Leukocyte Cell Types Research Articles

Unsupervised clustering of brain tumor leukocytes to reveal atypical lymphoid cell subsets.

25 Background: The brain tumor immune microenvironment (TiME) is characterised by suppression of tumor-infiltrating leukocytes. The lymphocyte niche activity is particularly downregulated within this TiME milieu. Methods: Here, using data-driven approaches in flow cytometry and RNA sequencing analysis, we aimed at dissecting leukocyte cell types infiltrating human brain tumor TiME. Results: Unsupervised clustering provided T lymphocyte subsets including double-negative (CD3+CD4- CD8-) T cells (DNT). Our DNT phenotype was validated with an independent dataset, suggesting DNT to be compatible with γδ T cell phenotype. In our cohort, myeloid immune cell frequencies were associated with malignancy type as previously reported, while a gliosarcoma notably presented unexpectedly high lymphocyte frequencies. Consistently, double-positive (CD4+ CD8+) T cells (DPT) had higher frequency in the gliosarcoma than other tumors. The flow cytometry results were supported by transcriptome patterns and the activity of immune-related genes in these tumors. Conclusions: Our findings feature the benefit of data-driven approaches for brain TiME lymphocyte analysis, and show that primary brain tumors can entail atypical lymphocyte subsets like DNTs and DPT cells. Unbiased lymphocyte characterisation can potentially outline novel targets for immune checkpoint blockade therapy.

IMMUNOSTIMULANT ACTIVITY OF HANTAP (STERCULIA COCCINEA JACK) LEAVES EXTRACT ON NON-SPECIFIC AND SPESIFIC IMMUNE RESPONSES

Objective: This study aims to determine the immunostimulant effect and the effective dose of hantap leaf ethanolic extract against non-specific and specific immune responses.
 Methods: The immunostimulant activity of the extract was tested by the carbon clearance method (non-specific response). The effect of the extract on delay-type hypersensitivity/DTH (cellular-specific response) was determined by the paw edema method. The number of leukocyte cells and the percentage of leukocyte cell types were also calculated. The antibody titer (humoral-specific response) test was carried out by the hemagglutination method.
 Results: The results of the non-specific immune response demonstrated that three doses of hantap leaf extract, when compared to the negative control, could accelerate the rate of carbon elimination and increase the phagocytosis index. Hantap leaf extract can provide an immunomodulatory effect by increasing the delayed-type hypersensitivity response by showing a greater volume of leg swelling than the negative control received CMC Na 0.5%, which was significantly different (p<0.05), increasing leukocyte count and leukocyte differential and increasing primary and secondary antibody titers. The effective dose of hantap leaves extract was 200 mg/kg body weight.
 Conclusion: This study proves that hantap leaf extract has an immunomodulatory effect that increases the immune system (immunostimulant)

Mouse methylation profiles for leukocyte cell types, and estimation of leukocyte fractions in inflamed gastrointestinal DNA samples.

Precise analysis of tissue DNA and RNA samples is often hampered by contaminating non-target cells whose amounts are highly variable. DNA methylation profiles are specific to cell types, and can be utilized for assessment of the fraction of such contaminating non-target cells. Here, we aimed 1) to identify methylation profiles specific to multiple types of mouse leukocytes, and 2) to estimate the fraction of leukocytes infiltrating inflamed tissues using DNA samples. First, genome-wide DNA methylation analysis was conducted for three myeloid-lineage cells and four lymphoid-lineage cells isolated by fluorescence-activated cell sorting after magnetic-activated cell sorting from leukocytes in the spleen. Clustering analysis using CpG sites within enhancers separated the three myeloid-lineage cells and four lymphoid-lineage cells while that using promoter CpG islands (TSS200CGIs) did not. Among the 266,108 CpG sites analyzed, one CpG site was specifically hypermethylated (β value ≥ 0.7) in B cells, and four, seven, 183, and 34 CpG sites were specifically hypomethylated (β value < 0.2) in CD4+ T cells, CD8+ T cells, B cells, and NK cells, respectively. Importantly, cell type-specific hypomethylated CpG sites were located at genes involved in cell type-specific biological functions. Then, marker CpG sites to estimate the leukocyte fraction in a tissue with leukocyte infiltration were selected, and an estimation algorithm was established. The fractions of infiltrating leukocytes were estimated to be 1.6-12.4% in the stomach (n = 10) with Helicobacter pylori-induced inflammation and 1.5-4.3% in the colon with dextran sulfate sodium-induced colitis (n = 4), and the fractions were highly correlated with those estimated histologically using Cd45-stained tissue sections [R = 0.811 (p = 0.004)]. These results showed that mouse methylation profiles at CpG sites within enhancers reflected leukocyte cell lineages, and the use of marker CpG sites successfully estimated the leukocyte fraction in inflamed gastric and colon tissues.

