CD63 Expression Research Articles

CD46 Is a Protein Receptor for Human Adenovirus Type 64

Certain species D human adenoviruses (HAdV-D19, -D37, and -D64) are causative agents of epidemic keratoconjunctivitis. HAdV-D37 has previously been shown to bind CD46 (membrane cofactor protein) and sialic acid as adhesion receptors. HAdV-D64 is genetically highly similar to HAdV-D37, with an identical fiber protein sequence, but differs substantially in its penton base and hexon proteins, two other major capsid components, due to genetic recombination. Here, we demonstrate that, like HAdV-D37, HAdV-D64 virions bind directly to CD46 and that CD46 and sialic acid also function as receptors for HAdV-D64 on multiple cell types. Expression of CD46 on CD46-negative cells conferred susceptibility to HAdV-D64 entry. Specifically blocking HAdV-D64 binding to CD46 on the host cell surface strongly inhibits viral entry and gene delivery into multiple cell lines that represent target tissues. We show that CD46 is expressed on human conjunctival epithelial cells and directly binds to the HAdV-D64 virion. Our results suggest that HAdV-D64 may be used to deliver genes to target conjunctival cells and that interrupting HAdV-D64 entry through its interaction with CD46 may prevent or lessen adenovirus-associated ocular disease.

Impact of UPF2 on the levels of CD81 on extracellular vesicles

Extracellular vesicles (EVs) are involved in cell-to-cell communication. Following uptake, EV cargo molecules, including DNA, RNA, lipids, and proteins, influence gene expression and molecular signaling in recipient cells. Although various studies have identified disease-specific EV molecules, further research into their biogenesis and secretion mechanisms is needed for clinical application. Here, we investigated the role of UPF2 in regulating the biogenesis and components of EVs. Notably, UPF2 promoted the expression of CD81, a membrane protein marker of EVs, as UPF2 silencing decreased CD81 levels in EVs, both inside the cell and secreted. In contrast, the expression levels of CD63 increased, without altering the size or numbers of EVs. In addition, reducing UPF2 levels did not affect the total number of EVs but lowered production of CD81-positive EVs and reduced the efficiency of uptake by recipient cells. Collectively, our findings uncover a novel function for UPF2 in regulating the production of CD81 and changing EV properties.

Enrichment protocols for human conjunctival extracellular vesicles and their characterization

The understanding of the role played by extracellular vesicles (EVs) in different tissues has improved significantly in the last years, but remains limited concerning the conjunctiva, a complex eye tissue whose role is pivotal for corneal protection. Here, we conducted a comparative study to isolate and characterize EVs from human conjunctival epithelial (IM-HConEpiC) and human conjunctival mesenchymal stromal cell (Conj-MSCs) secretomes using different isolation methods: differential ultracentrifugation (UC), and a combination of ultrafiltration (UF) with precipitation or size exclusion chromatography (SEC). EVs were characterized by total protein content, size, morphology, and expression of protein markers. EV functional effect was tested in an in vitro oxidative stress model. We successfully recovered EVs with the three methods, although significantly higher yields were obtained with UF-precipitation. Dynamic light scattering analysis confirmed the presence of nano-sized particles, being UC-isolated EVs larger than those isolated by UF-precipitation and UF-SEC. Atomic Force Microscopy showed EVs with a slightly ellipsoidal morphology. EVs enriched with UF-precipitation method were further analyzed, confirming the expression of Alix, CD63, TSG101, and Syntenin-1 by Western blotting and showing that Conj-MSC-derived EVs significantly reduced oxidative stress on IM-HConEpiC. Therefore, we conclude that UF-precipitation is the most efficient method for conjunctival EV enrichment.

