Cytokine Measurements Research Articles

Precise diagnosis and treatment of hemophagocytic lymphohistiocytosis guided by cytokine patterns

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening entity which presents as multi-organ dysfunction caused by overactivated T cell and macrophage and overwhelming hyperinflammation. Cytokine storm is the key pathophysiology of HLH. Due to the diversity of the underlying diseases, the difference of disease severity and the non-specific clinical manifestation, the early diagnosis and standardized treatment is a great challenge for clinicians. According to previous clinical studies, serum cytokine measurement is helpful for rapid diagnosis of HLH and the differentiation of HLH from other diseases caused by hypercytokinemia, and it perfoms well to clarify subtypes of HLH, to stratify patients with different risks and to predict the prognosis and outcome. Thus, serum cytokine measurement could be a useful tool for the precise diagnosis and treatment of HLH.

Rapid improvement in severe long COVID following perispinal etanercept

Background This study aimed to describe the neurological improvements in a patient with severe long COVID brain dysfunction following perispinal etanercept administration. Perispinal administration of etanercept, a novel method designed to enhance its brain delivery via carriage in the cerebrospinal venous system, has previously been shown to reduce chronic neurological dysfunction after stroke. Etanercept is a recombinant biologic that is capable of ameliorating two components of neuroinflammation: microglial activation and the excess bioactivity of tumor necrosis factor (TNF), a proinflammatory cytokine that is a key neuromodulator in the brain. Optimal synaptic and brain network function require physiological levels of TNF. Neuroinflammation, including brain microglial activation and excess central TNF, can be a consequence of stroke or peripheral infection, including infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19. Methods Standardized, validated measures, including the Montreal Cognitive Assessment, Beck Depression Index-II (BDI-II), Fatigue Assessment Scale, Controlled Oral Word Association Test, Trail Making Tests, Timed Finger-to-Nose Test, 20 m Self-Paced Walk Test, 5 Times Sit-to-Stand Test and Grip Strength measured with a Jamar Dynamometer were used to quantitate changes in cognition, depression, fatigue and neurological function after a single 25 mg perispinal etanercept dose in a patient with severe long COVID of 12 months duration. Results Following perispinal etanercept administration there was immediate neurological improvement. At 24 h, there were remarkable reductions in chronic post-COVID-19 fatigue and depression, and significant measurable improvements in cognition, executive function, phonemic verbal fluency, balance, gait, upper limb coordination and grip strength. Cognition, depression and fatigue were examined at 29 days; each remained substantially improved. Conclusion Perispinal etanercept is a promising treatment for the chronic neurologic dysfunction that may persist after resolution of acute COVID-19, including chronic cognitive dysfunction, fatigue, and depression. These results suggest that long COVID brain neuroinflammation is a potentially reversible pathology and viable treatment target. In view of the increasing unmet medical need, clinical trials of perispinal etanercept for long COVID are urgently necessary. The robust results of the present case suggest that perispinal etanercept clinical trials studying long COVID populations with severe fatigue, depression and cognitive dysfunction may have improved ability to detect a treatment effect. Positron emission tomographic methods that image brain microglial activation and measurements of cerebrospinal fluid proinflammatory cytokines may be useful for patient selection and correlation with treatment effects, as well as provide insight into the underlying pathophysiology.

840 Unsupervised learning reveals different degrees of heterogeneity as well as cell involvement in cutaneous lupus erythematous antimalarial treatment response subgroups

First-line treatment for cutaneous lupus erythematosus involves the use of antimalarials. Treatment response is highly variable with some patients responding well to hydroxychloroquine (HCQ), some necessitating the addition of quinacrine (QC) and others refractory to treatment from both (NR). Here we used unsupervised learning to highlight differences between the immune infiltrate composition of the three treatment groups. Infiltrate composition was characterized using imaging mass cytometry, where we stained biopsies (12 HCQ, 11 QC, 20 NR) with two separate panels of 37-metal conjugated antibodies, which enabled the measurement of different cell types, cytokines and pathway proteins within the tissue sample.

