Cluster Of Differentiation Antigens Research Articles

Exploration for sorting serum exosome as a measure of spinal cord injury severity

This study aimed to extract exosomes from the serum of rats with spinal cord injury and explore their potential as indicators for assessing the severity of spinal cord injury. Control rats were compared with rats in the mild and severe injury groups. Exosomes were isolated from rat serum using an optimized threshold condition with an ultra-high-speed flow cell sorter. Nanoparticle tracking analysis (NTA) was performed to determine the size distribution of exosomes, transmission electron microscopy (TEM) was used to observe the morphology of exosomes, and Western blot analysis was conducted to assess the expression of CD63(cluster of differentiation antigen 63) and CD9(cluster of differentiation antigen 9) proteins on the surface of exosomes. NanoFlow cytometry was utilized to evaluate the expression of CD63 and CD9. The optimal threshold condition for flow cytometry of exosomes was determined to be 0.002. Exosomes exhibited a cup-shaped morphology with a clean background. The percentages of exosomes in the control, mild injury, and severe injury groups were determined to be 6.4 × 1011 particles/mL, 6.2 × 1011 particles/mL, and 2.3 × 1011 particles/mL, respectively. The mean sizes of exosomes were 129.25 nm ± 18.3, 117.93 nm ± 39.9, and 116.38 nm ± 36.5, respectively. The percentages of CD9 were 15.5% ± 0.037, 10.4% ± 0.082, and 7.4% ± 0.051, while the percentages of CD63 were 1.2% ± 0.025, 2.6% ± 0.067, and 1.5% ± 0.038 for the control, mild injury, and severe injury groups, respectively. Comparison analysis revealed that the diameter of exosomes in rat serum ranged from 80 nm to 150 nm. The percentage of exosomes decreased, and CD9 expression significantly decreased as the severity of spinal cord injury increased. Flow cytometry allows for the isolation of exosomes from serum with high purity and uniform particle size. The percentage of serum exosomes and the expression of CD9 may serve as potential diagnostic indicators for spinal cord injury models.

Function and autophagy of monocyte-derived dendritic cells is affected by hepatitis B virus infection

BackgroundThe role of dendritic cells and the autophagy state of dendritic cells in the immune response of hepatitis B virus (HBV) infection was still controversial. In this study, we carefully examined the phenotype, function and autophagy pathway of dendritic cells in HBV infection.MethodsMonocyte-derived dendritic cells from healthy blood donors and patients with chronic HBV infection were stimulated by lipopolysaccharide, supernatant of HepG2.2.15 cells or supernatant of HepG2 cells respectively. Phenotype of dendritic cells was examined by flow cytometry and cytokines secretion was detected by enzyme-linked immunosorbent assay. Autophagy related proteins were detected by western blot and immunofluorescence analysis.ResultsOur results showed that the expression of both major histocompatibility complex II molecules and co-stimulated molecules including cluster of differentiation antigen 80, cluster of differentiation antigen 86 in the monocyte-derived dendritic cells from patients with chronic HBV infection was significantly higher than that from healthy donors when cultured with supernatant of HepG2.2.15 cells. The amount of cytokines, including tumour necrosis factor-α, interleukin-10 and interleukin-12, secreted by monocyte-derived dendritic cells from patients with chronic HBV infection was also significantly higher than that from healthy donors when stimulate by HBV. Interestingly, the expression level of autophagy-related proteins including autophagy-related protein5 and associated protein 1 light chain in dendritic cells from patients with chronic HBV infection was significantly increased when compared with that from healthy donors when re-exposed to HBV.ConclusionsOur results indicated that dendritic cells from patients with chronic HBV infection could intensively present antigen and express co-stimulatory molecules. The increased activation of dendritic cells might be related to the enhanced autophagy of dendritic cells in HBV infection.

