CD44 Protein Expression Research Articles

Investigation of CD133 and CD24 as candidate azoospermia markers and their relationship with spermatogenesis defects

Spermatogenesis is initiated when spermatogonial stem cells (SSCs) in the mature testes enter mitosis and trigger differentiation. Thus, spermatogenesis and the ability to maintain a continuous source of spermatogonia relies on the ability to differentiate SSCs. Many studies around the world have been performed to investigate the etiology of male infertility and recent studies have focused on the presence and identification of biomarkers. CD133 and CD24 are stem cell markers locating in the testis and spermatogonia. The aim of this study was to investigate the relationship of the CD133 and CD24 genes with spermatogenesis defects and examine them as a candidate a useful biomarker for azoospermia men. The association of CD133 and CD24 with spermatogenesis defects was investigated in patients with obstructive (O) and non-obstructive azoospermia (NOA). NOA cases were histopathologically classified into Hypospermatogenesis (HS), Maturation Arrest (MA), and Sertoli Cell Only Syndrome (SCO) groups. A qRT-PCR analysis of these genes was performed and protein expression levels were measured by Western blot analysis. CD133 expression in NOA group was found to be significantly different from OA and this was confirmed by immunohistochemistry and immunocytochemical assays.The qRT-PCR analysis revealed that gene expression of CD133 and CD24 had fold changes of 0.80 ± 0.34 and 1.59 ± 0.31 compared to controls, respectively in the HS group (p > 0.05) and 0.04 ± 0.01 and 0.54 ± 0.08 in the MA group (p < 0.05). In the SCO group, CD24 showed a 1.55 ± 0.35-fold increase (p > 0.05). CD133 gene expression was not detected at the transcriptional level in the SCO group. Western blot analysis of CD133 protein expression revealed 1.83, 4.11, and 11.4-fold decreases in the HS, MA and SCO groups, respectively, compared to controls (p < 0.05). CD24 showed fold changes of 1.18, 0.38, (p < 0.05), and 0.89 in the HS, MA, and SCO groups, respectively. Immunohistochemical analysis of CD133 revealed moderate, partial staining in the HS group, compared to substantial, wide-spread staining in the OA group. No staining was detected in either the MA or SCO groups. The localization of CD133 in healthy sperm was determined to be prominent in the tail and partly expressed in the head by confocal laser scanning microscopy analysis. It was also found that the expression of CD133 protein was high in healthy commercially-sourced Sertoli cells as well as in the Sertoli cells of OA individuals. Data from this study show that CD133 exhibits different profiles in infertile patient groups and thus may be considered as a candidate biomarker. CD24 can be associated with blockage of germ cell maturation in the MA group. Curative protocols for spermatogenesis defects may be possible with the use of these markers and thus their identification is extremely valuable in terms of human reproductive health.

Molecular Patterns Discriminate Accommodation and Subclinical Antibody-mediated Rejection in Kidney Transplantation

Accommodation in ABO-incompatible (ABOi) transplantation and subclinical antibody-mediated rejection in HLA-incompatible (HLAi) transplantation share several morphological similarities. Because the clinical long-term outcomes differ, we hypothesized different molecular processes involved in ABOi transplantation and subclinical antibody-mediated rejection. Using Illumina Human HT-12 v4 Expression BeadChips, the whole transcriptome was evaluated based on 3-month protocol C4d+ biopsies in otherwise stable ABOi and HLAi kidney grafts, as well as in C4d-negative HLA-compatible grafts exhibiting normal histological findings. Top differently regulated genes were further validated using real-time quantitative polymerase chain reaction in another patient cohort and complement regulatory proteins by immunohistochemistry. In the case of genes involved in immune response-related biological processes, ABOi and HLAi cohorts had similar transcriptomic profiles to C4d-negative controls. The majority of deregulated genes in the ABOi and HLAi groups consisted of metallothioneins and epithelial transporter genes. Increased expression of epithelial transporters (SLC4A1, SLC4A9, SLC17A3, SLC12A3, and SLC30A2) and class 1 metallothioneins (MT1F, MT1G, and MT1X) in HLAi transplantation was validated by real-time quantitative polymerase chain reaction. In comparison to controls, both incompatible cohorts were characterized by the upregulation of intrarenal complement regulatory genes. CD46 and CD59 transcripts were increased in the ABOi cohort, whereas CD46 solely in HLAi group, and CD59 protein expression was similar in both incompatible groups. Several epithelial transporters and metallothioneins discriminate subclinical antibody-mediated rejection in HLAi transplantation from accommodation in ABOi transplantation, which suggest different involved downstream mechanisms and increased risk of injury in HLAi settings. Metallothioneins with their antioxidative properties may help to attenuate the inflammation response induced by donor-specific anti-HLA antibody binding.

