Long-duration freewheel running and submandibular lymphocyte response to forced exercise in older mice

Submandibular lymph nodes (SLN) are crucial for immune surveillance of the anterior ocular chamber and upper respiratory tract; little is known about how training and exercise affect SLN lymphocytes. The intent of this study was to describe the impact of long term freewheel running followed by acute strenuous exercise on SLN lymphocytes in mice. Female C57BL/6 mice were assigned to running wheels or remained sedentary for 8 months, and further randomized to treadmill exercise and sacrifice immediately, treadmill exercise and sacrifice 24 h after exercise cessation, or no treadmill exposure. SLN lymphocytes were isolated and analyzed for CD3, CD4, CD8, and CD19 cell surface markers, phosphatidylserine externalization as a marker of apoptosis, and intracellular glutathione as a marker of oxidative stress. Compared with running wheel mice, older sedentary mice had a lower percent of T cells and higher percent of B cells (p < 0.05). Although intracellular glutathione did not differ between groups, running mice had a lower percent of Annexin V(+) SLN lymphocytes 24 h after treadmill exercise. Further research will be needed to determine if voluntary exercise translates into improved anterior ocular and upper respiratory tract health.     

Hypoxic exercise training causes erythrocyte senescence and rheological dysfunction by depressed Gardos channel activity

Despite enhancing cardiopulmonary and muscular fitness, the effect of hypoxic exercise training (HE) on hemorheological regulation remains unclear. This study investigates how HE modulates erythrocyte rheological properties and further explores the underlying mechanisms in the hemorheological alterations. Twenty-four sedentary males were randomly divided into hypoxic (HE; n = 12) and normoxic (NE; n = 12) exercise training groups. The subjects were trained on 60% of maximum work rate under 15% (HE) or 21% (NE) O(2) condition for 30 min daily, 5 days weekly for 5 wk. The results demonstrated that HE 1) downregulated CD47 and CD147 expressions on erythrocytes, 2) decreased actin and spectrin contents in erythrocytes, 3) reduced erythrocyte deformability under shear flow, and 4) diminished erythrocyte volume changed by hypotonic stress. Treatment of erythrocytes with H(2)O(2) that mimicked in vivo prooxidative status resulted in the cell shrinkage, rigidity, and phosphatidylserine exposure, whereas HE enhanced the eryptotic responses to H(2)O(2). However, HE decreased the degrees of clotrimazole to blunt ionomycin-induced shrinkage, rigidity, and cytoskeleton breakdown of erythrocytes, referred to as Gardos effects. Reduced erythrocyte deformability by H(2)O(2) was inversely related to the erythrocyte Gardos effect on the rheological function. Conversely, NE intervention did not significantly change resting and exercise erythrocyte rheological properties. Therefore, we conclude that HE rather than NE reduces erythrocyte deformability and volume regulation, accompanied by an increase in the eryptotic response to oxidative stress. Simultaneously, this intervention depresses Gardos channel-modulated erythrocyte rheological functions. Results of this study provide further insight into erythrocyte senescence induced by HE.     

Exercise training alleviates MCT1 and MCT4 reductions in heart and skeletal muscles of STZ-induced diabetic rats.

We compared the changes in monocarboxylate transporter 1 (MCT1) and 4 (MCT4) proteins in heart and skeletal muscles in sedentary control and streptozotocin (STZ)-induced diabetic rats (3 wk) and in trained (3 wk) control and STZ-induced diabetic animals. In nondiabetic animals, training increased MCT1 in the plantaris (+51%; P < 0.01) but not in the soleus (+9%) or the heart (+14%). MCT4 was increased in the plantaris (+48%; P < 0.01) but not in the soleus muscles of trained nondiabetic animals. In sedentary diabetic animals, MCT1 was reduced in the heart (-30%), and in the plantaris (-31%; P < 0.01) and soleus (-26%) muscles. MCT4 content was also reduced in sedentary diabetic animals in the plantaris (-52%; P < 0.01) and soleus (-25%) muscles. In contrast, in trained diabetic animals, MCT1 and MCT4 in heart and/or muscle were similar to those of sedentary, nondiabetic animals (P > 0.05) but were markedly greater than in the sedentary diabetic animals [MCT1: plantaris +63%, soleus +51%, heart +51% (P > 0.05); MCT4: plantaris +107%, soleus +17% (P > 0.05)]. These studies have shown that 1) with STZ-induced diabetes, MCT1 and MCT4 are reduced in skeletal muscle and/or the heart and 2) exercise training alleviated these diabetes-induced reductions.     

