Plasma Cytokine Research Articles

Modulation of monocyte activity by hepatocellular MicroRNA delivery through HBsAg particles: Implications for pathobiology of chronic hepatitis B.

HBsAg serves as an important immune-modulatory factor in chronic hepatitis B. One aspect of such modulation may act through monocytes, which are the major Ag-presenting cells taking up HBsAg. There is evidence for the encapsulation of hepatocellular microRNAs (miRNAs) by HBsAg particles, while its pathobiological significance is unclear. Here, we characterized the miRNA profile in patients with chronic hepatitis B and probed their association with liver inflammation. We collected plasma from patients that are treatment-naive with chronic hepatitis B (n = 110) and quantified total/HBsAg-enveloped miRNAs by qRT-PCR and plasma cytokines by ELISA. The biological effects of HBsAg-delivered miRNAs in monocytes were evaluated using multiple approaches. The clinical significance of candidate miRNAs and cytokines was corroborated in patients with HBV-associated advanced liver diseases. The plasma miRNA profile showed 2 major clusters, one significantly associated with HBsAg titer and the other correlated with liver inflammation. Among HBsAg-carried miRNAs, miR-939 displayed the most significant correlation with IL-8. Mechanistically, miR-939 in subviral particles enters monocytes and significantly augments IL-8 production through the mitogen-activated protein kinase (MAPK) p38 signaling pathway. Finally, the findings that miR-939 positively correlated with IL-8 level and inflammation/fibrosis stage in the cohort of HBV-associated advanced liver diseases support its causative role in the progression of liver diseases. HBsAg particles carry hepatocellular miRNAs, including miR-939, which enter monocytes and alter their functional status, such as IL-8 secretion. Our findings demonstrate that the HBsAg-miR-939-IL-8 axis may play a crucial role in HBV-induced hepatic necro-inflammation and the progression of advanced liver diseases.

Liposomal sodium clodronate mitigates radiation-induced lung injury through macrophage depletion

Radiation-induced lung injury (RILI) is a severe complication arising from thoracic tumor radiotherapy, which constrains the possibility of increasing radiation dosage. Current RILI therapies provide only limited relief and may result in undesirable side effects. Therefore, there is an urgent demand for effective and low-toxicity treatments for RILI. Macrophages play a pivotal role in RILI, promoting inflammation in the initial stages and facilitating fibrosis in the later stages. Sodium clodronate, a bisphosphonate, can induce macrophage apoptosis when encapsulated in liposomes. In this study, we explored the potential of liposomal sodium clodronate (LC) as a specific agent for depleting macrophages to alleviate acute RILI. We assessed the impact of LC on macrophage consumption both in vitro and in vivo. In a mouse model of acute RILI, LC treatment group led to a reduction in alveolar macrophage counts, mitigated lung injury severity, and lowered levels of pro-inflammatory cytokines in both plasma and bronchoalveolar lavage fluid. Additionally, we further elucidated the specific effects and mechanism of LC on macrophages in vitro. Alveolar macrophages MHS cells were subjected to varying concentrations of LC (0, 50, 100, 200 μg/ml), and the results demonstrated its dose-dependent inhibition of cell proliferation and induction of apoptosis. Moreover, LC decreased the secretion of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Conditioned media from LC-treated macrophages protected alveolar epithelial cells MLE-12 from radiation-induced damage, as demonstrated by reduced apoptosis and DNA damage. These findings imply that LC-mediated macrophage depletion may present a promising therapeutic strategy for alleviating radiation-induced lung injury.

Abstract PO-003: Peripheral blood and tumor tissue biomarkers associated with epcoritamab response in patients with relapsed or refractory diffuse large B-cell lymphoma: Data from the dose-expansion cohort of the phase 1/2 EPCORE NHL-1 trial (NCT03625037)

