Phagocytic Cell Function Research Articles

Methodological approaches to the comparative assessment of cellular reactions of innate immunity in mollusk <I>Pomacea</I> sp. (<I>Caenogastropoda, Ampullariidae</I>) in experimental aseptic inflammation

One of the significant methodological problems that arise in the study of cells of the innate immunity of invertebrates is the unsuitability of culture media and even salt solutions intended for mammals and humans. Another important methodological point is the significantly pronounced reactions of coagulation, which play an important protective role in all invertebrates, but manifest themselves in vitro in cellular suspensions prepared even with optimal saline solutions for this animal. To solve this problem, researchers widely use an anticoagulation buffer with ethylenediaminetetraacetic acid (EDTA) even in functional tests. From the standpoint of traditional approaches of mammalian immunology, the use of EDTA is possible only for the study of cell morphology, but not their functions. The aim of the work is to adapt methodological approaches to the comparative assessment of cellular reactions of innate immunity in Pomacea sp mollusks in experimental aseptic inflammation. To evaluate the functions of phagocytic cells in mollusk Pomacea sp., we used a complete salt solution (CSS) with optimal concentrations of glucose, calcium ions, magnesium with addition to prevent coagulation, cellular aggregation and degranulation instead of EDTA sodium salt of heparin at a concentration of 100 IU/mL (CSS-hep). As a result of a specially conducted experiment, it was shown that during incubation of hemolymph cells in this solution, their viability is preserved, evaluated by flow laser cytometry during incubation with annexin V-PE (PE Annexin V) and 7-aminoactinomycin D (7- AAD), as well as in a test with trypan blue. The possibility of studying the phagocytic activity and a phagosome acidification of leukocytes from hemolymph, hematopoiesis organ (kidney) and the focus of inflammation induced by intramuscular injection of sterile suspension of zymosan using CSS-hep is shown. Unlike the Limnaea stagnalis and Biomphalaria glabrata mollusks, in which the production of reactive oxygen species by phagocytes in the luminol-dependent chemiluminescence reaction is well documented, we were unable to induce this reaction in Pomacea sp. mollusks. In a specially conducted experiment, we did not reveal the effect of the sodium salt of heparin at a concentration of 100 IU/mL on the production of reactive oxygen species by rat phagocytes.

State of the Field: Cytotoxic Immune Cell Responses in C. neoformans and C. deneoformans Infection.

Cryptococcus neoformans is an environmental pathogen that causes life-threatening disease in immunocompromised persons. The majority of immunological studies have centered on CD4+ T-cell dysfunction and associated cytokine signaling pathways, optimization of phagocytic cell function against fungal cells, and identification of robust antigens for vaccine development. However, a growing body of literature exists regarding cytotoxic cells, specifically CD8+ T-cells, Natural Killer cells, gamma/delta T-cells, NK T-cells, and Cytotoxic CD4+ T-cells, and their role in the innate and adaptive immune response during C. neoformans and C. deneoformans infection. In this review, we (1) provide a comprehensive report of data gathered from mouse and human studies on cytotoxic cell function and phenotype, (2) discuss harmonious and conflicting results on cellular responses in mice models and human infection, (3) identify gaps of knowledge in the field ripe for exploration, and (4) highlight how innovative immunological tools could enhance the study of cytotoxic cells and their potential immunomodulation during cryptococcosis.

Microglial Melatonin Receptor 1 Degrades Pathological Alpha-Synuclein Through Activating LC3-Associated Phagocytosis InVitro.

Parkinson's disease (PD) is characterized by the formation of Lewy bodies (LBs), primarily constituted of α-synuclein (α-Syn). Microglial cells exhibit specific reactivity toward misfolded proteins such as α-Syn. However, the exact clearance mechanism and related molecular targets remain elusive. BV2 cells, primary microglia from wild-type and MT1 knockout mice, and primary cortical neurons were utilized as experimental models. The study investigated relevant mechanisms by modulating microglial MT1 expression through small RNA interference (RNAi) and lentiviral overexpression techniques. Furthermore, pathological aggregation of α-Syn was induced using pre-formed fibrils (PFF) α-Syn. Co-immunoprecipitation, immunofluorescence, Western blot (WB), and quantitative real-time PCR were used to elucidate the mechanisms of molecular regulation. In this study, we elucidated the regulatory role of the melatonin receptor 1 (MT1) in the microglial phagocytic process. Following MT1 knockout, the ability of microglial cells to engulf latex beads and zymosan particles decreased, subsequently affecting the phagocytic degradation of fibrillar α-Syn by microglial cells. Furthermore, the loss of MT1 receptors in microglial cells exacerbates the aggregation of α-Syn in neurons induced by pre-formed fibrils (PFF) α-Syn. Mechanistically, MT1 influences the phagocytic function of microglial cells by regulating the Rubicon-dependent LC3-associated phagocytosis (LAP) pathway. Taken together, the results suggest the neuroprotective function of microglial cells in clearing α-Syn through MT1-mediated LAP, highlighting the potential key role of MT1 in pathogenic mechanisms associated with α-Syn.

