Faculty Bibliography

Purpose: Previous studies have shown the potential of diffusion tensor imaging (DTI) in capturing damage of the cartilage. DTI indices, such as mean diffusivity (MD) and fractional anisotropy (FA) are potential biomarkers for cartilage composition and structure. Our hypothesis is that the DTI biomarkers can detect early changes in articular cartilage after mechanical injury. We use a model to replicate the mechanism of injury in the cartilage during ACL rupture, and compare the DTI biomarkers against histology and biomechanics. The study is designed to make the results translational to clinical practice, as we use a clinical scanner, employ the same imaging sequence, and use a protocol which can be optimized for clinical studies. Materials and Methods: Nine cylindrical articular cartilage-on-bone plugs from a knee replacement surgery were incubated at 37C with culture medium. Over a 3-week long protocol, cartilage MRI scanning is combined with biomechanical and histological tests. We first perform a baseline MRI and stress-strain test on each sample. MRI was performed at 3 Twith an in-house butterfly coil specifically designed for the extracted ex-vivo samples. The MRI protocol included a radial spin echo DTI (RAISED) sequence that we normally use for human in-vivo applications. We acquired two b = 0 and 12-direction b = 300 s/mm2 images at a resolution of 0.18 x 0.18 x 1.2 mm3 and derived FA and MD parameters. After MRI and the biomechanical tests, we imparted a zero (n=3), intermediate (n = 3), or high (n = 3) overload to simulate the traumatic injury of cartilage. Two weeks after, the samples underwent a third MRI and biomechanical testing. Cartilage samples were then processed for histology with safranin-O to show the PG density and distribution. Safranin-O slides were graded via OARSI scoring, varying from 0 (heathy) to 6 (bone remodelling). Results: The DTI biomarkers are sensitive to the changes on articular cartilage after injury. In severely injured samples, the MD increased by 15 % in the 2 weeks, from an average of 1.36 mum2/ms at the baseline, whereas the FAdecreased by 43%froma baseline of 0.25.Mildly injured samples showed a MD trend towards higher values, increasing by 9 % frombaseline 1.28 mum2/ms, whereas the FA did not change. As expected, the biomechanics tests reflect a trend of decreased stiffness (Young's modulus) with increasing injury. The change in MD is correlated with the changes in the biomechanics with rho = 0.75 (p = 0.04), and correlated with the OARSI (rho = 0.65, p = 0.07). The OARSI score of the baseline samples was 0.83 +/- 0.44. Severe injury samples increased their OARSI score by 2.53 +/- 1.30, while the mild injury had only a moderate increase in OARSI (0.62 +/- 1.30). Zero injury samples neither changed their score, nor their biomechanical parameters. Conclusion: DTI biomarkers do capture the early signs of damage in articular cartilage. The use of clinically feasible MRI protocols provide new biomarkers for the early diagnosis and monitoring of early stages of post-traumatic OA

Endoplasmic Reticulum (ER) is an organelle where most secretory and membrane proteins are synthesized, folded, and undergo further maturation. As numerous conditions can perturb such ER function, eukaryotic cells are equipped with responsive signaling pathways, widely referred to as the Unfolded Protein Response (UPR). Chronic conditions of ER stress that cannot be fully resolved by UPR, or conditions that impair UPR signaling itself, are associated with many metabolic and degenerative diseases. In recent years, Drosophila has been actively employed to study such connections between UPR and disease. Notably, the UPR pathways are largely conserved between Drosophila and humans, and the mediating genes are essential for development in both organisms, indicating their requirement to resolve inherent stress. By now, many Drosophila mutations are known to impose stress in the ER, and a number of these appear similar to those that underlie human diseases. In addition, studies have employed the strategy of overexpressing human mutations in Drosophila tissues to perform genetic modifier screens. The fact that the basic UPR pathways are conserved, together with the availability of many human disease models in this organism, makes Drosophila a powerful tool for studying human disease mechanisms.

