Phagocytosis Activity Research Articles

INFLUENCE OF LATENT TUBERCULOSIS INFECTION ON THE STATE OF THE AUTONOMIC NERVOUS SYSTEM AND SOMATOTROPIC HORMONE PRODUCTION

The influence of latent tuberculosis infection (LTBI) on the state of the autonomic nervous system (ANS) and the level of somatotropic hormone (STH) in adolescents (100 people) aged 11 to 13 years was studied. In patients with LTBI, there was an autonomic imbalance with a predominance of sympathicotonia, which was accompanied by hyperergic reactions to the recombinant tuberculosis allergen, while in vagotonic and eutonic patients the number of hyperergic reactions was significantly less. Insignificant dynamics of the immunological test, low levels of growth hormone, and low activity of phagocytosis in adolescents with vagotonia were established, which may indicate a lack of cellular immune response. A more pronounced inversion of the immunological test with the recombinant tuberculosis allergen was revealed against the background of preventive treatment in eutonics and sympathicotonics, who had a higher level of growth hormone and phagocytosis activity initially. The established relationship between the functional state of the ANS and the growth hormone indicator, which influence the activity of phagocytosis in patients with latent tuberculosis infection, can be used to predict the effectiveness of anti-tuberculosis preventive therapy and can serve as an additional prognostic criterion for assessing the risks of developing active tuberculosis.

Cytological and microbiological investigations of professional hygiene efficiency in patients with generalized periodontitis.

Aim: The purpose of this study is to assess the impact of occupational hygiene procedures for microbiological and cytological contents of periodontal pockets. Material and Methods: Cytological and microbiological content of the periodontal pockets before treatment and after professional hygiene procedures including scaling with hand instruments and root cementum polishing have been investigated in patients with periodontitis. Results: According to obtained data it can be resumed that in periodontitis patients with the depth of pockets 3-5,5 mm before professional hygiene all the pockets contain great number of Cocci, Spirochetes, Candida Albicans, Flagellated rods and Protozoa species. It was proved by revealing of small amount of Polymorphonuclear leukocytes with active phagocytosis. After scaling and planing of the roots, a decrease in the number of Protozoa and Candida Albicans was observed in 97% and 72% of the investigated cells, respectively. Conclusions: Cytological and microbiological content of periodontal pockets before treatment and after professional hygiene procedures including scaling and root planning testify to the level of local protective mechanisms, especially process of phagocytosis and virulence of microbial species in periodontal pockets.

Изучение состава фенольных соединений, антирадикальной активности и влияния на фагоцитоз сухого водного экстракта шишек сосны обыкновенной

Pine cone is a new promising type of plant raw material for using in a medical practice. Objective: determination of the phenolic compounds composition of pine cones dry aqueous extract, research of its toxicity, antiradical activity and effect on phagocytosis. Materials and methods. The research object was a collection of pine cones gathered in October 2020. A dry aqueous extract was obtained from the cones by extraction with hot water. The phenolic compounds composition of the pine cones dry aqueous extract was determined by ultra-efficient liquid chromatography/MS on a Waters Acquisition chromatograph with a diode-matrix UV detector and a tandem quadrupole MC detector TQD (Waters). To determine the antiradical activity, a reaction with a stable free radical, 2,2-diphenyl-1-picrylhydrazyl (DPPH), was used. The acute toxicity of the pine cones dry extract was researched by the V.V. Prozorovsky express method on white non-linear mice. The phagocytic activity of peripheral blood leukocytes was determined in vivo by a modified method. Results. The phenolic compounds composition of the extract was analysed. The research identified 12 substances present in the extract, representing at least 1% of the total area on the chromatogram. The following procyanidins were found in the pine cones extract: procyanidin B2, procyanidin B3, procyanidin C1, procyanidin D1. Additionally, flavonoids and phenolic carboxylic acids were identified. The pine cones dry extract demonstrated pronounced antiradical activity, comparable to that of ascorbic acid. The acute toxicity determination revealed that the LD50 of the researched compounds is in doses greater than 1260 mg/kg. Upon examination of the effect of pine cone extract on the phagocytic activity of peripheral blood leukocytes, an increase in the phagocytic index was observed, yet no change was noted in the activity of total leukocyte phagocytosis. Conclusion. The dry aqueous extract of pine cones contains biologically active components of phenolic nature, which suggests that further research into its pharmacological activity may be promising.

