Levels Of IFN-gamma Research Articles

A hypoallergenic peptide mix containing T cell epitopes of the clinically relevant house dust mite allergens.

BackgroundIn the house dust mite (HDM) Dermatophagoides pteronyssinus, Der p 1, 2, 5, 7, 21, and 23 have been identified as the most important allergens. The aim of this study was to define hypoallergenic peptides derived from the sequences of the six allergens and to use the peptides and the complete allergens to study antibody, T cell, and cytokine responses in sensitized and nonsensitized subjects.MethodsIgE reactivity of HDM‐allergic and non‐HDM‐sensitized individuals to 15 HDM allergens was established using ImmunoCAP ISAC technology. Thirty‐three peptides covering the sequences of the six HDM allergens were synthesized. Allergens and peptides were tested for IgE and IgG reactivity by ELISA and ImmunoCAP, respectively. Allergenic activity was determined by basophil activation. CD4+ T cell and cytokine responses were determined in PBMC cultures by CFSE dilution and Luminex technology, respectively.ResultsHouse dust mite allergics showed IgE reactivity only to complete allergens, whereas 31 of the 33 peptides lacked relevant IgE reactivity and allergenic activity. IgG antibodies of HDM‐allergic and nonsensitized subjects were directed against peptide epitopes and higher allergen‐specific IgG levels were found in HDM allergics. PBMC from HDM‐allergics produced higher levels of IL‐5 whereas non‐HDM‐sensitized individuals mounted higher levels of IFN‐gamma, IL‐17, pro‐inflammatory cytokines, and IL‐10.ConclusionIgG antibodies in HDM‐allergic patients recognize peptide epitopes which are different from the epitopes recognized by IgE. This may explain why naturally occurring allergen‐specific IgG antibodies do not protect against IgE‐mediated allergic inflammation. A mix of hypoallergenic peptides containing T cell epitopes of the most important HDM allergens was identified.

1195P - Safety, efficacy, and immune effects of intratumoral tilsotolimod in patients with refractory solid tumours: Updated results from ILLUMINATE-101

BackgroundTilsotolimod, a synthetic toll-like receptor 9 (TLR9) agonist, modulates the tumor immune microenvironment and has antitumor activity as monotherapy in preclinical models. In the ILLUMINATE-204 phase I/II study of tilsotolimod in combination with ipilimumab in patients with refractory melanoma, increased antitumor immune activity was observed in injected and uninjected tumors at 24hours following tilsotolimod treatment. The ILLUMINATE-101 phase Ib study explored the safety, efficacy, and immune effects of tilsotolimod monotherapy in multiple solid tumors. MethodsAdults with histologically or cytologically confirmed diagnosis of cancer not amenable to curative therapies received intratumoral tilsotolimod in doses escalating from 8mg to 32mg into a single lesion at weeks 1, 2, 3, 5, 8, and 11. Additional patients with advanced melanoma were enrolled into an expansion cohort at the 8-mg dose, the recommended phase II dose established for melanoma. Objectives included characterizing safety and efficacy, and immunological assessment. Blood samples and tumor biopsies of injected and distal lesions were obtained at baseline and 24hours post treatment for immune analyses. ResultsAs of May 6 2019, 54 patients have been enrolled, including 38 patients into the dose evaluation portion and 16 patients into the melanoma expansion cohort. No dose-limiting toxicities were observed. The most common treatment-related adverse events were fever, fatigue, and chills. Within 24hours, fresh tumor biopsies showed increased gene expression for multiple immune checkpoint pathways, increased IFN gamma levels, activation of the type 1 IFN pathway, and upregulation of MHC class I and II, compared to pretreatment biopsies. Of 43 evaluable patients, 15 (35%) had a RECIST v1.1 disease assessment of stable disease (duration 1.2 to 11.1 months, 1 patient ongoing). ConclusionsTilsotolimod was well tolerated and induced robust alterations in the tumor microenvironment, including immune checkpoint upregulation, activation of dendritic cells, and induction of Type 1 IFN signaling. A phase II study of tilsotolimod in combination with nivolumab and ipilimumab has been initiated for the treatment of multiple solid tumors (ILLUMINATE-206; NCT03865082). Clinical trial identificationNCT03052205. Editorial acknowledgementTed Everson, Idera Pharmaceuticals, Inc. Legal entity responsible for the studyIdera Pharmaceuticals, Inc. FundingIdera Pharmaceuticals, Inc. DisclosureH. Babiker: Advisory / Consultancy: Endocyte; Advisory / Consultancy: Celgene. V. Subbiah: Advisory / Consultancy: Idera Pharmaceuticals; Advisory / Consultancy: Novartis; Advisory / Consultancy: Bayer; Advisory / Consultancy: Blueprint Medicines; Advisory / Consultancy: Loxo Oncology. S. Rahimian: Shareholder / Stockholder / Stock options, Full / Part-time employment: Idera Pharmaceuticals. C. Haymaker: Research grant / Funding (institution): Idera Pharmaceuticals. C. Bernatchez: Research grant / Funding (institution): Idera Pharmaceuticals. G. Bindra: Shareholder / Stockholder / Stock options, Full / Part-time employment: Idera Pharmaceuticals. I. Iverson: Shareholder / Stockholder / Stock options, Full / Part-time employment: Idera Pharmaceuticals. S. Chunduru: Shareholder / Stockholder / Stock options, Full / Part-time employment: Idera Pharmaceuticals. A. Diab: Advisory / Consultancy: Array Biopharma; Advisory / Consultancy: Nektar Therapeutics; Honoraria (self): Bristol-Myers Squibb; Advisory / Consultancy: Jounce Therapeutics; Honoraria (self): Novartis; Advisory / Consultancy: Idera. All other authors have declared no conflicts of interest.

