Serum Cytokine Assay Research Articles

Macleaya cordata extract improves egg quality by altering gut health and microbiota in laying hens

This study investigated the effect of Macleaya cordata extract (MCE) on the performance, gut health, and microbiota of laying hens. A total of 192 thirty-wk-old Hyline brown laying hens were randomly divided into 4 treatment groups. The CON group received a basal diet, while the low (MCE250), medium (MCE350), and high (MCE450) dose groups were supplemented with 250, 350, and 450 mg/kg MCE, respectively. The egg weight and Haugh unit demonstrated a linear and quadratic increase with the MCE dose during the initial 4-wk period of the experiment (P < 0.05). Furthermore, the dietary supplementation of MCE led to a significant enhancement in eggshell thickness and Haugh unit at wk 8 and the data showed a statistically significant linear and quadratic increase (P < 0.05). Serum cytokine assay showed that dietary supplementation of MCE led to linear and quadratic increases in IL-4 and IL-10 level (P < 0.05). Dietary supplementation of 350 and 450 mg/kg MCE was observed to result in linear and quadratic increase in serum lysozyme levels (P < 0.05). The addition of MCE to the diet resulted in a linear and quadratic increase in the levels of sIgA in the jejunum and ileum (P < 0.05). In terms of gene expression, the addition of MCE to the diet resulted in linear and quadratic increases in the expression of IL-10, IgA, Serpinb14, Serpinb14B, and OIH (P < 0.05). The expression of jejunal genes pIgR and IL-4 was observed to increase in a linear and quadratic manner, respectively, following the dietary addition of 350 mg/kg MCE and IL-1β decreased in a linear manner (P < 0.05). Moreover, these favorable effects were maximized at medium dosage (350 mg/kg) of MCE addition, and intestinal microbial composition in the control and MCE350 groups was assessed. 350 mg/kg MCE increased the relative abundance of Bryobacter and Parasutterella and decreased the relative abundance of Erysipelatoclostridium in the cecum (P < 0.05). Spearman correlation analysis revealed that Bryobacter, Parasutterella, Skermanella, and Erysipelatoclostridium were associated with nonspecific immune functions (P < 0.05). In conclusion, 350 mg/kg MCE supplementation elevated the immune response, and upregulated the expression of genes related to protein production in eggs, thereby improving egg quality. These effects may be associated with changes in the microbiota, specifically Bryobacter, Parasutterella, and Erysipelatoclostridium.

Serum cytokine panels in pediatric clinical practice

BackgroundCytokines are soluble signaling proteins that regulate inflammation and coordinate immune responses. Serum cytokine assays are increasingly used in medical practice, yet our understanding of cytokines as biomarkers for disease remains limited. ObjectivesWe aimed to analyze real-world single center usage of a multiplexed cytokine panel, correlate its results with diagnosis and severity, and explore its utility in pediatric practice. MethodsA multiplexed cytokine panel, able to return same-day results, was implemented in April 2020 at our institution and its performance was validated for clinical use. Coded patient data were collected using a REDCap database, and correlations between cytokine levels and outcomes of interest were analyzed retrospectively. ResultsCytokine levels correlate with acuity of care, with patients admitted to the pediatric intensive care unit (PICU) having the highest cytokine values. Patients with familial HLH (fHLH) showed prominent peaks in IFNγ, IL-10, and TNF, while patients with sepsis exhibited high IL-6 and IL-8 with relatively modest IFNγ, and cytokine release syndrome (CRS) post-CAR T cell therapy often demonstrated pan-panel positivity at peak levels, with a similar pattern as that of fHLH. A ratio of [IFNγ]+[IL-10]/[IL-6]+[IL-8] levels was able to distinguish fHLH and CRS from severe sepsis. ConclusionsCytokine levels correlate with severity of illness and can help differentiate between syndromes that present similarly, including fHLH and CRS compared to sepsis. Cytokine panels can be used as biomarkers to inform diagnosis and management decisions, but significant work remains to dissect complex clinical patterns of disease.

The Roles of IL-18 in a Realistic Partial Body Irradiation with 5% Bone Marrow Sparing (PBI/BM5) Model.

IL-18 has been shown to play important roles in response to total body irradiation. However, homogenous total body irradiation is not a realistic model to reflect the radiation exposure in a real nuclear event. To further study the roles of IL-18 in a real nuclear scenario, we developed a mouse partial body irradiation with 5% bone marrow sparing (PBI/BM5) model to mimic the inhomogeneous radiation exposure. We established the dose response curves of PBI/BM5 using different radiation doses ranging from 12 to 16 Gy. Using the PBI/BM5 model, we showed that IL-18 knockout mice were significantly more radiation resistant than the wild-type mice at 14.73 Gy. We further studied the hematopoietic changes using a complete blood count, bone marrow colony-forming assays, and serum cytokine assays on the mice exposed to PBI/BM5 with IL-18BP treatment and wild-type/IL-18 knockout mice. In conclusion, our data suggest that IL-18 plays important roles in mouse survival in a realistic nuclear exposure model, potentially through the IL-18/IFNγ pathway.

