The lack of associations between alleles at the hypoxia-Inducible factor 1A C1772T loci and responses to acute hypoxia.

Objective The aim of this study was to investigate the associations between alleles of the hypoxia-inducible factor 1A (HIF1A) C1772T polymorphism and several physiological responses to hypoxia, including the hypoxic ventilatory response (HVR), and serum erythropoietin (EPO), arterial oxygen saturation (Sao2), and acute mountain sickness (AMS) responses during 8 hours of exposure to normobaric hypoxia. Methods A total of 76 males participated in the study; 52 participants completed an 8-hour exposure to 12.7% oxygen, during which time Sao2, EPO concentrations, and AMS scores were measured, while 62 individuals took part in an HVR trial (in total 38 individuals completed both protocols). DNA was obtained from leukocytes, and a 346-bp fragment of the HIF1A gene containing the C1772T polymorphism was amplified using polymerase chain reaction. Fragments were sequenced to reveal individual genotypes, and the associations between HIF1A genotype and EPO, Sao2, AMS responses to hypoxia and HVR were examined. Results The magnitude of the hypoxic responses was highly variable between individuals. The increase in participants' EPO responses ranged from 89% to 388% of baseline values following hypoxia, while Sao2 values during the exposure ranged from 71% to 89%. The HVR ranged from −0.04 to +2.18 L · min−1 · Sao2%−1 among participants. No significant differences in EPO, Sao2, AMS, or HVR results were observed between the HIF1A CC genotype and the combined CT/TT genotype group. Conclusion In this study, the HIF1A C1772T polymorphism does not appear to influence EPO, Sao2, or AMS responses during acute hypoxic exposure, or the magnitude of the HVR.

Influence of acute vitamin C and/or carbohydrate ingestion on hormonal, cytokine, and immune responses to prolonged exercise

Davison, G. and Gleeson, M. (2005). Influence of Acute Vitamin C and/or Carbohydrate Ingestion on Hormonal, Cytokine, and Immune Responses to Prolonged Exercise. International Journal of Sport Nutrition and Exercise Metabolism. 15(5), pp.465-479 RAE2008

FLT3-driven redox signalling in acute myeloid leukaemia

Acute myeloid leukaemia refers to cancer of the blood and bone marrow characterised by the rapid expansion of immature blasts of the myeloid lineage. The aberrant proliferation of these blasts interferes with normal haematopoiesis, resulting in symptoms such as anaemia, poor coagulation and infections. The molecular mechanisms underpinning acute myeloid leukaemia are multi-faceted and complex, with a range of diverse genetic and cytogenetic abnormalities giving rise to the acute myeloid leukaemia phenotype. Amongst the most common causative factors are mutations of the FLT3 gene, which codes for a growth factor receptor tyrosine kinase required by developing haematopoietic cells. Disruptions to this gene can result in constitutively active FLT3, driving the de-regulated proliferation of undifferentiated precursor blasts. FLT3-targeted drugs provide the opportunity to inhibit this oncogenic receptor, but over time can give rise to resistance within the blast population. The identification of targetable components of the FLT3 signalling pathway may allow for combination therapies to be used to impede the emergence of resistance. However, the intracellular signal transduction pathway of FLT3 is relatively obscure. The objective of this study is to further elucidate this pathway, with particular focus on the redox signalling element which is thought to be involved. Signalling via reactive oxygen species is becoming increasingly recognised as a crucial aspect of physiological and pathological processes within the cell. The first part of this study examined the effects of NADPH oxidase-derived reactive oxygen species on the tyrosine phosphorylation levels of acute myeloid leukaemia cell lines. Using two-dimensional phosphotyrosine immunoblotting, a range of proteins were identified as undergoing tyrosine phosphorylation in response to NADPH oxidase activity. Ezrin, a cytoskeletal regulatory protein and substrate of Src kinase, was selected for further study. The next part of this study established that NADPH oxidase is subject to regulation by FLT3. Both wild type and oncogenic FLT3 signalling were shown to affect the expression of a key NADPH oxidase subunit, p22phox, and FLT3 was also demonstrated to drive intracellular reactive oxygen species production. The NADPH oxidase target protein, Ezrin, undergoes phosphorylation on two tyrosine residues downstream of FLT3 signalling, an effect which was shown to be p22phox-dependent and which was attributed to the redox regulation of Src. The cytoskeletal associations of Ezrin and its established role in metastasis prompted the investigation of the effects of FLT3 and NADPH oxidase activity on the migration of acute myeloid leukaemia cell lines. It was found that inhibition of either FLT3 or NADPH oxidase negatively impacted on the motility of acute myeloid leukaemia cells. The final part of this study focused on the relationship between FLT3 signalling and phosphatase activity. It was determined, using phosphatase expression profiling and real-time PCR, that several phosphatases are subject to regulation at the levels of transcription and post-translational modification downstream of oncogenic FLT3 activity. In summary, this study demonstrates that FLT3 signal transduction utilises a NADPH oxidase-dependent redox element, which affects Src kinase, and modulates leukaemic cell migration through Ezrin. Furthermore, the expression and activity of several phosphatases is tightly linked to FLT3 signalling. This work reveals novel components of the FLT3 signalling cascade and indicates a range of potential therapeutic targets.

