Michelle Lacey

In Vitro High‐Capacity Assay to Quantify the Clonal Heterogeneity in Trilineage Potential of Mesenchymal Stem Cells Reveals a Complex Hierarchy of Lineage Commitment

In regenerative medicine, bone marrow is a promising source of mesenchymal stem cells (MSCs) for a broad range of cellular therapies. This research addresses a basic prerequisite to realize the therapeutic potential of MSCs by developing a novel high‐capacity assay to quantify the clonal heterogeneity in potency that is inherent to MSC preparations. The assay utilizes a 96‐well format to (1) classify MSCs according to colony‐forming efficiency as a measure of proliferation capacity and trilineage potential to exhibit adipo‐, chondro‐, and osteogenesis as a measure of multipotency and (2) preserve a frozen template of MSC clones of known potency for future use. The heterogeneity in trilineage potential of normal bone marrow MSCs is more complex than previously reported: all eight possible categories of trilineage potential were detected. In this study, the average colony‐forming efficiency of MSC preparations was 55–62%, and tripotent MSCs accounted for nearly 50% of the colony‐forming cells. The multiple phenotypes detected in this study infer a more convoluted hierarchy of lineage commitment than described in the literature. Greater cell amplification, colony‐forming efficiency, and colony diameter for tri‐ versus unipotent clones suggest that MSC proliferation may be a function of potency. CD146 may be a marker of multipotency, with ∼2‐fold difference in mean fluorescence intensity between tri‐ and unipotent clones. The significance of these findings is discussed in the context of the efficacy of MSC therapies. The in vitro assay described herein will likely have numerous applications given the importance of heterogeneity to the therapeutic potential of MSCs. STEM CELLS 2010;28:788–798

Epstein-Barr Virus Latent Membrane Protein 1 Induces Cellular MicroRNA miR-146a, a Modulator of Lymphocyte Signaling Pathways

ABSTRACT The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a functional homologue of the tumor necrosis factor receptor family and contributes substantially to the oncogenic potential of EBV through activation of nuclear factor κB (NF-κB). MicroRNAs (miRNAs) are a class of small RNA molecules that are involved in the regulation of cellular processes such as growth, development, and apoptosis and have recently been linked to cancer phenotypes. Through miRNA microarray analysis, we demonstrate that LMP1 dysregulates the expression of several cellular miRNAs, including the most highly regulated of these, miR-146a. Quantitative reverse transcription-PCR analysis confirmed induced expression of miR-146a by LMP1. Analysis of miR-146a expression in EBV latency type III and type I cell lines revealed substantial expression of miR-146a in type III (which express LMP1) but not in type I cell lines. Reporter studies demonstrated that LMP1 induces miR-146a predominantly through two NF-κB binding sites in the miR-146a promoter and identified a role for an Oct-1 site in conferring basal and induced expression. Array analysis of cellular mRNAs expressed in Akata cells transduced with an miR-146a-expressing retrovirus identified genes that are directly or indirectly regulated by miR-146a, including a group of interferon-responsive genes that are inhibited by miR-146a. Since miR-146a is known to be induced by agents that activate the interferon response pathway (including LMP1), these results suggest that miR-146a functions in a negative feedback loop to modulate the intensity and/or duration of the interferon response.

MicroRNA-155 Is an Epstein-Barr Virus-Induced Gene That Modulates Epstein-Barr Virus-Regulated Gene Expression Pathways

ABSTRACT The cellular microRNA miR-155 has been shown to be involved in lymphocyte activation and is expressed in Epstein-Barr virus (EBV)-infected cells displaying type III latency gene expression but not type I latency gene expression. We show here that the elevated levels of miR-155 in type III latency cells is due to EBV gene expression and not epigenetic differences in cell lines tested, and we show that expression in EBV-infected cells requires a conserved AP-1 element in the miR-155 promoter. Gene expression analysis was carried out in a type I latency cell line transduced with an miR-155-expressing retrovirus. This analysis identified both miR-155-suppressed and -induced cellular mRNAs and suggested that in addition to direct targeting of 3′ untranslated regions (UTRs), miR-155 alters gene expression in part through the alteration of signal transduction pathways. 3′ UTR reporter analysis of predicted miR-155 target genes identified the transcriptional regulatory genes encoding BACH1, ZIC3, HIVEP2, CEBPB, ZNF652, ARID2, and SMAD5 as miR-155 targets. Western blot analysis of the most highly suppressed of these, BACH1, showed lower expression in cells transduced with a miR-155 retrovirus. Inspection of the promoters from genes regulated in EBV-infected cells and in cells infected with an miR-155 retrovirus identified potential binding sequences for BACH1 and ZIC3. Together, these experiments suggest that the induction of miR-155 by EBV contributes to EBV-mediated signaling in part through the modulation of transcriptional regulatory factors.