Host CCR5 and HIV-1 env/pol polymorphisms: Role in transmission and virulence in the Kenyan population


 INTRODUCTION 
 HIV-1 env protein and host cluster of differentiation antigen 4 (CD4) and wild-CC chemokine receptor 5 (CCR5) proteins are responsible for the attachment and entry of HIV-1 into T lymphocytes, hence influencing its infectivity. However, information on the flow, distribution, ARVs resistance patterns and co-infections of the HIV-1 sub-types is limited. An up-to-date comprehensive review on the role of CCR5/CD4 and env/pol protein polymorphisms in the susceptibility to HIV-1 infection, virulence, transmission, disease progression and discordance in the Kenyan population is lacking. This study aimed to review the role of CCR5/CD4 and HIV-1 env/pol protein polymorphisms in the transmission and virulence of HIV-1 in the Kenyan population. 
 METHODOLOGY 
 Data was sourced by literature search on Google Scholar, PubMed, and ScienceDirect. Grey literature articles were manually reviewed. Study selection was based on relevance to the role of CCR5/CD4 and env/pol protein polymorphisms in the transmission and virulence of HIV-1 were included in the review. Data were extracted from individual studies or articles. Data were synthesized regarding the information on the role of CCR5/CD4 and env/pol protein polymorphisms in the transmission and virulence of HIV-1 is synchronized under the headings; introduction, molecular characterization of HIV-1, HIV-1 treatment and antiretrovirals (ARVs) resistance, influence of host genes on HIV-1 disease progression, CD4 as HIV-1 receptors, and characterization of genetic/antigenic variants circulating in a given population. 
 RESULTS 
 A total of 74 articles were screened and 66 eligible articles were reviewed, four (4) of them touching on the origin and evolutionary mechanisms of the human immunodeficiency virus (HIV), while 21 articles addressed molecular characterization of the HIV-1. A total of 11 articles addressed HIV-1 treatment and ARVs resistance whereas 13 articles addressed the influence of host genes on HIV/AIDS disease progression. Eleven (11) and 8 articles addressed the CD4 as HIV-1 receptors and characterization of HIV-1 genetic/antigenic variants circulating in a given population, respectively. 
 CONCLUSION 
 There are many HIV-1 subtypes in circulation in Kenya with uneven distribution and it is difficult to accurately predict future metamorphosis and determine and monitor the flow and ARVs resistance patterns of the sub-types. mutant allele of the β-chemokine receptor gene CCR5 prevents cell invasion and homozygotes for this mutation are resistant to infection with heterozygosity at this gene correlating with slowed HIV/AIDS disease progression. The C868T single nucleotide polymorphism (SNP) in CD4 increases the risk of HIV-1 acquisition. 
 RECOMMENDATIONS 
 There is a need for accurate prediction of the future metamorphosis and regular evaluation and monitoring of the flow, distribution, ARVs resistance patterns and co-infections of the HIV-1 sub-types and their variation over time. Detailed assessment of the role of CCR5/CD4 and env/pol protein polymorphisms in the susceptibility to HIV-1 infection, virulence, disease progression and discordance in some couples in the Kenyan population is important.

Revisiting the Role of CD63 as Pro-Tumorigenic or Anti-Tumorigenic Tetraspanin in Cancers and its Theragnostic Implications.

Cluster of differentiation antigen 63 (CD63) belongs to a superfamily of proteins, usually defined as tetraspanins which are known to transverse the bilayer membranes four times. The expression of CD63 has been shown to get altered in several cancers, where it has been demonstrated to act as both a tumor promoter and tumor suppressor. The present review describes the mechanism of how CD63 promotes tumor formation in certain cancer types while inhibiting in some other specific cancers. Glycosylation, a post-translational process plays a significant role in regulating the expression and function of these membrane proteins. Being a crucial exosomal flag protein, CD63 has been found to get involved in endosomal cargo sorting as well as the production of extracellular vesicles. Increased expression of exosomal CD63 derived from advanced tumors has demonstrated its role in promoting metastasis. CD63 also regulates the characteristic and function of stem cells on which they get expressed. This particular tetraspanin has been discovered to participate in gene fusion to perform distinctive roles in certain specific cancer types like breast cancer and pigmented epithelioid melanocytoma. Furthermore, this review mentions twelve different microRNAs obtained from miRDB that might target CD63. A few theragnostic uses of this membrane protein are also discussed. Thereby, the review indicates that further studies on CD63 might prove it to be an effective therapeutic target in different cancers in the coming future.

