Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice.

Senescent cells (SCs) accumulate with age and after genotoxic stress, such as total-body irradiation (TBI). Clearance of SCs in a progeroid mouse model using a transgenic approach delays several age-associated disorders, suggesting that SCs play a causative role in certain age-related pathologies. Thus, a 'senolytic' pharmacological agent that can selectively kill SCs holds promise for rejuvenating tissue stem cells and extending health span. To test this idea, we screened a collection of compounds and identified ABT263 (a specific inhibitor of the anti-apoptotic proteins BCL-2 and BCL-xL) as a potent senolytic drug. We show that ABT263 selectively kills SCs in culture in a cell type- and species-independent manner by inducing apoptosis. Oral administration of ABT263 to either sublethally irradiated or normally aged mice effectively depleted SCs, including senescent bone marrow hematopoietic stem cells (HSCs) and senescent muscle stem cells (MuSCs). Notably, this depletion mitigated TBI-induced premature aging of the hematopoietic system and rejuvenated the aged HSCs and MuSCs in normally aged mice. Our results demonstrate that selective clearance of SCs by a pharmacological agent is beneficial in part through its rejuvenation of aged tissue stem cells. Thus, senolytic drugs may represent a new class of radiation mitigators and anti-aging agents.

Immune Function and Muscle Adaptations to Resistance exercise in Older Adults: Study Protocol for a Randomized Controlled Trial of a Nutritional Supplement.

BACKGROUND: Immune function may influence the ability of older adults to maintain or improve muscle mass, strength, and function during aging. Thus, nutritional supplementation that supports the immune system could complement resistance exercise as an intervention for age-associated muscle loss. The current study will determine the relationship between immune function and exercise training outcomes for older adults who consume a nutritional supplement or placebo during resistance training and post-training follow-up. The supplement was chosen due to evidence suggesting its ingredients [arginine (Arg), glutamine (Gln), and ß-hydroxy ß-methylbutyrate (HMB)] can improve immune function, promote muscle growth, and counteract muscle loss. METHODS/DESIGN: Veterans (age 60 to 80 yrs, N = 50) of the United States military will participate in a randomized double-blind placebo-controlled trial of consumption of a nutritional supplement or placebo during completion of three study objectives: 1) determine if 2 weeks of supplementation improve immune function measured as the response to vaccination and systemic and cellular responses to acute resistance exercise; 2) determine if supplementation during 36 sessions of resistance training boosts gains in muscle size, strength, and function; and 3) determine if continued supplementation for 26 weeks post-training promotes retention of training-induced gains in muscle size, strength, and function. Analyses of the results for these objectives will determine the relationship between immune function and the training outcomes. Participants will undergo nine blood draws and five muscle (vastus lateralis) biopsies so that the effects of the supplement on immune function and the systemic and cellular responses to exercise can be measured. DISCUSSION: Exercise has known effects on immune function. However, the study will attempt to modulate immune function using a nutritional supplement and determine the effects on training outcomes. The study will also examine post-training benefit retention, an important issue for older adults, usually omitted from exercise studies. The study will potentially advance our understanding of the mechanisms of muscle gain and loss in older adults, but more importantly, a nutritional intervention will be evaluated as a complement to exercise for supporting muscle health during aging. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT02261961, registration date 10 June 2014, recruitment active.

Redox-regulated pathway of tyrosine phosphorylation underlies NF-κB induction by an atypical pathway independent of the 26S proteasome.

Alternative redox stimuli such as pervanadate or hypoxia/reoxygenation, induce transcription factor NF-κB by phospho-tyrosine-dependent and proteasome-independent mechanisms. While considerable attention has been paid to the absence of proteasomal regulation of tyrosine phosphorylated IκBα, there is a paucity of information regarding proteasomal regulation of signaling events distinct from tyrosine phosphorylation of IκBα. To delineate roles for the ubiquitin-proteasome pathway in the phospho-tyrosine dependent mechanism of NF-κB induction, we employed the proteasome inhibitor, Aclacinomycin, and the phosphotyrosine phosphatase inhibitor, pervanadate (PV). Results from these studies demonstrate that phospho-IκBα (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported. Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-κB. Consistent with previous reports, a significant redox imbalance leading to the activation of tyrosine kinases, as occurs with pervanadate, is required for the induction of NF-κB. Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated.