Abstract Introduction: Epcoritamab, a CD3xCD20 bispecific antibody, demonstrated deep and durable clinical responses in patients (pts) with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL). We evaluated the correlation of peripheral blood (PB) and tumor tissue biomarkers with clinical response to epcoritamab. Methods: Pts received priming (0.16 mg), intermediate (0.8 mg) and full epcoritamab doses (48 mg). Absolute lymphocyte count (ALC) was analyzed locally. Blood immunophenotyping was assessed by flow cytometry, plasma cytokines by MSD assay, and %CD20+ in tumors by immunohistochemistry (n=117). Gene expression in baseline tumor biopsies was evaluated using HTG EdgeSeq (n=70). Microenvironment scores were estimated from gene expression as a sum of immune (B/T, NK, dendritic, and myeloid cells) and stromal scores (adipocytes, fibroblasts, and endothelial cells). Best overall response as of April 21, 2023 was assessed by independent review committee using Lugano criteria. P values were based on Wilcoxon rank-sum test. Results: In PB at baseline, significantly higher ALC (P=0.031), CD4+ (P=0.0038), and CD8+ (P=0.043) T-cell levels were observed in responders (R) vs nonresponders (NR). Similarly, CD4+ (P=0.02) and CD8+ (P=0.03) signatures and immune and microenvironment scores were higher in baseline tumor tissue in R vs NR. In PB CD8+ T cells, NR showed higher %PD1+ (P=0.016), %TIM3+ (P=0.048), and %TIM3+TIGIT+ (P=0.045). High tumor %CD20+ (≥90%) was observed in most pts (n=97/117); lower %CD20+ (<50%) was observed only in 6 NRs and 1 R. Epcoritamab treatment caused a rapid decline in circulating B cells irrespective of clinical response. Transient reductions in PB CD4+ and CD8+ T cells (ie, margination) were observed within 24 hours (H) after each dose during cycle (C) 1 and were more pronounced in R vs NR. Magnitude of margination for CD8+ T cells after the priming and intermediate doses was significantly lower in NR (reduced by median 22 & 41%) vs R (57 & 70%), while the difference in CD4+ T cells was not as pronounced in NR vs R, resulting in a substantially lower CD4+/CD8+ ratio in NR vs R. In C1, epcoritamab led to an increase of PB CD4+ and CD8+ T cells expressing activation/proliferation markers, with significantly higher fold change from baseline in R vs NR, especially within 24H post-priming dose. Early cytokine peaks of IFNy and IL-10 were more pronounced 6–24H post-priming dose and 6–14H post-first full dose in complete R vs pts with progressive disease. Epcoritamab also appeared to modulate NK cell numbers (under further investigation). Conclusions: In pts with R/R DLBCL, T-cell abundance and increased margination, activation, proliferation, and modulation of cytokines differed in R vs NR. These findings are consistent with epcoritamab’s mechanism of action and demonstrate the dependency on proper T-cell engagement for clinical benefit. Our findings also revealed differences in the dynamics of CD4+ and CD8+ T-cell margination in R vs NR, warranting further investigation. Previously submitted in part to EHA 2024. Citation Format: Monica Wielgos-Bonvallet, Jimin Zhang, Han Si, Kubra Karagoz, Melih Acar, Yasmin Karimi, Herve Ghesquieres, Michael Roost Clausen, Iş?l Alt?ntaş, Binhuan Wang, Wissam Assaily, Matthew Loya, Gregg Masters, Anantharaman Muthuswamy, Brandon Higgs, Mariana Sacchi, Tahi Ahmadi, Mark Fereshteh, Edith Szafer-Glusman, David Soong, Maria Jure-Kunkel, Andrew J Steele. Peripheral blood and tumor tissue biomarkers associated with epcoritamab response in patients with relapsed or refractory diffuse large B-cell lymphoma: Data from the dose-expansion cohort of the phase 1/2 EPCORE NHL-1 trial (NCT03625037) [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-003.

Analysis of transition metals in glioblastoma tumor and plasma samples.

e14029 Background: Transition metals such as copper, iron, zinc, and manganese are known to play essential roles in human physiology as evidenced in conditions where these metals are dysregulated. The role of transition metals in glioblastoma biology has been largely understudied. Here we investigated whether plasma and tumor transition metal content is associated with glioblastoma patient outcomes. Methods: Transition metal content was measured in 31 glioblastoma tumor samples, 21 of which had matched plasma samples, using inductively coupled plasma mass spectroscopy (ICP-MS) or inductively coupled plasma atomic emission spectroscopy (ICP-AES). Concentrations of transition metals in plasma and tumor were then then correlated with clinical parameters including median overall survival (mOS) as well as plasma cytokines. Results: Spearman correlation analysis of transition metals in glioblastoma tumor samples found that tumor manganese and tumor zinc (R = 0.52, p = 0.0048) as well as tumor copper and tumor zinc (R = 0.36, p = 0.038) were positively correlated. Plasma transition metal content did not correlate with tumor transition metal content. Kaplan-Meier and Cox regression survival analysis between high and low expressors of each respective transition metal, separated at the median, revealed that high plasma iron content was associated with prolonged survival (median overall survival: 30.1 vs 12.4 months, P = 0.0036). Plasma from glioblastoma patients in the high plasma iron group had increased levels of IFN-β (10.91 pg/mL vs 7.43 pg/mL, P = 0.004). Plasma or tumor concentrations of copper, zinc, or manganese did not associate with survival. Conclusions: Increased plasma iron content is associated with prolonged survival as well as increased plasma IFN-β levels, suggesting a possible link between iron and immune responses in glioblastoma patients.

Maternal immune activation with toll-like receptor 7 agonist during mid-gestation alters juvenile and adult developmental milestones and behavior.

