Process Of Phagocytosis Research Articles

A novel small molecule phagocytosis inhibitor, KB-208, ameliorates ITP in mouse models with similar efficacy as IVIG.

The characteristic feature of immune cytopenias involves the process of extravascular phagocytosis, wherein macrophages in the spleen and/or liver engage in the destruction of blood cells that have been opsonized by auto- or alloantibodies. Therefore, new treatments that prevent phagocytosis will be advantageous, especially for short-term usage along with alternative options. KB-208, a small molecule drug, previously shown to be efficacious for the in vitro inhibition of phagocytosis was synthesized. A passive antibody mouse model of immune thrombocytopenia (ITP) was used. Three different mouse strains (BALB/c, C57BL/6, CD1) were used to determine the efficacy of KB-208 compared with IVIG to ameliorate the ITP. Toxicity was investigated after 60-day chronic administration of KB-208 by a biochemistry panel, gross necroscopy and histopathology. KB-208 showed similar efficacy to ameliorate the thrombocytopenia compared with IVIG in all three mouse strains. This small molecule drug was effective at 1 mg/kg in ameliorating ITP, in comparison with IVIG at 1000-2500 mg/kg. KB-208 did not affect other blood parameters or elevate serum biochemistry markers of toxicity nor were any abnormal histopathological findings found. KB-208 is similar to IVIG for the amelioration of ITP in multiple mouse strains. Chronic administration of KB-208 for 60 days did not demonstrate in vivo toxicity. These findings indicate that KB-208 is efficacious, without significant in vivo toxicities in mice, and is a potential small molecule candidate for further evaluation to be used in the treatment of ITP and possibly all immune cytopenias where phagocytosis is responsible for the pathophysiology.

The effect of chronic predatory stress on the histostructure of the rat liver

One of the topical issues of modern circulatory physiology is the study of the peculiarities of the influence of chronic emotional stress on the histostructure of the liver.Aim: To study changes in the histostructure of the liver in the simulation of chronic predatory stress.Methodology and Research Methods. The study was conducted on 60 male Wistar rats weighing 220–370 g, divided into control and experimental groups of 30 rats each. Modeling of chronic predatory stress was performed according to the method of V.E. Zeilikman et al. (2021). Histological examination of the liver was performed in animals of the control and experimental groups. The preparations were stained with hematoxylin-eosin.Results. Under conditions of chronic predatory stress, stress-induced changes develop in the liver of rats. Transformation of hepatocytes occurs with the development of dystrophic changes, expansion and fullness of sinusoidal capillaries with compression of hepatic beams and destruction of hepatic lobules, a decrease in the parenchymal-stromal ratio. Foci of foamy accumulation of macrophages appear without signs of necrosis, single or group apoptotic hepatocytes. Necrotic foci are formed. The number of Kupffer cells and binucleated hepatocytes increases.Conclusion. With chronic predatory stress, the processes of dystrophy, necrosis and apoptosis of hepatocytes, signs of phagocytosis and regenerative processes, and decreased immune protection develop in the liver of rats.

Sequential Immune Acquisition of Monoclonal Antibodies Enhances Phagocytosis of Acinetobacter baumannii by Recognizing ATP Synthase

Objectives: The aim of this study was to prepare monoclonal antibodies (mAbs) that broadly target Acinetobacter baumannii and protect against infection by multi-drug-resistant (MDR) A. baumannii from different sources. Methods: mAb 8E6 and mAb 1B5 were prepared by sequentially immunizing mice with a sublethal inoculation of three heterogeneous serotypes of pan-drug-resistant (PDR) A. baumannii, ST-208, ST-195, and ST-229. Results: The cross-recognition of heterogeneous bacteria (n = 13) by two mAbs and potential targets was verified, and the in vitro antibacterial efficacy of mAbs was assessed. The median killing rate of mAb 8E6 against A. baumannii in the presence of complement and dHL-60 cells was found to be 61.51%, while that of mAb 1B5 was 41.96%. When only dHL-60 cells were present, the killing rate of mAb 8E6 was 65.73%, while that of mAb 1B5 was 69.93%. We found that mAb 8E6 and mAb 1B5 broadly targeted MDR A. baumannii on the ATP synthase complex and were equipped with an antibacterial killing ability by enhancing the innate immune bacteriolytic effect of ST-208 and ST-195 strains. Both monoclonal antibodies were validated to protect against respiratory infection at 4 and 24 h via enhancing the release of innate immune substances and inflammatory cytokines, effectively shortening the disease period in mice. Conclusions: mAb 8E6 and mAb 1B5 significantly enhanced the opsonization process of phagocytosis against A. baumannii strains prevalent in southern China by targeting ATP synthase antigens thereof, resulting in protective effects in mice.