Immunophenotyping of Gastritis, Gastric Ulcer and Gastric Cancer using a Cluster of Differentiation (CD) Antibody Microarray

One of the factors that contribute to the development of gastric cancer is the host immune response. Extensive immunophenotype of gastric cancer can be identified by using antibody microarray technique that profiles more than 100 cluster of differentiation (CD) antigens in parallel. In this study, we used DotScanTM antibody microarray to profile CD antigen expression in patients with distinct digestive diseases for surface antigen disease-signatures. Patients’ blood samples with gastric disorders and healthy controls were taken and processed for leukocytes isolation using Histopaque density gradient centrifugation. Leukocytes were captured onto DotScanTM slides and cell binding densities were analyzed using DotReaderTM. Different groups of gastric diseases were found to be characterized by differentially expressed distinct CD antigens. Compared to normal healthy controls, 17, two and four highly expressed CD antigens were identified in gastritis, gastric ulcer and gastric cancer patients, respectively. The 17 CD antigens that show differential expression in gastritis were CD15, CD16, CD20, CD23, CD24, CD25, CD28, CD34, CD37, CD77, CD102, CD103, CD122, CD128, CD10, CD183, and CD184. High expression of CD64 and CD69 were found in gastric ulcer, whereas CD52, CD126, CD135, and CD121a were highly expressed in gastric cancer. CD antigens involve in T-cell functions had reduced expression in gastric cancer, while proinflammatory cytokines shows increased expression. These results demonstrate specific immunophenotype of CD antigens in patients with various gastric diseases and identification of differential expressed surface antigens may have clinical significance for diagnostic and therapeutic purposes.

The roles of BTG1 mRNA expression in cancers: A bioinformatics analysis.

BTG1 (B-cell translocation gene 1) may inhibit proliferation and cell cycle progression, induce differentiation, apoptosis, and anti-inflammatory activity. The goal of this study was to clarify the clinicopathological and prognostic significances of BTG1 mRNA expression and related signal pathways in cancers. Using the Oncomine, TCGA (the cancer genome atlas), xiantao, UALCAN (The University of ALabama at Birmingham Cancer data analysis Portal), and Kaplan-Meier plotter databases, we undertook a bioinformatics study of BTG1 mRNA expression in cancers. BTG1 expression was lower in gastric, lung, breast and ovarian cancer than normal tissue due to its promoter methylation, which was the opposite to BTG1 expression. BTG1 expression was positively correlated with dedifferentiation and histological grading of gastric cancer (p < 0.05), with squamous subtype and young age of lung cancer (p < 0.05), with infrequent lymph node metastasis, low TNM staging, young age, white race, infiltrative lobular subtype, Her2 negativity, favorable molecular subtyping, and no postmenopause status of breast cancer (p < 0.05), and with elder age, venous invasion, lymphatic invasion, and clinicopathological staging of ovarian cancer (p < 0.05). BTG1 expression was negatively correlated with favorable prognosis of gastric, lung or ovarian cancer patients, but the converse was true for breast cancer (p < 0.05). KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis showed that the top signal pathways included cytokine-cytokine receptor interaction, cell adhesion molecules, chemokine, immune cell receptor and NF (nuclear factor)-κB signal pathways in gastric and breast cancer. The top hub genes mainly contained CD (cluster of differentiation) antigens in gastric cancer, FGF (fibroblast growth factor)-FGFR (FGF receptor) in lung cancer, NADH (nicotinamide adenine dinucleotide): ubiquinone oxidoreductase in breast cancer, and ribosomal proteins in ovarian cancer. BTG1 expression might be employed as a potential marker to indicate carcinogenesis and subsequent progression, even prognosis.

New insight on the role of Helicobacter pylori cagA in the expression of cell surface antigens with important biological functions in gastric carcinogenesis.

Expression of cluster of differentiation (CD) antigens changes according to disease status and inflammation. Profiles of CD antigens expression in gastric cancer patients are different based on the status of H.pylori infection. We conducted this study to profile CD antigen markers in gastric adenocarcinoma cells (AGS cell line) infected with distinct cytotoxin-associated gene A (cagA) genotypes of H.pylori clinical isolates. The AGS cells were infected with H.pylori isolates with different cagA genotypes, and CD antigens expression was determined using DotScan™ antibody microarray. Formation of "hummingbird" phenotype was determined, and the percentage was calculated. H.pylori strains harboring cagA upregulated the expression of CD antigen involved in cancer stem cell formation (CD55), but downregulated CD antigens involved in immune regulation (CD40 and CD186) and cell adhesion (CD44). CD54 (neutrophil adhesion) and CD71 (iron transfer) were highly downregulated in the gastric cells infected with Western cagA isolates compared with East Asian isolates. CD antigen expression was different in the cells infected with H.pylori harboring different CagA EPIYA (Glu-Pro-Ile-Tyr-Ala) numbers, in which higher repression of CD54 and CD15 (Lewis x antigen) were observed in the isolate with the highest number of EPIYA motif. Furthermore, higher downregulation of CD15 was observed in the infected gastric cells with high percentage of "hummingbird" phenotype than that of low percentage of "hummingbird" phenotype. Our study demonstrated the critical roles of CD antigens in the CagA pathogenesis and should be investigated further.

