G2A Attenuates Propionibacterium acnes Induction of Inflammatory Cytokines in Human Monocytes.

Background:Acne vulgaris is a disease of the pilosebaceous unit characterized by increased sebum production, hyperkeratinization, and immune responses to Propionibacterium acnes (PA). Here, we explore a possible mechanism by which a lipid receptor, G2A, regulates immune responses to a commensal bacterium. Objective:To elucidate the inflammatory properties of G2A in monocytes in response to PA stimulation. Furthermore, our study sought to investigate pathways by which lipids modulate immune responses in response to PA. Methods:Our studies focused on monocytes collected from human peripheral blood mononuclear cells, the monocytic cell line THP-1, and a lab strain of PA. Our studies involved the use of enzyme-linked immunosorbent, Western blot, reverse transcription polymerase chain reaction, small interfering RNA (siRNA), and microarray analysis of human acne lesions in the measurements of inflammatory markers. Results:G2A gene expression is higher in acne lesions compared to normal skin and is inducible by the acne therapeutic, 13-cis-retinoic acid. In vitro, PA induces both the Toll-like receptor 2-dependent expression of G2A as well as the production of the G2A ligand, 9-hydroxyoctadecadienoic acid, from human monocytes. G2A gene knockdown through siRNA enhances PA stimulation of interleukin (IL)-6, IL-8, and IL-1β possibly through increased activation of the ERK1/2 MAP kinase and nuclear factor kappa B p65 pathways. Conclusion:G2A may play a role in quelling inflammatory cytokine response to PA, revealing G2A as a potential attenuator of inflammatory response in a disease associated with a commensal bacterium

Propionibacterium acnes bacteriophages display limited genetic diversity and broad killing activity against bacterial skin isolates.

UnlabelledInvestigation of the human microbiome has revealed diverse and complex microbial communities at distinct anatomic sites. The microbiome of the human sebaceous follicle provides a tractable model in which to study its dominant bacterial inhabitant, Propionibacterium acnes, which is thought to contribute to the pathogenesis of the human disease acne. To explore the diversity of the bacteriophages that infect P. acnes, 11 P. acnes phages were isolated from the sebaceous follicles of donors with healthy skin or acne and their genomes were sequenced. Comparative genomic analysis of the P. acnes phage population, which spans a 30-year temporal period and a broad geographic range, reveals striking similarity in terms of genome length, percent GC content, nucleotide identity (>85%), and gene content. This was unexpected, given the far-ranging diversity observed in virtually all other phage populations. Although the P. acnes phages display a broad host range against clinical isolates of P. acnes, two bacterial isolates were resistant to many of these phages. Moreover, the patterns of phage resistance correlate closely with the presence of clustered regularly interspaced short palindromic repeat elements in the bacteria that target a specific subset of phages, conferring a system of prokaryotic innate immunity. The limited diversity of the P. acnes bacteriophages, which may relate to the unique evolutionary constraints imposed by the lipid-rich anaerobic environment in which their bacterial hosts reside, points to the potential utility of phage-based antimicrobial therapy for acne.ImportancePropionibacterium acnes is a dominant member of the skin microflora and has also been implicated in the pathogenesis of acne; however, little is known about the bacteriophages that coexist with and infect this bacterium. Here we present the novel genome sequences of 11 P. acnes phages, thereby substantially increasing the amount of available genomic information about this phage population. Surprisingly, we find that, unlike other well-studied bacteriophages, P. acnes phages are highly homogeneous and show a striking lack of genetic diversity, which is perhaps related to their unique and restricted habitat. They also share a broad ability to kill clinical isolates of P. acnes; phage resistance is not prevalent, but when detected, it appears to be conferred by chromosomally encoded immunity elements within the host genome. We believe that these phages display numerous features that would make them ideal candidates for the development of a phage-based therapy for acne

BRED: A Simple and Powerful Tool for Constructing Mutant and Recombinant Bacteriophage Genomes