Anti-Inflammatory And Immunomodulating Activity of the 1(10) Β-Epoxy-5,7α,6β(Н)-Guai-3(4),11(13)-Dien-6,12-Olide

BACKGROUND: The article discusses the results of studies of the anti-inflammatory and immunomodulatory activity of the sesquiterpene lactone 1(10)β-epoxy-5,7α,6β(Н)-guai-3(4),11(13)-diene-6,12-olide (hereinafter referred to as epoxyguaianolide). AIM: The article discusses the results of studies of the anti-inflammatory and immunomodulatory activity of the sesquiterpene lactone 1(10)β-epoxy-5,7α,6β(Н)-guai-3(4),11(13)-diene-6,12-olide. MATERIALS AND METHODS: A non-steroidal anti-inflammatory drug diclofenac sodium at a dose of 25 mg/kg (ampoules, 2 mL each, manufactured by Germany) was used as a reference drug in models of acute aseptic inflammation and cotton ball granuloma. On the model of cyclophosphamide immunosuppression, the immunomodulator levamisole at a dose of 3 mg/kg, was used as a reference drug. The immunomodulating effect of the test samples was studied on outbred adult male rats and outbred mice. We calculated the arithmetic mean, the mean square error of the arithmetic mean, the significance of the difference between the mean Рt according to the student’s t-test (TTEST), and the significance of the difference in Pf variances according to the Fisher f-test. RESULTS: The results of the experiments indicate the presence of a pronounced immunomodulatory activity in epoxyguaianolide. When interacting with cells of the immune system, epoxyguaianolide activated the studied parameters in animals with immunosuppression, whereas in intact animals, it did not affect the above parameters. On the model of cyclophosphamide immunosuppression, it was found that epoxyguaianolide has a pronounced immunomodulatory effect, manifested in an increase in the number of T-lymphocytes and a subpopulation of theophylline-resistant T-lymphocytes, an increase in the number of theophylline-sensitive T- and B-lymphocytes, activation of phagocytosis processes, and increased cell migration. This action of epoxyguaianolide eliminates the minimal possibility of hyperactivation of the immune system, which is an important condition for its use as an immunotropic agent. CONCLUSIONS: The conducted experiments made it possible to recommend epoxyguaianolide for clinical trials as an immunomodulating agent in the treatment of chronic inflammatory diseases accompanied by secondary immunodeficiencies.

An AI-based imaging flow cytometry approach to study erythrophagocytosis.

Erythrophagocytosis is a process consisting of recognition, engulfment and digestion by phagocytes of antibody-coated or damaged erythrocytes. Understanding the dynamics that are behind erythrophagocytosis is fundamental to comprehend this cellular process under specific circumstances. Several techniques have been used to study phagocytosis. Among these, an interesting approach is the use of Imaging Flow Cytometry (IFC) to distinguish internalization and binding of cells or particles. However, this method requires laborious analysis. Here, we introduce a novel approach to analyze the phagocytosis process by combining Artificial Intelligence (AI) with IFC. Our study demonstrates that this approach is highly suitable to study erythrophagocytosis, categorizing internalized, bound and non-bound erythrocytes. Validation experiments showed that our pipeline performs with high accuracy and reproducibility.

Improved Liver Intravital Microscopic Imaging Using a Film-Assisted Stabilization Method.

Intravital microscopy (IVM) is a valuable method for biomedical characterization of dynamic processes, which has been applied to many fields such as neuroscience, oncology, and immunology. During IVM, vibration suppression is a major challenge due to the inevitable respiration and heartbeat from live animals. In this study, taking liver IVM as an example, we have unraveled the vibration inhibition effect of liquid bridges by studying the friction characteristics of a moist surface on the mouse liver. We confirmed the presence of liquid bridges on the liver through fluorescence imaging, which can provide microscale and nondestructive liquid connections between adjacent surfaces. Liquid bridges were constructed to sufficiently stabilize the liver after abdominal dissection by covering it with a polymer film, taking advantage of the high adhesion properties of liquid bridges. We further prototyped a microscope-integrated vibration-damping device with adjustable film tension to simplify the sample preparation procedure, which remarkably decreased the liver vibration. In practical application scenarios, we observed the process of liposome phagocytosis by liver Kupffer cells with significantly improved image and video quality. Collectively, our method not only provided a feasible solution to vibration suppression in the field of IVM, but also has the potential to be applied to vibration damping of precision instruments or other fields that require nondestructive ″soft″ vibration damping.