Kinetics of anti-Campylobacter jejuni monomeric and polymeric immunoglobulin A1 and A2 responses in serum during acute enteritis.

The intensity and kinetics of the serum polymeric and monomeric immunoglobulin A1 (IgA1) and IgA2 antibody responses to Campylobacter jejuni were analyzed. A rapid and marked serum IgA antibody response involving both the monomeric and polymeric components of IgA was observed after C. jejuni infections. IgA antibodies reached a peak of activity in serum during week 2 after the first symptoms of enteritis, about 10 days before the peak of IgG activity. Polymeric IgA accounted for most of the anti-C. jejuni activity at the peak of the IgA response (median, 90%; range, 44 to 98%) but rapidly disappeared from serum over a few weeks. In contrast, the serum monomeric IgA antibody response was low and was maintained over a prolonged period of time. Anti-C. jejuni IgA detected in the serum of healthy blood donors was mainly monomeric (median, 83%; range, 17 to 94%). In both the patients and the positive controls, IgA1 was the predominant (greater than 85%) subclass involved, even when the IgA antibody response was mainly polymeric. Our results suggest that polymeric IgA antibody responses are linked to a strong or persisting antigenic stimulation or both. Polymeric IgA antibodies appear to be a potential marker of acute C. jejuni infections, and their determination could provide a useful tool for the serological diagnosis of recent C. jejuni infections.

The humoral response to HIV-1: new insights, renewed focus.

During the past 2 decades, significant advances in our understanding of the humoral immune response to human immunodeficiency virus type 1 (HIV-1) infection have been made, yet a tremendous amount of work lies ahead. Despite these advances, strategies to reliably induce antibodies that can control HIV-1 infection are still critically needed. However, recent advances in our understanding of the kinetics, specificity, and function of early humoral responses offer alternative new approaches to attain this goal. These results, along with the new broadly neutralizing antibody specificities, the role for other antibody functions, the increased understanding of HIV-1-induced changes to B cell biology, and results from the RV144 "Thai" trial showing potential modest sterilizing protection by nonneutralizing antibody responses, have renewed focus on the humoral system. In this review, recent advances in our understanding of the earliest humoral responses are discussed, highlighting presentations from the meeting on the Biology of Acute HIV Infection.

Redox rhythm reinforces the circadian clock to gate immune response.

