Autophagy and ATP-induced anti-apoptosis in antigen presenting cells (APC) follows the cytokine storm in patients after major trauma

Severe trauma and the systemic inflammatory response syndrome (SIRS) occur as a result of a cytokine storm which is in part due to ATP released from damaged tissue. This pathology also leads to increased numbers of immature antigen presenting cells (APC) sharing properties of dendritic cells (DC) or macrophages (MΦ). The occurrence of immature APC appears to coincide with the reactivation of herpes virus infections such as Epstein Barr virus (EBV). The aim of this study was the comparative analysis of the ultrastructural and functional characteristics of such immature APC. In addition, we investigated EBV infection/ reactivation and whether immature APC might be targets for natural killers (NK). Significant macroautophagy, mitochondrial degradation and multivesicular body formation together with the identification of herpes virus particles were morphological findings associated with immature APC. Exogenous stressors such as ATP further increased morphological signs of autophagy, including LC3 expression. Functional tests using fluorescent bacteria proved impaired phagolysosome fusion. However, immature APC were susceptible to NK-92-mediated cytolysis. We found evidence for EBV latency state II infection by detecting EBV-specific LMP1 and EBNA2 in immature APC and in whole blood of these patients. In summary, trauma-induced cytokine storms may induce maturation arrest of APC, promote ATP-induced autophagy, support EBV persistence and impair the degradation of phagocytozed bacteria through inefficient phagolysosome fusion. The susceptibility to NK-mediated cytolysis supports the hypothesis that NK function is likely to contribute to immune reconstitution after major trauma by regulating immature APC, and ATP-induced autophagy and survival.     

Acidic preconditioning protects endothelial cells against apoptosis through p38- and Akt-dependent Bcl-xL overexpression

To analyze the underlying cellular mechanisms of adaptation to ischemia-induced apoptosis through short acidic pretreatment, i.e. acidic preconditioning (APC), Wistar rat coronary endothelial cells (EC) were exposed for 40 min to acidosis (pH 6.4) followed by a 14 h recovery period (pH 7.4) and finally treated for 2 h with simulated in vitro ischemia (glucose-free anoxia at pH 6.4). APC led to a transient activation of p38 and Akt kinases, but not of JNK and ERK1/2 kinases, which was accompanied by significant reduction of the apoptotic cell number, caspase-12/-3 cleavage and Bcl-xL overexpression. These effects of APC were completely abolished by prevention of Akt- or p38-phosphorylation during APC. Furthermore, knock-down of Bcl-xL by siRNA-transfection also abolished the anti-apoptotic effect of APC. Therefore, APC leads to protection of EC against ischemic apoptosis by activation of Akt and p38 followed by overexpression of Bcl-xL, which is a key anti-apoptotic mechanism of APC.     

After-Potentials and Large Depolarizations of Single Nodes of Ranvier Treated with Veratridine

Potential differences between normal nodes of Ranvier (single fiber from the sciatic nerve of the frog, air-gap method) and a node exposed to 1 to 2.5 x 10^-6 gm veratridine per ml were measured. Negative after-potentials occurred immediately after application of the alkaloid when spike configuration and resting potential were virtually unchanged. The after-potentials decreased in magnitude and their time constant increased as the resting membrane was depolarized either by outward currents or by a train of impulses. Increase of (Na)_o markedly increased the amplitude of the after-potential. After prolonged application of veratridine or with higher concentrations, a large slow depolarization (rate of potential change about 7 mv per second) could be triggered by a train of impulses or even a single spike. This depolarization could promptly be terminated by withdrawing Na. It is concluded that, once the nodal membrane has become permeable to Na (as during a spike), veratridine prevents the normal return of P_Na to its resting value.     

