Production Of Acid Metabolites Research Articles

PS-B11-9: LOW COLONIC PH AND PROTON-SENSOR GPR65: A MISSING BRIDGE LINKING DIET, GUT MICROBIOTA AND CARDIOVASCULAR HEALTH

Objective: Dietary fibre regulates blood pressure (BP) through gut microbial production of acidic metabolites called short-chain fatty acids (SCFAs). The acidic environment in the large intestine activates a proton-sensing G-protein coupled receptor, GPR65. We aimed to characterize the luminal pH in the intestine of hypertensive patients and normotensive individuals, and to validate in a murine model that dietary fibre intake is a critical determinant for intestinal pH. Furthermore, we aimed to determine whether pH-sensing by GPR65 mediates the cardiovascular protection conferred by dietary fibre. Design and method: 15 normotensive and 40 hypertensive (16 treated) participants had ambulatory BP profiles recorded using a Mobil-O-Graph device (CardioScan). Their intestinal luminal pH was measured in real-time by a SmartPillTM Motility Testing System (Medtronic). Analyses were performed on SPSS. Intestinal pH of C57BL/6J wild-type (WT) mice was measured after a 7-day dietary intervention of high-fibre (HF) or low-fibre (LF) diets (n = 5/group). Male WT and Gpr65-/- mice fed with HF or LF diets were implanted with minipumps containing angiotensin II (Ang-II, 0.5 mg/kg/day, 28 days, n = 11–12/group). BP was measured by tail-cuff, cardiac function by ultrasound, and fibrosis by Massons trichrome staining. Results: Compared to normotensive individuals, hypertensive participants had higher colonic minimum pH (mean ± SEM: 5.03 ± 0.08 vs 5.22 ± 0.34, the beta coefficient = 2.98, P = 0.024 adjusted for age, sex, BMI), driven by changes in the transverse colon where dietary fibre is fermented. In our murine model, HF fed mice exhibited lower colonic pH compared to LF fed mice (HF vs LF, 6.03 ± 0.42 vs 7.58 ± 0.31, P < 0.001). This was abolished when mice were treated with antibiotics. Under HF diet, Ang-II treated Gpr65-/- mice had significantly higher BP (mean arterial pressure, WT vs Gpr65-/-, 90.7 ± 2.22 vs 101.3 ± 1.82mmHg, P = 0.022), heart to tibia length ratio (0.0096 ± 0.0004 vs 0.0112 ± 0.0018 g/mm, P = 0.036), left ventricle posterior wall in the diastole (0.831 ± 0.016 vs 0.966 ± 0.020 mm, P = 0.010), cardiac fibrosis (1.44 ± 0.15% vs 2.30 ± 0.09%, P = 0.026) and lower ejection fraction (65.65 ± 1.94% vs 54.65 ± 1.60%, P < 0.001) and fractional shortening (22.70 ± 1.52% vs 15.63 ± 0.69%, P < 0.001) than Ang-II treated WT mice. The cardiovascular impact of Gpr65 genotype when Ang-II mice were fed with LF diet was negligible (All P > 0.05). Conclusions: This is the first evidence hypertensive participants have a higher luminal pH in the colon. This likely results from the difference in dietary fibre intake, which in a murine model profoundly determined the colonic pH. Low pH activates GPR65 signalling, which underlies the cardiovascular protection by dietary fibre. Our findings demonstrate a novel mechanism for BP regulation.

Abstract 14: An Anti-inflammatory Proton Sensing-receptor Contributes To Cardiovascular Protection Of Gut Microbiota-derived Metabolites

