cytochrome-c-t and Sepsis

Sepsis remains as a leading cause of death in critically ill patients. Unfortunately, there have been very few successful specific therapeutic agents that can significantly reduce the attributable mortality and morbidity of sepsis. Developing novel therapeutic strategies to improve outcomes of sepsis remains an important focus of ongoing research in the field of critical care medicine. Apoptosis has recently been identified as an important mechanism of cell death and evidence suggests that prevention of cell apoptosis can improve survival in animal models of sepsis and endotoxaemia. In this review article, we summarise the critical role of apoptosis of the immune cells in the pathophysiology of sepsis and propose that blocking cell-signaling pathways leading to apoptosis may present a promising specific therapy for sepsis. Various methods to inhibit apoptosis including the cell surface Fas receptor pathway inhibitors, caspase inhibitors, over-expression of anti-apoptotic genes and small interfering ribonucleic acid therapy are discussed.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; BH3 Interacting Domain Death Agonist Protein; Cytochromes c; Humans; Membrane Proteins; Proto-Oncogene Proteins; Sepsis

To present the recent findings obtained in clinical and experimental studies examining microcirculatory alterations in sepsis, their link to mitochondrial dysfunction, and current knowledge regarding the impact of these alterations on the outcome of septic patients.. Interlinked by a mutual cascade effect and driven by the host-pathogen interaction, microcirculatory and mitochondrial functions are impaired during sepsis. Mitochondrial respiration seems to evolve during the course of sepsis, demonstrating a change from reversible to irreversible inhibition. The spatiotemporal heterogeneity of microcirculatory and mitochondrial dysfunction suggests that these processes may be compartmentalized. Although a causal relationship between mitochondrial and microcirculatory dysfunction and organ failure in sepsis is supported by an increasing number of studies, adaptive processes have also emerged as part of microcirculatory and mitochondrial alterations. Treatments for improving or preserving microcirculatory, mitochondrial function, or both seem to yield a better outcome in patients.. Even though there is evidence that microcirculatory and mitochondrial dysfunction plays a role in the development of sepsis-induced organ failure, their interaction and respective contribution to the disease remains poorly understood. Future research is necessary to better define such relationships in order to identify therapeutic targets and refine treatment strategies.

Topics: Capillary Permeability; Cardiovascular Agents; Cytochromes c; Erythropoietin; Humans; Microcirculation; Mitochondria; Mitochondrial Diseases; Poly Adenosine Diphosphate Ribose; Recombinant Proteins; Sepsis

Sepsis, the principal cause of death in critically ill patients, is associated with impaired oxygen extraction by tissues. One possible explanation is the development of mitochondrial dysfunction and ineffective oxygen utilization. This abnormality has been termed cytopathic hypoxia. This may be caused by an abnormality in the transport of electrons down the cytochrome chain on the mitochondrial inner membrane. In this article we review our studies on abnormalities in the function of complex IV (cytochrome oxidase), the final electron acceptor in this chain. In addition, we provide evidence that administration of cytochrome c may overcome these abnormalities and provide a novel therapeutic alternative.

Topics: Animals; Critical Illness; Cytochromes c; Electron Transport Complex IV; Humans; Mitochondria, Heart; Muscle Relaxation; Myocardial Contraction; Myocardium; Sepsis

We previously found decreased levels of Coenzyme Q10 (CoQ10) in patients with septic shock. The objective of the current study was to assess whether the provision of exogenous ubiquinol (the reduced form of CoQ10) could increase plasma CoQ10 levels and improve mitochondrial function.. We performed a randomized, double-blind, pilot trial at a single, tertiary care hospital. Adults (age ≥18 years) with severe sepsis or septic shock between November 2012 and January 2014 were included. Patients received 200 mg enteral ubiquinol or placebo twice a day for up to seven days. Blood draws were obtained at baseline (0 h), 12, 24, 48, and 72 h. The primary outcome of the study was change in plasma CoQ10 parameters (total CoQ10 levels, CoQ10 levels relative to cholesterol levels, and levels of oxidized and reduced CoQ10). Secondary outcomes included assessment of: 1) vascular endothelial biomarkers, 2) inflammatory biomarkers, 3) biomarkers related to mitochondrial injury including cytochrome c levels, and 4) clinical outcomes. CoQ10 levels and biomarkers were compared between groups using repeated measures models.. We enrolled 38 patients: 19 in the CoQ10 group and 19 in the placebo group. The mean patient age was 62 ± 16 years and 47% were female. Baseline characteristics and CoQ10 levels were similar for both groups. There was a significant increase in total CoQ10 levels, CoQ10 levels relative to cholesterol levels, and levels of oxidized and reduced CoQ10 in the ubiquinol group compared to the placebo group. We found no difference between the two groups in any of the secondary outcomes.. In this pilot trial we showed that plasma CoQ10 levels could be increased in patients with severe sepsis or septic shock, with the administration of oral ubiquinol. Further research is needed to address whether ubiquinol administration can result in improved clinical outcomes in this patient population.. Clinicaltrials.gov identifier NCT01948063. Registered on 18 February 2013.

Topics: Cholesterol; Cytochromes c; Double-Blind Method; Female; Humans; Interleukins; Male; Micronutrients; Middle Aged; Pilot Projects; Sepsis; Shock, Septic; Ubiquinone; Vascular Cell Adhesion Molecule-1; Vascular Endothelial Growth Factor A

Background: The kidney is the most common extrapulmonary organ injured in sepsis. The current study examines the ability of aerosolized nanochemically modified tetracycline 3 (nCMT-3), a pleiotropic anti-inflammatory agent, to attenuate acute kidney injury (AKI) caused by intratracheal LPS. Methods: C57BL/6 mice received aerosolized intratracheal nCMT-3 (1 mg/kg) or saline, followed by intratracheal LPS (2.5 mg/kg) to induce acute lung injury-induced AKI. Tissues were harvested at 24 h. The effects of nCMT-3 and LPS on AKI were assessed by plasma/tissue levels of serum urea nitrogen, creatinine, neutrophil gelatinase-associated lipocalin, kidney injury molecule 1, and renal histology. Renal matrix metalloproteinase (MMP) level/activity, cytochrome C, Bax, Bcl-2, caspase-3, p38 mitogen-activated protein kinase activation, NLRP3, and caspase-1 were also measured. Apoptotic cells in kidney were determined by TUNEL assay. Renal levels of IL-1β and IL-6 were measured to assess inflammation. Results: Acute lung injury-induced AKI was characterized by increased plasma blood urea nitrogen, creatinine, injury biomarkers (neutrophil gelatinase-associated lipocalin, kidney injury molecule 1), and histologic evidence of renal injury. Lipopolysaccharide-treated mice demonstrated renal injury with increased levels of inflammatory cytokines (IL-1β, IL-6), active MMP-2 and MMP-9, proapoptotic proteins (cytochrome C, Bax/Bcl-2 ratio, cleaved caspase-3), apoptotic cells, inflammasome activation (NLRP3, caspase-1), and p38 signaling. Intratracheal nCMT-3 significantly attenuated all the measured markers of renal injury, inflammation, and apoptosis. Conclusions: Pretreatment with aerosolized nCMT-3 attenuates LPS-induced AKI by inhibiting renal NLRP3 inflammasome activation, renal inflammation, and apoptosis.

