Prevalence of Bacterial Contamination of Casting Material in a Pediatric Population.

Surgical site infection is a relatively common and devastating complication following pediatric orthopedic surgery. Many infections have been determined to be the result of settled airborne particles on surgical equipment and the sterile field. Fiberglass casts are commonly used orthopedic fixation devices before and after surgery; however, fiberglass casting material is expelled during the removal process and represents an uninvestigated area for the possibility of cast saw dust as a source of airborne bacterial contamination in an operating room setting. This study evaluates the prevalence and distribution of microbiota on 90 pediatric casts by collecting and culturing fiberglass cast material from 90 pediatric casts. Bacterial identification was performed using a Bruker Biotyper Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry device. 81 out of 90 casts (90%) showed evidence of microbial contamination. Isolated species were very diverse and ranged from normal skin flora to opportunistic pathogens. The 5 most commonly isolated organisms were Acinetobacter pittii, Enterobacter cloacae, Micrococcus luteus, Staphylococcus epidermidis, and Staphylococcus hominis. Further investigation is required to determine if casting material is truly a cause of surgical site infection.

Taurine enhances the growth of neural precursors derived from fetal human brain and promotes neuronal specification.

Taurine is present at high concentrations in the fetal brain and is required for optimal brain development. Recent studies have reported that taurine causes increased proliferation of neural stem/progenitor neural cells (neural precursor cells, NPCs) obtained from embryonic and adult rodent brain. The present study is the first to show that taurine markedly increases cell numbers in cultures and neuronal generation from human NPCs (hNPCs). hNPCs obtained from 3 fetal brains (14-15 weeks of gestation) were cultured and expanded as neurospheres, which contained 76.3% nestin-positive cells. Taurine (5-20 mM) increased the number of hNPCs in culture, with maximal effect found at 10 mM and 4 days of culture. The taurine-induced increase ranged from 57 to 188% in the 3 brains examined. Taurine significantly enhanced the percentage of neurons formed from hNPCs under differentiating conditions, with increases ranging from 172 to 480% over controls without taurine. Taurine also increased the cell number and neuronal generation in cultures of the immortalized human cell line ReNcell VM. These results suggest that taurine has a positive influence on hNPC growth and neuronal formation.

Wnt pathway activity confers chemoresistance to cancer stem-like cells in a neuroblastoma cell line.

Neuroblastoma is the most common solid tumor in infancy. We have shown that the neuroblastoma cell line SK-N-SH contains CD133+ cells that are more resistant than 133- cells to Doxorubicin (DOX), a common chemotherapeutic agent. We hypothesize that activation of wnt signaling pathway in CD133+ cells contributes to their chemoresistance. To test this hypothesis, CD133+ cells were positively selected using magnetic micro-beads. Subsequently, CD133+ and negatively selected CD133- cells were treated with 100 ng/ml of DOX for up to 72 h. Then, cells were either lysed for total RNA extraction or fixed for immunostaining. Wnt "SIGNATURE" PCR Array was used to determine if changes in wnt related gene expression levels occurred and to estimate a pathway activity score. Expression of wnt pathway proteins ß-Catenin and p-GSK3ß (S-9) was determined by immunocytochemistry. Two wnt pathway inhibitors were used to determine the changes in cell viability, using the MTT assay. Results showed that wnt related genes were differentially expressed in CD133+ cells as compared to CD133- cells, both with and without DOX treatment. Pathway activity scores showed that DOX treatment significantly suppressed the wnt pathway activity in CD133- cells. Expression of ß-catenin and p-GSK3ß (S-9) was significantly greater in DOX treated and untreated CD133+ cells. The presence of wnt inhibitors with DOX decreased the number of live cells in CD133+ group and the percentage of live cells in both groups were equal. These data suggest that higher wnt pathway activity could be responsible for the chemoresistance of CD133+ cells in neuroblastoma cell lines.

Ethanol affects differentiation-related pathways and suppresses Wnt signaling protein expression in human neural stem cells.

BACKGROUND: Prenatal exposure of the fetus to ethanol (EtOH) can be teratogenic. We previously showed that EtOH alters the cell fate of human neural stem cells (NSC). As Wnt signaling plays an important role in fetal brain development, we hypothesized that EtOH suppresses Wnt signaling protein expression in differentiating NSC and thereby contributes to fetal alcohol spectrum disorder. METHODS: NSC isolated from fetal human brains were cultured in mitogenic media to induce neurospheres, which were dissociated into single-cell suspensions and used for all experiments. Equal numbers of NSC were cultured on lysine/laminin-coated plates for 96 hours in differentiating media containing 0, 20, or 100 mM EtOH. Total mRNA was isolated from samples containing 0 or 100 mM EtOH and changes in expression of 263 genes associated with neurogenesis and NSC differentiation were determined by Oligo GEArray technology. The biological impact of gene changes was estimated using a systems biology approach with pathway express software and KEGG database. Based on the pathways identified, expression of Wnt proteins (Wnt3a and Wnt5a), Wnt-receptor complex proteins (p-LRP6, LRP6, DVL2, and DVL3), Wnt antagonist Naked-2 (NKD-2), and downstream Wnt proteins (ß-catenin, Tyr-p-GSK3ß, Ser-p-GSK3ß) were analyzed by Western blot. RESULTS: Of the 263 genes examined, the expressions of 22 genes in differentiating NSC were either upwardly or downwardly affected by EtOH. These genes are associated with 5 pathways/cellular processes: axon guidance; hedgehog signaling; TGF-ß signaling; cell adhesion molecules; and Wnt signaling. When compared to controls, EtOH, at both 20 and 100 mM concentrations, suppressed the expression of Wnt3a and Wnt5a, receptor complex proteins p-LRP6, LRP6 and DVL2, and cytoplasmic proteins Ser-p-GSK3ß and ß-catenin. Expression of NKD-2 and DVL3 remained unchanged and the expression of active Tyr-p-GSK3ß increased significantly. CONCLUSIONS: EtOH can significantly alter neural differentiation pathway-related gene expression and suppress Wnt signaling proteins in differentiating human NSC.