Thermosensitivity of the <Emphasis Type="Italic">Saccharomyces cerevisiae gpp1gpp2</Emphasis> double deletion strain can be reduced by overexpression of genes involved in cell wall maintenance

A Saccharomyces cerevisiae strain in which the GPP1 and GPP2 genes, both encoding glycerol-3-phosphate phosphatase isoforms, are deleted, displays both osmo- and thermosensitive (ts) phenotypes. We isolated genes involved in cell wall maintenance as multicopy suppressors of the gpp1gpp2 ts phenotype. We found that the gpp1gpp2 strain is hypersensitive to cell wall stress such as treatment with β-1,3-glucanase containing cocktail Zymolyase and chitin-binding dye Calcofluor-white (CFW). Sensitivity to Zymolyase was rescued by overexpression of SSD1, while CFW sensitivity was rescued by SSD1, FLO8 and WSC3—genes isolated as multicopy suppressors of the gpp1gpp2 ts phenotype. Some of the isolated suppressor genes (SSD1, FLO8) also rescued the lytic phenotype of slt2 deletion strain. Additionally, the sensitivity to CFW was reduced when the cells were supplied with glycerol. Both growth on glycerol-based medium and overexpression of SSD1, FLO8 or WSC3 had additive suppressing effect on CFW sensitivity of the gpp1gpp2 mutant strain. We also confirmed that the internal glycerol level changed in cells exposed to cell wall perturbation.     

Hemorrhagic disease in Kansas: enzootic stability meets epizootic disease

Kansas (USA) could represent a transition area between contrasting epidemiologic patterns of hemorrhagic disease (HD) in the midwestern United States. In this study, we compare the distribution of reported clinical HD with serologic data to determine whether the risk of HD in white-tailed deer (Odocoileus virginianus) is associated with geographic location corresponding to the reported distribution of two white-tailed deer subspecies. On the basis of a high prevalence of antibodies (91-100%) to multiple serotypes of epizootic hemorrhagic disease virus (EHDV) and bluetongue virus (BTV), with correspondingly few reports of clinical HD, it appears that a state of enzootic stability exists in central and western Kansas. This area corresponds to the reported range of O. virginianus texanus. In contrast, in the eastern third of the state, which corresponds to the reported range of O. virginianus macronurus, antibody prevalence is significantly lower (45%), EHDV serotypes appear to predominate, and HD, as confirmed by virus isolation, has been consistently reported. These results suggest an abrupt demarcation between enzootic stability in central and western Kansas to a pattern of epizootic HD within the eastern part of this state. Understanding host, vector, and environmental variables responsible for these contrasting patterns could have application to understanding the risk of HD in the midwestern United States.     

Soluble Adenylyl Cyclase Controls Mitochondria-dependent Apoptosis in Coronary Endothelial Cells

