokines and more frequently required biologic therapy. Focused analysis of WES data revealed that variants of unknown clinical significance (VUCS) were frequently identified in genes implicated in B cell development, immunodeficiencies, and inflammatory bowel disease risk. Treatments for SURF patients commonly included on-demand steroids, colchicine, and anti-IL1 therapy. Our findings suggest SURF is a heterogeneous group but has distinct clinical and immunologic features from disorders like PFAPA. Patients have frequent VUCS, which may have relevance to disease pathogenesis. A subset of patients showed more inflammation and increased need for biologic use. Further research is necessary to define whether there exist distinct SURF endotypes and to better predict treatment outcomes.

In human CD4+ T-Cells, omeprazole suppresses proliferation, downregulates V-ATPase, and promotes differentiation toward an autoimmunity-favoring phenotype

BackgroundProton pump inhibitors (PPIs) represent a commonly prescribed class of medications. Triggered by findings indicating a correlation between PPI usage and susceptibility to infectious or autoimmune diseases, we studied the impact of a pharmacological concentration of omeprazole on human CD4+ T-cells. MethodsIn mixed lymphocyte reactions (MLRs), we analyzed the proliferation index and measured the concentration of key cytokines representative of distinct CD4+ T-cell subsets. In CD4+ T-cells isolated from the MLRs, we evaluated proliferation markers and pathways, the expression of signature transcription factors of CD4+ T-cell subsets, vacuolar H+- ATPase (V-ATPase) levels, and the activation status of AMP-activated kinase (AMPK) and mammalian target of rapamycin complex-1 (mTORC1). ResultsOmeprazole reduced proliferation index in MLRs, and in isolated CD4+ T-cells, it downregulated the proliferation marker Ki-67, possibly mediated by the p53- p21 pathway. Analysis of cytokines and signature transcription factors of CD4+ T-cell subsets indicated that omeprazole decreased T helper 1 (Th1) differentiation, had negligible impact on Th2 differentiation, increased Th17 differentiation, and reduced regulatory T-cell (Treg) differentiation. Omeprazole also decreased V-ATPase, a known target of PPIs and a site for AMPK and mTORC1 activation. Consequently, this led to diminished activation of these kinases, potentially elucidating the mechanism by which omeprazole influences CD4+ T-cell differentiation. ConclusionOmeprazole downregulates V-ATPase and inhibits activation of AMPK and mTORC1. As a result, omeprazole suppresses CD4+ T-cell clonal expansion, potentially contributing to the observed association between PPIs and susceptibility to infections. Additionally, it modulates CD4+ T-cell differentiation in a manner that favors autoimmunity.

Artesunate alleviates radiation-induced submandibular gland epithelial cell damage in rats by reducing inflammation and apoptosis.

Salivary hypofunction is a common complication in patients with head and neck cancers following radiotherapy (RT). RT-induced inflammation in salivary gland cells leads to apoptosis and fibrosis. Artesunate (ART) is a bioactive compound with anti-inflammatory and anti-fibrosis properties. This study aimed to investigate the protective effects of ART on X-ray-induced injury of submandibular gland (SMG) epithelial cells in rats. Second-generation SMG epithelial cells were randomly divided into five groups: natural control group (NC), irradiated group (IR), and irradiated groups treated with ART at concentrations of 5, 10, and 20 μM. Cells were harvested 48 h postirradiation for analysis. The results demonstrated that ART attenuated the damage to AQP5, a crucial indicator of salivary gland function, as evidenced by the decreased expression of AQP5 at both mRNA and protein levels. Additionally, ART decreased the expression of inflammatory cytokines: IL-6 and TNF-α. TUNEL staining revealed reduced apoptosis in the ART groups, particularly the IR + 10 μM group. RT-PCR and Western blot analysis of apoptosis cytokines Bax/Bcl-2 and Caspase-3 confirmed these findings. Furthermore, ART inhibited the expression of NF-κB at both mRNA and protein levels. In conclusion, these results suggest that ART may reduce inflammation and apoptosis in SMG epithelial cells following radiation by inhibiting the NF-κB pathway.

