An important determinant of disease following Streptococcus pneumoniae (pneumococcus) lung infection is pulmonary inflammation mediated by polymorphonuclear leukocytes (PMNs). We found that upon intratracheal challenge of mice, recruitment of PMNs into the lungs within the first 3 hours coincided with decreased pulmonary pneumococci, whereas large numbers of pulmonary PMNs beyond 12 hours correlated with a greater bacterial burden. Indeed, mice that survived infection largely resolved inflammation by 72 hours, and PMN depletion at peak infiltration, i.e. 18 hours post-infection, lowered bacterial numbers and enhanced survival. We investigated host signaling pathways that influence both pneumococcus clearance and pulmonary inflammation. Pharmacologic inhibition and/or genetic ablation of enzymes that generate extracellular adenosine (EAD) (e.g. the ectoenzyme CD73) or degrade EAD (e.g. adenosine deaminase) revealed that EAD dramatically increases murine resistance to S. pneumoniae lung infection. Moreover, adenosine diminished PMN movement across endothelial monolayers in vitro, and although inhibition or deficiency of CD73 had no discernible impact on PMN recruitment within the first 6 hours after intratracheal inoculation of mice, these measures enhanced PMN numbers in the pulmonary interstitium after 18 hours of infection, culminating in dramatically elevated numbers of pulmonary PMNs at three days post-infection. When assessed at this time point, CD73 -/- mice displayed increased levels of cellular factors that promote leukocyte migration, such as CXCL2 chemokine in the murine lung, as well as CXCR2 and β-2 integrin on the surface of pulmonary PMNs. The enhanced pneumococcal susceptibility of CD73 -/- mice was significantly reversed by PMN depletion following infection, suggesting that EAD-mediated resistance is largely mediated by its effects on PMNs. Finally, CD73-inhibition diminished the ability of PMNs to kill pneumococci in vitro, suggesting that EAD alters both the recruitment and bacteriocidal function of PMNs. The EAD-pathway may provide a therapeutic target for regulating potentially harmful inflammatory host responses during Gram-positive bacterial pneumonia.

Recent studies have demonstrated that the function of glia is not restricted to the support of neuronal function. Especially, astrocytes are essential for neuronal activity in the brain. Astrocytes actively participate in synapse formation and brain information processing by releasing or uptaking gliotransmitters such as glutamate, D-serine, adenosine 5′-triphosphate (ATP) and adenosine. In the central nervous system, adenosine plays an important role in regulating neuronal activity as well as in controlling other neurotransmitter systems such as GABA, glutamate and dopamine. Ethanol increases extracellular adenosine levels, which regulates the ataxic and hypnotic/sedative (somnogenic) effects of ethanol. Adenosine signaling is also involved in the homeostasis of major inhibitory-excitatory neurotransmission (i.e. GABA or glutamate) through neuron-glial interactions, which regulates the effect of ethanol and sleep. Adenosine transporters or astrocytic SNARE-mediated transmitter release regulates extracellular or synaptic adenosine levels. Adenosine then exerts its function through several adenosine receptors and regulates glutamate levels in the brain. This review presents novel findings on how neuron-glial interactions, particularly adenosinergic signaling and glutamate uptake activity involving glutamate transporter 1 (GLT1), are implicated in alcoholism and sleep disorders.

Sleep impairments are comorbid with a variety of neurological and psychiatric disorders including depression, epilepsy, and alcohol abuse. Despite the prevalence of these disorders, the cellular mechanisms underlying the interaction between sleep disruption and behavior remain poorly understood. In this study, the impact of chronic sleep loss on sleep homeostasis was examined in C57BL/6J mice following 3 d of sleep restriction. The electroencephalographic power of slow-wave activity (SWA; 0.5-4 Hz) in nonrapid eye movement (NREM) sleep and adenosine tone were measured during and after sleep restriction, and following subsequent acute sleep deprivation. During the first day of sleep restriction, SWA and adenosine tone increased, indicating a homeostatic response to sleep loss. On subsequent days, SWA declined, and this was accompanied by a corresponding reduction in adenosine tone caused by a loss of one source of extracellular adenosine. Furthermore, the response to acute sleep deprivation (6 h) was significantly attenuated in sleep-restricted mice. These effects were long-lasting with reduced SWA and adenosine tone persisting for at least 2 weeks. To investigate the behavioral consequences of chronic sleep restriction, sensitivity to the motor-impairing effects of alcohol was also examined. Sleep-restricted mice were significantly less sensitive to alcohol when tested 24 h after sleep restriction, an effect that persisted for 4 weeks. Intracerebroventricular infusion of an adenosine A1 receptor antagonist produced a similar decrease in sensitivity to alcohol. These results suggest that chronic sleep restriction induces a sustained impairment in adenosine-regulated sleep homeostasis and consequentially impacts the response to alcohol.

The vulnerability of oligodendrocytes to ischemic injury may contribute to functional loss in diseases of central white matter. Immunocytochemical methods to identify oligodendrocyte injury in experimental models rely on epitope availability, and fail to discriminate structural changes in oligodendrocyte morphology. We previously described the use of a lentiviral vector (LV) carrying eGFP under the myelin basic protein (MBP) promoter for selective visualization of oligodendrocyte cell bodies and processes. In this study, we used LV-MBP-eGFP to label oligodendrocytes in rat cerebral white matter prior to transient focal cerebral ischemia, and examined oligodendrocyte injury 24 hours, 48 hours and one week post-reperfusion by quantifying cell survival and assaying the integrity of myelin processes. There was progressive loss of GFP+ oligodendrocytes in ischemic white matter at 24 and 48 hrs. Surviving GFP+ cells had non-pyknotic nuclear morphology and were TUNEL-negative, but there was marked fragmentation of myelin processes as early as 24 hours after stroke. One week after stroke, we observed a restoration of GFP+ oligodendrocytes in ischemic white matter, reflected both by cell counts and by structural integrity of myelin processes. Proliferating cells were not the main source of GFP+ oligodendrocytes, as revealed by BrdU incorporation. These observations identify novel transient structural changes in oligodendrocyte cell bodies and myelinating processes, which may have consequences for white matter function after stroke.

Lentiviral vectors transduce both dividing and non-dividing cells and can support sustained expression of transgenes. These properties make them attractive for the transduction of neurons and other neural cell types in vitro and in vivo. Lentiviral vectors can be targeted to specific cell-types by using different promoters in the lentiviral shuttle vector. Even with identical constructs, however, levels of expression can vary significantly in different types of neurons and different culture preparations; expression levels in the same neuronal subtypes can be very different in primary cell culture and in vivo. We systematically assessed the ability of different promoters to direct expression of foreign transgenes in primary murine neocortical neurons, cerebellar granule cells and in undifferentiated and differentiated neuroblastoma cells. In primary cortical neurons, constructs using the ubiquitin C promoter directed the highest level of transgene expression; the phosphoglycerate kinase (PGK) promoter also directed robust transgene expression, while the cytomegalovirus (CMV) and MND (a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer) promoters resulted in the expression of the transgenes in only limited number of neurons. In contrast, in cerebellar granule cells and in differentiated SH-SY5Y neuroblastoma cultures, the CMV promoter directed the most robust transgene expression. There was similar variability in transgene expression directed by these promoters in primary cultures of oligodendrocytes and astrocytes. These findings may prove useful in the design of lentiviral vectors for use in cell culture models of the nervous system.