Manipulating avocado fruit ripening with 1-methylcyclopropene

Previous investigations with 1-methylcyclopropene (1-MCP) on avocado (Persea americana Mill.) fruit have focussed mainly on improving storage life by reducing the severity of disorders causing discolouration of the flesh. Development of 1-MCP and ethylene treatments, which also help control the time to reach the eating ripe stage, may confer additional practical benefits. In this context, the current study investigated the potential of 1-MCP to accurately manipulate ripening of non-stored 'Hass' avocado fruit by treatment before or after ethylene and at different times during ripening. To investigate this, 500 nL L-1 1-MCP was applied within 1 day after harvest, followed by ethylene 0-14 days after 1-MCP. In addition, fruit were treated with ethylene, then 1-MCP 0-8 days after ethylene. Treatment of fruit with 500 nL L-1 1-MCP for 18 h at 20 degreesC provided the maximum effect by increasing the days from harvest to ripe (DTR) from 8 (with no 1-MCP) to 20. Fruit treated with 500 nL L-1 1-MCP for 18 h at 20 degreesC remained insensitive to 100 muL L-1 ethylene applied between 0 and 14 days after 1-MCP for 24 h at 20 degreesC. Ripening of fruit exposed to 100 muL L-1 ethylene for 24 h at 20 degreesC could be delayed by up to 3.3 days by applying 500 nL L-1 1-MCP for 18 h at 20 degreesC up to 2 days after ethylene treatment. However, once the fruit started to soften (sprung) there was little effect of 1-MCP on DTR, compared with no 1-MCP. 1-MCP treatment was associated with increased severity of body rots (caused mainly by Colletotrichum spp.) and stem-end rots (caused mainly by Dothiorella spp.), which was likely due to the increased DTR in these treatments. Significant differences in disease severity were found between orchards (replications), with replicates with low disease severity being less affected by 1-MCP treatment. These results indicate that 1-MCP can delay ripening, but careful sourcing of fruit is required to reduce the risk of diseases in ripe fruit. There is some capacity to delay ripening using 1-MCP after ethylene. There is little potential to control ripening using ethylene after treatment with 500 nL L-1 1-1-MCP, but lower concentrations may be more effective. (C) 2004 Elsevier B.V. All rights reserved.

Angiopoietin-1 promotes atherosclerosis by increasing the proportion of circulating Gr1 monocytes

Atherosclerosis is a chronic inflammatory disease occurring within the artery wall. A crucial step in atherogenesis is the infiltration and retention of monocytes into the subendothelial space of large arteries induced by chemokines and growth factors. Angiopoietin-1 (Ang-1) regulates angiogenesis and reduces vascular permeability and has also 15 been reported to promote monocyte migration in vitro. We investigated the role of Ang-1 in atherosclerosis-prone apolipoprotein-E (Apo-E) knockout mouse. Apo-E knockout (Apo-E-/-) mice fed a western or normal chow diet received a single iv injection of adenovirus encoding Ang-1 or control vector. Adenovirus-mediated systemic expression of Ang-1 induced a significant increase in early atherosclerotic lesion size and monocyte/macrophage accumulation compared with control animals receiving empty vector. Ang-1 significantly increased plasma MCP-1 and VEGF levels as measured by ELISA. FACS analysis showed that Ang-1 selectively increased inflammatory Gr1þmonocytes in the circulation, while the cell-surface 25 expression of CD11b, which mediates monocyte emigration, was significantly reduced. Ang-1 specifically increases circulating Gr1þinflammatory monocytes and increases monocyte/macrophage retention in atherosclerotic plaques, thereby contributing to development of atherosclerosis.