In vitro cyto- and geno-toxicity of asbestiform erionite from New Zealand

This work is an in vitro toxicity study of two asbestiform erionites from Kaipara and Gawler Downs in New Zealand. This study is the first, to the knowledge of the authors, to investigate the mechanisms that trigger adverse effects leading to carcinogenicity from New Zealand erionites. The effects induced by the erionite fibres from New Zealand were compared with those produced by positive (crocidolite) and negative (wollastonite) standards, and other erionite fibres described in the literature. The cytotoxicity/genotoxicity/inflammatory potential was determined by: (i) analysis of the cytotoxic potential by MTT tests on human cell lines mimicking primary cells making direct contact with fibres in the lungs, combined with apoptosis tests and cell membrane damage by fluorescence microscopy analyses; (ii) analysis of the genotoxic potential by quantification of DNA damage measuring double strand break foci by γ-H2AX nuclear staining in confocal microscopy analyses; (iii) analyses of the acute (24–72h) and early-chronic (7d) inflammatory effect by gene expression analyses of several cytokines, as well as of fibrotic and Epithelial to Mesenchymal transition (EMT) markers. The intensity of cell responses to these erionites are comparable to that of standard carcinogenic crocidolite, indicating that the two erionite fibres exhibit a significant acute toxic potential, with a particular alarming effect from the Gawler Downs sample from South Island. Our results confirm that the investigated erionites from New Zealand may represent an environmental hazard. However, further investigation is required to determine potential environmental exposure pathways by which erionite may become airborne and assess any environmental risks that may arise.

Contribution of IL-17C-mediated macrophage polarization to Type 17 inflammation in neutrophilic asthma

BackgroundIL-17C has been described in a variety of inflammatory diseases driven by neutrophils. However, the role of IL-17C in neutrophilic asthma has not been completely characterized.MethodsThe level of IL-17C in asthmatic patients and mice was assessed. Il-17c-deficient mice or mice treated with exogenous rmIL-17C were performed for OVA/CFA-induced asthmatic mice model. Pulmonary inflammation was evaluated by histological analysis, flow cytometry and cytokine analysis. Il-17re-overexpressed Raw264.7 were used in vitro to investigate the role of IL-17C in macrophage polarization.ResultsHere, we show IL-17C were increased in serum or plasma from asthmatic patients and OVA/CFA-induced asthma mice. In the OVA/CFA-induced model, exogenous rmIL-17C aggravated neutrophil- and Type 17-dominated inflammation and promoted M1 macrophage differentiation, whereas deficiency of Il-17c reversed the pro-inflammatory phenotypes and inhibited the expansion of M1 macrophages. In vitro, IL-17C in synergy with IFN-γ induced STAT1 activation in Il-17re overexpressed Raw264.7 to upregulate M1-related genes expression, and promoted pro-inflammatory M1 polymerization, whereas IL-17C in contrast to the effect of IL-4 inhibited STAT6 activation, to reduce Raw264.7 differentiation to M2 macrophage and functional M2-related genes expression.ConclusionsIL-17C promotes allergic inflammation via M1 polarization of pulmonary macrophages in neutrophilic asthma. Modulation of the IL-17C/IL-17RE axis represents a novel therapeutic target in neutrophilic asthma.

Templating of Monomeric Alpha-Synuclein Induces Inflammation and SNpc Dopamine Neuron Death in a Genetic Mouse Model of Synucleinopathy.

While the etiology of most cases of Parkinson's disease (PD) are idiopathic, it has been estimated that 5-10% of PD arise from known genetic mutations. The first mutations described that leads to the development of an autosomal dominant form of PD are in the SNCA gene that codes for the protein alpha-synuclein (α-syn). α-syn is an abundant presynaptic protein that is natively disordered and whose function is still unclear. In PD, α-syn misfolds into multimeric b-pleated sheets that aggregate in neurons (Lewy Bodies/neurites) and spread throughout the neuraxis in a pattern that aligns with disease progression. Here, using IHC, HC, HPLC, and cytokine analysis, we examined the sequelae of intraparenchymal brain seeding of pre-formed fibrils (PFFs) and monomeric α-syn in C57BL/6J (WT) and A53T SNCA mutant mice. We found that injection of PFFs, but not monomeric α-syn, into the striatum of C57BL/6J mice induced spread of aggregated α-syn, loss of SNpc DA neurons and increased neuroinflammation. However, in A53T SNCA mice, we found that both PFFs and monomeric α-syn induced this pathology. This suggests that the conformation changes in α-syn seen in the A53T strain can recruit wild-type α-syn to a pathological misfolded conformation which may provide a mechanism for the induction of PD in humans with SNCA duplication/triplication.

