Human Serum Albumin Misfolding in Aging and Disease.

Age-dependent conformational stability of human serum albumin was determined by the method of fluorescent bilayer liposome assay. After pre-heating at 80 °C, albumin in the sera of 74-year-old healthy subjects exhibited hydrophobic effects on liposomes and made liposomal membrane phospholipids more susceptible to hydrolysis by the lipolytic enzyme phospholipase A2. In contrast, albumin in the sera of 24-year-old individuals was stable at 80 °C and displayed no increased hydrophobic effects on liposomes. The results suggest that albumin in the sera of 74-year-old subjects is more easily converted to a misfolded form in which its protein structure is altered when compared to albumin in the sera of 24-year-old individuals. Misfolded albumin can lose its ability to carry out its normal homeostatic functions and may promote alterations in membrane integrity under inflammatory conditions. However, our investigation has limitations that include the lack of testing sera from large numbers of individuals across a broad range of age to validate our preliminary observations of age-dependent differences in albumin stability and its interactions with liposomes.

Neutrophil necrosis and annexin 1 degradation associated with airway inflammation in lung transplant recipients with cystic fibrosis.

BACKGROUND: Neutrophils sequestered in lower respiratory tract secretions in the inflamed lung may undergo apoptosis and/or necrosis and release toxic cellular contents that can injure airways or parenchyma. This study examined the viability of neutrophils retrieved from the proximal airways of lung transplant recipients with bacterial tracheobronchitis. METHODS: Integrity and stability of intracellular proteins in neutrophils from proximal airways and peripheral blood from lung transplant recipients with bacterial tracheobronchitis were analyzed via Western blot analysis and determination of neutrophil viability by morphologic appearance and flow cytometry. RESULTS: Neutrophils in tracheobronchial secretions from lung transplant recipients with cystic fibrosis who had normal chest radiographic imaging but bronchoscopic evidence of purulent tracheobronchitis post-transplant were necrotic and associated with degradation of intracellular protein annexin 1. The neutrophil influx was compartmentalized to large airways and not detected in peripheral bronchoalveolar airspaces sampled via bronchoalveolar lavage. Peripheral blood neutrophils from healthy subjects cultured in vitro demonstrated that annexin 1 degradation, particularly to a 33 kDa annexin 1 breakdown product (A1-BP), was associated with neutrophil necrosis, but not apoptosis. Although annexin 1 degradation was not specific to neutrophil necrosis, it was a sensitive marker of intracellular protein degradation associated with neutrophil necrosis. Annexin 1 degradation to 33 kDa A1-BP was not observed in peripheral blood neutrophils from healthy subjects, but annexin 1 appeared to be degraded in peripheral blood neutrophils of lung transplant recipients despite a normal morphologic appearance of these cells. CONCLUSIONS: Neutrophils were necrotic from the proximal airways of lung transplant recipients with bacterial tracheobronchitis, and this process may begin when neutrophils are still in the systemic circulation prior to sequestration in inflamed airways. Annexin 1 degradation to 33 kDa A1-BP may be useful as a sensitive marker to detect neutrophil necrosis.

Aging-Related and Gender Specific Albumin Misfolding in Alzheimer's Disease.

Aging-related protein misfolding and aggregation may play critical roles in the pathogenesis of numerous diseases. In the brain, extracellular aggregated amyloid-ß (Aß) is closely related to the death of neurons in individuals with Alzheimer's disease (AD). Albumin-Aß binding is important in preventing Aß fibril aggregation. However, because albumin is the most abundant and important antioxidant in the circulation, aging-related oxidative stress could have a significant effect on the molecular conformation and binding capacities of albumin. To investigate the link between misfolded albumin and AD, we developed fluorescent assays to determine the effects of misfolded albumin on membrane integrity in the presence of a lipolytic, inflammatory response-like enzyme, secretory phospholipase A2 (sPLA2). We found that misfolded albumin increased degradation of phospholipids in highly fluid bilayer membranes in the presence of sPLA2 due to hydrophobic effects of misfolded albumin. High amounts of misfolded albumin were present in sera of elderly (average 74 years) versus young (average 24 years) subjects (p < 0.0001). Albumin in cerebrospinal fluid (CSF) of elderly subjects, though present in small concentrations, had a 2- to 3-fold increased capacity to promote sPLA2-catalyzed membrane phospholipid degradation as compared with the same amount of albumin in serum (p < 0.0001). In addition, the fatty acid binding capacity of albumin in CSF from female subjects was considerably lower than values obtained for men, especially for individuals diagnosed with AD (p = 0.0006). This study suggests that inflammation, misfolded albumin and/or other dysfunctional proteins, and changes in membrane fluidity could alter cell membrane integrity and homeostasis and contribute to the pathogenesis of aging-related dementia and AD.