Infections during pregnancy are associated with increased risk for adult neuropsychiatric disease, such as major depressive disorder, schizophrenia, and autism spectrum disorder. In mouse models of maternal immune activation (MIA), different toll-like receptors (TLRs) are stimulated to initiate inflammatory responses in mother and fetus. The goal of this study was to determine sex-dependent aspects of MIA using a TLR7/8 agonist, Resiquimod (RQ), on neurodevelopment. RQ was administered to timed-pregnant mice on embryonic day (E) 12.5. At E15, maternal/fetal plasma cytokines were measured by enzyme-linked immunosorbent assay (ELISA). Maternal cytokines interleukin (IL)-6 and IL-10 were higher while tumor necrosis factor (TNF)-α and IL-17 were lower in pregnant dams exposed to RQ. Fetal cytokines (E15) were altered at the same timepoint with fetal plasma IL-6 and IL-17 greater after RQ compared to vehicle, while IL-10 and TNF-α were higher in male fetuses but not female. Other timed-pregnant dams were allowed to give birth. MIA with RQ did not alter the female to male ratio of offspring born per litter. Body weights were reduced significantly in both sexes at birth, and over the next 5 weeks. Offspring from RQ-injected mothers opened their eyes 5 days later than controls. Similarly, female offspring from RQ-injected mothers exhibited pubertal delay based on vaginal opening 2-3 days later than control females. On the behavioral side, juvenile and adult male and female MIA offspring exhibited less social-like behavior in a social interaction test. Anhedonia-like behavior was greater in MIA adult female mice. This study provides support for sex-dependent influences of fetal antecedents for altered brain development and behavioral outputs that could be indicative of increased susceptibility for adult disorders through immune mechanisms. Future studies are needed to determine neural cellular and molecular mechanisms for such programming effects.

Genetic variants in UNC93B1 predispose to childhood-onset systemic lupus erythematosus

Rare genetic variants in toll-like receptor 7 (TLR7) are known to cause lupus in humans and mice. UNC93B1 is a transmembrane protein that regulates TLR7 localization into endosomes. In the present study, we identify two new variants in UNC93B1 (T314A, located proximally to the TLR7 transmembrane domain, and V117L) in a cohort of east Asian patients with childhood-onset systemic lupus erythematosus. The V117L variant was associated with increased expression of type I interferons and NF-κB-dependent cytokines in patient plasma and immortalized B cells. THP-1 cells expressing the variant UNC93B1 alleles exhibited exaggerated responses to stimulation of TLR7/-8, but not TLR3 or TLR9, which could be inhibited by targeting the downstream signaling molecules, IRAK1/-4. Heterozygous mice expressing the orthologous Unc93b1V117L variant developed a spontaneous lupus-like disease that was more severe in homozygotes and again hyperresponsive to TLR7 stimulation. Together, this work formally identifies genetic variants in UNC93B1 that can predispose to childhood-onset systemic lupus erythematosus.

Correlation of distinct circulating cytokine/chemokine profiles with clinical benefits of Pexa-Vec (thymidine kinase-deactivated vaccinia virus plus GM-CSF) and cemiplimab (REGN2810; anti-PD-1) in metastatic or unresectable renal cell carcinoma (mRCC).

e14539 Background: This study explores the transformation of 'cold' tumour microenvironments into 'hot' ones in mRCC, focusing on the potential of Oncolytic Viruses (OVs) for personalized immunotherapy. Recognizing the variability in patient responses, the research underscores the necessity for reliable biomarkers. The objective is to examine the correlation between plasma cytokine/chemokine levels and the efficacy of Pexa-Vec and cemiplimab, proposing that these biomarkers could predict treatment outcomes and contribute to more personalized cancer treatment protocols. Methods: In this study, mRCC patients from the REN026 trial were categorized into three treatment groups: Pexa-Vec (Intratumoral and intravenous) and cemiplimab combination therapy (N=65), intravenous Pexa-Vec and cemiplimab combination therapy (N=54), and cemiplimab monotherapy (N=14). Blood samples were collected at baseline, during treatment, and at end of treatment (EOT) for analysis. Subsequently, the plasma samples underwent analysis using the Human Cytokine 96-Plex Discovery Assay through the Luminex 200 system. The statistical approach included the Mann–Whitney U-test or Wilcoxon signed-rank test, along with a Cox proportional hazards model. The False Discovery Rate (FDR) method was employed to control type I error. Results: During the follow-up period from June 2018 to February 2023, key outcomes were observed as follows (Table): In the Pexa-Vec and cemiplimab cohort, a lower baseline of MCP-3 or an increased rate of change in post-treatment levels of IFNβ, Eotaxin, IL-1α, and sFasL were associated with improved Progression-Free Survival (PFS). Specifically, a low baseline of MCP-3 or an increased level of Eotaxin and sFasL post-treatment correlated with enhanced Overall Survival (OS). In the intravenous Pexa-Vec and cemiplimab group, a rise in post-treatment levels of IFNβ, Eotaxin, IL-17F, and sFasL were indicative of better PFS, with elevated Eotaxin also suggesting improved OS. Within the cemiplimab monotherapy group, an increase in post-treatment IP-10 was linked to favourable PFS. Conclusions: Our findings suggest the potential utility of plasma cytokine and chemokine profiles in clinical benefits of mRCC patient to the combination of Pexa-Vec and cemiplimab, thereby aiding in future personalized treatment approaches.