Engineered CD147-Deficient THP-1 Impairs Monocytic Myeloid-Derived Suppressor Cell Differentiation but Maintains Antibody-Dependent Cellular Phagocytosis Function for Jurkat T-ALL Cells with Humanized Anti-CD147 Antibody.

CD147 is upregulated in cancers, including aggressive T-ALL. Traditional treatments for T-ALL often entail severe side effects and the risk of relapse, highlighting the need for more efficacious therapies. ADCP contributes to the antitumor response by enhancing the ability of phagocytic cells to engulf cancer cells upon antibody binding. We aimed to engineer CD147KO THP-1 cells and evaluated their differentiation properties compared to the wild type. A humanized anti-CD147 antibody, HuM6-1B9, was also constructed for investing the phagocytic function of CD147KO THP-1 cells mediated by HuM6-1B9 in the phagocytosis of Jurkat T cells. The CD147KO THP-1 was generated by CRISPR/Cas9 and maintained polarization profiles. HuM6-1B9 was produced in CHO-K1 cells and effectively bound to CD147 with high binding affinity (KD: 2.05 ± 0.30 × 10-9 M). Additionally, HuM6-1B9 enhanced the phagocytosis of Jurkat T cells by CD147KO THP-1-derived LPS-activated macrophages (M-LPS), without self-ADCP. The formation of THP-1-derived mMDSC was limited in CD147KO THP-1 cells, highlighting the significant impact of CD147 deletion. Maintaining expression markers and phagocytic function in CD147KO THP-1 macrophages supports future engineering and the application of induced pluripotent stem cell-derived macrophages. The combination of HuM6-1B9 and CD147KO monocyte-derived macrophages holds promise as an alternative strategy for T-ALL.

Taurine influences the phagocytic function of retinal pigment epithelium cells in healthy and dystrophic animals

Aims/Purpose: To investigate the influence of taurine on the phagocytic function of the retinal pigment epithelium (RPE) in normal and dystrophic rats.Methods: Normal albino Sprague–Dawley (SD) and pigmented dystrophic Royal College of Surgeons (RCS‐p+) rats that suffer an inherited photoreceptor degeneration due to RPE phagocytosis impairment were used for the study. Taurine depletion was induced in SD rats (n = 8) by providing β‐alanine in drinking water (3%) from postnatal day 21 (P21). Taurine supplementation was administered to RCS‐p+ rats (n = 8) in drinking water (0.2 M) from P21. Untreated RCSp+ (n = 8) and SD (n = 8) rats served as control groups. Rats were processed at P81 (SD) and at P45 (RCS) and received an intravitreal injection (IV) of. 1.5 μL of Fluorogold (FG, 3%) diluted in saline 24 h before processing. The RPE was dissected, flat‐mounted and immunodetected with anti‐S opsin antibodies to label the RPE phagosomes which were quantified using ImageJ and size criteria.Results: The mean numbers (± SD) of phagosomes per RPE whole mount was 414 ± 118 in control SD rats, 1034 ± 528 in taurine‐depleted SD rats 88 ± 12 in untreated RCS and 119 ± 9 in taurine‐treated RCS rats. The numbers of phagosomes were significantly higher in SD rats than in RCS‐p+ rats (t‐test, p < 0.0001) documenting the impaired phagocytic function of RCS‐p+ rats. In SD rats, the number of phagosomes were significantly higher in taurine‐depleted rats evidencing that taurine depletion decreases the RPE phagocytic capacity (t‐test, p < 0.0059). Finally, in RCSp+ rats, the numbers of phagosomes were significantly higher in taurine‐treated than in non‐treated RCS rats (t‐test, p < 0.0001) indicating that taurine supplementation ameliorates the phagocytic capacity of the RPE cells in RCS‐p+ rats.Conclusions: Taurine influences the phagocytic capacity of the RPE cells in normal rats and in dystrophic RCS‐p+ rats.

