What you should know about PR3-ANCA: Conformational requirements of proteinase 3 (PR3) for enzymatic activity and recognition by PR3-ANCA

The neutrophil azurophil granule constituent proteinase 3 (PR3) is the principal antigen for anti-neutrophil cytoplasmic antibodies (ANCA) in Wegener's granulomatosis. The conformation of the mature PR3 enzyme results from intracellular post-translational processing. The nascent molecule undergoes proteolytic cleavage of the amino-terminal signal peptide and activation dipeptide and of a carboxy-terminal peptide extension. The conformation of PR3 is stabilized by four disulfide bonds and, to a lesser extent, by asparagine-linked glycosylation. Most anti-neutrophil cytoplasmic antibodies directed against proteinase 3 (PR3-ANCA) recognize conformational epitopes. The expression of recombinant PR3 has provided a better understanding of the significance of the various intracellular processing steps for enzymatic activity and recognition by PR3-ANCA.     

What you should know about PR3-ANCA: An introduction

Anti-neutrophil cytoplasmic antibodies (ANCA) have become important diagnostic markers of small vessel vasculitides characterized by focal necrotizing lesions of vessel walls and accumulation of lymphocytes and macrophages around the affected vessels. IgG class ANCA directed to proteinase 3 (PR3) of neutrophils and monocytes seem to be directly involved in the pathophysiology of vascular damage by causing excessive neutrophil activation and vessel wall destruction. PR3 and elastase are important players in the mechanisms of vascular necrosis. Methods of detecting ANCA have now been defined but are not uniformly used, even though clinical decisions heavily depend on correct ANCA results.     

Criteria Used in Clinical Practice to Guide Immunosuppressive Treatment in Patients with Primary Sclerosing Cholangitis

Background & Aims

Current guidelines recommend immunosuppressive treatment (IT) in patients with primary sclerosing cholangitis (PSC) and elevated aminotransferase levels more than five times the upper limit of normal and elevated serum IgG-levels above twice the upper limit of normal. Since there is no evidence to support this recommendation, we aimed to assess the criteria that guided clinicians in clinical practice to initiate IT in patients with previously diagnosed PSC.

Methods

This is a retrospective analysis of 196 PSC patients from seven German hepatology centers, of whom 36 patients had received IT solely for their liver disease during the course of PSC. Analyses were carried out using methods for competing risks.

Results

A simplified autoimmune hepatitis (AIH) score >5 (HR of 36, p<0.0001) and a modified histological activity index (mHAI) greater than 3/18 points (HR 3.6, p = 0.0274) were associated with the initiation of IT during the course of PSC. Of note, PSC patients who subsequently received IT differed already at the time of PSC diagnosis from those patients, who did not receive IT during follow-up: they presented with increased levels of IgG (p = 0.004) and more frequently had clinical signs of cirrhosis (p = 0.0002).

Conclusions

This is the first study which investigates the parameters associated with IT in patients with PSC in clinical practice. A simplified AIH score >5 and a mHAI score >3, suggesting concomitant features of AIH, influenced the decision to introduce IT during the course of PSC. In German clinical practice, the cutoffs used to guide IT may be lower than recommended by current guidelines.     

MicroRNAs in Serum and Bile of Patients with Primary Sclerosing Cholangitis and/or Cholangiocarcinoma

Background and Aim

Patients with primary sclerosing cholangitis (PSC) are at high risk for the development of cholangiocarcinoma (CC). Analysis of micro ribonucleic acid (MiRNA) patterns is an evolving research field in biliary pathophysiology with potential value in diagnosis and therapy. Our aim was to evaluate miRNA patterns in serum and bile of patients with PSC and/or CC.

Methods

Serum and bile from consecutive patients with PSC (n = 40 (serum), n = 52 (bile)), CC (n = 31 (serum), n = 19 (bile)) and patients with CC complicating PSC (PSC/CC) (n = 12 (bile)) were analyzed in a cross-sectional study between 2009 and 2012. As additional control serum samples from healthy individuals were analyzed (n = 12). The miRNA levels in serum and bile were determined with global miRNA profiling and subsequent miRNA-specific polymerase chain reaction-mediated validation.

Results

Serum analysis revealed significant differences for miR-1281 (p = 0.001), miR-126 (p = 0.001), miR-26a (p = 0.001), miR-30b (p = 0.001) and miR-122 (p = 0.034) between patients with PSC and patients with CC. All validated miRNAs were significantly lower in healthy individuals. MiR-412 (p = 0.001), miR-640 (p = 0.001), miR-1537 (p = 0.003) and miR-3189 (p = 0.001) were significantly different between patients with PSC and PSC/CC in bile.