Cytokine alterations during paraneoplastic neutrophilia and leukemoid reaction in patients with advanced melanoma

BackgroundParaneoplastic leukemoid reaction (PLR) is a rare phenomenon in metastasized melanoma associated with poor prognosis and rapid disease progression. Currently, no specific therapeutic options exist other than treating the underlying malignancy.MethodsFive cases of paraneoplastic neutrophilia in patients with advanced-stage IV melanoma were enrolled in our study. Cytokine concentrations in patients’ serum samples were analyzed before and during PLR using a multiplex cytokine array. Further, immunohistochemical staining of tumor tissue biopsied during PLR was performed.Results and conclusionsWe observed a strong correlation between worsening of tumor burden and aggravation of neutrophilia. Cytokine measurements revealed an increase of proinflammatory cytokines (IL6, IFNγ), proangiogenic cytokines (VEGF) and immune stem cell growth factors (G-CSF) during PLR. Immunohistochemistry confirmed neutrophil infiltration of tumor tissue. The presented cytokine alterations provide a basis for further functional analysis, which is necessary for the development of targeted therapeutic approaches against PLR.

Effect of aqueous extract of seed of broccoli on inflammatory cytokines and Helicobacter pylori infection: a randomized, double-blind, controlled trial in patients without atrophic gastritis.

The purpose of this study was to investigate the anti-inflammatory effect of an aqueous extract of seed of broccoli (AESB) in Helicobacter pylori (HP)-infected patients without atrophic gastritis. This was a double-centre, randomized, double-blind, controlled study. A total of 110 HP-infected subjects were randomized to receive either AESB or placebo for 2months. Inflammatory cytokine (IL-8, IFN-γ, TNF-α, CRP, IL-17A, IL-1β, IL-18), pepsinogen I, II (PG I, PG II), and gastrin-17 (G-17) measurements and 13C-urea breath tests were performed at baseline and at 60days. At 60days, there was no significant difference in any of the inflammatory cytokines, pepsinogen or gastrin between the two groups. However, IL-8, IFN-γ, PG I, PG I/PG II ratio (PGR), and G-17 were reduced by 9.02pg/mL, 5.08pg/mL, 24.56ng/mL, 1.75 and 0.3pmol/L, respectively, in the AESB group compared with baseline (all P < 0.05). The HP eradication rates in the AESB group and placebo group were 11.11 and 3.70% at 60days, respectively (P > 0.05). No treatment-related adverse events were reported. Thus, AESB may reduce the risk of gastric mucosal lesions and decrease the risk of gastric cancer by relieving inflammatory cytokines. The safety profile of AESB was satisfactory. This study is registered with the Chinese Clinical Trials Registry (Registration No. ChiCTR2100054249).

Spontaneous NLRP3 inflammasome-driven IL-1-β secretion is induced in severe COVID-19 patients and responds to anakinra treatment

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may result in a severe pneumonia associated with elevation of blood inflammatory parameters, reminiscent of cytokine storm syndrome. Steroidal anti-inflammatory therapies have shown efficacy in reducing mortality in critically ill patients; however, the mechanisms by which SARS-CoV-2 triggers such an extensive inflammation remain unexplained.ObjectivesTo dissect the mechanisms underlying SARS-CoV-2–associated inflammation in patients with severe coronavirus disease 2019 (COVID-19), we studied the role of IL-1β, a pivotal cytokine driving inflammatory phenotypes, whose maturation and secretion are regulated by inflammasomes.MethodsWe analyzed nod-like receptor protein 3 pathway activation by means of confocal microscopy, plasma cytokine measurement, cytokine secretion following in vitro stimulation of blood circulating monocytes, and whole-blood RNA sequencing. The role of open reading frame 3a SARS-CoV-2 protein was assessed by confocal microscopy analysis following nucleofection of a monocytic cell line.ResultsWe found that circulating monocytes from patients with COVID-19 display ASC (adaptor molecule apoptotic speck like protein–containing a CARD) specks that colocalize with nod-like receptor protein 3 inflammasome and spontaneously secrete IL-1β in vitro. This spontaneous activation reverts following patient’s treatment with the IL-1 receptor antagonist anakinra. Transfection of a monocytic cell line with cDNA coding for the ORF3a SARS-CoV-2 protein resulted in ASC speck formation.ConclusionsThese results provide further evidence that IL-1β targeting could represent an effective strategy in this disease and suggest a mechanistic explanation for the strong inflammatory manifestations associated with COVID-19.

Metformin suppresses pro-inflammatory cytokines in vitreous of diabetes patients and human retinal vascular endothelium.