Compensatory increase in USP14 activity accompanies impaired proteasomal proteolysis during aging.

The deubiquitinating enzyme, USP14, found in association with the proteasome is essential in mediating ubiquitin trimming and in ensuring ubiquitin-homeostasis. As aging is accompanied by a significant decline in proteasomal proteolysis in primary human T lymphocytes, we evaluated the contributory role of USP14 in this decline. Our studies for the first time demonstrate that enzymatic activity of proteasome-associated USP14 is significantly higher in T cells obtained from elderly donors. Additionally, such an increase in USP14 activity could be mimicked by chemically inhibiting the proteasome, using lactacystin. Thus, USP14 activity appears to be reciprocally regulated by the catalytic function of the 26S proteasome. To determine whether the inhibition of USP14 activity counter regulates proteasomal proteolysis, T cells pretreated with a small molecule inhibitor of USP14, IU1, were activated and assessed for IκBα degradation as a measure of proteasomal proteolysis. While T cells obtained from young donors demonstrated increased degradation of IκBα, those from the elderly remained unaffected by IU1 pretreatment. Taken together, these results demonstrate that the decrease in proteolysis of proteasomal substrates during aging is independent of the increased USP14 activity and that the reciprocal regulation of USP14 and proteasomal catalytic activity may be necessary to maintain cellular ubiquitin homeostasis.

Altered regulation of CXCR4 expression during aging contributes to increased CXCL12-dependent chemotactic migration of CD4(+) T cells.

Chemokine-dependent migration of T lymphocytes assures recirculation of naïve T cells to secondary lymphoid organs and tissue-specific trafficking of memory-effector T cells. Previous studies carried out in rodents have demonstrated age-associated modulation of the expression of chemokine receptors such as CXCR4 and CCR5; however, little is known about the molecular mechanisms that regulate receptor expression and turnover in T cells, during advancing age in humans. Our recent results demonstrating increased chemotactic migration in response to CXCL12 in CD4(+) T cells obtained from the elderly, as compared to those from young donors, led us to hypothesize that increase in surface expression, because of altered endocytic regulation of CXCR4 on T cells during aging, might be directly responsible for increased migration toward CXCL12. Studies presented here demonstrate a significant increase in the surface expression of CXCR4 in CD4(+) T cells from elderly human donors, relative to those from the young. Additionally, CXCL12-mediated endocytosis of CXCR4 was differentially regulated during aging, which could be attributed to alterations in the ubiquitination of CXCR4. Thus, altered ubiquitination of CXCR4 may contribute to the increased surface expression and enhanced T-cell migration to chemotactic stimuli in the elderly.

Impairment of non-muscle myosin IIA in human CD4+ T cells contributes to functional deficits in the elderly.

Physiological aging imposes significant alterations in the repertoire of T cells and all associated functions. Although several studies have reported defects upon antigen-induced activation of T cells during aging, the molecular mechanisms that control T-cell receptor (TCR) downmodulation remain to be fully defined. While previous studies have assessed the role of F-actin in regulating activation-induced TCR internalization, few have delineated the roles of motor proteins, such as non-muscle myosin IIA (NMMIIA). In this study, we describe a series of experiments supporting the hypothesis that effective TCR downmodulation requires not only efficient reorganization of the actin cytoskeleton, but also functional NMMIIA. For the first time, we show that CD4(+) T cells from elderly human donors have dysfunctional NMMIIA that contributes to delaying activation-induced TCR internalization and impairing calcium mobilization. Additionally, our results demonstrate that chemical inhibition of NMMIIA in CD4(+) T cells from young donors also results in complete abrogation of TCR internalization, strongly supporting the fundamental role of NMMIIA in modulating this event. Recent observations that the generation of an efficient T-cell response requires migration prompted us to investigate whether NMMIIA also plays a regulatory role in CD4(+) T-cell migration. We show that chemical inhibition of NMMIIA downmodulates chemotactic migration in CD4(+) T cells from both young and elderly donors. Together, these data demonstrate a significant contribution of dysfunctional NMMIIA to TCR-mediated functional defects during aging.