Carbon nitride nanosheets induce pulmonary surfactant deposition via dysfunction of alveolar secretion and clearance

Graphitic carbon nitride is a new type of carbon-based nanomaterial with superior properties and great potential for application, which raised great concerns about their environmental and occupational exposure. Graphitic carbon nitride has been reported to accumulate in the lung potentially, however, the respiratory hazard effect of graphitic carbon nitride is still unknown. In the present study, we reported that graphitic carbon nitride (g-CN) and its doped variant sulfur doped graphitic carbon nitride (S-g-CN) nanosheets inhalation induced pulmonary inflammation, increased the production of pulmonary surfactant in alveolar type II epithelial cells, accompanied by the upregulation of lipids transporter expression levels in alveolar macrophages to clear excessive pulmonary surfactant in the alveolar. Further investigation found that the internalization of g-CN and S-g-CN prevented lipid phagocytosis and metabolic processes in alveolar macrophages, ultimately leading to the deposition of proteins and phospholipids in the lung. Furthermore, we found that g-CN was more robust in inhalation toxicity compared to sulfur doped form, which meant the doping of sulfur in graphitic carbon nitride reduced hazardous effects on the respiratory system. In summary, our study demonstrated that inhalation of graphitic carbon nitride nanosheets caused the deposition of pulmonary surfactants in lung, providing an insightful reference for pulmonary toxicity assessment of graphitic carbon nitride.

Screening and identification of DNA nucleic acid aptamers against F1 protein of Yersinia pestis using SELEX method.

Yersinia pestis is a bacterium that causes the disease plague. It has caused the deaths of many people throughout history. The bacterium possesses several virulence factors (pPla, pFra, and PYV). PFra plasmid encodes fraction 1 (F1) capsular antigen. F1 protein protects the bacterium against host immune cells through phagocytosis process. This protein is specific for Y. pestis. Many diagnostic techniques are based on molecular and serological detection and quantification of F1 protein in different food and clinical samples. Aptamers are small nucleic acid sequences that can act as specific ligands for many targets.This study, aimed to isolate the high-affinity ssDNA aptamers against F1 protein. In this study, SELEX was used as the main strategy in screening aptamers. Moreover, enzyme-linked aptamer sorbent assay (ELASA) and surface plasmon resonance (SPR) were used to determine the affinity and specificity of obtained aptamers to F1 protein. The analysis showed that among the obtained aptamers, the three aptamers of Yer 21, Yer 24, and Yer 25 were selected with a KD value of 1.344E-7, 2.004E-8, and 1.68E-8M, respectively. The limit of detection (LoD) was found to be 0.05, 0.076, and 0.033μg/ml for Yer 21, Yer 24, and Yer 25, respectively. This study demonstrated that the synthesized aptamers could serve as effective tools for detecting and analyzing the F1 protein, indicating their potential value in future diagnostic applications.

Anti-CAMP1 IgG promotes macrophage phagocytosis of Cutibacterium acnes type II

Among 5 types of the Christie-Atkins-Munch-Petersen factor (CAMP) of Cutibacterium acnes, CAMP1 is highly expressed in phylotype II as well as IB, and thought to be a virulence factor of opportunistic but fatal blood, soft tissue, and implant-related infections. The target of a human single-chain variable antibody fragment (scFv), recently isolated from a phage display library, has been identified as CAMP1 of phylotype II, using immunoprecipitation followed by mass spectrometry, phage display peptide biopanning, 3D-modelling, and ELISA. The IgG1 format of the antibody could enhance phagocytosis of C. acnes DMST 14916 by THP-1 human monocytes. Our results suggest that the antibody-dependent phagocytosis process is mediated by the caveolae membrane system and involves the induction of IL-1β. This is the first report on the study of a human antibody against CAMP1 of C. acnes phylotype II, of which a potential use as therapeutic antibody against virulence C. acnes infection is postulated.