Pemanfaatan Gel Ekstrak Kulit Buah Mangga Arumanis (Mangifera indica L.) Sebagai Antiinflamasi Dengan Metode Kantong Granuloma Secara In-Vivo

In previous research, mango rind extract has been studied as an antioxidant, antihypertensive, and antidiabetic, because it contains flavonoids and 17% mangiferin compounds. In this research, the effect of arumanis mango rind extract gel (Mangifera indica L.) as an anti-inflammatory was tested in male white mice induced by carrageenan using the granuloma pouch method. This experimental study used experimental white male mice which were grouped into 5 groups: the control group, the group with 2% mango rinf extract, the group with 4% mango rind extract, the group with 8% mango rind extract, and the comparison group. The parameters that were analyzed were exudate volume, total leukocytes, and type of leukocytes on days 3, 5, 7. The results showed that mango arumanis (Mangifera indica L.) rind extract had anti-inflammatory effectiveness topically as indicated by a decrease in the average exudate volume, total leukocytes, and leukocyte cell types (segmented neutrophil cells and lymphocyte cells) in each concentration group of mango rind extract, based on two-way ANOVA test and Duncan's test showed significant differences with respect to concentration and time (p&lt;0.05). It can be concluded that all gel concentrations of arumanis mango rind extract (Mangifera indica L.) 2%, 4%, and 8% have anti-inflammatory effects.

Evaluation of leukocytes cells types counts in blood from patients with different severities of periodontal diseases

Background: Periodontal diseases are inflammatory disorders caused by the accumulation of oral biofilm and the host response to this accumulation which characterized by exaggerated leukocytes and neutrophils attraction to the sites of inflammation by chemoattractants which are a very important part of the pathogenesis of periodontal diseases. This study aimed to determine and compare the clinical periodontal parameters and the leukocyte cell types in the peripheral blood between patients with gingivitis and periodontitis with different severities compared to healthy controls. Materials and methods: This study included 150 male subjects aged between 35-50 years. They were divided into three groups: gingivitis group (n=30), periodontitis patients (n=90) which subdivided into Mild =30 patients, Moderate =30 patients, Severe =30 patients and a control group (n=30) with clinically healthy periodontium. Clinical periodontal parameters were recorded ((plaque index (PLI), gingival index (GI), bleeding on probing (BOP), probing pocket depth (PPD) and clinical attachment level (CAL)). Blood samples were collected then an automated blood analyzer evaluated leukocyte cell types. Results: Significant differences in The counts of neutrophils and lymphocytes exhibited significant differences among the study groups and subgroups. On contrary, differences in monocytes, eosinophils, and basophils counts were not significant. Additionally, severity of periosontitis was significantly correlated with the mean counts of the various leukocyte cell types; however, clinical periodontal characteristics did not show such correlation with these inflammatory cells. Conclusion: This study demonstrated that periodontal disease with different severities is associated with possible episodes of bacteremia that originate from periodontal lesions which mediate inflammatory conditions that in turn causing changes in the systemic markers especially leukocytes cells types.

Effects of Transport and Holding Stress on Prussian Carp (Carassius gibelio, Bloch, 1782.) Leukogram Pattern