Relation between FMS-like tyrosine kinase 3 factor and hematological parameter in acute lymphoblastic leukemia patients by flow cytometry

BACKGROUND: Acute lymphoblastic leukemia (ALL) is a heterogeneous disorder that is caused by the clonal expansion of immature lymphoid cells with a high rate among children more than adults. FMS-like tyrosine kinase 3 (FLT3) is a cellular receptor belongs to the Class III receptor tyrosine kinase family. The main expression of FLT3 on bone marrow (BM) cells especially CD34+ hematopoietic stem cells, early progenitor cells, dendritic progenitor cells, and other cells of organs (brain, placenta, and testis). Activation of FLT3 results in increased cell proliferation, decreased cell apoptosis, and inhibition of differentiation of cells. This study aims to detect the expression of the FLT3 cluster of differentiation antigen 135 (CD135) in childhood B-ALL patients. Moreover, to correlate this expression with hematological parameters include a complete blood count and BM examination findings and clinical parameters. PATIENTS, MATERIALS AND METHODS: This study was conducted on 30 newly diagnosed pediatric ALL patients. Diagnosis of the disease was based on the blood film, BM examination findings, cytochemistry, and flowcytometry of peripheral blood (PB) and/or BM sample, 1 ml of PB and/or BM sample was collected in EDTA tubes for flowcytometry for detection of CD135. RESULTS: This study found that male patients were more than females with a male-to-female ratio (1.14:1) and a median age of 5 years. Most of the patients had a positive expression of the FLT3 receptor and according to NCI risk groups, 60% of patients fall in the standard risk and 40% in the high-risk group. There was a significant correlation between the level of FLT3 (CD135) and age but no significant correlation with hemoglobin, white blood count, platelets, and peripheral or BM blast percentage. CONCLUSION: In this study, the patients with positive FLT3 blast cells (which is a bad prognostic factor) were associated with good prognostic factors. This proves that FLT3 is an independent prognostic factor.

The prognostic significance of bone marrow histological evaluation in patients with multiple myeloma

Introduction: Recently, there has been enormous progress in the diagnosis and treatment of multiple myeloma (MM). Despite the importance of flow cytometry and cytogenetic, biomarkers are being sought to assess prognosis and response to treatment. The aim of this study was to determine the significance of histological evaluation and immunohistochemical markers in MM patients. Material and methods: A retrospective analysis was performed in 90 bone marrow (BM) biopsies collected during the diagnosis period and related to the recognized prognostic factors and response to treatment. The standardized staining methods and monoclonal antibodies were used to detect cluster of differentiation antigens — CD138, CD56, CD20, CD117, cyclin D1, p53, kappa and lambda light chains. Results: During the follow-up period of 49.6 (2–119.7) months in patients with increased levels of β 2 -microglobulin, higher International Prognostic Classification stage, with resistance to induction and higher relapse rate after first-line, a clonal lambda plasmocytes infiltration was observed. It also affected the response to induction with immunomodulatory drugs (IMiDs), but not bortezomib. The lowest response rate to IMiDs was noted in patients with CD117-lambda+ and CD56-lambda+ plasma cells. Clonal lambda hyperplasia connected with reduction in overall survival [hazard ratio (HR) 2.4; 95% confidence interval (CI): 0.96–5.92; p =0.047) and progression-free survival (PFS) (HR 4,32, 95% CI: 1.9–9.5; p =0.0002). The presence of plasmocytes with phenotype CD117-lambda+ (HR 3.37; 95% CI: 1.57–7.24; p =0.002) and CD56-lambda+ (HR 3,44; 95% CI: 1,63–7,27; p =0.001) contributed to PFS shortening. Conclusions: In conclusions, BM biopsy with immunohistochemistry allow the identification of poorly prognostic patients with MM.

Rapid Enzyme-Mediated Biotinylation for Cell Surface Proteome Profiling.