Advances in DNA sequencing technology have facilitated the determination of hundreds of complete genome sequences both for bacteria and their bacteriophages. Some of these bacteria have well-developed and facile genetic systems for constructing mutants to determine gene function, and recombineering is a particularly effective tool. However, generally applicable methods for constructing defined mutants of bacteriophages are poorly developed, in part because of the inability to use selectable markers such as drug resistance genes during viral lytic growth. Here we describe a method for simple and effective directed mutagenesis of bacteriophage genomes using Bacteriophage Recombineering of Electroporated DNA (BRED), in which a highly efficient recombineering system is utilized directly on electroporated phage DNA; no selection is required and mutants can be readily detected by PCR. We describe the use of BRED to construct unmarked gene deletions, in-frame internal deletions, base substitutions, precise gene replacements, and the addition of gene tags

Application of BRED technology to construct recombinant D29 reporter phage expressing EGFP

Bacteriophage Recombineering of Electroporated DNA (BRED) has been described for construction of gene deletion and point mutations in mycobacteriophages. Using BRED, we inserted a Phsp60-egfp cassette (1143 bp) into the mycobacteriophage D29 genome to construct a new reporter phage, which was used for detection of mycobacterial cells. The cassette was successfully inserted and recombinant mycobacteriophage purified. DNA sequencing of the cassette did not show any mutations even after several phage generations. Mycobacterium smegmatis mc2155 cells were infected with D29::Phsp60-egfp (MOI of 10) and evaluated for EGFP expression by microscopy. Fluorescence was observed at around 2 h after infection, but dissipated in later times because of cell lysis. We attempted to construct a lysis-defective mutant by deleting the lysA gene, although we were unable to purify the mutant to homogeneity even with complementation. These observations demonstrate the ability of BRED to insert c. 1 kbp-sized DNA segments into mycobacteriophage genomes as a strategy for constructing new diagnostic reporter phages.Fil: Silva, Joas L. da. Universidade de Sao Paulo; BrasilFil: Piuri, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Broussard, Gregory. University Of Pittsburgh; Estados UnidosFil: Marinelli, Laura J.. University Of Pittsburgh; Estados UnidosFil: Bastos, Gisele M.. Universidade de Sao Paulo; BrasilFil: Hirata, Rosario D. C.. Universidade de Sao Paulo; BrasilFil: Hatfull, Graham F.. University Of Pittsburgh; Estados UnidosFil: Hirata, Mario H.. Universidade de Sao Paulo; Brasi

Optimization of Single-Satellite Operational Schedules Towards Enhanced Communication Capacity

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/97112/1/AIAA2012-4610.pd

Serum amyloid A primes microglia for ATP-dependent interleukin-1\u3b2 release

Acute-phase response is a systemic reaction to environmental/inflammatory insults and involves production of acute-phase proteins, including serum amyloid A (SAA). Interleukin-1\u3b2 (IL-1\u3b2), a master regulator of neuroinflammation produced by activated inflammatory cells of the myeloid lineage, in particular microglia, plays a key role in the pathogenesis of acute and chronic diseases of the peripheral nervous system and CNS. IL-1\u3b2 release is promoted by ATP acting at the purinergic P2X7 receptor (P2X7R) in cells primed with toll-like receptor (TLR) ligands

A framework for the definition and interpretation of the use of surrogate endpoints in interventional trials