P5393Deficiency of the long noncoding RNA NEAT1 disturbs T cell and monocyte-macrophage lineage differentiation and functions and results in systemic inflammation with high circulating interferon levels

Abstract Background Inflammation is a key driver of atherosclerosis and myocardial infarction (MI), and beyond proteins and microRNAs, long noncoding RNAs (lncRNAs) are implicated in inflammation control. To obtain further information on the role of lncRNAs in the context of atherosclerosis, we analyzed transcriptome maps of circulating immune cells (PBMCs) of post-MI patients in whom the lncRNA NEAT1 was suppressed. Here, we report immune disturbances in murine NEAT1 knockout models with wildtype or ApoE−/− genetic background. Methods and results RNA-sequencing (RNA-seq) of PBMCs from post-MI patients revealed profound transcriptome disturbances compared to healthy controls. Among these, NEAT1 suppression was notable since it affected the most highly expressed lncRNA as part of a molecular circuit also encompassing chemokines and interleukins. We used NEAT1−/− mice to evaluate whether NEAT1 depletion per se may cause immune dysfunction. NEAT1−/− splenocytes displayed enhanced baseline ROS production, and RNA-seq identified anomalous expression and regulation of chemokines/ receptors, innate immunity genes, TNF and caspases. FACS revealed displayed anomalous Treg and TH cell differentiation in NEAT1−/− spleens vs. wildtype (WT). Beyond grossly altered transcriptome, NEAT1−/− bone marrow derived macrophages (BMDMs) responded to LPS with increased (p<0.001) ROS production, enhanced baseline phagocytic activity (p<0.001), and attenuated proliferation (p=0.001). FACS revealed deregulated monocyte-macrophage differentiation in NEAT1−/− bone marrow and blood. Further, NEAT1−/− mice displayed aortic wall CD68+ cell infiltration and there was evidence of myocardial inflammation which could lead to severe and potentially life-threatening structural damage in some of these animals. This observation suggests that even stochastic activation of the highly unstable NEAT1−/− immune system may trigger uncontrolled pathogenic cascades, explaining the survival disadvantage of NEAT1−/− mice. In addition to these studies on homozygous NEAT1−/− deficiency in WT background, we obtained data on mice with partial i.e. heterozygous NEAT1−/+ deficiency on ApoE−/− background. Analysis of this new NEAT1−/+ ApoE−/− strain indicates that even partial NEAT1 deficiency leads to systemic inflammation with high IFN-gamma levels, when the animals are exposed to immune stress e.g. high LDL cholesterol. Conclusions Regarding the monocyte-enriched NEAT1 suppressed in post-MI PBMCs, the data from NEAT1−/− and NEAT1−/+ ApoE−/− mice document NEAT1 as a key immune system coordinator whose deficiency affects monocyte-macrophage and T cell differentiation and functions and renders the immune system unstable and highly vulnerable to immune stress. Since in patients NEAT1 is part of a molecular circuit persistently deregulated post-MI, too, it appears reasonable to further search for new therapeutic targets within this circuit, taking advantage of the described genetic animal models.

Remote ischemic preconditioning protects against ischemic stroke in streptozotocin-induced diabetic mice via anti-inflammatory response and anti-apoptosis

ObjectiveIt has been shown that remote ischemic preconditioning (RIPreC) attenuates ischemic injury after stroke in healthy rats or mice. The present study aims to examine whether RIPreC offers neuroprotection against ischemic stroke in streptozotocin-induced diabetic mice. MethodsStreptozotocin (STZ, 120 mg/kg) was intraperitoneally injected into the mice to induce type 1 diabetic model. The immune and inflammatory changes were analyzed 2 days after reperfusion by flow cytometry and multiplex cytokine assay analysis, respectively. ResultsWe found that RIPreC reduced infarct sizes and alleviated neurological impairment in diabetic mice. RIPreC decreased CD8 T cells infiltrated into the brain, and attenuated the decreases of CD8 T cells in the blood, CD4 T cells and CD8 T cells in the spleen. Results from multiplex cytokine assay showed that RIPreC treatment decreased IL-6, IL-1 beta and TNF alpha levels in the cortex, while it inhibited IL-6 level in the hippocampus and striatum, and TNF alpha level in the hippocampus. RIPreC treatment also downregulated IL-6 and IFN gamma level in the blood, which increased after cerebral ischemic injury. In addition, RIPreC reduced pro-apoptotic protein BAX expression in the ischemic brain. ConclusionsOur results indicate that RIPreC attenuates cerebral injuries in streptozotocin-induced diabetic mice via anti-inflammatory response and anti-apoptosis in the ischemic brain.

Immuno-Stimulatory Effects of Mechanical and Thermal High Intensity Focused Ultrasound