High fibre (HF) diet protects against hypertension via the production of acidic metabolites, e.g. short-chain fatty acids, by the gut microbiota. While these metabolites have a direct role in blood pressure (BP) regulation, their acidic nature may activate proton-sensing receptors, which have anti-inflammatory functions. G-protein coupled receptor 65 (GPR65) is a proton-sensing receptor activated around pH 6.5 and is critical for gut homeostasis. We hypothesized that GPR65 is involved in the cardiovascular protection by dietary fibre. We first measured cecal pH of C57BL/6 (WT) mice after a 7-day dietary intervention with either HF or low fibre (LF) diets (n=6/group). HF diet lowered cecal pH to a level where GPR65 is highly activated, compared to the LF diet (6.5±0.1 vs 7.6±0.1, P<0.001). The impact of pH and GPR65 on T cell production of IFNγ, a pro-inflammatory cytokine, in vitro was measured by flow cytometry. Acidic pH inhibited the production of IFNγ by CD8+ T cells (pH 6.5 vs pH 7.5, P<0.001). Cells lacking GPR65 had higher IFNγ at both pH (P<0.001). To determine if GPR65 is involved in BP regulation by dietary fibre, WT and GPR65 knockout ( Gpr65 -/- ) mice were implanted with minipumps containing angiotensin II (Ang II, 0.5mg/kg/day, 28 days, n=8-9/group) and fed with HF diet. BP, cardiorenal function and immune cell infiltration were measured. Gpr65 -/- mice had higher BP compared to WT mice after 2 weeks (mean arterial pressure ± SEM; WT 79.8±2.4 vs Gpr65 -/- 95.8±1.6mmHg, P<0.001) and 4 weeks of Ang II infusion (WT 92.3±2.4 vs Gpr65 -/- 99.5±1.3, P=0.062). Gpr65 -/- mice developed cardiac (P=0.035) and renal (P=0.025) hypertrophy, and impaired renal natriuretic (P=0.054) and diuretic (P=0.056) function compared to WT mice. This was accompanied by higher macrophage (P=0.009) and γδ T cell (P=0.014) infiltration in the kidneys. In conclusion, our data suggest that pH-sensing by GPR65 contributes to the protection against hypertension by dietary fibre via inflammatory mechanisms. This is a novel mechanism that contributes to BP regulation via the gut microbiota.

DIETARY FIBRE REDUCES INTESTINAL PH AND EXHIBITS CARDIOVASCULAR‐PROTECTIVE EFFECTS THROUGH A PROTON‐SENSING RECEPTOR

Objective: Dietary fibre lowers blood pressure (BP) and risk of cardiovascular disease and death via production of acidic metabolites by the gut microbiota. The mechanisms involved, however, are still elusive. Here, we aimed to understand the role of intestinal pH and the proton-sensing receptor GPR65 in the cardiovascular protection by dietary fibre. Design and method: Intestinal pH of C57BL/6 (WT) mice was measured after a 7-day dietary intervention with different levels of dietary fibre. The impact of pH and GPR65 on production of an inflammatory cytokine, TNFa, was determined by flow cytometry. We also determined the cardiovascular phenotype of male and female naïve WT and Gpr65-/- mice, including BP, tissue weights, sodium handling and immune cell infiltration. Results: Compared to the control diet, high fibre diet significantly reduced pH in caecum (control 6.84 ± 0.12 vs high fibre 6.03 ± 0.07; P < 0.001) and colon (7.10 ± 0.12 vs 5.95 ± 0.12, P < 0.001), while a diet lacking fibre significantly elevated pH (caecum, 7.37 ± 0.05, P = 0.001; colon, 7.53 ± 0.07, P = 0.030). Acidic pH inhibited the production of the pro-inflammatory TNFa by both CD4+ and CD8+ T cells (pH 6 vs pH 7; P < 0.001). Deletion of GPR65 increased TNFa production, particularly at acidic pH (CD4+, P = 0.002; CD8+, P < 0.001). Compared to WT controls, male and female Gpr65-/- mice had higher BP (mean arterial pressure; males WT 76.57 ± 3.26 vs Gpr65-/- 87.69 ± 2.18, P = 0.013; females WT 73.40 ± 2.41 vs Gpr65-/- 82.27 ± 2.56, P = 0.018; mmHg), and significant cardiac (Males, P < 0.001; Females, P = 0.001), renal (Males, P = 0.003; Females, P = 0.003) and splenic (Males, P = 0.002; Females, P = 0.002) hypertrophies when adjusted to tibia length. Male Gpr65-/- mice exhibited compromised capability in handling sodium (P < 0.001) and water (P = 0.028). Furthermore, male Gpr65-/- mice had increased infiltrations of CD4+ T cells (P = 0.015) and gamma-delta T cells (P = 0.038) in the kidneys, suggesting an increased susceptibility to renal inflammation. Conclusions: We determined that dietary fibre reduces large intestine pH. Low pH inhibits TNFa production in a partially GPR65-dependent way. Gpr65-/- mice exhibited spontaneous cardiovascular disorders including higher BP. This supports that the cardiovascular protection by dietary fibre is likely to be through pH regulation and GPR65. This is a highly novel mechanism that regulates BP.