Cell surface is the primary site for sensing extracellular stimuli. The knowledge of the transient changes on the surfaceome upon a perturbation is very important as the initial changed proteins could be driving molecules for some phenotype. In this study, we report a fast cell surface labeling strategy based on peroxidase-mediated oxidative tyrosine coupling strategy, enabling efficient and selective cell surface labeling within seconds. With a labeling time of 1 min, 2684 proteins, including 1370 (51%) cell surface-annotated proteins (cell surface/plasma membrane/extracellular), 732 transmembrane proteins, and 81 cluster of differentiation antigens, were identified from HeLa cells. By comparison with the negative control experiment using quantitative proteomics, 500 (68%) out of the 731 significantly enriched proteins (p-value < 0.05, ≥2-fold) in positive experimental samples were cell surface-annotated proteins. Finally, this technology was applied to track the dynamic changes of the surfaceome upon insulin stimulation at two time points (5 min and 2 h) in HepG2 cells. Thirty-two proteins, including INSR, CTNNB1, TFRC, IGF2R, and SORT1, were found to be significantly regulated (p-value < 0.01, ≥1.5-fold) after insulin exposure by different mechanisms. We envision that this technique could be a powerful tool to analyze the transient changes of the surfaceome with a good time resolution and to delineate the temporal and spatial regulation of cellular signaling.

Risk factors of severe heart failure in HIV-positive patients

Aim . To study the relationship of N-terminal pro-brain natriuretic peptide (NT-proBNP) with clinical symptoms, structural and functional cardiac abnormalities in human immunodeficiency virus (HIV)-positive patients with heart failure (HF), as well as to identify risk factors for severe HF. Material and methods . During the year, 150 HIV-positive patients with typical symptoms and signs of stable HF were examined in the hospital. Among them, HF, confirmed by structural and functional cardiac changes and NT-proBNP increase above 125 pg/ml, was identified in 83 (55,3%) patients. These patients were divided into 3 groups depending on the blood concentration of NT-proBNP: group 1 (n=54) — patients with NT-proBNP of 125-700 pg/ml; group 2 (n=12) — patients with NT-proBNP of 701-1500 pg/ml; group 3 (n=17) — patients with NT-proBNP &gt;1500 pg/ml. Results . As NT-proBNP increased in HIV-positive patients with HF, the left ventricular (LV) ejection fraction (EF) significantly decreased (p=0,005). Also, the increase in the severity of HF symptoms (p&lt;0,001), prevalence of chronic kidney disease (CKD) (p&lt;0,001), chronic hepatitis B and/or C (p=0,011), prior infective endocarditis (p=0,002), thromboembolic events (p=0,007), chronic obstructive pulmonary disease (p=0,016), pneumonia (p=0,002) and inflammatory diseases during hospitalization (p=0,002), severe thrombocytopenia (p=0,032). We revealed significant differences between the groups in the frequency of decreased cluster of differentiation antigen 4 (CD4) &lt;200 cells/pl (p=0,013). Conclusion. In HIV-positive patients with clinical symptoms of HF, the prevalence of its verification in accordance with Russian guidelines (2020) was 55,3%. In 62,7% of HIV-positive patients with HF, preserved EF was detected. Among the comorbidities, 9 risk factors of severe HF in HIV-positive patients with NT-proBNP &gt;1500 pg/ml were identified. With CKD and inflammatory diseases during hospitalization, the relative risk of severe HF in patients with HIV infection is increased by more than 6 times, while with thromboembolic events — 5,3 times, infective endocarditis — 4,4 times, pneumonia during hospitalization and severe thrombocytopenia — more than 3,5 times, chronic obstructive pulmonary disease — 2,1 times, chronic hepatitis B and/or C — 1,7 times. As HIV infection progresses (CD4 &lt;200 cells/pL), the risk of severe HF increases 1,6 times.

The Selenoprotein MsrB1 Instructs Dendritic Cells to Induce T-Helper 1 Immune Responses.

Immune activation associates with the intracellular generation of reactive oxygen species (ROS). To elicit effective immune responses, ROS levels must be balanced. Emerging evidence shows that ROS-mediated signal transduction can be regulated by selenoproteins such as methionine sulfoxide reductase B1 (MsrB1). However, how the selenoprotein shapes immunity remains poorly understood. Here, we demonstrated that MsrB1 plays a crucial role in the ability of dendritic cells (DCs) to provide the antigen presentation and costimulation that are needed for cluster of differentiation antigen four (CD4) T-cell priming in mice. We found that MsrB1 regulated signal transducer and activator of transcription-6 (STAT6) phosphorylation in DCs. Moreover, both in vitro and in vivo, MsrB1 potentiated the lipopolysaccharide (LPS)-induced Interleukin-12 (IL-12) production by DCs and drove T-helper 1 (Th1) differentiation after immunization. We propose that MsrB1 activates the STAT6 pathway in DCs, thereby inducing the DC maturation and IL-12 production that promotes Th1 differentiation. Additionally, we showed that MsrB1 promoted follicular helper T-cell (Tfh) differentiation when mice were immunized with sheep red blood cells. This study unveils as yet unappreciated roles of the MsrB1 selenoprotein in the innate control of adaptive immunity. Targeting MsrB1 may have therapeutic potential in terms of controlling immune reactions.