Topics: Acute Kidney Injury; Acute Lung Injury; Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 1; Caspase 3; Creatinine; Cytochromes c; Inflammasomes; Inflammation; Interleukin-6; Lipocalin-2; Lipopolysaccharides; Mice; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Proto-Oncogene Proteins c-bcl-2; Sepsis; Tetracyclines

BACKGROUND Cellular immunity plays a crucial role in sepsis, and lymphocyte apoptosis is a key factor in immune homeostasis. Tumor necrosis factor-alpha (TNF-alpha)-induced protein 8-like 2 (TIPE2) is suggested to play a critical role in maintaining immune homeostasis. This study investigated the role of TIPE2 in CD4⁺ T lymphocyte apoptosis based on a mouse model of thermal injury. MATERIAL AND METHODS BALB/c male mice were randomized into 6 groups: sham, burn, burn with siTIPE2, burn with siTIPE2 control, burn with TIPE2, and burn with TIPE2 control groups. Splenic CD4⁺ T lymphocytes were collected by use of a magnetic cell sorting system. RESULTS We found that TIPE2 downregulation reduced the CD4⁺ T lymphocytes apoptosis in the burn with siTIPE2 group, and the protein expression of P-smad2/P-Smad3 were remarkably downregulated. In the burn with siTIPE2 group, Bcl-2 expression was increased compared with that in the sham group (P<0.05), and Bim expression was reduced (P<0.05). In the burn with TIPE2 group, the mitochondrial membrane potential was markedly reduced (P<0.01), while cytochrome C expression was clearly higher than that in the other groups (P<0.01). Activities of caspase-3, -8, and -9 were notably higher in the burn with TIPE2 group relative to those for other groups (P<0.05). CONCLUSIONS Downregulation of TIPE2 in vivo can reduce the apoptosis of CD4⁺ T lymphocytes following thermal damage, and activate the TGFß downstream signaling of Smad2/Smad3, upregulating Bim, and downregulating Bcl-2.

Topics: Animals; Apoptosis; Burns; CD4-Positive T-Lymphocytes; Cytochromes c; Hot Temperature; Intracellular Signaling Peptides and Proteins; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Models, Animal; RNA, Small Interfering; Sepsis; Signal Transduction; Smad2 Protein; Smad3 Protein

It was demonstrated that α1 adrenergic receptor (α1-AR) activation by phenylephrine (PE) attenuated cardiac dysfunction in lipopolysaccharide (LPS)-challenged mice. However, it is unclear whether PE suppresses sepsis-induced cardiomyocyte apoptosis. Here, we investigated the effects of PE on cardiomyocyte apoptosis in LPS-treated adult rat ventricular myocytes (ARVMs) and septic rats induced by cecal ligation and puncture. Cardiomyocyte apoptosis and caspase activity were detected by TUNEL and spectrophotometrical assay, respectively. Bax, Bcl-2 and cytochrome c (Cyt c) levels as well as IκBα, ERK1/2, p38 MAPK, JNK and cardiac troponin I (cTnI) phosphorylation were analyzed by Western blotting, and TNF-α concentration was analyzed by ELISA. PE inhibited LPS-induced caspase-3 activation in ARVMs, which was reversed by prazosin (a membrane permeable α1-AR antagonist), but not by CGP12177A (a membrane impermeable α1-AR antagonist). PE upregulated phosphorylated ERK1/2 and Bcl-2 contents, decreased TNF-α and Bax levels, Cyt c release, caspase-8/-9 activities as well as IκBα, p38MAPK and JNK phosphorylation in LPS-treated ARVMs, all of which were abolished by prazosin. Treatment with U0126 (a specific ERK1/2 inhibitor) reversed the effects of PE on IκBα, p38MAPK and JNK phosphorylation as well as caspase-3/-8/-9 activation in LPS-treated ARVMs. In septic rats, PE not only inhibited myocardial apoptosis as well as IκBα, p38MAPK, and JNK phosphorylation, but also upregulated myocardial phosphorylated ERK1/2. Furthermore, PE inhibited myocardial cTnI phosphorylation and improved cardiac function in septic rats. Taken together, our data suggest that α1-AR activation by PE inhibits sepsis-induced cardiomyocyte apoptosis and cardiac dysfunction via activating ERK1/2 signal pathway.

Topics: Adrenergic alpha-1 Receptor Agonists; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspases; Cytochromes c; Enzyme-Linked Immunosorbent Assay; In Situ Nick-End Labeling; Lipopolysaccharides; Male; Myocytes, Cardiac; NF-KappaB Inhibitor alpha; p38 Mitogen-Activated Protein Kinases; Phenylephrine; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Sepsis; Signal Transduction; Troponin T; Tumor Necrosis Factor-alpha

Sepsis is a life-threatening condition often associated with a high incidence of multiple organs injury. Several papers suggested the immune response by itself, with the production of humoral inflammatory mediators, is crucial in determining organ injury. However, little is known of how sepsis directly induces organ injury at the cellular levels. To assess this point, we set up an in vitro study to investigate the response of renal tubular cells (RTCs), monocytes (U937) and hepatocytes (HepG2) after 24 h-incubation with septic patients' plasma.. We enrolled 26 septic patients ("test" group). We evaluated cell viability, apoptosis and necrosis by flow cytometer. Caspase-3,-8,-9 and cytochrome-c concentrations have been analyzed using the Human enzyme-linked immunosorbent assay kit.. We found that a decrease of cell viability in all cell lines tested was associated to the increase of apoptosis in RTCs and U937 (p < 0.0001) and increase of necrosis in HepG2 (p < 0.5). The increase of apoptosis in RTCs and U937 cells was confirmed by higher levels of caspase-3 (p < 0.0001). We showed that apoptosis in both RTCs and U937 was triggered by the activation of the intrinsic pathway, as caspase-9 and cytochrome-c levels significantly increased (p < 0.0001), while caspase-8 did not change. This assumption was strengthened by the significant correlation of caspase-9 with both cytochrome-c (r = 0.73 for RTCs and r = 0.69 for U937) and caspase-3 (r = 0.69 for RTCs and r = 0.63 for U937).. Humoral mediators in septic patients' plasma induce apoptosis. This fact suggests that apoptosis inhibitors should be investigated as future strategy to reduce sepsis-induced organ damages.