Primary SARS-CoV-2 infection in children and adults results in similar Fc-mediated antibody effector function patterns.

Increasing evidence suggests that Fc-mediated antibody effector functions have an important role in protection against respiratory viruses, including SARS-CoV-2. However, limited data are available on the potential differences in the development, heterogeneity and durability of these responses in children compared to adults. Here, we assessed the development of spike S1-specific serum antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD) and natural killer cell activation (ADNKA), alongside specific antibody binding concentrations (IgG, IgA and IgM) and IgG avidity in healthy adults (n = 38, 18-56 years) and children (n = 21, 5-16 years) following primary SARS-CoV-2 infection, with a 10-month longitudinal follow-up. Differences between groups were assessed using a nonparametric Kruskal-Wallis test with Dunn's multiple comparisons test. We found similar (functional) antibody responses in children compared to adults, with a tendency for increased durability in children, which was statistically significant for ADCD (P < 0.05). While ADNKA was strongly reduced in both adults (P < 0.001) and children (P < 0.05) at the latest time point, ADCP remained relatively stable over time, possibly relating to an increase in avidity of the spike-specific antibodies (P < 0.001). Finally, the ADNKA capacity relative to antibody concentration appeared to decrease over time in both children and adults. In conclusion, our data provide novel insights into the development of SARS-CoV-2-specific antibody Fc-mediated effector functions in children and adults. An increased understanding of these characteristics in specific age populations is valuable for the future design of novel and improved vaccination strategies for respiratory viruses such as SARS-CoV-2.

Purinergic Receptor P2Y12-Mediated Tau Internalization in Microglia.

Microglia are the resident brain macrophage cells that are involved in constant surveillance of brain microenvironment. In Alzheimer's disease, microglia get over activated upon the accumulation of Tau and amyloid-β species in the extracellular space, ultimately leading to neurodegeneration. Microglia phagocytose the extracellular Tau species by several mechanisms among which P2Y12 receptor-mediated internalization of extracellular Tau is recently studied. Extracellular Tau activates microglia and directly interacts with the P2Y12 receptor. Tau-receptor complex is then internalized followed by perinuclear accumulation and lysosomal degradation. Upon microglial activation by extracellular Tau, P2Y12 receptor is also involved in membrane-associated actin remodeling which has its key role in active migration and phagocytosis.

New Frontiers in Monoclonal Antibodies for Relapsed/Refractory Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive lymphoma. Approximately 60% of patients are cured with R-CHOP as a frontline treatment, while the remaining patients experience primary refractory or relapsed disease (R/R). The prognosis for R/R DLBCL patients who are neither eligible for autologous stem-cell transplantations nor CAR-T-cell treatment is poor, representing an important unmet need. Monoclonal antibodies (mAbs) have dramatically improved therapeutic options in anti-cancer strategies, offering new opportunities to overcome chemo-refractoriness in this challenging disease, even in cases of primary non-responder DLBCL. Several novel mAbs, characterized by different mechanisms of action and targets, are now available for R/R DLBCL. Unbound mAbs induce an immune response against cancer cells, triggering different mechanisms, including antibody-dependent cellular cytotoxicity (ADCC), activation of antibody-dependent cell-mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). Antibody-drug conjugates (ADCs) and radioimmunotherapy (RIT), respectively, deliver a cytotoxic payload or a beta-emitter radionuclide to the targeted cells and nearby bystanders. Bispecific T-cell engagers (BiTes) and immune checkpoint inhibitors (ICIs) redirect and enhance the immune response against tumor cells. Here, we review therapeutic strategies based on monoclonal antibodies for R/R DLBCL.

RBD-Protein/Peptide Vaccine UB-612 Elicits Mucosal and Fc-Mediated Antibody Responses against SARS-CoV-2 in Cynomolgus Macaques.

Antibodies provide critical protective immunity against COVID-19, and the Fc-mediated effector functions and mucosal antibodies also contribute to the protection. To expand the characterization of humoral immunity stimulated by subunit protein-peptide COVID-19 vaccine UB-612, preclinical studies in non-human primates were undertaken to investigate mucosal secretion and the effector functionality of vaccine-induced antibodies in antibody-dependent monocyte phagocytosis (ADMP) and antibody-dependent NK cell activation (ADNKA) assays. In cynomolgus macaques, UB-612 induced potent serum-neutralizing, RBD-specific IgG binding, ACE2 binding-inhibition antibodies, and antibodies with Fc-mediated effector functions in ADMP and ADNKA assays. Additionally, immunized animals developed mucosal antibodies in bronchoalveolar lavage fluids (BAL). The level of mucosal or serum ADMP and ADNKA antibodies was found to be UB-612 dose-dependent. Our results highlight that the novel subunit UB-612 vaccine is a potent B-cell immunogen inducing polyfunctional antibody responses contributing to anti-viral immunity and vaccine efficacy.