New genetic predictors for abacavir tolerance in HLA-B*57:01 positive individuals

Abacavir hypersensitivity syndrome (ABC HSS) is strongly associated with carriage of human leukocyte antigen (HLA)-B*57:01, which has a 100% negative predictive value for the development of ABC HSS. However, 45% of individuals who carry HLA-B*57:01 can tolerate ABC. We investigated immune and non-immune related genes in ABC HSS (n = 95) and ABC tolerant (n = 43) HLA-B*57:01 + patients to determine other factors required for the development of ABC HSS. Assignment of phenotype showed that ABC HSS subjects were significantly less likely than tolerants to carry only ERAP1 hypoactive trimming allotypes (p = 0.02). An altered self-peptide repertoire model by which abacavir activates T cells is in keeping with observation that endoplasmic reticulum aminopeptidase 1 (ERAP1) allotypes that favour efficient peptide trimming are more common in ABC HSS patients compared to patients who tolerate ABC. Independently, non-specific immune activation via soluble cluster of differentiation antigen 14 (sCD14) may also influence susceptibility to ABC HSS.

Selective Protein Expression Changes of Leukocyte-Migration-Associated Cluster of Differentiation Antigens at the Blood-Brain Barrier in a Lipopolysaccharide-Induced Systemic Inflammation Mouse Model without Alteration of Transporters, Receptors or Tight Junction-Related Protein.

Leukocyte migration across the blood-brain barrier (BBB) is an important step in the progression of brain dysfunction in systemic inflammation. The purpose of this study was to identify the key regulatory molecule(s) at the BBB among the cluster of differentiation (CD) antigens involved in leucocyte migration in lipopolysaccharide (LPS)-induced systemic inflammation based on their absolute protein expressions. Here, we identified the absolute expression levels of 17 CD antigens in isolated brain capillaries (Bcap) of LPS-administered mice. Among them, the expression levels of CD54 and CD106 were dramatically increased in LPS-administered mice compared to the control by 6.21- and 3.67-fold, respectively. In peripheral blood mononuclear cells, the expression levels of CD11a/CD18, the counter-receptor for CD54, were similar to those of CD54 in Bcap of LPS-administered mice. On the other hand, the expression level of CD49d, part of CD29/CD49d complex, which is the counter-receptor for CD106, was under the limit of quantification. It is thus likely that CD54 at the BBB is predominantly involved in promoting leukocyte migration across the BBB in systemic inflammation. The expression levels of CD9, CD49c and CD97, which are thought to be involved in cell-to-cell interaction, were decreased by 40-60% in Bcap of LPS-administered mice. In contrast, the expression levels of 9 transporters, 2 receptors, and 1 tight junction-related protein in Bcap of LPS-administered mice were essentially unchanged compared to the control. These results suggest that enhancement of leucocyte migration in systemic inflammation involves dynamic changes of CD antigens without alterations of other major functional molecules.

Optimization of Cryopreserved Single Cell Suspension of Gastric Epithelial Cells for Successful DotscanTM Antibody Microarray

Cell cryopreservation plays a pivotal role for successful immunological assays. DotScanTM antibody microarray employs live cells with satisfactory cell viability for detection of clusters of differentiation (CD) antigens in various diseases. Effective cryopreservation contributes to successful capture of cells on DotScanTM antibody microarray. The purpose of this study was to evaluate the effect of cells viability on cells binding to specific CD antigens. Cells with similar concentration but different percentage of cell viabilities were captured on DotScanTM slides. Cells stored for 5 days at -80oC and added with freezing medium in stepwise manner had higher viabilities as compared to cells stored for 6 months at -80oC and added with freezing medium in direct manner (p=0.012). Cells with 88% viability had higher binding to CD44/CD29 control antibodies as compared to cells with 33% viability. Short-term storage of cells and the technique for freezing medium addition to cells play a critical role for successful cryopreservation of single cell suspension.

