Ischemic disruption of glutamate homeostasis in brain: quantitative immunocytochemical analyses

More than 10 years ago, it was shown by microdialysis that the excitatory transmitter glutamate accumulates in the interstitial space of brain subjected to ischemic insult. This was one of the key observations leading to the formulation of the `glutamate hypothesis' of ischemic cell death. It is now assumed that even a transient glutamate overflow may set in motion a number of events that ultimately cause cell loss in vulnerable neuronal populations. The aim of the present review is to discuss the intracellular changes that underlie the dysregulation of extracellular glutamate during and after ischemia, with emphasis on data obtained by postembedding, electron microscopic immunogold cytochemistry. While the time resolution of this approach is necessarily limited, it can reveal, quantitatively and at a high level of spatial resolution, how the intracellular pools of glutamate and metabolically related amino acids are perturbed during and after an ischemic insult. Moreover, this can be done in animals whose extracellular amino acid levels are monitored by microdialysis, allowing a direct correlation of extra- and intracellular changes. Immunogold analyses of brains subjected to ischemia have identified dendrites and neuronal somata as likely sources of glutamate efflux, probably mediated by reversal of glutamate uptake. The vesicular glutamate pool has been found to be largely unchanged after 20 min of ischemia. Ischemia causes an increased glutamate content and an increased glutamate/glutamine ratio in glial cells, as revealed by double immunogold labelling. This argues against the idea that glial cells contribute to the extracellular overflow of glutamate in the ischemic brain.     

Molecular and biochemical mechanisms ofPasteurella haemolyticaleukotoxin-induced cell death

Pasteurella haemolyticaleukotoxin (LKT) is a member of the RTX family of pore-forming toxins that kill bovine immune cells. Several studies have suggested that RTX toxins kill target cells by the induction of apoptosis. In the present study, BL3 bovine leukaemia cells were exposed to LKT and assessed by molecular and flow cytometric techniques that measure different aspects of apoptotic cell death. The intoxicated cells demonstrated morphological, light scatter and Hoechst 33258 staining characteristics consistent with cells undergoing apoptosis. The cells also exhibited internucleosomal DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage, both indicators of apoptosis. LKT-treated cells bound annexin-V-FITC indicating that phosphatidylserine groups were translocated from the inner to the outer leaflet of the cell membrane. The effect of LKT on cells was dose dependent and inhibitable by incubation with anti-LKT monoclonal antibody. Finally, an early step for induction of apoptosis appears to be the binding of LKT to a β2 integrin since pre-incubating cells with anti-β2 integrin antibodies inhibited LKT-induced apoptosis. This study provides new insights into understanding the pathogenesis of bovine pasteurellosis and could lead to the development of both preventative and therapeutic strategies for disease management.     

Distribution of glutamine-like immunoreactivity in the cerebellum of rat and baboon (Papio anubis) with reference to the issue of metabolic compartmentation

Summary The cellular and subcellular localization of glutamine, a major glutamate precursor, was studied by means of an antiserum raised against glutaraldehydefixed glutamine. Ultrathin sections from the cerebellar cortex of rat and baboon (Papio anubis) were incubated sequentially in the primary antiserum and in a secondary antibody coupled to colloidal gold particles. The labelling intensity was quantified by computer-aided calculation of gold particle densities. High levels of immunoreactivity occurred in glial cells (Bergmann fibres, astrocytes, and oligodendrocytes), intermediate levels in cell bodies and processes of granule cells, and low levels in terminals of presumed GABAergic or glutamatergic fibres (terminals of basket and Golgi cells, and of parallel, mossy, and climbing fibres). The labelling intensity of Purkinje cells showed some variation, but never exceeded that in glial cells. Within the nerve fibre terminals, the glutamine-like immunoreactivity showed some preference for mitochondria, but was otherwise evenly distributed. The predominant glial localization of glutamine was also obvious in light microscopic preparations processed according to the postembedding peroxidase-antiperoxidase procedure. Gold particle densities over different types of profile in glutamine immunolabelled sections were compared with particle densities over the corresponding types of profiles in neighbouring sections labelled with an antiserum to glutaraldehyde-fixed glutamate. The glutamate/glutamine ratio, expressed arbitrarily by the ratio between the respective gold particle densities, varied by a factor of about 6, with the highest ratio in the putative glutamatergic mossy and parallel fibre terminals, and the lowest ratio in glial elements. The remaining tissue components displayed intermediate ratios. The present study provides direct morphological evidence for the existence in the brain of distinct compartments with differing glutamate/glutamine ratios.This paper is dedicated to Professor Fred Walberg on the occasion of his 70th birthdayOn leave of absence from Department of Anatomy, Capital Institute of Medicine, You An Men Street, Beijing, China     

IL-15 enhances the function and inhibits CD95/Fas-induced apoptosis of human CD4+ and CD8+ effector-memory T cells

Although the effect of IL-15 has been described on murine cells in vitro and in vivo, its effect on human memory CD8(+) T cells is not well characterized. We show here that IL-15 preferentially enhances the activation and effector function of human effector-memory CD45RA(-)CD62L(-) and CD45RA(+)CD62L(-) CD4(+) and CD8(+) T cells in both healthy and HIV-infected individuals. We find that IL-15 increases 2- to 5-fold both the activation and secretion of the effector cytokines IFN-gamma and tumor necrosis factor (TNF)-alpha by anti-CD3-stimulated purified CD4(+) and CD8(+) T cells and peripheral blood mononuclear cells from healthy and HIV-infected individuals. Furthermore, IL-15 potently inhibits CD95/Fas-induced apoptosis of the effector-memory CD4(+) and CD8(+) T cells from HIV-infected individuals. These findings suggest that in addition to being a growth and survival factor for memory CD8(+) T cells, IL-15 is also a potent activator of human effector-memory CD8(+) T cells both in healthy and in HIV-infected individuals.     

