Changes In Serum Cytokine Levels Research Articles

Prevotella intermedia boosts OSCC progression through ISG15 upregulation: a new target for intervention

PurposePeriodontitis-associated bacteria, such as Porphyromonas gingivalis and Fusobacterium nucleatum, are closely linked to the risk of oral squamous cell carcinoma (OSCC). Emerging studies have indicated that another common periodontal pathogen, Prevotella intermedia (P. intermedia), is enriched in OSCC and could affect the occurrence and progression of OSCC. Our aim is to determine the effects of P. intermedia on the progression of OSCC and the role of antibiotics in reversing these effects.MethodsIn this study, a murine xenograft model of OSCC was established, and the mice were injected intratumorally with PBS (control group), P. intermedia (P.i group), or P. intermedia combined with an antibiotic cocktail administration (P.i + ABX group), respectively. The effects of P. intermedia and ABX administration on xenograft tumor growth, invasion, angiogenesis, and metastasis were investigated by tumor volume measurement and histopathological examination. Enzyme-linked immunosorbent assay (ELISA) was used to investigate the changes in serum cytokine levels. Immunohistochemistry (IHC) was adopted to analyze the alterations in the levels of inflammatory cytokines and infiltrated immune cells in OSCC tissues of xenograft tumors. Transcriptome sequencing and analysis were conducted to determine differential expression genes among various groups.ResultsCompared with the control treatment, P. intermedia treatment significantly promoted tumor growth, invasion, angiogenesis, and metastasis, markedly affected the levels of inflammatory cytokines, and markedly altered M2 macrophages and regulatory T cells (Tregs) infiltration in the tumor microenvironment. However, ABX administration clearly abolished these effects of P. intermedia. Transcriptome and immunohistochemical analyses revealed that P. intermedia infection increased the expression of interferon-stimulated gene 15 (ISG15). Correlation analysis indicated that the expression level of ISG15 was positively correlated with the Ki67 expression level, microvessel density, serum concentrations and tissue expression levels of inflammatory cytokines, and quantities of infiltrated M2 macrophages and Tregs. However, it is negatively correlated with the quantities of infiltrated CD4+ and CD8+ T cells.ConclusionIn conclusion, intratumoral P. intermedia infection aggravated OSCC progression, which may be achieved through upregulation of ISG15. This study sheds new light on the possible pathogenic mechanism of intratumoral P. intermedia in OSCC progression, which could be a prospective target for OSCC prevention and treatment.

Postbiotic Administration Ameliorates Colitis and Inflammation in Rats Possibly through Gut Microbiota Modulation.

Postbiotics are preparations of inanimate microorganisms and/or their components that are beneficial to host health. Compared with probiotics, the postbiotic dose required for exerting obvious protective effects is unknown. Thus, we conducted a dose-dependent postbiotic intervention study in dextran sulfate sodium (DSS)-induced colitis rats. The trial included five rat groups, including: control without DSS/postbiotic treatment, group C; 7-day DSS treatment, group D; 14-day low, medium, and high probiotic doses (0.1, 0.2, 0.4 g/kg; groups L, M, H, respectively) after DSS induction. We found that postbiotic intervention effectively mitigated the symptoms and inflammation in colitis rats, evidenced by the improved spleen index, less severe colon tissue damage, and changes in serum cytokine levels (decreases in tumor necrosis factor-α and interleukin-1β; increase in interleukin-10) in postbiotic groups compared with group D. Moreover, the therapeutic effect was dose-dependent. Fecal metabolomics analysis revealed that the postbiotic recipients had more anti-inflammatory metabolites, namely, salicyloyl phytophingosine, podophylloxin, securinine, baicalein, and diosmetin. Fecal metagenomics analysis revealed that the postbiotic recipients had more beneficial microbes and less pro-inflammatory bacteria. This study confirmed that postbiotics are effective in alleviating colitis in a dose-dependent manner. Our findings are of interest to food scientists, clinicians, and the health food industry.

Association of serum levels of inflammatory cytokines with retinopathy of prematurity in preterm infants.

Retinopathy of prematurity (ROP) is a retinal vascular developmental disease associated with risks factors such as supplementary oxygen use or low birth weight/early gestational age. Multiple studies have reported associations between ROP and systemic inflammation. In this study, we investigated serum cytokines associated with ROP development and severity and assessed their applicability as potential biomarkers of ROP. This prospective study was conducted at an institutional referral center between 2019 and 2021. To measure the serum levels of 40 inflammatory cytokines in eligible premature patients, we collected their serum samples during the enrollment of patients or the intravitreal injection of anti-vascular endothelial growth factor (VEGF) agents and after 2 and 4 weeks. Fifty patients were enrolled. In patients with type 1 ROP who received anti-VEGF agents (n = 22), the levels of serum intercellular adhesion molecule-1 decreased significantly (p < 0.05) at 4 weeks compared with the baseline level, whereas those of serum granulocyte-macrophage colony-stimulating factor increased significantly (p < 0.05). In patients with ROP who did not require any treatment (n = 14), no significant change was noted in the level of any of the 40 inflammatory cytokines. In control infants without ROP (n = 14), the serum levels of tumor necrosis factor-α, interleukin (IL)-15, and IL-12p40 increased significantly (p < 0.05) at 4 weeks. The changes in the levels of serum inflammatory cytokines did not vary significantly among the aforementioned three groups. A generalized estimating equation indicated that zone 1 ROP, stage 3 ROP, older postmenstrual age, respiratory distress syndrome, necrotizing enterocolitis, and sepsis were associated with the changes in serum cytokine levels. Although significant changes (compared with baseline) were observed in the serum levels of certain inflammatory cytokines in patients with type 1 ROP and infants without ROP, no significant difference in cytokine level fluctuations were noted among the three groups. Changes in serum inflammatory cytokine levels may not predict ROP development or severity. Additional comprehensive studies are warranted to establish their definitive role and significance in ROP, emphasizing the need for continued research in this area.

