A standardised protocol for the evaluation of small extracellular vesicles in plasma by imaging flow cytometry.

Flow cytometry provides robust, multi-parametric and quantitative information on single cells which also exhibits enormous potential as a tool for small particle characterisation. Small extracellular vesicle (sEV) detection by flow cytometry remains compromised due to the high prevalence of swarm detection, which is defined by the simultaneous illumination of more than one sEV, recorded as a single event. Detection of sEVs by imaging flow cytometry presents a major advantage by having the ability to resolve single particles from swarm detection based on the image features recorded for each event. In this study, we provide a simplified protocol that facilitates the removal of both swarm events and aggregated particles to improve the accuracy of sEV analysis. Our results indicate that imaging flow cytometry should be at the forefront as a robust and sensitive technique for sEV characterisation.

Morphology-dependent resonances in eccentrically stratified spheres.

Calculations based on an exact theory have been made of the scattering properties of spherical particles possessing arbitrarily placed spherical inhomogeneities. The inclusions are homogeneous but of otherwise arbitrary composition and are limited in size only by that of the host. It has been found that a single inclusion, though having a volume of only 10(-6) that of the host, can produce large fluctuations in light scattered by the system as the inclusion passes through regions of high local intensity. The resonance spectrum of a glycerol droplet with inclusions centered in a hot spot reveals that the inclusions cause a spectral shift of the resonance peak. The directions of the shifts are consistent with the phase-matching conditions that must be met by photons traversing an orbit inside the host in order for a resonance to be sustained. Absorption spectra of systems with inclusions that are also large enough to sustain resonances are examined as well.

T cells within germinal centers are specific for the immunizing antigen.

A normal antibody response to T cell-dependent Ag requires physical contact between Ag-specific B and T cells. Because such Ag-specific cells are rare in vivo, we sought to identify an in vivo site where they physically contact each other. We examined the Ag specificity of T cells in germinal centers (GC) in lymph nodes, where it is known that Ag-specific B cells proliferate and mature. We investigated the Ag specificity of GC T cells in situ by examining two characteristics: 1) expression of certain V alpha and V beta TCR families; and 2) incorporation of bromodeoxyuridine into T cell DNA after exposure to Ag as an index of Ag-induced proliferation. When GC were induced in mice with cytochrome c and myelin basic protein, the GC T cells were found to preferentially express V alpha 11 and V beta 8 TCR families, which are, respectively, the dominant TCR families in these two responses. Furthermore, GC T cells have proliferated upon exposure to the Ag that induced GC formation. Taken together, these results demonstrate that GC must recruit and retain Ag-specific T cells, thus implicating the GC as an in vivo site where Ag-specific T and B cells interact.

Scattering by two spheres in contact: comparisons between discrete-dipole approximation and modal analysis.

This paper applies two different techniques to the problem of scattering by two spheres in contact: modal analysis, which is an exact method, and the discrete-dipole approximation (DDA). Good agreement is obtained, which further demonstrates the utility of the DDA to scattering problems for irregular particles. The choice of the DDA polarizability scheme is discussed in detail. We show that the lattice dispersion relation provides excellent improvement over the Clausius-Mossoti polarizability parameterization.

Infrared optical properties of orthorhombic sulfur.

Because of recent developments in planetary astronomy, there has been a resurgence of interest in the optical and thermodynamic properties of elemental sulfur. An encounter between the space probe Galileo and the Jovian moons, particularly Io, is expected to contribute further to this interest. A thorough investigation of the optical properties of orthorhombic sulfur from 2 to 56 microm (5000-180 cm(-1)) is presented. Since less care was taken in many past studies of this element than was warranted, a critical review of some of the relevant literature is included. The near-normal specular reflectance of the (111) face of an orthorhombic sulfur crystal has been measured in both polarized and unpolarized radiation at room temperature. The reflectance of a cryptocrystalline melt freeze has also been obtained. Associated optical constants are determined from a Kramers-Kronig phase shift analysis of the reflectance data. The average reflectance and absolute refractive index n were found to vary with polarization from 0.100 to 0.125 and from 1.92 to 2.09, respectively. Between eight and eleven mostly weak absorption bands of the cyclo-S(8) molecule were discernible, but the attenuation index k remains small throughout most of the region studied. The crystal spectra were found to be quite sensitive to polarization in the neighborhood of the v(4) fundamental. Extrapolation of n to other temperatures and to the liquid phase through the use of the Lorentz-Lorenz relation is discussed.