The p38 mitogen-activated protein kinase regulates interleukin-1beta-induced IL-8 expression via an effect on the IL-8 promoter in intestinal epithelial cells

Several lines of evidence implicate the p38 mitogen-activated protein kinase (p38 MAPK) in the proinflammatory response to bacterial agents and cytokines. Equally, the transcription factor, nuclear factor (NF)-kappaB, is recognized to be a critical determinant of the inflammatory response in intestinal epithelial cells (IECs). However, the precise inter-relationship between the activation of p38 MAPK and activation of the transcription factor NF-kappaB in the intestinal epithelial cell (IEC) system, remains unknown. Here we show that interleukin (IL)-1beta activates all three MAPKs in Caco-2 cells. The production of IL-8 and monocyte chemotactic protein 1 (MCP-1) was attenuated by 50% when these cells were preincubated with the p38 MAPK inhibitor, SB 203580. Further investigation of the NF-kappaB signalling system revealed that the inhibitory effect was independent of the phosphorylation and degradation of IkappaBalpha, the binding partner of NF-kappaB. This effect was also independent of the DNA binding of the p65 Rel A subunit, as well as transactivation, determined by an NF-kappaB luciferase construct, using both SB 203580 and dominant-negative p38 MAPK. Evaluation of IL-8 and MCP-1 RNA messages by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the inhibitory effect of SB 203580 was associated with a reduction in this parameter. Using an IL-8-luciferase promoter construct, an effect of p38 upon its activation by both pharmacological and dominant-negative p38 construct co-transfection was demonstrated. It is concluded that p38 MAPK influences the expression of chemokines in intestinal epithelial cells, through an effect upon the activation of the chemokine promoter, and does not directly involve the activation of the transcription factor NF-kappaB

Porphyromonas gingivalis lipopolysaccharide alters atherosclerotic-related gene expression in oxidized low-density-lipoprotein-induced macrophages and foam cells

The molecular mechanism between atherosclerosis formation and periodontal pathogens is not clear although positive correlation between periodontal infections and cardiovascular diseases has been reported. Objective: To determine if atherosclerosis related genes were affected in foam cells during and after its formation by P. gingivalis lipopolysaccharide (LPS) stimulation. Methods: Macrophages from human THP-1 monocytes were treated with oxidized low density lipoprotein (oxLDL) to induce the formation of foam cells. P. gingivalis LPS was added to cultures of either oxLDL-induced macrophages or foam cells. The expression of atherosclerosis related genes was assayed by quantitative real time PCR and the protein production of granulocyte-macrophage colony-stimulating factor（GM-CSF）, monocyte chemotactic protein-1 (MCP-1), IL-1β, IL-10 and IL-12 was determined by ELISA. Nuclear translocation of NF-κB P65 was detected by immunocytochemistry and western blot was used to evaluate IKB-α degradation to confirm the NF-κB pathway activation. Results: P. gingivalis LPS stimulated atherosclerosis related gene expression in foam cells and increased oxLDL induced expression of chemokines, adhesion molecules, growth factors, apoptotic genes, and nuclear receptors in macrophages. Transcription of the pro-inflammatory cytokines IL-1β and IL-12 was elevated in response to LPS in both macrophages and foam cells, whereas the anti-inflammatory cytokine IL-10 was not affected. Increased NF-κB pathway activation was also observed in LPS and oxLDL co-stimulated macrophages. Conclusion: P. gingivalis LPS appears to be an important factor in the development of atherosclerosis by stimulation of atherosclerosis related gene expression in both macrophages and foam cells via activation of the NF-κB pathway.

Early inflammatory and myogenic responses to resistance exercise in the elderly

Introduction and Methods: This study compared changes in myokine and myogenic genes following resistance exercise (3 sets of 12 repetitions of maximal unilateral knee extension) in 20 elderly men (67.8 ± 1.0 years) and 15 elderly women (67.2 ± 1.5 years). Results: Monocyte chemotactic protein (MCP)-1, macrophage inhibitory protein (MIP)-1β, interleukin (IL)-6 and MyoD mRNA increased significantly (P < 0.05), whereas myogenin and myostatin mRNA decreased significantly after exercise in both groups. Macrophage-1 (Mac-1) and MCP-3 mRNA did not change significantly after exercise in either group. MIP-1β, Mac-1 and myostatin mRNA were significantly higher before and after exercise in men compared with women. In contrast, MCP-3 and myogenin mRNA were significantly higher before and after exercise in the women compared with the men. Conclusions: In elderly individuals, gender influences the mRNA expression of certain myokines and growth factors, both at rest and after resistance exercise. These differences may influence muscle regeneration following muscle injury

The effects of dietary fish oil on inflammation, fibrosis and oxidative stress associated with obstructive renal injury in rats.

