Jocelijn Meijerink

Identification of olfactory stimulants for Anopheles gambiae from human sweat samples

The behavioral and electroantennogram (EAG) responses of female Anopheles gambiae mosquitoes to pooled samples of freshly collected human sweat and human sweat incubated for 42–52 hr were tested. No behavioral or EAG response was obtained to pooled fresh sweat samples, whereas incubated pooled sweat samples produced a behavioral as well as an EAG response. GC-MS analysis of the headspace composition of the fresh sweat revealed ethanol (15.1% of the total amount of volatiles trapped), acetic acid (10.9%), and 3-hydroxy-2-butanone (9.5%) as the most abundant compounds; a wide range of ethyl esters was present as well. None of the ethyl esters was detected in the headspace collections from incubated sweat, while the relative amounts of ethanol, acetic acid, and 3-hydroxy-2-butanone were strongly reduced. In the latter collections, indole (27.9%), 1-dodecanol (22.4%), and 3-methyl-1-butanol (10%) were present in high amounts, while they were absent or present in only minor amounts in the headspace collections from fresh sweat. Geranyl acetone (6%) and 6-methyl-5-hepten-2-one (1.9%) were relatively abundant in both the fresh and incubated headspace samples. EAG responses were observed in response to indole, 6-methyl-5-hepten-2-one, and geranyl acetone.

Behavioural and electrophysiological responses of the female malaria mosquito Anopheles gambiae (Diptera: Culicidae) to Limburger cheese volatiles

Limburger cheese, previously shown to attract female Anopheles gambiae Giles, was solvent extracted and chemically fractionated into acid and non-acid fractions. The extracts and aliquots of headspace odour of the cheese were analysed by gas chromatography and electron impact mass spectrometry. Nineteen saturated and unsaturated aliphatic fatty acids, ranging in carbon chain length from C 2 to C 18 , were detected. The most abundant acids (>1 mg/g of cheese) identified in the acid extract were ethanoic, propanoic, butanoic, hexadecanoic and 9-octadecenoic acid. The same compounds were identified in analyses of headspace samples but only trace quantities of the less volatile acids (C 10 to C 16 ) were present, whilst C 18 acids were absent. Behavioural responses of female A. gambiae towards a range of dilutions of the acid extract (in diethyl ether) were recorded in a windtunnel bioassay. The undiluted extract was found to be repellent, but became highly attractive ( P «0.001) at lower doses, and was still significantly attractive ( P 6 times. A synthetic mixture of 12 of the more abundant aliphatic acids identified in the acid extract was found to be significantly attractive ( P 8 times. Electroantennographic (EAG) studies showed significant and reproducible responses to (saturated) Limburger cheese headspace. At doses higher than 0.1%, the synthetic mixture of 12 acids elicited significantly higher EAG amplitudes than the solvent control (paraffin oil). EAG responses were recorded from mosquitoes stimulated with C 5 to C 8 acids, that were characterized by significant dose-dependencies. Weaker, though significant EAG responses were obtained with the less volatile acids (C 9 to C 14 ). Only hexadecanoic acid did not elicit a detectable response. The electrophysiological and behavioural responses obtained with fatty acids isolated from Limburger cheese suggests that together they could act as a kairomone for female A. gambiae . The implications of this are discussed together with the occurrence and bacterial production of these compounds on human skin.

The response of the malaria mosquito, Anopheles gambiae, to two components of human sweat, ammonia and l‐lactic acid, in an olfactometer

In an olfactometer study on the response of the anthropophilic malaria mosquito Anopheles gambiae s.s. (Diptera, Culicidae) to human sweat it was found that freshly collected sweat, mostly of eccrine origin, was attractive, but that incubated sweat was significantly more attractive than fresh sweat. The behavioural response to l‐lactic acid and ammonia, the main constituents of sweat, was investigated. l‐lactic acid was attractive at one concentration only (11.11 mm) and removal of the l‐lactic acid from the sweat by enzymatic decomposition did not affect the attractiveness of sweat. Ammonia caused attraction over a range of 0.1–13.4 m on glass slides and at 0.84–8.40 μmol/min in an air stream. It is concluded that: human sweat contains kairomones for host‐seeking An. gambiae; ammonia is an important kairomone for this mosquito; and that l‐lactic acid is not a prerequisite in the attraction of An. gambiae to sweat.

