Differential engulfment of Staphylococcus aureus and Pseudomonas aeruginosa by monocyte-derived macrophages is associated with altered phagocyte biochemistry and morphology.

Knowledge of changes in macrophages following bacterial engulfment is limited. U937-derived macrophages were incubated with Staphylococcus aureus or Pseudomonas aeruginosa. Morphological and biochemical changes in macrophages following host-pathogen interactions were visualized using Scanning Electron Microscopy (SEM) and Fourier-Transform Infrared Spectroscopy (FTIR) respectively. Principal Component Analysis (PCA) was used to assess the variability in the FTIR spectra. Following host-pathogen interactions, survival of S. aureus was significantly lower than P. aeruginosa (P<0.05) and cellular morphology of macrophages was different after incubation with S. aureus compared to P. aeruginosa. Following incubation with S. aureus macrophages were more globular and amorphous in shape whereas long linear pseudopodia were observed following incubation with P. aeruginosa. Distinct FTIR spectra were identified in macrophages post interaction with the different bacteria and PCA analysis demonstrated distinct biochemical differences in the phagocytes following engulfment of the bacteria, with > 99 % of variability in the FTIR spectra explained by the first two principal components. These findings demonstrated that there were clear morphological and biochemical changes in macrophages following engulfment of two different bacterial types suggesting that the biochemical components of the bacterial cell wall influenced the biochemical characteristics and hence the morphology of macrophages in distinct ways.

A Pilot Study Investigating the Influence of Glucagon-Like Peptide-1 Receptor Single Nucleotide Polymorphisms on Gastric Emptying Rate in Caucasian Men.

Gastric emptying rate in humans is subject to large individual variability, but previous research on the influence of genetics is scarce. Variation in the glucagon-like peptide-1 receptor (GLP1R) gene is a plausible candidate gene to partially explain the high variance. This study aimed to investigate the influence of genetic variation in the GLP1R gene on gastric emptying rate of a glucose solution in humans. Forty eight healthy Caucasian males took part in this investigation. Gastric emptying rate of a 6% glucose solution was assessed using the 13C breath test method and a venous blood sample was obtained from each participant. Participants were genotyped for 27 Tag single nucleotide polymorphisms (SNPs) in the GLP1R locus using Sequenom MassARRAY iPLEX GOLD analysis and MALDI-TOF mass spectrometry. The time at which maximal emptying rate occurred (Tlag) was faster in participants with the CC genotype than in TT and TC genotypes for SNP rs742764: [median (quartiles) CC, 35 (30-36) min vs. TT, 43 (39-46) min, and TC, 41 (39-45) min; P < 0.01]. Tlag was also slower in participants with the AA genotype compared to the TT and TA genotypes for SNP rs2254336: [AA, 43 (39-49) min vs. TT, 36 (34-41) min, and TA, 39 (35-42) min; P < 0.05]. Analysis by phenotype also showed differences in half-emptying time (T12) and Tlag for SNPs rs9283907, rs2268657, and rs2254336. Several neighboring Tag SNPs within the GLP1R gene were found to be associated with gastric emptying rate, and should be further investigated.

The Effect of C-Reactive Protein Isoforms on Nitric Oxide Production by U937 Monocytes/Macrophages.

Inflammation is regulated by many endogenous factors including estrogen, a steroid hormone that declines with increasing age, leading to excessive inflammation in the elderly. C-reactive protein (CRP) is an acute phase inflammatory protein that exists in two forms, native CRP (nCRP) and monomeric CRP (mCRP), which mediate distinct biological activities. It is unclear how each CRP isoform mediates nitric oxide (NO), a signaling molecule generated by NO synthase (NOS). This study investigated whether CRP isoforms have distinct effects on NO production by unstimulated and lipopolysaccharide (LPS)-activated monocytes/macrophages and whether estrogen mediates CRP-induced NO production in an in vitro model of aging. NO and inducible NOS (iNOS) were measured (n = 12) by the Griess assay and an enzyme-linked immunosorbent assay, respectively following incubation (24 h) of human-derived U937 monocytes/macrophages with CRP isoforms [(nCRP) = 500 and 1,000 µg/ml; (mCRP) = 100 and 250 µg/ml] in the absence or presence of 17 beta-estradiol (1 × 10-7, 1 × 10-8, and 1 × 10-9 M). The response to each CRP isoform and estrogen was dependent on the differentiation and activation status of cells. Monocytes with or without prior LPS-activation significantly increased (P < 0.01) NO/iNOS production when treated with mCRP. The mCRP isoform had no effect (P > 0.05) on NO/iNOS production by unactivated or LPS-activated macrophages, whereas nCRP significantly (P < 0.05) reduced NO/iNOS production by macrophages, with or without prior LPS-activation. The nCRP isoform had opposing actions on monocytes, significantly (P < 0.01) increasing and reducing NO/iNOS by unactivated and LPS-activated monocytes, respectively. Estrogen significantly (P < 0.01) reversed nCRP-mediated NO inhibition by unactivated macrophages but decreased CRP-induced NO by unactivated monocytes treated with nCRP or mCRP and LPS-activated monocytes treated with mCRP. NO was differentially mediated by CRP isoforms in a cell-type/state-specific manner, with production corresponding to concomitant changes in iNOS levels. Collectively, the findings indicate nCRP and estrogen predominantly reduce NO production, whereas mCRP increases NO production. This supports growing evidence that mCRP exacerbates inflammation while nCRP and estrogen dampen the overall inflammatory response. Therapeutic strategies that restore estrogen levels to those found in youth and promote the stability of nCRP or/and prevent the formation of mCRP may reduce NO production in age-related inflammatory conditions.