Hyperpalatable Diet and Physical Exercise Modulate the Expression of the Glial Monocarboxylate Transporters MCT1 and 4

Hyperpalatable diets (HP) impair brain metabolism, and regular physical exercise has an apparent opposite effect. Here, we combined a prior long-term exposure to HP diet followed by physical exercise and evaluated the impact on some neuroenergetic components and on cognitive performance. We assessed the extracellular lactate concentration, expression of monocarboxylate transporters (MCTs), pyruvate dehydrogenase (PDH), and mitochondrial function in the hippocampus. Male C57BL/6J mice were fed 4 months with HP or a control diet. Subsequently, they were divided in the following groups: control diet sedentary (CDS), control diet exercise (CDE), HP diet sedentary (HPS), and HP diet exercise (HPE) (n = 15 per group) and were engaged for an additional 30-day period of voluntary exercise and HP diet. Relative to the control situation, exercise increased MCT1, MCT4, and PDH protein levels, while the HP diet increased MCT1 and MCT4 protein levels. The production of hydrogen peroxide (H₂O₂) and the mitochondrial membrane potential (??_m) stimulated by succinate in hippocampal homogenates were not significantly different between groups. ADP phosphorylation and the maximal respiratory rate induced by FCCP showed similar responses between groups, implying a normal mitochondrial function. Also, extracellular brain lactate levels were increased in the HPE group compared to other groups soon after performing the Y-maze task. However, such enhanced lactate levels were not associated with improved memory performance. In summary, hippocampal protein expression levels of MCT1 and 4 were increased by physical exercise and HP diet, whereas PDH was only increased by exercise. These observations indicate that a hippocampal metabolic reprogramming takes place in response to these environmental factors.     

T-Cell Autophagy Deficiency Increases Mortality and Suppresses Immune Responses after Sepsis

Background

Although the role of autophagy in sepsis has been characterized in several organs, its role in the adaptive immune system remains to be ascertained. This study aimed to investigate the role of autophagy in sepsis-induced T cell apoptosis and immunosuppression, using knockout mice with T cell specific deletion of autophagy essential gene Atg7.

Methods and Results

Sepsis was induced in a cecal ligation and puncture (CLP) model, with T-cell-specific Atg7-knockout mice compared to control mice. Autophagic vacuoles examined by electron microscopy were decreased in the spleen after CLP. Autophagy proteins LC3-II and ATG7, and autophagosomes and autolysosomes stained by Cyto-ID Green and acridine orange were decreased in CD4⁺ and CD8⁺ splenocytes at 18 h and 24 h after CLP. This decrease in autophagy was associated with increased apoptosis of CD4⁺ and CD8⁺ after CLP. Moreover, mice lacking Atg7 in T lymphocytes showed an increase in sepsis-induced mortality, T cell apoptosis and loss of CD4⁺ and CD8⁺ T cells, in comparison to control mice. This was accompanied by suppressed cytokine production of Th1/Th2/Th17 by CD4⁺ T cells, reduced phagocytosis in macrophages and decreased bacterial clearance in the spleen after sepsis.

Conclusion

These results indicated that sepsis led to down-regulation of autophagy in T lymphocytes, which may result in enhanced apoptosis induction and decreased survival in sepsis. Autophagy may therefore play a protective role against sepsis-induced T lymphocyte apoptosis and immunosuppression.     

Generation of human autologous melanoma-specific cytotoxic T cells from tumor-involved lymph nodes

Cytotoxic T lymphocytes (CTL) specific for autologous human melanoma have been successfully generated in vitro from tumor bearing lymph nodes without any stimulation by the autologous tumor. Tumor-involved lymph node cells (LNC) were cultured in serum free medium (AIM-V) containing 1,000 U/ml of recombinant interleukin-2. The best expansion and specific cytotoxicity of CTL were achieved in 4 to 6 weeks of culture. The predominant populations in cultured LNC-derived CTL were CD2+, CD3+, CD4-, CD8+, CD56-, and HLA-DR+ T cells. These data suggested that tumor-involved LNC may provide an alternative source for the generation of tumor-specific CTL in adoptive immunotherapy.     