Recent studies have shown that in addition to the transcriptional circadian clock, many organisms, including Arabidopsis, have a circadian redox rhythm driven by the organism's metabolic activities. It has been hypothesized that the redox rhythm is linked to the circadian clock, but the mechanism and the biological significance of this link have only begun to be investigated. Here we report that the master immune regulator NPR1 (non-expressor of pathogenesis-related gene 1) of Arabidopsis is a sensor of the plant's redox state and regulates transcription of core circadian clock genes even in the absence of pathogen challenge. Surprisingly, acute perturbation in the redox status triggered by the immune signal salicylic acid does not compromise the circadian clock but rather leads to its reinforcement. Mathematical modelling and subsequent experiments show that NPR1 reinforces the circadian clock without changing the period by regulating both the morning and the evening clock genes. This balanced network architecture helps plants gate their immune responses towards the morning and minimize costs on growth at night. Our study demonstrates how a sensitive redox rhythm interacts with a robust circadian clock to ensure proper responsiveness to environmental stimuli without compromising fitness of the organism.

The effect of nitric oxide surface flux on the foreign body response to subcutaneous implants.

Although the release of nitric oxide (NO) from biomaterials has been shown to reduce the foreign body response (FBR), the optimal NO release kinetics and doses remain unknown. Herein, polyurethane-coated wire substrates with varying NO release properties were implanted into porcine subcutaneous tissue for 3, 7, 21 and 42 d. Histological analysis revealed that materials with short NO release durations (i.e., 24 h) were insufficient to reduce the collagen capsule thickness at 3 and 6 weeks, whereas implants with longer release durations (i.e., 3 and 14 d) and greater NO payloads significantly reduced the collagen encapsulation at both 3 and 6 weeks. The acute inflammatory response was mitigated most notably by systems with the longest duration and greatest dose of NO release, supporting the notion that these properties are most critical in circumventing the FBR for subcutaneous biomedical applications (e.g., glucose sensors).

Improving Indwelling Glucose Sensor Performance: Porous, Dexamethasone-Releasing Coatings that Modulate the Foreign Body Response

Inflammation and the formation of an avascular fibrous capsule have been identified as the key factors controlling the wound healing associated failure of implantable glucose sensors. Our aim is to guide advantageous tissue remodeling around implanted sensor leads by the temporal release of dexamethasone (Dex), a potent anti-inflammatory agent, in combination with the presentation of a stable textured surface.

First, Dex-releasing polyurethane porous coatings of controlled pore size and thickness were fabricated using salt-leaching/gas-foaming technique. Porosity, pore size, thickness, drug release kinetics, drug loading amount, and drug bioactivity were evaluated. In vitro sensor functionality test were performed to determine if Dex-releasing porous coatings interfered with sensor performance (increased signal attenuation and/or response times) compared to bare sensors. Drug release from coatings monitored over two weeks presented an initial fast release followed by a slower release. Total release from coatings was highly dependent on initial drug loading amount. Functional in vitro testing of glucose sensors deployed with porous coatings against glucose standards demonstrated that highly porous coatings minimally affected signal strength and response rate. Bioactivity of the released drug was determined by monitoring Dex-mediated, dose-dependent apoptosis of human peripheral blood derived monocytes in culture.

The tissue modifying effects of Dex-releasing porous coatings were accessed by fully implanting Tygon® tubing in the subcutaneous space of healthy and diabetic rats. Based on encouraging results from these studies, we deployed Dex-releasing porous coatings from the tips of functional sensors in both diabetic and healthy rats. We evaluated if the tissue modifying effects translated into accurate, maintainable and reliable sensor signals in the long-term. Sensor functionality was accessed by continuously monitoring glucose levels and performing acute glucose challenges at specified time points.

Sensors treated with porous Dex-releasing coatings showed diminished inflammation and enhanced vascularization of the tissue surrounding the implants in healthy rats. Functional sensors with Dex-releasing porous coatings showed enhanced sensor sensitivity over a 21-day period when compared to controls. Enhanced sensor sensitivity was accompanied with an increase in sensor signal lag and MARD score. These results indicated that Dex-loaded porous coatings were able to elicit a favorable tissue response, and that such tissue microenvironment could be conducive towards extending the performance window of glucose sensors in vivo.

