Solid-state capture and real-time analysis of individual T cell activation via self-assembly of binding multimeric proteins on functionalized materials surfaces.

Polyfunctional T cell responses are increasingly underpinning new and improved vaccination regimens. Studies of the nature and extent of these T cell responses may be facilitated if specific T cell populations can be assessed from mixed populations by ligand-mediated capture in a solid-state assay format. Accordingly, we report here the development of a novel strategy for the solid-state capture and real-time activation analyses of individual cognate T cells which utilizes a spontaneous self-assembly process for generating multimers of biotinylated class I major histocompatibility-peptide complex (MHCp) directly on the solid-state assay surface while also ensuring stability by covalent interfacial binding. The capture surface was constructed by the fabrication of multilayer coatings onto standard slides. The first layer was a thin polymer coating with surface aldehyde groups, onto which streptavidin was covalently immobilized, followed by the docking of multimers of biotinylated MHCp or biotinylated anti-CD45.1 monoclonal antibody. The high binding strength at each step of this immobilization sequence aims to ensure that artefacts such as (partial) detachment, or displacement by proteins from solution, would not interfere with the intended biological assays. The multilayer coating steps were monitored by X-ray photoelectron spectroscopy; data indicated that the MHCp proteins self-assembled in a multimeric form onto the streptavidin surface. Immobilized multimeric MHCp demonstrated the capacity to bind and retain antigen-specific T cells from mixed populations of cells onto the solid carrier. Furthermore, real-time confocal microscopic detection and quantification of subsequent calcium flux using paired fluorescent ratiometric probes facilitated the analysis of individual T cell response profiles, as well as population analyses using a combination of individual T cell events.

Substance P immunoreactivity increases following human traumatic brain injury.

Recent experimental evidence suggests that neuropeptides, and in particular substance P (SP), are released following traumatic brain injury (TBI) and may play a significant role in the aetiology of cerebral edema and increased intracranial pressure. Whether SP may play a similar role in clinical TBI remains unknown and was investigated in the current study. Archival post-mortem material was selected from patients who had sustained TBI, had died and had undergone post-mortem and detailed neuropathological examination (n = 13). A second cohort of patients who had died, but who showed no neuropathological abnormality (n = 10), served as case controls. Changes in SP immunoreactivity were examined in the cerebral cortex directly beneath the subdural haematoma in 7 TBI cases and in proximity to contusions in the other 6 cases. Increased SP perivascular immunoreactivity was observed after TBI in 10/13 cases, cortical neurones in 12/13 and astrocytes in 10/13 cases. Perivascular axonal injury was observed by amyloid precursor protein (APP) immunoreactivity in 6/13 TBI cases. Co-localization of SP and APP in a small subset of perivascular fibres suggests perivascular axonal injury could be a mechanism of release of this neuropeptide. The abundance of SP fibres around the human cerebral microvasculature, particularly post capillary venules, together with the changes observed following TBI in perivascular axons, cortical neurones and astrocytes suggest a potentially important role for substance P in neurogenic inflammation following human TBI.

Co-expression of CYP27B1 enzyme with the 1.5kb CYP27B1 promoter-luciferase transgene in the mouse.

The renal enzyme 25-hydroxyvitamin D 1alpha-hydroxylase (CYP27B1), responsible for the synthesis of circulating. 1,25-dihydroxyvitamin D (1,25D), is also expressed in a number of non-renal tissues. The regulation of CYP27B1 expression by the short flanking promoter outside the kidney is, however, largely unknown. We have used a transgenic mice expressing the 1.5kb promoter of the human CYP27B1 gene fused to the firefly luciferase gene in order to investigate tissue-specific CYP27B1 expression. These transgenic animals demonstrated co-localised luciferase and endogenous CYP27B1 expression in kidney proximal convoluted tubular cells. Strong co-expression of luciferase and CYP27B1 also occurred in neurons and Purkinje cells of the cerebellum and in Leydig and Sertoli cells of the testes. Other tissues to exhibit CYP27B1-promoter directed luciferase activity included lung, prostate, trabecular bone and jejunum as well as the choroid epithelium. The tissue specific changes in luciferase activity were age-related. These findings demonstrate that the proximal 1.5kb 5' flanking region of the CYP27B1 gene directs the expression of CYP27B1 in a number of known and novel tissues in a specific manner.

Severity-dependent expression of pro-inflammatory cytokines in traumatic spinal cord injury in the rat.

The post-traumatic inflammatory response in acute spinal cord contusion injury was studied in the rat. Mild and severe spinal cord injury (SCI) was produced by dropping a 10 g weight from 3 and 12 cm at the T12 vertebral level. Increased immunoreactivity of TNF-alpha in mild and severe SCI was detected in neurons at 1 h post-injury, and in neurons and microglia at 6 h post-injury, with a less significant increase in mild SCI. Expression was short-lived and declined sharply by 1 d post-injury. RT-PCR showed an early significant up-regulation of IL-1 beta, IL-6 and TNF-alpha mRNAs, maximal at 6 h post-injury with return to control levels by 24 h post-injury, the changes being less statistically significantly in mild SCI. Western blot showed early transient increases of IL-1 beta, IL-6 and TNF-alpha proteins in severe SCI but not mild SCI. Immunocytochemical, western blotting and RT-PCR analyses suggest that endogenous cells (neurons and microglia) in the spinal cord, not blood-borne leucocytes, contribute to IL-1 beta, IL-6 and TNF-alpha production in the post-traumatic inflammatory response and that their up-regulation is greater in severe than mild SCI.

Early expression and cellular localization of proinflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in human traumatic spinal cord injury.

STUDY DESIGN: Post-traumatic inflammatory response was studied in 11 human cases of acute spinal cord contusion injury. OBJECTIVES: To examine the inflammatory cellular response and the immunocytochemical expression and localization of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in human spinal cord after contusion injury. SUMMARY OF BACKGROUND DATA: : The post-traumatic inflammatory response plays an important role in secondary injury mechanisms after spinal cord injury, and interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha are key inflammatory mediators. METHODS: : The study group comprised 11 patients with spinal cord contusion injury and 2 normal individuals. Histologic and immunocytochemical assessments were undertaken to evaluate the inflammatory cellular response and the immunoexpression of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in the injured human spinal cord. The cellular sources of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha were elucidated by immunofluorescence double-labeled confocal imaging. RESULTS: : Increased immunoreactivity of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha was detected in neurons 0.5 hour after injury, and in neurons and microglia 5 hours after injury, but the expression of these proinflammatory cytokines was short-lived and declined sharply to baseline by 2 days after injury. In the inflammatory cellular response, as early as 0.5 hour after spinal cord injury, activated microglia were detected, and axonal swellings and axons were surrounded by microglial processes. Numerous neutrophils appeared in the injured cord 1 day after injury, and then their number declined dramatically, whereas macrophages progressively increased after day 1. CONCLUSIONS: Endogenous cells (neurons and microglia) in the human spinal cord, not the blood-borne leukocytes, contribute to the early production of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in the post-traumatic inflammatory response, and microglia are involved the early response to traumatic axonal injury.