Topics: Apoptosis; Caspases; Cell Survival; Cytochromes c; Hep G2 Cells; Hepatocytes; Humans; Kidney Tubules, Proximal; Monocytes; Plasma; Sepsis; U937 Cells

Life-threatening cardiomyopathy is a severe, but common, complication associated with severe trauma or sepsis. Several signaling pathways involved in apoptosis and necroptosis are linked to trauma- or sepsis-associated cardiomyopathy. However, the underling causative factors are still debatable. Heparan sulfate (HS) fragments belong to the class of danger/damage-associated molecular patterns liberated from endothelial-bound proteoglycans by heparanase during tissue injury associated with trauma or sepsis. We hypothesized that HS induces apoptosis or necroptosis in murine cardiomyocytes. By using a novel Medical-

Topics: Algorithms; Animals; Apoptosis; Cardiomyopathies; Caspase 3; Cell Culture Techniques; Cells, Cultured; Cytochromes c; Heparitin Sulfate; Humans; Machine Learning; Mice; Myocytes, Cardiac; Necrosis; Receptor-Interacting Protein Serine-Threonine Kinases; Sepsis; Signal Transduction; Wounds and Injuries

A dedicated proteomic approach based on nano-Liquid Chromatography coupled with tandem mass spectrometry in ion trap is proposed for the analysis of proteins trapped in sorbent resin cartridges used to remove inflammatory mediators from blood by coupled plasma filtration adsorption (CPFA). The final purpose of the proposed proteomic approach was to obtain a reference map of plasma proteins trapped in CPFA sorbents used for the extracorporeal blood purification of healthy pigs, with the potential impact to design new bio-filters able to control the inflammatory imbalance under pathological conditions, such as severe sepsis. The five main steps of the proteomics analysis, (i) protein extraction from resin cartridges, (ii) two-dimensional gel electrophoresis (2D-PAGE) for protein separation and profiling, (iii) in-gel proteolytic digestion, (iv) tandem mass analysis of peptides resulting from enzymatic cleavage and (v) bioinformatics, for protein identification and post-processing validation of MS/MS data sets, have been carefully evaluated. Prior to electrophoresis, the efficiency of different extraction solutions and procedures to recovery plasma proteins trapped into the sorbents were tested. Then, a rapid one-step procedure for protein extraction was optimized. Protein bands corresponding to the main plasma proteins, namely porcine serum albumin, serotransferrin and immunoglobulins, were identified. In addition, the presence of haptoglobin, hemopexin, α-1 acid glycoprotein and fetuin-A, that are known as acute-phase reaction proteins, was observed, suggesting that CPFA resins led to a non-specifically protein depletion from plasma, rather than targeting specific molecules.

Topics: Acute-Phase Reaction; alpha-2-HS-Glycoprotein; Animals; Cattle; Chromatography, Liquid; Computational Biology; Cytochromes c; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Filtration; Haptoglobins; Hemopexin; Inflammation; Orosomucoid; Proteins; Proteolysis; Sepsis; Software; Sorption Detoxification; Swine; Tandem Mass Spectrometry

Cytochrome c is an essential component of the electron transport chain, and circulating cytochrome c might be an indicator of mitochondrial injury. The objective of this study was to determine whether cytochrome c levels are elevated in septic patients, whether there is an association between cytochrome c levels and lactate/inflammatory markers, and whether elevated levels of cytochrome c are associated with poor outcomes.. This was a single-center, prospective, observational, pilot study within a randomized, placebo-controlled trial. We enrolled adult patients in septic shock and with an elevated lactate (>3 mmol/L). Blood was collected at enrollment and at 12 and 24  h thereafter. Cytochrome c was measured in plasma using an electrochemiluminescence immunoassay.. We included 77 patients. Plasma cytochrome c levels were significantly higher in septic patients than in healthy controls (0.70  ng/mL [quartiles: 0.06, 1.99] vs. 0.19  ng/mL [quartiles: 0.03, 1.32], P = 0.008). Cytochrome c levels at enrollment were positively correlated with lactate levels (r(s) = 0.40, P < 0.001) but not with inflammatory markers. Patients who died before hospital discharge had significantly higher cytochrome c levels than survivors (0.99  ng/mL [quartiles: 0.36, 4.09] vs. 0.58  ng/mL [quartiles: 0.03, 1.64], P = 0.01). When analyzed over time, the difference between survivors and nonsurvivors remained significant (P < 0.001).. Cytochrome c levels are higher in septic patients than in controls. In unadjusted analysis, septic nonsurvivors had higher cytochrome c levels than survivors.

Topics: Aged; Aged, 80 and over; Cytochromes c; Cytokines; Female; Humans; Interleukin-2; Lactic Acid; Male; Middle Aged; Randomized Controlled Trials as Topic; Sepsis; Shock, Septic; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

The current study investigated the role of exogenous cytochrome c in sepsis-induced myocardial dysfunction (SIMD) using a mouse model and aimed to elucidate its effect on transforming growth factor-β1 (TGF-β1) expression during this process. A total of 75 male Kunming mice were randomly divided into the following five group: Normal (N, n=15); sham-operation (SHAM, n=15); sepsis (CLP, n=15); normal saline (NS, n=15); and cytochrome c (Cytc, n=15). Animals were sacrificed at 0, 6 or 12 h and the samples were analyzed using transmission electron microscopy, histopathological examination, reverse transcription-quantitative polymerase chain reaction, ELISA, protein analysis by western blotting. The SIMD model was developed and a significant downregulation of TGF-β1 gene expression, in addition to a reduction in the plasma and protein levels of TGF-β1 as well as the protein levels of TGF-β1-activated SMAD 1/5/8 were observed in the CLP group. The data from the current study indicate that using exogenous cytochrome c as a therapeutic strategy for SIMD is feasible, and may function via the downregulation of TGF-β1 expression through the SMAD 1/5/8 signaling pathway.