Abstract B070: Dissecting EV dynamics in the sequestration of doxorubicin from breast cancer cells

Abstract Background: Extracellular vesicles (EVs) are one of the three main analytes sought for in liquid biopsy development. EVs are released by all cell types including cancer cells and play a crucial role in cell-cell communication. Breast cancer cells have been shown to utilize EVs as a mechanism to sequester chemotherapeutic agents, thereby conferring resistance to treatment. This study investigates the role of EVs in doxorubicin (DOX) resistance in MDA-MB-231 breast cancer cells. Method: MDA-MB-231 cells were exposed to increasing dose of anthracycline for 6 and 12 months. Resistance was confirmed by cytotoxicity assay and western blot. Resistant and parent cells were labeled for immunohistochemistry and their EVs were isolated via ultracentrifugation. EVs were analysed using nanoscale flow cytometry and sent for mass spectrophotometry. Result: Using nanoscale flow cytometry, we optimized a method to detect DOX-carrying EVs based on the intrinsic fluorescence of the drug. We found that DOX-resistant cells exhibited lower intracellular DOX accumulation and higher expression of the EV marker CD63 compared to their parental counterparts. Conclusion: These results suggest that chemoresistant breast cancer cells may alter their EV production machinery to evade the lethal effects of DOX. Understanding this mechanism could lead to novel strategies to overcome chemoresistance in breast cancer. Citation Format: Sina Halvaei, Jing Xu, Suganthi Chittaranjan, Sharon Gorski, Karla Williams. Dissecting EV dynamics in the sequestration of doxorubicin from breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B070.

Differences in human and commercial hybrid pig platelet activation induced by borosilicate glass beads in a modified chandler loop-system.

The pig (Sus scrofa) is the most widely used large animal model in Europe, with cardiovascular research being one of the main areas of application. Adequate refinement of interventional studies in this field, meeting the requirements of Russell and Burch's 3 R concept, can only be performed if blood-contacting medical devices are hemocompatible. Because most medical devices for cardiovascular interventional procedures are developed for humans, they are tested only for compatibility with human blood. The aim of this study was therefore to determine whether there are differences in behavior of human and porcine platelets from commercial hybrid pigs when they come into contact with borosilicate glass, which was used as an exemplary thrombogenic material. For this purpose, changes in platelet count, platelet volume and platelet expression of the activation markers CD61, CD62P and CD63 were measured using a modified chandler loop-system simulating the fluidic effects of the bloodflow. Commercial hybrid pig and human platelets showed significant adhesions to borosilicate glass but the commercial hybrid pigs platelets showed a significantly higher tendency to adhere to borosilicate glass. In contrast to human platelets the platelets of commercial hybrid pigs showed significant activation after 4 to 8 minutes exposure to borosilicate glass and there were differences among the ratios of surface and activation markers in between the platelets of both species.

Diagnostic Implications of CD63 and CD64 Expression Levels and FcγRIIIA 158 V/F Gene Polymorphism in Primary Immune Thrombocytopenia Adult Patients.

Immune thrombocytopenic purpura (ITP) is an acquired autoimmune disease characterized by reduced platelet counts due to immune system dysregulation caused by many factors, including genetics, autoimmune diseases, infections, and inflammations. Therefore, the current study aimed to evaluate immunological markers such as the expression level of lysosomal associated membrane protein 3 (LAMP-3), also known as CD63, and the expression level of Fc-gamma receptor I (FcγRI), also known as CD64 and also investigate the association of Fc-gamma receptor IIIA (FcγRIIIA) 158 V/F polymorphism to the risk of ITP. A total of 180 subjects; 60 ITP patients, 60 patients with thrombocytopenia of other causes and 60 controls were enrolled into our study. The expression level of CD63 was done using reverse transcription quantitative PCR (RTqPCR), while CD64 expression level was done by flow cytometry. The polymorphism of FcγRIIIA 158 V/F gene was analyzed by polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) analysis. Finally, CD63 and CD64 protein-protein interactions were done by using the STRING online database. The expression of CD63 was significantly elevated in ITP patients than thrombocytopenia patients and healthy control. Also there was high expression level of CD64 on granulocytes and monocytes from ITP patients than other groups. Receiver operating characteristic curve (ROC curve) analysis of CD63 showed an area under the curve (AUC) revealed of 1.00, sensitivity of 100% and specificity of 100%; while for CD64 on granulocytes, AUC of 0.998 as well as a sensitivity of 96.66% and specificity of 93.33%. Regarding FcγRIIIa 158 V/F polymorphism, all patients and healthy volunteers included in this study showed the wild FF genotype. The expression of both CD63 and CD64 were significantly increased in ITP patients and could be good biomarkers to diagnose ITP. Additionally, there is no association between FcγRIIIa 158 V/F polymorphism and the risk of ITP disease.

Deciphering CD59: Unveiling Its Role in Immune Microenvironment and Prognostic Significance.

