Faculty Bibliography

Normal mammary morphogenesis involves transitions between simple and multilayered epithelial organization. We used electron microscopy and molecular markers to determine whether intercellular junctions and apico-basal polarity were maintained in the multilayer. We found that multilayered elongating ducts had polarized apical and basal tissue surfaces both in 3D culture and in vivo. However, individual cells were only polarized on surfaces in contact with the lumen or extracellular matrix. The basolateral marker Scribble and the apical marker atypical protein kinase C zeta localized to all interior cell membranes, while Par-3 displayed cytoplasmic localization, suggesting incomplete apico-basal polarity. Despite membrane localization of E-cadherin and beta-catenin, we did not observe a defined zonula adherens connecting interior cells. Instead, interior cells were connected through desmosomes and exhibited complex, interdigitating membrane protrusions. Single cell labeling revealed that individual cells were both protrusive and migratory within the epithelial multilayer. Inhibition of Rho kinase (ROCK) further reduced intercellular adhesion on apical and lateral surfaces, but did not disrupt basal tissue organization. Following morphogenesis, segregated membrane domains were re-established and junctional complexes reformed. We observed similar epithelial organization during mammary morphogenesis in organotypic culture and in vivo. We conclude that mammary epithelial morphogenesis involves a reversible, spatially limited, reduction in polarity and intercellular junctions and active, individualistic cell migration. Our data suggest that reductions in polarity and adhesion during breast cancer progression may reflect partial recapitulation of a normal developmental program.

Homeostasis of the protein-folding environment in the endoplasmic reticulum (ER) is maintained by signal transduction pathways that collectively constitute an unfolded protein response (UPR). These affect bulk protein synthesis and thereby the levels of ER stress, but also culminate in regulated expression of specific mRNAs, such as that encoding the transcription factor ATF4. Mechanisms linking eukaryotic initiation factor 2 (eIF2) phosphorylation to control of unfolded protein load in the ER were elucidated more than 10 years ago, but recent work has highlighted the diversity of processes that impinge on eIF2 activity and revealed that there are multiple mechanisms by which changes in eIF2 activity can modulate the translation of individual mRNAs. In addition, the potential for affecting this step of translation initiation pharmacologically is becoming clearer. Furthermore, it is now clear that another strand of the UPR, controlled by the endoribonuclease inositol-requiring enzyme 1 (IRE1), also affects rates of protein synthesis in stressed cells and that its effector function, mediated by the transcription factor X-box-binding protein 1 (XBP1), is subject to important mRNA-specific translational regulation. These new insights into the convergence of translational control and the UPR will be reviewed here.

Genomewide-association studies have revealed that SNPs (single nucleotide polymorphisms) in FTO (fat mass and obesity-associated) are robustly associated with BMI (body mass index) and obesity. FTO is an Fe(II) 2-OG (2-oxoglutarate)-dependent dioxygenase that can demethylate 3-meT (3-methylthymine) in single-stranded DNA, as well as 3-meU (3-methyluracil) and N6-methyl adenosine in RNA. In the present paper we describe the development of an RNase-cleavage assay measuring the demethylation activity of FTO on 3-meU. RNase A cleaves at the 3'-end of pyrimidines, including uracil, and a methyl group at position three of uracil inhibits cleavage. An oligonucleotide probe was designed consisting of a DNA stem, an RNA loop containing a single 3-meU as the only RNase A-cleavage site, a fluorescent reporter on one end and a quencher at the other end. FTO demethylation of the unique 3-meU enables RNase A cleavage, releasing the quencher and enabling a fluorescent signal. In the presence of excess RNase A, FTO activity is limiting to the development of fluorescent signal, which can be read continuously and is able to discriminate between wild-type and the catalytically dead R316Q FTO. 2-OG is a co-substrate of FTO and, as a metabolite in the citric acid cycle, is a marker of intracellular nutritional status. The assay described in the present paper was used to measure, for the first time, the K(m) of FTO for 2-OG. The K(m) of 2.88 muM is up to 10-fold lower than the estimated intracellular concentrations of 2-OG, rendering it unlikely that FTO functions as a sensor for 2-OG levels.

Trans-epithelial migration describes the ability of migrating cells to cross epithelial tissues and occurs during development, infection, inflammation, immune surveillance, wound healing and cancer metastasis. Here we investigate Drosophila primordial germ cells (PGCs), which migrate through the endodermal epithelium. Through live imaging and genetic experimentation we demonstrate that PGCs take advantage of endodermal tissue remodeling to gain access to the gonadal mesoderm and are unable to migrate through intact epithelial tissues. These results are in contrast to the behavior of leukocytes, which actively loosen epithelial junctions to migrate, and raise the possibility that in other contexts in which migrating cells appear to breach tissue barriers, they are actually exploiting existing tissue permeability. Therefore, the use of active invasive programs is not the sole mechanism to infiltrate tissues.

Tremendous global efforts have been made to collect data on the HIV/AIDS epidemic. Yet, significant challenges remain for generating and analysing evidence to allocate resources efficiently and implement an effective AIDS response. India offers important lessons and a model for intelligent and integrated use of data on HIV/AIDS for an evidence-based response. Over the past 15 years, the number of data sources has expanded and the geographical unit of data generation, analysis and use for planning has shifted from the national to the state, district and now subdistrict level. The authors describe and critically analyse the evolution of data sets in India and how they have been utilised to better understand the epidemic, advance policy, and plan and implement an increasingly effective, well-targeted and decentralised national response to HIV and AIDS. The authors argue that India is an example of how 'know your epidemic, know your response' message can effectively be implemented at scale and presents important lessons to help other countries design their evidence generation systems.

