Identification of conformational epitopes for human IgG on Chemotaxis inhibitory protein of Staphylococcus aureus

<p>Abstract</p> <p>Background</p> <p>The Chemotaxis inhibitory protein of <it>Staphylococcus aureus </it>(CHIPS) blocks the Complement fragment C5a receptor (C5aR) and formylated peptide receptor (FPR) and is thereby a potent inhibitor of neutrophil chemotaxis and activation of inflammatory responses. The majority of the healthy human population has antibodies against CHIPS that have been shown to interfere with its function <it>in vitro</it>. The aim of this study was to define potential epitopes for human antibodies on the CHIPS surface. We also initiate the process to identify a mutated CHIPS molecule that is not efficiently recognized by preformed anti-CHIPS antibodies and retains anti-inflammatory activity.</p> <p>Results</p> <p>In this paper, we panned peptide displaying phage libraries against a pool of CHIPS specific affinity-purified polyclonal human IgG. The selected peptides could be divided into two groups of sequences. The first group was the most dominant with 36 of the 48 sequenced clones represented. Binding to human affinity-purified IgG was verified by ELISA for a selection of peptide sequences in phage format. For further analysis, one peptide was chemically synthesized and antibodies affinity-purified on this peptide were found to bind the CHIPS molecule as studied by ELISA and Surface Plasmon Resonance. Furthermore, seven potential conformational epitopes responsible for antibody recognition were identified by mapping phage selected peptide sequences on the CHIPS surface as defined in the NMR structure of the recombinant CHIPS_31–121protein. Mapped epitopes were verified by <it>in vitro </it>mutational analysis of the CHIPS molecule. Single mutations introduced in the proposed antibody epitopes were shown to decrease antibody binding to CHIPS. The biological function in terms of C5aR signaling was studied by flow cytometry. A few mutations were shown to affect this biological function as well as the antibody binding.</p> <p>Conclusion</p> <p>Conformational epitopes recognized by human antibodies have been mapped on the CHIPS surface and amino acid residues involved in both antibody and C5aR interaction could be defined. This information has implications for the development of an effective anti-inflammatory agent based on a functional CHIPS molecule with low interaction with human IgG.</p

Clinical and Radiologic Predictors of Parastomal Hernia Development After End Colostomy

OBJECTIVE. Parastomal hernia (PSH) is a common complication that can occur after end colostomy and may result in considerable morbidity. To select the best candidates for prophylactic measures, knowledge of preoperative PSH predictors is important. This study aimed to determine the value of clinical parameters, preoperative CT-based body metrics, and size of the abdominal wall defect created during end colostomy and measured at postoperative CT for predicting PSH development. MATERIALS AND METHODS. Sixty-five patients who underwent permanent end colostomy with at least 1 year of follow-up were included. On preoperative CT, waist circumference, abdominal wall and psoas muscle indexes, rectus abdominis muscle diameter and diastasis, intra- and extraabdominal fat mass, and presence of other hernias were assessed. On postoperative CT, size of the abdominal wall defect and the presence of PSH were determined. To identify independent predictors of PSH development, univariate analysis with the Kaplan-Meier method and multivariate Cox regression analysis were performed. RESULTS. PSH developed after surgery in 30 patients (46%). Three independent risk factors were identified: chronic obstructive pulmonary disease (COPD) as a comorbidity (hazard ratio [HR], 6.4; 95% CI, 1.9-22.0; p = 0.003), operation time longer than 395 minutes (HR, 3.9; 95% CI, 1.5-10.0; p = 0.005), and maximum aperture diameter of more than 34 mm (HR, 5.2; 95% CI, 2.1-12.7; p <0.001). PSH developed in all nine patients with a maximum abdominal wall defect diameter of more than 50 mm at the ostomy site. CONCLUSION. COPD, longer operation time, and larger abdominal wall defect at the colostomy site can predict PSH development. Intraoperative creation of an abdominal wall ostomy opening that is more than 34 mm in diameter should be avoided

