Impaired ATP hydrolysis in blood plasma contributes to age-related neutrophil dysfunction.

BACKGROUND: The function of polymorphonuclear neutrophils (PMNs) decreases with age, which results in infectious and inflammatory complications in older individuals. The underlying causes are not fully understood. ATP release and autocrine stimulation of purinergic receptors help PMNs combat microbial invaders. Excessive extracellular ATP interferes with these mechanisms and promotes inflammatory PMN responses. Here, we studied whether dysregulated purinergic signaling in PMNs contributes to their dysfunction in older individuals. RESULTS: Bacterial infection of C57BL/6 mice resulted in exaggerated PMN activation that was significantly greater in old mice (64 weeks) than in young animals (10 weeks). In contrast to young animals, old mice were unable to prevent the systemic spread of bacteria, resulting in lethal sepsis and significantly greater mortality in old mice than in their younger counterparts. We found that the ATP levels in the plasma of mice increased with age and that, along with the extracellular accumulation of ATP, the PMNs of old mice became increasingly primed. Stimulation of the formyl peptide receptors of those primed PMNs triggered inflammatory responses that were significantly more pronounced in old mice than in young animals. However, bacterial phagocytosis and killing by PMNs of old mice were significantly lower than that of young mice. These age-dependent PMN dysfunctions correlated with a decrease in the enzymatic activity of plasma ATPases that convert extracellular ATP to adenosine. ATPases depend on divalent metal ions, including Ca2+, Mg2+, and Zn2+, and we found that depletion of these ions blocked the hydrolysis of ATP and the formation of adenosine in human blood, resulting in ATP accumulation and dysregulation of PMN functions equivalent to those observed in response to aging. CONCLUSIONS: Our findings suggest that impaired hydrolysis of plasma ATP dysregulates PMN function in older individuals. We conclude that strategies aimed at restoring plasma ATPase activity may offer novel therapeutic opportunities to reduce immune dysfunction, inflammation, and infectious complications in older patients.

Use of Serum Protein Measurements as Biomarkers that Can Predict the Outcome of Diabetic Foot Ulceration.

Objectives: To identify proteins that are prognostic for diabetic foot ulcer (DFU) healing and may serve as biomarkers for its management, serum samples were analyzed from diabetic mellitus (DM) patients. Approach: The serum specimens that were evaluated in this study were obtained from DM patients with DFU who participated in a prospective study and were seen biweekly until they healed their ulcer or the exit visit at 12 weeks. The group was divided into Healers (who healed their DFU during the study) and Non-Healers. Results: Interleukin (IL)-10, IL-4, IL-5, IL-6, and IL-13 and interferon-gamma were higher in the Healers while Fractalkine, IL-8, and TNFα were higher in the Non-Healers. The trajectory of IL-10 levels remained stable over time within and across groups, resulting in a strong prognostic ability for the prospective DFU healing course. Classification and Regression Tree analysis created an 11-node decision tree with healing status as the categorical response. Innovation: Consecutive measurements of proteins associated with wound healing can identify biomarkers that can predict DFU healing over a 12-week period. IL-10 was the strongest candidate for prediction. Conclusion: Measurement of serum proteins can serve as a successful strategy in guiding clinical management of DFU. The data also indicate likely superior performance of building a multiprotein biomarker score instead of relying on single biomarkers.

ATP release from influenza-infected lungs enhances neutrophil activation and promotes disease progression.

BACKGROUND: ATP enhances neutrophil responses, but little is known about the role of ATP in influenza infections. METHODS: We used a mouse influenza model to study if ATP release is associated with neutrophil activation and disease progression. RESULTS: Influenza infection increased pulmonary ATP levels 5-fold and plasma ATP levels 3-fold over the levels in healthy mice. Adding ATP at those concentrations to blood from healthy mice primed their neutrophils and enhanced CD11b and CD63 expression, CD62L shedding, and reactive oxygen species production in response to formyl peptide receptor (FPR) stimulation. Influenza infection also primed neutrophils in vivo, resulting in FPR-induced CD11b expression and CD62L shedding up to 3-times higher than that of uninfected mice. In infected mice, large numbers of neutrophils entered the lungs. These cells were significantly more activated than peripheral neutrophils of infected and pulmonary neutrophils of healthy mice. Plasma ATP levels of infected mice and influenza disease progression corresponded with the numbers and activation level of their pulmonary neutrophils. CONCLUSION: Our findings suggest that ATP release from the lungs of infected mice promotes influenza disease progression by priming peripheral neutrophils that become strongly activated and cause pulmonary tissue damage after their recruitment to the lungs.

Experimental treatments in clinical trials for diabetic foot ulcers: wound healers in the pipeline.