Pregnancy after heterotopic heart transplant removal.

Heterotopic heart transplants were introduced in 1974. The technique allows the patient's native heart to be preserved in situ, alongside the transplanted heterotopic donor heart. We present the case of a nulliparous woman who underwent heterotopic heart transplant in infancy, and subsequent explantation of the donor heart eleven years later, when her native heart function recovered. In adulthood the patient attended pre-pregnancy counselling and was awaiting cardiac magnetic resonance imaging when she presented pregnant at 6 weeks-of-gestation. She attended the joint cardiac obstetric and anaesthetic clinic, where she was reviewed monthly and had bi-monthly echocardiograms. At 35 weeks-of-gestation she was admitted to hospital with preeclampsia. After blood pressure control and steroid administration, a category 3 caesarean delivery under spinal anaesthesia was performed. To our knowledge this is the first case report describing pregnancy in a patient with a removed heterotopic heart transplant.

Predicting unknown species numbers using discovery curves.

A common approach to estimating the total number of extant species in a taxonomic group is to extrapolate from the temporal pattern of known species descriptions. A formal statistical approach to this problem is provided. The approach is applied to a number of global datasets for birds, ants, mosses, lycophytes, monilophytes (ferns and horsetails), gymnosperms and also to New World grasses and UK flowering plants. Overall, our results suggest that unless the inventory of a group is nearly complete, estimating the total number of species is associated with very large margins of error. The strong influence of unpredictable variations in the discovery process on species accumulation curves makes these data unreliable in estimating total species numbers.

A continuous fluorescence assay for the determination of calcium-dependent secretory phospholipase A2 activity in serum.

BACKGROUND: Calcium-dependent secretory phospholipase A(2)-IIA (sPLA(2)-IIA) in the circulation is a marker of inflammation, associated with acute and chronic disease processes. We describe a quick, sensitive and reliable microplate continuous fluorescence assay for determining sPLA(2) activity in serum. METHODS: Liposomes composed of a fluorescent probe and varying amounts of L-alpha-phosphatidylglycerol (PG) and 1,2-dioleoyl-L-alpha-phosphatidylcholine (DOPC) were used as substrates to determine the optimal protocol for sPLA(2) activity determination without interference from serum albumin and lipoproteins. RESULTS: Hydrolysis of the labeled substrate by sPLA(2)-IIA, characterized by increase in fluorescence intensity (FI) and confirmed by end-product analysis, occurred in a time-, calcium-, and protein-dependent manner. Liposomes containing 100% PG were most suitable for measurement of sPLA(2) activity without interference from serum components; LDL produced a Ca(2+)-independent increase in FI when liposomes containing DOPC were used. The assay determined that sPLA(2) activity in serum spiked with sPLA(2)-IIA and illustrated that endogenous sPLA(2) activity was markedly higher in sera from patients with sepsis than in healthy subjects. Intra-assay and inter-assay CVs were in the ranges of 1.6-8.8% and 3.0-11.5%, respectively. CONCLUSIONS: The described method has potential for rapid and sensitive screening of sPLA(2) activity in both clinical and research settings.

Defining and investigating occupational asthma: a consensus approach.