Abstract INTRODUCTION While immunotherapy through anti-PD1 checkpoint inhibition has demonstrated impressive clinical efficacy against a number of tumor types, its application has been ineffective against glioblastomas METHODS We wished to determine whether high intensity focused ultrasound (HIFU) augment the efficacy of anti-PD1 checkpoint inhibition against glioblastomas. HIFU can be administered through low-duty cycle to induce subcellular fragmentation without causing temperature elevation (termed mechanical HIFU). Alternatively, HIFU delivered through high-duty cycle can induce foci of temperature elevation and thermal ablation (termed thermal HIFU). We tested the impact of mechanical and thermal HIFU on the anti-glioblastoma effects of the immune checkpoint inhibitor, ipilimumab RESULTS In an in Vivo murine glioblastoma model, sites targeted by mechanical HIFU exhibited a 10 to 100 fold increase in accumulation in tumor infiltrating lymphocytes and interferon-gamma in the presence of PD-1 blockade. At least 75% of mice engrafted with glioblastomas achieved remission when treated with mechanical HIFU and PD-1 blockade. In contrast, none of the mice treated with single therapies achieved durable remission. Likelihood of remission correlated with the abundance of tumor infiltrating lymphocytes (P < .001) and IFN-gamma levels (P = .001). The synergy observed between mechanical HIFU and PD-1 blockade was not seen when combining thermal HIFU and PD-1 blockade, suggesting thermal protein denaturation is prohibitive to immune-stimulatory effects of HIFU CONCLUSION Mechanical HIFU, but not thermal HIFU, augments the anti-glioblastoma effects of PD-1 blockade. Our findings inform the utility of HIFU as an immune-adjuvant in glioblastoma therapy and provide a paradigm for conceptualizing the immune effects of laser thermal ablation

Effect of quercetin on E-NTPDase/E-ADA activities and cytokine secretion of complete Freund adjuvant-induced arthritic rats.

The effect of quercetin was assessed in rats induced with complete Freund adjuvant (CFA). Arthritis scores, paw oedema, latency, activities of myeloperoxidase (MPO), ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), and ectoadenosine deaminase (E-ADA) in lymphocytes were determined. Furthermore, nucleotide and nucleoside levels as well as the secretion of pro- and anti-inflammatory cytokines were evaluated. Animals were treated with saline and quercetin in doses of 5, 25, and 50mg/kg for 45days. The result revealed that quercetin (50mg/kg) reduced arthritis score and paw oedema, and increased the latency in the thermal hyperalgesia test. Histopathological analysis showed that all the doses of quercetin reduced infiltration of inflammatory cells. MPO activity was increased in the arthritis group; however, quercetin reduced this activity. E-NTPDase activity was increased in lymphocytes of arthritis rats, and treatment with quercetin reversed this increase. However, E-ADA activity was reduced in the arthritis group, and treatment with quercetin modulated the activity of this enzyme in arthritis rat groups. Serum adenosine levels were increased in arthritis, and the levels were lowered with quercetin treatment. Quercetin treatment in arthritis groups decreased the elevated levels of cytokines in the arthritis control group. Thus, quercetin demonstrated an anti-inflammatory effect, and this flavonoid may be a promising natural compound for the treatment of arthritis. SIGNIFICANCE OF THE STUDY: Quercetin may represent a potential therapeutic compound in the treatment of rheumatoid arthritis. Findings from this study indicate that quercetin suppresses swelling and attenuates the underlying inflammatory responses. This is the first report where quercetin was shown to modulate the immune response to arthritis via attenuation of the purinergic system (E-NTPDase and E-ADA activities) and the levels of IFN-gamma and IL-4. Thus, this work is relevant to basic research and may be translated into clinical practice.

Treatment with a Zinc Metalloprotease Purified from Bothrops moojeni Snake Venom (BmooMP-Alpha-I) Reduces the Inflammation in an Experimental Model of Dextran Sulfate Sodium-Induced Colitis.

It has been described that the metalloprotease BmooMP-alpha-I purified from Bothrops moojeni snake venom is able to hydrolyze the TNF molecule. However, this observation has been based mainly on in vitro investigation, in addition to molecular modeling and docking approaches. Considering that there is no in vivo study to demonstrate the biological effects of this enzyme, the major aim to the present work was to investigate whether the BmooMP-alpha-I has any anti-inflammatory efficacy by setting up a murine experimental design of colitis induced by dextran sulfate sodium (DSS). For this purpose, C57BL/6 mice were divided into six groups, as follows: (i) animals without intestinal inflammation, (ii) animals without intestinal inflammation treated with BmooMP-alpha-I (50 μg/animal/day), and (iii) animals with intestinal inflammation induced by 3% of DSS, (iv) mice with intestinal inflammation induced by DSS and treated with BmooMP-alpha-I enzyme at the 50, 25, or 12.5 μg/animal/day dosages by intraperitoneal route. Clinical signs of colitis were observed daily for calculating the morbidity scores, cytokine measurements, and histological features. We observed that the animals treated with different doses of the enzyme presented a remarkable improvement of colitis signs, as confirmed by a significant increase of the intestine length in comparison to the DSS group. Also, no difference was observed between the groups treated with the enzyme or vehicle, as the colon length of these animals was slightly lower than that of the group of healthy animals, without induction of intestinal inflammation. The cytokine quantification in supernatants of intestinal tissue homogenates showed a significant reduction of 38% in IFN-gamma levels, when the animals were treated with 50 μg of the BmooMP-alpha-I compared to the animals receiving DSS only. A significant reduction of 39% in TNF levels was also observed in all doses of treatment with BmooMP-alpha-I, in addition to a significant reduction of 35% in the amount of IL-12p40. Histological examinations revealed that the BmooMP-alpha-I 50 μg treated group preserved colon architecture and goblet cells and reduced the ulcer area, when compared with DSS mice, which showed typical inflammatory changes in tissue architecture, such as ulceration, crypt dilation, loss of tissue architecture, and goblet cell depletion, accompanied by a significant cell infiltration. In conclusion, our results suggest that the improvement of clinical scores and histological findings related to BmooMP-alpha-I treatment in this experimental model could be attributed to the metalloprotease ability to modulate cytokine production locally at the inflamed intestine. These findings highlight the potential anti-inflammatory role and effectiveness of this enzyme as a therapeutic alternative in this type of immunopathological condition.