CD59, a GPI-anchored membrane protein, protects cancer cells from complement-dependent cytotoxicity (CDC) by inhibiting the formation of the membrane attack complex (MAC). It has been demonstrated to be overexpressed in most solid tumors, where it facilitates tumor cell escape from complement surveillance. The role of CD59 in cancer growth and interactions between CD59 and immune cells that modulate immune evasion has not been well explored. Using cancer patient database from The Cancer Genome Atlas (TCGA) and other public databases, we analyzed CD59 expression, its prognostic significance, and its association with immune cell infiltration in the tumor microenvironment, identifying associated genomic and functional networks and validating findings with invitro cell-line experimental data. This article describes the abundant expression of CD59 in multiple tumors such as cervical squamous cell carcinoma (CESC), kidney renal cell carcinoma (KIRC), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), and stomach adenocarcinoma (STAD), as well as in pan-cancer, using The Cancer Genome Atlas (TCGA) database and confirmed using multiple cancer cell lines. The expression of CD59 significantly alters the overall survival (OS) of patients with multiple malignancies such as CESC, GBM, HNSC, and STAD. Further, the correlation between CD59 and Treg and/or MDSC in the tumor microenvironment (TME) has shown to be strongly associated with poor outcomes in CESC, GBM, HNSC, and STAD as these tumors express high FOXP3 compared to KIRC. Moreover, unfavorable outcomes were strongly associated with the expression of CD59 and M2 tumor-associated macrophage infiltration in the TME via the IL10/pSTAT3 pathway in CESC and GBM but not in KIRC. In addition, TGFβ1-dominant cancers such as CESC, GBM, and HNSC showed a high correlation between CD59 and TGFβ1, leading to suppression of cytotoxic T cell activity. Overall, the correlation between CD59 and immune cells predicts its prognosis as unfavorable in CESC, GBM, HNSC, and STAD while being favorable in KIRC.

Development of a Nomogram Based on Transcriptional Signatures, IFN-γ Response and Neutrophils for Diagnosis of Tuberculosis.

Tuberculosis (TB) is a major global health threat and its diagnosis remains challenging. This study aimed to develop a nomogram that incorporated peripheral blood transcriptional signatures and other blood tests for the diagnosis of tuberculosis. Patients with TB, patients with other definite pulmonary diseases (OPD), individuals with latent tuberculosis infection (LTBI), and healthy controls (HC) were retrospectively enrolled between May 2017 and April 2018. The results of the interferon-γ release assay (IGRA) and blood counts were obtained from medical records, and the transcripts of 10 genes were detected using reverse transcription polymerase chain reaction (RT-PCR). Variable selection was performed using least absolute shrinkage and selection operator regression (LASSO) and multivariate logistic regression was performed for the optimal prediction model with backward direction. The model was displayed as a nomogram, and its performance was evaluated for discrimination ability, calibration ability, and clinical usefulness. Internal validation of the prediction model was conducted using bootstrap resampling. A total of 185 participants were enrolled, including 84 patients with TB and 101 controls. A prediction nomogram composed of IGRA, percentage of neutrophils, and expression levels of CD64, granzyme A (GZMA), and PR/SET domain 1 (PRDM1) was established. The nomogram demonstrated good discrimination, with an unadjusted area under the curve (AUC) of 0.914 (95% CI: 0.875-0.954) and a bootstrap-corrected AUC of 0.914 (95% CI: 0.874-0.947). With a cutoff value of 0.519, the sensitivity and specificity for discriminating PTB from controls were 0.81 and 0.871, respectively. The nomogram also showed good calibration with the Hosmer-Lemeshow test (P=0.58) and good clinical practicality displayed by the decision curve analysis. A nomogram composed of IGRA, percentage of neutrophils, and expression of CD64, GZMA, and PRDM1 was established. The nomogram demonstrated a sensitivity and specificity of 81% and 87%, respectively, for differentiating TB from controls.

Hypoxic Stress Induces Complement-Mediated Lysis of Mesenchymal Stem Cells by Downregulating Factor H and CD59.