Recovery of the Decreased Phagocytic Function of Peripheral Monocytes and Neutrophil Granulocytes following Cytoreductive Surgery in Advanced Stage Epithelial Ovarian Cancer.

(1) Monocytes and neutrophil granulocytes are the phagocytic cells of the innate immune system, playing a crucial role in recognizing and eliminating tumor-transformed cells. Our objective was to assess the impact of advanced-stage epithelial ovarian cancer (EOC) and cytoreductive surgery on the phagocytic function of peripheral monocytes and neutrophil granulocytes. We aimed to compare the pre- and postoperative phagocytic function of these immune cells in EOC patients with healthy control women. Additionally, we aimed to examine the influence of surgery on phagocytic function by comparing pre- and postoperative samples from patients with benign gynecological tumors. (2) We examined peripheral blood samples from 20 patients with FIGO IIIC stage high-grade serous EOC and 16 patients with benign gynecological tumors as surgical controls, collected before and seven days after tumor removal surgery, and from 14 healthy women. After separation, the cells were incubated with Zymosan-A particles, and the phagocytic index (PI) was assessed using immunofluorescence microscopy. One-way ANOVA, the Kruskal-Wallis H-test, and the paired samples t-test were used for the statistical analysis of the data. A significance level of p < 0.05 was applied. (3) Peripheral monocytes and neutrophils from EOC patients exhibited significantly lower preoperative PI values compared to healthy controls (p < 0.001; p < 0.001, respectively). Following cytoreductive surgery, the PI values of immune cells in EOC patients significantly increased by the 7th postoperative day (p < 0.001; p < 0.001), reaching levels comparable to those of healthy controls (p = 0.700 and p = 0.991). In contrast, there was no significant disparity in the PI values of cells obtained from pre- and postoperative blood samples of surgical controls when compared to healthy women (monocytes: p = 0.361 and p = 0.303; neutrophils: p = 0.150 and p = 0.235). (4) EOC and/or its microenvironment may produce factors that reduce the phagocytic function of monocytes and neutrophils, and the production of these factors may be reduced or eliminated after tumor removal.

MiR-155 enhances phagocytosis of alveolar macrophages through the mTORC2/RhoA pathway.

Alveolar macrophage phagocytosis is significantly reduced in Chronic obstructive pulmonary disease, and cigarette smoke extract is one of the chief reasons for this decrease. Nevertheless, the specific underlying mechanism remains elusive. In this study, the role and possible mechanism of miR-155-5p/mTORC2/RhoA in the phagocytosis of mouse alveolar macrophages (MH-S) were explored. Our results revealed that cigarette smoke extract intervention reduced MH-S cell phagocytosis and miR-155-5p expression. Meanwhile, the dual-luciferase reporter assay validated that Rictor is a target of miR-155-5p. On the one hand, transfecting miR-155-5p mimic, mimic NC, miR-155-5p inhibitor, or inhibitor NC in MH-S cells overexpressing miR-155-5p increased the Alveolar macrophage phagocytotic rate, up-regulated the expression level of RhoA and p-RhoA, and down-regulated that of mTOR and Rictor mRNA and protein. On the other hand, inhibiting the expression of miR-155-5p lowered the phagocytotic rate, up-regulated the expression of mTOR, Rictor mRNA, and protein, and down-regulated the expression of RhoA and p-RhoA, which taken together, authenticated that miR-155-5p participates in macrophage phagocytosis via the mTORC2/RhoA pathway. Finally, confocal microscopy demonstrated that cells overexpressing miR-155-5p underwent cytoskeletal rearrangement during phagocytosis, and the phagocytic function of cells was enhanced, signaling that miR-155-5p participated in macrophage skeletal rearrangement and enhanced alveolar macrophage phagocytosis by targeting the expression of Rictor in the mTORC2/RhoA pathway.

