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Notes: Introduction: Ischemia-reperfusion (IR) injury is commonly associated with numerous pathologies including pressure sores, venous stasis ulcers, and lower extremity diabetic ulcers. The purpose of this study is to further investigate the relationship between age and ischemia-reperfusion skin injury in a rat model utilizing magnets for the purpose of injury creation.
Methods: Magnets were designed for subcutaneous placement and calibrated such that a second magnet placed externally over them would cause compression that exceeds capillary perfusion pressure (ischemia). Removing the external magnet results in reperfusion of the skin. After placing subcutaneous magnets in aged and young Fisher 344 rats, repeated cycles of external magnet placement and removal were performed. 
Results: Visual analysis of the skin revealed statistically significant greater areas of injury in the aged rats relative to their younger counterparts (37.4 ± 13.3% vs. 24.1 ± 14.8%, P 〈 .02)
Conclusions: Aged rats demonstrate an increased degree of injury relative to their younger counterparts in response to ischemia-reperfusion injury. Future studies will attempt to delineate differences in the markers of IR injury (such as myeloperoxidase and vitamin E levels) in aged versus young rats, giving insight to the mechanisms responsible for the impaired wound healing seen in the elderly.

Notes: Introduction: Telomeres are nucleoprotein structures at the ends of each chromosome. Due to the inability of DNA polymerase to replicate the full length of the chromosome, up to 50–200 base pairs of the telomere are lost during each successive round of cell division. In adult human somatic cells, telomerase is not active resulting in progressive loss of telomere length and entry into replicative senescence as observed in cell culture. hTERT is the catalytic subunit of telomerase, an enzyme which maintains telomere length. Transfection of human dermal fibroblasts (HDFs) by hTERT has been shown to reverse the senescent phenotype seen in aging HDFs in vitro. ERK (p44/42) is a MAP kinase which functions as a critical intermediary in the determination of cell growth and differentiation. Activation of ERK occurs through phosphorylation of threonine and tyrosine residues. 
Methods: In order to delineate some of the cellular mechanisms by which hTERT functions, we treated adenoviral hTERT (Ad-hTERT) transfected HDFs with TGFB1, and assayed phosphorylated ERK activity by Western blotting.
Results: Ad-hTERT treated HDFs demonstrated a 2–3 fold increase in phospho-ERK activity. In addition, our preliminary findings show that Ad-hTERT transfected HDFs have increased TGFB1, TGFB1-Receptor I and II, and COL1A1 gene expression by real-time rtPCR. 
Conclusions: Increased phosphor-ERK activity as well as increased TGFB1, TGFB1-Receptor I and II, and COL1A1 gene expression is seen in hTERT transfected HDF’s. Further studies will focus on defining other intermediary changes resulting from Ad-hTERT tranfection.Funding source: Geron Corporation

Notes: The scleractinian coral Oculina patagonica undergoes bleaching (loss of its endosymbiotic zooxanthellae) every summer in the eastern Mediterranean Sea when seawater temperatures rise. The causative agent of the disease is Vibrio shiloi. The pathogen adheres to a -galactoside-containing receptor in the coral mucus, penetrates into epithelial cells, differentiates into a viable-but-not-culturable form, multiplies, and produces a proline-rich peptide toxin that inhibits photosynthesis of the zooxanthellae in the presence of ammonia. Several of the virulence factors, such as adhesin, toxin, and superoxide dismutase, are produced only at the elevated summer seawater temperatures. The fireworm Hermodice carunculata is a winter reservoir and spring/summer vector for V. shiloi. The generality of the bacterial hypothesis of coral bleaching is discussed.

Notes: CD8 T cells are believed to play a key role in the immune control of human immunodeficiency virus-1 (HIV-1) infection in children as well as in adults. We have used an enhanced EliSpot (AmpliSpot) assay to quantitate CD8 T-cell responses directed to five human leucocyte antigen (HLA)-A2-presented HIV-1 epitopes derived from the key viral antigen Nef. Responses were assayed in one group of 21 children with vertically acquired HIV infection and one group of 19 adult subjects with chronic infection. The paediatric group displayed significantly weaker and more narrowly focused CD8 T-cell responses as compared with the adult subjects. Two epitopes stood out as the most frequently and strongly recognized, suggesting that they should be considered immunodominant in the CD8 T-cell response to HIV-1 Nef. Interestingly, the most frequently and strongly recognized epitope in both adults and children was previously identified in HLA-A2-transgenic mice, demonstrating the usefulness of such mice in finding natural viral epitopes. These findings indicate significant weakness in strength and breadth of the CD8 T-cell response to the target protein Nef in infected children and prompt renewed efforts into the immunology of vertically acquired HIV-1 infection.