Adaptive immune response in women from the Russian Arctic region after COVID-19 infection

BACKGROUND: The Arctic region of Russia is characterized by disproportionately high rates of morbidity and mortality from COVID-19 during the pandemic. The harsh climatic and environmental conditions in this area impede the development of self-regulation processes resulting in activation and strain of both cellular and humoral immunity. This leads to a depletion of the body’s reserve capacities. At present, there is lack of research examining how individuals who have recovered from COVID-19 are affected by the extreme conditions of Arctic Russia. AIM: To study the ratio of immunocompetent cells involved in the adaptive immune response following COVID-19 infection. MATERIAL AND METHODS: A total of 29 women aged 20 – 40 years were examined in Arkhangelsk as part of a comprehensive immunological study. This study involved assessment of the number of leukocytes, lymphocytes, and their phenotypes (CD5+, CD8+, CD10+, CD95+), as well as determination of phagocytic activity and phagocytic number. RESULTS: The cellular adaptive immune response in observed individuals 6 months after experiencing moderate COVID-19 disease was characterized by a very low concentration of T cells (CD5+) in all cases, CD10+ lymphocytes (44.83%) alongside with a high concentration of cytotoxic lymphocytes (CD8+) in 48.27% of individuals and lymphocytes with receptors for apoptosis (CD95+) in 51.72%, with relatively high phagocytic activity ranging from 90 to 100%. A correlation was found in 11.29% of women between the low content of CD10+ and CD95+ cells with the activity of phagocytosis. In 40% of women with high phagocytic activity, the concentrations of cytotoxic cells (CD8+) were found to be at a minimum level. CONCLUSIONS: Women with high phagocytic activity were found to have the lowest concentrations of cytotoxic cells, suggesting a potentially positive prognosis for reducing the risk of complications. This indicates that cellular immunity may play a role in determining the severity of COVID-19 infection in individuals with high phagocytic activity.

The Modulation of in Vitro Differentiation of Monocyte-derived Macrophage by Trypanosoma evansi Antigens in the Dromedary Camel

Studies on the camel immune response to Trypanosoma (T.) evansi, the causative agent of Surra, are very limited. In the present study, flow cytometry was employed to investigate the modulatory effects of different T. evansi antigens on the in vitro differentiation of camel blood monocytes into macrophages. For this, in vitro, separated camel monocytes were differentiated into monocyte-derived macrophages (MDM) in the presence or absence (control) of formalin-fixed (inactivated) T. evansi whole parasite (T. evansi group) or the purified Ro Tat 1.2 antigen (Ro Tat 1.2 group). The analysis of the antimicrobial functions of MDM (phagocytosis and reactive oxygen species (ROS) production) revealed reduced phagocytosis activity of camel MDM generated in the presence of T. evansi antigens. In addition, a lack of ROS-response was observed in camel MDM generated in the presence of T. evansi antigens after stimulation with PMA. These results indicated a compromising effect of T. evansi on the innate defense mechanisms in camels. Phenotypic analysis revealed the upregulation of major histocompatibility complex (MHC) class II molecules together with the lower abundance of the scavenger receptor for haptoglobin–hemoglobin complexes (CD163) on MDM generated in the presence of whole T. evansi parasites, indicating a polarizing effect of T. evansi on the differentiation of camel monocytes into an M1 phenotype. However, the reduced antimicrobial functions of these cells argue against their pro-inflammatory nature. Although both MDM generated in the presence of whole T. evansi antigens or their purified Ro Tat 1.2 proteins indicated similar expression levels of CD14 and MHCII molecules, the different abundance of the cell surface molecules CD172a, CD163, CD45, and CD44 indicated different phenotypes of the two MDMs. The results of the present study revealed compromising effects of T. evansi antigens on camel macrophages differentiated in vitro from blood monocytes. Whether these effects contribute to the in vivo pathogenesis of T. evansi in camels remains to be determined in future studies.