Effect of operative interval on efficacy of patients with open ocular trauma

Objective To investigate the effect of operative interval on efficacy of patients with open ocular trauma. Methods The clinical data of 102 patients with open ocular trauma from Jan. 2014 to Jan. 2018 were analyzed retrospectively.According to the timing of operation, all patients were divided into two groups, group A of 79 eyes in 52 cases and group B of 74 eyes in 50 cases.The operative interval between onset and operation in group A was 2-4 days, and in group B was 5-14 days.Vitrectomy was performed in both groups, and the efficacy were compared. Results The total cure rate in group A was 93.67%, which was higher than that in group B (82.43%)(P=0.031). The levels of serum IL-4, IL-6, IL-12, IL-17 and IFN-gamma in the two groups at 1 week after operation were lower than those before operation, and those in group A were lower than those in group B(P 0.05). Conclusion For the patients with open ocular trauma the sooner the operation is performed, the better the efficacy is. Key words: Trauma, ocular, open; Vitrectomy; Efficacy

Gene and protein delivery using four cell penetrating peptides for HIV-1 vaccine development.

Cell penetrating peptides (CPPs) can potently transport therapeutic molecules to target cells for treatment of a variety of diseases. Thus, their use is critical to improve therapeutic vaccines. Histidine-rich nona-arginine (HR9) and primary amphipathic peptide (MPG) showed the ability to transfer DNA into the cells. Moreover, the peptide derived from the C-terminal of the tumor suppressor protein p14ARF (M918) and arginine-rich peptide (penetratin) were utilized to deliver polypeptides and proteins into the living cells. In this study, the immunostimulatory properties of HIV-1 Nef DNA and protein constructs were evaluated using small heat shock protein 20 (sHsp20) and Freund's emulsion as an adjuvant, and four CPPs (HR9, MPG, M918, and penetratin) as a gene or protein carrier in BALB/c mice. Our data indicated that the HR9/DNA, MPG/DNA, M918/protein, and penetratin/protein complexes formed the stable nanoparticles that were effectively delivered in HEK-293T cell line at certain ratios. Moreover, a heterologous Hsp20-Nef DNA + MPG prime/rHsp20-Nef protein+M918 boost regimen significantly elicited higher levels of IgG2a, IgG2b, IFN-gamma, and Granzyme B directed toward Th1 responses in a long period (3 months) after the last immunization compared to other groups. Furthermore, the effective role of Hsp20 was detected as a natural adjuvant in enhancing immune responses against HIV-1 Nef antigen. These findings demonstrated that the simultaneous use of M918 and MPG CPPs as protein and gene carriers improves HIV-1 Nef-specific B- and T-cell immune responses as a promising approach for development of HIV-1 monovalent vaccine.

Relationships among stress, emotional intelligence, cognitive intelligence, and cytokines.

The brain has multiple functions, and its structures are very closely related to one another. Thus, the brain areas associated with stress, emotion, and intelligence are closely connected. The purpose of this study was to investigate the multiple associations between stress and emotional intelligence (EI), between EI and intelligence quotient (IQ), between cytokines and stress, and between cytokines and IQ. We measured the stress, EI, cognitive intelligence using IQ, and cytokine levels of 70 healthy subjects. We also analyzed the association of cytokines with IQ according to hemispheric dominance using the brain preference indicator (BPI). We found significant negative correlations between stress and the components of EI, such as emotional awareness and expression, emotional thinking, and emotional regulation. High levels of anger, which is a component of stress, were significantly related to poor emotional regulation. Additionally, emotional application was positively correlated with full-scale IQ scores and scores on the vocabulary, picture arrangement, and block design subtests of the IQ test. High IL-10 levels were significantly associated with low stress levels only in the right-brain-dominant group. High IL-10 and IFN-gamma levels have been associated with high scores of arithmetic intelligence. TNF-alpha and IL-6 were negatively associated with vocabulary scores and full-scale IQ, but IL-10 and IFN-gamma were positively associated with scores on the arithmetic subtest in left-brain-dominant subjects. On the other hand, IL-10 showed positive correlations with scores for vocabulary and for vocabulary and arithmetic in right-brain-dominant subjects. Furthermore, we found significant linear regression models which can show integrative associations and contribution on emotional and cognitive intelligence. Thus, we demonstrated that cytokines, stress, and emotional and cognitive intelligence are closely connected one another related to brain structure and functions. Also, the pro-inflammatory cytokines TNF-alpha and IL-6 had negative effects, whereas the anti-inflammatory cytokines (e.g., IL-10 and IFN-gamma) showed beneficial effects, on stress levels, and multiple dimensions of emotional and cognitive intelligence. Additionally, these relationships among cytokines, stress, and emotional and cognitive intelligence differed depending on right and left hemispheric dominance.

Protective role of secreted aspartyl proteinase in murine systemic candidiasis caused by non-albicans Candida species

Abstract Non-albicans Candida species are increasingly becoming more prevalent globally and emergence of drug resistance is another cause of concern. We investigated the protective potential of secreted aspartyl proteinase (Sap2), a leading candidate vaccine antigen identified from research based on C. albicans, in murine systemic candidiasis caused by non-albicans Candida species. The Sap2 genes were successfully cloned and expressed as recombinant proteins from C. albicans, C. tropicalis and C. parapsilosis. Groups of Balb/c mice were immunized with individual rSap2 protein along with alum as adjuvant, followed by systemic infection with lethal dose of C. tropicalis. The protective potential of each rSap2 protein was evaluated using survival analysis and estimation of organ fungal burden. Mice immunized with rSap2 cloned from C. parapsilosis showed highest increase in survival time (p=0.02) and maximum reduction in organ fungal burden (spleen, kidneys, lungs, brain) (p<0.05); compared to sham immunized controls. Immunization with rSap2 cloned from C. albicans did not improve survival in non-albicans C. tropicalis infection, despite the protein having ~60% homology across species. Mice immunized with rSap2 cloned from C. parapsilosis also exhibited significantly higher levels of IFN gamma and IL-4 cytokine levels just before infection, which correlated with protection. The anti-Sap2 specific antibody titres were measured. No correlation was observed between level of Sap2-specific IgG titres and survival outcome. The IgG response was primarily Th2, belonging to IgG1 subtype. Further cellular and histological studies are in progress to characterize the murine rSap2-parapsilosis specific protective immune response in detail.