The diabetic pilot animal study showed differences in wound healing patters between healthy and diabetic subjects. Diabetic rats showed lower levels of inflammation and vascularization of the tissue surrounding implants when compared to their healthy counterparts. Also, functional sensors treated with Dex-releasing porous coatings did not show enhanced sensor sensitivity over a 21-day period. Moreover, increased in sensor signal lag and MARD scores were present in porous coated sensors regardless of Dex-loading when compared to bare implants. These results suggest that the altered wound healing patterns presented in diabetic tissues may lead to premature sensor failure when compared to sensors implanted in healthy rats.

Exercise elicits phase shifts and acute alterations of melatonin that vary with circadian phase

To examine the immediate phase-shifting effects of high-intensity exercise of a practical duration (1 h) on human circadian phase, five groups of healthy men 20-30 yr of age participated in studies involving no exercise or exposure to morning, afternoon, evening, or nocturnal exercise. Except during scheduled sleep/dark and exercise periods, subjects remained under modified constant routine conditions allowing a sleep period and including constant posture, knowledge of clock time, and exposure to dim light intensities averaging (±SD) 42 ± 19 lx. The nocturnal onset of plasma melatonin secretion was used as a marker of circadian phase. A phase response curve was used to summarize the phase-shifting effects of exercise as a function of the timing of exercise. A significant effect of time of day on circadian phase shifts was observed (P < 0.004). Over the interval from the melatonin onset before exercise to the first onset after exercise, circadian phase was significantly advanced in the evening exercise group by 30 ± 15 min (SE) compared with the phase delays observed in the no-exercise group (-25 ± 14 min, P < 0.05). Phase shifts in response to evening exercise exposure were attenuated on the second day after exercise exposure and no longer significantly different from phase shifts observed in the absence of exercise. Unanticipated transient elevations of melatonin levels were observed in response to nocturnal exercise and in some evening exercise subjects. Taken together with the results from previous studies in humans and diurnal rodents, the current results suggest that 1) a longer duration of exercise exposure and/or repeated daily exposure to exercise may be necessary for reliable phase-shifting of the human circadian system and that 2) early evening exercise of high intensity may induce phase advances relevant for nonphotic entrainment of the human circadian system.

BRCA1 and GADD45 mediated G2/M cell cycle arrest in response to antimicrotubule agents

BRCA1 is a tumour suppressor gene implicated in the predisposition to early onset breast and ovarian cancer. We have generated cell lines with inducible expression of BRCA1 to evaluate its role in mediating the cellular response to various chemotherapeutic drugs commonly used in the treatment of breast and ovarian cancer. Induction of BRCA1 in the presence of Taxol and Vincristine resulted in a dramatic increase in cell death; an effect that was preceded by an acute arrest at the G2/M phase of the cell cycle and which correlated with BRCA1 mediated induction of GADD45. A proportion of the arrested cells were blocked in mitosis suggesting activation of both a G2 and a mitotic spindle checkpoint. In contrast, no specific interaction was observed between BRCA1 induction and treatment of cells with a range of DNA damaging agents including Cisplatin and Adriamycin. Inducible expression of GADD45 in the presence of Taxol induced both G2 and mitotic arrest in these cells consistent with a role for GADD45 in contributing to these effects. Our results support a role for both BRCA1 and GADD45 in selectively regulating a G2/M checkpoint in response to antimicrotubule agents and raise the possibility that their expression levels in cells may contribute to the toxicity observed with these compounds.

Truncated estrogen receptor alpha 46-kDa isoform in human endothelial cells: relationship to acute activation of nitric oxide synthase.