Factor H and membrane inhibitor of reactive lysis (CD59) are key regulators of complement activation. Mesenchymal stem cells (MSCs) secrete Factor H and express CD59 to protect themselves from complement-mediated damage. Severe hypoxia found to decrease the survival chances of MSCs after transplantation; however, little is known about the impact of severe hypoxia on modulating the complement system activity and its effect on MSCs survival. Our study seeks to explore the effect of severe hypoxia on modulating the complement cascade in MSCs. Human adipose tissue-derived MSCs (hAD-MSCs) were cultured under severe hypoxia using 400μM Cobalt Chloride (CoCl2) for 48h. The protein expressions of survival marker; Phosphoinositide 3-kinases (PI3K), and pro-apoptotic marker; Caspase-3 were assessed using western blotting. The level of complement system related factors; Factor H, CD59, C3b, iC3b, C5b, C9, and the complement membrane attack complex (MAC) were analyzed using Elisa assays, western blotting, and immunocytochemistry. Our results showed for the first time that severe hypoxia can significantly impair Factor H secretion and CD59 expression in MSCs. This has been associated with upregulation of MAC complex and increased level of cell lysis and apoptosis marked by downregulation of PI3K and upregulation of Annexin v and Caspase-3. The loss of Factor H and CD59 in hypoxic MSCs can initiate their lysis and apoptosis mediated by activating MAC complex. Preserving the level of Factor H and CD59 in MSCs has significant clinical implication to increase their retention rate in hypoxic conditions and prolong their survival.

Cleavable Novel Thienopyridine Derivatives as Potent Antithrombotic Agents

AbstractThienopyridine derivatives are specifically designed for utilization in antithrombotic therapy to help combat cardiovascular disorders linked to thrombosis. This study introduces new thienopyridine derivatives with superior therapeutic efficacy through synergies by dual functional compounds, developed as antithrombotic agents. Through computational design, we discovered 32 compounds out of a library of 100 compounds that exhibit efficacy. The compounds are synthesized by multiple steps and subsequently purified using chromatographic procedures to achieve a purity level of >99%. Using the ADP‐induced platelet aggregation assay, we have found 4 active test compounds (ACG‐0173‐19, ACG‐0173‐37, ACG‐A‐04, and ACG‐B‐03) out of a total of 32 compounds. On the other hand, ACG‐0173‐19, ACG‐0173‐37, ACG‐A‐04, and ACG‐B‐03 have demonstrated the highest percentage activity on factor Xa (FXa) inhibition. The ACG‐A‐04 chemical exhibited the highest hydrophilicity, specifically in terms of its aqueous solubility. It had a solubility of 0.44 mg/mL in pH 5.8, 0.29 mg/L in pH 6.2, and 0.145 mg/L in pH 7.4 buffers. In comparison, the standards APX‐01 (apixaban) and PRG‐01(prasugrel) had lower hydrophilicity. Based on cytotoxicity investigations, it was determined that the test substances do not exhibit harmful effects. The results indicated that the test chemicals can undergo hydrolysis in blood plasma rather than in other organs. Among the four test drugs, ACG‐1073‐19 exhibited an unbound fraction of 44.1%, which is twice as high as the standards (apixaban). ACG‐1073‐37 demonstrates superior plasma protein binding (33.2%) compared to all other test compounds. The compounds resulted in a higher expression of CD61, CD42b, and CD62P in platelets compared to the control. The ACG‐1073‐37 molecule had a Caco‐2 permeability of 51%, which is extremely close to the Caco‐2 permeability of the control medicines (55% for APX‐01 and 62% for PRG‐01). Through the assessment of microsomal stability, it was determined that ACG‐1073‐37 and ACG‐A‐04 exhibited metabolic stability for 42.4 and 34.14 min, respectively.

Comparison of the Characteristics of Circulating Small Extracellular Vesicles Isolated by Ultracentrifugation and a Commercial Kit.