Ghrelin protects against cobalt chloride-induced hypoxic injury in cardiac H9c2 cells by inhibiting oxidative stress and inducing autophagy

Ghrelin is a multifunctional peptide that actively protects against cardiovascular ischemic diseases, but the underlying mechanisms are unclear. We used CoCl₂ to mimic hypoxic conditions in cardiac H9c2 cells in order to study the mechanism by which ghrelin protects cardiac myocytes against hypoxic injury by regulating the content of intracellular ROS and autophagy levels. Cell apoptosis and necrosis were evaluated by the flow cytometry assay, Hoechst staining, and LDH activity. Cell viability was detected by the WST-1 assay; ROS levels were assessed using DCFH2-DA; and Nox1, catalase and Mn-SOD were assayed by real-time PCR and activity assays. LC3II was measured by Western blot analysis. We observed that CoCl₂ induced apoptosis and death of H9c2 cells in a dose- and time-dependent manner. This was characterized by an increase in cell apoptosis, LDH activity, ROS content, Nox1 expression, and autophagy levels and a decrease in cell viability, catalase, and Mn-SOD activities. Ghrelin treatment significantly attenuated CoCl₂-induced hypoxic injury by decreasing cell apoptosis, LDH activity, ROS content, and Nox1 expression and increasing cell viability, autophagy levels, catalase, and Mn-SOD mRNA levels and activities. Further experiments revealed that inhibiting autophagy using 3-MA or AMPK pathway with compound C almost abrogated the induction of ghrelin in autophagy. This was associated with a decrease in cell viability and an increase in LDH activity. Our results indicate that ghrelin protected cardiac myocytes against CoCl₂-induced hypoxic injury by decreasing Nox1 expression, increasing the expression and activity of endogenous antioxidant enzymes, and inducing protective autophagy in an AMPK-dependent manner.     

Individual Responses to Completion of Short-Term and Chronic Interval Training: A Retrospective Study

Alterations in maximal oxygen uptake (VO₂max), heart rate (HR), and fat oxidation occur in response to chronic endurance training. However, many studies report frequent incidence of “non-responders” who do not adapt to continuous moderate exercise. Whether this is the case in response to high intensity interval training (HIT), which elicits similar adaptations as endurance training, is unknown. The aim of this retrospective study was to examine individual responses to two paradigms of interval training. In the first study (study 1), twenty active men and women (age and baseline VO₂max = 24.0±4.6 yr and 42.8±4.8 mL/kg/min) performed 6 d of sprint interval training (SIT) consisting of 4–6 Wingate tests per day, while in a separate study (study 2), 20 sedentary women (age and baseline VO₂max = 23.7±6.2 yr and 30.0±4.9 mL/kg/min) performed 12 wk of high-volume HIT at workloads ranging from 60–90% maximal workload. Individual changes in VO₂max, HR, and fat oxidation were examined in each study, and multiple regression analysis was used to identify predictors of training adaptations to SIT and HIT. Data showed high frequency of increased VO₂max (95%) and attenuated exercise HR (85%) in response to HIT, and low frequency of response for VO₂max (65%) and exercise HR (55%) via SIT. Frequency of improved fat oxidation was similar (60–65%) across regimens. Only one participant across both interventions showed non-response for all variables. Baseline values of VO₂max, exercise HR, respiratory exchange ratio, and body fat were significant predictors of adaptations to interval training. Frequency of positive responses to interval training seems to be greater in response to prolonged, higher volume interval training compared to similar durations of endurance training.     