Background Estrogen acutely activates endothelial nitric oxide synthase (eNOS). However, the identity of the receptors involved in this rapid response remains unclear. Methods and Results We detected an estrogen receptor (ER) transcript in human endothelial cells that encodes a truncated 46-kDa ER (1a-hER-46). A corresponding 46-kDa ER protein was identified in endothelial cell lysates. Transfection of cDNAs encoding the full-length ER (ER-66) and 1a-hER-46 resulted in appropriately sized recombinant proteins identified by anti-ER antibodies. Confocal microscopy revealed that a proportion of both ER-66 and hER-46 was localized outside the nucleus and mediated specific cell-surface binding of estrogen as assessed by FITC-conjugated, BSA-estrogen binding studies. Both ER isoforms colocalized with eNOS and mediated acute activation of eNOS in response to estrogen stimulation. However, estrogen-stimulated transcriptional activation mediated by 1a-hER-46 was much less than with ER-66. Furthermore, 1a-hER-46 inhibited classical hER-66 mediated transcriptional activation in a dominant-negative fashion. Conclusions These findings suggest that expression of an alternatively spliced, truncated ER isoform in human endothelial cells confers a unique ability to mediate acute but not transcriptional responses to estrogen.

Ischemic preconditioning before lower limb ischemia-reperfusion protects against acute lung injury

Objective: Prolonged limb ischemia followed by reperfusion (I/R) is associated with a systemic inflammatory response syndrome and remote acute lung injury. Ischemic preconditioning (IPC), achieved with repeated brief periods of I/R before the prolonged ischemic period, has been shown to protect skeletal muscle against ischemic injury. The aim of this study was to ascertain whether IPC of the limb before I/R injury also attenuates systemic inflammation and acute lung injury in a fully resuscitated porcine model of hind limb I/R. Methods: This prospective, randomized, controlled, experimental animal study was performed in a university-based animal research facility with 18 male Landrace pigs that weighed from 30 to 35 kg. Anesthetized ventilated swine were randomized (n = 6 per group) to three groups: sham-operated control group, I/R group (2 hours of bilateral hind limb ischemia and 2.5 hours of reperfusion), and IPC group (three cycles of 5 minutes of ischemia/5 minutes of reperfusion immediately preceding I/R). Plasma was separated and stored at -70° C for later determination of plasma tumor necrosis factor-a and interleukin-6 with bioassay as markers of systemic inflammation. Circulating phagocytic cell priming was assessed with a whole blood chemiluminescence assay. Lung tissue wet-to-dry weight ratio and myeloperoxidase concentration were markers of edema and neutrophil sequestration, respectively. The alveolar-arterial oxygen gradient and pulmonary artery pressure were indices of lung function. Results: In a porcine model, bilateral hind limb (I/R) injury significantly increased plasma interleukin-6 concentrations, circulating phagocytic cell priming, and pulmonary leukosequestration, edema, and impaired gas exchange. Conversely, pigs treated with IPC before the onset of the ischemic period had significantly reduced interleukin-6 levels, circulating phagocytic cell priming, and experienced significantly less pulmonary edema, leukosequestration, and respiratory failure. Conclusion: Lower limb IPC protects against systemic inflammation and acute lung injury in lower limb I/R injury.

Bactericidal/permeability-increasing protein attenuates systemic inflammation and acute lung injury in porcine lower limb ischemia-reperfusion injury.