The cAMP signaling pathway plays an essential role in modulating the apoptotic response to various stress stimuli. Until now, it was attributed exclusively to the activity of the G-protein-responsive transmembrane adenylyl cyclase. In addition to transmembrane AC, mammalian cells possess a second source of cAMP, the ubiquitously expressed soluble adenylyl cyclase (sAC). However, the role of this cyclase in apoptosis was unknown. A mitochondrial localization of this cyclase has recently been demonstrated, which led us to the hypothesis that sAC may play a role in apoptosis through modulation of mitochondria-dependent apoptosis. To prove this hypothesis, apoptosis was induced by simulated in vitro ischemia or by acidosis, which is an important component of ischemia. Suppression of sAC activity with the selective inhibitor KH7 or sAC knockdown by small interfering RNA transfection abolished endothelial apoptosis. Furthermore, pharmacological inhibition or knockdown of protein kinase A, an important cAMP target, demonstrated a significant anti-apoptotic effect. Analysis of the underlying mechanisms revealed (i) the translocation of sAC to mitochondria under acidic stress and (ii) activation of the mitochondrial pathway of apoptosis, i.e. cytochrome c release and caspase-9 cleavage. sAC inhibition or knockdown abolished the activation of the mitochondrial pathway of apoptosis. Analysis of mitochondrial co-localization of Bcl-2 family proteins demonstrated sAC- and protein kinase A-dependent translocation of Bax to mitochondria. Taken together, these results suggest the important role of sAC in modulating the mitochondria-dependent pathway of apoptosis in endothelial cells.Increasing evidence suggests that apoptosis of endothelial cells (EC)³ may be responsible for acute and chronic vascular diseases, e.g. through atherogenesis (1), endothelial dysfunction (2), or thrombosis (3). Within several signaling mechanisms, a cAMP-dependent signaling pathway plays a substantial role in mediating apoptotic cell death induced by various stress factors. Elevation of the cellular cAMP either by forskolin-induced stimulation of the G-protein-responsive transmembrane adenylyl cyclase (tmAC) or by treatment with cAMP analogs has been shown to lead to both induction and suppression of apoptosis in different cell types (4–7). This discrepancy may be due to differences in cell types and experimental models. Alternatively, a lack of specificity of tmAC-induced signals, especially directed to distant intracellular targets like mitochondria, may be a cause of the discrepancy. Indeed, the classical model of cAMP signaling requires the diffusion of cAMP from plasma membrane-localized tmAC to targets localized throughout the cell. Diffusion of cAMP throughout the cytosol makes it difficult to selectively activate distally localized targets without also activating more proximal targets. Therefore, such diffusion of cAMP would likely diminish specificity, selectivity, and signal strength. This model is further complicated by the presence of phosphodiesterases, which degrade cAMP, thus preventing its diffusion.In addition to tmAC, a second source of cAMP, soluble adenylyl cyclase (sAC), was demonstrated for mammalian cells (8, 9). Cytosolic localization of sAC provides both specificity and selectivity by permitting generation of cAMP proximal to intracellular targets. Furthermore, this model for cAMP action incorporates phosphodiesterases, which would act to limit diffusion and prevent nonspecific effector activation.Whether sAC participates in apoptosis was unknown. A previous report demonstrated that sAC is co-localized with mitochondria (10). Because mitochondria play a fundamental role in apoptosis (11), we hypothesized that sAC may influence the development of apoptosis by modulating the mitochondrial pathway of apoptosis. Therefore, we aimed to examine the role of sAC in apoptotic cell death, especially its role in the modulation of the mitochondria-dependent pathway of apoptosis. For this purpose, apoptosis was induced in rat coronary EC by simulated in vitro ischemia or by acidosis. By applying pharmacological inhibition of sAC or small interfering RNA (siRNA)-mediated sAC knockdown, we found that sAC activity is required for the induction of apoptosis by ischemia or acidosis. Additionally, translocation of sAC to mitochondria and the sAC-dependent release of cytochrome c suggest that this cyclase specifically regulates the mitochondrial pathway of apoptosis.     

The Saanen goat as an animal model for post-laryngectomy research: practical implications

A modern way of voice rehabilitation after total laryngectomy includes the use of shunt valves and tracheostoma valves. Problems of fixation to the surrounding tissue are a major drawback in the use of the shunt valve, heat and moisture exchange (HME) filters and, especially, the tracheostoma valve. To solve these problems different tissue connectors were developed. The main objective was to test the feasibility of these prototypes in a new animal model. Here we discuss the results, problems and complications of the selected Saanen goat model. In this prospective laboratory study, 19 healthy adult female Saanen goats (Capra hircus) were used and observed post-surgically for 12 weeks. Selection criteria such as comparable anatomy to humans and easy handling were used for animal model development. Also a literature search using the Medline and the ISI Web of Science databases was performed. The anatomy of the Saanen goat was investigated in a separate postmortem study. Surgery consisted of a laryngotracheal separation and implantation of a tracheo-oesophageal and tracheostoma tissue connector with fibrin tissue glue. Postoperative care consisted of frequent stoma care, monitoring appetite, weight, vital signs and administration of antibiotics, analgesics and mucolytic agents. All animals survived the surgical procedure. However, postoperative care was extensive, labour intensive and was accompanied by several complications. Eleven animals died spontaneously before the end of the experiment. The tracheostoma tissue connector caused signs of local infection in all cases. There was no evidence of infection around the tracheo-oesophageal tissue connector in 18 cases. It was concluded that the use of goats in this tracheostoma model was associated with major complications and should, therefore, only be used for short-term experiments with intensive care. Additional research is needed to see if clinical application of the tissue connectors is possible in the future.