Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea

Acne rosacea is an inflammatory skin disease that affects 3% of the US population over 30 years of age and is characterized by erythema, papulopustules and telangiectasia. The etiology of this disorder is unknown, although symptoms are exacerbated by factors that trigger innate immune responses, such as the release of cathelicidin antimicrobial peptides. Here we show that individuals with rosacea express abnormally high levels of cathelicidin in their facial skin and that the proteolytically processed forms of cathelicidin peptides found in rosacea are different from those present in normal individuals. These cathelicidin peptides are a result of a post-translational processing abnormality associated with an increase in stratum corneum tryptic enzyme (SCTE) in the epidermis. In mice, injection of the cathelicidin peptides found in rosacea, addition of SCTE, and increasing protease activity by targeted deletion of the serine protease inhibitor gene Spink5 each increases inflammation in mouse skin. The role of cathelicidin in enabling SCTE-mediated inflammation is verified in mice with a targeted deletion of Camp, the gene encoding cathelicidin. These findings confirm the role of cathelicidin in skin inflammatory responses and suggest an explanation for the pathogenesis of rosacea by demonstrating that an exacerbated innate immune response can reproduce elements of this disease.     

Lysosomal thiol reductase negatively regulates autophagy by altering glutathione synthesis and oxidation

Redox regulation is critical for a number of cellular functions and has been implicated in the etiology and progression of several diseases, such as cardiovascular diseases, neurodegenerative diseases, and cancer. It has been shown that, in the absence of gamma-interferon inducible lysosomal thiol reductase (GILT), cells are under increased oxidative stress with higher superoxide levels and decreased stability, expression, and function of mitochondrial manganese superoxide dismutase (SOD2). Here, we further elucidate the role of GILT in the homeostatic regulation of oxidative stress. We show that GILT-deficient fibroblasts exhibit reduced glutathione levels, shift in GSSG/GSH ratio toward the oxidized form, and accumulate dysfunctional mitochondria. Redox-sensitive pathways involving Erk1/2 activation and nuclear high mobility group box 1 (HMGB1) protein cytosolic translocation are both activated and associated with increased autophagy in GILT−/− fibroblasts. We hypothesize that these events are responsible for degrading the damaged mitochondria and mitochondrial SOD2 in the absence of GILT. This is the first time to our knowledge that a lysosomal enzyme has been implicated in global effects within the cell.     

Helper B cells promote cytotoxic T cell survival and proliferation independently of antigen presentation through CD27/CD70 interactions

CD8-expressing cytotoxic T cell (CTL) interactions with APCs and helper T cells determine their function and ability to survive. In this study, we describe a novel interaction independent of Ag presentation between activated CTLs and bystander CD19-expressing B lymphocytes. Ag-stimulated CTLs serially engage autologous B lymphocytes through CD27/CD70 contact that promotes their survival and proliferation. Moreover, these interactions induce the release of proinflammatory cytokines that follows two general patterns: 1) an epitope-dependent enhancement of cytokine release, and 2) a previously undiscovered coordinate release of cytokines independent of epitope exposure. The latter includes chemoattractants targeting activated T cells. As a result, activated T cells are attracted to B cells, which exert a "helper" role in lymphatic organs or in areas of inflammation. This observation provides a mechanistic explanation to previously reported experimental observations suggesting that B cells are required for T cell priming in vivo.     

Label-free Quantitative Urinary Proteomics Identifies the Arginase Pathway as a New Player in Congenital Obstructive Nephropathy

Obstructive nephropathy is a frequently encountered situation in newborns. In previous studies, the urinary peptidome has been analyzed for the identification of clinically useful biomarkers of obstructive nephropathy. However, the urinary proteome has not been explored yet and should allow additional insight into the pathophysiology of the disease. We have analyzed the urinary proteome of newborns (n = 5/group) with obstructive nephropathy using label free quantitative nanoLC-MS/MS allowing the identification and quantification of 970 urinary proteins. We next focused on proteins exclusively regulated in severe obstructive nephropathy and identified Arginase 1 as a potential candidate molecule involved in the development of obstructive nephropathy, located at the crossroad of pro- and antifibrotic pathways. The reduced urinary abundance of Arginase 1 in obstructive nephropathy was verified in independent clinical samples using both Western blot and MRM analysis. These data were confirmed in situ in kidneys obtained from a mouse obstructive nephropathy model. In addition, we also observed increased expression of Arginase 2 and increased total arginase activity in obstructed mouse kidneys. mRNA expression analysis of the related arginase pathways indicated that the pro-fibrotic arginase-related pathway is activated during obstructive nephropathy. Taken together we have identified a new actor in the development of obstructive nephropathy in newborns using quantitative urinary proteomics and shown its involvement in an in vivo model of disease. The present study demonstrates the relevance of such a quantitative urinary proteomics approach with clinical samples for a better understanding of the pathophysiology and for the discovery of potential therapeutic targets.Congenital obstructive nephropathy is the main cause of end stage renal disease (ESRD) in children (1). The most frequently found cause of congenital obstructive nephropathy is ureteropelvic junction (UPJ)¹ obstruction with an estimated incidence of 1 in 1000–1500 births. Milder forms of UPJ obstruction often progress to the spontaneous resolution of the pathology over time. This has led to a watchful waiting approach with surgical intervention only if renal deterioration is detected (2). Although this medical surveillance prevents unnecessary surgery, it mostly relies on invasive follow-up. Consequently with the aim to reduce this invasive follow-up, several groups have initiated research to identify noninvasive urinary biomarkers of UPJ obstruction using both targeted and nontargeted (e.g. proteome analysis based) strategies. Targeted strategies including urinary cytokine expression analyses failed to clearly determine the need for surgery in UPJ obstruction (3, 4). On the other hand, untargeted strategies have been more successful and by using urinary proteomics, biomarkers for renal and non-renal diseases have been identified (5–9). Using urinary peptidome analysis, we identified and validated a urinary peptide panel that predicted the clinical outcome of newborns with UPJ obstruction with 97% accuracy several months in advance (3, 10). An independent small-scale study confirmed the efficiency of this biomarker panel (7). These studies indicate the potential of urinary proteomics to predict the clinical fate of patients with UPJ obstruction. Although these endogenous urinary peptide biomarkers are of great potential clinical value, sequencing of these biomarkers mainly identified collagen fragments that are less informative on the pathophysiology of the disease. In contrast, studies of the high molecular weight urinary proteome (i.e. proteins) might be more informative on the pathophysiology of disease. Different approaches have been used in the past to characterize the urinary proteome, either by 2D-gel electrophoresis coupled to mass spectrometry (11, 12) or reverse phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis (13–16). In-depth proteome analysis using extensive fractionation of the sample and high resolution, fast sequencing mass spectrometers have reported the identification of >2000 proteins in normal human urine (13, 15, 16). Here, we applied quantitative high-resolution label free LC-MS/MS analysis for the identification of urinary proteins associated to UPJ obstruction in newborns. Among a number of proteins uniquely associated with severe UPJ obstruction, we identified Arginase 1, not previously recognized in UPJ obstruction. Using an independent larger cohort, we further verified reduced urinary abundance of Arginase 1 using both Western blot and multiple reaction monitoring (MRM). Using the mouse model of obstructive nephropathy, we observed that the expression of arginases is modulated in situ in obstructed kidneys. Further gene expression analysis of the arginase pathway allowed us to hypothesize for a role of arginases in the development of fibrotic lesions in obstructive nephropathy.     