Effects of parity and week after calving on the metabolic, redox, and immune status of dairy cows

At the onset of lactation in dairy cows, inflammation and oxidative stress may occur and result in a risk of pathologies and lower milk yield. To propose an innovative management strategy for cows during this period, it is essential to better understand these physiological variations. Our objective was to evaluate the metabolic, redox and immune status of 7 primiparous and 8 multiparous Holstein cows during late gestation and the first months of lactation. Blood samples were collected between 3 weeks before calving until 12 weeks postpartum. Milk samples were also collected, but only at the time points after calving. The metabolic (nonesterified fatty acids (NEFA), BHB, glucose, urea, calcium) and redox (reactive oxygen metabolites (ROM), oxidative stress index (OSI), glutathione peroxidase activity, vitamin E) statuses were analyzed in plasma or erythrocytes. The expression of genes related to antioxidant functions was determined in leukocytes collected from milk. For immune status, plasma cytokine levels and the production of reactive oxygen species (ROS) in classical and regulatory neutrophils were measured in 2 whole blood ex vivo challenges. The data were analyzed using a mixed model that included the fixed effects of parity and week and their interaction. Milk yield, plasma NEFA and BHB in wk 2 and 4 after calving were higher in multiparous cows than in primiparous cows, whereas glucose and calcium tended to be lower. Plasma ROM and OSI levels in wk 8 were higher in multiparous than in primiparous cows. Multiparous cows also displayed higher glutathione peroxidase activity in erythrocytes, and antioxidant transcription factor and superoxide dismutase-1 expression levels in milk leukocytes. Moreover, multiparous cows had higher plasma concentrations of vitamin E but lower plasma levels of cytokines CXCL10, CCL2, IL1Rα and IFNγ. Following ex vivo whole blood stimulation with Escherichia coli, lower IL1α and TNFα levels were measured in multiparous than in primiparous cows. Intracellular ROS production by neutrophils was lower in multiparous than in primiparous cows. These results thus indicated marked physiological changes in wk 8 compared with wk 2 and 4 of lactation. These differences in the physiological status of primiparous and multiparous cows offer interesting perspectives for potential dietary strategies to prevent pathologies which take account of parity and week relative to calving.

ROLE OF CYTOKINES IN PRE-ECLAMPSIA: A REVIEW

Preeclampsia is a serious and life-threatening pregnancy complication. Reduced uteroplacental perfusion and oxygen tension impaired trophoblastic differentiation and invasion altered placental production of immunomodulators and growth factors are all considered important aspects of the aetiology of the condition. The placenta expresses a variety of pro and anti-inflammatory cytokines, adipokines and cytokine-like angiogenic growth factors, production of which is altered in preeclampsia, driven (at least in part) by hypoxia. Altered levels of cytokines have been measured in the circulation of women with preeclampsia. While the placenta undoubtedly makes an important contribution to plasma cytokine levels, production by maternal peripheral blood mononuclear cells (PBMCs) and other tissues is also likely to be significant, although to what extent remains undetermined. Increased placental expression of soluble receptors occurs with preeclampsia, resulting in elevated circulating concentrations which confer impaired angiogenesis, deficient placental vascularisation, increased placental apoptosis and endothelial dysfunction. The extent to which these changes reflect a response to the disorder, as opposed to being a causative factor in the development of maternal disease is a matter of some debate. Nevertheless, convincing evidence is now accruing that autocrine/paracrine interactions between placental cytokines/growth factors and the maternal endothelium plays a central role in the pathogenesis of preeclampsia.

Limosilactobacillus reuteri DSM 17938 relieves inflammation, endoplasmic reticulum stress, and autophagy in hippocampus of western diet-fed rats by modulation of systemic inflammation.

The consumption of western diets, high in fats and sugars, is a crucial contributor to brain molecular alterations, cognitive dysfunction and neurodegenerative diseases. Therefore, a mandatory challenge is the individuation of strategies capable of preventing diet-induced impairment of brain physiology. A promising strategy might consist in the administration of probiotics that are known to influence brain function via the gut-brain axis. In this study, we explored whether Limosilactobacillus reuteri DSM 17938 (L. reuteri)-based approach can counteract diet-induced neuroinflammation, endoplasmic reticulum stress (ERS), and autophagy in hippocampus, an area involved in learning and memory, in rat fed a high fat and fructose diet. The western diet induced a microbiota reshaping, but L. reuteri neither modulated this change, nor the plasma levels of short-chain fatty acids. Interestingly, pro-inflammatory signaling pathway activation (increased NFkB phosphorylation, raised amounts of toll-like receptor-4, tumor necrosis factor-alpha, interleukin-6, GFAP, and Haptoglobin), as well as activation of ERS (increased PERK and eif2α phosphorylation, higher C/EBP-homologous protein amounts) and autophagy (increased beclin, P62-sequestosome-1, and LC3 II) was revealed in hippocampus of western diet fed rats. All these hippocampal alterations were prevented by L. reuteri administration, showing for the first time a neuroprotective role of this specific probiotic strain, mainly attributable to its ability to regulate western diet-induced metabolic endotoxemia and systemic inflammation, as decreased levels of lipopolysaccharide, plasma cytokines, and adipokines were also found. Therapeutic strategies based on the use of L. reuteri DSM17938 could be beneficial in reversing metabolic syndrome-mediated brain dysfunction and cognitive decline.