Metformin is a traditional anti-hyperglycemic medication that has recently been shown to benefit vascular complications of diabetes via an anti-inflammatory mechanism other than glycemic control. This study aims to test the hypothesis that metformin suppresses diabetic retinopathy (DR) associated intraocular inflammation. Human vitreous from control and proliferative diabetic retinopathy (PDR) patients with or without long-term metformin treatment (> 5 years) were collected for multiple inflammatory cytokines measurements with a cytokine array kit. The vast majority of the measurable cytokines in PDR vitreous has a lower level in metformin group than non-metformin group. Although the p values are not significant due to a relatively small sample size and large deviations, the 95% confidence interval (CI) for the mean difference between the two groups shows some difference in the true values should not be neglected. Using quantitative ELISA, soluble intercellular adhesion molecule -1 (ICAM-1) and monocyte chemoattractant protein -1 (MCP-1) presented with significantly lower concentrations in metformin group versus non-metformin group. Metformin group also has significantly less up-regulated cytokines and diminished positive correlations among the cytokines when compared to non-metformin group. Possible role of AMP-activated protein kinase (AMPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in metformin’s anti-inflammatory effects were studied in human retinal vascular endothelial cells (hRVECs) cultured in normal glucose (NG) and high glucose (HG) conditions. Metformin inhibited HG-induced ICAM-1, IL-8, and MCP-1 via AMPK activation, whereas pharmacological AMPK inhibition had no effect on its inhibition of NF-κB p65, sICAM-1, and tumor necrosis factor-α (TNF-α). Metformin-induced suppression of the inflammatory cytokines could also be mediated through its direct inhibition of NF-κB, independent of AMPK pathway. This is a proof-of-concept study that found metformin treatment was associated with reduced inflammatory responses in vitreous of diabetes patients and retinal vascular endothelial cells, supporting the rationale for using metformin to treat DR at an early stage.

Hyperlipidemia is necessary for the initiation and progression of atherosclerosis by severe periodontitis in mice.

Hyperlipidemia is a major risk of atherosclerosis; however, systemic inflammatory diseases such as rheumatoid arthritis, psoriasis, systemic lupus erythematosus and systemic sclerosis are also known risks for the development of atherosclerosis. Periodontitis, a local and systemic inflammatory condition, has also been reported as a risk for atherosclerosis, but the specific link between periodontitis and atherosclerosis remains somewhat controversial. We previously reported that ligature-induced periodontitis exacerbates atherosclerosis in hyperlipidemic Apolipoprotein E-deficient (ApoE−/−) mice. To understand whether hyperlipidemia is necessary for the development and exacerbation of atherosclerosis associated with periodontitis, the present study created ligature-induced periodontitis in both wild-type (WT) and ApoE−/− mice. Subsequently, the status of local, systemic and vascular inflammation, serum lipid contents and arterial lipid deposition were examined with histological analysis, µCT, en face analysis, serum lipid and cytokine measurements, reverse transcription-quantitative PCR and immunohistochemical analysis. Ligature placement induced severe periodontitis in both WT and ApoE−/− mice at the local level as demonstrated by gingival inflammation, alveolar bone loss, increased osteoclastic activities and inflammation in alveolar bone. Systemic inflammation was also induced by ligature placement in both WT and ApoE−/− mice, albeit more so in ApoE−/− mice. The serum cholesterol levels were not altered by the ligature in both WT and ApoE−/− mice. However, the vascular inflammation and arterial lipid deposition were induced by ligature-induced periodontitis only in ApoE−/− mice, but not in WT mice. The present study indicated that the coupling of systemic inflammation and hyperlipidemia was necessary for the development and exacerbation of atherosclerosis induced by ligature-induced periodontitis in mice.

Therapeutic effects of a lipid transfer protein isolated from Morinda citrifolia L. (noni) seeds on irinotecan-induced intestinal mucositis in mice.

This work aimed to evaluate the activity of a lipid transfer protein isolated from Morinda citrifolia L. seeds, McLTP1, on the development of intestinal mucositis following irinotecan administration. McLTP1 (0.5, 2, and 8 mg/kg, i.v.) was injected into mice 1h before irinotecan administration (75 mg/kg, i.p.; 4 days), and then for additional 6 days. Seven days after the first dose of irinotecan, diarrhea was assessed, and the intestine was removed for histological evaluation, assessment of intestinal over-contractility, measurement of myeloperoxidase (MPO), proinflammatory cytokines and chemokine (IL-1, IL-6, and KC levels - a murine homolog of human IL-8 chemokine), analysis of cyclooxygenase 2 (COX-2), nuclear factor kappa B (NF-κB), and nitric oxide synthase (iNOS) expression. At the two highest doses, McLTP1 administration decreased mortality and diarrhea. McLTP1 (8 mg/kg, i.v.) significantly prevented irinotecan-induced intestinal damage and led to a reduction in over-contractility of the intestinal muscle (p < 0.05). Moreover, McLTP1 decreased the MPO, IL-1β, IL-6, and KC levels by 74.7%, 42%, 92.9%, and 95.9%, respectively. Also, the expression of COX-2, NF-κB, and iNOS was reduced. Our study provides a potential new therapeutic for preventing irinotecan-induced mucositis, improved clinical parameters, and reduced inflammation.