Hypoxic preconditioning‐induced autophagy enhances survival of engrafted endothelial progenitor cells in ischaemic limb

Recent clinical studies have suggested that endothelial progenitor cells (EPCs) transplantation provides a modest benefit for treatment of the ischaemic diseases such as limb ischaemia. However, cell‐based therapies have been limited by poor survival of the engrafted cells. This investigation was designed to establish optimal hypoxia preconditioning and evaluate effects of hypoxic preconditioning‐induced autophagy on survival of the engrafted EPCs. Autophagy of CD34⁺VEGFR‐2⁺ EPCs isolated from rat bone marrow increased after treatment with 1% O₂. The number of the apoptotic cells in the hypoxic cells increased significantly after autophagy was inhibited with 3‐methyladenine. According to balance of autophagy and apoptosis, treatment with 1% O₂ for 2 hrs was determined as optimal preconditioning for EPC transplantation. To examine survival of the hypoxic cells, the cells were implanted into the ischaemic pouch of the abdominal wall in rats. The number of the survived cells was greater in the hypoxic group. After the cells loaded with fibrin were transplanted with intramuscular injection, blood perfusion, arteriogenesis and angiogenesis in the ischaemic hindlimb were analysed with laser Doppler‐based perfusion measurement, angiogram and the density of the microvessels in histological sections, respectively. Repair of the ischaemic tissue was improved significantly in the hypoxic preconditioning group. Loading the cells with fibrin has cytoprotective effect on survival of the engrafted cells. These results suggest that activation of autophagy with hypoxic preconditioning is an optimizing strategy for EPC therapy of limb ischaemia.     

Effects of Intermittent Training on Anaerobic Performance and MCT Transporters in Athletes

This study examined the effects of intermittent hypoxic training (IHT) on skeletal muscle monocarboxylate lactate transporter (MCT) expression and anaerobic performance in trained athletes. Cyclists were assigned to two interventions, either normoxic (N; n = 8; 150 mmHg P_IO₂) or hypoxic (H; n = 10; ∼3000 m, 100 mmHg P_IO₂) over a three week training (5×1 h-1h30.week⁻¹) period. Prior to and after training, an incremental exercise test to exhaustion (EXT) was performed in normoxia together with a 2 min time trial (TT). Biopsy samples from the vastus lateralis were analyzed for MCT1 and MCT4 using immuno-blotting techniques. The peak power output (PPO) increased (p<0.05) after training (7.2% and 6.6% for N and H, respectively), but VO₂max showed no significant change. The average power output in the TT improved significantly (7.3% and 6.4% for N and H, respectively). No differences were found in MCT1 and MCT4 protein content, before and after the training in either the N or H group. These results indicate there are no additional benefits of IHT when compared to similar normoxic training. Hence, the addition of the hypoxic stimulus on anaerobic performance or MCT expression after a three-week training period is ineffective.     

Treatment of recurrent glioma with intracavitary alloreactive cytotoxic T lymphocytes and interleukin-2

 For a single-dose toxicity assessment, five patients with recurrent malignant glioma (ages 29–46 years) were treated with intracavitary alloreactive cytotoxic T lymphocytes (CTL) and interleukin-2 (IL-2). The trial tested the hypothesis that alloreactive CTL, sensitized to the major histocompatibility complex (MHC) proteins of the patient, offer selective, targeted killing of glioma cells that express MHC. Patient lymphocytes, which also express MHC, were irradiated and placed into CellMax artificial capillary systems with lymphocytes from MHC-disparate donors and CTL developed over a 2- to 3-week period with a low concentration of IL-2. The CTL largely expressed CD3 and CD11a/CD8 markers and lysed targets displaying patient MHC. CTL were implanted into the tumor bed at surgery and a catheter was used for subsequent infusions. Patients received one to five treatment cycles every other month; one cycle generally consisted of two or three CTL infusates administered within a 1- to 2-week period. Different unrelated donors were used for each cycle. Treatment was well tolerated; transient toxicity at grades 1–3 was recorded by NCI Common Toxicity Scale criteria. Two glioblastoma patients have died; one from tumor recurrence locally and the other from recurrence at a site distant from the treatment. Two of the five patients completed five cycles; one anaplastic oligodendroglioma patient shows no evidence of tumor 30 months from the start of immune therapy and an anaplastic astrocytoma patient shows stable disease 28 months after initiation of therapy. One anaplastic oligodendroglioma patient, who dropped the protocol during her second treatment cycle, has no evidence of tumor 28 months after recurrence. Received: 21 May 1997 / Accepted: 17 July 1997     