CD163 is a predictive biomarker for prognosis of classical Hodgkin's lymphoma in Saudi patients.

Hodgkin's lymphoma (HL) is a hematological malignancy characterized by a minority of neoplastic cells outnumbered by tumor-associated macrophages (TAMs). The overexpression of the CD163 antigen by TAMs is considered to be a significant predictive biomarker for risk stratification. This is likely caused by a genetic single-nucleotide polymorphism (SNP) at the gene promoter. The aim of the present retrospective case-control study was to establish a gene expression profile of a specific biomarker for classical HL (CHL) in order to predict the outcome and survival of CHL patients in Saudi Arabia. The protein expression of CD163 on TAMs was studied using immunohistochemistry (IHC). A prognosis index was calculated for the CD163 protein to assess the risk stratification of CHL. Genotyping of selected SNPs of this antigen was performed for 100 CHL cases and controls. The analysis revealed that the CD163 protein expression level was significantly correlated with disease relapse (DR) and overall survival (OS). In addition, the CD163 index threshold (15.0) was found to be significantly correlated with the relapse rate. Among the studied CD163 SNPs, rs75608120 exhibited a significant correlation with the DR rate of CHL patients, but not with OS. The findings of the present study confirmed that CD163 is a specific marker for TAMs, and its overexpression by TAMs is significantly associated with relapse and reduced survival post-therapy. In addition, a new methodology of indexing CD163 protein expression for HL risk stratification was proposed. Thus, the present study identified a specific predictive molecular and antigenic biomarker for CHL prognosis.

Dietary Turmeric Bisdemethoxycurcumin Suppresses Wilms’ Tumor 1 and CD34 Protein Expressions in KG-1a Leukemic Stem Cells

Leukemic cells remaining in the body is the main problem for cancer patients, and these cells are called Leukemic Stem Cells (LSCs). Many studies have revealed that the overexpression of the Wilms’ tumor 1 (WT1) protein is related to leukemogenesis. Curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin) from Thai turmeric (Curcuma longa Linn.) are the focus of this study because they have been previously been reported to show potent antileukemic activity. This study aims to investigate the cytotoxic effect of in-house curcuminoids on the human leukemic stem cell line (KG-1a) when compared to other leukemic cell lines (KG-1 and K562). MTT assays were used to determine the cytotoxicity of curcuminoids at various concentrations. Curcumin exhibited the strongest cytotoxic activity on KG-1a cells with IC50 values of 13.6 ± 1.9 µM. To determine the effect of curcuminoids on WT1 and CD34 protein expressions by Western blotting, KG-1a cells were treated with noncytotoxic concentrations (IC20 value). Bisdemethoxycurcumin showed the strongest suppression of WT1 and CD34 protein expressions with 73.3 ± 1.4 and 82.9 ± 2.0%, respectively. In summary, curcuminoids, especially bisdemethoxycurcumin, could inhibit WT1 and CD34 protein expressions. Thus, bisdemethoxycurcumin is a compound that calls for further studies of its process in the inhibition of WT1 and CD34 expressions in LSCs for the leukemia treatment.

Carboxypeptidase A4 promotes proliferation and stem cell characteristics of hepatocellular carcinoma.

Carboxypeptidase A4 (CPA4), a member of the metallo-carboxypeptidase family, is overexpressed in liver cancer and is associated with cancer progression. The role of CPA4 in hepatocellular carcinoma (HCC) remains unclear. In this study, we aimed to evaluate the relevance of CPA4 to the proliferation and expression of stem cell characteristics of hepatocellular carcinoma cells. Western blot analysis showed high CPA4 expression in the liver cancer cell line Bel7402 and low expression in HepG2 cells. Knock-down of CPA4 decreased cancer cell proliferation as detected by MTT and clone formation assays. The serum-free culture system revealed that downregulated CPA4 suppressed the sphere formation capacities of tumour cells. However, upregulated CPA4 increased the proliferation and sphere formation capacity. In addition, the protein expression of CD133, ALDH1 and CD44 also increased in cells with upregulated CPA4. In vivo, the overexpression of CPA4 in tumour cells that were subcutaneously injected into nude mice markedly increased the growth of the tumours. These data suggest that CPA4 expression leads to poor prognoses by regulating tumour proliferation and the expression of stem cell characteristics and may therefore serve as a potential therapeutic target of HCC.

CD44 Promotes Migration and Invasion of Docetaxel-Resistant Prostate Cancer Cells Likely via Induction of Hippo-Yap Signaling.