BACKGROUND: At present there is no internationally agreed definition of occupational asthma and there is a lack of guidance regarding the resources that should be readily available to physicians running specialist occupational asthma services. AIMS: To agree a working definition of occupational asthma and to develop a framework of resources necessary to run a specialist occupational asthma clinic. METHOD: A modified RAND appropriateness method was used to gain a consensus of opinion from an expert panel of clinicians running specialist occupational asthma clinics in the UK. RESULTS: Consensus was reached over 10 terms defining occupational asthma including: occupational asthma is defined as asthma induced by exposure in the working environment to airborne dusts vapours or fumes, with or without pre-existing asthma; occupational asthma encompasses the terms "sensitiser-induced asthma" and "acute irritant-induced asthma" (reactive airways dysfunction syndrome (RADS)); acute irritant-induced asthma is a type of occupational asthma where there is no latency and no immunological sensitisation and should only be used when a single high exposure has occurred; and the term "work-related asthma" can be used to include occupational asthma, acute irritant-induced asthma (RADS) and aggravation of pre-existing asthma. Disagreement arose on whether low dose irritant-induced asthma existed, but the panel agreed that if it did exist they would include it in the definition of "work-related asthma". The panel agreed on a set of 18 resources which should be available to a specialist occupational asthma service. These included pre-bronchodilator FEV1 and FVC (% predicted); peak flow monitoring (and plotting of results, OASYS II analysis); non-specific provocation challenge in the laboratory and specific IgE to a wide variety of occupational agents. CONCLUSION: It is hoped that the outcome of this process will improve uniformity of definition and investigation of occupational asthma across the UK.

Zn uptake behavior of rice genotypes and its implication on grain Zn biofortification.

Understanding Zn uptake dynamics is critical to rice grain Zn biofortification. Here we examined soil Zn availability and Zn uptake pathways as affected by genotype (high-grain Zn varieties IR69428 and IR68144), Zn fertilization and water management in two pot experiments. Results showed significant interactions (P < 0.05) between genotypes and Zn fertilization on DTPA (diethylenetriaminepentaacetic acid)-extractable soil Zn from early tillering to flowering. DTPA-extractable Zn in soils grown with IR69428 was positively correlated with stem (r = 0.78, P < 0.01), flagleaf (r = 0.60, P < 0.01) and grain (r = 0.67, P < 0.01) Zn concentrations, suggesting improved soil Zn availability and continued soil Zn uptake by IR69428 even at maturity. Conversely for IR68144, DTPA-extractable Zn was positively correlated only with leaf Zn uptake (r = 0.60, P < 0.01) at active tillering, indicating dependence on remobilization for grain Zn loading. Furthermore, the highest grain Zn concentration (P < 0.05) was produced by a combination of IR69428 and Zn fertilization applied at panicle initiation (38.5 µg g-1) compared with other treatments (P < 0.05). The results highlight that Zn uptake behavior of a rice genotype determines the fate of Zn from the soil to the grain. This has implications on overcoming Zn translocation barriers between vegetative parts and grains, and achieving grain Zn biofortification targets (30.0 µg g-1).

Domiciliary air filtration units, symptoms and lung function in atopic asthmatics.

Air infiltration units (AFUs) incorporating a high efficiency particulate air filter are theoretically able to remove almost all potential airborne allergens. This may have implications for subjects with allergic lower respiratory disease. AFUs were placed in the living room of 12 atopic asthmatics, and the internal filters were inserted and removed in a double-blind fashion. No difference in subjective symptom scoring, spirometry or bronchial reactivity was demonstrated. Peak expiratory flow rate (PEFR) variability was significantly improved from baseline readings, and there was a trend towards higher mean PEFRs when the filters were present in the AFU. Trends towards lower levels of airborne micro-organisms were also demonstrated when the filters were present, however no effect upon total airborne dust and airborne Der pI could be demonstrated.

Parenteral nutrition suppresses the bactericidal response of the small intestine.