Conclusions

Patients with PSC and/or CC have distinct miRNA profiles in serum and bile. Furthermore, miRNA concentrations are different in bile of patients with CC on top of PSC indicating the potential diagnostic value of these miRNAs.     

Complement receptor Mac-1 is an adaptor for NB1 (CD177)-mediated PR3-ANCA neutrophil activation

The glycosylphosphatidylinositol (GPI)-anchored neutrophil-specific receptor NB1 (CD177) presents the autoantigen proteinase 3 (PR3) on the membrane of a neutrophil subset. PR3-ANCA-activated neutrophils participate in small-vessel vasculitis. Since NB1 lacks an intracellular domain, we characterized components of the NB1 signaling complex that are pivotal for neutrophil activation. PR3-ANCA resulted in degranulation and superoxide production in the mNB1(pos)/PR3(high) neutrophils, but not in the mNB1(neg)/PR3(low) subset, whereas MPO-ANCA and fMLP caused similar responses. The NB1 signaling complex that was precipitated from plasma membranes contained the transmembrane receptor Mac-1 (CD11b/CD18) as shown by MS/MS analysis and immunoblotting. NB1 co-precipitation was less for CD11a and not detectable for CD11c. NB1 showed direct protein-protein interactions with both CD11b and CD11a by surface plasmon resonance analysis (SPR). However, when these integrins were presented as heterodimeric transmembrane proteins on transfected cells, only CD11b/CD18 (Mac-1)-transfected cells adhered to immobilized NB1 protein. This adhesion was inhibited by mAb against NB1, CD11b, and CD18. NB1, PR3, and Mac-1 were located within lipid rafts. In addition, confocal microscopy showed the strongest NB1 co-localization with CD11b and CD18 on the neutrophil. Stimulation with NB1-activating mAb triggered degranulation and superoxide production in mNB1(pos)/mPR3(high) neutrophils, and this effect was reduced using blocking antibodies to CD11b. CD11b blockade also inhibited PR3-ANCA-induced neutrophil activation, even when β2-integrin ligand-dependent signals were omitted. We establish the pivotal role of the NB1-Mac-1 receptor interaction for PR3-ANCA-mediated neutrophil activation.     

Exosome Analysis: A Promising Biomarker System with Special Attention to Saliva

Today, exosome-related studies have become a focus in science and technology. Recently, three scientists won the Nobel Prize for determining the mechanisms of exosomal transport, making exosomes a promising biomarker system for disease diagnosis and treatment. This review provides a general introduction of exosomes and explores the recent progress on the function, application, isolation, and identification of exosomes as biomarkers in blood and other body fluids, especially in saliva. Detailed information of exosomal proteins and RNAs is discussed in the paper because of their ability to determine the function of exosomes. Due to their noninvasive assessment for quick and convenient diagnosis of diseases, salivary exosomes may well be promising biomarkers.     

Collagen Peptides in Urine: A New Promising Biomarker for the Detection of Colorectal Liver Metastases

Introduction

For both patients and the outpatient clinic the frequent follow-up visits after a resection of colorectal cancer (CRC) are time consuming and due to large patient numbers expensive. Therefore it is important to develop an effective non-invasive test for the detection of colorectal liver metastasis (CRLM) which could be used outside the hospital. The urine proteome is known to provide detailed information for monitoring changes in the physiology of humans. Urine collection is non-invasive and urine naturally occurring peptides (NOPs) have the advantage of being easily accessible without labour-intensive sample preparation. These advantages make it potentially useful for a quick and reliable application in clinical settings. In this study, we will focus on the identification and validation of urine NOPs to discriminate patients with CRLM from healthy controls.

Materials and Methods

Urine samples were collected from 24 patients with CRLM and 25 healthy controls. In the first part of the study, samples were measured with a nano liquid chromatography (LC) system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific, Bremen, Germany). A discovery set was used to construct the model and consecutively the validation set, being independent from the discovery set, to check the acquired model. From the peptides which were selected, multiple reaction monitoring (MRM''s) were developed on a UPLC-MS/MS system.

Results

Seven peptides were selected and applied in a discriminant analysis a sensitivity of 84.6% and a specificity of 92.3% were established (Canonical correlation:0.797, Eigenvalue:1.744, F:4.49, p:0.005). The peptides AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGA P(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG were selected for further quantitative analysis which showed a sensitivity of 88% and a specificity of 88%.

Conclusion

Urine proteomic analysis revealed two very promising peptides, both part from collagen type 1, AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGAP(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG which could detect CRLM in a non-invasive manner.     