Role of C-Reactive Protein at Sites of Inflammation and Infection.

C-reactive protein (CRP) is an acute inflammatory protein that increases up to 1,000-fold at sites of infection or inflammation. CRP is produced as a homopentameric protein, termed native CRP (nCRP), which can irreversibly dissociate at sites of inflammation and infection into five separate monomers, termed monomeric CRP (mCRP). CRP is synthesized primarily in liver hepatocytes but also by smooth muscle cells, macrophages, endothelial cells, lymphocytes, and adipocytes. Evidence suggests that estrogen in the form of hormone replacement therapy influences CRP levels in the elderly. Having been traditionally utilized as a marker of infection and cardiovascular events, there is now growing evidence that CRP plays important roles in inflammatory processes and host responses to infection including the complement pathway, apoptosis, phagocytosis, nitric oxide (NO) release, and the production of cytokines, particularly interleukin-6 and tumor necrosis factor-α. Unlike more recent publications, the findings of early work on CRP can seem somewhat unclear and at times conflicting since it was often not specified which particular CRP isoform was measured or utilized in experiments and whether responses attributed to nCRP were in fact possibly due to dissociation into mCRP or lipopolysaccharide contamination. In addition, since antibodies for mCRP are not commercially available, few laboratories are able to conduct studies investigating the mCRP isoform. Despite these issues and the fact that most CRP research to date has focused on vascular disorders, there is mounting evidence that CRP isoforms have distinct biological properties, with nCRP often exhibiting more anti-inflammatory activities compared to mCRP. The nCRP isoform activates the classical complement pathway, induces phagocytosis, and promotes apoptosis. On the other hand, mCRP promotes the chemotaxis and recruitment of circulating leukocytes to areas of inflammation and can delay apoptosis. The nCRP and mCRP isoforms work in opposing directions to inhibit and induce NO production, respectively. In terms of pro-inflammatory cytokine production, mCRP increases interleukin-8 and monocyte chemoattractant protein-1 production, whereas nCRP has no detectable effect on their levels. Further studies are needed to expand on these emerging findings and to fully characterize the differential roles that each CRP isoform plays at sites of local inflammation and infection.

Health-related effects and improving extractability of cereal arabinoxylans.

Arabinoxylans (AXs) are major dietary fibers. They are composed of backbone chains of ß-(1-4)-linked xylose residues to which α-l-arabinose are linked in the second and/or third carbon positions. Recently, AXs have attracted a great deal of attention because of their biological activities such as their immunomodulatory potential. Extraction of AXs has some difficulties; therefore, various methods have been used to increase the extractability of AXs with varying degrees of success, such as alkaline, enzymatic, mechanical extraction. However, some of these treatments have been reported to be either expensive, such as enzymatic treatments, or produce hazardous wastes and are non-environmentally friendly, such as alkaline treatments. On the other hand, mechanical assisted extraction, especially extrusion cooking, is an innovative pre-treatment that has been used to increase the solubility of AXs. The aim of the current review article is to point out the health-related effects and to discuss the current research on the extraction methods of AXs.

The influence of angiotensin converting enzyme and bradykinin receptor B2 gene variants on voluntary fluid intake and fluid balance in healthy men during moderate-intensity exercise in the heat.