Sensitivities of antennal olfactory neurons of the malaria mosquito, Anopheles gambiae, to carboxylic acids.

Single sensillum recordings on the antennae of female Anopheles gambiae s.s. mosquitoes revealed neurons sensitive to aliphatic carboxylic acids within (a) subtype(s) sensilla trichodea. The aliphatic acids, acetic acid, propionic acid, butyric acid, iso-butyric acid and iso-valeric acid evoked an inhibition reaction in one of the cell types recorded from. A different cell type was excited in response to the former aliphatic acids, but showed a broader range of sensitivity, as acids with a longer carbon chain length, like caproic acid, elicited excitations as well. In addition, occasionally 1-octen-3-ol elicited an excitation reaction. This article focuses on the carboxylic acid inhibited cell type and its temporal pattern of response to different doses of the compounds. Furthermore, in order to compare the stimulatory effectiveness of the compounds on a per molecule basis, corrections were made for differences in volatility by determining the absolute number of molecules in the stimulus puff.

Xanthohumol from hop (Humulus lupulus L.) is an efficient inhibitor of monocyte chemoattractant protein-1 and tumor necrosis factor-α release in LPS-stimulated RAW 264.7 mouse macrophages and U937 human monocytes.

Activated macrophages in adipose tissue play a major role in the chronic inflammatory process that has been linked to the complications of overweight and obesity. The hop plant (Humulus lupulus L.) has been described to possess both anti-inflammatory and antidiabetic effects. In the present study, the chemical composition of a hop crude extract (HCE) was investigated by ultrahigh-performance liquid chromatography (UHPLC). Next, HCE and various fractions obtained by preparative HPLC were tested for their ability to inhibit production of two pro-inflammatory cytokines, monocyte chemoattractant protein-1 (MCP-1, CCL2) and tumor necrosis factor-alpha (TNF-alpha), which play crucial roles in the complications of obesity. The hop chalcone xanthohumol was found to be the most potent inhibitor of both cytokines in LPS-activated RAW 264.7 mouse macrophages and U937 human monocytes. Moreover, other constituents, namely, iso-alpha-acids, in combination with the beta-acid hulupone, showed a moderate but selective inhibitory activity only on MCP-1 release. These findings underscore the potential health effects of hop and support further optimization, selection, and use of this plant.

Comparative analyses of seeds of wild fruits of Rubus and Sambucus species from Southern Italy: Fatty acid composition of the oil, total phenolic content, antioxidant and anti-inflammatory properties of the methanolic extracts

Fruit seeds are byproducts from fruit processing. Characterisation of the bioactive compounds present in seeds and evaluation of their potential biological properties is therefore of particular importance in view of a possible valorisation of seeds as a source of health beneficial components. In this work, we have analysed the seeds of Sambucus and Rubus species in order to identify their bioactive components and to determine the antioxidant and anti-inflammatory activities of the extracts. We first analysed their oil content, in order to assess the fatty acid profile and tocopherol content. Moreover, the methanolic extracts of the seeds were analysed for their total phenolic contents and antioxidant capacities. Polyphenols were identified by HPLC-ESI-MS/MS analysis. Furthermore, extracts were evaluated for their inhibitory effects on the production of LPS-induced inflammatory mediators (NO, CCL-20) in RAW 264.7 cells. Our findings show that the methanolic extracts from Rubus seeds have strong antioxidant and anti-inflammatory properties and could therefore represent an attractive source of bioactive compounds for food, cosmetic, or pharmaceutical applications.

N-acyl amines of docosahexaenoic acid and other n–3 polyunsatured fatty acids – from fishy endocannabinoids to potential leads

N–3 long‐chain polyunsaturated fatty acids (n–3 LC‐PUFAs), in particular α‐linolenic acid (18:3n‐3), eicosapentaenoic acid (EPA; 20:5n‐3) and docosahexaenoic acid (DHA; 22:6n‐3) are receiving much attention because of their presumed beneficial health effects. To explain these, a variety of mechanisms have been proposed, but their interactions with the endocannabinoid system have received relatively little attention so far. However, it has already been shown some time ago that consumption of n–3 LC‐PUFAs not only affects the synthesis of prototypic endocannabinoids like anandamide but also stimulates the formation of specific n–3 LC‐PUFA‐derived conjugates with ethanolamine, dopamine, serotonin or other amines. Some of these fatty amides show overlapping biological activities with those of typical endocannabinoids, whereas others possess distinct and sometimes largely unknown receptor affinities and other properties. The ethanolamine and dopamine conjugates of DHA have been the most investigated thus far. These mediators may provide promising new leads to the field of inflammatory and neurological disorders and for other pharmacological applications, including their use as carrier molecules for neurotransmitters to target the brain. Furthermore, combinations of n–3 LC‐PUFA‐derived fatty acid amides, their precursors and FAAH inhibitors offer possibilities to optimise their effects in health and disease.

