CD59 Protects Primary Human Cerebrovascular Smooth Muscle Cells from Cytolytic Membrane Attack Complex.

Cerebral amyloid angiopathy is characterized by a weakening of the small and medium sized cerebral arteries, as their smooth muscle cells are progressively replaced with acellular amyloid ß, increasing vessel fragility and vulnerability to microhemorrhage. In this context, an aberrant overactivation of the complement system would further aggravate this process. The surface protein CD59 protects most cells from complement-induced cytotoxicity, but expression levels can fluctuate due to disease and vary between cell types. The degree to which CD59 protects human cerebral vascular smooth muscle (HCSM) cells from complement-induced cytotoxicity has not yet been determined. To address this shortcoming, we selectively blocked the activity of HCSM-expressed CD59 with an antibody and challenged the cells with complement, then measured cellular viability. Unblocked HCSM cells proved resistant to all tested concentrations of complement, and this resistance decreased progressively with increasing concentrations of anti-CD59 antibody. Complete CD59 blockage, however, did not result in total loss of cellular viability, suggesting that additional factors may have some protective functions. Taken together, this implies that CD59 plays a predominant role in HCSM cellular protection against complement-induced cytotoxicity. Over-expression of CD59 could be an effective means of protecting these cells from excessive complement system activity, with consequent reduction in the incidence of microhemorrhage. The precise extent to which cellular repair mechanisms and other complement repair proteins contribute to this resistance has yet to be fully elucidated.

Neutrophil Extracellular DNA Traps in Response to Infection or Inflammation, and the Roles of Platelet Interactions.

Neutrophils present the host's first line of defense against bacterial infections. These immune effector cells are mobilized rapidly to destroy invading pathogens by (a) reactive oxygen species (ROS)-mediated oxidative bursts and (b) via phagocytosis. In addition, their antimicrobial service is capped via a distinct cell death mechanism, by the release of their own decondensed nuclear DNA, supplemented with a variety of embedded proteins and enzymes. The extracellular DNA meshwork ensnares the pathogenic bacteria and neutralizes them. Such neutrophil extracellular DNA traps (NETs) have the potential to trigger a hemostatic response to pathogenic infections. The web-like chromatin serves as a prothrombotic scaffold for platelet adhesion and activation. What is less obvious is that platelets can also be involved during the initial release of NETs, forming heterotypic interactions with neutrophils and facilitating their responses to pathogens. Together, the platelet and neutrophil responses can effectively localize an infection until it is cleared. However, not all microbial infections are easily cleared. Certain pathogenic organisms may trigger dysregulated platelet-neutrophil interactions, with a potential to subsequently propagate thromboinflammatory processes. These may also include the release of some NETs. Therefore, in order to make rational intervention easier, further elucidation of platelet, neutrophil, and pathogen interactions is still needed.

Sialic acids Neu5Ac and KDN in adipose tissue samples from individuals following habitual vegetarian or non-vegetarian dietary patterns.

Sialic acids (Sias) are a class of sugar molecules with a parent nine-carbon neuraminic acid, generally present at the ends of carbohydrate chains, either attached to cellular surfaces or as secreted glycoconjugates. Given their position and structural diversity, Sias modulate a wide variety of biological processes. However, little is known about the role of Sias in human adipose tissue, or their implications for health and disease, particularly among individuals following different dietary patterns. The goal of this study was to measure N-Acetylneuraminic acid (Neu5Ac), N-Glycolylneuraminic acid (Neu5Gc), and 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) concentrations in adipose tissue samples from participants in the Adventist Health Study-2 (AHS-2) and to compare the abundance of these Sias in individuals following habitual, long-term vegetarian or non-vegetarian dietary patterns. A method was successfully developed for the extraction and detection of Sias in adipose tissue. Sias levels were quantified in 52 vegans, 56 lacto-vegetarians, and 48 non-vegetarians using LC-MS/MS with Neu5Ac-D-1,2,3-13C3 as an internal standard. Dietary groups were compared using linear regression. Vegans and lacto-ovo-vegetarians had significantly higher concentrations of Neu5Ac relative to non-vegetarians. While KDN levels tended to be higher in vegans and lacto-ovo-vegetarians, these differences were not statistically significant. However, KDN levels were significantly inversely associated with body mass index. In contrast, Neu5Gc was not detected in human adipose samples. It is plausible that different Neu5Ac concentrations in adipose tissues of vegetarians, compared to those of non-vegetarians, reflect a difference in the baseline inflammatory status between the two groups. Epidemiologic studies examining levels of Sias in human adipose tissue and other biospecimens will help to further explore their roles in development and progression of inflammatory conditions and chronic diseases.