Topics: Animals; Anti-Inflammatory Agents; Cardiomyopathies; Cytochromes c; Disease Models, Animal; Gene Expression Regulation; Male; Mice; Myocardium; Sepsis; Signal Transduction; Smad1 Protein; Smad5 Protein; Smad8 Protein; Transforming Growth Factor beta1

With both in vivo and in vitro experiments, the present study was conducted to investigate the effect of regulatory T cell (Treg) on promoting T-lymphocyte apoptosis and its regulatory mechanism through transforming growth factor-beta (TGF-β1) signaling in mice. A murine model of polymicrobial sepsis was reproduced by cecal ligation and puncture (CLP); PC61 and anti-TGF-β antibodies were used to decrease counts of CD4(+)CD25(+) Tregs and inhibit TGF-β activity, respectively. Splenic CD4(+)CD25(+) Tregs and CD4(+)CD25(-) T cells were isolated. Phenotypes, including cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), forkhead/winged helix transcription factor p3 (Foxp3), and TGFβ1(m+), as well as the apoptotic rate of CD4(+)CD25(-) T cell, were analyzed by flow cytometry. Real-time reverse transcription-polymerase chain reaction was performed to determine mRNA expression of TGF-β1, and the expressions of Smad2/Smad3, Bcl-2 superfamily members of Bcl-2/Bim, cytochrome C, the mitochondrial membrane potential, and caspases in CD4(+)CD25(-) T cells were simultaneously determined. After treatment with PC61 or anti-TGF-β antibody, CTLA-4, Foxp3, and TGFβ1(m+) expressions of CD4(+)CD25(+) Tregs were markedly decreased in comparison to that of the CLP group and the apoptosis rate of CD4(+)CD25(-) T cells was significantly positively correlated with the expression of TGF-β1. Meanwhile, levels of P-Smad2/P-Smad3, proapoptotic protein Bim, cytochrome C, and activity of caspase-3, -8, -9 were downregulated, whereas the mitochondrial membrane potential and antiapoptotic protein Bcl-2 expression were restored. Taken together, our data indicated that the TGF-β1 signal could be partly involved in the apoptosis of CD4(+)CD25(-) T cells promoted by CD4(+)CD25(+) Tregs, therefore inhibition of TGF-β1 expression may provide a novel strategy for the improvement of host immunosuppression following sepsis.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Caspases; CTLA-4 Antigen; Cytochromes c; Disease Models, Animal; Flow Cytometry; Forkhead Transcription Factors; Gene Expression; Gene Expression Profiling; Immunomodulation; Immunophenotyping; Male; Membrane Potential, Mitochondrial; Membrane Proteins; Mice; Phenotype; Phosphorylation; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Sepsis; Smad2 Protein; Smad3 Protein; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Transforming Growth Factor beta1

Our previous research demonstrated that sepsis produces mitochondrial dysfunction with increased mitochondrial oxidative stress in the heart. The present study investigated the role of mitochondria-localized signaling molecules, tyrosine kinase Src and tyrosine phosphatase SHP2, in sepsis-induced cardiac mitochondrial dysfunction using a rat pneumonia-related sepsis model. SD rats were given an intratracheal injection of Streptococcus pneumoniae, 4×10(6) CFU per rat, (or vehicle for shams); heart tissues were then harvested and subcellular fractions were prepared. By Western blot, we detected a gradual and significant decrease in Src and an increase in SHP2 in cardiac mitochondria within 24 hours post-inoculation. Furthermore, at 24 hours post-inoculation, sepsis caused a near 70% reduction in tyrosine phosphorylation of all cardiac mitochondrial proteins. Decreased tyrosine phosphorylation of certain mitochondrial structural proteins (porin, cyclophilin D and cytochrome C) and functional proteins (complex II subunit 30kD and complex I subunit NDUFB8) were evident in the hearts of septic rats. In vitro, pre-treatment of mitochondrial fractions with recombinant active Src kinase elevated OXPHOS complex I and II-III activity, whereas the effect of SHP2 phosphatase was opposite. Neither Src nor SHP2 affected complex IV and V activity under the same conditions. By immunoprecipitation, we showed that Src and SHP2 consistently interacted with complex I and III in the heart, suggesting that complex I and III contain putative substrates of Src and SHP2. In addition, in vitro treatment of mitochondrial fractions with active Src suppressed sepsis-associated mtROS production and protected aconitase activity, an indirect marker of mitochondrial oxidative stress. On the contrary, active SHP2 phosphatase overproduced mtROS and deactivated aconitase under the same in vitro conditions. In conclusion, our data suggest that changes in mitochondria-localized signaling molecules Src and SHP2 constitute a potential signaling pathway to affect mitochondrial dysfunction in the heart during sepsis.

Topics: Animals; Cyclophilins; Cytochromes c; Endopeptidase K; Male; Mitochondria; Myocardium; Oxidative Phosphorylation; Oxidative Stress; Peptidyl-Prolyl Isomerase F; Porins; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Sepsis; Signal Transduction; src-Family Kinases; Streptococcus pneumoniae; Subcellular Fractions

We tested the hypothesis that 5-Hydroxydecanoic acid (5HD), a putative mitoK(ATP) channel blocker, will reverse sepsis-induced cardiodynamic and adult rat ventricular myocyte (ARVM) contractile dysfunction, restore mitochondrial membrane permeability alterations and improve survival.. Male Sprague-Dawley rats (350-400 g) were made septic using 400 mg/kg cecal inoculum, ip. Sham animals received 5% dextrose water, ip. The Voltage Dependent Anion Channels (VDAC1), Bax and cytochrome C levels were determined in isolated single ARVMs obtained from sham and septic rat heart. Mitochondria and cytosolic fractions were isolated from ARVMs treated with norepinephrine (NE, 10 µmoles) in the presence/absence of 5HD (100 µmoles). A continuous infusion of 5HD using an Alzet pump reversed sepsis-induced mortality when administered at the time of induction of sepsis (-40%) and at 6 hr post-sepsis (-20%). Electrocardiography revealed that 5HD reversed sepsis-induced decrease in the average ejection fraction, Simpsons+m Mode (53.5±2.5 in sepsis and 69.2±1.2 at 24 hr in sepsis+5HD vs. 79.9±1.5 basal group) and cardiac output (63.3±1.2 mL/min sepsis and 79.3±3.9 mL/min at 24 hr in sepsis+5HD vs. 85.8±1.5 mL/min basal group). The treatment of ARVMs with 5HD also reversed sepsis-induced depressed contractility in both the vehicle and NE-treated groups. Sepsis produced a significant downregulation of VDAC1, and upregulation of Bax levels, along with mitochondrial membrane potential collapse in ARVMs. Pretreatment of septic ARVMs with 5HD blocked a NE-induced decrease in the VDAC1 and release of cytochrome C.. The data suggest that Bax activation is an upstream event that may precede the opening of the mitoK(ATP) channels in sepsis. We concluded that mitoK(ATP) channel inhibition via decreased mitochondrial membrane potential and reduced release of cytochrome C provided protection against sepsis-induced ARVM and myocardial contractile dysfunction.