High Fat High Sugar Diet Reduces Small Intestinal Secretion by Sex-Dependent Mechanisms.

The goal of this study was to determine the influence of high-fat high-sugar diet (Western diet) on intestinal function and subsequently to determine if there were any beneficial effects of exercise, genistein (a naturally occurring phytoestrogen) or both, on the intestine. We measured transepithelial short circuit current (Isc), across freshly isolated segments of jejunum from male and female C57Bl/6J mice randomly assigned to one of the following groups for the 12-week study duration: high-fat high-sugar diet (HFS), HFS with genistein (Gen), HFS with exercise (Ex), or HFS with both genistein and exercise (Gen+Ex) and compared them to lean controls. Genistein concentration was 600 mg genistein/kg diet. Exercise comprised of moderate intensity treadmill running (150 min per week). At the completion of the study, segments of jejunum were frozen for western blot determination of key proteins involved in secretory and absorptive functions, as well as senescence. Intestinal morphology was assessed. Serum cytokine assays were performed. Basal Isc was significantly decreased (by 70%, P<0.05) in HFS females and males versus leans. This decrease was partially mitigated by exercise in both sexes. In females, the HFS-induced decrease in Isc was attributed to a significant loss of CLC2, NKCC1 and CFTR expression whereas in males this was due to a significant loss of Na/K-ATPase, KCa and NKCC1 expression (indicating sex-dependent mechanisms). Exercise mitigated most of the loss of Isc in both sexes. Our data suggested that A2BR levels were dysregulated in HFS fed mice and that concomitant treatment with Gen or Gen+Ex prevented this disruption in females only. Inflammatory state was associated with body weight changes. Our data suggests that the reduced basal jejunal Isc in HFS mice is attributed to sex-dependent mechanisms and while exercise partially mitigated this, it's mechanism of action was unclear. Improved understanding of Western diet induced intestinal dysfunctions may allow for the development of novel drug targets to treat gastrointestinal disturbances in diabetic obesity.

Qingyi granules ameliorate severe acute pancreatitis in rats by modulating the gut microbiota and serum metabolic aberrations

Context Qingyi granules can be used to effectively treat patients with severe acute pancreatitis (SAP). Objective To elucidate the role of gut microbiota-mediated metabolism in the therapeutic effects of Qingyi granules. Materials and methods Sprague–Dawley rats were grouped into the sham operation, SAP model, Qingyi granule intervention (Q, 1.8 g/kg) and emodin intervention (E, 50 mg/kg) groups and observed for 24 h. H&E staining and ELISA were used for histopathological analysis and serum enzyme and cytokine assays. 16S rDNA sequencing and UHPLC-HRMS were used for gut microbiota analysis and untargeted metabolomics. Results In SAP rats, Qingyi granules decreased the pancreatic pathological score (Q, 7.4 ± 1.14; SAP, 11.6 ± 1.14, p < 0.01); serum amylase (Q, 121.2 ± 6.7; SAP, 144.3 ± 8.86, p < 0.05), lipase (Q, 566 ± 20.34; SAP, 656.7 ± 29.32, p < 0.01), and diamineoxidase (Q, 492.8 ± 26.08; SAP, 566.1 ± 26.83, p < 0.05) activities; and IL-1β (Q, 29.48 ± 0.88; SAP, 36.17 ± 1.88, p < 0.01), IL-6 (Q, 112.2 ± 3.57; SAP, 128.9 ± 9.09, p < 0.05) and TNF-α (Q, 215.3 ± 8.67; SAP, 266.4 ± 28.03, p < 0.05) levels. SAP induced Helicobacter and Lactobacillus overgrowth and suppressed Romboutsia and Allobaculum growth and caused aberrations in bacterial metabolites, which were partly reversed by Qingyi granules. Discussion and Conclusions Qingyi granules can modulate the gut microbiota and metabolic abnormalities to ameliorate SAP. Multi-omics approaches allow systematic study of the pharmacological mechanisms of compound prescriptions for critical illnesses.

