Topologically correct synthetic reconstruction of pathogen social behavior found during growth in deep tissue sites.

Within deep tissue sites, extracellular bacterial pathogens often replicate in clusters that are surrounded by immune cells. Disease is modulated by interbacterial interactions as well as bacterial-host cell interactions resulting in microbial growth, phagocytic attack and secretion of host antimicrobial factors. To overcome the limited ability to manipulate these infection sites, we established a system for ( growth in microfluidics-driven microdroplets that regenerates microbial social behavior in tissues.

Droplet Tn-Seq combines microfluidics with Tn-Seq for identifying complex single-cell phenotypes.

While Tn-Seq is a powerful tool to determine genome-wide bacterial fitness in high-throughput, culturing transposon-mutant libraries in pools can mask community or other complex single-cell phenotypes. Droplet Tn-Seq (dTn-Seq) solves this problem by microfluidics facilitated encapsulation of individual transposon mutants into growth medium-in-oil droplets, thereby enabling isolated growth, free from the influence of the population. Here we describe and validate microfluidic chip design, production, encapsulation, and dTn-Seq sample preparation.

Iron-Sulfur Cluster Repair Contributes to Yersinia pseudotuberculosis Survival within Deep Tissues.

To successfully colonize host tissues, bacteria must respond to and detoxify many different host-derived antimicrobial compounds, such as nitric oxide (NO). NO has direct antimicrobial activity through attack on iron-sulfur (Fe-S) cluster-containing proteins. NO detoxification plays an important role in promoting bacterial survival, but it remains unclear if repair of Fe-S clusters is also important for bacterial survival within host tissues.

Nasopharyngeal Exposure to Induces Extended Age-Dependent Protection against Pulmonary Infection Mediated by Antibodies and CD138 Cells.

commonly resides asymptomatically in the nasopharyngeal (NP) cavity of healthy individuals but can cause life-threatening pulmonary and systemic infections, particularly in the elderly. NP colonization results in a robust immune response that protects against invasive infections. However, the duration, mechanism, and cellular component of such responses are poorly understood.

Cytosolic Phospholipase Aα Promotes Pulmonary Inflammation and Systemic Disease during Streptococcus pneumoniae Infection.

Pulmonary infection by is characterized by a robust alveolar infiltration of neutrophils (polymorphonuclear cells [PMNs]) that can promote systemic spread of the infection if not resolved. We previously showed that 12-lipoxygenase (12-LOX), which is required to generate the PMN chemoattractant hepoxilin A (HXA) from arachidonic acid (AA), promotes acute pulmonary inflammation and systemic infection after lung challenge with As phospholipase A (PLA) promotes the release of AA, we investigated the role of PLA in local and systemic disease during infection. The group IVA cytosolic isoform of PLA (cPLAα) was activated upon infection of cultured lung epithelial cells and was critical for AA release from membrane phospholipids.

Histopathologic spectrum of hypersensitivity reactions associated with anti-CD52 therapy (alemtuzumab).

Background: Alemtuzumab is a humanized monoclonal antibody directed against CD52, a cell surface antigen on B and T lymphocytes, and used to treat B-cell chronic lymphocytic leukemia and cutaneous T-cell lymphoma. Skin rash is a common adverse reaction following treatment with alemtuzumab. However, the clinicopathologic features and immunologic basis for the reaction have not been previously reported.

Fis Is Essential for Yersinia pseudotuberculosis Virulence and Protects against Reactive Oxygen Species Produced by Phagocytic Cells during Infection.

All three pathogenic Yersinia species share a conserved virulence plasmid that encodes a Type 3 Secretion System (T3SS) and its associated effector proteins. During mammalian infection, these effectors are injected into innate immune cells, where they block many bactericidal functions, including the production of reactive oxygen species (ROS). However, Y.

Esophageal Swallowing Timing Measures in Healthy Adults During Videofluoroscopy.