Poster 101: Evaluation of Novel Treatment Agents on Rotator Cuff Tendon and Muscle Pathology in a Murine Subacromial Impingement Model

Poster 101: Evaluation of Novel Treatment Agents on Rotator Cuff Tendon and Muscle Pathology in a Murine Subacromial Impingement Model

Role of NCKAP1 in the Defective Phagocytic Function of Microglia-Like Cells Derived from Rapidly Progressing Sporadic ALS

Microglia plays a key role in determining the progression of amyotrophic lateral sclerosis (ALS), yet their precise role in ALS has not been identified in humans. This study aimed to identify a key factor related to the functional characteristics of microglia in rapidly progressing sporadic ALS patients using the induced microglia model, although it is not identical to brain resident microglia. After confirming that microglia-like cells (iMGs) induced by human monocytes could recapitulate the main signatures of brain microglia, step-by-step comparative studies were conducted to delineate functional differences using iMGs from patients with slowly progressive ALS [ALS(S), n = 14] versus rapidly progressive ALS [ALS(R), n = 15]. Despite an absence of significant differences in the expression of microglial homeostatic genes, ALS(R)-iMGs preferentially showed defective phagocytosis and an exaggerated pro-inflammatory response to LPS stimuli compared to ALS(S)-iMGs. Transcriptome analysis revealed that the perturbed phagocytosis seen in ALS(R)-iMGs was closely associated with decreased NCKAP1 (NCK-associated protein 1)-mediated abnormal actin polymerization. NCKAP1 overexpression was sufficient to rescue impaired phagocytosis in ALS(R)-iMGs. Post-hoc analysis indicated that decreased NCKAP1 expression in iMGs was correlated with the progression of ALS. Our data suggest that microglial NCKAP1 may be an alternative therapeutic target in rapidly progressive sporadic ALS.

HBSP improves kidney ischemia-reperfusion injury and promotes repair in properdin deficient mice via enhancing phagocytosis of tubular epithelial cells.

Phagocytosis plays vital roles in injury and repair, while its regulation by properdin and innate repair receptor, a heterodimer receptor of erythropoietin receptor (EPOR)/β common receptor (βcR), in renal ischaemia-reperfusion (IR) remains unclear. Properdin, a pattern recognition molecule, facilitates phagocytosis by opsonizing damaged cells. Our previous study showed that the phagocytic function of tubular epithelial cells isolated from properdin knockout (PKO) mouse kidneys was compromised, with upregulated EPOR in IR kidneys that was further raised by PKO at repair phase. Here, helix B surface peptide (HBSP), derived from EPO only recognizing EPOR/βcR, ameliorated IR-induced functional and structural damage in both PKO and wild-type (WT) mice. In particular, HBSP treatment led to less cell apoptosis and F4/80+ macrophage infiltration in the interstitium of PKO IR kidneys compared to the WT control. In addition, the expression of EPOR/βcR was increased by IR in WT kidneys, and furthered increased in IR PKO kidneys, but greatly reduced by HBSP in the IR kidneys of PKO mice. HBSP also increased PCNA expression in IR kidneys of both genotypes. Moreover, iridium-labelled HBSP (HBSP-Ir) was localized mainly in the tubular epithelia after 17-h renal IR in WT mice. HBSP-Ir also anchored to mouse kidney epithelial (TCMK-1) cells treated by H2O2. Both EPOR and EPOR/βcR were significantly increased by H2O2 treatment, while further increased EPOR was showed in cells transfected with small interfering RNA (siRNA) targeting properdin, but a lower level of EPOR was seen in EPOR siRNA and HBSP-treated cells. The number of early apoptotic cells was increased by EPOR siRNA in H2O2-treated TCMK-1, but markedly reversed by HBSP. The phagocytic function of TCMK-1 cells assessed by uptake fluorescence-labelled E.coli was enhanced by HBSP dose-dependently. Our data demonstrate for the first time that HBSP improves the phagocytic function of tubular epithelial cells and kidney repair post IR injury, via upregulated EPOR/βcR triggered by both IR and properdin deficiency.

Pulmonary Immunopathogenesis of Granulibacter bethesdensis

Abstract Granulibacter bethesdensis (Gb) is a Gram negative bacterial pathogen reported to cause disease exclusively in patients with Chronic Granulomatous Disease harboring mutations in one of the five subunits of NADPH oxidase complex. The pathogen has been shown to cause chronic as well as recurrent infections in CGD patients that can be fatal. Although systemic and septic infection have been reported, the pathogenesis of Gb in respiratory microenvironment is completely unknown. Here we compared the disease progression and infection outcome of wild-type (WT) and murine CGD model Gp91 −/−mice following intranasal infection with a clinical isolate of Gb. We found that compared to the WT mice, Gp91 −/−CGD mice are highly susceptible to respiratory infection with Gb. While the WT mice displayed transient increase in inflammatory cytokines and moderate leukocyte infiltration characteristic of a mild, resolving infection, CGD mice displayed hypercytokinemia but attenuated antimicrobial function of phagocytic cells. A significant upregulation of Type 2 inflammatory cytokine IFN-γ in lungs Gb infected gp91−/− prompted investigation the its role in GB pathogenesis. Neutralization of this cytokine mitigated the inflammatory response and reduced bacterial burden in infected Gp91−/− CGD mice resulting in significantly improved overall survival. Mechanistically, IFN-γ neutralization improved antimicrobial function of neutrophils. Taken together, our findings suggest that in the absence of a functional superoxide production machinery, G. bethesdensis is capable of causing a chronic respiratory infection with fulminant sepsis and IFN-γ produced during the infection has a detrimental effect on the bacterial clearance.