Local and Systemic Effects of Porphyromonas gingivalis Infection.

Porphyromonas gingivalis, a gram-negative anaerobe, is a leading etiological agent in periodontitis. This infectious pathogen can induce a dysbiotic, proinflammatory state within the oral cavity by disrupting commensal interactions between the host and oral microbiota. It is advantageous for P. gingivalis to avoid complete host immunosuppression, as inflammation-induced tissue damage provides essential nutrients necessary for robust bacterial proliferation. In this context, P. gingivalis can gain access to the systemic circulation, where it can promote a prothrombotic state. P. gingivalis expresses a number of virulence factors, which aid this pathogen toward infection of a variety of host cells, evasion of detection by the host immune system, subversion of the host immune responses, and activation of several humoral and cellular hemostatic factors.

A pilot study on the biochemical effects of repeated administration of 24% oral sucrose vs. 30% oral dextrose on urinary markers of adenosine triphosphate degradation.

OBJECTIVES: Premature neonates often receive oral sucrose or dextrose before tissue-damaging procedures (TDPs). Previous work showed that a single dose of sucrose, but not dextrose, increased cellular energy utilization and ATP degradation. This pilot study probes the effects of repeated administration of sucrose or dextrose on energy metabolism. METHODS: Urinary markers of ATP metabolism (hypoxanthine, xanthine, uric acid) are measured in premature neonates randomized to receive: (a) standard of care, (b) 0.2 ml 24% oral sucrose, or (c) 0.2 ml 30% oral dextrose, before every painful procedure on days-of-life 3-7. RESULTS: Standard of care is associated with highest xanthine/creatinine and uric acid/creatinine, likely because of fewer pain treatments. Benefits of repeated oral sucrose are unclear. Neonates receiving oral dextrose had lower xanthine/creatinine and uric acid/creatinine. CONCLUSIONS: Repeated treatments of neonatal procedural pain with 30% oral dextrose are less energetically demanding. Larger clinical studies are needed for comparison with sucrose treatments.

Platelet and neutrophil responses to Porphyromonas gingivalis in human whole blood.

Porphyromonas gingivalis is a causative agent for periodontal disease. Binding of platelets to this gram-negative anaerobe can regulate host hemostatic (thrombus forming) and immune (neutrophil interacting) responses during bacterial infection. Additionally, in response to bacterial pathogens neutrophils can release their DNA, forming highly prothrombotic neutrophil extracellular traps (NETs), which then further enhance platelet responses. This study evaluates the role of P. gingivalis on platelet expression of CD62P, platelet-neutrophil interactions, and labeled neutrophil-associated DNA. Human whole blood was preincubated with varying P. gingivalis concentrations, with or without subsequent addition of adenosine diphosphate (ADP). Flow cytometry was employed to measure platelet expression of CD62P using PerCP-anti-CD61 and PE-anti-CD62P, platelet-neutrophil interactions using PerCP-anti-CD61 and FITC-anti-CD16, and the release of neutrophil DNA using FITC-anti-CD16 and Sytox Blue labeling. Preincubation with a high (6.25 × 106  CFU/mL) level of P. gingivalis significantly increased platelet expression of CD62P in ADP treated and untreated whole blood. In addition, platelet-neutrophil interactions were significantly increased after ADP stimulation, following 5-22 min preincubation of blood with high P. gingivalis CFU. However, in the absence of added ADP, platelet-neutrophil interactions increased in a manner dependent on the preincubation time with P. gingivalis. Moreover, after ADP addition, 16 min preincubation of whole blood with P. gingivalis led to increased labeling of neutrophil-associated DNA. Taken together, the results suggest that the presence of P. gingivalis alters platelet and neutrophil responses to increase platelet activation, platelet interactions with neutrophils, and the level of neutrophil antimicrobial NETs.