Amiloride lowers plasma TNF and interleukin-6 but not interleukin-17A in patients with hypertension and type 2 diabetes.

Interleukin (IL)-17A contributes to hypertension in preclinical models. T helper 17 and dendritic cells are activated by NaCl, which could involve the epithelial Na+ channel (ENaC). We hypothesized that the ENaC blocker amiloride reduces plasma IL-17A and related cytokines in patients with hypertension. Concentrations of IL-17A, IFN-γ, TNF, IL-6, IL-1β, and IL-10 were determined by immunoassays in plasma from two patient cohorts before and after amiloride treatment: 1) patients with type 2 diabetes mellitus (T2DM) and treatment-resistant hypertension (n = 69, amiloride 5-10 mg/day for 8 wk) and 2) patients with hypertension and type 1 diabetes mellitus (T1DM) (n = 29) on standardized salt intake (amiloride 20-40 mg/day, 2 days). Plasma and tissue from ANG II-hypertensive mice with T1DM treated with amiloride (2 mg/kg/day, 4 days) were analyzed. The effect of amiloride and benzamil on macrophage cytokines was determined in vitro. Plasma cytokines showed higher concentrations (IL-17A ∼40-fold) in patients with T2DM compared with T1DM. In patients with T2DM, amiloride had no effect on IL-17A but lowered TNF and IL-6. In patients with T1DM, amiloride had no effect on IL-17A but increased TNF. In both cohorts, blood pressure decline and plasma K+ increase did not relate to plasma cytokine changes. In mice, amiloride exerted no effect on IL-17A in the plasma, kidney, aorta, or left cardiac ventricle but increased TNF in cardiac and kidney tissues. In lipopolysaccharide-stimulated human THP-1 macrophages, amiloride and benzamil (from 1 nmol/L) decreased TNF, IL-6, IL-10, and IL-1β. In conclusion, inhibition of ENaC by amiloride reduces proinflammatory cytokines TNF and IL-6 but not IL-17A in patients with T2DM, potentially by a direct action on macrophages.NEW & NOTEWORTHY ENaC activity may contribute to macrophage-derived cytokine release, since amiloride exerts anti-inflammatory effects by suppression of TNF and IL-6 cytokines in patients with resistant hypertension and type 2 diabetes and in THP-1-derived macrophages in vitro.

Direct assessment of leukocyte signalling and cytokine secretion reveals exercise intensity-dependent reductions in anti-inflammatory cytokine action.

Circulating interleukin (IL)-6 and IL-10 concentrations are widely used to evaluate the anti-inflammatory effects of exercise but do not capture cytokine action at the cellular level. Whether and how acute exercise impacts anti-inflammatory cytokine action in humans is unknown. To determine how exercise intensity and pattern impact IL-6 and IL-10 action in blood leukocytes, 16 active adults (eight males/eight females; age: 30±3years; body mass index: 22.8±2.3kg/m2; : 51±6mL/kg/min) completed a no-exercise control condition (CTL) or isocaloric bouts of cycling performed below (moderate continuous exercise; MCE) or above (heavy continuous or heavy intermittent exercise; HCE or HIE, respectively) lactate threshold. Venous blood (before, after, 30min after and 90min after exercise) was analysed for immune cell subpopulations, plasma cytokine concentrations, anti-inflammatory cytokine action and monocyte phenotype. Exercise induced rapid leukocytosis (P<0.001) and increased plasma IL-6 (P<0.001), IL-10 (P=0.0145) and tumour necrosis factor-⍺ (TNF-⍺) (P=0.0338) concentrations in an intensity-dependent manner (HCE and/or HIE vs. CTL). These systemic changes coincided with a diminished ability of IL-10/6 to phosphorylate STAT3 (P<0.001) and inhibit TNF-⍺ secretion (P=0.0238) in blood leukocytes following HCE and HIE. Monocyte polarization experiments revealed lower CD80 [MCE (P=0.0933) and HIE (P=0.0187) vs. CTL] and a tendency for higher CD163 expression (HCE vs. CTL, P=0.0985), suggesting that hyporesponsiveness to anti-inflammatory cytokine action does not impede the ability of exercise to promote an anti-inflammatory monocyte phenotype. These findings provide novel insights into the immunomodulatory effects of exercise in humans and highlight the importance of directly measuring cellular cytokine action when evaluating the anti-inflammatory effects of exercise. KEY POINTS: Circulating cytokine concentrations are frequently used to evaluate the anti-inflammatory effects of exercise but may not capture changes in cytokine action occurring at the cellular level. We directly assessed anti-inflammatory cytokine action - measured using a combination of intracellular signalling and cytokine secretion ex vivo - in distinct immune cell subpopulations after acute calorie-matched exercise bouts differing in intensity and pattern. Anti-inflammatory cytokine action was blunted following higher intensity exercise despite corresponding increases in circulating cytokine concentrations and immune cell counts. Changes in cytokine action were not explained by changes in cytokine receptor expression on circulating immune cells. Our findings provide new insights into the immunomodulatory effects of exercise in humans and highlight the importance of directly measuring cellular cytokine action when evaluating the anti-inflammatory effects of exercise.

