Faculty Bibliography

Introduction 2nd generation polymeric nanoparticles have shown significant advantages in drug delivery. They can be loaded with poorly water soluble drugs,1 their size can be judiciously controlled2 and their surface can be functionalized with a PEG coating and/or targeting ligands. Importantly, the polymeric core can be loaded with drugs and/or contrast agents for which the release rates can be controlled by the choice of polymer composition and molecular weight. High-density lipoprotein (HDL) is a natural nanoparticle that transports fats through the body, which has an inherent affinity for atherosclerotic plaques. HDL-like nanoparticles labeled with contrast agents have been shown suitable for molecular imaging as they effectively target atherosclerotic plaque. In the current study we developed a novel HDL-like hybrid nanoparticle using recently developed microfluidics technology.2 The nanoparticle is comprised of a lipid/apolipoprotein coating that encapsulates a poly(lactic-co-glycolic acid) (PLGA) core suitable for the delivery of drugs in a controlled manner. The versatility of the approach also allows the incorporation of functional lipids to render multifunctional nanoparticles with imaging, therapeutic and atherosclerosis targeting properties. Methods and Results Hybrid polymer-HDL nanoparticles with a PLGA core and a coating comprised of lipids and apolipoprotein A1 (PLGA-HDL) were synthesized using microfluidics. The synthetic approach consists of the rapid injection of the components in three different channels of a microfluidics chip. Amphiphilic phospholipids and PLGA were dissolved in a mixture of ethanol and acetonitrile. This solution was injected in the middle channel of the microfluidic chip and mixed with an aqueous apoprotein A1 (ApoA1) solution injected in the two outer channels. Inside the chip, controlled nanoprecipitation occurred through microvortices, resulting in the instantaneous and continuous production of hybrid PLGA-HDL nanoparticles with high reproducibility (!

BACKGROUND: A new HIV diagnostic algorithm has been proposed which replaces the use of the HIV-1 Western blot and HIV-1 immunofluorescence assays (IFA) as the supplemental test with an HIV-1/HIV-2 antibody differentiation assay. OBJECTIVES: To compare an FDA-approved HIV-1/HIV-2 antibody differentiation test (Multispot) as a confirmatory test with the HIV-1 Western blot and IFA. STUDY DESIGN: Participants were screened with an HIV-1/HIV-2 combination Antigen/Antibody (Ag/Ab) screening assay. Specimens with repeatedly reactive results were tested with Multispot and either Western blot or IFA. Specimens with discordant screening and confirmatory results were resolved with HIV-1 RNA testing. RESULTS: Individuals (37,876) were screened for HIV infection and 654 (1.7%) had a repeatedly reactive Ag/Ab assay result. On Multispot, 554 (84.7%) were HIV-1 reactive, 0 (0%) were HIV-2 reactive, 1 (0.2%) was reactive for both HIV-1 and HIV-2 (undifferentiated), 9 (1.4%) were HIV-1 indeterminate, and 90 (13.8%) were non-reactive. HIV-1 RNA was detected in 47/90 Multispot non-reactive (52.2%) specimens. Among specimens confirmed to have HIV infection (true positives), Multispot and Western blot detected HIV-1 antibody in a similar proportion of cases (93.7% vs. 94.4% respectively) while Multispot and IFA also detected HIV-1 antibody in a similar proportion of cases (84.5% vs. 83.4% respectively). CONCLUSIONS: In this study, Multispot confirmed HIV infections at a similar proportion to Western blot and IFA. Multispot, Western blot, and IFA, however, did not confirm all of the reactive Ag/Ab assay results and underscores the importance of HIV NAT testing to resolve discordant screening and confirmatory results.

