Influenza A virus (IAV) is a leading cause of respiratory tract disease worldwide. Anti-viral CD8+ T lymphocytes responding to IAV infection are believed to eliminate virally infected cells by direct cytolysis but may also contribute to pulmonary inflammation and tissue damage via the release of pro-inflammatory mediators following recognition of viral antigen displaying cells. We have previously demonstrated that IAV antigen expressing inflammatory cells of hematopoietic origin within the infected lung interstitium serve as antigen presenting cells (APC) for infiltrating effector CD8+ T lymphocytes; however, the spectrum of inflammatory cell types capable of serving as APC was not determined. Here, we demonstrate that viral antigen displaying neutrophils infiltrating the IAV infected lungs are an important cell type capable of acting as APC for effector CD8+ T lymphocytes in the infected lungs and that neutrophils expressing viral antigen as a result of direct infection by IAV exhibit the most potent APC activity. Our findings suggest that in addition to their suggested role in induction of the innate immune responses to IAV, virus clearance, and the development of pulmonary injury, neutrophils can serve as APCs to anti-viral effector CD8+ T cells within the infected lung interstitium.

Introduction Epithelia form intelligent, dynamic barriers between the external environment and an organism's interior. Intercellular cadherin-based adhesions adapt and respond to mechanical forces and cell density, while tight junctions flexibly control diffusion both within the plasma membrane and between adjacent cells. Epithelial integrity and homeostasis are of central importance to survival, and mechanisms have evolved to ensure these processes are maintained during growth and in response to damage. For instance, cell competition surveys the fitness of cells within epithelia and removes the less fit; extrusion or delamination can remove apoptotic or defective cells from the epithelial sheet, and can restore homeostasis when an epithelial layer become too crowded; spindle orientation ensures two-dimensional growth in simple epithelia and controls stratification in complex epithelia; and transition to a mesenchymal phenotype enables active escape from an epithelial layer. This review will discuss these mechanisms and consider how they are subverted in disease.

Brain fingerprinting detects concealed information stored in the brain by measuring brainwave responses. We compared P300 and P300-MERMER event-related brain potentials for error rate/accuracy and statistical confidence in four field/real-life studies. 76 tests detected presence or absence of information regarding (1) real-life events including felony crimes; (2) real crimes with substantial consequences (either a judicial outcome, i.e., evidence admitted in court, or a $100,000 reward for beating the test); (3) knowledge unique to FBI agents; and (4) knowledge unique to explosives (EOD/IED) experts. With both P300 and P300-MERMER, error rate was 0 %: determinations were 100 % accurate, no false negatives or false positives; also no indeterminates. Countermeasures had no effect. Median statistical confidence for determinations was 99.9 % with P300-MERMER and 99.6 % with P300. Brain fingerprinting methods and scientific standards for laboratory and field applications are discussed. Major differences in methods that produce different results are identified. Markedly different methods in other studies have produced over 10 times higher error rates and markedly lower statistical confidences than those of these, our previous studies, and independent replications. Data support the hypothesis that accuracy, reliability, and validity depend on following the brain fingerprinting scientific standards outlined herein.

Fluorescence activated cell sorting is the technique most commonly used to separate primary mammary epithelial sub-populations. Many studies incorporate this technique before analyzing gene expression within specific cellular lineages. However, to our knowledge, no one has examined the effects of fluorescence activated cell sorting (FACS) separation on short-term transcriptional profiles. In this study, we isolated a heterogeneous mixture of cells from the mouse mammary gland. To determine the effects of the isolation and separation process on gene expression, we harvested RNA from the cells before enzymatic digestion, following enzymatic digestion, and following a mock FACS sort where the entire cohort of cells was retained. A strict protocol was followed to minimize disruption to the cells, and to ensure that no subpopulations were enriched or lost. Microarray analysis demonstrated that FACS causes minimal disruptions to gene expression patterns, but prior steps in the mammary cell isolation process are followed by upregulation of 18 miRNA's and rapid decreases in their predicted target transcripts. V C 2015 International Society for Advancement of Cytometry Key terms FACS; mammary epithelial cells; miRNA MURINE mammary glands are highly branched tubular organs that develop mostly postnatally, within subcutaneous fat pads. The ducts that comprise the mammary gland arise from multipotent stem cells that generate all of the lineages found in the mature ductal tree (1,2). Differentiated mammary epithelial cells (MECs) can be broadly classified into two populations: luminal epithelial cells that line the ducts, and myoepithelial cells that form a layer surrounding the luminal cells. However, there are multiple cell types within each of these two populations; for example some luminal cells express the estrogen receptor and some do not.To identify the mechanisms that regulate lineage commitment, researchers have conducted transcriptional analysis of discrete mammary cell populations isolated by fluorescence activated cell sorting (FACS) (3,4). These studies all rely on the assumption that robust differences in gene expression between separate lineages are due to endogenous differences in gene expression. However, to our knowledge, this assumption has never been tested. Our study now demonstrates that FACS produces minimal short-term transcriptional artifacts, but also emphasizes the need for care in the methods used to isolate primary cells. MATERIALS AND METHODS MiceA 8-9 week old C3H mice were ordered from Harlan laboratories. Our handling and use of these mice strictly adhered to ACUC-approved protocol (M/12/080). Cell Preparation and RNA ExtractionMammary tissue from the 3rd, 4th, and 5th mammary fat pads was collected (minus the lymph nodes). Each replicate consisted of pooled tissue from three mice. Each mock sorting experiment contained three biological replicates from nine mice.

Alcohols and thiols were converted into alkyl chlorides, carboxylic acids were converted into acid chlorides, and mixtures of carboxylic acids and amines were converted into amides by reaction with carbon tetrachloride and 1% cross-linked polystyrenes containing phosphine residues. Some of these conversions were also effected by using a linear polymer containing phosphine residues. The reactions proceed in high yield, and isolation of the products is facilitated by the ready removal of all the polymer-supported species. The mechanism of the reactions between triphenylphosphine, carbon tetrachloride, and alcohols is complex, but the polymer-supported reactions appear to follow analogous pathways to the low molecular weight reactions as judged by the yields of chloroform and the number of equivalents of phosphine consumed per mole of alkyl chloride produced. The mechanism requires polymer-supported groups reacting together. The slow step in the reactions appears to be the generation of the chlorinating species. The polymer-supported reactions are faster than those using triphenylphosphine or 4-(diphenylphosphinyl)isopropylbenzene. It is suggested that this is due to a microenvironmental effect.