Time-dependent effect of in vivo inflammation on eicosanoid and endocannabinoid levels in plasma, liver, ileum and adipose tissue in C57BL/6 mice fed a fish-oil diet.

Eicosanoids and endocannabinoids/N-acylethanolamines (NAEs) are fatty acid derived compounds with a regulatory role in inflammation. Considering their complex metabolism, it is likely that inflammation affects multiple compounds at the same time, but how lipid profiles change in plasma and other tissues after an inflammatory stimulus has not been described in detail. In addition, dietary fish oil increases levels of several n-3 fatty acid derived eicosanoids and endocannabinoids, and this may lead to a broader change in the profiles of bioactive lipids. In the present study mice were fed a diet containing 3% w/w fish oil for 6 weeks before receiving i.p. saline or 3 mg/kg lipopolysaccharide (LPS) to induce an inflammatory response. Eicosanoid and endocannabinoid/NAE levels (in total 61 metabolites) in plasma, liver, ileum, and adipose tissue were quantified using targeted lipidomics after 2, 4, 8, and 24 h, respectively. Tissue- and time-dependent effects of LPS on bioactive lipid profiles were observed. For example, levels of CYP derived eicosanoids in the ileum were markedly affected by LPS, whereas this was less pronounced in the plasma and adipose tissue. For some compounds, such as 9,10-DiHOME, opposing effects of LPS were seen in the plasma compared to the other tissues, suggesting differential regulation of bioactive lipid levels after an inflammatory stimulus. Taken together, our results show that plasma levels do not always correlate with the effects found in the tissues, which underlines the need to measure profiles and pathways of mediators involved in inflammation, including endocannabinoid-like structures, in both plasma and tissues.

Measurement of palmitoylethanolamide and other N-acylethanolamines during physiological and pathological conditions

Palmitoylethanolamide (PEA) belongs to the N-acyl ethanolamines (NAEs), a group of endogenous compounds involved in a variety of physiological processes, including energy homeostasis and inflammation. This review focuses on the analysis of PEA in plasma and tissues and discusses effects of diet and some pathological processes on PEA levels. Originally isolated from egg yolk, PEA has been detected in a variety of tissues and plasma of different species. The compound is present at relatively high levels compared to other NAEs and now mostly analysed using liquid chromatography coupled to mass spectrometry. PEA plasma concentrations show marked fluctuations during the day. However, concentrations in tissues are likely to be more relevant than those in plasma. Most studies suggest that compared to other NAEs, tissue PEA tissue levels are not influenced by changes in dietary fatty acid composition. Effects of inflammation and disease on PEA tissue levels show differences between different models and studies. Therefore, more research is needed on the endogenous role and tissue kinetics of PEA during disease. The rediscovery of the therapeutic potential of PEA has fuelled research and the development of new pharmaceutical formulations. With regard to this there is a need for better kinetic data and models, preferably also on its tissue disposition. Moreover, it is important to learn more about effects of exogenous PEA on the kinetics of other NAEs (and endocannabinoids) and effects of inhibiting its breakdown using inhibitors of the degrading enzymes fatty acid amide hydrolase or N-acylethanolamine-hydrolyzing acid amidase.

Inhibition of COX-2-mediated eicosanoid production plays a major role in the anti-inflammatory effects of the endocannabinoid N-docosahexaenoylethanolamine (DHEA) in macrophages.

N‐docosahexaenoylethanolamine (DHEA) is the ethanolamine conjugate of the long‐chain polyunsaturated n‐3 fatty acid docosahexaenoic (DHA; 22: 6n‐3). Its concentration in animal tissues and human plasma increases when diets rich in fish or krill oil are consumed. DHEA displays anti‐inflammatory properties in vitro and was found to be released during an inflammatory response in mice. Here, we further examine possible targets involved in the immune‐modulating effects of DHEA.