Pathophysiological evaluations of initial plaque development after heart transplantation via serial multimodality imaging and cytokine assessments.

Detailed morphological characteristics of de novo and donor-transmitted plaques and the association of serum T-lymphocyte cytokine levels with plaque progression of coronary allograft vasculopathy within 1 year after heart transplantation are unknown. In this retrospective analysis of data in a prospectively maintained database, 40 heart transplant recipients were included. We performed serial 3 vessel optical coherence tomography and intravascular ultrasound analyses, at the 8 week (baseline) and 12 month post-transplantation follow-ups, and serum cytokine measurements (n=23). The correlation between serum cytokines and Δplaque burden (between baseline and follow-up) was evaluated depending on plaque morphology. Thirteen de novo plaques (maximum intimal thickness ≥0.5 mm at the 12 month follow-up without plaques at baseline) were identified in 8 recipients, and 31 donor-transmitted plaques (maximum intimal thickness ≥0.5 mm at baseline) were detected in 17 recipients. Compared with donor-transmitted plaques, the Δplaque burden in the de novo plaques, with mainly fibrous morphology, was high (38.8% [29.6%-41.2%] vs 8.7% [1.33%-13.6%], p < 0.001). Stratification of the morphology of donor-transmitted plaques revealed that the Δplaque burden in fibrous plaques (10.6% [7.0%-18.0%]) was similar to that in fibroatheroma (10.3% [8.7%-23.8%]). Serum interleukin-31 levels at baseline correlated with fibrous plaque proliferation (r=0.73, p=0.007) even under immunosuppressive conditions, whereas other cytokines (interleukin-1β, interleukin-17, and interferon-gamma) were mostly undetectable. Intimal fibrous proliferation contributed to the progression of donor-transmitted and de novo plaques. Serum interleukin-31 levels at baseline may contribute to intimal fibrous proliferation within 1 year after heart transplantation.

Molecular analysis of microorganisms in the semen and their impact on semen parameters.

Chronic genitourinary infections can alter male fertility and even promote carcinogenic processes. This study aimed to evaluate the effect of the presence in the semen of microorganisms on semen quality. Clinical symptoms and conventional and functional seminal parameters of eleven fertile donors and ten volunteers with prostatitis-like symptoms were evaluated. Nitric oxide, antioxidant capacity, and pro-inflammatory cytokines in semen and seminal plasma samples were also quantified. Finally, the expression of the ROR-γT, FoxP3, and T-bet genes in semen and the presence of DNA of microorganisms associated with prostatitis in urine and semen were evaluated. When compared with fertile donors, volunteers with chronic prostatitis-like symptoms reported erectile dysfunction (0% vs. 10%, p = 0.2825) and premature ejaculation (0% vs. 40%; p = 0.0190). No statistically significant differences were observed in seminal parameters, cytokine measurement, antioxidant capacity, nitric oxide concentration and ROR-γT, FoxP3, T-bet. Microorganisms responsible for sexually transmitted infections and some bacteria associated with the microbiota and infections in the prostate gland were detected. In the semen from the subjects with prostatitis-like symptoms T. vaginalis DNA was detected; in addition, N. gonorrhoeae DNA was also detected in semen and urine samples. S. pyogenes was detected in the urine samples from the control group. Prostatitis-like symptoms are a common finding in young men that affect sexual and reproductive health, but not always the seminal parameters or fertility. The presence of prostatitis- like symptoms does not affect seminal quality. However, it appears to be associated with an increased likelihood of erectile dysfunction and premature ejaculation. Thus, affecting the quality of life and sexual and reproductive health.

Skeletal Muscle Myofibers Directly Contribute to LPS-Induced Systemic Inflammatory Tone.

