An AscI boundary library for the studies of genetic and epigenetic alterations in CpG islands

Knudson's two-hit hypothesis postulates that genetic alterations in both alleles are required for the inactivation of tumor-suppressor genes. Genetic alterations include small or large deletions and mutations. Over the past years, it has become clear that epigenetic alterations such as DNA methylation are additional mechanisms for gene silencing. Restriction Landmark Genomic Scanning (RLGS) is a two-dimensional gel electrophoresis that assesses the methylation status of thousands of CpG islands. RLGS has been applied successfully to scan cancer genomes for aberrant DNA methylation patterns. So far, the majority of this work was done using NotI as the restriction landmark site. Here, we describe the development of RLGS using AscI as the restriction landmark site for genome-wide scans of cancer genomes. The availability of AscI as a restriction landmark for RLGS allows for scanning almost twice as many CpG islands in the human genome compared with using NotI only. We describe the development of an AscI-EcoRV boundary library that supports the cloning of novel methylated genes. Feasibility of this system is shown in three tumor types, medulloblastomas, lung cancers, and head and neck cancers. We report the cloning of 178 AscI RLGS fragments via two methods by use of this library.     

Hemolytic uremic syndrome after bone marrow transplantation: clinical characteristics and outcome in children.

Hemolytic uremic syndrome (HUS) is an uncommon but potentially life-threatening complication of hematopoietic stem cell transplantation. We retrospectively studied the medical records of 293 children who underwent allogeneic bone marrow transplantation at St. Jude Children's Research Hospital between 1992 and 1999 to describe the clinical course of and to identify risk factors for transplant-associated HUS. Conditioning regimens included cyclophosphamide, cytarabine, and total body irradiation for patients with hematologic malignancies (n = 244); patients with nonmalignant diseases (n = 49) received disease-specific regimens. Grafts from unrelated or mismatched related donors were depleted of T lymphocytes, whereas matched sibling grafts were unmanipulated. All patients received cyclosporine as prophylaxis for graft-versus-host disease. Recipients of grafts from matched siblings also received pentoxifylline or short-course methotrexate. HUS developed in 28 (9.6%) patients at a median of 171 days after transplantation. We identified older donor age (P = .029), use of antithymocyte globulin in the conditioning regimen (P = .008), and recipient CMV seronegativity (P = .011) as being associated with an increased risk of HUS. With a multiple regression analysis, the use of antithymocyte globulin (beta = .86; P = .04) and recipient cytomegalovirus seronegativity (beta = .93; P = .035) remained significant risk factors for the development of HUS.     

Increased circulating interleukin-7 levels in HIV-1-infected women

Sex-based differences in CD4 T-cell (CD4) counts are well recognized, but the basis for these differences has not been identified. Conceivably, homeostatic factors may play a role in this process by regulating T-cell maintenance and repletion. Interleukin (IL)-7 is essential for normal T-cell production and homeostasis. We hypothesized that differences in IL-7 might contribute to sex-based differences in CD4 counts. Circulating IL-7 levels were analyzed in 299 HIV-1-infected women and men. Regression analysis estimated that IL-7 levels were 40% higher in women than in men (P = 0.0032) after controlling for CD4 count, age, and race. Given the important role of IL-7 in T-cell development and homeostasis, these findings suggest that higher IL-7 levels may contribute to higher CD4 counts in women.     

Violence exposure,catecholamine excretion,and blood pressure nondipping status in African American male versus female adolescents

OBJECTIVE: Nondipping status (<10% decrease in blood pressure [BP] from awake to asleep) has been associated with end-organ disease (stroke and left ventricular hypertrophy) in adults. Nondipping status has also been observed in 30% of healthy African American adolescents, but little is known about the correlates of nondipping status in adolescents. This study examined the relationship between violence exposure, catecholamine excretion, and BP nondipping status in 56 healthy African American adolescents (27 boys, 29 girls; ages 11-18 years). METHODS: Participants completed the Survey of Exposure to Community Violence, wore an ambulatory BP monitor and provided one timed day and night urine collection for determination of epinephrine and norepinephrine excretion. RESULTS: Boys had higher daytime epinephrine (5.1 +/- 3.3 vs. 2.6 +/- 2.3 ng/min, p < .001) and norepinephrine excretion (29.2 +/- 25.1 vs. 16.5 +/- 14.9 ng/min, p < .05) and showed a greater prevalence of mean BP nondipping status than girls (37% vs. 10%, p < .03). Mean BP nondipping status was positively associated with victimization (r = 0.42, p < .0001). Regression analyses indicated a significant interaction between hearing about violence and sex for predicting daytime epinephrine (p < .02), with male nondippers showing a stronger positive association (partial correlation = 0.59, p < .05) than females (partial correlation = 0.03, p = NS). Logistic regressions also demonstrated a significant interaction between hearing about violence and sex for predicting mean BP dipping status, with male nondippers reporting the greatest exposure. CONCLUSIONS: Mean BP nondipping was associated with victimization in both boys and girls. Boys who reported higher levels of hearing about violence showed greater daytime epinephrine excretion and were more likely to be classified as nondippers.     