Patients receiving docetaxel developed a drug resistance within a few months. We generated docetaxel-resistant PC/DX25 and DU/DX50 CRPC cells from PC-3 and DU-145 PCa cells, respectively. We investigated the mechanism behind why PC/DX25 and DU/DX50 cells exhibited higher migration and invasion ability. Transwell assays were used to measure the migration and invasion of PCa cell. Fluorescence activated cell sorter (FACS) analysis was used to determine the population of cancer stem cell (CSC)-like cell. Micro-Western Array (MWA) was used to study the changes of the protein profile. FACS analysis revealed that PC/DX25 cells and DU/DX50 cells contain higher CD44+ population. MWA and Western blotting assay revealed that protein expression of CD44, YAP, CYR61, CTGF, phospho-ERK1/2 T202/Y204, ERK and vimentin was elevated in PC/DX25 cells. Knockdown of CD44 or YAP suppressed migration and invasion of PC/DX25 and DU/DX50 cells. Knockdown of CD44 decreased expression of YAP, CTGF and CYR61 but increased phosphorylation of S127 on YAP. CD44 knockdown also suppressed protein level of AKT, phospho-AKT T308, phospho-ERK1/2 T202/Y204 and vimentin. CD44 promotes migration and invasion of docetaxel-resistant PCa cells probably via induction of Hippo-Yap signaling pathway and CD44/YAP pathway may be a therapeutic target for docetaxel-resistant PCa.

ALDH1 expression correlates with an epithelial-like phenotype and favorable prognosis in lung adenocarcinoma: a study based on immunohistochemistry and mRNA expression data.

Cancer stem cells (CSC) and epithelial-mesenchymal transition (EMT) pathways are crucial for cancer progression. However, synergistic interactions between CSC and EMT are not clear in non-small cell lung cancer (NSCLC). The objective of this study was to investigate CSC markers such as CD44, NANOG, and ALDH1 expression and its correlation with EMT markers in NSCLC patients. Its association with survival was also determined. CD44, NANOG, and ALDH1 protein expression was evaluated in 267 resected NSCLC and its correlation with e-cadherin, β-catenin, p120 catenin, vimentin, SNAIL, and TWIST expressions was determined based on immunohistochemical and mRNA expression data from The Cancer Genome Atlas (TCGA) database. Survival analyses also were performed based on immunohistochemistry and mRNA expression data from Gene Expression Omnibus dataset. ALDH1 expression in lung adenocarcinoma was positively correlated with the epithelial-like phenotype, low vimentin and low TWIST in immunohistochemical and mRNA expression data. NANOG and ALDH1 expressions measured by immunohistochemical and mRNA expression profiling data of adenocarcinomas were associated with a favorable prognosis. ALDH1 was an independent favorable prognostic marker for overall survival or recurrence-free survival in adenocarcinoma (P = 0.026 and P = 0.033, respectively). The epithelial-like phenotype expressing P120-catenin and beta-catenin was associated with a favorable prognosis; however, the TWIST-expressing mesenchymal-like phenotype was correlated with an unfavorable prognosis. NANOG and ALDH1 protein or mRNA expression showed improved prognosis in adenocarcinoma alone. ALDH1 expression correlated with an epithelial-like phenotype.

An immunosuppressive macrophage profile attenuates the prognostic impact of CD20-positive B cells in human soft tissue sarcoma

BackgroundImmune cells can regulate disease progression and response to treatment in multiple tumor types, but their activities in human soft tissue sarcoma are poorly characterized.MethodsMarker-defined immune cell subsets were characterized from a tumor microenvironmental perspective in two independent cohorts of human soft tissue sarcoma by multiplex IHC, quantitative PCR and/or bioinformatics.ResultsB cell profiling revealed a prognostic role for CD20 protein (cohort 1, 33 patients) and MS4A1 gene expression (cohort 2, 265 patients). Multiplex IHC and gene correlation analysis supported a role in antigen presentation, immune cell differentiation and T cell activation. The prognostic role of MS4A1 expressing B cells was only observed in an IL10low, PTGS2low or CD163low tumor microenvironment according to the transcriptomic data. IL10 levels consistently correlated with the M2-like macrophage marker CD163, which also defined the majority of macrophages. A polarization of these cells toward a pro-tumoral phenotype was further supported by lack of correlation between CD163 and M1 markers like NOS2, as well as by low abundance of CD80 positive cells in tissue.ConclusionsAnalysis of CD20/MS4A1 expression in soft tissue sarcoma merits further attention as a promising candidate prognostic tool for survival, but not in patients with a pronounced immunosuppressive tumor microenvironment. Macrophages are ubiquitous and polarized toward a protumoral phenotype. This provides a rationale for further studies on B cell function and immunotherapy targeting M2-polarized macrophages.

Electrospun gelatin/PCL and collagen/PCL scaffolds for modulating responses of bone marrow endothelial progenitor cells.