The market offers a wide range of extracellular vesicles (EVs) isolation products, but their lack of standardization is a concern. Therefore, it is important to carefully assess the quality of the EVs obtained using these products. In this study, we compared the EXOCIB kit with the ultracentrifuge method, which is considered the gold standard for small EV isolation. After overnight fasting, small plasma EVs were extracted from four individuals using both the ultracentrifuge and the EXOCIB kit methods. The pooled EVs were then compared for the presence of the cluster of differentiation 63 (CD63) protein using the western blot analysis, and their size and zeta potential were performed by Dynamic Light Scattering (DLS). In addition, the size and morphology of small EVs were determined by using the Transmission Electron Microscopy (TEM) technique. An average hydrodynamic size of 135.7 nm and a zeta potential of -6.33 Mv at 25°C was found for small EVs isolated by the ultracentrifuge, whereas the kit method resulted in small EVs with a hydrodynamic size of 102.8 nm and a zeta potential of -0.907. Notably, the size of the particles in the kit samples was smaller compared to those obtained through the ultracentrifuge (P < 0.001). The western blot method confirmed the expression of CD63 in both methods, so the ultracentrifuge yielded small EVs with a higher level of purity compared to the kit-based approach (P = 0.036). The DLS findings revealed the existence of vesicles within the appropriate size range for small EVs like exosomes in both isolation techniques. The results of the western blot analysis, in conjunction with DLS, displayed that the ultracentrifuge method extracted small EVs with a greater degree of purity than the kit-based approach.

CD64 Expression on Neutrophils (nCD64) as a Biomarker in Adult Patients With Sepsis: A Cross-Sectional Study.

Introduction Neutrophils that are at rest exhibit very low expression of CD64, the high-affinity immunoglobulin fragment crystallizable γ receptor I, which is also found in monocytes. Neutrophils that have been exposed to endotoxins or are infected express more CD64. Objectives The aim of this study was to assess the CD64 biomarker expression on neutrophils using the flow cytometry method and its comparison with total leukocyte count, C-reactive protein (CRP) level, and blood culture sensitivity test in the diagnosis of sepsis in adults. Materials and methods Using the quick Sequential Organ Failure Assessment (qSOFA) scoring criteria, 94 blood samples from individuals with clinical indications of sepsis were included in this investigation. Samples were collected in K2 EDTA (ethylenediaminetetraacetic acid) vacutainers and analyzed for neutrophil CD64 (nCD64) levels using BD FACSLyricTM flow cytometer (BD, Franklin Lakes, NJ) within 24 hours of collection. Total leukocyte count, CRP levels, and blood culture sensitivity tests were also assessed simultaneously. Results Majority of the patients, i.e., 29 (30.9%), belonged to the age group of 19 to 35 years, with a female preponderance. The mean total leukocyte count was 19.49 ± 8.12 x 103/µL, mean CRP level was 81.19 ± 56.33 mg/dL, and the mean nCD64 expression - median fluorescence intensity was 197.26 ± 79.56. A positive blood culture was found in 59 (62.8%) cases. Neutrophils showed bright expression of nCD64 in 39 (41.5%) patients, dim expression in 35 (37.2%) patients, and moderate expression in 20 (21.3%) patients in the present study. The correlation between nCD64 expression with CRP, total leukocyte count, and blood culture sensitivity test was statistically significant (p=0.001). nCD64 expression showed a sensitivity of 93.2% and specificity of 91.4% for diagnosing sepsis, with an area under the curve (AUC) of 0.973. This proves nCD64 to be a highly effective biomarker compared to conventional methods. Conclusion nCD64 has a higher sensitivity and specificity as compared to total leukocyte count, CRP levels, and blood culture sensitivity test in the diagnosis of sepsis. It is an effective and rapid test that can enhance sepsis diagnostic accuracy, when combined with other biomarkers. Multicentric research needs to be conducted to validate these findings.

Biallelic PIGM Coding Variant Causes Intractable Epilepsy and Intellectual Disability Without Thrombotic Events.

During the past two decades, an emerging group of genes coding for proteins involved in glycosylphosphatidylinositol (GPI) anchor biosynthesis are being implicated in early-infantile epileptic encephalopathy. Amongst these, a hypomorphic promoter mutation in the mannosyltransferase-encoding PIGM gene was described in seven patients to date, exhibiting intractable absence epilepsy, portal and cerebral vein thrombosis and intellectual disability (ID). We describe here three siblings exhibiting intractable epilepsy and ID, found to harbor a homozygous c.224G>A p.(Arg75His) missense variant in PIGM, which segregated with the disease in the family. The variant is evolutionary conserved, extremely rare in general population databases and predicted to be deleterious. Structural modeling of the PIGM protein and the p.(Arg75His) variant indicates that it is located in a short luminal region of the protein, predicted to be hydrophilic. Functional prediction suggests that the entire local region is sensitive to mutations, with the p.(Arg75His) variant in particular. This is the first report of a PIGM coding variant, and the second variant altogether to be described affecting this gene. This phenotype differs from that of patients with the shared PIGM promoter mutation by lack of thrombotic events and no decrease in PIGM cDNA levels or CD59 expression on red blood cells.