Beneficial Effect of ACI-24 Vaccination on Aβ Plaque Pathology and Microglial Phenotypes in an Amyloidosis Mouse Model.

Amyloid-β (Aβ) deposition is an initiating factor in Alzheimer's disease (AD). Microglia are the brain immune cells that surround and phagocytose Aβ plaques, but their phagocytic capacity declines in AD. This is in agreement with studies that associate AD risk loci with genes regulating the phagocytic function of immune cells. Immunotherapies are currently pursued as strategies against AD and there are increased efforts to understand the role of the immune system in ameliorating AD pathology. Here, we evaluated the effect of the Aβ targeting ACI-24 vaccine in reducing AD pathology in an amyloidosis mouse model. ACI-24 vaccination elicited a robust and sustained antibody response in APPPS1 mice with an accompanying reduction of Aβ plaque load, Aβ plaque-associated ApoE and dystrophic neurites as compared to non-vaccinated controls. Furthermore, an increased number of NLRP3-positive plaque-associated microglia was observed following ACI-24 vaccination. In contrast to this local microglial activation at Aβ plaques, we observed a more ramified morphology of Aβ plaque-distant microglia compared to non-vaccinated controls. Accordingly, bulk transcriptomic analysis revealed a trend towards the reduced expression of several disease-associated microglia (DAM) signatures that is in line with the reduced Aβ plaque load triggered by ACI-24 vaccination. Our study demonstrates that administration of the Aβ targeting vaccine ACI-24 reduces AD pathology, suggesting its use as a safe and cost-effective AD therapeutic intervention.

Does taurine have a role in the phagocytic function of the retinal pigment epithelium?

AbstractPurpose: To study the role of taurine in the morphology and phagocytic function of the retinal pigment epithelium (RPE) in normal and dystrophic rats.Methods: Albino Sprague–Dawley (SD) rats and pigmented dystrophic Royal College of Surgeons (RCS‐p+) rats that suffer a severe form of inherited photoreceptor degeneration due to RPE phagocytosis impairment, were used for this study. SD rats received β‐alanine in drinking water (3%) for 2 months from P21 to induce taurine depletion and RCS‐p + rats received taurine supplementation in drinking water (0.2 M) for 24 days from P21. Groups of untreated RCS and SD rats were used as control. SD rats were processed 2 months after the start of treatment, while RCS rats were processed at P45. Twenty‐four hours before processing, the animals received an intravitreal injection of 1.5 μl of 3% fluorogold (FG) diluted in saline 24 to label the RPE cells. Animals were processed and the RPE was dissected flat‐mounted and observed using a confocal microscope (Leica SP8).Results: In control SD animals, Fluorogold (FG) accumulates homogeneously in the cytoplasm of the RPE cells. Taurine‐depleted SD animals showed less FG accumulation in the cytoplasm in all cells and, and some cells had FG accumulation only in part of the cytoplasm or no FG accumulation at all. Untreated RCS‐p + animals showed an inverted accumulation of FG because it appeared mainly in the membrane of the RPE but not in the cytoplasm. Finally, taurine‐treated RCS‐p + animals showed FG accumulation in the cytoplasm of the RPE cells, although the accumulation of FG was smaller than that found in SD rats.Conclusions: Taurine influences the phagocytic capacity of the RPE cells. Taurine depletion impairs the phagocytic capacity of the RPE cells in healthy retinas, and taurine supplementation improves the phagocytic function of RPE cells in dystrophic RCS‐p + rats.