Real-world improvement in ultra-low dose thoracic CT scores, systemic inflammatory markers, and patients reported outcome measures post elexacaftor/tezacaftor/ivacaftor treatment

BackgroundClinical trials with elexacaftor/tezacaftor/ivacaftor (ETI) in people with cystic fibrosis (PWCF) were associated with significant improvements in percent predicted forced expiratory volume in one second (ppFEV1), sweat chloride, weight and quality of life in the respiratory domain from the cystic fibrosis questionnaire revised (CFQ-R). Limited data exists on its effect on structural lung disease and inflammatory cytokines.MethodsIn a real-world setting with 61 PWCF, we prospectively recorded FEV1, sweat chloride, body mass index (BMI), and CFQ-R at baseline, 3- and 6-months post commencement of ETI. In addition, changes in ultra-low dose computed tomography (ULD-CT) Bhalla score, peripheral-blood and sputum inflammatory-cytokines, and patient-reported outcome measures (PROMs) including sino-nasal outcomes test-22 (SNOT-22), and fatigue scale (FACIT-Fatigue).ResultsSignificant improvements in ppFEV1 (p=0.0001), sweat chloride (p<0.0001), and BMI (p=0.0147) post-ETI treatment were noted. ULD-CT scores demonstrated reductions in peri-bronchial thickening, mucus plugging, and total Bhalla score (p<0.001), and improvements in emphysema extent (p<0.0027). Improvements in systemic inflammatory status were seen with a reduction in interleukin (IL)-1β (p=0.0049), IL-6 and IL-8 (p<0.0001), and increasing IL-10 (p=0.004). Sputum cytokine analysis was not performed as only 4 of 61 patients spontaneously expectorated sputum post ETI. PROMs improved significantly for the SNOT-22 (p<0.0001), FACIT-Fatigue score (p=0.0001), and CFQ-R domains including; respiratory(p<0.0001), physical (p=0.007), vitality (p=0.0004), treatment burden (p=0.0028), health (p=0.0007), social (p=0.0073), weight (p=0.0068), and role/school domain (p=0.0018).ConclusionETI responders, demonstrate significant improvements in CT imaging, circulating cytokines and PROMs which may be of further use evaluating CFTR modulation treatment response.

Pharmacodynamic effects and exploratory efficacy of afimetoran, a TLR7/8 inhibitor, in patients with cutaneous lupus erythematosus

Background: Cutaneous lupus erythematosus (CLE) is a chronic inflammatory skin condition occurring alone or as a manifestation of systemic lupus erythematosus (SLE). There is an unmet need for safe and effective CLE-focused treatments. Afimetoran is an investigational, first-in-class, potent oral inhibitor of toll-like receptors (TLRs) 7/8. Given the involvement of TLRs 7/8 in SLE, afimetoran may have therapeutic potential for CLE. A phase 1b randomized, double-blind, placebo-controlled study (NCT04493541) demonstrated preliminary safety and efficacy of afimetoran in patients with active CLE. We assessed the pharmacodynamics, safety, and efficacy of afimetoran and its impact on CLE pathobiology. Methods: Patients aged 18–65 years with SLE and cutaneous manifestations by EULAR/ACR 2019 classification criteria or biopsy-proven CLE, and modified CLE Disease Area and Severity Index-Activity (CLASI-A) score ≥6, were randomized 2:1 to once-daily afimetoran (30 mg) or placebo for 16 weeks with a 4-week post-treatment follow-up period. The primary endpoints were safety and tolerability; efficacy was exploratory. Transcriptomics and cytokine analyses were performed using peripheral whole blood and serum, respectively, at baseline (pre-dose) and each study visit (weeks 1, 2, 4, 8, 12, 16, and 20 [post-dose]). Statistical analyses compared data from the 13 randomized patients with thirteen age-, ethnicity-, and gender-matched healthy volunteers. Treatment responses were evaluated longitudinally in each treatment arm. The dataset was analyzed using a linear regression model. Gene set variation analysis (GSVA) was performed for pathway enrichment. Results: 13 patients with CLE were randomized (afimetoran, n=8; placebo, n=5); 12 patients completed 16 weeks of treatment and 1 discontinued after 6 weeks due to COVID-19. Compared with placebo, afimetoran demonstrated a favorable safety profile with no serious adverse events, and showed a greater reduction in CLASI-A scores as early as week 4, which persisted to week 20. Improvement in the molecular disease profile was rapid (week 1), sustained through the 16-week treatment period, and the 4-week post-treatment follow-up period. Expression of interferon (IFN) pathway genes was significantly reduced with afimetoran compared with baseline (ΔGSVA enrichment score [ES]=1.57, P<0.0001) and placebo (ΔGSVA ES=0.56, P<0.01); this effect was maintained through week 16 and ≥4 weeks post-treatment. Expression of TLR7 and TLR8 pathway genes and key cytokines (interleukin [IL]-6, tumor necrosis factor α, IL-18, IFNγ, macrophage inflammatory protein-1 alpha [CCL3/MiP1α] or beta [CCL4/MiP1β]) were greatly reduced with afimetoran treatment. GSVA demonstrated robust pharmacodynamic activity on immune cell populations, including immature and activated dendritic cells, macrophages, and inflammatory pathway signatures. Conclusion: In patients with CLE, afimetoran was safe, well tolerated, and demonstrated durable efficacy beyond the treatment period. Afimetoran’s clinical effects and potent pharmacodynamic impact on the molecular disease profile suggest a substantial therapeutic benefit for patients with CLE.