Previously, we showed an inverse correlation between HSP27 serum levels and experimental atherogenesis in ApoE(-/-) mice that over-express HSP27 and speculated that the apparent binding of HSP27 to scavenger receptor-A (SR-A) was of mechanistic importance in attenuating foam cell formation. However, the nature and importance of the interplay between HSP27 and SR-A in atheroprotection remained unclear. Treatment of THP-1 macrophages with recombinant HSP27 (rHSP27) inhibited acLDL binding (-34%; p<0.005) and uptake (-38%, p<0.05). rHSP27 reduced SR-A mRNA (-39%, p=0.02), total protein (-56%, p=0.01) and cell surface (-53%, p<0.001) expression. The reduction in SR-A expression by rHSP27 was associated with a 4-fold increase in nuclear factor-kappa B (NF-κB) signaling (p<0.001 versus control), while an inhibitor of NF-κB signaling, BAY11-7082, attenuated the negative effects of rHSP27 on both SR-A expression and lipid uptake. To determine if SR-A is required for HSP27 mediated atheroprotection in vivo, ApoE(-/-) and ApoE(-/-) SR-A(-/-) mice fed with a high fat diet were treated for 3weeks with rHSP25. Compared to controls, rHSP25 therapy reduced aortic en face and aortic sinus atherosclerotic lesion size in ApoE(-/-) mice by 39% and 36% (p<0.05), respectively, but not in ApoE(-/-)SR-A(-/-) mice. In conclusion, rHSP27 diminishes SR-A expression, resulting in attenuated foam cell formation in vitro. Regulation of SR-A by HSP27 may involve the participation of NF-κB signaling. Lastly, SR-A is required for HSP27-mediated atheroprotection in vivo.

Background: The actions of glucocorticoids and neurotrophins, such as BDNF, have been implicated in numerous psychiatric disorders. However, the mechanisms of how glucocorticoids and BDNF influence maladaptive actions are not well understood. We have previously shown that genetic disruption of glucocorticoid signaling in the hypothalamus resulted in disinhibition of the HPA axis, upregulation of hypothalamic levels of BDNF and increased CRH expression. Our present studies show there is a close relationship between BDNF signaling and the actions of the glucocorticoid receptor (GR), a ligand-activated transcription factor through post-transcriptional modifications by phosphorylation. Methods: Mass spectrometry analysis of the glucocorticoid receptor isolated from cortical neurons treated with BDNF revealed new phosphorylation sites. To test the significance of these events, we have examined the impact of BDNF signaling on glucocorticoid function using gene expression microarray and real time quantitative PCR in primary rat cortical neurons stimulated with the selective GR agonist dexamethasone (Dex) and BDNF, alone or in combination. Results: We found that BDNF treatment induces the phosphorylation of the glucocorticoid receptor (GR) at serine 155 (S155) and serine 287 (S287). Expression of a non-phosphorylatable alanine double mutant (S155A/ S287A) impaired the induction of a subset of BDNF and Dex regulated genes. Moreover, BDNF-induced GR phosphorylation increased GR occupancy and cofactor recruitment at the promoters of selective genes. Therefore, BDNF signaling acts to specify and amplify GR-mediated transcription by a phosphorylation-dependent mechanism. Conclusions: The interactions between BDNF and glucocorticoids include specific phosphorylation of GR by BDNF. We have identified several new serine phosphorylation sites in GR, which result in an amplification of transcriptional responses by BDNF signaling

Rhomboid proteases are a family of integral membrane proteins that have been implicated in critical regulatory roles in a wide array of cellular processes and signaling events. The determination of crystal structures of the prokaryotic rhomboid GlpG from Escherichia coli and Haemophilus influenzae has ushered in an era of unprecedented understanding into molecular aspects of intramembrane proteolysis by this fascinating class of protein. A combination of structural studies by X-ray crystallography, and biophysical and spectroscopic analyses, combined with traditional enzymatic and functional analysis has revealed fundamental aspects of rhomboid structure, substrate recognition and the catalytic mechanism. This review summarizes these remarkable advances by examining evidence for the proposed catalytic mechanism derived from inhibitor co-crystal structures, conflicting models of rhomboid-substrate interaction, and recent work on the structure and function of rhomboid cytosolic domains. In addition to exploring progress on aspects of rhomboid structure, areas for future research and unaddressed questions are emphasized and highlighted. This article is part of a Special Issue entitled: Intramembrane Proteases.

Abstract Adhesion G-protein-coupled receptors (GPCRs) are the most recently identified and least understood subfamily of GPCRs. Adhesion GPCRs are characterized by unusually long ectodomains with adhesion-related repeats that facilitate cell- cell and cell-cell matrix contact, as well as a proteolytic cleavage site-containing domain that is a structural hallmark of the family. Their unusual chimeric structure of adhesion-related ectodomain with a seven-pass transmembrane domain and cytoplasmic signaling makes these proteins highly versatile in mediating cellular signaling in response to extracellular adhesion or cell motility events. The ligand binding and cytoplasmic signaling modes for members of this family are beginning to be elucidated, and recent studies have demonstrated critical roles for Adhesion GPCRs in planar polarity and other important cell-cell and cell-matrix interactions during development and morphogenesis, as well as heritable diseases and cancer.