Investigation of the effects of oclacitinib maleate on clinical improvement and serum cytokine levels in dogs with atopic dermatitis

The aim of this study was to present allergens involved in the etiology of atopic dermatitis (AD) in dogs, to determine and classify the clinical severity of the disease using the CADESI-4 and pruritus scores, to investigate changes in serum cytokine levels in dogs with AD and their relationship with clinical findings, and to investigate the effects of oclacitinib maleate on clinical improvement and serum cytokine levels. The material of the study consisted of 20 dogs diagnosed with AD and 10 healthy dogs. CADESI and pVAS scores were obtained from the patients, and blood samples were taken. Hematological analyses, specific allergen tests (polycheck), and ELISA analyses were performed. Oclacitinib maleate (Apoquel, Zoetis) was administered orally to the patient dogs at a dose of 0.5 mg/kg twice a day for 7 days. It was found that the most common allergens involved in the etiology of AD in dogs were house dust mites type 1, house dust mites type 2, and mold fungi, and the mean CADESI and pVAS scores for clinical findings such as pruritus, alopecia, erythema, and lichenification in dogs with AD were 48 and 5.15 respectively. When compared to the control group, an increase in the percentage of eosinophils (p &lt; 0.05) and a decrease in serum IL-2 and IL-4 levels (p &lt; 0.05) were detected. When compared to other groups, it was found that the mean serum IL-4 levels (p &lt; 0.05) were low and IL-13 levels (p &lt; 0.05) were high in the group with severe clinical findings. It was determined that the use of oclacitinib maleate resulted in clinical improvement and caused an increase in the mean serum IL-2 levels (p &lt; 0.05) and a decrease in IL-31 and IL-33 levels (p &lt; 0.05). Oclacitinib maleate (Apoquel, Zoetis) was found to be an effective and reliable drug that can be used in the treatment of the disease, but it was concluded that the duration of use should be extended.

Fedratinib Treatment Reduces the Inflammatory Cytokine Profile and Decreases Exhausted T Cells Correlating with Clinical Response in Patients with Myelofibrosis: Biomarker Analysis from the Phase 3 FREEDOM2 Trial

Introduction: Myelofibrosis (MF) is a clonal myeloproliferative neoplasm (MPN) characterized by a hyperactive JAK-STAT pathway, splenomegaly, and constitutional symptoms. JAK inhibitors (JAKi) have been the mainstay in MF treatment over the past decade. Pro-inflammatory cytokines, the levels of which have prognostic value (Tefferi et al. J Clin Oncol 2011), have been shown to be upregulated in MF. However, there is paucity of data on chronic immune dysregulation in pathogenesis of MF. Here, we report the multiplatform biomarker analysis including immune changes from phase 3 (FREEDOM2) trial in patients with MF previously exposed to ruxolitinib (Rux) and treated with fedratinib vs. best available therapy (BAT) (primary efficacy: 35.8% vs 6% responders at the end of cycle 6 (EOC6) in fedratinib vs BAT, respectively). Of note, 78% of the patients on BAT continued RUX therapy until EOC6, allowing direct comparison between RUX and fedratinib. Methods: In the FREEDOM2 trial, patients (N=201) were randomized into two arms, fedratinib (400 mg/day) or BAT in a 2:1 ratio, respectively. The primary endpoint was the proportion of patients with ≥35% reduction of spleen volume (SVR35) on MRI at the end of cycle 6 (EOC6). In the FREEDOM2 biomarker cohort, mutational analysis was performed in enriched CD66b+ cells using the MLL myeloid panel targeting 74 genes. Rules-Based Medicine's (RBM) HumanMAP v2.0 panel targeting 85 cytokines was used to measure changes in serum cytokine levels from C1D1 to EOC6. Custom Q2 immune panels to detect myeloid and lymphoid subsets were used for immunophenotyping of peripheral blood samples. Results: Baseline mutations in FREEDOM2 were commonly found MPN mutations that include JAK2 V617F (71%), ASXL1 (43%), CALR (22%), TET2 (22%) and MPL (11%). However, baseline mutation status was not a predictor of response. Patients receiving fedratinib demonstrated significant down-regulation of several pro-inflammatory cytokines including CRP, ENRAGE, IL-18, IL-16, TIMP-1, and increase of anti-inflammatory cytokines including adiponectin, MMP2, and CEA (Wilcoxon signed rank test, BH adjusted p &amp;lt;0.001) at EOC6 vs C1D1. In addition, the change in levels of many cytokines correlated with the clinical endpoint (SVR35) demonstrating that the therapeutically relevant changes were mediated by fedratinib (Pearson rho P&amp;lt;0.05) (Figure 1). Importantly, fedratinib was superior to the BAT in its ability to alter levels of key cytokines (p &amp;lt;0.05) at EOC6. PD-1 expression has been shown to be upregulated in CD4+ and CD8+ T cells in MF patients and is associated with poor overall survival. However, RUX did not have a significant effect on PD1+ T cells (Veletic et al. Haematologica 2021). Interestingly, flow cytometric analysis revealed that fedratinib decreased exhausted CD8+ T cells (PD1+) up to 40% (∆ median change -11.4%,p&amp;lt;0.001)) (figure 2). Importantly, the reduction in exhausted T cells correlated with the reduction in spleen volume with fedratinib treatment ( R= -0.51, P=0.00011; Figure 2). Consistent with this, we also observed median increases in NK (+3%, p&amp;lt;0.001) and NK-T (+13.8%, p&amp;lt;0.001) populations when compared to the BAT arm. Conclusions: To our knowledge, this is the first comprehensive study detailing cytokine and immune changes in patients with MF in a clinical trial. In addition, our data for the first time demonstrates the impact of fedratinib on the immune system and its ability to reduce exhausted T cells, highlighting the immune modulatory mechanism of fedratinib. Notably, the cytokine and immune changes correlated with the primary endpoint and were not observed in the BAT arm demonstrating superior efficacy of fedratinib in previously RUX exposed patients. Furthermore, since majority of the patients in the BAT arm continued to receive RUX, these findings highlight the mechanistic differences between fedratinib and RUX on clinical response in MF (SVR35). Overall, our data highlight multiple novel avenues to achieve therapeutic efficacy in MF and positions fedratinib as an effective therapy with a distinctive mechanism of action. Additional analyses on the effect of fedratinib treatment on myeloid immune subsets and transcriptomic changes are underway to gain comprehensive mechanistic understanding.