BACKGROUND: Parenteral nutrition (PN) increases infectious risk in critically ill patients compared with enteral feeding. Previously, we demonstrated that PN feeding suppresses the concentration of the Paneth cell antimicrobial protein secretory phospholipase A2 (sPLA2) in the gut lumen. sPLA2 and other Paneth cell proteins are released in response to bacterial components, such as lipopolysaccharide (LPS), and they modulate the intestinal microbiome. Because the Paneth cell protein sPLA2 was suppressed with PN feeding, we hypothesized PN would diminish the responsiveness of the small bowel to LPS through reduced secretions and as a result exhibit less bactericidal activity. METHODS: The distal ileum was harvested from Institute of Cancer Research mice, washed, and randomized for incubation with LPS (0, 1, or 10 µg/mL). Culture supernatant was collected and sPLA2 activity was measured. Bactericidal activity of the ileum segment secretions was assessed against Pseudomonas aeruginosa with and without an sPLA2 inhibitor at 2 concentrations, 100 nmol/L and 1 µmol/L. Institute of Cancer Research mice were randomized to chow or PN for 5 days. Tissue was collected for immunohistochemistry (IHC) and ileal segments were incubated with LPS (0 or 10 µg/mL). sPLA2 activity and bactericidal activity were measured in secretions from ileal segments. RESULTS: Ileal segments responded to 10 µg/mL LPS with significantly greater sPLA2 activity and bactericidal activity. The bactericidal activity of secretions from LPS stimulated tissue was suppressed 50% and 70%, respectively, with the addition of the sPLA2-inhibitor. Chow displayed greater sPLA2 in the Paneth cell granules and secreted higher levels of sPLA2 than PN before and after LPS. Accordingly, media collected from chow was more bactericidal than PN. IHC confirmed a reduction in Paneth cell granules after PN. CONCLUSION: This work demonstrates that ileal segments secrete bactericidal secretions after LPS exposure and the inhibition of the Paneth cell antimicrobial protein sPLA2 significantly diminishes this. PN feeding resulted in suppressed secretion of the sPLA2 and resulted in increased bacterial survival. This demonstrates that PN significantly impairs the innate immune response by suppressing Paneth cell function.

Effect of prophylactic supplementation with grape polyphenolics on endotoxin-induced serum secretory phospholipase A2 activity in rats.

This study investigated whether dietary supplementation of polyphenolics-rich grape extract (GE) could attenuate endotoxin-induced serum secretory phospholipase A(2) (sPLA(2)) activity, a modulator of inflammation. Male Sprague-Dawley rats were fed a control diet or the diet supplemented with polyphenolic-rich GE (100 or 300 mg/kg daily) for 3 wk prior to intraperitoneal injection of 3 or 15 mg/kg LPS. A fluorometric assay was used to measure serum sPLA(2) activity during a 5-d period before and after LPS injection. Body weight, hematocrit, and serum C-reactive protein level were also measured. Administration of LPS induced a rapid increase in sPLA(2) activity, which peaked 1 to 2 d after LPS injection and resolved to near-baseline values on days 4 to 5. Marked declines in body weight and hematocrit, increases in C-reactive protein levels, and effects on health status also occurred. GE supplementation significantly attenuated the LPS-induced increase in sPLA(2) activity and decline in hematocrit, but its effects on the loss of body weight and C-reactive protein levels were not significant. Among the measurements, serum sPLA(2) was the only marker that showed a dose-dependent response to both LPS and GE supplementation. The current findings show that oral consumption of polyphenolic-rich GE suppresses endotoxin-induced sPLA(2) activity.

Route and type of nutrition and surgical stress influence secretory phospholipase A2 secretion of the murine small intestine.

BACKGROUND: The function of secretory phospholipase A2 (sPLA2) is site dependent. In tissue, sPLA2 regulates eicosanoid production; in circulation, sPLA2 primes neutrophils; and in the intestinal lumen, sPLA2 provides innate bactericidal immunity as a defensin-related protein. Since parenteral nutrition (PN) primes leukocytes while suppressing intraluminal mucosal immunity, the authors hypothesized that (1) PN would diminish luminal sPLA2 activity but increase activity in intestinal tissue and serum and (2) stress would accentuate these changes. METHODS: Mice received chow, a complex enteral diet (CED), intragastric PN (IG-PN), or PN in experiment 1 and chow, chow+stress, PN, or PN+stress in experiment 2. RESULTS: In experiment 1, luminal sPLA2 activity was greatest in chow and decreased in CED, IG-PN, and PN, with PN lower than CED and IG-PN. Compared to that after chow, serum sPLA2 activity dropped after CED, IG-PN, and PN. Serum sPLA2 was higher in portal than systemic serum. In experiment 2, PN lowered luminal sPLA2 activity vs chow. Stress lowered luminal sPLA2 activity in chow, without change in PN. Following stress, luminal immunoglobulin A increased in chow but not PN. Serum sPLA2 activity increased in PN. CONCLUSIONS: PN attenuates sPLA2 activity in intestinal fluid, consistent with suppressed innate mucosal defense. Stress suppresses luminal fluid sPLA2 activity in chow but not the immunoglobulin A response; PN impairs both. Stress significantly elevates serum sPLA2 in PN-fed mice, consistent with known increased neutrophil priming with PN. PN reduces innate bactericidal immunity of the gut but upregulates serum proinflammatory products poststress.