Genetic Requirements for the Survival of Tubercle Bacilli in Primates

BACKGROUND Tuberculosis (TB) leads to the death of 1.7 million people annually. The failure of the bacille Calmette-Guérin vaccine, synergy between AIDS and TB, and the emergence of drug resistance have worsened this situation. It is imperative to delineate the mechanisms employed by Mycobacterium tuberculosis to successfully infect and persist in mammalian lungs. METHODS Nonhuman primates (NHPs) are arguably the best animal system to model critical aspects of human TB. We studied genes essential for growth and survival of M. tuberculosis in the lungs of NHPs experimentally exposed to aerosols of an M. tuberculosis transposon mutant library. RESULTS Mutants in 108 M. tuberculosis genes (33.13% of all genes tested) were attenuated for in vivo growth. Comparable studies have reported the attenuation of only approximately 6% of mutants in mice. The M. tuberculosis mutants attenuated for in vivo survival in primates were involved in the transport of various biomolecules, including lipid virulence factors; biosynthesis of cell-wall arabinan and peptidoglycan; DNA repair; sterol metabolism; and mammalian cell entry. CONCLUSIONS Our study highlights the various virulence mechanisms employed by M. tuberculosis to overcome the hostile environment encountered during infection of primates. Prophylactic approaches aimed against bacterial factors that respond to such in vivo stressors have the potential to prevent infection at an early stage, thus likely reducing the extent of transmission of M. tuberculosis.

Adult human mesenchymal stem cells enhance breast tumorigenesis and promote hormone independence

Adult human mesenchymal stem cells (hMSCs) have been shown to home to sites of breast cancer and integrate into the tumor stroma. We demonstrate here the effect of hMSCs on primary breast tumor growth and the progression of these tumors to hormone independence. Co-injection of bone marrow-derived hMSCs enhances primary tumor growth of the estrogen receptor-positive, hormone-dependent breast carcinoma cell line MCF-7 in the presence or absence of estrogen in SCID/beige mice. We also show hormone-independent growth of MCF-7 cells when co-injected with hMSCs. These effects were found in conjunction with increased immunohistochemical staining of the progesterone receptor in the MCF-7/hMSC tumors as compared to MCF-7 control tumors. This increase in PgR expression indicates a link between MCF-7 cells and MSCs through ER-mediated signaling. Taken together, our data reveal the relationship between tumor microenvironment and tumor growth and the progression to hormone independence. This tumor stroma-cell interaction may provide a novel target for the treatment of estrogen receptor-positive, hormone-independent, and endocrine-resistant breast carcinoma.

Serum Cadmium Levels in Pancreatic Cancer Patients from the East Nile Delta Region of Egypt

The northeast Nile Delta region exhibits a high incidence of early-onset pancreatic cancer. It is well documented that this region has one of the highest levels of pollution in Egypt. Epidemiologic studies have suggested that cadmium, a prevalent pollutant in the northeast Nile Delta region, plays a role in the development of pancreatic cancer. Objective: We aimed to assess serum cadmium levels as markers of exposure in pancreatic cancer patients and noncancer comparison subjects from the same region in Egypt. Design and Participants: We assessed serum cadmium levels of 31 newly diagnosed pancreatic cancer patients and 52 hospital comparison subjects from Mansoura, Egypt. Evaluation/Measurements: Serum cadmium levels were measured using a novel immunoassay procedure. Results: We found a significant difference between the mean serum cadmium levels in patients versus comparison subjects (mean ± SD, 11.1 ± 7.7 ng/mL vs. 7.1 ± 5.0 ng/mL, respectively; p = 0.012) but not in age, sex, residence, occupation, or smoking status. The odds ratio (OR) for pancreatic cancer risk was significant for serum cadmium level [OR = 1.12; 95% confidence interval (CI), 1.04–1.23; p = 0.0089] and farming (OR = 3.25; 95% CI, 1.03–11.64; p = 0.0475) but not for age, sex, residence, or smoking status. Conclusions: The results from this pilot study suggest that pancreatic cancer in the East Nile Delta region is significantly associated with high levels of serum cadmium and farming. Relevance to Clinical Practice/Public Health: Future studies should further investigate the etiologic relationship between cadmium exposure and pancreatic carcinogenesis in cadmium-exposed populations.

DNA Hypomethylation and Hemimethylation in Cancer

In contrast to earlier views that there was much compartmentalization of the types of sequences subject to cancer-linked changes in DNA epigenetics, it is now clear that both cancer-associated DNA hypomethylation and hypermethylation are found throughout the genome. The hypermethylation includes promoters of tumor suppressor genes whose expression becomes repressed, thereby facilitating cancer formation. How hypomethylation contributes to carcinogenesis has been less clear. Recent insights into tissue-specific intra- and intergenic methylation and into cancer methylomes suggest that some of the DNA hypomethylation associated with cancers is likely to aid in tumor formation and progression by many different pathways, including effects on transcription in cis. Cancer-associated loss of DNA methylation from intergenic enhancers, promoter regions, silencers, and chromatin boundary elements may alter transcription rates. In -addition, cancer-associated intragenic DNA hypomethylation might modulate -alternative promoter usage, -production of intragenic noncoding RNA transcripts, cotranscriptional splicing, and transcription initiation or elongation. Initial studies of hemimethylation of DNA in cancer and many new studies of DNA demethylation in normal tissues suggest that active demethylation with spreading of hypomethylation can explain much of the cancer-associated DNA hypomethylation. The new discoveries that genomic 5-hydroxymethylcytosine is an intermediate in DNA demethylation, a base with its own functionality, and a modified base that, like 5-methylcytosine, exhibits cancer-associated losses, suggest that both decreased hydroxymethylation and decreased methylation of DNA play important roles in carcinogenesis.

