Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of chronic CNS disorders, there is an increasing demand for murine models with high validity at the behavioral level. However, multiple pathogenic mechanisms and complex functional deficits often impose challenges to reliably measure and interpret behavior of chronically sick mice. Therefore, the assessment of peripheral pathology and a behavioral profile at several time points using a battery of tests are required. Video-tracking, behavioral spectroscopy, and remote acquisition of physiological measures are emerging technologies that allow for comprehensive, accurate, and unbiased behavioral analysis in a home-base-like setting. This report describes a refined phenotyping protocol, which includes a custom-made monitoring apparatus (Integrated Behavioral Station, INBEST) that focuses on prolonged measurements of basic functional outputs, such as spontaneous activity, food/water intake and motivated behavior in a relatively stress-free environment. Technical and conceptual improvements in INBEST design may further promote reproducibility and standardization of behavioral studies.     

The chondrocyte-intrinsic circadian clock is disrupted in human osteoarthritis

Peripheral clocks are essential for driving cell differentiation. In osteoarthritis, loss of the normal differentiated chondrocyte (cartilage cell) phenotype is causative of disease. We investigated whether clock gene expression differed in osteoarthritic compared to “healthy” chondrocytes and used RNAi to determine whether the differences observed could affect chondrocyte phenotype. Following serum shock, PER2 expression was significantly higher, whereas BMAL1 expression was significantly lower, in osteoarthritic chondrocytes. Knockdown of BMAL1 in “healthy” chondrocytes was associated with higher cell proliferation and MMP13 expression, features characteristic of the osteoarthritic chondrocyte phenotype. Chondrocyte-intrinsic clock disruption may be a critical early step in osteoarthritis development.     

Natural variation in life history and aging phenotypes is associated with mitochondrial DNA deletion frequency in Caenorhabditis briggsae

Background  

Mutations that impair mitochondrial functioning are associated with a variety of metabolic and age-related disorders. A barrier to rigorous tests of the role of mitochondrial dysfunction in aging processes has been the lack of model systems with relevant, naturally occurring mitochondrial genetic variation. Toward the goal of developing such a model system, we studied natural variation in life history, metabolic, and aging phenotypes as it relates to levels of a naturally-occurring heteroplasmic mitochondrial ND5 deletion recently discovered to segregate among wild populations of the soil nematode, Caenorhabditis briggsae. The normal product of ND5 is a central component of the mitochondrial electron transport chain and integral to cellular energy metabolism.     

The LOC387715 gene, smoking, body mass index, environmental associations with advanced age-related macular degeneration

BACKGROUND AND AIMS: Age-related macular degeneration (AMD) is the leading cause of blindness in the Western World. It is now evident that both genetic and environmental factors contribute to disease susceptibility. We tested the hypotheses that (a) a common coding SNP in the LOC387715 gene is associated with advanced AMD (geographic atrophy or choroidal neovascularization), and (b) that modifiable environmental exposures alter AMD susceptibility associated with this SNP. METHODS: A case-control association analysis was performed on participants (530 advanced AMD cases and 280 controls) ascertained as part of the multi-center Age-Related Eye Disease Study. AMD status was determined by the reading center from fundus photographs using the AREDS AMD grading categorization. Environmental risk factor exposure data was collected from participants whose DNA was also genotyped for the LOC387715 gene SNP rs10490924. Multivariate logistic regression analyses were performed. RESULTS AND CONCLUSIONS: The number of risk alleles at the LOC387715 SNP was associated with advanced AMD, with odds ratios (OR) = 3.0 (95% confidence interval (CI) 2.1-4.3) for the GT heterozygous genotype and OR = 12.1 (5.6-26.5) for the homozygous TT risk genotype, after controlling for demographic and behavioral risk factors. The LOC387715 SNP was associated with both forms of advanced AMD. Current cigarette smoking and body mass index were independently related to AMD, controlling for genotype. However, there was no statistical interaction between LOC387715 genotype and smoking with regard to advanced AMD development.     

Persistence and growth of fecal Bacteroidales assessed by bromodeoxyuridine immunocapture

Extraintestinal growth of fecal bacteria can impair accurate assessment of watershed health. Anaerobic fecal bacteria belonging to the order Bacteroidales are attractive candidates for fecal source tracking because they have host-specific distributions and do not grow well in the presence of high oxygen concentrations. Growth of general and human-specific fecal Bacteroidales marker organisms in environmental samples (sewage) and persistence of the corresponding genetic markers were investigated using bromodeoxyuridine (BrdU) DNA labeling and immunocapture, followed by PCR detection. Background amplification of unlabeled controls occasionally occurred when a high number of PCR cycles was used. By using fluorescent detection of PCR products obtained after 15 cycles, which was determined to be quantitative, we enriched for BrdU-labeled DNA and did not detect unlabeled DNA. By using pure cultures of Bacteroides vulgatus, the ability of Bacteroidales bacteria to take up and incorporate BrdU into nascent DNA was confirmed. Fecal Bacteroidales organisms took up and incorporated BrdU into DNA during growth. In sewage incubated aerobically at the in situ temperature, Bacteroidales genetic marker sequences persisted for at least 24 h and Bacteroidales fecal bacteria grew for up to 24 h as well. Detection by PCR using a low, quantitative cycle number decreased the sensitivity of the assay such that we were unable to detect fecal Bacteroidales human-specific marker sequences in unlabeled or BrdU-labeled fractions, even when fluorescent detection was used. Using 30 PCR cycles with unlabeled fractions, human-specific Bacteroidales sequences were detected, and they persisted for up to 24 h in sewage. These data support the utility of BrdU labeling and immunocapture followed by length heterogeneity PCR or fluorescent detection using low numbers of PCR cycles. However, this method may not be sensitive enough to identify cells that are present at low densities in aquatic environments.     