Pembrolizumab in combination with epigenetic therapy is safe and active in heavily treated patients with peripheral T‐cell lymphoma and cutaneous T‐cell lymphoma

Introduction: Peripheral T-cell lymphomas are uniquely sensitive to epigenetic modifiers. We have shown that decitabine and 5-azacytidine (Marchi et al.; Br. J. Haematol. 171; 2015) induce the expression of cancer testis antigens in pre-clinical models of PTCL, and that pralatrexate modulates genes involved in cytokine production, viral response and apoptosis. These data suggest a role for the incorporation of the immune-checkpoint inhibitor, pembrolizumab, to epigenetic agents. Herein we report on the clinical activity of this approach in patients with relapsed or refractory (R/R) PTCL and CTCL. Methods: This is a phase 1b study of pembrolizumab combined with pralatrexate (Arm A), pralatrexate + decitabine (Arm B), or decitabine (Arm C) in patients with R/R PTCL and CTCL. A 3+3 dose-escalation/de-escalation is applied in Arms A and C while a DLT-adapted partial order continual reassessment method for dose-finding with combinations of agents is applied in Arm B. Changes in serum cytokine levels were assessed during cycle 1 using a Luminex-based immunoassay. T-cell subpopulation profiling in peripheral blood was performed using a multi-color flow cytometry assay (Cytek® Aurora). Results: Patient characteristics, toxicity data, and response analysis are shown in table 1. Serum levels of three cytokines, TNFα, MIP-3α and IL-10, measured at day 1 pre-treatment were significantly elevated (p = 0.0001, 0.011 and 0.0017, respectively) in trial patients compared to healthy controls. Patients who responded to treatment demonstrated a more consistent decline in levels of TNFα, MIP-3α, and IL-10 during cycle 1, whereas non-responders showed stable to increased levels of these cytokines. T-cell subpopulation analysis (n = 6) showed mean CD4:CD8 ratio of 0.5 in trial patients prior to treatment compared to 1.5 for healthy age and sex-matched controls (p = 0.016). No significant change in the proportion of PD1+CD8+ central memory cells was seen in trial patients between D0 and D28 of treatment (p = 0.12), and between D0 in comparison to healthy controls (p = 0.11). The proportion of PD1+CD8+ effector T cells was lower in trial patients relative to healthy controls both before and after treatment (p = 0.04 and 0.001, respectively). There was no difference in the proportion of PD1+CD8+ effector T cells among trial patients from D0 to D28 of treatment (p = 0.19). Patients who responded to treatment (n = 3), had a lower proportion of PD1+ CD8+ effector cells at the start of treatment relative to non-responders (p = 0.002). Conclusions: These clinical data suggest that the integration of pembrolizumab on an epigenetic backbone is safe and demonstrates encouraging responses in heavily treated patients with PTCL and CTCL. These correlative data suggest that specific cytokines (TNFα, MIP-3α, and IL-10) and changes in circulating T-cell subpopulations may have predictive value as biomarkers of disease response. The research was funded by: Merck and Co. Ongoing Trial Keywords: Aggressive T-cell non-Hodgkin lymphoma, Combination Therapies, Ongoing Trials Conflicts of interests pertinent to the abstract. N. N. Benani Consultant or advisory role: Vividion Therapeutics, Kymera, Secura Bio, Affirmed GmbH, Astellas Pharma, Acrotech Biopharma LLC M. E. Williams Consultant or advisory role: Astra Zeneca, Gilead, Janssen, Kite Pharma, Kymera, TG Therapeutics Honoraria: International Oncology Network, Research to Practice Research funding: Janssen, Kymera, Pharmacyclics J. S. Manavalan Research funding: Astex O. A. O'Connor Employment or leadership position: TG Therapeutics Stock ownership: TG Therapeutics E. Marchi Employment or leadership position: Myeloid Therapeutics Consultant or advisory role: Myeloid Therapeutics Honoraria: Kyowa Kirin Research funding: Merck and Co., Celgene/BMS, Astex, NomoCan Pharmaceuticals Other remuneration: University of Virginia (Patents & Royalties: 3062/170 PROV), Daiichi Sankyo (advisory board)

Oral squamous cell carcinoma-derived EVs promote tumor progression by regulating inflammatory cytokines and the IL-17A-induced signaling pathway

Inflammatory cytokines in the tumor microenvironment (TME) contribute to tumor growth, proliferation, and invasion, and tumor-derived extracellular vesicles (EVs) act as critical "messengers" of communication in the tumor microenvironment. The effects of EVs derived from oral squamous cell carcinoma (OSCC) cells on tumor progression and the inflammatory microenvironment are still unclear. Our study aims to investigate the role of OSCC-derived EVs in tumor progression, the imbalanced TME, and immunosuppression and their effect on the IL-17A-induced signaling pathway. EVs were isolated from the supernatant of a mouse OSCC cell line, SCC7. The effects of SCC7-EVs and the EV release-specific inhibitor GW4869 on the proliferation and migration of SCC7 cells were investigated in vitro by using CCK-8 and scratch wound healing assays. RT-qPCR and ELISA were performed to examine the alterations in cytokine levels. Then, a mouse xenograft model of OSCC was established by submucosal injection of SCC7 cells with or without SCC7-EV and GW4869 treatment. The effects of GW4869 and SCC7-EVs on xenograft tumor proliferation and invasion were investigated by tumor volume determination and histopathological examination. ELISA was used to investigate the changes in serum cytokine levels. Immunohistochemistry was adopted to analyze the alterations in the levels of inflammatory cytokines, immune factors, and crucial molecules in the IL-17A signaling pathway. SCC7-derived EVs increased the supernatant and serum levels of IL-17A, IL-10, IL-1β, and PD-L1, while GW4869 decreased those of TNF-α and IFN-γ. SCC7-EV treatment significantly increased xenograft tumor growth and invasion in mice but resulted in little liquefactive necrosis in tumors. However, GW4869 treatment significantly inhibited xenograft tumor growth but resulted in more liquefactive necrosis. SCC7-derived EVs decreased the expression level of PTPN2, suppressing the immune responses of CD8+T cells in vivo. Moreover, SCC7-EV treatment significantly enhanced the tumor expression levels of crucial molecules in the IL-17A pathway, including IL-17A, TRAF6 and c-FOS, whereas GW4869 treatment significantly reduced those levels in tumor tissues. Our results indicated that OSCC-derived EVs can promote tumor progression by altering the TME, causing an inflammatory cytokine imbalance, inducing immunosuppression, and contributing to overactivation of the IL-17A-induced signaling pathway. Our study might provide novel insights into the role of OSCC-derived EVs in tumor biological behavior and immune dysregulation.