The determination of potential transplantable substrates and substitution cells for corneal endothelium transplantation may compensate for the shortage of cornea donors. Appropriate biodegradable and biocompatible tissue-engineered substratum with seed cells for endothelial keratoplasty has been increasingly studied. In the present study, electrospun gelatin/polycaprolactone (PCL) and collagen/PCL scaffolds were successfully established. Bone marrow endothelial progenitor cells (BEPCs) were cultured on these scaffolds to determine whether the scaffolds may promote the proliferation of BEPCs as well as maintain stem cell characteristics. Two variations of hybrid scaffolds, collagen/PCL (70% collagen and 30% PCL) and gelatin/PCL (70% gelatin and 30% PCL), were established via electrospinning. Microscopic structure, hydrophilicity and wettability of the two scaffolds were subsequently investigated. BEPCs were separately cultured on the scaffolds and were also seeded on glass slides to establish the control group. Furthermore, cell morphology; adherence, as determined by investigation of F-actin expression levels; proliferation, as determined via Cell Counting Kit-8 assays, Ki-67 staining and bromodeoxyuridine (BrdU) staining; and stem cell markers, as determined by cluster of differentiation (CD)-34 and CD-133 protein expression levels; were investigated. In addition, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine gene expression. The two nanofiber scaffolds were established using electrospun techniques with expected hydrophilicity, wettability and biocompatibility. BEPCs were revealed to spread well on and strongly adhere to the collagen/PCL (70:30) and gelatin/PCL (70:30) scaffolds. Furthermore, Ki-67 and BrdU staining results revealed greater levels of positive dots on the two hybrid scaffolds compared with the control group. CD-34 and CD-133 protein staining demonstrated increased levels of fluorescence intensity on scaffolds compared with the control group. Furthermore, increased expression levels of differentiation markers, such as ATP binding cassette subfamily G member 2, leucine rich repeat containing G protein-coupled receptor 5 and CD166, were detected on both scaffolds. RT-qPCR results demonstrated that the expression of caspase-3, which is associated with apoptosis, was decreased on the two scaffolds compared with in the control group. The expression of inflammatory factors, including interleukin (IL)-1, exhibited a significant decrease on the gelatin/PCL scaffold compared with in the control group; whereas the difference between the expression level of IL-1 exhibited by the collagen/PCL group and the control group were not markedly different. Electrospun collagen/PCL and gelatin/PCL scaffolds exhibited the potential to enhance the adherence and proliferation of BEPCs. BEPCs cultured on the two scaffolds demonstrated increased stem cell characteristics and differentiation potential. Electrospun gelatin/PCL and collagen/PCL scaffolds may represent a promising substratum in tissue-engineered corneal endothelium.

Human Germinal Center B Cells Differ from Naïve and Memory B Cells in CD40 Expression and CD40L-Induced Signaling Response.

CD40 expression is required for germinal center (GC) formation and function, but the kinetics and magnitude of signaling following CD40 engagement remain poorly characterized in human B cells undergoing GC reactions. Here, differences in CD40 expression and signaling responses were compared across differentiation stages of mature human tonsillar B cells. A combination of mass cytometry and phospho-specific flow cytometry was used to quantify protein expression and CD40L-induced signaling in primary human naïve, GC, and memory B cells. Protein expression signatures of cell subsets were quantified using viSNE and Marker Enrichment Modeling (MEM). This approach revealed enriched expression of CD40 protein in GC B cells, compared to naïve and memory B cells. Despite this, GC B cells responded to CD40L engagement with lower phosphorylation of NFκB p65 during the first 30 min following CD40L activation. Before CD40L stimulation, GC B cells expressed higher levels of suppressor protein IκBα than naïve and memory B cells. Following CD40 activation, IκBα was rapidly degraded and reached equivalently low levels in naïve, GC, and memory B cells at 30 min following CD40L. Quantifying CD40 signaling responses as a function of bound ligand revealed a correlation between bound CD40L and degree of induced NFκB p65 phosphorylation, whereas comparable IκBα degradation occurred at all measured levels of CD40L binding. These results characterize cell-intrinsic signaling differences that exist in mature human B cells undergoing GC reactions. © 2019 International Society for Advancement of Cytometry.