OBJECTIVE: To investigate the role of recombinant bactericidal/permeability-increasing protein (rBPI21) in the attenuation of the sepsis syndrome and acute lung injury associated with lower limb ischemia-reperfusion (I/R) injury. SUMMARY BACKGROUND DATA: Gut-derived endotoxin has been implicated in the conversion of the sterile inflammatory response to a lethal sepsis syndrome after lower torso I/R injury. rBPI21 is a novel antiendotoxin therapy with proven benefit in sepsis. METHODS: Anesthetized ventilated swine underwent midline laparotomy and bilateral external iliac artery occlusion for 2 hours followed by 2.5 hours of reperfusion. Two groups (n = 6 per group) were randomized to receive, by intravenous infusion over 30 minutes, at the start of reperfusion, either thaumatin, a control-protein preparation, at 2 mg/kg body weight, or rBPI21 at 2 mg/kg body weight. A control group (n = 6) underwent laparotomy without further treatment and was administered thaumatin at 2 mg/kg body weight after 2 hours of anesthesia. Blood from a carotid artery cannula was taken every half-hour for arterial blood gas analysis. Plasma was separated and stored at -70 degrees C for later determination of plasma tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 by bioassay, and IL-8 by enzyme-linked immunosorbent assay (ELISA), as a markers of systemic inflammation. Plasma endotoxin concentration was measured using ELISA. Lung tissue wet-to-dry weight ratio and myeloperoxidase concentration were used as markers of edema and neutrophil sequestration, respectively. Bronchoalveolar lavage protein concentration was measured by the bicinclinoic acid method as a measure of capillary-alveolar protein leak. The alveolar-arterial gradient was measured; a large gradient indicated impaired oxygen transport and hence lung injury. RESULTS: Bilateral hind limb I/R injury increased significantly intestinal mucosal acidosis, intestinal permeability, portal endotoxemia, plasma IL-6 concentrations, circulating phagocytic cell priming and pulmonary leukosequestration, edema, capillary-alveolar protein leak, and impaired gas exchange. Conversely, pigs treated with rBPI21 2 mg/kg at the onset of reperfusion had significantly reduced intestinal mucosal acidosis, portal endotoxin concentrations, and circulating phagocytic cell priming and had significantly less pulmonary edema, leukosequestration, and respiratory failure. CONCLUSIONS: Endotoxin transmigration across a hyperpermeable gut barrier, phagocytic cell priming, and cytokinemia are key events of I/R injury, sepsis, and pulmonary dysfunction. This study shows that rBPI21 ameliorates these adverse effects and may provide a novel therapeutic approach for prevention of I/R-associated sepsis syndrome.

Chk1 Suppresses a Caspase-2 Apoptotic Response to DNA Damage that Bypasses p53, Bcl-2, and Caspase-3

Evasion of DNA damage-induced cell death, via mutation of the p53 tumor suppressor or overexpression of prosurvival Bcl-2 family proteins, is a key step toward malignant transformation and therapeutic resistance. We report that depletion or acute inhibition of checkpoint kinase 1 (Chk1) is sufficient to restore ?-radiation-induced apoptosis in p53 mutant zebrafish embryos. Surprisingly, caspase-3 is not activated prior to DNA fragmentation, in contrast to classical intrinsic or extrinsic apoptosis. Rather, an alternative apoptotic program is engaged that cell autonomously requires atm (ataxia telangiectasia mutated), atr (ATM and Rad3-related) and caspase-2, and is not affected by p53 loss or overexpression of bcl-2/xl. Similarly, Chk1 inhibitor-treated human tumor cells hyperactivate ATM, ATR, and caspase-2 after ?-radiation and trigger a caspase-2-dependent apoptotic program that bypasses p53 deficiency and excess Bcl-2. The evolutionarily conserved "Chk1-suppressed" pathway defines a novel apoptotic process, whose responsiveness to Chk1 inhibitors and insensitivity to p53 and BCL2 alterations have important implications for cancer therapy. © 2008 Elsevier Inc. All rights reserved.

High bax expression is a good prognostic indicator in acute myeloid leukaemia

Most cytotoxic drugs kill cells by instigating the process of apoptosis and it has been suggested that apoptotic markers may provide an indication of tumour chemosensitivity. The aim of this study was to determine if such a relationship exists in acute myeloid leukaemia (AML). The levels of spontaneous apoptosis, bcl-2 and bax were evaluated in 56 newly diagnosed AML patients to determine if they correlated with a response to cytotoxic therapy. Spontaneous apoptosis was lower, but bcl-2, bax and the bcl-2/bax ratio were higher in AML compared with normal individuals. AML patients with high bax expression at diagnosis had significantly better prognosis for disease-free survival, event-free survival and overall survival (P = 0.016). In the standard risk group, high bax expression was in keeping with significantly improved survival. Multivariate analysis revealed bax to be an independent predictor of survival. There was a significant reduction in bcl-2 and bax expression when AML patients entered complete remission and also in relapsed AML patients who entered a second remission. This study suggests that bax is a useful prognostic indicator in AML and may assist with therapeutic decision-making for patients in the standard risk category.