Endogenous Interferon-β-Inducible Gene Expression and Interferon-β-Treatment Are Associated with Reduced T Cell Responses to Myelin Basic Protein in Multiple Sclerosis

Autoreactive CD4⁺ T-cells are considered to play a major role in the pathogenesis of multiple sclerosis. In experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, exogenous and endogenous type I interferons restrict disease severity. Recombinant interferon-β is used for treatment of multiple sclerosis, and some untreated multiple sclerosis patients have increased expression levels of type I interferon-inducible genes in immune cells. The role of endogenous type I interferons in multiple sclerosis is controversial: some studies found an association of high expression levels of interferon-β-inducible genes with an increased expression of interleukin-10 and a milder disease course in untreated multiple sclerosis patients, whereas other studies reported an association with a poor response to treatment with interferon-β. In the present study, we found that untreated multiple sclerosis patients with an increased expression of interferon-β-inducible genes in peripheral blood mononuclear cells and interferon-β-treated multiple sclerosis patients had decreased CD4⁺ T-cell reactivity to the autoantigen myelin basic protein ex vivo. Interferon-β-treated multiple sclerosis patients had increased IL10 and IL27 gene expression levels in monocytes in vivo. In vitro, neutralization of interleukin-10 and monocyte depletion increased CD4⁺ T-cell reactivity to myelin basic protein while interleukin-10, in the presence or absence of monocytes, inhibited CD4⁺ T-cell reactivity to myelin basic protein. Our findings suggest that spontaneous expression of interferon-β-inducible genes in peripheral blood mononuclear cells from untreated multiple sclerosis patients and treatment with interferon-β are associated with reduced myelin basic protein-induced T-cell responses. Reduced myelin basic protein-induced CD4⁺ T-cell autoreactivity in interferon-β-treated multiple sclerosis patients may be mediated by monocyte-derived interleukin-10.     

Vigorous exercise acutely changes platelet and B-lymphocyte CD39 expression.

CD39/ATP diphosphohydrolase is expressed on B lymphocytes, cytotoxic T lymphocytes, monocytes, platelets, and endothelial cells, and it has a critical role in the inhibition of platelet responsiveness. To determine whether strenuous exercise could acutely change expression of CD39 in platelets and lymphocytes, eight healthy sedentary men, 34 yr old (SD 7), and eight physically active men, 34 yr old (SD 6), performed graded upright cycle ergometry to volitional exhaustion. Blood samples collected both at baseline and after exercise test were employed to measure CD39 expression in platelets and lymphocytes. The percentage of circulating platelet-platelet aggregates, the "in vitro" ADP and collagen-induced platelet aggregation, and the expression of both platelet glycoprotein IIb-IIIa (PAC-1) and P-selectin (CD62) were also considered markers of platelet activation. After strenuous exercise, all subjects demonstrated significant platelet activation as judged by the increased percentage of platelet-platelet aggregates. The in vitro ADP-induced platelet aggregation and the expression of CD62P on ADP-stimulated platelets significantly increased in sedentary but not in active subjects. After exercise, all of the subjects showed a significant reduction of CD39 expression in platelet [sedentary: from 2.2 (SD 0.8) to 1.1% (SD 0.8), P = 0.008; active: from 0.6 (SD 0.2) to 0.35% (SD 0.1), P = 0.009] and an increase of CD39 expression in B lymphocytes [sedentary: from 47 (SD 13) to 60% (SD 11), P = 0.0039; active: from 46 (SD 11) to 59% (SD 11), P = 0.0038]. Taken together, these findings confirm the critical role of this ADPase in inhibition of platelet responsiveness, also suggesting a possible role of B lymphocytes in thromboregulation mechanism.     