Serum cytokine levels and other associated factors as possible immunotherapeutic targets and prognostic indicators for lung cancer.

Lung cancer is one of the most prevalent cancer types and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for 80-85% of all cancer incidences. Lung cancer therapy and prognosis largely depend on the disease's degree at the diagnosis time. Cytokines are soluble polypeptides that contribute to cell-to-cell communication, acting paracrine or autocrine on neighboring or distant cells. Cytokines are essential for developing neoplastic growth, but they are also known to operate as biological inducers following cancer therapy. Early indications are that inflammatory cytokines such as IL-6 and IL-8 play a predictive role in lung cancer. Nevertheless, the biological significance of cytokine levels in lung cancer has not yet been investigated. This review aimed to assess the existing literature on serum cytokine levels and additional factors as potential immunotherapeutic targets and lung cancer prognostic indicators. Changes in serum cytokine levels have been identified as immunological biomarkers for lung cancer and predict the effectiveness of targeted immunotherapy.

Serum cytokine variations among inpatients with major depression, bipolar disorder, and schizophrenia versus healthy controls: a prospective ‘true-to-life’ study

There is increasing evidence of the association between chronic low-grade inflammation and severe mental illness (SMI). The objective of our study was to assess serum cytokine levels (SCLs) at admission and discharge in a true-to-life-setting population of inpatients with major depression (MD), bipolar disorder (BD), and schizophrenia (Sz), as well as of healthy controls. We considered MD, BD, and Sz to be SMIs. We evaluated 206 inpatients [MD, N = 92; BD, N = 26; mania (Ma), N = 44; Sz, N = 44). Generalized estimating equations were used to analyze variations in SCL [interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin (IL)-2, IL-4, IL-6, IL-10, and IL-17] at hospital admission and discharge. Results of 100 healthy controls were compared with those of SMI patients at both time points. We evaluated patients' improvement during in-hospital treatment in terms of general psychiatric symptoms, global clinical impression, functionality, and manic and depressive symptoms with validated scales. In all, 68.9% of patients completed the study. Overall, SMI inpatients had higher SCL when compared with controls regardless of diagnosis. There was a significant decrease in Brief Psychiatric Rating Scale (BPRS) and Clinical Global Impression-Severity Scale (CGI-S) scores, and an increase in Global Assessment of Functioning (GAF) scores for all disorders evaluated (p < 0.001), as well as a significant decrease in HDRS-17 scores among MD inpatients (p < 0.001) and in YMRS scores among Ma inpatients (p < 0.001). IL-2 and IL-6 levels decreased significantly between admission and discharge only among MD inpatients (p = 0.002 and p = 0.03, respectively). We found no further statistically significant changes in SCL among the remaining disorders (BD, Ma, and Sz). There was no significant decrease in IFN-γ (p = 0.64), TNF-α (p = 0.87), IL-4 (p = 0.21), IL-10 (p = 0.88), and IL-17 (p = 0.71) levels in any of the evaluated diagnoses. MD inpatients had a decrease in IL-2 and IL-6 levels during hospitalization, which was accompanied by clinical improvement. No associations were found for the remaining SMIs (BD, Ma, and Sz).

Short-term impact of glycaemic control and intravitreal ranibizumab treatment on serum cytokine levels and diabetic macular edema in patients with unregulated blood glucose

Objective: To evaluate the short-term effect of glycaemic control and intravitreal ranibizumab treatment on diabetic macular edema (DME) and to assess the correlation between HbA1c and certain serum cytokines. Design: A prospective study of 43 participants with HbA1c levels exceeding 53 mmol/mol (7%) and with DME, as detected by spectral domain optical coherence tomography (SDOCT). Subjects: Participants were grouped according to their initial best corrected distance visual acuity (BCVA). Group 1 was treated with three monthly doses of intravitreal ranibizumab (0.5 mg) injections, and Group 2 was followed without treatment. Methods: Serum cytokine levels, including interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1(MCP-1), and vascular endothelial growth factor (VEGF) were analysed at the beginning and at the end of 3 months, using enzyme-linked immunosorbent assays (ELISA). Results: A significant decrease in macular thicknesses (except for one quadrant) was observed in Group. Changes in serum cytokine levels were not correlated with HbA1c decrease. Serum VEGF level was significantly increased in Group 1, despite the intravitreal treatment. Conclusion: Short-term glycaemic control alone had limited value in the treatment of DME. The therapeutic effect of intravitreal treatment on DME supports the role of the local cytokine milieu in the pathophysiology.