Background: Interventional trials that evaluate treatment effects using surrogate endpoints have become increasingly common. This paper describes four linked empirical studies and the development of a framework for defining, interpreting and reporting surrogate endpoints in trials. Methods: As part of developing the CONSORT (Consolidated Standards of Reporting Trials) and SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) extensions for randomised trials reporting surrogate endpoints, we undertook a scoping review, e-Delphi study, consensus meeting, and a web survey to examine current definitions and stakeholder (including clinicians, trial investigators, patients and public partners, journal editors, and health technology experts) interpretations of surrogate endpoints as primary outcome measures in trials. Findings: Current surrogate endpoint definitional frameworks are inconsistent and unclear. Surrogate endpoints are used in trials as a substitute of the treatment effects of an intervention on the target outcome(s) of ultimate interest, events measuring how patients feel, function, or survive. Traditionally the consideration of surrogate endpoints in trials has focused on biomarkers (e.g., HDL cholesterol, blood pressure, tumour response), especially in the medical product regulatory setting. Nevertheless, the concept of surrogacy in trials is potentially broader. Intermediate outcomes that include a measure of function or symptoms (e.g., angina frequency, exercise tolerance) can also be used as substitute for target outcomes (e.g., all-cause mortality)-thereby acting as surrogate endpoints. However, we found a lack of consensus among stakeholders on accepting and interpreting intermediate outcomes in trials as surrogate endpoints or target outcomes. In our assessment, patients and health technology assessment experts appeared more likely to consider intermediate outcomes to be surrogate endpoints than clinicians and regulators. Interpretation: There is an urgent need for better understanding and reporting on the use of surrogate endpoints, especially in the setting of interventional trials. We provide a framework for the definition of surrogate endpoints (biomarkers and intermediate outcomes) and target outcomes in trials to improve future reporting and aid stakeholders' interpretation and use of trial surrogate endpoint evidence. Funding: SPIRIT-SURROGATE/CONSORT-SURROGATE project is Medical Research Council Better Research Better Health (MR/V038400/1) funded

A framework for the definition and interpretation of the use of surrogate endpoints in interventional trials

Background: Interventional trials that evaluate treatment effects using surrogate endpoints have become increasingly common. This paper describes four linked empirical studies and the development of a framework for defining, interpreting and reporting surrogate endpoints in trials. Methods: As part of developing the CONSORT (Consolidated Standards of Reporting Trials) and SPIRIT (Standard Protocol Items: Recommendations for Interventional Trials) extensions for randomised trials reporting surrogate endpoints, we undertook a scoping review, e-Delphi study, consensus meeting, and a web survey to examine current definitions and stakeholder (including clinicians, trial investigators, patients and public partners, journal editors, and health technology experts) interpretations of surrogate endpoints as primary outcome measures in trials. Findings: Current surrogate endpoint definitional frameworks are inconsistent and unclear. Surrogate endpoints are used in trials as a substitute of the treatment effects of an intervention on the target outcome(s) of ultimate interest, events measuring how patients feel, function, or survive. Traditionally the consideration of surrogate endpoints in trials has focused on biomarkers (e.g., HDL cholesterol, blood pressure, tumour response), especially in the medical product regulatory setting. Nevertheless, the concept of surrogacy in trials is potentially broader. Intermediate outcomes that include a measure of function or symptoms (e.g., angina frequency, exercise tolerance) can also be used as substitute for target outcomes (e.g., all-cause mortality)—thereby acting as surrogate endpoints. However, we found a lack of consensus among stakeholders on accepting and interpreting intermediate outcomes in trials as surrogate endpoints or target outcomes. In our assessment, patients and health technology assessment experts appeared more likely to consider intermediate outcomes to be surrogate endpoints than clinicians and regulators. Interpretation: There is an urgent need for better understanding and reporting on the use of surrogate endpoints, especially in the setting of interventional trials. We provide a framework for the definition of surrogate endpoints (biomarkers and intermediate outcomes) and target outcomes in trials to improve future reporting and aid stakeholders' interpretation and use of trial surrogate endpoint evidence

Multicentre observational study on multisystem inflammatory syndrome related to COVID-19 in Argentina