Angiotensin converting enzyme (ACE) and bradykinin receptor B2 (B2R) genetic variation may affect thirst because of effects on angiotensin II production and bradykinin activity, respectively. To examine this, 45 healthy Caucasian men completed 60 min of cycle exercise at 62% ± 5% peak oxygen uptake in a room heated to 30.5 ± 0.3 °C with ad libitum fluid intake. Blood samples were collected pre-, mid-, and immediately post-cycle. Fluid intake, body mass loss (BML), sweat loss (determined via changes in body mass and fluid intake), and thirst sensation were recorded. All participants were genotyped for the ACE insert fragment (I) and the B2R insert sequence (P). Participants were homozygous for the wild-type allele (WW or MM), heterozygous (WI or MP) or homozygous for the insert (II or PP). No differences between genotype groups were found in mean (±SD) voluntary fluid intake (WW: 613 ± 388, WI: 753 ± 385, II: 862 ± 421 mL, p = 0.31; MM: 599 ± 322, MP: 745 ± 374, PP: 870 ± 459 mL, p = 0.20), percentage BML or any other fluid balance variables for both the ACE and B2R genes, respectively. Mean thirst perception in the B2R PP group, however, was higher (p < 0.05) than both MM and MP at 30, 45, and 60 min. In conclusion, the results of this study suggest that voluntary fluid intake and fluid balance in healthy men performing 60 min of moderate-intensity exercise in the heat are not predominantly influenced by ACE or B2R genetic variation.

Tumor necrosis factor-alpha (TNF-α) is a therapeutic target for impaired cutaneous wound healing.

Impaired wound healing states lead to substantial morbidity and cost with treatment resulting in an expenditure of billions of dollars per annum in the U.S. alone. Both chronic wounds and impaired acute wounds are characterized by excessive inflammation, enhanced proteolysis, and reduced matrix deposition. These confounding factors are exacerbated in the elderly, in part, as we report here, related to increased local and systemic tumor necrosis factor-alpha (TNF-α) levels. Moreover, we have used a secretory leukocyte protease inhibitor (SLPI) null mouse model of severely impaired wound healing and excessive inflammation, comparable to age-related delayed human healing, to demonstrate that topical application of anti-TNF-α neutralizing antibodies blunts leukocyte recruitment and NFκB activation, alters the balance between M1 and M2 macrophages, and accelerates wound healing. Following antagonism of TNF-α, matrix synthesis is enhanced, associated with suppression of both inflammatory parameters and NFκB binding activity. Our data suggest that inhibiting TNF-α is a critical event in reversing the severely impaired healing response associated with the absence of SLPI, and may be applicable to prophylaxis and/or treatment of impaired wound healing states in humans.

The hormonal regulation of cutaneous wound healing.

Conditions of impaired wound healing in the elderly are associated with substantial morbidity and mortality and impose a significant financial burden upon the world's health services. The findings of a series of recent studies have served to highlight the contrasting contributions made by sex steroid hormones to the regulation of cutaneous repair processes. Although estrogens accelerate healing, the actions of the "male" sex hormones 5alpha-dihydrotestosterone and testosterone are primarily deleterious. The shift that occurs in the balance between serum estrogen and androgen levels as a normal feature of human aging may therefore have important consequences for fundamental tissue repair processes.

Closed-loop, multiobjective optimization of two-dimensional gas chromatography/mass spectrometry for serum metabolomics.

Metabolomics seeks to measure potentially all the metabolites in a biological sample, and consequently, we need to develop and optimize methods to increase significantly the number of metabolites we can detect. We extended the closed-loop (iterative, automated) optimization system that we had previously developed for one-dimensional GC-TOF-MS (O'Hagan, S.; Dunn, W. B.; Brown, M.; Knowles, J. D.; Kell, D. B. Anal. Chem. 2005, 77, 290-303) to comprehensive two-dimensional (GCxGC) chromatography. The heuristic approach used was a multiobjective version of the efficient global optimization algorithm. In just 300 automated runs, we improved the number of metabolites observable relative to those in 1D GC by some 3-fold. The optimized conditions allowed for the detection of over 4000 raw peaks, of which some 1800 were considered to be real metabolite peaks and not impurities or peaks with a signal/noise ratio of less than 5. A variety of computational methods served to explain the basis for the improvement. This closed-loop optimization strategy is a generic and powerful approach for the optimization of any analytical instrumentation.

Androgens modulate the inflammatory response during acute wound healing.

Impaired wound healing states in the elderly lead to substantial morbidity and mortality, and a cost to the health services of over 9 billion dollars per annum. In addition to intrinsic ageing processes that per se cause delayed healing, studies have suggested marked differences in wound repair between the sexes. We have previously reported that, castration of male mice results in a striking acceleration of local cutaneous wound healing and dampens the associated inflammatory response. In this study, we report that systemic 5alpha-reductase inhibition, which blocks the conversion of testosterone to its more active metabolite 5alpha-dihydrotestosterone, mimics the effects of castration in a rat model of cutaneous wound healing. The mechanisms underlying the observed effects involve a direct, cell-specific upregulation of pro-inflammatory cytokine expression by macrophages, but not fibroblasts, in response to androgens. Androgens require the transforming growth factor beta signalling intermediate Smad3 to be present in order to influence repair and local pro-inflammatory cytokine levels. That reducing 5alpha-dihydrotestosterone levels through 5alpha-reductase antagonism markedly accelerates healing suggests a specific target for future therapeutic intervention in impaired wound healing states in elderly males.