Fedratinib Induces Cytokine Changes Correlating with Clinical Response in Ruxolitinib Exposed Myelofibrosis Patients: Biomarker Analysis from the Freedom Trial

Background: Myelofibrosis (MF) is characterized by driver mutation (JAK2, CALR and MPL) mediated hyperactivity of the JAK-STAT signaling pathway, bone marrow fibrosis, impaired hematopoiesis, and splenomegaly /extramedullary hematopoiesis. Ruxolitinib (rux), a JAK inhibitor (JAKi), is the standard of care for higher risk MF patients. In 2019, the US FDA approved Fedratinib, a second JAKi that binds to both the ATP and substrate binding sites of the JAK2 kinase domain. The phase II JAKARTA2 study demonstrated that fedratinib is clinically efficacious in previously rux exposed patients. In the current work, through biomarker analysis of the phase 3b FREEDOM data, we sought to identify changes to the cytokine profile in fedratinib treated, previously rux exposed patients. In addition, we explored potential mechanism of fedratinib efficacy. Methods: In the FREEDOM study, patients (N=38) received fedratinib (400 mg/day) once daily in 28-day cycles until lack of efficacy, intolerance, or disease progression. Primary endpoint was proportion of subjects with ≥35% reduction of spleen volume assessed by magnetic resonance imaging (MRI) at the end of cycle 6 (SVR35). In the FREEDOM biomarker cohort, mutational detection was performed in immunomagnetically-enriched CD66+ myeloid cells using the MLL myeloid panel targeting 74 genes. Variants and indels were called using Pisces and Pindel, followed by annotation to COSMIC, ClinVar, gnomAd (non-cancer), and dbNSFP data bases. Clinically relevant somatic mutations were considered for analysis. Rules-Based Medicine's (RBM) HumanMAP v2.0 panel targeting 85 cytokines was used to measure changes in serum cytokine levels. 51 cytokines with variance > 0.3 on aggregated data were used for analysis. Pearson correlation analysis was used to compute Rho values between response and cytokine changes. Kinome screen was performed using the thermofisher Lanthascreen Eu kinase binding assay and the reaction biology kinome assay. Results and discussion Therapy related disease modifying effect in MF is currently being explored through ability to control variant allele frequency (VAF), reduce inflammation, minimize bone marrow fibrosis, in conjunction with restoration of normal hematopoiesis. In our study, JAK2 V617F mutation was detected in 48% (N=13/27) of total mutational biomarker cohort (N=27/38). However, response was independent of JAK2 V617F mutational status thereby decoupling this mutation with response/disease modification at cycle 6. As a next step, we evaluated if fedratinib treatment results in cytokine alterations that correlate with clinical response. Cytokine evaluation and paired analysis between cycle 1 and end of cycle 6 revealed increases in adiponectin, carcinoembryonic antigen (CEA), EPO and ferritin and decrease in EN-RAGE, IL-16, IL-18, IL1RA, TIMP-1, TNFR2, MPO, VCAM1 and VEGF (P < 0.001). Correlation analysis revealed adiponectin, CEA, ferritin, haptoglobin, erythropoietin and CCL18 to have significant correlation (P < 0.05) with response. The responders had the largest alteration in cytokine levels coupling these cytokines to response. Overall, fedratinib upregulates anti-inflammatory cytokines and reduces inflammatory cytokines which could contribute to symptom burden control and spleen volume reduction. These results in a rux exposed patient cohort suggests that fedratinib could have a differentiated profile from rux. To further investigate these differences, we performed kinome screens with two in vitro assays. Results indicate that fedratinib has a broader kinase inhibitory profile than rux. Thus, fedratinib could modulate additional signaling pathways explaining potential efficacy in rux exposed patients. Conclusion: Our biomarker analysis in previously rux exposed patients, shows that fedratinib triggers cytokine changes that correlate with response independent of mutational status. This suggests that at least at early timepoints, cytokine changes could be a potential biomarker for disease modifying effect. In addition, our in vitro analysis differentiates rux and fedratinib based on the inhibited kinases and altered biological pathways. Taken together our results indicate that while both rux and fedratinib are JAK inhibitors, they are well differentiated allowing fedratinib to be effective in previously rux exposed patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Hippocampal but Not Serum Cytokine Levels Are Altered by Traffic-Related Air Pollution in TgF344-AD and Wildtype Fischer 344 Rats in a Sex- and Age-Dependent Manner.

Epidemiological studies have demonstrated that air pollution is a significant risk factor for age-related dementia, including Alzheimer’s disease (AD). It has been posited that traffic-related air pollution (TRAP) promotes AD neuropathology by exacerbating neuroinflammation. To test this hypothesis, serum and hippocampal cytokines were quantified in male and female TgF344-AD rats and wildtype (WT) Fischer 344 littermates exposed to TRAP or filtered air (FA) from 1 to 15 months of age. Luminex™ rat 23-cytokine panel assays were used to measure the levels of hippocampal and serum cytokines in 3-, 6-, 10-, and 15-month-old rats (corresponding to 2, 5, 9, and 14 months of exposure, respectively). Age had a pronounced effect on both serum and hippocampal cytokines; however, age-related changes in hippocampus were not mirrored in the serum and vice versa. Age-related changes in serum cytokine levels were not influenced by sex, genotype, or TRAP exposure. However, in the hippocampus, in 3-month-old TgF344-AD and WT animals, TRAP increased IL-1ß in females while increasing TNF ɑin males. In 6-month-old animals, TRAP increased hippocampal levels of M-CSF in TgF344-AD and WT females but had no significant effect in males. At 10 and 15 months of age, there were minimal effects of TRAP, genotype or sex on hippocampal cytokines. These observations demonstrate that TRAP triggers an early inflammatory response in the hippocampus that differs with sex and age and is not reflected in the serum cytokine profile. The relationship of TRAP effects on cytokines to disease progression remains to be determined.

Treatment of back pain in active axial spondyloarthritis with serial locoregional water-filtered infrared A radiation: A randomized controlled trial.