Construction of eukaryotic expression vector for human platelet CD36 gene 220C>T and 429+4insg variants and analysis of their expressions in HEK293T cells

To construct eukaryotic expression vectors for human platelet CD36 gene 220 C>T and 429+4insg variants and analyze their expressions in HEK293T cells. RNA was isolated from human platelets and reversely transcribed into cDNA. Sequences of 220C>T and 429+4insg variants were derived by PCR amplification. The target sequence was ligated into a pcDNA3.1/V5-His-TOPO vector by TA cloning, which was transformed into TOP10 E. coli. Positive plasmids were screened by blue-white selection. After sequencing, plasmid DNA carrying 220C>T or 429+4insg variant was used to transfect HEK293T cells with the help of effectene. Expression of CD36 protein was then analyzed by flow cytometry and Western blotting. An eukaryotic expression vector pcDNA3.1/V5-His-CD36 (220C>T/429+4insg) was constructed by TA cloning. After transfected into HEK293T cells, the 220C>T and 429+4insg variants resulted in CD36 deficiency in HEK cells, which was confirmed by flow cytometry and Western blotting. The 220C>T and 429+4insg variants can cause CD36 deficiency in human platelets. This system may be used for assessing the effect of 220C>T, 429+4insg, and other variants on the expression of CD36.

Gestational exposure to PM2.5 impairs vascularization of the placenta

The placenta is the bridge that connects the developing fetus and mother and is the place where nutrient uptake, waste elimination and gas exchange occurs. Epidemiological studies indicate that fetal stress, such as gestational PM2.5 (particulate matter (PM) with diameter ≤ 2.5 μm) exposure, is a risk factor for abnormal placenta development that could induce adverse effects on the fetus. In the present study, PM samples were collected in Taiyuan. Then, pregnant C57BL/6 mice were treated with 3 mg/kg b.w. PM2.5 every other day starting on GD0.5, and were sacrificed at embryonic day 13.5 (E13.5), E15.5 and E18.5, and placentas were collected for placenta histopathological analysis and gene expression evaluation at the transcription or translation level. Our results showed that gestational exposure to PM2.5 significantly reduced the ratio of the mean area of labyrinthine (lab) layer/total area of placentas at E18.5. However, no significant difference was observed at E13.5 and E15.5. Interestingly, genes essential for this process were altered at E13.5 and E18.5 but not E15.5. Furthermore, gestational PM2.5 exposure decreased the mean area of maternal blood sinuses and fetal blood vessels and suppressed protein expression of CD31 in the PM2.5 group only at E18.5, while protein expression of CD34 in the PM2.5 group was similar to that in the control group at all time points. Finally, gestational exposure to PM2.5 activated mitogen-activated protein kinase (MAPK) pathways at E13.5 and E18.5 but not E15.5. Taking these results together, gestational exposure to PM2.5 leads to histopathological changes and vascularization injuries of the placenta, and this response may be associated with activation of the MAPK pathway.

Abstract WMP81: High-Dimensional Analysis of Immune Cell Characterization in Ischemic Postconditioning

Background: Ischemic postconditioning (IPostC), an emerging concept for stroke treatment, refers to a series of brief occlusions of the cerebral blood vessels after reperfusion. A precise understanding of immune response in IPostC is very important. We describe an approaching to identify immune cells and characterize their distinct functions within IPostC by using high parametric mass cytometry (CyTOF). Methods: Leukocytes were isolated from the ischemic brain and peripheral blood of ischemic stroke mice. These cell samples were simultaneously stained with 25 antibodies, including 16 cell surface antibodies, and an additional 9 antibodies against key molecules in cell signaling pathways. The samples were detected by CyTOF and analyzed using the Cytobank program. Results: We demonstrated that stroke resulted in significant increases of microglia, monocyte-derived macrophages (MoMΦs), natural killer (NK), conventional DCs (cDCs), B cells, CD4 + T, CD8 + T cells, effector memory CD4 T (CD4Tem) cells, effector memory CD8 T (CD8Tem) cells and regulatory T (Treg) cells (p&lt;0.05). Additionally, the increase of brain-infiltrating leukocyte subsets was accompanied with a decrease of corresponding leukocyte subsets in the peripheral blood. IPostC decreased the counts of brain-infiltrating MoMΦs, CD4 + T, CD4Tem, CD8Tem and NK cells (p&lt;0.05). In addition, SAPDE analysis showed that individual leukocytes in the ischemic brain had higher protein expressions of CD45, CD11b, CD11c, B220, CD4, CD8, CD44, and CD49b than sham brains, suggesting that these proteins may be actively involved in brain injury. We can see that IPostC downregulated these protein expressions. Our results revealed differences in endogenous signaling tone within ischemic stroke and IPostC, including increased AKT, EGFR, P38 phosphorylation and IκBa in microglia and MoMΦs, and decreased in IPostC. Conclusions: We revealed the phenotypic ranges of immune cells and identified cell surface receptors in individual leukocytes. The approach enabled us to know more about the phenotypical and functional characteristics of immune populations in the brain and peripheral blood after IPostC. It is expected to serve as a resource for understanding immune response within IPostC.

Matrix Metalloproteinase 9 and Osteopontin Interact to Support Synaptogenesis in the Olfactory Bulb after Mild Traumatic Brain Injury.