TMEM176B regulates phagosome maturation of glial cells and its increased activity contributes to the therapy of Alzheimer’s disease

AbstractBackgroundGiven recent interest in using amyloid b (Aβ)‐targeted antibodies as a therapy for patients with Alzheimer’s disease (AD), removal of Aβ by phagocytosis likely protective early in AD. Impaired phagocytic function of glial cells surrounding Aβ plaques during later stages in AD likely contributes to worsened disease outcomes, therefore enhancing phagocytic activity of glial cells could be a potent strategy for AD therapy.MethodChanges in TMEM176B expression according to the progression of AD pathology was assessed by measuring TMEM176B protein in human AD brain lysates and TMEM176B transcripts in Ab‐treated primary rat astrocytes. The roles of TMEM176B in phagosome maturation of glial cells was investigated by measuring Rab5, Rab7, LAMP1, LAMP2, cathepsin D, and LC3B in presence of Aβ. Intracellular pH of glial cells was detected using fluorescent dye‐based pHrodo Red AM intracellular pH indicator (Invitrogen, Carlsbad, CA). In 5xFAD and 3xTg AD mice model, therapeutic effects of AAV‐TMEM176B was examined. AAV (adenovirus‐associated virus)‐TMEM176B (1 – 2×1010 GC) was intracerebroventricularly injected, and Aβ plaque and p‐tau in brains were assessed 3 months later. Behavioral studies such as Y‐maze, Morris water maze, and novel‐object‐recognition were conducted to exam the effect of TMEM176B overexpression in alleviation of cognition decline.ResultIn human AD brains, Tmem176B expression was increased in moderate AD (Braak III‐IV) was decreased in severe AD (Braak V‐VI). TMEM176B was upregulated by acute Aβ treatment in primary rat astrocytes, and was gradually downregulated concomitantly with impairment of Aβ phagocytosis activity by chronic exposure to Aβ. The TMEM176B agonistic compound augmented phagosome maturation and phagosomal acidification, which led to increased Aβ phagocytosis and lysosomal degradation. Studies confirmed that intracerebroventrical administration of AAV carrying TMEM176B in AD mice results in enhanced glial phagocytosis followed by lowered Aβ burden in brain. Moreover, overexpression of TMEM176B by AAV injection significantly improved cognition impairment of AD mice.ConclusionData from these studies suggest that TMEM176B plays a direct and critical role in phagolysosmal function of glial cells and AD pathogenesis and highlight this ion channel as a potential therapeutic target for treating AD.

Advantage of extracellular vesicles in hindering the CD47 signal for cancer immunotherapy.

The cluster of differentiation 47 (CD47) protein is abundantly expressed on various malignant cells and suppresses the phagocytic function of macrophages and dendritic cells. High CD47 expression levels are correlated with poor cancer survival. Antagonizing CD47 antibodies with potent antitumor effects have been developed in clinical trials, but have critical side effects, inducing anemia and thrombocytopenia. To develop a safe and potent CD47 blockade, we designed extracellular vesicles (EVs) harboring signal regulatory protein alpha (SIPRα)-EV-SIRPα (EVs that express SIPRα). EV-SIRPα showed minimal toxic effects on hematologic parameters and utilized RBCs as delivery vehicles to tumors rather than inducing anemia. EV-SIRPα inhibited ligation of residual CD47 molecules, which attribute to the EV-endocytosis-mediated CD47 depletion and steric hindrance of EV. In an immunologically cold tumor model, EV-SIRPα induced tumor-specific T-cell-mediated antitumor effects. When directly administered to the accessible lesions, EV-SIRPα monotherapy elicited an abscopal effect in the B16F10 tumor model by increasing immune cell infiltration and CD8⁺-mediated immunity against non-treated tumors. The combinational approach by loading doxorubicin into the EV-SIRPα dramatically reduced the tumor burden and led to 80% complete remission rate. Thus, a potent EV-based CD47 blockade that is hematologically safe, has efficient signaling blocking efficacy, and has systemic antitumor immunity against cancer is recommended.

Targeting Phospholipase D Pharmacologically Prevents Phagocytic Function Loss of Retinal Pigment Epithelium Cells Exposed to High Glucose Levels.

We previously described the participation of canonical phospholipase D isoforms (PLD1 and PLD2) in the inflammatory response of retinal pigment epithelium (RPE) cells exposed to high glucose concentrations (HG). Here, we studied the role of the PLD pathway in RPE phagocytic function. For this purpose, ARPE-19 cells were exposed to HG (33 mM) or to normal glucose concentration (NG, 5.5 mM) and phagocytosis was measured using pHrodo™ green bioparticles® or photoreceptor outer segments (POS). HG exposure for 48 and 72 h reduced phagocytic function of ARPE-19 cells, and this loss of function was prevented when cells were treated with 5 μM of PLD1 (VU0359595 or PLD1i) or PLD2 (VU0285655-1 or PLD2i) selective inhibitors. Furthermore, PLD1i and PLD2i did not affect RPE phagocytosis under physiological conditions and prevented oxidative stress induced by HG. In addition, we demonstrated PLD1 and PLD2 expression in ABC cells, a novel human RPE cell line. Under physiological conditions, PLD1i and PLD2i did not affect ABC cell viability, and partial silencing of both PLDs did not affect ABC cell POS phagocytosis. In conclusion, PLD1i and PLD2i prevent the loss of phagocytic function of RPE cells exposed to HG without affecting RPE function or viability under non-inflammatory conditions.