Ethanol alters cell fate of fetal human brain-derived stem and progenitor cells.

BACKGROUND: Prenatal ethanol (ETOH) exposure can lead to fetal alcohol spectrum disorder (FASD). We previously showed that ETOH alters cell adhesion molecule gene expression and increases neurosphere size in fetal brain-derived neural stem cells (NSC). Here, our aim was to determine the effect of ETOH on the cell fate of NSC, premature glial-committed precursor cells (GCP), and premature neuron-committed progenitor cells (NCP). METHODS: NSC, GCP, and NCP were isolated from normal second-trimester fetal human brains (n = 3) by positive selection using magnetic microbeads labeled with antibodies to CD133 (NSC), A2B5 (GCP), or PSA-NCAM (NCP). As a result of the small percentage in each brain, NSC were cultured in mitogenic media for 72 hours to produce neurospheres. The neurospheres from NSC and primary isolates of GCP and NCP were used for all experiments. Equal numbers of the 3 cell types were treated either with mitogenic media or with differentiating media, each containing 0 or 100 mM ETOH, for 120 hours. Expression of Map2a, GFAP, and O4 was determined by immunoflourescence microscopy and western blot analysis. Fluorescence intensities were quantified using Metamorph software by Molecular Devices, and the bands of western blots were quantified using densitometry. RESULTS: ETOH in mitogenic media promoted formation of neurospheres by NSC, GCP, and NCP. Under control conditions, GCP attached and differentiated, NSC and NCP formed neurospheres that were significantly smaller in size than those in ETOH. Under differentiating conditions, Map2a expression increased significantly in NSC and GCP and reduced significantly in NCP, and GFAP expression reduced significantly in GCP and NCP, and Gal-C expression reduced significantly in all 3 cell types in the presence of ETOH compared to controls. CONCLUSIONS: This study shows that ETOH alters the cell fate of neuronal stem and progenitor cells. These alterations could contribute to the mechanism for the abnormal brain development in FASD.

Resistance of stem-like cells from neuroblastoma cell lines to commonly used chemotherapeutic agents.

BACKGROUND: Cancer stem cell theory suggests that the presence of tumor initiating stem-like cells in cancers may be responsible for cancer progression and relapse. CD133 cell surface maker expression has been used to identify stem-like cells in cancer cell lines. Our goal was to identify such cells in neuroblastoma cell lines and to study the cytotoxicity of common anticancer drugs for those cells. MATERIALS AND METHODS: CD133+ cells from SK-N-SH and SK-N-BE cell lines were isolated using magnetic microbeads. Cytotoxicity of four anticancer drugs was studied on CD133+ and CD133- populations. The percentage of live, apoptotic, and dead cells in each population after drug treatment was estimated by MTT and PI/Annexin-binding assays. Western blot analyses were used to identify differences in the expression of kinases. RESULTS: Eight to 10% of SK-N-SH and 3-5% of SK-N-BE cells were CD133+. These cells were more resistant than CD133- cells to all four chemotherapeutic agents tested in the MTT assay. Decreased apoptosis was observed in CD133+ cells compared to CD133- cells by PI/Annexin V-binding assay. Western blot analysis showed that CD133+ cells expressed less MKP-1. Phosphorylated forms of both ERK and P-38 kinases were expressed at higher levels in CD133+ cells than in CD133- cells. CONCLUSIONS: This study suggests that CD133+ cells are more resistant to anticancer drugs than CD133- cells. Differences in the expression and phosphorylation of kinases could be partially responsible for this difference. Targeting CD133-expressing cells could be a strategy to develop more effective treatments for neuroblastoma.

Adipogenic cascade can be induced without adipogenic media by a human adenovirus.