Phase 1 dose expansion and biomarker study assessing first-in-class tumor microenvironment modulator VT1021 in patients with advanced solid tumors

BackgroundPreclinical studies have demonstrated that VT1021, a first-in-class therapeutic agent, inhibits tumor growth via stimulation of thrombospondin-1 (TSP-1) and reprograms the tumor microenvironment. We recently reported data from the dose escalation part of a phase I study of VT1021 in solid tumors. Here, we report findings from the dose expansion phase of the same study.MethodsWe analyzed the safety and tolerability, clinical response, and biomarker profile of VT1021 in the expansion portion of the phase I study (NCT03364400). Safety/tolerability is determined by adverse events related to the treatment. Clinical response is determined by RECIST v1.1 and iRECIST. Biomarkers are measured by multiplexed ion beam imaging and enzyme-linked immunoassay (ELISA).ResultsFirst, we report the safety and tolerability data as the primary outcome of this study. Adverse events (AE) suspected to be related to the study treatment (RTEAEs) are mostly grade 1–2. There are no grade 4 or 5 adverse events. VT1021 is safe and well tolerated in patients with solid tumors in this study. We report clinical responses as a secondary efficacy outcome. VT1021 demonstrates promising single-agent clinical activity in recurrent GBM (rGBM) in this study. Among 22 patients with rGBM, the overall disease control rate (DCR) is 45% (95% confidence interval, 0.24-0.67). Finally, we report the exploratory outcomes of this study. We show the clinical confirmation of TSP-1 induction and TME remodeling by VT1021. Our biomarker analysis identifies several plasmatic cytokines as potential biomarkers for future clinical studies.ConclusionsVT1021 is safe and well-tolerated in patients with solid tumors in a phase I expansion study. VT1021 has advanced to a phase II/III clinical study in glioblastoma (NCT03970447).

Anti-neurofascin-155 antibody mediated a distinct phenotype of chronic inflammatory demyelinating polyradiculoneuropathy.

To investigate Ranvier's autoantibodies prevalence and isotypes in various peripheral neuropathy variants, compare clinical features between seronegative and seropositive patients, and elucidate immune mechanisms underlying antibody generation. Antibodies against anti-neurofascin-155 (NF155), NF186, contactin-1 (CNTN1), CNTN2, contactin-associated protein 1 (CASPR1), and CASPR2 were identified through cell-based assays. Plasma cytokines were analyzed in anti-NF155 antibody-positive chronic inflammatory demyelinating polyneuropathy (NF155+ CIDP) and Ranvier's antibodies-negative CIDP (Ab- CIDP) patients using a multiplexed fluorescent immunoassay, validated in vitro in a cell culture model. In 368 plasma samples, 50 Ranvier's autoantibodies were found in 45 individuals, primarily in CIDP cases (25 out of 69 patients) and in 10 out of 122 Guillain-Barré syndrome patients. Anti-NF155 and CNTN1-IgG were exclusive to CIDP. Fourteen samples were NF155-IgG, primarily IgG4 subclass, linked to CIDP features including early onset, tremor, sensory disturbance, elevated CSF protein, prolonged motor latency, conduction block, and poor treatment response. NF155-IgG had low sensitivity (20.28%) but high specificity (100%) for CIDP, rising to 88.88% with tremor and prolonged motor latency. Cytokine profiling in NF155+ CIDP revealed distinct immune responses involving helper T cells, toll-like receptor pathways. Some NF155+ CIDP patients had circulating NF155-specific B cells producing NF155-IgG without antigen presence, suggesting therapeutic potential. The study emphasizes the high specificity and sensitivity of NF155-IgG for diagnosing CIDP characterized by distinctive features. Further investigation into circulating NF155-specific B cell phenotypes may pave the way for B cell directed therapy.

Identification of plasma and urinary inflammatory markers in severe knee osteoarthritis: Relations with synovial fluid markers