Platelet plug formation in whole blood is enhanced in the presence of Porphyromonas gingivalis.

Porphyromonas gingivalis is a gram-negative anaerobic bacterium and an etiologic agent of adult periodontitis. By inducing a dysbiotic state within the host microbiota it contributes to a chronic inflammatory environment in the oral cavity. Under some circumstances, the oral bacteria may gain access to systemic circulation. While the most widely recognized function of platelets is to reduce hemorrhage in case of vascular damage, it is known that platelets are also involved in the hematologic responses to bacterial infections. Some pathogenic bacteria can interact with platelets, triggering their activation and aggregation. The aim of this study was to assess platelet responses to the presence of P. gingivalis in whole blood. Human whole blood was pretreated with P. gingivalis and then platelet plug formation was measured under high shear conditions using the PFA-100. In the presence of P. gingivalis, time for a platelet plug to occlude the aperture in the collagen/ADP cartridge was shortened in a manner dependent on bacterial concentration and the duration of bacterial preincubation of blood. P. gingivalis enhances thrombus forming potential of platelets in whole blood.

Oral dextrose reduced procedural pain without altering cellular ATP metabolism in preterm neonates: a prospective randomized trial.

OBJECTIVE: To examine the effects of 30% oral dextrose on biochemical markers of pain, adenosine triphosphate (ATP) degradation, and oxidative stress in preterm neonates experiencing a clinically required heel lance. STUDY DESIGN: Utilizing a prospective study design, preterm neonates that met study criteria (n = 169) were randomized to receive either (1) 30% oral dextrose, (2) facilitated tucking, or (3) 30% oral dextrose and facilitated tucking 2 min before heel lance. Plasma markers of ATP degradation (hypoxanthine, uric acid) and oxidative stress (allantoin) were measured before and after the heel lance. Pain was measured using the premature infant pain profile-revised (PIPP-R). RESULTS: Oral dextrose, administered alone or with facilitated tucking, did not alter plasma markers of ATP utilization and oxidative stress. CONCLUSION: A single dose of 30% oral dextrose, given before a clinically required heel lance, decreased signs of pain without increasing ATP utilization and oxidative stress in premature neonates.

Oxidative Stress Biomarker Decreased in Preterm Neonates Treated With Kangaroo Mother Care.

OBJECTIVE: Due to physiological and metabolic immaturity, prematurely born infants are at increased risk because of maternal separation in many neonatal intensive care units (NICUs). The stress induced from maternal-infant separation can lead to well-documented short-term physiologic instability and potentially lifelong neurological, sociological, or psychological sequelae. Based on previous studies of kangaroo mother care (KMC) that demonstrated improvement in physiologic parameters, we examined the impact of KMC on physiologic measures of stress (abdominal temperature, heart rate, oxygen saturation, perfusion index, near-infrared spectrometry), oxidative stress, and energy utilization/conservation in preterm infants. METHODS: In this randomized, stratified study of premature neonates, we compared the effects on urinary concentrations of biomarkers of energy utilization and oxidative stress of 1 hr of KMC versus incubator care on Day 3 of life in intervention-group babies (n = 26) and control-group babies (n = 25), respectively. On Day 4, both groups received 1 hr of KMC. Urinary samples were collected 3 hr before and 3 hr after intervention/incubator care on both days. Energy utilization was assessed by measures of adenosine triphosphate (ATP) degradation (i.e., hypoxanthine, xanthine, and uric acid). Oxidative stress was assessed using urinary allantoin. Mixed-models analysis was used to assess differences in purine/allantoin. RESULTS: Mean allantoin levels over Days 3 and 4 were significantly lower in the KMC group than in the control group (p = .026). CONCLUSIONS: Results provide preliminary evidence that KMC reduces neonatal oxidative stress processes and that urinary allantoin could serve as an effective noninvasive marker for future studies.

Platelet-Neutrophil Interactions Are Lower in Cord Blood of Premature Newborns.