Abstract Stress inevitably occurs during any fish handling and manipulation in culturing, research, or clinical examination situations that require capture and removal of fish from water. Different stress factors can affect the changes in the relative numbers and function of cells of the fish immune system. Catching, transportation and over-crowding caused stress-induced changes in the total number of leukocytes and thrombocytes, as well as changes in the leukocyte formula in Prussian carp (Carassius gibelio, Bloch, 1782) presented here. Cytochemical characterization of leukocyte cell types was performed by applying Myeloperoxidase (MPO), Periodic Acid Schiff (PAS) and Sudan Black B (SBB) staining of Prussian carp blood smears. Cytochemical characterization is a rapid and efficient method for white leukocyte differentiation and insight in their functional status. Comparison and analysis in Prussian carp hematological parameters from fish with and without exposure to stressful conditions such as capture, manipulation, transport and holding, revealed significant differences between stressed and non-stressed fish. Significant reduction in the total number of thrombocytes and lymphocytes and the increase in total neutrophil count were observed in stressed animals. However, differences in total leukocyte number and the number of monocytes were not observed. Deviations from the estimated reference intervals for Prussian carp hematological parameters clearly indicated the presence/absence of a stress reaction and to some extent its intensity. Estimated reference intervals and characterization of morphological and cytochemical appearance of blood cells form a solid basis for further research of the cellular immune function and hematology changes in Prussian carp.

Hematologic parameters of captive Bothrops atrox (Squamata: viperidae)

The breeding of venomous snakes in captivity for research purposes and mainly as a source of pharmaceutical products highlights the need to determine hematological parameters for monitoring and ensuring a healthy breeding populationThe complete blood count is used to help diagnose alterations such as anemia, inflammatory diseases, parasitemia, hematopoietic disorders, hemostatic and toxicological changes, as well as bacterial and viral inclusions. Thus, the objective of this study was to define reference parameters for complete blood count in Bothrops atrox snakes. Blood samples were collected from 20 specimens of B. atrox from the Pentapharm do Brasil commercial breeding facility for laboratory examination. Mean values and standard deviation were: hematocrit 33.6 ± 5.47%, hemoglobin 10.81 ± 2.07g/dL, total number of erythrocytes 0.59 ± 0.1 x 106/mm3, leukocytes 11387.5 ± 3279.2/mm3 and thrombocytes 28175 ± 6320/mm3. No significant difference was observed between males and females and heterophils were the predominant leukocyte cell type.

The effect of induced lymphatic circulation on lymphangiogenesis and inflammatory mediators in rats with adjuvant induced arthritis

Abstract Rheumatoid arthritis (RA) is a chronic inflammatory disease involving dysfunctional lymphatic circulation leading to edema. The adjuvant-induced arthritis (AIA) rat model was used to study the effect of lymphatic pump treatment (LPT) on the expression levels of inflammatory cytokines and lymphangiogenesis within the draining popliteal lymph nodes (PLN). PLN from both control and LPT treatment groups were harvested, and their cDNA were analyzed by qPCR to determine the levels of cytokines, VEGF-C, and VEGFR-3. Additionally, immunohistochemistry was used to visualize the expression of VEGF-C and VEGFR-3 and flow cytometry was used to quantify leukocyte cell types in the PLN. The LPT group showed a decrease in ankle circumference and arthritis grade a few days after the initiation of treatment. Flow cytometry of popliteal lymph nodes showed an increase in CD8, CD4, and CD3 cells in arthritic rats, however cell populations were not significantly different between treatment groups. The qPCR data indicated that the LPT group had a slight reduction in levels of IL-17a, IL-4, IL-6, and IL-1β. IHC showed that the PLN from the LPT group had the highest expression area of VEGF-C and the lowest expression area of VEGFR-3 compared to control. In conclusion, The LPT group showed a reduction in ankle circumference and arthritis score, suggesting that LPT helped alleviate edema and inflammation. When compared to control, the LPT group had lower expression of pro-inflammatory cytokines and elevated levels of VEGF-C, an important lymphatic vessel growth factor, in PLN. Thus, the data suggests that LPT may elevate the swelling and inflammation in rat AIA through the reduction of inflammatory cytokines and the elevation of a lymphatic growth factor.

Deep phenotyping of surface stimulatory and inhibitory co-receptors on cancer-resident T and NK cells reveals cell subsets within the tumor-reactive CTL population that are uniquely defined by NKG2A expression.