Specific Features of Pulmonary Tuberculosis Patients Treatment with Interferon-Gamma Depending on the Nature of the Tissue Inflammatory Reaction

The objective: to evaluate effectiveness of treatment of infiltrative pulmonary tuberculosis patients with exudative or productive types of the tissue inflammatory reaction when additional immunotherapy with recombinant IFN-γ is used.Subjects and Methods. 127 infiltrative tuberculosis patients were enrolled in the study, of them 66 had an exudative type of the inflammatory reaction (2 Groups OG-1 and GS-1 were formed), 61 had a proliferative type (2 Groups OG-2 and GS-2 were formed). Patients from OG-1 and OG-2 Groups additionally received different regimens with recombinant interferon-γ.Result: Comprehensive treatment of infiltrative tuberculosis including intramuscular administration (according to a certain regimen) of recombinant IFN-γ contributes to achievement of pronounced positive changes of clinical and radiological symptoms in patients with both exudative and productive types of the tissue inflammatory reaction. In the groups treated with recombinant IFN-γ, restoration of the cellular immune response, cellular cooperation, normalization of the cytokine profile and increased phagocytosis activity were confirmed by the results of laboratory tests.

Quantitative analysis of microglia morphological changes in the hypothalamus of chronically stressed rats

Based on the successful establishment of a rat model of chronic restraint stress, we used multiple algorithms to quantify the morphological changes of rat hypothalamic microglia from various perspectives, providing a pathomorphological basis for the subsequent study of molecular mechanisms of hypothalamic stress injury, such as neuroinflammation. To verify the successful establishment of the chronic stress model, an enzyme-linked immunosorbent assay was performed to detect serum glucocorticoid levels. Microglia labeled with Iba1 in frozen sections of rat hypothalamus were scanned and photographed at multiple levels using confocal microscopy. Subsequently, images were processed for external contouring and skeletonization, and morphological indices of microglia were calculated and analyzed using fractal, skeleton, and Sholl analysis. In addition, the co-expression of CD68 (a marker that can reflect phagocytic activity) and Iba1 was observed by immunofluorescence technique. Compared with the control group, microglia in the chronic stress group displayed reduced fractal dimension and lacunarity, increased density and circularity, enlarged soma areas, and shortened and reduced branches. Sholl analysis confirmed the reduced complexity of microglia following chronic stress. Meanwhile, microglia CD68 increased significantly, indicating that the microglia in the chronic stress group have greater phagocytosis activity. In summary, chronic restraint stress promoted the conversion of microglia in the rat hypothalamus to a less complex form, manifested as larger soma, shorter and fewer branches, more uniform and dense texture, and increased circularity; indeed, the shape of these microglia resembled that of amoeba and they displayed strong phagocytosis activity.

Bioinformatics analyses and experimental validation of the role of phagocytosis in low-grade glioma.

Phagocytosis is of vital importance in tumor immune response. The alteration of phagocytosis in low-grade glioma (LGG) has not been investigated. The mRNA, copy number variation, single nucleotide variation, and methylation levels of phagocytosis-related genes were summarized in pan-cancer. Non-negative matrix factorization clustering was utilized to identify two LGG subtypes. LASSO regression analysis was performed to construct a phagocytosis-related prognostic signature (PRPS). Immune characteristics, immunotherapy response, and targeted-drug sensitivity were further explored. The phagocytosis activity in glioma was evaluated using scRNA-seq data. Multiplex immunohistochemical (m-IHC) technology was performed to identify the tumor-infiltrating immune cells in LGG. The phagocytosis-related genes altered obviously in pan-cancer compared with corresponding normal tissues. Two LGG subtypes were obtained and the subtype with poor prognosis was combined with lower tumor purity, more active immune-related pathways, increasing infiltration of CD4+ T cells, CD8+ T cells, and natural killer (NK) cells, decreasing infiltration of macrophages, mast cells, and neutrophils, distinct pathway activity and cell death status, greater response to immunotherapy, and higher sensitivity to cyclophosphamide, erlotinib, gefitinib, lapatinib, and sorafenib. In addition, a PRPS involving 10 genes (i.e., SLC11A1, CAMK1D, PLA2G5, STAP1, ALOX15, PLCG2, SFTPD, AZU1, RAB27A, and LAMTOR2) was constructed to estimate the risk level of each LGG sample and high risk LGG patients had poor prognosis, upregulated infiltration of neutrophil, macrophage, Treg, and myeloid dendritic cell, down regulated infiltration of monocyte and NK cell, and increasing expression of large number of immune checkpoint genes. The phagocytosis activity is notably active in monocyte/macrophage. The m-IHC results confirmed increased infiltration of macrophages and neutrophils in LGG samples with high SLC11A1 expression. The molecular characteristics of phagocytosis were revealed and the PRPS laid the foundation for personalized therapy in LGG.