Peptides of endogenous retrovirus Gag gene variants are not agonistic ligands for autoreactive T cells in non-obese diabetic mice

Abstract Endogenous retroviruses (ERV) have been isolated from the pancreatic islets of non-obese diabetic (NOD) mice. We previously demonstrated that a murine leukemia retrovirus–like ERV Gag protein was secreted together with exosomes by an insulinoma and by primary islet mesenchymal stem cells. Here, we further demonstrated that Gag is an effective antigen to stimulate IFN-gamma production by autoreactive T cells using recombinant Gag protein or virus-like particles (VLPs) expressing the Gag gene. However, candidate peptides of this Gag protein were not effective in stimulating the T cells. Upon analyses of ERV Gag genes expressed in the islets of prediabetic NOD mice, we were surprised to find multiple Gag gene variants with complete open-reading frames. These results suggested the expression of different endogenous proviruses, or alternatively, the detection of point mutations incurred during viral replication. A new batch of altered peptides was synthesized reflecting the variations among the Gag gene variants, and a few of them induced detectable amount of cytokine secretion from the T cells. But, the levels of IFN-gamma induced by the peptides were much lower than that induced by the Gag protein and the VLPs, suggesting a low affinity activation. This weak T-cell-stimulating activity of the Gag peptides was further confirmed by testing diabetogenic T-cell clones, as well as by using MHC tetramers loaded with candidate peptides. The data suggest that ERV Gag or its gene variants may trigger autoimmunity via a pathway that could be independent from direct presentation of viral peptides to autoreactive T cells.

Interferon-alpha 2 but not Interferon-gamma serum levels are associated with intramuscular fat in obese patients with nonalcoholic fatty liver disease

BackgroundIntramuscular triglycerides (IMTGs) represent an important energy supply and a dynamic fat-storage depot that can expand during periods of elevated lipid availability and a fatty acid source. Ultrasonography (US) of human skeletal muscles is a practical and reproducible method to assess both IMTG presence and entity. Although a crosstalk between cytokines in skeletal muscle and adipose tissue has been suggested in obesity, condition leading to hepatic steatosis (HS) or better defined as nonalcoholic fatty liver disease and cancer, there are still questions to be answered about the role of interferons (IFNs), alpha as well as gamma, and IMTG in obesity. We aimed at discovering any correlation between IFNs and IMTG.MethodsWe analysed anthropometric data, metabolic parameters and imaging features of a population of 80 obese subjects with low-prevalence of co-morbidities but HS in relation to IFNs serum levels. A population of 38 healthy subjects (21 males) served as controls. The levels of serum IFNs were detected by a magnetic bead-based multiplex immunoassays.ResultsSerum concentrations of IFN-alpha 2 were increased, while serum levels of IFN-gamma were decreased confronted with those of controls; the severity of IMTG, revealed at US as Heckmatt scores, was inversely predicted by IFN-alpha 2 serum concentrations; IMTG scores were not predicted by serum levels of IFN-gamma; IMTG scores were predicted by HS severity, ascertained at US; HS severity was predicted by visceral adipose tissue, assessed by US, but the latter was not instrumental to IMTG.Discussion and conclusionThis study has added some pieces of observation about the cytokine network regulating the interplay between IMTG and obesity in obese patients with HS.

Evaluation of serum interferons in patients with age-related macular degeneration.

Background:Environmental, genetic, and immunological factors may play a role in the pathogenesis of age-related macular degeneration (AMD). In an attempt to better understand the pathogenesis of AMD, in this study, we evaluated the serum interferon (IFN) levels in patients with AMD and compared it with persons without AMD.Materials and Methods:In this case–control study, 42 patients with AMD and 42 healthy individuals (without AMD) were enrolled as the case and control groups, respectively. The two groups were matched regarding their age and sex. We classified the case group as dry-type and wet-type AMD. Blood samples were obtained and the serum was collected and frozen at −20°C. Alpha-, beta-, and gamma-IFN levels were measured using the sandwich ELISA method and compared between and within the groups.Results:The mean beta IFN levels in both case and control groups were 46.88 ± 27.25 pg/ml and 34.90 ± 18.81 pg/ml (P = 0.021), respectively. Regarding gamma and alpha IFN, the serum levels were not detectable in most of the patients and no significant difference was detected between the case and control groups.Conclusion:We found that serum beta IFN levels are higher in patients with AMD. This finding may have diagnostic, therapeutic, and prognostic value in AMD patients and can be a beginning for further evaluation.

The Protective Effects of 2,3,5,4'-Tetrahydroxystilbene-2-O-β-d-Glucoside in the OVA-Induced Asthma Mice Model.