Long non-coding RNAs as novel therapeutic targets in juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia (JMML) treatment primarily relies on hematopoietic stem cell transplantation and results in long-term overall survival of 50-60%, demonstrating a need to develop novel treatments. Dysregulation of the non-coding RNA transcriptome has been demonstrated before in this rare and unique disorder of early childhood. In this study, we investigated the therapeutic potential of targeting overexpressed long non-coding RNAs (lncRNAs) in JMML. Total RNA sequencing of bone marrow and peripheral blood mononuclear cell preparations from 19 untreated JMML patients and three healthy children revealed 185 differentially expressed lncRNA genes (131 up- and 54 downregulated). LNA GapmeRs were designed for 10 overexpressed and validated lncRNAs. Molecular knockdown (>= 70% compared to mock control) after 24 h of incubation was observed with two or more independent GapmeRs in 6 of them. For three lncRNAs (lnc-THADA-4, lnc-ACOT9-1 and NRIR) knockdown resulted in a significant decrease of cell viability after 72 h of incubation in primary cultures of JMML mononuclear cells, respectively. Importantly, the extent of cellular damage correlated with the expression level of the lncRNA of interest. In conclusion, we demonstrated in primary JMML cell cultures that knockdown of overexpressed lncRNAs such as lnc-THADA-4, lnc-ACOT9-1 and NRIR may be a feasible therapeutic strategy

Enhanced Activity and Altered Specificity of Phospholipase A2 by Deletion of a Surface Loop

Protein engineering and x-ray crystallography have been used to study the role of a surface loop that is present in pancreatic phospholipases but is absent in snake venom phospholipases. Removal of residues 62 to 66 from porcine pancreatic phospholipase A2 does not change the binding constant for micelles significantly, but it improves catalytic activity up to 16 times on micellar (zwitterionic) lecithin substrates. In contrast, the decrease in activity on negatively charged substrates is greater than fourfold. A crystallographic study of the mutant enzyme shows that the region of the deletion has a well-defined structure that differs from the structure of the wild-type enzyme. No structural changes in the active site of the enzyme were detected

LaeA-dependent production of small molecules of Aspergillus niger that compete with specific antibodies that bind to human immune receptors

Microorganisms secrete a variety of compounds into their environment such as proteins, carbohydrates, and secondary metabolites. These molecules play diverse roles in the interaction of microbes with their abiotic and biotic environment. Little is known about secreted fungal molecules mediating immune evasion. Here we screened culture media of three Aspergilli to assess whether these fungi secrete molecules that can compete with specific antibodies that bind to human immune receptors. Culture media of Aspergillus fumigatus Af293, Aspergillus tubingensis CBS 133792 and the non-acidifying mutant strain Aspergillus niger D15#26 contained components that showed competition for binding to a total of 13 receptors, of which PSGL-1, CXCR1, and CXCR2, were shared between the three species. Filtration experiments showed that most, if not all, interacting components were [&le;] 3 kDa. Production of the components that competed with antibodies to bind to CD88 and CXCR2 was shown to be regulated by LaeA. The component(s) that competed for binding to CXCR1 was not only produced in the non-acidifying strain Aspergillus niger D15#26 but also in the non-acidifying oahA deletion strain of Aspergillus niger. Together, these data show that Aspergillus species might produce small molecules that interact with human immune receptors

Altered Fc glycosylation of anti-HLA alloantibodies in hemato-oncological patients receiving platelet transfusions

Background: The formation of alloantibodies directed against class I human leukocyte antigens (HLA) continues to be a clinically challenging complication after platelet transfusions, which can lead to platelet refractoriness (PR) and occurs in approximately 5%–15% of patients with chronic platelet support. Interestingly, anti-HLA IgG levels in alloimmunized patients do not seem to predict PR, suggesting functional or qualitative differences among anti-HLA IgG. The binding of these alloantibodies to donor platelets can result in rapid clearance after transfusion, presumably via FcγR-mediated phagocytosis and/or complement activation, which both are affected by the IgG-Fc glycosylation. Objectives: To characterize the Fc glycosylation profile of anti-HLA class I antibodies formed after platelet transfusion and to investigate its effect on clinical outcome. Patients/Methods: We screened and captured anti-HLA class I antibodies (anti-HLA A2, anti-HLA A24, and anti-HLA B7) developed after platelet transfusions in hemato-oncology patients, who were included in the PREPAReS Trial. Using liquid chromatography-mass spectrometry, we analyzed the glycosylation profiles of total and anti-HLA IgG1 developed over time. Subsequently, the glycosylation data was linked to the patients' clinical information and posttransfusion increments. Results: The glycosylation profile of anti-HLA antibodies was highly variable between patients. In general, Fc galactosylation and sialylation levels were elevated compared to total plasma IgG, which correlated negatively with the platelet count increment. Furthermore, high levels of afucosylation were observed for two patients. Conclusions: These differences in composition of anti-HLA Fc-glycosylation profiles could potentially explain the variation in clinical severity between patients