Olfactory receptor axons reinnervate the olfactory bulb (OB) after chemical or transection lesion. Diffuse brain injury damages the same axons, but the time course and regulators of OB reinnervation are unknown. Gelatinases (matrix metalloproteinase [MMP]2, MMP9) and their substrate osteopontin (OPN) are candidate mediators of synaptogenesis after central nervous system (CNS) insult, including olfactory axon damage. Here, we examined the time course of MMP9, OPN, and OPN receptor CD44 response to diffuse OB injury. FVBV/NJ mice received mild midline fluid percussion insult (mFPI), after which MMP9 activity and both OPN and CD44 protein expression were measured. Diffuse mFPI induced time-dependent increase in OB MMP9 activity and elevated the cell signaling 48-kD OPN fragment. This response was bimodal at 1 and 7 days post-injury. MMP9 activity was also correlated with 7-day reduction in a second 32-kD OPN peptide. CD44 increase peaked at 3 days, delayed relative to MMP9/OPN response. MMP9 and OPN immunohistochemistry suggested that deafferented tufted and mitral neurons were the principal sites for these molecular interactions. Analysis of injured MMP9 knockout (KO) mice showed that 48-kD OPN production was dependent on OB MMP9 activity, but with no KO effect on CD44 induction. Olfactory marker protein (OMP), used to identify injured olfactory axons, revealed persistent axon damage in the absence of MMP9. MMP9 KO ultrastructure at 21 days post-injury indicated that persistent OMP reduction was paired with delayed removal of degenerated axons. These results provide evidence that diffuse, concussive brain trauma induces a post-injury interaction between MMP9, OPN, and CD44, which mediates synaptic plasticity and reinnervation within the OB.

Expression of MMP-14 and CD44 associated with proliferation of retinoblastoma cells

Retinoblastoma is a the most common primary intraocular malignant tumor in childhood. The eexpressions of CD44 and MMP-14 and Their role in proliferation and migration cells appear crucial for retinoblastoma invasion. Thirty-five paraffin blocks from retinoblastoma patients with tumor material were compared with eight paraffin blocks of normal retina. Retinoblastoma patients were selected from patients diagnosed with retinoblastoma at Dr. Soetomo General Hospital, Surabaya. Immunohistochemical method was used to evaluate the expression of CD44, MMP-14, and cell proliferation (Ki-67). The expression of CD44, MMP-14, and Ki-67 protein was positive in all retinoblastoma samples. In retinoblastoma samples, 54,3% (19/35) of CD44 showed high expression (score 3). The majority (40%) of MMP-14 expression was low (score 1) in retinoblastoma samples. The majority (60%) of Ki-67 (21/35) showed high expression (score 2) in retinoblastoma samples. In normal retina, 100% CD44 was moderately expressed (score 1), Ki-67 and MMP-14 were not expressed. Their expression in retinoblastoma was significant when compared with normal retina. In retinoblastoma sample, positive staining of CD44 showed high expression in the cytoplasm of the cell. This suggests that hyaluronic acid, as a ligand to CD44 in cellÂ’s cytoplasm, has low molecular weight, expresses angiogenesis properties, and promotes tumor cell proliferation. MMP-14 in this study was reported to be expressed mostly on low level group. The expression of CD44 and MMP-14 is thought to play a role in inducing cell proliferation and invasion of retinoblastoma cells.

Thymopentin promotes ovarian angiogenesis in mice by activating N6-methyladenosine (m6A) RNA modification of key factors in the Notch/Tie1 pathway

Abstract: BACKGROUND: Premature ovarian failure (POF) is a typical condition of pathological ovarian ageing. Injuries and atrophies of blood vessels in the ovaries can lead to ovarian insufficiency, but the underlying mechanism remains unclear. AIMS: This study investigated the epigenetic mechanism by which thymopentin (TP-5) activated ovarian angiogenesis to achieve its effects on POF. MATERIALS AND METHODS: The qPCR, western blot and immunofluorescence staining were used to detecte the expression levels of gene. The hematoxylin and eosin stainingwas used to histopathological examination. The RNA-Seq sequencing was used to transcriptome detection. RESULTS: First, pathological examination revealed that TP-5 significantly increased ovary weight in the cyclophosphamide-induced POF mouse model. The proportion of atretic follicles was reduced, and the level of E2 in the peripheral blood was elevated. Meanwhile, immunofluorescence staining showed that TP-5 increased protein expression of CD31 and Tie1 in the ovarian tissues of mice with POF, suggesting that TP-5 could induce ovarian angiogenesis. RNA-Seq sequencing results suggested that TP-5 could induce the high level of Notch/Tie2 pathway expression that is associated with angiogenesis in ovarian tissues. qPCR and western blotting showed significantly increased expression of METTL3, a methyltransferase that catalyses the formation of N6-methyladenosine (m6A) in RNA. Meanwhile, the results of the dot blot suggested that the overall RNA methylation level was significantly higher in the ovarian tissues of mice in the TP-5 group than in the control group. Finally, RIP-PCR analysis showed that specific sites in the 3'-untranslated region (3'UTR) of the mRNA of key factors in the Notch/Tie2 pathway were hypermethylated in the ovaries of mice in the TP-5 treatment group. CONCLUSION: Therefore, this study demonstrated that TP-5 exerted its therapeutic effect on POF by stimulating angiogenesis. The mechanism of action is the promotion of the expression of RNA m6A methyltransferases by TP5, which increases the overall RNA m6A methylation level in mouse ovarian tissue. Specifically, the methylation ofspecific m6A sites in the 3'UTR of the RNA of key factors in the Notch/Tie2 pathway increased, which enhanced their stability and expression levels, leading to the induction of angiogenesis.