Two-component macrophage model for active phagocytosis with pseudopod formation

Macrophage phagocytosis is critical for the immune response, homeostasis regulation, and tissue repair. This intricate process involves complex changes in cell morphology, cytoskeletal reorganization, and various receptor-ligand interactions controlled by mechanical constraints. However, there is a lack of comprehensive theoretical and computational models that investigate the mechanical process of phagocytosis in the context of cytoskeletal rearrangement. To address this issue, we propose a novel coarse-grained mesoscopic model that integrates a fluid-like cell membrane and a cytoskeletal network to study the dynamic phagocytosis process. The growth of actin filaments results in the formation of long and thin pseudopods, and the initial cytoskeleton can be disassembled upon target entry and reconstructed after phagocytosis. Through dynamic changes in the cytoskeleton, our macrophage model achieves active phagocytosis by forming a phagocytic cup utilizing pseudopods in two distinct ways. We have developed a new algorithm for modifying membrane area to prevent membrane rupture and ensure sufficient surface area during phagocytosis. In addition, the bending modulus, shear stiffness, and cortical tension of the macrophage model are investigated through computation of the axial force for the tubular structure and micropipette aspiration. With this model, we simulate active phagocytosis at the cytoskeletal level and investigate the mechanical process during the dynamic interplay between macrophage and target particles.

Portable dielectrophoresis for biology: ADEPT facilitates cell trapping, separation, and interactions

Dielectrophoresis is a powerful and well-established technique that allows label-free, non-invasive manipulation of cells and particles by leveraging their electrical properties. The practical implementation of the associated electronics and user interface in a biology laboratory, however, requires an engineering background, thus hindering the broader adoption of the technique. In order to address these challenges and to bridge the gap between biologists and the engineering skills required for the implementation of DEP platforms, we report here a custom-built, compact, universal electronic platform termed ADEPT (adaptable dielectrophoresis embedded platform tool) for use with a simple microfluidic chip containing six microelectrodes. The versatility of the open-source platform is ensured by a custom-developed graphical user interface that permits simple reconfiguration of the control signals to address a wide-range of specific applications: (i) precision positioning of the single bacterium/cell/particle in the micrometer range; (ii) viability-based separation by achieving a 94% efficiency in separating live and dead yeast; (iii) phenotype-based separation by achieving a 96% efficiency in separating yeast and Bacillus subtilis; (iv) cell–cell interactions by steering a phagocytosis process where a granulocyte engulfs E. coli RGB-S bacterium. Together, the set of experiments and the platform form a complete basis for a wide range of possible applications addressing various biological questions exploiting the plug-and-play design and the intuitive GUI of ADEPT.

PEGylated nanoparticles interact with macrophages independently of immune response factors and trigger a non-phagocytic, low-inflammatory response

Poly-ethylene-glycol (PEG)-based nanoparticles (NPs) - including cylindrical micelles (CNPs), spherical micelles (SNPs), and PEGylated liposomes (PLs) - are hypothesized to be cleared in vivo by opsonization followed by liver macrophage phagocytosis. This hypothesis has been used to explain the rapid and significant localization of NPs to the liver after administration into the mammalian vasculature. Here, we show that the opsonization-phagocytosis nexus is not the major factor driving PEG-NP – macrophage interactions. First, mouse and human blood proteins had insignificant affinity for PEG-NPs. Second, PEG-NPs bound macrophages in the absence of serum proteins. Third, lipoproteins blocked PEG-NP binding to macrophages. Because of these findings, we tested the postulate that PEG-NPs bind (apo)lipoprotein receptors. Indeed, PEG-NPs triggered an in vitro macrophage transcription program that was similar to that triggered by lipoproteins and different from that triggered by lipopolysaccharide (LPS) and group A Streptococcus. Unlike LPS and pathogens, PLs did not increase transcripts involved in phagocytosis or inflammation. High-density lipoprotein (HDL) and SNPs triggered remarkably similar mouse bone-marrow-derived macrophage transcription programs. Unlike opsonized pathogens, CNPs, SNPs, and PLs lowered macrophage autophagosome levels and either reduced or did not increase the secretion of key macrophage pro-inflammatory cytokines and chemokines. Thus, the sequential opsonization and phagocytosis process is likely a minor aspect of PEG-NP – macrophage interactions. Instead, PEG-NP interactions with (apo)lipoprotein and scavenger receptors appear to be a strong driving force for PEG-NP – macrophage binding, entry, and downstream effects. We hypothesize that the high presence of these receptors on liver macrophages and on liver sinusoidal endothelial cells is the reason PEG-NPs localize rapidly and strongly to the liver.