OBJECTIVE: To quantify platelet-neutrophil interaction by flow cytometry, in newborn cord blood, as a function of gestational age. RATIONALE: Little is known about platelet function markers in the newborn, and developmental variations in these markers are not well described. METHODS: Cord blood samples were obtained from 64 newborns between 23 and 40 weeks' gestation. The neonates were grouped into three categories: preterm (< 34 weeks' gestation, n = 21), late preterm (34 to < 37 weeks' gestation, n = 22), and term (≥37 weeks' gestation, n = 21). We monitored the expression of P-selectin and the formation of platelet-neutrophil aggregates (PNAs) by flow cytometry while using adenosine 5'-diphosphate (ADP) or thrombin receptor-activating peptide (TRAP) as agonists. RESULTS: PNAs were significantly lower in preterm compared to term neonates after TRAP or ADP stimulations (11.5 ± 5.2% vs. 19.9 ± 9.1%, p < 0.001, or 24.0 ± 10.1% vs. 39.1 ± 18.2%, p = 0.008, respectively). The expression of P-selectin also tended to be lower in preterm neonates, with significant positive correlations between P-selectin expression and PNA formation. CONCLUSIONS: The potential formation of PNAs correlates with gestational age. This suggests that the development of functional competencies of platelets and neutrophils continues throughout gestation, progressively enabling interactions between them.

The Energy Costs of Prematurity and the Neonatal Intensive Care Unit (NICU) Experience.

Premature neonates are in an energy deficient state due to (1) oxygen desaturation and hypoxia events, (2) painful and stressful stimuli, (3) illness, and (4) neurodevelopmental energy requirements. Failure to correct energy deficiency in premature infants may lead to adverse effects such as neurodevelopmental delay and negative long-term metabolic and cardiovascular outcomes. The effects of energy dysregulation and the challenges that clinicians in the Neonatal Intensive Care Unit (NICU) face in meeting the premature infant's metabolic demands are discussed. Specifically, the focus is on the effects of pain and stress on energy homeostasis. Energy deficiency is a complex problem and requires a multi-faceted solution to promote optimum development of premature infants.

The relationship of red blood cell transfusion to intestinal mucosal injury in premature infants.

OBJECTIVE: To determine the incidence of intestinal mucosal injury before and after transfusions in premature infants. STUDY DESIGN: Urine was collected throughout the hospital stay of 62 premature infants and specimens obtained within 24h before and after transfusion were assayed for intestinal fatty acid binding protein (iFABP). A urinary iFABP:creatinine ratio (iFABPu:Cru) of 2.0pg/nmol was considered elevated. RESULT: Forty-nine infants were transfused. iFABPu:Cru was elevated following 71 (75.6%) of 94 transfusions for which urine was available. In 51 (71.8%) of these, iFABPu:Cru was also elevated prior to the transfusion. Among four cases of transfusion-associated NEC, iFABPu was elevated following every sentinel transfusion and prior to three of them. CONCLUSION: Subclinical intestinal mucosal injury is frequent following blood transfusions in premature infants and, when present, usually precedes transfusion. This suggests that transfusion may not be a primary mediator of intestinal injury so much as anemia and its associated conditions. LEVEL OF EVIDENCE: Prognosis study/level 3.

Urinary Allantoin Is Elevated in Severe Intraventricular Hemorrhage in the Preterm Newborn.

Germinal matrix intraventricular hemorrhage (IVH) is the most common type of intracranial hemorrhage observed in preterm neonates. It is a precursor of poor neurocognitive development, cerebral palsy, and death. The pathophysiology is not well defined, but damage to the fragile germinal matrix vasculature may be due to free radicals generated during inflammation and as a consequence of ischemia followed by reperfusion. Assessment of the oxidative stress status in these infants is therefore important. Urinary allantoin concentration was measured in preterm neonates as a marker of oxidative stress associated with IVH. Urine was collected from 44 preterm neonates at four time points between 24 and 72 hours of life (HOL), and the allantoin content was determined by gas chromatography mass spectrometry (GCMS). Records were retrospectively reviewed, and the incidence and severity of IVH was categorized as follows: no IVH (n = 24), mild (grade 1-2) IVH (n = 13), and severe (grade 3-4) IVH (n = 7). Neonates with severe IVH showed significantly elevated allantoin levels vs subjects with no IVH from 36 HOL (0.098 ± 0.013 µmol and 0.043 ± 0.007 µmol, respectively, p = 0.002). The allantoin concentration remained elevated even at 72 HOL (0.079 ± 0.014 µmol and 0.033 ± 0.008 µmol, respectively, p = 0.021). There were no significant differences in allantoin levels in the no IVH and mild IVH groups. IVH was diagnosed by head imaging on average at about 11th postnatal day. Urinary allantoin levels were significantly elevated during the first 3 days of life in the neonates subsequently diagnosed with severe IVH, suggesting that oxidative stress might be a crucial factor in IVH pathogenesis. Further studies are needed to assess the usefulness of urinary allantoin in early identification of preterm infants at risk for or with severe IVH and monitoring of the response to interventions designed to prevent or treat it.