ULK1: A Promising Biomarker in Predicting Poor Prognosis and Therapeutic Response in Human Nasopharygeal Carcinoma

Plenty of studies have established that dysregulation of autophagy plays an essential role in cancer progression. The autophagy-related proteins have been reported to be closely associated with human cancer patients’ prognosis. We explored the expression dynamics and prognostic value of autophagy-related protein ULK1 by immunochemistry (IHC) method in two independent cohorts of nasopharygeal carcinoma (NPC) cases. The X-tile program was applied to determine the optimal cut-off value in the training cohort. This derived cutoff value was then subjected to analysis the association of ULK1 expression with patients’ clinical characteristics and survival outcome in the validation cohort and overall cases. High ULK1 expression was closely associated with aggressive clinical feature of NPC patients. Furthermore, high expression of ULK1 was observed more frequently in therapeutic resistant group than that in therapeutic effective group. Our univariate and multivariate analysis also showed that higher ULK1 expression predicted inferior disease-specific survival (DSS) (P<0.05). Consequently, a new clinicopathologic prognostic model with 3 poor prognostic factors (ie, ULK1 expression, overall clinical stage and therapeutic response) could significantly stratify risk (low, intermediate and high) for DSS in NPC patients (P<0.001). These findings provide evidence that, the examination of ULK1 expression by IHC method, could serve as an effective additional tool for predicting therapeutic response and patients’ survival outcome in NPC patients.     

What you should know about PR3-ANCA: Evidence for the role of T cells in the pathogenesis of systemic vasculitis

The pathogenesis of systemic vasculitis is complex and is likely to involve many mechanisms. There is a growing body of evidence that T cells may contribute to the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides. Predominantly, T cells and monocytes are found in inflammatory infiltrates in patients with Wegener's granulomatosis (WG). The production of ANCA appears to be T-cell-dependent. T lymphocytes from the peripheral blood of patients with ANCA-associated vasculitis have been shown to proliferate in response to proteinase 3 (PR3). These and other findings outlined in this review indicate T-cell involvement, although further studies are still needed to elucidate the exact contribution of T cells to the pathogenesis of systemic vasculitis.     

Poly(3-Hydroxypropionate): a Promising Alternative to Fossil Fuel-Based Materials

Polyhydroxyalkanoates (PHAs) are storage compounds synthesized by numerous microorganisms and have attracted the interest of industry since they are biobased and biodegradable alternatives to fossil fuel-derived plastics. Among PHAs, poly(3-hydroxypropionate) [poly(3HP)] has outstanding material characteristics and exhibits a large variety of applications. As it is not brittle like, e.g., the best-studied PHA, poly(3-hydroxybutyrate) [poly(3HB)], it can be used as a plasticizer in blends to improve their properties. Furthermore, 3-hydroxypropionic acid (3HP) is considered likely to become one of the new industrial building blocks, and it can be obtained from poly(3HP) by simple hydrolysis. Unfortunately, no natural organism is known to accumulate poly(3HP) so far. Thus, several efforts have been made to engineer genetically modified organisms capable of synthesizing the homopolymer or copolymers containing 3HP. In this review, the achievements made so far in efforts to obtain biomass which has accumulated poly(3HP) or 3HP-containing copolymers, as well as the properties of these polyesters and their applications, are compiled and evaluated.     

What you should know about PR3-ANCA. Evidence for the role of T cells in the pathogenesis of systemic vasculitis

The pathogenesis of systemic vasculitis is complex and is likely to involve many mechanisms. There is a growing body of evidence that T cells may contribute to the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides. Predominantly, T cells and monocytes are found in inflammatory infiltrates in patients with Wegener's granulomatosis (WG). The production of ANCA appears to be T-cell-dependent. T lymphocytes from the peripheral blood of patients with ANCA-associated vasculitis have been shown to proliferate in response to proteinase 3 (PR3). These and other findings outlined in this review indicate T-cell involvement, although further studies are still needed to elucidate the exact contribution of T cells to the pathogenesis of systemic vasculitis.     

Complete genome sequence of Halogeometricum borinquense type strain (PR3)

Halogeometricum borinquense Montalvo-Rodríguez et al. 1998 is the type species of the genus, and is of phylogenetic interest because of its distinct location between the halobacterial genera Haloquadratum and Halosarcina. H. borinquense requires extremely high salt (NaCl) concentrations for growth. It can not only grow aerobically but also anaerobically using nitrate as electron acceptor. The strain described in this report is a free-living, motile, pleomorphic, euryarchaeon, which was originally isolated from the solar salterns of Cabo Rojo, Puerto Rico. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the halobacterial genus Halogeometricum, and this 3,944,467 bp long six replicon genome with its 3937 protein-coding and 57 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Contradictory functions (activation/termination) of neutrophil proteinase 3 enzyme (PR3) in interleukin-33 biological activity