The role of TLR2 in vivo following challenge with Staphylococcus aureus and prototypic ligands

Based on a wealth of in vitro macrophage studies, immunity to Staphylococcus aureus cell wall-derived peptidoglycan (PGN) and lipoteichoic acid has been attributed to TLR2. We investigated whether the in vitro paradigm of TLR2 dominance would hold true in vivo. Using an experimental peritonitis model, we challenged mice with PGN or lipoteichoic acid and found that only PGN resulted in significant leukocyte (primarily neutrophil) accumulation in the peritoneum at 4 h. PGN-mediated leukocyte recruitment was P-/E-selectin dependent but only partially TLR2 dependent, and also involved the C5aR. Concomitant inhibition of TLR2 and C5aR resulted in a further reduction in PGN-induced peritonitis. Peritoneal neutrophilia was partially mast cell dependent; however, the defect could not be reconstituted with TLR2(-/-) or C5aR(-/-) mast cells. Interestingly, macrophage-deficient mice did not have defective neutrophil recruitment. By 24 h, the response to PGN involved primarily monocytes and was TLR2 and C5aR independent. Finally, we challenged mice with live S. aureus and found a similar degree of TLR2 involvement in leukocyte recruitment to that observed with PGN. Most importantly, bacterial clearance from the spleen and peritoneum was not altered in TLR2(-/-) mice vs wild-type mice. Morbidity was only significantly increased in S. aureus-infected mice treated with a blocking Fab against C5aR. Taken together, these studies indicate that in vivo responses to prototypic TLR2 ligands do not necessarily recapitulate the absolute necessity for TLR2 observed in vitro, and additional receptors contribute, in a significant manner, to PGN and S. aureus-mediated immune responses.     

The significance of p16INK4a in cell defenses against transformation

Human cells, including fibroblast strains that have been immortalized by telomerase, are much more resistant to transformation than rodent cells. Most of the experimental evidence suggests that transformation of human fibroblasts requires inactivation of both the retinoblastoma (pRb) and p53 tumor suppressors as well as the addition of one or more dominant oncogenes. By starting with strains of primary fibroblast (Leiden and Q34 cells) that are genetically deficient for p16INK4a, we have been able to generate anchorage independent colonies simply by addition of telomerase (hTERT) and either Ras or Myc. Importantly, the transformed cells appear to retain pRb and p53 functions and are essentially diploid. Whereas Leiden cells expressing the individual oncogenes did not form tumors in mice, the combination of hTERT, Myc and Ras enabled them to become tumorigenic, albeit at a frequency suggestive of an additional genetic event. Significantly, we have obtained karyotypically stable tumors without the need to use DNA tumor virus oncoproteins and without deliberate ablation of p53.     

Erythropoietin-dependent autocrine secretion of tumor necrosis factor-alpha in hematopoietic cells modulates proliferation via MAP kinase--ERK-1/2 and does not require tyrosine docking sites in the EPO receptor

Primary erythroid cells and erythroid cell lines may synthesize and secrete tumor necrosis factor-alpha (TNF-alpha) following stimulation with erythropoietin (EPO). The effect of triggering TNF-alpha synthesis and secretion was investigated in erythroleukemia and myeloid cell lines: HCD57, DA3-EPOR, and BAF3-EPOR. The EPO-induced, membrane-bound form of autocrine TNF-alpha seemed to enhance proliferation of HCD57 and DA3-EPOR cells; however, the concentration of secreted autocrine/paracrine TNF-alpha was never sufficient to have an effect. Autocrine TNF-alpha acts through TNFRII receptors to stimulate proliferation. Modulation of mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK-1/2) activity by the membrane-bound form of autocrine TNF-alpha apparently played a central role in the control of EPO-dependent proliferation of HCD57 and DA3-EPOR cells. Primary erythroid cells and DA3-EPOR cells were found to express similar, high levels of both TNFRI and TNFRII, showing that differential expression of TNF-alpha receptors does not explain why primary cells are inhibited and DA3-EPOR cells are stimulated by autocrine TNF-alpha. BAF3 cells expressing a mutant EPOR with no cytoplasmic tyrosine residues were capable of triggering EPO-dependent TNF-alpha synthesis and secretion, indicating that tyrosine-docking sites in the EPOR were not required for EPO-dependent TNF-alpha secretion.     

ARTEMIS stabilizes the genome and modulates proliferative responses in multipotent mesenchymal cells

Background  

Unrepaired DNA double-stranded breaks (DSBs) cause chromosomal rearrangements, loss of genetic information, neoplastic transformation or cell death. The nonhomologous end joining (NHEJ) pathway, catalyzing sequence-independent direct rejoining of DSBs, is a crucial mechanism for repairing both stochastically occurring and developmentally programmed DSBs. In lymphocytes, NHEJ is critical for both development and genome stability. NHEJ defects lead to severe combined immunodeficiency (SCID) and lymphoid cancer predisposition in both mice and humans. While NHEJ has been thoroughly investigated in lymphocytes, the importance of NHEJ in other cell types, especially with regard to tumor suppression, is less well documented. We previously reported evidence that the NHEJ pathway functions to suppress a range of nonlymphoid tumor types, including various classes of sarcomas, by unknown mechanisms.