CDP-choline significantly restores phosphatidylcholine levels by differentially affecting phospholipase A2 and CTP: phosphocholine cytidylyltransferase after stroke.

Phosphatidylcholine (PtdCho) is a major membrane phospholipid, and its loss is sufficient in itself to induce cell death. PtdCho homeostasis is regulated by the balance between hydrolysis and synthesis. PtdCho is hydrolyzed by phospholipase A2 (PLA2), PtdChospecific phospholipase C (PtdCho-PLC), and phospholipase D (PLD). PtdCho synthesis is rate-limited by CTP:phosphocholine cytidylyltransferase (CCT), which makes CDP-choline. The final step of PtdCho synthesis is catalyzed by CDP-choline:1,2-diacylglycerol cholinephosphotransferase. PtdCho synthesis in the brain is predominantly through the CDP-choline pathway. Transient middle cerebral artery occlusion (tMCAO) significantly increased PLA2 activity, secretory PLA2 (sPLA2)-IIA mRNA and protein levels, PtdCho-PLC activity, and PLD2 protein expression following reperfusion. CDP-choline treatment significantly attenuated PLA2 activity, sPLA2-IIA mRNA and protein levels, and PtdCho-PLC activity, but did not affect PLD2 protein expression. tMCAO also resulted in loss of CCT activity and CCTalpha protein, which were partially restored by CDP-choline. No changes were observed in cytosolic PLA2 or calcium-independent PLA2 tMCAO. protein levels after Up-regulation of PLA2, PtdCho-PLC, and PLD and regulation of CCT collectively down-resulted in loss of PtdCho, which was significantly restored by CDP-choline treatment. CDP-choline treatment significantly attenuated the infarction volume by 55 +/- 5% after 1 h of tMCAO and 1 day of reperfusion. Taken together, these results suggest that CDP-choline significantly restores Ptd-Cho levels by differentially affecting sPLA2-IIA, PtdCho-PLC, and CCTalpha after transient focal cerebral ischemia. A hypothetical scheme is proposed integrating results from this study and from other reports in the literature.

Endotoxin exposure in asthmatic children and matched healthy controls: results of IPEADAM study.

UNLABELLED: Children spend increasing time indoors. Exposure to environmental factors may contribute to the development or exacerbation of the asthmatic phenotype. Inter-relationships between these factors might influence the manifestation of asthma. Endotoxin exposure has been shown to have pro-inflammatory and protective effects in different situations. We investigated the exposure to several indoor pollutants (endotoxin, Der p 1, damp, ETS, PM2.5) in asthmatic and healthy children. The children were recruited from two primary care centers according to their response to a validated questionnaire. Asthmatic children were matched for sex, age and sib-ship size with children living in asthma free households. Of 90 matched pairs, higher levels of endotoxin were found in the living room carpets, but not the bedroom carpet or mattresses of the asthma compared with the control homes (STATA analysis OR: 1.88 (1.11-3.18); P=0.018). Asthmatic children were also more likely to live as part of a single parent family, in a house where the parents self-reported the presence of damp, and where the living room had been redecorated in the 12 months prior to the sampling visits. This study suggests that endotoxin in urban homes is a risk factor for the development of asthma. Moreover, this study found that there were no statistically significant interactions between environmental factors. PRACTICAL IMPLICATIONS: This study has demonstrated that the home environments of English children (4-17) with asthma and without the disease do not differ greatly. With the exception of endotoxin, the parameters examined in this study, including house dust mite allergens, nitrogen dioxide, ETS and damp are unlikely to be related to the development of asthma. Avoidance of these pollutants may not be beneficial in preventing asthma in this age group.