Topics: Animals; Anti-Arrhythmia Agents; bcl-2-Associated X Protein; Body Temperature; Cytochromes c; Decanoic Acids; Hemodynamics; Hydroxy Acids; Male; Membrane Potential, Mitochondrial; Mitochondria; Myocardial Contraction; Myocardium; Myocytes, Cardiac; Potassium Channel Blockers; Potassium Channels; Random Allocation; Rats; Rats, Sprague-Dawley; Sepsis; Tumor Necrosis Factor-alpha; Voltage-Dependent Anion Channel 1

To investigate cell apoptosis, mitochondrial membrane potential, cytochrome C and mechanisms of mitochondria in lymphocyte apoptosis of sepsis.. In the research, female C57BL/6 mice whose body weight ranged from 17 to 25 grams were utilized and assigned randomly to two groups: sham operated group (Control), cecal ligation and puncture group (CLP). The present study was undertaken by using the mice splenic lymphocyte to investigate cell apoptosis, mitochondrial membrane potential, cytochrome C. The apoptosis alteration was evaluated by Annexin V-FITC/PI double staining with flow cytometry. The alteration of mitochondrial membrane potential was investigated by Rhodamine-123 staining of cells. Cytochrome C of mitochondria and cytosol was investigated by Western blot methods. Statistical analysis was performed using SPSS 11.5 for Windows software. Experiment data was indicated with mean ± standard.. The splenic lymphocyte apoptosis was significantly accelerated in the CLP group when compared with that in control group (17.3% ± 2.2% vs. 3.5% ± 0.5%, P < 0.05). The Rhodamine-123 fluorescent intensity in splenic lymphocyte apoptosis was reduced in CLP group (76.2% ± 1.6%). Comparison between sham group (99.6% ± 0.4%) and CLP group had statistical significance (P < 0.05). Apoptosis could induce mitochondrial cytochrome C release into cytoplasm. In the CLP group, elevation of cytochrome C in cytosol was concurrently in accordance with decline in mitochondrial cytochrome C content.. These data suggest that mitochondria and mitochondria signal pathway play an important role in lymphocyte apoptosis of sepsis.

Topics: Animals; Apoptosis; Cells, Cultured; Cytochromes c; Disease Models, Animal; Female; Lymphocytes; Mice; Mice, Inbred C57BL; Mitochondria; Sepsis

Activated Protein C renders anti-apoptotic properties in neurons and endothelial cells. The aim of the present study was to evaluate the in vivo cytoprotective role of Protein C zymogen (PC) administration in septic rat brain. Male Wistar rats (n=60) were subjected to sepsis via Cecal Ligation and Puncture (CLP). Animals were randomly divided either to receive 100 IU/kg human PC concentrate at 1, 7 and 13 h post CLP (CLP+PC group) or placebo treatment (CLP group). At pre-specified time points (6, 12, 24, 36, 48 and 60 h post CLP) five animals from either group were euthanized and the brain tissue was removed. Apoptosis in both neurons (Neu-N+) and astroglia (GFAP+) was assessed by flow cytometry using 7-aminoactinomycin D (7AAD). Immunohistochemical detection of cleaved caspase 3, bax, bcl-2, cytochrome c and caspase 8 was also performed. PC treated animals had significantly reduced apoptosis in neurons at 6 and 24 h post CLP (p=0.04 and p=0.016 respectively) and necrosis at 6, 12 and 60 h post CLP (p=0.008, p=0.012 and p=0.032 respectively). Astrocyte necrosis was also decreased in septic rats receiving PC (6, 12 and 60 h post CLP p=0.008, p=0.016 and p=0.008 respectively). In addition, active caspase 3, bax, cytochrome c and caspase 8 expression was significantly decreased during early sepsis (6-36 h) while bcl-2 expression was increased (24 h p=0.001 and 60 h p=0.001) in the PC treated animals compared to placebo. PC concentrate administration in experimental sepsis produced a time dependent inhibition of apoptosis in rat neurons and astrocytes. The inhibition of sepsis related apoptosis concerned both the mitochondrial and caspase 8 dependent pathways.

Topics: Animals; Apoptosis; Astrocytes; bcl-2-Associated X Protein; Brain; Caspases; Cytochromes c; Cytoprotection; Flow Cytometry; Immunohistochemistry; Male; Neurons; Protein C; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Sepsis; Time Factors

Renal dysfunction attributable to sepsis was long considered a result of hemodynamic instability and subsequent local ischemia. Recent data show that apoptosis may be implicated also. The purpose of this study was to evaluate the role of apoptosis and the expression of the bax, bcl-2, caspase-8, and cytochrome c proteins in the renal parenchymal cells of rats with sepsis.. Sepsis was induced using cecal ligation and puncture (CLP) in 62 male Wistar rats, which were euthanized 6, 12, 24, 36, 48, or 60 h later. Ten sham-treated animals served as a control group. Another group of 50 animals were subjected to CLP and then supervised for 60 h. Renal apoptosis was evaluated using light and transmission electron microscopy, in situ nick-end labeling (TUNEL), and flow cytometry using 7-amino-actinomycin D (7-AAD). Caspase-mediated apoptosis was assessed using M30 antibody. The expression of the apoptosis-regulator proteins B-cell lymphoma 2 (bcl-2), bcl-2-associated x protein (bax), caspase-8, and cytochrome c was detected immunohistochemically.. Sepsis increased inflammatory infiltration (p < 0.001) and necrosis (p < 0.001) in renal parenchyma. Apoptosis was significantly more common than in the kidneys of control animals (p = 0.02). Nuclei stained by the TUNEL technique were predominant in the tubular cells of non-survivors (p = 0.05). The time distribution of all types of cell death was increased significantly 6 h after the induction of sepsis, and declined subsequently. Caspase-generated cytokeratin 18 (CK18) new epitope (M30) was significantly more abundant in the kidneys of animals with sepsis than in control rats, with peaks at 6 h and 60 h post-procedure (p < 0.001). In addition, cells initiating apoptosis were significantly more common at 6 h than at 48 h post-CLP (p = 0.014). Caspase-8 protein immunodetection followed the same time pattern as cell death, increasing as early as 6 h post-CLP and decreasing thereafter (p = 0.013). Bax protein expression was elevated significantly early in the course of sepsis (p = 0.037), whereas the other members of the mitochondrial-dependent pathway remained constant. Animals dying from sepsis had a significantly greater prevalence of bax- (p = 0.037) and caspase-8- (p = 0.031) immunoreactive renal cells.. Apoptosis in renal tissue was significantly more common in animals with sepsis than in controls. The time distribution of cell death markers showed a consistent pattern, making early sepsis the likely initiator of the apoptotic events.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 8; Cytochromes c; Flow Cytometry; Immunohistochemistry; In Situ Nick-End Labeling; Keratin-18; Kidney Tubules; Male; Microscopy, Electron; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Sepsis; Specific Pathogen-Free Organisms

Mitochondrial dysfunction is thought to play a role in the pathogenesis of a variety of disease states, including sepsis. An acquired defect in oxidative phosphorylation potentially causes sepsis-induced organ dysfunction. Cytochrome oxidase (CcOX), the terminal oxidase of the respiratory chain, is competitively inhibited early in sepsis and progresses, becoming noncompetitive during the late phase. We have previously demonstrated that exogenous cytochrome c can overcome myocardial CcOX competitive inhibition and improve cardiac function during murine sepsis at the 24-h point. Here, we evaluate the effect of exogenous cytochrome c on CcOX activity and survival in mice at the later time points. Exogenous cytochrome c (800 microg) or saline was intravenously injected 24 h after cecal ligation and puncture (CLP) or sham operation. Steady-state mitochondrial cytochrome c levels and heme c content increased significantly 48 h post-CLP and remained elevated at 72 h in cytochrome c-injected mice compared with saline injection. Cecal ligation and puncture inhibited CcOX at 48 h in saline-injected mice. However, cytochrome c injection abrogated this inhibition and restored CcOX kinetic activity to sham values at 48 h. Survival after CLP to 96 h after cytochrome c injection approached 50% compared with only 15% after saline injection. Thus, a single injection of exogenous cytochrome c 24 h post-CLP repletes mitochondrial substrate levels for up to 72 h, restores myocardial COX activity, and significantly improves survival.