BACKGROUND: Axial spondyloarthritis (axSpA) is an inflammatory rheumatic disease primarily affecting the axial skeleton.OBJECTIVE: To evaluate the short-term effects of locoregional water-filtered infrared A radiation (sl-wIRAR) in the treatment of lower back pain in patients with axSpA.METHODS: Patients with active axSpA with non-steroidal anti-inflammatory drug (NSAID) therapy undergoing a 7-day multimodal rheumatologic complex treatment in an in-patient setting were eligible. Patients were randomly assigned to the intervention group (IG) receiving sl-wIRAR treatment of the back (2 treatments/day for 30 min each for 6 days) or to the control group (CG) receiving no treatment. Primary outcome was a between-group difference in pain after sl-wIRAR therapy measured on a numeric rating scale (NRS) (0 no pain, 10 worst pain). Secondary outcomes included an assessment of i) the onset and development of analgesic effects and an evaluation of whether sl-wIRAR ii) improved axSpA-specific well-being and iii) influenced serum cytokine levels.RESULTS: Seventy-one patients were enrolled, completed the trial and were analyzed (IG: 36 patients, CG: 35 patients). In the IG, there was a statistically significant change ( 0.0005) in pain level [NRS] (1.6 1.9 [5; 2]) from baseline (4.1 2.4 [0; 8]) to trial completion (2.6 2.0 [0; 7]) and a significant difference to the CG ( 0.006). In the IG there was a significant improvement in axSpA-specific well-being (BAS-G) ( 0.006). A physiologically relevant change in serum cytokine levels could not be observed.CONCLUSION: sl-wIRAR treatment can be useful in the treatment of patients with active axSpA as it leads to a rapid reduction of pain.

Changes in dead space components during pressure-controlled inverse ratio ventilation: A secondary analysis of a randomized trial.

BackgroundWe previously reported that there were no differences between the lung-protective actions of pressure-controlled inverse ratio ventilation and volume control ventilation based on the changes in serum cytokine levels. Dead space represents a ventilation-perfusion mismatch, and can enable us to understand the heterogeneity and elapsed time changes in ventilation-perfusion mismatch.MethodsThis study was a secondary analysis of a randomized controlled trial of patients who underwent robot-assisted laparoscopic radical prostatectomy. The inspiratory to expiratory ratio was adjusted individually by observing the expiratory flow-time wave in the pressure-controlled inverse ratio ventilation group (n = 14) and was set to 1:2 in the volume-control ventilation group (n = 13). Using volumetric capnography, the physiological dead space was divided into three dead space components: airway, alveolar, and shunt dead space. The influence of pressure-controlled inverse ratio ventilation and time factor on the changes in each dead space component rate was analyzed using the Mann-Whitney U test and Wilcoxon’s signed rank test.ResultsThe physiological dead space and shunt dead space rate were decreased in the pressure-controlled inverse ratio ventilation group compared with those in the volume control ventilation group (p < 0.001 and p = 0.003, respectively), and both dead space rates increased with time in both groups. The airway dead space rate increased with time, but the difference between the groups was not significant. There were no significant changes in the alveolar dead space rate.ConclusionsPressure-controlled inverse ratio ventilation reduced the physiological dead space rate, suggesting an improvement in the total ventilation/perfusion mismatch due to improved inflation of the alveoli affected by heterogeneous expansion disorder without hyperinflation of the normal alveoli. However, the shunt dead space rate increased with time, suggesting that atelectasis developed with time in both groups.

Absence of TNF-α Promotes Anemia of Inflammation and Inflammatory Bowel Disease

Anemia of inflammation (AI) is a hallmark of a multitude of disorders including infections, autoimmune diseases and malignancies. The pathophysiology of AI is currently under investigation. While the hematological aspect of AI and the involvement of cytokines such as IL6, IL1α, IL1 β has been subject to extensive study, the inflammatory and immunological arm has only recently begun to be explored. This involves a complex interplay of cytokine mediators both pro and anti-inflammatory, mediating a broader innate and ultimately adaptive immune response that could potentially be linked to the hematological deficiencies seen under AI. In particular, highly elevated levels of the cytokine TNF a have been noted in inflammatory conditions, including an established acute mouse model of AI induced by a single injection of the heat killed pathogen Brucella abortus (BA). The latter results observed by us prompted us to furtherinvestigate the role of this cytokine in BA induced AI.To study the above we injected mice lacking TNFα (TNFα-/-) with BA and studied hematological, inflammatory and immunological changes in our model. Following a single injection of BA, we found that TNFα -/- mice develop an irreversible macrocytic, hyperchromic anemia with hemoglobin levels dropping as low as 7 g/dL within two weeks of BA injection and not returning to normal even after eight weeks unlike their WT counterparts. This is further characterized by a complete abolition of Bone Marrow (BM) erythropoiesis and extra medullary erythropoiesis in the spleen (SPL). We also found elevated serum levels of erythropoietin and iron, indicating that the resulting anemia is not caused by a lack of these factors. The anemia was accompanied by a fall in the lifespan of RBCs in peripheral blood and a concurrent increase in production of reactive oxygen species by progenitors and terminally differentiated RBCs. Moreover, our experiments using WT GFP+ RBCs in BA treated TNFα-/- mice indicate that even the healthy GFP+ RBCs are cleared faster in the latter animals. Preliminary data, suggest that macrophages derived from BA treated TNFα-/- express pro-inflammatory markers and that the same cells can consume more RBCs than macrophages from control animals, suggesting increased macrophagocytosis. Subsequently, we looked at changes in serum cytokine levels over time after injection of BA. We found that unlike WT animals injected with BA, TNFα-/- mice show a sustained production of IFNγ, IL12-p40 and IL1β. We also found an expansion of splenic resident macrophages and T cells which are cell types implicated in the production of the cytokines mentioned previously. Finally, to extend the reach of this model to other disorders we treated TNFα-/- mice with low doses of dextran sodium sulfate (DSS) and found that only these animals developed inflammatory bowel disease (IBD) characterized by blood in the feces and severe anemia, unlike WT animals treated the same way.Our observations on the upregulation of IFNγ and IL12 is leading us to study macrophage activation and T helper cell polarization in the BM, SPL and lymph nodes. Moreover, we are also investigating the potential crosstalk between these effectors, cytokines and erythropoiesis looking at oxidative stress, survival and cell differentiation of erythroid progenitors. As a first step, we are investigating the recovery from BA of IFNγ-/- mice, as TNFα-/- mice with AI show a significant upregulation of IFNγ. Preliminary complete blood count of BA treated IFNγ -/- mice indicates that these animals do recover from an inflammatory insult and the anemia is reversible, unlike the TNFα-/- mice. These results suggest that absence of IFNγ could potentially rescue the severely impaired phenotype that we found in our TNFα-/- mice and the recovery in BA treated TNFα/IFNγ double knock out mice.Given the history of TNFα in enabling inflammatory reactions and promoting cell death, the view of this cytokine in supporting recover under AI as inferred from our results is relatively novel and suggest caution in the use of TNFα inhibitors. Furthermore, our data point to an important role in the crosstalk between TNFα and INFγ and in their effect on erythropoiesis. DisclosuresNo relevant conflicts of interest to declare.