CD40 ligation restores cytolytic T lymphocyte response and eliminates fibrosarcoma in the peritoneum of mice lacking CD4+ T cells

Absence of CD4⁺ T cell help has been suggested as a mechanism for failed anti-tumor cytotoxic T lymphocytes (CTL) response. We examined the requirement for CD4⁺ T cells to eliminate an immunogenic murine fibrosarcoma (6132A) inoculated into the peritoneal cavity. Immunocompetent C3H mice eliminated both single and repeat intraperitoneal (IP) inoculums, and developed high frequency of 6132A-specific interferon-γ (IFNγ)-producing CTL in the peritoneal cavity. Adoptive transfer of peritoneal exudate cells (PEC) isolated from control mice, protected SCID mice from challenge with 6132A. In contrast, CD4 depleted mice had diminished ability to eliminate tumor and succumbed to repeat IP challenges. Mice depleted of CD4⁺ T cells lacked tumor-specific IFNγ producing CTL in the peritoneal cavity. Adoptive transfer of PEC from CD4 depleted mice failed to protect SCID mice from 6132A. However, splenocytes isolated from same CD4 depleted mice prevented tumor growth in SCID mice, suggesting that 6132A-specific CTL response was generated, but was not sustained in the peritoneum. Treating CD4 depleted mice with agonist anti-CD40 antibody, starting on days 3 or 8 after initiating tumor challenge, led to persistence of 6132A-specific IFNγ producing CTL in the peritoneum, and eliminated 6132A tumor. The findings suggest that CTL can be activated in the absence of CD4⁺ T cells, but CD4⁺ T cells are required for a persistent CTL response at the tumor site. Exogenous stimulation through CD40 can restore tumor-specific CTL activity to the peritoneum and promote tumor clearance in the absence of CD4⁺ T cells.Supported in part by grants from Children’s Hospital of Wisconsin Foundation, Society of University Surgeons Foundation, Florence and Marshall Schwid Foundation, Elsa Pardee Foundation, Kathy Duffy Fogarty Fund of the Greater Milwaukee Foundation (JS) and NIH grant RO1-CA-37156 (HS); Andrew Lodge and Ping Yu have contributed equally to this work.     

Diverse Host Responses and Outcomes following Simian Immunodeficiency Virus SIVmac239 Infection in Sooty Mangabeys and Rhesus Macaques

Sooty mangabeys naturally infected with simian immunodeficiency virus (SIV) do not develop immunodeficiency despite the presence of viral loads of 10⁵ to 10⁷ RNA copies/ml. To investigate the basis of apathogenic SIV infection in sooty mangabeys, three sooty mangabeys and three rhesus macaques were inoculated intravenously with SIVmac239 and evaluated longitudinally for 1 year. SIVmac239 infection of sooty mangabeys resulted in 2- to 4-log-lower viral loads than in macaques and did not reproduce the high viral loads observed in natural SIVsmm infection. During acute SIV infection, polyclonal cytotoxic T-lymphocyte (CTL) activity coincident with decline in peak plasma viremia was observed in both macaques and mangabeys; 8 to 20 weeks later, CTL activity declined in the macaques but was sustained and broadly directed in the mangabeys. Neutralizing antibodies to SIVmac239 were detected in the macaques but not the mangabeys. Differences in expression of CD38 on CD8⁺ T lymphocytes or in the percentage of naive phenotype T cells expressing CD45RA and CD62L-selection did not correlate with development of AIDS in rhesus macaques. In macaques, the proportion of CD4⁺ T lymphocytes expressing CD25 declined during SIV infection, while in mangabeys, CD25-expressing CD4⁺ T lymphocytes increased. Longitudinal evaluation of cytokine secretion by flow cytometric analysis of unstimulated lymphocytes revealed elevation of interleukin-2 and gamma interferon in a macaque and only interleukin-10 in a concurrently infected mangabey during acute SIV infection. Differences in host responses following experimental SIVmac239 infection may be associated with the divergent outcome in sooty mangabeys and rhesus macaques.     