Mobilisation of hazardous elements from arsenic-rich mine drainage ochres by three Aspergillus species

Natural ferric ochres that precipitate in streambeds at abandoned mining sites are natural scavengers of various metals and metalloids. Thus, their chemical and structural modification via microbial activity should be considered in evaluation of the risks emerging from probable spread of contamination at mining sites. Our results highlight the role of various aspergilli strains in this process via production of acidic metabolites that affect mobility and bioavailability of coprecipitated contaminants. The Mössbauer analysis revealed subtle structural changes of iron in ochres, while the elemental analysis of non-dissolved residues of ochres that were exposed to filamentous fungi suggest coinciding bioextraction of arsenic and antimony with extensive iron mobilisation. However, the zinc bioextraction by filamentous fungi is less likely dependent on iron leaching from ferric ochres. The strain specific bioextraction efficiency and subsequent bioaccumulation of mobilised metals resulted in distinct tolerance responses among the studied soil fungal strains. However, regardless the burden of bioextracted metal(loid)s on its activity, the Aspergillus niger strain has shown remarkable capability to decrease pH of its environment and, thus, bioextract significant and environmentally relevant amounts of potentially toxic elements from the natural ochres.

Assessment of Aspergillus niger Strain's Suitability for Arsenate-Contaminated Water Treatment and Adsorbent Recycling via Bioextraction in a Laboratory-Scale Experiment.

In this work, the viability of bioaccumulation and bioextraction processes for arsenic removal from contaminated waters, as well as the recycling of arsenate-treated amorphous ferric oxyhydroxide adsorbent (FeOOH) were evaluated using the common soil microscopic filamentous fungus Aspergillus niger. After treating the contaminated arsenate solution (100 mg As L−1) with FeOOH, the remaining solution was exposed to the growing fungus during a static 19-day cultivation period to further decrease the arsenic concentration. Our data indicated that although the FeOOH adsorbent is suitable for arsenate removal with up to 84% removal efficiency, the fungus was capable of accumulating only up to 13.2% of the remaining arsenic from the culture media. This shows that the fungus A. niger, although highly praised for its application in environmental biotechnology research, was insufficient for decreasing the arsenic contamination to an environmentally acceptable level. However, the bioextraction of arsenic from arsenate-treated FeOOH proved relatively effective for reuse of the adsorbent. Due to its production of acidic metabolites, which decreased pH below 2.7, the fungal strain was capable of removing of up to 98.2% of arsenic from the arsenate-treated FeOOH adsorbent.

Acidic tumor microenvironment in human melanoma

One characteristic of solid tumors such as malignant melanoma is the acidification of the tumor microenvironment. The deregulation of cancer cell metabolism is considered a main cause of extracellular acidosis. Here, cancer cells utilize aerobic glycolysis instead of oxidative phosphorylation even under normoxic conditions, as originally described by Otto Warburg. These metabolic alterations cause enhanced acid production, especially of lactate and carbon dioxide (CO2 ). The extensive production of acidic metabolites and the enhanced acid export to the extracellular space cause a consistent acidification of the tumor microenvironment, thus promoting the formation of an acid-resistant tumor cell population with increased invasive and metastatic potential. As melanoma is one of the deadliest and most metastatic forms of cancer, understanding the effects of this extracellular acidosis on human melanoma cells with distinct metastatic properties is important. The aim of this review was to summarize recent studies of the acidification of the tumor microenvironment, focusing on the specific effects of the acidic milieu on melanoma cells and to give a short overview of therapeutic approaches.

Chili Peppers, Curcumins, and Prebiotics in Gastrointestinal Health and Disease.

There is growing evidence for the role of several natural products as either useful agents or adjuncts in the management of functional GI disorders (FGIDs). In this review, we examine the medical evidence for three such compounds: chili, a culinary spice; curcumin, another spice and active derivative of a root bark; and prebiotics, which are nondigestible food products. Chili may affect the pathogenesis of abdominal pain especially in functional dyspepsia and cause other symptoms. It may have a therapeutic role in FGIDs through desensitization of transient receptor potential vanilloid-1 receptor. Curcumin, the active ingredient of turmeric rhizome, has been shown in several preclinical studies and uncontrolled clinical trials as having effects on gut inflammation, gut permeability and the brain-gut axis, especially in FGIDs. Prebiotics, the non-digestible food ingredients in dietary fiber, may serve as nutrients and selectively stimulate the growth and/or activity of certain colonic bacteria. The net effect of this change on colonic microbiota may lead to the production of acidic metabolites and other compounds that help to reduce the production of toxins and suppress the growth of harmful or disease-causing enteric pathogens. Although some clinical benefit in IBS has been shown, high dose intake of prebiotics may cause more bloating from bacterial fermentation.

