Faculty Bibliography

OBJECTIVES: Cartilage is a highly mechano-responsive tissue. Chondrocytes undergo a series of complex changes, including proliferation and metabolic alteration as the target of external biomechanical and biochemical stimuli. IL-1beta is known to regulate chondrocyte metabolism and play an important role in the pathogenesis of osteoarthritis. The objective of this study was to employ low-intensity pulsed ultrasound (LIPUS) as a localized mechanical stimulus and assess its effects on chondrocyte migration, proliferation, metabolism, and differentiation, as well as its ability to suppress IL-1beta mediated catabolism in cartilage. METHODS: Human cartilage explants and chondrocytes were stimulated by LIPUS in presence and absence of of IL-1beta to asses cartilage degradation, chondrocytes metabolism, migration and proliferation. Western blot analyses were conducted to study IL-1beta the associated NFkappaB pathway in chondrocytes. RESULTS: LIPUS stimulation increased the proteoglycan content in human cartilage explants and inhibited IL-1beta induced loss of proteoglycans. LIPUS stimulation increased rates of chondrocyte migration and proliferation, and promoted chondrogenesis in mesenchymal stem cells. Further, LIPUS suppressed IL-1beta induced activation of phosphorylation of NFkappaB-p65 and IkBalpha leading to reduced expression of MMP13 and ADAMT5 in chondrocytes. CONCLUSIONS: Collectively, these data demonstrate the potential therapeutic effects of LIPUS in preventing cartilage degradation and treating osteoarthritis via a mechanical stimulation that inhibits the catabolic action of IL-1beta and stimulates chondrocyte migration, proliferation, and differentiation.

Gaucher disease (GD), the most common lysosomal storage disease, is caused by mutations in GBA1 encoding of beta-glucocerebrosidase (GCase). Recently it was reported that progranulin (PGRN) insufficiency and deficiency associated with GD in human and mice, respectively. However the underlying mechanisms remain unknown. Here we report that PGRN binds directly to GCase and its deficiency results in aggregation of GCase and its receptor LIMP2. Mass spectrometry approaches identified HSP70 as a GCase/LIMP2 complex-associated protein upon stress, with PGRN as an indispensable adaptor. Additionally, 98 amino acids of C-terminal PGRN, referred to as Pcgin, are required and sufficient for the binding to GCase and HSP70. Pcgin effectively ameliorates the disease phenotype in GD patient fibroblasts and animal models. These findings not only demonstrate that PGRN is a co-chaperone of HSP70 and plays an important role in GCase lysosomal localization, but may also provide new therapeutic interventions for lysosomal storage diseases, in particular GD.

Rheumatoid arthritis is an autoimmune disease that is characterized by chronic inflammation and destruction of joints. Netrin-1, a chemorepulsant, laminin-like matrix protein, promotes inflammation by preventing macrophage egress from inflamed sites and is required for osteoclast differentiation. We asked whether blockade of Netrin-1 or its receptors [Unc5b and DCC (deleted in colorectal carcinoma)] may be useful therapeutic targets for treatment of inflammatory arthritis. Arthritis was induced in 8-wk-old C57Bl/6 mice by intraperitoneal injection of K/BxN serum. Murine monoclonal antibodies against Netrin-1, Unc5b, or DCC (10 microg/mouse) were injected weekly for 4 wk (n = 10). Paw swelling and thickness were assessed and following euthanasia 2-4 wk after serum transfer, paws were prepared for micro-computed tomography and histology. Paw inflammation was maximal 2 wk after injection. Anti-Netrin-1 or anti-Unc5b, but not anti-DCC, antibodies significantly reduced paw inflammation (clinical score: 9.8 +/- 0.8, 10.4 +/- 0.9, and 13.5 +/- 0.5, respectively vs 16 +/- 0 for control; P < 0.001). Micro-computed tomography showed bony erosions in untreated or anti-DCC-treated mice, whereas there were no erosions in anti-Netrin-1/anti-Unc5b-treated-animals. Tartrate-resistant acid phosphatase staining demonstrated a marked decrease in osteoclasts in anti-Netrin-1/anti-Unc5b-treated animals. Immunofluorescence staining revealed a decrease in cathepsin K+ and CD68+ cells in anti-Netrin-1/anti-Unc5b-treated animals. Blockade of Netrin-1/Unc5b by monoclonal antibodies prevents bone destruction and reduces the severity of K/BxN serum transfer-induced arthritis. Netrin-1 may be a novel therapeutic target for treatment of inflammatory bone destruction.-Mediero, A., Wilder, T., Ramkhelawon, B., Moore, K. J., Cronstein, B. N. Netrin-1 and its receptor Unc5b are novel targets for the treatment of inflammatory arthritis.