Autophagy and CD4+ T lymphocyte destruction by HIV-1

The first step of HIV-1 infection is mediated by the binding of envelope glycoproteins (Env) to CD4 and two major coreceptors, CCR5 or CXCR4. The HIV-1 strains that use CCR5 are involved in primo-infection whereas those HIV-1 strains that use CXCR4 play a major role in the demise of CD4+ T lymphocytes and a rapid progression toward AIDS. Notably, binding of X4 Env expressed on cells to CXCR4 triggers apoptosis of uninfected CD4+ T cells. We now have just demonstrated that, independently of HIV-1 replication, transfected or HIV-1-infected cells that express X4 Env induce autophagy and accumulation of Beclin 1 in uninfected CD4+ T lymphocytes via CXCR4. Moreover, autophagy is a prerequisite to Env-induced apoptosis in uninfected bystander T cells, and CD4+ T cells still undergo an Env-mediated cell death with autophagic features when apoptosis is inhibited. To the best of our knowledge, these findings represent the first example of autophagy triggered through binding of virus envelope proteins to a cellular receptor, without viral replication, leading to apoptosis. Here, we proposed hypotheses about the significance of Env-induced Beclin 1 accumulation in CD4+ T cell death and about the role of autophagy in HIV-1 infected cells depending on the coreceptor involved.     

Activating Nrf-2 Signaling Depresses Unilateral Ureteral Obstruction-Evoked Mitochondrial Stress-Related Autophagy,Apoptosis and Pyroptosis in Kidney

Exacerbated oxidative stress and inflammation may induce three types of programmed cell death, autophagy, apoptosis and pyroptosis in unilateral ureteral obstruction (UUO) kidney. Sulforaphane activating NF-E2-related nuclear factor erythroid-2 (Nrf-2) signaling may ameliorate UUO-induced renal damage. UUO was induced in the left kidney of female Wistar rats. The level of renal blood flow, cortical and medullary oxygen tension and reactive oxygen species (ROS) was evaluated. Fibrosis, ED-1 (macrophage/monocyte) infiltration, oxidative stress, autophagy, apoptosis and pyroptosis were evaluated by immunohistochemistry and Western blot in UUO kidneys. Effects of sulforaphane, an Nrf-2 activator, on Nrf-2- and mitochondrial stress-related proteins and renal injury were examined. UUO decreased renal blood flow and oxygen tension and increased renal ROS, 3-nitrotyrosine stain, ED-1 infiltration and fibrosis. Enhanced renal tubular Beclin-1 expression started at 4 h UUO and further enhanced at 3d UUO, whereas increased Atg-5-Atg12 and LC3-II expression were found at 3d UUO. Increased renal Bax/Bcl-2 ratio, caspase 3 and PARP fragments, apoptosis formation associated with increased caspase 1 and IL-1β expression for pyroptosis formation were started from 3d UUO. UUO reduced nuclear Nrf-2 translocation, increased cytosolic and inhibitory Nrf-2 expression, increased cytosolic Bax translocation to mitochondrial and enhanced mitochondrial Cytochrome c release into cytosol of the UUO kidneys. Sulforaphane significantly increased nuclear Nrf-2 translocation and decreased mitochondrial Bax translocation and Cytochrome c release into cytosol resulting in decreased renal injury. In conclusion, sulforaphane via activating Nrf-2 signaling preserved mitochondrial function and suppressed UUO-induced renal oxidative stress, inflammation, fibrosis, autophagy, apoptosis and pyroptosis.     

Effects of live high, train low hypoxic exposure on lactate metabolism in trained humans.

We determined the effect of 20 nights of live high, train low (LHTL) hypoxic exposure on lactate kinetics, monocarboxylate lactate transporter proteins (MCT1 and MCT4), and muscle in vitro buffering capacity (betam) in 29 well-trained cyclists and triathletes. Subjects were divided into one of three groups: 20 consecutive nights of hypoxic exposure (LHTLc), 20 nights of intermittent hypoxic exposure [four 5-night blocks of hypoxia, each interspersed with 2 nights of normoxia (LHTLi)], or control (Con). Rates of lactate appearance (Ra), disappearance (Rd), and oxidation (Rox) were determined from a primed, continuous infusion of l-[U-14C]lactic acid tracer during 90 min of steady-state exercise [60 min at 65% peak O2 uptake (VO(2 peak)) followed by 30 min at 85% VO(2 peak)]. A resting muscle biopsy was taken before and after 20 nights of LHTL for the determination of betam and MCT1 and MCT4 protein abundance. Ra during the first 60 min of exercise was not different between groups. During the last 25 min of exercise at 85% VO(2 peak), Ra was higher compared with exercise at 65% of VO(2 peak) and was decreased in LHTLc (P < 0.05) compared with the other groups. Rd followed a similar pattern to Ra. Although Rox was significantly increased during exercise at 85% compared with 65% of VO(2 peak), there were no differences between the three groups or across trials. There was no effect of hypoxic exposure on betam or MCT1 and MCT4 protein abundance. We conclude that 20 consecutive nights of hypoxia exposure decreased whole body Ra during intense exercise in well-trained athletes. However, muscle markers of lactate metabolism and pH regulation were unchanged by the LHTL intervention.     