INTRODUCTION: Diabetes affects 400 million people globally and patients and causes nephropathy, neuropathy, and vascular disease. Amongst these complications, diabetic foot ulcers remain a substantial problem for patients and clinicians. Aggressive wound care and antibiotics remain important for the healing of these chronic wounds, but even when treated these chronic ulcers can lead to infection and amputations. AREAS COVERED: This paper reviews the pathophysiology of diabetic foot ulcers and the current management strategies. Then, it discusses novel therapeutics such as topical oxygen therapy as well as autologous patches and macrophage creams. EXPERT OPINION: Diabetic foot ulcers are a substantial problem for patients and clinicians. Early identification, aggressive wound care, and normoglycemia remain the standard of care, however when these fail it is important to adapt. Since each patient and wound vary drastically we believe they should be treated as such. For patient with intact perfusion, topical ON101 and sucrose octasulfate creams can help. While patient with peripheral arterial disease should consider topical oxygen therapy as an adjunct. However, as scientists gain a better understanding of the pathophysiology behind DFUs, the hope is that this new wave of therapeutics will emerge.

ATP breakdown in plasma of children limits the antimicrobial effectiveness of their neutrophils.

Neutrophils (PMNs) require extracellular ATP and adenosine (ADO) to fight bacterial infections, which often have life-threatening consequences in pediatric patients. We wondered whether the ATP and ADO levels in the plasma of children change with age and if these changes influence the antimicrobial efficacy of the PMNs of these children. We measured plasma concentrations of ATP and ADO and the activities of the enzymes responsible for the breakdown of these mediators in plasma samples from healthy children and adolescents (n = 45) ranging in age from 0.2 to 15 years. In addition, using blood samples of these individuals, we compared how effective their PMNs were in the phagocytosis of bacteria. In an experimental sepsis model with young (10 days) and adolescent mice (10 weeks), we studied how age influenced the resilience of these animals to bacterial infections and whether addition of ATP could improve the antimicrobial capacity of their PMNs. We found that plasma ATP levels correlated with age and were significantly lower in infants (< 1 year) than in adolescents (12-15 years). In addition, we observed significantly higher plasma ATPase and adenosine deaminase activities in children (< 12 years) when compared to the adolescent population. The activities of these ATP and ADO breakdown processes correlated inversely with age and with the ability of PMNs to phagocytize bacteria. Similar to their human counterparts, young mice also had significantly lower plasma ATP levels when compared to adolescent animals. In addition, we found that mortality of young mice after bacterial infection was significantly higher than that of adolescent mice. Moreover, bacterial phagocytosis by PMNs of young mice was weaker when compared to that of older mice. Finally, we found that ATP supplementation could recover bacterial phagocytosis of young mice to levels similar to those of adolescent mice. Our findings suggest that rapid ATP hydrolysis in the plasma of young children lowers the antimicrobial functions of their PMNs and that this may contribute to the vulnerability of pediatric patients to bacterial infections.

Optimized flow cytometry assays to monitor neutrophil activation in human and mouse whole blood samples.

Polymorphonuclear neutrophils (PMNs) protect the host from invading microorganisms. However, excessively activated PMNs can also cause damage to host tissues under inflammatory conditions. Here we developed simple assays to determine the activation state of PMNs in human whole blood that contains soluble mediators known to influence PMN functions. Because mouse models are widely used to study the role of PMNs in infectious and inflammatory diseases, we adapted these assays for the rapid and reliable assessment of PMN functions in murine blood samples. Freshly collected whole blood samples were stimulated with agonists of the formyl peptide receptors (FPR) of PMNs and changes in reactive oxygen species (ROS) production and the expression of CD11b, CD62L (L-selectin), CD66b, and CD63 on the cell surface were analyzed with flow cytometry. We optimized these assays to minimize inadvertent interferences such as cell stress generated during sample handling and the loss of plasma mediators that regulate PMN functions. Human PMNs readily responded to the FPR agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP). The most sensitive responses of human PMNs to fMLP were CD11b, CD62L, and CD66b expression with half maximal effective concentrations (EC50) of 5, 8, and 6 nM fMLP, respectively. CD63 expression and ROS production required markedly higher fMLP concentrations with EC50 values of 19 and 50 nM fMLP, respectively. Mouse PMNs did not respond well to fMLP and required significantly higher concentrations of the FPR agonist WKYMVm (W-peptide) to achieve equivalent cell activation. The most sensitive response of mouse PMNs was ROS production with an EC50 of 38 nM W-peptide. Because mice do not express CD66b, we only assessed the expression of CD62L, CD11b, and CD63 with EC50 values of 54, 119, and 355 nM W-peptide, respectively. Validation of our optimized assays showed that they sensitively detect the responses of human PMNs to priming with endotoxin in vitro as well as the corresponding responses of murine PMNs to bacterial infection in a sepsis model. We conclude that these optimized assays could be useful tools for the monitoring of patients with infections, sepsis, and other inflammatory conditions as well as for the design and interpretation of preclinical studies of these diseases in mouse models.

