Requirements for passage of T lymphocytes across non-inflamed retinal microvessels

Although activated T lymphocytes can migrate through unstimulated neural endothelium to perform immune surveillance or initiate inflammation, the precise mechanism by which this occurs is not clear. In this study, we have used intravital scanning laser ophthalmoscopy to show that circulating, activated T cells induce early changes in the retinal venules that enable T cell diapedesis in the absence of cell rolling, and without any reduction in shear stress within the venules. Concanavalin A (Con A)-activated T cells, but not naive T cells, were able to penetrate the normal blood-retinal barrier (BRB) 8-16 h after adoptive transfer. A minimum number (> or =1 x 10(5) cells/mouse) of Con A-activated T cells needed to be transferred before lymphocytes crossed the normal BRB. Cell rolling and reduction of shear stress did not occur in normal retinal venules and post-capillary venules. In contrast, in mice with experimental autoimmune uveoretinitis (EAU), in which the BRB has broken down, 45% of blast cells were rolling in retinal venules. Cell rolling correlated with significantly reduced shear stress. Both naive and Con A-activated T cells could cross the disabled barrier, with Con A-activated T cells migrating faster and in greater numbers than naive cells. Adoptive transfer of Con A-activated cells into normal recipient mice induced limited and transient breakdown of the BRB and up-regulation of ICAM-1 but not P-selectin. Pretreatment of Con A-activated cells with anti-LFA-1 significantly suppressed T cell infiltration in normal recipient mice. Our data indicate that critical to immune surveillance in the central nervous system (CNS) is the ability of activated T cells to interact with the endothelium, up-regulating ICAM-1 and inducing transient breakdown of the barrier.     

New Insights into Hydrocyclone Operation

Hydrocyclones form an important stage within conveying systems for dewatering or size classification. Over the past few decades they have been subject to significant modeling and experimental investigations. Yet, it is only with modern developments in computational fluid dynamics (CFD) and nonintrusive measurements such as tomography and laser techniques that a proper understanding of the internal flow dynamics is being developed. This paper presents new insight into the mechanisms governing the air-core dynamics and particle separation within the hydrocyclone that have been achieved by means of CFD. In particular, the flow field is identified to be distinctly asymmetric, such that the commonly assumed deterministic and symmetric assumptions of particle separation are inaccurate. In fact, radial particle transport is identified to rely upon the positive correlation of the mean and fluctuating components of radial velocity. The mechanism for air-core development during start-up is also inferred and for the first time related to experimental observation. High-speed video and radiographic measurements have provided validation for the predicted flowfield. In addition, the potential for application of tomographic techniques to the challenging verification of high-solids CFD has been demonstrated.     

High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus

Adeno-associated virus (AAV) is a potential vector for in vivo gene therapy. A critical analysis of its utility has been hampered by methods of production that are inefficient, difficult to scale up, and that often generate substantial quantities of replication-competent AAV. We describe a novel method for producing AAV that addresses these problems. A cell line, called B50, was created by stably transfecting into HeLa cells a rep/cap-containing plasmid utilizing endogenous AAV promoters. Production of AAV occurs in a two-step process. B50 is infected with an adenovirus defective in E2b, to induce Rep and Cap expression and provide helper functions, followed by a hybrid virus in which the AAV vector is cloned in the E1 region of a replication-defective adenovirus. This results in a 100-fold amplification and rescue of the AAV genome, leading to a high yield of recombinant AAV that is free of replication-competent AAV. Intramuscular injection of vector encoding erythropoietin into skeletal muscle of mice resulted in supraphysiologic levels of hormone in serum that was sustained and caused polycythemia. This method of AAV production should be useful in scaling up for studies in large animals, including humans.     

High resolution multichannel fluorescence detection for capillary electrophoresis. Application to multicomponent analysis

A wavelength-resolved fluorescence detector for laser-induced fluorescence detection in capillary electrophoresis (CE) is described that uses a charge injection device (CID) array detector Post-column fluorescence detection occurs using a sheath flow cell. The limit of detection for fluorescein is 4.8 x 10(-11) M (29,000 molecules), the spectral resolution is 0.56 nm/pixel, and the spectrograph/CID monitors a 250 nm spectrum throughout the 250-875 nm range. Custom array readout, data manipulation and data processing methods are described to convert wavelength/spatial CID images into electropherograms. The application of the system to characterizing bilirubins in human serum is described, demonstrating the ability to match electrophoretic peaks to standards using spectral information.     

Measurement of carbonic anhydrase activity using a sensitive fluorometric assay

The dehydration reaction of bicarbonate was measured using the fluorescent pH indicator, 8-hydroxypyrene-1,3,6-trisulfonate (pyranine), in combination with stopped-flow spectrofluorometry. The initial rate of bicarbonate dehydration was measured after mixing a pH 6.0 solution with a pH 8.0 solution containing bicarbonate. Addition of carbonic anhydrase to the pH 6.0 solution enabled the measurement of the initial rate of activity at physiological temperatures with resolution times of 2 ms. This assay was used to resolve differences in activity and sensitivity to sulfonamides by comparing mammalian carbonic anhydrase isoforms. The fluorescent technique used in this study is very sensitive, allowing the determination of initial rates with a protein concentration as little as 65 ng/ml. Pyranine can also be loaded into membrane vesicles to follow carbonic anhydrase activity within vesicles. The change in pH within vesicles is dependent on the concentration of externally added bicarbonate and the presence of carbonic anhydrase on either side of the membrane. Therefore, this assay can be used to measure carbon dioxide flux across membranes and to assess the contribution of carbonic anhydrase to this flux.     

Models of the self: self-construals and gender

The authors first describe individual differences in the structure of the self. In the independent self-construal, representations of others are separate from the self. In the interdependent self-construal, others are considered part of the self (H. Markus & S. Kitayama, 1991). In general, men in the United States are thought to construct and maintain an independent self-construal, whereas women are thought to construct and maintain an interdependent self-construal. The authors review the psychological literature to demonstrate that many gender differences in cognition, motivation, emotion, and social behavior may be explained in terms of men's and women's different self-construals. Recognition of the interdependent self-construal as a possible alternative conception of the self may stimulate new investigations into the ways the self influences a person's thinking, feeling, and behaving.