Abstract From generating the TOP-GAL mouse to pioneering high-throughput RNAi, and small molecule chemical genetic screens in Drosophila and mammalian cells, Ram DasGupta has consistently developed innovative technological tools of immense value to the fields in which he has chosen to work.

Abstract On a recent visit Richard O Hynes, FRS, HHMI, Daniel K. Ludwig Professor for Cancer Research at the Koch Institute for Integrative Cancer Research, MIT, graciously agreed to be interviewed in person for the first in Cell Communication and Adhesion's series on "Leaders in Cell Adhesion". In this interview we discussed three things: 1) the early role of family, mentors, and luck on his career path; 2) his major discoveries of fibronectin, integrins and the evolution of extracellular matrix proteins; and 3) his role in, and thoughts on, current science policy. This interview reveals his characteristic calmness and infectious optimism, his spontaneous and down to earth sense of humor, and his great ability to place scientific questions in perspective. The interview, carried out on April 30(th) 2013 is reported here verbatim with only minor editing for clarity.

Inflammation-associated pigmentation changes are extremely common, but the etiology underlying this clinical observation remains elusive. Particularly, it is unclear how the myriad of cytokines known to be involved in inflammatory skin processes affect epidermal melanocytes. We sought to determine how IL-17 and tumor necrosis factor (TNF) influence normal human melanocytes, as these two cytokines have been implicated in various skin diseases. IL-17 and TNF jointly stimulated broad inductions of cytokines, including melanoma mitogens CXCL1 and IL-8. Moreover, IL-17 and TNF synergistically inhibited pigmentation-related signaling and melanin production, and induced keratinocyte production of beta-defensin 3, an antagonist for melanocortin 1 receptor. When analyzing psoriasis lesions that are known to overexpress IL-17 and TNF, we observed an increase in melanocyte number and a simultaneous decrease in pigmentation signaling. Furthermore, therapeutic neutralization of TNF and IL-17 with mAbs resulted in a rapid recovery of pigment gene expression in psoriasis lesions. These results demonstrate that IL-17 and TNF can affect both the growth and pigment production of melanocytes, which may contribute to the pigmentation changes associated with psoriasis. These findings may allow the development of novel therapeutics for pigmentary disorders and bring new insights into the immune milieu surrounding melanocytes and related neoplasms.

OBJECTIVE: ANXA6, the gene for annexin A6, is highly expressed in osteoarthritic (OA) articular chondrocytes but not in healthy articular chondrocytes. This study was undertaken to determine whether annexin A6 affects catabolic events in these cells. METHODS: Articular chondrocytes were isolated from Anxa6-knockout mice, wild-type (WT) mice, and human articular cartilage in which ANXA6 was overexpressed. Cells were treated with interleukin-1beta (IL-1beta) or tumor necrosis factor alpha (TNFalpha), and expression of catabolic genes and activation of NF-kappaB were determined by real-time polymerase chain reaction and luciferase reporter assay. Anxa6(-/-) and WT mouse knee joints were injected with IL-1beta or the medial collateral ligament was transected and partial resection of the medial meniscus was performed to determine the role of Anxa6 in IL-1beta-mediated cartilage destruction and OA progression. The mechanism by which Anxa6 stimulates NF-kappaB activity was determined by coimmunoprecipitation and immunoblot analysis of nuclear and cytoplasmic fractions of IL-1beta-treated Anxa6(-/-) and WT mouse chondrocytes for p65 and Anxa6. RESULTS: Loss of Anxa6 resulted in decreased NF-kappaB activation and catabolic marker messenger RNA (mRNA) levels in IL-1beta- or TNFalpha-treated articular chondrocytes, whereas overexpression of ANXA6 resulted in increased NF-kappaB activity and catabolic marker mRNA levels. Annexin A6 interacted with p65, and loss of Anxa6 caused decreased nuclear translocation and retention of the active p50/p65 NF-kappaB complex. Cartilage destruction in Anxa6(-/-) mouse knee joints after IL-1beta injection or partial medial meniscectomy was reduced as compared to that in WT mouse joints. CONCLUSION: Our data define a role of annexin A6 in the modulation of NF-kappaB activity and in the stimulation of catabolic events in articular chondrocytes.