Aging and immune function: molecular mechanisms to interventions.

Abstract The immune system of an organism is an essential component of the defense mechanism aimed at combating pathogenic stress. Age-associated immune dysfunction, also dubbed "immune senescence," manifests as increased susceptibility to infections, increased onset and progression of autoimmune diseases, and onset of neoplasia. Over the years, extensive research has generated consensus in terms of the phenotypic and functional defects within the immune system in various organisms, including humans. Indeed, age-associated alterations such as thymic involution, T cell repertoire skewing, decreased ability to activate naïve T cells and to generate robust memory responses, have been shown to have a causative role in immune decline. Further, understanding the molecular mechanisms underlying the generation of proteotoxic stress, DNA damage response, modulation of ubiquitin proteasome pathway, and regulation of transcription factor NFκB activation, in immune decline, have paved the way to delineating signaling pathways that cross-talk and impact immune senescence. Given the role of the immune system in combating infections, its effectiveness with age may well be a marker of health and a predictor of longevity. It is therefore believed that a better understanding of the mechanisms underlying immune senescence will lead to an effective interventional strategy aimed at improving the health span of individuals. Antioxid. Redox Signal. 14, 1551-1585.

Proteasome inhibition up-regulates inflammatory gene transcription induced by an atypical pathway of NF-kappaB activation.

Proteasome inhibition has become synonymous with inhibition of NF-kappaB activity. However, hyperactive NF-kappaB responses often accompany physiological conditions marked by proteasomal defects, i.e. advancing age, geriatric diseases, and bortezomib resistance. These paradoxical NF-kappaB responses are likely to be impervious to proteasomal defects because they stem from atypical NF-kappaB signaling induced by upstream mechanisms which are proteasome-independent. While this atypical pathway does not require proteasome for NF-kappaB nuclear translocation, a role for proteasome in regulating nuclear NF-kappaB remains unexplored. We now demonstrate that proteasome stringently controls transcription of inflammatory mediators regulated by this atypical NF-kappaB pathway. Proteolytic activity of the proteasome mediates the removal of the NF-kappaB subunit, p65/RelA, from inflammatory genes, thereby terminating atypical NF-kappaB-dependent transcriptional responses. For the first time, we demonstrate that both 19S and 20S components of the 26S proteasome complex are recruited to an inflammatory gene promoter; additionally, the 19S and 20S complexes appear to play distinct roles in the negative regulation of NF-kappaB-dependent transcription. By demonstrating that proteasome regulates the termination of atypical NF-kappaB-dependent transcriptional responses, these studies clearly indicate a novel, regulatory role for proteasome in atypical NF-kappaB signaling. Moreover, these results signal a potential interplay between lowered proteasomal function and increased inflammation and may explain why inflammation accompanies physiological conditions under which proteasomal function is compromised, such as during advancing age or following bortezomib treatment. Given this role for proteasome in inflammation resolution, restoration of proteasome function may constitute a novel mechanism for intervening in chronic inflammatory diseases.

Killing of Bacillus spores is mediated by nitric oxide and nitric oxide synthase during glycoconjugate-enhanced phagocytosis.