The abundance, anatomical distribution, and vascularity of skeletal muscle make it a potentially important contributor to local cytokine production and systemic cytokine abundance during inflammatory events. An orchestrated balance between the production of pro- and anti-inflammatory mediators is necessary for proper immune function, yet the contribution of the body’s largest organ system, comprised primarily of skeletal muscle myocytes that fuse to form myofibers, to this process is largely unknown. Endotoxin (lipopolysaccharide, LPS) stimulates toll-like receptor 4 (TLR4) to induce the production of several pro-inflammatory cytokines, including interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2), by a of myriad cell types. We sought to quantify the influence of myofibers on systemic cytokine concentrations following an acute endotoxemia challenge. To accomplish this, we generated muscle specific conditional knockouts for TLR4 (TLR4SMKO), IL-6 (IL6SMKO), and CCL2 (CCL2SMKO). We administered low concentrations of intravenous LPS (IV LPS) to these receptor and effector knockout mice and collected samples after 3 h. Using gene expression analysis of gastrocnemius muscle and serum cytokine measurements after IV LPS, we determined that deletion of myofiber IL-6 or CCL2 led to a 93% and 57% reduction of these specific cytokines in the systemic circulation, respectively. Myofiber specific TLR4 deletion decreased the expression of IL-6, CCL2, and C-X-C motif chemokine ligand 1 (CXCL1) in the gastrocnemius muscle. These data indicate the critical involvement and direct contribution of myofibers during the early systemic inflammatory cytokine response to endotoxin.

The alterations of cerebrospinal fluid TNF-alpha and TGF-beta2 levels in early relapsing-remitting multiple sclerosis.

This study aimed to analyze serum and cerebrospinal fluid (CSF) concentrations of proinflammatory and anti-inflammatory cytokines produced by T regulatory (Treg) cells in early RRMS according to the 2017 McDonald criteria. Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS) with the cytokine network playing an important role. However, there is a continual lack of data regarding the immunopathogenesis of early RRMS, especially according to the 2017 McDonald criteria. The study groups included early RRMS patients during relapse (n = 18), remission (n = 14), and the control group. The MS diagnosis was established according to the 2017 McDonald criteria. Patients were studied up to 1year after diagnosis was made. A quantitative test kit based on ELISA was used for cytokine measurement in the serum and CSF. Comparative and correlation analyses between the levels of TNF-α, TGF-β2, IgG index, and relapse duration were performed. Significantly higher CSF concentrations of TNF-α in both RRMS-relapse and RRMS-remission groups were found compared to the controls (p &lt; 0.01). The CSF levels of TGF-β2 in the RRMS-relapse group were significantly lower in comparison to the control group (p = 0.01). An inappropriate inflammatory response seems to occur in early RRMS and includes the production of TNF-α and a decrease in TGF-β2 release suggesting a significant Treg cells role. Further studies on the topic may contribute to developing new disease-modifying drugs and biochemical markers of the disorder.

Pinus sylvestris L. and Syzygium aromaticum (L.) Merr. & L. M. Perry Essential Oils Inhibit Endotoxin-Induced Airway Hyperreactivity despite Aggravated Inflammatory Mechanisms in Mice.

Scots pine (SO) and clove (CO) essential oils (EOs) are commonly used by inhalation, and their main components are shown to reduce inflammatory mediator production. The aim of our research was to investigate the chemical composition of commercially available SO and CO by gas chromatography–mass spectrometry and study their effects on airway functions and inflammation in an acute pneumonitis mouse model. Inflammation was evoked by intratracheal endotoxin and EOs were inhaled three times during the 24 h experimental period. Respiratory function was analyzed by unrestrained whole-body plethysmography, lung inflammation by semiquantitative histopathological scoring, myeloperoxidase (MPO) activity and cytokine measurements. α-Pinene (39.4%) was the main component in SO, and eugenol (88.6%) in CO. Both SO and CO significantly reduced airway hyperresponsiveness, and prevented peak expiratory flow, tidal volume increases and perivascular edema formation. Meanwhile, inflammatory cell infiltration was not remarkably affected. In contrast, MPO activity and several inflammatory cytokines (IL-1β, KC, MCP-1, MIP-2, TNF-α) were aggravated by both EOs. This is the first evidence that SO and CO inhalation improve airway function, but enhance certain inflammatory parameters. These results suggest that these EOs should be used with caution in cases of inflammation-associated respiratory diseases.