Antibacterial Activity of Partially Oxidized Ag/Au Nanoparticles against the Oral Pathogen Porphyromonas gingivalis W83.

Advances in nanotechnology provide opportunities for the prevention and treatment of periodontal disease. While physicochemical properties of Ag containing nanoparticles (NPs) are known to influence the magnitude of their toxicity, it is thought that nanosilver can be made less toxic to eukaryotes by passivation of the NPs with a benign metal. Moreover, the addition of other noble metals to silver nanoparticles, in the alloy formulation, is known to alter the silver dissolution behavior. Thus, we synthesized glutathione capped Ag/Au alloy bimetallic nanoparticles (NPs) via the galvanic replacement reaction between maltose coated Ag NPs and chloroauric acid (HAuCl4) in 5% aqueous triblock F127 copolymer solution. We then compared the antibacterial activity of the Ag/Au NPs to pure Ag NPs on Porphyromonas gingivalis W83, a key pathogen in the development of periodontal disease. Only partially oxidized glutathione capped Ag and Ag/Au (Au:Ag≈0.2) NPs inhibited the planktonic growth of P. gingivalis W83. This effect was enhanced in the presence of hydrogen peroxide, which simulates the oxidative stress environment in the periodontal pocket during chronic inflammation.

Oral sucrose for heel lance enhances adenosine triphosphate use in preterm neonates with respiratory distress.

OBJECTIVE: To examine the effects of oral sucrose on procedural pain, and on biochemical markers of adenosine triphosphate utilization and oxidative stress in preterm neonates with mild to moderate respiratory distress. STUDY DESIGN: Preterm neonates with a clinically required heel lance that met study criteria (n = 49) were randomized into three groups: (1) control (n = 24), (2) heel lance treated with placebo and non-nutritive sucking (n = 15) and (3) heel lance treated with sucrose and non-nutritive sucking (n = 10). Plasma markers of adenosine triphosphate degradation (hypoxanthine, xanthine and uric acid) and oxidative stress (allantoin) were measured before and after the heel lance. Pain was measured using the Premature Infant Pain Profile. Data were analyzed using repeated measures analysis of variance, chi-square and one-way analysis of variance. RESULTS: We found that in preterm neonates who were intubated and/or were receiving ⩾30% FiO2, a single dose of oral sucrose given before a heel lance significantly increased markers of adenosine triphosphate use. CONCLUSION: We found that oral sucrose enhanced adenosine triphosphate use in neonates who were intubated and/or were receiving ⩾30% FiO2. Although oral sucrose decreased pain scores, our data suggest that it also increased energy use as evidenced by increased plasma markers of adenosine triphosphate utilization. These effects of sucrose, specifically the fructose component, on adenosine triphosphate metabolism warrant further investigation.

Urinary Hypoxanthine as a Measure of Increased ATP Utilization in Late Preterm Infants.

OBJECTIVE: To examine the effect of neonatal morbidity on ATP breakdown in late preterm infants. STUDY DESIGN: Urinary hypoxanthine concentration, a marker of ATP breakdown, was measured from 82 late preterm infants on days of life (DOL) 3 to 6 using high-performance liquid chromatography. Infants were grouped according to the following diagnoses: poor nippling alone (n = 8), poor nippling plus hyperbilirubinemia (n = 21), poor nippling plus early respiratory disease (n = 26), and respiratory disease alone (n = 27). RESULTS: Neonates with respiratory disease alone had significantly higher urinary hypoxanthine over DOL 3 to 6 when compared with neonates with poor nippling (P = .020), poor nippling plus hyperbilirubinemia (P < .001), and poor nippling plus early respiratory disease (P = .017). Neonates with poor nippling who received respiratory support for 2 to 3 days had significantly higher hypoxanthine compared with infants who received respiratory support for 1 day (P = .017) or no days (P = .007). CONCLUSIONS: These findings suggest that respiratory disorders significantly increase ATP degradation in late premature infants.