Abstract C009: MK2 knockout and radiation enhances immune cell influx in a mouse model of HPV+ head and neck squamous cell carcinoma

Abstract Introduction: HPV mediated head and neck squamous cell carcinoma (HNSCC) has an immunosuppressive microenvironment which contributes to local and distant failures. We have previously shown that MAPKAPK2 (MK2) inhibition (MK2i) in HNSCC results in improved response to irradiation (RT). MK2i was synergistic with RT resulting in better control than monotherapy. To understand the mechanism behind the improvement of RT in MK2i treated mice we tested the hypothesis that MK2 ablation can enhance the radiotherapy mediated immunogenic response in a solid tumor. Methods: We used CRISPR gene editing to knockout the MK2 gene in the highly metastatic murine cell line, MLM3 (KO). We implanted tumors into c57bl6 and NSG mice and compared tumor growth compared to wild type (WT) with and without radiation. At end point, tumors were excised, digested and immunophenotyped utilizing labelled antibodies and flow cytometry. Before immunophenotyping, 8 mgs of tumor were cut off and incubated in media for 24 hours to assess cytokine release by cytokine array analysis. Results: We found that the differences in tumor growth between WT and KO cells in c57bl6 mice were significantly greater than those seen in NSG mice. Survival studies with c57bl6 mice implanted with WT or KO tumors with and without RT showed that MLM3 KO implanted mice had significantly greater survival (58.5 days) compared to WT (35.5 days). WT tumors treated with RT improved median overall survival with the WT + RT group surviving longer than WT (56 days) and the MK2 KO + RT had the longest survival overall (65 days). Immunophenotyping WT v KO tumors grown in c57bl6 mice showed an increase in CD45 cells in the KO tumors compared to WT. The cells that were increased in number in the KO tumors were labelled by Ly6G (neutrophils), F4/80 (macrophages), CD14+MD11c (dendritic cells), CD4 (Th), CD8 (Tc), CD3+NK1.1 (NKt) and NK1.1 (NK cells). Irradiating the tumors accentuated the influx of immune cells causing a greater influx of CD45 cells at 3 days post-RT which included increases in F4/80+CD80 (M1 macrophages), CD14 (monocytes), CD11c+MHCII, NK1.1, NK1.1+CD3 and CD4 cells. However, the influx of CD8 cells were reduced at 3 days. At 8 days post RT, many cell types remained elevated in the KO tumors compared to wildtype and there was a partial recovery of CD8 cell influx. However, there was also an increase in immune suppressor cells into the tumors including Ly6C+arginase (M-MDSCs), Ly6C+arginase (PMN-MDSCs) and CD4+T-bet (Th1) cells. The cytokine analysis showed that six cytokines were differentially secreted from tumors: Dkk-1, IL-2, IGFBP-5, IL-3, IL-17a and CD14. All these cytokines were suppressed in the KO with respect to the WT tumors. Conclusion: Here we show evidence that genetic ablation of MK2 in a mouse model of HPV mediated HNSCC results in an increase in immune cells infiltrate into tumors. Irradiation enhances this difference by stimulating a greater influx of immune cells. These data suggest that tumoral MK2 may aid in the immunosuppression of tumors in HNSCC. Citation Format: Deri Morgan, Colby Spiess, Alyssa Schmidt, Dakota D.D. Okuwone, Harmony Saunders, Chris E. Lominska, Mary A. Markiewicz, Yelder Tonia, Devin Shrock, Yuting Lin, Hao Gao, Gregory N Gan. MK2 knockout and radiation enhances immune cell influx in a mouse model of HPV+ head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C009.