Dynamic Serial Cytokine Measurements During Intravenous Ca-DTPA Chelation in Gadolinium Deposition Disease and Gadolinium Storage Condition: A Pilot Study.

The aim of this study was to investigate the feasibility of measuring early changes in serum cytokine levels after intravenous diethylenetriaminepentaacetic acid (Ca-DTPA) chelation in patients manifesting either gadolinium deposition disease (GDD) or gadolinium storage condition (GSC) and the possible usefulness of this method in further research. Four patients with recent-onset GDD (≤1 year) and 2 patients with long-standing GSC (4 and 9 years) underwent chelation with intravenous bolus administration of Ca-DTPA. Multiple blood draws were performed to measure serum cytokines: at T = 0 (before Ca-DTPA injection) and 1, 5, 10, 30, 60 minutes, and 24 hours after Ca-DTPA injection. Patients rated the severity of GDD symptom flare at 24 hours. The 24-hour urine Gd amounts were measured prechelation and for the 24 hours after chelation. Serum samples were analyzed blind to whether patients had GDD or GSC but with knowledge of the time points characterizing each sample. Urine samples for both GDD and GSC patients showed increases in Gd postchelation. All GDD patients experienced flare reactions postchelation; the 2 GSC patients did not. Two cytokines, EGF and sCD40L, peaked at 30 minutes postchelation in at least 4 of the 6 participants. Three cytokines, ENA78/CXCL5, EOTAXIN/CCL11, and LEPTIN, peaked at 24 hours in at least 4 of the 6 participants. Two participants were high outliers for a large number of cytokines across time points. No clear distinction between GDD and GSC was apparent from the cytokine patterns, although differences were present. This pilot study describes precise temporal resolution (in the range of minutes) after a cytokine-inciting event. Select cytokines exhibited peak values at different time points. At this preliminary stage of investigation, peak cytokine release seems to reflect the amount of Gd mobilized rather than the severity of the patient symptomatic reaction. Too few subjects were studied to support statistical analysis between GDD and GSC groups, although differences were observed through visual data analysis.

OP0045 DELINEATION OF A PROINFLAMMATORY CYTOKINE PROFILE TARGETED BY JAK1/2 INHIBITION USING BARICITINIB IN A PHASE 2 SLE TRIAL

Background:Given the unmet clinical needs in systemic lupus erythematosus (SLE), including poor disease control and drug toxicities, new therapies are needed. In a phase 2, randomized, placebo-controlled, double-blind study (JAHH), once-daily baricitinib (bari) resulted in significant clinical improvement in patients (pts) with active SLE versus PBO. Bari inhibits JAK1 and JAK2 signalling, and in turn may affect STAT1, STAT2, STAT4 pathways. Therefore, bari has the potential to simultaneously impact several pro-inflammatory immune cytokines implicated in the pathogenesis of SLE, including IFN-α, IFN-γ, IL-6, IL-12, and IL-23.Objectives:The objectives of the current study were: 1) to examine baseline serum cytokines in the JAHH phase 2 clinical trial for correlations with clinical or immunologic assessments; 2) to determine if changes in serum cytokine levels were associated with bari treatment.Methods:Pts enrolled in the JAHH phase 2 trial received daily treatment with PBO, bari 2 mg, or bari 4 mg through week 24. Serum samples were collected at baseline (week [wk] 0), wk 12, and wk 24) from SLE pts (n=270) and 50 sex- and age-matched controls. Samples were analyzed for: IL-2, IL-3, IL-5, IL-6, IL-10, IL-17A, IL-21, IL-12/23p40, IL-12p70, GM-CSF, IFN-α and IFN-γ using ultrasensitive quantitative assays. IFN gene signature, autoantibodies, C3 and C4 were measured as previously described [1].Results:At wk 0, serum IL-17A, IL-12/23p40, IL-6, IFN-γ and IFN-α were readily detectable. IL-12/23p40 was detectable in 100% of pts vs. 100% of controls, IFN-γ in 89% of pts vs. 66% of controls, IL-6 in 53% of pts vs. 12% of controls and in IFN-α 41% of pts vs. 2% of controls; detection of serum IL-2, GM-CSF, IL-5, IL-10 and IL-17A was variable (Fig 1). At baseline (wk 0), IL-12/23p40 was positively correlated with SLEDAI and IFN gene signature and negatively correlated with serum C4. IL-6 was positively correlated with joint swelling, joint tenderness, IFN-γ and C3. Serum IFN-α was positively correlated with serum IFN-γ, anti-Sm and anti-RNP, and the IFN gene signature (Fig 2). Treatment with bari 4 mg (Fig 1B) significantly decreased serum IL12/23p40 and IL-6 cytokine levels at wk 12 (p&lt;0.05) but not serum IFN-α or IFN-γ levels (Fig 1B).Figure 1.* p = 0.015; ** p = 0.001; Abbreviations: LLOQ, Lower limit of quantification.Figure 2.Abbreviations: Anti-dsDNA, Anti-double stranded DNA; Anti-RNP, Anti-ribonucleoprotein; CLASI, Cutaneous lupus erythematosus disease area and severity index; SLEDAI, SLE disease activity index.Conclusion:Bari 4 mg treatment was associated with statistically significant decreases of serum IL-12/23p40 and IL-6 at week 12 which continued through week 24. Serum IFN-α or IFN-γ were not reduced with bari treatment. Thus, bari 4 mg simultaneously impacted multiple pro-inflammatory cytokines implicated in the pathogenesis of SLE.