Abstract 2059: Immunotherapy testing in 3D Ex vivo Patient Tissue Platform with preserved tumor microenvironment

Abstract Introduction Broad application of immunotherapy to treat cancer has been hampered by low response rates in the clinics (~20%) and high failure rates of clinical trials. The lack of translational preclinical models that accurately replicate human immunological complexity is one of the reasons leading to ineffective candidates in clinical trials. There is an urgent need of advanced preclinical models that can enhance clinical trial success rate and improve clinical impact of immunotherapies. We present a novel 3D Ex vivo Patient Tissue platform combining short-term 3D ex vivo tumor culture system with high content image (HCI)-based analysis. The platform preserves tumor heterogeneity and tumor immune context, including (exhausted) tumor infiltrating lymphocytes. We report a quantification of ex vivo tumor responses to a panel of immunotherapies, including checkpoint inhibitors, tested on patient tissues from various cancers. Methods Patient tumor tissues were obtained from hospitals and tissue providers and processed within 24 hours to preserve the tumor microenvironment (TME). Tumor clusters from ovarian, cervical, breast, and non-small cell lung cancer patients were embedded in a protein-rich hydrogel and exposed to panels of immunomodulatory drugs in a 384-well format for 5-7 days. Phenotypic effects of the drugs on physiologically relevant morphological changes, such as tumor cell killing and immune cell proliferation, were measured using our proprietary automated HCI analysis platform. In addition, IHC and FACS analysis of primary samples as well as cytokine measurements were performed. Results Responses of ex vivo tissues to immunotherapies targeting various pathways (e.g., ipilimumab, pembrolizumab and STING agonists), combination treatments and selected controls were evaluated by automated phenotypic analysis platform. Based on their sensitivity, patient tissues were classified as immunotherapy responders or non-responders. A deeper investigation of the responder tissues was performed with IHC and FACS to pinpoint the critical TME components necessary for immunotherapy sensitivity. In addition, cytokine profiling was done in supernatants from treated ex vivo cultures to confirm the functional HCI readouts. Accurate and reproducible response evaluation demonstrated the feasibility of preclinical immunotherapy drug testing on primary patient material using this platform. Conclusion The 3D ex vivo patient tissue platform successfully combined drug testing protocols using fresh patient tumor tissue with preserved TME and advanced 3D HCI analysis. The platform offers a rapid, reliable and patient-relevant approach to test (clinical) immunotherapies for different solid tumours. It has the potential to significantly improve the preclinical evaluation of immunotherapies and support the decision-making process during progression of drug candidates to the clinic. Citation Format: Nataliia Beztsinna, Niels Meesters, Lidia Daszkiewicz, Fanny Grillet, Donny van der Meer, Kuan Yan, Emma Spanjaard, Willemijn Vader, Leo Price. Immunotherapy testing in 3D Ex vivo Patient Tissue Platform with preserved tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2059.

The immuno-behavioural covariation associated with the treatment response to bumetanide in young children with autism spectrum disorder

Bumetanide, a drug being studied in autism spectrum disorder (ASD) may act to restore gamma-aminobutyric acid (GABA) function, which may be modulated by the immune system. However, the interaction between bumetanide and the immune system remains unclear. Seventy-nine children with ASD were analysed from a longitudinal sample for a 3-month treatment of bumetanide. The covariation between symptom improvements and cytokine changes was calculated and validated by sparse canonical correlation analysis. Response patterns to bumetanide were revealed by clustering analysis. Five classifiers were used to test whether including the baseline information of cytokines could improve the prediction of the response patterns using an independent test sample. An immuno-behavioural covariation was identified between symptom improvements in the Childhood Autism Rating Scale (CARS) and the cytokine changes among interferon (IFN)-γ, monokine induced by gamma interferon and IFN-α2. Using this covariation, three groups with distinct response patterns to bumetanide were detected, including the best (21.5%, n = 17; Hedge’s g of improvement in CARS = 2.16), the least (22.8%, n = 18; g = 1.02) and the medium (55.7%, n = 44; g = 1.42) responding groups. Including the cytokine levels significantly improved the prediction of the best responding group before treatment (the best area under the curve, AUC = 0.832) compared with the model without the cytokine levels (95% confidence interval of the improvement in AUC was [0.287, 0.319]). Cytokine measurements can help in identifying possible responders to bumetanide in ASD children, suggesting that immune responses may interact with the mechanism of action of bumetanide to enhance the GABA function in ASD.