Nitric oxide (NO) is a signaling and defense molecule of major importance. NO endows macrophages with bactericidal, cytostatic as well as cytotoxic activity against various pathogens. Bacillus spores can produce serious diseases, which might be attenuated if macrophages were able to kill the spores on contact. Present research was carried out to study whether glycoconjugates stimulated NO and nitric oxide synthase (NOS2) production during phagocytosis killing of Bacillus spores. Murine macrophages exposed to glycoconjugate-treated spores induced NOS2 and NO production that was correlated with high viability of macrophages and killing rate of bacterial spores. Increased levels of inducible NOS2 and NO production by macrophages in presence of glycoconjugates suggested that the latter provide an activation signal directed to macrophages. Glycoconjugates were shown to exert a protective influence, sparing macrophages from spore-induced cell death. In presence of glycoconjugates, macrophages efficiently kill the organisms. Without glycoconjugate activation, murine macrophages were ineffective at killing Bacillus spores. These results suggest that glycoconjugates promote killing of Bacillus spores by blocking spore-induced macrophage cell death, while increasing their activation level and NO and NOS2 production. Glycoconjugates suggest novel antimicrobial approaches to prevention and treatment of infection caused by bacterial spores.

Catalytic activity of the proteasome fine-tunes Brg1-mediated chromatin remodeling to regulate the expression of inflammatory genes.

The induction of key pro-inflammatory genes is regulated by the SWI/SNF class of ATP-dependent remodeling complexes. In particular, the catalytic ATPase subunit, Brg1, is distinctly involved in the chromatin remodeling required for activating pro-inflammatory genes in a temporally, ordered fashion. Despite advances in our understanding of the role for Brg1 in the kinetics of inflammatory responses, little is known about the precise mechanisms which down-regulate Brg1 activity. Biochemical studies implicate a role for the proteasome in the regulation of SWI/SNF assembly and function; however, it is unclear if proteasome-dependent mechanisms modulate its remodeling activity or recruitment to chromatin in order to regulate inflammatory gene transcription. We now demonstrate for the first time that proteasome function represents an important mechanism for limiting inducible association of Brg1 with promoters of SWI/SNF-regulated, inflammatory genes. As a result, catalytic activity of the proteasome fine-tunes the kinetics of inflammatory gene transcription by inhibiting both premature and persistent chromatin remodeling at SWI/SNF-regulated genes. These results provide mechanistic insight into the interplay between nucleosome remodeling, inflammation and proteasome, and underscore the critical role of the proteasome in controlling both extent and duration of inflammatory responses.

Ca2+ homeostasis regulates Xenopus oocyte maturation.

In contrast to the well-defined role of Ca2+ signals during mitosis, the contribution of Ca2+ signaling to meiosis progression is controversial, despite several decades of investigating the role of Ca2+ and its effectors in vertebrate oocyte maturation. We have previously shown that during Xenopus oocyte maturation, Ca2+ signals are dispensable for entry into meiosis and for germinal vesicle breakdown. However, normal Ca2+ homeostasis is essential for completion of meiosis I and extrusion of the first polar body. In this study, we test the contribution of several downstream effectors in mediating the Ca2+ effects during oocyte maturation. We show that calmodulin and calcium-calmodulin-dependent protein kinase II (CAMK2) are not critical downstream Ca2+ effectors during meiotic maturation. In contrast, accumulation of Aurora kinase A (AURKA) protein is disrupted in cells deprived of Ca2+ signals. Since AURKA is required for bipolar spindle formation, failure to accumulate AURKA may contribute to the defective spindle phenotype following Ca2+ deprivation. These findings argue that Ca2+ homeostasis is important in establishing the oocyte's competence to undergo maturation in preparation for fertilization and embryonic development.

Lower expression of catalytic and structural subunits of the proteasome contributes to decreased proteolysis in peripheral blood T lymphocytes during aging.

