Huseyin Mehmet

The AC133 Epitope, but not the CD133 Protein, Is Lost upon Cancer Stem Cell Differentiation

Colon cancer stem cells (CSC) can be identified with AC133, an antibody that detects an epitope on CD133. However, recent evidence suggests that expression of CD133 is not restricted to CSCs, but is also expressed on differentiated tumor cells. Intriguingly, we observed that detection of the AC133 epitope on the cell surface decreased upon differentiation of CSC in a manner that correlated with loss of clonogenicity. However, this event did not coincide with a change in CD133 promoter activity, mRNA, splice variant, protein expression, or even cell surface expression of CD133. In contrast, we noted that with CSC differentiation, a change occured in CD133 glycosylation. Thus, AC133 may detect a glycosylated epitope, or differential glycosylation may cause CD133 to be retained inside the cell. We found that AC133 could effectively detect CD133 glycosylation mutants or bacterially expressed unglycosylated CD133. Moreover, cell surface biotinylation experiments revealed that differentially glycosylated CD133 could be detected on the membrane of differentiated tumor cells. Taken together, our results argue that CD133 is a cell surface molecule that is expressed on both CSC and differentiated tumor cells, but is probably differentially folded as a result of differential glycosylation to mask specific epitopes. In summary, we conclude that AC133 can be used to detect cancer stem cells, but that results from the use of this antibody should be interpreted with caution.

Mg2+ regulates cytotoxic functions of NK and CD8 T cells in chronic EBV infection through NKG2D.

Magnesium to the Rescue Individuals with X-linked immunodeficiency with Mg2+ defect, Epstein-Barr virus (EBV) infection, and neoplasia (XMEN) disease are genetically deficient for expression of MAGT1, a magnesium transporter. Chaigne-Delalande et al. (p. 186) sought to better understand why these individuals are chronically infected with EBV at high viral loads and are susceptible to the development of lymphomas. CD8+ T cells and natural killer cells, which help to keep EBV infection in check, exhibited reduced cytotoxicity owing to their lower expression of the cell surface receptor NKG2D, which triggers cytolysis upon ligation. Magnesium supplementation in vitro and also in two XMEN patients restored levels of free Mg2+, increased NKG2D expression, and resulted in reduced amounts of EBV+ cells, suggesting that this may be an effective therapeutic approach for XMEN patients. Magnesium supplementation in patients with a primary immunodeficiency restores immune responses to Epstein-Barr virus. The magnesium transporter 1 (MAGT1) is a critical regulator of basal intracellular free magnesium (Mg2+) concentrations. Individuals with genetic deficiencies in MAGT1 have high levels of Epstein-Barr virus (EBV) and a predisposition to lymphoma. We show that decreased intracellular free Mg2+ causes defective expression of the natural killer activating receptor NKG2D in natural killer (NK) and CD8+ T cells and impairs cytolytic responses against EBV. Notably, magnesium supplementation in MAGT1-deficient patients restores intracellular free Mg2+ and NKG2D while concurrently reducing EBV-infected cells in vivo, demonstrating a link between NKG2D cytolytic activity and EBV antiviral immunity in humans. Moreover, these findings reveal a specific molecular function of free basal intracellular Mg2+ in eukaryotic cells.