IL-1 family ligand does not possess a typical hydrophobic signal peptide and needs a processing enzyme for maturation. The maturation process of IL-33 (IL-1F11), a new member of the IL-1 family ligand, remains unclear. Precursor IL-33 ligand affinity column isolates neutrophil proteinase 3 (PR3) from human urinary proteins. PR3 is a known IL-1 family ligand-processing enzyme for IL-1β (IL-1F2) and IL-18 (IL-1F4), including other inflammatory cytokines. We investigated PR3 in the maturation process of precursor IL-33 because we isolated urinary PR3 by using the precursor IL-33 ligand affinity column. PR3 converted inactive human and mouse precursor IL-33 proteins to biological active forms; however, the increase of PR3 incubation time abrogated IL-33 activities. Unlike caspase-1-cleaved precursor IL-18, PR3 cut precursor IL-33 and IL-18 at various sites and yielded multibands. The increased incubation period of PR3 abated mature IL-33 in a time-dependent manner. The result is consistent with the decreased bioactivity of IL-33 along with the increased PR3 incubation time. Six different human and mouse recombinant IL-33 proteins were expressed by the predicted consensus amino acid sequence of PR3 cleavage sites and tested for bioactivities. The human IL-33/p1 was highly active, but human IL-33/p2 and p3 proteins were inactive. Our results suggest the dual functions (activation/termination) of PR3 in IL-33 biological activity.     

Interaction of purified human proteinase 3 (PR3) with reconstituted lipid bilayers.

Proteinase 3 (PR3), the major target autoantigen in Wegener's granulomatosis is a serine proteinase that is normally stored intracellularly in the primary granules of quiescent neutrophils and monocytes. Upon cell activation, a significant portion of this antigen is detected on the cell surface membrane. The nature of the association of PR3 with the membrane and its functional significance are unknown. We investigated the interaction of purified human PR3 with mixtures of zwitterionic (dimyristoyl-L-alpha-phosphatidylcholine, DMPC) and anionic (dimyristoyl-L-alpha-phosphatidylglycerol, DMPG) phospholipids in reconstituted lipid bilayers using differential scanning calorimetry and lipid photolabeling, and measured the affinity of this interaction using spectrophotometry. Two other primary granule constituents, human neutrophil elastase (HNE) and myeloperoxidase (MPO) were investigated for comparison. In calorimetric assays, using lipid vesicles of mixed DMPC/DMPG, increasing PR3 concentrations (protein/lipid molar ratio from 0 to 1 : 110) induced a significant decrease of the main chain transition enthalpy and a shift in chain melting temperatures which is indicative of partial insertion of PR3 into the hydrophobic region of the lipid membranes. This was confirmed by hydrophobic photolabeling using liposomes containing trace amounts of the photoactivable [125I]-labeled phosphatidylcholine analog TID-PC/16. The molar affinity of PR3, HNE, and MPO to lipid vesicles of different DMPC/DMPG ratios was then determined by spectrophotometry. At a DMPC/DMPG ratio of 1 : 1, molar affinities of PR3, Kd = 4.5 +/- 0.3 microm; HNE, 14.5 +/- 1.2 microm; and MPO, 50 +/- 5 microm (n = 3) were estimated. The lipid-associated PR3 exhibited two-fold lower Vmax and Km values, and its enzyme activity was slightly more inhibited (Ki) by the natural alpha1-proteinase inhibitor (alpha1-PI) or an autoantibody to PR3.     

The inheritance of pseudo-self-compatibility (PSC) in Nemesia strumosa Benth

Summary Pseudo-self-compatibility (PSC) in Nemesia strumosa was determined by recessive modifying genes which interferred with the normal stylar incompatibility reaction. The PSC levels ranged from none or low to amounts resembling full self-compatibility. PSC within a progeny could be maintained at low or high levels by selecting parents either high or low in PSC. Pollen from plants of any PSC level failed to produce seed when placed on other incompatible styles bearing the same S alleles.Scientific Journal Series Paper Number 9420 of the Minnesota Agricultural Experiment Station.     

Epidemiology of Subacute Sclerosing Panencephalitis (SSPE) in Germany from 2003 to 2009: A Risk Estimation

Subacute sclerosing panencephalitis (SSPE) is a fatal long-term complication of measles infection. We performed an estimation of the total number of SSPE cases in Germany for the period 2003 to 2009 and calculated the risk of SSPE after an acute measles infection. SSPE cases were collected from the Surveillance Unit for Rare Paediatric Diseases in Germany and the Institute of Virology and Immunobiology at the University of Würzburg. The total number of SSPE cases was estimated by capture-recapture analysis. For the period 2003 to 2009, 31 children with SSPE who were treated at German hospitals were identified. The capture-recapture estimate was 39 cases (95% confidence interval: 29.2–48.0). The risk of developing SSPE for children contracting measles infection below 5 years of age was calculated as 1∶1700 to 1∶3300. This risk is in the same order of magnitude as the risk of a fatal acute measles infection.