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.

Live imaging of photoreceptor outer segment phagocytosis in zebrafish

Retina depends on retinal pigment epithelium (RPE) for its survival. To maintain normal functionality, photoreceptors renew 10% of their outer segments every day and this process requires efficient phagocytosis of the aged photoreceptor fragments by the RPE. Phagocytosis is a dynamic process with strong circadian regulation, but detailed understanding on the regulation of the process and differences between photoreceptor types is presently inadequate. Previous studies have been carried out mainly with in vitro models and cell cultures, and therefore, the dynamics of the phagocytosis process in the context of a living animal has remained elusive. Here, we present an approach that utilizes transgenic zebrafish lines with fluorescently tagged photoreceptor and RPE cells for in vivo imaging of their interactive functions in a living animal.Zebrafish is one of the most accessible animal models to study retinal physiology in vivo and currently the only vertebrate model that can be used to image retinal tissue dynamics in real time. By utilizing already existing low‐pigmented zebrafish lines and microinjecting plasmids coding for photoreceptor and RPE cell membrane targeted tags, we have created low‐pigmented fish with fluorescent RPE cells and the outer segments of the different photoreceptor types. Here, we first show with histological sections how the plasmids are expressed in the correct photoreceptor types and RPE cells in larval zebrafish eyes. Then, we present our in vivo imaging approach where we anesthetise and mount the zebrafish to a confocal microscope. Our results using time‐lapse imaging demonstrate how we can follow the interaction between photoreceptors and RPE in live animals in resolution that enables detecting the movement of photoreceptor outer segment fragments in the subretinal space. Taken together, our study introduces a new approach to investigate the dynamics of phagocytosis of different photoreceptor types by the RPE in real time in a living animal.

Updates in molecules associated with pathogenesis of amoebiasis: A mini review

Amoebiasis caused due to the protozoan parasite Entamoeba histolytica has been ranked as the third major reason of mortality after Malaria and Schistosomiasis. The pathogen adversely impacts the health of the children and hinders their development and can lead to intestinal and extra-intestinal manifestation, leading to more than 100,000 deaths annually. The most common route of transmission is via faecal oral route, but recent reports also suggest sexual transmission among homosexual men. Recent experiments in in-vitro, in-vivo and in-silico studies have contributed to a better understanding of pathogenesis by filling in the knowledge gaps related to adhesion, invasion, phagocytosis, encystation, trogocytosis and many other processes. Experimental studies evaluating the pathogenic potential of Entamoeba dispar also highlights the importance of considering other species under this genus. Despite the recent advances, there is a need for new drug targets due to the lack of a successful vaccine yet. This review therefore summarizes the recent molecules that have been identified and assessed to play a role in various pathogenic mechanisms of the parasite, further contributing to the progression of the disease.

#82 : Does Apoptotic Body Impair Pregnancy Outcome

Background: Morphologically suboptimal blastocyst is often associated with poorer pregnancy outcomes. Apoptotic body is one of the abnormal findings in blastocysts characterised by cellular shrinkage and a darker appearance. The presence of apoptotic bodies may be suggestive of ongoing cellular degeneration. Cellular degeneration in the blastocyst is part of its self-corrective mechanism. The degenerated cells will deplete through the phagocytosis process. Aims: This is a retrospective study that aims to analyse whether the presence of apoptotic body has a significant impact on pregnancy outcomes. Method: We compare the outcome of the frozen-thawed embryo transfer (FET) cycle of blastocysts with apoptotic body and without it as a control. We further divide the cases into single blastocyst transfer and double blastocyst transfer. The data was collected from Vistana Fertility Centre from January to December 2022. Results: Conclusion: Data analysis showed that there is no significant difference between the study group and the control group. Hence, the presence of dead cells did not negatively impact the pregnancy outcomes.