A fluorescent immunosensor on optical fibre for the multiplex detection of proinflammatory cytokines

Cytokines are typical mediators of the immune response. Single cytokine measurement is unable to reflect the true complexity of these physiological processes and multiplex detection is required. In this work, we developed an optical fibre based biosensor for multiplex detection of cytokines. This multiplex biosensor has been successfully applied for the measurement of proinflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the linear range of 12.5–200 pg mL−1 with the limit of detection (LOD) of 12.5 pg mL−1. Additionally, it has compatible performance with ELISA kit for the monitoring of cytokines from rat PBMC cell culture mediums stimulated by LPS or UV radiation. Moreover, this universal platform could be applied for multiplex detection of other proteins by simply changing the capture/detection antibody pairs. This fibre based multiplex biosensor is designed for in-situ measurement of target cytokines, which is significant for the investigation of inflammatory processes or disease prognosis and treatment monitoring.

Preliminary data from an ongoing phase 1 dose-escalation study of CCX559, an orally administered small molecule PD-L1 inhibitor, in patients with advanced solid tumors.

2593 Background: The novel small molecule CCX559 is a highly potent and selective PD-L1 inhibitor that induces the dimerization and internalization of cell-surface PD-L1. CCX559, when orally administered in animal models, demonstrated anti-tumor efficacy, including the ability to induce complete responses (Li C, et al. Cancer Res 2021;81(13_Suppl): Abstract nr 1274). Safety pharmacology and toxicology studies in animals demonstrated an acceptable safety profile for CCX559. Taken together, the preclinical data supports the initiation of human trials in patients with advanced solid tumors. PD-(L)1 therapies have been shown to increase peripheral T cell activation and cytokines such as IFNγ and CXCL9 in patients (Herbst RS, et al. Nature.2014; 515(7528):563–567). Methods: This phase 1, first-in-patient, multicenter, open-label, dose-escalation study evaluates safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary anti-tumor activity of CCX559 in patients with refractory, locally advanced or metastatic solid tumors. CCX559 is dosed orally once daily with a starting dose level of 30 mg. Dose escalation/de-escalation is based on the Bayesian Optimal Interval (BOIN) design. PBMC and plasma samples were collected from patients over the first 2 cycles (6 weeks) of treatment, and PD assays were performed, including measurement of cytokines and T cell proliferation. Results: As of February 1, 2022, one patient per cohort was enrolled at the 30 mg and 60 mg levels, and 9 patients were enrolled in the 120 mg dose cohort. Of the 11 patients enrolled, 5 patients (all 120 mg cohort) remain on treatment. No DLTs, treatment-related SAEs, or severe (Grade 3 or higher) treatment-related AEs have been reported. PD assays have been performed with samples from the 30 mg (n=1), 60 mg (n=1), and 120 mg (n=3) cohorts. Patients in all three cohorts showed increased peripheral CD4 and CD8 T cell proliferation starting in the first cycle (21 days) of treatment, as measured by Ki67 positivity. Increases in plasma IFNγ, CXCL9, CXCL10, and CXCL11 levels were observed, in particular in one patient (120 mg cohort) starting 15 days after treatment initiation. Conclusions: Initial results from the phase 1 dose-escalation study of CCX559 indicate on-target PD effects consistent with PD-L1 inhibition. Updated PD data, together with the safety and PK profile, will be presented. Clinical trial information: ACTRN12621001342808.

N‑acetyl cysteine prevents ambient fine particulate matter‑potentiated atherosclerosis via inhibition of reactive oxygen species‑induced oxidized low density lipoprotein elevation and decreased circulating endothelial progenitor cell.

Ambient fine particulate matter (PM) serves an important role in the development of cardiovascular disease, including atherosclerosis. Antioxidant N-acetyl cysteine (NAC) has protective effects in the cardiovascular system. However, it is unknown if NAC prevents PM-potentiated atherosclerosis in hyperlipidemia. Low-density lipoprotein (LDL) receptor knockout mice were pretreated with 1 mg/ml NAC in drinking water for 1 week and continued to receive NAC, high-fat diet and intranasal instillation of PM for 1 week or 6 months. Blood plasma was collected for lipid profile, oxidized (ox-)LDL, blood reactive oxygen species (ROS) and inflammatory cytokine (TNF-α, IL-1β and IL-6) measurement. Blood cells were harvested for endothelial progenitor cell (EPC) population and intracellular ROS analysis. Murine aorta was isolated for atherosclerotic plaque ratio calculation. NAC treatment maintained circulating EPC level and significantly decreased blood ox-LDL and ROS, inflammatory cytokines, mononuclear and EPC intracellular ROS levels as well as aortic plaque ratio. NAC prevented PM-potentiated atherosclerosis by inhibiting plasma ROS-induced ox-LDL elevation, mononuclear cell and EPC intracellular ROS-induced circulating EPC reduction and inflammatory cytokine production.