Comparative study of the modulation of fructose/sucrose-induced hepatic steatosis by mixed lipid formulations varying in unsaturated fatty acid content.

BackgroundNon-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in developed countries. NAFLD encompasses a spectrum of diseases, ranging from hepatic steatosis to non-alcoholic steatohepatitis (NASH), cirrhosis, and liver failure. The etiology of NAFLD remains unclear but is thought to relate to increased fatty acid flux within the liver that results in toxic fatty acid metabolite production. One source of increased fatty acid flux is fructose/sucrose-induced hepatic lipogenesis. Current treatment for NAFLD encompasses dietary modifications. However, little scientific evidence exists on which to base many dietary recommendations, especially the intake of different types of carbohydrates and fats. We hypothesized that lipid mixtures of unsaturated fatty acids would inhibit lipogenesis and subsequent hepatic steatosis induced by high carbohydrate diets. The aim of this study was to examine the effects of different complex mixtures of fatty acids upon the development of fructose/sucrose-induced hepatic steatosis.MethodsC57BL/6 mice were randomized to normocaloric chow-based diets that varied in the type of carbohydrate (starch, sucrose, fructose). Animals in each carbohydrate group were further randomized to diets that varied in lipid type (no additional lipid, soybean oil, fish oil, olive/soybean oil, macadamia nut oil). These oils were chosen based upon their content of omega-6 polyunsaturated fatty acids, omega-3 polyunsaturated fatty acids, omega-9 monounsaturated fatty acids, or omega-7 monounsaturated fatty acids. Fatty acid flux in the liver was determine by assessing hepatic lipid content (steatosis). We also assessed fatty acid levels in the plasma and liver of the animals, hepatic lipogenesis activity, hepatic stearoyl-CoA-1 desaturase activity, and hepatic elongase activity.ResultsAnimals consumed similar amounts of the diets and maintained normal body weights throughout the study. Both sucrose and fructose induced hepatic lipogenesis and steatosis, with fructose being more potent. All mixed lipids similarly inhibited steatosis, limiting lipid content to levels found in the control (starch) animals. Lipogenesis and stearoyl-CoA-1 desaturase activity were increased in the sucrose and fructose groups. Levels of these enzymatic processes remained at baseline in all of the lipid groups.ConclusionThis is the first study to compare various complex lipid mixtures, based upon dietary oils with different types of long-chain fatty acids, upon development of sucrose/fructose-induced steatosis. Both carbohydrate source and lipid content appear important for the modulation of steatosis. Moderate intake of complex lipids with high unsaturated to saturated fatty acid ratios inhibited both lipogenesis and steatosis.

PWE-247 Slow transit constipation is associated with altered colonic ph and increased motility: novel findings from studies using the wireless motility capsule