Background: The impact of the pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS) in low- and middle-income countries remains poorly understood. Our aim was to understand the characteristics and outcomes of PIMS-TS in Argentina. Methods: This observational, prospective, and retrospective multicenter study enrolled patients younger than 18 years-old manifesting PIMS-TS, Kawasaki disease (KD) or Kawasaki shock syndrome (KSS) between March 2020 and May 2021. Patients were followed-up until hospital discharge or death (one case). The primary outcome was pediatric intensive care unit (PICU) admission. Multiple logistic regression was used to identify variables predicting PICU admission. Results: Eighty-one percent, 82%, and 14% of the 176 enrolled patients fulfilled the suspect case criteria for PIMS-TS, KD, and KSS, respectively. Temporal association with SARS-CoV-2 was confirmed in 85% of the patients and 38% were admitted to the PICU. The more common clinical manifestations were fever, abdominal pain, rash, and conjunctival injection. Lymphopenia was more common among PICU-admitted patients (87% vs. 51%, p < 0.0001), who also showed a lower platelet count and higher plasmatic levels of inflammatory and cardiac markers. Mitral valve insufficiency, left ventricular wall motion alterations, pericardial effusion, and coronary artery alterations were observed in 30%, 30%, 19.8%, and 18.6% of the patients, respectively. Days to initiation of treatment, rash, lymphopenia, and low platelet count were significant independent contributions to PICU admission. Conclusion: Rates of severe outcomes of PIMS-TS in the present study agreed with those observed in high-income countries. Together with other published studies, this work helps clinicians to better understand this novel clinical entity.Fil: Vainstein, Eduardo. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Baleani, Silvia. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Urrutia, Luis. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Affranchino, Nicolás. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Ackerman, Judith. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños Pedro Elizalde (ex Casa Cuna); ArgentinaFil: Cazalas, Mariana. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Goldsman, Alejandro. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Sardella, Angela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Tolin, Ana Laura. Gobierno de la Provincia de Mendoza. Hospital Pediátrico Humberto Notti; ArgentinaFil: Goldaracena, Pablo. Provincia de Buenos Aires. Ministerio de Salud. Hospital de Niños "Sor María Ludovica" de La Plata; ArgentinaFil: Fabi, Mariana. Provincia de Buenos Aires. Ministerio de Salud. Hospital de Niños "Sor María Ludovica" de La Plata; ArgentinaFil: Cosentino, Mariana. Hospital Británico de Buenos Aires; ArgentinaFil: Magliola, Ricardo. Hospital Británico de Buenos Aires; ArgentinaFil: Roggiero, Gustavo. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; ArgentinaFil: Manso, Paula. Provincia de Buenos Aires. Ministerio de Salud. Hospital Alta Complejidad en Red El Cruce Dr. Néstor Carlos Kirchner Samic; ArgentinaFil: Triguy, Jésica. Gobierno de la Provincia de Mendoza. Hospital Pediátrico Humberto Notti; ArgentinaFil: Ballester, Celeste. Gobierno de la Provincia de Mendoza. Hospital Pediátrico Humberto Notti; ArgentinaFil: Cervetto, Vanesa. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños Pedro Elizalde (ex Casa Cuna); ArgentinaFil: Vaccarello, María. Sanatorio de la Trinidad; ArgentinaFil: De Carli, Domingo Norberto. Clínica del Niño de Quilmes; ArgentinaFil: De Carli, Maria Estela. Clínica del Niño de Quilmes; ArgentinaFil: Ciotti, Ana Laura. Hospital Nacional Profesor Alejandro Posadas; ArgentinaFil: Sicurello, María Irene. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Rios Leiva, Cecilia. Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal de Agudos "Eva Perón"; ArgentinaFil: Villalba, Claudia. Gobierno de la Ciudad de Buenos Aires. Hospital de Pediatría "Juan P. Garrahan"; ArgentinaFil: Hortas, María. Sanatorio de la Trinidad; ArgentinaFil: Peña, Sonia. Gobierno de la Provincia de Mendoza. Hospital Pediátrico Humberto Notti; ArgentinaFil: González, Gabriela. Gobierno de la Provincia de Mendoza. Hospital Pediátrico Humberto Notti; ArgentinaFil: Zold, Camila Lidia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Murer, Mario Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Fisiología y Biofísica Bernardo Houssay. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Fisiología y Biofísica Bernardo Houssay; ArgentinaFil: Grippo, M.. No especifíca;Fil: Vázquez, H.. No especifíca;Fil: Morós, C.. No especifíca;Fil: Di Santo, M.. No especifíca;Fil: Villa, A.. No especifíca;Fil: Lazota, P.. No especifíca;Fil: Foti, M.. No especifíca;Fil: Napoli, N.. No especifíca;Fil: Katsikas, M. M.. No especifíca;Fil: Tonello, L.. No especifíca;Fil: Peña, J.. No especifíca;Fil: Etcheverry, M.. No especifíca;Fil: Iglesias, D.. No especifíca;Fil: Alcalde, A. L.. No especifíca;Fil: Bruera, M.J.. No especifíca;Fil: Bruzzo, V.. No especifíca;Fil: Giordano, P.. No especifíca;Fil: Pena Acero, F.. No especifíca;Fil: Netri Pelandi, G.. No especifíca;Fil: Pastaro, D.. No especifíca;Fil: Bleiz, J.. No especifíca;Fil: Rodríguez, M. F.. No especifíca;Fil: Laghezza, L.. No especifíca;Fil: Molina, M. B.. No especifíca;Fil: Patynok, N.. No especifíca;Fil: Chatelain, M. S.. No especifíca;Fil: Aguilar, M. J.. No especifíca;Fil: Gamboa, J.. No especifíca;Fil: Cervan, M.. No especifíca;Fil: Ruggeri, A.. No especifíca;Fil: Marinelli, I.. No especifíca;Fil: Checcacci, E.. No especifíca;Fil: Meregalli, C.. No especifíca;Fil: Damksy Barbosa, J.. No especifíca;Fil: Fernie, L.. No especifíca;Fil: Fernández, M. J.. No especifíca;Fil: Saenz Tejeira, M.M.. No especifíca;Fil: Cereigido, C.. No especifíca;Fil: Nunell, A.. No especifíca;Fil: Villar, D.. No especifíca;Fil: Mansilla, A. D.. No especifíca;Fil: Darduin, M. D.. No especifíca