Notes: Reactive nitrogen species are thought to be involved in both hypoxic-ischemic and cytokine-induced brain injury, including periventricular leukomalacia (PVL), the major pathological substrate of cerebral palsy in premature infants. PVL appears to be the result of perinatal inflammatory events and hypoxic-ischemic injury to the cerebral white matter. The chronic disturbance of myelination resulting from PVL suggests that developing oligodendrocytes (OLs) are involved in its pathogenesis. We hypothesized that nitric oxide (NO) could participate in the pathogenesis of PVL through a toxic effect on developing OLs. Using primary cultures of highly enriched OLs we found that NO is toxic to developing OLs (O4+, O1–, MBP–), with an EC50 value of 236 ± 125 µm of DETANOnoate. Peroxynitrite formation does not appear to be involved in NO toxicity in developing OLs, as determined by the failure of peroxynitrite scavengers as well as superoxide dismutase overexpression to prevent NO-induced toxicity. Similarly, several pathways involving PARP, excitotoxicity, guanylyl cyclase and caspase activation were not related to NO toxicity to developing OLs. NO toxicity to OLs resulted in ATP depletion and loss of mitochondrial membrane potential (ΔΨ) in developing OLs. Apoptosis-inducing factor (AIF) has been shown to be involved in caspase-independent cell death, and we found that AIF translocated from mitochondria into the nucleus upon NO exposure. In conclusion, we suggest that the vulnerability of developing OLs to NO involves mitochondrial dysfunction and translocation of AIF from mitochondria to nuclei.

Notes: We investigated the mechanism of 3-morpholinosyndnomine (SIN-1) neurotoxicity in nearly pure neuronal cultures. In a simple saline solution, SIN-1 neurotoxicity was found to be mediated by peroxynitrite and independent of glutamate receptor activation [Y. Zhang & P.A. Rosenberg (2002) Eur. J. Neurosci, 16, 1015–1024]. To further study the mechanism of peroxynitrite toxicity to neurons we investigated the role of caspases and poly (ADP-ribose) polymerase (PARP) in this model system. Ac-Tyr-Val-Ala-Asp-chloromethyl ketone (Ac-YVAD-cmk), a specific caspase-1 inhibitor, completely blocked neurotoxicity as well as ATP depletion induced by SIN-1. However, a caspase-3 inhibitor and a pan-caspase inhibitor were both without effect. These results suggested that the protection of Ac-YVAD-cmk might not be due to its inhibition of caspase-1. Indeed, Western blot analysis and assay of caspase activity indicated that caspase activation was not involved in SIN-1 toxicity. Ac-YVAD-cmk also completely blocked in vitro protein nitration induced by SIN-1 or peroxynitrite, suggesting that Ac-YVAD-cmk may interact with peroxynitrite directly. Similarly, although activation of PARP is thought to be a major cause of peroxynitrite-induced ATP depletion, and two PARP inhibitors, 1,5-dihydroxyisoquinoline (DHQ) and 3-aminobenzamide (3-AB), completely prevented ATP depletion and neurotoxicity induced by SIN-1, SIN-1 did not increase poly (ADP-ribosyl)ation and PARP activity. Furthermore, DHQ and 3-AB completely prevented in vitro protein nitration induced by peroxynitrite, indicating that DHQ and 3-AB directly interact with peroxynitrite. Taken together, these results suggest that in the model system used here peroxynitrite neurotoxicity is independent of caspase and PARP activation, and therefore implicate a novel mechanism.

Notes: Adenosine is an important regulator of neuronal excitability. Zaprinast is a cyclic nucleotide phosphodiesterase inhibitor, and has been shown in the hippocampal slice to suppress excitation. This action can be blocked by an adenosine receptor antagonist, and therefore is presumably due to adenosine release stimulated by exposure to zaprinast. To explore the mechanism of this phenomenon further, we examined the effect of zaprinast on adenosine release itself in cultured rat forebrain neurons. Zaprinast significantly stimulated extracellular adenosine accumulation. The effect of zaprinast on adenosine appeared to be mediated by increasing intracellular cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA): (i) zaprinast stimulated intracellular cAMP accumulation; (ii) a cAMP antagonist (Rp-8-Br-cAMP) significantly reduced the zaprinast effect on adenosine; (iii) an inhibitor of phosphodiesterase (PDE)1 (vinpocetine) and an activator of adenylate cyclase (forskolin) mimicked the effect of zaprinast on adenosine. We also found that zaprinast had no effect on adenosine in astrocyte cultures, and tetrodotoxin completely blocked zaprinast-evoked adenosine accumulation in neuronal cultures, suggesting that neuronal activity was likely to be involved. Consistent with a dependence on neuronal activity, NMDA receptor antagonists (MK-801 and D-APV) and removal of extracellular glutamate by glutamate–pyruvate transaminase blocked the effect of zaprinast. In addition, zaprinast was shown to stimulate glutamate release. Thus, our data suggest that zaprinast-evoked adenosine accumulation is likely to be mediated by stimulation of glutamate release by a cAMP- and PKA-dependent mechanism, most likely by inhibition of PDE1 in neurons. Furthermore, regulation of cAMP, either by inhibiting cAMP–PDE activity or by stimulating adenylate cyclase activity, may play an important role in modulating neuronal excitability. These data suggest the existence of a homeostatic negative feedback loop in which increases in neuronal activity are damped by release of adenosine following activation of glutamate receptors.