Role of Tumor Microenvironment in the Risk of Thromboembolic Complications in Ovarian Cancer

Introduction. Incidence of ovarian cancer remains high in the overall prevalence of oncological pathology. Adjuvant chemotherapy refers to its treatment options. Patients with oncological pathology are faced with a high risk of thrombosis and thromboembolism, with up to 30% lethal outcome within a month of its development. A number of cancer cells are known to induce platelet aggregation, contributing to thrombosis and metastasis as a result of this interaction. Accordingly, the paper is aimed at presenting a clinical case for demonstrating the role of P-selectin expression in the complications in a patient with ovarian cancer. Materials and methods. The present paper evaluates platelet activation marker in a patient undergoing chemotherapy courses after cytoreductive surgery. Following the case conference and in accordance with the clinical recommendations of the Russian Oncology Association (AOR) and Russian Society of Clinical Oncology (RUSSCO), cytoreduction (CC-0), radical hysterectomy, transverse colectomy, left hemicolectomy with rectum resection were performed. The interventions included ascendostomy, pelvic, lateral right-sided and left-sided peritonectomy, pelvic lymphoadenectomy, total omentectomy, Renape-French HIPEC (hyperthermic intraperitoneal chemotherapy), abdominal and pelvic drainage. Expression of P-selectin on the platelet surface was measured as a marker of platelet activation. Results and discussion. At the time of admission, the patient had high CD62 expression activity compared to healthy volunteers (CD62 ADP- — 11.2%, CD62 ADP — 24.7% vs CD62 ADP- — 1.3%, CD62 ADP — 17.2%). During the complex treatment of ovarian cancer, the platelet activation increased (CD62 ADP- — 21.8 %, CD62 ADP+ — 30.1 %). At discharge, CD62 expression values reached the conditional norm, presumably indicating thrombosis development. Conclusion. Tumor microenvironment influences the hemostasis system. Detailed study into this issue obtains a high potential for the prevention of primary and secondary thromboembolic complications in oncologic patients.

Rs2564978(T) allele associated with severe influenza a disrupts binding site for myeloid differentiation factor PU.1 and reduces &lt;i&gt;CD55/DAF&lt;/i&gt; gene promoter activity in macrophages

An inhibitor of the complement system CD55/DAF is expressed on many cell types. Dysregulation of CD55 expression is associated with increased disease severity during influenza A infection, as well as with vascular complications in pathologies involving excessive activation of the complement system. Using a luciferase reporter system, we performed functional analysis of the single nucleotide polymorphism rs2564978 located in the promoter of the CD55 gene in human pro-monocytic cell line U937. We have shown a decreased activity in activated U937 cells of the CD55 gene promoter carrying minor rs2564978(T) allele associated with the severe course of influenza A(H1N1)pdm09. Using bioinformatic resources, we determined that transcription factor PU.1 can potentially bind to the CD55 promoter region containing rs2564978 in an allele-specific manner. The involvement of PU.1 in modulating CD55 promoter activity was determined by genetic knockdown of PU.1 using small interfering RNAs under specific monocyte activation conditions.

Neutrophil Diversity (Immature, Aged, and Low-Density Neutrophils) and Functional Plasticity: Possible Impacts of Iron Overload in β-Thalassemia.

Neutrophil dysfunction is a form of immune suppression in patients with β-thalassemia (Beta-thal), although data on this are limited. In this study, blood from patients and healthy volunteers was analyzed. Flow cytometry analysis demonstrated an increase in immature neutrophils (CD16- CD62L+) and aged (senescent) neutrophils (CD16+ CD62L-) in Beta-thal patients compared to healthy volunteers. The Beta-thal neutrophils demonstrated less prominent chemotaxis and phagocytosis than healthy neutrophils at the baseline. With phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) stimulations, some of the indicators, including the flow cytometry markers (CD11b, CD62L, CD66b, CD63, apoptosis, and reactive oxygen species) and neutrophil extracellular traps (NETs; detected by anti-citrullinated histone 3 immunofluorescence), were lower than the control. Additionally, low-density neutrophils (LDNs), which are found in the peripheral blood mononuclear cell (PBMC) fraction, were observed in Beta-thal patients but not in the control group. The expression of CD11b, CD66b, CD63, arginase I, and ROS in LDNs was higher than the regular normal-density neutrophils (NDNs). The proliferation rate of CD3+ T cells isolated from the PBMC fraction of healthy volunteers was higher than that of the cells from patients with Beta-thal. The incubation of red blood cell (RBC) lysate plus ferric ions with healthy NDNs transformed the NDNs into the aged neutrophils (decreased CD62L) and LDNs. In conclusion, iron overload induces neutrophil diversity along with some dysfunctions.