Conditional knockout of AIM2 in microglia ameliorates synaptic plasticity and spatial memory deficits in a mouse model of Alzheimer's disease.

Synaptic dysfunction is a hallmark pathology of Alzheimer's disease (AD) and is strongly associated with cognitive impairment. Abnormal phagocytosis by the microglia is one of the main causes of synapse loss in AD. Previous studies have shown that the absence of melanoma 2 (AIM2) inflammasome activity is increased in the hippocampus of APP/PS1 mice, but the role of AIM2 in AD remains unclear. Injection of Aβ1-42 into the bilateral hippocampal CA1 was used to mimic an AD mouse model (AD mice). C57BL/6 mice injected with AIM2 overexpression lentivirus and conditional knockout of microglial AIM2 mice were used to confirm the function of AIM2 in AD. Cognitive functions were assessed with novel object recognition and Morris water maze tests. The protein and mRNA expression levels were evaluated by western blotting, immunofluorescence staining, and qRT-PCR. Synaptic structure and function were detected by Golgi staining and electrophysiology. The expression level of AIM2 was increased in AD mice, and overexpression of AIM2 induced synaptic and cognitive impairments in C57BL/6 mice, similar to AD mice. Elevated expression levels of AIM2 occurred in microglia in AD mice. Conditional knockout of microglial AIM2 rescued cognitive and synaptic dysfunction in AD mice. Excessive microglial phagocytosis activity of synapses was decreased after knockout of microglial AIM2, which was associated with inhibiting complement activation. Our results demonstrated that microglial AIM2 plays a critical role in regulating synaptic plasticity and memory deficits associated with AD, providing a new direction for developing novel preventative and therapeutic interventions for this disease.

The potential mechanism of concentrated mannan-oligosaccharide promoting goldfish’s (Carassius auratus Linnaeus) resistance to Ichthyophthirius multifiliis invasion

Because of the low host specificity, Ichthyophthirius multifiliis (Ich) can widely cause white spot disease in aquatic animals, which is extremely difficult to treat. Prior research has demonstrated a considerable impact of concentrated mannan-oligosaccharide (cMOS) on the prevention of white spot disease in goldfish, but the specific mechanism is still unknown. In this study, transcriptome sequencing, histological analysis, immunofluorescence analysis, phagocytosis activity assay and qRT-PCR assay were used to systematically reveal the potential mechanism of cMOS in supporting the resistance of goldfish (Carrasius auratus) to Ich invasion. According to the transcriptome analysis, the gill tissue of goldfish receiving the cMOS diet showed greater expression of mannose-receptor (MRC) related genes, higher phagocytosis activity, up-regulated expression of phagocytosis-related genes and inflammatory-related genes compared with the control, indicating that cMOS can have an effect on phagocytosis and non-specific immunity of goldfish. After the Ich challenge, transcriptome analysis revealed that cMOS fed goldfish displayed a higher level of phagocytic response, whereas non-cMOS fed goldfish displayed a greater inflammatory reaction. Besides, after Ich infection, cMOS-fed goldfish displayed greater phagocytosis activity, a stronger MRC positive signal, higher expression of genes associated with phagocytosis (ABCB2, C3, MRC), and lower expression of genes associated with inflammation (IL-1β, IL-17, IL-8, TNF-α, NFKB). In conclusion, our experimental results suggest that cMOS may support phagocytosis by binding to MRC on the macrophage cell membrane and change the non-specific immunity of goldfish by stimulating cytokine expression. The results of this study provide new insights for the mechanism of cMOS on parasitic infection, and also suggest phagocytosis-related pathways may be potential targets for prevention of Ich infection.