BackgroundThis study aimed to identify plasma and urinary cytokines as potential biomarkers for severe knee osteoarthritis (OA). It also investigated associations between these cytokines and cartilage markers, as well as their connections with synovial fluid (SF) markers.MethodsSamples of plasma, urine, and SF were obtained from patients (n = 40) undergoing total knee arthroplasty (TKA) or unicompartmental knee arthroplasty (UKA) due to severe knee OA. Control samples of plasma and urine were collected from non-OA individuals (n = 15). We used a Luminex immunoassay for the simultaneous measurement of 19 cytokines, MMP-1, and MMP-3 levels. COMP, CTX-II, and hyaluronan (HA) levels were quantified using enzyme-linked immunosorbent assay (ELISA) kits. Receiver operating characteristic (ROC) curves were utilized to analyze each biomarker’s performance. Correlations among these biomarkers were evaluated via Spearman’s correlation.ResultsThe levels of plasma (p)CCL11, pCXCL16, pIL-8, pIL-15, pHA, urinary (u)CCL2, uCCL11, uCCL19, uCXCL16, uIL-1β, uIL-6, uIL-8, uIL-12p70, uIL-15, uIL-33, uMMP-3, uHA, uCTX-II, and uCOMP were significantly elevated in individuals with severe knee OA. Notably, specific correlations were observed between the plasma/urine biomarkers and SF biomarkers: pCCL11 with sfHA (r = 0.56) and sfTNF-α (r = 0.58), pIL-15 with sfCCL19 (r = 0.43) and sfCCL20 (r = 0.44), and uCCL19 with sfCCL11 (r = 0.45) and sfIL-33 (r = 0.51). Positive correlations were also observed between uCCL11 and its corresponding sfCCL11(r = 0.49), as well as between sfCCL11 and other cytokines, namely sfCCL4, sfCCL19, sfCCL20, sfIL-33, and sfTNF-α (r = 0.46–0.63).ConclusionThis study provides an extensive profile of systemic inflammatory mediators in plasma of knee OA and identified four inflammatory markers (pCCL11, pIL-15, uCCL11, and uCCL19) reflecting joint inflammation.

Exercise does not cause post-exertional malaise in Veterans with Gulf War Illness: A randomized, controlled, dose–response, crossover study

Chronic multisymptom illnesses (CMI) such as Myalgic Encephalomyelitis/Chronic Fatigue Syndrome, Long-COVID, and Gulf War Illness (GWI) are associated with an elevated risk of post-exertional malaise (PEM), an acute exacerbation of symptoms and other related outcomes following exercise. These individuals may benefit from personalized exercise prescriptions which prioritize risk minimization, necessitating a better understanding of dose–response effects of exercise intensity on PEM. MethodsVeterans with GWI (n = 40) completed a randomized controlled crossover experiment comparing 20 min of seated rest to light-, moderate-, and vigorous-intensity cycling conditions over four separate study visits. Symptoms, pain sensitivity, cognitive performance, inflammatory markers (C-reactive protein and plasma cytokines) were measured before and within 1 h after exercise and seated rest. Physical activity behavior was measured ≥ 7 days following each study visit via actigraphy. Linear mixed effects regression models tested the central hypothesis that higher intensity exercise would elicit greater exacerbation of negative outcomes, as indicated by a significant condition-by-time interaction for symptom, pain sensitivity, cognitive performance, and inflammatory marker models and a significant main effect of condition for physical activity models. ResultsSignificant condition-by-time interactions were not observed for primary or secondary measures of symptoms, pain sensitivity, cognitive performance, and a majority of inflammatory markers. Similarly, a significant effect of condition was not observed for primary or secondary measures of physical activity. ConclusionsUndesirable effects such as symptom exacerbation were observed for some participants, but the group-level risk of PEM following light-, moderate-, or vigorous-intensity exercise was no greater than seated rest. These findings challenge several prior views about PEM and lend support to a broader body of literature showing that the benefits of exercise outweigh the risks.

Abstract IA008: Dissecting innate immune mechanisms of resistance to immune checkpoint blockade therapy in bladder cancer

Abstract We have explored the TME of bladder cancer (BC) leveraging single-cell RNA sequencing (scRNAseq) and clinical trial data to ascertain the complexity of tumor-infiltrated immune ecllls in BC. As a first step we identify distinct tumor-associated macrophage (TAM) populations associated with immune checkpoint blockade (ICB) response and resistance. Through plasma cytokine profiling, in vitro assays, and spatial analyses, we uncover novel cytokine-driven axes influencing these TAM populations and their impact on T cells, offering insights into enhancing anti-tumor T cell immunity in BC. Second, we investigate the potential of urine-derived cells (UDCs) as surrogates for monitoring the BC TME. Our scRNAseq atlas of BC patient UDCs reveals a diverse composition of immune cell populations, strikingly transcriptionally similar to tumor immune cells and containing immune subsets absent or rare in peripheral blood, suggesting their utility as biomarkers for response to therapeutic intervention. Collectively, these findings provide new insights into the immune landscape of BC and provide putative targets to enhance existing immunotherapy strategies in BC. Citation Format: Nina Bhardwaj. Dissecting innate immune mechanisms of resistance to immune checkpoint blockade therapy in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr IA008.

Effects of esketamine and fluoxetine on depression-like behaviors in chronic variable stress: a role of plasma inflammatory factors.