Influence of photobiomodulation and radiofrequency on the healing of pressure lesions in mice.

The objective of this study was to ascertain the impact of photobiomodulation and radiofrequency on the healing of pressure injuries in mice. A total of 70 animals were randomly assigned to seven experimental groups. A pressure injury was induced in the dorsal region of the mice by the application of two magnets. The photobiomodulation treatment was administered at a dosage of 3.6J per session. In the radiofrequency group, the treatment time was four minutes and the power was 22 watts. The analyses included the lesion area, infrared thermography, and the collection of material for cytokine, histological, and histochemical analyses following euthanasia. In the macroscopic analyses, the 660nm photobiomodulation group demonstrated superior outcomes in comparison to the control group. With regard to the microscopic analyses, the greatest difference between the groups was observed when TNF-α was evaluated in the photobiomodulation group. It can be observed that the groups irradiated by electrophysical means (i.e., a combination of radiofrequency with PBM 830nm-660nm) exhibited a positive influence on the repair process, with the greatest impact observed in the group irradiated by a combination of radiofrequency and 660nm photobiomodulation.

Formulated chitosan microspheres remodelled the altered gut microbiota and liver miRNA in diet-induced Type-2 diabetic rats

Chitosan was formulated into a microsphere and comprehensively characterized and evaluated for its anti-inflammatory potential and anti-diabetic properties against the high sugar fat diet-induced diabetic animals. The diabetic model was induced through feeding with a high-sugar fat diet. Metformin, a standard antidiabetic drug, and CMS (chitosan microspheres) were administered orally for 90 days as reversal strategies. Upon completion of the study, the following parameters, such as serum biochemistry, cytokine analysis, tissue histology, liver miRNA sequencing, and Shotgun metagenomics studies from stool samples, were performed. SEM images of the microsphere indicated a smooth morphology, while FTIR and DSC respectively, confirmed the presence of functional groups of chitosan and the thermal stability of the formulation. Following HSFD induction, all the parameters analyzed were altered compared to the control group. In both reversal groups, serum biochemical parameters were restored, which was at par with the control. A significant increase in the anti-inflammatory cytokine IL-10, and a remarkable reduction in TNF-α and MCP-1 inflammatory cytokines were observed in both reversal groups. Tissue histology indicated improvements in low-grade inflammation, induced in the diabetic group. miR-203 was upregulated in the CMS-treated group, while miR-103 was downregulated. The study further delved into the impact on gut microbiota and KEGG. Major phyla i.e., Bacteroidetes, Cyanobacteria, Firmicutes, Proteobacteria, and Verrucomicrobia showed restoration, while upregulation of DNA polymerase zeta in T2D showed reversal after the treatment. The formulation showed reversal at par with metformin and also confirms its anti-diabetic and anti-inflammatory activities of CMS, with microfloral and miR regulatory functions.

HSP90 Complex From OLP Lesion Induces T-Cell Polarization via Activation of Dendritic Cells.

To investigate the effects of the heat shock protein 90 (HSP90) complex from oral lichen planus (OLP) lesion tissues on dendritic cell (DC) activation and the polarization of naïve T cells. Expression of HSP90 in OLP lesions and healthy control (HC) mucosa was evaluated by single-cell RNA sequence, IHC, qRT-PCR, and immunoblotting. HSP90 complex was extracted by immunoprecipitation from oral mucosa as the agonist of DCs. Expression of IFN-α and MHC was detected by flow cytometry. After cocultured with pre-stimulated DCs, polarization of naïve T cells was investigated by cytokine analysis. HSP90 was significantly higher in the lamina propria of OLP lesion and closely related to lymphocyte infiltration. HSP90 complex of OLP lesion activated DCs via TLR9 and increased their IFN-α secretion and MHC II expression. Pre-stimulated DCs increased the proportion of Th17 cells. HSP90 complex isolated from OLP lesion activated TLR9/IFN-α of DCs and further promoted the polarization of naïve T cells toward Th17 immunity.