Scope: We examined whether dietary supplementation with fish oil modulates inflammation, fibrosis and oxidative stress following obstructive renal injury. Methods and results: Three groups of Sprague-Dawley rats (n = 16 per group) were fed for 4 wk on normal rat chow (oleic acid), chow containing fish oil (33 g eicosapentaenoic acid and 26 g docosahexaenoic acid per kg diet), or chow containing safflower oil (60 g linoleic acid per kg diet). All diets contained 7% fat. After 4 wk, the rats were further subdivided into four smaller groups (n = 4 per group). Unilateral ureteral obstruction was induced in three groups (for 4, 7 and 14 days). The fourth group for each diet did not undergo surgery, and was sacrificed as controls at 14 days. When rats were sacrificed, plasma and portions of the kidneys were removed and frozen; other portions of kidney tissue were fixed and prepared for histology. Compared with normal chow and safflower oil, fish oil attenuated collagen deposition, macrophage infiltration, TGF-beta expression, apoptosis, and tissue levels of arachidonic acid, MIP-1 alpha, IL-1 beta, MCP-1 and leukotriene B(4). Compared with normal chow, fish oil increased the expression of HO-1 protein in kidney tissue. Conclusions: Fish oil intake reduced inflammation, fibrosis and oxidative stress following obstructive renal injury.

Cytokine responses to carbohydrate ingestion during recovery from exercise-induced muscle injury.

We investigated the effect of carbohydrate ingestion after maximal lengthening contractions of the knee extensors on circulating concentrations of myocellular proteins and cytokines, and cytokine mRNA expression in muscle. Using a cross-over design, 10 healthy males completed 5 sets of 10 lengthening (eccentric) contractions (unilateral leg press) at 120% 1 repetition-maximum. Subjects were randomized to consume a carbohydrate drink (15% weight per volume; 3 g/kg BM) for 3 h after exercise using one leg, or a placebo drink after exercise using the contralateral leg on another day. Blood samples (10 mL) were collected before exercise and after 0, 30, 60, 90, 120, 150, and 180 min of recovery. Muscle biopsies (vastus lateralis) were collected before exercise and after 3 h of recovery. Following carbohydrate ingestion, serum concentrations of glucose (30-90 min and at 150 min) and insulin (30-180 min) increased (P < 0.05) above pre-exercise values. Serum myoglobin concentration increased (similar to 250%; P < 0.05) after both trials. In contrast, serum cytokine concentrations were unchanged throughout recovery in both trials. Muscle mRNA expression for IL-8 (6.4-fold), MCP-1 (4.7-fold), and IL-6 (7.3-fold) increased substantially after carbohydrate ingestion. TNF-alpha mRNA expression did not change after either trial. Carbohydrate ingestion during early recovery from exercise-induced muscle injury may promote proinflammatory reactions within skeletal muscle.

The effects of acute exercise-induced cortisol on CCR2 expression on human monocytes

CC-chemokine receptor 2 (CCR2) and its ligand, monocyte chemotactic protein-1 (MCP-1, also known as CCL2), are crucial for the recruitment of monocytes/macrophages to sites of inflammation. We conducted a series of experiments to investigate the relationship between stress, monocyte CCR2 expression and migration activity. First, we collected peripheral blood mononuclear cells (PBMC) from untrained subjects (n=8) and measured CCR2 expression on CD14(+) monocytes cultured with cortisol, epinephrine and norepinephrine. Second, we collected PBMC from the subjects before and after they cycled for 60 min at 70% peak O(2) uptake (VO2(peak)), and measured alterations in CCR2 expression on monocytes following exercise. Third, we cultured PBMC with serum obtained before and after exercise and the glucocorticoid antagonist RU-486 to determine the effect of cortisol on CCR2 expression in vitro. Last, we measured the ability of PBMC treated with serum or cortisol to migrate through membrane filters in response to CCL2. Cortisol (but not epinephrine or norepinephrine) increased CCR2 expression on monocytes in a dose- and time-dependent manner. Exercise did not influence CCR2 expression on PBMC, whereas incubation of PBMC with post-exercise serum significantly increased CCR2 expression. Both cortisol and post-exercise serum increased the migration of PBMC toward CCL2. The increase in CCR2 expression on PBMC following stimulation with cortisol and serum was blocked by the glucocorticoid receptor antagonist RU-486. In conclusion, cortisol released during exercise increased monocyte CCR2 expression and migration activity in vitro. These alterations may influence inflammation and regeneration of damaged tissue after acute stress.