Cellular systems biology identifies dynamic trophoblast populations in early human placentas

IntroductionThe human placenta is accessible in early developmental stages and affords a unique opportunity to investigate human organogenesis and the dynamics of transitory cell populations in human placenta development. MethodsThe cell surface proteomic profile of early trophoblast cells of first trimester human placentas was quantified using a high throughput flow cytometry screen of 370 Cluster of Differentiation (CD) antigens. Targeted investigation of candidate trophoblast progenitor populations was done through immunohistochemistry, multi-color flow cytometry, and genome wide expression analysis. ResultsUsing a novel batch correction and normalization methodology, we identified 23 increasing and 13 decreasing markers of dynamic populations between the week 6 and week 10 of placenta development. We identified and characterized two transient populations expressing either EpCAM (CD326) or CDCP1 (CD318). Immunohistochemistry revealed these CD antigens are expressed by discrete cells with EpCAM localized to the proximal villi columns and CDCP1 to distal columns. Flow analysis confirmed independence of these populations and identified EpCAM cells as positive for EGFR. Microarray analysis indicated EpCAM+/EGFR+ and EFGR + cells showed high degree of gene expression similarities to villus cytotrophoblast but loss of EpCAM expression was concomitant with exit from the cell cycle. Similarly, CDCP1 positive trophoblast are enriched in cell cycle gene sets and expressed genes with significant similarity to extravillous cytotrophoblast. DiscussionOur study indicates at least two distinct subpopulations of cytotrophoblasts exist in the early first trimester within the column that likely maintains pools of actively dividing progenitor cells giving rise to the developing placenta villous tree.

Early PREdiction of sepsis using leukocyte surface biomarkers: the ExPRES-sepsis cohort study

Reliable biomarkers for predicting subsequent sepsis among patients with suspected acute infection are lacking. In patients presenting to emergency departments (EDs) with suspected acute infection, we aimed to evaluate the reliability and discriminant ability of 47 leukocyte biomarkers as predictors of sepsis (Sequential Organ Failure Assessment score ≥ 2 at 24h and/or 72h following ED presentation). In a multi-centre cohort study in four EDs and intensive care units (ICUs), we standardised flow-cytometric leukocyte biomarker measurement and compared patients with suspected acute infection (cohort-1) with two comparator cohorts: ICU patients with established sepsis (cohort-2), and ED patients without infection or systemic inflammation but requiring hospitalization (cohort-3). Between January 2014 and February 2016, we recruited 272, 59 and 75 patients to cohorts 1, 2, and 3, respectively. Of 47 leukocyte biomarkers, 14 were non-reliable, and 17 did not discriminate between the three cohorts. Discriminant analyses for predicting sepsis within cohort-1 were undertaken for eight neutrophil (cluster of differentiation antigens (CD) CD15; CD24; CD35; CD64; CD312; CD11b; CD274; CD279), seven monocyte (CD35; CD64; CD312; CD11b; HLA-DR; CD274; CD279) and a CD8 T-lymphocyte biomarker (CD279). Individually, only higher neutrophil CD279 [OR 1.78 (95% CI 1.23-2.57); P = 0.002], higher monocyte CD279 [1.32 (1.03-1.70); P = 0.03], and lower monocyte HLA-DR [0.73 (0.55-0.97); P = 0.03] expression were associated with subsequent sepsis. With logistic regression the optimum biomarker combination was increased neutrophil CD24 and neutrophil CD279, and reduced monocyte HLA-DR expression, but no combination had clinically relevant predictive validity. From a large panel of leukocyte biomarkers, immunosuppression biomarkers were associated with subsequent sepsis in ED patients with suspected acute infection. NCT02188992.

Aberrant Expression of CD Markers in Cases with Acute Leukemia in Sohag University Hospital