Human OTULIN haploinsufficiency impairs cell-intrinsic immunity to staphylococcal alpha-toxin

The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded by a gene on chromosome 5p. Patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infection. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but tumor necrosis factor receptor-mediated nuclear factor kappa B signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts, but not leukocytes, facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor alpha-toxin. Naturally elicited antibodies against alpha-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to alpha-toxin in nonleukocytic cells.Peer reviewe

The diagnosis and treatment of invasive aspergillosis in Dutch haematology units facing a rapidly increasing prevalence of azole-resistance

Patients with haematological malignancies are at risk for invasive fungal diseases (IFD). A survey was conducted in all Dutch academic haematology centres on their current diagnostic, prophylactic and therapeutic approach towards IFD in the context of azole-resistance. In all 8 centres, a haematologist and microbiologist filled in the questionnaire that focused on different subgroups of haematology patients. Fungal prophylaxis during neutropaenia was directed against Candida and consisted of fluconazole and/or amphotericin B suspension. Mould-active prophylaxis was given to acute myeloid leukaemia patients during chemotherapy in 2 of 8 centres. All centres used azole prophylaxis in a subset of patients with graft-versus-host disease. A uniform approach towards the diagnosis and treatment of IFD and in particular azole-resistant Aspergillus fumigatus was lacking. In 2017, all centres agreed to implement a uniform diagnostic and treatment algorithm regarding invasive aspergillosis with a central role for comprehensive diagnostics and PCR-based detection of azole-resistance. This study (DB-MSG 002) will re-evaluate this algorithm when 280 patients have been treated. A heterogeneous approach towards antifungal prophylaxis, diagnosis and treatment was apparent in the Netherlands. Facing triazole-resistance, consensus was reached on the implementation of a uniform diagnostic approach in all 8 centres

Penilaian Kinerja Keuangan Koperasi di Kabupaten Pelalawan

This paper describe development and financial performance of cooperative in District Pelalawan among 2007 - 2008. Studies on primary and secondary cooperative in 12 sub-districts. Method in this stady use performance measuring of productivity, efficiency, growth, liquidity, and solvability of cooperative. Productivity of cooperative in Pelalawan was highly but efficiency still low. Profit and income were highly, even liquidity of cooperative very high, and solvability was good

Juxtaposing BTE and ATE – on the role of the European insurance industry in funding civil litigation

One of the ways in which legal services are financed, and indeed shaped, is through private insurance arrangement. Two contrasting types of legal expenses insurance contracts (LEI) seem to dominate in Europe: before the event (BTE) and after the event (ATE) legal expenses insurance. Notwithstanding institutional differences between different legal systems, BTE and ATE insurance arrangements may be instrumental if government policy is geared towards strengthening a market-oriented system of financing access to justice for individuals and business. At the same time, emphasizing the role of a private industry as a keeper of the gates to justice raises issues of accountability and transparency, not readily reconcilable with demands of competition. Moreover, multiple actors (clients, lawyers, courts, insurers) are involved, causing behavioural dynamics which are not easily predicted or influenced. Against this background, this paper looks into BTE and ATE arrangements by analysing the particularities of BTE and ATE arrangements currently available in some European jurisdictions and by painting a picture of their respective markets and legal contexts. This allows for some reflection on the performance of BTE and ATE providers as both financiers and keepers. Two issues emerge from the analysis that are worthy of some further reflection. Firstly, there is the problematic long-term sustainability of some ATE products. Secondly, the challenges faced by policymakers that would like to nudge consumers into voluntarily taking out BTE LEI