Nicotinamide mononucleotide as a therapeutic agent to alleviate multi-organ failure in sepsis

BackgroundSepsis-caused multi-organ failure remains the major cause of morbidity and mortality in intensive care units with limited therapeutics. Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD+), has been recently reported to be protective in sepsis; however, its therapeutic effects remain to be determined. This study sought to investigate the therapeutic effects of NMN in septic organ failure and its underlying mechanisms.MethodsSepsis was induced by feces-injection-in-peritoneum in mice. NMN was given after an hour of sepsis onset. Cultured neutrophils, macrophages and endothelial cells were incubated with various agents.ResultsWe demonstrate that administration of NMN elevated NAD+ levels and reduced serum lactate levels, oxidative stress, inflammation, and caspase-3 activity in multiple organs of septic mice, which correlated with the attenuation of heart dysfunction, pulmonary microvascular permeability, liver injury, and kidney dysfunction, leading to lower mortality. The therapeutic effects of NMN were associated with lower bacterial burden in blood, and less ROS production in septic mice. NMN improved bacterial phagocytosis and bactericidal activity of macrophages and neutrophils while reducing the lipopolysaccharides-induced inflammatory response of macrophages. In cultured endothelial cells, NMN mitigated mitochondrial dysfunction, inflammation, apoptosis, and barrier dysfunction induced by septic conditions, all of which were offset by SIRT3 inhibition.ConclusionNAD+ repletion with NMN prevents mitochondrial dysfunction and restrains bacterial dissemination while limiting inflammatory damage through SIRT3 signaling in sepsis. Thus, NMN may represent a therapeutic option for sepsis.

Comparison Study of Phagocytosis Activity among Live Candida albicans, Live and Heat-treated Saccharomyces cerevisiae

Comparison Study of Phagocytosis Activity among Live Candida albicans, Live and Heat-treated Saccharomyces cerevisiae

PEMBERIAN EKSTRAK DAUN KETAPANG UNTUK MENINGKATKAN IMUN – NON SPESIFIK PADA IKAN (ARTIKEL REVIEW)

This review article focuses on the administration of ketapang leaf extract to enhance non-specific immunity in fish. Ketapang leaves have been of interest as a potential immunostimulant in aquaculture to boost the non-specific immune response in fish. In this review, various studies exploring the effects of ketapang leaf extract on the non-specific immune response of fish have been examined. The findings suggest that the administration of ketapang leaf extract can lead to an increase in non-specific immune parameters, such as phagocytosis activity, lysozyme activity, and total protein content. The potential use of ketapang leaf extract as an immunostimulant in aquaculture shows promising results in enhancing the non-specific immune response of fish. Strengthening the non-specific immune system can help fish combat various pathogens and diseases, thereby improving their overall health and survival in aquaculture settings. However, further research is needed to fully understand the mechanisms of action and optimal dosage of ketapang leaf extract as an immunostimulant. Additionally, considering its potential positive impact on fish health, the use of ketapang leaf extract in aquaculture warrants comprehensive studies to ensure its safe and effective implementation.

Establishment and application of a microglial phagocytosis/cell health high content assay for Alzheimer’s disease drug discovery targeting microglia

AbstractBackgroundTargeting microglial phagocytosis has been proposed as a therapeutic strategy for Alzheimer’s disease drug discovery. A reliable cellular assay capable of measuring drug‐regulated phagocytosis activity while simultaneously measuring treatment‐caused cell stress and toxicity is needed for drug discovery research targeting microglia. This need motivated us to develop and establish a high content microglial phagocytosis/cell health assay to meet the challenge.MethodThe assay uses 384‐well cellular imaging plates, microglial cell lines (HMC3 and BV2) and mouse primary microglia, pHrodo‐myelin/membrane debris as ligand, and a high content imaging system for imaging and analysis. Assay procedures include 1) plate cells on day 1 and incubate cell plates overnight in 37oC; 2) treat cells with compounds on day 2; 3) add pHrodo‐myelin/membrane debris to cell plates on day 3 and incubate in 37oC for 20 hours; 4) stain cell nuclei with Hoechst‐33342 for 30 min before performing high content imaging and analysis on day 4. Three selected parameters are measured from cell images: 1) mean total fluorescence intensity per cell of pHrodo‐myelin/membrane debris in phagocytosis vesicles to quantify phagocytosis; 2) cell counts per well (assessing effects on proliferation and cell death); and 3) average nuclear intensity (detecting induced apoptosis).ResultVarious treatments targeting different mechanisms regulating microglial phagocytosis were tested; we observed that Cytochalasin D inhibited phagocytosis, LPS stimulated phagocytosis in BV2 cells but not HMC3 cells, Idelalisib (PI3Kd inhibitor) inhibited HMC3 cell phagocytosis, and Saracatinib (Src/abl family of kinases inhibitor) stimulated HMC3 phagocytosis. These results validated the assay for use in drug discovery targeting microglial phagocytosis. With the assay, we identified some INPP5D inhibitors and some PLCG2 activators stimulated phagocytosis in a concentration range without significant cell stress/toxicity with all three cell types tested.ConclusionA microglial phagocytosis/cell health assay was established. The simultaneous measurement of phagocytosis and cell health allows for the distinction of drug effects on regulation of phagocytosis from cellular stress/toxicity‐related changes, a distinguishing feature of the assay, which helped compound selection of drug discovery projects targeting INPP5D and PLCG2 respectively.

