KLK5 Inactivation Reverses Cutaneous Hallmarks of Netherton Syndrome

Netherton Syndrome (NS) is a rare and severe autosomal recessive skin disease which can be life-threatening in infants. The disease is characterized by extensive skin desquamation, inflammation, allergic manifestations and hair shaft defects. NS is caused by loss-of-function mutations in SPINK5 encoding the LEKTI serine protease inhibitor. LEKTI deficiency results in unopposed activities of kallikrein-related peptidases (KLKs) and aberrantly increased proteolysis in the epidermis. Spink5 ^-/- mice recapitulate the NS phenotype, display enhanced epidermal Klk5 and Klk7 protease activities and die within a few hours after birth because of a severe skin barrier defect. However the contribution of these various proteases in the physiopathology remains to be determined. In this study, we developed a new murine model in which Klk5 and Spink5 were both knocked out to assess whether Klk5 deletion is sufficient to reverse the NS phenotype in Spink5 ^-/- mice. By repeated intercrossing between Klk5 ^-/- mice with Spink5 ^-/- mice, we generated Spink5 ^-/- Klk5 ^-/- animals. We showed that Klk5 knock-out in Lekti-deficient newborn mice rescues neonatal lethality, reverses the severe skin barrier defect, restores epidermal structure and prevents skin inflammation. Specifically, using in situ zymography and specific protease substrates, we showed that Klk5 knockout reduced epidermal proteolytic activity, particularly its downstream targets proteases KLK7, KLK14 and ELA2. By immunostaining, western blot, histology and electron microscopy analyses, we provide evidence that desmosomes and corneodesmosomes remain intact and that epidermal differentiation is restored in Spink5 ^-/- Klk5 ^-/-. Quantitative RT-PCR analyses and immunostainings revealed absence of inflammation and allergy in Spink5 ^-/- Klk5 ^-/- skin. Notably, Il-1β, Il17A and Tslp levels were normalized. Our results provide in vivo evidence that KLK5 knockout is sufficient to reverse NS-like symptoms manifested in Spink5 ^-/- skin. These findings illustrate the crucial role of protease regulation in skin homeostasis and inflammation, and establish KLK5 inhibition as a major therapeutic target for NS.     

Increased breast density correlates with the proliferation-seeking radiotracer (99m)Tc(V)-DMSA uptake in florid epithelial hyperplasia and in mixed ductal carcinoma in situ with invasive ductal carcinoma but not in pure invasive ductal carcinoma or in mild epithelial hyperplasia

The purpose of this study was to assess the relationship of mammographic breast density (BD) and cell proliferation/focal adhesion kinase activation-seeking radiotracer technetium 99m pentavalent dimercaptosuccinic acid (99mTc(V)-DMSA) uptake in women with different breast histologies, that is, mild epithelial hyperplasia (MEH), florid epithelial hyperplasia (FEH), mixed ductal carcinoma in situ with invasive ductal carcinoma (DCIS + IDC), and pure IDC. Fifty-five women with histologically confirmed mammary pathologies were submitted preoperatively to mammography and 99mTc(V)-DMSA scintimammography. The percentage and intensity of 99mTc(V)-DMSA uptake and the percentage of BD were calculated by computer-assisted methods and compared (t-test) between the breast pathologies.?In breasts with increased BD, FEH and DCIS + IDC were found. On the contrary, pure IDC and MEH were identified in breasts with significantly lower BD values. In breasts with increased 99mTc(V)-DMSA area and intensity of uptake, FEH was the main lesion found compared to all other histologies. Linear regression analysis between BD and 99mTc(V)-DMSA uptake area and intensity revealed significant coefficients of correlation (r = .689, p < .001 and r = .582, p < .001, respectively). Increased BD correlates with the presence of FEH and mixed DCIS + IDC but not with pure IDC or MEH. Its close relationship to 99mTc(V)-DMSA, which also showed an affinity to FEH, indicates that stromal microenvironment may constitute a specific substrate leading to progression to different subtypes of cancerous lesions originating from different pathways.     

Metabolic response of perfused livers to various oxygenation conditions

Isolated liver perfusion systems have been used to characterize intrinsic metabolic changes in liver as a result of various perturbations, including systemic injury, hepatotoxin exposure, and warm ischemia. Most of these studies were done using hyperoxic conditions (95% O(2)) but without the use of oxygen carriers in the perfusate. Prior literature data do not clearly establish the impact of oxygenation, and in particular that of adding oxygen carriers to the perfusate, on the metabolic functions of the liver. Therefore, herein the effects of oxygen delivery in the perfusion system on liver metabolism were investigated by comparing three modes of oxygenation. Rat livers were perfused via the portal and hepatic veins at a constant flow rate of 3 mL/min/g liver in a recirculating perfusion system. In the first group, the perfusate was equilibrated in a membrane oxygenator with room air (21% O(2)) before entering the liver. In the second group, the perfusate was equilibrated with a 95% O(2)/5% CO(2) gas mixture. In the third group, the perfusate was supplemented with washed bovine red blood cells (RBCs) at 10% hematocrit and also equilibrated with the 95% O(2)/5% CO(2) gas mixture. Oxygen and CO(2) gradients across the liver were measured periodically with a blood gas analyzer. The rate of change in the concentration of major metabolites in the perfusate was measured over time. Net extracellular fluxes were calculated from these measurements and applied to a stoichiometric-based optimization problem to determine the intracellular fluxes and active pathways in the perfused livers. Livers perfused with RBCs consumed oxygen at twice the rate observed using hyperoxic (95% O(2)) perfusate without RBCs, and also produced more urea and ketone bodies. At the flow rate used, the oxygen supply in perfusate without RBCs was just sufficient to meet the average oxygen demand of the liver but would be insufficient if it increased above baseline, as is often the case in response to environmental perturbations. Metabolic pathway analysis suggests that significant anaerobic glycolysis occurred in the absence of RBCs even using hyperoxic perfusate. Conversely, when RBCs were used, glucose production from lactate and glutamate, as well as pathways related to energy metabolism were upregulated. RBCs also reversed an increase in PPP fluxes induced by the use of hyperoxic perfusate alone. In conclusion, the use of oxygen carriers is required to investigate the effect of various perturbations on liver metabolism.     