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

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

Chimeric Antigen Receptor Engineering of Mesenchymal Stromal Cells (CAR-MSC) Enhance Immunosuppression and Outcomes in Graft Versus Host Disease (GvHD) Preclinical Models

Mesenchymal stromal cells (MSCs) have attracted attention as a treatment for graft vs host disease (GvHD) given their immunosuppressive and off-the-shelf properties. Although MSCs are well-tolerated in GvHD patients, therapeutic efficacy is inconsistent due to insufficient immunosuppression at inflamed tissue sites. We therefore proposed engineering MSCs with chimeric antigen receptors (CAR) to enhance immunosuppressive efficacy at sites of inflammation. In GvHD, donor T cells attack host epithelial tissues in the skin, liver, and intestinal tract in part through the interaction of T cell integrins with E-cadherin (Ecad) expressed on epithelia. We thus hypothesized that redirecting MSCs with an Ecad-targeted CAR (EcCAR-MSCs) would lead to enhanced antigen-specific immunosuppression at these sites of inflammation. Therefore, we transduced adipose-derived MSCs with CAR containing anti-Ecad (human/canine cross-reactive) scFv with CD28ζ signaling domain generating EcCAR-MSCs at high efficiency (>90%). CD28 signal was selected for its ability to activate downstream immunosuppressive factors To test their immunosuppressive function, we cocultured EcCAR-MSCs for 24 hours with activated donor T cells with or without CAR antigen-specific soluble Ecad stimulation. Cocultures with antigen-stimulated EcCAR-MSCs led to significant T cell suppression (p=.0023) as compared to unstimulated EcCAR-MSCs and untransduced MSCs (UTD-MSCs). To verify enhanced immunosuppression found in vitro and test therapeutic efficacy in vivo, we tested EcCAR-MSCs in GvHD xenograft models. GvHD was induced in NOD-SCID-γ-/- mice via intravenous injection of human peripheral blood mononuclear cells (25 x 106 cells/mouse). We then treated mice with EcCAR-MSCs (1x106 cells), UTD-MSCs, or vehicle control intraperitoneally. Mice were monitored for weight loss, clinical GvHD symptom score (diarrhea, activity, posture, fur, and skin integrity), human T cell counts in peripheral blood, and survival outcomes. EcCAR-MSCs ameliorated weight loss (Fig.1), clinical GvHD score (3 vs 5.2 score, p=.0014), T cell suppression (60 vs 450cells/uL, p=.0039), and Treg induction (6.7 vs 2.1% in CD4+ cells, p=.0009), leading to improved overall survival (Fig. 2). Mechanisms underlying superior immunosuppression in EcCAR-MSCs upon CAR stimulation were interrogated via RNA sequencing, serum cytokine assays, and immunophenotype analyses. RNA sequencing evaluated gene signatures across Ecad-stimulated and unstimulated EcCAR-MSC and UTD-MSC groups. CAR CD28-specific stimulation was supported by the upregulation of downstream immunosuppressive gene targets such as NFkB (p=.0002) and JUN (p=.0082) found only in Ecad-stimulated EcCAR-MSCs. Furthermore, gene set enrichment analysis supported TNFαactivation via NFκB (p<.0001) and IL-10 anti-inflammatory pathway (p=.036) enrichment in EcCAR-MSCs upon Ecad stimulation. To corroborate transcriptional enrichments, serum cytokines were analyzed. Here, mice treated with stimulated EcCAR-MSCs vs UTD-MSCs revealed elevated anti-inflammatory serum cytokines, such as IL-10 (76 vs 13pg/mL, p<.0001), TNFα (35 vs 10pg/mL, p<.0001), and G-CSF (95 vs 50pg/mL, p=.0064). Immunophenotypic analyses revealed upregulated T cell inhibitory receptor expression on stimulated EcCAR-MSCs vs UTD-MSCs, including programmed death-1 (12 vs 0.7%, p=.0087) and galectin-9 (67 vs 17%, p<.0001). Collectively, our mechanistic studies support enhanced EcCAR-MSC immunosuppression through immunosuppressive gene pathway enrichment, increased anti-inflammatory cytokine secretion, and upregulated inhibitory receptors upon antigen-specific stimulation. Finally, to demonstrate EcCAR-MSC safety, we ruled out unwanted MSC differentiation by flow cytometry (Maintenance of CD105+, CD90+, CD73+, CD34-, and CD14- stemness phenotype) and RNAseq (stem cell pathway enrichment, p<.0001) . Safety of EcCAR-MSCs were also verified in large animal models using healthy canines through intraperitoneal EcCAR-MSC injection (2x106 cells/kg), with no reported weight loss, hematological toxicity, or organ damage observed 28 days following administration. In summary, our findings position CAR-MSCs as a safe and novel therapeutic platform to enhance MSC antigen-specific immunosuppressive functionality for improved treatment outcomes in GvHD and other autoimmune diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparative analysis of the immune response to RFA and cryoablation in a colon cancer mouse model

