Faculty Bibliography

Wound healing is a complex biological process involving the interaction of many cell types to replace lost or damaged tissue. Although the biology of wound healing has been extensively investigated, few studies have focused on the role of mast cells. In this study, we investigated the possible role of mast cells in wound healing by analyzing aspects of cutaneous excisional wound healing in three types of genetically mast cell-deficient mice. We found that C57BL/6-Kit(W-sh/W-sh), WBB6F1-Kit(W/W-v), and Cpa3-Cre; Mcl-1(fl/fl) mice re-epithelialized splinted excisional skin wounds at rates very similar to those in the corresponding wild type or control mice. Furthermore, at the time of closure, scars were similar in the genetically mast cell-deficient mice and the corresponding wild type or control mice in both quantity of collagen deposition and maturity of collagen fibers, as evaluated by Masson's Trichrome and Picro-Sirius red staining. These data indicate that mast cells do not play a significant non-redundant role in these features of the healing of splinted full thickness excisional cutaneous wounds in mice.

Hepatic steatosis is a global epidemic that is thought to contribute to the pathogenesis of type 2 diabetes. MicroRNAs (miRs) are regulators that can functionally integrate a range of metabolic and inflammatory pathways in liver. We aimed to investigate the functional role of miR-155 in hepatic steatosis. Male C57BL/6 wild-type (WT) and miR-155(-/-) mice were fed either normal chow or high fat diet (HFD) for 6 months then lipid levels, metabolic and inflammatory parameters were assessed in livers and serum of the mice. Mice lacking endogenous miR-155 that were fed HFD for 6 months developed increased hepatic steatosis compared to WT controls. This was associated with increased liver weight and serum VLDL/LDL cholesterol and alanine transaminase (ALT) levels, as well as increased hepatic expression of genes involved in glucose regulation (Pck1, Cebpa), fatty acid uptake (Cd36) and lipid metabolism (Fasn, Fabp4, Lpl, Abcd2, Pla2g7). Using miRNA target prediction algorithms and the microarray transcriptomic profile of miR-155(-/-) livers, we identified and validated that Nr1h3 (LXRalpha) as a direct miR-155 target gene that is potentially responsible for the liver phenotype of miR-155(-/-) mice. Together these data indicate that miR-155 plays a pivotal role regulating lipid metabolism in liver and that its deregulation may lead to hepatic steatosis in patients with diabetes.

Tight control of antigen-receptor gene rearrangement is required to preserve genome integrity and prevent the occurrence of leukaemia and lymphoma. Nonetheless, mistakes can happen, leading to the generation of aberrant rearrangements, such as Tcra/d-Igh inter-locus translocations that are a hallmark of ataxia telangiectasia-mutated (ATM) deficiency. Current evidence indicates that these translocations arise from the persistence of unrepaired breaks converging at different stages of thymocyte differentiation. Here we show that a defect in feedback control of RAG2 activity gives rise to bi-locus breaks and damage on Tcra/d and Igh in the same T cell at the same developmental stage, which provides a direct mechanism for generating these inter-locus rearrangements. Both the RAG2 C-terminus and ATM prevent bi-locus RAG-mediated cleavage through modulation of three-dimensional conformation (higher-order loops) and nuclear organization of the two loci. This limits the number of potential substrates for translocation and provides an important mechanism for protecting genome stability.

BACKGROUND: Obsessive-compulsive disorder (OCD) is a severe condition with varied symptom presentations. Currently, the cognitive-behavioral treatment with the most empirical support is exposure and ritual prevention (EX/RP); however, clinical impression and some empirical data suggest that certain OCD symptoms are more responsive to treatment than others. METHODS: Prior work identifying symptom dimensions within OCD is discussed, including epidemiological findings, factor analytic studies, and biological findings. Symptom dimensions most reliably identified include contamination/cleaning, doubt about harm/checking, symmetry/ordering, and unacceptable thoughts/mental rituals. The phenomenology of each of these subtypes is described and research literature is summarized, emphasizing the differential effects of EX/RP and its variants on each of these primary symptom dimensions. RESULTS: To date it appears that EX/RP is an effective treatment for the various OCD dimensions, although not all dimensions have been adequately studied (i.e. symmetry and ordering). CONCLUSIONS: Modifications to treatment may be warranted for some types of symptoms. Clinical implications and directions for future research are discussed.

In vivo wound healing experiments remain the most predictive models for studying human wound healing, allowing an accurate representation of the complete wound healing environment including various cell types, environmental cues, and paracrine interactions. Small animals are economical, easy to maintain, and allow researchers to take advantage of the numerous transgenic strains that have been developed to investigate the specific mechanisms involved in wound healing and regeneration. Here we describe three reproducible murine wound healing models that recapitulate the human wound healing process.