BMSCs-derived exosomes carrying miR-668-3p promote progression of osteoblasts in osteonecrosis of the femoral head: Expression of proteins CD63 and CD9

Recently, exosomes that are derived from bone marrow mesenchymal stem cells (BMSCs) have garnered considerable interest due to their significant roles in the processes of bone regeneration and repair. Among the various molecular components present within these exosomes, miR-668-3p has emerged as a pivotal microRNA that may be instrumental in modulating the function and proliferation of osteoblasts, the cells responsible for bone formation. The primary objective of this research was to examine the enhancing effects of BMSC-derived exosomes that are enriched with miR-668-3p on the advancement of osteoblasts in the context of osteonecrosis of the femoral head. Furthermore, the study aimed to analyze how the expression of specific exosomal proteins, namely CD63 and CD9, influences this biological process. To conduct the investigation, BMSCs were isolated from healthy rat models, followed by the extraction of their secreted exosomes. The subsequent phase of the study involved assessing the proliferation and differentiation of osteoblasts by introducing the exosomes enriched with miR-668-3p into an experimental setup representing osteonecrosis of the femoral head. The findings revealed that exosomes derived from BMSCs, which contained miR-668-3p, significantly enhanced the proliferation of osteoblasts as well as the expression of key osteogenic marker genes. Notably, the levels of CD63 and CD9 proteins were markedly increased in the treated groups, indicating that the mechanisms underlying this promotion might involve cell adhesion and the endocytic uptake of exosomes.

SARS-CoV-2 ORF3a Protein Impairs Syncytiotrophoblast Maturation, Alters ZO-1 Localization, and Shifts Autophagic Pathways in Trophoblast Cells and 3D Organoids.

SARS-CoV-2 infection poses a significant risk to placental physiology, but its impact on placental homeostasis is not well understood. We and others have previously shown that SARS-CoV-2 can colonize maternal and fetal placental cells, yet the specific mechanisms remain unclear. In this study, we investigate ORF3a, a key accessory protein of SARS-CoV-2 that exhibits continuous mutations. Our findings reveal that ORF3a is present in placental tissue from pregnant women infected with SARS-CoV-2 and disrupts autophagic flux in placental cell lines and 3D stem-cell-derived trophoblast organoids (SC-TOs), impairing syncytiotrophoblast differentiation and trophoblast invasion. This disruption leads to protein aggregation in cytotrophoblasts (CTB) and activates secretory autophagy, increasing CD63+ extracellular vesicle secretion, along with ORF3a itself. ORF3a also compromises CTB barrier integrity by disrupting tight junctions via interaction with ZO-1, mediated by its PDZ-binding motif, SVPL. Co-localization of ORF3a and ZO-1 in SARS-CoV-2-infected human placental tissue supports our in vitro findings. Deleting the PDZ binding motif in the ORF3a protein (ORF3a-noPBM mutant) restored proper ZO-1 localization at the cell junctions in an autophagy-independent manner. Lastly, we demonstrate that constitutive ORF3a expression induces SC-TOs to transition towards a secretory autophagy pathway likely via the PBM motif, as the ORF3a-NoPBM mutants showed a significant lack of CD63 expression. This study demonstrates the functional impact of ORF3a on placental autophagy and reveals a new mechanism for the activation of secretory autophagy, which may lead to increased extracellular vesicle secretion. These findings provide a foundation for exploring therapeutic approaches targeting ORF3a, specifically focusing on its PBM region to block its interactions with host cellular proteins and limiting placental impact.