Mechanistic investigation of seeded growth in triblock copolymer stabilized gold nanoparticles.

We report the seeded synthesis of gold nanoparticles (GNPs) via the reduction of HAuCl4 by (L31 and F68) triblock copolymer (TBP) mixtures. In the present study, we focused on [TBP]/[Au(III)] ratios of 1-5 (≈1 mM HAuCl4) and seed sizes ~20 nm. Under these conditions, the GNP growth rate is dominated by both the TBP and seed concentrations. With seeding, the final GNP size distributions are bimodal. Increasing the seed concentration (up to ~0.1 nM) decreases the mean particle sizes 10-fold, from ~1000 to 100 nm. The particles in the bimodal distribution are formed by the competitive direct growth in solution and the aggregative growth on the seeds. By monitoring kinetics of GNP growth, we propose that (1) the surface of the GNP seeds embedded in the TBP cavities form catalytic centers for GNP growth and (2) large GNPs are formed by the aggregation of GNP seeds in an autocatalytic growth process.

Oral sucrose for heel lance increases adenosine triphosphate use and oxidative stress in preterm neonates.

OBJECTIVE: To examine the effects of sucrose on pain and biochemical markers of adenosine triphosphate (ATP) degradation and oxidative stress in preterm neonates experiencing a clinically required heel lance. STUDY DESIGN: Preterm neonates that met study criteria (n = 131) were randomized into 3 groups: (1) control; (2) heel lance treated with placebo and non-nutritive sucking; and (3) heel lance treated with sucrose and non-nutritive sucking. Plasma markers of ATP degradation (hypoxanthine, xanthine, and uric acid) and oxidative stress (allantoin) were measured before and after the heel lance. Pain was measured with the Premature Infant Pain Profile. Data were analyzed by the use of repeated-measures ANOVA and Spearman rho. RESULTS: We found significant increases in plasma hypoxanthine and uric acid over time in neonates who received sucrose. We also found a significant negative correlation between pain scores and plasma allantoin concentration in a subgroup of neonates who received sucrose. CONCLUSION: A single dose of oral sucrose, given before heel lance, significantly increased ATP use and oxidative stress in premature neonates. Because neonates are given multiple doses of sucrose per day, randomized trials are needed to examine the effects of repeated sucrose administration on ATP degradation, oxidative stress, and cell injury.

Procedural pain and oxidative stress in premature neonates.

UNLABELLED: Preterm neonates exposed to painful procedures in the neonatal intensive care unit exhibit increased pain scores and alterations in oxygenation and heart rate. It is unclear whether these physiological responses increase the risk of oxidative stress. Using a prospective study design, we examined the relationship between a tissue-damaging procedure (TDP; tape removal during discontinuation of an indwelling central arterial or venous catheter) and oxidative stress in 80 preterm neonates. Oxidative stress was quantified by measuring uric acid (UA) and malondialdehyde (MDA) concentration in plasma before and after neonates (n = 38) experienced a TDP compared to those not experiencing any TDP (control group, n = 42). Pain was measured before and during the TDP using the Premature Infant Pain Profile (PIPP). We found that pain scores were higher in the TDP group compared to the control group (median scores, 11 and 5, respectively; P < .001). UA significantly decreased over time in control neonates but remained stable in TDP neonates (132.76 to 123.23 µM versus 140.50 to 138.9 µM; P = .002). MDA levels decreased over time in control neonates but increased in TDP neonates (2.07 to 1.81 µM versus 2.07 to 2.21 µM, P = .01). We found significant positive correlations between PIPP scores and MDA. Our data suggest a significant relationship between procedural pain and oxidative stress in preterm neonates. PERSPECTIVE: This article presents data describing a significant relationship between physiological markers of neonatal pain and oxidative stress. The method described in this paper can potentially be used to assess the direct cellular effects of procedural pain as well the effectiveness of interventions performed to decrease pain.