Several metabolic abnormalities are associated with relative excess or deficiency of adipose tissue. Identifying the regulators of adipogenic differentiation is critical for its successful manipulation. Ad36, a human adenovirus, is a novel factor that promotes adipogenesis. We exploited the adipogenic potential of Ad36 to reveal exogenous modifiers of adipogenesis in rodent preadipocyte cell line in the presence or absence of differentiation inducers methyl-isobutyl-xanthine, dexamethasone, and insulin (M, D, and I; MDI). A nonadipogenic human adenovirus Ad2 was used as a negative control for viral infection. First, we confirmed that, Ad36, but not Ad2, increases lipid accumulation in the presence or absence of MDI. Time-course studies for expression of key genes of adipogenic cascade showed that it is Ad36, but not Ad2, which downregulated preadipocyte marker gene Wnt10b, and upregulated expression of early (C/EBPDelta and C/EBPbeta), intermediate (PPARgamma2), and late genes (aP2 and G3PDH) of adipogenic cascade even in the absence of MDI. In the presence of MDI, onset of expression of adipogenic genes coincided for Ad36 and control groups, but the expressions were significantly greater for the Ad36 group. Next, we observed that attenuation of Ad36 mRNA expression by an antiadenoviral agent reduced 3T3-L1 differentiation, indicating that viral mRNA expression is required for the process. Furthermore, with or without MDI or its components, Ad36 significantly increased lipid accumulation in 3T3-L1 cells. Cell confluency at the time of Ad36 infection positively influenced lipid accumulation. The results reveal that Ad36 is an MDI-independent exogenous regulator of the adipogenic process. Elucidating the molecular pathways involved may reveal novel regulatory controls of adipogenesis.

Ethanol increases fetal human neurosphere size and alters adhesion molecule gene expression.

BACKGROUND: Ethanol (ETOH) consumption by pregnant women can result in Fetal Alcohol Spectrum Disorder (FASD). To date, the cellular targets and mechanisms responsible for FASD are not fully characterized. Our aim was to determine if ETOH can affect fetal human brain-derived neural progenitor cells (NPC). METHODS: Neural progenitor cells were isolated by positive selection from normal second trimester fetal human brains (n = 4) and cultured, for up to 72 hours, in mitogenic media containing 0, 1, 10, or 100 mM ETOH. From 48 to 72 hours in culture, neurospheres generated in these conditions were filmed using time-lapse video microscopy. At the end of 72 hours, neurosphere diameter and roundness were measured using videographic software. Mitotic phase analysis of cell-cycle activity and apoptotic cell count were also performed at this time, by flow cytometry using propidium iodide (PI) staining. Real-time PCR was used to estimate expression of genes associated with cell adhesion pathways. RESULTS: Neurosphere diameter correlated positively (r = 0.87) with increasing ETOH concentrations. There was no significant difference in cell-cycle activity and no significant increase in apoptosis with increasing ETOH concentrations. Time-lapse video microscopy showed that ETOH (100 mM) reduced the time for neurosphere coalescence. Real-time PCR analysis showed that ETOH significantly altered the expression of genes involved in cell adhesion. There was an increase in the expression of alpha and beta Laminins 1, beta Integrins 3 and 5, Secreted phosphoprotein1 and Sarcoglycan epsilon. No change in the expression of beta Actin was observed while the expression of beta Integrin 2 was significantly suppressed. CONCLUSIONS: ETOH had no effect on NPC apoptosis but, resulted in more rapid coalescence and increased volume of neurospheres. Additionally, the expression of genes associated with cell adhesion was significantly altered. ETOH induced changes in NPC surface adhesion interactions may underlie aspects of neurodevelopmental abnormalities in FASD.

A human adenovirus enhances preadipocyte differentiation.

OBJECTIVE: Adenovirus 36 (Ad-36) has been shown to increase adiposity in experimentally infected chickens, mice, and marmosets (nonhuman primates). Neutralizing antibodies to Ad-36 are associated with obesity in humans. The metabolic and molecular mechanisms responsible for Ad-36-induced adipogenesis are unknown. As a potential adipogenic mechanism, this study examined if Ad-36 enhanced differentiation of preadipocytes. RESEARCH METHODS AND PROCEDURES: To determine the suitability of 3T3-L1 cells (murine preadipocyte cell line) as a model, the first experiment determined if Ad-36 attaches and initiates replication in the cells. Next, effects of Ad-36 on the number of differentiated adipocytes, glycerol 3-phosphate dehydrogenase (GPDH) levels, and cellular lipid accumulation were determined. The last experiment determined the effect of Ad-36 on human primary preadipocyte differentiation. Ad-2, a known nonadipogenic human adenovirus, was used as a negative control in these experiments. RESULTS: Immunofluorescence studies showed adenoviral attachment to 3T3-L1 cells, and reverse transcriptase-polymerase chain reaction showed expression of the Ad-36 E1A gene in the infected cells. Ad-36, but not Ad-2, increased the number of differentiated adipocytes, GPDH enzyme levels, and the total cellular lipid content. Also, Ad-36, but not Ad-2, increased GPDH levels in human preadipocytes. DISCUSSION: Taken together, these experiments showed that Ad-36 enhanced differentiation of preadipocytes, which may be a contributory mechanism to its adipogenic effect in vivo. The lack of effect of Ad-2 on differentiation demonstrated that the observed findings were not a common characteristic of all adenoviruses. Future understanding of the molecular interactions of cellular and viral genes responsible for enhanced differentiation may reveal novel signaling pathways and controls of preadipocyte differentiation.