Asthma is an inflammatory disease caused by an imbalance of Th1 and Th2 cells. In general, asthma is characterized by a stronger Th2 response. Most conventional asthma treatment focuses on improving airway flow or suppression of airway inflammation. To reduce the side effects of currently used asthma medicines, we have conducted studies on natural products that have no side effects. 2,3,5,4′-tetrahydroxystilbene-2-O-β-d-glucoside (TSG), the main compound of Polygonum multiflorum (PM), has various biological activities, including anti-inflammation and anti-oxidation activities. However, the effect of TSG on asthma has not been studied yet. We examined the effects of TSG on Th2 immune responses using an OVA-induced asthma animal model. OVA-sensitized mice were treated with TSG. 24 h after the last intranasal challenge, airway hyperresponsiveness (AHR) was measured or serum and bronchoalveolar lavage fluid (BALF) were harvested. We measured typical Th1 and Th2 cytokines in serum and BALF. As a result, TSG suppressed Th2 responses, as shown by the lower levels of IL-4, IL-5, total IgE, OVA-specific IgE, and OVA-specific IgG1. On the other hand, TSG increased Th1 responses, as shown by the levels of IFN-gamma. Collectively, these results confirm the potential of TSG for asthma treatment through modulation of inflammatory responses. Considering that the cytotoxic effect of PM extract is due to the cis isomer of TSG, if the effect of TSG on asthma treatment is found to be non-toxic in clinical trials, it would be more effective to use it as a purified component than PM extract as an asthma treatment agent.

Tandom Autologous Transplantation and Combined Infusion of CD19 and Bcma-Specific Chimeric Antigen Receptor T Cells for High Risk MM: Initial Safety and Efficacy Report from a Clinical Pilot Study

Background: Multiple myeloma (MM) is an incurable plasma cell malignancies despite the advent of numerously new drugs. Survival was poor particularly for high risk patients such as R-ISS stage III. The preliminary data from our center showed that the median PFS after auto-HSCT was 24 months for patients with R-ISS III stage, while 17 months for patients that achieved PR or less after induction. Chimeric antigen receptor (CAR)-transduced T cells is a promising strategy for cancer immunotherapy. Our previous study showed good response for RRMM patients after CD19 and BCMA-specific CART therapy without severe CRS and other deadly side effects. To improve the survival of high risk patients, this study was designed to observe the safety and efficacy of combined infusion of CD19 and BCMA-specific CART cells after autologous transplantation (SZ-MM-CART02 study, NCT 03455972).Methods:18-65y NDMM in R-ISS stage III, or who only achieved PR or less after 4 cycles of PAD triplet induction were enrolled with serum creatinine (Cr) <2.0 mg/dL, and adequate hepatic, cardiac and pulmonary function. BCMA and CD19 expression on MM cells were analyzed by flow cytometry. Lymphocytes were collected from PBSCs and cultured with an anti-CD3 monoclonal antibody to activate T-cell proliferation. The cells were transduced with recombinant lentiviral verctors which respectively contained the anti-BCMA or anti-CD19 single chain variable fragment (scFv), the cytoplasmic portion of the OX40 and CD28 costimulatory moiety, and the CD3z T-cell activation domain. This is the new third generation CAR technique applied in clinic. BUCY were used as conditioning, followed by infusion of autologous stem cells. Median time to engraftment were 10 days for nutrophils. CART-19 (1×107/kg on d0) and CART-BCMA cells as split-dose (40% on d1 and 60% on d2) were infused derectly on d14 to d20 after autologous transplantation. Levels of CAR-transduced cells are measured by qPCR. The cytokine release syndrome (CRS) was graded according to the UPen cytokine release syndrome grading system. Neurotoxic side effects and other toxicities were assessed according to the CTCAE v 4.03. Plasma levels of IL-2, IL-4, IL-6, IL-10, TNF-alpha, IFN-gamma, and IL-17A proteins were determined with a cytokine kit. Imids alone were given as maintenance therapy. Responses were assessed by IMWG criteria. 10-color flow cytometry was used to monitor MRD regularly after CART treatment. The median of follow-up was 3 (2∼11) months.Results: To date, 9 patients have completed the CART cells infusion (cohort 1). All cases expressed BCMA >50% without CD19 expression on MM cells. CRS occurred in 9 patients (100%) grade1 or 2 associated with fever(n=9), fatigue (n=9), elevated IL-6 and CRP (n=9), elevated ALT (n=1, grade 1). Two patients needed to use low-dose vascular active drugs (pts 04 and 07). Other toxicities to date included coagulopathy (n=6, grade 1 for 4 pts and grade 2 for 2 pts), elevated troponin T (n=4, grade 1), and atrial flutter (n=1). There was no serious CRS or neurologic complications occurred in this group of patients.The ORR was 100% with all patients were monitored for a period of more than 2 months, which may be eventually further improved. There were 2 CR, 1 VGPR, 4 PR, 2 SD after induction; 3 CR, 2 VGPR, 4 PR after APBSCT; 3 CR, 6 VGPR after CART therapy. MRD negativity in BM increased from 37.5% after transplantation to 66.7% after CART therapy latest. Four patients (pts 02, 03, 06 and 07) obtained partial PR after transplantation, and got VGPR after CART cells infusion. We found dramatic in vivo CART expansion that median of peak value of CART copies was 1059.54 folds (ranged from 536.90 to 10997.93 folds) which was 100 folds to that with RRMM patients in our previous study.Conclusions: Tandom autologous transplantation and combined infusion of CART-19 and CART-BCMA cells could be another choice of consolidation treatment for high risk MM patients. Toxicities to date including CRS and organ function impairment seemed to be mild and reversable. It is worthy of further study to compare DFS, OS between single autologous transplantation and tandom transplantation with CART therapy. Immune environment in high risk patients with multiple myelomaI remodelled by auto-HSCT may contribute to more rapid expansion of CART cells than that in RRMM patients, suggesting that the extent of CART expansion depends more than tumor burden. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bioengineered Autologous Dendritic Cells Enhance CAR T Cell Cytotoxicity By Providing Cytokine Stimulation and Intratumoral Dendritic Cells