Low CD46 expression on activated CD4+ T cells predict improved Th1 cell reactivity to calcitriol in majority of patients with allergic eosinophilic asthma and healthy donors.

Previous research showed that the intracellular complement system, with CD46 as its central molecule, regulates the Th1 response associated with IFN-γ production and transition to a type 1 regulatory response (Tr1) characterized by IL-10 production. This transition can be influenced by a vitamin D (calcitriol), favouring a shift towards Tr1 cells and increased IL-10 production, as described in some autoimmune diseases. It is unknown whether calcitriol modulates CD46-induced Th1 response towards regulatory type 1 T cells (Tr1) in allergic eosinophilic asthma and its value in relation to reducing inflammatory response. CD4+ T cells from 58 patients with allergic eosinophilic asthma (AEA) and 49 healthy donors (HDs) were stimulated with αCD3/αCD46/IL-2 or αCD3/αCD46/IL-2/Calcitriol in vitro for 60 h and analyzed by flow cytometry. IFN-γ and IL-10 levels in cell culture supernatants were measured using ELISA. CD4+ T cells from patients with AEA demonstrated elevated CD46 expression in both the non-activated state and under stimulation conditions with αCD3/αCD46/IL-2 or αCD3/αCD46/IL-2/Calcitriol. Moreover, CD46 expression in AEA patients fluctuated with the pollen season, showing a significant increase during period of low pollen exposure. Calcitriol further induced CD4+Tr1 cells from in vitro generated CD4+Th1 cells in both HDs and AEA patients. However, in both cohorts were individuals (HDs: 35/49, AEA: 40/58) who responded to calcitriol with a more pronounced regulatory response. The calcitriol-induced regulatory effect manifested by a stronger surface decrease of CD46 on activated CD4+ T cells (by 40% in HDs and by 26% in AEA), accompanied by a significant inhibition of IFN-γ and increased IL-10 production (by 31% in HDs and by 85% in AEA). These individuals were termed as the CD46D group. Contrary to this, calcitriol induced an increase in CD46 expression at the CD4+ T cell surface in a minor group of HDs (14/49), and AEA patients (18/58), who were termed as the CD46I group. In CD46I group, CD4+ T cells produced less IFN-γ in comparison with CD46D group (by 33% in HDs and by 43% in AEA) and were unable to upregulate IL-10 production following stimulation with αCD3/αCD46/IL-2/Calcitriol. Our results suggest the potential existence of a key for stratifying individuals suitable for calcitriol treatment in the context of low serum vitamin D levels. After validation in clinical studies, this key could be used as an adjunctive therapy not only for patients with allergic eosinophilic asthma, but also for other diseases.

The Study of Pro-Inflammatory Molecules Induced by Genetically and Phenotypically Diverse Strains of Haemophilus influenzae Type a in an in vitro Infection Model

Introduction: Haemophilus influenzae type a (Hia) has recently emerged as a cause of invasive disease in North American Indigenous children. Factors determining the outcomes of exposure to the pathogen, from asymptomatic carriage to fatal disease, are poorly understood. The role of innate immune activation in the pathogenesis of invasive Hia disease remains unexplored. We used clinical Hia isolates to determine whether innate immune responses depended on the presence of the capsule, strain genetic background, and abilities to cause invasive disease. Methods: Differentiated THP-1 cells and HL-60 neutrophil-like cells were stimulated with four Hia strains (invasive or noninvasive; encapsulated or nonencapsulated), in comparison to 1 invasive and 1 noninvasive non-typeable H. influenzae. Surface expression of ICAM-1 and CD64 and release of pro-inflammatory cytokines TNF-α and IL-1β were quantified. Results: In vitro Hia infection resulted in robust activation of inflammatory responses in terms of expression of ICAM-1 and release of TNF-α and IL-1β, irrespective of the presence or absence of the capsule, or abilities to cause invasive disease. Inhibition of TLR4 decreased TNF-α release by THP-1 cells stimulated by Hia. Conclusion: Powerful activation of pro-inflammatory responses induced by Hia may contribute to the pathogenesis of invasive Hia disease. As the activation of macrophages and neutrophils did not depend on encapsulation or source of Hia isolation, the functional abilities of phagocytic cells unlikely represent a limiting factor in host defenses. The development of invasive versus noninvasive disease may depend on the functional abilities of the adaptive immune system.