Differences in Gastric Carcinoma Microenvironment Stratify According to EBV Infection Intensity: Implications for Possible Immune Adjuvant Therapy

Epstein-Barr virus (EBV) is associated with roughly 10% of gastric carcinomas worldwide (EBVaGC). Although previous investigations provide a strong link between EBV and gastric carcinomas, these studies were performed using selected EBV gene probes. Using a cohort of gastric carcinoma RNA-seq data sets from The Cancer Genome Atlas (TCGA), we performed a quantitative and global assessment of EBV gene expression in gastric carcinomas and assessed EBV associated cellular pathway alterations. EBV transcripts were detected in 17% of samples but these samples varied significantly in EBV coverage depth. In four samples with the highest EBV coverage (hiEBVaGC – high EBV associated gastric carcinoma), transcripts from the BamHI A region comprised the majority of EBV reads. Expression of LMP2, and to a lesser extent, LMP1 were also observed as was evidence of abortive lytic replication. Analysis of cellular gene expression indicated significant immune cell infiltration and a predominant IFNG response in samples expressing high levels of EBV transcripts relative to samples expressing low or no EBV transcripts. Despite the apparent immune cell infiltration, high levels of the cytotoxic T-cell (CTL) and natural killer (NK) cell inhibitor, IDO1, was observed in the hiEBVaGCs samples suggesting an active tolerance inducing pathway in this subgroup. These results were confirmed in a separate cohort of 21 Vietnamese gastric carcinoma samples using qRT-PCR and on tissue samples using in situ hybridization and immunohistochemistry. Lastly, a panel of tumor suppressors and candidate oncogenes were expressed at lower levels in hiEBVaGC versus EBV-low and EBV-negative gastric cancers suggesting the direct regulation of tumor pathways by EBV.

DNA methylation and differentiation: silencing, upregulation and modulation of gene expression.

Differentiation-related DNA methylation is receiving increasing attention, partly owing to new, whole-genome analyses. These revealed that cell type-specific differential methylation in gene bodies is more frequent than in promoters. We review new insights into the functionality of DNA methylation during differentiation, with emphasis on the methylomes of myoblasts, myotubes and skeletal muscle versus non-muscle samples. Biostatistical analyses of data from reduced representation bisulfite sequencing are discussed. Lastly, a model is presented for how promoter and intragenic DNA hypermethylation affect gene expression, including increasing the efficiency of polycomb silencing at some promoters, downmodulating other promoters rather than silencing them, counteracting enhancers with heterologous specificity, altering chromatin conformation by inhibiting the binding of CTCF, modulating mRNA transcript levels by inhibiting overlapping promoters of noncoding RNA genes or by regulating the use of alternative mRNA promoters, modulating transcription termination, regulating alternative splicing and acting as barriers to the spread of activating chromatin.

Decompressive craniectomy for elevated intracranial pressure and its effect on the cumulative ischemic burden and therapeutic intensity levels after severe traumatic brain injury.

BACKGROUNDIncreased intracranial pressure (ICP) can cause brain ischemia and compromised brain oxygen (PbtO2 ≤ 20 mm Hg) after severe traumatic brain injury (TBI). OBJECTIVEWe examined whether decompressive craniectomy (DC) to treat elevated ICP reduces the cumulative ischemic burden (CIB) of the brain and therapeutic intensity level (TIL). METHODSTen severe TBI patients (mean age, 31.4 ± 14.2 years) who had continuous PbtO2 monitoring before and after delayed DC were retrospectively identified. Patients were managed according to the guidelines for the management of severe TBI. The CIB was measured as the total time spent between a PbtO2 of 15 to 20, 10 to 15, and 0 to 10 mm Hg. The TIL was calculated every 12 hours. Mixed-effects models were used to estimate changes associated with DC. RESULTSDC was performed on average 2.8 days after admission. DC was found to immediately reduce ICP (mean [SEM] decrease was 7.86 mm Hg [2.4 mm Hg]; P = .005). TIL, which was positively correlated with ICP (r = 0.46, P ≤ .001), was reduced within 12 hours after surgery and continued to improve within the postsurgical monitoring period (P ≤ .001). The duration and severity of CIB were significantly reduced as an effect of DC in this group. The overall mortality rate in the group of 10 patients was lower than predicted at the time of admission (P = .015). CONCLUSIONThese results suggest that a DC for increased ICP can reduce the CIB of the brain after severe TBI. We suggest that DC be considered early in a patients clinical course, particularly when the TIL and ICP are increased.