CRISPR/Cas9 Screens Reveal Multiple Layers of B Cell CD40 Regulation

CD40 has major roles in B-cell development, activation and germinal center responses. CD40 hypoactivity causes immunodeficiency, whereas its overexpression causes autoimmunity and lymphomagenesis. To systematically identify B-cell autonomous CD40 regulators, we performed CRISPR/Cas9 genome-wide screens. These found CD40 pathway components and revealed mechanisms strongly influencing CD40 responses. At the transcriptional level, the ubiquitin ligase FBXO11 supported CD40 abundance by targeting the repressor CTBP1. FBXO11 knockout decreased primary B-cell CD40 expression and impaired class-switch recombination. Frequent lymphoma monoallelic FBXO11 mutations may therefore balance BCL6 increase with CD40 loss. At the mRNA level, CELF1 controlled exon splicing critical for CD40 protein expression, while the N6-adenosine methyltransferase WTAP controlled CD40 message abundance. At the protein level, ESCRT negatively-regulated activated CD40 levels while the negative feedback phosphatase DUSP10 downmodulated MAPK. These results reveal multiple layers of CD40 regulation, serve as a resource for future studies of this key co-receptor, and highlight potential therapeutic targets. CD40 has major roles in B-cell development, activation and germinal center responses. CD40 hypoactivity causes immunodeficiency, whereas its overexpression causes autoimmunity and lymphomagenesis. To systematically identify B-cell autonomous CD40 regulators, we performed CRISPR/Cas9 genome-wide screens. These found CD40 pathway components and revealed mechanisms strongly influencing CD40 responses. At the transcriptional level, the ubiquitin ligase FBXO11 supported CD40 abundance by targeting the repressor CTBP1. FBXO11 knockout decreased primary B-cell CD40 expression and impaired class-switch recombination. Frequent lymphoma monoallelic FBXO11 mutations may therefore balance BCL6 increase with CD40 loss. At the mRNA level, CELF1 controlled exon splicing critical for CD40 protein expression, while the N6-adenosine methyltransferase WTAP controlled CD40 message abundance. At the protein level, ESCRT negatively-regulated activated CD40 levels while the negative feedback phosphatase DUSP10 downmodulated MAPK. These results reveal multiple layers of CD40 regulation, serve as a resource for future studies of this key co-receptor, and highlight potential therapeutic targets.

Overexpression of the cancer stem cell marker CD133 confers a poor prognosis in invasive breast cancer.

CD133/ prominin 1 is a cancer stem cell marker associated with cancer progression and patient outcome in a variety of solid tumours, but its role in invasive breast cancer (BC) remains obscure. The current study aims to assess the prognostic value of CD133 expression in early invasive BC. CD133 mRNA was assessed in the METABRIC cohort and at the proteomic level using immunohistochemistry utilising a large well-characterised BC cohort. Association with clinicopathological characteristics, expression of other stem cell markers and patient outcome were evaluated. High expression of CD133 either in mRNA or protein levels was associated with characteristics of poor prognosis including high tumour grade, larger tumour size, high Nottingham Prognostic Index, HER2 positivity and hormonal receptor negativity (all; p < 0.001). High CD133 expression was positively associated with proliferation biomarkers including p16, Cyclin E and Ki67 (p < 0.01). Tumours expressing CD133 showed higher expression of other stem cell markers including CD24, CD44, SOX10, ALDHA3 and ITGA6. High expression of CD133 protein was associated with shorter BC-specific survival (p = 0.026). Multivariate analysis revealed that CD133 protein expression was an independent risk factor for shorter BC-specific survival (p = 0.038). This study provides evidence for the prognostic value of CD133 in invasive BC. A strong positive association of BC stem cell markers is observed at the protein level. Further studies to assess the value of stem cell markers individually or in combination in BC is warranted.