The field of Immuno-Oncology (IO) is evolving to utilise novel antibody backbones that can co-target multiple cell-surface stimulatory and inhibitory co-receptors (SICR). This approach necessitates a better understanding of SICR co-expression at the single-cell level on IO-relevant tumor-infiltrating leukocyte (TIL) cell types such as T and natural killer (NK) cells. Using high-dimensional flow cytometry we established a comprehensive SICR profile for tumor-resident T and NK cells across a range of human solid tumors where there is a clear need for improved immunotherapeutic intervention. Leveraging the power of our large flow panel, we performed deep-phenotyping of the critical CD8+CD39+ Cytotoxic T Lymphocyte (CTL) population that is enriched for tumor-reactive cytotoxic cells, revealing subsets that are differentiated by their SICR profile, including three that are uniquely defined by NKG2A expression. This study establishes a comprehensive SICR phenotype for human TIL T and NK cells, providing insights to guide the design and application of the next generation of IO molecules.

ANTI INFLAMMATORY ACTIVITY OF SUNGKAI LEAVES (Peronema canescens JACK) ETHANOL EXTRACT IN CARRAGEENAN INDUCED MICE

Anti-inflammatory compounds play a role by inhibiting the formation of prostaglandin mediators, inhibiting the migration of leukocytes to the area of ​​inflammation, and inhibiting the release of prostaglandins from the cells where they are located. Several plant-derived compounds that can act as anti-inflammatory candidates are flavonoids, saponins, alkaloids, and phenols. This study aims to determine the anti-inflammatory activity of the ethanol extract of Sungkai leaves and to determine the effect of the concentration given on the level of anti-inflammatory activity. The method used was the formation of granuloma pockets and edema on the mice's backs by inducing subcutaneous carrageenan using ethanol extract of Sungkai leaves with various concentrations of 5%, 10%, and 15%, respectively. The results of anti-inflammatory activity showed that the ethanol extract of Sungkai leaves had a significant effect on the average exudate volume and the percentage of inflammation inhibition. At a concentration of 15%, the extract was able to reduce the volume of exudate by 46,67 ± 5,506 µl and inhibition of inflammation by 87.78%. In addition, the ethanol extract of Sungkai leaves significantly affected lymphocytes, stem neutrophils, and segment neutrophils, but did not significantly affect the number of monocyte cells. At a concentration of 15%, the ethanol extract of Sungkai leaves showed the smallest number of leukocyte cell types, namely 50.11 ± 2.389 lymphocyte cells; stem neutrophil cells 10,44 ± 0,475; segmented neutrophil cells were 19.78 ± 0.596 and monocyte cells 2,0 ± 0,236. It can be concluded that the ethanol extract of Sungkai leaves has anti-inflammatory activity but has not approached the effect of anti-inflammatory drugs in general.

Abstract MP221: Early Recruitment Of Neutrophils To The Ischemic Heart Is Orchestrated By Catecholamine-induced Demargination

Myocardial infarction (MI) induces a rapid and robust inflammatory response characterized by infiltration of different leukocyte cell types to the infarcted heart. Neutrophils are the first cells to arrive at the infarct where they release a plethora of proteolytic enzymes, exacerbate tissue injury, expand infarct size and promote cardiac dysfunction/ failure. We previously have shown that granulopoiesis (in the bone marrow, BM) is the major source of neutrophils during MI. However, the increase in the number of neutrophils at the infarct begins much earlier than the peak response in the BM (~ 24 hours) suggesting that sources other than granulopoiesis may contribute to the overall neutrophil pool. Because the marginated pool of neutrophils represents the same size of circulating pool, we hypothesized that demargination of neutrophils from the vascular wall contribute to the neutrophil burden in the heart, particularly during the early hours after MI.Using a mouse model of the permanent ligation of the left anterior descending coronary artery, BM ablation of hematopoietic stem cells, flow cytometry and BM transplant techniques, we found that the first wave of neutrophils recruited to the ischemic heart are exclusively sourced from the vasculature and not granulopoiesis in the BM/ spleen. The neutrophils recruited at the heart bore all hallmarks of demargination induced by dexamethasone including decreased F-actin, CD62L, and increased Adam17 expression. The recruitment of neutrophils is orchestrated by catecholamine stress as experimental strategies aimed at blockade of β1 and β2 adrenergic receptors (AR) reduced neutrophil burden in the ischemic heart. Interestingly, despite a decrease in neutrophil burden, the cardiac function did not improve but rather declined. However, short-term inhibition (6-12 hours post-MI) of β AR system either by receptor blockade (propranolol) or inhibition of catecholamine synthesis (AMPT) not only reduced neutrophil burden but also significantly improved cardiac function. Together these data suggest that catecholamine stress induced-demargination constitute the major source of neutrophils during the early hours post-MI. Pharmacological strategies aimed at suppressing the initial onslaught of neutrophils on the ischemic heart may represent a novel and viable approach towards a better resolution of the injury. Currently, studies are underway to define the signaling mechanisms that drive demargination and, to optimize the dose/ duration of a specific β receptor blocker to achieve better functional outcomes.