The immune response to radiofrequency ablation (RFA) and cryoablation (CRA) was characterized and compared in a colon cancer mouse model. All studies were conducted under a research protocol approved by the National Institutes of Health, Clinical Center, Animal Care and Use Committee. BALB/cJ mice were inoculated with CT26 cells, and randomized to RFA, CRA, or sham treatment. Mice were sacrificed 3 days post-treatment, and tumor, spleen, and serum were harvested. Cell death was determined by Caspase-3 immunohistochemical and TUNEL stains. Immune response was analyzed using flow cytometry, serum cytokine assay and immunohistochemistry. Cell death, necrosis, and apoptosis induced by ablation were comparable in RFA and CRA. Decreased frequency of systemic T-regulatory cells was found in the CRA group. Both RFA and CRA reduced frequencies of several myeloid-derived suppressor cell (MDSC) subpopulations. RFA induced pro-inflammatory cytokine secretion including TNF-α and IL-12 as well as anti-inflammatory cytokines IL-5, and IL-10. CRA augmented secretion of a wider array of cytokines compared to RFA with both pro- and anti-inflammatory properties including IL-1β, IL-5, IL-6, IL-10, and KC GRO. In the tumor microenvironment, RFA reduced the number of T-regulatory cells, a finding not observed with CRA. Reduction of immune suppression via decreases in T-regulatory cells and MDSC was found to be induced by RFA or CRA. CRA augmented a wider range of cytokines than RFA, which were mainly pro-inflammatory, but also anti-inflammatory. In the tumor microenvironment, RFA demonstrated more pronounced anti-tumoral immunity. Further delineation of specific immunomodulation induced by ablation could inform drug-device development and may play a role in future hypothesis-driven immunomodulatory paradigms that combine immunotherapy drugs with tumor destruction for the treatment of metastatic colon cancer.

Longitudinal Immune Cell Profiling in Patients With Early Systemic Lupus Erythematosus.

To investigate the immune cell profiles of patients with systemic lupus erythematosus (SLE), and to identify longitudinal changes in those profiles over time. We employed mass cytometry with 3 different panels of 38-39 markers (an immunophenotyping panel, a Tcell/monocyte panel, and a B cell panel) in cryopreserved peripheral blood mononuclear cells (PBMCs) from 9 patients with early SLE, 15 patients with established SLE, and 14 controls without autoimmune disease. We used machine learning-driven clustering, flow self-organizing maps, and dimensional reduction with t-distributed stochastic neighbor embedding to identify unique cell populations in early SLE and established SLE. We used mass cytometry data of PBMCs from 19 patients with early rheumatoid arthritis (RA) and 23 controls to compare levels of specific cell populations in early RA and SLE. For the 9 patients with early SLE, longitudinal mass cytometry analysis was applied to PBMCs at enrollment, 6 months after enrollment, and 1 year after enrollment. Serum samples were also assayed for 65 cytokines using Luminex multiplex assay, and associations between cell types and cytokines/chemokines were assessed. Levels of peripheral helper T cells, follicular helper T (Tfh) cells, and several Ki-67+ proliferating subsets (ICOS+Ki-67+ CD8 T cells, Ki-67+ regulatory T cells, CD19intermediate Ki-67high plasmablasts, and PU.1high Ki-67high monocytes) were increased in patients with early SLE, with more prominent alterations than were seen in patients with early RA. Longitudinal mass cytometry and multiplex serum cytokine assays of samples from patients with early SLE revealed that levels of Tfh cells and CXCL10 had decreased 1 year after enrollment. Levels of CXCL13 were positively correlated with levels of several of the expanded cell populations in early SLE. Two major helper T cell subsets and unique Ki-67+ proliferating immune cell subsets were expanded in patients in the early phase of SLE, and the immunologic features characteristic of early SLE evolved over time.

CSF-1R inhibitor, pexidartinib, sensitizes esophageal adenocarcinoma to PD-1 immune checkpoint blockade in a rat model.

Esophageal adenocarcinoma (EAC) is a leading cause of cancer deaths. Pexidartinib, a multi-gene tyrosine kinase inhibitor, through targeting colony-stimulating factor 1 (CSF-1) receptor (CSF-1R), down modulates macrophage-mediated pro-survival tumor signaling. Previously, CSF-1R inhibitors have successfully shown to enhance antitumor activity of PD-1/PD-L1 inhibitors by suppressing tumor immune evasion, in solid tumors. In this study, we investigated the antitumor activity of pexidartinib alone or in combination with blockade of PD-1 in a de novo EAC rat model. Here, we showed limited toxicity with significant tumor shrinkage in pexidartinib treated animals compared to controls, single agent and in combination with a PD-1 inhibitor, AUNP-12. Suppression of CSF-1/CSF-1R axis resulted in enhanced infiltration of CD3 + CD8 + T cells with reduced M2 macrophage polarization, in the tumor microenvironment (TME). Endpoint tissue gene expression in pexidartinib treated animals demonstrated upregulation of BAX, Cas3, TNFα, IFNγ and IL6 and downregulation of Ki67, IL13, IL10, TGFβ and Arg1 (P &lt; 0.05). Additionally, among the pexidartinib treated animals responders compared to nonresponders demonstrated a significant upregulation of pretreatment CSF-1 gene, confirming that tumor-associated macrophage suppression directly translates to clinical benefit. Moreover, a posttreatment serum cytokine assay exhibited similar systemic trends as the gene expression in the TME, depicting increases in proinflammatory cytokines and decreases in anti-inflammatory cytokines. In conclusion, our study established a promising combinatorial strategy using a CSF-1R inhibitor to overcome resistance to PD-1/PD-L1 axis blockade in an EAC model, providing the rationale for future clinical strategies.