Topics: Animals; Cytochromes c; Electron Transport Complex IV; Heart; Heme; Kinetics; Male; Mice; Mice, Inbred C57BL; Mitochondria; Myocardium; Oxidative Stress; Phosphorylation; Sepsis

Evidence from the literature has demonstrated that reactive oxygen species (ROS) play an important role in the development of multiple organ failure and septic shock. In addition, mitochondrial dysfunction has been implicated in the pathogenesis of multiple organ dysfunction syndrome (MODS). The hypothesis of cytopathic hypoxia postulates that impairment in mitochondrial oxidative phosphorylation reduces aerobic adenosine triphosphate (ATP) production and potentially induces MODS. In this work, our aim was to evaluate the effects of antioxidants on oxidative damage and energy metabolism parameters in liver of rats submitted to a cecal ligation puncture (CLP) model of sepsis. We speculate that CLP induces a sequence of events that culminate with liver cells death. We propose that mitochondrial superoxide production induces mitochondrial oxidative damage, leading to mitochondrial dysfunction, swelling and release of cytochrome c. These events occur in early sepsis development, as reported in the present work. Liver cells necrosis only occurs 24 h after CLP, but all other events occur earlier (6-12 h). Moreover, we showed that antioxidants may prevent oxidative damage and mitochondrial dysfunction in liver of rats after CLP. In another set of experiments, we verified that L-NAME administration did not reverse increase of superoxide anion production, TBARS formation, protein carbonylation, mitochondrial swelling, increased serum AST or inhibition on complex IV activity caused by CLP. Considering that this drug inhibits nitric oxide synthase and that no parameter was reversed by its administration, we suggest that all the events reported in this study are not mediated by nitric oxide. In conclusion, although it is difficult to extrapolate our findings to human, it is tempting to speculate that antioxidants may be used in the future in the treatment of this disease.

Topics: Animals; Antioxidants; Aspartate Aminotransferases; Cecum; Cytochromes c; Disease Models, Animal; Energy Metabolism; Hepatocytes; Intestinal Perforation; Ligation; Male; Mitochondria; Necrosis; Oxidative Stress; Protein Carbonylation; Rats; Rats, Wistar; Sepsis; Superoxides; Thiobarbituric Acid Reactive Substances; Time Factors

Intestinal epithelial cell apoptosis has been reported in sepsis as a mechanism of organ failure. The aim of this study was to clarify the role of apoptosis-regulating proteins (bcl-2, bax, cytochrome-c, and caspase-8) in septic rats by studying their expression in gastric and intestinal epithelial cells.. Adult Wistar rats were subjected to the cecal ligation and puncture (CLP) model of sepsis and randomly divided into two study groups. Sixty-two animals were sacrificed 6, 12, 24, 36, 48, and 60 h post-procedure, and 50 animals served as the survival study group. Sham-operated animals (n = 40) were used as controls. Gastric and intestinal tissue was excised, and immunohistochemical detection of bcl-2, bax, cytochrome-c, and caspase-8 protein expression was performed.. In gastric mucosa, sepsis induced upregulation of bax and downregulation of caspase-8 expression (p = 0.053 and p = 0.05, respectively). Both bax and caspase-8 were upregulated as early as 6 h post CLP and progressively decreased (p = 0.001, p = 0.004 respectively). In contrast, the expression of the anti-apoptotic bcl-2 was upregulated progressively during the sepsis syndrome (p = 0.03). In intestine, sepsis induced a fourfold upregulation of the cytoprotective bcl-2 (p = 0.0001), accompanied by a remarkable increase in bax (p = 0.002) and caspase-8 (p = 0.0001) expression and a decrease in cytochrome-c expression (p = 0.02). The time distribution of the apoptosis regulators followed the same pattern as in gastric tissue, showing an upregulation of the proapoptotic bax and cytochrome c (p = 0.04) during the early phases and a progressively increased expression of bcl-2 during the late phases (p = 0.0001). Bax expression in gastric epithelium of subjects with septic syndrome was detrimental to survival (p = 0.0001), whereas the expression of the cytoprotective bcl-2 in intestinal epithelium appeared to favor a good prognosis (p = 0.0001).. Sepsis results in alterations of apoptosis regulators in gastrointestinal cells. Alterations of bax and bcl-2 expression in gastric and intestinal epithelial cells may predict the outcome in septic rats.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Caspase 8; Cytochromes c; Cytokines; Enzyme-Linked Immunosorbent Assay; Gastric Mucosa; Immunoenzyme Techniques; Male; Prognosis; Proportional Hazards Models; Random Allocation; Rats; Rats, Wistar; Sepsis; Up-Regulation

Studies in sepsis suggest that mitochondria mediate multiple organ dysfunction, including cardiac failure; however, the underlying molecular mechanisms remain elusive. This study examined changes in mitochondrial membrane integrity, antioxidant activities, and oxidative stress in the heart after infectious challenge (intratracheal Streptococcus pneumoniae, 4 x 10(6) colony-forming units). Inflammation responses also were examined.. Cardiac tissues were harvested from Sprague-Dawley rats 4, 8, 12, and 24 h after bacterial challenge (or intratracheal vehicle for sham-treated animals) and homogenized, followed by preparation of subcellular fractions (mitochondrial, cytosol, and nuclei) or whole-tissue lysate. We examined mitochondrial outer membrane damage and cytochrome C translocation to evaluate mitochondrial integrity, mitochondrial lipid and protein oxidation to assess oxidative stress, and mitochondrial superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities to estimate antioxidant defense. In addition, we measured nuclear factor-kappa B (NF-kappaB) activation in myocardium and cytokine production to investigate inflammatory responses to septic challenge.. Oxidation of mitochondrial protein and lipid was evident 4 h through 24 h after bacterial challenge. Mitochondrial outer membrane damage and cytochrome C release were accompanied by down-regulation of mitochondrial SOD and GPx activity. After bacterial challenge, systemic and myocardial cytokine production increased progressively, and NF-kappaB was activated gradually.. Sepsis impaired cardiac mitochondria by damaging membrane integrity, increasing oxidative stress, and altering defenses against reactive oxygen species. These alterations occur earlier than or simultaneously with inflammatory responses in myocardium after infectious challenge, suggesting that mitochondria play a role in modulating inflammation in sepsis.