Mounting evidence has identified the rapid and sustained antidepressive and anxiolytic-like effects of esketamine. However, the underlying mechanism of this no-monoamine target rapid-onset antidepressant is still underexplored. Immune-inflammatory pathways and cell-mediated immune activation, mainly including inflammatory cytokines in plasma, play a pivotal role in the pathogenesis of major depressive disorder and are also a potential therapeutic target for MDD. The current study was designed to clarify the role of esketamine on the expression of plasma cytokines in a depressive-like model introduced by chronic variable stress (CVS). In this study, a 21-day consecutive CVS protocol was applied to produce depressive- and anxiety-like behaviors. After the single dose or 7-day repeated administration of esketamine or fluoxetine, the depressive- and anxiety-like behaviors and the expression of inflammatory cytokines in plasma were examined. Both a single dose of esketamine and 7-days repeated fluoxetine administration elicited anti-depressive and anxiolytic effects in mice exposed to CVS. Additionally, CVS produced significant changes in the plasma inflammatory factors, notably increasing the expression of IL-1β, IL-6, IL-8, IL-17A, TNFα, IL-4, IL-9, IL-24, IL-37, IFN-β, and CXCL12, while reducing IL-10 and IL-33. With the administration of esketamine and fluoxetine, CVS-produced inflammatory disturbances were partially normalized. Together, our findings provide a novel insight that acute esketamine treatment could rescue CVS-produced depressive-like and anxiety-like behaviors in mice by normalizing the expression of inflammatory cytokines; this effect was similar to the repeated administration of fluoxetine. These results contributed to the understating of rapid anti-depressant effects elicited by esketamine.

Phase II trial of vaccination with autologous, irradiated melanoma cells engineered by adenoviral mediated gene transfer to secrete granulocyte-macrophage colony stimulating factor in patients with stage III and IV melanoma.

In the era of immune checkpoint blockade, the role of cancer vaccines in immune priming has provided additional potential for therapeutic improvements. Prior studies have demonstrated delayed type hypersensitivity and anti-tumor immunity with vaccines engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF). The safety, efficacy and anti-tumor immunity of GM-CSF secreting vaccine in patients with previously treated stage III or IV melanoma needs further investigation. In this phase II trial, excised lymph node metastases were processed to single cells, transduced with an adenoviral vector encoding GM-CSF, irradiated, and cryopreserved. Individual vaccines were composed of 1x106, 4x106, or 1x107 tumor cells, and were injected intradermally and subcutaneously at weekly and biweekly intervals. The primary endpoints were feasibility of producing vaccine in stage III patients and determining the proportion of patients alive at two years in stage IV patients. GM-CSF vaccine was successfully developed and administered in all 61 patients. Toxicities were restricted to grade 1-2 local skin reactions. The median OS for stage III patients (n = 20) was 71.1 (95% CI, 43.7 to NR) months and 14.9 (95%CI, 12.1 to 39.7) months for stage IV patients. The median PFS in stage III patients was 50.7 (95%CI, 36.3 to NR) months and 4.1 (95% CI, 3.0-6.3) months in stage IV patients. In the overall population, the disease control rate was 39.3% (95%CI, 27.1 to 52.7%). In stage III patients, higher pre-treatment plasma cytokine levels of MMP-1, TRAIL, CXCL-11, CXCL-13 were associated with improved PFS (p<0.05 for all). An increase in post-vaccination levels of IL-15 and TRAIL for stage III patients was associated with improved PFS (p=0.03 for both). Similarly, an increase in post-vaccination IL-16 level for stage IV patients was associated with improved PFS (p=0.02) and clinical benefit. Vaccination with autologous melanoma cells secreting GM-CSF augments antitumor immunity in stage III and IV patients with melanoma, is safe, and demonstrates disease control. Luminex data suggests that changes in inflammatory cytokines and immune cell infiltration promote tumor antigen presentation and subsequent tumor cell destruction. Additional investigation to administer this vaccine in combination with immune checkpoint inhibitors is needed.

Feasibility of hemoperfusion using extracorporeal therapy in the horse.

Develop, implement, and monitor for adverse effects of, a novel hemoperfusion therapy in adult horses. A prospective, observational feasibility study using three healthy adult horses from the North Carolina State University teaching herd. Health status was determined by physical exam, complete blood count, coagulation panel, and serum biochemistry. Each horse was instrumented with a 14 Fr × 25 cm double-lumen temporary hemodialysis catheter and underwent a 240 min polymer-based hemoperfusion session. Horses were administered unfractionated heparin to maintain anti-coagulation during the session. Given the novelty of this therapy in horses, each horse was treated as a learning opportunity that informed an iterative process of protocol development and modification. Our long-term goal is to investigate potential clinical applications of hemoperfusion in horses, including cytokine reduction in horses with severe SIRS/sepsis. Horses were monitored for changes in clinical exam, biochemistry and hematology parameters. Additionally, cytokines were quantified to determine whether extracorporeal hemadsorption therapy alone caused an inflammatory response. Our results show that hemoperfusion therapy was associated with decreased platelet counts and serum albumin concentration. There was no significant change in plasma cytokine concentrations with hemoperfusion therapy. In one horse, the cytokine concentrations decreased, as previously reported with hemoperfusion therapy in humans. We hypothesized that hemoperfusion therapy could be performed in healthy adult horses without significant adverse effects. Polymer-based hemoperfusion is a feasible extracorporeal therapy (ECT) modality for adult horses. Additional studies are needed to further establish clinical protocols, as well as establish efficacy of polymer-based hemoperfusion for treatment of various conditions in horses, including intoxications, immune-mediated conditions, and sepsis.