Optimized HPLC method to elucidate the complex purinergic signaling dynamics that regulate ATP, ADP, AMP, and adenosine levels in human blood.

ATP released into the bloodstream regulates immune responses and other physiological functions. Excessive accumulation of extracellular ATP interferes with these functions, and elevated plasma ATP levels could indicate infections and other pathological disorders. However, there is considerable disagreement about what constitutes normal plasma ATP levels. Therefore, we optimized a method to accurately assess ATP concentrations in blood. We found that rapid chilling of heparinized blood samples is essential to preserve in vivo ATP levels and that differential centrifugation minimizes inadvertent ATP release due to cell damage and mechanical stress. Plasma samples were stabilized with perchloric acid, etheno-derivatized, and delipidated for sensitive analysis of ATP and related compounds using high-performance liquid chromatography (HPLC) and fluorescence detection. We measured 33 ± 20 nM ATP, 90 ± 45 nM ADP, 100 ± 55 nM AMP, and 81 ± 51 nM adenosine in the blood of healthy human adults (n = 10). In critically ill patients, ATP levels were 6 times higher than in healthy subjects. The anticoagulant greatly affected results. ATP levels were nearly 8 times higher in EDTA plasma than in heparin plasma, while AMP levels were 3 times lower and adenosine was entirely absent in EDTA plasma. If EDTA blood was not immediately chilled, ATP, ADP, and AMP levels continued to rise, which indicates that EDTA interferes with the endogenous mechanisms that regulate plasma adenylate levels. Our optimized method eliminates artifacts that prevent accurate determination of plasma adenylates and will be useful for studying mechanisms that regulate adenylate levels and for monitoring of pathological processes in patients with infections and other diseases.

Characterization of a novel herbicide and antibiotic-resistant Chlorella sp. with an extensive extracellular matrix.

A herbicide and antibiotic-resistant microalgal strain, isolated from a eutrophic site at Giofyros river (Heraklion, Crete, Greece) was extensively characterized. In the presence of relatively high concentrations of common photosynthesis inhibitors (DCMU and atrazine), as well as various antibiotics (spectinomycin, kanamycin, and chloramphenicol), the green microalga was able to increase its biomass in approximately equal levels compared to the control. Despite the high concentrations of the inhibitors, photosynthetic efficiency and chlorophyll a amount per dry cell biomass were comparable to those of control cultures in almost all cases. 18S rDNA analysis showed that this microalga belongs to the Chlorella genus. Optical and electron microscopy studies revealed the presence of an extensive extracellular matrix (EM) that surrounds the cells and plays an important role in colony formation and cell-cell interactions. Fourier transform infrared spectroscopy provided evidence that the EM consists of a polysaccharide. This matrix could be separated from the cells with a simple centrifugation. Depending on growth conditions, the dry cell biomass of this Chlorella strain was found to contain 35-39% proteins and 27-42% carbohydrates. The results of this study have demonstrated that the EM plays a protective role for cell homeostasis maintenance against the various chemical agents. This green microalga is a suitable candidate for further studies regarding sustainable biomass production in waste waters for a series of applications.

The effect of nitrogen starvation on membrane lipids of Synechocystis sp. PCC 6803 investigated by using easy ambient sonic-spray ionization mass spectrometry.

BACKGROUND: Glycerolipids are important components of membranes in cyanobacteria that possess vital roles in biological processes. The effect of nitrogen deprivation on membrane lipids of Synechocystis sp. PCC 6803 lipids has not been previously examined. METHODS: Easy ambient sonic-spray ionization mass spectrometry (EASI-MS) was used for the analysis of Synechocystis 6803 cells with minimal sample preparation, providing rapid qualitative and relative quantitative information on the lipid content of their membranes. RESULTS: Changes in the degree of unsaturation of membrane lipids were observed for cells grown under normal conditions during different growth phases. This physiological remodeling was disrupted when nitrogen was withdrawn from the cultivation medium. However, this disruption was reversed when the cells were resuspended in normal N-containing medium. Mass spectrometric data were supported by examination of cells by electron microscopy. CONCLUSIONS: EASI-MS was applied for the first time in the analysis of Synechocystis 6803 cells grown under nitrogen deprived conditions and was found to be a powerful technique operating in a high-throughput manner for the rapid lipid profiling of cells at different growth stages and under different growth conditions. GENERAL SIGNIFICANCE: The effect of nitrogen deprivation on membrane lipids of Synechocystis 6803 cells was revealed using an ambient ionization technique which enabled high-throughput cell analysis with minimal sample preparation. The results obtained have the potential to be used in future studies to decipher the involvement of enzymes in the observed lipid profile changes.