Exposure to house dust mite allergens and the clinical activity of asthma.

BACKGROUND: House dust mite allergens play an important role in inducing IgE-mediated sensitization and the development of bronchial hyperresponsiveness (BHR) and asthma. This study investigated the relationship between mite allergen exposure and the clinical activity and severity of asthma. METHODS: Nonsmoking adult patients with asthma (n = 53) were randomly recruited from the asthma registry of two large family practitioner surgeries. Each participant underwent skin testing with common inhalant allergens, a methacholine bronchoprovocation test, and pulmonary function testing on up to 3 separate occasions over a 4-week period. BHR was expressed both as PD20 and dose-response ratio (DRR), and the patients with patients with PD20 of less than 12.25 mumol methacholine were classified as methacholine reactors. Patients were also asked to record peak expiratory flow rate (PEFR) values at 2-hour intervals during waking hours for 1 month. Daily PEFR variability was calculated as amplitude percent mean. Dust samples were collected by vacuuming bedding, bedroom carpets and mattresses. In addition, in the homes of 32 subjects with positive skin test responses to mites, airborne samples were taken overnight for 8 hours with a personal sampler attached to each subject's pillow. Der p 1 and Der p 2 levels were determined by a two-site monoclonal antibody-based ELISA. RESULTS: No difference in mite exposure was found between subjects who were sensitive to mites and those who were not. However, mite-sensitive methacholine reactors were exposed to significantly higher concentrations of Der p 1 in beds than mite-sensitive methacholine nonreactors (13.2 micrograms/gm and 1.45 micrograms/gm, respectively; p < 0.02). Der p 1 and Der p 2 were undetectable in 30 of 32 airborne samples. In mite-sensitive patients both Der p 1 and Der p 2 in beds significantly correlated with BHR (PD20: r = -0.49, DRR, r = 0.49; PD20: r = -0.46, DRR: r = 0.43) and amplitude percent mean PEFR (r = 0.38, r = 0.41) for Der p 1 and Der p 2, respectively. There was a significant negative correlation between exposure to Der p 1 and percent predicted FEV1 (r = -0.43). The correlation between Der p 2 and percent predicted FEV1 just failed to reach a significant level but showed a clear trend ( r = -0.35, p = 0.068). CONCLUSIONS: Clinical activity and severity of asthma (measured by the level of BHR, PEFR variability, and percent predicted FEV1) in mite-sensitive patients is related to exposure to mite allergens in the dust reservoir, with levels in bed being an important indicator that correlated with disease activity.

Mainstream and sidestream cigarette smoke exposure increases Ca2+-dependent phospholipid binding proteins in guinea pig alveolar type II cells.

We have earlier identified the presence of a 36 kDa Ca2+-dependent phospholipid-binding protein (PLBP) in guinea pig alveolar type II cells. PLBP has been suggested to act as a mediator in facilitating and regulating intracellular surfactant assembly and delivery to the plasma membrane of type II cells for secretion into alveolar space. It has been reported that cigarette smoke exposure (CSE) causes a decrease in the surfactant activity in bronchial washings. We have also reported earlier that mainstream (MS) and sidestream (SS) CSE causes desensitization of beta-adrenoreceptors in guinea pig alveolar type II cells. Since both Ca2+ and beta-adrenoreceptors are involved in surfactant secretion and PLBP is involved in surfactant delivery, it is important to know whether CSE causes any change in the PLBP level in alveolar type II cells. In the present study, we have demonstrated that MS and SS CSE causes a significant increase in the levels of PLBP in alveolar type II cells (107 and 150%, respectively) and in lung lavage (42 and 125%, respectively) in comparison to that in sham control (430 ng/mg protein in alveolar type II cells and 780 ng/mg protein in lung lavage). The mechanism by which smoke exposure causes an elevation in the levels of PLBP in alveolar type II cells and lung lavage remains to be investigated.