The Streptococcus virulence protein PepO triggers anti-tumor immune responses by reprograming tumor-associated macrophages in a mouse triple negative breast cancer model

BackgroundThe efficacy of current surgery and chemotherapy for triple negative breast cancer (TNBC) is limited due to heterogenous and immunosuppressive tumor microenvironment (TME). Tumor associated macrophages (TAMs), which are regarded as an M2 tumor-promoting phenotype, are crucial in the development of the immunosuppressive TME. Targeting TAM reprograming is a promising strategy in anti-tumor therapy since reprogramming techniques provide the opportunity to actively enhance the antitumor immunological activity of TAM in addition to eliminating their tumor-supportive roles, which is rarely applied in TNBC clinically. However, how to drive M2 macrophages reprogramming into M1 with high potency remains a challenge and the molecular mechanisms how M2 macrophages polarized into M1 are poorly understood. Here, we identified a new immunoregulatory molecular PepO that was served as an immunoregulatory molecule governed the transformation of tumor-promoting M2 to tumor-inhibitory M1 cells and represented an effective anti-tumor property.ResultsAt the present study, we identified a new immunoregulatory molecular PepO, as a harmless immunoregulatory molecule, governed the transformation of tumor-promoting M2 to tumor-inhibitory M1 cells efficiently. PepO-primed M2 macrophages decreased the expression of tumor-supportive molecules like Arg-1, Tgfb, Vegfa and IL-10, and increased the expression of iNOS, Cxcl9, Cxcl10, TNF-α and IL-6 to inhibit TNBC growth. Moreover, PepO enhanced the functions of macrophages related to cell killing, phagocytosis and nitric oxide biosynthetic process, thereby inhibiting the development of tumors in vivo and in vitro. Mechanistically, PepO reprogramed TAMs toward M1 by activating PI3K-AKT-mTOR pathway via TLR4 and suppressed the function of M2 by inhibiting JAK2-STAT3 pathway via TLR2. The PI3K inhibitor LY294002 abrogated the role of PepO in switching M2 macrophages into M1 and in inhibiting TNBC growth in vivo. And PepO failed to govern the M2 macrophages to reprogram into M1 macrophages and inhibit TNBC when TLR2 or TLR4 was deficient. Moreover, PepO enhanced the antitumor activity of doxorubicin and the combination exerted a synergistic effect on TNBC suppression.ConclusionsOur research identified a possible macrophage-based TNBC immunotherapeutic approach and suggested a novel anticancer immunoregulatory molecular called PepO.

Expression of Tie2 (angiopoietin receptor) on the monocyte subpopulations from ischemic stroke patients: Histological and flowcytometric studies.

Different subpopulations of monocytes play roles in phagocytosis, inflammation, and angiogenic processes e.g., Tie2-expressing monocytes (TEMs). The brain is flooded with macrophages that are derived from monocytes within 3-7 days after a stroke. This study aimed to determine the expression level of Tie2 (an angiopoietin receptor) on monocytes and their subpopulations in ischemic stroke patients using the histological and immunohistological study of bone marrow biopsies and blood flow cytometry examination. Ischemic stroke patients within two days were selected. Participants in the control group were healthy volunteers of matched age and gender. Sample collection was performed within 24 to 48 hours after medical consultants confirmed the stroke diagnosis. An iliac crest bone marrow biopsy was obtained and fixed for histological and immunohistological staining with antiCD14 and antiCD68. Flow cytometry was used to determine the total monocyte population, monocyte subpopulations, and TEMs after staining with monoclonal antibodies to CD45, CD14, CD16, and Tie2. Post-stroke patients' bone marrow cells were hypercellular. There was an apparent increase in CD68 and CD14-positive cells. Ischemic stroke patients exhibited low percentages of nonclassical monocytes CD14lowCD16++, with an increase in intermediate monocytes CD14highCD16+. Moreover, ischemic stroke patients had significantly higher levels of TEMs than control group. The results of this study demonstrate dysregulation of angiogenesis in monocyte subsets in ischemic stroke patients, which could be used as an early diagnostic marker of neurovascular damage and may need angiogenic therapy or improved medications to prevent further damage of blood vessels.

A novel quantification method for retinal pigment epithelium phagocytosis using a very-long-chain polyunsaturated fatty acids-based strategy.