Aging, in the immune system, is characterized by a decreased ability to respond to exogenous insults, resulting in increased susceptibility to infections and blunted response to vaccination. While significant age-associated deficits in immune function have been documented, the underlying molecular mechanisms are still being investigated. A consistent decline in the proteolytic activity of the proteasome has been demonstrated with advancing age, implicating an important role for the proteasome in immune senescence, by studies that largely employed proteasome-enriched preparations from cell lysates. With the availability of novel cell permeable active site probes designed specifically for assaying proteasomal activity in live cells, we now confirm our earlier data demonstrating lower catalytic activity of the proteasome in primary human T cells obtained from the elderly when compared to those from young donors. Loss in proteasomal catalytic activity translated into a loss in functional activity, as was observed in a degradation assay employing an ubiquitinated protein substrate, Ub-IkappaBalpha. Unlike fluorogenic peptide substrates, use of ubiquitinated protein substrates not only confer greater stringency in terms of proteasomal hydrolysis, but also involve the participation of the 19S regulatory component. This age-associated loss in proteasomal activity is accompanied by alteration in the levels of catalytic, structural and regulatory subunits, with no change in that of the 11S activator or the inhibitor PAAF1. Oxidative modification, such as carbonylation and lipid-peroxidation, of proteasomal subunits was also detected in T cells from the elderly. Thus, oxidative modification and lower levels of proteasomal subunits contribute to decreased proteolytic activity during immune-senescence.

Redox regulation of the proteasome in T lymphocytes during aging.

Proteasome is a major cellular organelle responsible for the regulated turnover of both normal and misfolded proteins. Recent reports from our laboratory have implicated lowered proteasomal chymotryptic activity to be responsible for decreased induction of the transcription factor NFkappaB in T lymphocytes during aging. In this study, we have further analyzed the basis for this decline in proteasomal function, by focusing on the role of oxidative stress. On exposure to the prooxidant BSO, both ATP-stimulatable 26S and ATP-independent 20S proteasomal catalytic activity could be down-regulated in T cells from young donors, mimicking the decline observed in T cells from the elderly. Loss in these catalytic activities, following exposure to prooxidant stimulus, also resulted in a decline in both activation-induced proliferation and degradation of the inhibitor IkappaBalpha, with concomitant increase in the accumulation of carbonylated proteins, mimicking responses seen in T cells from the elderly. Pretreatment with an antioxidant, NAC, could override prooxidant-mediated, but not age-associated, decrease in both 20S and 26S proteasomal activities. These results suggest that the decrease in proteasomal activities observed during aging may be secondary to oxidative stress and underlie immune senescence.

Constitutive degradation of IkappaBalpha in human T lymphocytes is mediated by calpain.

BACKGROUND: Activation-induced induction of transcription factor NFkappaB in T lymphocytes is regulated by its inhibitor IkappaBalpha. NFkappaB activation has been demonstrated to occur either by phosphorylation on serine residues 32 and 36 of the inhibitor, IkappaBalpha, followed by ubiquitination and degradation of the inhibitor by the 26S proteasome, or by a proteasome-independent mechanism involving tyrosine phosphorylation, but not degradation. However, the mechanism underlying constitutive regulation of the levels of the inhibitor, IkappaB, in primary human T lymphocytes, remains to be fully delineated. RESULTS: We demonstrate here, the involvement of a proteasome-independent pathway for constitutive regulation of IkappaBalpha levels in primary human T lymphocytes. Pretreatment with a cell permeable calpain inhibitor, E64D, but not with a proteasome specific inhibitor, lactacystin, blocks stimulus-independent IkappaBalpha degradation in primary human T cells. However, E64D pre-treatment fails to impact on IkappaBalpha levels following stimulation with either TNFalpha or pervanadate. Other isoforms of the inhibitor, IkappaBbeta, and IkappaBgamma, appear not to be subject to a similar ligand-independent regulation. Unlike the previously reported decline in ligand-induced degradation of IkappaBalpha in T cells from the elderly, constitutive degradation does not exhibit an age-associated decline, demonstrating proteasome-independent regulation of the activity. CONCLUSION: Our studies support a role for an E64D sensitive protease in regulating constitutive levels of IkappaBalpha in T cells, independent of the involvement of the 26S proteasome, and suggests a biological role for constitutive degradation of IkappaBalpha in T cells.

Soy immunotherapy for peanut-allergic mice: modulation of the peanut-allergic response.