Objectives: Establishing the range of normal esophageal bolus transit times (ETT) is valuable when distinguishing pathology from normal variance, especially in elderly patients, and has not been documented for paste or pill. The aim of this study was to measure esophageal transit of liquid, paste, and pill during upright videofluoroscopy.

Methods: One hundred eighteen healthy adults (mean age 54; range 20-98 years; SD = 21.

Extracellular Adenosine Protects against Streptococcus pneumoniae Lung Infection by Regulating Pulmonary Neutrophil Recruitment.

An important determinant of disease following Streptococcus pneumoniae (pneumococcus) lung infection is pulmonary inflammation mediated by polymorphonuclear leukocytes (PMNs). We found that upon intratracheal challenge of mice, recruitment of PMNs into the lungs within the first 3 hours coincided with decreased pulmonary pneumococci, whereas large numbers of pulmonary PMNs beyond 12 hours correlated with a greater bacterial burden. Indeed, mice that survived infection largely resolved inflammation by 72 hours, and PMN depletion at peak infiltration, i.

The α-tocopherol form of vitamin E reverses age-associated susceptibility to streptococcus pneumoniae lung infection by modulating pulmonary neutrophil recruitment.

Streptococcus pneumoniae infections are an important cause of morbidity and mortality in older patients. Uncontrolled neutrophil-driven pulmonary inflammation exacerbates this disease. To test whether the α-tocopherol (α-Toc) form of vitamin E, a regulator of immunity, can modulate neutrophil responses as a preventive strategy to mitigate the age-associated decline in resistance to S.

The ability of an attaching and effacing pathogen to trigger localized actin assembly contributes to virulence by promoting mucosal attachment.

Enterohaemorrhagic Escherichia coli (EHEC) colonizes the intestine and causes bloody diarrhoea and kidney failure by producing Shiga toxin. Upon binding intestinal cells, EHEC triggers a change in host cell shape, generating actin 'pedestals' beneath bound bacteria. To investigate the importance of pedestal formation to disease, we infected genetically engineered mice incapable of supporting pedestal formation by an EHEC-like mouse pathogen, or wild type mice with a mutant of that pathogen incapable of generating pedestals.

Refining a brief decision aid in stable CAD: cognitive interviews.

Background: We describe the results of cognitive interviews to refine the "Making Choices©" Decision Aid (DA) for shared decision-making (SDM) about stress testing in patients with stable coronary artery disease (CAD).

Methods: We conducted a systematic development process to design a DA consistent with International Patient Decision Aid Standards (IPDAS) focused on Alpha testing criteria. Cognitive interviews were conducted with ten stable CAD patients using the "think aloud" interview technique to assess the clarity, usefulness, and design of each page of the DA.

Determination of absolute counts of circulating regulatory T cells in cynomolgus macaques using an optimized flow cytometric method.

Regulatory T cells (Tregs) are a rare subset of lymphocytes that inhibit the activation and effector functions of T cells and are important regulators of immune responses. Although Tregs are well characterized in humans and rodents, little is known about their immunophenotyping (IP) profile in cynomolgus macaques (Macaca fascicularis), which is an important species for pharmacological and toxicological evaluation of potential immune modulators because of their similar physiologic, genetic, and metabolic response patterns to humans. The authors have developed an immunophenotyping panel using a high-throughput 96-well microtiter plate-based assay to detect circulating Tregs (CD3(+)CD4(+)CD25(hi)FoxP3(+)) and have determined the normal range for the number of Tregs in naive healthy cynomolgus macaques to be 56.

A small animal model for mother-to-fetus transmission of ts1, a murine retrovirus.

Infection with a murine retrovirus, MoMuLV-TB, ts1 in BALB/c mice has been established as a small animal model for retroviral neurodegenerative disease as shown with infections such as HIV. However, mother-to-pup transmission has never been demonstrated in this model. The current investigation examines vertical transmission of ts1 in this mouse model.