Plasma cytokine changes in relation to exercise intensity and muscle damage

The purpose of this study was to compare the effects of exercise intensity and exercise-induced muscle damage on changes in anti-inflammatory cytokines and other inflammatory mediators. Nine well-trained male runners completed three different exercise trials on separate occasions: (1) level treadmill running at 60% VO2max (moderate-intensity trial) for 60 min; (2) level treadmill running at 85% VO2max (high-intensity trial) for 60 min; (3) downhill treadmill running (-10% gradient) at 60% VO2max (downhill running trial) for 45 min. Blood was sampled before, immediately after and 1 h after exercise. Plasma was analyzed for interleukin-1 receptor antagonist (IL-1ra), IL-4, IL-5, IL-10, IL-12p40, IL-13, monocyte chemotactic protein-1 (MCP-1), prostaglandin E(2), leukotriene B(4) and heat shock protein 70 (HSP70). The plasma concentrations of IL-1ra, IL-12p40, MCP-1 and HSP70 increased significantly (P<0.05) after all three trials. Plasma prostaglandin E(2) concentration increased significantly after the downhill running and high-intensity trials, while plasma IL-10 concentration increased significantly only after the high-intensity trial. IL-4 and leukotriene B(4) did not increase significantly after exercise. Plasma IL-1ra and IL-10 concentrations were significantly higher (P<0.05) after the high-intensity trial than after both the moderate-intensity and downhill running trials. Therefore, following exercise up to 1 h duration, exercise intensity appears to have a greater effect on anti-inflammatory cytokine production than exercise-induced muscle damage

Packed red blood cells suppress myeloid dendritic cell and monocyte inflammatory responses in a whole blood model of transfusion

Purpose/Objective: The basis for poor outcomes in some patients post transfusion remains largely unknown. Despite leukodepletion, there is still evidence of immunomodulatory effects of transfusion that require further study. In addition, there is evidence that the age of blood components transfused significantly affects patient outcomes. Myeloid dendritic cell (DC) and monocyte immune function were studied utilising an in vitro whole blood model of transfusion. Materials and methods: Freshly collected (‘recipient’) whole blood was cultured with ABO compatible leukodepleted PRBC at 25% blood replacement-volume (6hrs). PRBC were assayed at [Day (D) 2, 14, 28and 42 (date-of expiry)]. In parallel, LPS or Zymosan (Zy) were added to mimic infection. Recipients were maintained for the duration of the time course (2 recipients, 4 PRBC units, n = 8).Recipient DC and monocyte intracellular cytokines and chemokines (IL-6, IL-10, IL-12,TNF-a, IL-1a, IL-8, IP-10, MIP-1a, MIP-1b, MCP-1) were measured using flow cytometry. Changes in immune response were calculated by comparison to a parallel no transfusion control (Wilcoxin matched pairs). Influence of storage age was calculated using ANOVA. Results: Significant suppression of DC and monocyte inflammatory responses were evident. DC and monocyte production of IL-1a was reduced following exposure to PRBC regardless of storage age (P < 0.05 at all time points). Storage independent PRBC mediated suppression of DC and monocyte IL-1a was also evident in cultures costimulated with Zy. In cultures co-stimulated with either LPS or Zy, significant suppression of DC and monocyte TNF-a and IL-6 was also evident. PRBC storage attenuated monocyte TNF-a production when co-cultured with LPS (P < 0.01 ANOVA). DC and monocyte production of MIP-1a was significantly reduced following exposure to PRBC (DC: P < 0.05 at D2, 28, 42; Monocyte P < 0.05 all time points). In cultures co-stimulated with LPS and zymosan, a similar suppression of MIP-1a production was also evident, and production of both DC and monocyte MIP-1b and IP-10 were also significantly reduced. Conclusions: The complexity of the transfusion context was reflected in the whole blood approach utilised. Significant suppression of these key DC and monocyte immune responses may contribute to patient outcomes, such as increased risk of infection and longer hospital stay, following blood transfusion.