Impact of persistent antigenic challenges and splenectomy on immune cells in β-Thalassemic patients.

Infection is common in thalassemia patients and is one of the leading causes of death. It's still unclear why these individuals are so sensitive to infection. There is strong evidence that a deficiency in the functioning of phagocytic cells plays a key role in the weakened resistance to pathogenic bacteria. The purpose of this study was to investigate the function of phagocytic cells by comparing the serum levels of granulocyte macrophage-colony stimulating factor (GM-CSF) and Neopterin in thalassemia patients to healthy people. The study included 50 thalassemia patients and 30 healthy controls. Enzyme-linked immunosorbent assay (ELISA) was applied to estimate the serum levels of GM-CSF and Neopterin. Serum levels of GM-CSF were significantly elevated in thalassemia patients when compared to healthy people (p &lt; 0.05), while serum levels of Neopterin showed no significant change between thalassemia patients and healthy controls. Both serum levels of GM-CSF and Neopterin showed no significant difference between Splenectomized and healthy controls. Total leukocyte counts, lymphocytes, MID (monocytes), platelets, and RBC were all significantly higher in thalassemia patients compared to healthy controls. But, granulocyte counts showed no significant difference between the thalassemia patients and the healthy controls. On the other hand, total leukocytes, monocytes, lymphocytes and platelets counts were significantly raised in splenectomized patients when compared to healthy controls and non-splenectomized patients, respectively. We came to the conclusion that thalassemia patients have a high immune cell count, which is most likely due to the antigenic difficulties posed by blood transfusions. On the other hand, these patients have an impaired immune system.

Influence of nanocrystalline cerium dioxide on antigens of non-specific protection of quails

Intensive poultry farming technologies are closely linked to a variety of environmental, technological, feed and biological stressors, which tend to negatively affect their productivity and immune defenses. In the pathogenesis of such detrimental effects, the leading role belongs to the overproduction of oxygen free radicals - oxidative stress. The latter activates a number of transcription factors, including Nrf-2 and Nf-kB, which modulate the antioxidant defense network and participate in the organization of infection control. NDC is able to reduce the activation of Nf-kB and thus maintain antioxidant balance, but the response of the immune system to this factor is insufficiently studied. The aim of our work is to investigate the effect of NDC on the antigen of non-specific immunity of quails by adding it to drinking water. The object of study were the quail of the Pharaoh breed, experimental and control groups formed at the age of one day on the principle of analogues. The birds were kept in cages with free access to food and water. Quails of the experimental group in drinking water was added to the feed additive Nanocerium at a dose of 8.6 mg per liter of drinking water. This additive is an aqueous dispersion of NDC with an average nanoparticle size of 2-7 nm. The average weight of quails at the end of the experiment (56 days) in the experimental group was 20.2 g greater than in the control. Morphometric studies of the thymus, bursa and spleen showed no effect of NDC on the central and peripheral immune systems. Morphological parameters of the blood were within the physiological norm, but in the experimental group the number of erythrocytes and hemoglobin content were higher. The leukocyte count showed an increase in the leukocyte count (according to Garkavi LH) in the experimental group. Humoral performance was identical in both groups. Studies of cellular defense indicate no effect on the phagocytic function of peripheralblood cells. Functional and metabolic activity under the influence of NDC in the spontaneous test probably did not differ and increased in the stimulated. Therefore, quails that received nanocerium feed additive with water had a higher immunoresistance. Key words: birds, nanocerium, morphometry, thymus, natural immunity, morphological parameters of blood, hematopoiesis, humoral immunity, cellular immunity, hematological parameters. Accepted abbreviations: NDC – nanocrystalline cerium dioxide, ROS – reactive oxygen species, Nf-kB – nuclear factor - kV, Nrf-2 – nuclear factor - erythroid 2 and related factor 2.

Phytochemical Analysis and Immunomodulatory Potential on Diabetic-Infected Tuberculosis by Fruit Etlingera rubroloba A.D. Poulsen.