No Difference Among Inhaled Anesthetics on the Growth and Metastasis of Murine 4T1 Breast Cancers in a Mouse Model of Spontaneous Metastasis.

Objective: This study evaluates the effect of the commonly used inhaled anesthetics isoflurane, sevoflurane, and desflurane on the viability and migration of murine 4T1 breast cancer cells, the growth, and lung metastasis in a syngenetic model of spontaneous metastasis. Methods: The murine 4T1 breast cancer cells were exposed to isoflurane (2%), sevoflurane (3.6%), or desflurane (10.3%) for 3 h. Cell viability was measured using the MTT assay. The migratory capacity of 4T1 cells was assessed using a scratch assay after 24 h incubation. Female balb/c mice were subjected to orthotopic implantation of 4T1 cells under anesthesia with one of the inhaled anesthetics: 2% isoflurane, 3.6% sevoflurane, or 10.3% desflurane. Subsequently, resection of primary tumors was performed under the identical anesthetic used during implantation for 3 h. Three weeks later, the mice were euthanized to harvest lungs for ex vivo bioluminescent imaging and histological analysis. Blood was collected for serum cytokine assays by ELISA. Results: There was no difference in cell viability among isoflurane, sevoflurane, desflurane, and control groups (n = 180 for each group, P = 0.648). Sevoflurane but not isoflurane or desflurane significantly increased the migration of 4T1 cells compared to the control group (n = 18, P = 0.024). There was no difference in the growth of the orthotopically implanted primary tumors (n = 12 for the isoflurane group, n = 11 for the sevoflurane group, and for the desflurane group, P = 0.879). Surgical dissection of primary tumors in mice under anesthesia with isoflurane, sevoflurane, or desflurane led to no difference in lung metastasis following surgery (P = 0.789). No significant difference was observed among isoflurane, sevoflurane, and desflurane groups in the serum levels of IL-6 (P = 0.284), CCL-1 (P = 0.591), MCP-1 (P = 0.135), and VEGF (P = 0.354). Conclusion: Our study demonstrated that sevoflurane increased the migration of 4T1 breast cancer cells in vitro. Inhaled anesthetics isoflurane, sevoflurane, and desflurane had no difference on the growth of primary tumor and the lung metastasis of 4T1 cells in the mouse model of spontaneous metastasis with surgical removal of primary tumors.

CSF-1R inhibitor, pexidartinib, sensitizes esophageal cancer to PD-1 immune checkpoint blockade in a rat model.

333 Background: Esophageal adenocarcinoma (EAC) is a leading cause of cancer deaths. Pexidartinib, is a multi-gene tyrosine kinase inhibitor, targeting FLT3, KIT and CSF1R. In particular, pexidartinib by targeting CSF-1R down modulates macrophage mediated pro-survival tumor signaling. Recently, CSF-1R inhibitors have successfully shown to enhance antitumor activity of PD-1/PD-L1 inhibitors by suppressing tumor immune evasion, in multiple solid tumors. Methods: Seventy-two rats underwent an end-to-side esophagojejunostomy to induce gastroesophageal reflux, resulting in EAC carcinogenesis. At 32 weeks postoperatively, tumor bearing animals were randomized to a dose of 30mg/kg of pexidartinib or vehicle control, 5 days on and 2 days off (14 day cycle), for a total of 3 cycles. A PD-1 inhibitor, AUNP-12, at a dose of 3mg/kg or placebo, was administered on day 12 of each cycle. At 38 weeks postoperatively animals were euthanized. Safety and efficacy was evaluated by objective health assessments, pre and posttreatment MRI, RT-PCR, immunofluorescent labeling and a serum cytokine assay. Results: The pexidartinib +/- PD-1 inhibitor (Px+P and Px-P) groups demonstrated no significant difference in daily weight change (p = 0.98) or in mortality (p = 0.59) when compared to vehicle control (V+P and V-P) groups. On post-mortem serum analysis AST (p = 0.01) and ALT (p = 0.001) were elevated, while albumin (p = 0.57) and BUN/creatinine ratio (p = 0.09) were reported within normal ranges, when comparing treatment to placebo groups. Pre to posttreatment, mean MRI tumor volume decreased by 37% and 54% in the Px-P and Px+P animals and increased by 147% and 88% in the V+P and V-P animals, respectively (p = &lt; 0.0001). Overall response rate was 69% and 87% in Px-P and Px+P animals compared to 21% and 13% in V-P and V+P animals, respectively (p = &lt; 0.0001). Downstream gene expression demonstrated upregulation of TNF-α (p = 0.003), IFN-γ (p = 0.002) and IL-6 (p = 0.0001) and downregulation of IL-13 (p = 0.051), IL-10 (p = 0.051), TGF-β (p = 0.024) and Arg-1 (p = 0.0003), in treatment vs. placebo animals. Increased apoptosis (Cas-3 p = 0.0005) and reduced proliferation (Ki-67 p = &lt; 0.0001) were reported in the treatment animals. Higher CD3+CD8+ T cell densities and CD86/CD206 macrophage ratio was reported in treatment groups compared to placebo (p = &lt; 0.0001). Serum cytokine levels significantly displayed upregulation of TNF-α and IFN-γ and downregulation of IL-4 and IL-10 in the treatment animals compared to the placebo. Conclusions: Overall, this study established a promising combinatorial strategy using a CSF-1R inhibitor to overcome resistance to PD-1/PD-L1 axis blockade in an esophageal cancer model, providing the rationale for future clinical strategies.