Bridging the biology of the ovine TRALI model – human inflammatory cell responses to TRALI inducing supernatants

Background Transfusion-related acute lung injury (TRALI) is a serious and potentially fatal consequence of transfusion. A two-event TRALI model demonstrated date-of-expiry - day (D) 5 platelet (PLT) and D42 packed red blood cell (PRBC) supernatants (SN) induced TRALI in LPS-treated sheep. We have adapted a whole blood transfusion culture model as an investigative bridge between the ovine TRALI model human responses to transfusion. Methods A whole blood transfusion model was adapted to replicate the ovine model - specifically +/- 0.23μg/mL LPS as the first event and 10% SN volume (transfusion) as the second event. Four pooled SN from blood products, previously used in the TRALI ovine model, were investigated: D1-PLT, D5-PLT, D1-PRBC, and D42-PRBC. Fresh human whole blood (recipient) was mixed with combinations of LPS and BP-SN stimuli and incubated in vitro for 6 hrs. Addition of golgi plug enabled measurement of monocyte cytokine production (IL-6, IL-8, IL-10, IL-12, TNF-α, IL-1α, CXCL-5, IP-10, MIP-1α, MCP-1) using multi-colour flow cytometry. Responses for 6 recipients were assessed. Results In the presence of LPS, D42-PRBC-SN significantly increased monocyte IL-6 (P=0.031), IL-8 (P=0.016) and IL-1α (P=0.008) production compared to D1-PRBC-SN. This response to D42-PRBC-SN was LPS-dependent, and was not evident in non-LPSstimulated controls. This response was also specific to D42-PRBC-SN, as similar changes were not evident for the D5-PLT-SN, compared to the D1-PLT-SN, regardless of the presence of LPS. D5-PLT-SN significantly increased IL-12 production (P=0.024) compared to D1-PLT-SN. This response was again LPS-dependent. Conclusions These data demonstrate a novel two-event mechanism of monocyte inflammatory response that was dependent upon both the presence of date-of-expiry blood product SN and LPS. Further, these results demonstrate different cytokines responses induced by date-of-expiry PLT-SN and PRBC-SN. These data are consistent with the evidence from the ovine TRALI model, and enhancing its relevance to transfusion related changes in humans.

Stored blood products augment inflammation in a two-event model of Transfusion Related Acute Lung Injury (TRALI)

Aim/Background TRALI is hypothesised to develop via a two-event mechanism involving both the patieint's underlying morbidity and blood product factors. The storage of cellular products has been implicated in cases of non-antibody mediated TRALI, however the pathophysiological mechanisms are undefined. We investigated blood product storage-related modulation of inflmmatory cells and medicators involved in TRALI. Methods In an in vitro mode, fresh human whole blood was mixed with culture media (control) or LPS as a 1st event and "transfused" with 10% (v/v) pooled supernatant (SN) from Day 1 (d1, n=75) or Day 42 (D42, n=113) packed red blood cells (PRBCs) as a 2nd event. Following 6hrs, culture SN was used to assess the overall inflammatory response (cytometric bead array) and a duplicate assay containing protein transport inhibitor was used to assess neutrophil- and monocyte-specific inflmamatory responses using multi-colour flow cytometry. Panels: IL-6, IL-8, IL-10, IL-12, IL-1, TNF, MCP-1, IP-10, MIP-1. One-way ANOVA 95% CI. Results In the absence of LPS, exposure to D1 or D42 PRBC-SN reduced monocyte expression of IL-6, IL-8 and Il-10. D42 PRBC-SN also reduced monocyte IP-10, and the overall IL-8 production was increased. In the presence of LPS, D1-PRBC SN only modified overall IP-10 levels which were reduced. However, cf LPS alone, the combination of LPS and D42 PRBC-SN resulted in increased neutrophil and monocyte productionof IL-1 and IL-8 as well as reduced monocyte TNF production. Additionally, LPS and D42 PRBC-SN resulted in overall inflmmatory changes: elevated IL-8,

Effect of exercise training on skeletal muscle cytokine expression in the elderly