Major alteration of lung microbiome and the host responses in critically ill COVID-19 patients with high viral load

Patients with COVID-19 under invasive mechanical ventilation are at higher risk of developing ventilator-associated pneumonia (VAP), associated with increased healthcare costs, and unfavorable prognosis. The underlying mechanisms of this phenomenon have not been thoroughly dissected. Therefore, this study attempted to bridge this gap by performing a lung microbiota analysis and evaluating the host immune responses that could drive the development of VAP. In this prospective cohort study, mechanically ventilated patients with confirmed SARS-CoV-2 infection were enrolled. Nasal swabs (NS), endotracheal aspirates (ETA), and blood samples were collected initially within 12 h of intubation and again at 72 h post-intubation. Plasma samples underwent cytokine and metabolomic analyses, while NS and ETA samples were sequenced for lung microbiome examination. The cohort was categorized based on the development of VAP. Data analysis was conducted using RStudio version 4.3.1. In a study of 36 COVID-19 patients on mechanical ventilation, significant differences were found in the nasal and pulmonary microbiome, notably in Staphylococcus and Enterobacteriaceae, linked to VAP. Patients with VAP showed a higher SARS-CoV-2 viral load in respiratory samples, elevated neutralizing antibodies, and reduced inflammatory cytokines, including IFN-δ, IL-1β, IL-12p70, IL-18, IL-6, TNF-α, and CCL4. Metabolomic analysis revealed changes in 22 metabolites in non-VAP patients and 27 in VAP patients, highlighting D-Maltose-Lactose, Histidinyl-Glycine, and various phosphatidylcholines, indicating a metabolic predisposition to VAP. This study reveals a critical link between respiratory microbiome alterations and ventilator-associated pneumonia in COVID-19 patients with higher SARS-CoV-2 viral loads in respiratory samples, elevated neutralizing antibodies, and reduced inflammatory cytokines, including IFN-δ, IL-1β, IL-12p70, IL-18, IL-6, TNF-α, and CCL4. These findings provide novel insights into the underlying mechanisms of VAP, with potential implications for management and prevention.

CTIM-02. PIONEERING QUAD-TARGETING CAR T CELL THERAPY IN PEDIATRIC CNS TUMORS – ANALYSIS FROM THE INITIAL PATIENTS TREATED ON THE FIRST-IN-HUMAN PHASE 1 TRIAL BRAINCHILD-04

Abstract BACKGROUND BrainChild-04 is a first-in-human clinical trial of quad-chimeric antigen receptor (CAR) T cell therapy for children and young adults with central nervous system (CNS) tumors. This trial administers repeated locoregional CAR T cells targeting B7-H3, EGFR, HER2, and IL-13Ralpha2 (“quad-CAR T cells”), leveraging multi-antigen targeting to address the tumor heterogeneity of high-grade CNS tumors. METHODS The primary endpoints are feasibility and safety, and secondary endpoints are disease response and correlative studies of CAR T cell activity. The trial utilized a Bayesian Optimal Interval (BOIN) statistical design with three planned dose regimens(DR). There are 2 arms with weekly delivery for 3 weeks of a 4-week cycle:(A) –diffuse intrinsic pontine glioma (DIPG), (B) –non-pontine diffuse midline glioma (DMG) or other relapsed CNS tumors. RESULTS Enrolled patients include DMG N=6, DIPG N=5, other CNS tumors N=4. Of the 15 enrolled patients, 7 have been infused with 8 awaiting infusions. A total of 33 intracranial CAR T doses have been delivered, including Arm A DR 1 (N=1), Arm B DR 1 (N=3), DR2 (N=3). The most common adverse events have been grade 1-2 fever (7/7 patients), grade 1-3 headache (6/7), and grade 1-2 nausea/vomiting (6/7). There have been no DLTs and no cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity (ICANS). CTCAE and tumor inflammation-associated neurotoxicity are both being captured. All treated patients are alive with median follow up time post-initial infusion of 93(14-198) days. CAR T cells were detected by flow cytometric analysis of CSF post-infusion in 14/24 CSF samples. Radiographic response, CSF circulating tumor DNA, targeted mass spectrometry, and cytokine analysis will be presented. CONCLUSIONS Our preliminary experience suggests tolerability of locoregional delivery of quad-CAR T cells at doses currently evaluated. The trial remains ongoing and these data support continued evaluation of this product to better assess anti-tumor activity in patients with CNS tumors, including DIPG/DMG.