Hepatitis B virus middle surface antigen loss promotes clinical variant persistence in mouse models

ABSTRACT Hepatitis B virus (HBV) middle surface antigen (MHBs) mutation or deletion occurs in patients with chronic HBV infection. However, the functional role of MHBs in HBV infection is still an enigma. Here, we reported that 7.33% (11/150) isolates of CHB patients had MHBs start codon mutations compared with 0.00% (0/146) in acute hepatitis B (AHB) patients. Interestingly, MHBs loss accounted for 11.88% (126/1061) isolates from NCBI GenBank, compared with 0.09% (1/1061) and 0.00% (0/1061) for HBV large surface antigen (LHBs) loss and HBV small surface antigen (SHBs) loss, respectively. One persistent HBV clone of genotype B (B56, MHBs loss) from a CHB patient was hydrodynamically injected into BALB/c mice. B56 persisted for >70 weeks in BALB/c mice, whereas B56 with restored MHBs (B56M+) was quickly cleared within 28 days. Serum cytokine assays demonstrated that CXCL1, CXCL2, IL-6 and IL-33 were significantly increased during rapid HBV clearance in B56M+ mice. Furthermore, the enhancers and promoters of B56 were proved to be required for B56 persistence in mice. Ablating MHBs expression improved the persistence of a new clone (HBV1.3, genotype B) which was recreated by using enhancers and promoters of B56. These data demonstrated that MHBs deletion can promote the persistence of specific HBV variants in a hydrodynamic mouse model. MHBs re-expression restored a rapid clearance of HBV, which was accompanied by cytokine responses including the elevation of CXCL1, CXCL2, IL-6 and IL-33.

Molecular correlates of response to nivolumab at baseline and on treatment in patients with RCC

BackgroundNivolumab is an immune checkpoint inhibitor targeting the programmed death-1 receptor that improves survival in a subset of patients with clear cell renal cell carcinoma (ccRCC). In contrast to other...

Acute Chelation Therapy-Associated Changes in Urine Gadolinium, Self-reported Flare Severity, and Serum Cytokines in Gadolinium Deposition Disease.

The aim of this study was to determine the following in patients who have undergone magnetic resonance imaging with gadolinium-based contrast agents (GBCAs) and meet the proposed diagnostic criteria for gadolinium deposition disease (GDD): (1) the effectiveness of chelation therapy (CT) with intravenous Ca-diethylenetriaminepentaacetic acid in removing retained gadolinium (Gd) and factors affecting the amount removed; (2) the frequency of CT-induced Flare, that is, GDD diagnostic symptom worsening, and factors affecting Flare intensity; (3) whether, as reported in a separate cohort, GDD patients' serum cytokine levels differ significantly from those in healthy normal controls and change significantly in response to CT; and (4) whether urine Gd, Flare reaction, and serum cytokine findings in GDD patients are mimicked in non-ill patients described as having gadolinium storage condition (GSC). Twenty-one GDD subjects and 3 GSC subjects underwent CT. Patients provided pre-CT and post-CT 24-hour urine samples for Gd content determination along with pre-CT and 24-hour post-CT serum samples for cytokine analysis. Patients rated potential Flare 24 hours after CT. Pre-CT and post-CT 24-hour urine Gd analyses and Luminex serum cytokine assays were performed blind to patients' GDD and GSC status and all other data except age and sex. Serum cytokine levels in a healthy normal control group of age- and sex-matched subjects drawn from Stanford influenza vaccination studies were measured once, contemporaneously with those of GDD and GSC patients, using the same Luminex assay. Urine Gd amounts increased post-CT by 4 times or more after 87% of the 30 CT sessions. The most important factors appeared to be the time since the last GBCA dose and the cumulative dose received. Urine Gd amounts for GDD and GSC patients fell in the same ranges. All GDD patients, and no GSC patient, reported a Flare 24 hours post-CT. Linear regression found that Flare intensity was significantly predicted by a model including pre- and post-CT Gd amounts and the number of GBCA-enhanced magnetic resonance imaging. Post-CT, multiple cytokines showed strong positive relationships with GDD patients' Flare intensity in multivariable models. The pre-CT serum levels of 12 cytokines were significantly different in GDD patients compared with healthy flu vaccine controls. The small number of GSC patients precluded analogous statistical testing. Post-CT, GDD patients' serum levels of 20 cytokines were significantly decreased, and 2 cytokines significantly increased. These cytokines did not exhibit the same change pattern in the 3 GSC patients. The small number of GSC patients precluded statistical comparisons of GSC to GDD patients' results. In this preliminary study, 24-hour urine Gd content increased markedly and similarly in GDD and GSC patients after Ca-diethylenetriaminepentaacetic acid CT. Post-CT Flare reaction developed only in GDD patients. The current study is the second finding significantly different serum cytokine levels in GDD patients compared with healthy normal controls. These differences and the difference between GDD and GSC patients' Flare and cytokine responses to CT suggest some inflammatory, immunologic, or other physiological differences in patients with GDD. Further research into the treatment and physiological underpinnings of GDD is warranted.