OBJECTIVES: Obesity and obesity-associated inflammation is central to a variety of end-organ sequelae including atherosclerosis, a leading cause of death worldwide. Although mouse models have provided important insights into the immunopathogenesis of various diseases, modeling atherosclerosis in mice has proven difficult. Specifically, wild-type (WT) mice are resistant to developing atherosclerosis, while commonly used genetically modified mouse models of atherosclerosis are poor mimics of human disease. The lack of a physiologically relevant experimental model of atherosclerosis has hindered the understanding of mechanisms regulating disease development and progression as well as the development of translational therapies. Recent evidence suggests that housing mice within their thermoneutral zone profoundly alters murine physiology, including both metabolic and immune processes. We hypothesized that thermoneutral housing would allow for augmentation of atherosclerosis induction and progression in mice. METHODS: ApoE-/- and WT mice were housed at either standard (TS) or thermoneutral (TN) temperatures and fed either a chow or obesogenic "Western" diet. Analysis included quantification of (i) obesity and obesity-associated downstream sequelae, (ii) the development and progression of atherosclerosis, and (iii) inflammatory gene expression pathways related to atherosclerosis. RESULTS: Housing mice at TN, in combination with an obesogenic "Western" diet, profoundly augmented obesity development, exacerbated atherosclerosis in ApoE-/- mice, and initiated atherosclerosis development in WT mice. This increased disease burden was associated with altered lipid profiles, including cholesterol levels and fractions, and increased aortic plaque size. In addition to the mild induction of atherosclerosis, we similarly observed increased levels of aortic and white adipose tissue inflammation and increased circulating immune cell expression of pathways related to adverse cardiovascular outcome. CONCLUSIONS: In sum, our novel data in WT C57Bl/6 mice suggest that modulation of a single environmental variable, temperature, dramatically alters mouse physiology, metabolism, and inflammation, allowing for an improved mouse model of atherosclerosis. Thus, thermoneutral housing of mice shows promise in yielding a better understanding of the cellular and molecular pathways underlying the pathogenesis of diverse diseases.

Computational models are an invaluable tool in modern biology. They provide a framework within which to summarize existing knowledge, enable competing hypotheses to be compared qualitatively and quantitatively, and to facilitate the interpretation of complex data. Moreover, models allow questions to be investigated that are difficult to approach experimentally. Theories can be tested in context, identifying the gaps in our understanding and potentially leading to new hypotheses. Models can be developed on a variety of scales and with different levels of mechanistic detail, depending on the available data, the biological questions of interest, and the available mathematical and computational tools. The goal of this review is to provide a broad picture of how modeling has been applied to reproductive biology. Specifically, we look at four uses of modeling: i) comparing hypotheses, ii) interpreting data, iii) exploring experimentally challenging questions, and iv) hypothesis evaluation and generation. We present examples of each of these applications in reproductive biology, drawing from a range of organisms - including Drosophila, Caenorhabditis elegans, mouse, and humans. We aim to describe the data and techniques used to construct each model, and to highlight the benefits of modeling to the field, as complementary to experimental work

Vitiligo, characterized by progressive skin depigmentation, results from autoimmune-mediated melanocyte loss. The mechanisms underlying disease onset are poorly delineated. Triggers, including exposure to phenols such as monobenzone (MB), are thought to disrupt melanocyte homeostasis and ultimately instigate an autoimmune reaction. We have shown that MB disrupts cellular homeostasis and induces endoplasmic reticulum (ER) stress that leads to activation of the unfolded protein response (UPR). Three proteins, including PERK, each activate UPR arms that orchestrate the restoration of homeostasis. When activated, PERK phosphorylates eIF2a, a translation initiation factor, thus reducing protein synthesis and ER stress. In this study, we investigated the impact of the PERKeIF2a cascade on melanocyte viability and sensitivity to MB. Basal levels of phospho-eIF2a are higher in melanocytes compared to cutaneous fibroblasts or keratinocytes. When PERK expression is downregulated by RNAi, there is a significant reduction in melanocyte viability (88% decrease, p < 0.05 shPERK versus non-target/ shNT; n = 3). Some melanocytes (shPERKLT) can however survive despite prolonged PERK downregulation. Survival correlated with a paradoxical increase in phospho-eIF2a levels and reduced sensitivity to MB (Cleaved/c-PARP levels lower in shPERKLT cells treated with 400 muM MB compared to shNT cells). Chemical inhibition of PERK kinase activity, using GSK2606414, prevented eIF2a phosphorylation and sensitized melanocytes to MB (c-PARP observed with 250 muM MB+ GSK2606414, compared to 400 muM MB + vehicle). PERK-eIF2a axis activity contributes to melanocyte viability and determines sensitivity to MB. Pathways, such the UPR, which has also been implicated in autoimmune diabetes, may link exposure to vitiligo-inducing triggers and onset of autoimmunity. These pathways represent novel therapeutic targets to prevent vitiligo progression or improve efficacy of repigmentation protocols