BACKGROUND: Allergen-specific immunotherapy (IT) is an effective therapeutic modality to prevent further anaphylactic episodes in patients with insect sting hypersensitivity and is being investigated for peanut allergy. So far, peanut-specific IT has been unsuccessful because of the side effects of therapy. Soybean seed storage proteins share significant homology with the respective peanut allergens. OBJECTIVE: This study was undertaken in mice to investigate whether specific doses of soybean would desensitize peanut-allergic mice. METHODS: C3H/HeJ mice were sensitized to peanut with 3 intraperitoneal (IP) injections of crude peanut extract. The mice were desensitized by IP injections with either crude peanut or soybean extract for 4 weeks, 3 times a week. Controls included placebo desensitization with PBS and naive mice. After 2 weeks of rest, mice were challenged IP with crude peanut extract. Thirty minutes later, symptom scores and body temperatures were recorded. Serum immunoglobulins, peanut-induced splenocyte proliferation, and secreted cytokines were measured before and after desensitization. RESULTS: The clinical symptoms in the soybean- and peanut-desensitized animals were markedly reduced compared with the placebo-treated mice. Specific IgG1 levels to crude peanut were significantly lower in the soy IT group than in the peanut IT group. The cellular response to crude peanut was also downregulated in the soy IT group, as shown by decreased peanut-specific stimulation indices and a cytokine profile skewed toward a T H 1 response. CONCLUSIONS: Soy IT can be used to desensitize/downregulate peanut-specific response in peanut-allergic mice and could provide a new therapeutic intervention for peanut allergy.

Inhaled isobutyl nitrite inhibited macrophage inducible nitric oxide by blocking NFkappaB signaling and promoting degradation of inducible nitric oxide synthase-2.

We previously reported that inhaled isobutyl nitrite inhibited macrophage tumoricidal activity by inhibiting inducible nitric oxide (NO) production. In the present study, a much shorter inhalant exposure regimen (five daily exposures) inhibited inducible NO and the NO synthase (NOS2). One of the ways in which NO and NOS2 are regulated is by ubiquitin-dependent NOS2 degradation. Immunoprecipitated NOS2 showed increased poly-ubiquitination, following exposure to the inhalant. In addition, Western blots of macrophage nuclear extracts for the NFkappaB subunit, p65, showed that exposure to the inhalant inhibited NFkappaB signaling, necessary for induction of NOS2. The inhalant blocked phosphorylation of the NFkappaB inhibitor, IkappaBalpha. The inhibition of NFkappaB signaling following inhalant exposure was confirmed using mice transgenic for the kappaB-dependent promoter of the HIV 5' LTR linked to luciferase. The data suggested that inhalant exposure likely inhibited macrophage NO production by blocking NFkappaB-mediated activation signaling and promoting poly ubiquitination of NOS2.

Tyrosine phosphorylation-dependent activation of NFkappaB is compromised in T cells from the elderly.

NFkappaB induction and gene regulation are compromised in T lymphocytes during aging. This has been attributed to altered proteasomal function resulting in decreased ubiquitin-mediated degradation of IkappaBalpha. However, little is known about the impact of aging on the mechanisms that lead to the release of active NFkappaB employing pro-oxidant pathways. Oxidant-mediated activation of NFkappaB has been previously shown to involve proteasome independent mechanisms and hence may be an important alternate conduit to the induction of this central transcription factor in aging. Employing H(2)O(2) and pervanadate we not only demonstrate lowered tyrosine phosphorylation of IkappaBalpha, but also compromised induction of nuclear NFkappaB in T cells from the elderly. Lowered tyrosine phosphorylation of IkappaBalpha may be due to a decrease in activity of p56(lck) and ZAP-70, since treatment with piceatannol, an inhibitor of syk and src family kinases, mimics age associated decline in tyrosine phosphorylation of IkappaBalpha in T cells from young donors. Thus, alternate pathways of NFkappaB induction are also impaired in T cells from the elderly and may underlie immune-deficit accompanying aging.