The Application of Cytokine Expression Assays to Differentiate Active From Previously Treated Syphilis.

To investigate the role of serum cytokine assays to distinguish between active from treated syphilis among serofast patients, we recruited individuals into a prospective cohort study. Participants underwent routine syphilis screening. We selected specimens from a majority cohort of serofast participants with treated and active syphilis. We analyzed specimens with a 62-cytokine multiplex bead-based enzyme-linked immunosorbent assay. Cytokines, brain-derived neurotrophic factor and tumor necrosis factor β, were most predictive. We built a decision tree that was 82.4% accurate, 100% (95% confidence interval, 82%-100%) sensitive, and 45% (18%-75%) specific. Our decision tree differentiated between serum specimens from serofast participants with treated syphilis versus active syphilis.

An initial investigation of serum cytokine levels in patients with gadolinium retention.

ObjectiveTo determine whether individuals with proposed gadolinium deposition disease (GDD) have elevated serum levels of pro-inflammatory and pro-fibrotic cytokines, and whether specific cytokines are correlated with certain symptoms.Materials and MethodsTwenty-four participants recruited between May 2016 and June 2017 met GDD diagnostic criteria. The 64 control subjects provided serum samples before prophylactic flu vaccination. Serum cytokine levels were obtained with Luminex serum cytokine assay using eBiosciences/Affymetrix human 62-plex kits. Wilcoxon rank-sum tests were performed to assess the difference between the median fluorescence intensity values for the participants and the control group. Generalized linear models were built to evaluate the association between each cytokine of interest and selected participant symptoms.ResultsSerum levels of 14 cytokines, including nine pro-inflammatory cytokines, were statistically significantly elevated compared to controls (p ≤ 0.05). Hypotheses regarding pro-fibrotic cytokines and cytokine links to specific symptoms' intensity were not confirmed.ConclusionThe statistically significantly elevated cytokines may be markers of susceptibility to GDD or agents of symptom induction. These findings suggest that individuals developing symptoms characteristic of GDD after a contrast-assisted magnetic resonance imaging should be studied to investigate whether gadolinium retention and elevated cytokines may be related to their symptoms.

Eupatorin Suppressed Tumor Progression and Enhanced Immunity in a 4T1 Murine Breast Cancer Model.

Eupatorin is a polymethoxy flavone extracted from Orthosiphon stamineus and was reported to exhibit cytotoxic effects on several cancer cell lines. However, its effect as an anti–breast cancer agent in vivo has yet to be determined. This study aims to elucidate the potential of eupatorin as an anti–breast cancer agent in vivo using 4T1 challenged BALB/c mice model. In this article, BALB/c mice (20-22 g) challenged with 4T1 cells were treated with 5 mg/kg or 20 mg/kg eupatorin, while the untreated and healthy mice were fed with olive oil (vehicle) via oral gavage. After 28 days of experiment, the mice were sacrificed and blood was collected for serum cytokine assay, while tumors were harvested to extract RNA and protein for gene expression assay and hematoxylin-eosin staining. Organs such as spleen and lung were harvested for immune suppression and clonogenic assay, respectively. Eupatorin (20 mg/kg) was effective in delaying the tumor development and reducing metastasis to the lung compared with the untreated mice. Eupatorin (20 mg/kg) also enhanced the immunity as the population of NK1.1+ and CD8+ in the splenocytes and the serum interferon-γ were increased. Concurrently, eupatorin treatment also has downregulated the expression of pro-inflammatory and metastatic related genes (IL-1β. MMP9, TNF-α, and NF-κB). Thus, this study demonstrated that eupatorin at the highest dosage of 20 mg/kg body weight was effective in delaying the 4T1-induced breast tumor growth in the animal model.