▪Background: The combination of antigen recognition, costimulatory ligands, and dendritic cells (DC)-derived cytokines (IL-12 or type I IFNs) stimulate T cells upon antigen presentation of DC. Chimeric antigen receptor (CAR) T cells induce anti-tumor cytotoxicity independent of DC by employing antigen recognition portion (single chain variable fragment)/CD3zeta and costimulatory signaling domain. However, the clinically available CARs are not engineered to provide DC-derived cytokine stimulation to T cells. This deficiency may prevent the CAR T cells from developing optimal effector functions, surviving, and forming a responsive memory T cell population. DC can enhance CAR T cell functionality by producing T cell stimulating cytokines. Intratumoral DC, marked by the expression of CD141/CLEC9A, play a critical role in recruiting T cells into the intratumoral area and inducing T cell cytotoxicity against the tumor. IRF8 is an essential transcription factor in developing intratumoral DCs. A co-stimulatory protein, 4-1BB is expressed on activated T cells and is a part of a CAR construct. 4-1BB has been suggested to stimulate IRF8 through the NF-kB signaling and could participate in the generation of intratumoral DC. Therefore, we hypothesized that autologous DCs transduced with 4-1BB CAR would enhance the efficacy of anti-CD33 CAR T cell therapy against acute myelogenous leukemia (AML) by providing DC-derived cytokines and recruiting CAR T cells in bone marrow microenvironment.Methods: We sorted bone marrow CD34+ progenitors and T cells. Cells were transduced with an anti-CD33 41BBz CAR lentivector (pCCL-HP67.6-4-1BB-CD3z). We sorted transduced T (CAR T), and CD34+ progenitors three days after transduction. While expanding transduced CAR T cells further, we induced the differentiation of transduced CD34+ cells to DC (CAR-DC) in vitro by incubating cells with Flt3L/GM-CSF/IL-4 and AML cell lysate. After an additional four days of culture, we analyzed CAR-DC using flow cytometry. We co-cultured a human AML cell line, Kasumi-1 cells with CAR T +/- CAR-DC (E/T ratio=1), or mock control, and quantified cell death in different CAR T to Kasumi-1 ratios (10, 5, and 2) using CytoTox 96 NonRadioactive Cytotoxicity Assay and Annexin V. We also utilized multiplex cytokine immunoassays to quantify cytokine production. For in vivo studies, we injected luciferase-GFP tagged Kasumi-1 cells (10X106) into NSG mice, followed by injection of CAR T (5X105) +/- CAR-DC (1.5X105) or control T cells (5X105). We monitored the NSG mice using serial bioluminescence imaging and compared the survival of each group.Results: On phenotypic analysis using flow cytometry, we found that frequencies of cells expressing CD141/CLEC9A+ were significantly higher in CAR-DC vs. control DC (35.2 +/- 4.1 % vs. 9.0 +/- 1.7 % of HLA-DR+ cells), which suggest 4-1BB activation induce CD34+ progenitors to intratumoral DCs. The cytotoxicity assay showed 63.2 +/- 0.6 % Kasumi-1 death with CAR T/CAR-DC compared to 46.5 +/- 3.5 % with CAR T cells alone. CAR T/CAR-DC also demonstrated more Annexin V positive Kasumi-1 cells compared to CAR T and control T cells (78.4 +/- 5.1 % vs 39.9 +/- 7.7 % vs 17.6 +/- 2.2 %). These cytotoxicity assays demonstrated that CAR-DC enhanced the anti-Kasumi-1 cytotoxicity of anti-CD33 CAR T cells. CAR T cells co-cultured with CAR-DC produced a two-fold higher IFN-gamma and TNF-alpha than CAR T cells alone (p<0.01). The IFN-gamma and TNF-alpha production increases in correlation with the counts of CAR T cells. However, CAR T/CAR-DC group produced a four-fold higher IL-12 throughout different E/T ratios compared to CAR T alone group (p<0.01), which suggest DCs are the major source of IL-12 production and CAR T cells produce a higher level of IFN-gamma and TNF-alpha in response to DCs. In vivo NSG mice experiments demonstrated that CAR T/CAR-DC group had increased survival (p<0.01) and decreased AML burden than CAR T alone group.Conclusions: Our data show that 1) in vitro differentiation of DCs with 4-1BB stimulation increases intratumoral CD141/CLEC9A+ DCs, 2) interaction between CAR-DC and CAR T cells enhances cytotoxic cytokine production in response to DC-derived IL-12. These combined effects resulted in improved anti-CD33 CAR T cytotoxicity in vitro and in vivo NSG AML mice model. Our findings implicate the development of a new strategy of CAR T therapy combined to CAR-DC to increase the efficacy of cancer immunotherapy. DisclosuresNo relevant conflicts of interest to declare.

