ObjectiveIdentify autoantibodies in anti-Ro/SS-A negative primary Sjögren’s syndrome (SS).MethodsThis is a proof-of-concept, case-control study of SS, healthy (HC) and other disease (OD) controls. A discovery dataset of plasma samples (n=30 SS, n=15 HC) was tested on human proteome arrays containing 19 500 proteins. A validation dataset of plasma and stimulated parotid saliva from additional SS cases (n=46 anti-Ro+, n=50 anti-Ro–), HC (n=42) and OD (n=54) was tested on custom arrays containing 74 proteins. For each protein, the mean+3 SD of the HC value defined the positivity threshold. Differences from HC were determined by Fisher’s exact test and random forest machine learning using 2/3 of the validation dataset for training and 1/3 for testing. Applicability of the results was explored in an independent rheumatology practice cohort (n=38 Ro+, n=36 Ro–, n=10 HC). Relationships among antigens were explored using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) interactome analysis.ResultsRo+SS parotid saliva contained autoantibodies binding to Ro60, Ro52, La/SS-B and muscarinic receptor 5. SS plasma contained 12 novel autoantibody specificities, 11 of which were detected in both the discovery and validation datasets. Binding to ≥1 of the novel antigens identified 54% of Ro–SS and 37% of Ro+SS cases, with 100% specificity in both groups. Machine learning identified 30 novel specificities showing receiver operating characteristic area under the curve of 0.79 (95% CI 0.64 to 0.93) for identifying Ro–SS. Sera from Ro–cases of an independent cohort bound 17 of the non-canonical antigens. Antigenic targets in both Ro+and Ro–SS were part of leukaemia cell, ubiquitin conjugation and antiviral defence pathways.ConclusionWe identified antigenic targets of the autoantibody response in SS that may be useful for identifying up to half of Ro seronegative SS cases.

Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern (PAMP) contributing to anthrax pathology, including organ dysfunction and coagulopathy. Increases in apoptotic lymphocytes are a late-stage feature of anthrax and sepsis, suggesting there is a defect in apoptotic clearance. Here, we tested the hypothesis that B. anthracis PGN inhibits the capacity of human monocyte-derived, tissue-like macrophages (Mφ) to efferocytose apoptotic cells. Exposure of CD206+CD163+ Mφ to PGN for 24h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the pro-efferocytic signaling receptors MERTK, TYRO3, AXL, integrin αVβ5, CD36 and TIM-3, whereas TIM-1, αVβ5, CD300b, CD300f, STABILIN-1 and STABILIN-2 were unaffected. Soluble forms of MERTK, TYRO3, AXL, CD36, and TIM-3 were increased in PGN-treated supernatants, suggesting involvement of proteases. ADAM17 is a major membrane-bound protease implicated in mediating efferocytotic receptor cleavage. ADAM17 inhibitors TAPI-0 and Marimastat abolished TNF release, indicating effective protease inhibition, modestly increased cell-surface levels of MerTK and TIM-3 but only partially restored efferocytic capacity by PGN-treated Mφ. We conclude that human serum factors are required for optimal recognition of PGN by human Mφ and that B. anthracis PGN inhibits efferocytosis in part by reducing cell surface expression of efferocytic receptors.

Phage ScienceWizSam was isolated from soil using Arthrobacter sp. strain ATCC 21022. The phage genome is 58,217 bp with 96 open reading frames (ORFs). All of the ORFs are transcribed rightwards. Based on gene content similarity, ScienceWizSam is assigned to phage subcluster AU1.

Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen associated molecular pattern contributing to anthrax pathology including coagulopathy in late stage disease. Lymphocyte apoptosis is an established phenomenon in bacterial sepsis, including fulminant anthrax. Clearance of apoptotic cells (efferocytosis) is thought to be mediated by tissue-resident macrophages (MF), modeled in vitro as M2-like MF. Circulating innate cells need serum opsonization of PGN for cellular activation, but opsonization requirements for human MF activation have not been reported. Herein, we tested the hypothesis that PGN requires human serum opsonization for MF activation and inhibits human M2-like MF efferocytosis of apoptotic human neutrophils. MF were differentiated in vitro with M-CSF and polarized to M2 phenotype with dexamethasone. Cytokine and soluble receptor levels were assessed by ELISA and Luminex assays. Cell surface markers and fraction of MF containing fluorescently-labeled apoptotic human neutrophils were assessed by flow cytometry. M2-like MF recognition of PGN was enhanced in the presence of human but not bovine serum, noted by increased secretion of TNF-a and IL-10 in PGN-treated culture supernatants. Efferocytosis, and cell surface expression of MerTK, Tyro3, Axl, αVβ5, CD36 and Tim-3, all pro-efferocytic receptors, was significantly downregulated by pre-treatment of MF with PGN. Increases in soluble pro-efferocytic receptors were detected in PGN-treated MF culture supernatants. We conclude that PGN impairs efferocytosis by human M2-like macrophages, likely by down-regulating the expression of cell surface receptors known to signal apoptotic cell engulfment. Supported by NIH (U19AI062629).

The Genus Mycobacterium includes pathogens known to cause disease in mammals such as tuberculosis (Mycobacterium tuberculosis) and skin infections (M. abscessus). M. smegmatis is a model bacterium that can cause opportunistic infections in human tissues and, rarely, a respiratory disease. Due to the emergence of multidrug-resistant bacteria, phage therapy is potentially an alternative way of treating these bacterial infections. As bacteriophages are specific to their bacterial host, it ensures that the normal flora is unharmed. Fulbright is a mycobacteriophage that infects the host bacteria M. smegmatis. The main goal of this study is to incorporate Mycobacteriophage Fulbright into a polycaprolactone (PCL) nanofiber and test its antimicrobial effect against the host bacteria, M. smegmatis. Stability tests conducted over 7 days showed that the phage titer does not decrease when in contact with PCL, making it a promising vehicle for phage delivery. Antimicrobial assays showed that PCL_Fulbright effectively reduces bacterial concentration after 24 h of contact. In addition, when stored at −20 °C, the phage remains viable for up to eleven months in the fiber. Fulbright addition on the nanofibrous mats resulted in an increase in water uptake and decrease in the mechanical properties (strength and Young’s modulus) of the membranes, indicating that the presence of phage Fulbright can greatly enhance the physical and mechanical properties of the PCL. Cytotoxicity assays showed that PCL_Fulbright is not cytotoxic to Balbc/3T3 mouse embryo fibroblast cell lines; thus, phage-incorporated PCL is a promising alternative to antibiotics in treating skin infections.