MerCASBA: an updated and refined database of caspase substrates

In this correspondence, we describe a significant update and expansion of the original CASBAH (CAspase Substrate dataBAse Homepage) created by Luthi and Martin [1]. CASBAH contained both putative (not formally identified in the cited reference(s)) and experimentally confirmed caspase substrates. In MerCASBA (MERck CAspase Substrate dataBAse), we discarded putative entrants and reviewed the literature sources of all other entrants to retain only experimentally confirmed sites. Additionally, minor errors inadvertently introduced in CASBAH, including mistyped substrate sequences, amino acid position numbers, and UNIPROT IDs were corrected. We also added caspase recognition sequences identified in recent papers using unbiased proteomic approaches [2, 3]. Finally, we undertook a comprehensive analysis of scientific literature published since CASBAH to identify novel substrates. The focus on experimentally validated cleavage sites distinguishes this study from other recent work [4]. The updated database, MerCASBA, comprises 724 caspase cleavage sites from 558 unique substrates and represents a significant (485 %) increase over the 149 experimentally confirmed caspase recognition sites in the original version of CASBAH [1]. Importantly, we have included extended information about the flanking regions: two additional amino acids at the N-terminal side (P5 and P6) and 4 amino acids after the cleavage site (P10–P40) (Fig. 1a). We compared the amino acid frequency at every position between P6–P40 in MerCASBA versus their distribution in the human proteome (Fig. 1b). One striking observation is the paucity of either Glu or Pro at position P10—each is under-represented more than tenfold (p \ 0.001). Our analysis of all 724 caspase cleavage sites within intact proteins confirmed an earlier study [5], which also found that Pro and Glu were the least preferred amino acids at P10. The inefficient processing of potential caspase substrates containing Pro at P10 is supported by experimental data: caspase 3 does not cleave in vitro between Asp and Pro of DMMDP in syntaxin-5, despite its location in an exposed loop region (Fig. 4 of [6]). Our observation that Pro is not favored at the P10 position similarly argues against it being a recognition site for caspase cleavage. Salvesen et al., adopting a peptide/purified caspase model [5], reported that small amino acids are preferred at the P10 position of caspase substrates. Analysis of the MerCASBA database confirms this; for example Gly at position P10 is Arthur Fridman, Irene Pak and Brent Butts have contributed equally to this work.

TLR4, TLR7/8 agonist-induced miR-146a promotes macrophage tolerance to MyD88-dependent TLR agonists

TLRs facilitate the recognition of pathogens by immune cells and the initiation of the immune response, leading to the production of proinflammatory cytokines and chemokines. Production of proinflammatory mediators by innate immune cells, such as macrophages, is tightly regulated to facilitate pathogen clearance while limiting an adverse impact on host tissue. Exposure of innate immune cells to TLR ligands induces a state of temporary refractoriness to a subsequent exposure of a TLR ligand, a phenomenon referred to as “tolerance.” This study sought to evaluate the mechanistic regulation of TLR4 and TLR7/8 ligand‐induced tolerance to other TLRs by microRNA‐146a. With the use of THP‐1 macrophages, as well as human classic and alternative macrophages, we demonstrate that priming with a TLR4 agonist (LPS) or a TLR7/8 agonist (R848) induces homologous and heterologous tolerance to various TLR ligands in macrophages, leading to the impaired production of cytokines and chemokines. We also demonstrate that overexpression of microRNA‐146a is sufficient to mimic LPS or R848‐induced hyporesponsiveness. Conversely, inhibition of microRNA‐146a activity leads to LPS‐ or R848‐induced TLR hyper‐responsiveness in TLR signaling tolerance. Furthermore, we demonstrate that microRNA‐146a dampens cytokine production following a primary stimulus with MyD88‐dependent but not MyD88‐independent TLR pathways. Collectively, these data provide comprehensive evidence of the central role of microRNA‐146a in TLR signaling tolerance to plasma membrane, as well as endosomal TLR ligands in human macrophages.

Insulin-like growth factor-II and heparin are anti-apoptotic survival factors in human villous cytotrophoblast.

OBJECTIVE This study aimed to determine the effects of insulin-like growth factors (IGF-I and IGF-II), heparin, aspirin and vitamin C on the proliferation and apoptosis of human villous cytotrophoblast from first trimester and term placentae. STUDY DESIGN Villous cytotrophoblast cells were isolated from uncomplicated first trimester (n=12) and term placental tissues (n=12) using negative immunoselection with an antibody to HLA class I antigens. Cells were incubated with IGF-I, IGF-II, heparin, aspirin and vitamin C either alone, or in combination with either TNF-α/IFN-γ or staurosporine. Proliferation was determined by measurement of Ki67 expression using immunocytochemistry. Trophoblast apoptosis was determined by TUNEL staining. Finally RT-PCR was carried out to identify IGF-binding insulin receptor isoforms. Data were expressed as means±SEM. One way analysis of variance (ANOVA) with Bonferroni correction was used to determine if differences between groups were statistically significant. RESULTS Following negative immunoselection >98% of cells were positively stained for cytokeratin 7, a marker for cytotrophoblasts, and <1% were vimentin positive. First trimester and term trophoblasts underwent spontaneous apoptosis which was inhibited by approximately 50% in the presence of IGF-II or heparin. Apoptosis was significantly increased following incubation with a combination of TNF-α and IFN-γ or staurosporine. Apoptosis was decreased to basal levels following coincubation with IGF-II or heparin. Incubation with IGFs or heparin resulted in a small, but significant increase in Ki67 expression. Insulin receptor isoform A, which binds IGF-II with high affinity, was present in all trophoblast samples tested. CONCLUSION These results suggest that heparin and IGF-II, but not IGF-I are important regulators of villous cytotrophoblast survival in early and late pregnancy.