Effect of Zataria multiflora on serum cytokine levels and pulmonary function tests in sulfur mustard-induced lung disorders: A randomized double-blind clinical trial.

Zataria multiflora (Z. multiflora) belongs to the Lamiaceae family and has several traditional uses owing to its antiseptic, aesthetic, antispasmodic, analgesic, and antidiarrheal properties. We aimed to investigate the effect of Z. multiflora on serum cytokine levels and pulmonary function tests (PFT) in patients exposed to sulfur mustard (SM) for a long term (27-30 years). Thirty-five patients were randomly assigned to the placebo group (P) and two experimental groups treated with Z. multiflora extracts, i.e., 5 and 10 mg/kg/day (Z5 and Z10). Serum levels of cytokines including IL (2, 4, 6, 8, and 10) and IFN-γ as well as PFT indices such as maximum mid-expiratory flow (MMEF) and maximum expiratory flow at 25, 50, and 75% of vital capacity (VC) (MEF25, 50, and 75) were assessed at the beginning (phase 0) and at the end of 4 and 8 weeks (phases I and II, respectively) after starting the treatment. Serum levels of IL-2, IL-6, and IL-8 were significantly decreased, while serum levels of IL-10 and IFN-γ were significantly increased in the Z5 and Z10 treatment groups in phases I and II as compared to those in phase 0 (p < 0.05 to p < 0.001). MMEF and MEF25, 50, and 75 values were significantly increased in the Z5 group in phase II and in the Z10 group in phases I and II compared to those in phase 0 (p < 0.05 to p < 0.001). The percent change in serum cytokine levels and the change in MEF25, 50, and 75 during the two-month treatment period were significantly higher in the treatment groups than in the placebo group. Two months of treatment with Z. multiflora reduced inflammation, while it enhanced anti-inflammatory cytokines and improved PFT indices in SM-exposed patients.

Accelerated elimination of human cancer cells by a CD40 agonist antibody combined with a PD-1 antagonist in CD4-depleted mice.

The elimination of residual microscopic cancer cells is important cancer treatment. The immunoediting theory describes the balance between the immune system and cancer cells. The current study investigated changes in the immune system during the elimination of cancer cells and evaluated the influence of cluster of differentiation (CD)4 or CD8 depletion. A human squamous cell cancer cell line (SNU1041) was injected in the lateral tongue of immunocompetent mice and the changes in the CD4, CD8, CD11b, CD19, CD40 and CD40 ligand (L) populations in the blood, lymph nodes and spleen were evaluated using flow cytometry, and changes in serum cytokine levels were evaluated using a magnetic bead panel. Cancer cell elimination was delayed by CD4 depletion but not by CD8 depletion. The CD8-depleted group indicated increased levels of CD40L, interferon-gamma, interleukin (IL)-10, IL-6, and tumor necrosis factor-α. It was concluded that CD4 served a crucial role in the elimination of human cancer cells. Furthermore, the efficacies of CD40 agonist and programmed cell death protein 1 (PD1) antagonist treatments were assessed in CD4-depleted mice. CD40 agonist treatment resulted in faster cancer cell elimination and increased cytokine excretion. In conclusion, CD4 or CD40L significantly influenced cancer elimination. CD40 agonist antibodies may be potent adjuvant agents that can be used in patients with reduced CD4 or CD40L expression.

The Effect of a 30-Min Water-Pipe Smoking Session on Cognitive Measures and Cardio-Pulmonary Parameters.

One session of water-pipe tobacco smoking (WPS) can increase carboxyhemoglobin (COHb) to levels comparable to those reported in carbon monoxide poisoning, which may cause memory impairment and confusion. A prospective study evaluating healthy volunteers pre- and post-30 min of WPS session. Primary outcome parameters were executive cognitive measures [digit span test and Paced Auditory Serial Addition Test (PASAT)]. The effect of repeated cognitive testing 30 min apart without WPS was evaluated in age- and sex-matched healthy volunteers. Secondary outcome parameters included cardio-pulmonary, COHb, serum nicotine, and cytokine changes. Thirty-five subjects aged 25.6 ± 4.5 years smoked water-pipe for a 30-min session. Control group included 20 subjects aged 25.2 ± 5.1 years. Digit span test median score decreased after WPS (16 and 15, respectively, p = .003), insignificant decrease in controls. Median PASAT score increased after WPS (49 and 52, respectively, p = .009); however, a much larger significant increase was observed in controls (p ≤ .001). One WPS session resulted in significant increases in heart and respiratory rates and significant decrease in FEF25-75%. Post WPS, median COHb levels increased (from 2.2% to 10.7%, p < .0001) as did median serum nicotine levels (from 1.2 to 26.8 ng/mL, p < .0001). Serum cytokines levels: IL-2 and IL-6 increased (p < .0001 for each), and IL-10 and IL-5 decreased (p < .0001 and p = .04, respectively). One session of WPS resulted in significant negative effects on cognitive executive measures, significant increases in COHb and serum nicotine levels, and significant changes in serum cytokines. Our findings call for increasing awareness towards the possible consequences of cognitive alterations following a 30-min session of WPS. One 30-min session of water-pipe smoking resulted in negative effects on executive cognitive measures, increased carboxyhemoglobin and serum nicotine, and significant changes in serum cytokine levels. This study adds to the accumulating evidence on the harmful effects of water-pipe smoking, a growing epidemic, and calls for awareness of its possible consequences of acute cognitive alterations.