Aging is associated with increased circulating pro-inflammatory and lower anti-inflammatory cytokines. Exercise training, in addition to improving muscle function, reduces these circulating pro-inflammatory cytokines. Yet, few studies have evaluated changes in the expression of cytokines within skeletal muscle after exercise training. The aim of the current study was to examine the expression of cytokines both at rest and following a bout of isokinetic exercise performed before and after 12 weeks of resistance exercise training in young (n = 8, 20.3 ± 0.8 yr) and elderly men (n = 8, 66.9 ± 1.6 yr). Protein expression of various cytokines was determined in muscle homogenates. The expression of MCP-1, IL-8 and IL-6 (which are traditionally classified as ‘pro-inflammatory’) increased substantially after acute exercise. By contrast, the expression of the anti-inflammatory cytokines IL-4, IL-10 and IL-13 increased only slightly (or not at all) after acute exercise. These responses were not significantly different between young and elderly men, either before or after 12 weeks of exercise training. However, compared with the young men, the expression of pro-inflammatory cytokines 2 h post exercise tended to be greater in the elderly men prior to training. Training attenuated this difference. These data suggest that the inflammatory response to unaccustomed exercise increases with age. Furthermore, regular exercise training may help to normalize this inflammatory response, which could have important implications for muscle regeneration and adaptation in the elderly.

An adipoinductive role of inflammation in adipose tissue engineering : key factors in the early development of engineered soft tissues

Tissue engineering and cell implantation therapies are gaining popularity because of their potential to repair and regenerate tissues and organs. To investigate the role of inflammatory cytokines in new tissue development in engineered tissues, we have characterized the nature and timing of cell populations forming new adipose tissue in a mouse tissue engineering chamber (TEC) and characterized the gene and protein expression of cytokines in the newly developing tissues. EGFP-labeled bone marrow transplant mice and MacGreen mice were implanted with TEC for periods ranging from 0.5 days to 6 weeks. Tissues were collected at various time points and assessed for cytokine expression through ELISA and mRNA analysis or labeled for specific cell populations in the TEC. Macrophage-derived factors, such as monocyte chemotactic protein-1 (MCP-1), appear to induce adipogenesis by recruiting macrophages and bone marrow-derived precursor cells to the TEC at early time points, with a second wave of nonbone marrow-derived progenitors. Gene expression analysis suggests that TNFα, LCN-2, and Interleukin 1β are important in early stages of neo-adipogenesis. Increasing platelet-derived growth factor and vascular endothelial cell growth factor expression at early time points correlates with preadipocyte proliferation and induction of angiogenesis. This study provides new information about key elements that are involved in early development of new adipose tissue.

Effect of tocopherol on atherosclerosis, vascular function and inflammation in Apolipoprotein E knockout mice with subtotal nephrectomy

Background/Aims: Inflammation and endothelial dysfunction contribute to cardiovascular disease, prevalent in chronic kidney disease (CKD). Antioxidant supplements such as tocopherols may reduce inflammation and atherosclerosis. This study aimed to investigate the effect of tocopherol supplementation on vascular function, aortic plaque formation, and inflammation in apolipoprotein E−/− mice with 5/6 nephrectomy as a model of combined cardiovascular and kidney disease. Methods: Nephrectomized mice were assigned to a normal chow diet group (normal chow), a group receiving 1000 mg/kg diet of α-tocopherol supplementation or a group receiving 1000 mg/kg diet mixed-tocopherol (60% γ-tocopherol). Results: Following 12 weeks, in vitro aortic endothelial-independent relaxation was enhanced with both α-tocopherol and mixed-tocopherol (P < 0.05), while mixed-tocopherol enhanced aortic contraction at noradrenaline concentrations of 3 × 10−7 M to 3 × 10−5 M (P < 0.05), when compared to normal chow. Supplementation with α- and mixed-tocopherol reduced systemic concentrations of IL-6 (P < 0.001 and P < 0.001, respectively) and IL-10 (P < 0.05 and P < 0.001, respectively), while α-tocopherol also reduced MCP-1 (P < 0.05) and tumor necrosis factor (TNF)-α (P < 0.05). Aortic sinus plaque area was significantly reduced with α-tocopherol supplementation when compared to normal chow (P < 0.01). Conclusion: Tocopherol supplementation favorably influenced vascular function and cytokine profile, while it was also effective in reducing atherosclerosis in the apolipoprotein E−/− mouse with CKD.