Lactoferrin (Lf) is a bi-lobed, iron-binding protein found on mucosal surfaces and at sites of inflammation. Gram-negative pathogens from the Neisseriaceae and Moraxellaceae families are capable of using Lf as a source of iron for growth through a process mediated by a bacterial surface receptor that directly binds host Lf. This receptor consists of an integral outer membrane protein, lactoferrin binding protein A (LbpA), and a surface lipoprotein, lactoferrin binding protein B (LbpB). The N-lobe of the homologous transferrin binding protein B, TbpB, has been shown to facilitate transferrin binding in the process of iron acquisition. Currently there is little known about the role of LbpB in iron acquisition or how Lf interacts with the bacterial receptor proteins. No structural information on any LbpB or domain is available. In this study, we express and purify from Escherichia coli the full-length LbpB and the N-lobe of LbpB from the bovine pathogen Moraxella bovis for crystallization trials. We demonstrate that M. bovis LbpB binds to bovine but not human Lf. We also report the crystal structure of the N-terminal lobe of LbpB from M. bovis and compare it with the published structures of TbpB to speculate on the process of Lf mediated iron acquisition.

BACKGROUND: Nasal polyposis (NP) is a Th2-skewed inflammatory disorder, but it is unclear what role regulatory T cells (T-reg) play in disease pathology. We investigated the expression profiles of T-reg and T-helper-cell-associated genes and their response to glucocorticosteroid (GC) treatment in Chinese patients with NP. METHODS: Biopsies were obtained from 29 non-treated NP patients for comparison with inferior turbinates collected from healthy controls. In 13 patients, NP samples were collected both before and after short-term oral GC treatment. Levels of mRNA for T-cell markers were determined by microarray and quantitative PCR. Cellular infiltrates were assessed by histo- and immunohistochemistry. RESULTS: FOXP3(+) T-reg were increased in GC-naive NP, and numbers were negatively correlated with eosinophil infiltration. Helios staining was not detected, suggesting that FOXP3(+) cells in NP are not thymus-derived T-reg. Compared with controls, mRNA levels corresponding to T-reg genes were significantly increased in NP (FOXP3, TGFB1, IL10, SMAD3, IL2RA, and JAK3), but transcription factors associated with Th2 (GATA3) or Th17 responses (RORc) were significantly reduced. FOXP3 mRNA levels positively correlated with other T-reg cell markers. Microarray analysis showed that most Th2-related markers (e.g., Eotaxin-1, CCL13, and CCL18) were upregulated in GC-naive NP vs controls. GC therapy significantly suppressed eosinophilic inflammation in NP, but did not significantly alter the expression levels of T-reg/Th2-associated genes. CONCLUSIONS: Upregulation of FOXP3(+) -inducible T-reg cells and downregulation of Th2 and Th17 markers in NP indicate a regulatory response occurring at a site of persistent mucosal inflammation. However, immune regulation fails to control the underlying tissue pathology. Expression of T-reg/Th2 markers after GC treatment was unaltered, suggesting that T-cell-driving NP inflammatory mediators are GC resistant.

OBJECTIVES: The reported long-term safety of kidney donation is inconsistent with the impairment of kidney function observed following nephrectomy for renal cell cancer. We aimed to investigate if indication for nephrectomy (kidney cancer vs. living donation) was an independent risk factor for kidney function deterioration. MATERIALS AND METHODS: Between 1985 and 2008, 124 patients with localized renal cell carcinoma who meet the criteria used for living donation, underwent radical nephrectomy (group 1) at our institution. Group 1 was retrospectively compared with 124 consecutive living donor nephrectomies (group 2) performed from 2004 to 2008. Kidney function evaluation was performed preoperatively and at 1, 2, 3, and 4 years postoperatively with calculation of estimated glomerular filtration rate through the Modification of Diet in Renal Disease (MDRD-eGFR) and the adjusted Cockroft and Gault (CG-eGFR) formula. Multivariate logistic regression included patients' characteristics and indication for nephrectomy as predictors of kidney function deterioration. RESULTS: Mean decrease in MDRD-eGFR was 30.4% and 32.4% in groups 1 and 2 (P = 0.30). Prevalence of chronic kidney disease (CKD), defined by MDRD-eGFR < 60 mL/min/m(2), varied from 42.3% to 71% in group 1 and from 41.6% to 56% in group 2 at different time points (P = 0.073). Prevalence of CKD at 4 years defined by MDRD-eGFR < 45 mL/min/m(2) was significantly increased in group 1 compared with group 2 (16.2% and 5.3%, P < 0.005, respectively). Linear regression analysis showed only baseline kidney function and patient age predicted a significant decrease in postoperative kidney function (P < 0.001 and P = 0.04). CONCLUSIONS: Renal cell carcinoma is not an independent risk factor for kidney function impairment following nephrectomy. Selected kidney cancer patients with few morbidities face the same deterioration of meanly 30% of kidney function compared with living donors, but their lower baseline function results in an increased risk for CKD.