THE EFFECT OF MANGO LEAVES EXTRACT VAR ARUM MANIS (Mangifera indica L.) AS IMUNOSTIMULANTS ON MICE MODEL

Background : Every time our body is always exposed to microorganisms that can cause infection. Usually we are immune to infection because of the immune system that protects us, but when the body is exposed to antigens while the immune system activity decreases, then we need immunostimulants. One of traditional plant always get to use as the traditional drug is mango plant, Previous research it have effect as antioxidant, antidiabetes type II, antiinflammatory, anticancer, antihypertensive, and antimicrobial. It contains the flavonoid, alkaloid, phenolic,and steroid compounds. This study aims to determine the effect of mango leaves extract var arum manis against the activity and phagocytosis capacity of macrophage cells, and the percentage of leukocyte cell types, as well as the effect of dose variations on the administration of var arum manis mango leaves extract. Methods :Twenty mice were divided into 4 groups. Consisting of group I getting 0.5% Na.CMC, groups II, III and IV getting mango leaves extract var arum manis with consecutive doses of 30 mg / kgBW, 60 mg / kgBW, and 120 mg / kgBW which were given respectively. orally once a day for 7 days. On the eighth day, Staphylococcus aureus bacteria were injected intraperitoneally and after one hour, the activity and capacity of macrophage cell phagocytosis and the percentage of leukocyte cell types were calculated. Result and Discussion: The activity and phagocytosis capacity of macrophage cell was determined by calculating the phagocytosis index where the results obtained were the higher the dose, the increased activity and phagocytosis capacity of macrophage cell which affected the percentage of leukocyte cell types. The data were then processed by statistical analysis of one-way ANOVA. Conclusion of research showed that the mango leaves extract var arum manis at a dose of 30 mg / KgBW, 60 mg / KgBW, 120 mg / KgBW was significant (p ≤ 0.05) and affected activity and phagocytosis capacity of macrophage cells and the percentage of leukocyte cell types significantly (p ≤ 0.05), where the most effective result was a dose of 120 mg / KgBW

Time-dependent hematological responses of Nile tilapia Oreochromis niloticus exposed to an estuarine contaminated water

We aimed to study hematological responses of Oreochromis niloticus experimentally exposed to the contaminated water of the Santos-São Vicente Estuary, testing hypotheses that exposure time to estuarine water promotes deleterious effects on hematological parameters and evaluating the use of erythrocytes and leukocytes alterations as environmental biomarkers. Estuarine water was collected from Largo da Pompeba. For the biological assay, 28 juveniles of O. niloticus (red strain) of both genders were randomly selected from commercial pisciculture. For the biological assay, 28 juveniles of O. niloticus of both sexes were randomly selected from commercial fish farms. The juveniles were kept in estuarine water for 72 and 120 hours and, after exposure, blood was collected by puncture of the caudal vein to determine total erythrocytes, hemoglobin concentration, hematocrit, hematimetric indices and total leukocytes, as lymphocyte, neutrophils, monocytes, eosinophils, and basophils were quantified by blood extensions. To test exposure overtime on hematological variables, we performed a two-factor Multivariate Analysis of Variance. Exposure for 72 hours resulted in immunosuppression as seen by the reduced counts of neutrophils, monocytes, and lymphocytes in the bloodstream, whereas after 120 hours the immune system was stimulated with the increase of all leukocyte cell types. Exposure to estuarine water resulted in marked changes in the leukocyte count of O. niloticus, demonstrating that alterations in white blood cells might be more sensitive biomarkers than red blood parameters.

Impact of copper and zinc mixture on haematological parameters of rainbow trout (Oncorhynchus mykiss): acute exposure and recovery.