Statins increase thrombomodulin expression and function in human endothelial cells by a nitric oxide-dependent mechanism and counteract tumor necrosis factor alpha-induced thrombomodulin downregulation.

Expression of functionally active thrombomodulin (TM) on the luminal surface of endothelial cells is critical for vascular thromboresistance. TM maintains thrombohemorrhagic homeostasis by forming a complex with thrombin, which subsequently loses its procoagulant properties and instead activates protein C. Acquired deficiency of endothelial TM is of particular pathophysiological significance in sepsis and related disorders. We show here that two different 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), atorvastatin and simvastatin, strongly increase the expression and functional activity of TM in human umbilical vein endothelial cells, human coronary artery endothelial cells, and EA.hy926 endothelial cells. The increase in endothelial TM conferred by statin was prevented by the addition of mevalonic acid, geranylgeranyl-pyrophosphate, and nitric oxide scavenger, and was mimicked by the addition of a specific inhibitor of geranylgeranyl transferase, as well as by nitric oxide donors. Moreover, statin counteracted tumor necrosis factor alpha-induced downregulation of endothelial cell TM. The increase in endothelial cell TM activity in response to statin constitutes a novel pleiotropic (non-lipid-related) effect of these commonly used compounds, and may be of clinical significance in disorders where deficient endothelial TM and protein C activation play a pathophysiological role.

Cytotoxicity by nitrite inhalants is not related to peroxynitrite formation.

Nitrite inhalant abuse has been correlated with HIV and Kaposi's sarcoma. Mouse models of inhalant exposure show immunosuppression and loss of immune cells. In the present study, isobutyl nitrite caused a dose-dependent loss of viability of a macrophage cell line. In the absence of cells, isobutyl nitrite reacted with hydrogen peroxide to form peroxynitrite. However, assays of mitochondrial respiration and nitration that detect peroxynitrite indicated that very little was present in cell cultures following exposure to the inhalants. Isobutyl, isoamyl, and butyl nitrites inhibited mitochondrial respiration, but only at high concentrations. Similarly, the nitrating activity of isobutyl nitrite occurred only at high concentrations and was not affected by the presence of hydrogen peroxide. Western blots showed that the inhalant did not increase nitrotyrosine formation in RAW cells or in peritoneal exudate macrophages (PEM) from exposed mice. Thus, the toxicity induced by isobutyl nitrite was probably not due to peroxynitrite formation.

Ubiquitin-proteasome pathway is compromised in CD45RO+ and CD45RA+ T lymphocyte subsets during aging.

Recent reports from our laboratory have demonstrated that CD45RO+ and CD45RA+ T lymphocytes from the elderly are compromised in their response to activation-induced IL-2 receptor expression, IkappaB-alpha degradation, as well as nuclear translocation of NFkB. To understand the basis of this activation-induced dysfunction in the elderly, we have examined the role of the ubiquitin-proteasome pathway. Our results demonstrate that both CD45RO+ and CD45RA+ T lymphocytes from the elderly show significant reduction in the constitutive 26S proteasome-associated chymotryptic activity, when compared to those in the young. Additionally, anti-CD3-CD28 treatment induced enhancement of proteasome-associated enzymatic activity in cells from the young, but not in cells from the elderly. Lowered proteasome-associated activity and its effect on reduced immune responses in the elderly could be mimicked by experiments which involved pretreatment of T cells from young donors with a proteasome specific inhibitor, lactacystin. These data demonstrate that IL-2 receptor induction is clearly compromised in T cells from the young when proteasomes are inhibited by pretreatment with lactacystin. An examination of ubiquitin specific hydrolase activity, demonstrated a decrease in activated CD45RA+ and CD45RO+ T cell subsets from the elderly when compared to young. These results suggest that lowered proteasome-associated enzymatic activity in combination with compromised de-ubiquitinating activity may be responsible for lowered activation-induced NFkB and NFkB-mediated gene expression in elderly subjects.