<h3>Introduction</h3> The wireless motility capsule (WMC) allows investigation of regional gut transit times, intraluminal pH and pressure changes as it traverses the gastrointestinal tract. It is a useful diagnostic tool to confirm slow transit constipation (STC), which recent studies suggest is allied to dysregulated rather than impaired colonic motility (Dinning <i><i>et al</i></i>. NGM 2010). Whether luminal pH differs in STC is unknown. AIM: to compare colonic pressure and pH profiles between healthy volunteers (HV) and STC. Method data from 18 WMC studies in STC (15 female, median age 37; colon transit time (CTT) &gt;72h) were compared to those of 112 HV (48 female; median age 43). Pressure and pH profiles were analysed for: (1) the whole colon; (2) within the caecum and proximal colon (first 60 min following passage of WMC through the ICJ); and (3) within the distal colon (last 60 min prior to WMC expulsion). Results overall frequency of colonic contractions was similar between STC and HV (STC: 3.0 ± 1.3/min vs. HV: 2.8 ± 1.2/min; <i>P</i>=NS). However, the colonic motility index (CMI) was significantly higher in STC than HV (STC: 330 ± 166 vs. HV: 184 ± 104; <i>P &lt; 0</i>.0001). CMI and CTT were positively correlated (<i>r = 0</i>.38; <i>P = 0</i>.003). The proximal and distal colonic pressure profiles were similar between groups. For colonic pH profile, STC had a significantly more alkaline pH, both overall (STC: 7.7 ± 1.0 vs. HV: 6.8 ± 0.7; <i>P &lt; 0</i>.0001), and in the distal colon (STC: 8.1 ± 1 vs. HV: 7.2 ± 0.9; <i>P = 0</i>.0004). Using linear regression modelling, alkaline pH was associated with an increase in the CMI when controlling for age and gender between groups (beta=62.1; <i>P = 0</i>.023). Conversely, proximal colonic pH was more acidic in STC, manifest as a greater difference in pH across the ICJ than in HV (STC delta ICJ: 1.8 ± 0.6 vs. HV: 1.3 ± 0.8; P &lt; 0.005). Conclusion the CMI was found to be higher in STC than HV, supporting a more contemporary acceptance of underlying dysregulation of colonic contractility rather than “inertia”. Differences in colonic pH in STC may reflect alterations in regional gut microbiota (i.e. dysbiosis), resulting in excessive production of acidic metabolites in the proximal colon (e.g. short-chain fatty acids) of STC patients, and more alkaline metabolites in the distal colon (perhaps related to methanogenic activity). The observed associations between colonic pH, CMI and CCT warrants further investigation; conceivably, this may represent a biomarker to sub-classify constipated patients, and identify those who may benefit from antibiotic and/or dietary interventions <h3>Disclosure of interest</h3> None Declared.

Reducing activity, glucose metabolism and acid tolerance response of Bacillus cereus grown at various pH and oxydo-reduction potential levels

Bacillus cereus is a major foodborne bacterial pathogen able to survive a large number of physical-chemical stresses. B. cereus encounters different pH and redox potential (Eh7) levels during its passage through the gastrointestinal tract. Analysis of the combined influence of pH and redox stresses on B. cereus F4430/73 physiology found that B. cereus F4430/73 growth at pH 7.0 at 37 °C had strong reducing capacities, with a total change of 315 mV from an initial redox value of +214 ± 17 mV. The combination of low Eh7 and low pH led to a drastic reduction of growth parameters compared to oxidative Eh7 and neutral pH. Metabolic analysis showed that low pH significantly modifies glucose fermentative metabolism, with changes including decreased production of acid metabolite (acetate, lactate, formate) and increased production of 2,3-butanediol. Low Eh7 slightly enhanced the acid-tolerance response of B. cereus whereas low pH pre-adaptation led to thermal stress cross-protection. These results highlight new mechanisms that bring fresh insight into B. cereus pH and redox stress adaptations.

JJK694, a Synthesized Obovatol Derivative, Inhibits Platelet Activation by Suppressing Cyclooxygenase and Lipoxygenase Activities

Obovatol has various biological activities, including anti-proliferative, neurotrophic, anti-fibrillogenic, anti-platelet, anti-fungal and anti-inflammatory activities. In this study, we investigated the effects of JJK694, a synthesized obovatol derivative, on rabbit platelet activation and its molecular mechanisms. JJK694 significantly inhibited washed rabbit platelet aggregation and serotonin secretion induced by collagen and arachidonic acid, but had little effect on thrombin- or U46619-induced aggregation. These results suggest that JJK694 selectively inhibits collagen- and arachidonic acid-mediated signaling. JJK694 also showed a concentration-dependent decrease in cytosolic Ca(2+) mobilization, but it had no effect on arachidonic acid liberation. On the other hand, it significantly inhibited the formation of arachidonic acid metabolites, including thromboxane A(2) (TXA(2)), prostaglandin D(2), and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), by suppression of cyclooxygenase (COX)-1 and lipoxygenase (LOX) activities. These results indicate that JJK694 hasanti-platelet activities through inhibition of arachidonic acid metabolite production by suppression of COX-1 and LOX activities.