HEMATOLOGY OF Osphronemus goramy FED CONTAINING FERMENTED TURI (Sesbania grandiflora) LEAF MEAL and CHALLENGED WITH Aeromonas hydrophila

Turi leaf (Sesbania grandiflora) is a natural ingredient with antibacterial compounds. This research was conducted from April to June 2022 at the Fish Nutrition Laboratory and the Laboratory of Parasites and Fish Diseases, Faculty of Fisheries and Marine, Universitas Riau. This study aimed to obtain the best dose of fermented turi leaves in feed seen from hematology before and after the challenge test with A.hydrophila. The method used in this study is an experimental method using a one-factor Completely Randomised Design (CRD) with five treatment levels, namely Kn: (no treatment), Kp: (no cure and infected with A.hydrophila), P1: (fermented feed containing 15% turi leaves, 85% soy flour), P2: (30% turi leaves, 70% soy flour), and P3: (45% turi leaves, 55% soy flour). Fish were reared for 46 days. The challenge test was conducted on day 32 with A.hydrophila bacteria at a density of 108 CFU/mL, as much as 0.1 mL/head. The results showed that fermented feed contained 45% turi leaves and 55% soybeans (P3). Wheat flour obtained total erythrocytes 2.51x106 cells/mm3, hematocrit 40.33%, hemoglobin 10.40 g/dL, total leukocytes 10.13x104 cells/mm3, phagocytosis activity 21.13%, survival rate 90%, and absolute weight growth 9.52g. Water quality parameters were temperature ranging from 27-300C, pH 5.9-6.2, DO 5.5-5.9 mg/L, and NH3 0.015-0.017 m g/L. Based on the results of the study, it can be concluded that there is an effect of adding fermented turi leaves to feed on the hematology of gouramy before and after the A.hydrophila bacterial challenge test has a significant impact

Preliminary study of immunomodulatory effects of fermented garlic honey on non-specific immune responses

Introduction: Honey and Garlic is recognized to offer several health advantages due to its numerous constituents, one of which being flavonoids. Flavonoids’ pharmacological activity includes antibacterial, antioxidant, antifungal, antiviral, and immunomodulatory properties. Honey's immunomodulatory action plays a vital role in immune system boosting. The purpose of this study is to determine the immunomodulatory effect of fermented garlic honey, which is considered to have a synergistic effect, using two alternative techniques. Methods: Macrophage phagocytic activity is the first approach, while the delayed type hypersensitivity is the second. Each method used 30 male rats (Rattus norvegicus) divided into 5 groups, where each group consisted of 6 rats. Group I negative (Placebo 0,005 g/kg BW), group II positive (Stimuno 0,005 g/kg BW), group III FGH dose I (1,5 g/kg BW), group IV FGH dose II (3 g/kg BW), group V FGH dose III (4,5 g/kg BW). The dosage form was orally given for 21 days. The Macrophage phagocytic activity method measured %activity of macrophage phagocytosis using a microscope. The rats were injected with an antigen which is Staphylococcus aureus by intraperitoneal route. Results: The Delayed Type Hypersensitivity was measured volume change of the rat’s feet at the 4, 24, and 48 hours using a plethysmometer. Based on the results, fermented garlic honey had an immunomodulatory effect. The dose 2 (3 g/kg BW) has the highest effectiveness as an immunomodulator. Fermented Garlic Honey may be concluded to be efficient as an immunomodulator by enhancing the macrophage phagocytic activity indicator and lowering the edema volume of the rat. Conclusion: Fermented garlic honey can increase the immune system and can be used as an immunostimulant. The most effective dose of fermented garlic honey as immunomodulator is dose 2 was 3 g/kg BW.