Topics: Animals; Cytochromes c; Cytokines; Disease Models, Animal; Glutathione Peroxidase; Inflammation; Male; Membrane Lipids; Mitochondria, Heart; Mitochondrial Membranes; Mitochondrial Proteins; Myocardium; NF-kappa B; Oxidation-Reduction; Oxidative Stress; Pneumococcal Infections; Rats; Rats, Sprague-Dawley; Sepsis; Shock; Streptococcus pneumoniae; Superoxide Dismutase

We hypothesized that progressive decline in myocardial performance would correlate with upregulation of markers for apoptotic mechanisms following increased duration of polymicrobial sepsis in the rat. Male Sprague-Dawley rats (350-400 g) were randomized into sham, 1-, 3- and 7-day sepsis groups. Each septic rat received 200 mg/kg cecal inoculum intraperitoneally (i.p). The post-mortem analysis showed a severely inflamed peritoneum with the presence of pus in all septic animals that was directly proportional to the duration of sepsis. We observed 10, 33 and 42% mortality in the 1-, 3- and 7-day sepsis groups, respectively. Septic animals at 3 and 7 days exhibited an increased wet lung/total body weight and heart weight/total body weight. A significant increase in total cardiac troponin I (cTnI) and C Reactive Protein (CRP) and endothelin-1 (ET-1) was also observed with an increased duration of sepsis. Myocardial ET-1 concentration in the 7-day post-sepsis group was significantly elevated compared to the sham and 1-day post-sepsis groups. Sepsis also produced a significant decrease in the mean arterial pressure in the 7-day post-sepsis group and tachycardia in the 1-, 3-, and 7-day post-sepsis groups compared to the sham group. A significant prolongation of the left ventricular isovolumic relaxation rate constant, tau, and left ventricular end-diastolic pressure in the 1-, 3- and 7-day post-sepsis groups compared to the sham group was observed. In addition, a significant decrease in the rates of left ventricular relaxation (-dP/dt) and contraction (+dP/dt) in the 3- and 7-day post-sepsis groups compared to the sham and 1-day post-sepsis group was observed. Sepsis produced a significant upregulation in the expression of myocardial TRADD, cytosolic active caspase-3, the Bax/Bcl(2) ratio, and the mitochondrial release of cytochrome C in the 3- and 7-day post-sepsis groups. We observed a progressive increase in the number of TUNEL positive nuclei, cytosolic caspase-3 activation and co-localization of PARP in the nuclei at 1, 3 and 7 days post-sepsis. These data suggest that the progression of sepsis from 1 day to 3-7 days produce distinct cardiodynamic characteristics with a more profound effect during later stages. The sepsis-induced decline in myocardial performance correlates with the induction of myocardial apoptosis.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Biomarkers; C-Reactive Protein; Caspase 3; Cytochromes c; Disease Models, Animal; DNA Fragmentation; Endothelins; Heart Failure; Heart Rate; Hypotension; In Situ Nick-End Labeling; Male; Myocardium; p38 Mitogen-Activated Protein Kinases; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Random Allocation; Rats; Rats, Sprague-Dawley; Sepsis; Suppuration; Survival Rate; Tachycardia; TNF Receptor-Associated Death Domain Protein; Troponin I; Ventricular Dysfunction

Topics: Animals; Cell Hypoxia; Cytochromes c; Humans; Mitochondria; Sepsis

Mitochondrial dysfunction may play a role in the pathogenesis of sepsis-induced organ dysfunction. Respiratory-chain deficiencies that occur in sepsis, however, have never been shown to cause organ failure or to be reversible. Cytochrome oxidase uses electrons donated by its substrate, cytochrome c, to reduce oxygen to H2O. In the septic heart, cytochrome oxidase is competitively inhibited. We hypothesized that cytochrome oxidase inhibition coupled with reduced substrate availability is a reversible cause of sepsis-associated myocardial depression.. Prospective observational study aimed to overcome myocardial cytochrome oxidase inhibition with excess cytochrome c and improve cardiac function.. University hospital-based laboratory.. Seventy-five C57Bl6 male mice.. Mice underwent cecal ligation and double puncture, sham operation, or no operation. Exogenous cytochrome c or an equal volume of saline was intravenously injected at the 24-hr time point. All animals were evaluated 30 mins after injection.. Exogenous cytochrome c readily repleted cardiac mitochondria with supranormal levels of substrate (>1.6 times baseline), restored heme c content, and increased cytochrome oxidase kinetic activity. This increased left ventricular pressure and increased pressure development during isovolumic contraction (dP/dtmax) and relaxation (dP/dtmin) by >45% compared with saline injection.. Impaired oxidative phosphorylation is a cause of sepsis-associated myocardial depression, and mitochondrial resuscitation with exogenous cytochrome c overcomes cytochrome oxidase inhibition and improves cardiac function.

Topics: Animals; Cytochromes c; Electron Transport Complex IV; Heme; Male; Mice; Mice, Inbred C57BL; Mitochondria, Heart; Oxidative Phosphorylation; Prospective Studies; Sepsis

This issue's recently published papers concentrates on early goal directed therapy, starting with new data from the original study through to new studies that may have a major bearing on the treatment of septic shock in years to come. A timely reminder about talking, walking and teaching clinical medicine completes the roundup.

Topics: Animals; Critical Care; Cytochromes c; Disease Models, Animal; Education, Medical; Humans; Hydrogen; Hypoxia; Inflammation; Mice; Mitochondria; Rats; Sepsis; Time Factors; Vasoconstrictor Agents

This study examined the effects of antioxidant vitamins on several aspects of sepsis-related myocardial signaling cascades. Sprague-Dawley rats were divided into four groups: group 1, vehicle-treated shams; group 2, sham-operated rats given antioxidant vitamins (vitamin C, 24 mg/kg; vitamin E, 20 U/kg; vitamin A, 417 U/kg; and zinc, 3.7 ng/kg) by oral gavage in 0.5 ml water twice daily for 3 days and no septic challenge (vitamin-treated, sham-operated rats); group 3, intratracheal delivery of Streptococcus pneumoniae, 4 x 10(6) colony forming units in a volume of 0.3 ml phosphate buffer solution; group 4, S. pneumonia challenge as described for group 3 plus antioxidant vitamins (as described for group 2). Hearts collected 24 h after septic challenge were used to examine several aspects of cell signaling and ventricular function. As a result, when compared with sham-operated rats, sepsis in the absence of antioxidant therapy promoted NF-kappaB activation, increased mitochondrial cytochrome c release, increased myocyte cytokine secretion, increased caspase activation, and impaired left ventricular function. Antioxidant vitamin therapy plus septic challenge prevented NF-kappaB activation, reduced mitochondrial cytochrome c release, decreased caspase activity, abrogated cardiomyocyte secretion of inflammatory cytokines, and improved myocardial contractile function. In conclusion, antioxidant vitamin therapy abrogated myocardial inflammatory cytokine signaling and attenuated sepsis-related contractile dysfunction, suggesting that antioxidant vitamin therapy may be a potential approach to treat injury and disease states characterized by myocardial dysfunction.