Progranulin (PGRN) is a growth factor with a unique beads-on-a-string structure that is involved in multiple pathophysiological processes, including anti-inflammation, tissue repair, wound healing, neurodegenerative diseases, and tumorigenesis. This review presents up-to-date information concerning recent studies on the role of PGRN in inflammatory arthritis and osteoarthritis, with a special focus on the involvement of the interactions and interplay between PGRN and tumor necrosis factor receptor (TNFR) family members in regulating such musculoskeletal diseases. In addition, this paper highlights the applications of atsttrin, an engineered protein comprising three TNFR-binding fragments of PGRN, as a promising intervention in treating arthritis.

There is little evidence on prevalence or risk factors for soil transmitted helminth infections in Timor-Leste. This study describes the epidemiology, water, sanitation and hygiene, and socioeconomic risk factors of STH and intestinal protozoa amongst communities in Manufahi District, Timor-Leste. As part of a cluster randomised controlled trial, a baseline cross-sectional survey was conducted across 18 villages, with data from six additional villages. Stool samples were assessed for soil transmitted helminth and protozoal infections using quantitative PCR (qPCR) and questionnaires administered to collect water, sanitation and hygiene and socioeconomic data. Risk factors for infection were assessed using multivariable mixed-effects logistic regression, stratified by age group (preschool, school-aged and adult). Overall, soil transmitted helminth prevalence was 69% (95% Confidence Interval 67-71%), with Necator americanus being most common (60%; 95% Confidence Interval 58-62%) followed by Ascaris spp. (24%; 95% Confidence Interval 23-26%). Ascaris-N. americanus co-infection was common (17%; 95% Confidence Interval 15%-18%). Giardia duodenalis was the main protozoan identified (13%; 95% Confidence Interval 11-14%). Baseline water, sanitation and hygiene infrastructure and behaviours were poor. Although risk factors varied by age of participants and parasite species, risk factors for N. americanus infection included, generally, age in years, male sex, and socioeconomic quintile. Risk factors for Ascaris included age in years for children, and piped water to the yard for adults. In this first known assessment of community-based prevalence and associated risk factors in Timor-Leste, soil transmitted helminth infections were highly prevalent, indicating a need for soil transmitted helminth control. Few associations with water, sanitation and hygiene were evident, despite water, sanitation and hygiene being generally poor. In our water, sanitation and hygiene we will investigate implications of improving WASH on soil transmitted helminth infection in impoverished communities.

The Ras-related (R-Ras) isoforms TC21, R-Ras and M-Ras are members of the Ras superfamily of small GTPases. R-Ras family proteins are frequently overexpressed in human cancers, and expression of activated mutants of these GTPases is sufficient to induce cell transformation. Unlike Ras, few activating mutations of R-Ras proteins have been reported in human cancer, and very little is known about the regulation of their activity. In this study, we report that TC21 and R-Ras are phosphorylated on a conserved serine, Ser186 and Ser201, respectively, in intact cells. This residue is located in the C-terminal hypervariable region of the proteins and is not conserved in M-Ras. We show that the MAP kinases ERK1/2 phosphorylate TC21 and R-Ras on this C-terminal serine residue both in vitro and in vivo. Phosphorylation of R-Ras proteins does not affect their subcellular localization or stability but rather stimulates their activation. Phosphorylation-defective mutants of R-Ras and TC21 are compromised in their ability to promote cancer cell adhesion and migration/invasion, respectively. Importantly, we show that phosphorylation of TC21 and R-Ras potentiates their tumorigenic activity in immunodeficient mice. Our results identify a novel regulatory mechanism of the small GTPases TC21 and R-Ras that controls their oncogenic potential.