2279. A Randomized Open-Label Trial of 2-Dose or 3-Dose Primary Rabies Immunization Among Thai Children

BackgroundThe World Health Organization (WHO) recently recommended 2-dose primary rabies immunization instead of the 3-dose standard regimen. Given limited data of 2-dose regimens in pediatric population, this study was conducted. The objective was to compare the immunogenicity between 2-dose and 3-dose primary rabies immunization.MethodsThis study was an open-label clinical trial. Inclusion criteria were children aged 2–12 years with rabies virus neutralizing antibody (RVNA) titers < 0.5 IU/ml at baseline. The participants were divided into 2-dose vaccination (2D) on days 0 and 28 and 3-dose vaccination (3D) on days 0, 7, and 28 with a 2:1 ratio. A dose of 0.5 ml purified vero cell rabies vaccine (PVRV) was administered intramuscularly. RVNA titers were measured at 14-day post primary immunization. RVNA titers ≥ 0.5 IU/ml were considered seroprotective against rabies. Geometric mean titers (GMT) were calculated. T cell specific response to rabies vaccine antigen were measured from peripheral blood mononuclear cells (PBMCs) using the interferon-gamma enzyme-linked immunospot (IFN-gamma ELISpot) assay.ResultsFrom September to October 2017, 105 participants (52% male), 76 in 2D group and 29 in 3D group were enrolled. Median age and body weight was 70 months (IQR 53–88) and 19.2 kilograms (IQR 15.9–24.3), respectively. All participants had seroprotection at 14-day post primary immunization with GMT of 18.6 (95% CI 15.8-21.9) and 16.3 (95% CI 13.1–20.0) in 2D and 3D groups, respectively (P = 0.35). Median IFN-gamma level at 14-day post primary immunization was 60 spot forming cells (SFC) per 106PBMCs and 132 SFC per 106PBMCs in the 2D and 3D groups, respectively (P = 0.15).ConclusionThe immunogenicity of 2-dose primary rabies immunization at 14-day post primary vaccination is comparable to the 3-dose regimen. Participants are currently being followed for 1-year results.Disclosures All authors: No reported disclosures.

Production of recombinant TSA-1 and evaluation of its potential for the immuno-therapeutic control of Trypanosoma cruzi infection in mice

ABSTRACTA therapeutic vaccine for human Chagas disease (American trypanosomiasis caused by Trypanosoma cruzi) is under development based on the success of vaccinating mice with DNA constructs expressing the antigens Tc24 and Tc-TSA-1. However, because DNA and nucleic acid vaccines produce less than optimal responses in humans, our strategy relies on administering a recombinant protein-based vaccine, together with adjuvants that promote Th1-type immunity. Here we describe a process for the purification and refolding of recombinant TSA-1 expressed in Escherichia coli. The overall yield (20–25%) and endotoxin level of the purified recombinant TSA-1 (rTSA-1) is suitable for pilot scale production of the antigen for use in phase 1 clinical trials. Mice infected with T. cruzi were treated with rTSA-1, either alone or with Toll-like receptor 4 (TLR-4) agonist adjuvants including monophosphoryl lipid A (MPLA), glucopyranosyl lipid A (GLA, IDRI), and E6020 (EISEI, Inc). TSA-1 with the TLR-4 agonists was effective at reducing parasitemia relative to rTSA-1 alone, although it was difficult to discern a therapeutic effect compared to treatment with TLR-4 agonists alone. However, rTSA-1 with a 10 ug dose of MPLA optimized reductions in cardiac tissue inflammation, which were significantly reduced compared to MPLA alone. It also elicited the lowest parasite burden and the highest levels of TSA-1-specific IFN-gamma levels and IFN-gamma/IL-4 ratios. These results warrant the further evaluation of rTSA-1 in combination with rTc24 in order to maximize the therapeutic effect of vaccine-linked chemotherapy in both mice and non-human primates before advancing to clinical development.

Endometrium infection by human herpesvirus-6A: implication in female idiopathic infertility

We first reported HHV-6A DNA presence in 43% of endometrial cells from women with idiopathic infertility, whereas no fertile control women harbored the virus1. Growing evidence confirms this implication1-6. We investigated the effect of HHV-6A infection on the immunological status of the endometrium. This is a case-control study. The sample size guarantees to achieve a 0.8 power with 5% alpha error in detecting differences between groups. Endometrial biopsies, uterine flushing and whole blood samples were collected during the implantation (mid secretory phase) windows in 67 idiopathic infertile women. The presence of HHV-6A infection was evaluated by DNA and mRNA analysis (U22, U42, U94 genes) on endometrial cellular subsets1. We analyzed the endometrial immunological status evaluating: i) the levels of immune-regulatory HLA-G and HLA-E molecules, innate inflammatory (IL-6, IL-8, TNF-alpha), Th1 pro-inflammatory (IFN-gamma, IL-1alpha, IL-1beta, IL-12) and Th2 anti-inflammatory (IL-4, IL-10) cytokines by multiplex ELISA in uterine flushing samples; ii) endometrial receptivity to cytotrophoblasts in an endometrial 3D in vitro model; iii) natural killer (NK) cells and regulatory T cells percentage and immune-phenotype in endometrial biopsies and peripheral blood by flow cytometry. We confirmed the presence of HHV-6A infection in a 40% of idiopathic infertile women. We observed increased levels of IFN-gamma, IL-1beta and IL-12 (p<0.001; Student t test), decreased levels of both IL-4 and IL-10 (p<0.001) in uterine flushing samples from HHV-6A positive women. HLA-G and HLA-E molecules were not expressed on the surface of endometrial epithelial cells from HHV-6A positive women. As a proof of principle, endometrial biopsis positive for HHV-6A infection presented a lower permittivity to cytotrophoblasts invasion. We observed a lower amount of endometrial (e)NK cells in the endometrium of women positive for HHV-6A infection compared with endometrium negative for HHV-6A (p<0.0001; Student T test). In particular, when we analyzed (e)NK subpopulations, we observed a lower percentage of CD56brightCD16- (e)NK cells in women positive for HHV-6A infection (p<0.0001). When we looked at peripheral blood cell subsets, we observed a decrease in CD4+CD25+CD127dim/- regulatory T cells in women positive for HHV-6A infection (p=0.016). The identification of an effect of HHV-6A infection on endometrial immune status opens new perspectives in idiopathic infertile women care management. In addition, it would be possible to select antiviral therapies as novel therapeutic approaches to those idiopathic infertile women characterized by the presence of endometrial HHV-6A infection, to increase their pregnancy rate.