Discovery of thiadiazoles as a novel structural class of potent and selective PDE7 inhibitors. Part 2: metabolism-directed optimization studies towards orally bioavailable derivatives

The synthesis and optimization of pharmacokinetic parameters of structurally novel small PDE7 inhibitors is discussed.     

Remodeling of chromatin loops does not account for specification of replication origins during Xenopus development

We have investigated the possible relationship between replicons and chromatin loops during Xenopus development. In early embryos, replication of the ribosomal RNA genes (rDNA) can initiate at apparently any sequence. Nevertheless, the need for a regular spacing of replication origins suggests that some periodic chromatin folding might dictate which sites are actually used for initiation. After the midblastula transition, replication initiation is restricted to the rDNA intergenic spacers. A remodeling of chromatin folding could account for this change in origin usage. Here, it is reported that nuclear matrix anchorage of the Xenopus rDNA occurs at multiple, apparently random sequences, throughout embryonic development as well as in adult cells. In vitro matrix rebinding assays confirmed the lack of specific anchoring sequences in the rDNA, before as well as after specific replication origins are established. Thus, no change in loop attachment sites could explain the change in origin usage at this locus. Nonspecific loop anchorage was a special feature of the rDNA locus, since the same nuclear matrices were able selectively to bind the scaffold attachment region (SAR) of the Drosophila histone gene cluster in vitro. Blastula and gastrula nuclear matrices bound a higher amount of SAR sequences than matrices from later stages or adult cells. This developmental change in SAR binding might explain the increase in size of the bulk of genomic DNA loops that occurs after the gastrula stage. However, no change in chromatin loop organization that could explain the midblastula stage transition from small to large replicons was observed. Received: 15 January 1998; in revised form: 4 March 1998 / Accepted: 9 March 1998     

Association between testosterone, estradiol and sex hormone binding globulin levels in men with type 1 diabetes with nephropathy

Male sex is a risk factor for development and progression of diabetic nephropathy; however, the relationship between sex hormone levels and diabetic nephropathy in type 1 diabetic men is unknown. This was a prospective follow-up study as part of the nationwide Finnish Diabetic Nephropathy (FinnDiane) Study; 297 patients were followed for 5.9 ± 1.5 years. Serum total testosterone (Tt) and estradiol (Te), calculated free testosterone (cFt) and estradiol (cFe) and sex hormone binding globulin were measured at baseline and correlated with urinary albumin excretion rate, estimated glomerular filtration rate and markers of metabolic syndrome. Diabetes without renal disease was associated with decreased Tt (p < 0.001), Te (p < 0.001) and cFt (p = 0.001) levels compared with healthy non-diabetic men. With progression of renal disease from micro- to macroalbuminuria, this decrease in serum Tt was even more pronounced. Cox regression showed that cFt and cFe were independent predictors of the progression from macroalbuminuria to end-stage renal disease. Our study shows that men with type 1 diabetes exhibit dysregulated sex hormone levels, which is most pronounced in men with progressive renal disease, suggesting that sex hormones may play a role in the pathogenesis of diabetic nephropathy associated with type 1 diabetes.     

Gene electrotransfer: from biophysical mechanisms to in vivo applications

Gene electrotransfer can be obtained not just on single cells in diluted suspension. For more than 10 years, this is a quasi routine strategy in tissue on the living animal and a few clinical trials have now been approved. New problems have been brought by the close contacts of cells in tissue both on the local field distribution and on the access of DNA to target cells. They need to be solved to provide a further improvement in the efficacy and safety of protein expression. There is a competition between gene transfer and cell destruction. Nevertheless, present results are indicative that electrotransfer is a promising approach for gene therapy. High level and long-lived expression of proteins can be obtained in muscles. This is used for a successful method of electrovaccination.