Topics: Animals; Antioxidants; Apoptosis; Calcium; Cardiomyopathies; Caspases; Cytochromes c; Cytokines; Inflammation; Male; Myocardial Contraction; Myocardium; Myocytes, Cardiac; NF-kappa B; Pneumococcal Infections; Rats; Rats, Sprague-Dawley; Sepsis; Signal Transduction; Sodium; Streptococcus pneumoniae; Ventricular Function, Left; Vitamins

Neutropenia is a common sequela of neonatal sepsis. Recent clinical trials have shown the beneficial effects of colony-stimulating factors (CSFs) on outcome in this group, but the exact mechanism remains unknown. Neonates and mothers who were at high-risk for infection were recruited for cord blood sampling in a university tertiary referral maternity hospital. Neonatal and adult neutrophils were evaluated for their ability to combat bacterial infection by examining their functional activity (CD11b and reactive oxygen intermediates) and their persistence at inflammatory sites (apoptosis). The mechanism for altered apoptotic responses was assessed by caspase activation assays, X chromosome-linked inhibitor of apoptosis protein expression, and cytosolic cytochrome c release. Although granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) significantly delayed neutrophil apoptosis in normal adults, only G-CSF had a similar effect in normal neonates. Neutrophils from neonates who are at high risk for infection are unresponsive to the antiapoptotic effects of G-CSF or GM-CSF, unlike maternal neutrophils, which have delayed apoptosis in response to GM-CSF. However, CD11b expression and reactive oxygen intermediate production were significantly increased in normal neonatal neutrophils that were incubated with GM-CSF versus controls but not G-CSF or lipopolysaccharide. Decreased cytosolic cytochrome c release and caspases 3 and 9 activity are associated with the CSF-mediated delay in apoptosis in adults but not in newborns. The antiapoptotic X chromosome-linked inhibitor of apoptosis protein is up-regulated in neonates compared with adults and may mediate their differential spontaneous apoptosis. These results have important implications for the use of CSFs in neonatal sepsis, as responses differ from those seen in adults. Further delineation of neonatal neutrophil responses to CSFs may improve their therapeutic potential.

Topics: Adult; Apoptosis; Base Sequence; Caspase 3; Caspase 9; Caspases; Cell Survival; Cytochromes c; Female; Fetal Blood; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; In Vitro Techniques; Infant, Newborn; Male; Neutrophils; Pregnancy; Proteins; Recombinant Proteins; RNA, Messenger; Sepsis; X-Linked Inhibitor of Apoptosis Protein

Oxidative damage is one of the major factors that lead to cell damage, organ dysfunction and death in sepsis. Thus, an attractive candidate for the pharmacologic treatment of the septic syndrome is desferoxamine (DFX), an antioxidant iron chelator used for the removal of iron and a potential free radical scavenger.. The impact of DFX administration on the survival of septic animals. The effect on cell integrity and cycle of vital organs.. Sepsis was induced in 40 rats using the cecal ligation and puncture method (CLP) and 20 rats randomly received twice subcutaneously DFX (total dose: 40 mg/kg). Rats were monitored for 36 h and all vital organs were harvested for pathology examination and immunohistochemical detection of Bax, Bcl-2, cytochrome c and caspase-8 apoptosis regulating proteins.. Mean survival in the DFX group was 34.2 h (median 36.0, S.D. 4.4) and 30.2 h (median 36.0, S.D. 9.1) in the control group (p=0.04), while 36 h after follow up 85% of the DFX-treated rats and 55% of placebo rats were alive (p=0.04). Expression of pro-apoptotic bax protein was significantly increased in the heart, liver and kidney of animals in the DFX group compared to the control group.. Treatment with the polymeric iron chelator DFX significantly increases survival of septic subjects and alters the expression of bax, an apoptosis regulating protein in certain organs (heart, liver and kidney).

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 8; Caspases; Cytochromes c; Deferoxamine; Free Radical Scavengers; Gene Expression; Genes, bcl-2; Iron Chelating Agents; Kidney; Liver; Male; Myocardium; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Sepsis; Survival Analysis

To investigate whether apoptosis is a possible mechanism of brain dysfunction occurring in septic syndrome.. Experimental prospective study.. Laboratory of Surgical Research at the University of Athens.. Male pathogen-free Wistar rats.. Rats (n = 112) were subjected to sepsis by cecal ligation and puncture. Sham-operated animals (n = 40) underwent the same procedure but without ligation or puncture. Septic animals were either randomly divided (n = 62) in six groups and studied at 6, 12, 24, 36, 48, and 60 hrs after the operation or monitored (n = 50) for 48 hrs as a survival study group. Sham-operated animals were killed at 6, 12, 24, 36, 48, and 60 hrs after the procedure. Brain and cecum were then removed and postfixed in paraffin sections. Apoptosis was evaluated by light microscopy in hematoxylin and eosin-stained specimens and by transmission electron microscopy. In paraffin-embedded sections, immunostaining for bax, bcl-2, cytochrome c, and caspase-8 was done.. In septic rats, increased apoptosis was detected in neurons of the CA1 region of the hippocampus, in choroid plexus, and in Purkinje cells of the cerebellum. Bax immunopositivity was found decreased after the septic insult (p =.03). Bax immunoreactivity was altered as the septic syndrome evolved; it was up-regulated in the early stages (6-12 hrs) and progressively decreased in the late phases (p =.001). Cytochrome c presented a similar regional pattern of expression and was found to be the sole gene marker carrying an independent prognostic role (p =.03). Both bcl-2 and caspase-8 expression remained at constant levels at all times evaluated.. There is evidence that more neurons undergo apoptosis during sepsis than in normal brain tissue in certain sites where the blood-brain barrier is compromised. In this phenomenon, mitochondrial gene regulators such as bax and products such as cytochrome c seem to play important regulating and prognostic roles, respectively.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 8; Caspases; Cecum; Choroid Plexus; Cytochromes c; Cytoplasm; Disease Models, Animal; Genes, bcl-2; Hippocampus; Male; Mitochondria; Neurons; Prognosis; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Purkinje Cells; Rats; Rats, Wistar; Sepsis; Survival Analysis; Time Factors