IMMU-24. GLIOBLASTOMA ENRICHED GLYCOSPHINGOLIPIDS MODULATE THE FUNCTION OF HUMAN INKT CELLS

Abstract Glioblastoma is an aggressive primary brain tumor highly resistant to currently available immunotherapies. A deeper understanding of its underlying immunoregulatory mechanisms is paramount to developing future immunotherapeutic treatments. Invariant natural killer T (iNKT) cells are unconventional T cells that recognize lipid antigens presented by an MHC-like molecule called CD1d. Although iNKT cells have been shown to regulate tumor immunity in other cancer types, their role in glioblastoma is not well characterized. Given the lipid-rich nature of the brain and the unique metabolic activity of glioblastoma cells, we hypothesized that interactions between glioblastoma and iNKT cells through glioblastoma-produced lipids could contribute to the immunosuppressive nature of the disease. We report multiple lipid species enriched in aggressive human glioblastoma stem-like cell (GSC) lines that activate human iNKT cells and modulate their functions. Lipidomic LC-MS analysis of GSCs, low-grade glioma stem-like cell lines, and normal human astrocytes revealed various glycosphingolipid species, including sulfatides, highly enriched in the GSCs. Multiple enriched sulfatide species, when presented by CD1d, were recognized by and activated iNKT cells in a dose-dependent manner. Additionally, cytokine analysis of stimulated human PBMC-derived iNKT cells demonstrated that the enriched sulfatides did not induce Th1 cytokine production, but rather many non-Th1 cytokines that potentially counteract Th1-type immune responses. This modulation of iNKT cell function by glioblastoma-enriched glycosphingolipids may contribute to the immunosuppression of glioblastoma and could highlight sulfatide production as a potential therapeutic target for glioblastoma treatment.

Inflammatory stress determines the need for chemotherapy in patients with HER2-positive esophagogastric adenocarcinoma receiving targeted and immunotherapy.

Anti-PD-1, trastuzumab, and chemotherapy are used in the treatment of patients with advanced HER2-positive esophagogastric adenocarcinoma (EGA), but long-term survival remains limited. Herein, we report extended follow-up data from the INTEGA trial (NCT03409848), which investigated the efficacy of the anti-PD-1 nivolumab, trastuzumab, and FOLFOX chemotherapy (FOLFOX arm) in comparison to a chemotherapy-free regimen involving nivolumab, trastuzumab, and the anti-CTLA-4 ipilimumab (Ipi arm) in the first-line setting for advanced disease. The 12-month overall survival (OS) showed no statistical difference between the arms, with 57% OS (95% CI: 41%-71%) in the Ipi arm and 70% OS (95% CI: 54%-82%) in the FOLFOX arm. Crossing of the survival curves indicated a potential long-term benefit for some patients within the Ipi arm, but early progressors in the Ipi arm underlined the need for biomarker-guided strategies to optimize treatment selection. To this end, metabolomic and cytokine analysis demonstrated elevated levels of normetanephrine, cortisol, and interleukin 6 (IL-6) in immunotherapy-unresponsive patients in the Ipi arm, suggesting a role for systemic inflammatory stress in modulating antitumor immune responses. Patients with this signature also showed an increased neutrophil-to-lymphocyte ratio (NLR) that persisted in the Ipi arm, but not in the FOLFOX arm, and strongly correlated with survival. Furthermore, a low NLR characterized patients benefiting from immune- and targeted therapy without the need for additional chemotherapy. This data suggests that patient selection based on inflammatory stress-driven immune changes could help to customize first-line treatment in patients with advanced HER2-positive EGA to potentially improve long-term survival.