OP0024 DIFFERENTIATION BETWEEN IL-6 AND IL-17 PATHWAY INHIBITION IN RELATIONSHIP WITH CLINICAL OUTCOMES IN NON-BIOLOGICAL DMARD-IR AND BIOLOGICAL DMARD-IR PSORIATIC ARTHRITIS PATIENTS TREATED WITH UPADACITINIB IN SELECT-PsA 1 AND SELECT-PsA 2 STUDIES

BackgroundThe differential contribution of IL-6 and IL-17 pathways to the pathogenesis of psoriatic arthritis (PsA) is not fully understood. Upadacitinib (UPA), an oral JAK inhibitor, was more effective than placebo (PBO) in improving key clinical manifestations of PsA in two global phase 3 trials, SELECT-PsA 1 (non-biological DMARD-IR, nbDMARD-IR) and SELECT-PsA 2 (biological DMARD-IR, bDMARD-IR).1,2 Targeted proteomic analysis suggested that UPA modulates multiple biological pathways in innate and adaptive immune systems via direct and indirect inhibition of key regulators, including IL-6 and IL-17 pathways, with a possible shift from Th1 predominance in nbDMARD-IR PsA to a more Th17 bias in bDMARD-IR PsA.3ObjectivesWe assessed the relationship between IL-6 and IL-17 pathway modulation and different clinical outcomes after UPA treatment in nbDMARD-IR and bDMARD-IR PsA patients.MethodsA subset of patients was randomly selected from SELECT-PsA 1 (n=74 of UPA 15 mg QD, n=74 of PBO) and PsA 2 studies (n=90 of UPA 15 mg QD, n=81 of PBO). Serum levels of IL-6, IL-17A, IL-17F, and beta-defensin 2 (BD2) proteins were measured at baseline, week 2, and week 12 by validated immunoassays. The quantitative cytokine measurements were transformed as log10, and PASI score was transformed as log10 (x+1) prior to analysis. A Repeated Measure Mixed Linear Model was used to compare UPA versus PBO treatment effects in overall selected patients and between responders and non-responders defined by PASDAS score ≤ 3.6 (Minimal Disease Activity, MDA)4 and PASI75 at week 12, respectively. The relationships between cytokines and clinical outcomes (PASI and DAS28-CRP) were assessed by Pearson’s correlation at baseline and after treatment.ResultsIn nbDMARD-IR PsA patients, baseline IL-17A, IL-17F, and BD2 levels correlated with each other and with PASI, while IL-6 appeared independent from the IL-17 pathway and correlated with DAS28-CRP. At week 12, UPA treatment significantly decreased IL-6 and BD2. The decrease of IL-6 was more pronounced in PASDAS MDA responders and correlated with DAS28-CRP improvement, but the decrease of BD2 was significant in PASI75 responders and correlated with PASI improvement. In contrast, IL-17A and IL-17F were not significantly changed after UPA treatment, neither correlated with clinical outcomes at week 12.In bDMARD-IR PsA patients, baseline IL-17A level was significantly elevated compared to nbDMARD-IR patients but weakly correlated with other cytokines and show no correlation with PASI. At week 12, the reduction of IL-6 after UPA treatment was not different between responders and non-responders (PASDAS MDA or PASI75) and did not correlate with DAS28-CRP improvement, while the reduction of BD2 remained significant in PASI75 responders and correlated with PASI improvement. Further, UPA treatment significantly reduced IL-17A in PASDAS MDA responders and IL-17F in PASI75 responders compared to non-responders, respectively. The reduction of IL-17F correlated with PASI improvement at week 12.ConclusionIL-6 and IL-17 pathway inhibition after UPA treatment showed different profiles in relationship with clinical outcomes in nbDMARD-IR versus bDMARD-IR PsA patients. IL-6 decrease was more pronounced in nbDMARD-IR PsA patients and associated with joint manifestation improvement, while IL-17A and IL-17F decreases were only observed in bDMARD-IR PsA patients and associated with psoriasis improvement. BD2, a biomarker of Th17-associated skin pathology, significantly decreased after UPA treatment in both nbDMARD-IR and bDMARD-IR PsA studies, which likely contributed to UPA effects on psoriasis improvement in a broad range of PsA patients.