The sex steroid precursor DHEA accelerates cutaneous wound healing via the estrogen receptors.

Age-related impaired wound healing states lead to substantial morbidity and cost, with treatment in the USA resulting in an expenditure of over $9 billion per annum. Dehydroepiandrosterone (DHEA) is a ubiquitous adrenal hormone with immunomodulatory properties whose levels decline significantly with advanced age in humans. Conversion of DHEA locally to downstream steroid hormones leads to estrogenic and/or androgenic effects which may be important in age-related skin homeostasis, and which would avoid systemic adverse effects related to estrogen. We report that systemic DHEA levels are strongly associated with protection against chronic venous ulceration in humans. DHEA accelerated impaired healing in an impaired healing model (mice rendered hypogonadal) associated with increased matrix deposition and dampens the exaggerated inflammatory response. Such effects were mediated by local conversion of DHEA to estrogen, acting through the estrogen receptor, and vitro studies suggest a direct effect on specific pro-inflammatory cytokine production by macrophages via mitogen activated kinase (MAP) and phosphatidylinositol 3 (PI3) kinase pathways. In addition, we show that local injection of DHEA accelerates impaired healing in an ageing mouse colony. We suggest that exogenous application of DHEA accelerates impaired wound repair, results which may be applicable to the prophylaxis and treatment of human impaired wound healing states.

The dinucleotide (CA) repeat polymorphism of estrogen receptor beta but not the dinucleotide (TA) repeat polymorphism of estrogen receptor alpha is associated with venous ulceration.

Venous ulcers are the predominant form of chronic wound in the elderly, accounting for around 70% of all cases. The steroid sex hormone estrogen plays a crucial role in normal human skin maintenance and during cutaneous wound repair following injury. Estrogen can reverse age-related impaired wound healing by dampening the inflammatory response and increasing matrix deposition at the wound site. The molecular actions of estrogen are mediated through two nuclear sex steroid hormone receptors, estrogen receptor alpha (ERalpha) and beta (ERbeta). We have conducted a case-control study to investigate whether dinucleotide repeat polymorphisms in the estrogen receptor genes are associated with venous ulceration in the UK Caucasian population. Genomic fragments containing the ERalpha dinucleotide (TA)(n) repeat polymorphism or the ERbeta dinucleotide (CA)(n) repeat polymorphism were amplified by polymerase chain reaction in subject DNA samples and genotyped according to fragment length by capillary electrophoresis. There was no evidence to suggest that the TA repeat polymorphism of ERalpha was associated with venous ulceration. However, the CA*18 allele of the ERbeta CA repeat polymorphism was significantly associated with venous ulceration (n = 120, OR = 1.8, 95% CI = 1.1-2.8, P = 0.02). When the CA repeats alleles were grouped together into either low (L < or = 18) or high (H > 18) numbers of CA repeats, the low (L) repeat allele was significantly associated with venous ulceration (OR = 1.5, 95% CI = 1.0-2.2, P = 0.03). Our results show that a specific ERbeta variant is associated with impaired healing in the elderly, predisposing individuals to venous ulceration.

Potential role of estrogens in wound healing.

Estrogens play a vital role in the development of sexually dimorphic characteristics essential for reproduction. In recent years, insight has been gained into the role of estrogens in non-reproductive pathophysiological processes, including neoplasia, vascular disease and osteoporosis. Intriguingly, the skin appears to act as an end-organ target for estrogenic action; marked structural and functional skin changes occurring after the menopause can be related to altered hormonal profiles. One of the most important consequences of such hormonal changes is the age-related delay in cutaneous wound healing, leading to substantial morbidity and mortality, and increased costs to health services. Reduced estrogen levels have major downstream effects on cellular and tissue responses to injury; such downstream effects include impaired cytokine signal transduction, unchecked inflammation, and altered protein balance, and have a major impact on the rate of wound healing. Further understanding of the complex interaction between aging cells and the hormonal micro-environment is essential to develop focused therapeutic strategies to improve cutaneous wound healing in hypogonadal individuals, including the elderly.

Ageing and wound healing.

Cutaneous wound healing is a complex process encompassing a number of overlapping events including leukocyte recruitment, matrix deposition, epithelialization, and ultimately resolution of inflammation with the formation of a mature scar. Morbidity associated with age-related delayed wound healing imposes an enormous social and financial burden; unless improved wound care strategies are developed the projected relative and absolute increase in the elderly population will further exacerbate this problem. In recent years insight has been gained into the impact of ageing on cellular and tissue responses, resulting from impaired cytokine signal transduction, unchecked inflammation, an altered balance of protein synthesis and degradation, and subsequent downstream effects on the rate and quality of the wound healing response. Further understanding of the complex interaction between the ageing cell and its microenvironment is essential in order to develop focussed therapeutic strategies to improve healing in the elderly.