Significant changes in composition of rainbow trout Oncorhynchus mykiss blood cells types were induced after 4-days exposure with mixture of Cu2+ and Zn2+ at 0.25, 0.125 and 0.06 parts of LC50 in comparison to control group. The highest concentration of metal mixture (0.25 of LC50) significantly induced elevation of the number of monocytes and poly-segmented neutrophils. Treatment with 0.125 parts of LC50 concentration increased the number of thrombocytes, monocytes and non-segmented neutrophils. The most diluted mixture resulted in significant induction of thrombocytes, monocytes, non- and poly segmented neutrophils. Analysis of leucocyte cell types in the O. mykiss blood samples after 4-days of exposure at all applied mixture parts showed signs of monocytosis and neutrophilia. Comparison of different types of leucocytes' percentages (leukogram) in fish after 4-days exposure to metal mixture and after 4, 8, and 12-days recovery periods showed that, values of neutrophils even after the 12-days recovery period at all tested parts of LC50, and monocytes after exposure with the highest (0.25) used part of LC50 were not restored to control group levels. Depuration and recovery processes in treated fish are concentration and recovery period dependent.

Sensitive Immunopeptidomics by Leveraging Available Large-Scale Multi-HLA Spectral Libraries, Data-Independent Acquisition, and MS/MS Prediction

Mass spectrometry (MS) is the state-of-the-art methodology for capturing the breadth and depth of the immunopeptidome across human leukocyte antigen (HLA) allotypes and cell types. The majority of studies in the immunopeptidomics field are discovery driven. Hence, data-dependent tandem MS (MS/MS) acquisition (DDA) is widely used, as it generates high-quality references of peptide fingerprints. However, DDA suffers from the stochastic selection of abundant ions that impairs sensitivity and reproducibility. In contrast, in data-independent acquisition (DIA), the systematic fragmentation and acquisition of all fragment ions within given isolation m/z windows yield a comprehensive map for a given sample. However, many DIA approaches commonly require generating comprehensive DDA-based spectrum libraries, which can become impractical for studying noncanonical and personalized neoantigens. Because the amount of HLA peptides eluted from biological samples such as small tissue biopsies is typically not sufficient for acquiring both meaningful DDA data necessary for generating comprehensive spectral libraries and DIA MS measurements, the implementation of DIA in the immunopeptidomics translational research domain has remained limited. We implemented a DIA immunopeptidomics workflow and assessed its sensitivity and accuracy by matching DIA data against libraries with growing complexity—from sample-specific libraries to libraries combining 2 to 40 different immunopeptidomics samples. Analyzing DIA immunopeptidomics data against a complex multi-HLA spectral library resulted in a two-fold increase in peptide identification compared with sample-specific library and in a three-fold increase compared with DDA measurements, yet with no detrimental effect on the specificity. Furthermore, we demonstrated the implementation of DIA for sensitive personalized neoantigen discovery through the analysis of DIA data with predicted MS/MS spectra of clinically relevant HLA ligands. We conclude that a comprehensive multi-HLA library for DIA approach in combination with MS/MS prediction is highly advantageous for clinical immunopeptidomics, especially when low amounts of biological samples are available.

EPCO-25. AN IMMUNOMETHYLOMIC PLATFORM INTEGRATING SYSTEMIC IMMUNE PROFILES AND EPIGENETIC AGE IN NEURO-ONCOLOGY

Abstract Lineage-specific DNA methylation marks differentiate leukocyte cell types while individual biological aging mechanisms impact other methylation alterations. Human glioma incidence and survival times have been shown to be associated with aberrant immune profiles and have a strong dependency on age. Here we developed a single epigenetic analysis framework to evaluate both immune cell fractions and epigenetic age in peripheral blood. We examined these measures in archived blood from 197 triple-negative glioma patients (TNG; IDH wildtype, 1p19q intact and TERT wildtype) and 312 frequency-matched controls from the SF Bay Area Adult Glioma Study (AGS). Significant differences were observed with TNG cases having lower CD4 and CD8 T cell, natural killer, and B cell fractions, and higher neutrophil fractions than controls. TNG cases were significantly older than controls in two of three epigenetic age estimates; however, there was no difference in epigenetic age acceleration once immune cell proportions were considered. For the TNG cases, we augmented results from several machine learning methods to delineate risk groups of TNG patients with significantly different overall survival. We compared survival models built by recursive partitioning, random forest, and elastic net methods. The final model was chosen by repeated bootstrap sampling via the Brier score loss function and validated in an independent set of 72 IDH-mutant only or TERT-mutant only glioma patients also from the AGS. The final model indicated important interactions between immune cell fractions (including CD4 and CD8 T cells and neutrophils) and treatment, age, and dexamethasone status when adjusted for the main effects of epigenetic age, glioblastoma status, and the neutrophil-to-lymphocyte ratio. The capacity of immunomethylomics to capture diverse, clinically relevant information and the simplicity of its implementation make this a powerful tool for personalized patient evaluation in the neuro-oncology clinic.