Specific genomic aberrations in primary colorectal cancer are associated with liver metastases

Background: Accurate staging of colorectal cancer (CRC) with clinicopathological parameters is important for predicting prognosis and guiding treatment but provides no information about organ site of metastases. Patterns of genomic aberrations in primary colorectal tumors may reveal a chromosomal signature for organ specific metastases. Methods: Array Comparative Genomic Hybridization (aCGH) was employed to asses DNA copy number changes in primary colorectal tumors of three distinctive patient groups. This included formalin-fixed, paraffin-embedded tissue of patients who developed liver metastases (LM; n = 36), metastases (PM; n = 37) and a group that remained metastases-free (M0; n = 25). A novel statistical method for identifying recurrent copy number changes, KC-SMART, was used to find specific locations of genomic aberrations specific for various groups. We created a classifier for organ specific metastases based on the aCGH data using Prediction Analysis for Microarrays (PAM). Results: Specifically in the tumors of primary CRC patients who subsequently developed liver metastasis, KC-SMART analysis identified genomic aberrations on chromosome 20q. LM-PAM, a shrunken centroids classifier for liver metastases occurrence, was able to distinguish the LM group from the other groups (M0&PM) with 80% accuracy (78% sensitivity and 86% specificity). The classification is predominantly based on chromosome 20q aberrations. Conclusion: Liver specific CRC metastases may be predicted with a high accuracy based on specific genomic aberrations in the primary CRC tumor. The ability to predict the site of metastases is important for improvement of personalized patient management.MediamaticsElectrical Engineering, Mathematics and Computer Scienc