Comments on methods and results in: Sherman et al., "Segregation analysis of balanced pericentric inversions in pedigree data"

The analysis by Sherman et al. (1986), its basis and results have been examined. The analysis relies on general methods, which may give acceptable results under the special conditions considered by the authors, but will usually produce more or less misleading results. The program POINTER (Lalouel & Morton 1981) is shown to be based partly on a chain of irrelevant arguments for the actual context. The so-called "conventional ascertainment rules" (Morton et al. 1983) are shown to produce misleading results in cases where their assumptions are not satisfied. The mean risk for unbalanced offspring is underestimated because of an erroneous ascertainment correction. The segregation frequencies are found to be different in three national samples, contrary to the claim by Sherman et al. Only a small proportion of all information available in the data has been utilized. Alternative and more appropriate models, hypotheses and procedures have been suggested. The frequent use of packages with computer programmes of standard statistical procedures in nonstandard situations with data from collaborative studies in human cytogenetics is discussed.     

The unusual transmembrane electron transporter DsbD and its homologues: a bacterial family of disulfide reductases

The bacterial membrane protein DsbD transfers electrons across the cytoplasmic membrane to reduce protein disulfide bonds in extracytoplasmic proteins. Its substrates include protein disulfide isomerases and a protein involved in cytochrome c assembly. Two membrane-embedded cysteines in DsbD alternate between the disulfide-bonded (oxidized) and reduced states in this process.     

Inhibition of C5 or absence of C6 protects from sepsis mortality

Inhibiting complement anaphlytoxin C5a during sepsis may prevent sepsis mortality. Although human anti-C5 antibodies exist, their therapeutic use in microbial sepsis has been avoided because of the hypothesis that inhibiting C5b will prevent formation of the bactericidal membrane attack complex (MAC) and worsen clinical outcome. We wished to test the hypothesis that inhibition of C5 would improve outcomes in sepsis. Sepsis was induced in rats by laparotomy and cecal ligation and puncture (CLP) by an IACUC-approved protocol. Sham animals underwent laparotomy without CLP. Following CLP rats were randomized to receive a single IV dose of purified IgG ant-C5 antibody (Ab) or control IgG Ab. Anti-C5 Ab treated rats (n = 20) had significantly lower mortality vs. controls (n = 21), 20% vs. 52% (P = 0.019, log-rank). Analysis of bacterial load by culture of spleen and liver homogenates showed a reduction in colony forming units in anti-C5 Ab treated rats vs. control IgG (P = 0.003 and 0.009, respectively). Anti-C5 treatment reduced lung injury as measured by total MPO content of lung tissue (P = 0.024). Finally, rats genetically deficient in C6 production, unable to form MAC but capable of producing C5a and C5b, were protected from CLP-induced sepsis mortality. Our results show that in anti-C5 antibody therapy prevents CLP sepsis-induced mortality and improves lung injury. Inhibition of the complement MAC does not increase bacterial load or mortality, therefore, the use of anti-C5 therapy may be beneficial rather than detrimental in sepsis.     

How can the evaluation of genetic tests be enhanced? Lessons learned from the ACCE framework and evaluating genetic tests in the United Kingdom.

Advances in genetic technology are increasing the availability of genetic tests, not only for rare single gene disorders, but also for common diseases such as breast and colo-rectal cancer. Before there can be widespread uptake of these tests, they must be evaluated to confirm the benefits of their use. But how should genetic tests be evaluated, given the speed at which new tests are emerging? One highly influential approach is the analytic validity, clinical validity, clinical utility and ethical, legal and social issues (ACCE) framework, which has provided a benchmark for the evaluation of genetic tests. The approach has been adopted and adapted by the United Kingdom Genetic Testing Network, with the help of the Public Health Genetics Unit in Cambridge, to evaluate new genetic tests for use in the National Health Service. We discuss a number of conceptual, methodological, and practical issues concerning the evaluation of genetic tests, based on lessons learned from applying the ACCE framework and from the UK experience, and make a number of recommendations to further strengthen the evaluation of genetic tests.     

Longitudinal Split of the Peroneus Brevis Tendon and Lateral Ankle Instability: Treatment of Concomitant Lesions

OBJECTIVE: To describe the clinical picture, pathophysiology, and treatment of concomitant lesions of the peroneus brevis tendon and lateral ligament injuries to the ankle. BACKGROUND: In some cases, chronic lateral ankle instability is associated with a longitudinal partial tear in the peroneus brevis tendon. Patients who suffer from this lesion usually have atypical posterolateral ankle pain combined with signs of recurrent ligament instability ("giving way"). The tendon injury is often overlooked because it is combined with the ligament injury, and the injury mechanisms are similar. DESCRIPTION: Tears or laxity in the superior peroneal retinaculum allow the anterior part of the injured peroneus brevis tendon to ride over the sharp posterior edge of the fibula, leading to a longitudinal tear in the tendon. This combined injury should be suspected in patients with recurrent giving way of the ankle joint and retromalleolar pain. The diagnosis can be established using either ultrasonography or magnetic resonance imaging. DIFFERENTIAL DIAGNOSIS: Ligament injury, tenosynovitis, peroneus longus tendon lesion, os peroneum fracture, distal peroneus brevis tendon tear, or anomalous peroneus tertius tendon. TREATMENT: The tendon injury and the ligament insufficiency should be repaired at the same time. CONCLUSIONS: We recommend reconstruction of the superior peroneal retinaculum, combined with repair of the tendon, using side-to-side sutures and anatomical reconstruction of the lateral ankle ligaments.