Cytosolic DNA Sensors and CNS Responses to Viral Pathogens.

Viral central nervous system (CNS) infections can lead to life threatening encephalitis and long-term neurological deficits in survivors. Resident CNS cell types, such as astrocytes and microglia, are known to produce key inflammatory and antiviral mediators following infection with neurotropic DNA viruses. However, the mechanisms by which glia mediate such responses remain poorly understood. Recently, a class of intracellular pattern recognition receptors (PRRs), collectively known as DNA sensors, have been identified in both leukocytic and non-leukocytic cell types. The ability of such DNA sensors to initiate immune mediator production and contribute to infection resolution in the periphery is increasingly recognized, but our understanding of their role in the CNS remains limited at best. In this review, we describe the evidence for the expression and functionality of DNA sensors in resident brain cells, with a focus on their role in neurotropic virus infections. The available data indicate that glia and neurons can constitutively express, and/or can be induced to express, various disparate DNA sensing molecules previously described in peripheral cell types. Furthermore, multiple lines of investigation suggest that these sensors are functional in resident CNS cells and are required for innate immune responses to viral infections. However, it is less clear whether DNA sensormediated glial responses are beneficial or detrimental, and the answer to this question appears to dependent on the context of the infection with regard to the identity of the pathogen, host cell type, and host species. Defining such parameters will be essential if we are to successfully target these molecules to limit damaging inflammation while allowing beneficial host responses to improve patient outcomes.

Abstract 4434: Comprehensive bioinformatic analysis of immune composition and genes for survival prediction in sarcoma

Abstract Introduction Sarcomas, a broad family of mesenchymal malignancies, exhibit remarkable histologic diversity. Previous study has reported that the infiltration of immune and stromal cells in tumor microenvironment contribute significantly to prognosis. ESTIMATE, an algorithm to calculate immune and stromal scores, predicts the infiltration of non-cancer components. The Cancer Genome Atlas (TCGA) database is available to grasp potential correlations between gene set prolife and overall survival of malignancies, including sarcomas. To better understand the proportions of immune cells in the tumor microenvironment, We used CIBERSORT deconvolution software and ssGSEA to infer the relative proportions of several distinct leukocyte cell types in the tumors from microarray gene expression data of sarcoma patients. By taking advantage of both TCGA database of sarcomas cohorts and ESTIMATE algorithm, we extracted a list of genes that predict poor outcomes in sarcoma patients. Finally, we validated these genes in an independent sarcoma cohort from the GSE17679. Thus, we obtained a list of tumor microenvironment-related genes that predict poor outcomes in sarcoma patients. Methods Gene expression profile and clinical data for sarcoma patients was obtained from the TCGA data portal. Immune scores and stromal scores were calculated by applying the ESTIMATE algorithm to the downloaded database. Batch adjusted data was subsequently analyzed using CIBERSORT to resolve the immune composition. For validation, gene expression profiles for Ewing sarcoma patients were obtained from the Gene Expression Omnibus dataset GSE17679. Results The immune and stromal score are associated with prognosis. The type 2 macrophages in sarcoma makes up the largest composition of all immune cells in the tumor microenvironment of sarcoma (except synovial sarcoma), which predicts poor outcomes. From functional enrichment analysis of TCGA database applied by ESTIMATE algorithm-based immune scores, we extracted that NR1H3, VAMP5, GIMAP2, GBP2, HLA-E and CRIP1 are highly expressed in the immune microenvironment, predicting good outcomes in sarcoma patients. Conclusion We extracted a list of tumor microenvironment related genes. These genes were validated in an independent sarcoma cohort and that may represent promising novel signatures for the diagnosis and prognosis prediction of sarcoma. The immune composition analysis could be useful for outlining the prognosis. Citation Format: Hongmin Chen. Comprehensive bioinformatic analysis of immune composition and genes for survival prediction in sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4434.