The vertebrate retinal pigment epithelium (RPE) lies adjacent to the photoreceptors and is responsible for the engulfment and degradation of shed photoreceptor outer segment fragments (POS) through receptor-mediated phagocytosis. Phagocytosis of POS is critical for maintaining photoreceptor function and is a key indicator of RPE functionality. Popular established methods to assess RPE phagocytosis rely mainly on quantifying POS proteins, especially their most abundant protein rhodopsin, or on fluorescent dye conjugation of bulk, unspecified POS components. While these approaches are practical and quantitative, they fail to assess the fate of POS lipids, which make up about 50% of POS by dry weight and whose processing is essential for life-long functionality of RPE and retina. We have developed a novel very-long-chain polyunsaturated fatty acids (VLC-PUFA)-based approach for evaluating RPE phagocytic activity by primary bovine and rat RPE and the human ARPE-19 cell line and validated its results using traditional methods. This new approach can be used to detect in vitro the dynamic process of phagocytosis at varying POS concentrations and incubation times and offers a robust, unbiased, and reproducible assay that will have utility in studies of POS lipid processing.

MicroRNA-30a inhibits cell proliferation in a sepsis-induced acute kidney injury model by targeting the YAP-TEAD complex

BackgroundAcute kidney injury (AKI) is a primary feature of renal complications in patients with sepsis. MicroRNA (miRNA/miR)-30a is an essential regulator of cardiovascular diseases, tumors, phagocytosis, and other physical processes, but whether it participates in sepsis-induced AKI (sepsis-AKI) is unknown. We aimed to elucidate the functions and molecular mechanism underlying miR-30a activity in sepsis-AKI. MethodsThe classical cecal ligation and puncture (CLP) method and lipopolysaccharide (LPS)-induced Human Kidney 2 (HK-2) cells were used to establish in vivo and in vitro sepsis-AKI models. Specific pathogen-free and mature male Sprague-Dawley (SD) rats, aged 6–8 weeks (weight 200–250 g), were randomly divided into five-time phase subgroups. Fluid resuscitation with 30 mL/kg 37 °C saline was administered after the operation, without antibiotics. Formalin-fixed, paraffin-embedded kidney sections were stained with hematoxylin and eosin. SD rat kidney tissue samples were collected for analysis by real-time quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. HK-2 cells were transfected with hsa-miR-30a-3p mimics or inhibitors, and compared with untreated normal controls. RNA, protein, and cell viability were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blot, and cell counting kit-8 methods. A Dual-Luciferase Assay Kit (Promega) was used to measure luciferase activity 48 h after transfection with miR-30a-3p mimics. ResultsExpression levels of miR-30a-3p and miR-30a-5p in renal tissues of the sepsis group were significantly reduced at 12 h and 24 h (P <0.05). Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were significantly increased in renal tissue 3 h after the operation in rats (P <0.05), and gradually decreased 6 h, 12 h, and 24 h after CLP. Levels of miR-30a-5p and miR-30a-3p were significantly down-regulated at 3 h after LPS treatment (P <0.05), and gradually decreased in HK-2 cells. One hour after LPS (10 µg/mL) treatment, TNF-α and IL-1β levels in HK-2 cells were significantly up-regulated (P < 0.05), and they were markedly down-regulated after 3 h (P <0.05). IL-6 expression levels began to rise after LPS treatment of cells, peaked at 6 h (P <0.05), and then decreased to the initial level within a few hours. Stimulation with 10 µg/mL LPS promoted HK-2 cells proliferation, which was inhibited after miR-30a-3p-mimic transfection. Bioinformatics prediction identified 37 potential miR-30a-3p target genes, including transcriptional enhanced associate domain 1 (TEAD1). After transfection of HK-2 cells with miR-30a-3p mimics and miR-30a-3p inhibitor, TEAD1 transcript was significantly up- and down-regulated, respectively (both P <0.05). After LPS treatment (24 h), expression of TEAD1 in the inhibitors group was significantly increased (P <0.01), while that in the mimics group was significantly suppressed (P <0.01). In the dual luciferase reporter experiment, miR-30a-3p overexpression decreased fluorescence intensity (P <0.01) from TEAD1-wt-containing plasmids, but did not influence fluorescence intensity from TEAD1-muta-containing plasmids. LPS may promote HK-2 cells proliferation through the miR-30a-3p/TEAD1 pathway. ConclusionIn a background of expression of inflammatory factors, including TNF-α, IL-1β, and IL-6, which were transiently increased in the sepsis-AKI model, miR-30a was down-regulated. Down-regulated miR-30a-3p may promote cell proliferation by targeting TEAD1 in LPS-induced HK-2 cells, demonstrating its potential as a biomarker for early sepsis-AKI diagnosis.