Expression of Matrix Metalloproteinase-2, Tissue Inhibitor of Matrix Metalloproteinase-2 and CD147 in the Traditional Chinese Medicine “Compound T11” for Treatment of Chronic Liver Injury

Objectives: To measure the expression of matrix metalloproteinase (MMP)-2, tissue inhibitor of matrix metalloproteinase inhibitor (TIMP)-2, and CD147 in mice with chronic liver injury induced by carbon tetrachloride after treatment with the traditional Chinese medicine (TCM) “Compound T11”. Method: Sixty male ICR mice were divided randomly into 6 groups of 10: control (C), model (M), low-dose treatment (LT; 50 mg/mL of Compound T11), medium-dose treatment (MT, 100 mg/mL), high-dose treatment (HT, 150 mg/mL), and positive drug treatment (YT, 67.5 mg/mL). Each group was modeled for 7 weeks. Groups M, LT, MT, HT, and YT were injected (s.c.) with 20% carbon tetrachloride diluted with olive oil, and group C was given olive oil in the same way twice a week. After modeling, the treatment groups were administered Compound T11 at the concentrations shown above by oral gavage daily for 2 weeks, while group C was given 0.5% carboxymethyl cellulose sodium. After the final treatment, mice were killed and their liver tissues were excised. Immunohistochemical staining was performed to measure the protein expression of MMP-2, TIMP-2, and CD147, and western blotting was used to measure the protein expression of MMP-2, TIMP-2, CD147, and α-smooth muscle actin (SMA). MMP-2, TIMP-2, and CD147 mRNA expression was determined by quantitative fluorescence real-time PCR. Results: Compound T11 increased the protein expression of MMP-2 and CD147 and decreased the protein expression of TIMP-2 and α-SMA. Conclusions: Treatment of chronic liver injury by TCM Compound T11 may be associated with changes to the expression of MMP-2 and CD147, and the inhibition of TIMP-2 expression.

CD47 but Not Calreticulin Is over Expressed in Patients with Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are chronic myeloid cancers characterized by the overproduction of mature blood cells, and the tendency to evolve into acute leukaemia. In solid tumours, calreticulin (CALR) overexpression on the cell surface, produces a pro-phagocytic signal and is counteracted by concomitant expression of anti- phagocytic CD47, reflecting an apoptosis vs survival mechanism. In this study, we investigated the expression of CALR and CD47 in patients with MPN and potential changes induced by treatment.CALR and CD47 gene and protein expression were measured by Real Time PCR and western blotting, in K562 and in mononuclear cells obtained by FICOLL separation, from peripheral blood of 13 MPN patients [4 Polycythaemia Vera, 8 Essential Thrombocythemia, and 1 myelofibrosis; 7 treated (IFN or Hydroxyurea) and 6 untreated] and compared with 4 healthy controls. Cells were also fractionised into 4 compartments: membrane, cytoplasm, cytosol and nucleus and protein fractions analysed.We found a significant increase in CD47 protein expression into the membrane of MPN patients comparing with controls (91.9 % vs 19.7%). Interestingly in treated MPN the CD47 expression increases even further in comparison with untreated (92.6% vs 91.9%). No significant differences were found in total CALR expression comparing with controls (3.23, vs 2.95-fold), however in treated patients we observed a reduction in the expression in the membrane comparing with untreated (26.4 % vs 39.5 %) and an increase into cytoplasm (69.4 % vs 54.6 %). These findings suggest CD47 exposure as a mechanism of defence in MPN cells during treatment.To better understand the potential effects of therapy on CALR and CD47 expression, we incubated K562 with 0.05μM/ml of Ruxolitinib (RUXO), re-dosed at 24 hours and harvested at 48 hours. RUXO induced CALR internalization, reducing its expression into the membrane (RUXO 8.2% vs Untreated: 13.3%), but increasing its expression in cytosol (RUXO 51% vs Untreated 48.4%) and in the cytoplasm (RUXO 13.5% vs Untreated 2.9%). In contrast, CD47 expression remains relatively unmodified, with only slight increases on the membrane (RUXO 43.1% vs Untreated 40.4%) and in the cytoplasm (RUXO 34.5% vs UT 32.5%).In this study we demonstrated that CD47 but not CALR, is overexpressed on the membrane of patients with MPN. This opposes previous studies in solid tumours, which show significant increases of both CALR and CD47. This suggests a role for CD47 as a strong anti-phagocytic signal responsible for immune survival in MPN. We have also shown a potential mechanism adopted by MPN cells in response to therapy, with the internalization of CALR and the enhanced membrane expression of CD47 which remains strongly expressed on cell surface. The addition of Anti-CD47 compounds in combination with conventional therapies might represent a future therapeutically strategy to counteract MPN cells immune-escape mechanism. DisclosuresNo relevant conflicts of interest to declare.