Glucocorticoid receptor sensitivity in early pregnancy in an African American cohort.

Disruption in homeostatic feedback loops between inflammatory mediators and the hypothalamic-pituitary-adrenal (HPA) axis is a key mechanism linking chronic stress to inflammation and adverse health outcomes, including those occurring during pregnancy. In particular, alterations in glucocorticoid sensitivity may occur as a result of chronic stress, including that due to racial discrimination, and may be implicated in the persistent adverse maternal and infant health outcomes experienced by African Americans. While there are a few large-scale studies in human pregnancy that measure both cytokines and HPA axis hormones, to our knowledge, none directly measure glucocorticoid sensitivity at the cellular level, especially in an African American population. We measured the full range of the dexamethasone (DEX) dose-response suppression of TNF-α in first-trimester blood samples from 408 African American women and estimated leukocyte cell type contribution to the production of TNF-α. The mean (SD) DEX level needed to inhibit TNF-α production by 50% (ie, DEX IC50 ) was 9.8 (5.8) nmol/L. Monocytes appeared to be the main driver of Uninhibited TNF-α production, but monocyte counts explained only 14% of the variation. Monocyte counts were only weakly correlated with the DEX IC50 (r=-.11, P<.05). Moreover, there was no statistically significant correlation between the DEX IC50 and circulating pro-inflammatory (CRP, IL-6, IFN-γ) or anti-inflammatory (IL-10) mediators (P>.05). These findings challenge some prior assumptions and position this comprehensive study of glucocorticoid sensitivity as an important anchor point in the growing recognition of interindividual variation in maternal HPA axis regulation and inflammatory responses.

Differential white blood cell numbers in response to lipopolysaccharide exposure in marine and terrestrial mammals

Marine mammals, such as bottlenose dolphins (Tursiops truncatus) are often seen with wounds, bruises or shark bites down to the muscle, apparently with no signs of infection. In terrestrial mammals, including humans, similar injuries would be associated with infections, inflammation and potentially death. The objective of this study was to compare the differential leukocyte numbers in marine and terrestrial mammals under control conditions and following exposure to lipopolysaccharide (LPS). Blood samples were collected following routine procedures from healthy human anonymous volunteers from the blood bank at Instituto Mexicano del Seguro Social, La Paz, Baja California Sur, Mexico, and from healthy dolphins at CaboDolphin. All samples were taken with ethylenediaminetetraacetic acid (EDTA) as anticoagulant, transported to the laboratory on ice and processed within 24 h. Leukocytes were separated by centrifugation with ficoll hypaque and subjected to cell culture at 35°C with 5% CO2 and constant humidity. Leukocytes (106 cells) from both species were exposed to 10 μg LPS μL−1 for 24 or 48 h and number of each leukocyte cell type was registered; cell type was assigned based on morphological characteristics. Under control (no LPS) conditions, number of lymphocytes from both species was higher at 24 and 48 h as compared to the initial numbers. In humans, but not in dolphins, lymphocyte numbers were lower in cells exposed to LPS for 24 or 48 h as compared to untreated cells. In both species, at all times and regardless of exposure to LPS, an inverse relationship was observed between lymphocyte and neutrophil numbers. In humans, basophil number was lower in cells exposed to LPS for 24 and 48 h as compared to untreated cells. In T. truncatus only one cell with morphology similar to basophils was observed in only one individual. These differences in leukocyte types between species in response to LPS exposure could contribute to explain the apparent lack of infection or inflammation in injured marine mammals. This research was conducted in accordance with institutional ethical standards and following the 1964 Declaration of Helsinki and later amendments and in accordance with the FASEB Statement of Principles for the use of Animals in Research and Education. The research protocol (2018‐785‐010) was approved by the Ethics Committee of Instituto Mexicano del Seguro Social (CONBIOÉTICA‐09‐CEI‐009‐2016061).Support or Funding InformationConsejo Nacional de Ciencia y Tecnología (CONACYT CB‐2016‐01‐283669)