Abstract 528: Radioembolization followed by durvalumab and tremelimumab does not induce immune responses against liver-metastasized MSS colorectal cancer

Abstract Introduction: PD-1 inhibitors have been ineffective in microsatellite stable (MSS) metastatic colorectal cancer (mCRC). Preclinical models suggest that radiation therapy may sensitize MSS CRC to PD-1 blockade. This constitutes the rationale for combining radioembolization to the liver in patients (pts) with MSS CRC with liver metastasis. Methods: Pts with MSS mCRC with liver predominant disease who progressed following at least 1 prior line of treatment, were eligible for study treatment. Treatment consisted of Y90 radioembolization to the liver (SIRTEX®) followed 2-3 weeks later by the intravenous (IV) combination of durvalumab (D) at 1500 mg and tremelimumab (T) at 75 mg Q4W for 4 months, followed by D 1500 mg Q4W x 8 cycles, or until disease progression (PD). Tumor biopsies were obtained at baseline, 1-2 weeks post SIR-Spheres®, and 2-3 weeks after D+T. A Simon 2-stage design was implemented, with a planned expansion to 18 patients if at least 1 response is noted in the 1st 9 pts. Correlative studies included tumor and peripheral blood flow cytometry, serum cytokine assays, and tumor IHC multiplex assay for CD8, CD4, CD68 and Cytokeratin20 expression. Immune and cancer related gene expression of the tumor microenvironment was analyzed via NanoString. Results: 9 pts enrolled in the 1st stage of the study, all with PD within or after their first 2 cycles of treatment. Per pre-planned design, the study was closed for futility. Here we report our correlative study for this trial. Based on IHC, intratumoral TILs (CD4 and CD8 T cells) were not detectable on any of the serial tumor biopsies (pre-Y90, post-Y90, and post D+T), while heavy CD68+ macrophage infiltration was consistently observed. Such observations were statistically validated by comparing paired serial samples using NanoString. Increased expression of collagen genes, such as COL1A1, COL1A2, and COL3A1, following Y90 was noted, as reported in literatures for cases with chemo and radiation resistance. MDM2, known to associate with resistance to PD-1/PD-L1 inhibitors, was also upregulated following Y90. Furthermore, flow cytometry results showed no difference in CD4+ T cells, CD8+T cells, CD20+B cells, CD33+HLA-DR-MDSCs, and CD4+Foxp3+ regulatory T cells based on paired serial PBMC samples. However, we observed a significant increase in PD-1+CD4+ and PD-1+CD8+ T cell subpopulations in PBMC following D+T, which agrees with other reports that PD-1/PD-L1 targeting leads to the expansion of PD-1+ T cells. In addition, CD3-CD56+ NK cell population was increased following D+T when compared with Y90. Conclusion: Y90 radioembolization can be added safely to D+T but did not promote tumor-directed immune responses against liver-metastasized MSS CRC. The associated correlative studies do not support a role for Y90 radioembolization to convert immunologically ‘cold’ tumors into ‘hot’ tumors. Citation Format: Chongkai Wang, John Park, Ching Ouyang, Raju Pillai, Jeffrey Longmate, Holly Yin, Christian Avalos, Maricel Gozo, Colt Egelston, Peter P. Lee, Marwan G. Fakih. Radioembolization followed by durvalumab and tremelimumab does not induce immune responses against liver-metastasized MSS colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 528.

Restoration of Decreased T Helper 1 and CD8+ T Cell Subsets Is Associated With Regression of Lymphoproliferative Disorders Developed During Methotrexate Treatment.

Lymphoproliferative disorder (LPD), including malignant lymphoma, is a relatively rare but life-threatening complication in RA patients under methotrexate (MTX) therapy. Spontaneous regression of LPD after MTX withdrawal is regarded as a distinct characteristic in part of such LPDs. The present study aimed to investigate the immunological difference in regressive LPD and persistent LPD. We studied RA patients who developed LPD during MTX administration (n = 35) and clinically matched controls (n = 35). The time of MTX cessation was defined as week 0, and LPD patients were divided into two groups according to LPD status at week 12: regressive group (n = 22) and persistent group (n = 13). Flow cytometric analysis of whole blood samples and serum cytokine assays were conducted for LPD (n = 10) and control patients (n = 10) at weeks 0, 4, and 12. There was a significant decrease in peripheral lymphocytes and the proportion of T helper 1 cells (Th1 cells), effector memory CD8+ T cells (EMCD8+ T) and Epstein-Barr virus (EBV)-specific CD8+ T cells at the time of LPD diagnosis, and a significant increase after MTX cessation was observed in the regressive group but not in the persistent group. The expansion of Th1 cells and EMCD8+ T cells significantly correlated with an increase in serum interferon (IFN)-γ concentration. Changes in Th1 cells, EMCD8+ T cells and EBV-specific CD8+ T cells, which coincided with an increase in IFN-γ, were significantly different between regressive LPD and persistent LPD after MTX cessation.