&lt;b&gt;Background and Objective:&lt;/b&gt; &lt;i&gt; Etlingera rubroloba&lt;/i&gt; (&lt;i&gt;E. rubroloba&lt;/i&gt;) A.D. Poulsen is an endemic plant in South-East Sulawesi and is a newly discovered species. This plant is expected to have the potential as an immunomodulator in patients with diabetes mellitus (DM), which can prevent tuberculosis infection by increasing the phagocytic function of macrophage cells and interleukin-12 (IL-12) levels. &lt;b&gt;Materials and Methods:&lt;/b&gt; Phytochemical analysis of the ethanolic extract of the fruit of &lt;i&gt;E. rubroloba&lt;/i&gt; A.D. Poulsen using Liquid Chromatography-Mass Spectrometry (LC-MS/MS) was carried out. The immunomodulatory potential &lt;i&gt;in vivo&lt;/i&gt; on BALB/c mice model DM was carried out by oral induction of TB antigen with extract dose, control positive, negative and normal groups. Furthermore, the phagocytic activity of macrophage cells can be seen with a microscope and the levels of IL-12 with the Elisa kit. &lt;b&gt;Results:&lt;/b&gt; The results showed the ethanol extract of the fruit of &lt;i&gt;E. rubroloba&lt;/i&gt; contained eight chemical compounds and had potential as immunomodulators in BALB/c DM mice induced by TB antigen by increasing the phagocytic activity of macrophage cells and levels of IL-12, which were significantly different from the negative control (p<0.05). &lt;b&gt;Conclusion:&lt;/b&gt; The chemical composition of the ethanol extract of the fruit of &lt;i&gt;E. rubroloba&lt;/i&gt; has the potential as an immunomodulator in TB antigen-induced DM &lt;i&gt;in vivo&lt;/i&gt;.

Abnormal Complex Karyotyping in A Patient Suspected of Acute Myeloblastic Leukemia (AML-M5): A Case Study

Hemophagocytic Lymphohistiocytosis (HLH) is a condition of immune dysregulation characterized by severe organ damage induced by a hyperinflammatory response and uncontrolled T-cell and macrophage activation. Patients with Acute Myeloblastic Leukemia (AML) may be prone to develop HLH. Hemophagocytic lymphohistiocytosis syndrome in AMLpatients with an abnormal complex karyotyping can worsen the patients' prognosis and outcome. A 47-year-old-female presented with prolonged fever, chills, fatigue, weight loss, productive cough, and anemia (blood transfusion (+)). Laboratory findings: hemoglobin 8.5 g/dL, WBC 151.99x103/μL, and platelet count 28x103/μL, peripheral blood 13% blast like cells, 19% promonocytes, 43% atypical (bizarre) monocytes, 25% neutrophils. Levels of CRP&gt;150 mg/L and procalcitonin 82.67 ng/mL, negative HBsAg, and positive IGRA test. Bone marrow morphology showed hypercellularity, decreased thrombopoiesis and erythropoiesis, increased granulopoiesis, macrophages, and hemophagocytosis. Karyotyping results: abnormal karyotypes: 46: XX (9 cells), 44: X (-18), 45: XX (-4), 45: XX (+7, -2, -16), 46: XX (chtb (3), chtb (4), chtb (5), chtb (9), chtb (12), chtb (22)), 46: XX (chtb (5), chtb (7)), 46: XX (chtb (6), chtb (12)), 46: XX (dic 2), 46: XX (chtb (1) (q12), chtb (3) (p21)), 46: XX (chtb (X) (q25) ), 46: XX (der (9), dic (9)), t (9:22)), 46: XX ((+ 21), (-13) chtb (2), p (23), t ( 9:22)). The conclusion was abnormal complex karyotyping. High concentrations of inflammatory cytokines (interleukin-1, interleukin-6, TNF-alpha, and interferon-gamma) secreted by malignant cells and increased phagocytic function of leukemic cells play an important role in the pathogenesis of HLH. Monocytic components (subtypes AML4 and AML5 of the FAB classification) are predisposing factors in cases of AML-related HLH. Cytogenetic abnormalities involving 8p11 and 16p13 are more common in AML-related HLH. Complex genetic abnormalities exacerbate the prognosis of AML, especially in treatment failure. A concluded that was diagnosed with HLH due to AML-M5 with genetic abnormalities of BCR ABL (+), monosomy, trisomy, and multiple chromatid breakage with high mortality. Karyotyping examination is important to determine the prognosis of the disease.