Gaucher disease (GD) is the most common of the lysosomal storage disorders and is caused by defects in the GBA gene encoding glucocerebrosidase (GlcCerase). The accumulation of its substrate, glucocylceramide (GlcCer) is considered the main cause of GD. We found here that the expression of human mutated GlcCerase gene (hGBA) that is associated with neuronopathy in GD patients causes neurodevelopmental defects in Drosophila eyes. The data indicate that endoplasmic reticulum (ER) stress was elevated in Drosophila eye carrying mutated hGBAs by using of the ER stress markers dXBP1 and dBiP. We also found that Ambroxol, a potential pharmacological chaperone for mutated hGBAs, can alleviate the neuronopathic phenotype through reducing ER stress. We demonstrate a novel mechanism of neurodevelopmental defects mediated by ER stress through expression of mutants of human GBA gene in the eye of Drosophila.

Phospholamban (PLN) is a small integral membrane protein, which binds and inhibits in a yet unknown fashion the Ca(2+)-ATPase (SERCA) in the sarcoplasmic reticulum. When reconstituted in planar lipid bilayers PLN exhibits ion channel activity with a low unitary conductance. From the effect of non-electrolyte polymers on this unitary conductance we estimate a narrow pore with a diameter of ca. 2.2 A for this channel. This value is similar to that reported for the central pore in the structure of the PLN pentamer. Hence the PLN pentamer, which is in equilibrium with the monomer, is the most likely channel forming structure. Reconstituted PLN mutants, which either stabilize (K27A and R9C) or destabilize (I47A) the PLN pentamer and also phosphorylated PLN still generate the same unitary conductance of the wt/non-phosphorylated PLN. However the open probability of the phosphorylated PLN and of the R9C mutant is significantly lower than that of the respective wt/non-phosphorylated control. In the context of data on PLN/SERCA interaction and on Ca(2+) accumulation in the sarcoplasmic reticulum the present results are consistent with the view that PLN channel activity could participate in the balancing of charge during Ca(2+) uptake. A reduced total conductance of the K(+) transporting PLN by phosphorylation or by the R9C mutation may stimulate Ca(2+) uptake in the same way as an inhibition of K(+) channels in the SR membrane. The R9C-PLN mutation, a putative cause of dilated cardiomyopathy, might hence affect SERCA activity also via its inherent low open probability.

Chemotaxis assays are an invaluable tool for studying the biological activity of inflammatory mediators such as CC chemokines, which have been implicated in a wide range of chronic inflammatory diseases. Conventional chemotaxis systems such as the modified Boyden chamber are limited in terms of the data captured given that the assays are analysed at a single time-point. We report the optimisation and validation of a label-free, real-time cell migration assay based on electrical cell impedance to measure chemotaxis of different primary murine macrophage populations in response to a range of CC chemokines and other chemoattractant signalling molecules. We clearly demonstrate key differences in the migratory behavior of different murine macrophage populations and show that this dynamic system measures true macrophage chemotaxis rather than chemokinesis or fugetaxis. We highlight an absolute requirement for Galphai signaling and actin cytoskeletal rearrangement as demonstrated by Pertussis toxin and cytochalasin D inhibition. We also studied the chemotaxis of CD14(+) human monocytes and demonstrate distinct chemotactic profiles amongst different monocyte donors to CCL2. This real-time chemotaxis assay will allow a detailed analysis of factors that regulate macrophage responses to chemoattractant cytokines and inflammatory mediators.

Coronary artery disease, resulting from atherosclerosis, is the leading cause of death in the Western world. Most previous studies have subjected atherosclerotic arteries, a tissue of mixed cellular composition, to homogenization in order to identify the factors in plaque development, thereby obscuring information relevant to specific cell types. Because macrophage foam cells are critical mediators in atherosclerotic plaque advancement, we reasoned that performing gene analysis on those cells would provide specific insight in novel regulatory factors and potential therapeutic targets. We demonstrated for the first time in vascular biology that foam cell-specific RNA can be isolated by laser capture microdissection (LCM) of plaques. As expected, compared to whole tissue, a significant enrichment in foam cell-specific RNA transcripts was observed. Furthermore, because regression of atherosclerosis is a tantalizing clinical goal, we developed and reported a transplantation-based mouse model. This involved allowing plaques to form in apoE-/- mice and then changing the plaque's plasma environment from hyperlipidemia to normolipidemia. Under those conditions, rapid regression ensued in a process involving emigration of plaque foam cells to regional and systemic lymph nodes. Using LCM, we were able to show that under regression conditions, there was decreased expression in foam cells of inflammatory genes, but an up-regulation of cholesterol efflux genes. Interestingly, we also found that increased expression of chemokine receptor CCR7, a known factor in dendritic cell migration, was required for regression. In conclusion, the LCM methods described in this chapter, which have already lead to a number of striking findings, will likely further facilitate the study of cell type-specific gene expression in animal and human plaques during various stages of atherosclerosis, and after genetic, pharmacologic, and environmental perturbations.