Detecting danger--or just another odorant? Olfactory sensitivity for the fox odor component 2,4,5-trimethylthiazoline in four species of mammals

2,4,5-trimethylthiazoline (TMT) is a volatile component of the anal gland secretion of the red fox and elicits behavioral and physiological fear responses in the rat. Using instrumental conditioning paradigms, we determined olfactory detection thresholds for TMT in three rats, a natural prey species of the red fox, and compared their performance to that of three squirrel monkeys, three spider monkeys and four pigtail macaques, all non-prey species of the red fox. We found that the rats were able to discriminate concentrations between 0.04 and 0.10 ppt (parts per trillion) of TMT from the odorless solvent which is by far the lowest olfactory detection threshold for an odorant reported in rats so far. In contrast, the spider monkeys needed 0.14-1.38 ppb (parts per billion), the pigtail macaques 0.41-4.07 ppb, and the squirrel monkeys 4.07-13.80 ppb to detect TMT which does not rank among the lowest olfactory thresholds reported for these three primate species. Thus, these results support the assumption that the behavioral relevance of an odorant may be an important determinant of a species' olfactory sensitivity.     

Adsorption of serum fetuin to hydroxylapatite does not contribute to osteoblast phenotype modifications

Osteoblasts exhibit enhanced differentiation and altered gene profiles when cultured on hydroxyapatite (HA) compared to plastic surfaces. To begin determining mechanisms for this response, we used proteomics to identify proteins predominantly found in osteoblasts on HA but not plastic surfaces. Two-dimensional gel electrophoresis and Western analyses indicate that fetuin is abundant in extracts from HA, but not plastic surfaces. Incubation of HA and plastic surfaces with cell culture medium (containing 10% serum) under cell-free conditions shows that fetuin is predominantly derived from the culture medium serum and readily adsorbs to the HA surface. However, we did detect low levels of fetuin B mRNA in osteoblasts. Serum albumin, actin-beta, apolipoprotein-AI, and vimentin also adsorbed to HA. To determine the role of fetuin in the HA-induced osteoblast phenotype changes, osteoblasts were seeded onto fetuin-coated or uncoated HA under serum-free conditions. Osteoblast morphology was similar on both HA surfaces, suggesting that HA alone (without adsorbed serum proteins) is sufficient for cell attachment and spreading. Similarly, genes previously reported to be modulated by HA (glvr-1, DMP-1, osteoglycin, and proliferin 3) were modulated even in the absence of fetuin or other serum proteins. These data show that HA surface can be enriched selectively with fetuin from serum; however, neither fetuin or other serum proteins are required to mediate HA-induced osteoblast attachment, spreading, or changes in expression of genes examined. This finding suggests that factors intrinsic to HA are required for the response.     

Analysis of aberrant somatic hypermutation (SHM) in non-Hodgkin's lymphomas of patients with chronic HCV infection

Hepatitis C virus (HCV) and aberrant somatic hypermutation (SHM) have each been suggested to contribute to the development of B-cell non-Hodgkin's lymphoma (NHL). The incidence of PIM-1, PAX-5, RhoH/TTF, and c-MYC mutations in tumour biopsy specimens from 32 HCV-infected B-cell NHL patients was analysed to determine whether the extent of aberrant SHM among these patients differed from that previously reported for HCV-negative B-cell NHL patients. Mutation of PIM-1, PAX-5, RhoH/TTF, and c-MYC was detected in 4 (13%), 5 (16%), 4 (13%), and 4 (13%) of 32 samples, respectively. In HCV-positive B-cell NHL patients, the frequency of aberrant SHM was lower than that already found in HCV-negative B-cell NHL patients. This indicates that, unlike B-cell lymphomas from HCV-negative patients, aberrant SHM may not contribute significantly to malignant transformation in HCV-associated B-cell lymphomas.     

Homeostasis and the age-associated defect of CD4 T cells

Survival and homeostatic division of naive CD4 T cells is regulated by the cellular and non-cellular milieu and together these processes ensure that a population of naive CD4 T cells persists into old age. However, the naive CD4 T cells from aged animals show reduced IL-2 production, proliferation, helper function and effector generation and memory function. We explore here whether the age-related defects in naive CD4 T cells are due to the aged environment from which they come or to intrinsic defects that are caused by homeostasis and their long lifespan.     

Activation of transforming growth factor-beta1/p38/Smad3 signaling in stromal cells requires reactive oxygen species-mediated MMP-2 activity during bone marrow damage

Dose-escalated chemotherapy has proven utility in a variety of treatment settings, including preparative regimens before bone marrow or hematopoietic stem cell transplantation. However, the potential damage imposed by aggressive regimens on the marrow microenvironment warrants further investigation. In the present study, we tested the hypothesis that dose-escalated chemotherapy, with etoposide as a model chemotherapeutic agent, activates the transforming growth factor beta-1 (TGF-beta1) signaling pathway in bone marrow stromal cells. After high-dose etoposide exposure in vitro, Smad3 protein was phosphorylated in a time-and dose-dependent manner in marrow-derived stromal cells, coincident with the release of active and latent TGF-beta1 from the extracellular matrix. Phosphorylation was modulated by p38 kinase, with translocation of Smad3 from the cytoplasm to the nucleus subsequent to its phosphorylation. Etoposide induced activation of TGF-beta1 followed the generation of reactive oxygen species and required matrix metalloproteinase-2 (MMP-2) protein availability. Chemotherapy effects were diminished in MMP-2(-/-) knockout stromal cells and TGF-beta1 knockdown small interfering RNA-transfected stromal cells, in which phosphorylation of Smad3 was negligible after etoposide exposure. Stable transfection of a human MMP-2 cDNA into bone marrow stromal cells resulted in elevated phosphorylation of Smad3 during chemotherapy. These data suggest TGF-beta1/p38/Smad3 signaling cascades are activated in bone marrow stromal cells after dose-escalated chemotherapy and may contribute to chemotherapy-induced alterations of the marrow microenvironment.