Introduction: Acute leukemias are a group of malignancies with clinical, morphologic, immunologic, and molecular patterns and display patterns of surface antigen expression (CD antigens). Precursor cells from different blast cells in acute leukemia express different subdivisions of surface molecules many of which are now defined as cluster of differentiation (CD) antigens. Every leukemia type has specific set of CD markers, what constitute nowadays current classification of hematopoietic malignancies and the basis of diagnosis. The expression of CD markers on leukocytes can be determined by flowcytometry. Immunophenotyping improves both accuracy and reproducibility of acute leukemia classification. Flow cytometry is a technique used to analyze multiple phenotypic and functional parameters simultaneously within a single cell or a population of cells. Aim of the work: Assess the prevalence of aberrant expression of CD markers in cases with acute leukemia in Sohag University Hospitals. Patients and Methods: This study was carried out in the Clinical Pathology Department, faculty of medicine, Sohag university hospital on fifty (50) newly diagnosed acute leukemia patients who were subjected to bone marrow aspiration, their ages ranged between 1.5 to 71 years, 29 (58%) of them were males and 21 (42%) were females with a male to female ratio of 1.4:1, during the period from November 2015 to October 2016. Results: B-cell acute lymphoblastic leukemia positive for the following CD markers CD10, CD19, TdT, HLA-DR and expressing CD33 and case of AML (M4-M5) expressing CD7 lymphoid marker. Conclusion: Aberrant expression of CD markers is seen in several cases of acute leukemia. The frequency of aberrant antigen expression in acute leukemia was comparable with most published international data. The presence of aberrancy helps to identify a neoplastic process.

Plasmacytoid Dendritic Cell Function and Cytokine Network Profiles in Patients with Acute or Chronic Hepatitis B Virus Infection.

Background:Plasmacytoid dendritic cells (pDCs) and cytokines play an important role in occurrence and recovery of hepatitis B virus (HBV) infection. The aim of this study was to explore the frequency and function of pDC and serum cytokine network profiles in patients with acute or chronic HBV infection.Methods:The healthy individuals (HI group), hepatitis B envelope antigen (HBeAg)-positive chronic HBV patients in immune tolerance (IT) phase (IT group), HBeAg-positive chronic HBV patients (CHB group), and acute HBV patients (AHB group) were enrolled in this study. The frequency of cluster of differentiation antigen 86 (CD86) + pDC and the counts of CD86 molecular expressed on surface of pDC were tested by flow cytometer. The quantitative determinations of cytokines, including Fms-like tyrosine kinase 3 ligand (Flt-3L), interferon (IFN)-α2, IFN-γ, interleukin (IL)-17A, IL-6, IL-10, transforming growth factor (TGF)-β1 and TGF-β2, were performed using Luminex multiplex technology.Results:In this study, there were 13 patients in HI group, 30 in IT group, 50 in CHB group, and 32 in AHB group. Compared with HI group, HBV infected group (including all patients in IT, CHB and AHB groups) had significantly higher counts of CD86 molecular expressed on the surface of pDC (4596.5 ± 896.5 vs. 7097.7 ± 3124.6; P < 0.001). The counts of CD86 molecular expressed on the surface of pDC in CHB group (7739.2 ± 4125.4) was significantly higher than that of IT group (6393.4 ± 1653.6, P = 0.043). Compared with IT group, the profile of cytokines of Flt-3L, IFN-γ, and IL-17A was decreased, IFN-α2 was significantly increased (P = 0.012) in CHB group. The contents of IL-10, TGF-β1, and TGF-β2 in AHB group were significantly increased compared with IT and CHB groups (all P < 0.05).Conclusions:This study demonstrated that the function of pDC was unaffected in HBV infection. The enhanced function of pDC and IFN-α2 might involve triggering the immune response from IT to hepatitis active phase in HBV infection. Acute patients mainly presented as down-regulation of the immune response by enhanced IL-10 and TGF-β.

Porcine CD14 gene silencing partially inhibited the bacterial immune response mediated by TLR4 signaling pathway

Cluster of differentiation antigen 14 (CD14) is the membrane receptor protein in Toll-like Receptor 4 (TLR4) signaling pathway, which plays an important regulation role in not only innate immune response but also adaptive immune response. In this study, the pig kidney epithelial cell (PK15) line with CD14 gene silencing mediated by lentivirus was established and cells of CD14-RNAi and NC group were exposed to three kinds of Escherichia coli (E. coli F18ab, E. coli F18ac and E. coli K88ac) and LPS. Then qPCR and western blot were used to detect expression levels of TLR4 signaling pathway-related genes. Finally, ELISA was used to detect the level of proinflammatory cytokines in the cell culture supernatant. The results showed that the expression level of TLR4 signaling pathway-related genes in the entire signal pathway had obvious increases when cells were exposed to the stimulation induced by E. coli and LPS. In addition, the expression levels of CD14-RNAi group were overall significantly lower than NC group (P<0.05 or P<0.01), which was the same with the release levels of proinflammatory cytokines. This study revealed that pig CD14 gene silencing partially inhibited immune response to E. coli F18 invasion mediated by TLR4 signaling pathway.