Pharmacological Properties of Myrtacine® and Its Potential Value in Acne Treatment

This study aimed at evaluating the antiproliferative, antibacterial, and anti-inflammatory properties of an ethanolic myrtle extract (Myrtacine®) in vitro, characterising its potential active compounds (myrtucommulones A and B') by structural analysis, and evaluating their biological activity. Antiproliferative activity was assessed by the BrdU incorporation assay in HaCat keratinocytes and inhibitory and bactericidal activities against P. ACNES strains by measuring the minimal inhibitory concentration (MIC) and D value. Anti-inflammatory effect was evaluated by measuring 6-keto-prostaglandin F1 α and [³H]-arachidonic acid metabolite production in keratinocytes stimulated for inflammation. Myrtacine® inhibited keratinocyte proliferation by 27 % and 76 % at 1 and 3 µg/mL, respectively (p < 0.001). A comparable effect, though less marked, was observed with 5 µg/mL myrtucommulones A and B' (-36 % and -28 %, respectively). Myrtacine® inhibited erythromycin-sensible and -resistant P. ACNES strains growth with MICs of 4.9 µg/mL and 2.4 µg/mL, respectively. Myrtucommulone B' and myrtucommulone A displayed a similar inhibitory activity against both strains (for both strains, MIC = 1.2 µg/mL and about 0.5 µg/mL, respectively). At 3 and 10 µg/mL, Myrtacine® significantly decreased all metabolite production from cyclooxygenase (81 % and 107 %, p < 0.0001) and lipoxygenase (52 % and 95 %, p < 0.001) pathways. Finally, Myrtacine® exhibited a concentration-dependent anti-lipase activity at 100 µg/mL and 1 mg/mL, as it decreased lipase activity by respectively 53 % and 100 % (p < 0.01 for both). In conclusion, in vitro, Myrtacine® demonstrated antiproliferative, antibacterial, and anti-inflammatory properties that may be of value to exert a global action in the treatment of acne lesions.

Effects of high pressure treatment on glycolytic enzymes of Lactococcus lactis subsp. lactis, Streptococcus thermophilus and Lactobacillus acidophilus

High pressure processing (HPP) reduces the glycolytic activity of lactic acid bacteria (LAB) and provides a means to control further production of acidic metabolites in fermented dairy products during storage. However, there is limited information on the effects of HPP on specific enzymes of dairy starter bacteria responsible for the metabolism of lactose. The aim of this study was to determine pressure-induced inactivation of glycolytic enzymes in Lactococcus lactis subsp. lactis C10, Streptococcus thermophilus TS1 and Lactobacillus acidophilus 2400. Cultures were grown for 16 h in M17 or MRS broth containing 5% (w/v) lactose at pH 6.5 (maintained by addition of 10 M NaOH). The cells were harvested by centrifugation, washed and resuspended in 100 mM phosphate buffer (pH 6.5) and pressure-treated at 300 and 600 MPa (≤ 22 °C, 5 min). The ability of pressure-treated resting cells of Lactococcus, incubated with 5% (w/v) lactose at 30 °C, to ferment lactose was evaluated by determining titratable acidity (TA) during incubation. The activities of phospho-β-galactosidase (P-β-gal), β-galactosidase (β-gal) and lactate dehydrogenase (LDH) were determined in cell-free extracts of untreated and pressure-treated cells. Resting cells of Lactococcus treated at 600 MPa had a substantially lower rate of acidification than the controls and those treated at 300 MPa. Both P-β-gal and β-gal were significantly inactivated (p < 0.01) in the starter cultures treated at 300 or 600 MPa. The LDH in Lactococcus and Lactobacillus was highly resistant to pressure treatment at 300 MPa. In contrast, the LDH in Streptococcus was almost completely inactivated at ≥ 300 MPa. Industrial relevance Continuing production of acidic metabolites in fermented dairy products during storage can be a technological challenge that adversely affects product quality. The current study demonstrates that high pressure processing (HPP) offers the potential of controlling this problem by inactivation of glycolytic enzymes in various mesophilic and thermophilic starter cultures. The findings of this research will assist in establishing optimised operating parameters for HPP treatment of cultured products to extend shelf-life, by reducing acid production during storage.