Tumor-associated macrophages (TAMs) are increasingly investigated in cancer immunology and are considered a promising target for better and tailored treatment of malignant growth. Although TAMs also have high diagnostic and prognostic value, TAM imaging still remains largely unexplored. Here, we describe the development of reconstituted high-density lipoprotein (rHDL)-facilitated TAM PET imaging in a breast cancer model. METHODS: Radiolabeled rHDL nanoparticles incorporating the long-lived positron-emitting nuclide (89)Zr were developed using 2 different approaches. The nanoparticles were composed of phospholipids and apolipoprotein A-I (apoA-I) in a 2.5:1 weight ratio. (89)Zr was complexed with deferoxamine (also known as desferrioxamine B, desferoxamine B), conjugated either to a phospholipid or to apoA-I to generate (89)Zr-PL-HDL and (89)Zr-AI-HDL, respectively. In vivo evaluation was performed in an orthotopic mouse model of breast cancer and included pharmacokinetic analysis, biodistribution studies, and PET imaging. Ex vivo histologic analysis of tumor tissues to assess regional distribution of (89)Zr radioactivity was also performed. Fluorescent analogs of the radiolabeled agents were used to determine cell-targeting specificity using flow cytometry. RESULTS: The phospholipid- and apoA-I-labeled rHDL were produced at 79% +/- 13% (n = 6) and 94% +/- 6% (n = 6) radiochemical yield, respectively, with excellent radiochemical purity (>99%). Intravenous administration of both probes resulted in high tumor radioactivity accumulation (16.5 +/- 2.8 and 8.6 +/- 1.3 percentage injected dose per gram for apoA-I- and phospholipid-labeled rHDL, respectively) at 24 h after injection. Histologic analysis showed good colocalization of radioactivity with TAM-rich areas in tumor sections. Flow cytometry revealed high specificity of rHDL for TAMs, which had the highest uptake per cell (6.8-fold higher than tumor cells for both DiO@Zr-PL-HDL and DiO@Zr-AI-HDL) and accounted for 40.7% and 39.5% of the total cellular DiO@Zr-PL-HDL and DiO@Zr-AI-HDL in tumors, respectively. CONCLUSION: We have developed (89)Zr-labeled TAM imaging agents based on the natural nanoparticle rHDL. In an orthotopic mouse model of breast cancer, we have demonstrated their specificity for macrophages, a result that was corroborated by flow cytometry. Quantitative macrophage PET imaging with our (89)Zr-rHDL imaging agents could be valuable for noninvasive monitoring of TAM immunology and targeted treatment.

Synaptic roles for neurofilament (NF) proteins have rarely been considered. Here, we establish all four NF subunits as integral resident proteins of synapses. Compared with the population in axons, NF subunits isolated from synapses have distinctive stoichiometry and phosphorylation state, and respond differently to perturbations in vivo. Completely eliminating NF proteins from brain by genetically deleting three subunits (alpha-internexin, NFH and NFL) markedly depresses hippocampal long-term potentiation induction without detectably altering synapse morphology. Deletion of NFM in mice, but not the deletion of any other NF subunit, amplifies dopamine D1-receptor-mediated motor responses to cocaine while redistributing postsynaptic D1-receptors from endosomes to plasma membrane, consistent with a specific modulatory role of NFM in D1-receptor recycling. These results identify a distinct pool of synaptic NF subunits and establish their key role in neurotransmission in vivo, suggesting potential novel influences of NF proteins in psychiatric as well as neurological states.