Depletion of L-arginine induces autophagy as a cytoprotective response to endoplasmic reticulum stress in human T lymphocytes

L-arginine (L-Arg) deficiency results in decreased T-cell proliferation and impaired T-cell function. Here we have found that L-Arg depletion inhibited expression of different membrane antigens, including CD247 (CD3ζ), and led to an ER stress response, as well as cell cycle arrest at G₀/G₁ in both human Jurkat and peripheral blood mitogen-activated T cells, without undergoing apoptosis. By genetic and biochemical approaches, we found that L-Arg depletion also induced autophagy. Deprivation of L-Arg induced EIF2S1 (eIF2α), MAPK8 (JNK), BCL2 (Bcl-2) phosphorylation, and displacement of BECN1 (Beclin 1) binding to BCL2, leading to autophagosome formation. Silencing of ERN1 (IRE1α) prevented the induction of autophagy as well as MAPK8 activation, BCL2 phosphorylation and XBP1 splicing, whereas led T lymphocytes to apoptosis under L-Arg starvation, suggesting that the ERN1-MAPK8 pathway plays a major role in the activation of autophagy following L-Arg depletion. Autophagy was required for survival of T lymphocytes in the absence of L-Arg, and resulted in a reversible process. Replenishment of L-Arg made T lymphocytes to regain the normal cell cycle profile and proliferate, whereas autophagy was inhibited. Inhibition of autophagy by ERN1, BECN1 and ATG7 silencing, or by pharmacological inhibitors, promoted cell death of T lymphocytes incubated in the absence of L-Arg. Our data indicate for the first time that depletion of L-Arg in T lymphocytes leads to a reversible response that preserves T lymphocytes through ER stress and autophagy, while remaining arrested at G₀/G₁. Our data also show that the L-Arg depletion-induced ER stress response could lead to apoptosis when autophagy is blocked.     

Depletion of L-arginine induces autophagy as a cytoprotective response to endoplasmic reticulum stress in human T lymphocytes

L-arginine (L-Arg) deficiency results in decreased T-cell proliferation and impaired T-cell function. Here we have found that L-Arg depletion inhibited expression of different membrane antigens, including CD247 (CD3ζ), and led to an ER stress response, as well as cell cycle arrest at G₀/G₁ in both human Jurkat and peripheral blood mitogen-activated T cells, without undergoing apoptosis. By genetic and biochemical approaches, we found that L-Arg depletion also induced autophagy. Deprivation of L-Arg induced EIF2S1 (eIF2α), MAPK8 (JNK), BCL2 (Bcl-2) phosphorylation, and displacement of BECN1 (Beclin 1) binding to BCL2, leading to autophagosome formation. Silencing of ERN1 (IRE1α) prevented the induction of autophagy as well as MAPK8 activation, BCL2 phosphorylation and XBP1 splicing, whereas led T lymphocytes to apoptosis under L-Arg starvation, suggesting that the ERN1-MAPK8 pathway plays a major role in the activation of autophagy following L-Arg depletion. Autophagy was required for survival of T lymphocytes in the absence of L-Arg, and resulted in a reversible process. Replenishment of L-Arg made T lymphocytes to regain the normal cell cycle profile and proliferate, whereas autophagy was inhibited. Inhibition of autophagy by ERN1, BECN1 and ATG7 silencing, or by pharmacological inhibitors, promoted cell death of T lymphocytes incubated in the absence of L-Arg. Our data indicate for the first time that depletion of L-Arg in T lymphocytes leads to a reversible response that preserves T lymphocytes through ER stress and autophagy, while remaining arrested at G₀/G₁. Our data also show that the L-Arg depletion-induced ER stress response could lead to apoptosis when autophagy is blocked.