High-resolution analysis of the human retina miRNome reveals isomiR variations and novel microRNAs

MicroRNAs play a fundamental role in retinal development and function. To characterise the miRNome of the human retina, we carried out deep sequencing analysis on sixteen individuals. We established the catalogue of retina-expressed miRNAs, determined their relative abundance and found that a small number of miRNAs accounts for almost 90% of the retina miRNome. We discovered more than 3000 miRNA variants (isomiRs), encompassing a wide range of sequence variations, which include seed modifications that are predicted to have an impact on miRNA action. We demonstrated that a seed-modifying isomiR of the retina-enriched miR-124-3p was endowed with different targeting properties with respect to the corresponding canonical form. Moreover, we identified 51 putative novel, retina-specific miRNAs and experimentally validated the expression for nine of them. Finally, a parallel analysis of the human Retinal Pigment Epithelium (RPE)/choroid, two tissues that are known to be crucial for retina homeostasis, yielded notably distinct miRNA enrichment patterns compared to the retina. The generated data are accessible through an ad hoc database. This study is the first to reveal the complexity of the human retina miRNome at nucleotide resolution and constitutes a unique resource to assess the contribution of miRNAs to the pathophysiology of the human retina.     

A novel monoclonal antibody against human Argonaute proteins reveals unexpected characteristics of miRNAs in human blood cells

Argonaute (Ago) proteins bind to microRNA (miRNAs) and short interfering RNAs (siRNAs) and form the core components of effector complexes that mediate miRNA and siRNA function. Currently, there is a paucity of reliable antibodies against mammalian Ago proteins, thus precluding studies of endogenous Ago proteins from tissues. Here we report the development of 2A8, a novel anti-Ago monoclonal antibody that recognizes human and mouse Ago proteins and efficiently immunoprecipitates miRNAs. We report the characterization of 2A8 and its use to clone miRNAs from human brain and from preparations of human polymorphonuclear leukocytes (neutrophils), which revealed a prevalent miRNA with unusual features.     

Gene symbol disambiguation using knowledge-based profiles

MOTIVATION: The ambiguity of biomedical entities, particularly of gene symbols, is a big challenge for text-mining systems in the biomedical domain. Existing knowledge sources, such as Entrez Gene and the MEDLINE database, contain information concerning the characteristics of a particular gene that could be used to disambiguate gene symbols. RESULTS: For each gene, we create a profile with different types of information automatically extracted from related MEDLINE abstracts and readily available annotated knowledge sources. We apply the gene profiles to the disambiguation task via an information retrieval method, which ranks the similarity scores between the context where the ambiguous gene is mentioned, and candidate gene profiles. The gene profile with the highest similarity score is then chosen as the correct sense. We evaluated the method on three automatically generated testing sets of mouse, fly and yeast organisms, respectively. The method achieved the highest precision of 93.9% for the mouse, 77.8% for the fly and 89.5% for the yeast. AVAILABILITY: The testing data sets and disambiguation programs are available at http://www.dbmi.columbia.edu/~hux7002/gsd2006     

A potential role for multiple tissue kallikrein serine proteases in epidermal desquamation

Desquamation of the stratum corneum is a serine protease-dependent process. Two members of the human tissue kallikrein (KLK) family of (chymo)tryptic-like serine proteases, KLK5 and KLK7, are implicated in desquamation by digestion of (corneo)desmosomes and inhibition by desquamation-related serine protease inhibitors (SPIs). However, the epidermal localization and specificity of additional KLKs also supports a role for these enzymes in desquamation. This study aims to delineate the probable contribution of KLK1, KLK5, KLK6, KLK13, and KLK14 to desquamation by examining their interactions, in vitro, with: 1) colocalized SPI, lympho-epithelial Kazal-type-related inhibitor (LEKTI, four recombinant fragments containing inhibitory domains 1-6 (rLEKTI(1-6)), domains 6-8 and partial domain 9 (rLEKTI(6-9')), domains 9-12 (rLEKTI(9-12)), and domains 12-15 (rLEKTI(12-15)), secretory leukocyte protease inhibitor, and elafin and 2) their ability to digest the (corneo)desmosomal cadherin, desmoglein 1. KLK1 was not inhibited by any SPI tested. KLK5, KLK6, KLK13, and KLK14 were potently inhibited by rLEKTI(1-6), rLEKTI(6-9'), and rLEKTI(9-12) with Ki values in the range of 2.3-28.4 nm, 6.1-221 nm, and 2.7-416 nm for each respective fragment. Only KLK5 was inhibited by rLEKTI(12-15) (Ki = 21.8 nm). No KLK was inhibited by secretory leukocyte protease inhibitor or elafin. Apart from KLK13, all KLKs digested the ectodomain of desmoglein 1 within cadherin repeats, Ca2+ binding sites, or in the juxtamembrane region. Our study indicates that multiple KLKs may participate in desquamation through cleavage of desmoglein 1 and regulation by LEKTI. These findings may have clinical implications for the treatment of skin disorders in which KLK activity is elevated.