Pharmacological Inhibition of O-GlcNAcase Does Not Increase Sensitivity of Glucocorticoid Receptor-Mediated Transrepression

Glucocorticoid signaling regulates target genes by multiple mechanisms, including the repression of transcriptional activities of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) though direct protein-protein interactions and subsequent O-GlcNAcylation of RNA polymerase II (pol II). Recent studies have shown that overexpression of O-linked β-N-acetylglucosamine transferase (OGT), which adds an O-linked β-N-acetylglucosamine (O-GlcNAc) group to the C-terminal domain of RNA pol II, increases the transrepression effects of glucocorticoids (GC). As O-GlcNAcase (OGA) is an enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, we hypothesized that the potentiation of GC effects following OGT overexpression could be similarly observed via the direct inhibition of OGA, inhibiting O-GlcNAc removal from pol II. Here we show that despite pharmacological evidence of target engagement by a selective small molecule inhibitor of OGA, there is no evidence for a sensitizing effect on glucocorticoid-mediated effects on TNF-α promoter activity, or gene expression generally, in human cells. Furthermore, inhibition of OGA did not potentiate glucocorticoid–induced apoptosis in several cancer cell lines. Thus, despite evidence for O-GlcNAc modification of RNA pol II in GR-mediated transrepression, our data indicate that pharmacological inhibition of OGA does not potentiate or enhance glucocorticoid-mediated transrepression.

Comparison of the In Vivo Distribution of Four Different Annexin A5 Adducts in Rhesus Monkeys

Annexin A5 has been used for the detection of apoptotic cells, due to its ability to bind to phosphatidylserine (PS). Four different labeled Annexin A5 adducts were evaluated in rhesus monkey, with radiolabeling achieved via 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Of these adducts differing conjugation methods were employed which resulted in nonspecific radiolabeling (AxA5-I), or site-specific radiolabeling (AxA5-II). A nonbinding variant of Annexin A5 was also evaluated (AxA5-IINBV), conjugation here was site specific. The fourth adduct examined had both specific and nonspecific conjugation techniques employed (AxA5-IImDOTA). Blood clearance for each adduct was comparable, while appreciable uptake was observed in kidney, liver, and spleen. Significant differences in uptake of AxA5-I and AxA5-II were observed, as well as between AxA5-II and AxA5-IINBV. No difference between AxA5-II and AxA5-IImDOTA was observed, suggesting that conjugating DOTA nonspecifically did not affect the in vivo biodistribution of Annexin A5.

Emerging therapies in the treatment of rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease with a significant morbidity defined by marked destruction and deformity of joints. It is characterized by autoantibody production, synovial inflammation, and erosion of the cartilage and bone. The current first‐line treatment for RA is methotrexate (MTX), an orally active disease‐modifying anti‐rheumatic drug (DMARD). Biologic DMARDs that target tumor necrosis factor (TNF) or other molecules have emerged as potent alternative therapies for patients with inadequate response to MTX therapy. Despite the huge success of MTX and/or biologics, there is still a significant unmet medical need in RA. Approximately one‐third of RA patients are nonresponsive to currently available therapies. With their critical roles in mediating multiple inflammatory pathways, small‐molecule tyrosine kinase (TK) inhibitors are gaining attention as candidates for oral RA drugs with positive outcomes for a number of late‐stage clinical trials of small‐molecule Jak (Tasocitinib) or Syk (fostamatinib) inhibitors. With the potential for attenuating multiple inflammatory pathways activated in RA, tasocitinib and fostamatinib may represent new and welcome additions to the RA therapeutic landscape. Drug Dev Res 72:805–816, 2011.