Acidic pH stimulates the production of the angiogenic CXC chemokine, CXCL8 (interleukin‐8), in human adult mesenchymal stem cells via the extracellular signal‐regulated kinase, p38 mitogen‐activated protein kinase, and NF‐κB pathways

Blood vessel injury results in limited oxygen tension and diffusion leading to hypoxia, increased anaerobic metabolism, and elevated production of acidic metabolites that cannot be easily removed due to the reduced blood flow. Therefore, an acidic extracellular pH occurs in the local microenvironment of disrupted bone. The potential role of acidic pH and glu-leu-arg (ELR(+)) CXC chemokines in early events in bone repair was studied in human mesenchymal stem cells (hMSCs) treated with medium of decreasing pH (7.4, 7.0, 6.7, and 6.4). The cells showed a reciprocal increase in CXCL8 (interleukin-8, IL-8) mRNA levels as extracellular pH decreased. At pH 6.4, CXCL8 mRNA was induced >60x in comparison to levels at pH 7.4. hMSCs treated with osteogenic medium (OGM) also showed an increase in CXCL8 mRNA with decreasing pH; although, at a lower level than that seen in cells grown in non-OGM. CXCL8 protein was secreted into the medium at all pHs with maximal induction at pH 6.7. Inhibition of the G-protein-coupled receptor alpha, G(alphai), suppressed CXCL8 levels in response to acidic pH; whereas phospholipase C inhibition had no effect on CXCL8. The use of specific mitogen-activated protein kinase (MAPK) signal transduction inhibitors indicated that the pH-dependent increase in CXCL8 mRNA is due to activation of ERK and p38 pathways. The JNK pathway was not involved. NF-kappaB inhibition resulted in a decrease in CXCL8 levels in hMSCs grown in non-OGM. However, OGM-differentiated hMSCs showed an increase in CXCL8 levels when treated with the NF-kappaB inhibitor PDTC, a pyrrolidine derivative of dithiocarbamate.

Development of in vitro biocompatibility assays for surgical material.

This work reports the development of a test to evaluate the biologic effects of implant material used in orthopaedics and traumatology based on the examination of inflammation and allergic reactions at the cellular level. The variation in arachidonic acid metabolite production by murine peritoneal macrophage cultures was studied using different powders of implant material. Macrophage activation by zymosan served as a control. Mouse peritoneal macrophages were labeled with 14C-arachidonic acid, and the synthesis of cyclooxygenase products (6-keto-prostaglandin F1 alpha; prostaglandins F2 alpha, E2, D2; and thromboxane B2) and lipoxygenase products (hydroxyeicosatetraenoic acids) was analyzed and quantified by chromatography. Results obtained through these assays support the reported clinical data that chrome and nickel increase the production of hydroxyeicosatetraenoic acids by mouse peritoneal macrophages. HXPATRI, titan oxide, and monoclinic zircon also increase the production of hydroxyeicosatetraenoic acids in contrast to other powders tested (alumina, HXPBL, chrome cobalt alloy, stainless steel 316L, titan, quadratic zircon), which have little effect on the production of arachidonic acid metabolites by the lipoxygenase pathway. It is concluded that determination of arachidonic acid metabolite production by murine peritoneal macrophage cultures is appropriate for evaluating implant material.

Effects of a thromboxane synthetase inhibitor on platelet function; possible risks of use in pregnancy

It has been proposed that thromboxane synthetase inhibitors may be of use in the treatment of hypertensive disorders of pregnancy. A patient in whom aspirin did not prevent the development of pre-eclampsia in a previous pregnancy was treated with a thromboxane synthetase inhibitor (dazmegrel, Pfizer) in addition to low-dose aspirin. Increased urinary levels of 6-keto-prostaglandin Flα were found throughout pregnancy, which is consistent with the mode of action. At 17–18 weeks of gestation urinary prostaglandin E2 and FDα levels were increased compared with control pregnancies. These increases in PGE2 and PGF2α production were associated with mid-trimester abortion. In vitro studies were carried out to determine the effects of dazmegrel on platelet eicosanoid production. In whole blood from non-pregnant female volunteers this compound inhibited thromboxane B2 production and significantly enhanced prostaglandin E2 production and slightly increased prostacyclin production, demonstrating a redirection of prostaglandin endoperoxides. This suggested that similar changes in arachidonic acid metabolite production may occur in vivo and in vitro , and that thromboxane synthetase inhibitors should not be used during early pregnancy, since increased production of prostaglandins E2 and F2α may result in preterm labour or abortion.