Optical resonances and two-sphere systems.

After a historical review of previous research into cooperative scattering, a theoretical investigation of the effects of interparticle coupling on morphology-dependent resonances of spheres is conducted. Bispheres composed of identical, slightly dissimilar, and very different monomers are considered. Calculations are presented of resonance spectra for selected orientations of the bispheres relative to the incident wave vector along with spectra that should prove useful in describing the scattering properties of a monodisperse ensemble of randomly oriented bispheres. (The bispheres in this dispersion may be constituted from dissimilar monomers.) Normalized source functions for regions inside and near the scatterers are also provided. Finally a numerical simulation of an interesting experiment is carried out in which a resonating aerosol passes through the focal volume of a relatively large, spherical microlens.

Oral tolerance in experimental autoimmune encephalomyelitis: serum and salivary antibody responses.

We have recently reported that the oral administration of myelin basic protein (MBP) prior to encephalitogenic challenge results in suppression of experimental autoimmune encephalomyelitis (EAE). We examined the serum and salivary antibody responses to MBP in orally tolerant rats using an avidin-biotin enzyme-linked immunosorbent assay. Serum anti-MBP IgA and IgG, but not IgM levels are suppressed in orally tolerant versus control rats. This suppression is time dependent and is confined to the period when animals would otherwise be manifesting EAE clinical signs. In contrast, there is an increase in salivary anti-MBP IgA levels in MBP-fed rats relative to vehicle-fed controls. Thus, MBP-induced unresponsiveness is demonstrable at the humoral level, and moreover, a discrete compartmentalization between the serum and salivary anti-MBP responses exists.

A technique for quantitative assessment of three-dimensional motion with applications to human joints.

A technique is developed for quantitative measurement of general three-dimensional motion, and this technique is applied to the kinematics of anatomical joints. The spatial locations of three orthogonal points representing coordinate frames on each member of the joint are measured during motion of the joint by photo encoders of a three-dimensional mechanical pointer. Kinematic calculations are used to derive, from the experimentally collected data, the six orthogonal components of the motion of one member relative to the other. The accuracy of this technique is presented. Applications to the knee and ankle are discussed.

Some novel features of morphology dependentresonances of bispheres.

On aggregation, two identical spheres produce split resonant structures so broadened as to be observable even if their (single) progenitor resonances are narrow beyond detectability.

Consummate solution to the problem of classical electromagnetic scattering by an ensemble of spheres. II: Clusters of arbitrary configuration.

The order-of-scattering approach developed earlier [Opt. Lett. 13,90 (1988)] and applied there to the case of linear chains of spheres is extended to the more difficult problem of scattering by clusters of spheres, the centers of which no longer need lie on a common axis. To help establish the validity of this most general calculation, comparisons are made between theoretical and experimental results for triangular and tetrahedral arrays of spheres. We also perform calculations based on an older method that requires the inversion of matrices, and we find that for the cases considered here the order-of-scattering method is substantially faster.

Oral ingestion of Legionella pneumophila.

Guinea pigs were fed L. pneumophila through an orogastric tube. Gastric acid was rapidly cidal to the organisms. Serial necropsies demonstrated the organisms in the colon and blood stream at 1 hr. Guinea pigs fed large doses of L. pneumophila seroconverted. Previously fed guinea pigs were then challenged with a lethal intraperitoneal dose of L. pneumophila and were protected in a dose-dependent fashion. Pretreatment of the guinea pigs with cimetidine lowered the dose of oral L. pneumophila required for protection. This model may be useful in studying the immune response after oral ingestion of L. pneumophila.

Intratracheal infection with L. pneumophila subtypes.

Two strains of Legionella pneumophila serogroup 1 (UH1 and RH1) were injected intratracheally into guinea pigs. There were no differences in infectivity or mortality, but there was evidence of disseminated infections in more guinea pigs infected with the UH1 strain. Guinea pigs that received sublethal inocula were then rechallenged intratracheally and had a lower mortality rate than did previously uninfected controls.

Serum bactericidal activity against Legionella pneumophila.

Two strains of Legionella pneumophila serogroup 1 (UH1 and RH1) were incubated in fresh human serum. The UH1 strain was serum resistant, whereas the RH1 strain was serum susceptible. The bactericidal activity of fresh serum was abrogated by heating. Serum resistance of L. pneumophila strains may correlate with increased virulence.