Current leishmaniasis treatment is strangled due to concealed residence of parasite and reduced host cell mediated immune response. To circumvent above challenges, novel macrophage targeted oily core polymeric shell based doxorubicin (DOX) loaded nanocapsules (NCAPs) were fabricated employing chondroitin sulphate (CHD) for complimentary immunotherapy coupled chemotherapy against leishmaniasis. Excellent encapsulation efficiency along with pH dependent drug release was demonstrated by NCAPs. Improved cell cycle arrest at G1-S phase (1.56 folds) and apoptosis against promastigotes (6.26 folds), support the remarkable in-vitro antileishmanial activity of NCAPs (IC50: 0.254+/-0.038mug/ml) compared to free DOX (IC50: 0.543+/-0.012mug/ml). In-vivo antileishmanial activity in hamsters represented a significantly enhanced parasitic inhibition by NCAPs (1.42 folds). Improved activity was mediated via immunotherapeutic activity of NCAPs which up-regulated Th1 immune response (IL-12, INF-gamma and TNF-alpha) and down-regulated Th2 immune response (IL-4, IL-10 and TGF-beta). In conclusion, current novel nano-formulation could be a viable option against leishmaniasis.

The urinary tract exits to a body surface area that is densely populated by a wide range of microbes. Yet, under most normal circumstances, it is typically considered sterile, i.e., devoid of microbes, a stark contrast to the gastrointestinal and upper respiratory tracts where many commensal and pathogenic microbes call home. Not surprisingly, infection of the urinary tract over a healthy person's lifetime is relatively infrequent, occurring once or twice or not at all for most people. For those who do experience an initial infection, the great majority (70% to 80%) thankfully do not go on to suffer from multiple episodes. This is a far cry from the upper respiratory tract infections, which can afflict an otherwise healthy individual countless times. The fact that urinary tract infections are hard to elicit in experimental animals except with inoculum 3-5 orders of magnitude greater than the colony counts that define an acute urinary infection in humans (105 cfu/ml), also speaks to the robustness of the urinary tract defense. How can the urinary tract be so effective in fending off harmful microbes despite its orifice in a close vicinity to that of the microbe-laden gastrointestinal tract? While a complete picture is still evolving, the general consensus is that the anatomical and physiological integrity of the urinary tract is of paramount importance in maintaining a healthy urinary tract. When this integrity is breached, however, the urinary tract can be at a heightened risk or even recurrent episodes of microbial infections. In fact, recurrent urinary tract infections are a significant cause of morbidity and time lost from work and a major challenge to manage clinically. Additionally, infections of the upper urinary tract often require hospitalization and prolonged antibiotic therapy. In this chapter, we provide an overview of the basic anatomy and physiology of the urinary tract with an emphasis on their specific roles in host defense. We also highlight the important structural and functional abnormalities that predispose the urinary tract to microbial infections.

Cellular metabolism is increasingly recognized to control immune cell fate and functions. miR-33 is a regulator of cellular lipid metabolism that represses genes involved in cholesterol efflux, HDL biogenesis and fatty acid oxidation. We demonstrate that by altering the balance of aerobic glycolysis and mitochondrial oxidative phosphorylation, miR-33 instructs macrophage inflammatory polarization and shapes innate and adaptive immune responses. Targeted deletion of miR-33 in macrophages increases oxidative respiration, enhances spare respiratory capacity, and induces the expression of genes that define M2 macrophage polarization. We show that these changes are independent of effects on cholesterol efflux, but instead require miR-33 targeting of the energy sensor AMP-activated protein kinase. Notably, inhibition of miR-33 also increases macrophage expression of the retinoic acid-producing enzyme Aldh1a2 and retinal dehydrogenase activity both in vitro and in vivo. Consistent with the ability of retinoic acid to foster inducible regulatory T cells, anti-miR33-treated macrophages have an enhanced capacity to induce FoxP3 expression in naive CD4+ T cells. Finally, treatment of western diet-fed Ldlr-/- mice with miR-33 inhibitors for 8 weeks (conditions that do not alter HDL cholesterol levels) promoted the accumulation of inflammation suppressing M2 macrophages and FoxP3+ T regulatory cells in plaques, and reduced atherosclerosis progression by 40 %. Collectively, these results identify a novel role for miR-33 in the regulation of macrophage inflammation and show that antagonism of miR-33 reduces atherosclerotic inflammation by promoting M2 macrophage polarization and regulatory T cell induction