Cholinergic stimulation of the Na+/K+ adenosine triphosphatase as revealed by microphysiometry

The activation of a wide range of cellular receptors has been detected previously using a novel instrument, the microphysiometer. In this study microphysiometry was used to monitor the basal and cholinergic-stimulated activity of the Na+/K+ adenosine triphosphatase (ATPase) (the Na+/K+ pump) in the human rhabdomyosarcoma cell line TE671. Manipulations of Na+/K+ ATPase activity with ouabain or removal of extracellular K+ revealed that this ion pump was responsible for 8.8 +/- 0.7% of the total cellular energy utilization by those cells as monitored by the production of acid metabolites. Activation of the pump after a period of inhibition transiently increased the acidification rate above baseline, corresponding to increases in intracellular [Na+] ([Na+]i) occurring while the pump was off. The amplitude of this transient was a function of the total [Na+]i excursion in the absence of pump activity, which in turn depended on the duration of pump inhibition and the Na+ influx rate. Manipulations of the mode of energy metabolism in these cells by changes of the carbon substrate and use of metabolic inhibitors revealed that, unlike some other cells studied, the Na+/K+ ATPase in TE671 cells does not depend on any one mode of metabolism for its adenosine triphosphate source. Stimulation of cholinergic receptors in these cells with carbachol activated the Na+/K+ ATPase via an increase in [Na+]i rather than a direct activation of the ATPase.

Influence of Dietary Fish Oil on Granulomatous Inflammation in the Lung Following Inhalation of Silica and Infection with Bacillus Calmelte-Guerin

AbstractRecent evidence has suggested that fish oil diets, in addition to purported beneficial effects on the cardiovascular system, may also act to modulate inflammatory and immunologic reactions. These actions may be due to an increased content in fish oils of the n-3 fatty acid, eicosapentaenoic acid, which can competitively inhibit the production of arachidonic acid metabolites, forming 3-series prostaglandins and 5-series leukotrienes. In many cases these metabolites are not as biologically active as those formed from arachidonic acid. We examined the effects of dietary fish oil on granu-lomatous lesion development, pulmonary adherent cell (macrophage) activity, and arachidonic acid metabolite production following a regimen of silica inhalation and infection with Bacillus Calmette-Guerin (BCG). Mice on diets containing 10% corn oil or a mixture of 9% fish and 1% corn oils were placed in silica inhalation chambers for 6 wk, infected with BCG, and returned to inhalation chambers. Control animals, on ei...

The effects of (R)-N-(1-methyl-2-phenylethyl) adenosine (L-PIA), a standard A1-selective adenosine agonist on rat acute models of inflammation and neutrophil function

L-PIA, a standard A1-selective adenosine agonist, was evaluated orally in carrageenan (CRG)- and reverse passive arthus-pleurisy. White blood cell (WBC) and exudate accumulation were assessed four hours after induction of the inflammatory response. L-PIA inhibited WBC accumulation in both models with ID50's of 4.37 and 4.42 mg/kg, respectively. In contrast, exudate was inhibited by L-PIA only in the CRG pleurisy model (ID50 = 1.01 mg/kg). In mechanistic studies, L-PIA reversed the drop in circulating neutrophil count which occurred within 15 minutes after CRG injection, suggesting that L-PIA may inhibit adhesion of the cells to the endothelium. The effects of L-PIA on several parameters of rat neutrophil function were determined. Enzyme release, O2-, TXB2, and LTB4 production were monitored in response to FMLP and opsonized zymosan (SOZ) stimulation. At high concentrations, L-PIA had a mild inhibitory effect on O2- release in response to FMLP and had a moderate effect on arachidonic acid metabolite production in response to both stimuli. The other response were unaffected. These results suggest that L-PIA may prevent diapedisis or neutrophil adhesion to the endothelium, but has a minimal effect on enzyme release, O2-, LTB4 and TXB2 production.

A study on the biological behavior of human brain tumors Part II: Steroid receptors and arachidonic acid metabolism

The significance of steroid receptors (SR) in human brain tumors is presently a field of intense investigation in order to clarify some aspects of the biological behavior of these neoplasms. We studied the relationship between the presence of steroid receptors and the production of metabolites of the arachidonic acid cascade which have been reported to have a role in the biological behavior of some human tumors. We found that some metabolites of arachidonic acid are produced in different amounts in brain tumors which either did or did not express some steroid receptors. In particular the PGE2 were higher in estrogen receptors (ER) positive meningiomas than in ER negative ones and 6-keto-PGF1 alpha, the stable metabolite of prostacyclin, is significantly higher in androgen receptors (AR) negative meningiomas than in AR positive ones. In neuroepithelial tumors the glucocorticoid receptors (GR) positive cases synthesized more TxB2 and less PGE2 than the GR negative ones. Our data seem to suggest that some correlations exist between the presence of some steroid receptors and arachidonic acid metabolite production.