Notes: Oxidative injury to premyelinating oligodendrocytes (preOLs) in developing white matter has been implicated in the pathogenesis of periventricular leukomalacia, the lesion underlying most cases of cerebral palsy in premature infants. In this study, we investigated the pathways of OL death induced by intracellular glutathione (GSH) depletion. We found that the lipoxygenase (LOX) inhibitors AA-861 and BMD-122 (N-benzyl-N-hydroxy-5-phenylpentamide; BHPP), but not the cyclooxygenase (COX) inhibitor indomethacin, fully protected the cells from GSH depletion caused by cystine deprivation. Arachidonic acid (AA), the substrate for 12-LOX, potentiated the toxicity of mild cystine deprivation and at higher concentration was itself toxic. This toxicity was also blocked by 12-LOX inhibitors. Consistent with a role for 12-LOX in the cell death pathway, 12-LOX activity increased following cystine deprivation in OLs. Blocking 12-LOX with AA-861 effectively inhibited the accumulation of reactive oxygen species (ROS) induced by cystine deprivation. These data suggest that, in OLs, intracellular GSH depletion leads to activation of 12-LOX, ROS accumulation and cell death. Mature OLs were more resistant than preOLs to cystine deprivation. The difference in sensitivity was not due to a difference in 12-LOX activity but rather appeared to be related to the presence of stronger antioxidant defense mechanisms in mature OLs. These results suggest that 12-LOX activation plays a key role in oxidative stress-induced OL death.

Notes: Periventricular leukomalacia, the predominant pathological lesion underlying cerebral palsy in premature infants, is thought to be the result of hypoxic–ischemic injury to the cerebral white matter. The main cell type injured is the developing oligodendrocyte (OL), which has been shown to be more sensitive than mature OLs to both excitotoxic and oxidative mechanisms of injury. A maturation dependence of OL vulnerability to cystine deprivation-induced glutathione depletion has been previously demonstrated in culture. We hypothesized that mitochondria could be involved in this toxicity by generating superoxide and that increased superoxide dismutase (SOD) activity in mature OLs may account for their greater resistance. Cystine deprivation toxicity was found to be associated with mitochondrial dysfunction and intracellular superoxide accumulation in developing OLs. CuZnSOD protein expression and enzyme activity was similar along the OL lineage. In contrast, MnSOD was up-regulated in mature OLs, as manifested by a 53% increase in its expression and a four-fold increase in its activity. Overexpressing MnSOD in developing OLs was associated with a protective effect on mitochondrial membrane potential and a decrease in cell death induced by mild cystine deprivation. The greater challenge presented by total cystine deprivation was resistant to MnSOD overexpression and appeared to be related to hydrogen peroxide toxicity. These data suggest a primary involvement of superoxide in glutathione depletion toxicity in developing OLs, and suggest an important role for MnSOD in the resistance observed in mature OLs.

Notes: Background Allergens are common non-infectious antigens to which people will mount T cell dependent humoral responses. Among genetically susceptible individuals, an antigen-specific response results involving the production of allergen-specific IgE (atopy).Objective Determine if this susceptibility is manifested as an inherited, allergen-specific trait or a random response to allergens among susceptible people.Methods We evaluated allergen-specific outcomes in 1099 members of families with positive atopic history (26 multi-generation and 112 nuclear families). Each was tested for sensitivity to 14 common allergens by standardized skin prick test (SPT), a marker of specific IgE production. Over 15 000 individual SPT's were evaluated. Among five randomly selected multi-generation families (N=163), semi-quantitative determinations of Amb a 1-specific IgA1,2 and IgG1–4 were determined in three groups: (A) Amb a SPT+/Amb a 1-IgE+, (B) Amb a SPT−/Amb a 1-IgE+, (C) Amb a SPT−/Amb a 1-IgE−.Results By rank correlation statistics, there were no discernible ‘patterns’ of specific SPT outcomes among any of the multi-generation families, suggesting that environmental exposure rather than allergen-specific inheritance determined the responses. This was confirmed among the nuclear families since the conditional SPT outcomes among children were independent of the SPT responses of their parents. Among five randomly selected multi-generation families, the relative proportionate concentrations of the Amb a 1-specific IgA and IgG subclasses were comparable, regardless of atopic sensitization to the ragweed allergen Amb a.Conclusion While the general propensity for atopy may be inherited, an individual's specific atopic outcome is a random variable independent of familial sensitization patterns.